Case Report Rapport de cas Septicemic pasteurellosis in farmed elk (Cervus canadensis) in Alberta Pritpal S. Malhi, Musangu Ngeleka, Murray R. Woodbury Abstract — Septicemic pasteurellosis is a bacterial disease of domestic and wild animals including bison, elk, and pronghorn antelope caused by Pasteurella multocida. Here we report 2 cases of septicemic pasteurellosis in farmed elk. Pasteurella multocida serogroup B was isolated from multiple tissues in both animals. Gene sequencing (16S ribosomal RNA) and BLAST query confirmed that the sequence is 99% to 100% homologous to the P. multocida sequences in the database. Résumé — Pasteurellose septicémique chez des wapitis d’élevage (Cervus canadensis) en Alberta. La pasteurellose septicémique est une maladie bactérienne des animaux domestiques et sauvages, dont le bison, le wapiti et l’antilocarpe, qui est causée par Pasteurella multocida. Dans le présent article, nous présentons un rapport sur 2 cas de pasteurellose septicémique chez les wapitis d’élevage. Le sérogroupe B de Pasteurella multocida a été isolé dans des plusieurs tissus des deux animaux. Le séquençage des gènes (ARN ribosomique16S) et une recherche BLAST a confirmé que la séquence est de 99 % à 100 % homologue aux séquences de P. multocida dans la base de données. (Traduit par Isabelle Vallières) Can Vet J 2016;57:961–963
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epticemic pasteurellosis is a fatal, sometimes epidemic bacterial disease of domestic and wild animals including free-ranging bison (Bison bison), elk (Cervus canadensis), and pronghorn antelope (Antilocapra americana) (1–3). The disease is caused by various types of Pasteurella multocida classified according to the Carter-Heddleston system of classification by capsular group letter and somatic type number (4,5) (e.g., A:2). Pasteurella multocida serotypes B:2 and E:2 have been identified with hemorrhagic septicemia (septicemic pasteurellosis) in domestic water buffalo (Bubalis bubalis) and cattle (Bos taurus) (4) and types A:2, A:3,4, B:1, and B:3,4 have caused epidemics in various wild ruminants (6). Outbreaks have been reported in farmed fallow (Dama dama) and chital deer (Axis axis) in Denmark, the United Kingdom, and Australia (7). However, the published literature contains no references to septicemic pasteurellosis in North American cervids except for wild elk (2,8). Prairie Diagnostic Services (Malhi, Ngeleka), Saskatoon, Saskatchewan; Department of Large Animal Clinical Sciences (Woodbury), Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan. Address all correspondence to Dr. Murray Woodbury; e-mail: [email protected] The authors received no financial support for the research, authorship, and/or publication of this article. Use of this article is limited to a single copy for personal study. Anyone interested in obtaining reprints should contact the CVMA office ([email protected]) for additional copies or permission to use this material elsewhere. CVJ / VOL 57 / SEPTEMBER 2016
Case description An outbreak of septicemic pasteurellosis in farmed elk caused by Pasteurella multocida serogroup B is described. The affected animals were from a closed herd of 95 elk that were divided into 3 adjacent enclosures (Table 1). Three horses were allowed to co-mingle with the cow/calf group but electrified perimeter fences prevented fence-line contact with other domestic or wild ruminants. The elk were free-grazed on tame grass and alfalfa pasture and the 240-acre enclosure holding the mature cows with calves had approximately 120 acres of 25% spruce/75% poplar bush. The cows and calves had access to a small dugout and were provided with water from a well that serves the house on the property. In addition to what was growing in the enclosures during the outbreak, the bull and the yearlings were fed a daily portion of commercial elk velvet ration containing undetermined levels of protein, vitamins, and minerals. There was no other mineral supplementation or forage feeding before or during the outbreak. Mortalities (Table 1) were observed over a period of high environmental temperatures but no extreme weather events occurred during this time. Psychological stress from predators such as bears in the vicinity of the herd may have been present. The sequence of events during the outbreak is uncertain because of the difficulty in observing morbidity in large enclosures with bush and finding dead animals when terminally sick animals seek cover. After observing the index case on July 18, 2014 (Table 1), the producer found autolyzed carcasses in the cow-calf enclosure that were likely the first outbreak mortalities, presumably from some time in the first 3 wk of July. 961
Table 1. Description of outbreak mortalities
R A P P O R T D E CA S
Diagnostic N = 95 Mortalities Necropsy testing Cows (n = 38) with calves (n = 28) (in a 240-acre enclosure)
Case #1: July 18, 2014 Case #2: July 21, 2014 Cows (n = 8) in July and August Calves (n = 10) in July and August
July 18, 2014 July 21, 2014 None None
Yes Yes None None
Yearlings (n = 28) (in a 30-acre enclosure)
n = 4 in September
None
None
Mature bull (n = 1) (in a 30-acre enclosure)
None
Mortality continued among the cows for approximately 2 wk after observation of the index case. Based on an initial presumptive diagnosis of acute-stage disease from clostridial infection, the producer handled and vaccinated the yearling group and the herd bull. They were given an 8-way clostridial vaccine (Ultrachoice 8 vaccine, Zoetis Canada, Kirkland, Quebec), a single valent C. haemolyticum vaccine (Colorado Serum, Denver, Colorado, USA) and a cattle dose of long acting penicillin (Derapen SQ/LA injectable penicillin; Zoetis Canada). The animals were brought in and handled again in the first wk of September. Two or 3 days subsequent to this event there were mortalities (Table 1) in the yearling group but no necropsy or diagnostic testing was performed on these carcasses. The cows were not handled and vaccinated because they had very young (2 mo) calves at foot. The calf losses in the cow-calf group were possibly due to the death of their mothers and subsequent starvation. On gross examination of carcass #1 by the local veterinarian on July 18, 2014, there were fibrin tags on abdominal organs and serosanguinous effusion with floating fibrin was observed in the pleural cavity although the lungs appeared normal. There were petechiae on the pericardium. There was slight enlargement of the liver. The urinary bladder was reddened in appearance and empty. There was remarkable subcutaneous edema around the vulva, perineum, and udder. The cow was lactating but the mammary tissue appeared normal. On July 21, a second necropsy (case #2) was performed. This cow was in good body condition and no subcutaneous edema was evident. There was serosanguinous fluid in the abdominal and chest cavities with congestion in the lungs and liver. There were gas bubbles in the spleen and diaphragm; intestines were autolyzed. The rumen was full but contents appeared normal. Fresh and formalin-fixed tissues from both cases (case #1 — liver, kidney, urinary bladder, mammary gland, and skeletal muscle; case #2 — liver, kidney, spleen, heart, lung, and intestines) were received at the Prairie Diagnostic Services laboratory on July 22, 2014. Formalin-fixed samples were submitted for histopathology. Fresh tissues from both animals (case #1 — liver and mammary tissue; case #2 — liver, spleen and kidney) were submitted for bacterial culture. Fluorescent antibody testing (FAT) for clostridia (C. chauvoei, C. novyi, C. septicum and C. sordellii) was also requested on unfixed heart tissue from the second animal. Histological examination of submitted tissues from case #1 revealed inflammation of the mammary gland and skeletal 962
muscle. The interlobular septa of the mammary glandular parenchyma were expanded with edema fluid, fibrin, and abundant neutrophils. The glandular parenchyma was also diffusely infiltrated by neutrophils. Blood vessels were distended with neutrophils and fibrin and there was vasculitis. The skeletal muscle (specific location unknown from sampling) also had severe inflammation consisting of multifocal to coalescing infiltrates of large numbers of neutrophils. Multifocally, there was fibrinoid necrosis of the vessel walls and there was vasculitis. Numerous Gram-negative bacteria were present within or around vessels. There was necrosis of myofibers characterized by fragmented sarcoplasm, pyknotic nuclei, and loss of cross-striations. The renal capillaries and hepatic sinusoids multifocally contained small numbers of bacteria. Morphological diagnoses of suppurative mastitis and suppurative myositis with vasculitis were established. No significant inflammation was detected in any of the submitted tissues from case #2. A few glomerular capillaries contained bacteria within their lumen. The fresh tissues submitted for bacteriology testing were inoculated on blood and MacConkey agar plates and incubated at 37°C for 48 h. Pasteurella multocida was isolated in large numbers from both cases (Case #1: 41 from liver and mammary tissue; Case #2: 41 from the kidney and 21 from liver and spleen). Other organisms including Clostridium perfringens, Enterobacter species, and Escherichia coli were isolated in small numbers (1 to 21) from some tissues and were considered as postmortem invaders. Tests for Bacillus anthracis and for other histotoxic clostridia (C. chauvoei, C. septicum, C. sordellii, and C. novyi) were negative. Based on these findings Pasteurella multocida was considered significant. In order to determine the capsular antigen group of the organism, isolates from both cases were typed by PCR, as described previously (9) and were identified as P. multocida serogroup B. Gene sequencing (16S ribosomal RNA) and a Basic Local Alignment Search Tool (BLAST) query (www.ncbi.nlm.nih.gov/blast) showed 99% to 100% homology of the 2 isolates to P. multocida sequences in the database (GenBank accession number AF169324). To conclude, a diagnosis of septicemic pasteurellosis was made based on the isolation of P. multocida serogroup B from multiple tissues in both cases.
Discussion To the authors’ knowledge, this is the first peer-reviewed report of septicemic pasteurellosis in farmed Canadian elk. This disease generally occurs in epidemic form and is likely transmitted by CVJ / VOL 57 / SEPTEMBER 2016
CVJ / VOL 57 / SEPTEMBER 2016
single peer-reviewed report of this disease in Canada. Veterinary practitioners and diagnosticians should include septicemic pasteurellosis in a list of their differentials in similar circumstances. This may lead to a more detailed description of this disease in the future.
Acknowledgment The authors thank Dr. Sandy Gaube for the case referral and provision of diagnostic material for this case. CVJ
References 1. Dunbar MR, Wolcott MJ, Rimler RB, Berlowski BM. Septicemic pasteurellosis in free-ranging neonatal pronghorn in Oregon. J Wildl Dis 2000; 36:383–388. 2. Franson JC, Smith BL. Septicemic pasteurellosis in elk (Cervus elaphus) on the United States National Elk Refuge, Wyoming. J Wildl Dis 1988; 24:715–717. 3. Heddleston KL, Gallagher JE. Septicemic pasteurellosis (hemorrhagic septicemia) in the American bison: A serologic survey. J Wildl Dis 1969;5: 206–207. 4. Carter GR. Studies on Pasteurella multocida. I. A hemagglutination test for the identification of serological types. Am J Vet Res 1955;16: 481–484. 5. Heddleston KL, Gallagher JE, Rebers PA. Fowl cholera: Gel diffusion precipitin test for serotyping Pasteurella multocida from avian species. Avian Dis 1972;16:925–936. 6. Miller MW. Pasteurellosis. In: Infectious Diseases of Wild Animals. Ames, Iowa: Iowa State University Press, 2001. 7. Eriksen L, Aalbaek B, Leifsson PS, et al. Hemorrhagic septicemia in fallow deer (Dama dama) caused by Pasteurella multocida multocida. J Zoo Wildl Med 1999;30:285–292. 8. Newsletter CCWHC. Septicemic pasteurellosis — Elk, 1999. Available from: http://www2.cwhc-rcsf.ca/newsletters/newsletter6-1.pdf Last accessed June 20, 2016. 9. Townsend KM, Boyce JD, Chung JY, Frost AJ, Adler B. Genetic organization of Pasteurella multocida cap Loci and development of a multiplex capsular PCR typing system. J Clin Microbiol 2001;39:924–929.
963
CA S E R E P O R T
ingestion or inhalation during direct contact with affected animals or contaminated feed or water. Infected animals shed the organisms through nasal secretion when stressed and animals carrying a pathogenic or new strain are likely responsible for its introduction into vulnerable populations (6). However, infection from another species is also possible. Bacterial exposure dose, strain virulence, proliferation rate, and host immune competency affect the ultimate outcome of infection but environmental stressors or concomitant infections may be a prerequisite to development of clinical disease (6). Clinical signs include severe depression, profuse salivation, edema of the head, neck and brisket, and severe respiratory distress with foamy nasal discharge, leading to death. Chronic cases of hemorrhagic septicemia or septicemic pasteurellosis have not been reported. Treatment with antibiotics such as penicillins, tetracyclines, and cephalosporins is possible but impractical in most farmed cervid populations. Preventive strategies in farmed cervids are aimed at reduction of nutritional and social stress as well as general disease prevention such as regular anthelmintic treatment. Environmental stressors such as extreme weather conditions are more difficult to manage. The diagnosis of septicemic pasteurellosis in this case report is mainly based on the isolation of Pasteurella multocida serogroup B from multiple tissues in both animals. The information on spectrum of lesions of this disease is limited and is mostly based on field necropsy observations. There were only a limited number of formalin-fixed tissues submitted for histopathology and no clinical signs were observed in the present cases. Based on the evidence of pasteurellosis, no virology testing was done. Hence we could not rule out a predisposing or concurrent viral infection. Therefore, within these limitations, we have documented an outbreak of septicemic pasteurellosis, which is uncommonly reported in North America. To our knowledge, there is not a
S
epticemic pasteurellosis is a fatal, sometimes epidemic bacterial disease of domestic and wild animals including free-ranging bison (Bison bison), elk (Cervus canadensis), and pronghorn antelope (Antilocapra americana) (1–3). The disease is caused by various types of Pasteurella multocida classified according to the Carter-Heddleston system of classification by capsular group letter and somatic type number (4,5) (e.g., A:2). Pasteurella multocida serotypes B:2 and E:2 have been identified with hemorrhagic septicemia (septicemic pasteurellosis) in domestic water buffalo (Bubalis bubalis) and cattle (Bos taurus) (4) and types A:2, A:3,4, B:1, and B:3,4 have caused epidemics in various wild ruminants (6). Outbreaks have been reported in farmed fallow (Dama dama) and chital deer (Axis axis) in Denmark, the United Kingdom, and Australia (7). However, the published literature contains no references to septicemic pasteurellosis in North American cervids except for wild elk (2,8). Prairie Diagnostic Services (Malhi, Ngeleka), Saskatoon, Saskatchewan; Department of Large Animal Clinical Sciences (Woodbury), Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan. Address all correspondence to Dr. Murray Woodbury; e-mail: [email protected] The authors received no financial support for the research, authorship, and/or publication of this article. Use of this article is limited to a single copy for personal study. Anyone interested in obtaining reprints should contact the CVMA office ([email protected]) for additional copies or permission to use this material elsewhere. CVJ / VOL 57 / SEPTEMBER 2016
Case description An outbreak of septicemic pasteurellosis in farmed elk caused by Pasteurella multocida serogroup B is described. The affected animals were from a closed herd of 95 elk that were divided into 3 adjacent enclosures (Table 1). Three horses were allowed to co-mingle with the cow/calf group but electrified perimeter fences prevented fence-line contact with other domestic or wild ruminants. The elk were free-grazed on tame grass and alfalfa pasture and the 240-acre enclosure holding the mature cows with calves had approximately 120 acres of 25% spruce/75% poplar bush. The cows and calves had access to a small dugout and were provided with water from a well that serves the house on the property. In addition to what was growing in the enclosures during the outbreak, the bull and the yearlings were fed a daily portion of commercial elk velvet ration containing undetermined levels of protein, vitamins, and minerals. There was no other mineral supplementation or forage feeding before or during the outbreak. Mortalities (Table 1) were observed over a period of high environmental temperatures but no extreme weather events occurred during this time. Psychological stress from predators such as bears in the vicinity of the herd may have been present. The sequence of events during the outbreak is uncertain because of the difficulty in observing morbidity in large enclosures with bush and finding dead animals when terminally sick animals seek cover. After observing the index case on July 18, 2014 (Table 1), the producer found autolyzed carcasses in the cow-calf enclosure that were likely the first outbreak mortalities, presumably from some time in the first 3 wk of July. 961
Table 1. Description of outbreak mortalities
R A P P O R T D E CA S
Diagnostic N = 95 Mortalities Necropsy testing Cows (n = 38) with calves (n = 28) (in a 240-acre enclosure)
Case #1: July 18, 2014 Case #2: July 21, 2014 Cows (n = 8) in July and August Calves (n = 10) in July and August
July 18, 2014 July 21, 2014 None None
Yes Yes None None
Yearlings (n = 28) (in a 30-acre enclosure)
n = 4 in September
None
None
Mature bull (n = 1) (in a 30-acre enclosure)
None
Mortality continued among the cows for approximately 2 wk after observation of the index case. Based on an initial presumptive diagnosis of acute-stage disease from clostridial infection, the producer handled and vaccinated the yearling group and the herd bull. They were given an 8-way clostridial vaccine (Ultrachoice 8 vaccine, Zoetis Canada, Kirkland, Quebec), a single valent C. haemolyticum vaccine (Colorado Serum, Denver, Colorado, USA) and a cattle dose of long acting penicillin (Derapen SQ/LA injectable penicillin; Zoetis Canada). The animals were brought in and handled again in the first wk of September. Two or 3 days subsequent to this event there were mortalities (Table 1) in the yearling group but no necropsy or diagnostic testing was performed on these carcasses. The cows were not handled and vaccinated because they had very young (2 mo) calves at foot. The calf losses in the cow-calf group were possibly due to the death of their mothers and subsequent starvation. On gross examination of carcass #1 by the local veterinarian on July 18, 2014, there were fibrin tags on abdominal organs and serosanguinous effusion with floating fibrin was observed in the pleural cavity although the lungs appeared normal. There were petechiae on the pericardium. There was slight enlargement of the liver. The urinary bladder was reddened in appearance and empty. There was remarkable subcutaneous edema around the vulva, perineum, and udder. The cow was lactating but the mammary tissue appeared normal. On July 21, a second necropsy (case #2) was performed. This cow was in good body condition and no subcutaneous edema was evident. There was serosanguinous fluid in the abdominal and chest cavities with congestion in the lungs and liver. There were gas bubbles in the spleen and diaphragm; intestines were autolyzed. The rumen was full but contents appeared normal. Fresh and formalin-fixed tissues from both cases (case #1 — liver, kidney, urinary bladder, mammary gland, and skeletal muscle; case #2 — liver, kidney, spleen, heart, lung, and intestines) were received at the Prairie Diagnostic Services laboratory on July 22, 2014. Formalin-fixed samples were submitted for histopathology. Fresh tissues from both animals (case #1 — liver and mammary tissue; case #2 — liver, spleen and kidney) were submitted for bacterial culture. Fluorescent antibody testing (FAT) for clostridia (C. chauvoei, C. novyi, C. septicum and C. sordellii) was also requested on unfixed heart tissue from the second animal. Histological examination of submitted tissues from case #1 revealed inflammation of the mammary gland and skeletal 962
muscle. The interlobular septa of the mammary glandular parenchyma were expanded with edema fluid, fibrin, and abundant neutrophils. The glandular parenchyma was also diffusely infiltrated by neutrophils. Blood vessels were distended with neutrophils and fibrin and there was vasculitis. The skeletal muscle (specific location unknown from sampling) also had severe inflammation consisting of multifocal to coalescing infiltrates of large numbers of neutrophils. Multifocally, there was fibrinoid necrosis of the vessel walls and there was vasculitis. Numerous Gram-negative bacteria were present within or around vessels. There was necrosis of myofibers characterized by fragmented sarcoplasm, pyknotic nuclei, and loss of cross-striations. The renal capillaries and hepatic sinusoids multifocally contained small numbers of bacteria. Morphological diagnoses of suppurative mastitis and suppurative myositis with vasculitis were established. No significant inflammation was detected in any of the submitted tissues from case #2. A few glomerular capillaries contained bacteria within their lumen. The fresh tissues submitted for bacteriology testing were inoculated on blood and MacConkey agar plates and incubated at 37°C for 48 h. Pasteurella multocida was isolated in large numbers from both cases (Case #1: 41 from liver and mammary tissue; Case #2: 41 from the kidney and 21 from liver and spleen). Other organisms including Clostridium perfringens, Enterobacter species, and Escherichia coli were isolated in small numbers (1 to 21) from some tissues and were considered as postmortem invaders. Tests for Bacillus anthracis and for other histotoxic clostridia (C. chauvoei, C. septicum, C. sordellii, and C. novyi) were negative. Based on these findings Pasteurella multocida was considered significant. In order to determine the capsular antigen group of the organism, isolates from both cases were typed by PCR, as described previously (9) and were identified as P. multocida serogroup B. Gene sequencing (16S ribosomal RNA) and a Basic Local Alignment Search Tool (BLAST) query (www.ncbi.nlm.nih.gov/blast) showed 99% to 100% homology of the 2 isolates to P. multocida sequences in the database (GenBank accession number AF169324). To conclude, a diagnosis of septicemic pasteurellosis was made based on the isolation of P. multocida serogroup B from multiple tissues in both cases.
Discussion To the authors’ knowledge, this is the first peer-reviewed report of septicemic pasteurellosis in farmed Canadian elk. This disease generally occurs in epidemic form and is likely transmitted by CVJ / VOL 57 / SEPTEMBER 2016
CVJ / VOL 57 / SEPTEMBER 2016
single peer-reviewed report of this disease in Canada. Veterinary practitioners and diagnosticians should include septicemic pasteurellosis in a list of their differentials in similar circumstances. This may lead to a more detailed description of this disease in the future.
Acknowledgment The authors thank Dr. Sandy Gaube for the case referral and provision of diagnostic material for this case. CVJ
References 1. Dunbar MR, Wolcott MJ, Rimler RB, Berlowski BM. Septicemic pasteurellosis in free-ranging neonatal pronghorn in Oregon. J Wildl Dis 2000; 36:383–388. 2. Franson JC, Smith BL. Septicemic pasteurellosis in elk (Cervus elaphus) on the United States National Elk Refuge, Wyoming. J Wildl Dis 1988; 24:715–717. 3. Heddleston KL, Gallagher JE. Septicemic pasteurellosis (hemorrhagic septicemia) in the American bison: A serologic survey. J Wildl Dis 1969;5: 206–207. 4. Carter GR. Studies on Pasteurella multocida. I. A hemagglutination test for the identification of serological types. Am J Vet Res 1955;16: 481–484. 5. Heddleston KL, Gallagher JE, Rebers PA. Fowl cholera: Gel diffusion precipitin test for serotyping Pasteurella multocida from avian species. Avian Dis 1972;16:925–936. 6. Miller MW. Pasteurellosis. In: Infectious Diseases of Wild Animals. Ames, Iowa: Iowa State University Press, 2001. 7. Eriksen L, Aalbaek B, Leifsson PS, et al. Hemorrhagic septicemia in fallow deer (Dama dama) caused by Pasteurella multocida multocida. J Zoo Wildl Med 1999;30:285–292. 8. Newsletter CCWHC. Septicemic pasteurellosis — Elk, 1999. Available from: http://www2.cwhc-rcsf.ca/newsletters/newsletter6-1.pdf Last accessed June 20, 2016. 9. Townsend KM, Boyce JD, Chung JY, Frost AJ, Adler B. Genetic organization of Pasteurella multocida cap Loci and development of a multiplex capsular PCR typing system. J Clin Microbiol 2001;39:924–929.
963
CA S E R E P O R T
ingestion or inhalation during direct contact with affected animals or contaminated feed or water. Infected animals shed the organisms through nasal secretion when stressed and animals carrying a pathogenic or new strain are likely responsible for its introduction into vulnerable populations (6). However, infection from another species is also possible. Bacterial exposure dose, strain virulence, proliferation rate, and host immune competency affect the ultimate outcome of infection but environmental stressors or concomitant infections may be a prerequisite to development of clinical disease (6). Clinical signs include severe depression, profuse salivation, edema of the head, neck and brisket, and severe respiratory distress with foamy nasal discharge, leading to death. Chronic cases of hemorrhagic septicemia or septicemic pasteurellosis have not been reported. Treatment with antibiotics such as penicillins, tetracyclines, and cephalosporins is possible but impractical in most farmed cervid populations. Preventive strategies in farmed cervids are aimed at reduction of nutritional and social stress as well as general disease prevention such as regular anthelmintic treatment. Environmental stressors such as extreme weather conditions are more difficult to manage. The diagnosis of septicemic pasteurellosis in this case report is mainly based on the isolation of Pasteurella multocida serogroup B from multiple tissues in both animals. The information on spectrum of lesions of this disease is limited and is mostly based on field necropsy observations. There were only a limited number of formalin-fixed tissues submitted for histopathology and no clinical signs were observed in the present cases. Based on the evidence of pasteurellosis, no virology testing was done. Hence we could not rule out a predisposing or concurrent viral infection. Therefore, within these limitations, we have documented an outbreak of septicemic pasteurellosis, which is uncommonly reported in North America. To our knowledge, there is not a
