Course: VPM 201 Fall, 2010Lecturer: C. Anne Muckle

Small, aerobic, non-motile gram-negative coccobacilli

Catalase positive, oxidase positive, most produce urease

Gram-stain better with carbol fuchsin than with safranin

Are Modified Acid-Fast (stain pink) using the Modified Ziehl-Neelsen stain (ZN stain used for mycobacterium)

Require enriched media & prolonged incubation

High potential for lab-acquired infections

Primary isolation can be done in Level 2 diagnostic labs,

but identification requires Biosafety Level 3 Reference Lab ◦ Canadian Food Inspection Agency Lab, Nepean, Ont. Canada

◦ National Microbiology Lab, Winnipeg, Manitoba

Level 3 pathogen: Classified as potential bioterrorism agent because of aerosol transmission

risk and no human vaccines available

Brucella are modified acid-fast

Six traditional Brucella species & their primary host:

B. abortus – cattle, bison

B. canis – dogs

B. melitensis - goats & sheep

B. neotomae – wood rats (nonpathogenic) in Utah, USA

B. ovis - sheep

B. suis – pigs, hares, caribou, reindeer, rodents

Newer Brucella species:

B. cetaceae, B. pinnipediae, B. maris in marine mammals

Brucella species cause brucellosis

Cause disease in several animal species: humans, ruminants, swine, dogs, horses, cervids, rodents, marine mammals Cats resistant to infection

Infect reticuloendothelial system (RES) & reproductive tract, generally resulting in late term abortion in females & inflammatory lesions in the male reproductive tract.

Mature animals most susceptible and remain infected for life, young animals are more resistant

Can survive in environment a long time (frozen, dry, or moist), Fortunately, they are susceptible to disinfectants, heat + pasteurisation

Are invasive FIPS; survival in phagocytes leads to granulomatous disease of the RES and reproductive tract

Virulence factors ???

Are urease-positive (except B. ovis)

But no capsule, flagella, fimbriae, spores, toxins, no plasmids, PAIs, no drug resistance

*yet are very invasive & survive inside host cells

Penetrate mucosal surfaces or Peyer’s patches, regional L.N.’s, +/-spleen, bacteremia

*Preferential localisation of brucella organisms in uterus of pregnant females due to erythritol & other “allantoic fluid factors” which stimulate their growth

Brucella - liver granuloma with necrosis

Brucellosis is a zoonotic disease and foodborne infection of historical significance

Brucellosis is considered by FAO, WHO, and 0IE as the most widespread zoonosis in the world.

An occupational hazard for farmers, vets, animal handlers, abattoir workers, kennel workers & breeders, hunters.

Human infection is chronic & debilitating

Consumption of unpasteurised dairy products is a risk

Pasteurisation of milk has been important in the control of human brucellosis

*B. abortus is a Reportable Disease in Canada & USA*

http://www.inspection.gc.ca/english/sci/bio/anima/disemala/brucelle.shtml#il

Transmission is by ingestion, inhalation of aerosols, contact on nasal and conjunctival mucosa, skin abrasions, venereal (not all Brucellaspecies)

Primary sources of transmission:

contact with abortion tissues & fluids (fetus, placenta, vaginal exudate), milk, semen, urine, & blood

cows also eat placental tissues & lick calves after calving

Venereal transmission of B. suis, B. ovis, B. melitensis, & B. canis from infected males to females

*Accidental self- inoculation of Brucella vaccines by vets

.

B. abortus infects primarily cattle and bison◦ also dogs, horses, sheep, goats, pigs, cervids, camels, humans

Cows: localise/replicate in trophoblasts of placenta ulceration of chorioallantoic membrane (necrotic placentitis). Fetus infected by blood and/or ingestion of amniotic fluid.

Abortion in 3rd trimester; no warning; still birth/weak calf , retained placenta (RP); mechanism fetal death? (anoxia, endotoxemia, fetal stress..?)

Placenta leathery with thickened intercotyledonary regions, necrotic yellow-grey cotyledons with brown exudate, ◦ aborted fetus either no overt signs of infection or autolytic/edematous

Most return to normal fertility, but shed bacteria in milk for life. Brucellae bacteria remain in uterus and supramammary L.N.

◦ Calves may be born weak or healthy (“healthy” carrier heifer calves)

B. abortus – bovine placentitis;necrotic cotyledons

Bulls: orchitis, epididymitis, ’d fertility; (no venereal transmission?)

Chronic infection can cause hygromas of bursa, tendon sheaths, as well as arthritis.

Brucellosis - bovine vertebrae. Purulent exudate within a vertebra extends into the adjacent spinal canal. © 2004-2010 Iowa State University

Hygroma

An Old World Disease introduced into the New World

*The general consensus is that B. abortus was introduced into NA wildlife by domestic cattle*

Ref. On the Origin of Brucellosis in Bison of Yellowstone National Park: A Review .Mary Meagher and Margaret E. Meyer

Conservation Biology, Vol. 8, No. 3 (Sep., 1994), pp. 645-653 )

B. abortus is established in bison & elk in Canada’s Wood Bison National Park; & in Yellowstone National Park (YNP) in USA→ ongoing problems with surrounding cattle herds; State of Montana lost its brucellosis free status in 2007 & downgraded to Brucellosis Class A State; but opposition to suggested creation of a brucellosis buffer (“hot”) zone around YNP, requiring all cattle entering zone to be tested for brucellosis.

The southern YNP elk were originally infected with B. abortus from ranch cattle in 1880s during co-mingling at winter feed grounds; source of infection of YNP bison is ?? but is thought to have come from park cows prior to 1917.

History of Canadian Wood bison herds - http://www.pc.gc.ca/eng/pn-np/ab/elkisland/natcul/natcul1/b/ii.aspx

Elk and moose: Some wild elk in Canada & USA are infected with B. abortus.

Moose are very susceptible to B. abortus & die quickly so that no sero-positive free-ranging moose have been found in NA. Predators (wolves/coyotes, crows, bears, vultures) spread carcass and abortion tissues.

A case report of B. suis biovar 4 infection in a moose→ chronic carpal bursitis & osteomyelitis (Ref . Honour S & MH Hickling. J Wildlife Diseases . 1993;29:596-598)

Deer are susceptible to B. abortus, but disease is relatively rare in Canada, USA.

Farmed reindeer & elk (cervids): CFIA requires testing prior to importation of animals and herds must be tested every two years.

Brucellosis could be spread by wolves eating infected moose carcass

Horses:

Considered resistant, but rare infection occurs from contact with infected cattle (B. abortus) or swine (B. suis);

Brucellae localise in joints, bursae, & tendon sheaths causing supraspinous bursitis known as “fistulous withers” & “poll evil”, and hygroma (subcutaneous swelling over a bursa)

Still something to keep in mind as a differential diagnosis:

Today, the highest prevalence of equine brucellosis is in Texas that has Class B status for brucellosis, the incidence is also higher in some Class A states, such as Florida, Alabama, and Louisiana, as compared to the brucellosis-free states. Indiana is a brucellosis-free state and has not had a reported case of equine brucellosis in the past two years. However, Brucella abortus should be a differential diag­nosis for Indiana horses with fistulous withers, especially if they have been transported from brucellosis-positive regions. www.horsetech.com Read more: http://www.articlesbase.com/diseases-and-conditions-

articles/equine-brucellosis-in-india-787600.html#ixzz11IbyBaAk

Brucellosis in horses – fistulous withers and poll evil

*B. melitensis is the most virulent of the Brucella species & least host -specific*

B. melitensis is most virulent species for humans*

causes disease in sheep and goats, also camels, alpacas; goats very susceptible

Transmission to offspring by dam’s infected milk

Endemic in many countries of Europe, Middle East, Africa, China, Latin & South America

Causes most cases of human brucellosis from ingestion of raw milk

(local delicacies – raw goat, sheep or camel milk or cheeses)

S. suis and swine:

*Canada is officially free of B. suis (except for B. suis biovar 4)

Sows: abortion in sows (normal, weak, stillborn or mummified piglets) with temporary or permanent sterility

Boars: orchitis and infertility, venereal transmission by infected boars to sows

Also lameness, spondylitis, & paralysis, granulomas in organs

B. suis has five biovars: biovars 1 & 3 - domestic and feral swine - biovar 2 - wild hares in Europe, are reservoir for swine biovar 4 - (B. rangifur) reindeer & caribou throughout the Arctic

region (Alaska, Canada, Siberia) biovar 5 - rodents in Europe

Caribou carpus, B. suis biovar 4.Credit: Dr. G. Wobeser,Canadian Cooperative Wildlife Health Centre

B. suis – abortion in sows

Feral pigs are a source of B. suis infection for humans and domestic pigs in USA

A conservation group believes the threat wild hogs pose to Michigan’s farms, forests and wildlife is so grave that the state should pay bounty hunters up to $75 apiece to eliminate the 3,000 to 5,000 feral pigs roaming in 63 counties

“If we turn a blind eye, we’ll have 50,000 in two years,” said Dennis Fijalkowski, executive director of the non-profit Michigan Wildlife Conservancy.

The hogs, including European boars, generally are escapees from commercial game ranches.

“They eat anything that moves or doesn’t move,” he said. Wild hogs have caused great damage in other states, destroying habitat

including in the Everglades and killing other animals, including ground-nesting birds and their young and fawn deer. The bounty system Fijalkowski proposes would pay the most for females weighing more than 100 pounds and less for large males. Smaller pigs would bring a bounty of just $20 under his proposal.

Currently, Michigan hunters are encouraged to shoot any wild hogs they see. Anyone holding a valid Michigan hunting license can kill the wild hogs.

Courtesy of Detroit Free Press

Only in sheep, but worldwide distribution, least pathogenic of Brucella species

Important cause of epididymitis & orchitis in rams, causing infertility, also nephritis

Less frequently a cause of abortion in ewes

Venereal transmission (ram to ram, or ram to ewe)

B. canis infects dogs, foxes, coyotes (rarely humans, but is considered a zoonotic)

Females: infection of pregnant bitch results in abortion, stillbirths, splenitis, endometritis, sublumbar L.N. lympadenopathy, bacteremia

Males: prostatitis, sublumbar lymphadenopathy, epididymitis with testicular atrophy & scrotal swelling, infertility; scrotal dermatitis (licking scrotum)

Transmission from ingestion or inhalation from aborted puppies, vaginal discharges, milk, semen, urine. Nonpregnant females shed organisms in saliva, nasal, vaginal secretions

Occasionally: osteomyelitis, endophthalmitis, meningitis, discospondylitis, and glomerulonephritis

B. canis reported from several countries including Canada, USA, Mexico

(see Iowa State University reference, next page)

Using the Canine Brucellosis Antibody Test Kit a rapid slide agglutination test (RSAT) D-Tec® CB, Synbiotics Corporation

RSAT useful for testing breeding dogs

B. canis more prevalent in USA than in Canada (6% in southern USA); B. canis is reportable in some states in USA

Occasionally, normal dogs will react + in RSAT due to non-specific agglutinins. These are removed by 2- mercaptoethanol, so the kit includes a second test =

2ME RSAT.

If a dog is positive with the 2ME RST, then the serum should be retested by agar gel immunodiffusion (AGID) at a Brucellosis Reference Lab

Definitive diagnosis is by culture of tissues or blood at a Brucellosis Reference Lab.

◦ Brucellosis Reference Labs: In Canada - CFIA, Ottawa, Ontario.◦ In USA: Animal Health Diagnostic Center, Cornell University, Ithaca NY.

A good B. canis reference by Iowa State University, 2009: http://www.cfsph.iastate.edu/Factsheets/pdfs/brucellosis_canis.pdf

An old disease; contagious undulant fevers described in the Mediterranean area with different names since the 1500s

1887 - Micrococcus melitensis described by Dr. David Bruce as cause of Malta/Maltese fever of the island of Malta; discovered goats as main reservoir of infection & association of infection with drinking raw milk.

1897- Brucella abortus discovered by Dr. Bernhard Bang (vet/physician) in Denmark as cause of contagious abortion in cattle, & infections in sheep, goats, horses; known as “Bang’s disease”

Sir Temi Zammit

Dr. David Bruce

Dr. Bernhard Bang

In humans, brucellosis can be caused by B. abortus, B. melitensis, B. suis and, rarely, B. canis

*B. melitensis is most virulent species for humans*

A RES disease with lymphadenopathy, splenomegaly, hepatomegaly,

Nonspecific signs: undulating fever, night sweats, fatigue, muscle & joint pain, backaches, depression and insomnia

Specific signs: arthritis, osteomyelitis (vertebra) & endocarditis, orchitis

Chronic & debilitating; tough to diagnose/easy to misdiagnose (→ suicide)

Treatment: doxycycline and rifampin for 6 weeks and longer

Bovine brucellosis disease control & elimination is based on federal government programs :

“Test and slaughter”

Involves detection by serologic testing; quarantine of positive herds; identification and slaughter of infected cattle; screening of bulk milk of dairy herds for antibodies using the Brucella Milk

ring test (and the use of vaccines in USA, but not in Canada)

Test and slaughter Brucella milk ring test –Detects Brucella bacteriain bulk tank milk

There are many serodiagnostic tests for bovine brucellosis:

Use screening tests with high sensitivity and low specificity: Serum agglutination tests: RBT (rose bengal plate test), BPAT ( buffered acidified plate antigen test) Cross-reactions in cattle exposed to Yersinia, Francisella, & E. coli

O157:H7

Follow-up of positive reactors using confirmatory tests with high specificity:

(rivanol agglutination, complement fixation, ELISA, fluorescence immunoassays)

ELISA for screening B. ovis in sheep

The PrioCHECK® Brucella Ab - an indirect ELISA that detects antibodies against Brucella abortus and Brucella melitensis in

blood and milk samples of cattle, sheep and goats.

Rose bengal plate test

B. abortus strain 19, live attenuated:

first vaccine, introduced in 1939 in USA

can cause abortions in cattle and orchitis in bulls,& be shed in milk,

also infects humans by accidental self-administration

vaccinated animals have positive reactions in serodiagnostic tests

B. abortus strain RB51, live attenuated:

Introduced in USA in 1996; does not cause animals to shed

also infectious for humans

vaccinated animals do not react on serodiagnostic tests

B. melitensis REV 1, a live attenuated vaccine used for sheep and goats; safe & protective, but causes positive reactions in serodiagnostic tests

BRUDOLL-M is a live, attenuated and freeze-dried vaccine against Brucellosis (Brucella melitensis Rev-1) disease of sheep and goats.

*Brucellosis (B. abortus) is a reportable disease in Canada, you must contact a CFIA district veterinarian*

Canada’s cattle herd was declared officially brucellosis-free in 1985. Disease monitoring continues, but milk ring testing and market cow testing of slaughter cattle ended in 1999.

B. abortus and B. suis are officially eradicated except for geographically restricted wildlife reservoirs in Northern Canada

*B. abortus and bovine tuberculosis exist in Wood Bison National Park, in Alberta /NWT

This is an ongoing political issue involving aboriginal rights/ wildlife conservation & environmentalists/ cattle industry/ federal government

B. suis biovar 4 is endemic in Arctic region

B. ovis and B. canis - not subject to control; are indigenous/endemic

B. melitensis - has never been found in Canada

The Italian government has set up an emergency commission to try and stop the spread of the disease, which affects milk production. The plains of Campania, around Naples, are home to large buffalo herds.

The area is the exalted home of mozzarella di bufala: not only an essential part of the Italian diet, but one of Italy's most important exports.

But the Neapolitan farmers who make this famous milky cheese are facing a crisis.

In the next two months, the Italian government will start the slaughter of 32,000 buffalo, infected with Brucellosis - a contagious bacterial disease that in livestock leads to abortion, infertility and reduced milk production.

It can be transmitted through food to humans, causing severe intermittent fever - though the milk which produces the cheese is perfectly safe when it is pasteurised.

Brucellosis has been present in the herd for 10 years.

But the Italian papers say the local vets who are supposed to test and put down infected animals have been intimidated by the local mafia - the Camorra - who also control some of the farms.

Consequently, it is reported, the disease has been allowed to spread to almost 30% of the herd.

Caserta, one of two key mozzarella-producing areas, is the worst affected.

In the coming weeks, armed police will accompany government vets to help with the cull.

They say every infected animal destroyed will be burnt - and it is estimated the cost to the Italian government will be 66m euros ($97m; £49m).

BBC News, Rome, Jan. 17, 2008

Occupational hazard for veterinarians prior to the use of rectal gloves for dystocia in cattle, sheep, goats, pigs

Occupational hazard for veterinarians vaccinating cattle with B. abortus Strain 19 & RB51

Many vets have suffered from or died of brucellosis