Toxoplasma gondii and Neospora caninum in wildlife: common parasites in Belgian foxes and Cervidae?

Sera from Cervidae were tested for the presence of antibodies against Neospora caninum using ELISA; and against Toxoplasma gondii using SAG1-ELISA and a commercially available agglutination test. The T. gondii seroprevalence was 52% (38/73) in roe deer (Capreolus capreolus), 0% in bred fallow deer (0/4) (Dama dama) and red deer (0/7) (Cervus elaphus). We found 2.7% of the roe deer samples and none of the bred deer samples positive for N. caninum. Brain samples from wild roe deer, red deer and red foxes (Vulpes vulpes) were tested for the presence of T. gondii and N. caninum DNA using multiplex real-time PCR. We detected T. gondii in 18.8% (57/304) of the red foxes and in 1 of the 33 deer samples. N. caninum was found in 6.6% of the red foxes and in 2 roe deer samples. Twenty-six of the T. gondii positive DNA extracts from the red fox samples were genotyped. Twenty-five were type II and only one was found to be type III.     

Red foxes (Vulpes vulpes) are a natural intermediate host of Neospora caninum

The present study was undertaken to determine if red foxes are natural intermediate and/or definitive host for Neospora caninum and to study the importance of infection of N. caninum in this species in North-eastern Spain. Faecal samples and brain tissues were obtained from 122 foxes from 21 rural areas of Catalonia. Faeces collected were examined for parasite eggs and coccidian oocysts using sucrose flotation. For PCR-based diagnosis of N. caninum in brain tissues, the specific genomic Nc5 region was selected as the target sequence for DNA amplification. To control for PCR failure and facilitate identification of truly negative samples, the competitor pNc5C molecule was added to all negative samples in a second round of PCR reactions. Of the 122 foxes analysed, 13 (10.7%) were positive by PCR for N. caninum. Signal intensities of all positive samples were relatively weak with the exception of one sample from a 3-month male animal, that also showed the highest repeatability. No differences were observed by sex, age or area of sampling analysis. Detection of stages of N. caninum in brain from naturally infected red foxes demonstrated that red foxes are a natural intermediate host for N. caninum. Faecal samples were analysed for the presence of N. caninum oocysts, however, no oocysts compatible with N. caninum were found. A widespread latent infection of red foxes in North-eastern Spain found in the present study indicates that red foxes could have a very important role in the epidemiology of neosporosis in our area.     

Experimental infection of dogs (Canis familiaris) with sporulated oocysts of Neospora caninum

Neospora caninum is widely distributed in the world and this parasite is one of the major causes of abortion in cattle. Dogs and coyotes are definitive hosts of N. caninum and several species of domestic and wild animals are intermediate hosts. Dogs can become infected by the ingestion of tissues containing cysts and then excrete oocysts. It is not yet known whether sporulated oocysts are able to induce a patent infection in dogs, i.e. a shedding of N. caninum oocysts in feces. The objective of this study was to experimentally examine the infection of dogs by sporulated oocysts. The oocysts used in the experiment were obtained by feeding dogs with brain of buffaloes (Bubalus bubalis) positive for anti-N. caninum antibodies by indirect fluorescent antibody test (IFAT ≥200). Oocysts shed by these dogs were confirmed to be N. caninum by molecular methods and by bioassay in gerbils, and sporulated N. caninum oocysts were used for the oral infection of four dogs. The dogs were 8 weeks old and negative for antibodies to N. caninum and Toxoplasma gondii. Dogs 1 and 4 received an inoculum of 10,000 sporulated oocysts each; dog 2 an inoculum of 5000 sporulated oocysts and dog 3 received 1000 sporulated oocysts of N. caninum. The total feces excreted by these dogs were collected and examined daily for a period of 30 days. No oocysts were found in their feces. The dogs were monitored monthly for a 6-month period to observe a possible seroconversion and when this occurred the animals were eliminated from the experiment. Dogs 1 and 4 seroconverted 1 month after the infection with titer, in the IFAT, of 1600 and 800, respectively; the other two dogs presented no seroconvertion during the 6-month period. Dogs 1 and 2 were euthanized 180 days after infection and were examined for the detection of N. caninum in tissues (brain, muscle, lymph node, liver, lung, heart and bone marrow) by immunohistochemistry and PCR with negative results in both techniques. Bioassay in gerbils with brain of these dogs was also performed and again the results were negative. In conclusion, dogs infected with sporulated oocysts of N. caninum were not able to shed oocysts in feces. However, a higher dose of infection stimulated the production of antibodies against N. caninum in the dogs.     

In vitro activity of neem (Azadirachta indica) and cassava (Manihot esculenta) on three pre-parasitic stages of susceptible and resistant strains of Teladorsagia (Ostertagia) circumcincta

Canines are definitive hosts of Neospora caninum (Apicomplexa). For horizontal transmission from canines to occur, viable oocysts of N. caninum must occur in the environment of susceptible intermediate hosts. Canids in Australia include wild dogs and Aboriginal community dogs. Wild dogs are those dogs that are not dependent on humans for survival and consist of the dingo, feral domestic dog and their hybrid genotypes. Aboriginal community dogs are dependent on humans, domesticated and owned by a family, but are free-roaming and have free access throughout the community. In this study the extent of N. caninum infection was determined in a total of 374 dogs (75 wild dogs and 299 Aboriginal community dogs) using a combination of microscopic, molecular and serological techniques. Oocysts of N. caninum were observed in the faeces of two juvenile Aboriginal community dogs (2/132; 1.5%). To estimate N. caninum prevalence, a new optimised cut-off of 18.5% inhibition for a commercial competitive ELISA was calculated using a two-graph receiver-operating characteristic (TG-ROC) analysis and IFAT as the gold standard resulting in equal sensitivity and specificity of 67.8%. Of the 263 dog sera tested the true prevalence of N. caninum antibodies was 27.0% (95% confidence limit: 10.3-44.1%). The association between the competitive ELISA results in dogs less than 12 month old and older dogs was significant (P=0.042). To our knowledge this is the first large scale parasitological survey of the Aboriginal community dogs and wild dogs from Australia. The high prevalence of N. caninum infection in Aboriginal community dogs illustrates that horizontal transmission of N. caninum is occurring in Australia. These results demonstrated that N. caninum in dogs is widespread, including the semi-arid to arid regions of north-western New South Wales and the Northern Territory. The populations of free-ranging dogs are likely to be important contributors to the sylvatic life cycle of N. caninum.     

Prevalence of antibodies against Toxoplasma gondii and Neospora caninum in moose (Alces alces) and roe deer (Capreolus capreolus) in Sweden

Toxoplasma gondii and Neospora caninum are two coccidian parasites with a worldwide distribution. T. gondii is one of the more common parasitic zoonoses in the world and in young children and immunocompromised persons, infection can lead to severe disease and death. N. caninum is an important cause of abortions in cattle. Wildlife have been identified as reservoirs and transmitters for both parasites. The purpose of this study was to investigate the seroprevalences of T. gondii, and N. caninum in moose (Alces alces), and roe deer (Capreolus capreolus) in Sweden. Blood samples were collected from 417 moose during 2000-2005 and from 199 roe deer during 1990-2007. The samples were investigated for presence of antibodies by a T. gondii direct agglutination test and a N. caninum iscom ELISA. Because the iscom ELISA has not been validated for moose or roe deer, sera that gave a positive result were further investigated by immunoblot analysis to verify presence of antibodies. Antibodies to T. gondii were detected in 85 (20%) and 68 (34%) moose and roe deer sera, respectively. In moose the seroprevalence was higher in south and central Sweden than in the north, whereas there was no difference between the regions for roe deer. Adult moose and roe deer had higher odds of being seropositive than young animals but there were no difference in seroprevalence between males and females. One roe deer was positive by immunoblotting and was regarded as N. caninum positive, whereas all moose sera were negative. The results show that T. gondii infection is widely spread in the Swedish moose and roe deer populations. Precautions should therefore be taken when handling internal organs and carcasses of harvested cervids. Proper handling and cooking of game meat also is important to prevent toxoplasmosis in humans.     

Seroprevalence of Neospora caninum in non-carnivorous wildlife from Spain

Serum samples from 1034 non-carnivorous wildlife from Spain were tested for antibodies to Neospora caninum by competitive screening enzyme linked immunosorbent assay (ELISA) and confirmed by an indirect fluorescent antibody test (IFAT). High agreement was observed between results in both techniques (kappa value higher than 0.9). Prevalences of N. caninum antibodies positive by both techniques were 11.8% of 237 red deer (Cervus elaphus), 7.7% of 13 barbary sheep (Ammotragus lervia), 6.1% of 33 roe deer (Capreolus capreolus) and 0.3% of 298 wild boar (Sus scrofa). In one of 53 hares (Lepus granatensis), antibodies were found in the ELISA but could not be confirmed by IFAT due to lack of sample. Antibodies to N. caninum were not found in any of 251 wild rabbits (Oryctolagus cuniculus), 79 fallow deer (Dama dama), 27 mouflon (Ovis ammon), 40 chamois (Rupicapra pyrenaica) and three Spanish ibex (Capra pyrenaica). Statistically significant differences were observed between N. caninum seroprevalence in red deer and management of hunting estates (open versus fenced) with higher prevalence in fenced estates, and among sampling sites. Seroprevalence was particularly high in some areas (MO estate in South-Central Spain or some estates of Catalonia, North-East Spain), while no contact with N. caninum was observed in others. Results indicate that in certain areas of Spain, N. caninum is present in wildlife, especially in red deer. These results have important implications in both sylvatic cycles and may influence the prevalence of infection in cattle farms in those areas. To our knowledge, this is the first report of antibodies to N. caninum in wildlife from Spain and the first report of N. caninum antibodies in barbary sheep and wild boar.     

Incidence of Neospora caninum and other intestinal protozoan parasites in populations of Swiss dogs

The protozoan parasite Neospora caninum is one of the most important abortifacient organisms in cattle worldwide. The dog is known to act as definitive host although its potential role as infection source for bovines still remains unelucidated. The aim of the present study was to compile initial epidemiological data on the prevalence and incidence of N. caninum in Swiss dogs acting as definitive hosts. Thus, 249 Swiss dogs were investigated coproscopically in monthly intervals over a period of 1 year. A total of 3289 fecal samples was tested by the flotation technique. Among these, 202 were shown to contain Sarcocystis sp. (6.1%), 149 Cystoisospora sp. (=Isospora sp.; 4.5%) and 25 Hammondia/Neospora-like oocysts (HNlO) (0.7%). All but one sample containing HNlO were from different dogs; one dog shed HNlO at two subsequent time points. Calculation of the yearly incidence for HNlO resulted in the surprisingly high value of 9.2%. Farm dogs exhibited a higher incidence for HNlO than urban family dogs. Thirteen out of the 25 HNlO-samples showed sporulation after 5 days incubation at room temperature. HNlO were further differentiated by species-specific PCR. However, all HNlO-samples were negative for N. caninum, Hammondia heydorni and Toxoplasma gondii. One reason may be the low oocyst density found in most fecal samples, which did not permit us to carry out PCR under optimal conditions. Three out of the 25 HNlO-cases contained enough oocysts to allow further enrichment and purification by the flotation technique. Subsequently, twenty to fifty sporulated HNlO-oocysts were orally administered to Meriones unguiculatus. All gerbils were seronegative for N. caninum at 5 weeks p.i. A N. caninum-seroprevalence of 7.8% was determined by ELISA upon 1132 serum samples collected from dogs randomly selected by veterinarians among their clinical patients.     

The role of the dog in the epidemiology of neosporosis in cattle

Neospora caninum is an intracellular protozoan parasite that was discovered in a dog in 1988. Since then, N. caninum has been demonstrated in a variety of animal species and it has been recognized as an important cause of abortion in cattle. An infection with N. caninum can be maintained in cattle herds for several generations by transplacental transmission from cow to calf. Recently, it was demonstrated that dogs can act as definitive hosts of N. caninum and therefore may be a source of infection for other species by shedding oocysts. Further evidence of a role of the dog in spreading the infection to cattle has been derived from epidemiological studies. The present state of knowledge is reviewed in this paper.     

Prevalence of antibodies to Neospora caninum and Toxoplasma gondii in gray foxes (Urocyon cinereoargenteus) from South Carolina

Little is known about the epidemiology of Neospora caninum in wild mammal populations. It has been suggested that a sylvatic cycle exists for N. caninum. Dogs and potentially other canids are a definitive host for N. caninum. The present study was done to determine the prevalence of antibodies to N. caninum in a population of gray foxes (Urocyon cinereoargenteus) from a nonagricultural setting in South Carolina. We also determined the prevalence of antibodies to Toxoplasma gondii in these animals. Antibody levels were measured in direct agglutination tests using either N. caninum or T. gondii formalin-fixed tachyzoites as antigen. Four (15.4%) of the 26 gray foxes had titers to N. caninum. Titers to N. caninum were low being 1:25 in three gray foxes and 1:50 in the fourth gray fox. Antibodies to T. gondii were observed in 16 (61.5%) gray foxes. Titers to T. gondii were usually >1:50 and two gray foxes had titers of 1:1600. Results of this study indicate that gray foxes have more exposure to T. gondii than to N. caninum in this environment.     

Spatial associations among density of cattle, abundance of wild canids, and seroprevalence to Neospora caninum in a population of beef calves

OBJECTIVE: To determine the epidemiologic plausibility of a sylvatic transmission cycle for Neospora caninum between wild canids and beef cattle. DESIGN: Spatial analysis study. ANIMALS: 1,009 weaned beef steers from 94 beef herds in Texas. PROCEDURE: Calves were grouped on the basis of seroprevalence for N caninum and ecologic region in Texas. The Morans I test was used to evaluate spatial interdependence for adjusted seroprevalence by ecologic region. Cattle density (Number of cattle/259 km2 [Number of cattle/100 mile2] of each ecologic region) and abundance indices for gray foxes and coyotes (Number of animals/161 spotlight-transect [census] km [Number of animals/100 census miles] of each ecologic region) were used as covariates in spatial regression models, with adjusted seroprevalence as the outcome variable. A geographic information system (GIS) that used similar covariate information for each county was used to validate spatial regression models. Results-Spatial interdependence was not detected for ecologic regions. Three spatial regression models were tested. Each model contained a variable for cattle density for the ecologic regions. Results for the 3 models revealed that seroprevalence was associated with cattle density and abundances of gray foxes, coyotes, or both. Abundances of gray foxes and coyotes were collinear. Results of a GIS-generated model validated these spatial models. CONCLUSIONS AND CLINICAL RELEVANCE: In Texas, beef cattle are at increased risk of exposure to N caninum as a result of the abundance of wild canids and the density of beef cattle. It is plausible that a sylvatic transmission cycle for neosporosis exists.     

Recent advances in understanding the epidemiology of Neospora caninum in cattle

INTRODUCTION: Neospora caninum is the most frequently diagnosed cause of cattle abortion in New Zealand and is an important pathogen worldwide. The parasite has been found in aborted bovine foetuses and in puppies with neurological disease. Recent developments have provided new insights into the epidemiology of the disease that are reviewed in this article. TRANSMISSION: Transplacental infection is of major importance in the spread of N. caninum overseas. Most congenital infections result in the birth of apparently healthy calves. Seroepidemiological studies indicate introduced point-source infections are also likely, as do investigations of abortion outbreaks in cattle herds attributed to N. caninum. Horizontal transmission is an important determinant of the stability of infection prevalence in a herd. Two potential sources of horizontal transmission that have recently been reported are: via a canine definitive host and; cow-to-calf transmission through pooled colostrum or milk. Transmission parameters for the organism have been estimated using mathematical modeling, to explore the epidemiology and options for control of N. caninum infection in dairy cattle in the absence of precise epidemiological data. LIFE-CYCLE: Seroepidemiologic studies, combined with the knowledge that dogs can be a definitive host, provide evidence supporting a dog-cattle life-cycle. The importance of dogs in the epidemiology of bovine neosporosis is not yet clear. It is likely that N. caninum oocysts in dog faeces could serve as a source of infection for cattle and recent studies have shown that the canine and bovine isolatesflare the same organism. Infection of cattle orally exposed to oocysts has been demonstrated experimentally but not in nature. It is not known if other wild carnivores may serve as definitive hosts of N. caninum. CONCLUSION: Despite active research for over a decade, very little is known about the mechanisms of transmission of N. caninum. Vertical transmission is well documented but horizontal spread and the pathogenesis of abortion from this disease need further investigation. It is evident that, even with a very high probability of vertical transmission, some form of horizontal transmission is required for the disease to persist in a herd.     

Failure of dogs to shed oocysts after being fed bovine fetuses naturally infected by Neospora caninum

Neospora caninum is a protozoan that causes abortion in cattle. The dog has recently been identified as a definitive host for N. caninum. To verify if bovine fetuses can infect dogs, nine 2-4-month-old dogs were fed bovine fetuses naturally infected by N. caninum. None of the dogs excreted oocysts, seroconverted, had clinical signs or lesions compatible with N. caninum infection. Additional studies will be necessary to determine the natural mode of infection of dogs by N. caninum.     

Shedding of Neospora caninum oocysts by dogs fed different tissues from naturally infected cattle

Neospora caninum is one of the most important causes of abortion in dairy cattle worldwide. The distribution of N. caninum in tissues of adult cattle is unknown and the parasite has not been demonstrated histologically in tissues of cows. In the present study the distribution of N. caninum in different tissues of adult cattle was evaluated by bioassays in dogs. Seventeen dogs (2-3 month-old) were fed different tissues of 4 naturally exposed adult cattle (indirect fluorescent antibody test N. caninum titer ≥ 400): 5 were fed with masseter; 5 with heart, 3 with liver, 4 with brain, and 3 pups were used as non-infected control. Two dogs fed masseter, 2 fed heart, 1 fed liver, and 3 fed brain shed oocysts, and all dogs presented no seroconvertion to N. caninum during the observation period of 4 weeks. The oocysts were confirmed as N. caninum based on the detection of N. caninum-specific DNA by PCR and sequencing. The results indicate that dogs can be infected by N. caninum with different tissues of infected cattle.     

Prevalence of antibodies to Neospora caninum in dogs from Amazon,Brazil

Neospora caninum is an important cause of abortion in dairy cattle worldwide. Dogs are important in the epidemiology of this parasite because they are the only hosts known to excrete N. caninum oocysts. Antibodies to N. caninum were assayed in serum samples from 157 dogs from Monte Negro, Rond?nia, Amazon, Brazil using the indirect fluorescent antibody test. Antibodies to N. caninum were found in 13 (8.3%) of dogs in titers of 1:50 in 1, 1:100 in 2, 1:200 in 5, 1:800 in 1, 1:1600 in 2, and 1:3200 in 2 dogs. These data indicate that N. caninum infection is prevalent even in remote areas of the Amazon.     

Neospora caninum antibodies in wild carnivores from Spain

Serum samples from 251 wild carnivores from different regions of Spain were tested for antibodies to Neospora caninum by the commercial competitive screening enzyme linked immunosorbent assay (c-ELISA) and confirmed by Neospora agglutination test (NAT) and/or by indirect fluorescent antibody test (IFAT). Samples with antibodies detected by at least two serological tests were considered seropositive. Antibodies to N. caninum were found in 3.2% of 95 red foxes (Vulpes vulpes); in 21.4% of 28 wolves (Canis lupus); in 12.0% of 25 Iberian lynx (Lynx pardinus); in 16.7% of 6 European wildcats (Felis silvestris); in 6.4% of 31 Eurasian badgers (Meles meles); in 21.4% of 14 stone martens (Martes foina); in 66.7% of 3 pine martens (M. martes) and in 50% of 2 polecats (Mustela putorius). Antibodies to N. caninum in common genets (Genetta genetta) and Egyptian mongooses (Herpestes ichneumon) were only observed by c-ELISA but were not confirmed by IFAT and/or NAT. No antibodies were detected in 5 Eurasian otters (Lutra lutra) by any technique. Statistically significant differences were observed among species and among geographical areas. The highest seroprevalence of N. caninum infection was observed in the Cantabric Coastal region characterized by high humidity. To our knowledge, this is the first report of antibodies to N. caninum in free ranging wild carnivores, other than wild canids, in Europe. The existence of a possible sylvatic cycle could have important implications in both sylvatic and domestic cycles since they might influence the prevalence of infection in cattle farms in those areas.     

Potential existence of a sylvatic cycle of Taenia ovis krabbei in Patagonia, Argentina

Red deer (Cervus elaphus) were introduced in southern Latin America about a century ago and characteristics of the invasion raise concerns over their epidemiological role for various diseases. We report on the possible occurrence of Taenia ovis krabbei established in a sylvatic cycle in Patagonia. Hook characters, size, appearance, and location of a cysticercus from a wild red deer are consistent with Taenia ovis ovis or T. o. krabbei. Although it is not possible to differentiate between T. o. ovis and T. o. krabbei on morphological grounds with certainty, several biological characteristics indicate the cysticercus may belong to T. o. krabbei. Red deer have been reported to be refractory to T. o. ovis infection whereas other potential intermediate hosts like cattle, goats, pigs and sheep have been shown to be refractory to T. o. krabbei. Other native ungulates sympatric with red deer in Patagonia include Lama guanicoe and the endangered huemul deer (Hippocamelus bisulcus). Possible or known definitive hosts include native felids like Puma concolor, Felis colocolo, F. guigna and canids like Dusicyon griseus, D. culpaeus, and domestic dogs.     

Seroprevalence of Neospora caninum and Toxoplasma gondii in black-tailed deer (Odocoileus hemionus columbianus) and mule deer (Odocoileus hemionus hemionus)

Deer are considered important intermediate hosts for the coccidian parasites, Toxoplasma gondii and Neospora caninum. Antibodies to N. caninum and T. gondii were determined in sera of 42 mule deer (Odocoileus hemionus hemionus) and 43 black-tailed deer (Odocoileus hemionus columbianus) from Washington state, USA, using direct agglutination test with specific antigens. A titer of 1:25 was considered diagnostic for both parasites. N. caninum antibodies were found in 7 of 42 mule deer and 8 of 43 black-tailed deer. T. gondii antibodies were found in 14 black-tailed deer but not in any of the mule deer. This is probably the first report of seroprevalence of N. caninum in these hosts.     

Detection of specific antibodies to Neospora caninum and Toxoplasma gondii in naturally infected alpacas (Lama pacos), llamas (Lama glama) and vicunas (Lama vicugna) from Peru and Germany

Sera of an experimentally Neospora caninum infected llama and a non-infected control llama were used to establish an immunoblot, an ELISA and an IFAT to detect antibodies against N. caninum tachyzoites. Subsequently, serum samples collected from a total of 871 South American Camelids (SAC: Lama glama, Lama pacos, Lama vicugna) of two farms in Peru and from 32 SAC of a farm in central Germany were examined for antibodies against N. caninum and Toxoplasma gondii. Based on the recognition of specific bands in the immunoblot, sera of SAC from Peru were differentiated into N. caninum-positive (n = 18) and T. gondii-positive (n = 30) samples and into samples negative or inconclusive for both parasites. Using the immunoblot results as the reference, a modified version of the p38-ELISA and the IFAT were evaluated for detecting N. caninum antibodies in SAC sera. Applying a cut-off as determined by two graph-receiver operating characteristic analysis both, the ELISA and the IFAT, exhibited a sensitivity and specificity of about 95% in the SAC sera from Peru. Serological testing confirmed that SAC may become infected with N. caninum under field conditions in Peru. In addition to alpacas and llamas also 114 wild living vicunas had been examined for antibodies against N. caninum. However, only the alpacas and llamas but no vicunas were found N. caninum-positive. In contrast, T. gondii-seropositive animals were detected in all three SAC species. The lack of N. caninum-seropositive vicunas indicates that in the study area in Peru wild canids might not serve as definitive hosts of N. caninum while for T. gondii a life cycle including wild felids is likely. On the German farm no N. caninum- but only T. gondii-seropositive SAC (n = 14) were detected. The seroprevalence of T. gondii infection was significantly higher in adult SAC (alpacas in Peru, llamas in Germany) than in crias (i.e. < 12 months old foals) indicating that the predominant route of infection is post natal. Since the present study was restricted to a few farms, the seroprevalences determined are not representative. However, our results confirm natural infections with N. caninum and T. gondii in SAC. Whether these infections are linked to any disease, e.g. reproductive losses, has to be clarified in further studies.     

Gray wolf (Canis lupus) is a natural definitive host for Neospora caninum

The gray wolf (Canis lupus) was found to be a new natural definitive host for Neospora caninum. Neospora-like oocysts were found microscopically in the feces of three of 73 wolves from Minnesota examined at necropsy. N. caninum-specific DNA was amplified from the oocysts of all three wolves. Oocysts from one wolf were infective for the gamma interferon gene knock out (KO) mice. Viable N. caninum (designated NcWolfUS1) was isolated in cell cultures seeded with tissue homogenate from the infected mouse. Typical thick walled tissue cysts were found in outbred mice inoculated with the parasite from the KO mouse. Tissue stages in mice stained positively with N. caninum-specific polyclonal antibodies. Our observation suggests that wolves may be an important link in the sylvatic cycle of N. caninum.     

Mycobacterium avium subsp. paratuberculosis in wild animal species and cattle in Styria/Austria

Infections with Mycobacterium ovium ssp. paratuberculosis (M. paratuberculosis) are increasingly recognised worldwide. In addition to an increased prevalence of paratuberculosis in Austrian cattle herds, recent years have also shown a rise in infections with M. paratuberculosis in wild red and roe deer, chamois and mouflon. During the period from June 2002 to September 2004, mesenteric lymph nodes were taken from a total of 483 wild animals hunted or found dead and from 338 deceased cattle. Samples were analysed using PCR and cultivation methods. In the case of pathomorphological changes or anamnestic indications, investigations also included an analysis of organ samples (e.g. liver, lung) or foetuses. The tests revealed that 129 wild animal samples (red deer, roe deer, chamois, mouflon, fallow deer, ibex, foxes, mountain hare, yellow-necked field mouse, and capercaillie) contained M. paratuberculosis. The major symptoms in the wild aninodes. Evidence of diarrhoea was only observed in about 15% of the positive cases. The study for the first time provided evidence of intrauterine transmission of M. paratuberculosis in red deer (3 cases) and chamois (1 case) and succeeded in the isolation of the pathogen from the liver, lung and subcutaneous granulomas of wild animals. Of the total of 338 mesenteric lymphnodes of cattle from 303 herds, 80 samples from 77 herds tested positive for paratuberculosis. Twenty-two wild animal and 3 cattle isolates have so far been molecularly typed using IS900-RFLP and RAPD analyses in order to prove epidemiological relationships between occurrences in cattle and wild animals. The increase of paratuberculosis in wild animal species is assumed to have been caused by the purchase of animals, a strong increase in suckler cow farming (cow-calf herds) with a concentration of pathogens in the environment and by inadequate feed hygiene for wild animals.