Investigations on airborne microorganisms in animal stables. 2. Report: further characterization of airborne Clostridium perfringens.

Toxovars of 97 airborne C. perfringens isolates and 10 C. perfringens isolates from fecal samples of a calf stable were determined by an EIA procedure. Most airborne and fecal isolates belonged to toxovar A (88.7% and 80.0% respectively). Eight point two% of airborne C. perfringens were identified as toxovar C and 3.1% as toxovar D. Toxovar B was not found in the airborne state. Twenty% of fecal C. perfringens belonged to toxovar D. Toxovar B and C was not isolated from fecal samples. In addition, all fecal and air-borne isolates of C. perfringens toxovar D strains were analyzed in SDS-PAGE for their polypeptide pattern. All isolates from both sources exhibited the same polypeptide pattern after electrophoretic analysis in SDS-PAGE. Both results, determination of toxovars as well as polypeptide pattern analysis in SDS-PAGE, suggest that a major source of airborne C. perfringens in animal stables is animal feces.     

The impact of various browse feeds with different tannin content on the fecal shedding of Clostridium perfringens in West African dwarf sheep

In 1994 and 1995 leaves from eight browse feeds, containing tannins in different amounts (BF), were fed to West African Dwarf Sheep in Benin to evaluate their impact on Clostridium perfringens in the intestinal tract. An inhibitory impact of various BF on the growth of C. perfringens was assessed in in-vitro assays before, and thus a potential use of these leaves as a preventive diet against C. perfringens enterotoxemia in small ruminants was assumed. Surprisingly, an inhibitory impact of the BF on the shedding of C. perfringens in the feces of West African Dwarf Sheep could not be shown in seven of the eight BF examined. However, the pattern of inhibition of unlike C. perfringens toxovars may differ and a selective inhibitory impact of the BF Dialium guineense on C. perfringens toxovar D may be assumed.     

Toxin types of Clostridium perfringens isolated from free-ranging,semi-domesticated reindeer in Norway

Samples of faeces were taken from 166 healthy domesticated reindeer (Rangifer tarandus tarandus) from three flocks in different reindeer husbandry districts in northern Norway and examined bacteriologically for the presence of Clostridium perfringens. The organism was isolated from 98 (59 per cent) of the reindeer. The isolates were classified into C perfringens toxin types by PCR analysis specific for the genes encoding the four major toxins (alpha, beta, epsilon and tau) and were subclassified by the detection of the genes encoding C perfringens beta2-toxin and enterotoxin. All the isolates belonged to C perfringens toxin type A. In addition, 15 of the 98 isolates were PCR-positive for the beta2-toxin gene, and two of the isolates had the the gene encoding for enterotoxin.     

不同方法对牛舍气载魏氏梭菌型别的研究

分别采用ELISA、多重PCR方法对从一个犊牛舍分离到的魏氏梭菌进行型别研究,结果显示:ELISA检测结果为88%的菌种属于A型,3.4%为C型,8.6%是D型;而多重PCR结果均为A型。     

The expression of Clostridium perfringens consensus beta2 toxin is associated with bovine enterotoxaemia syndrome

Clostridium perfringens has been implicated in a broad array of enteric infections including the fatal haemorrhagic enteritis/enterotoxaemia syndrome in cattle. The beta2 toxin (CPB2), encoded by cpb2, is suspected to be implicated in this syndrome. However, among C. perfringens isolates from cattle suspected of clostridial disease, an atypical allele was recently found to predominate at the cpb2 locus and atypical corresponding CPB2 proteins were shown to be poorly expressed, thus arguing against a biologically significant role of the beta2 toxin in clostridial diseases in cattle. This study compared genotype and phenotype of the beta2 toxin between C. perfringens isolates from a group of healthy calves (n=14, 87 isolates) and from a group of enterotoxaemic calves (n=8, 41 isolates). PCR results revealed the exclusive presence of the typical "consensus"cpb2 in the enterotoxaemic group. Western blot analysis demonstrated that the typical variant of CPB2 was often expressed in isolates from enterotoxaemic calves (43.9%) and infrequently in isolates from healthy cattle (6.9%). These data suggest that the typical variant of the CPB2 toxin may play a role in the pathogenesis of cattle enterotoxaemia.     

ELISA对动物舍气载魏氏梭菌型别的研究

采用ELISA方法对从一个犊牛舍空气中分离到的魏氏梭菌进行型别的鉴定。结果显示：88％的菌种属于A型，3.4％为C型，8.6％是D型，B型未能确认。     

Cerebrospinal elaphostrongylosis in dairy goats in northern Norway.

Ten carcasses and three vertebral columns from north Norwegian dairy goats, which had been killed due to clinical signs of severe neurologic disease, were received for necropsy. Pathological examination revealed nematodes and nematode ova in the central nervous system (CNS) of nine goats. Worms found by gross examination were identified as Elaphostrongylus rangiferi Mitskevich, 1960. Focal traumatic encephalomyelomalacia, apparently caused by migrating worms, perivascular cuffing, eosinophilic leptomeningitis and perineural infiltrations and granulomas, could be demonstrated in CNS sections from all 13 animals examined. Clinical signs reported were initial pruritus followed by motor weakness, lameness, paresis, reduced vision, circling, abnormal head position, bulging eyes and scoliosis. The disease occurred from September to January in regions with a considerable migrant reindeer population. It was concluded that the reported outbreaks of neurologic disease represented seasonal occurrence of cerebrospinal elaphostrongylosis caused by Elaphostrongylus rangiferi, the elaphostrongyloid nematode of reindeer (Rangifer tarandus tarandus).     

Differences in the seroprevalence of Salmonella spp. in free-ranging and corralled semi-domesticated reindeer (Rangifer tarandus tarandus) in Finland

Serum samples from 1032 semi-domesticated reindeer (Rangifer tarandus tarandus) from Finland were examined for the occurrence of Salmonella-antibodies by use of an indirect ELISA. The majority of samples originated from clinically healthy slaughter reindeer, kept extensively (n = 802; year of sampling: 1996). The remaining samples (n = 230) came from a research herd, permanently kept intensively, with repeated outbreaks of diarrhoea. In this study, 29 of the examined serum samples showed an OD above the determined cut-off. The prevalence in the clinically healthy slaughter reindeer was 0.9%, in the research herd 4.2% in 1996, 10.5% in 1997 and 12.9% in 1998. It must be assumed that the intensive husbandry in the corralled research herd may favour the spreading of infectious agents and eventually outbreaks of crowding diseases in the herd. This investigation is complemented by a review on the occurrence of Salmonella in wild and semi-domesticated cervids.     

A role for the Clostridium perfringens beta2 toxin in bovine enterotoxaemia?

Non-enterotoxigenic type A Clostridium perfringens are associated with bovine enterotoxaemia, but the alpha toxin is not regarded as responsible for the production of typical lesions of necrotic and haemorrhagic enteritis. The purpose of this study was to investigate the putative role of the more recently described beta2 toxin. Seven hundred and fourteen non-enterotoxigenic type A C. perfringens isolated from 133 calves with lesions of enterotoxaemia and high clostridial cell counts (study population) and 386 isolated from a control population of 87 calves were tested by a colony hybridisation assay for the beta2 toxin. Two hundred and eighteen (31%) C. perfringens isolated from 83 calves (62%) of the study population and 113 (29%) C. perfringens isolated from 51 calves (59%) of the control population tested positive with the beta2 probe. Pure and mixed cultures of four C. perfringens (one alpha+beta2+, one alpha+enterotoxin+ and two alpha+) were tested in the ligated loop assay in one calf. Macroscopic haemorrhages of the intestinal wall, necrosis and haemorrhages of the intestinal content, and microscopic lesions of necrosis and polymorphonuclear and mononuclear cell infiltration of the intestinal villi were more pronounced in loops inoculated with the alpha and beta2-toxigenic C. perfringens isolate. These results suggest in vivo synergistic role of the alpha and beta2 toxins in the production of necrotic and haemorrhagic lesions of the small intestine in cases of bovine enterotoxaemia. However, isolation of beta2-toxigenic C. perfringens does not confirm the clinical diagnosis of bovine enterotoxaemia and a clostridial cell counts must still be performed.     

Parelaphostrongylus tenuis in captive reindeer and sable antelope

Parelaphostrongylus tenuis caused neurologic disease in 6 reindeer (Rangifer tarandus tarandus) and 2 sable antelope (Hippotragus niger) that were housed at the National Zoological Park Conservation and Research Center in Front Royal, Va. Progressive hindlimb ataxia and weakness were seen in all affected animals. The parasite was recovered from the subdural space of one reindeer and was seen histologically within the neuropil of another reindeer and a sable antelope. In the other reindeer and sable antelope, diagnoses of parelaphostrongylosis were based on clinicoepidemiologic findings and microscopic lesions. Because parelaphostrongylosis is a density-dependent disease and because the number of white-tailed deer probably will increase, the incidence of P tenuis infections in domestic and nondomestic animals probably will increase as well. Zoos and wildlife parks especially should be cautious when introducing exotic hoofstock to areas with Parelaphostrongylus tenuis-infected white-tailed deer.     

Experimental infection of the deer ked (Lipoptena cervi) has no negative effects on the physiology of the captive reindeer (Rangifer tarandus tarandus)

The deer ked (Lipoptena cervi) is a haematophagous parasitic fly of cervids that spread to Finland in the early 1960's. Presently its northern distribution limit lies at approximately 65°N and it is gradually spreading northwards. In Finland the principal host species has been the moose (Alces alces), but the deer ked is about to establish contact with another potential host, the semi-domesticated reindeer (Rangifer tarandus tarandus) causing possible threats to reindeer health and management. The aim of this study was to investigate if the deer ked would have an influence on the welfare of the reindeer. Eighteen adult reindeer were divided into three experimental groups: the control group and two infected groups with 300 deer keds per reindeer introduced in August-September. One of the infected groups was treated with subcutaneous ivermectin in November. To gather comprehensive data on potential health hazards caused by the deer ked a wide array of physiological variables was measured during and at the end of the experiment in December. The keds caused no clear changes in the complete blood count, plasma clinical chemistry, amino acids, endocrinology, energy stores, enzyme activities or tissue fatty acid profiles of the host. The haematological, clinical chemical and endocrinological values displayed changes that could be related to the seasonal physiological adaptations of the species. In conclusion, at the duration and intensity of infection that were employed, the effects of the deer ked on the measured physiological variables of the reindeer were insignificant.     

Real-time multiplex PCR assays for reliable detection of Clostridium perfringens toxin genes in animal isolates

Typing of Clostridium perfringens strains by PCR-based determination of toxin genes proved to be a reliable method for diagnosis of enterotoxaemia in various animal species. We report the establishment and validation of three real-time fluorogenic (TaqMan) multiplex PCRs for the detection of C. perfringens alpha-, beta-, beta2-, epsilon-, entero- and iota-toxin genes. The composition of the PCRs was chosen with regard to robustness of the assays and in order to increase sensitivity compared to the conventional simplex PCRs. The combination of probe dyes selected for the real-time assays (FAM/TAMRA, Cy-5/BHQ-2 and VIC/TAMRA) as well as the designation of the chromosome-borne alpha-toxin as internal positive control allowed the creation of highly specific and sensitive, as well as time and cost effective PCRs. One hundred and three strains of C. perfringens isolated in Switzerland derived from clinical or suspected cases of enterotoxaemia in 10 different animal species were tested. The toxin genotypes were in agreement in both the conventional PCRs and the newly designed multiplex PCRs. Furthermore, the real-time PCR carried out as simplex allows to quantitate the copy numbers of plasmid-borne toxin genes in relation to the chromosomally located alpha-toxin gene.     

Gamma herpesvirus carrier status of captive artiodactyls

Between 1998 and 2000, 103 individuals of 19 species of the order Artiodactyla at Whipsnade Wild Animal Park were tested for evidence of infection with gamma herpesviruses in order to distinguish between species which are susceptible to malignant catarrhal fever (MCF), caused by alcelaphine herpesvirus-1 (AlHV-1) of wildebeest (Connochaetes sp.) or ovine herpesvirus-2 (OvHV-2) of domestic sheep, and species which carry related viruses sub-clinically. Gamma herpesvirus DNA was detected in the known, or suspected, carrier species: roan antelope (Hippotragus equinus), scimitar-horned oryx (Oryx dammah), gemsbok (Oryx gazella), musk ox (Ovibos muschatus) and mouflon (Ovis musimon). In six other species: lowland anoa (Bubalus depressicornis) yak (Bos grunniens), sitatunga (Tragelaphus spekei), greater kudu (Tragelaphus strepsiceros), waterbuck (Kobus ellipsiprymnus) and Nile lechwe (Kobus megaceros), DNA was present in some newborn calves and over 30% of adults, strongly suggesting a carrier state. In contrast five Père David's deer (Elaphurus davidianus) and two swamp deer (Cervus duvauceli) died of MCF during the study. A virus isolated from scimitar-horned oryx calves produced cytopathic effects in scimitar-horned oryx kidney cell-culture and caused MCF in a rabbit.     

A 12-month survey of gastrointestinal helminth infections of cervids kept in two zoos in Belgium.

Infections with helminths are a major health issue in captive and wild deer. In this study, fecal egg count patterns and clinical signs associated with gastrointestinal nematodes were assessed for 12 mo in nine cervid herds kept under different husbandry conditions at two sites. At site 1, an urban zoo, fecal egg counts remained low and no clinical signs of parasitic gastroenteritis were seen in the herds of fallow deer (Dama dama), Dybowski's deer (Cervus nippon dybowski), pudu (Pudu pudu), and reindeer (Rangifer tarandus tarandus). Helminth infection at this site may have been successfully prevented by daily dung removal of the small sandy-soil enclosures, and applying routine anthelmintic treatment was not justified. At site 2, a wild animal park, involved species were red deer (Cervus elaphus hippelaphus), Nelson's elk (Cervus elaphus nelsoni), Père David's deer (Elaphurus davidianus), European elk (Alces alces alces), and reindeer (Rangifer tarandus tarandus). Nematode eggs were frequently encountered in herds of red deer, Nelson's elk, and European elk, which were kept on larger, grassy enclosures that were irregularly cleaned. The trimodal pattern of fecal egg counts in herds from the wild animal park, consisting of a small spring rise in June, a peak in October, and a small rise in February, indicates that infective larvae on pastures are the main source of infection. In addition, routine anthelmintic treatment with fenbendazole in April and July limited egg shedding, but reinfection rapidly occurred. In two European elk and one reindeer, increasing fecal egg counts were associated with loss of fecal consistency and reduced appetite. Three genera and three species of nematodes were recovered at necropsy of one red deer and three Nelson's elk: Spiculopteragia spiculoptera, Trichostrongylus spp., Nematodirus filicollis, Capillaria spp., Oesophagostomum radiatum, and Trichuris spp., with total worm counts between 950 and 8,700.     

Necrotic enteritis in collared (Pecari tajacu) and white-lipped (Tayassu pecari) peccaries

An outbreak of necrotic enteritis caused by Clostridium perfringens type C was diagnosed in captive collared (Pecari tajacu) and white-lipped (Tayassu pecari) peccaries housed in the Laboratory of Applied Ethology of Universidade Estadual de Santa Cruz located in Ilhéus, State of Bahia, Brazil. Four collared peccaries and three white-lipped peccaries, all juveniles (25-105 days old), were affected. For all affected animals, lethargy and inappetance were followed by sudden death within 24 hours. Histopathology of intestinal wall, culture of C. perfringens type C, and the identification of beta-toxin from intestinal content confirmed the diagnosis.     

Bacterial intestinal flora associated with enterotoxaemia in Belgian Blue calves

The enterotoxaemia syndrome in Belgian Blue calves is characterised by a high case fatality rate, sudden death, lesions of haemorrhagic enteritis of the small intestine and, quite often an absence of other clinical signs but its cause has not been yet identified. As a first step in this identification, the aerobic and anaerobic intestinal flora of a population of 78 calves, originating from farms located in southern Belgium and that died in circumstances defined as "calf enterotoxaemia" (study population) and of 64 calves that died in other circumstances (control population) were studied qualitatively and quantitatively. The colonies were identified after subcultures with appropriate API sugar sets. Anaerobically Clostridium perfringens was isolated in higher numbers (mean values of 10(7)-10(7.5) colony forming units (CFU) versus 10(4)-10(5) CFU per ml of intestinal content) and from more animals (79 versus 19%) in the study population than in the control population, although individual results from both populations could overlap. Other clostridial species, i.e. mainly urease-negative C. sordellii and C. bifermentans, were isolated in high numbers (>10(6) CFU per ml of intestinal content) from a few animals in the study population only. All but one of the 705 C. perfringens isolates from both populations belonged to the A toxin type and none of the urease-negative C. sordellii was toxigenic. Gram-negative anaerobes were not isolated in high numbers from any of the samples. Aerobically beta-haemolytic E. coli were significantly more frequent among the study population, but were isolated from only 25% of the animals. Salmonella Typhimurium was isolated from only two animals in the study population. Less than 1% of the E. coli isolated were verotoxigenic and one-third were necrotoxigenic. At this stage only non-enterotoxigenic type A C. perfringens are thus statistically associated with the enterotoxaemia syndrome in Belgian Blue calves and fulfil the first of the Koch's postulates.     

Significance of beta 2-toxigenic clostridium perfringens infections in animals and their predisposing factors--a review

The novel beta 2-toxin of Clostridium perfringens has recently been described as the cause of enteric diseases in animals. The biological activity of beta 2-toxin is similar to that of the beta1-toxin with a possibly weaker cytotoxic activity. However, the production of beta 2-toxin in vitro is not seen in all beta 2-toxin-gene (cpb2)-positive C. perfringens strains, and to deduce a clinical importance solely from the detection of cpb2 is difficult. Detection of cpb2-positive C. perfringens from various animal species with and without enteric diseases demonstrates the wide distribution of cpb2 in nature, and the presence of cpb2 gene is therefore not considered a risk by itself. Predisposing factors like low trypsin activity in the intestinal tract, antibiotic and/or antiphlogistic treatment or changes in diet can result in the selection of beta 2-toxigenic C. perfringens which may lead to enteritis or enterotoxaemia.     

Echinococcus granulosus in Finland

Echinococcus granulosus is shown to occur in eastern Finland in a sylvatic cycle involving wolves (Canis lupus) as the definitive host and reindeer (Rangifer tarandus tarandus) and probably also elk (Alces alces) and the wild forest reindeer (Rangifer tarandus fennicus) as intermediate hosts. Even though the prevalence of E. granulosus in reindeer has increased in recent years, it is still very low (<0.013%). The results suggest, however, that the reindeer is a good sentinel animal to show the contamination of soil with Echinococcus eggs. This is the first report of wolves acting as a definitive host for E. granulosus in northern Europe. The parasite seems to be quite common in the Finnish wolf population, with a prevalence of approximately 30% in both intestinal and faecal samples. The present results and previous ones indicate that E. granulosus infection has not spread to dogs in the reindeer herding area.     

Rabies Antibody Titres in Vaccinated Reindeer

One dose of inactivated, adjuvanted rabies vaccine of cell culture origin (Rabisin) induced good but short-duration immunity in close to 100% of the 50 semi-domesticated reindeer (Rangifer tarandus tarandus L.) vaccinated. Most of the animals (44) had rabies virus antibody titre ≥1.5 IU/ml at 38 days after vaccination. Five animals had titre 0.5 IU. Antibody titres were not, however, present 1 year after primary vaccination in most animals. About 1 year (360-413 days) after primary vaccination, 22 of the 39 reindeer that could be sampled had rabies virus antibody titre <0.5 IU/ml.     

An SEM Study of the Morphology of the Lower Respiratory-tract Surface of the Reindeer (Rangifer tarandus tarandus L.)

The morphological features of the surface of the lower respiratory tract (trachea, bronchus, bronchiolus, distal airways, and alveoli) from 10 reindeer (Rangifer tarandus tarandus L.), differing in age and sex, were studied using scanning-electron microscopy. The respiratory surface of the reindeer generally resembles that reported previously in similar studies for other mammalian species. Ciliated epithelial cells, goblet cells, microvillous cells, Clara cells, alveolar epithelial cells of type 1 and type 2, and alveolar macrophages could be distinguished by their universally characteristic surface morphologies. The rarity of Kohn pores in the alveolar walls of reindeer was considered to be the most striking difference in comparison to most other species.