Correction: Exposure of bighorn sheep to domestic goats colonized with Mycoplasma ovipneumoniae induces sub-lethal pneumonia.

[This corrects the article DOI: 10.1371/journal.pone.0178707.].

Exposure of bighorn sheep to domestic goats colonized with Mycoplasma ovipneumoniae induces sub-lethal pneumonia.

BACKGROUND: Bronchopneumonia is a population limiting disease of bighorn sheep (Ovis canadensis) that has been associated with contact with domestic Caprinae. The disease is polymicrobial but is initiated by Mycoplasma ovipneumoniae, which is commonly carried by both domestic sheep (O. aries) and goats (Capra aegagrus hircus). However, while previous bighorn sheep comingling studies with domestic sheep have resulted in nearly 100% pneumonia mortality, only sporadic occurrence of fatal pneumonia was reported from previous comingling studies with domestic goats. Here, we evaluated the ability of domestic goats of defined M. ovipneumoniae carriage status to induce pneumonia in comingled bighorn sheep. METHODOLOGY/PRINCIPAL FINDINGS: In experiment 1, three bighorn sheep naïve to M. ovipneumoniae developed non-fatal respiratory disease (coughing, nasal discharge) following comingling with three naturally M. ovipneumoniae-colonized domestic goats. Gross and histological lesions of pneumonia, limited to small areas on the ventral and lateral edges of the anterior and middle lung lobes, were observed at necropsies conducted at the end of the experiment. A control group of three bighorn sheep from the same source housed in isolation during experiment 1 remained free of observed respiratory disease. In experiment 2, three bighorn sheep remained free of observed respiratory disease while comingled with three M. ovipneumoniae-free domestic goats. In experiment 3, introduction of a domestic goat-origin strain of M. ovipneumoniae to the same comingled goats and bighorn sheep used in experiment 2 resulted in clinical signs of respiratory disease (coughing, nasal discharge) in both host species. At the end of experiment 3, gross and histological evidence of pneumonia similar to that observed in experiment 1 bighorn sheep was observed in both affected bighorn sheep and domestic goats. CONCLUSIONS/SIGNIFICANCE: M. ovipneumoniae strains carried by domestic goats were transmitted to comingled bighorn sheep, triggering development of pneumonia. However, the severity of the disease was markedly milder than that seen in similar experiments with domestic sheep strains of the bacterium.

Role of carriers in the transmission of pneumonia in bighorn sheep (Ovis canadensis).

In the absence of livestock contact, recurring lamb mortality in bighorn sheep (Ovis canadensis) populations previously exposed to pneumonia indicates the likely presence of carriers of pneumonia-causing pathogens, and possibly inadequate maternally derived immunity. To investigate this problem we commingled naïve, pregnant ewes (n=3) with previously exposed rams (n=2). Post-commingling, all ewes and lambs born to them acquired pneumonia-causing pathogens (leukotoxin-producing Pasteurellaceae and Mycoplasma ovipneumoniae), with subsequent lamb mortality between 4-9 weeks of age. Infected ewes became carriers for two subsequent years and lambs born to them succumbed to pneumonia. In another experiment, we attempted to suppress the carriage of leukotoxin-producing Pasteurellaceae by administering an antibiotic to carrier ewes, and evaluated lamb survival. Lambs born to both treatment and control ewes (n=4 each) acquired pneumonia and died. Antibody titers against leukotoxin-producing Pasteurellaceae in all eight ewes were 'protective' (>1:800 and no apparent respiratory disease); however their lambs were either born with comparatively low titers, or with high (but non-protective) titers that declined rapidly within 2-8 weeks of age, rendering them susceptible to fatal disease. Thus, exposure to pneumonia-causing pathogens from carrier ewes, and inadequate titers of maternally derived protective antibodies, are likely to render bighorn lambs susceptible to fatal pneumonia.

Serologic, trace element, and fecal parasite survey of free-ranging, female mule deer (Odocoileus hemionus) in eastern Washington, USA.

Blood and fecal samples collected from 97 free-ranging mule deer (Odocoileus hemionus), from four distinct herds during the spring of 2000 or 2001 in eastern Washington, US, were tested for exposure to selected pathogens, concentrations of trace elements, and presence of parasites in feces. Antibodies were detected to the following: Leptospira interrogans serovar Bratislava (4%), Leptospira interrogans serovar Canicola (1%), Leptospira interrogans serovar Grippotyphosa (13%), Bovine viral diarrhea virus 1 (57%), Bovine respiratory syncytial virus (71%), Infectious bovine rhinotracheitis virus (51%), Bovine parainfluenza virus 3 (61%), Bluetongue virus (25%), and Epizootic hemorrhagic disease virus (25%); 3 of 63 (5%) samples had antibody to Neospora spp. All samples tested for antibody to Brucella abortus and L. interrogans serovar Icterohaemorrhagiae, L. interrogans serovar Pomona, and L. interrogans serovar Hardjo samples were negative. Trace element concentrations from 97 sera were deficient for selenium (17%), copper (19%), iron (34%), calcium (3%), and phosphorus (2%) compared with thresholds established for domestic livestock. Parasites detected in 97 fecal samples included dorsal-spined larvae (probably Parelaphostrongylus sp.) (40%), abomasal nematode eggs (1%), Capillaria sp. eggs (1%), Nematodirus sp. eggs (26%), Moniezia sp. eggs (1%), and Eimeria sp. (2%).

Epizootic pneumonia of bighorn sheep following experimental exposure to Mycoplasma ovipneumoniae.

BACKGROUND: Bronchopneumonia is a population limiting disease of bighorn sheep (Ovis canadensis). The cause of this disease has been a subject of debate. Leukotoxin expressing Mannheimia haemolytica and Bibersteinia trehalosi produce acute pneumonia after experimental challenge but are infrequently isolated from animals in natural outbreaks. Mycoplasma ovipneumoniae, epidemiologically implicated in naturally occurring outbreaks, has received little experimental evaluation as a primary agent of bighorn sheep pneumonia. METHODOLOGY/PRINCIPAL FINDINGS: In two experiments, bighorn sheep housed in multiple pens 7.6 to 12 m apart were exposed to M. ovipneumoniae by introduction of a single infected or challenged animal to a single pen. Respiratory disease was monitored by observation of clinical signs and confirmed by necropsy. Bacterial involvement in the pneumonic lungs was evaluated by conventional aerobic bacteriology and by culture-independent methods. In both experiments the challenge strain of M. ovipneumoniae was transmitted to all animals both within and between pens and all infected bighorn sheep developed bronchopneumonia. In six bighorn sheep in which the disease was allowed to run its course, three died with bronchopneumonia 34, 65, and 109 days after M. ovipneumoniae introduction. Diverse bacterial populations, predominantly including multiple obligate anaerobic species, were present in pneumonic lung tissues at necropsy. CONCLUSIONS/SIGNIFICANCE: Exposure to a single M. ovipneumoniae infected animal resulted in transmission of infection to all bighorn sheep both within the pen and in adjacent pens, and all infected sheep developed bronchopneumonia. The epidemiologic, pathologic and microbiologic findings in these experimental animals resembled those seen in naturally occurring pneumonia outbreaks in free ranging bighorn sheep.

Role of Bibersteinia trehalosi, respiratory syncytial virus, and parainfluenza-3 virus in bighorn sheep pneumonia.

Pneumonic bighorn sheep (BHS) have been found to be culture- and/or sero-positive for Bibersteinia trehalosi, respiratory syncytial virus (RSV), and parainfluenza-3 virus (PI-3). The objective of this study was to determine whether these pathogens can cause fatal pneumonia in BHS. In the first study, two groups of four BHS each were intra-tracheally administered with leukotoxin-positive (Group I) or leukotoxin-negative (Group II) B. trehalosi. All four animals in Group I developed severe pneumonia, and two of them died within 3 days. The other two animals showed severe pneumonic lesions on euthanasia and necropsy. Animals in Group II neither died nor showed gross pneumonic lesions on necropsy, suggesting that leukotoxin-positive, but not leukotoxin-negative, B. trehalosi can cause fatal pneumonia in BHS. In the second study, two other groups of four BHS (Groups III and IV) were intra-nasally administered with a mixture of RSV and PI-3. Four days later, RSV/PI-3-inoculated Group IV and another group of four BHS (Group V, positive control) were intra-nasally administered with Mannheimia haemolytica, the pathogen that consistently causes fatal pneumonia in BHS. All four animals in group III developed pneumonia, but did not die during the study period. However all four animals in Group IV, and three animals in Group V developed severe pneumonia and died within two days of M. haemolytica inoculation. The fourth animal in Group V showed severe pneumonic lesions on euthanasia and necropsy. These findings suggest that RSV/PI-3 can cause non-fatal pneumonia, but are not necessary predisposing agents for M. haemolytica-caused pneumonia of BHS.

Survival of bighorn sheep (Ovis canadensis) commingled with domestic sheep (Ovis aries) in the absence of Mycoplasma ovipneumoniae.

To test the hypothesis that Mycoplasma ovipneumoniae is an important agent of the bighorn sheep (Ovis canadensis) pneumonia that has previously inevitably followed experimental commingling with domestic sheep (Ovis aries), we commingled M. ovipneumoniae-free domestic and bighorn sheep (n=4 each). One bighorn sheep died with acute pneumonia 90 days after commingling, but the other three remained healthy for >100 days. This unprecedented survival rate is significantly different (P=0.002) from that of previous bighorn-domestic sheep contact studies but similar to (P>0.05) bighorn sheep survival following commingling with other ungulates. The absence of epizootic respiratory disease in this experiment supports the hypothesized role of M. ovipneumoniae as a key pathogen of epizootic pneumonia in bighorn sheep commingled with domestic sheep.

A multivalent Mannheimia-Bibersteinia vaccine protects bighorn sheep against Mannheimia haemolytica challenge.

Bighorn sheep (BHS) are more susceptible than domestic sheep (DS) to Mannheimia haemolytica pneumonia. Although both species carry M. haemolytica as a commensal bacterium in the nasopharynx, DS carry mostly leukotoxin (Lkt)-positive strains while BHS carry Lkt-negative strains. Consequently, antibodies to surface antigens and Lkt are present at much higher titers in DS than in BHS. The objective of this study was to determine whether repeated immunization of BHS with multivalent Mannheimia-Bibersteinia vaccine will protect them upon M. haemolytica challenge. Four BHS were vaccinated with a culture supernatant vaccine prepared from M. haemolytica serotypes A1 and A2 and Bibersteinia trehalosi serotype T10 on days 0, 21, 35, 49, and 77. Four other BHS were used as nonvaccinated controls. On the day of challenge, 12 days after the last immunization, the mean serum titers of Lkt-neutralizing antibodies and antibodies to surface antigens against M. haemolytica were 1:160 and 1:4,000, respectively. Following intranasal challenge with M. haemolytica A2 (1 × 10(5) CFU), all four control BHS died within 48 h. Necropsy revealed acute fibrinonecrotic pneumonia characteristic of M. haemolytica infection. None of the vaccinated BHS died during the 8 weeks postchallenge observation period. Radiography at 3 weeks postchallenge revealed no lung lesions in two vaccinated BHS and mild lesions in the other two, which resolved by 8 weeks postchallenge. These results indicate that if BHS can be induced to develop high titers of Lkt-neutralizing antibodies and antibodies to surface antigens, they are likely to survive M. haemolytica challenge which is likely to reduce the BHS population decline due to pneumonia.

Defective bacterial clearance is responsible for the enhanced lung pathology characteristic of Mannheimia haemolytica pneumonia in bighorn sheep.

The molecular and cellular basis for the enhanced lung pathology and mortality caused by Mannheimia haemolytica in bighorn sheep (BHS, Ovis canadenesis), in comparison to domestic sheep (DS, Ovis aries), is not clear. Polymorphonuclear leukocytes (PMNs) of BHS are four- to eight-fold more susceptible to M. haemolytica leukotoxin-induced cytolysis, which is likely to reduce the number of functional phagocytes in the lung. We hypothesized that enhanced lung pathology is due to defective clearance of M. haemolytica from the lungs of BHS. To test this hypothesis, M. haemolytica (1 × 10(7) colony forming units [cfu]) were inoculated intra-tracheally into three groups each of BHS and DS, which were euthanized and necropsied at 4, 12, and 18 h post-inoculation (hpi). Bacterial and leukocyte counts were performed on broncho-alveolar lavage fluid (BALF) collected at necropsy. BALF from BHS euthanized at 4 and 12 hpi contained a significantly higher number of M. haemolytica than that from DS. More importantly, DS did not have any bacteria in BALF at 18 hpi, while the BHS still had significant numbers. As expected, the BHS did exhibit more extensive lung lesions at 12 and 18 hpi when compared to DS. At 18 hpi, necrotic PMNs were observed in the lesional lung tissues of BHS, but not DS. Furthermore, BALF from BHS had significantly lower titers of antibodies to Lkt and surface antigens of M. haemolytica, than that of DS. These findings suggest that the enhanced pathology in BHS lungs is due to defective clearance of M. haemolytica from the lungs.

Co-expression of ovine LPS receptor CD14 with Mannheimia haemolytica leukotoxin receptor LFA-1 or Mac-1 does not enhance leukotoxin-induced cytotoxicity.

Leukotoxin (Lkt) and LPS are the major virulence determinants of Mannheimia haemolytica that contribute to the pathogenesis of bovine and ovine pneumonic pasteurellosis. We have previously identified bovine and ovine CD18 as the functional receptor for Lkt. LPS complexes with Lkt resulting in increased thermal stability and enhanced cytotoxic activity of Lkt. Cellular recognition of LPS involves several different molecules including CD14. We hypothesized that expression of ovine CD14 together with LFA-1 or Mac-1 would enhance Lkt-induced cytotoxicity. Ovine cDNA for CD14 was amplified by PCR and cloned into mammalian expression vectors. The 1122 bp cDNAs for bighorn sheep (BHS) and domestic sheep (DS) CD14 encode 373 amino acids which exhibit 99% identity with each other. Ovine CD14 plasmids were transfected either into HEK-293 cells, or previous HEK-293 transfectants stably expressing ovine LFA-1 or Mac-1. Flow cytometric analysis of transfectants confirmed the cell surface expression of CD14. The transfectants expressing LFA-1 or Mac-1 and the transfectants co-expressing CD14 with LFA-1 or Mac-1 did not show any significant difference in Lkt-induced cytotoxicity when incubated with LPS complexed Lkt. In contrast, incubation of the LFA-1 or Mac-1 and LFA-1/CD14 or Mac-1/CD14 transfectants with Lkt which lacks LPS, resulted in reduced cytotoxicity. None of the above transfectants showed any difference in [Ca²+](i) elevation when incubated with both types of Lkt preparations. Lkt did not induce any cytotoxicity or [Ca²+](i) elevation in ovine CD14 transfectants or parent HEK-293 cells. Based on these findings, we conclude that expression of CD14 together with LFA-1 or Mac-1 does not enhance Lkt-induced cytotoxicity, whereas LPS enhances cytotoxicity by complexing with Lkt.

Transmission of Mannheimia haemolytica from domestic sheep (Ovis aries) to bighorn sheep (Ovis canadensis): unequivocal demonstration with green fluorescent protein-tagged organisms.

Previous studies demonstrated that bighorn sheep (Ovis canadensis) died of pneumonia when commingled with domestic sheep (Ovis aries) but did not conclusively prove that the responsible pathogens were transmitted from domestic to bighorn sheep. The objective of this study was to determine, unambiguously, whether Mannheimia haemolytica can be transmitted from domestic to bighorn sheep when they commingle. Four isolates of M. haemolytica were obtained from the pharynx of two of four domestic sheep and tagged with a plasmid carrying the genes for green fluorescent protein (GFP) and ampicillin resistance (AP(R)). Four domestic sheep, colonized with the tagged bacteria, were kept about 10 m apart from four bighorn sheep for 1 mo with no clinical signs of pneumonia observed in the bighorn sheep during that period. The domestic and bighorn sheep were then allowed to have fence-line contact for 2 mo. During that period, three bighorn sheep acquired the tagged bacteria from the domestic sheep. At the end of the 2 mo of fence-line contact, the animals were allowed to commingle. All four bighorn sheep died 2 days to 9 days following commingling. The lungs from all four bighorn sheep showed gross and histopathologic lesions characteristic of M. haemolytica pneumonia. Tagged M. haemolytica were isolated from all four bighorn sheep, as confirmed by growth in ampicillin-containing culture medium, PCR-amplification of genes encoding GFP and Ap(R), and immunofluorescent staining of GFP. These results unequivocally demonstrate transmission of M. haemolytica from domestic to bighorn sheep, resulting in pneumonia and death of bighorn sheep.

Molecular cloning of interleukin-1ß, interleukin-8, and tumor necrosis factor-α of bighorn sheep (Ovis canadensis) and comparison with those of other species.

The susceptibility to, and pathology induced by, Mannheimia haemolytica infection in bighorn sheep (BHS) and domestic sheep (DS) are distinctly different. Bighorn sheep are particularly susceptible to pneumonia caused by M. haemolytica, and the pneumonic lesions in infected BHS are more severe than those in DS. The molecular basis for this disparity has not been elucidated. Proinflammatory cytokines have been implicated in the pathogenesis of multiple lung diseases of humans and animals. It is possible that the enhanced pathology observed in the pneumonic lungs of M. haemolytica-infected BHS, in comparison to that of DS, is due to comparatively higher levels of proinflammatory cytokine expression in BHS. As the first step towards elucidating this concept, we have cloned and sequenced the cDNA encoding the cytokines interleukin-1ß (IL-1ß), interleukin-8 (IL-8), and tumor necrosis factor-α (TNF-α) of BHS. The cDNA of BHS IL-1ß, IL-8, and TNF-α consists of 801, 306, and 705 base pairs encoding 266, 101, and 234 amino acids, respectively. The availability of cDNA encoding IL-1ß, IL-8, and TNF-α of BHS should facilitate the elucidation of the role of these cytokines in the differential pathology induced by M. haemolytica infection in BHS and DS.

Differential expression of interleukin-8 by polymorphonuclear leukocytes of two closely related species, Ovis canadensis and Ovis aries, in response to Mannheimia haemolytica infection.

The pneumonic lesions and mortality caused by Mannheimia haemolytica in bighorn sheep (BHS; Ovis canadensis) are more severe than those in the related species, domestic sheep (DS; Ovis aries), under both natural and experimental conditions. Leukotoxin (Lkt) and lipopolysaccharide (LPS) are the most important virulence factors of this organism. One hallmark of pathogenesis of pneumonia is the influx of polymorphonuclear leukocytes (PMNs) into the lungs. Lkt-induced cytolysis of PMNs results in the release of cytotoxic compounds capable of damaging lung tissue. Interleukin-8 (IL-8) is a potent PMN chemoattractant. The objective of the present study was to determine if there is differential expression of IL-8 by the macrophages and PMNs of BHS and DS in response to M. haemolytica. Macrophages and PMNs of BHS and DS were stimulated with heat-killed M. haemolytica or LPS. IL-8 expression by the cells was measured by enzyme-linked immunosorbent assays and real-time reverse transcription-PCR (RT-PCR). The PMNs of BHS expressed severalfold higher levels of IL-8 than those of DS upon stimulation. Lesional lung tissue of M. haemolytica-infected BHS contained significantly higher levels of IL-8 than nonlesional tissue. The bronchoalveolar lavage (BAL) fluid of infected BHS also contained higher levels of IL-8 than that of infected DS. Depletion of IL-8 reduced migration of PMNs toward BAL fluid by approximately 50%, indicating that IL-8 is integral to PMN recruitment to the lung during M. haemolytica infection. Excessive production of IL-8, enhanced recruitment of PMNs, and PMN lysis by Lkt are likely responsible for the severity of the lung lesions in M. haemolytica-infected BHS.

Mycoplasma ovipneumoniae can predispose bighorn sheep to fatal Mannheimia haemolytica pneumonia.

Mycoplasma ovipneumoniae has been isolated from the lungs of pneumonic bighorn sheep (BHS). However experimental reproduction of fatal pneumonia in BHS with M. ovipneumoniae was not successful. Therefore the specific role, if any, of M. ovipneumoniae in BHS pneumonia is unclear. The objective of this study was to determine whether M. ovipneumoniae alone causes fatal pneumonia in BHS, or predisposes them to infection by Mannheimia haemolytica. We chose M. haemolytica for this study because of its isolation from pneumonic BHS, and its consistent ability to cause fatal pneumonia under experimental conditions. Since in vitro culture could attenuate virulence of M. ovipneumoniae, we used ceftiofur-treated lung homogenates from pneumonic BHS lambs or nasopharyngeal washings from M. ovipneumoniae-positive domestic sheep (DS) as the source of M. ovipneumoniae. Two adult BHS were inoculated intranasally with lung homogenates while two others received nasopharyngeal washings from DS. All BHS developed clinical signs of respiratory infection, but only one BHS died. The dead BHS had carried leukotoxin-positive M. haemolytica in the nasopharynx before the onset of this study. It is likely that M. ovipneumoniae colonization predisposed this BHS to fatal infection with the M. haemolytica already present in this animal. The remaining three BHS developed pneumonia and died 1-5 days following intranasal inoculation with M. haemolytica. On necropsy, lungs of all four BHS showed lesions characteristic of bronchopneumonia. M. haemolytica and M. ovipneumoniae were isolated from the lungs. These results suggest that M. ovipneumoniae alone may not cause fatal pneumonia in BHS, but can predispose them to fatal pneumonia due to M. haemolytica infection.

Experimental infection of liver flukes, Fasciola hepatica and Fascioloides magna, in Bison (Bison bison).

This experimental study was conducted to evaluate the susceptibility of American bison (Bison bison) to liver flukes, Fascioloides magna and Fasciola hepatica. Six bison were each experimentally inoculated with 600 metacercariae of Fascioloides magna, and three were later treated with triclabendazole suspension at 40 mg/kg of body weight. Four additional bison were each experimentally inoculated with 600 metacercariae of Fasciola hepatica. Five control bison were placebo controls. Two controls and all inoculated bison were euthanized 10 mo (Fascioloides magna) and 7 mo (Fasciola hepatica) after inoculation. None of the control bison or the bison inoculated with Fascioloides magna had flukes or lesions characteristic of fluke infection at necropsy. All four bison inoculated with Fasciola hepatica had characteristic liver fluke lesions at necropsy, and three of four bison contained four, 103, and 111 adult flukes, respectively. Fluke eggs were detected in feces of all Fasciola hepatica-inoculated bison during the experiment, but not from the Fascioloides magna-infected bison or control bison. Clinical signs of infection were not observed during the experiment, but hemoglobin and packed cell volumes were lower in the Fasciola hepatica bison when compared to controls, and eosinophil levels were increased. Triclabendazole at 40 mg/kg of body weight appeared to be safe in bison because no toxic reactions were observed. Results from this study indicated bison are susceptible to infection with Fasciola hepatica and are efficient definitive hosts. Because no Fascioloides magna were recovered, bison may have a decreased susceptibility or innate resistance to Fascioloides magna infection, which may account for a lack of reported infections in this host.

Echinococcus granulosus in gray wolves and ungulates in Idaho and Montana, USA.

We evaluated the small intestines of 123 gray wolves (Canis lupus) that were collected from Idaho, USA (n=63), and Montana, USA (n=60), between 2006 and 2008 for the tapeworm Echinococcus granulosus. The tapeworm was detected in 39 of 63 wolves (62%) in Idaho, USA, and 38 of 60 wolves (63%) in Montana, USA. The detection of thousands of tapeworms per wolf was a common finding. In Idaho, USA, hydatid cysts, the intermediate form of E. granulosus, were detected in elk (Cervus elaphus), mule deer (Odocoileus hemionus), and a mountain goat (Oreamnos americanus). In Montana, USA, hydatid cysts were detected in elk. To our knowledge, this is the first report of adult E. granulosus in Idaho, USA, or Montana, USA. It is unknown whether the parasite was introduced into Idaho, USA, and southwestern Montana, USA, with the importation of wolves from Alberta, Canada, or British Columbia, Canada, into Yellowstone National Park, Wyoming, USA, and central Idaho, USA, in 1995 and 1996, or whether the parasite has always been present in other carnivore hosts, and wolves became a new definitive host. Based on our results, the parasite is now well established in wolves in these states and is documented in elk, mule deer, and a mountain goat as intermediate hosts.

Experimental infection of bighorn sheep with liver flukes (Fasciola hepatica).

Nine Rocky Mountain bighorn sheep (Ovis canadensis canadensis) were each inoculated orally with 250 metacercariae of Fasciola hepatica. Blood and fecal samples were collected at the time of inoculation and at 5, 10, 14, and 20 wk after inoculation. Numbers of fluke eggs in feces, hemoglobin, packed cell volume, and eosinophil values were determined. Five of the bighorn sheep were treated with triclabendazole at 40 mg/kg of body weight 14 wk after inoculation. Based on fecal evaluations, all bighorns developed patent infections. Six weeks after treatment, fluke eggs were not detected in feces from the five treated animals but were present in two of four untreated animals. One untreated bighorn sheep was euthanized 20 wk after inoculation, and 57 adult F. hepatica were recovered from the liver. Results from this experiment indicated that bighorn sheep are efficient hosts for F. hepatica. Triclabendazole at 40 mg/kg of body weight was safe and, based on fluke egg recovery in feces, apparently an effective treatment. To my knowledge, this is the first published report of F. hepatica in bighorn sheep.

Transmission of lungworms (Muellerius capillaris) from domestic goats to bighorn sheep on common pasture.

Four domestic goats (Capra hircus) that were passing first-stage dorsal-spined larvae of Muellerius capillaris were copastured on a 0.82-ha pasture for 11 mo from May 2003 to April 2004 with seven Rocky Mountain bighorn sheep (Ovis canadensis) that were not passing dorsal-spined larvae. During the 11-mo experiment, two bighorn sheep died from pneumonia caused by Mannheimia (Pasteurella) haemolytica biotype A, serotype 2. The remaining five bighorn sheep and the four domestic goats remained healthy throughout the experiment. Muellerius larvae were detected from all domestic goats on a monthly basis throughout the experiment and were first detected from all five surviving bighorn sheep approximately 5 mo after the copasturing began. Once the bighorn sheep began passing Muellerius larvae, larvae were detected in low numbers from all bighorn sheep every month thereafter for the 6 mo the goats were still in the enclosure and continued to pass larvae for more than 3 yr after the goats were removed from the experiment. Six bighorn sheep in two similar enclosures that did not contain goats did not pass Muellerius larvae before, during, or after the experimental period. Results of this experiment indicate that M. capillaris from domestic goats is capable of infecting bighorn sheep when animals are copastured together on a common range.

Mannheimia haemolytica serotype A1 exhibits differential pathogenicity in two related species, Ovis canadensis and Ovis aries.

Mannheimia haemolytica causes pneumonia in both bighorn sheep (BHS, Ovis canadensis) and domestic sheep (DS, Ovis aries). Under experimental conditions, co-pasturing of BHS and DS results in fatal pneumonia in BHS. It is conceivable that certain serotypes of M. haemolytica carried by DS are non-pathogenic to them, but lethal for BHS. M. haemolytica serotypes A1 and A2 are carried by DS in the nasopharynx. However, it is the serotype A2 that predominantly causes pneumonia in DS. The objectives of this study were to determine whether serotype A1 exhibits differential pathogenicity to BHS and DS, and to determine whether leukotoxin (Lkt) secreted by this organism is its primary virulence factor. Three groups each of BHS and DS were intra-tracheally administered either 1 x 10(9)cfu of serotype A1 wild-type (lktA-Wt group), Lkt-deletion mutant of serotype A1-(lktA-Mt group), or saline (control group), respectively. In the lktA-Wt groups, all four BHS died within 48h while none of the DS died during the 2-week study period. In the lktA-Mt groups, none of the BHS or DS died. In the control groups, one DS died due to an unrelated cause. Necropsy and histopathological findings revealed that death of BHS in the lktA-Wt group was due to bilateral, fibrinohemorrhagic pneumonia. Although the A1-Mt-inoculated BHS were clinically normal, on necropsy, lungs of two BHS showed varying degrees of mild chronic pneumonia. These results indicate that M. haemolytica serotype A1 is non-pathogenic to DS, but highly lethal to BHS, and that Lkt is the primary virulence factor of M. haemolytica.

Bighorn sheep beta2-integrin LFA-1 serves as a receptor for Mannheimia haemolytica leukotoxin.

Mannheimia haemolytica is an important cause of pneumonia in bighorn sheep (BHS; Ovis canadensis). Leukotoxin (Lkt), the primary virulence determinant of M. haemolytica, induces cytolysis of all subsets of leukocytes. Previously, we have shown that CD18, the beta subunit of beta2-integrins, mediates Lkt-induced cytolysis. However, it is not clear whether CD18 of all three beta2-integrins, LFA-1, Mac-1, and CR4, mediates Lkt-induced cytolysis. The objective of this study was to determine whether BHS LFA-1 (CD11a/CD18) serves as a receptor for Lkt. Plasmids encoding cDNA for BHS CD11a and CD18 were cotransfected into Lkt-resistant HEK-293 cells. Flow cytometric analysis of transfectants confirmed cell surface expression of BHS LFA- 1, Lkt-LFA-1 binding and Lkt-induced intra-cellular calcium elevation. More importantly, the transfectants were efficiently lysed by Lkt in a concentration-dependent manner. Collectively, these results indicate that BHS LFA-1 serves as a functional receptor for M. haemolytica Lkt.