Turid Vikøren

Campylobacter spp., Salmonella spp., Verocytotoxic Escherichia coli, and Antibiotic Resistance in Indicator Organisms in Wild Cervids

AbstractFaecal samples were collected, as part of the National Health Surveillance Program for Cervids (HOP) in Norway, from wild red deer, roe deer, moose and reindeer during ordinary hunting seasons from 2001 to 2003. Samples from a total of 618 animals were examined for verocytotoxic E. coli (VTEC); 611 animals for Salmonella and 324 animals for Campylobacter. A total of 50 samples were cultivated from each cervid species in order to isolate the indicator bacterial species E. coli and Enterococcus faecalis/E. faecium for antibiotic resistance pattern studies. Salmonella and the potentially human pathogenic verocytotoxic E. coli were not isolated, while Campylobacter jejuni jejuni was found in one roe deer sample only. Antibiotic resistance was found in 13 (7.3%) of the 179 E. coli isolates tested, eight of these being resistant against one type of antibiotic only. The proportion of resistant E. coli isolates was higher in wild reindeer (24%) than in the other cervids (2.2%). E. faecalis or E. faecium were isolated from 19 of the samples, none of these being reindeer. All the strains isolated were resistant against one (84%) or more (16%) antibiotics. A total of 14 E. faecalis-strains were resistant to virginiamycin only. The results indicate that the cervid species studied do not constitute an important infectious reservoir for either the human pathogens or the antibiotic resistant microorganisms included in the study.SammendragUndersøkelse av fecesprøver fra hjortevilt for Campylobacter spp., Salmonella spp., verocytotoksike Escherichia coli og antibiotikaresistens hos indikatororganismer. Fecesprøver ble samlet inn i regi av Helseovervåkingsprogrammet for hjortevilt (HOP) fra kronhjort, rådyr, elg og villrein i løpet av jaktsesongene fra 2001 til 2003. Prøver fra i alt 618 dyr ble undersøkt for verocytotoksiske E. coli (VTEC), 611 dyr for Salmonella og 324 dyr for Campylobacter. For å studere antibiotikaresistens- mønstre ble indikatorbakteriene E. coli og Enterococcus faecalis / E. faeciumi forsøkt isolert fra til sammen 50 prøver fra hver dyreart. Salmonella og de potensielt humanpatogene verocytotoksiske E. coli ble ikke isolert, mens Campylobacter jejuni jejuni ble funnet i prøven fra ett eneste rådyr. Antibiotikaresistens ble påvist hos 13 (7,3%) av de 179 E. coli isolatene som ble testet. Av disse var åtte resistente mot bare en type antibiotika. Andelen resistente E. coli isolater var høgere hos villrein (24%) enn hos det øvrige hjorteviltet (2,2%). E. faecalis eller E. faeciumi ble isolert fra 19 prøver, men ingen av disse var fra villrein. Alle stammene var resistente mot ett (84%) eller flere (16%) antibiotika. Til sammen 14 E. faecalis-stammer var resistente mot bare virginiamycin. Resultatene indikerer at hjortevilt ikke utgjør et smittereservoar av betydning verken for de humanpatogene bakteriene som inngikk i studien, eller for antibiotikaresistente bakterier.

ANTIBODIES TO RUMINANT ALPHA-HERPESVIRUSES AND PESTIVIRUSES IN NORWEGIAN CERVIDS

A serologic survey revealed that Norwegian populations of free-ranging reindeer (Rangifer tarandus tarandus), roe deer (Capreolus capreolus), red deer (Cervus elaphus), and moose (Alces alces) have been exposed to alpha-herpesviruses and pestiviruses. A total of 3,796 serum samples collected during the period 1993–2000 were tested in a neutralization test for antibodies against bovine herpesvirus 1 (BHV-1) or cervid herpesvirus 2 (CerHV-2), and 3,897 samples were tested by a neutralization test and/or enzyme-linked immunosorbent assay for antibodies against bovine viral diarrhea virus (BVDV). Antibodies against alpha-herpesvirus were found in 28.5% of reindeer, 3.0% of roe deer, and 0.5% of red deer, while all moose samples were negative. In reindeer, the prevalence of seropositive animals increased with age and was higher in males than females. Antibodies against BVDV were detected in 12.3% of roe deer, 4.2% of reindeer, 2.0% of moose and 1.1% of red deer. The results indicate that both alpha-herpesvirus and pestivirus are endemic in reindeer and pestivirus is endemic in roe deer in Norway. The viruses may be specific cervid strains. Seropositive red deer and moose may have become exposed as a result of contact with other ruminant species.

First case of chronic wasting disease in Europe in a Norwegian free-ranging reindeer

Chronic wasting disease (CWD) is a fatal contagious prion disease in cervids that is enzootic in some areas in North America. The disease has been found in deer, elk and moose in the USA and Canada, and in South Korea following the importation of infected animals. Here we report the first case of CWD in Europe, in a Norwegian free-ranging reindeer in Southern Norway. The origin of the disease is unknown. Until now a low number of cervids, and among them a few reindeer, have been tested for CWD in Norway. Therefore the prevalence of CWD is unknown.

EPIDEMIOLOGIC AND PATHOLOGIC ASPECTS OF SALMONELLA TYPHIMURIUM INFECTION IN PASSERINE BIRDS IN NORWAY

Septicemic salmonellosis caused by Salmonella Typhimurium 4, 12: i : 1, 2 was diagnosed in 94 (64.8%) of 145 small passerines comprising nine species, examined in Norway during 1999–2000. The birds were found dead at private feeding places throughout the country. The bullfinch (Pyrrhula pyrrhula), Eurasian siskin (Carduelis spinus), common redpoll (Carduelis flammea), and Eurasian greenfinch (Carduelis chloris) were the most frequently affected species. Pathologic findings in 94 carcasses included poor body condition (84%), enlarged spleen (73%), and necrosis of crop/esophagus (78%), liver (53%), spleen (46%), proventriculus (13%), and intestine (5.3%). Histologically, necrosis consisted of debris, fibrin, inflammatory cells, and aggregates of Gram-negative bacteria and occasionally giant cells. Based on information from questionnaires sick and dead birds were observed at feeding places from December to June, with a distinct peak during February and March. The duration of recorded outbreaks varied from less than 1 wk to 4 mo. In a separate study, 1,990 apparently healthy passerines caught at feeding places established for bird-ringing purposes were surveyed for cloacal carriage of Salmonella spp. Forty (2.0%) of the birds examined, representing sampling sites both in southern and northern parts of the country, harbored S. Typhimurium 4, 12: i : 1, 2 in their intestines. The carrier species largely reflected the species most often suffering from fatal infection.

MALIGNANT CATARRHAL FEVER IN FREE-RANGING CERVIDS ASSOCIATED WITH OVHV-2 AND CPHV-2 DNA

Pathologic lesions were summarized in 18 free-ranging cervids (15 moose [Alces alces], two roe deer [Capreolus capreolus], and one red deer [Cervus elaphus[) diagnosed with malignant catarrhal fever (MCF) after examination at the National Veterinary Institute, Oslo 1982–2005. Eye lesions (conjunctivitis, corneal opacity, fibrin clots in the anterior eye chamber) were the most frequent gross finding. Erosive-ulcerative mucosal lesions in the nose and mouth were also commonly found. Histopathology revealed a nonpurulent vasculitis and perivasculitis in the central nervous system (CNS) typical of MCF in 16 of the cases. The diagnosis in the remaining two animals was based upon histologic eye lesions consistent with MCF (CNS not available for examination). Polymerase chain reaction was run on samples from 15 individuals for evidence of MCF-virus DNA, and ovine herpesvirus-2 (OvHV-2) DNA was detected in five moose, one roe deer, and one red deer, and caprine herpesvirus-2 (CpHV-2) DNA was detected in two moose and one roe deer. Sera from 1,000 free-ranging cervids were tested for specific antibodies to MCF-associated viruses (MCFV) by competitive inhibition enzyme-linked immunosorbent assay. The seroprevalences were: red deer 5%, reindeer (Rangifer tarandus) 4%, roe deer 2%, and moose 0.4% (n = 250 for all four species). The results indicate that sheep and goat MCFV may cause serious disease in wild moose, roe deer, and red deer. The seropositive cervids most likely represent individuals infected with either OvHV-2 or CpHV-2, but may also reflect infections with other related MCFV.

LIVER CONCENTRATIONS OF COPPER, COBALT, AND SELENIUM IN WILD NORWEGIAN RED DEER (CERVUS ELAPHUS)

Liver samples from 245 wild red deer (Cervus elaphus) collected during the licensed hunting season in 2001 from five different locations in western Norway were analyzed for copper (Cu), cobalt (Co), and selenium (Se). The associations between these trace elements and geographical location, age group, and sex were studied. The median (and range of) liver concentrations (μg/g wet weight) for all the examined deer were: Cu 20 (1.7–103), Co 0.08 (<0.01–0.18), and Se 0.09 (0.04–1.0). The results indicate a generally low status of Cu and Se. In total, 15 (6%) red deer had deficient Cu levels (<4 μg/g). For all three elements, the liver concentrations showed a significant geographic variation. The geographic difference was most distinct for Cu. The lowest median Cu concentration was found in deer from the island Hitra, where 13% of the animals had deficient Cu levels. Significant differences between age groups were found for all elements, and generally, the adults (≥2.5 yr) had the highest levels. No significant sex differences within the various age groups were found, with three exceptions: female calves and adults had significantly higher Co levels than male deer, and adult males had significantly higher Se levels than adult females. The Cu and Se status of wild red deer in parts of Norway is low; however, the significance of this needs to be explored further.

OSTEOPOROSIS, BONE MINERALIZATION, AND STATUS OF SELECTED TRACE ELEMENTS IN TWO POPULATIONS OF MOOSE CALVES IN NORWAY

This study was conducted to clarify the etiology of a high frequency of bone fractures and osteoporosis in the moose (Alces alces) population in southern Norway. Liver samples, both metacarpi, and carcass data were collected from 21 and 22 moose calves shot in 1994 in Birkenes (southern Norway), and Nærøy (central Norway), respectively. The liver samples were analyzed for copper, manganese, zinc, cobalt, chromium, molybdenum, and selenium. Bone samples were subject to histologic, radiologic, and chemical examinations. Three of the calves from Birkenes and one calf from Nærøy showed histologic and radiologic evidence of generalized osteoporosis consistent with osteoporosis due to starvation. The calves with osteoporosis had the lowest carcass weights and radio-opacities recorded. There was a positive correlation between carcass weight and bone radio-opacity. Density, ash content, phosphorus, and calcium contents and phosphorous/calcium ratio in bone samples, as well as hepatic trace element status, were within the normal range for all calves in both populations. Two of the osteoporotic calves, were reported to have been orphaned. Our results indicate that the high frequency of bone fractures reported in moose in southern Norway is not associated with regional differences in trace element status or bone mineral balance. We propose that the occurrence of osteoporosis in moose calves in Birkenes may have resulted from inadequate nutrition following general overcrowding and high pressure on feed resources in the southernmost part of Norway.

FLUORIDE EXPOSURE IN CERVIDS INHABITING AREAS ADJACENT TO ALUMINUM SMELTERS IN NORWAY. II. FLUOROSIS

Mandibles from 1104 red deer (Cervus elaphus), 147 moose (Alces alces), and 453 roe deer (Capreolus capreolus), collected between 1990 and 1993 in the vicinity of seven Norwegian aluminum smelters, were examined for dental fluorotic and osteofluorotic lesions. The metacarpal or metatarsal bones from 214 of these cervids also were evaluated. Dental fluorotic lesions occurred in all three cervid species. Prevalence of dental fluorosis was generally low at the various locations, with the exception of Årdal, where 15% of the cervids examined were affected. Only sporadic cases of severe dental fluorotic lesions were diagnosed. All red deer yearlings (1.5 yr) with mandibular fluorine (F) levels exceeding 2,000 ppm F, had dental fluorosis. However, the lowest skeletal fluorine level found in a fluorotic animal of this age was 1,355 ppm F. Gross osteofluorosis occurred in only three cervids, all with mandibular fluorine residues >8,000 ppm F. Hence, generalized fluorosis was not a prominent feature in the material studied.

Natural and experimental Salmonella Typhimurium infections in foxes (Vulpes vulpes)

The red fox (Vulpes vulpes) can be considered as a relevant indicator species for Salmonella in the local environment and Salmonella faecal carriage was investigated in 215 red foxes in Norway shot during the winters 2002/2003 and 2003/2004. Fourteen (6.5%) of the foxes carried Salmonella. Four isolates were determined as serovars Kottbus (n=2) and Hessarek (n=2) of Salmonella enterica subspecies enterica, and one as S. enterica subspecies IIIb:61:k:1,5,(7). The remaining nine isolates were S. enterica subspecies enterica serovar Typhimurium 4,12:i:1,2 and all displayed the same pulsed-field gel electrophoresis (PFGE) profile designated A2. This serovar regularly causes disease outbreaks amongst small passerine birds during winter and most of these outbreaks are associated with the PFGE profile A2. The results strongly indicated that the Salmonella Typhimurium infections in red foxes were primarily acquired through ingestion of infected small passerines. To investigate the capability of the A2 strain to establish a true intestinal infection in the fox an inoculation experiment with an A2 isolate from small passerines was carried out in farmed silver foxes (V. vulpes). The experiment also included one strain with an uncommonly occurring profile (X201) from small passerines. To highlight possible differences in capability of the two inoculation strains to pass the acid gastric juice in the fox, in vitro studies of their acid tolerance was carried out. Also their catalase activity and biofilm production were studied. All three foxes inoculated with the A2 strain developed sub-clinical intestinal infection of 2 weeks duration, whereas none of the three foxes inoculated with the X201 strain shed this bacterium. The X201 strain displayed a much lower capability, than the A2 strain, to survive at pH 3 in vitro. The low acid tolerance probably made it difficult for the X201 strain to pass the stomach and establish an intestinal infection in the experimental foxes. Reduced catalase activity and biofilm production were found for the X201 strain, indicating that the low acid tolerance was caused by a defect in the stationary-phase stress response system.

SEROLOGIC SURVEY FOR ANTIBODIES AGAINST MYCOBACTERIUM AVIUM SUBSP. PARATUBERCULOSIS IN FREE-RANGING CERVIDS FROM NORWAY

Affinity between protein-G and immunoglobulins from red deer (Cervus elaphus), moose (Alces alces), roe deer (Capreolus capreolus), and reindeer (Rangifer tarandus tarandus) was tested in a competition binding assay. Sera from red deer, reindeer, and moose inhibited the assay less than sera from cattle (less affinity), whereas sera from roe deer showed a slightly higher affinity to protein-G than did sera from cattle. The conclusion was made that protein-G could be used instead of anti-species antibodies for these cervid species, where the aim of the screening was to look for exposure or lack of exposure to mycobacteria in the tested populations. Serologic screening of 1,373 free-ranging cervids for antibodies against Mycobacterium avium subsp. paratuberculosis was conducted. All sera were tested by a protein-G–based antigen-absorbed enzymelinked immunosorbent assay (ELISA). Seropositive moose (10/537; 1.9%), red deer (14/371; 3.8%), roe deer (6/49; 12.2%), and semidomesticated reindeer (11/325; 3.4%) were found, whereas wild reindeer (n=91) were seronegative. In addition, the red deer sera were tested with a commercial ELISA, by which two animals tested positive and nine were suspicious of having M. avium subsp. paratuberculosis antibodies. Tissue samples and feces from 10 moose originating from a population with a clustering of seropositive animals were investigated by histology and bacteriology with negative results. Paratuberculosis has never been diagnosed in free-ranging or farmed cervid species in Norway. Thus, further studies are indicated to prove that the present findings reflect an infection with M. avium subsp. paratuberculosis.