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i EPIDEMIOLOGY, DIAGNOSIS AND CHEMOTHERAPY OF STRANGLES IN EQUINES By Muhammad Ijaz 2005-VA-150 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE Of DOCTOR OF PHILOSOPHY In CLINICAL MEDICINE Department of Clinical Medicine and Surgery FACULTY OF VETERINARY SCIENCE UNIVERSITY OF VETERINARY AND ANIMAL SCIENCES, LAHORE 2010
Transcript
Page 1: CLINICAL MEDICINE - prr.hec.gov.pk

i

EPIDEMIOLOGY, DIAGNOSIS AND CHEMOTHERAPY OF STRANGLES IN EQUINES

By

Muhammad Ijaz 2005-VA-150

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE

Of

DOCTOR OF PHILOSOPHY

In

CLINICAL MEDICINE

Department of Clinical Medicine and Surgery FACULTY OF VETERINARY SCIENCE

UNIVERSITY OF VETERINARY AND ANIMAL SCIENCES, LAHORE

2010

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In the name of Lord who created, Created man from clot,

And thy Lord is the most Bounteous, Who taught by pen,

Taught man when he knew not.

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To

The Controller of Examinations, University of Veterinary and Animal Sciences, Lahore.

We, the Supervisory Committee, certify that the contents and form of the

thesis, submitted by Mr. Muhammad Ijaz, Regd No, 2005-VA-150, have been found

satisfactory and recommend that it be processed for the evaluation of External

Examiner(s) for the award of degree.

Chairman___________________________

Prof. Dr. Muhammad Sawar Khan

Member_____________________________

Prof. Dr. Muhammad Arif Khan

Member ______________________________

Prof. Dr. Azhar Maqbool

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WISDOM IS THE PART AND PARCEL

OF MY RELIGION,

KNOWLEDGE MY WEAPON,

PATIENCE MY DRESS,

FAITH MY DIET, AND SINCERITY

MY COMPANION

HADIS – E – NABVI (PEACE BE UPON HIM)

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I

DEDICATE THE FRUIT OF THIS HUMBLE EFFORT TO

THE HOLY PROPHET

(PEACE BE UPON HIM)

THE GREAT SOCIAL REFORMER

AND MY

MAGNIFICENTLY PRECIOUS

BELOVED PARENTS, MY SUPERVISER, MY BROTHERS & MY FRIENDS

WHO ALWAYS APPRECIATE AND PRAY FOR ME TO ACHIEVE HIGHER GOALS OF LIFE.

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AACCKKNNOOWWLLEEDDGGEEMMEENNTTSS

I am thankful to most gracious and ALMIGHTY ALLAH (AZAWAJAL)

who gave me the health and opportunity to complete this work, I bow before my

compassionate endowments, Peace be upon HOLY PROPHET MUHAMMAD

(PEACE BE UPON HIM) who is ever an ember of guidance and knowledge for

humanity.

I feel great honor to place on the record my sincere thanks to my

supervisor Prof. Dr. Muhammad Sarwar Khan, Department of Clinical Medicine

and Surgery, University of Veterinary and Animal Sciences, Lahore. He

supervised my research lightheartedly and proficiently made the dispatch of

intimidating work load possible by persistent guidance and scholarly criticism

communicated to me during the course of this study and execution of this

manuscript.

The co-operation extended by the members of my supervisory committee,

Prof. Dr. Muhammad Arif Khan, Chairman, Department of Clinical Medicine

and Surgery, University of Veterinary and Animal Sciences Lahore and Prof. Dr.

Azhar Maqbool, Department of Parasitology, University of Veterinary and

Animal Sciences, Lahore is very sincerely appreciated for their skillful

suggestions during the whole span of this investigation.

I wish to express my gratitude to Prof. Dr. John F. Timoney, for his

guidance, advice and support during this work. I am very thankful to him for

providing me the research facilities which helped in timely completion of this

work. I would like to thank the valuable contribution of Dr. Sridhar Vilineni. I

also wish to thank my lab members, Dr. Sergey Artiushin and Mike Fettinger for

their valuable input.

I have great sense of obligation to reverend Dr. Muhammad Muddassir

Ali, Dr. Muhammad Avais and Dr Muhammad Hassan Saleem, Department of

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Clinical Medicine and Surgery, University of Veterinary and Animal Sciences

Lahore, for their keen interest, propitious guidance, enlightened views and

valuable suggestions for the successful accomplishment of present study. I am also

thankful to the Laboratory staff Department of Clinical Medicine and Surgery.

I am whole heartedly thankful to Dr. Muhammad Arshad Shad (Lt. Col)

being my elder brother, his keen interest and indefatigable help with anything,

anytime, any where and computing exhaustive statistical evaluation from raw

data, made it all possible for me to undertake and complete this project

successfully.

I am extremely grateful to Higher Education Commission of Pakistan for

providing me financial support for this study.

I affectionally like to revive and appreciate the sincerity, help and

encouragement of my friends Dr. Zafar ullah Khan, Dr. Arslan Farooq, Dr. Agha

Shahzad, Dr. Zeshan M. Iqbal, Dr. Sohail Akbar, Dr. Muhammad Farooq, Dr.

Asim Munawar, Dr. Abdul Rehman, Dr Umair Iftikhar, Dr Manuchahar Ali, Dr.

Tanveer Hussain, Dr. Mir Ahmad, Dr Abdul Qadus, Dr. Khalid Mehmood, Dr.

Ahmad Jawad Sabir, Samuel Shahzad, Dr. Salman Khalid and all of other friends.

Last but not least, I must acknowledge my indebtedness to my loving

parents, and brothers for the motivation to take up this program of studies,

financial support, and their hands in prayers for my success, and great patience

and goodwill throughout the fairly long period of training at this institution.

Finally, I hope that inadvertent errors will be forgiven by the readers.

MUHAMMAD IJAZ

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TABLE OF CONTENTS

Dedication………………………………………………………........v

Acknowledgements ……….……………………………………..…..vi

List of Tables .……..………………………………………………...ix

List of Figures……………………………………………………….xiv

SR. NO. CHAPTERS PAGE NO.

1 INTRODUCTION 1

2 REVIEW OF LITERATURE 7

3 MATERIALS AND METHODS 32

4 RESULTS 46

5 DISCUSSION 127

6 SUMMARY 145

LITERATURE CITED 151

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LIST OF TABLES

Table No. Title Page No.

3.1 Details of all the primers used in the study 37

3.2 Geographic origin, year of isolation and strain characteristics of S.equi

40

4.1 Prevalence of Strangles in nasal discharge and pus samples of sub-mandibular lymph nodes of horses on the basis of culture.

48

4.2 Prevalence of Strangles in nasal discharge of horses on the basis of culture.

50

4.3 Prevalence of Strangles in pus samples of sub-mandibular lymph node of horses on the basis of culture.

51

4.4 Prevalence of Strangles in nasal discharge and pus samples of sub-mandibular lymph nodes of horses on the basis of PCR.

52

4.5 Prevalence of Strangles in nasal discharge of horses on the basis of PCR.

54

4.6 Prevalence of Strangles in pus samples of sub-mandibular lymph nodes of horses on the basis of PCR.

55

4.7 Prevalence of Strangles in nasal discharge and pus samples of sub-mandibular lymph nodes of mules on the basis of culture.

56

4.8 Prevalence of Strangles in nasal discharge of mules on the basis of culture.

58

4.9 Prevalence of Strangles in pus samples of sub-mandibular lymph nodes of mules on the basis of culture.

59

4.10 Prevalence of Strangles in nasal discharge and pus samples of sub-mandibular lymph nodes of mules on the basis of PCR.

60

4.11 Prevalence of Strangles in nasal discharge of mules on the basis of PCR.

61

4.12 Prevalence of Strangles in pus samples of sub-mandibular lymph nodes of mules on the basis of PCR.

62

4.13 Mortality rate in horses under 5 years of age 64

4.14 Mortality rate in mules under 5 years of age 65

4.15 Details of all amplicons used in the present study. 67

4.16 SeM alleles in S. equi isolated over a period of 40 years in N. America, Europe and Asia

68

4.17 Identification of six new alleles by BLAST analysis against www.pubmlst.org/szooepidemicus/

70

4.18 Frequency of single nucleotide polymorphism (SNPs) in SeM, SzPSe and Se18.9 in 25 isolates of S. equi.

71

4.19 Details of SzPSe of all isolates of S. equi 72

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Table No. Title Page No.

4.20 Comparison of culture and PCR for identification of carrier of S. equi in naturally infected horse ≤ 5 years of age.

74

4.21 Comparison of culture and PCR for identification of carrier of S. equi in naturally infected mule ≤ 5 years of age.

76

4.22 Total white blood cell count (x109/L) in healthy and diseased horses and mules suffering from strangles. (Mean ± SE)

78

4.23 Mean segmented Neutrophilic count (x109/L) in healthy and diseased horses and mules suffering from strangles. (Mean ± SE)

80

4.24 Total Lymphocytic count (x109/L) in healthy and diseased horses and mules suffering from strangles. (Mean ± SE)

82

4.25 Total Monocytic count (x109/L) in healthy and diseased horses and mules suffering from strangles. (Mean ± SE)

83

4.26 Total Eosinophil count (x109/L) in healthy and diseased horses and mules suffering from strangles. (Mean ± SE)

84

4.27 Total Basophilic count (x109/L) in healthy and diseased horses and mules suffering from strangles. (Mean ± SE)

86

4.28 Erythrocytes count (X 1012/L) in healthy and diseased horses and mules suffering from strangles. (Mean ± SE)

88

4.29 Packed cell volume (%) in healthy and diseased horses and mules suffering from strangles. (Mean ± SE)

89

4.30 Haemoglobin concentration (g/L) in healthy and diseased horses and mules suffering from strangles. (Mean ± SE)

91

4.31 Total white blood cell count (x109/L) in healthy and carrier horses and mules from strangles. (Mean ± SE)

93

4.32 Mean segmented Neutrophils count (x109/L) in healthy and carrier horses and mules from strangles. (Mean ± SE)

95

4.33 Total Lymphocytic count (x109/L) in healthy and carrier horses and mules from strangles. (Mean ± SE)

96

4.34 Total Monocyte count (x109/L) in healthy and carrier horses and mules from strangles. (Mean ± SE)

98

4.35 Total Eosinophil count (x109/L) in healthy and carrier horses and mules from strangles. (Mean ± SE)

100

4.36 Total Basophil count (x109/L) in healthy and carrier horses and mules from strangles. (Mean ± SE)

102

4.37 Erythrocytes count (X 1012/L) in healthy and carrier horses and mules from strangles. (Mean ± SE) 104

4.38 Packed cell volume (%) in healthy and carrier horses and mules from strangles. (Mean ± SE)

105

4.39 Haemoglobin concentration (g/L) in healthy and carrier horses and mules from strangles. (Mean ± SE)

107

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Table No. Title Page No.

4.40 Total serum protein values (g/L) of healthy and diseased horses and mules suffered from strangles. (Mean ± SD)

109

4.41 Serum albumin values (g/L) of healthy and diseased horses and mules suffered from strangles. (Mean ± SD)

110

4.42 Serum globulin values (g/L) of healthy and diseased horses and mules suffered from strangles. (Mean ± SD)

112

4.43 Fibrinogen values (g/L) of healthy and diseased horses and mules suffered from strangles. (Mean ± SD)

114

4.44 In-vitro Antibiotic sensitivity against S. equi in horses 116

4.45 In-vitro Antibiotic sensitivity against S. equi in mules 117

4.46 In-vivo Antibiotic sensitivity against S. equi in horses 119

4.47 In-vivo Antibiotic sensitivity against S. equi mules 120

4.48 In-vitro efficacy of Phenol as disinfectant against S. equi by using Phenol Coefficient test

121

4.49 In-vitro efficacy of Dettol as disinfectant against S. equi by using Phenol Coefficient test

122

4.50 In-vitro efficacy of Povidone Iodine as disinfectant against S. equi by using Phenol Coefficient test

123

4.51 In-vitro efficacy of 0.6% Sulfuric acid as disinfectant against S. equi by using Phenol Coefficient test

124

4.52 In-vitro efficacy of Bleach as disinfectant against S. equi by using Phenol Coefficient test

124

4.53 Overall comparison of Different Disinfectants used against S. equi

125

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LIST OF FIGURES

Fig No. Title Page No.

4.1 Prevalence of Strangles in horses and mules 47

4.2 Month wise prevalence of Strangles in horses on the basis of culture. 49

4.3 Month wise prevalence of Strangles in horses on the basis of PCR. 53

4.4 Month wise prevalence of Strangles in mules on the basis of culture 57

4.5 Month wise prevalence of Strangles in mules on the basis of PCR. 60

4.6 PCR amplification of DNAs from equine isolates of S. equi with specific primers.

63

4.7 PCR amplification of DNAs from equine isolates of S. equi to analyse variation in SeM (a), SzPSe (b), Se18.7 (c) and EqbE (d).

69

4.8 Week wise comparison of Total white blood cell count (x109/L) in healthy and diseased horses and mules suffering from strangles. (Mean ± SE)

80

4.9 Week wise comparison of Mean segmented Neutrophilic count (x109/L) in healthy and diseased horses and mules suffering from strangles. (Mean ± SE)

82

4.10 Week wise comparison of Total Lymphocytic count (x109/L) in healthy and diseased horses and mules suffering from strangles. (Mean ± SE)

83

4.11 Week wise comparison of Total Monocytic count (x109/L) in healthy and diseased horses and mules suffering from strangles. (Mean ± SE)

85

4.12 Week wise comparison of Total Eosinophil count (x109/L) in healthy and diseased horses and mules suffering from strangles. (Mean ± SE)

86

4.13 Week wise comparison of Total Basophilic count (x109/L) in healthy and diseased horses and mules suffering from strangles. (Mean ± SE)

88

4.14 Week wise comparison of Erythrocytes count (X 1012/L) in healthy and diseased horses and mules suffering from strangles. (Mean ± SE)

89

4.15 Week wise comparison of packed cell volume (%) in healthy and diseased horses and mules suffering from strangles. (Mean ± SE)

91

4.16 Week wise comparison of Haemoglobin concentration (g/L) in healthy and diseased horses and mules suffering from strangles. (Mean ± SE)

93

4.17 Week wise comparison of Total white blood cell count (x109/L) in healthy and carrier horses and mules. (Mean ± SE)

95

4.18 Week wise comparisons of Neutrophils count (x109/L) in healthy and carrier horses and mules. (Mean ± SE)

96

4.19 Week wise comparisons of total lymphocytic count (x109/L) in healthy 98

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and carrier horses and mules. (Mean ± SE)

4.20 Week wise comparisons of total Monocytic count (x109/L) in healthy and carrier horses and mules. (Mean ± SE)

100

4.21 Week wise comparisons of total eosinophilic count (x109/L) in healthy and carrier horses and mules. (Mean ± SE)

102

4.22 Week wise comparisons of total Basophilic count (x109/L) in healthy and carrier horses and mules. (Mean ± SE)

104

4.23 Week wise comparisons of Erythrocytes count (X 1012/L) in healthy and carrier horses and mules. (Mean ± SE)

105

4.24 Week wise comparisons of packed cell volume (%) in healthy and carrier horses and mules. (Mean ± SE)

107

4.25 Week wise comparisons of Hb concentration (g/L) in healthy and carrier horses and mules. (Mean ± SE)

108

4.26 Age wise comparison of total serum protein values (g/L) of healthy and diseased horses and mules suffered from strangles. (Mean ± SD)

110

4.27 Age wise comparison of serum albumin values (g/L) of healthy and diseased horses and mules suffered from strangles. (Mean ± SD)

112

4.28 Age wise comparison of serum globulin values (g/L) of healthy and diseased horses and mules suffered from strangles. (Mean ± SD)

113

4.29 Age wise comparison of fibrinogen values (g/L) of healthy and diseased horses and mules suffered from strangles. (Mean ± SD)

116

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Chapter-01

INTRODUCTION

According to the Agriculture Census Organization, the total equine population

in Pakistan was 4.8 million in 2006. This has risen to 5.1 million (Horses 0.4, Asses

4.5 and Mules 0.2) as per the census report for 2008-2009. (Anonymous 2008-09)

Strangles is named from the air restriction in late stages of the disease where

the horse breathes as if it is being strangled because of the restriction of the trachea

due to swollen lymph nodes. It is considered to be one of the top three most

significant and feared respiratory diseases in horses (Natarajan and Langohr, 2003). It

accounts for close to 30% of all equine infections reported worldwide, making it the

most frequently encountered single horse illness (Harrington et al., 2002).

The mechanism and route of entrance of Streptococcus equi subsp. equi (S.

equi) into the lymphoid system has not so far been properly elucidated. An in vitro

trial revealed adherence of S. equi to equine epithelial cheek cells, tongue and nasal

epithelial cells (Srivastava and Barnum, 1983b; Valentin Weigand et al., 1988).

Timoney, (1988) reported adherence of large numbers of S. equi on the soft palate

and adjacent tonsillar tissue of two experimentally infected ponies one hour post

inoculation. He further reported presence of S. equi on the soft palate, tonsils and the

retropharyngeal lymph nodes of a pony necropsy five days post infection. This

information is suggestive of sites of colonisation, subsequent to infection.

A number of researchers have investigated the link between Strangles and S.

equi. Todd, (1910) and Shultz (1888) discovered the relationship between Strangles

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and a chain forming coccus. Bazeley and Battle (1940) found that this coccus, S. equi,

was consistently associated with strangles and Bazeley (1943) showed that

inoculation of horses with pure cultures of S. equi resulted in the reproduction of the

classic disease. Bryans et al., (1964) also confirmed the relationship in a later study.

He was also able to induce clinical disease in horses by infecting with a broth

containing S. equi.

The symptoms become apparent after an incubation period of 3 to 8 days, and

the clinical course usually lasts 3 to 4weeks (Nara et al., 1983). Marked fever (103-

106°F) develops during the acute phase and may subside until the lymph nodes

abscess, this is the time when a second wave of fever may develop. Affected horses

become anorexic, depressed, and develop bilateral, serous to mucoid nasal discharge

within 24 hours of fever. The discharge becomes mucopurulent as the disease

progresses, and a moist cough may develop in some cases. Plasma fibrinogen

concentration and leukocyte counts usually increase at this time. The submandibular

lymph nodes are involved most oftenly and become enlarged, firm, and painful. The

retropharyngeal lymph nodes may also be affected, and if they become markedly

enlarged may induce dysphagia. The abscessed lymph nodes typically rupture 7 to 10

days after the onset of clinical signs and, in uncomplicated cases; recovery is

complete 1 to 2 weeks thereafter (Reed, 2004).

The highly host adapted S. equi of Lancefield group C causes equine

strangles, a highly contagious purulent rhino-tonsillitis and lymphadenitis of the head

and neck. Isolates of S. equi constitute a clone or biovar of an ancestral S.

zooepidemicus with which it shared greater than 97% DNA homology. Its anti-

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phagocytic SeM protein is an important protective antigen and virulence factor that

functions by binding fibrinogen thereby masking C3b binding sites on the surface of

S. equi and by directly inhibiting deposition of the opsonic forms of C3 on the

bacterial surface (Boschwitz and Timoney, 1994). Although early studies carried out

by Anzai et al. (2005) and Kelly et al. (2006) indicated that SeM was highly

conserved but variation in the N-terminal sequences from aa 37 to 143 has recently

been reported in isolates from N. America, Europe and Japan DNA codon changes

associated with non-synonymous substitutions of amino acids throughout the N-

terminus are strongly suggestive of immune selection pressure. However, allelic

variation in SeM does not significantly affect susceptibility of S. equi to opsonization

or ability to bind fibrinogen (Timoney et al., 2009). Epitopes on the variable region

reactive with convalescent mucosal IgA are more frequent than epitopes reactive with

convalescent IgG suggesting that IgA is a more significant selection pressure. There

is also evidence that N17 terminal variation affects the conformational and not the

linear epitope (Timoney et al., 2009).

SeM allelic variants of S. equi from the same animal including those with

chronic guttural pouch or cranial sinus infection have been shown to emerge over a

short period (Anzai et al., 2005; Kelly et al., 2006). Interestingly, some isolates from

the guttural pouch have large in frame deletions of the SeM N-terminus (Chanter et

al., 2000). It suggests that loss of this region has survival value in the face of local

acquired immune responses that target the N-terminus. SeM. is one of a small number

of immunoreactive 1 surface exposed and secreted proteins of S. equi that elicit

convalescent serum and mucosal antibody responses (Timoney et al., 2007). The

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surface exposed SzPSe and secreted Se18.9 proteins are of particular interest, because

they elicit strong serum and mucosal antibody responses and bind to tonsillar

epithelial cells (Timoney et al., 2007; Tiwari et al., 2007; Fan et al., 2008). They are

therefore potentially subject to the same selection pressure as SeM during the

acquired immune response of the horse. SzPSe is a homologue of the variable Moore

and Bryans typing antigen SzP (Walker and Timoney, 1998), which has been shown

to elicit opsonic and protective antibodies. The anti-phagocytic Se18.9 is uniquely

expressed and secreted by S. equi. It also becomes associated with the bacterial

surface (Tiwari et al., 2007). Incubation of this protein with equine neutrophils causes

a significant reduction in their bactericidal activity for both S. zooepidemicus and S.

equi, an effect partially neutralized by Se18.9 specific antibody.

Once infected, the majority of animals recover and eliminate S. equi over a

period of 4–6 weeks. However, in 10% of clinically recovered cases S. equi may

continue to be shed intermittently for prolonged periods. This carrier status is

probably caused by incomplete drainage of exudate from the guttural pouches

(empyaema) and/or sinuses following rupture of abscesses formed in the

retropharyngeal lymph nodes (Newton et al., 1997).

Changes in hematological parameters, such as total and differential white cell

counts have been recorded in horses naturally and experimentally infected with S.

equi (Hamlen et al., (1994). In another study Dalgleish et al. (1993) performed blood

neutrophil count on a group of naturally infected horses. Knight et al. (1975) and

Nara et al. (1983) have also carried out hematological studies including total white

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cell counts in horses on days 2, 5, 10, 15, and 20 following experimental infection on

horses experimentally infected with S. equi

The use of antibiotics in the treatment of strangles with early clinical signs of

infection can be of great value. By following this regime, the course of the disease is

shortened, lymph node abscesses and their complications are prevented. Degree of

environmental contamination from infected discharges and secretions is also greatly

reduced. Early identification of new cases during outbreaks, through careful

observation of exposed horses and recording of rectal temperatures twice daily, will

facilitate initiation of antimicrobial treatment early in the disease course and

maximize the effectiveness of this approach. Wilson, (1988) recommends use of

Procaine penicillin G at dose rate of 22,000 IU/kg IM twice a day for at least 10 to 14

days. He further suggests continuing it for 5 days beyond resolution of clinical signs.

The organism is also susceptible to ampicillin, ceftiofur, erythromycin, rifampin,

tetracycline, and trimethoprim/sulfonamides, although the later appears to be less

effective than beta-lactam antimicrobials in treating field cases. Inadequate doses of

antibiotics and/or an inadequate length of treatment may result in treatment failure or

recurrence. No evidence reported that early treatment with antibiotics increases the

risk for development of internal abscesses (Sonea, 1984).

Outbreaks of strangles may last for months or years within large horse

populations. It will perpetuate itself with frequent new arrivals in the horse facility

providing a continuous supply of susceptible animals (Harrington et al., 2002).

Although it is a potentially fatal disease, even then it can be contained and treated by

paying due attention.

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Keeping in view the importance and utilization of equines in our country and

the significant losses rendered by Strangles, the present project was designed to

achieve the following objectives

To study the epidemiology of disease.

To determine whether immune selection pressure that resulted in N-terminal

sequence diversity in SeM also affected SzPSe and Se18.9 or otherwise.

PCR based diagnosis of S. equi from carrier animals.

To study the effect of Strangles on Haemogram, Leucogram and Serum

proteins.

To evaluate the efficacy of various drugs under laboratory and field

conditions.

To evaluate the efficacy of various disinfectants under laboratory conditions

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7

Chapter-02

REVIEW OF LITERATURE

Van Dorssen, (1939) isolated S. equi from horses that were affected from

strangles. A total of 80 samples were collected for the study. S. equi was isolated

from the abscesses of above mentioned animals and suggested that S. equi was the

causative agent of strangles in horses and measures to be taken against this organism

to combat the disease. Bazeley and Battle, (1940) studied various types of

Strepotococci in equine infections. They isolated 457 strains of haemolytic

Streptococci from 415 cases of horses. All strains belong to group C Lancefield by

appropriate precipitin tests. On the basis of fermentation tests and colony type

Streptococci were divided into five types. The colonies of Streptococci were dom-

shaped and honey coloured and grew up to about 3 mm in diameter in 24 hours.

Tenacious strands were observed when a platinum loop was withdrawn after touching

a colony. Hignett and King, (1940) investigated the Streptococcal infection in horses

and summarized that horses are highly susceptible to Streptococci than any other

domestic animal. Streptococci group caused a wide variety of local and generalized

disease conditions in horses such as strangles, pneumonia, metritis, and sinusitis and

as commonest secondary invaders in wounds such as poll evil, fistulous withers and

quitter. Namikawa et al. (1940) used an anaerobic technique for its growth to study

the microbial characteristics of S. equi. The colony features of the bacteria presented

a “Medusa Head” formation after the 18 hours and the distinction was lost after few

hours because the bacteria growing in this form were non-encapsulated. Medusa Head

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8

colonies were produced by 43 out of 44 strains of C type S. equi and 34 out of 36

strains of G type S. equi. Tuji and Sato, (1940) examined 88 cases of diseased horses

and also 6 healthy horses. From these they isolated 100 different strains of

Streptococci. They concluded that two strain of S. equi, C type strains were isolated

from a case of coryza and from strangles complication and infectious anaemia. In the

other cases, C and G type strains of S.equi were isolated from spleen and lungs

respectively. Castagnoli and Balboni, (1942) reported of S. equi in two 4 year old,

horses and two, 4 year mares in cerebrum and developed cerebral signs. From the

brain of each of the two mares S. equi was isolated. Eberbeck and Halswick, (1943)

studied the pathogenesis of strangles over a period of three years at Army Horses

under constant veterinary supervision and concluded that many of the sequelae of

strangles were of an allergic nature. Minett, (1944) studied the incidence of strangles

hardly fell below 50% in remount depot and stud farm of sub-continent during that

period 1914-1942. Mona and Sargodha are two important army remount depots in

Pakistan and reported annual incidence of strangles based on the mean annual

percentage of 1917-1940 was 76.2% at Mona and similarly the annual incidence of

strangles based on the mean annual percentage of 1927-1940 at Sargodha was 72.6%

reported. While this incidence of strangles among mules at these stations was lower,

with mean annual rates of 53.2% at Mona and 46.8% at Sargodha. When we talk

about the stud farms the yearly incidence varied from zero to 50%. Climatic factors

including rainfall, temperature, relative humidity, wind velocity and dust storms were

also considered important in occurrence of the strangles. In a more equable climate,

the attack rate was higher in the cold season than in the hot and was very much less in

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the summer rainy season. Monsoon conditions seemed unfavorable for the spread of

strangles. Peatt, (1945) reviewed veterinary services in India and Burma in the

Second World War and reported that strangles was recorded as very virulent in two

big remount depots where the mortality was reached upto16%. Menninger, (1949)

studied the causative agent of strangles. He stated that four mares contracted typical

strangles 2-6 days and he isolated S. equi from the mandibular lymph node abscesses.

He determined that strangles in equine is due to a pathogenic bacterium, S.equi. He

also observed some predisposing factors which may either be stress of a virus

infection and or a nonspecific weakening resulting from natural causes such as fatigue

and exposure to harsh weather. Tajima and Ueda, (1953) reported his observation on

the four cases of purulent encephalomyelitis in strangles affected horses and showed

nervous signs including depression or excitement, incoordination, rigidity of the neck

during the course of strangles. Paunovic, (1957) reported an abdominal form of

strangles while having rectal examination of four horses completely with a history of

colic. Vukovic, (1961) diagnosed strangles in 114 of 1853 horses examined during

that specified period 1952-59 in Sarajevo. Out of these 114 horses only nine showed

the infection in the mesenteric lymph nodes. Mahaffey, (1962) observed that strangles

was commonest disease in large stud farms and army remounts depots where the

horses are kept together in large numbers. Strangles is highly infectious and

contagious disease of all equines and causative agent of strangles is very resistant

which may remain viable for weeks or months in dried pus. Natural infection was

acquired by direct or indirect contact between healthy, susceptible and carrier

animals, diseased animals or the products of the latter. In almost every case acute

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10

inflammation of the submandibular lymph nodes commenced early during the course

of disease. The submandibular lymph nodes showed the cardinal signs of inflamation

in about 4-5 days. In the beginning they were hard but after few days they began to

fluctuate with pus inside of them. As the abscesses matured, the overlying skin

became denuded of hair and small amounts of sticky fluid oozed out over the surface.

Pharyngitis was very common along with abscessation of adjacent lymph nodes.

Roaring and some other respiratory diseases were usually seen as sequelae of

strangles. Bryans et al. (1964) mentioned the etiology of strangles. In his

experiments using 23 horses they indicated that S. equi is the only causative agent of

equine strangles. Strangles was set up by intranasal inoculation of horses with pus

from abscesses and cultures with abscesses. Wagenaar and Schaaf, (1965) discussed

the clinical picture, etiology and active immunization of strangles and concluded that

antibiotics ought not be used in cases running a normal course. In complicated cases

treatment with penicillin for 5-10 days would be useful but isolation and

identification of diseased animals is important. Wisecup et al. (1967) studied

strangles in a herd of donkeys in which the lesions consisted of mainly caseation and

calcification of the abdominal lymph nodes. S. equi was isolated from these lesions.

Culturing S. equi from mare exudates appeared to be the most reliable method for

identification of the infected animals. The clinical syndromes caused by S. equi in

donkeys showed a clear discrimination from those of strangles in horses. Roberts,

(1971) noted chorioretinitis in six mares and seven geldings after recovery from an

attack of strangles. He described the lesions grossly and histopathologicaly and

discussed their relationship with the previous infection of strangles. Woolcock,

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(1975) studied an atypical variety of S. equi. It was shown to be deficient in capsular

material, to be very virulent for mice and possess a cell-wall protein similar to M-like

protein of classical S. equi. Antiserum prepared against classical S. equi effectively

opsonised and induced the formation of long chains by these atypical strains. It is

possible to use this variant of S. equi to overcome many of the current problems link

with the manufacture and use of strangles vaccines. Woolcock, (1975) studied the

epidemiology of equine Streptococci. Three different types of samples were collected

from equine tonsillar tissue, draining regional lymph nodes and as well as deep nasal

swabs and examined bacteriologically. Group C Streptococci, predominantly S.

zooepidemicus, was found in all types of samples. One of the most frequent sites for

isolation was the tonsil. S. equi was not found in any of the tissues sampled. Niebauer

et al. (1979) reported a case of brain metastatic strangles abscess in three year old

horse. The horse was showing signs included ataxia, swelling of the mandibular

lymph nodes and eye disorders. During post mortem examination, a tumour was

found in the subdura of the left frontal lobe and in caudate nucleus encephalitis was

established. S. equi was isolated from that tumour. Ford and Lokai, (1980) tried to

find out the method of transmission of various diseases in weanling horses. They

gathered 300 weanling horses on equal number of acres of grossly overgrazed

pasture. That pasture was previously grazed by horses, out of which some of them

had suffered from strangles. Management of the horses was so poor that when

strangles appeared; it affected all weanling horses and killed 10% of them.

Postmortem examination showed that S. equi could cause abscesses in any organ of

the body including the brain. Prescott et al. (1982) discussed a mild and weak form of

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strangles caused by S. equi. A mild form of strangles caused by S. equi was identified

on a massive type of stud farm that was for breeding purpose. The organism varied

from most of S. equi isolates because of absence of the mucoid capsule even after 24

hours of culture, leaving a matt-type colony. Typically, the clinical signs were a

transient (24-48 hours) fever, anorexia and profuse nasal discharge. Half of the

diseased horses expressed restrained mandibular lymph node enlargement, and these

glands usually ruptured or were drained. The use of a PHAT (passive

hemagglutination antibody test) showed that subclinical infection was widespread in

horses on the farm. George et al. (1983) identified the carriers of S. equi in a naturally

infected herd. During an epidemic of strangles in a population of research horses, out

of which four mares were identified as carriers of S. equi infection. Three of the

mares were paradigm of strangles. They showed intense regional lymphadenitis with

or without rupture of abscessed lymph nodes. The 4th mare showed occurence of

serous to mucopurulent nasal discharge, but never had more than a mild degree of

lymph node enlargement. From the abscessed lymph nodes and nasopharyngeal swab

specimens S. equi was isolated from the first 3 mares from 6 to 19 weeks after rupture

of infected lymph nodes. From the nasopharynx of the 4th mare S. equi was isolated

and intermittently over the ensuing 6 months. After that, 4th mare was kept in

isolation during the 7th month, where she continued to shed S. equi for 4 more

months. An integral and meticulous physical examination during the 10th month,

including radiography of the head and thorax, did not show any pertinent

abnormalities, but a pharyngeal swab was culture-positive for S. equi. This isolate

after taking the 4th mare was used to inoculate 2 yearling colts, which developed

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strangles and from which S. equi was reisolated. Shedding of S. equi by mare 4 was

stopped in the 11th month, and at necropsy 2 months later, S. equi was not found in

any organ or tissue. Corticosteroids administration 3 weeks prior to necropsy had

persuaded neither shedding of the S. equi nor clinical signs of strangles. This study

concluded clinical, epidemiologic, and bacteriologic documentation to support the

existence of a carrier state following natural infection with S. equi. Nara et al. (1983)

designed the experimental study of S. equi infection in horses and found out its

correlation with in vivo and in vitro immune responses. They conducted an

experiment on 14 horses, divided into 2 groups based on 18 or 24 hours skin-test

reactions to S. equi, and inoculated virulent strain of S. equi nasopharyngeally.

Animals (n = 6, group I) with confirmation of previous exposure to S. equi, with one

exception, developed very few or no signs of disease after inoculation. Whereas S.

equi skin-test negative and seronegative horses (n = 8, group II) showed predictable

and severe clinical signs of infection after their inoculation, including shedding of the

pathogen from nasal discharge and ruptured submandibular lymph nodes. It was

concluded that resistance to virulent strain of S. equi infection is correlated with

existing humoral and cellular immune responses to Streptococcal antigens. The

horses that were prone, their recovery from infection were accompanied by the

appearance of antibodies and the positive skin-test response to S. equi antigens. Piche,

(1984) discussed the clinical observations during an epidemic of strangles that

occurred during the spring, summer and fall of 1980 on a Standardbred stud farm in

Eastern Alberta. S. equi might be introduced by a mare that was brought to the stud

form for breeding pupose. All of the horses on the farm were affected and for the

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most part of the study that the disease was allowed to run its natural course. Only

badly affected horses were treated. During this outbreak, the foals were

prophylactically treated with penicillin to prevent them from contracting the disease.

Out of them 10 horses died due to complications of strangles infection. Timoney and

Trachman, (1985) studied the immunologically reactive proteins in acid extracts.

Then they adopt combination of chromatographic and immunologic procedures to

identify S.equi from culture supernatants. Both high and low molecular weight

components of each of these protein preparations were vigilant for mice. It was

surmised that there were variety of hydrolytic fragments of the M protein of S. equi

were present. Convalescent horse sera that exhibited strong bactericidal activity for S.

equi always reacted with polypeptides in the molecular weight range of 24,000 to

29,000, whereas pre infection sera did not. Rabbit anti sera to affinity-purified S. equi

protein also reacted with these polypeptides, as well as with a polypeptide of about

36,000 to 37,000 molecular weight. While M protein in acid extract and culture

supernatant did not cross-react in immunodiffusion, rabbit antiserum to affinity-

purified M protein from an acid extract of S. equi reacted aggressively with culture

supernatant proteins of approximate molecular weights of 67,000, 58,000, and

43,000. We suggest, therefore, that the M protein in culture supernatant is covered by

other sequences that are removed by hot acid during acid extracts preparation.

Evermann et al. (1987) find out the frequency of respiratory diseases in the horses in

North Western USA. The samples from clinically affected horses showed that both

equine influenza and S. equi were involved. Endemic strangles which occurred in all

horses that were susceptible, but mainly foals were affected showed pyrexia,

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inflammation of the respiratory tract and lymphadenitis. Clabough, (1987)

investigated various aspects of strangles infection. He described 100% morbidity and

1-2% mortality. He concluded that mortality was due to dissemination of the infection

to other parts of the body. Once introduced, S. equi was a consistent problem due to

persistent ambient contamination. The infective organism was present in nasal and

abscess discharge from the infected horses. The IP of the disease was usually 4-8

days and there was a sudden onset of anorexia and fever. Initially Nasal discharge

was serous and developed within 24 hours, then it became purulent as the disease

attained its peak. Control of the disease was best accomplished by isolating the

affected horse and adopting strict hygienic measures by personnel handling the

animals. Gumbrell, (1987) reported that incidence rate of strangles had increased

significantly in Canterbury around Christchurch and normally 1-5 cases were

confirmed every month at the Lincoln Animal Health Laboratory. Mayr, (1987)

reviewed the major respiratory infectious diseases in horses and found that among

major diseases of horses, respiratory diseases pose the greatest threat to horses. The

most important respiratory infections of horses were caused by S. equi. Yelle, (1987)

published an article dealing with clinical aspects of S. equi. He concluded that the

incubation period of the disease and the course of the disease was 3-8 days and 3-4

weeks respectively the course of disease was usually 3-4 weeks reported. Morbidity

rate was too high 100% in susceptible population and the mortality rate was 2-3% if

the appropriate treatment was given. He described various risk factors and observed

that overcrowding and parasitism may increase the risk of the disease. Muhktar and

Timoney, (1988) observed the chemotactic response of leukocytes to S. equi infection

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in horses. They concluded that the response was due to intense infiltration of lymph

node with polymorphonuclear leukocytes which suggested an influential chemotactic

response to the S. equi. Wilson, (1988) reviewed various aspects of strangles. He

found that S. equi is the causative organism and confirmed it as the major cause of

economic losses to the horse industry throughout the world. Because of the current

control measures were not completely effective in preventing the disease. The disease

affects only equines (horse, mules and donkeys). It was common in all horse-raising

areas especially young ones. Strangles was commonly seen in weanlings, yearlings

and young horses; very less in foals under one month and horses over 5 years of age.

This reflected very specific immunity, and horses of any age were susceptible if not

previously exposed or vaccinated. Overcrowding, movement and mixing of horses

from different sources are important predisposing factors. Sweeney et al. (1989)

studied the epizootic and infections of S. equi in horses. The age-specific attack rates

of S. equi infections in horses for the different age groups were calculated 17.6% for

broodmares, 47.5% for 1-year-old horses, and 37.5% for foals. S. equi was isolated

from different nasal swab, pharyngeal, or lymph node specimens in 31 (60.8%) of 51

disease horses. A male 1-year-old horse that had been taken from Kentucky to farm

A, was considered the index case. Six (19.4%) out of 31 horses with strangles

remained carriers for S. equi after clinical signs of the disease had ended. These

horses were not found positive for S.equi. After that they were kept with those horses

that were infected with S equi but strangles was not developed. Jorm, (1990) studied

Strangles in horse studs and calculated incidence, risk factors and effect of

vaccination. 179 horse studs in New South Wales was conducted to estimate the

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incidence of strangles during that specified period 1985 to 1988, to identify risk

factors associated for strangles epidemics. During this particular period of time, forty-

nine studs (27.4%) had at least a single epidemic of strangles and 62 stud farms

(34.6%) had only a case of strangles. The average incidence rate of strangles was

calculated as 2.1 cases per 100 horses per year. The risk of strangles increased with

the increased horse population and rose markedly when more than 100 mares had

been served in the 1988-89 season. Different types of feeders, fences and water

sources were also significantly increased the chance of strangles outbreaks. Boyes et

al. (1991) reported a case of S.equi infection in which panophthalmitis also occurred.

The S. equi was isolated from corneal ulcer. The ulcer developed due to an extension

of septic uveitis subsequent to submandibular lymphadenopathy. The condition was

refractory to treatment and panophthalmitis ensued. S. equi was isolated from the

anterior chamber of infected eye through enucleation. It was observed that uveitis was

associated with S.equi, especially in those cases which had the history of strangles.

Hamlen et al. (1992) studied the hematologic parameters of 23 foals at weeks 0, 2, 4,

6, and 10 following the onset of a strangles epizootic. The epizootic was initiated by

group exposure with S, equi to a foal experimentally. The group was consisted of 12

foals, 6 months of exposure with S. equi epizootic, and 11 foals, previously

unexposed. It was observed that 91% of the unexposed and 17% of the previously

exposed foals developed clinical signs of strangles. Significantly increase in mean

WBC count, neutrophil cell count, fibrinogen concentration and plasma protein

concentration were seen in strangles cases as compared to foals not classified as cases

and were associated with clinical signs. Similarly, animals suffered from strangles

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showed decreased PCV, Hg concentration, and RBC count, although statistically

insignificant, as compared to those without strangles during 4th, 6th, and 10th week that

may have biological significance. Similar, but more pronounced, changes in the

hematological parameters of the foal were observed in in those cases where S. equi

was inoculated experimentally. Besides of this effect of S. equi infection on the

hematology of foals should be considered in their convalescent care. Zadeh et al.

(1992), in Tehran, investigated the epizootology of strangles in equine. According to

an epidemiological survey 89-100% of horses were affected with the strangles. In

young horses the clinical signs were very severe but mortality rate was

approximately zero. All of the animals responded to these antibiotics

penicillin/streptomycin. Wood et al. (1993) studied the persistent infection with that

organism S. equi. They detected S. equi in the nasopharynx from one horse in the

severe outbreaks. They concluded that effective control measures of outbreaks of

strangles required monitoring of horses through swabbing. Dalgleish et al. (1993)

studied an outbreak of strangles infection in young ponies. A natural outbreak of

strangles occurred in 19 young experimental ponies. The disease was diagnosed in 11

of them within just two days of their arrival at Glasgow University veterinary school

and five others ponies were developed clinical signs within four days, a morbidity

rate reached upto 84%. All of the affected ponies had typical signs of strangles

disease including pyrexia, dullness, anorexia, regional lymphadenitis, occasionally

seen with rupture of the lymph node, conjunctivitis and mucopurulent nasal

discharge. 9 out of 19 ponies were destroyed during the clinical phase of the disease

for seeing post mortem changes. The clinical disease in the remaining animals lasted

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upto 21 days although one pony had to be destroyed 10 days after the onset of clinical

finding because of the development of septic arthritis. All 16 affected animals

exhibited blood neutrophilia and high plasma fibrinogen levels. Beta haemolytic

streptococci were then isolated by nasopharyngeal swabbing from 18 out of 19

ponies. While the S. equi was confirmed in three animals within the first four days of

the outbreak. The majority of the other isolates identified to species were S

zooepidemicus. Beta haemolytic streptococci were still present in 6 ponies after 40

days and they had clinically recovered and were isolated on regular basis from these

three ponies which did not develop clinical strangles but remained in contact with

affected animals throughout the study. Timoney, (1993) illustrated the etiology;

epizootiology, pathogenesis, and clinical presentation of strangles. Streptococcus equi

is highly host specific to equine and shows no antigenic variation. Apparently

protective immunity is produced by a combination of serum opsonic and

nasopharyngeal mucosal antibodies responses. Vaccines based on M protein or

attenuated bacterial suspensions abate the clinical attack rate up to 50%, much lower

level of protection than that produced during recovery from strangles. Hamlen et al.

(1994) studied epidemiological and immunological features of S. equi infection in

different foals. A multiphase study was done to indicate the effects of S. equi

infection in previously exposed and unexposed foals. In phase I, they observed 22

weanling foals involved in a naturally occurring S equi epizootic, along with 11

unexposed foals for comparison, matched for sex, age, and breed. After six months

(phase II), an epizootic was experimentally induced in previously exposed and

unexposed foals from phase I and studied the prevalence, duration of clinical signs,

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the relative risk of developing disease, bacteriologic culture results, hematologic

responses, mucosal and serum immunologic responses were determined. The

protection from disease in phase-I and -II foals was associated with high values for

serum S equi M protein-specific IgG at the onset of the epizootic (for phase 1 P <

0.02 and for phase II P < 0.01), and with a rapid (within 2 weeks of exposure)

mucosal S equi M protein-specific IgG response (for phase I P < 0.05 and P = for

phase II 0.01). Dwyer, (1995) compared the disinfectants of equine ambience that

provoke managers to take this challenge. For this purpose variety of surfaces which

may be contaminated with wide range of horse pathogens were used. The most

frequently occurring infectious diseases for which disinfection and disease control are

very important are strangles, salmonellosis and rotavirus diarrhoea. These are the

most difficult to control. The phenolic disinfectants have been demonstrated to be

effective in the presence of organic matter and are also have property of being

virucidal. When used after thorough cleaning and rinsing of stall surfaces, phenolic

disinfectants have proved effective in controlling outbreaks of different diseases.

Inspite of this, 10% iodophors used for hand washing and cleaning equipment is also

bactericidal and virucidal. Quaternary ammonium compounds like bleach,

chlorhexidine and pine oil are available commercially, but these disinfectants have no

consequence in the presence of the organic matter. Golland et al. (1995) carried out a

retrospective study on 46 horses suffering from retropharyngeal lymph node

infection. Horses aged <1year were observed oftenly affected (46%). A high

percentage of cases (39%) then exposed to horses and results were confirmed or

suspected strangles. The most frequent signs were unilateral or bilateral swelling of

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throat region (65%), respiratory dyspnoea (38%), purulent nasal discharge (20%),

inappetence and signs of depression (15%) and dysphagia (9%). Along with

rhinopharyngoscopy, ultrasonography, haematology, cytological and microbial

analysis of material, aspirated from swelling of soft tissue, also helped in diagnosis of

the disease. Anzai et al. (1997) isolated the organism S. equi from thoroughbred

horses from racehorse-breeding area of Japan. During the breeding season in 1995 it

was observed whether strangles has spread in Hidaka district of Hokkaido and the

main racehorse-breeding area of Japan. For this aim an epizootiological survey with

bacterial isolation was conducted. S. equi was isolated from two Thoroughbred horses

showing signs of submandibular lymphadenitis. Then these isolates were

characterized and classified by serological grouping, different biochemical tests and

analysis of cell surface proteins by Western immunoblotting. From that survey, it is

end noted that S. equi has invaded the Hidaka district and in racehorse-breeding farms

in this area that strangles has become prevalent. Timoney et al. (1997) compared the

sequences and functions of, S. equi M-Like proteins, SeM and SzPSe. S. equi, a

Lancefield group C Streptococcus, causes strangles, contagious purulent

lymphadenitis and pharyngitis of members of the Equidae family. The antiphagocytic

M-like protein (58-kDa) SeM is a prominant virulence factor and protective antigen.

The amino acid sequence and structure of SeM has been known and equated to that of

a second, M-like protein SzPSe (40-kDa) of S. equi and along with other

Streptococcal proteins.It was shoen that SeM and SzPSe have no homology except

their signal and anchor sequences of the membrane and are alpha helical fibrillar

molecule. It was also observed that they have clear heterozygosity with other

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Streptococcus M and M like proteins. Present study tells us that SzPSe is an allele of

SzP protein that encodes the mutable protective M-like and typing antigens of S.

zooepidemicus. Opsonogenicity of SeM only for S. equi. In contrast to SeM protein

the SzPSe is highly opsonogenic for S. zooepidemicus but not for S. equi. In the blood

SeM and SzPSe bind with equine fibrinogen. When they measured the size of SeM

and SzPSe of geographically and temporally separated isolates of S. equi they found

that there is no difference in size. This syudy shows that the S.equi is a clonal

pathogen of S. zooepidemicus. Al-Ghamdi et al. (2000) evaluated the use of repetitive

sequence-based PCR for molecular epidemiological analysis of S. equi. Inception of

the study was with 63 S. equi isolates from different areas of the world and 17 S. equi

isolates were collected during epidemics of S. equi. Then an aliquot of S. equi

genomic DNA was processed through PCR, using the specified enterobacterial

repetitive intergenic consensus primers. After this they run the samples on 1.5%

agarose gel and used software to equate these rep-PCR results. When they used these

primers to analyze 100ng genomic DNA of S. equi they observed the pattern of 6 to

14 bands. The initial isolates of 32 were segregated into 7 rep-PCR subtypes. They

also found 30 rep-PCR subtypes among 29 S equi isolates collected from Michigan,

Minnesota, Australia, Canada and 34 S equi isolates obtained from Kentucky State

and other sources when the epidemic of disease occurred, the same clone was

identified in several horses. All infected horses on the same farm had a single clone of

S equi. They concluded that this rep-PCR was most authentic for depicting S. equi

into various rep-PCR subtypes. Besides of this the results further disclosed that

isolates with the same geographic source and date of collection, did not have the same

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rep-PCR subtype. A single clone of S equi usually predominated in an epidemic.

Chanter et al. (2000) examined S. equi with truncated M-proteins isolated from

outwardly healthy horses. The M-protein genes of S. equi isolated from 17 healthy

horses after four strangles outbreaks, including a quarantined animal, were compared

with S. equi isolates from 167 active cases of strangles across four countries. Sixteen

most occuring S. equi carriers were included in healthy horses, one from each of the

four outbreaks. These outwardly healthy carriers had empyema of the guttural pouch,

an increase in the size of equine Eustachian tube. A persistent carrier from two of

these outbreaks, the healthy animal and quarantined animal with normal guttural

pouches, from which S. equi was isolated only once, were colonized by variant S.

equi with truncated M-protein genes. The truncated M-protein genes had in-frame

deletions between the signal sequence and the central repeat region in slightly

different positions, equivalent to approximately 20% of the expressed protein. It was

end noted that immunoblotting with antibody to recombinant M-protein finalized that

the variants showed a truncated form of the M-protein. In contrast to the outwardly

healthy carriers of S. equi, only 1/167 of S. equi isolates from strangles cases

possessed a truncated M-protein gene. Comparing the isolates of healthy horses with

a truncated M-protein, much more of the N terminal of the truncated M-protein was

retained in the variant S. equi from a strangles case. Variant S. equi from outwardly

healthy horses were more susceptible to phagocytosis by neutrophils in vitro than

typical isolates. This was the first report of identification of truncated M-protein in S.

equi. The distribution of these variants between infected cases and carriers proposed

that the 80% of the M-protein retained in the variants that may contribute to

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colonization whilst the deleted portion of the gene may be cause of full virulence.

Newton et al. (2000) studied the control of strangles outbreaks by isolation of guttural

pouch carriers through PCR and culture of S equi. From the previous study the use of

repeated nasopharyngeal swabbing and culture of Streptococcus equi visualized that

healthy carriers developed in more than 50% of strangles outbreaks. The guttural

pouches were the only site where S. equi colonisation on endoscopic examination of

horses during one of these outbreaks and sometimes S. equi was not identified by

culture of nasopharyngeal swabs for carriers up to 2 or 3 months before nasal

shedding resumed sporadically. Therefore more sensitive way of detecting S. equi on

swabs from established guttural pouch carriers was required. Strangles outbreaks

were reported in detail using endoscopy, in order to make development and

assessment of a suitable PCR test. From 3 protracted strangles outbreaks on different

kinds of establishment’s ranges between 29 and 52% of sampled horses were infected

as noticed by culture and PCR. Out of these, between 9 and 44% were identified as

carrying S. equi after clinical signs had abolished and the most predominant site of

carriage was the guttural pouch. Prolonged carrier of S. equi, which lasted up to 8

months, did not stop spontaneously before treatment was incepted to remove the

infections. For identification and separation of the carrier animals, along with strict

hygienic conditions, apparently resulted in the control of strangles outbreaks and

allowed the premises to come to normal activity. To compare PCR and culture, many

more swabs were found to be positive using Polymerase Chain Reaction (56 vs. 30%

of 61 swabs). Similarly for guttural pouch samples from 12 established carriers (PCR

76% and culture 59%). PCR can only identify dead organisms and is, thus, liable to

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produce false positive results. Verheyen et al. (2000) studied to treat guttural pouch

infection and inflammation in asymptomatic carriers from this S. equi. Three

outbreaks of strangles were diagnosed by endoscopic study and a total of 14

asymptomatic carriers of S. equi were noticed out of which 13 showed evidence of

carrier state in the guttural pouch. Then treatment was started to eliminate S. equi.

Two other horses were referred to them with severe guttural pouch pathology and

from which S. equi was also cultured, and treatment of these cases was also

described. In the first instance treatment was directed towards removal of gross

guttural pouch pathology as seen on endoscopic examination. This was done by using

a combination of irrigation of the pouch with moderate to large amounts of saline

then suction of fluid and endoscopic manipulation of chondroids. Antibiotic treatment

was used to cure S. equi infection. Systemic antibiotics were given to all animals, but

in some cases along with antibiotics topical antimicrobial treatment was also given.

Treatment was generally considered as successful when the guttural pouches

appeared normal and S. equi was not noticed in nasopharangeal swabs and completed

the pouch lavages on 3 consecutive occasions. Successful treatment of one carrier

required surgical intervention due to occlusion of both guttural pouch pharyngeal

openings. 14 out of 15 carriers were successfully treated by endoscopic elimination of

inflammatory material and antibiotic treatment, without any surgical intervention. 5

carriers originally given potentiated sulphonamide (33%) required for further therapy

with combination of penicillin or ceftiofur, given systemically and topically, before

whole S. equi infection and associated inflammation of the guttural pouches were

completely removed. Harrington et al. (2002) studied Streptococcus equi is

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26

responsible for strangles, one of the most dominated diseases of the equine. The

animal suffering and economic burden associated with this disease need effective

treatment. Current antibiotic treatment is often ineffective and thus recent attention

has focused on vaccine production. A systematic understanding of S. equi virulence,

leading to the identification of targets to which protective immunity can be directed,

is a prerequisite of the development of such a vaccine. Here, the virulence factors of

S. equi are studied. Ensink et al. (2003) evaluated the efficacy of

trimethoprim/sulfadiazine and procaine penicillin G against S. equi subsp.

zooepidemicus in ponies. Tissue chambers, implanted s/c on both sides of the neck,

were inoculated with S. zooepidemicus to compare the efficacy in a purulent

infection. The TMP/SDZ treatment consisted of one i.v. injection of 5 mg/kg TMP

and 25 mg/kg SDZ and the same dose of TMP/SDZ per os both given 20 h after

inoculation. Then the oral dose was repeated every 12 h for 21 days. The penicillin

treatment was given i.v. injection of 20 000 IU/kg sodium penicillin G and i.m.

injection of 20 000 IU/kg procaine penicillin G, both given 20 h after infection. Then

the i.m. dose was repeated after every 24 h for 21 days. TMP/SDZ resulted in a

limited reduction of viable bacterial count in the TC but did not remove the infection,

thus resulting in abscess formation in 10-42 days in all eight ponies.While, penicillin

eliminated the streptococci in 7 of 8 ponies, and only one pony suffered from abscess

formation on day 10. It is concluded that penicillin showed significantly better

efficacy than TMP/SDZ. Therefore, it is recommended that TMP/SDZ should not be

used to treat purulent infections in secluded sites in horses. Masakazu et al. (2003)

observed fever and enlarged submandibular lymph nodes were seen during quarantine

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27

inspection in 3 out of 9 quarter horses imported from the USA. They were found to be

negative in bacterial examinations during the quarantine period then these horses

were released. Riding club was their destination; however, S. equi was isolated from 3

of them. Then strangles infection spread to other horses in the riding club. Nasal

swabs were obtained from all horses at the club on weekly basis until the disease

subsided 22 weeks later. S. equi was isolated from 25 of 58 horses (43.1%). It was

concluded that the imported carrier horses were responsible for the spreading of

infection throughout the riding club. Dwyer, (2004) compared the different

environmental disinfectants to control equine infectious diseases. Therefore it is

recommended that cleaning and disinfection is essential to the environmental control

of infectious diseases of all animals. To see the types of pathogens, environment,

disinfection process and success can be attained in effectively by stopping disease

outbreaks. Timoney, (2004) studied the important and potent pathogenic Streptococci

for horse which includes S. equi, S. zooepidemicus, S. dysgalactiae subsp. equisimilis

and S. pneumoniae capsule Type III. S. zooepidemicus strain is the ancestor of S.equi,

both shares more than 98% DNA homology and that is why expresses many common

proteins and virulence factors. Strategic progress has been made for identification of

different virulence factors and proteins which are only expressed by S. equi. Most of

these proteins and virulence factors are expressed on bacterial surface. The notable

examples include antiphagocytic SeM, the secreted pyrogenic superantigens SePE-I

and H. The genomic DNA sequence of S. equi will greatly help in identification and

characterization of more virulence factors and vaccine targets. S. equi is the most

frequently isolated opportunist organism of the horse. Gronbaek et al. (2006)

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28

evaluated a nested PCR test and bacterial culture from nasal swabs and abscesses for

the diagnosis of S. equi infection (strangles). When he used nested PCR all 45 S.equi

were positive and not a single amplicon was formed when testing the other 120

Lancefield group C isolates. He collected 43 samples from 11 horses that were

showing typical clinical signs of strangles. He investigated the diagnostic sensitivity

for PCR test was 45% and 80% for samples from the nasal passages and abscesses,

respectively; whereas diagnostic sensitivity for cultivation was 18% and 20%.

Therefore it is stated that diagnostic sensitivity was significantly higher for PCR than

for bacterial cultivation. Along with this, they evaluated the shedding of S. equi in 2

infected horse populations. They observed the intermittent shedding period of S.equi

which was up to 15 days. Shedding of S. equi was seen in both from horses with and

without clinical signs. They finally concluded that nested PCR test is highly species-

specific and is very sensitive method for detection of S. equi from clinical samples.

Kelly et al. (2006) observed the sequence variation of the SeM gene of S. equi allows

discrimination of the source of Strangles outbreaks. Improved understanding

regarding, the epidemiology of S. equi transmission demands high sensitivity and sub

typing methods that can rationally differentiate between strains. S. equi is highly

homogeneous and cannot be distinguished by multilocus sequence-typing or

multilocus enzyme electrophoretic methods that use housekeeping genes.

Nevertheless, sequential analysis of the N-terminal region of the SeM genes of 60 S.

equi isolates from 27 outbreaks of strangles, find out 21 DNA codon changes. These

resulted in the non synonymous replacement of 18 amino acids and allowed the

assignment of S. equi strains to 15 distinct subtypes. The findings of the present study

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29

propose the presence of multiple epitopes across this region that is subjected to

selective immune pressure, especially in the establishment of chronic S. equi

infection. They further pointed out the the application of SeM gene sub typing

procedure to examine potential cases related to administration of a live attenuated S.

equi vaccine. SeM gene sub typing discriminated between the vaccinal strain and field

strains of S. equi the actual cause of disease. The results were confirmed by the

establishment and application of a PCR test, which identifies the aroA partial gene

deletion present in the Equilis StrepE vaccine strain. It was concluded that the

injection site lesions were due to vaccinal strain, all seven outbreaks of strangles

investigated in recently vaccinated horses were found to be due to agreeing infection

with wild-type S. equi and not reversion of the vaccine strain. Tiwari et al. (2007)

examined Se18.9, an anti-phagocytic factor H binding protein of S. equi. The escape

from phagocytosis is of great significance in virulence determination of S. equi, the

cause of strangles in equine and discriminate it from the closely related S.

zooepidemicus. They explained Se18.9, a novel H factor binding protein which is

only secreted by S. equi and not by S. zooepidemicus that reduces accumulation of C3

on the surface of bacteria and in consequence reduces the bactericidal activity of

neutrophils significantly suspended in normal serum for both S. equi and S.

zooepidemicus. Se18.9 is produced and released in large quantity by actively dividing

cells abundantly and is also bound to the surface of bacteria. Strong mucosal and

serum antibody responses are elicited in S. equi infected horses. Although there was

no gene identical to Se18.9 in S. zooepidemicus, sequences encoding proteins of same

size with closely related signal peptide sequences were found in 3 of 12 randomly

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30

selected strains. As Se18.9 is specific to S. equi, and immunoreactive with mucosal

IgA and convalescent sera, so it could be used for diagnostic purpose. Waller and

Jolley, (2007) investigated getting a grip on strangles. Strangles, that remained one of

the most commonly diagnosed and a high ranked infectious disease of horses through

world-wide. This review article elaborated the diagnosis and pathogenesis of

strangles and is fastidious to the importance of prolonged infection with particular

attention to the significance of persistent infections. Now it is possible to combine

recent sequence data obtained from the N-terminal site of the SeM to reallocate the

SeM alleles with the help of on-line database. Hypotheses concerning the inception of

this variation and the capability of being exploit for the epidemiological investigation

of outbreaks are suggested. Zadeh et al. (2007) concluded that strangles is an acute

disease of horses caused by S. equi and characterized by inflammation of the URT

and abscess formation in the lymph nodes, distributed worldwide. Outbreak occurs

mostly in a large population of young horses and affected young ones upto 100%.

High incidence is observed soon after the gathering of large number of susceptible

horses, which may have come from different areas and are stabled together. The

infection source is the nasal discharge from infected animals, which contaminates the

pasture, feed and water troughs. Infection occurs either by ingestion or by inhalation

of droplets. Two cases of strangles were diagnosed at Veterinary Faculty Teaching

Hospital in Tehran during April (1990). Then an epizootological survey immediately

started and showed 89-100% of horses were in incubation stage. The clinical signs

were intense in younger horses but the mortality rate was zero. All of the infected

horses gave good response to the treatment with i/m injection of

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penicillin/streptomycin. Jannatabadi et al. (2008) checked the existence of S. equi as

possible causative agent of upper respiratory tract infection in Mashhad area. Nasal

swabs samples were collected from 30 horses with URT infections. Then different

types of bacteria were isolated from samples S. equi (1 isolate), S. zooepidemicus (25

isolates) and others. Confirmation of these isolates of S. equi and S. zooepidemicus

were done by different biochemical tests and Polymerase chain reaction. For further

molecular identification of S. equi and S. zooepidemicus, two genomic region SeM

and sodA were amplified. Knowles et al. (2010) tested 30 horses having no external

clinical signs of strangles for exposure to S. equi using a latest serological test. Inspite

of this, carrier state of S. equi was also checked by using endoscopy of the guttural

pouches and PCR. Serological results were showed that four horses had been recently

exposed to S. equi and started non-specific clinical signs of respiratory disease. One

asymptomatic horse out of four was also positive for S. equi by PCR.

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Chapter-03

MATERIALS AND METHODS

The present study was conducted on strangles in equines of Lahore and

Sargodha districts of the Punjab province of Pakistan. The samples collected were

processed at Medicine and Microbiology Laboratories of the University of Veterinary

and Animal Sciences, Lahore. Pakistan and Gluck equine research center, Department

of Veterinary Science, University of Kentucky, USA. The study was comprised of

five phases as under.

PHASE I:

Epidemiology of Disease:

In this phase of epidemiology, prevalence of the disease, variations in SeM,

SzPSe, Se18.9 proteins and mortality rate were studied. For prevalence a total of 500

equines (n=250 horses; n=250 mules) was examined from Lahore and Sargodha

districts of Punjab province. The data related to equines were collected in a data

capture form. The entries in data capture form included location, species, age, sex,

breed, season, previous disease history and mortality.

Prevalence:

Prevalence was referred to the amount of disease in each district for a period

of one year, without distinction between old and new cases were calculated as per

formula described by Thrusfield, (2002).

No. of individual having a disease at a particular point in time P = --- ------------------------------------------------------------------------------

No. of individuals in the population at risk at that point in time

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Mortality Rate

The measure of the number of deaths in each district was calculated as per

procedure described by Thrusfield, (2002) as given below.

No. of deaths due to disease that occur in a Population during a particular period of time

Mortality Rate = ------------------------------------------------------------- The sum, overall individuals, of the length of time at risk of developing disease

Collection of Samples:

Following two types of samples were collected aseptically for identification of

causative agent (S. equi) of strangles.

a) Nasal discharge was collected from nasal chambers using sterile cotton swabs.

b) Pus from affected lymph nodes was collected aseptically using sterile

disposable syringes (Merchant and Packer, 1983).

The collected samples were processed at Medicine & Microbiology

Laboratories, University of Veterinary and Animal Sciences (UVAS), Lahore

Pakistan and Gluck equine research center, Department of Veterinary Science,

University of Kentucky, USA.

Culture and isolation of S. equi:

The samples were cultured on blood agar plates and incubated anaerobically

for 24 hours to optimize the isolation of the organism preferentially against the other

organisms present in the nasal passages (Jorm, 1990).

Confirmation of S. equi by using biochemical test:

The above isolates were identified on the basis of cultural, morphological and

biochemical characteristics following the techniques as described by Buxton and

Fraser (1975) and Merchant and Packer (1983).The biochemical tests to be carried

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34

out were include, Catalase reaction, Methyle blue reduction and Sugar fermentation

test ( Trehalose, Latose, Manitol, Salicin and Maltose). Isolates identified as S. equi

fermented salicin and sucrose, but not lactose, sorbitol, trehalose. (Quinn et al.,

1994).

Colony characteristics of S.equi Beta hemolytic Pattern of S. equi

Selection of beta haemolytic colonies:

Pure β-hemolytic colonies on blood agar were selected for DNA extraction.

Genomic DNA purification kit method:

1. Take 10-20mg of bacterial (S. equi) culture and resuspend in 1.5ml eppendorf

containing 200µl TE buffer.

2. Add 400µl of Lysis solution and mix it.

3. Incubate at 65ºC for 5 minutes.

4. Add 600µl Chloroform and gently emulsify by inversion.

5. Centrifuge at 10,000 rpm for 2 minutes.

6. Transfer the upper aqueous phase containing DNA to a fresh tube.

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7. Add 800µl precipitation solution (To prepare fresh working precipitation

solution, mix 720µl nuclease free water with 80µl of supplied 10X

concentrated precipitation solution.).

8. Mix at room temperature for 1-2 minutes.

9. Centrifuge at 10,000 rpm for 2 minutes.

10. Remove the supernatant completely.

11. Dissolve the DNA pellet in 100µl of 1.2M NaCl solution. Make sure that the

pellet is completely dissolved.

12. Add 300µl cold absolute ethanol and let the DNA precipitated (10 minutes at -

20ºC). This time can be extended upto overnight stay at -20ºC.

13. Centrifuge at 10,000 rpm for 6-8 minutes.

14. Discard the supernatant and keep the pellet.

15. Wash the pellet once with 100µl 70% cold ethanol.

16. Let the pellet dry completely.

17. Dissolve the DNA pellet in 100µl of autoclaved double distilled deionized

water.

Quantification of Extracted Genomic DNA:

Quantification of extracted DNA was done using 0.8% Agarose gel. A

standard DNA ladder (50bp) was run with the sample for quantification. Picture was

taken by gel documentation system for record. All samples were brought to same

concentration.

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POLYMERASE CHAIN REACTION:

Primer Selection and Optimization:

Primers for SeM gene of S. equi were showed in table 3.1. These primers

were optimized by gradient PCR using thermocycler. The PCR product was run on

1.2% Agarose Gel.

PCR amplification:

PCR amplification was carried out using S. equi specific primers. These

primers amplify 677bp and 325bp region of SeM gene of S. equi. The PCR was

carried out with the reaction mixture of 25 μl and the quantities of other components

are as under.

2x Taq PCR buffer 11 µl

ddH2O 07 µl

Primer F (3µM) 2.5 µl

Primer F (3µM) 2.5 µl

Sample DNA 02 µl

Total 25 µl

The PCR was carried out in four steps. 1st step was initial denaturation at

950C for 4 min then for each of 30 cycles, the denaturation at 950C for 1min,

annealing for 1 min and 3rd stage extension at 720C for 2 min. PCR cycles were

followed by 10min of final extension at 720C. And at the end, final holding

temperature was 40C until the PCR tubes were taken out of thermal cycler and placed

in refrigerator or run on Agarose Gel.

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Table 3.1: Details of all the primers used in the study.

4. Analysis of PCR products (Agarose gel electrophoresis):

1. The amplified PCR products were analyzed by electrophoresis at 80V and

300mA in 1.2 % agarose gel for one hour and thirty minutes using 1X TAE

buffer.

2. Desired amount of agarose (1.2g) was taken in flask containing the

electrophoresis buffer 1X TAE (100ml), melted in microwave oven for 2

minutes and swirled to ensure even mixing.

Primer Sequence Amplicon

(bp)

Annealing

Temp. Source

SeMF

SeMR

TGCATAAAGAAGTTCCTGTC

GATTCGGTAAGAGCTTGACG 677 560C

Jannatabadi et al., 2008

SeMF

SeMR

CATACCTATCTCCATCAGCA

CGAACTCTGAGGTTAGTCGT 325 570C

Jannatabadi et al., 2008)

IGSzMF

IGSzMR

AAA GTG TGC CCA TAA CGG GTA

CGG CTA TTG TCC ATT GGG GAA 1812 650C

Present study

IGSZPF IGSZPR

CTT GCT AAA GTA ATG GTT GAC

GTT TGT GAG CAA GGC TTA GTC 1212 500C

Present study

SzPIPF SzPIPR

ATG GCA AAA AAA GAA ATG AAG

TTA GTT TTC TTT GCG TCT TGT TGA 1152 590C

Present study

IG18.7F

IG18.7R

ATA ACC ACT CGT TTA CAT GAG TG

GGT CCA AAA ATA CTA TTC TGA ACC 735 600C

Present study

Se18.7F

Se18.7R

AGT TTT AGC CAG TGC AGC AGC

TTA ATT CTC CAG ACT TTT CAA G 481 550C

Present study

EqbE -F

EqbE-R

AAG ATA TAG CAG CAT CGT ATC G

TCT AAA TCT CTA TTA AAT AGC GGT ATA TTG

130 550C Heather et al., 2008

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3. Casting tray was prepared by wrapping meshing tape on its both sides and a

suitable comb was adjusted on it for making the wells

4. The melted agarose was cooled to 45°C and Ethidium Bromide (5μl) was

added into it, before pouring on the gel casting tray.

5. The gel thickness was kept in the range of 0.5-1.0cm. The air bubbles were

removed and it was kept at room temperature for solidification.

6. After solidification the comb was removed carefully to avoid tearing of wells.

The mesh tape was also removed from the sides of the casting tray.

7. The gel casting tray containing the gel was placed in the electrophoresis tank,

having 1X TAE buffer.

8. PCR products (10-15μl) were loaded on the gel in the respective wells after

mixing with appropriate amount (4μl) of Invitrogen’s Blue Juice (6X gel

loading buffer).

9. 100 base pair DNA marker was run along with, as reference.

10. Electric current (80V, 300mA) was applied until the blue dye was about the

three quarters the way down the gel (approximately 90 minutes).

11. Gel was taken out of the electrophoresis tank when the dye had reached near

the anode part of the gel.

12. Gel was then photographed under the UV transilluminator at a wavelength of

254nm with Eagle Eye Gel Documentation System (Stratagene, USA).

Variations in SeM, SzPSe and Se 18.9 proteins of S.equi:

Twenty-five isolates of S. equi including CF32 (S. equi) as reference strain

from outbreaks of equine strangles in N. America, Europe, Australia and Pakistan

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during the years 1969 to 2009 were selected for the study (Table 3.2). These included

pairs of sequential isolates from horse JB with a chronic infection of the maxillary

sinus and one horse 836 that experienced prolonged shedding. The collected samples

were processed in the Gluck equine research center, Department of Veterinary

Science, University of Kentucky, USA. All isolates of S. equi were negative for acid

production from lactose, sorbitol and trehalose. Each was propagated from single

colonies in Todd-Hewitt broth (THB) for DNA isolation. DNA from Pakistani

isolates S24 and L32 were extracted using a Genomic Purification Kit (Fermentas #

K0512). DNAs from the remaining S. equi isolates were obtained as follows. Pellet

from overnight culture in 10 ml THB was suspended in 150 μl digestion buffer (10

mM Tris HCl, 1 mM EDTA, pH 8.0) to which was added 10 μl lysozyme solution (5

mg/ml digestion buffer). Following incubation at 37ºC for 30 min, 20 μg proteinase K

(Sigma) in 5 μl digestion buffer was added followed by heating at 56ºC for 30 min.

Finally each bacterial suspension was boiled for 10 min, centrifuged at 10,000 x g for

5 min and the supernatant stored at -20ºC. Primers designed for amplification and

sequencing of SeM, SzPSe, and Se18.9 are listed in Table 3.1. The PCR protocol

consisted of 30 cycles of 94ºC for 1 min, annealing for 1 min, and 72ºC for 2 min.

PCR products were purified using GeneJET PCR purification kit (Fermentas) and

sequences (Eurofins MWG Operon) obtained using primers from the initial

amplification.

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Table 3.2: Geographic origin, year of isolation and strain characteristics of S.equi .

Strai

n

Year

isolated

Geographic

Origin Strain Characteristics/Comments

S24 2010 Pakistan Isolated S.equi from mandibular abscess

L32 2009 Pakistan Isolated S.equi from nasal wash

MN08 1996 USA(MN) Mucoid S.equi from mild case of strangles

119 1191 Scotland Mucoid S.equi from typical strangles case.

Jba 1998 USA (NY) Mucoid S.equi from persistently infected ethmoid sinus

JBb 1996 USA (KY) Mucoid S.equi from persistently infected ethmoid sinus

FF 1998 USA (KY) Mucoid S.equi

836a 1997 USA (KY) Mucoid S.equi isolated from pony 836,experimentally infected with S.equi CF32

836b 1998 USA (KY) Mucoid S.equi isolated from pony experimentally infected with S.equi CF32

LEX 1990 USA (KY) Non mucoid S.equi from mandibular abscess

E33 1976 USA (NY) Non mucoid S.equi

181 1990 AUSTRALIA Mucoid S.equi from lung abscess 331 1982 Swedish Mucoid S.equi from typical strangles.

BM 1983 Ireland Matte colony of S.equi from case of strangles

M4 1991 Germany Mucoid S.equi

E9 1975 USA (NY) Matte S.equi from strangles case (nasal wash)

FT 2003 USA (IA) Mucoid S.equi from horse with post strangles myopathy

JP 2010 Denmark Mucoid S.equi from guttural pouch

NM 2007 USA (KY) Non mucoid S.equi

MER7

2007 USA (VA) Non mucoid S.equi

15C 2001 USA (UT) Mucoid S.equi from strangles case in captured wild horse

Lou 2010 USA (ME) Non mucoid raised colony of S.equi from mandibular abscess

107 1991 Scotland Mucoid S.equi from nasal wash from typical strangles

K58 1969 USA (KY) Mucoid S.equi from case of strangles

W60 1976 USA (NY) Non mucoid, prototype US strain

CF32 1981 USA (NY) Mucoid, prototype US strain

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PHASE II:

Study of carrier animals:

A total of 40 equines (n=20 horses; n=20 mules) affected with strangles and

showed clinical sign of lymph node enlargement that remained positive after one

week of infection were monitored for 12 weeks to study the carrier status. Samples

were collected on weekly basis from these equines and were processed by using

culture and Polymerase Chain Reaction.

PHASE III:

Haematological Examination:

For haematological examination, 40 horses (n=20 healthy horses; n= 20

diseased horses) and 40 mules (n=20 healthy mules; n= 20 diseased mules) under 5

years of age suffering from natural outbreak of strangles and similarly for carrier

animals 40 horses (n=20 healthy horses; n= 20 carrier horses) and 40 mules (n=20

healthy mules; n= 20 carrier mules) recovered from strangles from Lahore and

Sargodha districts of Punjab province were included. The blood samples were

collected at weekly intervals from affected equines and monthly intervals form carrier

animals, directly from jugular vein, in sterilized plastic bottles coated with EDTA @

1mg/ml of blood. Following haematological parameters, total white blood cell count,

mean segmented neutrophils count, total lymphocytic count, total monocytic count,

total eosinophilic count, total basophilic count, total erythrocytic count, packed cell

volume and haemoglobin concentration were studied.

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Protein Analysis:

For protein analysis 40 equids (n=20 horses, n= 20 mules) under 5 years of

age suffering from natural outbreak of strangles from Lahore and Sargodha districts

of Punjab province were included. Blood samples were collected from Jugular vein in

clean dried centrifugal tube without anticoagulant and brought to laboratory for the

separation of serum. Coagulated blood was centrifuged at 3000 rpm for 20 minutes

and with the help of pipette serum was transferred into clean dried bottles. Samples

were stored 40C for further analysis (Weichselbaum, 1946). Following parameters,

total serum protein, serum albumin, serum globulin and fibrinogen were studied by

using serum chemistry analyzer.

PHASE IV:

THERAPEUTIC TRIALS:

In-vitro testing of Antibiotics:

In this phase, in-vitro antibiotic sensitivity of S. equi to various antibiotics

(Procaine penicillin, ceftiofur Na, cephradine, erythromycin, ampicillin, tetracycline,

chloramphenicol, sulfamethoxazole, trimethoprim+sulfadiazine and gentamycin.) was

determined.

Procedure of Kirby-Bauer Antibiotic Sensitivity Test:

1. Inoculate all blood agar plates with S. equi.

2. Using the swab, streak the entire agar surface horizontally, vertically and

around the outer edge of plate to ensure heavy growth over the entire surface.

3. Allow the culture plates to dry for about 5 minutes.

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4. Using the Sensi-disc dispenser, apply the antibiotic discs by placing the

dispenser over the blood agar surface.

5. Then gently pressed each disc so that they adhere to the surface of blood agar.

6. Incubated all cultured plates for 24 hours at 370C.

7. Measured zone of inhibition of each disc.

In-vivo Antibiotic Trials:

Based on the above in-vitro sensitivity test four top ranking antibiotics were

selected and administered to diseased animals and their efficacy were studied. Detail

of groups is as follows:

Groups No of animals

Antibiotic Horse Mules

A 10 10 1

B 10 10 2

C 10 10 3

D 10 10 4

The efficacy of antibiotics was checked on the basis of disappearance of

clinical signs.

PHASE V:

In-vitro Disinfectant Trial:

In vitro testing of S. equi with disinfectants to determine susceptibility of

bacteria to the disinfectants was performed. The disinfectants used in the study were

Dettol, Povidone iodine, 0.6% H2SO4 and Bleach. The efficacy of disinfectants was

compared by using the Phenol Co-efficient Test (Cappuccino and Sherman 2004).

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Procedure of Phenol Co-efficient Test:

1- 45 nutrient broth tubes were labelled with the names and dilution of the

disinfectant and time interval of subculturing.

2- In test tube rack, six serial dilution 1:50, 1:100, 1:150, 1:150, 1:200, 1:250

and 1:300 of each disinfectant used in the present study (phenol, dettol,

povidone iodine, 0.6% H2SO4 and bleach) were made in normal saline in

separate tubes.

3- Rapidly inoculated one drop of the S. equi culture into each tube of

disinfectants and noted the time of inoculation.

4- All the test tubes were agitated to ensure contact between the disinfectant

and the microbes.

5- Using sterile technique, at intervals of 5, 10 and 15 minutes one loopful

was transferred from each of the test tubes into the appropriately labeled

sterile tube of nutrient broth.

6- All tubes were incubated for 48 hours at 370C.

The phenol coefficient was then calculated by using following formula.

Highest dilution of disinfectant that kill microorganism in 10 min. not in 5 min. -------------------------------------------------------------------------------------------------------

Highest dilution of phenol that kill microorganism in 10 min. not in 5 min.

Statistical Analysis

Data on prevalence of the diseases on the basis of culture and PCR were

analyzed by Chi-square test using statistical software package STATA 9.1 college

station T×77845, USA while data on haematological and biochemical examination

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45

were analyzed with one way ANOVA. The difference between diseased and healthy

animal along different weeks was tested by Tukey’s test. A P-value <0.05 was used to

reject the null hypothesis that the model is not significant. Whereas sequences of

genes were compared with S. equi CF32 and with the database using multiple

sequence alignment by CLUSTALW. SeM alleles were identified using S. equi SeM

database www.pubmlst.org/szooepidemicus/ using BLAST. New unlisted alleles were

assigned numbers 71 to 76. Frequencies of single nucleotide polymorphism (SNP) in

SeM, SzPSe and Se18.9 were obtained by aligning DNA sequences of each isolate and

counting SNPs relative to those sequences in S. equi CF32. Inserted and deleted

sequences were not included in computation of SNPs. G-C percentages were obtained

using G-C Calculator.

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Chapter- 04

RESULTS

To study strangles in equines present project was conducted in Lahore and

Sargodha districts of the Punjab province of Pakistan. The samples collected were

processed at the Medicine and Microbiology Laboratories of the University of

Veterinary and Animal Sciences, Lahore and Gluck Equine Research Centre,

Department of Veterinary Science, University of Kentucky, USA. Data on various

parameters were collected and analysed. Results are given below.

EPIDEMIOLOGY OF STRANGLES:

To study overall prevalence of strangles in equines a total of 500 equines

(n=250 horses; n=250 mules) were examined. On the basis of culture 113 horses and

99 mules were found positive for strangles. An overall prevalence of strangles was

recorded as 42.4% whereas prevalence of strangles in horses and mules was recorded

as 45.2% and 39.6 respectively. Prevalence once evaluated through PCR 122 horses

and 113 mules tested positive for strangles resulting in overall prevalence rate as

47%. Prevalence rate for horses and mules remained 48.8% and 45.2% respectively

shown in fig.4.1. It is obvious from the results that prevalence rate in both the species

in response to PCR were higher than those in response to culture.

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Fig 4.1: Prevalence of Strangles in horses and mules

PREVALENCE OF STRANGLES IN HORSES:

Prevalence of Strangles in horses on the basis of culture:

The results of prevalence of strangles in nasal discharge and pus from sub-

mandibular lymph nodes of horses in different age groups and in different months of

the year are shown in table 4.1. The prevalence rate in horses under 1 year of age was

45(90%), between 1-2 years of age was 42(84%), between 2-3 years of age was

14(28%), between 3-4 years of age 8(16%) and 4-5 years of age was 4(8%). The

highest prevalence was recorded in the group having less than 1 year of age and then

between 1-2 years and decreased with increase in age. Prevalence in horses of

different age groups was observed to be significantly different from each other

(p<0.05). It can be concluded from the results that the horses under 2 years of age are

the most susceptible to strangles infection.

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Table 4.1: Prevalence of Strangles in nasal discharge and pus samples of sub-mandibular lymph nodes of horses on the basis of culture.

Chi-square analysis showed significant difference in prevalence of strangles among all age groups (Chi-square value 122.02, P- value 0.0001)

Similarly, when the prevalence of strangles in horses during different months

of year was calculated, it was found to be the highest during the months of February,

March, April and May as shown in fig. 4.2. While few cases were seen during the

months of January, June and July and no cases were seen during other months of the

year.

Fig 4.2: Month wise prevalence of Strangles in horses on the basis of culture.

Age Groups

n Number positive for S. Equi Total

(%) Jan Feb Mar Apr May Jun July Aug Sep Oct Nov Dec <1year 50 02 09 11 13 07 02 01 00 00 00 00 00 45(90)

1-2year 50 01 08 15 12 02 04 00 00 00 00 00 00 42(84)

2-3year 50 00 03 05 03 03 00 00 00 00 00 00 00 14(28)

3-4year 50 00 01 02 03 02 00 00 00 00 00 00 00 08(16)

4-5year 50 00 01 00 02 01 00 00 00 00 00 00 00 04(08)

Total 250 03 22 33 33 15 06 01 00 00 00 00 00 113(45.2)

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A significant difference was observed (p<0.05) among prevalence rates during

different months of the year. It was concluded from the results reported here, that the

strangles season, in Pakistan lasts from January through the month of July. Similarly

when compared the prevalence of strangles in different seasons of Pakistan. The

highest prevalence rates were recorded during the spring months. The reason might

be the maximum exposure and movement of horses during the months of spring as

equestrian activities are at the peak during these months.

Prevalence of Strangles in nasal discharges:

The prevalence of strangles in nasal discharges of horses on the basis of

culture in different age groups and in different months of year is shown in table 4.2.

The prevalence rate in less than 1 year of age was 29(87.88%), 1-2 years of age was

30(81.08%), 2-3 years of age was 6(17.14%), 3-4 years of age 6(13.33%) and 4-5

years of age was 3(6.82%) on the basis of culture. The highest prevalence rates in

nasal discharges of horses were recorded between 1-2 years of age and then under 1

year of age while the lowest prevalence rate was recorded in 4-5years of age.

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Table 4.2: Prevalence of Strangles in nasal discharge of horses on the basis of culture.

Age

Groups n

Number positive for S. equi Total (%) Jan Feb Mar Apr May Jun July Aug Sep Oct Nov Dec

<1year 33 01 04 08 09 04 02 01 00 00 00 00 00 29(87.88)

1-2year 37 01 04 11 09 01 04 00 00 00 00 00 00 30(81.08)

2-3year 35 00 00 03 01 02 00 00 00 00 00 00 00 06(17.14)

3-4year 45 00 01 01 02 02 00 00 00 00 00 00 00 06(13.33)

4-5year 44 00 01 00 01 01 00 00 00 00 00 00 00 03(06.82)

Total 194 02 10 23 22 10 06 01 00 00 00 00 00 74(38.14)

Chi-square analysis showed significant difference in prevalence of strangles among all age groups (Chi-square value 100.09, P- value 0.0001) Prevalence of Strangles in pus samples of sub-mandibular lymph nodes:

The prevalence of strangles in pus samples of sub-mandibular lymph nodes of

horses on the basis of culture in different age groups and in different months of year

was recorded and is shown in table 4.3. The prevalence rate on the basis of culture in

horses under 1 year of age was 16(94.12%), 1-2 years of age 12(92.31%), 2-3 years

of age 8(53.33%), 3-4 years of age 2(40.00%) and 4-5 years of age 1(16.67%). The

highest prevalence rates were recorded in pus samples of sub-mandibular lymph

nodes of horses in less than 1 year of age then 1-2 years of age while the lowest

prevalence rate was calculated in 4-5years of age.

When comparing the prevalence rate of strangles in nasal discharges and pus

samples of sub-mandibular lymph nodes in different age groups of horses, it was

observed that number of strangles cases were significantly higher (p<0.05) in pus

samples from sub-mandibular lymph nodes as compared to nasal discharges.

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Table 4.3: Prevalence of Strangles in pus samples of sub-mandibular lymph node of horses on the basis of culture.

Age

Groups n

Number positive for S. equi Total (%) Jan Feb Mar Apr May Jun July Aug Sep Oct Nov Dec

<1year 17 01 05 03 04 03 00 00 00 00 00 00 00 16(94.12)

1-2year 13 00 04 04 03 01 00 00 00 00 00 00 00 12(92.31)

2-3year 15 00 03 02 02 01 00 00 00 00 00 00 00 08(53.33)

3-4year 05 00 00 01 01 00 00 00 00 00 00 00 00 02(40.00)

4-5year 06 00 00 00 01 00 00 00 00 00 00 00 00 01(16.67)

Total 56 01 12 10 11 05 00 00 00 00 00 00 00 39(69.64)

Chi-square analysis showed significant difference in prevalence of strangles among all age groups (Chi-square value 19.90, P- value 0.0001) Prevalence of Strangles on the basis of Polymerase Chain Reaction:

Likewise, the prevalence of strangles on the basis of Polymerase Chain

Reaction in different age groups of horses and in different months of year is shown in

table 4.4. The prevalence rate under 1 year of age was 46(92%), 1-2 years of age was

43(86%), 2-3 years of age was 17(34%), 3-4 years of age 10(20%) and 4-5 years of

age was 6(12%). The highest prevalence was recorded in the group of horses having

less than 1 year of age then 1-2 years and decreased with increase in age. The

significant differences were observed (p<0.05) among horses of different age groups.

Therefore it is concluded from the present study that the sensitivity of PCR appears to

be much greater than culture for nasal and pus samples from affected sub-mandibular

lymph nodes. As PCR can be completed in 4-5 hours hence is considered to be an

effective tool in diagnosis, control and management of strangles infection.

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Table 4.4: Prevalence of Strangles in nasal discharge and pus samples of sub-mandibular lymph nodes of horses on the basis of PCR.

Chi-square analysis showed significant difference in prevalence of strangles among all age groups (Chi-square value 113.12, P- value 0.0001)

Similarly the year round prevalence of strangles in horses on the basis of PCR

was recorded and it was found to be the highest during the months of February,

March, April and May as shown in fig. 4.3. While few cases were seen during the

months of January, June and July and no cases were seen during the remaining

months. The differences among prevalence rates during different months of year were

found to be significant (p<0.05). It was concluded from the present study, that the

strangles season in Pakistan from January to July is at the peak. When we compared

strangles in different seasons (summer, winter, spring and autumn), the highest

prevalence was recorded during spring season. The reason may be due to maximum

exposure of horses during the spring time.

Age Groups

n Number positive for S. equi Total

(%) Jan Feb Mar Apr May Jun July Aug Sep Oct Nov Dec <1year 50 02 09 11 14 07 02 01 00 00 00 00 00 46(92)

1-2year 50 01 09 15 12 02 04 00 00 00 00 00 00 43(86)

2-3year 50 00 03 06 04 04 00 00 00 00 00 00 00 17(34)

3-4year 50 00 01 04 03 02 00 00 00 00 00 00 00 10(20)

4-5year 50 00 01 01 03 01 00 00 00 00 00 00 00 06(12)

Total 250 03 23 37 36 16 06 01 00 00 00 00 00 122(48.8)

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Fig 4.3: Month wise prevalence of Strangles in horses on the basis of PCR.

Prevalence of Strangles in nasal discharge:

The prevalence of strangles in nasal discharge of horses on the basis of PCR

in different age groups and in different months of year is shown in table 4.5. The

prevalence rate in less than 1 year of age was 30(90.91%), 1-2 years of age was

31(83.78%), 2-3 years of age was 7(20%), 3-4 of years age 6(13.33%) and 4-5 years

of age was 3(6.82%). The highest prevalence in nasal discharge samples was recorded

in less than 1 year of age then 1-2 years and so on.

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Table 4.5: Prevalence of Strangles in nasal discharge of horses on the basis of PCR.

Age

Groups n

Number positive for S. equi Total (%) Jan Feb Mar Apr May Jun July Aug Sep Oct Nov Dec

<1year 33 01 04 08 10 04 02 01 00 00 00 00 00 30(90.91)

1-2year 37 01 05 11 09 01 04 00 00 00 00 00 00 31(83.78)

2-3year 35 00 01 03 01 02 00 00 00 00 00 00 00 07(20.00)

3-4year 45 00 01 01 02 02 00 00 00 00 00 00 00 06(13.33)

4-5year 44 00 01 00 01 01 00 00 00 00 00 00 00 03(06.82)

Total 194 02 12 23 23 10 06 01 00 00 00 00 00 77(39.69)

Chi-square analysis showed significant difference in prevalence of strangles among all age groups (Chi-square value 104.81, P- value 0.0001)

Prevalence of Strangles in pus samples of sub-mandibular lymph nodes:

The prevalence of strangles in pus samples of the sub-mandibular lymph

nodes of horses on the basis of PCR in different age groups and in different months of

year is shown in table 4.6. The prevalence rate under 1 year of age 16(94.12%), 1-2

years of age 12(92.31%), 2-3 years of age 10(66.67%), 3-4 years of age 4(80%) and

4-5 years of age was 3(50%). The highest prevalence in pus samples of sub-

mandibular lymph nodes of horses was recorded in less than 1 year of age and then

between 1-2 years and so on.

When compared the prevalence of strangles in nasal discharge and pus

samples of sub-mandibular lymph nodes in different age groups. It was observed that

the number of cases of strangles were significantly higher (p<0.05) in pus samples

from sub-mandibular lymph nodes as compare to nasal discharge samples.

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Table 4.6: Prevalence of Strangles in pus samples of sub-mandibular lymph nodes of horses on the basis of PCR.

Age

Groups n

Number positive for S. equi Total (%) Jan Feb Mar Apr May Jun July Aug Sep Oct Nov Dec

<1year 17 01 05 03 04 03 00 00 00 00 00 00 00 16(94.12)

1-2year 13 00 04 04 03 01 00 00 00 00 00 00 00 12(92.31)

2-3year 15 00 03 03 02 02 00 00 00 00 00 00 00 10(66.67)

3-4year 05 00 00 02 02 00 00 00 00 00 00 00 00 04(80.00)

4-5year 06 00 00 01 02 00 00 00 00 00 00 00 00 03(50.00)

Total 56 01 12 13 13 06 00 00 00 00 00 00 00 45(80.36)

Chi-square analysis showed non significant difference in prevalence of strangles among all age groups (Chi-square value 08.50, P- value 0.075)

Comparison of Culture and PCR:

From the results of present study it was observed that the sensitivity of the

Polymerase Chain Reaction appears to be much greater, than the culture for both

nasal and pus samples taken from the affected submandibular lymph nodes. Since the

test is completed in four to five hours it can be an effective tool in the control and

management of outbreaks. Finally it was concluded that culture along with PCR is the

best technique for the diagnosis of strangles, because the culture is of value as it

definitively establishes infection and can conveniently be performed on the same

samples used for PCR.

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PREVALENCE OF STRANGLES IN MULES:

Prevalence of Strangles in mules on the basis of culture:

The prevalence of strangles in mules on the basis of culture in different age

groups and in different months of year is shown in table 4.7. The overall prevalence

rate in less than 1 year of age 41(82%), 1-2 years of age 39(78%), 2-3 years of age

10(20%), 3-4 years of age 6(12%) and 4-5 years of age was 3(6%). The highest

prevalence was recorded in less than 1 year of age then 1-2 years and decreased with

increase in age. The difference between prevalence rates among mules of different

age groups was significant (p<0.05). Therefore it is concluded from the result of

present study that the mules under 2 years of age were highly susceptible to strangles

infection as compared to over 2 years of age.

Table 4.7: Prevalence of Strangles in nasal discharge and pus samples of sub-mandibular lymph nodes of mules on the basis of culture.

Chi-square analysis showed significant difference in prevalence of strangles among all age groups (Chi-square value 115.96, P- value 0.0001)

Year round prevalence of strangles in mules was also recorded and it was

found to be the highest during the months of February, March, April and May.

Age Groups

n Number positive for S. equi Total

(%) Jan Feb Mar Apr May Jun July Aug Sep Oct Nov Dec <1year 50 00 07 11 17 06 00 00 00 00 00 00 00 41(82%)

1-2year 50 00 06 10 19 04 00 00 00 00 00 00 00 39(78%)

2-3year 50 00 01 02 05 02 00 00 00 00 00 00 00 10(20%)

3-4year 50 00 00 01 04 01 00 00 00 00 00 00 00 06(12%)

4-5year 50 00 00 00 03 00 00 00 00 00 00 00 00 03(06%)

Total 250 00 14 24 48 13 00 00 00 00 00 00 00 99(39.6)

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Results are shown in fig. 4.4. While few cases were seen during the months of

January, June and July and no cases were seen during remainding months.

The significant difference was also observed (p<0.05) among prevalence rates

of different months of year. It was concluded that the strangles season in Pakistan

starts from month of January through the month of July, which was similar to the

horses, results. Similarly, when comparing the prevalence of strangles in different

seasons of Pakistan (summer, winter, spring and autumn), the highest prevalence rates

were recorded during the months of spring.

Fig 4.4: Month wise prevalence of Strangles in mules on the basis of culture.

Prevalence of Strangles in nasal discharge:

The prevalence of strangles in nasal discharge of mules on the basis of culture

in different age groups and in different months of year is shown in table 4.8. The

prevalence rate under 1 year of age 23(74.2%), 1-2 years of age 26(74.3%), 2-3 years

of age 4(10.30%), 3-4 years of age 4(9.80%) and 4-5 years of age was 2(4.80%). The

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58

highest prevalence rate in nasal discharges of mules was recorded in 1-2 years of age

and then under 1 year of age while the lowest prevalence rate was recorded in 4-

5years of age that is also similar to horses.

Table 4.8: Prevalence of Strangles in nasal discharge of mules on the basis of culture.

Age

Groups n

Number positive for S. equi Total (%) Jan Feb Mar Apr May Jun July Aug Sep Oct Nov Dec

<1year 31 00 03 07 11 02 00 00 00 00 00 00 00 23(74.2)

1-2year 35 00 03 07 14 02 00 00 00 00 00 00 00 26(74.3)

2-3year 39 00 00 01 02 01 00 00 00 00 00 00 00 04(10.3)

3-4year 41 00 00 01 02 01 00 00 00 00 00 00 00 04(09.8)

4-5year 42 00 00 00 02 00 00 00 00 00 00 00 00 02(04.8)

Total 188 00 06 16 31 06 00 00 00 00 00 00 00 59(31.4)

Chi-square analysis showed significant difference in prevalence of strangles among all age groups (Chi-square value 87.11, P- value 0.0001)

Prevalence of Strangles in pus samples of sub-mandibular lymph nodes:

The prevalence of strangles in pus samples of sub-mandibular lymph nodes of

mules on the basis of culture in different age groups and in different months of year is

shown in table 4.9. The prevalence rate under 1 years of age 18(94.70%), 1-2 years of

age was 13(86.70%), 2-3 years of age was 6(54.5%), 3-4 years of age 2(22.20%) and

4-5 years of age was 1(12.50%) on the basis of culture. The highest prevalence in pus

samples of sub-mandibular lymph nodes of mules was recorded in the group having

less than 1 year of age and then between 1-2 years and so on.

The prevalence of strangles in nasal discharge and pus samples of sub-

mandibular lymph nodes was compared in different age groups. It was observed that

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59

number of cases of strangles were significantly higher (p<0.05) in pus samples from

sub-mandibular lymph nodes as compared to nasal discharge samples.

Table 4.9: Prevalence of Strangles in pus samples of sub-mandibular lymph nodes of mules on the basis of culture.

Age Groups

n Number positive for S. equi Total

(%) Jan Feb Mar Apr May Jun July Aug Sep Oct Nov Dec <1year 19 00 04 04 06 04 00 00 00 00 00 00 00 18(94.7)

1-2year 15 00 03 03 05 02 00 00 00 00 00 00 00 13(86.7)

2-3year 11 00 01 01 03 01 00 00 00 00 00 00 00 06(54.5)

3-4year 09 00 00 00 02 00 00 00 00 00 00 00 00 02(22.2)

4-5year 08 00 00 00 01 00 00 00 00 00 00 00 00 01(12.5)

Total 62 00 08 08 17 07 00 00 00 00 00 00 00 40(64.5)

Chi-square analysis showed significant difference in prevalence of strangles among all age groups (Chi-square value 27.75, P- value 0.0001)

Prevalence of Strangles on the basis of Polymerase Chain Reaction:

The overall prevalence of strangles in mules on the basis of Polymerase Chain

Reaction in different age groups and in different months of year is shown in table

4.10. The prevalence rate under 1 year of age was 44(88%), 1-2 years of age

42(84%), 2-3 years of age 13(26%), 3-4 years of age 8(16%) and 4-5 years of age was

6(12%). The highest prevalence was recorded in less than 1 year of age and then

between 1-2 year and decreased with increase in age. A significant difference was

observed (p<0.05) between prevalence rates, among mules of different age groups.

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Table 4.10: Prevalence of Strangles in nasal discharge and pus samples of sub-mandibular lymph nodes of mules on the basis of PCR.

Chi-square analysis showed significant difference in prevalence of strangles among all age groups (Chi-square value 114.26, P- value 0.0001) Prevalence of strangles in mules, during different months of the year, was also

recorded. It was found to be the highest during the months of February, March, April

and May and is shown in fig. 4.5. While few cases were seen during the months of

January, June and July and no cases were seen during rest of the months. The results

of different months were significantly different (p<0.05) from each other.

Fig 4.5: Month wise prevalence of Strangles in mules on the basis of PCR.

Age Groups

n Number positive for S. equi Total

(%) Jan Feb Mar Apr May Jun July Aug Sep Oct Nov Dec <1year 50 01 07 12 18 06 00 00 00 00 00 00 00 44(88%)

1-2year 50 00 08 11 19 04 00 00 00 00 00 00 00 42(84%)

2-3year 50 00 02 04 05 02 00 00 00 00 00 00 00 13(26%)

3-4year 50 00 01 02 04 01 00 00 00 00 00 00 00 08(16%)

4-5year 50 00 02 01 03 00 00 00 00 00 00 00 00 06(12%)

Total 250 01 20 30 49 13 00 00 00 00 00 00 00 113(45.2)

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Prevalence of Strangles in nasal discharge:

The prevalence of strangles in nasal discharge of mules on the basis of PCR in

different age groups and in different months of year is shown in table 4.11. The

prevalence rate under 1 year of age was 26(83.9%), between 1-2 years of age was

28(80.0%), between 2-3 years of age was 7(17.90%), between 3-4 years of age

4(9.80%) and 4-5 years of age was 4(9.50%) on the basis of PCR. The highest

prevalence in nasal discharge of mules was recorded in the group having less than 1

year of age and then 1-2 years of age.

Table 4.11: Prevalence of Strangles in nasal discharge of mules on the basis of PCR.

Age

Groups n

Number positive for S. Equi Total (%) Jan Feb Mar Apr May Jun July Aug Sep Oct Nov Dec

<1year 31 01 03 08 12 02 00 00 00 00 00 00 00 26(83.9)

1-2year 35 00 05 07 14 02 00 00 00 00 00 00 00 28(80.0)

2-3year 39 00 01 02 02 02 00 00 00 00 00 00 00 07(17.9)

3-4year 41 00 00 01 02 01 00 00 00 00 00 00 00 04(09.8)

4-5year 42 00 01 01 02 00 00 00 00 00 00 00 00 04(09.5)

Total 188 01 10 19 32 07 00 00 00 00 00 00 00 69(36.7)

Chi-square analysis showed significant difference in prevalence of strangles among all age groups (Chi-square value 90.04, P- value 0.0001)

Prevalence of Strangles in pus samples of sub-mandibular lymph nodes:

The prevalence of strangles in pus samples of sub-mandibular lymph nodes of

mules on the basis of PCR in different age groups and in different months of year is

shown in table 4.12. The prevalence rate under 1 year of age was 18(94.70%),

between 1-2 years of age was 14(93.3%), between 2-3 years of age was 6(54.5%),

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62

between 3-4 years of age 4(44.40%) and 4-5 years of age was 2(25.0%) on the basis

of PCR. The highest prevalence in pus samples of sub-mandibular lymph nodes of

mules was recorded in the group aging less than 1 year and then between 1-2 years

and so on.

The prevalence of strangles in nasal discharge and pus samples of sub-

mandibular lymph nodes was compared in different age groups. It was observed that

number of cases of strangles were significantly higher (p<0.05) in pus samples from

sub-mandibular lymph nodes as compared to nasal discharge samples.

Table 4.12: Prevalence of Strangles in pus samples of sub-mandibular lymph nodes of mules on the basis of PCR

. Age

Groups n

Number positive for S. Equi Total (%) Jan Feb Mar Apr May Jun July Aug Sep Oct Nov Dec

<1year 19 00 04 04 06 04 00 00 00 00 00 00 00 18(94.7)

1-2year 15 00 03 04 05 02 00 00 00 00 00 00 00 14(93.3)

2-3year 11 00 01 01 03 01 00 00 00 00 00 00 00 06(54.5)

3-4year 09 00 01 00 03 00 00 00 00 00 00 00 00 04(44.4)

4-5year 08 00 01 00 01 00 00 00 00 00 00 00 00 02(25.0)

Total 62 00 10 09 18 07 00 00 00 00 00 00 00 44(71.0)

Chi-square analysis showed significant difference in prevalence of strangles among all age groups (Chi-square value 21.57, P- value 0.0001)

Comparison of Culture and PCR in Mules:

The results of present study showed that the sensitivity of the Polymerase Chain

Reaction appears to be much greater than culture for both nasal and pus samples

taken from affected submandibular lymph nodes. The PCR can be completed in four

to five hours, might be an effective tool in the control and management of outbreaks.

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63

Finally it was concluded that culture along with PCR is the best technique for the

diagnosis of strangles because culture is of value as it definitively establishes

infection and can conveniently be performed on the same samples used for PCR.

Fig.4.6: PCR amplification of DNAs from equine isolates of S. equi with specific primers. Lanes 1-9 shows the PCR products of S. equi with sized 677 and 325 bp by

using SeM primers. Lane 10 and 11 serve as -ve control and +ve control (CF32, S. equi, USA,) respectively.

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64

MORTALITY:

Mortality rate in horses:

Mortality rate in horses suffering from strangles is shown in table 4.13, out of

122 horses only 2(1.64%) horses died, one from less than one year of age and one

from 1-2 years of age. The difference in mortality rate of affected horses among

different age groups was not significant (P>0.05) however it is concluded from the

present study that the severity of strangles is greater in horses of less than 2 years of

age, as compared to over two year of age.

Table 4.13: Mortality rate in horses under 5 years of age

Age groups Number affected

Mortality Number %age

<1year 46 01 2.17

1-2year 43 01 2.32

2-3year 17 00 00.0

3-4year 10 00 00.0

4-5year 06 00 00.0

Total 122 02 1.64

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65

Mortality rate in Mules:

Similarly mortality rate in mules with strangles is shown in table 4.14, out of

113 mules, only 1(0.88%) mule that was from less than one year of age died. The non

significant difference (P>0.05) was observed in mortality rate of affected mules of

different age groups. It is concluded from the results of the present study that severity

of strangles was greater in the mules of less than 2 year of age as compared to over

two years of age.

Table 4.14: Mortality rate in mules under 5 years of age

Age groups Number affected

Mortality Number %age

<1year 44 01 2.27

1-2year 42 00 00.0

2-3year 13 00 00.0

3-4year 08 00 00.0

4-5year 06 00 00.0

total 113 01 0.88

When comparing the mortality rate in horses and mules no significant

difference (P>0.05) was observed among all age groups however it was known from

the present study that the severity of diseases was seen more in horses as compared to

mules.

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66

Variations in SeM, SzPSe and Se 18.9 proteins of S.equi:

SeM.

Results of PCR of the SeM genes of 24 selected isolates of S. equi and the US

prototype strain CF32 generated products of 1812 bp is shown in fig. 4.7. Resulting

DNA sequences encoded 19 different SeM alleles including numbers 71-76 not

previously included in the database (www.pubmlst.org/szooepidemicus/) (Table

4.16). Residues most frequently subjected to substitution were located at position 58,

63, 108 and 143. Single nucleotide polymorphisms (SNPs) in SeM were found at 93

loci and totalled 181. Fifty-eight of these were non-synonymous, that is, mutations

were resulting in amino acid replacements in SeM. Non-synonymous SNPs were 15.7

times more frequent in the N-terminal region (positions 114 to 629) than in the

remainder of the SeM sequence (Table 4.18). Phylogenies generated by

neighbourhood joining indicate that SeM allele 71 identified in isolates S24 and L32

from Pakistan was the most distantly related allele of the 25 isolates in the study. This

is explained by the remote and isolated Pakistani equid population, which

hypothetically favors preservation and continued divergence of a specific SeM allele.

Other newly identified alleles (72 - 76) in N. America isolates showed 96 - 99%

similarity with alleles 62, 59, 57 and 37 in the SeM database.

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67

Table 4.15: Details of all amplicons used in the present study.

Strain ID Year isolated Geographic OriginAmplicon detected

SeM SzPSe Se18.9 EqbES24 2010 Pakistan + + + +

L32 2009 Pakistan + + + +

MN08 1998 USA(MN) + + + +

119 1191 Scotland + + + +

JBa 1998 USA (NY) + + + +

JBb 1996 USA (KY) + + + +

FF 1998 USA (KY) + + + +

836a 1997 USA (KY) + + + +

836b 1998 USA (KY) + + + +

LEX 1990 USA (KY) + + + +

E33 1976 USA (NY) + + + +

181 1990 AUSTRALIA + + + +

331 1982 Swedish + + + +

BM 1983 Ireland + + + +

M4 1991 Germany + + + +

E9 1976 USA (NY) + + + +

FT 2003 USA (IA) + + + +

JP 2010 Denmark + + + +

NM 2007 USA (KY) + + + +

MER7 2007 USA (VA) + + + +

15C 2001 USA (UT) + + + +

Lou 2010 USA (ME) + + + +

107 1991 Scotland + + + +

K58 1969 USA (KY) + + + +

W60 1976 USA (NY) + + - -

CF32 1981 USA (NY) + + + +

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Table 4.16: SeM alleles in S. equi isolated over a period of 40 years in N. America, Europe and Asia.

Isolate Allele No.

Amino acid residues s 38 47 51 52 53 56 57 58 60 62 63 65 69 90 99 102 103 104 106 107 108 110 111 113 122 125 127 143

TW 1 S R D L K L S E A S R A Q L R Y Y N L M H S S L R S A S CF32 2 * * * * * * * D * * G * * * * * * * * * * * * * * * * R 4047 3 * * * * * * * * * * * * * * * * * * * V * * * * * * * * S24 71 * T * F * * N * * T G * * * * * * * * I Q * * P S N S * L32 71 * T * F * * N * * T G * * * * * * * * I Q * * P S N S *

MN08 2 * * * * * * * D * * G * * * * * * * * * * * * * * * * * 119 1 * * * * * * * * * * * * * * * * * * * * * * * * * * * * JBa 26 * * * * * * * D * * * * * * * * * * * * R * L * * * * R JBb 42 * K N * R F * * * * * * * * * * N * * * R P P * S * * * FF 27 * K N * R F * * * * * * * * * * N * * * R P P * S * * R

836a 31 * * * * * * * D * * G * * * * F * * * * * * * * * * * R 836b 32 * * * * * * * D * * G * * * * F H * * * R * * * * * * R

Lex90 2 * * * * * * * D * * G * * * * * * * * * * * * * * * * R E33 22 P * * * * * * * * * G * * * * * * * * K * P * * * * * R 181 15 * * * * * * * * * * * * * * * * * * * * Q * * * * * * * 331 36 * * * * * * * D * * * * L * * * * * * * * * * * * * * * BM 23 P * * * * * * * * * G * * * * * H * * * * * * * * * * R M4 72 * * * * * * * D * G * * * * * * * * * * * * * * * * * * E9 22 P * * * * * * * * * G * * * * * * * * K * P * * * * * R FT 28 * * * * * * * D * * * * * * * * * * * * * * * * * * * R JP 73 * * * * * * * * D * * T * F * * * * * * R * * * * * * *

NM 39 * * * * * * N D * * * * * * K * * * * * * * * * * * * * MER7 74 * * * * * * * * * * * * * * * * H Y S * * P * S * * * R 15C 75 * * * * * * N D * * * * * * K * * * * * R * * * * * * * Lou 2 * * * * * * * D * * G * * * * * * * * * * * * * * * * R 107 76 * * * * * * * * * * * * * * * * * * * R A F * * * * * * K58 2 * * * * * * * D * * G * * * * * * * * * * * * * * * * R

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Fig.4.7:. PCR amplification of DNAs from equine isolates of S. equi to analyse variation in SeM (a), SzPSe (b), Se18.7 (c) and EqbE (d). Lanes 1 -11 included PAK24 (Pakistan), 1079 (Scotland), K-58 (USA), Boldmani (Ireland), 181093SYD (Australia), German Martin-4 (Germany), John Paul (Denmark), Lexington 90 (Lexington, KY, USA), 331(81) (Sweden), S. zooepidemicus W60 (-ve control) and S. equi CF32 (+ve control).

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Table 4.17: Identification of six new alleles by BLAST analysis against www.pubmlst.org/szooepidemicus/

Strain Peptide having allelic

residues

Homology Result

Pak24

SEVSRTATPTLSRDFKNRLNEIAITGDHAS

SAQKVRNLLKGASVRDLQALLRGLDSARAA

YGRDDYYNLLIQLSSMPNDKPDGDRSQLNL

SSLLVDEIEKRIADGDSYAK

allele 41 - 90.00%

allele 40 - 90.00%

allele 35 - 88.18%

New allele 1

Pak32

SEVSRTATPTLSRDFKNRLNEIAITGDHAS

SAQKVRNLLKGASVRDLQALLRGLDSARAA

YGRDDYYNLLIQLSSMPNDKPDGDRSQLNL

SSLLVDEIEKRIADGDSYAK

allele 41 - 90.00%

allele 40 - 90.00%

allele 35 - 88.18%

New allele 1

German

Martin4

SEVSRTATPRLSRDLKNRLSDIAIGRDAS

SAQKVRNLLKGASVGDLQALLRGLDSARA

AYGRDDYYNLLMHLSSMLNDKPDGDRRQL

SLASLLVDEIEKRIADGDS

allele 62 - 99.06%

allele 51 - 98.11%

allele 69 - 98.11%

New allele 2

John Paul

SEVSRTATPRLSRDLKNRLSEIDISRDTS

SAQKVRNLLKGASVGDLQALLRGFDSARA

AYGRDDYYNLLMRLSSMLNDKPDGDRRQL

SLASLLVDEIEKRIADGDS

allele 59 - 99.06%

allele 6 - 98.11%

allele 47 - 97.17%

New allele 3

Mergana

Aug 07

SEVSRTATPRLSRDLKNRLSEIAISRDAS

SAQKVRNLLKGASVGDLQALLRGLDSARA

AYGRDDYHNLSMHLPSMSNDKPDGDRRQL

SLASLLVDEIEKRIADGDR

allele 57 - 96.23%

allele 25 - 96.23%

allele 54 - 96.19%

New allele 4

15C

SEVSRTATPRLSRDLKNRLNDIAISRDAS

SAQKVRNLLKGASVGDLQALLRGLDSARA

AYGKDDYYNLLMRLSSMLNDKPDGDRRQL

SLASLLVDEIEKRIADGDS

allele 39 - 99.06%

allele 38 - 98.11%

allele 61 - 97.17%

New allele 5

1079

SEVSRTATPRLSRDLKNRLSEIAISRDASS

AQKVRNLLKGASVGDLQALLRGLDSARAAY

GRDDYYXFLRAFFSMLKDKPAGDFRQLSLA

‘SLLVDEIEKRIADGDS

allele 15 - 91.51%

allele 43 - 91.51%

allele 1 - 91.51%

New allele 6

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Table 4.18: Frequency of single nucleotide polymorphism (SNPs) in SeM, SzPSe and Se18.9 in 25 isolates of S. equi.

SzPSe.

PCR of the SzPSe genes of 24 selected isolates of S. equi and the US

prototype strain CF32 generated products of 1152 bp is shown in fig. 4.7. With one

exception, sequence analysis of the SzPSe genes provided no instances of variation.

The single exception, Australian isolate 181, had a deletion of one PEPK repeat.

Remarkably, although 92 SNPs were found at 48 loci in SzPSe of the 25 S. equi

isolates including CF32 S. equi, no SNPs encoding non-synonymous substitutions

were found (Table 4.18). Thus, there is evidence of a high rate of recombination in

the SzP gene.

Moreover, the HV region appears to have been horizontally acquired since its

G-C % (38.3) differs significantly (p<0.01) from that (47.0) of the remaining SzPSe

sequence. Recombination and the presence of exogenous DNA sequence are factors

that would favour occurrence of SNPs. The biological/immunological significance of

this variation is not understood, but does not appear to involve opsonogenic epitopes.

Future work might logically address the effect of variation on the conformational

adhesion epitope on host cell specificity.

Se 18.9.

PCR of the Se18.9 genes of 24 selected isolates of S. equi and the US

prototype strain CF32 generated products of 481 bp is shown in fig. 4.7. Se 18.9

Gene Bases SNP Loci SNPs Non-synonymous

SNPs (%)SeM Nucleotide 114 - 429 315 44 97 46 (47) Nucleotide 430 - 1605 1290 49 84 12 (14) SzPSe 1140 48 92 0 (0) Se18.9 492 02 04 0 (0)

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72

protein has a proven virulence function in common with SeM, it is logically a target

of immune selection pressure yet only 2 SNP loci were found in the DNA sequences

of the Se18.9 genes in 25 isolates. The unexpected absence of variants of Se18.9 in a

population of SeM allelic variants of S. equi argues either for an immutable and

essential structure or virulence function that is minor compared to that of SeM. It

might also be argued that as a secreted protein, Se18.9 might have less survival value

for S. equi than a protein anchored on its surface. The much lower frequency of SNP

loci in Se18.9 compared to SeM and SzPSe is unexplained.

Table 4.19: Details of SzPSe of all isolates of S. equi S. equi strain ID N-terminus HV region PE(P)K repeats

S24 N2 HV4 18 L32 N2 HV4 18

MN08 N2 HV4 18 119 N2 HV4 18 JBa N2 HV4 18 JBb N2 HV4 18 FF N2 HV4 18

836a N2 HV4 18 836b N2 HV4 18 LEX N2 HV4 18 181 N2 HV4 17 E33 N2 HV4 18 331 N2 HV4 18 BM N2 HV4 18 M4 N2 HV4 18 E9 N2 HV4 18 FT N2 HV4 18 JP N2 HV4 18

NM N2 HV4 18 MER7 N2 HV4 18 15C N2 HV4 18 Lou N2 HV4 18 107 N2 HV4 18 K58 N2 HV4 18 W60 N2 HV4 18 CF32 N2 HV4 18

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STUDY OF CARRIER STATUS OF HORSES AND MULES:

Carrier status of Horses:

Out of 122 horses, 20 horses (10<2 years and 10 between 2 and 5 years of

age) positive for strangles agent after one week of infection and were monitored for

12 weeks to study their carrier status is shown in table 4.20. After the end of the 3rd

week all horses < 2 years of age were positive but at the end of 4th to 7th weeks there

remained 5, 2, 1 and zero out of 10, respectively on the basis of culture, whereas

through polymerase chain reaction at the end of the 4th week all horses <2 years of

age were positive, but at the end of 5th to 10th weeks there remained 7, 5, 4, 2, 1 and

zero out of 10, respectively. While in 2 and 5 years old horses, all were positive up to

the 1st week but at the end of 2nd to 8th weeks there were 9, 7, 6, 3, 1, 1 and zero

horses positive, respectively out of 10, on the basis of culture. Through PCR all

horses were positive up to 4th week but at the end of 5th to 9th weeks there were 9, 7,

6, 3, 2 and zero. Horses were declared free of infection on the basis of three

consecutive negative samples through culture and PCR.

Therefore it is concluded from the findings of present study that sensitivity of

Polymerase Chain Reaction appears to be much greater than culture for detection of

carrier status of horses. According to present study, recovered horses should be kept

in quarantine period at least for 9 weeks because the recovered horses remain in

shedder state for a prolonged period of time, and through periodic shedding of S equi

can be a source of infection for susceptible equines.

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Table 4.20: Comparison of culture and PCR for identification of carrier of S. equi in naturally infected horses ≤ 5 years of age.

Weeks post infection n=20 Number positive for S.equi

<2year 2-

5year <2year 2-5year

Culture PCR Culture PCR Zero/Infection day 10 10 10 10 10 10

1st week post

infection 10 10 10 10 10 10

2nd week post

infection 10 10 10 10 09 10

3rd week post

infection 10 10 10 10 07 10

4th week post

infection 10 10 05 10 06 10

5th week post

infection 10 10 02 07 03 09

6th week post

infection 10 10 01 05 01 07

7th week post

infection 10 10 - 04 01 06

8th week post

infection 10 10 - 02 - 03

9th week post

infection 10 10 - 01 - 02

10th week post

infection 10 10 - - - -

11th week post

infection 10 10 - - - -

12th week post

infection 10 10 - - - -

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Carrier status of Mules:

Similarly, out of 113 mules, 20 mules (10<2 years and 10 between 2 and 5

years of age) remaining positive after one week of infection were monitored for 12

weeks to study their carrier status. Results are shown in table 4.21. After the end of

2nd week all mules < 2 years of age were positive but at the end of 3rd to 6th weeks

there remained 7, 3, 1 and zero mules out of 10, respectively on the basis of culture.

Through the polymerase chain reaction at the end of the 5th week all mules <2 years

of age were positive, but at the end of 6th to 10th weeks there remained 9, 7, 3, 2 and

zero mules out of 10, respectively.

While in the group having 2 to 5 year old mules, all were positive up to the 2nd

week but at the end of 3rd to 7th weeks there were 6, 4, 2, 1, 1 and zero mules out of

10 mules, respectively on the basis of culture. Through PCR, all mules were positive

up to 5th week but at the end of 6th to 10th weeks there were 8, 5, 2, 1 and zero

respectively. Mules were declared free of infection on the basis of three consecutive

negative samples through culture and PCR.

From the results of the present study, it can therefore be concluded that

sensitivity of Polymerase Chain Reaction is much greater than culture for the study of

carrier status of mules. Moreover, recovered mules should be kept in the quarantine at

least for a period of 9 weeks, because the recovered mules remains carrier for

prolonged period of time and through periodic shedding of S equi can cause infection

in apparently healthy but susceptible animals.

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Table 4.21: Comparison of culture and PCR for identification of carrier of S. equi in naturally infected mules ≤ 5 years of age.

Weeks post infection n=20 Number positive for S.equi

<2year 2-

5year <2year 2-5year

Culture PCR Culture PCR Zero/Infection day 10 10 10 10 10 10

1st week post

infection 10 10 10 10 10 10

2nd week post

infection 10 10 10 10 10 10

3rd week post

infection 10 10 07 10 06 10

4th week post

infection 10 10 03 10 04 10

5th week post

infection 10 10 01 10 02 10

6th week post

infection 10 10 - 09 01 08

7th week post

infection 10 10 - 07 01 05

8th week post

infection 10 10 - 03 - 02

9th week post

infection 10 10 - 02 - 01

10th week post

infection 10 10 - - - -

11th week post

infection 10 10 - - - -

12th week post

infection 10 10 - - - -

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PHASE 4:

HAEMATOLOGICAL EXAMINATION:

Haematological studies were conducted to examine the effect of strangles on

various blood parameters during active infection and carrier state in horses and mules.

1-Haematological Examination of Diseased horses and mules:

For haematological examination 40 horses (n=20 healthy horses; n= 20

diseased horses) and 40 mules (n=20 healthy mules; n= 20 diseased mules) under 5

years of age suffering from natural outbreak of strangles from Lahore and Sargodha

districts of Punjab province of Pakistan were included. Parameters like total white

blood cell count, mean segmented neutrophils count, total lymphocytic count, total

monocytic count, total eosinophilic count, total basophilic count, total erythrocytic

count, packed cell volume and haemoglobin concentration were studied.

Total white blood cell count:

Total white blood cell count in horses and mules suffering for strangles is

shown in table 4.22. In horses total WBCs were 14.99±0.22x109/L at 1st week,

14.07±0.19x109/L at 2nd week, 12.80±0.16x109/L at 3rd week and 11.91±0.16x109/L

at 4th week post infection. When compared total WBCs of diseased and healthy

horses on weekly basis significant increase was observed up to 3rd week of infection

while during the 4th week of infection there was no significant increase (P>0.05).

Among all four weeks total WBCs were according to the following order 4th

<3rd<2nd<1st week. It was also observed that at the end of 1st week post infection

total white blood cell count was highest which significantly decreased to the normal

values during the subsequent weeks (P<0.05).

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In mules total white blood cell count was 13.27±0.22x109/L at the end of 1st

week, 12.98±0.22x109/L at 2nd week, 12.18±0.17x109/L at 3rd week and

10.89±0.19x109/L at 4th week post infection. Similar pattern was observed in mules

as in horses when compared diseased mules with healthy mules on weekly basis

significant increase was observed up to 3rd week of infection while during the 4th

week of infection there was no significant increase (P>0.05). It was also observed that

rise in total white blood cell count was rapid and much higher in horses as compared

to mules.

Table 4.22: Total white blood cell count (x109/L) in healthy and diseased horses and mules suffering from strangles. (Mean ± SE)

Mean in a column followed by the same letter were not significantly different at P≤0.05, by Tukey HSD test. * indicates significant difference (p<0.05) among healthy and diseased groups ** indicates non-significant difference (p>0.05) between healthy and diseased group

When diseased horses compared with diseased mules, little difference was

observed, while increase in total white blood cell count was significantly higher

(P<0.05) in affected horses and mules than healthy animals as shown in fig. 4.8. It

was also observed that the level of total WBCs remained increased in affected

Post Infection Weeks

n Horses Mules

Horses Mules Healthy

n=20 Diseased

n=20 Healthy

n=20 Diseased

n=20

1st 40 40 *11.78±0.17a *14.99±0.22a *09.85±0.16b *13.27±0.22a

2nd 40 40 *10.71±0.16b *14.07±0.19b *10.73±0.09a *12.98±0.22a

3rd 40 40 *09.93±0.09c *12.80±0.16c *09.28±0.13c *12.18±0.17b

4th 40 40 **11.16±0.19b **11.91±0.16d **10.13±0.12b **10.89±0.19c

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79

animals than healthy animals during all the four weeks. This indicates the

significance of total WBCs during disease condition and is one of the diagnostic

points of strangles disease.

Fig 4.8: Week wise comparison of Total white blood cell count (x109/L) in healthy and diseased horses and mules suffering from strangles. (Mean ± SE)

Mean Segmented Neutrophilic Count (MSNC):

Data regarding mean segmented neutrophilic count in horses and mules

suffered from strangles is shown in table 4.23. In horses mean segmented neutrophilic

count was 07.07±0.11x109/L at 1st week, 06.98±0.07x109/L at 2nd week,

05.08±0.08x109/L at 3rd week and 04.55±0.07x109/L at 4th week post infection.

When compared diseased and healthy horses on weekly basis significant increase

(P<0.05) in the values was observed in all four weeks of infection. It was also seen

that at the end of 1st week post infection mean segmented neutrophilic count was

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80

highest which significantly decreased to the normal values during the subsequent

weeks (P<0.05).

In mules mean segmented neutrophilic count was 06.80±0.10x109/L at 1st

week, 06.03±0.05x109/L at 2nd week, 05.45±0.07x109/L at 3rd week and

04.98±0.06x109/L at 4th week post infection. When the result of diseased mules

compared with healthy mules on weekly basis significant increase in the values was

observed up to 3rd week of infection while on the 4th week of infection the increase

was non significant (P>0.05).

When compared diseased horses with diseased mules, there were no

significant differences observed (P>0.05), while mean segmented neutrophilic count

significantly increased (P<0.05) in affected horses and mules as compared to healthy

animals as shown in fig. 4.9. It was also observed that the level of MSNC remained

increased in diseased animals than healthy animals during all the four weeks that

indicate significance of MSNC during disease condition and can also form one of the

diagnostic points of strangles disease.

Table 4.23: Mean segmented Neutrophilic count (x109/L) in healthy and diseased horses and mules suffering from strangles. (Mean ± SE)

Mean in a column followed by the same letter were not significantly different at P≤0.05, by Tukey HSD test. * indicates significant difference (p<0.05) among healthy and diseased groups ** indicates non-significant difference (p>0.05) between healthy and diseased group

Post Infection Weeks

n Horses Mules

Horses Mules Healthy

n=20 Diseased

n=20 Healthy

n=20 Diseased

n=20

1st 40 40 *04.54±0.07a *07.07±0.11a *04.68±0.10a *06.80±0.10a

2nd 40 40 *04.72±0.08a *06.98±0.07a *03.98±0.04b *06.03±0.05b

3rd 40 40 *03.96±0.05b *05.08±0.08b *04.11±0.04b *05.45±0.07c

4th 40 40 *03.78±0.07b *04.55±0.07c **04.60±0.07a **04.98±0.06d

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Fig 4.9: Week wise comparison of Mean segmented Neutrophilic count (x109/L) in healthy and diseased horses and mules suffering from strangles. (Mean ± SE)

Total Lymphocytic count:

The result of total lymphocytic count in horses and mules suffering from

strangles is shown in table 4.24. Total lymphocytic count in horses were recorded as

02.71±0.05x109/L at the end of 1st week, 03.98±0.07x109/L at the end of 2nd week,

04.76±0.08x109/L at the end of 3rd week and 02.56±0.04x109/L at the end of 4th

week post infection. When diseased horses compared with healthy horses on weekly

basis there, no significant increase or decrease (P>0.05) during all four weeks was

observed.

In mules the values of total lymphocytic count was 03.89±0.05x109/L at 1st

week, 02.97±0.05x109/L at 2nd week, 03.03±0.04x109/L at 3rd week and

03.71±0.06x109/L at 4th week post infection. When compared diseased mules with

healthy mules on weekly basis a significant decrease (P<0.05) was observed in all

four weeks of infection and this was opposite to horses. It was further noticed that at

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82

the end of 2nd week post infection total lymphocytic count was lowest which

significantly increased to the normal values during the subsequent weeks (P<0.05).

When data of diseased horses compared with that of diseased mules on weekly basis

there was no significant increase or decrease (P>0.05) observed among all four weeks

as shown in fig. 4.10.

Table 4.24: Total Lymphocytic count (x109/L) in healthy and diseased horses and mules suffering from strangles. (Mean ± SE)

Mean in a column followed by the same letter were not significantly different at P≤0.05, by Tukey HSD test. * indicates significant difference (p<0.05) among healthy and diseased groups ** indicates non-significant difference (p>0.05) between healthy and diseased group

Fig 4.10: Week wise comparison of Total Lymphocytic count (x109/L) in healthy and diseased horses and mules suffering from strangles. (Mean ± SE)

Post Infection Weeks

n Horses Mules

Horses Mules Healthy

n=20 Diseased

n=20 Healthy

n=20 Diseased

n=20 1st 40 40 **02.89±0.05c **02.71±0.05c *05.53±0.05a *03.89±0.05a

2nd 40 40 **03.48±0.06b **03.98±0.07b *05.40±0.03a *02.97±0.05b

3rd 40 40 **03.98±0.04a **04.76±0.08a *04.98±0.06b *03.03±0.04b

4th 40 40 **02.93±0.05c **02.56±0.04c *05.02±0.07b *03.71±0.06a

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83

Total Monocytic count (TMC):

Data on total monocytic count in horses and mules suffered from strangles is

shown in table 4.25. Total monocytic count in horses was 00.53±0.021 x109/L at the

end of 1st week, 00.45±0.014 x109/L at the end of 2nd week, 00.49±0.011 x109/L at

the end of 3rd week and 00.47±0.011 x109/L at the end of 4th week. Comparison of

diseased horses with healthy horses revealed a non significant increase (P>0.05)

among all four weeks.

In mules the result of total monocytic count was 00.80±0.021x109/L at 1st

week, 00.87±0.014x109/L at 2nd week, 00.53±0.017 x109/L at 3rd week and

00.43±0.012 x109/L at 4th week post infection. When compared diseased mules with

healthy mules on weekly basis a significant increase (P<0.05) was observed in all

four weeks of infection, while in horses this increase was non significant (P>0.05).

When diseased horses compared with diseased mules on weekly basis the difference

of total monocytic count was observed non significant (P>0.05) among all four weeks

is shown in fig. 4.11. It was also observed that the value of total monocytic count

remained increased among all four weeks in diseased animals than healthy animals.

Table 4.25: Total Monocytic count (x109/L) in healthy and diseased horses and mules suffering from strangles. (Mean ± SE)

Mean in a column followed by the same letter were not significantly different at P≤0.05, by Tukey HSD test. * indicates significant difference (p<0.05) among healthy and diseased groups ** indicates non-significant difference (p>0.05) between healthy and diseased group

Post Infection Weeks

N Horses Mules

Horses Mules Healthy

n=20 Diseased

n=20 Healthy

n=20 Diseased

n=20

1st 40 40 **00.36±0.018a **00.53±0.021a *00.17±0.010ab *00.80±0.021b

2nd 40 40 **00.38±0.020a **00.45±0.014b *00.20±0.009a *00.87±0.014a

3rd 40 40 **00.38±0.016a **00.49±0.011ab *00.18±0.010ab *00.53±0.017c

4th 40 40 **00.36±0.015a **00.47±0.011b *00.15±0.007b *00.43±0.012d

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Fig 4.11: Week wise comparison of Total Monocytic count (x109/L) in healthy and diseased horses and mules suffering from strangles. (Mean ± SE)

Total Eosinophilic count:

The results of total eosinophilic count in horses and mules suffering from

strangles is shown in table 4.26. Total eosinophilic count in horses was

00.48±0.010x109/L at 1st week, 00.32±0.022x109/L at 2nd week, 00.45±0.011 x109/L

at 3rd week and 00.50±0.014 x109/L at 4th week post infection. On comparison of

diseased horses with healthy horses on weekly basis a non significant difference

(P>0.05) of total eosinophilic count was observed.

Table 4.26: Total Eosinophil count (x109/L) in healthy and diseased horses and mules suffering from strangles. (Mean ± SE)

Mean in a column followed by the same letter were not significantly different at P≤0.05, by Tukey HSD test. * indicates significant difference (p<0.05) among healthy and diseased groups ** indicates non-significant difference (p>0.05) between healthy and diseased group

Post Infection Weeks

N Horses Mules

Horses Mules Healthy

n=20 Diseased

n=20 Healthy

n=20 Diseased

n=20 1st 40 40 **00.53±0.016a **00.48±0.010a **00.27±0.014b **00.51±0.015a

2nd 40 40 **00.46±0.010c **00.32±0.022b **00.34±0.014a **00.54±0.015a

3rd 40 40 **00.51±0.014ab **00.45±0.011a **00.29±0.007b **00.49±0.007a

4th 40 40 **00.48±0.007bc **00.50±0.014a **00.28±0.007b **00.42±0.011b

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Total eosinophilic count in mules was 00.51±0.015x109/L at 1st week,

00.54±0.015x109/L at 2nd week, 00.49±0.007 x109/L at 3rd week and 00.42±0.011

x109/L at 4th week. Similarly as in horses, the difference of total eosinophilic count in

diseased and healthy mules was observed non significant (P>0.05) among all four

weeks. When compared diseased horses with diseased mules on weekly basis the

difference of total eosinophilic count was also found non significant (P>0.05) as

shown in fig. 4.12.

Fig 4.12: Week wise comparison of Total Eosinophil count (x109/L) in healthy and diseased horses and mules suffering from strangles. (Mean ± SE)

Total Basophil count:

Data regarding total basophilic count in horses and mules suffered from

strangles is shown in table 4.27. The results of total basophilic count in horses were

00.43±0.012x109/L at 1st week, 00.48±0.010x109/L at 2nd week, 00.20±0.007x109/L

at 3rd week and 00.07±0.003x109/L at 4th week of post infection. When compared

diseased and healthy horses on weekly basis significant increase was observed up to

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3rd week of infection while on the 4th week of infection there was non significant

increase (P>0.05).

Table 4.27: Total Basophilic count (x109/L) in healthy and diseased horses and mules suffering from strangles. (Mean ± SE)

Mean in a column followed by the same letter were not significantly different at P≤0.05, by Tukey HSD test. * indicates significant difference (p<0.05) among healthy and diseased groups ** indicates non-significant difference (p>0.05) between healthy and diseased group It was also observed that at the end of 2nd week post infection total basophilic

count was highest which significantly decreased to the normal values during the

subsequent weeks (P<0.05). Total basophilic count in mules were 00.17±0.010x109/L

at the end of 1st week, 00.13±0.007x109/L at the end of 2nd week, 00.20±0.007

x109/L at the end of 3rd week and 00.11±0.012x109/L at the end of 4th week post

infection. When diseased mules compared with healthy mules on weekly basis the

difference of total basophilic count was non significant (P>0.05). When the results of

diseased horses compared with diseased mules on weekly basis no significant

increase (P>0.05) was observed as shown in fig. 4.13.

Post Infection Weeks

N Horses Mules

Horses Mules Healthy

n=20 Diseased

n=20 Healthy

n=20 Diseased

n=20

1st 40 40 *00.05±0.004a *00.43±0.012b **00.05±0.003b **00.17±0.010b

2nd 40 40 *00.03±0.003b *00.48±0.010a **00.04±0.003b **00.13±0.007c

3rd 40 40 *00.04±0.003ab *00.20±0.007c **00.07±0.003a **00.20±0.007a

4th 40 40 **00.01±0.001c **00.07±0.003d **00.02±0.004c **00.11±0.012c

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Fig 4.13: Week wise comparison of Total Basophilic count (x109/L) in healthy and diseased horses and mules suffering from strangles. (Mean ± SE)

Total Erythrocytic count:

Data on total erythrocytes count in horses and mules suffering for strangles is

shown in table 4.28. Total erythrocyte count in horses was 5.97±0.04 x 1012/L at 1st

week, 6.03±0.04 x1012/L at 2nd week, 5.91±0.05 x 1012/L at 3rd week and 5.87±0.04

x 1012/L at 4th week. When compared diseased horses with healthy horses on weekly

basis the decrease of total erythrocytes count was observed non significant (P>0.05)

among all four weeks.

Total erythrocytic count in mules was 6.07±0.05 x 1012/L at the end of 1st

week, 6.35±0.05 x 1012/L at the end of 2nd week, 6.56±0.06 x 1012/L at the end of 3rd

week and 6.37±0.05 x 1012/L at the end of 4th week post infection. When compared

diseased mules with healthy mules on weekly basis the decreases of total erythrocytes

count was observed to be non significant (P>0.05) among all four weeks. Similarly

when diseased horses compared with diseased mules on weekly basis difference of

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88

total erythrocytes count was observed non significant (P>0.05) among all four weeks

as shown in fig. 4.14.

Table 4.28: Erythrocytes count (X 1012/L) in healthy and diseased horses and mules suffering from strangles. (Mean ± SE)

Mean in a column followed by the same letter were not significantly different at P≤0.05, by Tukey

HSD test.

* indicates significant difference (p<0.05) among healthy and diseased groups

** indicates non-significant difference (p>0.05) between healthy and diseased group

Fig 4.14: Week wise comparison of Erythrocytes count (X 1012/L) in healthy and diseased horses and mules suffering from strangles. (Mean ± SE)

Post Infection Weeks

N Horses Mules

Horses Mules Healthy

n=20 Diseased

n=20 Healthy

n=20 Diseased

n=20

1st 40 40 **6.27±0.03a **5.97±0.04ab **6.79±0.07a **6.07±0.05c

2nd 40 40 **6.13±0.05ab **6.03±0.04a **6.81±0.07a **6.35±0.05b

3rd 40 40 **6.07±0.06b **5.91±0.05ab **6.77±0.06a **6.56±0.06a

4th 40 40 **6.02±0.04b **5.87±0.04b **6.80±0.07a **6.37±0.05ab

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Packed cell volume:

Data regarding packed cell volume in horses and mules suffered from

strangles is shown in table 4.29. In horses packed cell volume was 29.14±0.14% at

the 1st week, 32.98±0.06% at the 2nd week, 30.67±0.17b % at the 3rd week

and28.10±0.09% at the end of 4th week post infection. When compared diseased and

healthy horses on weekly basis the decrease of packed cell volume was observed non

significant (P>0.05) among all four weeks.

In mules packed cell volume was 28.45±0.14% at the 1st week, 33.04±0.10%

at the 2nd week, 29.70±0.14% at the 3rd week and 31.13±0.23% at the end of 4th

week. When compared the data of diseased and healthy mules on weekly basis a non

significant (P>0.05) decrease of packed cell volume was observed among all four

weeks. Similarly the difference of values of packed cell volume in diseased horses

and diseased mules on weekly basis was also found to be non significant (P>0.05)

among all four weeks as shown in fig. 4.15.

Table 4.29: Packed cell volume (%) in healthy and diseased horses and mules suffering from strangles. (Mean ± SE)

Mean in a column followed by the same letter were not significantly different at P≤0.05, by Tukey HSD test. * indicates significant difference (p<0.05) among healthy and diseased groups ** indicates non-significant difference (p>0.05) between healthy and diseased group

Post Infection Weeks

N Horses Mules

Horses Mules Healthy

n=20 Diseased

n=20 Healthy

n=20 Diseased

n=20 1st 40 40 **35.67±0.24b **29.14±0.14c **37.56±0.14a **28.45±0.14d

2nd 40 40 **34.89±0.17c **32.98±0.06a **36.70±0.07b **33.04±0.10a

3rd 40 40 **34.88±0.17c **30.67±0.17b **35.97±0.24c **29.70±0.14c

4th 40 40 **36.45±0.07a **28.10±0.09d **36.01±0.20c **31.13±0.23b

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Fig 4.15: Week wise comparison of packed cell volume (%) in healthy and diseased horses and mules suffering from strangles. (Mean ± SE)

Haemoglobin concentration:

The results of haemoglobin concentration in horses and mules suffering for

strangles are shown in table 4.30. Haemoglobin concentration in horses was

109.45±0.21g/L at 1st week, 100.67±0.11g/L at 2nd week, 99.12±0.20g/L at 3rd

week and 104.61±0.17g/L at 4th week post infection. When diseased and healthy

horses were compared on weekly, the decrease of haemoglobin concentration was

observed to be non significant (P>0.05) among all four weeks. It was also noted that

this decrease of haemoglobin concentration was more in mules than in horses.

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Table 4.30: Haemoglobin concentration (g/L) in healthy and diseased horses and mules suffering from strangles. (Mean ± SE)

Mean in a column followed by the same letter were not significantly different at P≤0.05, by Tukey HSD test. * indicates significant difference (p<0.05) among healthy and diseased groups ** indicates non-significant difference (p>0.05) between healthy and diseased group

Similarly haemoglobin concentration in mules was 123.48±0.61g/L at 1st

week, 126.11±0.13g/L at 2nd week, 122.22±0.17 g/L at 3rd week and

125.85±0.17g/L at 4th week post infection. When diseased and healthy mules

compared on weekly basis, the decrease of haemoglobin concentration was observed

as non significant (P>0.05) among all four weeks. When compared the diseased

horses with diseased mules on weekly basis, haemoglobin concentration was

observed non significant (P>0.05) as shown in fig. 4.16.

Post Infection Weeks

N Horses Mules

Horses Mules Healthy

n=20 Diseased

n=20 Healthy

n=20 Diseased

n=20

1st 40 40 **123.47±0.61a **109.45±0.21a **145.76±0.17a **123.48±0.61b

2nd 40 40 **109.89±0.20d **100.67±0.11c **139.68±0.10b **126.11±0.13a

3rd 40 40 **115.73±0.23c **099.12±0.20d **135.95±0.17c **122.22±0.17c

4th 40 40 **117.24±0.17b **104.61±0.17b **133.97±0.14d **125.85±0.17a

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92

Fig 4.16: Week wise comparison of Haemoglobin concentration (g/L) in healthy and diseased horses and mules suffering from strangles. (Mean ± SE)

2- Haematological examination of carrier horses and mules:

For haematological examination of carrier animals, 40 horses (n=20 healthy

horses; n= 20 carrier horses) and 40 mules (n=20 healthy mules; n= 20 carrier mules)

recovered from strangles from Lahore and Sargodha districts of Punjab province of

Pakistan were selected and kept under test upto 8th week of recovery. Parameters like

total white blood cell count, mean segmented neutrophils count, total lymphocytic

count, total monocytic count, total eosinophilic count, total basophilic count, total

erythrocytic count, packed cell volume and haemoglobin concentration were studied.

Total white blood cell count:

Total white blood cell count in carrier horses and mules for strangles is shown

in table 4.31. In horses total WBCs were 14.31±0.19x109/L at 2nd week,

14.07±0.19x109/L at 4th week, 11.69±0.23x109/L at 6th week and 10.88±0.17x109/L

at 8th week of recovery. When compared total WBCs of carrier and healthy horses on

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93

weekly basis a significant increase was observed up to 4th week after recovery while

on the 6th and 8th week of recovery this increase was non significant (P>0.05).

In carrier mules total white blood cell count was 13.03±0.2x109/L at 2nd

week, 12.67±0.18x109/L at 4th week, 11.18±0.17x109/L at 6th week and

10.69±0.18x109/L at 8th week of recovery. Similar pattern was observed in carrier

mules as in carrier horses while on comparison of carrier mules with healthy mules

on weekly basis a significant increase was observed up to 4th week of recovery while

on the 6th and 8th of recovery this increase was non significant (P>0.05). It was also

noticed that rise in total white blood cell count was rapid and much more in carrier

horses as compared to carrier mules. When carrier horses compared with carrier

mules, a little difference was observed while total white blood cell count was

significantly less (P<0.05) in normal horses and mules than carrier animals upto 4th

week after recovery as shown in fig. 4.17.

Table 4.31: Total white blood cell count (x109/L) in healthy and carrier horses and mules from strangles. (Mean ± SE)

Mean in a column followed by the same letter were not significantly different at P≤0.05, by Tukey HSD test. * indicates significant difference (p<0.05) among healthy and diseased groups ** indicates non-significant difference (p>0.05) between healthy and diseased group

Weeks After

Recovery

N Horses Mules

Horses Mules Healthy

n=20 Carrier

n=20 Healthy

n=20 Carrier

n=20

2nd 40 40 *11.08±0.13a *14.31±0.19a *10.64±0.12ab *13.03±0.21a

4th 40 40 *10.98±0.17a *14.07±0.19a *10.84±0.09a *12.67±0.18a

6th 40 40 **11.23±0.16a **11.69±0.23b **10.35±0.16b **11.18±0.17b

8th 40 40 **11.20±0.18a **10.88±0.17c **10.26±0.13b **10.69±0.18b

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Fig 4.17: Week wise comparison of Total white blood cell count (x109/L) in healthy and carrier horses and mules. (Mean ± SE)

Mean Segmented Neutrophilic Count (MSNC):

Data regarding mean segmented neutrophilic count in carrier horses and

carrier mules from strangles is shown in table 4.32. In carrier horses mean segmented

neutrophilic count was 07.13±0.09x109/L at 2nd week, 06.67±0.09x109/L at 4th

week, 04.97±0.13x109/L at 6th week and 04.49±0.07x109/L at 8th week of recovery.

When compared carrier horses with healthy horses on weekly basis significant

increase was observed up to 4th week after recovery while on the 6th and 8th after

recovery this increase was non significant (P>0.05).

In carrier mules mean segmented neutrophilic count was 06.93±0.07x109/L at

2nd week, 05.79±0.11x109/L at 4th week, 05.32±0.07x109/L at 6th week and

04.59±0.08x109/L at 8th week of recovery. When the result of carrier mules

compared with healthy mules on weekly basis significant increase was observed up to

6th week of recovery while on the 8th week of recovery this increase was non

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95

significant (P>0.05). The result of carrier mules was different from the result of

carrier horses. When carrier horses compared with carrier mules, no significant

difference was observed (P>0.05) while mean segmented neutrophilic count was

significantly less (P<0.05) in healthy horses and mules than carrier animals upto 4th

week after recovery is shown in fig. 4.18.

Table 4.32: Mean segmented Neutrophils count (x109/L) in healthy and carrier horses and mules from strangles. (Mean ± SE)

Mean in a column followed by the same letter were not significantly different at P≤0.05, by Tukey HSD test. * indicates significant difference (p<0.05) among healthy and diseased groups ** indicates non-significant difference (p>0.05) between healthy and diseased group

Fig 4.18: Week wise comparisons of Neutrophils count (x109/L) in healthy and carrier horses and mules. (Mean ± SE)

Weeks After

Recovery

N Horses Mules

Horses Mules Healthy

n=20 Carrier

n=20 Healthy

n=20 Carrier

n=20

2nd 40 40 *04.37±0.33a *07.13±0.09a *04.71±0.10a *06.93±0.07a

4th 40 40 *04.66±0.40ab *06.67±0.09b *04.62±0.11a *05.79±0.11b

6th 40 40 **04.81±0.52a **04.97±0.13c *04.42±0.10a *05.32±0.07c

8th 40 40 **04.92±0.67a **04.49±0.07d **04.60±0.08a **04.59±0.08d

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96

Total Lymphocytic count:

The result of total lymphocytic count in carrier horses and carrier mules for

strangles is shown in table 4.33. Total lymphocytic count in carrier horses was

02.62±0.05x109/L at the end of 2nd week, 03.68±0.09x109/L at the end of 4th week,

03.49±0.13x109/L at the end of 6th week and 02.61±0.04x109/L at the end of 8th

week of recovery. When carrier horses compared with healthy horses there were no

significant increase or decrease (P>0.05) observed among all eight weeks after

recovery.

Table 4.33: Total Lymphocytic count (x109/L) in healthy and carrier horses and mules from strangles. (Mean ± SE)

Mean in a column followed by the same letter were not significantly different at P≤0.05, by Tukey HSD test. * indicates significant difference (p<0.05) among healthy and diseased groups ** indicates non-significant difference (p>0.05) between healthy and diseased group

In mules the values of total lymphocytic count was 03.73±0.06x109/L at 2nd

week, 02.81±0.05x109/L at 4th week, 03.14±0.06x109/L at 6th week and

03.65±0.06x109/L at 8th week of recovery. When compared carrier mules with

healthy mules on weekly basis a significant decrease (P<0.05) was observed in all 8

weeks after recovery and this was opposite to carrier horses. It was also observed that

Weeks After

Recovery

N Horses Mules

Horses Mules Healthy

n=20 Carrier

n=20 Healthy

n=20 Carrier

n=20

2nd 40 40 **02.69±0.07c **02.62±0.05b *05.59±0.04a *03.73±0.06a

4th 40 40 **03.27±0.06b **03.68±0.09a *05.43±0.03a *02.81±0.05c

6th 40 40 **03.65±0.08a **03.49±0.13a *04.77±0.07b *03.14±0.06b

8th 40 40 **03.07±0.05b **02.61±0.04b *04.91±0.07b *03.65±0.06a

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97

at the end of 4th week post infection total lymphocytic count was lowest which

significantly increased to the normal values during the subsequent weeks (P<0.05).

When diseased horses compared with diseased mules on weekly basis there was no

significant increase or decrease (P>0.05) observed among all eight weeks after

recovery as shown in fig. 4.19.

Fig 4.19: Week wise comparisons of total lymphocytic count (x109/L) in healthy and carrier horses and mules. (Mean ± SE)

Total Monocytic count (TMC):

Data on total monocytic count in carrier horses and carrier mules from

strangles is shown in table 4.34. Total monocytic count in carrier horses was

00.51±0.019x109/L at the end of 2nd week, 00.44±0.014x109/L at the end of 4th

week, 00.41±0.012x109/L at the end of 6th week and 00.39±0.013x109/L at the end of

8th week of recovery. When compared carrier horses with healthy horses the

observed increase was non significant (P>0.05) among all eight weeks after recovery.

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Table 4.34: Total Monocyte count (x109/L) in healthy and carrier horses and mules from strangles. (Mean ± SE)

Mean in a column followed by the same letter were not significantly different at P≤0.05, by Tukey HSD test. * indicates significant difference (p<0.05) among healthy and diseased groups ** indicates non-significant difference (p>0.05) between healthy and diseased group

In mules the result of total monocytic count was 00.78±0.02x109/L at 2nd

week, 00.83±0.01x109/L at 4th week, 00.49±0.01x109/L at 6th week and

00.38±0.012x109/L at 8th week of recovery. When compared carrier mules with

healthy mules on weekly basis significant increase was observed up to 4th week of

recovery while on the 6th and 8th of recovery this increase was non significant

(P>0.05). Weekly comparison of carrier horses with carrier mules revealed that

difference of total monocytic count was non significant (P>0.05) among all eight

weeks after recovery as shown in fig. 4.20.

Weeks After

Recovery

N Horses Mules

Horses Mules Healthy

n=20 Carrier

n=20 Healthy

n=20 Carrier

n=20

2nd 40 40 **00.38±0.017a **00.51±0.019a *00.21±0.007ab *00.78±0.024a

4th 40 40 **00.41±0.015a **00.44±0.014b *00.23±0.005a *00.83±0.014a

6th 40 40 **00.37±0.014a **00.41±0.012b **00.19±0.008bc **00.49±0.014b

8th 40 40 **00.38±0.013a **00.39±0.013b **00.17±0.006c **00.38±0.012c

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99

Fig 4.20: Week wise comparisons of total Monocytic count (x109/L) in healthy and carrier horses and mules. (Mean ± SE)

Total Eosinophilic count:

The result of total eosinophilic count in carrier horses and carrier mules for

strangles is shown in table 4.35. Total eosinophilic count in carrier horses was

00.47±0.015x109/L at 2nd week, 00.30±0.017x109/L at 4th week, 00.41±0.010x109/L

at 6th week and 00.45±0.017x109/L at 8th week of recovery. When compared carrier

horses with healthy horses the difference of total eosinophilic count was observed non

significant (P>0.05).

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100

Table 4.35: Total Eosinophil count (x109/L) in healthy and carrier horses and mules from strangles. (Mean ± SE)

Mean in a column followed by the same letter were not significantly different at P≤0.05, by Tukey HSD test. * indicates significant difference (p<0.05) among healthy and diseased groups ** indicates non-significant difference (p>0.05) between healthy and diseased group

Total eosinophilic count in carrier mules was 00.49±0.014x109/L at 2nd week,

00.51±0.013x109/L at 4th week, 00.47±0.010x109/L at 6th week and

00.40±0.011x109/L at 8th week of recovery. Similarly, the difference of total

eosinophilic count of carrier horses and carrier mules was observed non significant

(P>0.05) among all eight weeks after recovery. Difference between the values of

carrier and healthy mules was also non significant. When compared carrier horses

with carrier mules the difference of total eosinophilic count was observed non

significant (P>0.05) as shown in fig. 4.21.

Weeks After

Recovery

N Horses Mules

Horses Mules Healthy

n=20 Carrier

n=20 Healthy

n=20 Carrier

n=20

2nd 40 40 **00.49±0.017a **00.47±0.015a **00.29±0.011a **00.49±0.014a

4th 40 40 **00.44±0.010b **00.30±0.017c **00.31±0.012a **00.51±0.013a

6th 40 40 **00.48±0.015ab **00.41±0.010b **00.31±0.008a **00.47±0.010a

8th 40 40 **00.47±0.007ab **00.45±0.017ab **00.30±0.007a **00.40±0.011b

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101

Fig 4.21: Week wise comparisons of total eosinophilic count (x109/L) in healthy and carrier horses and mules. (Mean ± SE)

Total Basophil count:

Data regarding total basophilic count in carrier horses and carrier mules from

strangles is shown in table 4.36. The result of total basophilic count in carrier horses

was 00.39±0.011x109/L at 2nd week, 00.46±0.016x109/L at 4th week,

00.09±0.015x109/L at 6th week and 00.04±0.005x109/L at 8th week of recovery.

When compared carrier and healthy horses, significant increase was observed up to

4th week of recovery while on the 6th and 8th week of recovery this increase was non

significant (P>0.05).

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102

Table 4.36: Total Basophil count (x109/L) in healthy and carrier horses and mules from strangles. (Mean ± SE)

Mean in a column followed by the same letter were not significantly different at P≤0.05, by Tukey HSD test. * indicates significant difference (p<0.05) among healthy and diseased groups ** indicates non-significant difference (p>0.05) between healthy and diseased group

Total basophilic count in carrier mules was 00.19±0.010x109/L at the end of

2nd week, 00.14±0.011x109/L at the end of 4th week, 00.21±0.009x109/L at the end

of 6th week and 00.07±0.012x109/L at the end of 8th week of recovery. When carrier

mules compared with healthy mules, the difference of total basophilic count was

observed non significant (P>0.05). When the result of carrier horses compared with

carrier mules no significant increase (P>0.05) was observed as shown in fig. 4.22.

Weeks After

Recovery

N Horses Mules

Horses Mules Healthy

n=20 Carrier

n=20 Healthy

n=20 Carrier

n=20

2nd 40 40 *00.06±0.005a *00.39±0.011b **00.04±0.004b **00.19±0.010a

4th 40 40 *00.04±0.005b *00.46±0.016a **00.05±0.005ab **00.14±0.011b

6th 40 40 **00.03±0.004bc **00.09±0.015c **00.06±0.005a **00.21±0.009a

8th 40 40 **00.02±0.006c **00.04±0.005d **00.01±0.003c **00.07±0.012c

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103

Fig 4.22: Week wise comparisons of total Basophilic count (x109/L) in healthy and carrier horses and mules. (Mean ± SE)

Table 4.30: Total Erythrocytic count:

Data on total erythrocytes count in carrier horses and carrier mules for

strangles is shown in table 4.37. Total erythrocyte count in carrier horses was

5.88±0.15x 1012/L at 2nd week, 5.84±0.09x1012/L at 4th week, 5.76±0.11x 1012/L at

6th week and 5.70±0.06x 1012/L at 8th weeks of recovery. When compared carrier

horses with healthy horses, the decrease of total erythrocytes count was observed non

significant (P>0.05) among all eight weeks of recovery.

Total erythrocytic count in carrier mules was 6.17±0.08x 1012/L at the end of

2nd week, 6.11±0.07x 1012/L at the end of 4th week, 6.50±0.05x 1012/L at the end of

6th week and 6.59±0.08x 1012/L at the end of 8th week of recovery. When carrier

mules compared with healthy mules, the decreases of total erythrocytes count were

observed non significant (P>0.05) as in carrier horses. Similarly when carrier horses

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104

compared with carrier mules, the difference of total erythrocytes count was observed

non significant (P>0.05) upto 8th week of recovery as shown in fig. 4.23.

Table 4.37: Erythrocytes count (X 1012/L) in healthy and carrier horses and mules from strangles. (Mean ± SE)

Mean in a column followed by the same letter were not significantly different at P≤0.05, by Tukey HSD test. * indicates significant difference (p<0.05) among healthy and diseased groups ** indicates non-significant difference (p>0.05) between healthy and diseased group

Fig 4.23: Week wise comparisons of Erythrocytes count (X 1012/L) in healthy and carrier horses and mules. (Mean ± SE)

Weeks After

Recovery

N Horses Mules

Horses Mules Healthy

n=20 Carrier

n=20 Healthy

n=20 Carrier

n=20

2nd 40 40 **6.14±0.05a **5.88±0.15a **6.67±0.08a **6.17±0.08b

4th 40 40 **6.08±0.06a **5.84±0.09a **6.59±0.10a **6.11±0.07b

6th 40 40 **6.01±0.05a **5.76±0.11a **6.61±0.09a **6.50±0.05a

8th 40 40 **5.98±0.06a **5.70±0.06a **6.57±0.09a **6.59±0.08a

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Packed cell volume:

Data regarding packed cell volume in carrier horses and carrier mules from

strangles is shown in table 4.38. In carrier horses packed cell volume was

28.87±0.23% at the 2nd week, 33.08±0.09% at the 4th week, 31.54±0.25% at the 6th

week and 29.22±0.31% at the end of 8th week of recovery. When carrier horses

compared with healthy horses, the decrease of packed cell volume was observed non

significant (P>0.05).

In carrier mules packed cell volume was 29.63±0.23% at the 2nd week,

34.15±0.26% at the 4th week, 32.11±0.51% at the 6th week and 33.57±0.39% at the

end of 8th week of recovery. When compared carrier and healthy mules the difference

of packed cell volume was observed as non significant (P>0.05) in all weeks after

recovery. Similarly the difference of carrier horses with carrier mules packed cell

volume was observed alsonon significant (P>0.05) as shown in fig. 4.24.

Table 4.38: Packed cell volume (%) in healthy and carrier horses and mules from strangles. (Mean ± SE)

Mean in a column followed by the same letter were not significantly different at P≤0.05, by Tukey HSD test. * indicates significant difference (p<0.05) among healthy and diseased groups ** indicates non-significant difference (p>0.05) between healthy and diseased group

Weeks After

Recovery

N Horses Mules

Horses Mules Healthy

n=20 Carrier

n=20 Healthy

n=20 Carrier

n=20

2nd 40 40 **34.98±0.37a **28.87±0.23c **36.82±0.21ab **29.63±0.23c

4th 40 40 **35.75±0.24a **33.08±0.09a **37.10±0.18a **34.15±0.26a

6th 40 40 **33.60±0.33b **31.54±0.25b **36.09±0.26bc **32.11±0.51b

8th 40 40 **35.48±0.29a **29.22±0.31c **35.81±0.26c **33.57±0.39a

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106

Fig 4.24: Week wise comparisons of packed cell volume (%) in healthy and carrier horses and mules. (Mean ± SE)

Haemoglobin concentration:

The result of haemoglobin concentration in carrier horses and carrier mules

for strangles is shown in table 4.39. Haemoglobin concentration in carrier horses was

111.32±0.53g/L at 2nd week, 106.87±0.34g/L at 4th week, 105.34±0.29at 6th week

and 110.91±0.60at 8th week of recovery. When carrier and healthy horses compared,

the decrease of haemoglobin concentration was observed non significant (P>0.05). It

was also noted that this decrease of haemoglobin concentration was more in carrier

mules than in carrier horses.

Similarly haemoglobin concentration in carrier mules was 125.53±0.41g/L at

2nd week, 129.20±0.46g/L at 4th week, 130.61±0.42g/L at 6th week and

131.12±0.55g/L at 8th week of recovery. When carrier and healthy mules compared

the decrease of haemoglobin concentration was observed non significant (P>0.05)

during all weeks after recovery. When carrier horses compared with carrier mules the

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107

haemoglobin concentration was observed non significant (P>0.05) as shown in fig.

4.25.

Table 4.39: Haemoglobin concentration (g/L) in healthy and carrier horses and

mules from strangles. (Mean ± SE)

Mean in a column followed by the same letter were not significantly different at P≤0.05, by Tukey HSD test. * indicates significant difference (p<0.05) among healthy and diseased groups ** indicates non-significant difference (p>0.05) between healthy and diseased group

Fig 4.25: Week wise comparisons of Hb concentration (g/L) in healthy and carrier horses and mules. (Mean ± SE)

Weeks After

Recovery

N Horses Mules

Horses Mules Healthy

n=20 Carrier n=20

Healthy n=20

Carrier n=20

2nd 40 40 **124.51±0.37a **111.32±0.53a **141.81±0.79a **125.53±0.41c

4th 40 40 **115.43±0.84c **106.87±0.34b **137.73±0.40bc **129.20±0.46b

6th 40 40 **110.89±0.52d **105.34±0.29b **139.48±0.59b **130.61±0.42ab

8th 40 40 **119.37±0.47b **110.91±0.60a **136.22±0.26c **131.12±0.55a

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108

Protein Analysis:

For protein analysis 40 equids (n=20 horses, n= 20 mules) under 5 years of

age suffering from natural outbreak of strangles from Lahore and Sargodha districts

of Punjab province of Pakistan were selected for recording of observations.

Parameters studied included, total serum protein, serum albumin, serum globulin and

fibrinogen.

Total Serum Proteins: Total serum protein values in horses and mules suffering from strangles are

shown in table 4.40. In horses total serum protein values 82.7±3.3g/L under one year

of age, 80.9±5.7g/L between 1-2 year of age, 79.3±6.4g/L between 2-3 year of age,

78.8±5.3g/L between 3-4 year of age and78.5±4.7g/L between 4-5 year of age were

observed. When compared total serum protein values of diseased and healthy horses

on age basis, a significant increase (P<0.05) was observed in all age groups. Among

all age groups total serum protein values were according to the following order 5th <

4th < 3rd< 2nd < 1st week. It was also observed that total serum protein value was

highest in group which was under one year of age, it significantly decreased to the

normal during the subsequent weeks (P<0.05).

In mules total serum protein values 81.5±5.3g/L under one year of age,

83.1±6.3g/L between 1-2 years of age, 80.1±6.4g/L between 2-3 years of age,

79.3±5.5g/L between 3-4 years of age and78.4±4.8g/L between 4-5 years of age were

observed. When compared total serum protein values of diseased and healthy mules

of different age groups, a significant increase (P<0.05) was observed in all age

groups. Among all age groups total serum protein values were according to the

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109

following order 5th < 4th < 3rd< 2nd < 1st week. It was also observed that total serum

protein values were highest in group having age under one year which significantly

decreased to the normal in the subsequent age groups (P<0.05).When diseased horses

compared with diseased mules, no significant difference was observed while total

serum protein values were significantly less (P<0.05) in healthy horses and mules

than diseased as shown in fig. 4.26.

Table 4.40: Total serum protein values (g/L) of healthy and diseased horses and mules suffered from strangles. (Mean ± SD)

Mean in a row followed by the same letter were not significantly different at P≤0.05, by paired T test.

Fig 4.26: Age wise comparison of total serum protein values (g/L) of healthy and diseased horses and mules suffered from strangles. (Mean ± SD)

Groups N

Horses Mules Horses Mules

Healthy Diseased Healthy Diseased Healthy Diseased Healthy Diseased

< 1year 10 10 10 10 64.9a ±3.4

82.7b ±3.3

65.8a ± 3.5

81.5b ± 5.3

1-2 year

10 10 10 10 66.5a ±3.1

80.9b ±5.7

68.0a ± 3.8

83.1b ± 6.3

2-3 year

10 10 10 10 62.7a ±1.2

79.3b ±6.4

67.3a ± 4.4

80.1b ±6.4

3-4 year

10 10 10 06 63.6a ±5.8

78.8b ±5.3

64.7a ± 4.8

79.3b ±5.5

4-5 year

10 10 10 03 64.1a ±4.1

78.5b ±4.7

65.0a ± 4.4

78.4b ±4.8

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110

Serum Albumin:

Data on serum albumin in horses and mules suffering from strangles are

shown in table 4.41. In horses serum albumin values 25.4±2.9g/L under one year of

age, 27.1±3.9g/L between 1-2 years of age, 24.8±4.4g/L between 2-3 years of age,

26.2±4.5g/L between 3-4 years of age and28.4±5.4g/L between 4-5 years of age were

observed. When compared serum albumin values of diseased and healthy horses on

age basis significant decrease (P<0.05) was observed in all age groups.

In mules serum albumin values 26.3±4.2g/L under one year of age,

26.1±4.2g/L between 1-2 years of age, 27.5±4.0g/L between 2-3 years of age,

28.3±5.1g/L between 3-4 years of age and28.7±5.4g/L between 4-5 years of age were

observed. Age based comparison of serum albumin values of diseased and healthy

horses revealed a significant decrease (P<0.05).

Table 4.41: Serum albumin values (g/L) of healthy and diseased horses and mules suffered from strangles. (Mean ± SD)

Mean in a row followed by the same letter were not significantly different at P≤0.05, by paired T test.

Groups N

Horses Mules Horses Mules

Healthy Diseased Healthy Diseased Healthy Diseased Healthy Diseased

< 1year 10 10 10 10 33.8a

±2.2

25.4b

±2.9

34.4a

± 2.6

26.3b

±4.2

1-2 year 10 10 10 10 36.3a

±2.4

27.1b

±3.9

34.3a

± 3.0

26.1b

±4.2

2-3 year 10 10 10 10 34.7a

±4.4

24.8b

±4.4

36.1a

± 5.2

27.5b

±4.0

3-4 year 10 10 10 06 35.3a

±5.3

26.2b

±4.5

35.2a

± 5.1

28.3b

±5.1

4-5 year 10 10 10 03 34.5a

±4.1

28.4b

±5.4

36.5a

± 4.9

28.7b

±5.4

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111

Fig 4.27: Age wise comparison of serum albumin values (g/L) of healthy and diseased horses and mules suffered from strangles. (Mean ± SD)

When diseased horses compared with diseased mules, no significant

difference was observed while serum albumin values were significantly less (P<0.05)

in diseased horses and mules than healthy as shown in fig. 4.27.

Serum Globulin:

Serum globulin in horses and mules suffering from strangles are shown in

table 4.42. In horses serum globulin values 37.1±3.4g/L under one year of age,

36.7±4.5g/L between 1-2 years of age, 35.2±4.0g/L between 2-3 years of age,

36.7±2.7g/L between 3-4 years of age and 32.9±1.9g/L between 4-5 years of age

were observed. When compared serum albumin values of diseased and healthy horses

on age basis, a significant increase (P<0.05) was observed in group having age under

one year, while rest of the age groups showed a non significant increase.

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112

Table 4.42: Serum globulin values (g/L) of healthy and diseased horses and mules suffered from strangles. (Mean ± SD)

Mean in a row followed by the same letter were not significantly different at P≤0.05, by paired T test.

Fig 4.28: Age wise comparison of serum globulin values (g/L) of healthy and diseased horses and mules suffered from strangles. (Mean ± SD)

Groups N

Horses Mules Horses Mules

Healthy Diseased Healthy Diseased Healthy Diseased Healthy Diseased

< 1year 10 10 10 10 32.9a

±1.8

37.1b

±3.4

33.7a

± 1.2

36.2b

±3.8

1-2 year 10 10 10 10 34.5a

±3.0

36.7a

±4.5

33.6a

± 2.9

37.3b

±4.3

2-3 year 10 10 10 10 33.2a

±6.5

35.2a

±4.0

33.9a

± 5.6

35.3a

±4.0

3-4 year 10 10 10 06 34.4a

±3.2

36.7a

±2.7

33.5a

± 2.7

34.9a

±3.5

4-5 year 10 10 10 03 33.7a

±4.4

32.9a

±1.9

33.8a

± 4.5

33.1a

±1.9

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113

In mules serum albumin values 36.2±3.8g/L under one year of age,

37.3.1±4.3g/L between 1-2 years of age, 35.3±4.0g/L between 2-3 years of age,

34.9±3.5g/L between 3-4 years of age and33.1±1.9g/L between 4-5 years of age were

observed. When compared serum albumin values of diseased and healthy horses on

age basis, a significant increase (P<0.05) was observed in group having age from one

to two years. It was different from the horses where significant increase was observed

only in the group which was under one year of age. When diseased horses compared

with diseased mules, no significant difference was observed while serum globulin

values were increased but not significantly in diseased horses and mules than healthy

as shown in fig. 4.28.

Fibrinogen values:

Data on fibrinogen values in horses and mules suffering from strangles are

shown in table 4.43. In horses fibrinogen values 7.2±2.3g/L under one year of age,

6.7±2.4g/L between 1-2 years of age, 6.3±2.2g/L between 2-3 years of age,

5.7±1.0g/L between 3-4 years of age and 5.1±0.7g/L between 4-5 years of age were

observed. Among all age groups fibrinogen values were according to the following

order 5th < 4th < 3rd< 2nd < 1st week. It was also observed that fibrinogen values

were highest in under one year of age group which significantly decreased to the

normal values in the subsequent age groups (P<0.05). When compared fibrinogen

values of diseased and healthy horses on age basis significant increase (P<0.05) was

observed in all age groups that indicate significance of hyperfibrinegemia for

diagnosis of diseased.

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114

In mules fibrinogen values 6.5±3.1g/L under one year of age, 7.0±2.6g/L

between 1-2 years of age, 6.0±2.3g/L between 2-3 years of age, 5.0±1.4g/L between

3-4 years of age and 4.5±1.2g/L between 4-5 years of age were observed. Similarly in

mules fibrinogen values were according to the following order 5th < 4th < 3rd< 2nd <

1st week in all age groups. It was also observed that fibrinogen values were highest

under one year of age group which significantly decreased to the normal in the

subsequent age groups (P<0.05).When compared fibrinogen values of diseased and

healthy horses on age basis significant increase (P<0.05) was observed in all age

groups that was similar to horses. When diseased horses compared with diseased

mules, no significant difference was observed while fibrinogen values were

significantly increased (P<0.05) in diseased horses and mules than healthy as shown

in fig. 4.29.

Table 4.43: Fibrinogen values (g/L) of healthy and diseased horses and mules suffered from strangles. (Mean ± SD)

Mean in a row followed by the same letter were not significantly different at P≤0.05, by paired T test.

Groups N

Horses Mules Horses Mules

Healthy Diseased Healthy Diseased Healthy Diseased Healthy Diseased

< 1year 10 10 10 10 03.4a

±1.0

07.2b

±2.3

03.3a

±1.0

06.5b

±3.1

1-2 year 10 10 10 10 03.8a

±1.0

06.7b

±2.4

03.6a

±1.2

07.0b

±2.6

2-3 year 10 10 10 10 03.5a

±1.1

06.3b

±2.2

03.9a

±1.5

06.0b

±2.3

3-4 year 10 10 10 06 03.7a

±0.7

05.7b

±1.0

03.6a

±0.9

05.0b

±1.4

4-5 year 10 10 10 03 03.6a

±1.4

05.1b

±0.7

03.8a

±1.4

04.5b

±1.2

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115

Fig 4.29: Age wise comparison of fibrinogen values (g/L) of healthy and diseased horses and mules suffered from strangles. (Mean ± SD)

THERAPEUTIC TRIALS:

In-vitro Antibiotic sensitivity test of horses:

Data on twenty randomly selected β hemolytic colonies of horses

subjected to sensitivity of ten antibiotics is shown in table 4.44. It was observed that

higher number of field samples of S. equi were sensitive to Procaine penicillin

followed by ceftiofur Na, cephradine, erythromycin, ampicillin, tetracycline,

chloramphenicol, sulfamethoxazole, trimethoprim +sulphadiazine and gentamycin,

respectively. It is concluded from the result of present study that field isolate of S.

equi is still more sensitive to Procaine penicillin, ceftiofur Na, cephradine and

erythromycin as compared to other antibiotics.

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116

Table 4.44: In-vitro Antibiotic sensitivity against S. equi in horses

ZI= zone of inhibition

In-vitro Antibiotic sensitivity test of mules:

Data regarding, twenty randomly selected β hemolytic colonies of S. Equi of

mules subjected to sensitivity of ten antibiotics is shown in table 4.45. In the present

study it was also observed that higher number of field isolates of S. equi were

sensitive to Procaine penicillin followed by ceftiofur Na, erythromycin, cephradine,

ampicillin, tetracycline, chloramphenicol, sulfamethoxazole, trimethoprim +

sulphadiazine and gentamycin that was similar to horses.

Antibiotics Sensitive Discs No. of Horses

Remarks Sensitive Intermediate Resistant

No. of isolates

ZI (mm)

No. of isolates

ZI (mm)

No. of isolates

ZI (mm)

Procaine Penicillin 20 19 >30 01 28-29 - <28

Ceftiofur Na 20 18 >27 01 25-26 01 <25

Cephradine 20 16 >25 02 23-24 02 <23

Erythromycin 20 17 >22 02 20-21 01 <20

Tetracycline 20 12 >16 06 14-15 02 <14

Sulfamethoxazole 20 13 >14 04 12-13 03 <12

Trimethoprim+sulphadiazine 20 12 >12 03 10-11 05 <10

Chloramphenicol 20 13 >15 05 12-14 02 <12

Ampicillin 20 14 >19 04 15-18 02 <15

Gentamycin 20 12 >10 06 7-9 02 <7

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117

Table 4.45: In-vitro Antibiotic sensitivity against S. equi in mules

Antibiotics Sensitive Discs No. of Mules

Remarks Sensitive Intermediate Resistant

No. of isolates

ZI (mm)

No. of isolates

ZI (mm)

No. of isolates

ZI (mm)

Procaine Penicillin 20 19 >30 01 28-29 - <28

Ceftiofur Na 20 18 >27 02 25-26 - <25

Cephradine 20 15 >25 04 23-24 01 <23

Erythromycin 20 16 >22 02 20-21 02 <20

Tetracycline 20 13 >16 04 14-15 03 <14

Sulfamethoxazole 20 15 >14 03 12-13 02 <12

Trimethoprim+sulphadiazine 20 14 >12 04 10-11 02 <10

Chloramphenicol 20 12 >15 04 12-14 04 <12

Ampicillin 20 15 >19 02 15-18 03 <15

Gentamycin 20 14 >10 05 7-9 01 <7

ZI= zone of inhibition

In-vivo Antibiotic sensitivity test of horses:

Out of ten antibiotics used in-vitro antibiotic sensitivity test against S. equi,

four top ranking antibiotics were selected for in-vivo trials in horses.

Group A:

The ten horses (n=5 without abscess, n=5 with abscess) of group A were

treated with rank 1 antibiotic i.e. Procaine Penicillin is shown in table 4.46. The

efficacy of Procaine Penicillin was measured on the basis of disappearance of clinical

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118

signs. Response to the antibiotic in the horses without abscess was excellent while the

horses with abscess exhibited a poor response.

Group B:

The ten horses (n=5 without abscess, n=5 with abscess) of group B were

treated with rank 2 antibiotic i.e. ceftiofur Na is shown in table 4.46. The criteria for

measuring the efficacy of ceftiofur Na was same as in Group A. The results in horses

without abscess were very good while the abscessed horses showed poor results.

Group C:

The ten horses (n=5 without abscess, n=5 with abscess) of group C were

treated with rank 3 antibiotic i.e. cephradine is shown in table 4.46. The efficacy of

cephradine was measured on similar basis as in above mentioned groups. The results

showed good response in horses without abscess whereas the response was poor in

horses with abscess.

Group D:

The ten horses (n=5 without abscess, n=5 with abscess) of group D were

treated with rank 4 antibiotic i.e. erythromycin is shown in table 4.46. The efficacy of

erythromycin was measured on the basis of disappearance of clinical signs in horses.

Like the other groups, the results in horses without abscess were good while the

horses with abscess showed poor results.

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119

Table 4.46: In-vivo Antibiotic sensitivity against S. equi in horses

Groups Antibiotics used No. of horses (n=10)

Without abscess(n=5)

With abscess(n=5)

A Procaine Penicillin Excellent poor

B Ceftiofur Na Very good poor

C Cephradine Good poor

D Erythromycin Good poor

In-vivo Antibiotic sensitivity test of mules:

Similarly in mules out of ten antibiotics, four top ranked antibiotics were

selected on the basis of in-vitro antibiotic sensitivity test and used as in-vivo trials in

mules.

Group A:

A group of ten mules comprised of n=5 without abscess, n=5 with abscess of

group A were treated with rank 1 antibiotic i.e. Procaine Penicillin is shown in table

4.47. The efficacy of procaine penicillin was measured on the basis of disappearance

of clinical signs in the mules. The results of present study showed excellent response

in mules without abscess while the response was poor in mules with abscess.

Group B:

A group of ten mules comprised of n=5 without abscess, n=5 with abscess of

group B were treated with rank 2 antibiotic i.e. Ceftiofur Na, results are shown in

table 4.47. The efficacy of Ceftiofur Na was measured on similar basis as in group A.

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120

The results in mules without abscess were very good while in mules with abscess

were poor.

Group C:

A group of ten mules (n=5 without abscess, n=5 with abscess) of group C

were treated with rank 3 antibiotic i.e. Cephradine, results are shown in table 4.47.

The criteria for measuring the efficacy of antibiotics was the disappearance of clinical

signs and results obtained in mules without abscess were good while poor in

abscessed mules.

Group D:

A group of ten mules (n=5 without abscess, n=5 with abscess) of group D

were treated with rank 4 antibiotic i.e. erythromycin, results are shown in table 4.47.

The efficacy of erythromycin was measured on the basis of disappearance of clinical

signs in the mules and similar results were obtained as in Group C.

Table 4.47: In-vivo Antibiotic sensitivity against S. equi mules

Groups Antibiotics used No. of mules (n=10)

Without abscess(n=5)

With abscess(n=5)

A Procaine Penicillin Very good poor

B Ceftiofur Na Excellent poor

C Cephradine Good poor

D Erythromycin Good poor

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121

It is concluded from the result of present study that treatment should be started

as early as possible before the appearance of abscess in both horses and mules.

In-vitro Disinfectant Trial:

In-vitro testing of S. equi with various disinfectants was performed by using

the Phenol Co-efficient Test to determine susceptibility of bacteria. For that purpose,

four disinfectants Dettol, Povidone iodine, 0.6% H2SO4 and Bleach were used in the

study.

In-vitro Disinfectant trial of Phenol:

Data regarding the growth of S. equi after using phenol as disinfectant in

different interval of time is shown in table 4.48. In-vitro disinfectant trial of Phenol

was made to compare with other disinfectants used in the study. For that purpose six

serial dilutions of phenol were made in separate tubes as follows 1:50, 1:100, 1:150,

1:200, 1:250 and 1:300. Samples from these tubes were then plated on blood agar

plates after 5, 10 and 15 minutes. The highest dilution of phenol was recorded as

1:200.

Table 4.48: In-Vitro efficacy of Phenol as disinfectant against S. equi

Phenol

Dilution 5 minutes 10 minutes 15minutes

1 to 50 No growth No growth No growth

1 to 100 Growth No growth No growth

1 to 150 Growth No growth No growth

1 to 200 Growth No growth No growth

1 to 250 Growth Growth No growth

1 to 300 Growth Growth No growth

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In-vitro Disinfectant trial of Dettol:

The result on the growth of S. equi after using dettol as disinfectant is given in

table 4.49. For in-vitro disinfectant trial of dettol, six serial dilutions were made as

follows 1:50, 1:100, 1:150, 1:200, 1:250 and 1:300 in separate tubes. The time used to

take the samples from the tubes and to check the growth on the blood agar was same

as in phenol. After that highest dilution of dettol was recorded as 1:100 while phenol

was 1:200. The phenol coefficient was calculated by dividing the highest dilution of

dettol over highest dilution of phenol.

Table 4.49: In-Vitro efficacy of Dettol as disinfectant against S. equi

Dettol

Dilution 5 minutes 10 minutes 15minutes

1 to 50 No growth No growth No growth

1 to 100 Growth No growth No growth

1 to 150 Growth Growth No growth

1 to 200 Growth Growth Growth

1 to 250 Growth Growth Growth

1 to 300 Growth Growth Growth

Phenol Coefficient=100/200=0.5

In-vitro Disinfectant trial of Povidone Iodine:

Data on the growth of S. equi after using povidone iodine as disinfectant is

given in table 4.50. For povidone iodine in-vitro disinfectant trial six serial dilutions

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were made as were in phenol and dettol. The time used to take samples was 5, 10 and

15 minutes. The highest dilution of povidone iodine was recorded as 1:250 and the

phenol coefficient was calculated as 1.25.

Table 4.50: In-Vitro efficacy of Povidone Iodine as disinfectant against S. equi

Povidone

Iodine

Dilution 5 minutes 10 minutes 15minutes

1 to 50 No growth No growth No growth

1 to 100 Growth No growth No growth

1 to 150 Growth No growth No growth

1 to 200 Growth No growth No growth

1 to 250 Growth No growth No growth

1 to 300 Growth Growth No growth

Phenol Coefficient=250/200=1.25

In-vitro Disinfectant trial of 0.6%H2SO4:

The result on the growth of S. equi after using 0.6% H2SO4 as disinfectant is

given in table 4.51. For in-vitro disinfectant trial of 0.6% H2SO4 six serial dilutions

were made as in above disinfectants. The time used to take samples from the dilutions

was same as in above disinfectants. The highest dilution was recorded as 1:200 and

phenol coefficient was 1.00.

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Table 4.51: In-Vitro efficacy of 0.6% Sulfuric acid as disinfectant against S. equi

Phenol Coefficient=200/200=1.00

In-vitro Disinfectant trial of Bleach:

The results on the growth of S. equi after using bleach as disinfectant are

given in table 4.52. For in-vitro disinfectant trial of bleach serial dilutions were made

as in above disinfectants. Samples from different dilutions were taken after the same

time as in other disinfectants used in the study. Highest dilution was 1:150. The

phenol coefficient was calculated 0.75.

Table 4.52: In-Vitro efficacy of Bleach as disinfectant against S. equi

Bleach

Dilution 5 minutes 10 minutes 15minutes

1 to 50 No growth No growth No growth

1 to 100 Growth No growth No growth

1 to 150 Growth No growth No growth

1 to 200 Growth Growth No growth

1 to 250 Growth Growth No growth

1 to 300 Growth Growth No growth

Phenol Coefficient=150/200=0.75

0.6%H2SO4

Dilution 5 minutes 10 minutes 15minutes

1 to 50 No growth No growth No growth

1 to 100 Growth No growth No growth

1 to 150 Growth No growth No growth

1 to 200 Growth No growth No growth

1 to 250 Growth Growth No growth

1 to 300 Growth Growth No growth

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Comparison of Disinfectants:

When compared all four disinfectants used against S. equi in the present study

with phenol by using the Phenol Co-efficient Test, povidone iodine was found to be

the best one because its phenol coefficient was 1.25 that was greater than phenol 1.00

while 0.6% H2SO4 showed similar phenol coefficient as that of phenol. The dettol and

bleach showed phenol coefficient lesser than phenol as shown in table 4.53. Therefore

use of povidone iodine and 0.6% H2SO4 on priority basis against S. equi is

recommended as compared to other disinfectants.

Table 4.53: Overall comparison of Different Disinfectants used against S. equi

Disinfectants Highest Dilution of disinfectant

Highest dilution of

Phenol

Phenol Coefficient

Remarks

Dettol 1:100 1:200 100/200=0.5 Fair

Povidone Iodine 1:250 1:200 250/200=1.25 Very good

Bleach 1:150 1:200 150/200=0.75 Satisfactory

0.6%H2SO4 1:200 1:200 200/200=1.00 Good

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Chapter-05

DISCUSSION

EPIDEMIOLOGICAL STUDIES:

PREVALENCE OF HORSES AND MULES:

Data on the overall prevalence of S. equi on the basis of culture in horses and

mules is shown in table 4.1 and 4.7. Out of 250 horses and 250 mules, 113(45.2%)

horses and 99 (39.6%) mules tested positive for S. equi. Minett, 1944 and Peat, 1945

reported the incidence of strangles in both horses and mules at Remount depot Mona

and Sargodha, Pakistan. The number of S. equi isolates were significantly higher

(P<0.05) in pus samples collected from sub-mandibular lymph nodes as compared to

nasal discharge samples. The difference in number was found significant (P<0.05)

among horses and mules of different age groups. The highest prevalence of strangles

was recorded in horses and mules aging less than 2 years as compared to those having

age more than 2 years. Similar findings were reported by Fallon, 1969. He reported

that the disease is more prevalent in young animals, especially in populations with a

prior history of strangles outbreaks. Results of our study also correlate with the

findings of Timoney, 1993 who also reported that horses of all ages may be affected,

but the disease is most common and most severe in young horses. It is therefore

mostly prevalent on breeding farms. Outbreaks are often initiated by the introduction

of an animal to the farm that is either incubating the disease or is still shedding the

organism during the recovery phase. Sweeny, 1987 and Piche, 1984 observed that

yearlings and young adults are most at risk, followed by weanlings and then adults.

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Typically, yearlings are most severely affected with a longer duration of clinical signs

(Piche, 1984). During the breeding season, nursing mares brought of suckling foals

may introduce S. equi in this manner. Findings of our study are also in line with the

findings of Sweeny, 1990 who reported that infection occurs primarily in 1 to 5 years

old horses, but is not restricted to age groups. As regards incidence of strangles from

the end of January to the beginning of May was found to be the highest (2.6%) in

foals of 9 months–2 years compared to adult mules 2–5 years old, confirming that

once an animal is infected with strangles it attains lifelong immunity (Walker and

Timoney, 1998). Our results were also broadly consistent with the findings of Ashraf

(2000) who reported 33.2% strangles in Pakistan in mules less than 2 year of age and

35.4% in mules of more than 2 year of age. Similarly, results of the present study are

in agreement with the findings of Manzoor et al. 2008 who reported 54% infection in

foals in Punjab, Pakistan. Our findings are also in agreement with the results of

Sweeney et al., 1989 who found rates of S. equi infections of the upper respiratory

tract and lymph nodes (strangles) in horses to be 47.5% for 1-year-old horses, and

37.5% for foals. S. equi was isolated from nasal, pharyngeal, or lymph node

specimens in 31 (60.8%) of 51 sick horses. Our results also correlate with the study of

Hamlen et al., 1994 who reported that foals were highly susceptible to developing

strangles following S. equi exposure as shown by attack rates of 86% (19/22) and

91% (10/11) respectively.

In the present study the prevalence of strangles in horses and mules were also

calculated round the year, it was found to be the highest during the months of

February, March, April and May, while few cases were seen during the months of

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January, June and July and no cases were seen during rest of the months. The

significant difference was observed (p<0.05) among different months of year.

Similarly when compared the prevalence of strangles in different seasons of Pakistan,

the highest prevalence rate was recorded during the spring months. Our study

correlates with the findings of Manzoor et al., 2008 who recorded incidence of

strangles in foals of 9 months to 2 years of age and it was found to be the highest

during the spring season (Mid of January to Start of May).

Data regarding the overall prevalence of strangles on the basis of Polymerase

chain reaction of S. equi in equines is also shown in table 4.4 and 4.10. Out of Equine

groups of 250 horses and mules each, 122(48.8%) horses and 113(45.2%) mules

tested positive for S. equi. On comparison of the prevalence rate on the basis of PCR

and culture, (nasal swabs or pus samples from affected submandibular lymph nodes)

the sensitivity of Polymerase chain reaction appeared to be much greater than culture.

Laboratory experimentation has demonstrated a detection sensitivity of 10 or fewer

colony forming units (Timoney and Artiushin, 1997). The culture along with PCR is

best techniques for diagnosing S. equi. Culture is of value because it definitely

establishes infection and can conviently be performed on the same samples used for

PCR. Since PCR can be completed in four to five hours it should be an effective tool

in the management of outbreaks and in screening equines before and after

transportation (Timoney and Artiushin, 1997). Newton et al., 2000 reported that PCR

has been developed to detect the DNA sequence of the S. equi SeM gene, and can be

used to confirm the diagnosis of strangles within hours of sample submission.

Because this test does not differentiate between dead and live bacteria, a positive test

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may not correlate with active infection; therefore, a positive culture may be necessary

to confirm the diagnosis. Timoney and Artiushin, 1997 also reported that PCR is

approximately three times more sensitive than the culture in detecting S. equi. The

relative insensitivity of the culture may be due to an inadequate number of collected

organisms, overgrowth of contaminants, or slow growth.

MORTALITY RATE:

Data regarding the mortality rate in horses and mules are shown in table 4.13

and 4.14. In horses and mules the mortality was recorded 1.64% and 0.88%,

respectively under 5 years of age. The non significant (P>0.05) difference was

observed in mortality rate of affected equines among different age groups. From the

present study it was concluded that the severity of disease is greater in animals less

than 2 years of age as compared to over two year of age. The results of the present

study were also in line with the findings of Higgins and Snyder (2006) who reported

mortality rates between 1-5%. Mortality is generally low (1-2%) in uncomplicated

cases (McAllister, 1982; Radostits et al., 2000; Bryans and Moore, 1972; McGee,

1969). Radostits et al. (2000) also reported that most deaths are due to the

dissemination of organisms to the other organs. Various research workers reported

wide range of mortality in equines due to this disease which include Minett, 1944

who recorded mortality as 5% at Mona and 6.1% at Sargodha in horses and 1% in

mules at both the depots. Similarly Wilson, 1988 recorded mortality rates as 1-5%. A

2.6% mortality was recorded by Sweeny et al., 1989 whereas 1-2%, 4.4% and zero

percent mortality was recorded by Clabough, 1987, Vukovic, 1961 and Zedeh et al.,

1992 respectively.

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VARIATIONS IN SeM, SzPSe & Se 18.9 PROTEINS OF S.equi:

SeM:

PCR of the SeM genes of 24 selected isolates of S. equi and the US prototype

strain CF32 generated products of 1812 bp whereas resulting DNA sequences

encoded 19 different SeM alleles including numbers 71-76 not previously included in

the database (www.pubmlst.org/szooepidemicus/) (Table 4.16). The results confirmed

the great variability of the N-terminus of SeM noted in previous studies (Anzai et al.,

2005; Kelly et al., 2006). Residues most frequently subject to substitution were

located at position 58, 63, 108 and 143. Single nucleotide polymorphisms (SNPs) in

SeM were found at 93 loci and totaled 181. Fifty-eight of these were non-

synonymous, that is, were mutations resulting in amino acid replacements in SeM.

Non-synonymous SNPs were 15.7 times more frequent in the N-terminal region

(positions 114 to 629) than in the remainder of the SeM sequence (Table 4.18). These

results are consistent with a previous estimate (3.054) of the ratio of non-synonymous

to synonymous amino acid substitutions (dN/dS) in the SeM N-terminus (Kelly et al.,

2006). Discovery of non-synonymous substitutions distal to the N-terminus indicates

this region is also under diversifying selection pressure albeit of lower magnitude

than in the N-terminus. Substitution of amino acids in this region has been shown to

alter a conformational epitope, and is more likely due to immune selection of mucosal

IgA or local T cell rather than serum IgG responses (Timoney et al., 2009). Since

emergence of variants has been detected in chronically infected guttural pouches and

cranial sinuses, alteration in conformation of the N-terminus may serve as a

mechanism of immune escape in these niches as well as implicating an unknown

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interaction of the N-terminus of SeM with mucosal IgA or T-cell responses that

reduces survival of the bacterium. Fibrinogen binding or opsonization of S.equi by

SeM specific antibody are not significantly affected by N1 terminal variation

(Timoney et al., 2009). The survival value 1 of allelic variation seems to be restricted

to the mucosal compartment, since horses with chronic guttural pouch infections do

not experience recurrence of clinical strangles caused by the newly emerging SeM

allelic variants. In these cases, the acquired systemic immune response continues to

protect the animal’s regional lymph nodes and tonsils. Phylogenies generated by

neighborhood joining indicate that SeM allele 71 identified in isolates S24 and L32

from Pakistan was the most distantly related allele of the 25 isolates in the study. This

is explained by the remote and isolated Pakistani equid population, which

hypothetically favors preservation and continued divergence of a specific SeM allele.

Other newly identified alleles (72 - 76) in N. America isolates showed 96 - 99%

similarity with alleles 62, 59, 57 and 37 in the SeM database.

SzPSe:

With one exception, sequence analysis of the SzPSe genes provided no

instances of variation. The single exception, Australian isolate 181, had a deletion of

one PEPK repeat remarkably, although 92 SNPs were found at 48 loci in SzPSe of the

25 S. equi isolates including S. equi CF32, no SNPs encoding non-synonymous

substitutions were found (Table 4.19). In the horses, SzPSe elicits strong serum IgG

and mucosal IgA responses during recovery from strangles and so is exposed to

immune selection pressures similar to those exerted on SeM (Timoney et al., 2007)

although the results indicate that selection is purifying and not diversifying, moreover

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survival of S. equi in the horses requires conservation of SzPSe. This may be related

to a binding function of SzP to receptors in the crypts of the lingual and palatine

tonsils, sites of entry of S. equi (Kumar et al., 2007). The epitope responsible for

binding of SzP requires a specific conformation (Fan et al., 2008). Unlike S. equi, SzP

alleles in S. zooepidemicus are highly variable and are the basis for the Moore and

Bryans typing scheme (Moore and Bryans, 1969). The SzP family of proteins shows 2

forms of N-terminal variation, N1 and N2, at least 5 variants of a central non–alpha-

helical hypervariable region, HV1 to 5 and a variable number of carboxyterminal

PEPK repeats (Walker and Timoney, 1998). Thus, there is evidence of a high rate of

recombination in the SzP gene.

Moreover, the HV region appears to have been horizontally acquired since its

G-C % (38.3) differs significantly (p<0.01) from that of the remaining SzPSe

sequence (47.0). Recombination and the presence of exogenous DNA sequence are

factors that would favor occurrence of SNPs. The biological/immunological

significance of this variation is not understood, but does not appear to involve

opsonogenic epitopes. Future work might logically address the effect of variation on

the conformational adhesion epitope on host cell specificity.

Se18.9:

The action of highly conserved Se18.9 is anti-phagocytic in nature. It is

accomplished by binding complement control factor H and reducing the bactericidal

activity of neutrophils by an unknown mechanism (Tiwari et al., 2007). Uniquely

expressed by S. equi, Se18.9 binds to tonsillar epithelium and stimulates strong

mucosal IgA and serum IgG responses. These antibodies neutralize the bactericidal

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activity of Se18.9 of neutrophils. Since this protein has a proven virulence function in

common with SeM, it is logically a target of immune selection pressure yet only 2

SNP loci have been found in the DNA sequences of the Se18.9 genes in 25 isolates.

The unexpected absence of variants of Se18.9 in a population of SeM allelic variants

of S. equi argues either for an immutable and essential structure or virulence function

that is minor compared to that of SeM. It might also be argued that as a secreted

protein, Se18.9 might have less survival value for Se than a protein anchored on its

surface. The much lower frequency of SNP loci in Se18.9 compared to SeM and

SzPSe is unexplained.

Mutation plays an important role in a pathogen’s accommodation to adaptive

immune responses, which in turn favors its persistence and transmission to new hosts

(Aguileta et al., 2009). The remarkable genetic and phenotypic conservation of the

almost colonial S. equi compared to the closely related S. zooepidemicus is consistent

with its adaptation to a specific host. Variations in virulence proteins such as SzPSe

and Se18.9 may reduce its fitness to infect and replicate and so colonies of S. equi

with mutations that affect these proteins do not survive. In contrast, changes in the N7

terminal sequence of SeM provide a survival advantage in niches such as the guttural

pouch or cranial sinuses where mucosal responses dominate the host – parasite

interaction.

STUDY OF CARRIER STATUS OF HORSES & MULES:

Data regarding carrier state of naturally infected horses and mules are shown

in table 4.20 and 4.21. Out of 122 positive horses and 113 positive mules for

strangles, 20 horses and 20 mules (10 < 2 year and 10 between 2 and 5 years of age)

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remaining positive after one week of infection were monitored for 12 weeks to study

their carrier status. In horses after the end of 3rd week all horses < 2 years of age

were found positive but at the end of 4th to 7th weeks there remained 5, 2, 1 and zero

out of 10, whereas in mules after the end of 2nd week all mules < 2 years of age were

positive but at the end of 3rd to 6th weeks there remained 7, 3, 1 and zero mules out

of 10, respectively on the basis of culture. But when compared the results with PCR,

at the end of the 4th week all horse <2 years of age were positive, but at the end of 5th

to 10th weeks there remained 7, 5, 4, 2, 1 and zero horses out of 10, while in mules at

the end of the 5th week all mules < 2 years of age were positive, but at the end of 6th

to 10th weeks there remained 9, 7, 3, 2 and zero mules out of 10, respectively. The

carrier status in 2 and 5 year old horses and mules was also evaluated. All horses

were found positive up to the 1st week but at the end of 2nd to 8th weeks there

remained 9, 7, 6, 3, 1, 1 and zero out of 10, whereas in mules all were positive up to

the 2nd week but at the end of 3rd to 7th weeks there were 6, 4, 2, 1, 1 and zero out of

10 mules, respectively on the basis of culture. But through PCR, all horses were

found positive up to 4th week but at the end of 5th to 9th weeks there were 9, 7, 6, 3,

2 and zero, in comparison with horses all mules were positive up to 5th week but at

the end of 6th to 10th weeks there were 8, 5, 2, 1 and zero. Horses and mules were

declared free of infection on the basis of three consecutive negative samples through

culture and PCR. Our findings are broadly consistent with the findings of Timoney,

1988 who reported that a 6 week course of shedding may be more typical. The

organism survives only for a short period in the environment unless protected in

moist discharges. Our study also correlates with the study of Timoney and Artiushin,

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1997 who reported that the sensitivity of PCR appears to be much greater than the

culture. Our findings are in agreement with the results of Kahn, 2005 who reported

that most horses continue to shed organism up to one month following recovery.

Three negative nasopharyngeal swabs, at 4-7 days intervals, should be obtained prior

to release from quarantine, and a minimum isolation period should be one month.

Prolonged bacterial shedding has been identified in a small number of horses. Our

results were also broadly consistent with the findings of Sweeny et al., 1989 who

reported that the shedder state for S. equi implies that the equine harbors the S. equi

organism without manifesting overt clinical signs of strangles. Georage et al., (1983)

reported that 3 of 20 mares with strangles shed S. equi organism for at least 6 weeks

after lymph node rupture and a fourth mare never had lymphadenopathy, but on

arrival to the herd, it had cultures that were positive for S. equi and it continued to

shed S. equi intermittently over the next 10 months. Before this the longest reported

time between the disappearance of clinical signs of strangles and a culture positive

specimen of S. equi was 4 months. Our study correlates with the finding of Sweeny,

1990 who reported that horses with strangles may shed the organism for several

weeks following clinical recovery, with one survey detecting the organism for up to

10 month after exposure. Similarly Woolcock, 1975 suggested that the clinical

disease within a population might be a pre-requisite for development of the shedder

state because he was unable to isolate S. equi from horses on farms with strangles but

without active cases at the time of study. Sweeny et al., 1989 also reported failure to

isolate S. equi from horses which never developed strangles. It suggests that shedders

of S. equi among horses that never manifest clinical signs of strangles are rare. We

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believe that S. equi shed in nasal secretions in horses recently recovered from

strangles is the most likely source of the organism for susceptible horses. Newton et

al., (2000) identified that after the clinical signs are abolished from the animals, the

animal remains in the carrier state and the most predominant site for S. equi was the

guttural pouch. He also reported that the prolonged carrier of S. equi, which lasted

upto 8 months, was again symmetrical with his study. Our results were also broadly

consistent with the findings of Sweeny et al., 2005 who reported that healthy horses

recovering from recent strangles disease might continue to harbor the S equi after a

full clinical recovery. There is evidence that a moderate proportion of horses continue

to harbor S equi for several weeks after clinical signs have disappeared, even though

the organism is no longer detectable in the majority 4 to 6 weeks after total recovery.

A recovered horse may be a potential source of infection for at least 6 weeks after its

clinical signs of strangles have resolved. Our results were also in line with the results

of Georage et al., (1983) who recorded that infected horses can shed S equi at least 4

weeks after the onset of clinical signs and the premises might harbor the organism for

a period of one year or longer. Although outbreaks may be initiated by the

introduction of clinically normal animals into a herd, diseases may become enzootic

on premises resulting in periodic outbreaks when the number of susceptible animals

increases.

It is concluded that do not mix recovered animals from strangles with healthy

animals at least for 9 weeks because the recovered animals remain carriers for

prolonged period of time (6-9 weeks). Periodic shedding of S equi can be a source of

infection for susceptible animals.

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HAEMATOLOGICAL STUDIES:

The results of present study has revealed a significant increase (P < .05) of

total WBCs, MSNC, and basophils in strangles affected horses, while a non

significant difference was observed (P > .05) among values of lymphocytes,

eosinophils, basophils, erythrocytes, hemoglobin and packed cell volume. Whereas in

strangles affected mules the total WBCs, MSNC, and monocytes were significantly

increased (P < .05), while the values of lymphocytes significantly decreased (P < .05)

.Hemoglobin and Packed cell volume decreased but difference was non significant (P

> .05). A non significant difference (P>0.05) was observed in eosinophils, basophils

and erythrocytes. Our results correlate with the findings of Timoney, 2010 who

reported that hematological evidence of an acute phase response in the earlier stages

of strangles includes elevations in the white cell count (20750 ± 1583 cells/µl),

neutrophils (15058 ± 1604 cells/µl) and monocytes (6200 ± 1600 cells/µl) while

hemoglobin and pack cell volume may also be slightly or moderately reduced during

recovery. Gomez, 1990 also recorded hematlogic changes which include leukocytosis

with counts up to 30,000/µL, a segmented neutophil count that may be in excess of

25,000/µL. Hamlen et al., 1994 reported that strangles cases experienced leukocytosis

and neutrophilia in association with an increase in the health index but neutrophilia

was not associated with a left shift. Leukocytosis and neutophilia were also observed

in the foal experimentally inoculated with S .equi and have been reported by others

following experimental S. equi infections (Evers, 1968; Knight et al., 1975; Nara et

al., 1983). Our results were also in line with the findings of Higgins and Snyder

(2006) who reported pronounced leukocytosis with neutrophilia. Our results were

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also broadly consistent with the findings of Hamlen et al., 1994 who reported that the

total leukocyte count increases quickly in horses in the first week post infection,

whereas this increase was delayed into the 2nd week. Other blood parameters

including total eosinophilic count, total basophilic count, total erythrocyte count,

packed cell volume, and hemoglobin concentration remained non significant (P > .05)

during the four weeks post infection. The results of this study were also in line with

Mahaffey, 1962 and Hamlen et al., 1994 who reported that packed cell volume in

mules did not differ in all the weeks. However it was less than the normal mules, as in

the present study. Higgins and Snyder, 2006 also reported leukocytosis and

neutrophilia. Our findings were also in the same line with the finding of Hamlen et

al., 1994 who reported that strangles cases had lower mean PCV and hemoglobin

concentration than non infected animals during weeks 4, 6 and 10 of the outbreak.

The erythrocytes count was also lower in +ve cases compared to non infected animals

during weeks 6 and 10. Collins, 1999 reported that a complete blood count proved to

be a useful adjunctive test to support a diagnosis of S equi and may help differentiate

horses with acute S equi infection from those with acute viral infection. Our findings

are also supported by the results of Evers, 1968 and Knight et al., 1975 who observed

decreases in PCV, hemoglobin concentration and erythrocytes counts have been

reported in horses following experimental inoculation with S. equi. Evers, 1968 also

observed that mean PCV, hemoglobin concentration and RBC count of 20 horses

decreased to 15%, 18% and 22%, respectively, 14 days post S. equi inoculation.

Knight et al., 1975 also reported that mean PCV and hemoglobin concentration

decreased to14% and 12%, respectively in 20 horses 6 days post infection. Evers,

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1968 and Knight et al., 1975 both attributed these changes to in vivo S. equi

associated hemolysis. The most probable mechanisms responsible for the observed

mild to moderate decreases in PCV, hemoglobin concentration and erythrocytes count

in naturally exposed strangles and a more pronounced decrease seen in the

experimentally infected foals include factors affecting erythrocyte survival,

production and dilution. Hemodilution or increases in plasma volume, may account

for decreases in hematologic parameters of foals, particularly in the neonate and at

weaning (Harvey et al. 1987; Harvey et al. 1984; Jeffcott et al. 1982). Golland et al.,

1995 reported that hematological data was recorded for 8 horses. Leukocytosis with a

marked neutrophilia was present in 7 horses, with total nucleated cell counts of up to

23.2×109 cells/L. This was typically followed by the appearance of band neutrophils

and marked lymphocytosis. Seven horses were anemic (mean packed cell volume

0.32L/L). Evers, 1968 reported that temporary reduction of numbers of erythrocytes

and the amount of hemoglobin during the course of disease supports the theory that S.

equi exerts a hemolytic effect. Our findings are in agreement with the results of

Canfield et al., 2000 who reported that all experimentally infected horses with S. equi

showed a consistent leukocytosis as a consequence of a mature neutrophilia. These

changes developed within two days of infection and in some individuals persisted up

to 35 days. Neutrophilia in all the horses were mature and an increase in band

neutrophils was not detected in any of the horses. Other abnormalities in individual

horses included a mild lymphocytosis and a monocytosis.

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BIOCHEMICAL STUDIES:

The result of the present study revealed that values of total serum protein,

serum globulin, and fibrinogen were significantly increased (P<0.05), whereas the

value of serum albumin significantly decreased (P<0.05) in strangles affected horses

and mules. Our study correlates with the study of Radostits et al. (2000) who reported

that hyperfibrinogenaemia is characteristic of both acute and chronic phase of disease.

Similarly hyperproteinemia attributable to a polyclonal gammaglobulinaemia is

characteristic of chronic abscess. Our studies also correlate with the findings of

Timoney, 2010 who reported that plasma protein (7.08±0.17g/dl and fibrinogen

(560±48mg/dL) are also increased. Fibrinogenaemia has also been reported by

Higgins and Snyder (2006). Gomez, 1990 also recorded fibrinogen level of 6.0mg/dL.

Our findings of fibrinogen correlate with the finding of Golland et al., 1995 who

recorded hyperfibrinogenaemia was present in 2 horses, with values of 7 and 8 g/L.

The results of the present study are in complete agreement with the findings of Taylor

and Wilson, 2006 who reported elevated concentration of globulin and fibrinogen,

and anemia of chronic inflammation are typical findings. Collins, 1999 reported that

plasma fibrinogen concentration frequently prove to be a useful adjunctive test to

support a diagnosis of S equi and may help differentiate horses with acute S equi

infection from those with acute viral infection.

THERAPEUTIC EFFICACY:

The resulst of in-vitro antibiotic sensitivity test revealed, that S equi was

sensitive to Procaine penicillin followed by ceftiofur Na, cephradine, erythromycin,

ampicillin, tetracycline, chloramphenicol, sulfamethoxazole, trimethoprim +

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sulfanomides and gentamycin in equines whereas the results of in-vivo antibiotic

trials revealed that horses and mules suffering from strangles without abscess

formation were sensitive to Procaine penicillin followed by ceftiofur Na, cephradine

and erythromycin whereas those animals who developed abscess were ineffective.

From the results of of present study it is concluded that Procaine penicillin is most

effective in-vitro and in-vivo antibiotic followed by ceftiofur Na and cephradine.

These antibiotics might be used for the treatment of strangles infection. Our resulst

correlate with the study of George et al., 1983 who examined the sensitivity of in-

vitro antibiotics against S equi and found that all isolates were sensitive to following

antibiotics, penicillin, methicillin, erythromycin, tetracycline and ampicillin but

resistant to streptomycin and kanamycin. Our findings are also in agreement with the

results of Radostits et al. 2000 who recommended penicillin as a drug of choice for

treatment of strangles. Similarly, results of the present study are in agreement with

the findings of Higgins and Snyder, 2006 who reported that streptococci are very

sensitive to penicillin, ampicillin, erythromycin, chloramphenicol, cephalosporin and

tetracycline. Our results were also broadly consistent with the findings of Timoney,

2009 who reported that S. equi is highly sensitive to a wide range of antibiotics,

including Procaine penicillin, and there is no evidence of emerging drug resistance.

Penicillin treatment in the early part of the acute phase is often curative. Similarly,

results of the present study are in agreement with the findings of Manzoor et al., 2008

who reported antibiotic susceptibility of each of three Streptococcal species revealed

that in vitro Penicillin G and cefotoxime were very effective against Streptococci.

McAllister, 1982 and Swerezek, 1979 reported that antibiotic therapy is most

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beneficial prior to the development of abscessation. McAllister, 1982 also observed

that penicillin is the drug of choice as there are no documented cases of S equi

resistance which is well in line with the results of present study. Our results were also

in agreement with the findings of Timoney, 1993 who reported that S. equi is very

sensitive to penicillin, chloramphenicol, erythromycin, tetracycline and lincomycin.

Procaine penicillin G is the antibiotic of choice and will show a quick clinical

improvement with reduction in fever and lymph node enlargement.

DISINFECTANT TRIALS:

Data regarding the comparison of different disinfectants is shown in table

4.53. Among four disinfectants, povidone iodine was found to be the best because its

phenol coefficient is 1.25 that is greater than phenol i.e. 1.00 while 0.6%solution of

H2SO4 showed a similar phenol coefficient as that of phenol. The phenol coefficients

of dettol and bleach were 0.5 and 0.75 respectively which are less than phenol i.e.

1.00. Therefore it is recommended that S. equi is highly sensitive to povidone iodine

and 0.6% H2SO4. Our results were also broadly consistent with the findings of

Higgins and Snyder (2006) who reported that all containers used for feed or water

should be cleaned and disinfected. Surfaces of stalls contaminated with discharges

should be similarly cleaned and disinfected. Effective disinfectants are povidone

iodine, chlorhexidine gluconate, 0.6 sulfuric acid, glutaraldehyde and phenol (1:200).

Our study also correlates with the study of Kahn, 2005 who used chlorhexidine

gluconate or gluteraldehyde for cleaning of contaminated equipment with S. equi.

Taylor and Wilson, 2006 reported that attempts should be made to disinfect areas that

have been occupied by infected horses. Organic material, such as feed and manure,

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should be removed from the contaminated area and should be disposed of or

composted in an area that is not used for horses. Stall walls, feeders, waterers and

impervious floors should then be washed thoroughly to remove as much organic

material as possible before applying an effective disinfectant (eg, povidone iodine,

chlorhexidine and glutaraldehyde). Our results were also broadly consistent with the

findings of Jorm, 1992 who reported that organism is quickly killed at 560C and is

killed within 90 minutes by 0.6% sulfuric acid, a 1:200 dilution of phenol and by

disinfectants such as povidone iodine, chlorhexidine gluconate and glutaraldehyde.

Other disinfectants are less effective. Results of the present study are in agreement

with the findings of Wilson, 1988 and Reed et al., 2004 who reported that organism

does not survive for a prolonged period in the environment because of its

susceptibility to heat, sunlight, desiccation and many disinfectants including povidone

iodine, chlorhexidine gluconate and glutaraldehyde.

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Chapter-06

SUMMARY

Strangles is an infectious malady of equidae characterized by upper

respiratory tract infection, dysponea, anorexia, regional suppurative lymphadenitis

and causes high morbidity and low mortality. Considering the significance and

utilization of equines in our country and the substantial losses rendered by Strangles,

the present project was designed to study epidemiology, diagnosis and chemotherapy

of strangles in Lahore and Sargodha districts of the Punjab province in Pakistan.

The present study comprised of five phases. In phase-I, epidemiology of the

disease including prevalence, variations in SeM, SzPSe and Se18.9 proteins and

mortality rate were studied in Lahore and Sargodha districts. For epidemiology, nasal

swabs and pus samples from the affected lymph nodes of 500 equines (n=250 horses,

n=250 mules) suspected for strangles were collected and cultured for identification of

S. equi. The collected samples were processed at Medicine and Microbiology

Laboratories of the University of Veterinary and Animal Sciences, Lahore, Pakistan

and Gluck equine research center, Department of Veterinary Science, University of

Kentucky, USA. Out of 250 horses and 250 mules, 113(45.2%) horses and 99

(39.6%) mules tested positive for S. equi. on the basis of culture. Number of S. equi

isolates were significantly higher (P<0.05) in pus samples taken from sub-mandibular

lymph nodes as compared to nasal discharge samples. The difference was significant

(P<0.05) among mules of different age groups. The highest prevalence of strangles

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was recorded in horses and mules less than 2 year of age as compared to those having

age more than 2 years.

In the present study, prevalence of strangles round the year in horses and

mules were also calculated and it was found to be the highest during the months of

February, March, April and May while few cases were seen during the months of

January, June and July and no cases were observed during others months. The

significant difference was observed (p<0.05) among the prevalence levels of strangles

in different months of the year. Similarly when compared the prevalence of strangles

in different seasons of Pakistan i.e. summer, winter, spring and autumn. The highest

prevalence rate was recorded during the spring season.

The prevalence on the basis of Polymerase chain reaction (PCR) of S. equi in

horses and mules was also recorded. Out of 250 horses and 250 mules tested,

122(48.8%) horses and 113(45.2%) mules were positive for S. equi. When compared

the prevalence rate on the basis of PCR and culture of nasal and pus samples from

affected submandibular lymph nodes it revealed that the sensitivity of Polymerase

chain reaction appears to be much greater than culture. The culture along with PCR is

the best diagnostic technique for S. equi as PCR test does not differentiate between

dead and live bacteria, hence a positive test may not correlate with active infection;

therefore, a positive culture may be necessary to confirm the diagnosis.

In this phase of epidemiological study of disease, effect of selective pressure

of allelic diversity in SeM of S. equi on immunoreactive proteins SzPSe and Se18.9

was also studied. The aim of this study was to determine whether variations in SeM

are accompanied by variations in the immunoreactive surface of exposed SzPSe and

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secreted Se18.9. Sequences of genes of 25 S. equi alleles isolated from different

countries of the world over a period of 40 years were compared. Twenty different

SeM alleles were identified including 6 not included in the data base (http://

pubmlst.org/szooepidemicus). Amino acid variation was also detected distal to the N-

terminus of SeM. No variation was observed in SzPSe except for an Australian isolate

which showed a deletion of one PEPK repeat. The Se18.9 protein in all 25 isolates of

S. equi did not exhibit any variation. Interestingly, only 2 SNP loci were detected in

Se18.9 compared to 93 and 49 in SeM and SzPSe respectively. The greater frequency

of mutation in SzPSe compared to Se18.9 may be related to a high rate of

recombination of SzPSe and the inclusion of exogenous DNA sequence based on the

atypical GC percentage of its central hyper variable region.

In horses the mortality rate was recorded as 1.64% whereas the mortality rate

in mules having less than 5 years of age was found to be 0.88%. No significant

difference (P>0.05) in mortality rate among horses and mules of different age groups

affected with strangles was observed.

In phase-II of the present study, carrier status of the horses and mules were

studied. Out of 122 horses found positive to PCR, 20 horses (10<2 years and 10

between 2 and 5 years of age) were selected and monitored for 12 weeks. Their nasal

swab samples were used for identification of bacteria through culture and PCR on

weekly basis. Till the end of 3rd week all horses < 2 years of age remained positive

but at the end of 4th to 7th weeks there remained positive only 5, 2, 1 and zero horses

out of 10, respectively on the basis of culture whereas through PCR at the end of the

4th week all horse <2 years of age were found positive, but at the end of 5th to 10th

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weeks there remained 7, 5, 4, 2, 1 and zero horses out of 10, respectively. While all

the horses aging between 2 to 5 year, were positive up to the 1st week but at the end

of 2nd to 8th week out of 10 there were 9, 7, 6, 3, 1, 1 and zero horses respectively

positive on the basis of culture but through PCR, all horses were positive till 4th week

but at the end of 5th to 9th week number was reduced to 9, 7, 6, 3, 2 and zero.

Similarly, out of 113 mules, 20 mules (10<2 year and 10 between 2 and 5 years of

old) were also monitored for 12 weeks to study their carrier status. After the end of

2nd week all mules < 2 years of age were positive but at the end of 3rd to 6th weeks

there remained 7, 3, 1 and zero mules out of 10, respectively on the basis of culture

but through PCR at the end of the 5th week all mules <2 years of age were positive,

but at the end of 6th to 10th weeks there remained 9, 7, 3, 2 and zero mules out of 10,

respectively. While in 2 and 5 year old mules, all were positive up to the 2nd week

but at the end of 3rd to 7th weeks there were 6, 4, 2, 1, 1 and zero mules out of 10,

respectively on the basis of culture but through PCR, all mules were positive up to 5th

week but at the end of 6th to 10th weeks there were 8, 5, 2, 1 and zero. Horses and

mules were declared free of infection on the basis of three consecutive negative

samples through culture and PCR.

From the result of present study, it may be concluded that sensitivity of

Polymerase Chain Reaction appears to be much greater than culture for study of

carrier status of equines. Moreover, recovered animals should be kept in quarantine

period at least upto 9th week because the recovered horses and mules remain carrier

for prolonged period of time and can act as source of infection for susceptible animals

through periodic shedding of S equi.

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In phase-III of the present study, effect of strangles on various haematological

parameters (Hemoglobin, Erythrocyte sedimentation rate, Total erythrocyte count,

Total leukocyte count, Differential leukocyte count, Packed cell volume) were

determined. Present study revealed that, total WBCs, MSNC, and basophils were

significantly increased (P < .05) in strangles affected horses, while the difference of

the values of lymphocytes, eosinophils, basophils, erythrocytes, hemoglobin and

Packed cell volume were non significant (P > .05). It was further found that in mules

total WBCs, MSNC, and monocytes in strangles affected mules were significantly

increased (P < .05), while the values of lymphocytes significantly decreased (P < .05).

Values of Hemoglobin and Packed cell volume were found to be decreased but

difference was non significant (P > .05). The difference of values of other blood

parameters like eosinophils, basophils and erythrocytes was observed non significant

(P>0.05).

The effect of strangles on total serum proteins, serum albumin, serum globulin

and fibrinogen were also studied in this phase. In the present study, the levels of total

serum protein, serum globulin, and fibrinogen were found to be significantly

increased (P<0.05), whereas a significant decrease (P<0.05) was observed in the

value of serum albumin in strangles affected horses and mules.

Phase-IV encompassed in-vitro and in-vivo antibiotic trials. In-vitro

antibiotic sensitivity of S. equi was determined against procaine penicillin,

erythromycin, chloramphenicol, ampicillin, cephradine, ceftiofur Na, tetracycline,

sulfamethoxazole, gentamycin and trimethoprim + sulfdiazine through disc diffusion

method. Four top ranking antibiotics were then administered to four groups

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(comprising 10 horses and10 mules) for in-vivo trials. Efficacy of the antibiotics was

assessed weekly on the basis of negative nasal swab culture. Results of in-vitro

antibiotic sensitivity revealed that in horses and mules, S equi was most sensitive to

Procaine penicillin followed by ceftiofur Na, cephradine, erythromycin, ampicillin,

tetracycline, chloramphenicol, sulfamethoxazole, trimethoprim + sulfdiazine and

gentamycin whereas the result of in-vivo antibiotic trials revealed that horses and

mules suffered from strangles without abscess formation were most sensitive to

Procaine penicillin followed by ceftiofur Na, cephradine and erythromycin whereas

animals which developed abscess showed no response. It is concluded from the result

of present study that Procaine penicillin is most effective in-vitro and in-vivo

antibiotic followed by ceftiofur Na and cephradine. These antibiotics might be used

for the treatment of strangles infection.

Phase-V, comprised over in-vitro trials of disinfectants. Efficacy of

disinfectants, like povidone iodine, 0.6% H2SO4, dettol and bleach was assessed.

Phenol Co-efficient Test was applied, to ascertain efficacy of these disinfectants, used

in, in-vitro trials. Among four disinfectants, povidone iodine was found to be the best

one with a phenol coefficient of 1.25 that is greater than phenol i.e. 1.00 while 0.6%

H2SO4 showed similar phenol coefficient as that of phenol. The phenol coefficient of

dettol and bleach were observed as 0.5 and 0.75 respectively. Therefore it is

recommended that S. equi is highly sensitive to povidone iodine and 0.6% H2SO4.

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