Serological investigation of aborted sheep and pigs for infection by Neospora caninum

Serology for Neospora caninum was undertaken using direct ELISAs on sera from 660 aborted sheep and 454 breeding sows, which had aborted or were considered infertile. All ovine sera were further tested by indirect fluorescent antibody test (IFAT) for N. caninum, and a latex agglutination test (LAT) for Toxoplasma gondii was performed on 423 of the samples, including all those positive by ELISA. ELISA-positive porcine sera were tested by IFAT and an inhibition ELISA for antibodies to N. caninum and by LAT for T. gondii. Only 3 (0.45%) of the ovine sera were seropositive for N. caninum by both ELISA and IFAT whereas although 40 porcine sera were seropositive by ELISA all were negative by IFAT. The results suggest that environmental exposure to N. caninum occurs rarely in sheep and pigs.     

The diagnosis of Neospora abortions in cattle

An indirect immunofluorescent antibody test (IFAT) and an enzyme-linked immunosorbent assay (ELISA) for specific anti-Neospora antibodies in bovine sera and foetal fluids were compared with histological examination results on aborted foetal material. The agreement between serological and histological examination results was poor, while the two serological tests showed a high degree of agreement. Serological testing of diagnostic serum samples and foetal fluids suggests that the prevalence of anti-Neospora antibodies in cattle which recently aborted is around 40%, in line with previous estimates of the number of abortions in dairy cattle caused by Neospora sp. A sero-epidemiological approach to the diagnosis of Neospora abortions in cattle may be suggested from these data.     

Prevalence of antibodies to Neospora caninum in Korean native beef cattle

A total of 438 sera from Korean native beef cattle in 9 provinces were tested for Neospora caninum antibodies using an immunofluorescent antibody test (IFAT). Eighteen (4.1%) cattle were positive by IFAT. The titers ranged from 1:200 (10 animals), 1:400 (5 animals), 1:800 (2 animals) to 1:1,600 (1 animal). Although the seroprevalence was slightly higher in Chungnam (8.9%), this was not significantly different from those noted in Kyunggi, Kangwon, Kyungbuk, Kyungnam, and Cheju provinces. Sera obtained from beef cattle in the provinces of Chungbuk, Jeonbuk and Jeonnam were all negative. Neospora positive sera were also tested for anti-Toxoplasma gondii antibodies using a commercial latex agglutination test (LAT). Antibody to T. gondii was detected in only 1 (5.6%) of 18 N. caninum positive sera. These results indicate that N. caninum and T. gondii infection are present at a low level in the Korean native beef cattle.     

Seroprevalence of Neospora caninum in dairy cattle in Bahia, Brazil.

Sera collected from 447 dairy cattle on 14 dairy farms were tested for Neospora caninum antibodies by use of an immunofluorescent antibody technique. Positive reactions with titres > or =1:200 were found in 63 (14.09%) of animals. Neospora positive sera were also tested for Toxoplasma gondii antibodies by using a commercial latex agglutination test. Antibodies to T. gondii were detected in 3 (4.76%) of 63 N. caninum positive sera. These results indicate that N. caninum infection is widespread among dairy cattle in Bahia state.     

Serologic immunoreactivity to Neospora caninum antigens in dogs determined by indirect immunofluorescence, western blotting and dot-ELISA

Neospora caninum, is a coccidian protozoan known as a major cause of bovine abortion and canine neuropathies. The aim of the present study was to develop a reliable and quick test to detect antibodies to N. caninum in dog sera. Sixty-five serum samples from dogs, including 35 positive and 30 negative for N. caninum antibodies were used for standardization of the test. In parallel, immunoreactivity of the sera to Toxoplasma gondii antigens was investigated using a passive agglutination test. A dot-ELISA test, using soluble extract of N. caninum tachyzoites on nitrocellulose ester membranes, was developed and standardized. SDS-PAGE and complementary analysis of reactivity by Western blotting were used for the characterization of the immunoreactive fractions of all tested sera. The sensitivity and specificity of the dot-ELISA were 94 and 73%, respectively, compared to IFAT at a cut-off of 1:50, and 87 and 100% compared to IFAT at a cut-off of 1:25. Among the sera that tested positively for both IFAT and dot-ELISA, only 8.6% were reactive to T. gondii. The most immunoreactive fractions in Western blots were the 14-, 33-, 42- and 55 kDa bands, with percentages of 42, 60, 42 and 37%, respectively. The 60 kDa band showed a non-specific reaction in 43% of neosporosis-negative animals by both dot-ELISA and IFAT. These results indicate that the dot-ELISA using N. caninum antigen present good sensitivity and specificity, and might be used as a screening test to detect antibodies to N. caninum in dogs.     

Seroprevalence of Neospora caninum in dogs in south-western Poland

The prevalence of anti-Neospora caninum antibodies was determined in serum samples from 110 dogs in south-western Poland, using the indirect fluorescent antibody test (IFAT). Antibodies to N. caninum were found in 18 dogs (16.36%), of which 7 were also positive to Toxoplasma gondii. The titres of anti-N. caninum antibodies varied from 1:50 to 1:200.     

Granulomatous meningoencephalitis due to toxoplasma gondii in a black-headed night monkey (Aotus nigriceps)

This report describes toxoplasmosis infection in a black-headed night monkey (Aotus nigriceps) from the Zoological Park of the Federal University of Mato Grosso, Brazil. The animal was submitted alive to the Veterinary Hospital of the Federal University of Mato Grosso presenting neurologic clinical signs. There were no improvements on the animal's clinical condition after treatment, so it was euthanized. At necropsy, the most significant gross lesions observed were hemorrhages diffusely scattered throughout the cerebral hemispheres. Histologic findings were characterized by nonsuppurative encephalitis and mild multifocal necrotizing nonsuppurative myocarditis. Groups of tachyzoites and cysts were observed surrounding the inflamed areas of cerebral parenchyma. Sections of the brain were submitted for immunohistochemistry examination for Toxoplasma gondii and Neospora caninum and also for polymerase chain reaction (PCR) to determine the presence of Toxoplasma gondii. The samples were positive for Toxoplasma gondii by PCR; however, cross reactivity was observed between Toxoplasma gondii and Neospora caninum antibodies. These findings were consistent with a toxoplasmosis infection in this animal.     

Detection of Nesopora caninum-Specific DNA from Cerebrospinal Fluid by Polymerase Chain Reaction in a Dog with Confirmed Neosporosis

A one-month male Greyhound dog presented with a swinging gait of the hindlimbs, and later developed muscular atrophy of the femoral region and hyperextension of hindlimbs. The dog had positive serum IFAT titers to Neospora caninum, but a negative titer in the cerebrospinal fluid (CSF). N. caninum-specific DNA was amplified from the CSF using a semi-nested polymerase chain reaction assay. Clusters of protozoa in biopsied muscle fibers were subsequently confirmed as N. caninum tachyzoites by immunohistochemical examination. Early recognition and treatment are necessary for effective recovery of clinical canine neosporosis, but antemortem diagnosis is difficult. We suggest that the detection of parasite deoxyribonucleic acid in the CSF is a useful antemortem diagnostic method in facilitating treatment of this disease.     

Activities of adenine nucleotide and nucleoside degradation enzymes in platelets of rats infected by Trypanosoma evansi

Pigeons (Columba livia) cohabit with humans in urban and rural areas, representing a public health problem since microorganisms are transmitted through the inhalation of dust from their dry feces (chlamydiosis) and through ingestion of their undercooked or poorly refrigerated meat (toxoplasmosis). This study aimed to evaluate the presence of Chlamydophila psittaci and Toxoplasma gondii in pigeons from four cities in São Paulo State, Brazil. C. psittaci was evaluated through hemi-nested polymerase chain reaction (hnPCR) using cloacal and tracheal swabs, whereas T. gondii specific antibodies were assessed by means of modified agglutination test (MAT), mouse brain and muscle bioassay, and polymerase chain reaction (PCR). To confirm the infection in mice, T. gondii antibodies were assayed by using indirect fluorescent antibody test (IFAT). Considering C. psittaci, 40/238 (16.8%; 95%CI 12.6–22.1%) samples were positive according to hnPCR, especially for the cities of São Paulo (42.5%) and Bauru (35%). As regards T. gondii, 12/238 (5%; 95%CI 2.9–8.6%) serum samples were positive according to MAT. Of these, five samples had titer equal to 1:8; six samples, 1:16; and one sample, 1:32. Bioassay, IFAT and PCR were negative for mouse toxoplasmosis. The absence of T. gondii antibodies suggests that pigeons may be infected with a low concentration of the agent, not detected by the antigen test. Thus, C. psittaci represents an actual problem concerning bird health.     

Transplacental toxoplasmosis in a reindeer (Rangifer tarandus) fetus

Toxoplasma gondii infection was diagnosed in a full term stillborn reindeer (Rangifer tarandus) fetus. The fetus had encephalitis and placentitis associated with T. gondii. Tissue cysts were identified histologically in sections of brain and tachyzoites were present in placenta and the myocardium. Protozoa in the brain, heart, and placenta stained positively with T. gondii antibodies, but not with Neospora caninum antibodies in an immunohistochemical test. The dam of the fetus had a 1:12,800 titer to T. gondii in the modified agglutination test employing whole tachyzoites and mercaptoethanol. This is the first confirmed report of T. gondii infection in reindeer.     

Serological survey and first finding of Neospora caninum in Taiwan, and the detection of its antibodies in various body fluids of cattle

A serological survey for antibodies against Neospora caninum in cattle, goats and farm dogs in Taiwan was carried out. Sera of 613 cattle from 25 dairy farms, 24 goats from six goat farms and 13 dogs from six dairy cattle farms were tested for antibodies against N. caninum using indirect fluorescence antibody test (IFAT). The same sera were also tested for antibodies against Toxoplasma gondii using latex agglutination test. Of the 613 cattle sera, 44.9% (275/613) were found to have antibodies against N. caninum. Among these 275 positive cattle, 77 also possessed antibodies against T. gondii. Nevertheless, 92 cattle which were negative for N. caninum showed antibodies against T. gondii. Of the 24 goat sera tested, none was found to be positive for N. caninum but 50% (12/24) were positive for T. gondii. Of the 13 farm dogs tested, three were found to possess antibodies against N. caninum, two of which tested negative for T. gondii antibodies. Besides sera, antibodies to N. caninum in cattle could be observed in the milk, vaginal secretion and saliva. However, the order of higher frequency of antibodies detection is in sera, milk, vaginal secretion and saliva. This is the first demonstration of the presence of antibodies to N. caninum in vaginal secretion and saliva of cattle. A 50microm cyst was observed in the brain of one of the 13 prednisolone-treated SPF ICR mice which had been peritoneally inoculated 4 months earlier with the brain homogenate of a serologically N. caninum positive but T. gondii negative cattle. Thus, we have confirmed for the first time the presence of N. caninum in Taiwan and also observed that it is widespread among dairy cattle and farm dogs.     

Acute toxoplasmosis in three wild arctic foxes (Alopex lagopus) from Svalbard; one with co-infections of Salmonella Enteritidis PT1 and Yersinia pseudotuberculosis serotype 2b

Acute disseminated toxoplasmosis was diagnosed in three wild arctic foxes (Alopex lagopus) that were found dead in the same locality on Svalbard (Norway). The animals included one adult female and two 4-months-old pups. The adult fox was severely jaundiced. Necropsy revealed multifocal, acute, necrotizing hepatitis, acute interstitial pneumonia, and scattered foci of brain gliosis, often associated with Toxoplasma tachyzoites. One pup also had Toxoplasma-associated meningitis. In addition, the latter animal was infected with Yersinia pseudotuberculosis serotype 2b and Salmonella Enteritidis phage type 1 (PT1), which may have contributed to the severity of the Toxoplasma infection in this animal. The diagnosis of toxoplasmosis was confirmed by positive immunohistochemistry and detection of anti-Toxoplasma gondii antibodies in serum of all foxes. The animals were negative for Neospora caninum, canine distemper virus, canine adenovirus, and rabies virus on immunolabelling of tissue sections and smears.     

Toxoplasmosis in Tammar wallabies (Macropus eugenii) in the Budapest Zoo and Botanical Garden (2006-2010)

Smaller macropodid species (commonly referred to as wallabies) are extremely susceptible to toxoplasmosis: in most cases, infection with Toxoplasma gondii leads to death within a short time. Between June 2006 and July 2010, T. gondii was detected by immunohistochemical examination in six Tammar wallabies (Macropus eugenii) that died in the Budapest Zoo and Botanical Garden; in another four specimens histopathology revealed T. gondii-like organisms (which could not be differentiated from Neospora caninum solely by morphology), and in another 11 animals toxoplasmosis as the possible cause of death could not be excluded. The current zoo population of 12 Tammar wallabies was tested for T. gondii IgG antibodies by the modified agglutination test (MAT), with negative results. We suppose that most of the deaths were due to acute toxoplasmosis resulting from a recent infection.     

Isolation and molecular characterisation of Neospora caninum in cattle in New Zealand

AIM: To isolate Neospora caninum from the brains of naturally infected cattle and use molecular techniques to characterise the isolates. METHODS: Neospora caninum tachyzoites were isolated in Vero cell culture from the brains of a cow and two calves. The isolates were characterised using polymerase chain reaction (PCR) methods, DNA sequencing, an immunofluorescent antibody test (IFAT), transmission electron microscopy (TEM), and immunohistochemistry (IHC). The brains of the three cattle were subjected to histopathological examination. A pathogenicity study was conducted in 120 BALB/c mice. RESULTS: Neospora caninum tachyzoites were isolated from all three cases and first observed in vitro between 14 and 17 days post-inoculation. Parasites were sub-cultured and maintained in Vero cell culture for more than 6 months. PCR products were generated for all three isolates, using two different primers. Sequencing of the PCR products and a subsequent BLAST search identified the isolates as N. caninum. In addition, the isolates tested positive using IFAT and IHC, and ultrastructure revealed by TEM was characteristic of N. caninum. Histopathological examination revealed lesions characteristic of N. caninum in 1/3 brains. In the pathogenicity study using BALB/c mice, the mortality rate was 3-7%. CONCLUSION: This was the first successful isolation of N. caninum in New Zealand confirmed using molecular characterisation tests.     

Serological investigation of an outbreak of Neospora caninum-associated abortion in a dairy herd in southeastern United States

An outbreak of Neospora caninum-associated abortion occurred in a South Carolina dairy wherein greater than 10% of the herd aborted over a 4-month period. Of the total number of cows at mid-late gestation, nearly 40% (28/71) aborted while the remaining 60% (43/71) gave birth to normal calves. Immunohistochemical examination of brain tissue from a subset of aborted fetuses confirmed N. caninum as the causative agent of abortion in these animals. A variety of serological assays, including indirect fluorescence antibody test (IFAT), recombinant enzyme-linked immunosorbent assay (rELISA), ISCOM-ELISA, avidity ELISA, and Neospora agglutination test (NAT), were used to evaluate sera collected during the outbreak from 240 cows for antibodies to N. caninum. IFAT and ISCOM-ELISA testing showed that nearly 80% of the dairy cows had antibodies to N. caninum. NAT and rELISA had similar levels of seropositivity relative to IFAT and ISCOM-ELISA, but the percentage of positive sera was dependent on the cut-off value chosen. As indicated by kappa coefficient statistical analysis, ISCOM-ELISA and IFAT exhibited the highest level of agreement in identifying N. caninum-positive and -negative cows. A decrease in the percentage of seropositive cows as determined by ISCOM-ELISA and IFAT with increasing cow age was noted. However, no significant difference was observed between cow age and abortion status. In addition to these tests, an avidity ELISA was performed on all sera with high (> or =0.4) ISCOM-ELISA readings. Avidity index (AI) increased with time post-abortion suggesting that most abortions were due to recent N. caninum infection. Of the cows at risk for abortion, the mean serological AI of aborting cows was significantly lower (P<0.05) than mean serological AI of non-aborting cows.     

Toxoplasmosis in Pallas' cats (Otocolobus felis manul) at the Denver Zoological Gardens.

In May 1996 the Denver Zoological Gardens obtained two male and two female Pallas' cats (Otocolobus felis manul) that were wild-caught in the Ukraine. These animals were part of a group of 16 wild-caught adults (eight male and eight female) imported to the United States and Canada between 1995 and 1996. The Denver Zoological Gardens cats were quarantined at the zoo hospital for approximately I mo. During the quarantine period they were immobilized for physical examination, and sera were obtained from them to evaluate for exposure to Toxoplasma gondii. All cats were positive for T. gondii antibodies by latex agglutination (titers from 1:512 to 1:1,024). After being paired for breeding, one pair produced two litters, and another pair produced four litters, a total of 17 kittens between 1997 and 2001. Four kittens and two young adults died from a disseminated granulomatous and necrotizing inflammation consistent with toxoplasmosis. Toxoplasma gondii infection was confirmed in all six deceased cats by polymerase chain reaction performed on formalin-fixed tissues. An additional five kittens disappeared and were not available for necropsy. The fatality rate from toxoplasmosis was 35.3% (6/17) for cats that were available for necropsy and could have been as high as 64.7% (11/17) if it were assumed that the disappeared kittens were also affected. The Pallas' kitten survival rate at the Denver Zoological Gardens was 35.3%. This article describes the clinical and pathologic features of toxoplasmosis in a group of Pallas' cats at the Denver Zoological Gardens.     

Serologic prevalence of Sarcocystis neurona, Toxoplasma gondii, and Neospora caninum in horses in Brazil.

OBJECTIVE: To determine serologic prevalence of Sarcocystis neurona, Toxoplasma gondii, and Neospora caninum in horses in Brazil. DESIGN: Prevalence survey. ANIMALS: 101 Thoroughbreds in Brazil. PROCEDURE: Blood samples were obtained from horses and tested for serum antibodies against S neurona by use of an immunoblot procedure with culture-derived S neurona merozoites as antigen, and for serum antibodies against T gondii and N caninum by use of a modified agglutination test with formalin-preserved tachyzoites and mercaptoethanol. RESULTS: Antibodies against S neurona and T gondii were detected in 36 and 16 of 101 horses, respectively. Cross-reactivity between antibodies against T gondii and S neurona was not detected. Antibodies against N caninum were not detected in any samples. CONCLUSIONS AND CLINICAL RELEVANCE: The high prevalence of antibodies against S neurona detected in clinically normal horses emphasizes the importance of examining CSF for antibodies when establishing a diagnosis of equine protozoal myeloencephalitis.     

Investigation for presence of Neospora caninum, Toxoplasma gondii and Brucella-species infection in killer whales (Orcinus orca) mass-stranded on the coast of Shiretoko, Hokkaido, Japan

Twelve killer whale (Orcinus orca) were hemmed in by ice floes, and nine died on the Aidomari coast in the Nemuro Strait in Rausu, Shiretoko, Hokkaido, Japan on 8 February 2005. Tissue samples collected from 8 whales were tested for Neospora caninum, Toxoplasma gondii, and Brucella species DNA by polymerase chain reaction (PCR) assay. Gamma-globulin isolated from blood samples by ammonium sulfate precipitation was tested for antibodies to these pathogens by means of agglutination tests and immunoblotting. None of the 8 tissue samples had antibodies to the pathogens, when subjected to agglutination tests. In immunoblotting, one sample (sample No.5) showed antibody binding to N. caninum antigens. In the PCR assay, none of the samples was positive. Further study is necessary to examine the prevalence of the pathogens in marine mammals inhabiting this area.     

Investigation of Neospora sp. antibodies in aborted mares from Normandy, France

Neospora caninum, an apicomplexan protozoan parasite, is recognized as a major cause of abortion in cattle while limited information is presently available on association between equine Neospora infections and abortions. The aim of the present study was to document prevalence of antibodies against Neospora sp. in aborted mares as a clue to the role of N. caninum in mare reproductive failure in Normandy, France. Using an agglutination test, the number of animals with elevated (>80) anti-Neospora sp. antibody titer was higher in a group of 54 aborted mares than in randomly chosen groups of 45 mares and 76 horses sampled for equine arteritis virus and Fasciola hepatica antibodies, respectively (P<0.001). N. caninum DNA was found in 3/91 fetal brains, 2/77 fetal hearts, and 1/1 placenta, and present in both brains and hearts of two fetuses. In 13 cases for which both mare serum and fetus were available, no fetal N. caninum amplification product was present while a large variation of maternal antibody titers was found. Data prompt at additional surveys of association between equine reproductive failure and Neospora sp. infection.     

Serodiagnosis of ovine toxoplasmosis: an assessment of the latex agglutination test and the value of IgM specific titres after experimental oocyst-induced infections

The antibody response of 20 pregnant ewes to oocyst infection with Toxoplasma gondii was determined by the latex agglutination test (LAT) and compared with the indirect fluorescent antibody test (IFAT) and a commercially available indirect haemagglutination test (IHAT). The LAT and IFAT showed a similar rapid response with antibody first appearing by two to three weeks after infection and titres that correlated closely (r = 0.81, P less than 0.001). The IHAT response was slower and less consistent up to seven weeks after infection. The LAT response was biphasic in seven of the sheep. Sera were fractionated using a minicolumn gel filtration technique and specific IgM and IgG titres determined by LAT. IgM titres peaked three weeks after infection and IgG titres exceeded IgM titres at a mean time of 4.7 weeks after infection (range 3 to 7). Eleven sheep exhibited fetopathy with abortion/parturition 12 to 53 days after infection; in nine of them IgG titres exceeded IgM at that time. A non-specific anti-toxoplasma reaction associated with IgM antibody occurred at low titre in one sheep. The results indicate that used from a dilution of 1/64 the LAT is a sensitive, reliable and rapidly responsive serological test for toxoplasma infection in ewes and it may be utilised with sample fractionation techniques to determine IgM titres. It is suggested that the best time to examine ewe sera to assist diagnosis of toxoplasma abortion is one week after abortion. While the determination of specific IgM titres in ewe sera may assist epidemiological studies and, sometimes, diagnosis, in the majority of aborting ewe sera it is unlikely to aid diagnosis.