Kinetic ELISA for detection of antibodies to Psoroptes sp. (Acari: Psoroptidae) in bighorn sheep (Ovis canadensis)

A kinetic enzyme-linked immunosorbent assay (ELISA) was developed using antigenic extracts prepared from Psoroptes cuniculi mites and sera from 37 Psoroptes sp.-infested and 43 uninfested bighorn sheep (Ovis canadensis). Serial dilutions of these serum samples, representing 3 bighorn sheep subspecies and 9 geographic areas, gave parallel responses when plotted as log dilution versus log kinetic rate. Therefore, all 80 samples were run at a single dilution (1:100) and positive/negative cutoff values were established as the mean kinetic rate of all negative sera plus either 2, 3, or 4 standard deviations. The resulting ELISA was highly reproducible and accurate with sensitivities and specificities of 100% and 97.7%, 94.6% and 97.7%, and 94.6% and 100%, respectively. This immunoassay will be useful for prospective and retrospective studies assessing the distribution and prevalence of Psoroptes sp. infestations in bighorn sheep.     

Exposure to Psoroptes sp. mites is common among bighorn sheep (Ovis canadensis) populations in California.

Sera (n = 806) from 50 populations of bighorn sheep (Ovis canadensis) in California (USA) were evaluated for antibodies to Psoroptes sp. mites using a kinetic enzyme-linked immunosorbent assay (ELISA). Test values for each sample were determined to be either positive or negative at each of two ELISA cutoff values that provided either 100% sensitivity (low cutoff) or 100% specificity (high cutoff), respectively. One hundred sixty-eight (20.8%) sera were seropositive at the low cutoff value, and 87 (10.8%) of these sera also were seropositive at the high cutoff value. Eleven populations were designated as scabies-suspect and 25 populations were designated as scabies-positive because they had at least one seropositive animal at the low and the high cutoff values, respectively. Based on these results, exposure to Psoroptes sp. mites appeared to be widely distributed among bighorn sheep populations from 1980 to 1990 and infested animals may have been present prior to 1980.     

The rise and fall of psoroptic scabies in bighorn sheep in the San Andres Mountains, New Mexico

Between 1978 and 1997, a combination of psoroptic scabies (Psoroptes spp.), mountain lion (Puma concolor) predation, and periodic drought reduced a population of native desert bighorn sheep (Ovis canadensis) in the San Andres Mountains (SAM), New Mexico, from >200 individuals to a single ewe. In 1999, this ewe was captured, ensured to be Psoroptes-free, and released back into the SAM. Eleven radio-collared rams were translocated from the Red Rock Wildlife Area (RRWA) in New Mexico into the SAM range and monitored through 2002 to determine whether Psoroptes spp. mites were still in the environment. None of these sentinel rams acquired scabies during this period, and no additional native sheep were found to be present in the range. In 2002, 51 desert bighorn sheep were translocated into the SAM from the Kofa National Wildlife Refuge in Arizona (n = 20) and the RRWA in New Mexico (n = 31). Twenty-one bighorn sheep have died in the SAM since that time, but Psoroptes spp. mites have not been detected on any of these animals, nor have they been found on mule deer (Odocoileus hemionus) sampled since 2000. We conclude that psoroptic scabies is no longer present in the San Andres bighorn sheep population and that psoroptic scabies poses a minimal to nonexistent threat to the persistence of this population at this time.     

Antigenic characterization of Psoroptes spp. (Acari: Psoroptidae) mites from different hosts

Immunoblotting with defined antigens and antisera revealed extensive and nearly complete antigenic cross-reactivity between Psoroptes spp. mites from a bighorn sheep, a mule deer, a cow, and a rabbit. Antigenic differences were not detected between mites from the sympatric bighorn sheep and mule deer. However, minor antigenic differences between mites from the cow and rabbit suggested that these mites were distinct from each other, as well as from the mites from the bighorn sheep and mule deer. These results are consistent with earlier morphologic studies of these populations of mites and provide additional support for the hypothesis that putative populations and/or species of Psoroptes mites may not be reproductively or ecologically isolated, particularly when their hosts are sympatric.     

Sharing of Pasteurella spp. between free-ranging bighorn sheep and feral goats

Pasteurella spp. were isolated from feral goats and free-ranging bighorn sheep (Ovis canadensis canadensis) in the Hells Canyon National Recreation Area bordering Idaho, Oregon, and Washington (USA). Biovariant 1 Pasteurella haemolytica organisms were isolated from one goat and one of two bighorn sheep found in close association. Both isolates produced leukotoxin and had identical electrophoretic patterns of DNA fragments following cutting with restriction endonuclease HaeIII. Similarly Pasteurella multocida multocida a isolates cultured from the goat and one of the bighorn sheep had D type capsules, serotype 4 somatic antigens, produced dermonecrotoxin and had identical HaeIII electrophoretic profiles. A biovariant U(beta) P.haemolytica strain isolated from two other feral goats, not known to have been closely associated with bighorn sheep, did not produce leukotoxin but had biochemical utilization and HaeIII electrophoretic profiles identical to those of isolates from bighorn sheep. It was concluded that identical Pasteurella strains were shared by the goats and bighorn sheep. Although the direction of transmission could not be established, evidence suggests transmission of strains from goats to bighorn sheep. Goats may serve as a reservoir of Pasteurella strains that may be virulent in bighorn sheep; therefore, goats in bighorn sheep habitat should be managed to prevent contact with bighorn sheep. Bighorn sheep which have nose-to-nose contact with goats should be removed from the habitat.     

Psoroptic scabies in Rocky Mountain bighorn sheep (Ovis canadensis canadensis) from Wyoming

Thirteen Rocky Mountain bighorn sheep (Ovis canadensis canadensis) with clinical signs of psoroptic scabies were captured in Wyoming. Signs included droopy ears, depilation on the head and neck, and exudate in the ears. Mites were identified as either Psoroptes cervinus or P. equi. Two ewes with scabies at the time of original capture had no clinical signs of mite infection 1 and 2 yr later.     

Effects of delivery method on serological responses of bighorn sheep to a multivalent Pasteurella haemolytica supernatant vaccine

The safety and efficacy of a remotely delivered multivalent Pasteurella haemolytica supernatant vaccine (serotypes A2 and T10) were examined in captive Rocky, Mountain bighorn sheep (Ovis canadensis canadensis). Twenty bighorn sheep were grouped according to baseline leukotoxin neutralizing antibody titers (< or =2 or >2 log2(-1)) and vaccination history (previously vaccinated or unvaccinated). Within these groups, animals were randomly assigned to one of two delivery treatments: hand injection (control) or biobullet implantation. All bighorns received a single dose from the same lot of vaccine (n = 10/treatment); four additional animals were injected intramuscularly with 0.9% saline as unvaccinated sentinels. Mild, transient lameness one day after hand injection or biobullet implantation was the only adverse effect. Serum neutralizing antibody titers to P. haemolytica leukotoxin differed between delivery treatments (P = 0.009) and among baseline titer/vaccination history groups (P = 0.013). Neutralizing titers were higher among hand-injected bighorns. Although neutralizing titers were lower among implanted bighorns than hand-injected controls at 1 wk (P = 0.002) and 2 wk (P = 0.021) after vaccination, seroconversion rates in response to implantation (6/10) and hand injection (9/10) did not differ (P = 0.303). Agglutinating antibody titers to T10 were high and did not vary over time or between delivery treatments. Agglutinating antibody titers to A2 in the hand-injected controls were not different (P > or = 0.07) than those in bighorns vaccinated with biobullet implantation. These data demonstrate that although hand injection elicits higher absolute titers, biobullet implantation may also stimulate effective antibody responses to P. haemolytica supernatant vaccine. Further evaluation of biobullet vaccination against pneumonic pasteurellosis in free-ranging populations of wild bighorn sheep is warranted.     

Hemorrhagic disease in bighorn sheep in Arizona

Two bighorn sheep from Arizona (USA) were submitted for necropsy. One was a Rocky Mountain bighorn (Ovis canadensis canadensis) and the other was a desert bighorn (Ovis canadensis mexicana). Both had lesions consistent with those of hemorrhagic disease (HD). Epizootic hemorrhagic disease virus (EHDV) type-2 and bluetongue virus (BTV) type-17, respectively, were isolated from the sheep tissues. To our knowledge, HD caused by either EHDV or BTV infection has not been documented previously in Arizona bighorn sheep.     

Response of vaccinated and unvaccinated bighorn sheep (Ovis canadensis canadensis) to experimental respiratory syncytial virus challenge

Five Rocky Mountain bighorn sheep (Ovis canadensis canadensis), approximately 5 mo old and without detectable antibody titers to respiratory syncytial virus (RSV), were assigned to two groups to study the effects of RSV challenge inoculation in vaccinated (n = 3) and unvaccinated (n = 2) bighorns. The three lambs vaccinated with a modified live bovine RSV vaccine developed a detectable antibody response to the vaccine. Vaccinated and unvaccinated lambs challenged with an ovine isolate of RSV developed increased levels of neutralizing antibody, but clinical signs of disease were not observed. Neutralizing antibody titers to RSV remained higher (2-4-fold) in vaccinated lambs over time when compared to unvaccinated lambs.     

Brucellosis in captive Rocky Mountain bighorn sheep (Ovis canadensis) caused by Brucella abortus biovar 4

Nine (four female, five male) captive adult Rocky Mountain bighorn sheep (Ovis canadensis) contracted brucellosis caused by Brucella abortus biovar 4 as a result of natural exposure to an aborted elk (Cervus elaphus) fetus. Clinical signs of infection were orchitis and epididymitis in males and lymphadenitis and placentitis with abortion in females. Gross pathologic findings included enlargement of the testes or epididymides, or both, and yellow caseous abscesses and pyogranulomas of the same. Brucella abortus biovar 4 was cultured in all bighorn sheep from a variety of tissues, including testes/epididymides, mammary gland, and lymph nodes. All bighorn sheep tested were positive on a variety of standard Brucella serologic tests. This is the first report of brucellosis caused by B. abortus in Rocky Mountain bighorn sheep. It also provides evidence that bighorn sheep develop many of the manifestations ascribed to this disease and that infection can occur from natural exposure to an aborted fetus from another species. Wildlife managers responsible for bighorn sheep populations sympatric with Brucella-infected elk or bison (Bison bison) should be cognizant of the possibility of this disease in bighorn sheep.     

Susceptibility of two Rocky Mountain bighorn sheep to experimental infection with Anaplasma ovis.

In North America, the role of wild ruminants in the epidemiology of anaplasmosis has not been clearly defined. Such information is particularly meager in regard to bighorn sheep. We report the susceptibility of two Rocky Mountain bighorn sheep (Ovis canadensis canadensis) to experimental infection with a well characterized field isolate of Anaplasma ovis obtained from domestic sheep in Idaho. Both bighorn sheep developed infection resulting in severe clinical disease, with relatively high parasitemias, icterus and anemia. One animal required tetracycline therapy and responded well to treatment, while the other recovered uneventfully without treatment. Both bighorn sheep were spleen-intact, a condition that in A. ovis-exposed domestic sheep typically is associated with mild infection. The results indicate that bighorn sheep may be adversely affected if exposed to the organism in nature.     

Seroprevalence of Toxoplasma gondii in Rocky Mountain bighorn sheep (Ovis canadensis)

Serum samples from 697 Rocky Mountain bighorn sheep (Ovis canadensis) from North America were examined for antibodies to Toxoplasma gondii by the modified agglutination test incorporating mercaptoethanol and formalin-fixed tachyzoites. Antibodies to T. gondii were found in 25 of 697 (3.6%) sheep in titers of 1:25 (8 sheep), 1:50 (4 sheep), 1:100 (7 sheep), 1:200 (1 sheep), 1:400 (1 sheep), 1:800 (1 sheep), and 1:1,600 (3 sheep). This is the first record of T. gondii exposure in bighorn sheep.     

New ruminant hosts and wider geographic range identified for Babesia odocoilei (Emerson and Wright 1970)

Babesia odocoilei was found to infect two previously unknown host species, desert bighorn sheep (Ovis canadensis nelsoni) and musk oxen (Ovibos moschatus), both of which are members of the family Bovidae. Previously, B. odocoilei has been reported in only Cervidae hosts. New geographic regions where B. odocoilei infections have not been reported previously include Pennsylvania and New York, where fatal babesiosis has occurred in reindeer (Rangifer tarandus tarandus); New Hampshire, where elk (Cervus elaphus canadensis) have been affected; and California, home of the infected desert bighorn sheep. Infection with B. odocoilei in these hosts was confirmed by parasite small subunit ribosomal RNA gene sequence analysis. A serosurvey for B. odocoilei antibody activity in New Hampshire showed prevalence rates of 100% at two elk farms and 12% at another farm. Control of potential vector ticks, Ixodes scapularis, especially when translocating livestock, is imperative to prevent outbreaks of babesiosis in managed herds of potential host species.     

Transplacental transmission of Protostrongylus sp. in California bighorn sheep (Ovis canadensis californiana) in Oregon

Transplacental transmission of Protostrongylus sp. was documented for the first time in California bighorn sheep (Ovis canadensis californiana) by recovery of third stage larvae from two fetuses.     

Experimental infection of some North American wild ruminants and domestic sheep with Mycobacterium paratuberculosis: clinical and bacteriological findings

Mycobacterium paratuberculosis originally isolated from bighorn sheep (Ovis canadensis) with spontaneous paratuberculosis was used to orally inoculate Rocky Mountain elk (Cervus elaphus nelsoni) calves, mule deer (Odocoileus hemionus) fawns, white-tailed deer (Odocoileus virginianus) fawns, bighorn X mouflon (Ovis musimon) hybrid lambs, and domestic lambs. All experimentally exposed animals became infected. During the first year of infection, hybrid and domestic sheep were able to control the infection but infection was progressive in elk and deer. Clinical paratuberculosis occurred only in mule deer.     

Toxoplasmic encephalitis in a free-ranging Rocky Mountain bighorn sheep from Washington

A 4-mo-old free-ranging Rocky Mountain bighorn sheep (Ovis canadensis canadensis) from the Hells Canyon area (Washington, USA) was diagnosed with encephalitis associated with Toxoplasma gondii infection. The sheep had concurrent pneumonic pasteurellosis and resided in a geographic area with endemic Pasteurella-associated pneumonia and mortality in bighorn sheep. The brain had multifocal necrotizing and nonsuppurative encephalitis with intralesional protozoa. The protozoa were identified as T. gondii by immunohistochemistry. To our knowledge, this is the first report of T. gondii infection in a Rocky Mountain bighorn sheep.     

Detecting nonhemolytic Pasteurella haemolytica infections in healthy Rocky Mountain bighorn sheep (Ovis canadensis canadensis): influences of sample site and handling

Effects of sampling procedures on ability to culture Pasteurella spp. from Rocky Mountain bighorn sheep (Ovis canadensis canadensis) were examined experimentally. Sample site influenced (P less than 0.0001) recovery of P. haemolytica in adult bighorn sheep. We isolated nonhemolytic P. haemolytica from 18 of 19 tonsillar swabs and 18 of 19 tonsillar biopsies from adult sheep, yet only four of 19 nasal swabs yielded isolates. Sample handling also affected (P less than 0.0001) recovery of P. haemolytica. Nonhemolytic P. haemolytica was cultured from 14 of 19 tonsillar swabs plated directly onto blood agar, but from only two of 19 swabs stored for 24 hr in modified Stuart's medium. We detected nonhemolytic P. haemolytica at least once in bronchial aspirates from four and in nasal swabs from three of six bighorn lambs. Based on direct cultures of tonsillar swabs and/or biopsies, all 26 bighorn sheep (seven lambs, 19 adults) sampled were infected with nonhemolytic P. haemolytica; only two lambs developed pneumonia during the study period. Thirty-four of 37 nonhemolytic P. haemolytica isolates tested were biotype T; three were biotype A. Serotypes 3; 4; 3, 4 and 3, 4, 10 were identified in a subsample of 17 isolates. Our data suggest tonsillar swabs or biopsies plated directly onto blood agar and incubated immediately offer the greatest probability of recovering nonhemolytic P. haemolytica from health bighorn sheep.     

Comparison of pulmonary defense mechanisms in Rocky Mountain bighorn (Ovis canadensis canadensis) and domestic sheep

Alveolar macrophages were obtained from Rocky Mountain bighorn sheep (Ovis canadensis canadensis) and domestic sheep for the purpose of comparing pulmonary host defense mechanisms in the two species. Specific variables studied included (1) characterization of the cell types present in the lung, (2) alveolar macrophage phagocytic and bactericidal functions, (3) measurement of protein levels in lavage fluid, and (4) measurement of cortisol levels in lavage fluid. While phagocytic cell populations were similar between bighorn and domestic sheep, a significantly higher percentage of lymphocytes were present in bighorns than domestics (20% in bighorn versus 6% in domestic sheep). Significant differences were not observed in the phagocytic or bactericidal functions of macrophages between the two species. Significant differences were not observed in either lavage fluid protein levels or in cortisol levels.     

Elaeophorosis in bighorn sheep in New Mexico

Two bighorn sheep (Ovis canadensis) in New Mexico (USA) were found to be naturally infected with Elaeophora schneideri. An adult ram examined in 1997 in the Fra Cristobal Mountains had 26 nematodes in the carotid and iliac arteries, and microfilariae were present in the skin, nasal mucosa, brain, and lungs. This ram was markedly debilitated prior to euthanasia and extensive crusty, scabby lesions were observed on its head. In 1998, a yearling ewe found dead adjacent to Watson Mountain near the Gila Wilderness area was found to have 13 nematodes present in its heart. This is the first report of E. schneideri in bighorn sheep, and we suggest that bighorn sheep are susceptible to E. schneideri infection wherever they coexist with mule deer (Odocoileus hemionus hemionus) and appropriate tabanid vectors.     

Occurrence, diagnosis, and strain typing of Mycobacterium avium subspecies paratuberculosis infection in Rocky Mountain bighorn sheep (Ovis canadensis canadensis) in southwestern Alberta

The role that wildlife may play in the transmission of Mycobacterium avium subspecies paratuberculosis (Map), the causative agent of Johne's disease (JD), and the potential consequences of infection in these populations are being given increasing consideration. A yearling male Rocky Mountain bighorn sheep (Ovis canadensis canadensis) from southwestern Alberta, Canada, was found infected with Map in August 2009. Clinical signs of emaciation and diarrhea and histologic findings of diffuse granulomatous enteritis of the distal ileum, lymphadenitis of the mesenteric lymph nodes, and lymphangitis of the ileum were similar to previously described cases of JD in bighorn sheep. Infection with Map was confirmed by bacterial isolation through fecal culture, acid-fast staining, and polymerase chain reaction (PCR) of IS900. The Map1506 gene was sequenced, and the isolate was identified as a Cattle (Type II) strain. In a follow-up herd-level survey, three of 44 fecal samples (7%) from individual bighorn sheep from the same herd as the index case were PCR-positive and identified as Type II Map strains. Twenty-five samples from a distant bighorn population were negative. Additional strain typing of the isolates from the index case and the positive fecal samples was done by sequencing three discriminatory short sequence repeat (SSR) regions. All four SSR profiles differed from one another, suggesting multiple introductions or a long-existing circulation of Map within this bighorn population. Detailed molecular analyses are essential for understanding and managing diseases at the wildlife-livestock interface.