Fatal Pasteurella haemolytica pneumonia in bighorn sheep after direct contact with clinically normal domestic sheep

Six Rocky Mountain bighorn sheep were raised in captivity from birth (n = 5) or taken from the wild as a lamb (n = 1). After the bighorn sheep were in captivity for over a year, 6 clinically normal domestic sheep were placed on the 2 ha of pasture on which the bighorn sheep were kept. Nasal swab specimens were obtained from all sheep at the time the domestic sheep were introduced. Pasteurella haemolytica was isolated from swab specimens obtained from 4 of 6 domestic sheep, but not from specimens obtained from the bighorn sheep. All 6 bighorn sheep died of acute hemorrhagic pneumonia after exposure to domestic sheep. Death in the bighorn sheep occurred on days 4, 27, 27, 29, 36, or 71 after initial exposure to domestic sheep. Pasteurella haemolytica was isolated from respiratory tract tissue specimens of all bighorn sheep at the time of death. None of the domestic sheep were clinically ill during the study. At the end of the study, 3 of 6 domestic sheep were euthanatized, and at necropsy, P haemolytica was isolated from 2 of them. The most common serotypes in bighorn and domestic sheep were P haemolytica T-3 and A-2. Other serotypes isolated included P haemolytica A-1, A-9, and A-11 in bighorn sheep and A-1 in domestic sheep. On the basis of results of this study and of other reports, domestic sheep and bighorn sheep should not be managed in proximity to each other because of the potential fatal consequences in bighorn sheep.     

Microorganisms associated with a pneumonic epizootic in Rocky Mountain bighorn sheep (Ovis canadensis canadensis).

A comprehensive study of a pneumonic epizootic was initiated when the first signs of disease were noted in a metapopulation of bighorn sheep inhabiting Hells Canyon, bordering Idaho, Oregon, and Washington. A total of 92 bighorn sheep were tested for etiologic agents during the following 6-mo study period. The study population included bighorn sheep believed to be the subpopulation in which disease was first noted, and these sheep were translocated to a holding facility in an effort to contain the disease (group A1, n = 72); bighorn sheep in other subpopulations (group A2) with evidence of clinical disease were captured, sampled, given antibiotics, and released (n = 8) and those that were found dead were necropsied (n = 12). Samples, including oropharyngeal and nasal swabs, and lung and liver tissue were collected from the bighorn sheep identified above. Tissue was collected at necropsy from 60 group A1 bighorn sheep that died following translocation, and samples were cultured for bacteria and viruses. Blood samples were tested for antibodies against known respiratory viruses, and histopathology was conducted on tissue samples. The major cause of death in both group A1 and group A2 bighorn sheep was a rapidly developing fibrinous bronchopneumonia. Multiple biovariants of Pasteurella were isolated from oropharyngeal and nasal samples from both groups, and Mycoplasma ovipneumonia was isolated from five group A1 oropharyngeal samples. Organisms isolated from lung tissue included Pasteurella multocida multocida a and Pasteurella trehalosi, both of which differentiated into multiple strains by restriction enzyme analysis, and parainfluenza-3 virus (PI-3). Paired serum samples revealed > fourfold increases in titers against PI-3 and bovine respiratory syncytial viruses. It was concluded that this epizootic resulted from a complex of factors including multiple potential respiratory pathogens, none of which were identified as a primary pathogen, and possible stress factors.     

Survey of desert bighorn sheep in California for exposure to selected infectious diseases

From February 1983 to June 1985, 188 desert bighorn sheep (Ovis canadensis nelsoni, = 161 and Oc cremnobates, = 27) from 18 herds in 17 mountain ranges and one captive herd were caught, marked, and had blood, fecal, and nasal mucus samples collected. Nasal swab specimens were cultured bacteriologically and virologically specifically for parainfluenza-3 (PI-3) virus. Bacterial flora differed from herd to herd. Pathogenic pneumophilic bacteria (eg, Pasteurella sp) seldom were found. Parainfluenza-3 virus was isolated from 6 bighorn sheep in 3 herds. Fecal specimens were examined for parasite ova and low numbers of lungworm (Protostrongylus sp) larvae were found in feces from 2 herds. Sera were evaluated for antibodies against respiratory syncytial virus, ovine progressive pneumonia, infectious bovine rhinotracheitis, PI-3, bovine viral diarrhea, brucellosis, leptospirosis, contagious ecthyma, bluetongue, and epizootic hemorrhagic disease. Blood clots were cultured virologically for bluetongue and epizootic hemorrhagic disease. Serologic evidence of bluetongue and/or epizootic hemorrhagic disease was found in 9 herds, and bluetongue virus (serotypes 10,11,13 and 17) was isolated from 3 herds. Antibody titers against PI-3 and respiratory syncytial virus were found in 9 and 13 herds, respectively. Evidence of bovine viral diarrhea infection was found in 6 herds, whereas infectious bovine rhinotracheitis was found in only 1 herd. Antibody titers against contagious ecthyma were found in 9 of 18 herds in California, and active lesions were seen occasionally. Evidence of ovine progressive pneumonia, leptospirosis, or brucellosis was not found.     

Susceptibility of Rocky Mountain bighorn sheep and domestic sheep to pneumonia induced by bighorn and domestic livestock strains of Pasteurella haemolytica.

Bighorn sheep were inoculated intratracheally with suspensions of nonhemolytic Pasteurella haemolytica biotype T (10(12) organisms) unique to wild bighorns, with beta-hemolytic P. haemolytica biotype T (10(12) organisms) isolated from clinically normal domestic sheep or intradermally with half a dose of a cattle vaccine containing P. haemolytica biotype A (10(5) organisms). The bighorn strain caused lobar necrotizing bronchopneumonia whereas both domestic livestock strains precipitated fatal septicemia and fibrinous bronchopneumonia. The serotypes given were T3, T4, T15 and A1 and these were recovered from lung lesions and other organs. In three trials, domestic sheep were inoculated intratracheally with suspensions of bighorn sheep pneumonic lungs, and two concentrations of the P. haemolytica bighorn strain (10(4) and 10(12) organisms). One of these sheep was inoculated intrabronchially. The domestic sheep experienced a transient fever and elevated white blood cell counts. After six days, none of the sheep had lung lesions and inoculated organisms could not be recovered. It is suggested that bighorn sheep are very susceptible to P. haemolytica from domestic livestock and should not be allowed in contact with sheep or cattle.     

Study on the etiologic role of bovine respiratory syncytial virus in pneumonia of dairy calves

Bovine respiratory syncytial virus (BRSV) was the viral agent most commonly identified in 14 epizootics of pneumonia in dairy calves. A microtiter serum-virus neutralization test proved to be the best means of identifying involvement of BRSV; seroconversion (fourfold or greater rise in titer) was demonstrated in 10 of the 14 epizootics. Only limited involvement of bovine viral diarrhea virus, infectious bovine rhinotracheitis virus, parainfluenza 3 virus, and bovine adenovirus type 3 was recognized. Pasteurella multocida was isolated in 12 of 14 epizootics, and Pasteurella haemolytica in 4 of 14 epizootics. Mycoplasmal and ureaplasmal agents were isolated in all 14 epizootics.     

Parainfluenza 3 vaccination of sheep

Outbreaks of pneumonia associated with Pasteurella haemolytica have occurred in sheep in the area of Perthshire served by this practice, and on some farms the disease has been an important annual cause of loss. Serological evidence was obtained that parainfluenza 3 (PI3) virus might be implicated as a predisposing factor to pasteurellosis. A live attenuated PI3 virus vaccine licensed for use in cattle was given intranasally to ewes on one farm. Many sheep seroconverted and outbreaks of pneumonia were negligible around the subsequent lambing time. The protection of the flock appeared to last for one season only. Subsequently ewes and lambs on other farms were vaccinated and on these farms there were fewer deaths than expected due to pasteurellosis.     

Properties of a respiratory syncytial virus isolated from a sheep with rhinitis

A virus isolated from a yearling cross-bred ewe was identified as respiratory syncytial virus (RSV) by indirect immunofluorescence and by virus neutralization with bovine RSV antisera. The virus caused a mild conjunctivitis in 3-month-old lambs when inoculated alone. Although clinical signs of pneumonia were not observed, there was gross and microscopic evidence of pulmonary inflammation in the lungs of lambs inoculated with either the sheep RSV isolate alone or in conjunction with Pasteurella haemolytica. Lung lesions in the dual infection were more severe, with approximately 10% of the total lung mass affected. Lavage fluids from lambs inoculated with virus and bacteria contained approximately 3 times more inflammatory cells than from control lambs or lambs inoculated with virus only. The sheep RSV isolate was classified as a mild respiratory pathogen in lambs of this age. Speculations on the potential importance of this virus in interspecies transmission to cattle and goats were discussed.     

Haemophilus somnus (Histophilus somni) in bighorn sheep.

Respiratory disease and poor lamb recruitment have been identified as limiting factors for bighorn-sheep populations. Haemophilus somnus (recently reclassified as Histophilus somni) is associated with respiratory disease in American bison, domestic sheep, and cattle. It is also harbored in their reproductive tracts and has been associated with reproductive failure in domestic sheep and cattle. Therefore, reproductive tract and lung samples from bighorn sheep were evaluated for the presence of this organism. Organisms identified as H. somnus were isolated from 6 of 62 vaginal but none of 12 preputial swab samples. Antigen specific to H. somnus was detected by immunohistochemical study in 4 of 12 formalin-fixed lung tissue samples of bighorn sheep that died with evidence of pneumonia. Notably, H. somnus was found in alveolar debris in areas of inflammation. The 6 vaginal isolates and 2 H. somnus isolates previously cultured from pneumonic lungs of bighorn sheep were compared with 3 representative isolates from domestic sheep and 2 from cattle. The profiles of major outer membrane proteins and antigens for all of the isolates were predominantly similar, although differences that may be associated with the host-parasite relationship and virulence were detected. The DNA restriction fragment length profiles of the bighorn-sheep isolates had similarities not shared with the other isolates, suggesting distinct phylogenetic lines. All of the isolates had similar antimicrobial profiles, but the isolates from the bighorn sheep produced less pigment than those from the domestic livestock, and growth of the former was not enhanced by CO2. Wildlife biologists and diagnosticians should be aware of the potential of these organisms to cause disease in bighorn sheep and of growth characteristics that may hinder laboratory detection.     

A spatial risk assessment of bighorn sheep extirpation by grazing domestic sheep on public lands

Bighorn sheep currently occupy just 30% of their historic distribution, and persist in populations less than 5% as abundant overall as their early 19th century counterparts. Present-day recovery of bighorn sheep populations is in large part limited by periodic outbreaks of respiratory disease, which can be transmitted to bighorn sheep via contact with domestic sheep grazing in their vicinity. In order to assess the viability of bighorn sheep populations on the Payette National Forest (PNF) under several alternative proposals for domestic sheep grazing, we developed a series of interlinked models. Using telemetry and habitat data, we characterized herd home ranges and foray movements of bighorn sheep from their home ranges. Combining foray model movement estimates with known domestic sheep grazing areas (allotments), a Risk of Contact Model estimated bighorn sheep contact rates with domestic sheep allotments. Finally, we used demographic and epidemiologic data to construct population and disease transmission models (Disease Model), which we used to estimate bighorn sheep persistence under each alternative grazing scenario. Depending on the probability of disease transmission following interspecies contact, extirpation probabilities for the seven bighorn sheep herds examined here ranged from 20% to 100%. The Disease Model allowed us to assess the probabilities that varied domestic sheep management scenarios would support persistent populations of free-ranging bighorn sheep.     

Cloning and comparison of bighorn sheep CD18 with that of domestic sheep, goats, cattle, humans and mice

Previously, we have shown that CD18, the beta-subunit of beta(2)-integrins, serves as a receptor for leukotoxin (Lkt) secreted by Mannheimia (Pasteurella) haemolytica on bovine leukocytes. Anti-CD18 monoclonal antibodies (mAbs) inhibit Lkt-induced cytolysis of bighorn sheep (Ovis canadensis) leukocytes suggesting that CD18 may serve as a receptor for Lkt on the leukocytes of this species as well. Confirmation of bighorn sheep CD18 as a receptor for Lkt, and elucidation of the enhanced Lkt-susceptibility of bighorn sheep polymorphonuclear leukocytes (PMNs), necessitates the cloning and sequencing of cDNA encoding bighorn sheep CD18. Hence, in this study we cloned and sequenced the cDNA encoding CD18 of bighorn sheep, and compared with that of other animal species. The cDNA of bighorn sheep CD18 has an open reading frame (ORF) of 2310bp. CD18 sequences obtained individually from peripheral blood mononuclear cells (PBMCs) and PMNs were identical to each other. Comparison of the deduced 770-amino acid sequence of CD18 of bighorn sheep with that of domestic sheep, goats, cattle, humans and mice revealed 99, 98, 95, 82 and 80% identity, respectively. Availability of cloned bighorn sheep CD18 cDNA should allow the molecular characterization of M. haemolytica Lkt-receptor interactions in bighorn sheep and other ruminants that are susceptible to this disease.     

Clinical study of the disease of calves associated with Mycoplasma bovis infection

Clinical, bacteriological and serological examination of 35 calves from the age of 5 to 26 days was performed in a Holstein-Friesian dairy herd endemically infected with Mycoplasma bovis. M. bovis was isolated from 48.6% of nasal swabs taken from the calves at the age of 5 days, and from 91.4% of the same calves at the age of 26 days, indicating the gradual spread of infection. The isolation rate of Pasteurella multocida did not change much, and varied from 28.6 to 25.7%. No P. haemolytica could be detected. In addition to M. bovis and P. multocida, the herd was also infected with different viruses (including bovine viral diarrhoea virus, infectious bovine rhinotracheitis virus, bovine adenoviruses, parainfluenza-3 virus, and bovine respiratory syncytial virus) as a large proportion of the sera of newborn calves contained colostral antibodies against these viruses. In most of the newborn calves severe clinical signs (fever, depression, inappetence, hyperventilation, dyspnoea, nasal discharge and coughing) due to M. bovis infection developed. The clinical signs appeared already on the fifth day of life, and their incidence was the highest at the age of 10 to 15 days. Three calves (8.6%) died as a result of severe serofibrinous pneumonia. The surviving calves showed very poor weight gain (ranging from 1.5 to 3.5 kg) during the first two weeks of life.     

Evaluation of bluetongue virus diagnostic tests in free-ranging bighorn sheep

Five bluetongue virus (BTV) diagnostic tests were evaluated for use in free-ranging bighorn sheep. We sampled one bighorn sheep population four times between 1989 and 1995. The tests evaluated included virus isolation (VI), polymerase-chain reaction (PCR), serum neutralization (SN), agar-gel immunodiffusion (AGID), and competitive enzyme-linked immunosorbent assay (c-ELISA). The c-ELISA, AGID and SN tests had high levels of agreement in determining serogroup exposure in bighorn sheep. We used maximum-likelihood algorithms to estimate the parameters of each diagnostic test used. Although the c-ELISA and AGID had high sensitivity and specificity, the SN had perfect specificity but lower apparent sensitivity. Due to the potential of cross-reactions among multiple serotypes, results of the SN must be interpreted with caution when assessing serotype exposure in an area where multiple serotypes are endemic. The PCR assay delineated convalescent antibody titers from more-recent infections, and consequently, was pivotal in distinguishing a different exposure pattern between the bighorn sheep and cattle in an adjacent herd. Based on an increasing seroprevalence (50% to 100%), BTV circulated through this bighorn sheep population between 1989 and 1993. This increase in seroprevalence coincided with a bighorn die-off due to BTV infection in June, 1991. An adjacent cattle herd was sampled in 1995 for comparison. The bighorn sheep and adjacent cattle had different patterns of exposure to BTV between 1994 and 1995. There was no evidence that BTV circulated through the bighorn sheep population from 1994 to 1995. In 1995, seroprevalence to BTV decreased to 72%, none of yearling bighorn was seropositive, and all of the 39 bighorn sheep were PCR-negative. In contrast, all adult cattle were seropositive to BTV by c-ELISA and SN, and 4 of the calves were seropositive; 11 of the 24 cattle were PCR-positive, including all five calves. Overall, the pattern of temporal herd immunity in the bighorn sheep appeared to follow a classic epidemic curve, with the appearance and subsequent disappearance of herd immunity coinciding with the 1991 die-off in this population. As low levels of herd immunity and high proportions of susceptible animals are key factors in the development of epidemics, this population of bighorn sheep may be at increased risk for a BTV epidemic in the future.     

Neospora caninum in sheep: a herd case report

Neospora caninum was detected by means of PCR in the brain of 4 out of 20 aborted fetuses in a flock of 117 sheep exhibiting a persistent abortion problem, and N. caninum tissue cysts were furthermore found in encephalitic lesions in one of the PCR-positive fetuses. Toxoplasma gondii was detected as aborting agent in another 3 out of 20 fetuses. Antibodies to N. caninum (by indirect fluorescence antibody test (IFAT)) were found in 10.3% of 117 ewes and antibodies for T. gondii were found in 97.4% of 117 ewes. Other organisms associated with abortion were Chlamydia psittaci in three fetuses and Pasteurella multocida in one fetus. This is the first report of N. caninum associated abortion in naturally infected sheep.     

Eradication of ovine progressive pneumonia from sheep flocks

Two management methods for controlling ovine progressive pneumonia in sheep were evaluated. By a test and cull method, wherein seropositive sheep were culled from the flock, the causative virus was eradicated from a closed flock and controlled in an open flock. By an isolate-and-test method, wherein lambs were removed from infected ewes before nursing and reared in isolation, the virus was eradicated in 1 of 2 attempts to establish virus-free flocks. It was concluded that either method should be effective in controlling ovine progressive pneumonia. Results indicated that annual serologic monitoring of a virus-free flock would be necessary to ensure that the virus-free status is maintained.     

Pathological and microbiological studies on pneumonic lungs from Danish calves

During 1 year, the association between microbiological and pathological findings in 72 lungs from calves submitted to the Danish Veterinary Laboratory for diagnostic purposes was studied. All cases were evaluated pathologically and bacteriologically, whereas only 68 cases were examined for the presence of bovine respiratory syncytial virus (BRSV), parainfluenza-3 virus (PI-3 virus) and bovine coronavirus, 62 cases for bovine viral diarrhoea virus (BVD), 45 cases for bovine adenovirus and 51 cases for mycoplasmas. Based on histopathological examination, the cases were diagnosed as fibrinous and/or necrotizing bronchopneumonia, suppurative bronchopneumonia, embolic pneumonia and others. The diagnoses were based on the dominating and most severe lesions in each lung. Haemophilus somnus, Pasteurella multocida, Actinomyces pyogenes, P. haemolytica and BRSV were the most commonly found bacterial and viral lung pathogens, respectively. Pasteurella spp. and H. somnus were often associated with the more severe fibrinonecrotizing type of bronchopneumonia, whereas BRSV was primarily detected in cases of suppurative bronchopneumonia. Mycoplasma bovis was isolated from one case only, whereas M. dispar, M. bovirhinis and Ureaplasma diversum were present, often concomitantly, in the majority of cases. Aspergillus fumigatus was isolated from one case.     

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.     

Characterization of an avian cholera epizootic in wild birds in western Nebraska

Avian cholera killed an estimated 2500 birds in western Nebraska and eastern Wyoming from 28 November 1985 to late January 1986. Wild mallards (Anas platyrhynchos) suffered the most losses. Other wild waterfowl, wild turkeys (Meleagris gallopavo), a few domestic fowl, and a bald eagle (Haliaeetus leucocephalus) also died. Pasteurella multocida serotype 1 was the predominant isolate from these carcasses. Cold, wet weather persisted throughout the outbreak, but daily losses in the flock of 50,000 mallards using the area were low. Pasteurella multocida was isolated from nasal swabs of 35 of 37 cattle from a feedlot in which many of these mallards were feeding. Eighty percent of the cattle isolates had antigenic characteristics of serotype 3 or serotype 3 with cross-reactivity. Isolates from wild mallards, wild turkeys, and the bald eagle were virulent to game-farm mallards when inoculated subcutaneously, but P. multocida isolates from cattle were not.     

Polymerase chain reaction techniques for differentiating cytotoxic and noncytotoxic Pasteurella trehalosi from Rocky Mountain bighorn sheep.

OBJECTIVE: To evaluate 2 polymerase chain reaction (PCR)-based methods for differentiating cytotoxic and noncytotoxic Pasteurella trehalosi from Rocky Mountain bighorn sheep (Ovis canadensis canadensis). SAMPLE POPULATION: 23 isolates of P. trehalosi from bighorn sheep in Colorado, including 18 from free-ranging herds and 5 from a captive herd. PROCEDURE: Using a sequence of the leukotoxin gene region of P. haemolytica serotype 1, 7 PCR primers were designed. A PCR amplification was performed on a sample of bacterial cell suspensions from pure cultures of P. trehalosi with known in vitro cytotoxic effects. The 2 most promising primer pairs were used in a study of 23 P. trehalosi isolates. Results were analyzed for association with cytotoxicity and 3 distinct ribotypes (Eco, Aco, and Bco). RESULTS: Significant associations were observed between in vitro cytotoxicity and PCR results for coding region, between ribotype Eco classification and PCR results for coding region, and between ribotype Eco classification and PCR results for promoter region. There was a negative association between ribotype Aco classification and PCR results for coding and promoter regions. CONCLUSIONS AND CLINICAL RELEVANCE: The PCR for the leukotoxin A coding region may be useful in differentiating cytotoxic from noncytotoxic P. trehalosi isolates recovered from bighorn sheep. It may be useful for studying epidemiologic features of pasteurellosis in bighorn sheep and for designing vaccines to protect wild sheep against pneumonia caused by P. trehalosi and P. haemolytica.     

Bighorn sheep fetal lung cell line for detection of respiratory viruses

Pneumonia is an important disease of bighorn sheep (BHS) that is primarily responsible for the drastic decline in numbers of these animals in North America. Members of the genus Mannheimia and Pasteurella have frequently been isolated from the pneumonic lungs of BHS. Antibodies to several respiratory viruses, including bovine parainfluenza virus 3 (BPIV-3), bovine respiratory syncytial virus (BRSV), bovine viral diarrhea virus (BVDV), and bovine herpesvirus 1 (BoHV-1), have been detected in herds of BHS. The availability of BHS fetal lung cell lines is likely to enhance the chances of isolation of these viruses. Here we report the development of such a cell line. This line is permissive for BPIV-3, BRSV, BVDV, and BoHV-1 infection, as revealed by an enzyme immunoassay of virus-infected cells with antibodies specific for each of these viruses. This cell line should be valuable for detecting these 4, and possibly other, respiratory viruses in BHS.     

犊牛呼吸道合胞体病毒和巴氏杆菌混合感染的调查研究

为了对疑似牛呼吸道合胞体病毒和巴氏杆菌混合感染的犊牛进行病原鉴定,本研究采用常规病毒经细菌分离鉴定和PCR方法分别进行分离与鉴定。结果表明,该病毒株能在BT细胞上增殖并产生特征性合胞体形态的细胞病变;无血凝性和血吸附特性;能被牛呼吸道合胞体病毒(bovine respiratory syncytial virus,BRSV)标准阳性血清中和;分离的病毒经RT-PCR鉴定为牛呼吸道合胞体病毒;根据菌落形态、细菌染色特性及生化特性,鉴定分离的细菌为巴氏杆菌。提示,该牛场为牛呼吸道合胞体病毒和巴氏杆菌混合感染。