Indian Journal of Experimental Biology Vol. 40, March 2002, pp. 296-303
Evaluation of guinea pig model for experimental Salmonella serovar Abortusequi infection in reference to infertility
B R Singh, Javed Alarn, D Hansda, J C Verma, V P Singh & M P Yadav
National Salmonella Centre (Vet), Division of Bacteriology and Mycology, Indian Veterinary Research Institute, Izatnagar 243 122, India Received 1 August 2001; revised 12 December 2001
The present study conclusively revealed the role for Salmonella enterica subspecies enterica serovar Abortusequi in conception failure. None of the 1 2 guinea pigs conceived when orally exposed to sublethal dose of the pathogen during breeding, while 66.67% of animals in control group were found pregnant during same period of observation under similar conditions. Salmonella carrier animals also had drastic reduction in conception rate ( 16.67%). During mid pregnancy, S. Abortusequi exposure to guinea pigs through intravaginal, intramuscular and subcutaneous routes induced fetal death followed by resorption. While 2 out of 6 orally inoculated and 3 out of 6 intraperitonially inoculated guinea pigs aborted, in rest of the animals fetal death was followed by meceration and resorption. It was interesting to note that S. Abortusequi could not persist longer than a week in males while in pregnant females it could be detected for >10 weeks after inoculation. In late pregnancy, most of the exposed animals aborted and non aborting animals though had normal parturition, survival rate of their babies was nearly zero in comparison to the control group. The study revealed role for S. Abortusequi in impairing conception, abortion, early fetal deaths, fetal meceration and resorption. Further studies are required to identify factors responsible for increased susceptibility of females particularly during pregnancy.
Infertility is a complex syndrome either caused by defects in anatomy, disturbed physiology or pathology of the genital tract. Among the pathological causes, infectious diseases are important. Of these, bacterial infections viz. listeriosis, brucellosis and campylobacteriosis are quite frequent and well studied I . Salmonellosis, specially caused by host adapted serovars has often been reported to be associated with infertility. However, firm evidences are lacking to prove the role for Salmonella in infertility in absence of evidences to reveal affinity of this pathogen to tissues of the genital tract. Although S. enterica subspecies enterica serovar Abortusequi (written as S. Abortusequi) 1 has been shown to affect the gravid uterus of equines, it has rarely b�en shown to be associated with nongravid uteri2•3 . Some studies have indirectly revealed that salmonellosis due to wide host range serovars viz. S. typhimurium may also be associated with infertility4.5. Therefore, to determine the role for salmonellosis in infertility, this study was conducted with S. Abortusequi, an equine host adapted Salmonella, in guinea pigs, used earlier as an experimental model6 for S. Abortusequi induced abortions.
Materials and Methods Bacterial strain-Reference S. Abortusequi strain
(E 1 56), pathogenic to mouse (MLD50 _ 107 CFU) and
resistant to nalidixic acid (MIC-50 Ilg mrl), maintained at the National Salmonella Centre (Vet) was used throughout the study.
Animal model-Adult (5 to 6 months old, -500g in weight) male and female guinea pigs were procured from the Laboratory Animal Resource (LAR) Section of the Institute, having no detectable (with ELISA and micro-agglutination test) S. Abortusequi antibodies, were used in the experiment. All the animals were maintained in pans (75 x 1 25 x 35 cm) on sterilised rice husk padding, in groups of 6-7, on sufficient green fodder and concentrate ration recommended for breeding animals by LAR Section. All the animals were screened for faecal excretion of Salmonella for 7 dIlys continuously by processing faecal swabs for selective enrichment and plating (described later) . Salmonella isolates were characterized through morphological, cultural, biochemical and serological characteristics7• Only Salmonella free animals were included in the study.
Breeding -Six adult females were kept with an adult male for 1 7 days and thereafter male was separated. Females were palpated for pregnancy at 40th
and 55th day of breeding. Non-pregnant animals were sacrificed to collect visceral organs (liver, spleen, gall bladder), contents from intestine (ileum-jejunum) and uterus for isolation of Salmonella7•
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SINGH et al. : EVALUATION OF GUINEA PIG MODEL FOR EXPERIMENTAL SALMONELlA SEROVAR 29'7
Salmonella carriers-Forty-two adult female guinea pigs were orally inoculated with 4.3 x 107 CFU of S. Abortusequi (E- 1 56) overnight broth culture and were monitored for Salmonella excretion as above for 75 days of inoculation by taking faecal swabs, twice weekly . Animals excreting Salmonella, even after a month of inoculation but having no sign of salmonellosis were designated as carriers.
Experimental plan for breeding efficiencyThree groups of adult female guinea pigs consisting of 12 Salmonella free animals in group 1 and 2 each and 12 Salmonella- carrier animals in group 3 were set to breed as above. Group 1 of Salmonella free animals was exposed to oral infection of S. Abortusequi (-4.3 x 107 CFU per animal) on the first day of breeding while the other group 2 was kept as control.
Effect of Salmonella on pregnant guinea pigsTo assess the effect of S. Abortusequi infection on pregnant guinea pigs, groups of 6 pregnant guinea pigs (-40 days and -55 days), procured from LAR Section, were exposed to S. Abortusequi (E- 156) through oral (4.3 x 109CFU per animal), intra vaginal (4.3 x 109 CFU per animal), intra muscular (4.2 x 107
CFU per animal), intra peritoneal (4.2 x 107 CFU per animal) and subcutaneous (4.2 x 107 CFU per animal) routes. Control group was kept separately without any exposure to Salmonella. Each group was observed for 75 days for parturition, resorption of fetus, live births (litter size), faecal Salmonella excretion and survival of baby guinea pigs up to one month of age.
Three aborted animals were monitored for faecal and vaginal excretion of Salmonella by processing rectal and vaginal swabs, respectively, taken on
. 0, 1 ,3,7 , 15,21 ,30 and 45th day of abortion. Salmonella carriage in male guinea pigs-Male
animals inoculated with S. Abortusequi through oral (4.3 x 109 CFU per animal), subcutaneous (4.2 x 1 07
CFU per animal) or intra muscular (4.2 x 107 CFU per animal) routes were also monitored for faecal excretion of the pathogen up to 2 1 days of infection and then sacrificed to isolate Salmonella from visceral organs.
Salmonella carriage in female guinea pigs-To assess the carriage and excretion of S. Abortusequi by adult female guinea pigs, groups of 1 2 adult female guinea pigs were exposed to S. Abortusequi (E- 1 56) through oral (4.3 x. 109CFU per animal), intra vaginal (4.3 x 1 09 CFU per animal), intra muscular (4.2 x 107
CFU per animal), intra peritoneal (4.2 x 1 07 CFU per animal) and subcutaneous (4.2 x 1 07 CFU per animal) routes while control group was kept separately without any exposure to Salmonella. Each group was observed for 75 days for faecal excretion and survival of Salmonella in liver, spleen, gall b1addt!r, kidneys and uteri.
Isolation of Salmonella-To isolate Salmonella from faeces and different visceral organs of experimental animals, faecal swabs/tissue homogenates were enriched in Rappaport Vassiliadis (RV) broth containing nalidixic acid (30 ).l.g ml-I ) and incubated at 42°C for 1 8 hr and then plated on to Enteric hektoen agar (Hi-Media, Mumbai), containing nalidixic acid (30 ).l.g mn) plates and suspected isolated colonies were characterised through biochemical and serological methods7 •
ELISA for Salmonella antibodies-The test was conducted as described by Hudson and Hal using sonicated antigen (200 ).l.g rnI-l) prepared from overnight culture of S. Abortusequi (E156) according to Singh and Sharma9.
Micro agglutination test for Salmonella agglutinins-The test was conducted in V well microtitre plates8 using Salmonella (E156) '0' 4, 12 and 'H' enx antigens7 •
Results ]'lone of the guinea pigs, received from LAR Section
had detectible '0'4, 1 2 and 'H'enx agglutinin titre in their serum samples. Background opacity was detectable with ELISA in undiluted serum (0.22±0.0 13) but at 1 : 100 dilution all samples induced opacity similar to blank control (0.03 ± 0.0 1 1 ).
Table I -Faecal excretion of S.Abortusequi in adult male guinea pigs experimentally infected through various routes
Mode of inoculation No. of animals No. of animals excreting S.Abortusegui o DPI I DPI 3 DPI 7 DPI 15 DPI 21 DPI
Oral 6 0 •
5 2 0 0 0 sc 6 0 2 0 I 0 0 ip 6 0 3 0 0 0
DPI-days post inoculation; sc-subcutaneous; ip-intraperitoneal.
298 INDIAN J EXP BIOL, MARCH 2002
Carriage of Salmonella Abortusequi in experimentally inoculated male guinea pigs- Observations on S. Abortusequi inoculated males revealed (Table 1 ) faecal excretion of Salmonella only up to 7 days in subcutaneously inoculated animals and only up to 3 days in orally and intra muscularly inoculated animals. None of the animal harbored S. Abortusequi in visceral organs when sacrificed on day 21 of infection.
Carriage of Salmonella Abortusequi in experimentally inoculated female guinea pigs-Results of faecal excretion of S. Abortusequi in non pregnant female animals (Fig. I ) revealed faecal excretion of Salmonella up to 75 days of inoculation through either of the route except oral route. Salmonella could not be isolated from any of the orally inoculated animals from rectal swabs on or after 55 days of inoculation. On sacrificing the animals on day 75 of inoculation, S. Abortusequi could be isolated from spleen of
� .8 � (,) x Q) '0 CD .0 E ::l Z
1 4
1 2
1 0
8
6
4
2
o o 3 7 1 5 21
Days post inoculation
1 , 3, 3, 2 and 2 animals inoculated through oral, intra vaginal, intra muscular, intra peritoneal and subcutaneous routes, respectively. Salmonella could not be isolated from any of the liver, gall bladder or kidneys. However, one uterus each from intra vaginally and subcutaneously inoculated animals contained S. Abortusequi.
Salmonella Abortusequi in experimentally inoculated breeding female guinea pigs-Of the 1 2 control animals, 8 were palpated positive for pregnancy at day 40 of breeding while only 2 of the 1 2 animals Salmonella carrier became pregnant. None of the 1 2 animals exposed to S. Abortusequi during breeding was found pregnant even after 55 days of observation.
Salmonella Abortusequi in experimentally inoculated guinea pigs with -40 days of gestationResults of Salmonella isolation from rectal swabs of pregnant guinea pigs (Fig. 2) showed faecal excretion
D Oral • Intra vaginal
II Intra muscular • Intra peritoneal
• Subcutaneous
28 37 45 55 65 75
Fig. I -Faecal excretion of S. Abortusequi by adult female guinea pigs experimentally inoculated through different routes.
Table 2 - lmpact of Salmonella Abortusequi infection on guinea pigs at about 40 days of gestation
Observations Control Route of inoculation (Animals ex�sed) Oral IVG 1M IP SC
Animals aborted 0 2 0 0 3 0 Neither aborted nor parturited 0 3 6 6 3 6 Faecal excretors at 75 days post infection 0 0 0 1 0 0 Harboring Salmonella in spleen 0 1 0 2 2 2 Li ve babies born 1 8 2 0 0 0 0 Babies died in 21 days of life 1 0 0 0 0 0 Dead babies having Salmonella in spleen 0 2 0 0 0 0
IVG, intra-vaginal; 1M, intra-muscular; IP, intra-peritoneal; SC, subcutaneous. Note, None of the animal yielded Salmonella from uteruslliverl gall bladder or kidneys when sacrificed at 75th day of inoculation. For each treatment 6 animals were taken.
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SINGH et al.: EVALUATION OF GUINEA PIG MODEL FOR EXPERIMENTAL SALMONELLA SEROVAR 299
of the pathogen by animals inoculated through parenteral (subcutaneous, intra muscular and intra peritoneal) routes but rectal swabs from those inoculated either through oral or intra vaginal routes had no Salmonella on day 75 of inoculation. Salmonella could not be isolated from, uteri liver, gall bladder or kidneys. S. Abortusequi could be isolated from spleen of 1 , 3, 3, 3 and 3 animals inoculated through oral, intravaginal, intra-muscular, intra-peritoneal and subcutaneous routes, respectively. Results of abortion and post abortion observations (Table 2) revealed either resorption (Fig. 3) or abortion in most of the animals with no living baby guinea pigs from S. Abortusequi inoculated animals. However, control group of guinea pigs had 17 healthy babies in the end of experiment.
7
6
5
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o +------,-L-o 3 7 1 5 21
Days post inoculation
Both the babies born live from orally inoculated guinea pigs died on day 4 of parturition having watery diarrhoea. Salmonella could be isolated from feces as well as from spleen of the dead baby guinea pigs. In contrast, the only baby died in control group had no Salmonella in its feces or in spleen.
Salmonella Abortusequi in experimentally inoculated guinea pigs with -55 days of gestationSalmonella could be isolated from rectal swabs of pregnant guinea pigs inoculated through intra vaginal, intra-muscular and intra peritoneal routes but rectal swabs from those inoculated either through oral or subcutaneous routes had no Salmonella on day 75 of inoculation. Salmonella could be isolated from one uterus each from animals inoculated through intra
D Oral
fi htra rruscular
• Subcutaneous
28 37 45
• ntra vaginal
• ntra peritoneal
55 65 75
Fig. 2-Faecal excretion of S. Abortusequi by adult female guinea pigs experimentally inoculated through different routes during pregnancy.
Table 3-lmpact of Salmonella Abortusequi infection on guinea pigs with -55 days of gestation Observations Control Route of inoculation (Animals exposed)
Oral IVG 1M IP SC
Animals died before parturition 0 0 0 I I 2 Animals aborted 0 3 5 4 5 4 Neither aborted nor parturited 0 0 0 0 0 0 Faecal excretors at 75 days post infection 0 0 I I 2 0 Harboring Salmonella in spleen on 75lh day 0 2 3 4 4 4 Harboring Salmonella in uterus on 75th day 0 0 I I I 0 Live babies born 2 1 1 2 3 4 0 0 Babies died in 2 1 days of life 3 8 I 2 0 0 Dead babies having Salmonella in spleen 0 7 1 2 0 0
IVG, intra-vaginal; 1M, intra-muscular; IP, intra-peritoneal; SC, subcutaneous Note, None of the animal yielded Salmonella from liver! gall bladder or kidneys when sacrificed at 75th day of inoculation. For each treatment 6 animls were taken.
300 INDIAN J EXP BIOL, MARCH 2002
Table 4-Faecal/vaginal excretion of S. Abortusequi in aborted guinea pigs following experimental infection through various routes at -55 days of pregnancy
Days post Salmonella positive rectal swabs from animals inoculated Salmonella positive vaginal swabs from animals inoculated abortion through through
Oral Iva 1M IP SC Oral Iva 1M IP SC (3) (5) (4) (5) (4) (3) (5) (4) (5) (4)
0 I 3 0 0 I 3 5 4 5 4 I 3 5 4 2 2 2 4 4 4 4 3 2 5 4 5 3 3 5 4 4 3 7 2 5 4 5 4 3 4 4 5 4
15 3 2 4 5 4 3 5 4 5 4 2 1 2 2 4 5 4 3 5 4 5 4 30 0 I 2 5 4 3 5 3 3 4 38 2 3 4 2 2 4 2 3 3 45 2 0 0 0 1 3 2 2 1
Iva, intra-vaginal; 1M, intra-muscular; IP, intra-peritoneal; SC, subcutaneous Figures in parenthesis indicate the total number of animals swabbed
Fig. 3-Normal (left most), gravid (55 days gestation, right most) and uteri showing resorption of foetuses (3 in the centre) on oral Salmonella Abortusequi inoculation in pregnant (around 40 days of gestation) guinea pigs. All infected and control pregnant animals were sacrificed at 55'h day of gestation.
vaginal, intra-muscular and intra peritoneal routes but not from liver, gall bladder or kidneys. S. Abortusequi could be isolated from spleen of 2, 3, 4, 4 and 4 animals inoculated through oral, intra-vaginal, intramuscular, intra-peritoneal and subcutaneous routes, respectively. Results of inoculation of Salmonella in -55 days pregnant guinea pigs (Table 3) revealed either death or abortion in most animals inoculated parenterally. However, orally and intra-vaginally inoculated with Salmonella and control groups had 4, 2 and 18 healthy babies, respectively in the end of the experiment. All the babies born live but died early in infancy, had diarrhoea. Salmonella could be isolated from spleen of all dead baby guinea pigs except one born from orally inoculated and 3 from control group. From feces of all the babies born of Salmonella inocu-
- lated mothers, the pathogen could be isolated either in pure culture (in one baby each from intra-vaginally and intra-muscularly inoculated groups) or as mixed infection in association with E. coli or Klebsiella
aerogenes (in babies of orally and intra-muscularly inoculated groups). None of the 3 babies died in control group had Salmonella either in feces or in spleen. However, Enterobacter agglumerans could be isolated from feces but not from spleen.
S. Abortusequi could be isolated from rectal as well as vaginal swabs of all the aborted animals up to a month of abortion and thereafter variably from rectal swabs of animals inoculated through oral and intra vaginal routes but rarely from parenterally inoculated animals. Vaginal swabs were positive for S. Abortusequi for longer period than rectal/faecal swabs (Table 4)
Discussion Salmonella enterica subspecies enterica compris
ing around 1 500 serovars includes most of the Salmonella which cause disease in humans and animals. Pathology of Salmonella is largely an outcome of host-bacteria interaction and varies with specific serovar in different host species 10. Salmonella Abortusequi, though an equine host adapted serovar, may infect other animals and humansl l . In equines, it often causes abortion in late pregnancy and perinatal foal mortality due to enteritis, omphalophlebitis, polyarthritis and septicemia12• 1 3 • It rarely affects non breeding adult females and males to cause only mild infection or orchitis, respectively. In earlier studies in different laboratory animals, it rarely produces classical abortion, the main outcome in natural infection in equines 14• It is of utmost importance to standardize a laboratory model because i'n most places experimentation on horses is not permissible or feasible. Moreover, cost of the horse also interferes the studies,
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SINGH et al. : EV ALVA TION OF GUINEA PIG MODEL FOR EXPERIMENTAL SALMONELLA SEROVAR 301
which are essential, particularly for determining safety and potency of vaccines. Besides, mere isolation of S. Abortusequi from equines or other sources may not be enough to conclude it as a primary cause of disease. Therefore, pathogenicity testing in a suitable model is important I 5 . Most of the rodents and lagomorphs are born comparatively immature and abortions are rare even in late pregnancy. Therefore guinea pigs which give birth to quite mature babies and have gestational physiology comparable to equine l4, 16 appears to be a suitable model for S. Abortusequi. Therefore, this study was undertaken to standardise the guinea pig model for future studies.
Absence of '0' 4, 1 2 and ' H' enx agglutinating antibodies in serum samples of guinea pigs before inoculation of Salmonella, not only reveals absence of S. Abortusequi infection but also that of related serovars in the guinea pig colony. Positive results using cell lysate antigen in ELISA with many of the undiluted sera or diluted below 1 : 1 00, indicated either over-sensivity of the ELISA or presence of cross reacting antibodies to some antigens, widely present in nature like enterobacterial common antigen present in many of the intestinal commensals? Therefore, for ELISA, all serum samples were diluted with PBS to 1 :200 to avoid the detection of cross-reacting anti- . bodies.
Under this study, male guinea pigs appeared to be quite resistant to experimental S. Abortusequi infection not only with respect to clinical or overt disease but also for colonization of the pathogen in gastrointestinal tract. None of the male guinea pig, either inoculated orally or parenterally with pathogenic S. Abortusequi, excreted the pathogen for more than 7 days, while many of the females continued to excrete S. Abortusequi for more than 2.5 months. S. Abortusequi was also absent from visceral organs of the male guinea pigs indicating some sex factors, yet to be identified, playing role in establishment of S. Abortusequi in females. Adult male guinea pigs appear to behave similar to stallions which are often
f I · +' • I I re ractory to natura InlectlOn . Oral as well as parenteral inoculation of S. Abor
tusequi in female guinea pigs led to development of Salmonella carriage state in the host up to the level of >30% which is common in host adapted Salmonella in their favoured hosts17, therefore guinea pigs appears to mimic as right type of model for S. Abortusequi.
In general, parenteral infection was more long lasting than oral infection and converted more animals to carrier of S. Abortusequi which may be due to lack of
competition for parenterally inoculated Salmonella encountered in intestine by orally inoculated S. Abortusequi.
Not only sex but the age and physiological state of animals also appeared to be important factors to decide the course of S. Abortusequi infection in guinea pigs. Pregnant animals were found to be more sensitive, as comparatively more animals turned carrier and died particularly in advance pregnancy probably due to in utero death of fetuses not leading to abortions. Such deaths were more common with parenteral inoculation of the pathogen. Similar susceptibility in
h b b d . . 1 8·20 late pregnancy as een 0 serve In equmes too
Besides, during pregnancy gastrointestinal tract appears to be more receptive to S. Abortusequi as on
.oral infection >66% females inoculated either in mid or late pregnancy kept on excreting Salmonella even after 2 months of infection while only 16% of those infected during non-pregnant stage turned excretor in the same period of observation under similar maintenance and environmental conditions. S. Abortusequi could be isolated from uterus of those animals only inoculated late in gestation. This difference of susceptibility to S. Abortusequi in breeding and nonbreeding animals was less significant on parenteral infection. Alam2 1 and Hansda22 have reported similar observations on aroA and aroA-htrA mutants of S. Abortusequi in guinea pigs.
S. Abortusequi infection in mid gestation led to death of fetuses and resorption while abortions took place in guinea pigs infected in late pregnancy. It may be either due to absence of hard tissues in fetuses of mid pregnancy which might have made them easy subject for lysis and resorption or due to acuteness of infection as few of those animals fnfected through oral or intra-peritoneal routes aborted even in mid pregnancy. However, more detailed studies, to quantitate infection are necessary to reach at final conclusion.
Few baby guinea pigs delivered alive by Salmonella infected mothers either at early or late pregnancy, died of salmonellosis and S. Abortusequi could be isolated from their spleens. Though, babies (5.5-14 . 1 %) of control groups died with similar clinical sign of diarrhoea and prostration as in S. Abortusequi infected groups, Salmonella could not be isolated from their faeces or spleens. Similar deaths have been reported in foals born on farms with prevalence of S. Abortusequi infection in mares 1 3 revealing suitability of the experimental host systems.
302 INDIAN J EXP BIOL, MARCH 2002
Most of the aborted guinea pigs continued for long to excrete Salmonella not only in faeces but in vaginal discharges too, indicating the danger of spread of Salmonella in their environment and possibly to mating males. This post abortion vaginal and faecal excretion has commonly been reported in S. Abortusequi infected mares. Thus female guinea pig seems to behave similar to the natural host, the pregnant mares.,
From the results, i t is evident that S. Abortusequi can cause infertility in carrier animals as well as in those exposed to sublethal infection without any clinical sign of salmonellosis. Normal conception rate in control group was 66.67% (8/ 12) while i t remained nil in those infected orally during breeding and 1 6.67% in Salmonella carriers. However, further investigations are necessary to reveal the mechanism of causing infertility as Salmonella could not be isolated from the uterus of those females which failed to breed and sacrificed after 55 days of breeding. Conclusion can be drawn that S. Abortusequi play a significant role in causing infertility and repeat-breeding. The same may be true for salmonellosis induced by other Salmonella strains in domestic animals and should be considered while treating and handling repeatbreeding problem, quite prevalent in all types of l ivestock similar to prevalence of salmonellosis.
Variations in susceptibility may be associated with immune response. It has frequently been reported that antibody titres in pregnancy goes down with advancing of gestation9, either it is low serum level of antibodies or immunomodulatory effect of sex and pregnancy hormones23,24 or something else leading to precipitation of Salmonella infection, only further investigations can pinpoint the exact factor(s) making animals more susceptible during pregnancy. Earlier studies too have shown difference i n susceptibility to S. Abortusequi at different stages of breeding and in different sexes. Alam21 and Hansda22 have shown that male animals mount more pronounced delayed type of hypersensitivity response to S. Abortusequi than females, which may be an important factor in early clearance of S. Abortusequi by males. The study concludes that S. Abortusequi is an i mportant pathogen which affects breeding in guinea pigs and probably in its natural host, the equine mares too.
Acknowledgement Authors are thankful to Dr M C Saxena, Incharge,
Laboratory Animal Research, for providing Salmonella free guinea pigs and helping in diagnosis of pregnancy.
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