Hoofstock & Megavertebrate Bacteriology

Question 1

Describe Brucella infections in North American wildlife.

Describe the morphology and survival strategy of Brucella spp.

What type of disease does it cause in people?

What did the Federal Brucellosis Eradication Program entail?

What are the four main brucella infections in NA wildlife?

Answer 1

Chapter 45 – Brucellosis in North American Wildlife

• Brucella sp bacterial organisms are small, nonspore-forming gram negative coccobacilli living inside host cells
• Subdivided into biovars and genotypes
• Most cause zoonotic disease
  
  • Humans -undulant fever – persistent and recurrent fever, chills, night sweats, headache, arthralgia, arthritis, anorexia, nausea, weight loss, malaise, dementia
  • Responsive to antibiotic therapy, recrudescence not uncommon
• Different organisms have been found in humans, cattle, swine, dogs, sheep, woodrats, cetaceans, seals, frogs, voles, foxes, boars, baboons, rodents
• Cooperative State-Federal Brucellosis Eradication Program
  
  • 80 year eradication effort in the US
  • Early 1900s – most economically devastating disease of livestock
  • 1934 – Cooperative State-Federal Brucellosis Eradication program begun
  • Quarantine, test and slaughter, calfhood vaccination, adult vaccination
  • When began, 50% of cattle herds tested had seropositive/suspect animals
  • By 1990s, <200 affected herds remained
  • In 1950s, efforts to control swine brucellosis begun, currently is eradicated from commercial swine
• Brucellosis in Wildlife
  
  • Four ecosystem infections in NA wildlife
    
    • B. abortus in bison and elk in Greater Yellowstone Area and in bison in Wood Buffalo National Park in Canada
    • B. suis in invasive wild swine
    • B suis biovar 4 in caribou and other artic wildlife
    • B ceti and B pinnipedialis in marine mammals

Question 2

Describe brucellosis in Yellowstone - what species are affected?

How prevalent is brucella in the Wood Buffalo National Park Bison?

How is brucella transmitted in Bison? What risk are there to domestic cattle?

How does it affect their reproductive success? What reproductive lesions are observed in bison?

What nonreproductive lesions are seen?

Answer 2

• Brucellosis in Bison/Elk in Yellowstone
  
  • First diagnosed in 1917 in bison in Yellowstone, in elk in 1930-31 in Yellowstone and national Elk Refuge in Wyoming
  • 5000 bison and 100,000 elk in greater Yellowstone area, with bison having ~50-60% seroprevalence and elk having 0-40% seroprevalence
  • Many cases of transmission to cattle and captive bison herds
  • Elk have become a maintenance host due to increasing population, land use changes causing larger congregations of elk, reintroduction of wolves changing elk behavior
• Brucellosis in Wood Buffalo National Park Bison
  
  • Canada’s only known wildlife reservoir is several subpopulations of bison in and near WBNP.
  • Population ~4500 animals, with ~25% of examined having evidence of infection
• Brucellosis in Bison
  
  • Transmitted through ingestion/mucosal contact with organisms shed in placenta, fetus, birth fluids. Milk – but calves appear to clear any infection this way.
  • Seroconversion to positive rates (indicating at least exposure) are 20% per year for calves up to 3yrs, then 10% per year.
  • Latent infection or “heifer syndrome” is rare in cattle, not been documented yet in bison. Calves born to infected dams latently infected, seroconvert after first parturition where they abort or shed organisms.
  • Some female bison infected abort several times, some do not. Some shed at parturition, some have normal calves.
  • Infection causes metritis and mastitis
  • In bulls, causes seminal vesiculitis, epididymitis, ampullitis, and orchitis – may affect fertility
  • Does not seem to have venereal transmission
  • Nonreproductive tract lesions – arthritis, abscesses, bursitis, hyrgroma formation

Question 3

How does Brucella affect cervids?

What species affects them? What species of cerivds are affected?

What lesions are seen in each species?

Why are some cervids less effective transmitters of this disease?

Do all cervids follow that same pattern or are others more affected by the disease?

Answer 3

• Brucellosis in Elk
  
  • B abortus, elk less efficient disease transmitters – birthing process more isolated
  • Bursa, joint, tendon sheath infections not uncommon
  • Less frequently affects mammary gland
• Brucellosis in moose
  
  • B abortus
  • Pericarditis, myocarditis, pleuritis, peritonitis, arteritis, lymphadenitis, orchitis, hepatitis, arthritis
  • Persistent bacteremia, generally fatal, dead end host
• Brucellosis in Caribou
  
  • Hundreds of thousands of free ranging caribou in boreal, montane, artic environments, as well as herds of reindeer and domestic caribou
  • Brucellosis serologically detected in humans in Alaska in 1939, B suis biovar 4 was isolated in 1963 from caribou.
  • Current seroprevalence is <5% in Alaskan reindeer and variable in caribou (can exceed 40%)
  • Transmission by ingestion or contact with prodcts of partuition, unknown if venereal or vertical transmission through milk
  • Abortion, weak calves, retained placentas, lameness, sterility, orchitis, epididymitis, seminal vesiculitis, metritis, mastitis, nephritis, bursitis.
  • Vaccination and test/cull practiced in reindeer

Question 4

Describe the effects of brucella in bighorn sheep.

What lesions have been observed?

Answer 4

• Brucellosis in Bighorn Sheep
  
  • B abortus infection seen at research facility – fence line contact with infected elk
  • Abortion, placentitis, orchitis, epididymitis, lymphadenitis, death of two rams
  • Serological titers to B ovis detected. In experimental infection, showed seroconversion, abortion, epididymitis, ampullitis, seminal vesiculitis, prostatitis, necrotizing and pyogranulomatous orchitis

Question 5

Describe brucellosis in North American feral swine.

What species and serovar predominantly affects them?

What clinical signs and lesions are seen in feral swine?

How is thies disease transmitted? What risks are their to the domestic population?

Answer 5

• Brucellosis in Wild Pigs
  
  • Brucella suis – isolated in invasive pig in 1974 in South Carolina, has been isolated in 8 states since
  • Biovar 1 predominant in NA, biovar 3 found in Hawaii
  • 38 states and 3 canadian provinces harbor invasive wild pigs, little information on epidemiology
  • B suis infection – weak/stillborn piglets, abortion, orchitis, lameness, posterior paralysis due to arthritis. Associated lesions – placentitis, seminal vesiculitis, epididymitis, arthritis, abscessation
  • Transmission venereal route and exposure to aborted fetuses or other products of parturition
  • B suis in humans occurs through aerosol and oral routes and breaks in the skin

Question 6

Describe the etiologic agent of Johne’s Disease.

How does strain affect species susceptibility?

Is it stable in the environment?

Answer 6

Key Points:

• Must distinguish between presence of organism and true infection in free-ranging wildlife
• Mycobacterium avium subsp. Paratuberculosis (MAP)
• Ubiquitous opportunistic pathogen for humans and animals
• Acid-fast, slow- to very slow-growing, nonpigmented, rod-shaped bacteria with complex, lipid-rich cell walls
• MAP strains are not restricted to host species
  
  • Any strain of MAP may infect any ruminant host
• Shed by infected animal in milk and feces
• Resistant to heat, cold, drying and acidic conditions
• Does not replicate in the environment
• Eventually dies off completely, but very slowly
• Study - diurnal temperature fluctuations more relevant than UV for complete MAP die-off
• Contaminated drinking water may be reservoir for new infections longer than contaminated ground
• In the US, genome is highly conserved
  
  • Most (78%) isolates from cattle belong to same genetic node
  • Cattle strain- now Type 1

Question 7

How do Johne’s infections differ between ruminant and nonruminant hosts?

Describe the epidemiology of Johne’s.

What industry has the highest incidence of Johne’s infections?

How is this disease transmitted?

Answer 7

Significant differences in ruminant vs nonruminant hosts

Ruminant Infections-

• Johne’s disease primarily a concern for ruminant species
• Disease and death reported for captive and free-ranging ungulates of all taxonomic groups
• All ruminant species thought to be susceptible, clearing infection is unlikely

Nonruminant Infections-

• Evidence of MAP infection reported in nonruminant wildlife species on occasion, but not clinical disease
• Histopath changes are mild to nonexistent (exception – lagamorphs)
• Greater likelihood for presence of MAP in carnivorous predators or scavengers or species in close proximity to infected domestic agricultural animals

Epidemiology-

• Primary route wildlife encounters MAP- sharing range with infected domestic agricultural ruminant species
• Global diary industry reports the highest prevalence of Johne’s disease
  
  • USDA- organism detected on 68% of US dairy premises
• Environmental (soil and water) burden of MAP highest in intensively farmed areas
  
  • Cow produces 100 pounds manure/day
  • Manure spreading on field common agricultural practice
• Fecal-oral route primary means of transmission
  
  • MAP ingested by susceptible animal (<6 months old)
  • Sufficient organisms taken up by M cells in distal ileal Peyer’s patches to reside and replicate in phagosomes of subepithelial macrophages
  • Months to years- animal’s immune system ignores intracellular pathogen
  • Animal appears healthy but can still shed MAP in feces
• In utero transmission possible
• At some point during adulthood, infection spreads to regional LNs and disseminates throughout the body
• Most young acquire organism via manure-soiled teats, contaminated water, colostrum or milk, or in utero
  
  • Transmission rate increases as dam reaches further stages of infection
• Experimentally- infective oral dose for our cattle, deer, and goats is 4 x 10^6 CFUs (200 mg wet weight pelleted MAP)
  
  • Infected cattle may shed 10^6 CFUs/g of manure

Question 8

What are the clinical signs of Johne’s disease in ruminants?

What lesions are seen on necropsy?

Answer 8

Clinical Signs-

• Clinical disease known as paratuberculosis- slow to develop
• Affected ruminants usually >= 1 year
• Vague, nonspecific primary clinical signs- loss of body condition, poor hair coat, +_/-diarrhea in later stages

Pathology-

• Lesions range from inapparent to florid, depending on species and stage of infection
• Some cases of sheep, bison, nondomestic species- GI appears normal
• Ileum may be thickened, corrugated, reddened, enlarged mesenteric LNs
• Total absence or necrosis of abdominal fat stores
• Histo (Fig 81-2)- granulomatous infiltrate- intra-cytoplasmic, acid-fast, rod-shaped organisms in macrophages of lamina propria

Question 9

Johnes is maintained in what wild NA ruminants?

How prevalent is this disease?

How does the course of disease differ in these animals from that in cattle?

Answer 9

Bison-

• ELISA to detect antibody
• Strain isolated and characterized in other countries same as detected in US species
• Clinical signs, pathologic lesions same as in cattle and transmission is the same
• Canada- several herds PCR positive, but organism not recovered
• Serologic survey of banked sera- ELISA results for more than 1200 samples negative

Deer and Elk-

• Infected tule elk (Cervus elaphus nannodes) -first detected in 1979- Point Reyes Ntl Seashore, Marin County, CA
  
  • Confirmed in adults since then, but no obvious effect on herd health or reproduction
  • No evidence of MAP infection in native black-tailed deer at PRNS
  • Axis deer and fallow deer sharing range with tule deer- infection prevalence of 8-9% 20 years ago- no obvious effect on herds
• Surveys in Arkansas, Montana, and Wyoming elk- no indicators of MAP infection
• Free-ranging white-tailed deer- causative organisms isolated from clinically normal animals
  
  • Clinical cases in free-ranging WTD rare, several reports in captive WTD
• Florida Key deer- (O. virginianus clavium- endangered) paratuberculosis first diagnosed in 1996, organism persists in population and environment at low prevalence
• White-tailed deer-
• Virginia- single case reported – 2 yr old male
• No further cases found on surveillance
• Multistate survey in southern US- low prevalence of infection (0.3%)
• Clinical Johne’s disease reported occasionally in other free-ranging ungulates-
  
  • Rocky Mountain bighorn sheep
  • Rocky Mountain goats
  • Free-ranging red and fallow deer in Europe
• Quicker progression from infection to clinical signs in farmed cervids than bovids

Question 10

Johnes has been detected in what nonruminant species?

Were lesions observed in any cases?

Do they shedd the bacteria?

Answer 10

Rabbits-

• Research focused on rabbits sharing herd of cattle with Johne’s disease in Tayside, Scotland
• GI lesions with intracellular acid-fast bacilli in many cases
• Organism shed in the fecal pellets in one study
• Horizontal and vertical transmission in this population
• Population may serve as a reservoir for domestic and wildlife species
• Isolation of MAP from other lagomorph species in US (Sylvilagus floridanus) and Chile (Lepus europaeus), but no lesions or organisms detected

Camelids-

• Free-ranging guanacos (Lama guanicoe) on Tierra del Fuego Island, Chile
  
  • Shedding MAP, cattle-type strain
  • Low prevalence (4.2%, 21/501 fecal samples)
• Paratuberculosis of domestic alpacas in Australia and camels from Egypt and Saudi Arabia reported

Question 11

Describe the diagnosis of Johne’s.

What samples can be submitted for culture?

What are some complications with PCR?

What are some complications with using serologic testing?

What lesions are typically seen on necropsy? What tissues are best to examine?

Answer 11

Diagnosis-

• Recommend wildlife managers use labs experienced in MAP diagnosis specifically
  
  • In US labs can show competency in serologic, culture, and PCR detection in MAP infection in samples from cattle
  • Annual assay-specific check tests provided by USDA-APHIS (www.aphis.usda.gov/animal_health/lab_info_services/approved_labs.shtml)
• Two approaches to MAP diagnosis:
  
  • Look for evidence of organism (culture or direct PCR) – available for all species
  • Screen for response to infection (limited to cattle, sheep, and goats)
• ELISA for deer in Australia

Culture-

• Samples- blood, milk, water, soil, forage, fecal and tissue samples
• Pool fecal or environmental samples to decrease costs
• Liquid media more sensitive than solid media
• Samples incubated for at least 7 wks before considered MAP free
• Identification of organism- acid-fast staining, PCR or colony characteristics
• Quantification of number of MAP in sample useful for assessing level of environmental contamination
• Isolation of MAP from fecal sample may be organism passing through GI and not true infection

PCR-

• Used to ID acid-fast organism from culture, or directly from fecal without culture
• Target- MAP gene insertion sequence IS900, also other targets for multiple target PCR assay
• Prone to molecular contamination, or confounding by suboptimal processing protocols
• Most commercial direct PCR kits validated for bovine feces only
  
  • Can get false negative in feces from other species
• Reliance on PCR confirmation without proof of the living organism or other indicator of infection not recommended
  
  • Do not make culling decisions on the basis of direct PCR fecal results only
• Can also be used on paraffin-embedded tissues or an adjunct at necropsy
  
  • Higher sensitivity for tissues with visible intracellular MAP

Serologic Testing-

• Antibodies produced at late and clinical stages of MAP infection
• Does not clear or slow progress of Johne’s disease since intracellular pathogen
• Can detect herd prevalence via ELISA or AGID
• Sensitivity highest for clinically ill animals
• Assays validated for domestic (primarily bovine) species
  
  • USDA approved assay for use in small ruminants
• False negatives if infected animal not yet producing antibodies
• False positives if cross-reaction with other mycobacterial species such as M. bovis or Corynebacterium pseudotuberculosis
• Neither AGID or ELISA validated for nondomestic hoofstock
  
  • Can’t use in individual animal, but can be used for trends in herds

Postmortem Testing-

• Best tissues- ileum and mesenteric LNs, even if no gross lesions
• Two sets of tissues- one for culture, one for staining
• Lesions typically noncaseating granulomatous infection (caseation noted in red deer)
• Epitheliod macrophages within intestinal lamina propria and submucosa

Question 12

How is Johne’s treated?

How is it managed?

Answer 12

Treatment and Vaccines-

• Few treatment trials for infected ruminants- high cost and infeasibility
• Drugs tested used for other mycobacterioses- rifampin, clarithromycin, isoniazid
• One study in cattle-signs abated during treatment, but returned once stopped
• Killed vaccines-
  
  • Approved in numerous countries for cattle or small ruminants
• Other vaccines under development for domestic agrigultural species
• Transmission or organism continues, will have new cases in vaccinated herd or flock
• Only organism detection methods can be used in vaccinated herds or flocks

Management and Control

• Should be directed at preventing introduction to new populations
• Do not use infected wild animals for reintroduction or translocation programs
• Capture and release programs- move to uncontaminated location some months before birthing season begins
• Minimize contact between wildlife and domestic species
• MAP infection may become a greater threat in free-ranging wildlife health with rising prevalence of domestic livestock and shrinking wildlife habitat