Domestic Bighorn Sheep Interface Problem Overview and Research American Sheep Industry Annual Convention Reno, NV January 27-31, 2015 Maggie Highland, DVM, PhDc, Dipl. ACVP PhD Veterinary Training Program USDA-ARS ADRU Veterinary Microbiology and Pathology Washington State University Pullman, WA PLC
Bighorn pneumonia and the domestic sheep industry Captive commingling studies and anecdotal field reports association 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
What do we know about bighorn sheep pneumonia? Polymicrobial (more than 1 bacteria involved) Multifactorial (the presence of the bacteria in BHS alone does NOT = disease/death) Incompletely understood disease phenomenon
Overview of bacterial pneumonia in sheep BHS (wild) DS 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 Lambs >> Adults Etiology Polymicrobial (bacteria, viruses) or Single microbial Multifactorial (colostrum, air quality, environmental stressors)
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 Confounding the matter. DS and BHS pneumonic agents as commensals 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) 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 disease and 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
Captive interspecies commingling studies BHS commingled w/ Mo-free DS natural exposure BHS develop serologic nlkta 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 (personal communication T. Besser; assay performed in Srikumaran laboratory - WSU)
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)
mg IgG /ml sample Passive transfer Total IgG Lamb serum 100.0 Ewe serum colostrum 24 hr 10.0 * * * * 3 wk 6 wk 9 wk * * 12 wk 16 wk 20 wk 1.0 Domestic Bighorn 24 wk Sheep IgG (total) ELISA kit; Alpha Diagnostics (Highland, unpublished data)
Cytotoxicity Assay (LktA+)Mh supernatant LDH release assay, neutrophils 100 90 80 70 DS BHS % 60 50 40 30 20 10 0 Grans Neuts 256 512 1024 Cell Purity (LktA+)Mh supernatant dilutions
% cytotoxicity Cytotoxicity Assay with supernatant from (LktA+)Fusobacterium necrophorum LDH release assay, neutrophil enriched WBCs 100.0 90.0 80.0 70.0 60.0 50.0 DS 40.0 30.0 BHS 20.0 10.0 0.0 % neuts 128 256 512 1024 2048 4096 (LktA+) F. necrophorum supernatant dilution
% bacteristasis/killing Lkt+ M. haemolytica growth inhibition/killing by neutrophils 75.0 70.0 65.0 60.0 55.0 50.0 45.0 40.0 35.0 30.0 25.0 20.0 15.0 10.0 5.0 DS Sera, diff neuts DS Sera, equalized neuts HI-DS Sera, equalized neuts Naïve DS sera Naïve BHS sera 0.0 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 Commensal bacteria screening in DS and BHS Mycoplasma ovipneumoniae strain typing Pasteurellaceae (Mh, Bt, Pm) Disease documentation in DS and BHS Dates Number affected Symptoms Pathogens identified and by whom Human interactions with BHS Wildlife agencies Other gov t and private activities Hunting Permits issued Herd size # Harvested Environment Weather Feeding stations Natural disasters (ie. fire) Non-human predators
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 Willow Isabella Denise Kenny Lyle 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: Maggie.Highland@ars.usda.gov