10/3/2011

Course: VPM 201 Fall, 2011

Lecturer: 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 Brucella are
modified acid-fast
◦ National Microbiology Lab, Winnipeg, Manitoba

 Level 3 pathogen:
 Classified as potential bioterrorism agent because of aerosol
transmission risk and no human vaccines available

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 Six classical Brucella species & their primary hosts:

 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

 Marine Brucella species & their primary hosts:

 B. ceti (was B. cetaceae – see S&P text) - cetaceans (whales,
dolphins, & porpoises)
 B. pinnipedialis (was B. pinnipediae - see S&P text) - seals

 Brucella species cause brucellosis

 Cause disease in several animal species: humans, ruminants, swine,

dogs, horses, cervids (deer, elk, reindeer, moose), rodents, marine
mammals
 Cats are resistant to infection

 Infect reticuloendothelial system (RES ) (= mononuclear phagocyte

system) & 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 +
pasteurization

Louis Pasteur invented pasteurization in 1862

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 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.

 Human infection is chronic & debilitating

 An occupational hazard for farmers, vets, animal handlers,

abattoir workers, kennel workers & breeders, hunters.

 Consumption of unpasteurized dairy products is a risk

 Pasteurization of milk has been important in the control of
human brucellosis
"If you're on a farm, feces and other things have a risk of getting
into the milk and causing contamination. That's why pasteurization
was invented - to get rid of harmful bacteria.“ – Fergus Clydesdale.

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

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

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Transmission is by ingestion, inhalation of aerosols, contact on

nasal and conjunctival mucosa, skin abrasions, and venereal (not
all Brucella species have venereal transmission)

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 abortions in dairy cattle

 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. Brucella 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

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 Bulls: orchitis, epididymitis, decreased fertility; (no venereal

transmission?)

 Chronic infection can cause hygromas of bursa, tendon sheaths,

as well as arthritis.

Hygroma

Brucellosis - bovine vertebrae.

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

An Old World Disease introduced into the New World

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 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 resulting in ongoing problems
with surrounding cattle herds; State of Montana lost its brucellosis free
status in 2007 & was downgraded to Brucellosis Class A State; but there is
opposition to the suggested creation of a brucellosis buffer (“hot”) zone
around YNP, requiring all cattle entering zone to be tested for brucellosis.
 Update 2011: Beginning Sept. 1, Colorado will require that all sexually intact female cattle that have
spent any time near Yellowstone carry a Colorado-approved ear tag. Wyoming opted not to go with an
animal identification system. Nebraska has published draft rules that could take effect as early as
April 1, 2011.

 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

The USA Yellowstone National Park Buffalo

Story

Buffalo Field Campaign

West Yellowstone, Montana
Working in the field every day to stop the
slaughter of Yellowstone's wild free roaming buffalo

Total Yellowstone
Buffalo Killed
Winter 2007/2008
1616

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 Bison within the park are infected with both tuberculosis and
brucellosis. In 1995, a Bison Research and Containment Program (BRCP)
was initiated to ensure the diseases were properly researched and
contained within WBNP. In 1999, a Risk Assessment was carried out to
determine the risk of infection with tuberculosis or brucellosis to
cattle and disease-free captive and free-ranging bison.

To prevent the spread of these diseases to uninfected herds outside

the park, a Bison Control Area was created. Any bison found within the
Bison Control Area are quickly removed.

Dr. Todd Shury immobilizes a bull wood bison to help Parks

Canada determine how TB and brucellosis are affecting the
Dr. Scott McBurney, Clinical Veterinary
bison herd at Wood Buffalo National Park. Professional-Wildlife Pathologist, Dept. Pathology &
© Parks Canada, Dr. Todd Shury, 1999 Microbiology, AVC

 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

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 Horses:
 Are considered resistant, but rare infection occurs from contact with
infected cattle (B. abortus) or swine (B. suis);
 Brucella organisms localise in joints, bursae, & tendon sheaths causing
supraspinous bursitis known as “fistulous withers” & “poll evil”, and
hygroma (subcutaneous swelling over a bursa)

 Still brucellosis is 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, B. abortus should be a differential
diagnosis for Indiana horses with fistulous withers, especially if they have been
transported from brucellosis-positive regions.

Brucellosis in horses – fistulous withers and poll evil

 B. melitensis is the most virulent of the Brucella species &

least host -specific
 B. melitensis is the most virulent species for humans

 Causes disease in sheep and goats, also camels, alpacas

 Goats are very susceptible
 Transmission to offspring by dam’s infected milk

 Endemic in many countries of Europe, Middle East, Africa, China,

Latin & South America
 B. melitensis causes most cases of human brucellosis from the
ingestion of raw milk
 (local delicacies – raw goat, sheep or camel milk or cheeses)

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B. suis – abortion in sows

 B. 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

 Feral pigs are a source of B. suis infection for humans

and domestic pigs in USA.

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
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)

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 B. canis infects dogs, foxes, coyotes (rarely humans, but is

considered a zoonosis

 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 (higher prevalence in USA and 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)
 The RSAT is 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

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 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”

Dr. David Bruce

Sir Temi Zammit- in 1905 demonstrated contaminated milk

as source of brucellosis in humans by isolating B. melitensis Dr. Bernhard Bang
from the blood of the goat.

 In humans, brucellosis can be caused by B. abortus, B.

melitensis, B. suis and, rarely, B. canis
 Remember: B. melitensis is most virulent Brucella 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

(→ depression and suicide)
 Treatment: doxycycline and rifampin for 6 weeks and longer

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 Bovine brucellosis disease control & elimination are 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 bacteria
in 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

Rose bengal plate test 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.

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 B. abortus strain 19, live attenuated:

 Was the 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

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 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 pasteurized.
 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

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