Abstract

Monitoring circulating pathogens in wildlife populations is important in evaluating causes and sources of disease as well as understanding transmission between wild and domestic animals. In spring 2010, a sudden die-off in a chamois (Rupicapra rupicapra) population sharing habitat with livestock occurred in northeastern Austria. Nineteen animals were submitted for examination. Necropsy and pathohistologic and bacteriologic results yielded lesions associated with Pasteurellaceae species. Additional testing included enterobacterial repetitive intergenic consensus and random amplification of polymorphic DNA PCR analysis to evaluate the circulating strains. The isolated strains were most closely related to Mannheimia glucosida and Bibersteinia trehalosi. Reports of mass mortalities in chamois due to pneumonia have been reported previously in the northern Alpine area of Italy. To the authors' knowledge, this is the first report of acute mortality due to strains of Mannheimia and Bibersteinia in Austrian chamois.

The Alpine chamois (Rupicapra rupicapra) shares Alpine habitat with various livestock species. Research has demonstrated the possibility of pathogen transmission between wildlife and livestock (Palmer et al. 2012). Species of Pasteurellaceae are the most common cause for bacterial bronchopneumonia in domestic and wild ungulates (Richard et al. 1992; Caswell and Williams 2007). Because Pasteurellaceae are a part of the normal upper respiratory tract flora in healthy animals, a positive bacteriologic sample of Pasteurellaceae without pathologic signs must be evaluated with caution (Donachie 2007). However, if immune response is compromised, as in times of stress, or they occur secondary to a virus or parasite infection, Pasteurellaceae species can cause local or systemic disease (Caswell and Williams 2007).

In spring 2010, a sudden die-off in chamois occurred in northeastern Austria (between 48°7′N, 14°52′E and 48°1′N, 15°36′E). Hunters reported a decrease of up to approximately 30% in the chamois populations affecting all age classes. Because most carcasses were too scavenged or decomposed to examine, only 19 animals were submitted for necropsy. Eight male (five adult, three subadult) and 11 female chamois (six adult, five subadult) were evaluated. Initial bacteriologic examinations identified Pasteurella-like strains. We conducted further examinations to specifically identify the pathogens.

All except one of the 19 chamois originated from two neighboring hunting grounds in northeastern Austria (districts of Amstetten and Lilienfeld). The remaining chamois came from an area further west (district of Salzburg-Umgebung), was in no contact with the other animals, and is thought to represent an independent event. Necropsy and pathohistologic, bacteriologic, parasitologic, and (in 16 cases) virologic examinations were carried out.

Bacterial isolations were accomplished directly from the submitted lung samples in all but four samples. The predominant isolates were pre-identified by routine microbiologic methods or using commercial identification kits (Quinn et al. 1994; Murray 2007; Loncaric et al. 2011). For the genotypic characterization, random amplification of polymorphic DNA (RAPD)-PCR, enterobacterial repetitive intergenic consensus (ERIC)-PCR, and the BOX element-PCR, and 16S rDNA sequence analysis were done (Versalovic et al. 1994; Mahenthiralingam et al. 1996; Loncaric et al. 2009, 2011). Phylogenetic analyses were conducted using MEGA 5 (Tamura et al. 2011). All isolates were screened for the gene encoding leukotoxin (lktA) by PCR (Davies et al. 2001).

Macroscopically, all animals were emaciated. The lungs showed severe multifocal to coalescing bronchopneumonic pattern, especially the cranial and lateral portions. Two animals revealed a severe diffuse suppurative pleuropneumonia. Several approximately walnut-sized, dorsally located, whitish firm foci due to lung-worm infestation were noted in eight animals. Microscopically, the lungs of 15 animals showed a severe subacute multifocal to coalescing fibrino-suppurative, partly necrotizing bronchopneumonia. In four animals (three adult females, one subadult female), the lesions were chronic. The infestation of the gastrointestinal tract with parasites varied highly. The most common parasites found were Strongyloides sp., Haemonchus sp., and coccidia. The virologic analyses (PCR, cell-culture, and electron microscopy) for Pestivirus and Parainfluenza 3 were negative and therefore are not discussed further.

Pasteurella-like colony types were predominant in all samples tested and, if possible, were further analyzed. Isolates 476_10 and 588_10 were oxidase and catalase positive, showing hemolysis on blood agar; isolates 379_10, 55_10, 679_10, 303_10, 306_10, and 563_10 were weakly oxidase positive and catalase negative. Employing the API 20NE test system, all eight isolates were characterized as Pasteurellaceae without precise species identification. Four different RAPD, BOX, und ERIC banding patterns were observed (Fig. 1). The isolates with different genomic fingerprints (representative isolates) were further analyzed by 16S rDNA sequence analysis. Isolate 476_10 shared highest similarity scores with the type strain of Mannheimia glucosida CCUG 38457T (98.64%). Other representative strains (379_10, 306_10, and 563_10) were most closely related to the type strain of Bibersteinia trehalosi NCTC 10370T (98.23%, 98.64%, and 99.40%). The sequences have been deposited to GenBank (JQ936477–JQ936480). These relationships are confirmed by phylogenetic analysis (Fig. 2). Two isolates, 476_10 and 588_10, had detectable lktA.

Figure 1.

Electrophoretic profiles of chamois (Rupicapra rupicapra) isolates by BOX element-PCR (top panel), enterobacterial repetitive intergenic consensus-PCR (center panel), and random amplification of polymorphic DNA-PCR (lower panel). Lane M: Lambda DNA/Eco91I (BstEII) Marker; lanes 1 and 2 (isolates 476_10 and 588_10): isolates most closely related to Mannheimia glucosida CCUG 38457T; lanes 3–8 (379_10; 55_10; 679_10; 303_10; 306_10; 563_10): isolates most closely related to Bibersteinia trehalosi NCTC 10370T.

Figure 1.

Electrophoretic profiles of chamois (Rupicapra rupicapra) isolates by BOX element-PCR (top panel), enterobacterial repetitive intergenic consensus-PCR (center panel), and random amplification of polymorphic DNA-PCR (lower panel). Lane M: Lambda DNA/Eco91I (BstEII) Marker; lanes 1 and 2 (isolates 476_10 and 588_10): isolates most closely related to Mannheimia glucosida CCUG 38457T; lanes 3–8 (379_10; 55_10; 679_10; 303_10; 306_10; 563_10): isolates most closely related to Bibersteinia trehalosi NCTC 10370T.

Figure 2.

Maximum-likelihood phylogenetic tree generated from partial 16S rRNA gene sequences of four chamois (Rupicapra rupicapra) representative isolates (379_10, 476_10, 306_10, 563_10), and closest relatives. Bootstrap values (expressed as percentages of 100 replications) greater than 75% are shown at branching points. The tree was rooted using Escherichia coli ATCC 11775T as an outgroup.

Figure 2.

Maximum-likelihood phylogenetic tree generated from partial 16S rRNA gene sequences of four chamois (Rupicapra rupicapra) representative isolates (379_10, 476_10, 306_10, 563_10), and closest relatives. Bootstrap values (expressed as percentages of 100 replications) greater than 75% are shown at branching points. The tree was rooted using Escherichia coli ATCC 11775T as an outgroup.

The family Pasteurellaceae contains several genera, including six species of Mannheimia (Euzéby 2013), and the genus Bibersteinia (Blackall et al. 2007). We demonstrate the occurrence of Bibersteinia and Mannheimia in wild chamois from the Austrian Alps. Angen et al. (2009) showed that members of the genera Pasteurella and Mannheimia occur at a high prevalence in healthy animals. Nonetheless, they may cause severe disease and great economic losses (Radostits et al. 2000). In cattle (Bos taurus), Mannheimia haemolytica is one of the most common pathogens associated with respiratory tract diseases, especially in young or recently transported animals (Rice et al. 2007). Mannheimia glucosida is frequently isolated from ovine species (Caprinae), can be isolated from bovine species (Bovinae), and is similarly associated with respiratory diseases (Angen et al. 1999; Kehrenberg et al. 2001). Villard et al. (2006) isolated Mannheimia glucosida from a pneumonic lung of a chamois. Bibersteinia trehalosi has been identified in domestic (Ovis aries) and bighorn sheep (Ovis canadensis), as well as in chamois, and is acknowledged as a respiratory tract pathogen (Villard et al. 2006; Wolfe et al. 2010).

Different animal species show varying susceptibility to different strains of Mannheimia. For example, bighorn sheep show a higher morbidity and mortality following infection with M. haemolytica than do domestic sheep (Herndon et al. 2011). Given the acute die-off in this chamois population, we suspect that M. glucosida and B. trehalosi result in high morbidity and mortality in chamois. Because retrieving dead animals from the wild is difficult due to scavenging (Wobeser 2006) and inaccessibility of Alpine terrain, we assume that many deceased chamois were not recovered. The infestation with gastrointestinal and respiratory parasites probably caused poor nutritional state and potentially made the animals more prone to severe respiratory lesions. Chamois are more prone to developing suppurative pneumonia when infested with lung worms due to the lesions caused by the parasites (Deutz and Deutz 2011). No other pathogen (virus or bacteria) was detected, and we are confident that the Pasteurellaceae isolated was the most likely cause of disease. This conclusion is supported by two lktA-positive isolates most closely related to the type strain of M. glucosida; lktA is a fundamental virulence factor in the pathogenesis of pneumonic pasteurellosis (Davies et al. 2001).

Infection with Pasteurellaceae can cause great losses in wild and domestic animal populations, and transmission is possible from one to the other (Wolfe et al. 2010). As has been shown elsewhere (Richomme et al. 2006), interspecific transmission of pathogens such as Pasteurella spp. is increased when mountain ungulates and domestic ungulates are allowed to move freely. This occurs on Austrian Alpine pastures during summer when cattle move around freely and share common food resources with chamois. It is therefore important to monitor pathogens in livestock and wild animal populations that share habitat.

LITERATURE CITED

LITERATURE CITED
Angen
Ø
,
Quirie
M
,
Donachie
W
,
Bisgaard
M
.
1999
.
Investigations on the species specificity of Mannheimia (Pasteurella) haemolytica serotyping
.
Vet Microbiol
65
:
283
290
.
Angen
Ø
,
Thomsen
J
,
Larsen
LE
,
Larsen
J
,
Kokotovic
B
,
Heegaard
PMH
,
Enemark
JMD
.
2009
.
Respiratory disease in calves: Microbiological investigations on trans-tracheally aspirated bronchoalveolar fluid and acute phase protein response
.
Vet Microbiol
137
:
165
171
.
Blackall
PJ
,
Bojesen
AM
,
Christensen
H
,
Bisgaard
M
.
2007
.
Reclassification of [Pasteurella] trehalosi as Bibersteinia trehalosi gen. nov., comb. nov
.
Int J Syst Evol Microbiol
57
:
666
674
.
Caswell
JL
,
Williams
K
.
2007
.
Respiratory system
.
In:
Jubb, Kennedy & Palmer's pathology of domestic animals
,
Maxie
MG
,
editor
.
Elsevier
,
Philadelphia, Pennsylvania
, pp.
523
524
.
Davies
RL
,
Whittam
TS
,
Selander
RK
.
2001
.
Sequence diversity and molecular evolution of the leukotoxin (lktA) gene in bovine and ovine strains of Mannheimia (Pasteurella) haemolytica
.
J Bacteriol
183
:
1394
1404
.
Deutz
A
,
Deutz
U
.
2011
.
Wildkrankheiten–Hundekrankheiten–Zoonoses. Erkennen–Vermeiden– (Be)Handeln
.
Leopold Stocker Verlag GmbH
,
Graz, Austria
,
264
pp.
Donachie
W
.
2007
.
Pasteurellosis
.
In:
Diseases of sheep
,
Aitken
ID
,
editor
.
Blackwell Publishing Ltd.
,
Oxford, UK
, pp.
224
231
.
Euzéby
JP
.
2013
.
LPSN—List of prokaryotic names with standing in nomenclature, http://www.bacterio.cict.fr/index.html. Accessed February 2014
.
Herndon
CN
,
Shanthalingam
S
,
Knowles
DP
,
Call
DR
,
Srikumaran
S
.
2011
.
Comparison of passively transferred antibodies in bighorn and domestic lambs reveals one factor in differential susceptibility of these species to Mannheimia haemolytica–induced pneumonia
.
Clin Vaccine Immunol
18
:
1133
1138
.
Kehrenberg
C
,
Salmon
SA
,
Watts
JL
,
Schwarz
S
.
2001
.
Tetracycline resistance genes in isolates of Pasteurella multocida, Mannheimia haemolytica, Mannheimia glucosida and Mannheimia varigena from bovine and swine respiratory disease: Intergeneric spread of the tet(H) plasmid pMHT1
.
J Antimicrob Chemother
48
:
631
640
.
Loncaric
I
,
Oberlerchner
JT
,
Heissenberger
B
,
Moosbeckhofer
R
.
2009
.
Phenotypic and genotypic diversity among strains of Aureobasidium pullulans in comparison with related species
.
Antonie van Leeuwenhoek
95
:
165
178
.
Loncaric
I
,
Ruppitsch
W
,
Licek
E
,
Moosbeckhofer
R
,
Busse
HJ
,
Rosengarten
R
.
2011
.
Characterization of selected Gram-negative non-fermenting bacteria isolated from honey bees (Apis mellifera carnica)
.
Apidologie
42
:
312
325
.
Mahenthiralingam
E
,
Campbell
ME
,
Foster
J
,
Lam
JS
,
Speert
DP
.
1996
.
Random amplified polymorphic DNA typing of Pseudomonas aeruginosa isolates recovered from patients with cystic fibrosis
.
J Clin Microbiol
34
:
1129
1135
.
Murray
PR
.
2007
.
Manual of clinical microbiology, 9th Ed
.
ASM Press
,
Washington, DC
,
2256
pp.
Palmer
MV
,
Thacker
TC
,
Waters
WR
,
Gortazar
C
,
Corner
LAL
.
2012
.
Mycobacterium bovis: A model pathogen at the interface of livestock, wildlife, and humans
.
Vet Med Int
2012
:
236205
.
Quinn
PJ
,
Carter
ME
,
Markey
BK
,
Carter
GR
.
1994
.
Clinical veterinary microbiology
.
Mosby
,
Edinburgh, UK
,
648
pp.
Radostits
OM
,
Gay
CC
,
Blood
DC
,
Hinchcliff
K
.
2000
.
Veterinary medicine: A textbook of the diseases of cattle, sheep, pigs, goats and horses
.
W.B. Saunders Company Limited
,
Philadelphia, Pennsylvania
,
1877
pp.
Rice
JA
,
Carrasco-Medina
L
,
Hodgins
DC
,
Shewen
PE
.
2007
.
Mannheimia haemolytica and bovine respiratory disease
.
Anim Health Res Rev
8
:
117
128
.
Richard
Y
,
Borges
E
,
Gauthier
D
,
Favier
C
,
Sanchis
R
,
Oudar
J
.
1992
.
Isolation from chamois of 33 Pasteurella beta hemolytic strains of the haemolytica-trehalosi group [biotype, serotype, SDS polyacrylamide gel profile]
.
Gibier Faune Sauvage
9
:
71
85
.
Richomme
C
,
Gauthier
D
,
Fromont
E
.
2006
.
Contact rates and exposure to inter-species disease transmission in mountain ungulates
.
Epidemiol Infect
134
:
21
30
.
Tamura
K
,
Peterson
D
,
Peterson
N
,
Stecher
G
,
Nei
M
,
Kumar
S
.
2011
.
MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods
.
Mol Biol Evol
28
:
2731
2739
.
Versalovic
J
,
Schneider
M
,
De Bruijn
FJ
,
Lupski
JR
.
1994
.
Genomic fingerprinting of bacteria using repetitive sequence-based polymerase chain reaction
.
Methods Mol Cell Biol
5
:
25
40
.
Villard
L
,
Gauthier
D
,
Lacheretz
A
,
Abadie
G
,
Game
Y
,
Maurin
F
,
Richard
Y
,
Borges
E
,
Kodjo
A
.
2006
.
Serological and molecular comparison of Mannheimia haemolytica and Pasteurella trehalosi strains isolated from wild and domestic ruminants in the French Alps
.
Vet J
171
:
545
550
.
Wobeser
GA
.
2006
.
How disease is detected, described, and measured
.
In:
Essentials of disease in wild animals
,
Wobeser
GA
,
editor
.
Blackwell Publishing
,
Ames, Iowa
, pp.
45
61
.
Wolfe
LL
,
Diamond
B
,
Spraker
TR
,
Sirochman
MA
,
Walsh
DP
,
Machin
CM
,
Bade
DJ
,
Miller
MW
.
2010
.
A bighorn sheep die-off in southern Colorado involving a Pasteurellaceae strain that may have originated from syntopic cattle
.
J Wildl Dis
46
:
1262
1268
.