Domestic – Bighorn Sheep Interface
Problem Overview and Research
Maggie Highland, DVM, PhDc, Dipl. ACVPPhD Veterinary Training Program
USDA-ARS ADRUVeterinary Microbiology and Pathology
Washington State UniversityPullman, WA
PLC
American Sheep Industry Annual Convention
Reno, NV
January 27-31, 2015
Bighorn pneumonia and the domestic sheep industry
Captive commingling studies and anecdotal field reportsassociation between interspecies contact and BHS pneumonia
Captive experiments (infecting BHS and DS; blood cell testing)BHS are more susceptible to pneumonia-associated bacteria
Profound economic and ecologic impacts
DS grazing restrictions on public land allotments
• ~48% of DS in the U.S.A. spend time on public lands
• $232M @ farm gate + $576M in supported economic activity(personal communication: Margaret Soulen Hinson, 2012)
Resources invested in wild BHS herds & >3 decades of research
Pneumonic disease continues to impact wild BHS
Polymicrobial (more than 1 bacteria involved)
What do we know about bighorn sheep pneumonia?
Incompletely understood disease phenomenon
Multifactorial(the presence of the bacteria in BHS
alone does NOT = disease/death)
BHS (wild)
Reports of respiratory disease date back to the 1920’s
All age outbreaks often followed by year(s) of disease in lambs
→ population-limiting disease
Cause
• Long been debated……debate continues
• Polymicrobial (multiple bacteria in lungs) and multifactorial
• Viruses sometimes found
DS
Lambs >> Adults
Etiology
• Polymicrobial (bacteria, viruses) or Single microbial
• Multifactorial (colostrum, air quality, environmental stressors)
Overview of bacterial pneumonia in sheep
DS and BHS pneumonia-associated bacteria
Mycoplasma ovipneumoniae (Mo)
Pasteurellaceae
Mannheimia haemolytica (Mh)• Pasteurella haemolytica biotype A (prior to 1999)
Bibersteinia trehalosi (Bt)• P. haemolytica biotype T and 3 (prior to 1990)
• P. trehalosi (1990-2007)
Pasteurella multocida
Anaerobic bacteria – Fusobacterium necrophorum
Other aerobic bacteria
Mycoplasma ovipneumoniae
1° respiratory pathogen → 2° pulmonary bacterial infections
Colonizes respiratory epithelium → impede mucociliary clearance
Chronic pneumonia in young DS (“coughing syndrome” )
Documented as species specific = sheep and goats (wild and domestic)
High association with pneumonia in wild BHS supported by multiple studies
Infection of BHS in historic captive commingling studies identified
Historic infrequent detection (difficult to culture)
Mannheimia haemolytica
Pasteurellaceae (“Pasteurella”) family member
Easily cultured by standard laboratory methods
Historically most commonly reported bacteria in BHS pneumonia
(along with Bibersteinia trehalosi……
remember both use to be called “Pasteurella”)
Only certain strains cause disease (leukotoxin-expressing strains)
Acute bronchopneumonia in compromised ruminants
Infection with a 1° pathogen (such as Mycoplasma spp.)
Environmental stressors (air quality, crowding, shipping, other?)
“Shipping fever”
Fusobacterium necrophorum
Anaerobic (difficult to culture by standard methods)
Found in the environment, oral cavity, rumen (“commensal”)
Disease in ruminants: hepatic abscesses, foot rot, necrotic laryngitis
Secondary infections due to epithelial compromise (opportunistic infections)
Leukotoxin (Lkt)
Secreted leukotoxin
Recently reported in high association with polymicrobial BHS pneumonia
Non-culture methods – 16s sequencing and cloning(Besser, Highland, et al. Emerg. Infect. Dis. 2012)
M. ovipneumoniae
Healthy DS herds: 87% positive (453 tested) (National Animal Health Monitoring System-Sheep201; personal communication T. Besser)
Healthy BHS herds: 4 of 32 positive Pneumonic BHS herds: healthy carriers present (disease w/in last 10 yrs) (Besser, et al. Prev. Vet. Med. 2012)
Confounding the matter….
DS and BHS pneumonic agents as “commensals”
“Pasteurella” bacteria (including disease causing forms)
Upper respiratory/throat in both DS and BHS Multiple publications support this fact
In other words….infection does NOT equal diseaseand
transmission does NOT equal disease
Past Research
How did we get here?
Captive interspecies commingling studies
Species commingled Bighorn sheep
(died/total)
% death # of studies
Bacteria
DS (39) 41/43 95% 7 Mh, Bt, Mo, A. pyogenes,
Corynebacterium
Mo-free DS (4) 1/4 25% 1 Mh, Bt (@day 90)
Goat (7) 2/10 20% 2 Mh
Horse (3) 1/6 17% 1 Pm, Strep zoo
Cattle 1/9 11% 2 Mh
(Foreyt: 1982, 1989, 1990, 1994, 1996, 1998, 2009; Onderka1988; Besser2012)
Death in BHS between 8 days and 3 months
BHS – DS Comparative Experiments
Neutrophils (a type of white blood cell) collected from BHS & DS- BHS neutrophils 4 – 8 x more sensitive than DS to toxin of “Pasteurellas”
(Silflow, et al. J Wildl Dis. 1989; 1994)
Inoculation studies (intranasal and intratracheal)- Multiple studies reported death in BHS and no disease in DS
- no consideration of the immune status of the animals prior to study or BHS had “low” titers (Dassanayake, et al. Vet. Microbiol. 2009; 2013)
- BHS have “defective pulmonary clearance” of M. haemolytica- BHS had significantly lower lung and blood antibody titers to bacteria(Subramanian, et al. Vet Microbiol. 2011)
Passive transfer study indicating low transfer of antibodies - Looked specifically at antibodies for one bacteria (M. haemolytica)
- also reported that the bighorn ewes had low/no M. haemolytica titers(Herndon, Clin Vaccine Immunol, 2011)
Leading to a belief (misconception?) that bighorn sheep are “immunocompromised”Unable to handle even natural infection with leukotoxin positive “Pasteurellas”
Neutralizing serologic LktA titers
Pre-commingling Day 104
BHS – P1 0 5.3 (day 90)
BHS – A 0 7.6
BHS – B 0 8.9
BHS - C 5.3 8.3
DS - A 7.6 8.3
DS – B 7.6 7.6
DS – C 6.0 8.3
DS – D 8.3 7.6
“natural” exposure BHS develop serologic nLktA
(personal communication T. Besser; assay performed in Srikumaran laboratory - WSU)
Captive interspecies commingling studiesBHS commingled w/ Mo-free DS
Current research at ADRU-ARS-USDA Pullman, WA
If domestic and bighorn sheep are raised equally, are their immune systems equally protective?
Domestic sheep and bighorn sheep were taken at birth
• No contact with the ewe or any other sheep
• Hand-raised (collected colostrum from ewe then milk replacer after)
• Separated by species
• Remain free of virulent (Lkt+) Pasteurella bacteria and M. ovipneumoniae
What we are investigating?
• Passive transfer of antibodies from ewe to lamb
• Repeat toxicity assays on neutrophils from SPF animals
• Comparative look at immune cell response to pathogen exposure (in vitro)- Innate and acquired immune responses
• Intranasal immunization (atomization)
heat killed Mycoplasma ovipneumoniae and Mannheimia haemolytica
(Bacteria from lung tissue of BHS that died from pneumonia)
1.0
10.0
100.0 Eweserumcolostrum
24 hr
3 wk
6 wk
9 wk
12 wk
16 wk
20 wk
24 wk
mg
IgG
/m
l sam
ple
Lam
b
seru
m
**
*
Passive transferTotal IgG
(Highland, unpublished data)
* **
Sheep IgG (total) ELISA kit; Alpha Diagnostics
Domestic Bighorn
0
10
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100
DS
BHS
Grans Neuts 256 512 1024
Cell Purity (LktA+)Mh supernatant dilutions
%
Cytotoxicity Assay – (LktA+)Mh supernatantLDH release assay, neutrophils
0.0
10.0
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% c
yto
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BHS
% neuts 128 256 512 1024 2048 4096
(LktA+) F. necrophorum supernatant dilution
Cytotoxicity Assay with supernatant from(LktA+)Fusobacterium necrophorum
LDH release assay, neutrophil enriched WBCs
0.0
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% b
act
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s/k
illi
ng
Lkt+ M. haemolytica growth inhibition/killing by neutrophils
DS Sera, diff neuts
DS Sera, equalized neuts
HI-DS Sera, equalized neuts
Naïve DS sera
Naïve BHS sera
DS BHS
Additional Research
Investigating other potential wildlife reservoirs/carriers of Mycoplasma ovipneumonie
• Rabbits (wild cottontail)
• Deer/other wild ungulates
Data Base for Record and Risk Assessment
Land use• GIS documentation or mapping of BHS herd locations and herd size• Mapping of all public DS rangelands• Survey all private lands within and surrounding known BHS herd ranges
Map locations of private lands that have DS and goats
Human interactions with BHS• Wildlife agencies• Other gov’t and private activities• Hunting
Permits issued Herd size # Harvested
Disease documentation in DS and BHS• Dates• Number affected• Symptoms• Pathogens identified and by whom
Environment• Weather• Feeding stations• Natural disasters (ie. fire)• Non-human predators
Commensal bacteria screening in DS and BHS•Mycoplasma ovipneumoniae strain typing•Pasteurellaceae (Mh, Bt, Pm)
Points of Interest
• No evidence of epizootic spread of M. haemolytica– Vaccine feasibility?
• Different strains of Mh are not cross protective (problem with shipping fever)
• M. ovipneumoniae: same strain identified in BHS has not yet been reported in “associated” or “nearby” DS– Do DS carry M. ovipneumoniae? YES
– Are they only source? NO (BHS carry this bacterium too; other animals?)
• Time between BHS infection w/ M. ovipneumoniae to disease onset
• Be critical of published research
Conclusion• Polymicrobial disease – focus for years was narrowed to M. haemolytica
(“Pasteurella”), with no strong evidence to support this as the primary cause for epizootic pneumonia (when present…different strains)
• Mycoplasma ovipneumoniae – remember this bacteria
• USDA research: Alternative approach to comparatively evaluate the immune systems of BHS and DS to advance the basic understanding of immune responses to pulmonary disease agents
• Database: attempt to sort out the multifactorial component
Kenny Lyle
IsabellaWillow
Denise
Polly
Thank You
• Donald Knowles
• Tom Besser
• Stephen White
• Jim Reynolds
• Nic Durfee
• David Herndon
• Frances Cassirer
• Dave Schneider
• USDA-ADRU Pullman Staff
• WSU-VMP staff
• Washington Animal Disease Diagnostic Laboratory
Questions?
Contact info: [email protected]
