Potential application of serological tests on fluids from carcasses: detection of antibodies against Toxoplasma gondii and Sarcoptes scabiei in red foxes (Vulpes vulpes)

Background

Serological surveys for disease investigation of wild animal populations require obtaining blood samples for analysis, which has logistic, ethic and economic difficulties. Applying serological test to fluids collected from dead animals is an alternative. The aim of this study was to assess if antibodies could be detected in two types of fluids collected from 56 carcasses of red foxes (Vulpes vulpes): pleural fluid and lung extract.

Findings

In 22 (39%) foxes antibodies against Sarcoptes scabiei were detected in both fluid types by ELISA and Western blot. In 46 (82%) foxes, antibodies against Toxoplasma gondii were detected in pleural fluid and in 41 (73%) in lung extract applying a Toxo-screen test (DAT). Antibodies were still detectable in the same fluids kept at room temperature for 28 days, although in fewer foxes (16 and 14 foxes tested for T. gondii in lung extract and pleural fluid respectively; and 1 and 4 tested for S. scabiei in lung extract and pleural fluid respectively.

Conclusions

These results indicate the potential utility of using fluids from carcasses for antibody screening of wild animals at the population level.     

Seroprevalences of Toxoplasma gondii and Neospora sp. infections in Swedish horses

Sera from 414 Swedish horses were investigated for the presence of antibodies to Toxoplasma gondii and Neospora sp. by the T. gondii direct agglutination test (DAT), and an Neospora caninum iscom-ELISA. Five sera (1%) had a titre >1:40 in DAT, but when analysed by immunoblotting against T. gondii antigens only two of them were positive, giving a seroprevalence of 0.5%. Since the Neospora iscom ELISA had not been validated for equine sera it was used for an initial screening, and all sera with an optical density exceeding 0.200 absorbance units were selected for further investigation by immunoblot analysis. Of the 39 sera tested by immunoblotting, four reacted with at least two of the immunodominant Neospora antigens recognized by the positive control sera and were judged as positive, resulting in a seroprevalence of 1%. This is the first evidence of Neospora infection in Swedish horses. The study illustrates the necessity of critically evaluating results of serological analyses performed by methods that are not validated for the animal species under investigation.     

Seroprevalence of Neospora caninum in free living and farmed red deer (Cervus elaphus) in Poland

Serum samples from 47 free living and 106 farmed red deer (Cervus elaphus) from the Mazurian Lake District in north-east Poland were investigated for the presence of antibodies to Neospora caninum. A modified Neospora iscom-ELISA was used for initial screening. All sera with optical density (OD) values exceeding 0.400 absorbance units were further investigated by Western blot analysis. Eighteen sera were positive in both tests. Six of these were from free living and 12 from farmed animals giving prevalence of 13 and 11%, respectively. This is the first report of N. caninum infection in farmed and free-living red deer living in the same region where neosporosis was confirmed in cattle and the first evidence of exposure to the parasite in red deer in Poland.     

Isolation of Neospora caninum from a calf in Malaysia

In order to attempt isolate the protozoan parasite Neospora caninum, an N. caninum seropositive pregnant Sahiwal Friesian cross heifer from a large-scale dairy farm in Malaysia was kept for observation until parturition at the Veterinary Research Institute, Ipoh. The heifer gave birth to a female calf that was weak, underweight and unable to rise. Precolostral serum from the calf had an N. caninum indirect fluorescent antibody test titre of 1:3200. It died 12 h after birth and necropsy was performed. Brain homogenate from the calf was inoculated into 10 BALB/c mice that were kept for 3 months after which brain tissue from the mice was inoculated onto 24 h fresh monolayer Vero cell lines. The cell cultures were examined daily until growth of intracellular protozoa was observed. DNA of the organisms from the cell cultures was analyzed by PCR and DNA sequencing. DNA fragments of the expected size were amplified from the isolate using N. caninum-specific primers, and sequence analysis of ITS1 clearly identified the isolate as N. caninum. This is the first successful isolation of N. caninum from a bovine in Malaysia, and the isolate is designated Nc-MalB1.     

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.     

Persistence of Neospora caninum-specific immunoglobulin G antibodies in bovine blood and lung tissue stored at room temperature.

Blood and tissue samples obtained at necropsy from carcasses showing varying degrees of postmortem decomposition are sometimes used as sources of antibodies for serological tests, primarily in studies involving wild animals or in examination of aborted fetuses. To test the persistence of antibodies in blood and lung tissue kept at room temperature (approximately 20degreesC) for up to 48 and 118 days, respectively, serum and lung fluid from Neospora caninum-infected and uninfected cattle were tested using an indirect N. caninum immunostimulating complex enzyme-linked immunosorbent assay (ELISA). Testing of selected samples was also performed using a commercial Neospora competitive ELISA. Despite severe changes in the gross appearance of the samples, the blood collected through venipuncture from live animals remained essentially sterile, whereas the pieces of lung tissue showed a variety of bacterial and fungal growth. Nevertheless, there was no obvious reduction in antibody levels in either the blood or the tissue samples during the 2 observation periods. This study indicates that specific immunoglobulin G antibodies may be remarkably stable in sterile blood and decomposed lung tissue.     

Prevalence of antibodies against Toxoplasma gondii and Neospora caninum in Hungarian red foxes (Vulpes vulpes)

In the present study we have investigated the seroprevalence to the protozoan parasites Toxoplasma gondii and Neospora caninum in 337 red foxes (Vulpes vulpes) from 16 out of 19 counties in Hungary. The foxes were originally collected within a National vaccination program against rabies. Antibodies to T. gondii were detected in as many as 228 (68%) of the foxes using a commercial direct agglutination test (DAT). In an indirect iscom ELISA, five foxes (1.5%) were positive for antibodies against N. caninum. The high prevalence of foxes positive for T. gondii might be explained by the widespread occurrence of the parasite in the diet of foxes. As a contrast, latent infections of N. caninum among red foxes in Hungary are much less common.     

Seroprevalences of Toxoplasma gondii and Neospora caninum in Swedish red foxes (Vulpes vulpes).

The prevalences of antibodies to the protozoan parasites Toxoplasma gondii and Neospora caninum were investigated by the direct agglutination test (DAT) and ELISA, respectively, in 221 red foxes (Vulpes vulpes) from different parts of Sweden. A total of 84 (38%) of the analysed sera had antibodies to T. gondii, but none of the foxes had antibodies to N. caninum. The results indicate that T. gondii infection is fairly common in Swedish red foxes and that the infection is present in most parts of the country. They also show that N. caninum is not widespread as a latent infection among red foxes in Sweden.