Diagnosis of Sarcocystis cruzi, Neospora caninum, and Toxoplasma gondii infections in cattle

The aim of the study was to diagnose Sarcocystis sp. infections in cattle and to detect coinfections by Toxoplasma gondii and/or Neospora caninum. Blood, diaphragm, esophagus, and myocardium from 90 beef cattle from Argentina were collected. Histopathological, immunohistochemical, polymerase chain reaction assays, and direct microscopical examination were carried out. Sarcocysts from myocardium were measured and counted. Indirect fluorescent antibody test (IFAT) for the three protozoans was performed. Sarcocystis cruzi sarcocysts were found in 100% of myocardium samples. Sarcocysts per gram ranged from 8 to 380 with higher values found in adult cattle (p < 0.001). T. gondii and N. caninum were not detected by immunohistochemistry. T. gondii DNA was found in myocardium of 2/20 seropositive animals, while N. caninum DNA was not found. Antibodies against S. cruzi were detected in all samples, those against N. caninum in 73% and against T. gondii in 91% of the samples (IFAT titer ≥25). It is concluded that serology by IFAT is a suitable method to diagnose these protozoan infections due to its specific IgG detection; therefore, IFAT may be a useful tool to evaluate the impact of each protozoan infection in coinfected animals. Financial support for this study was provided by SeCyT through BID 1728 PICT No. 10858/8.     

Seroprevalence of Toxoplasma gondii and Neospora caninum in dogs from Korea

Toxoplasma gondii and Neospora caninum are closely related protozoan parasites, they share many common hosts, and can cause neurological diseases in dogs. Dogs can have close contacts with humans and livestock and therefore they can act as reservoirs of these parasites. The aim of this study was to survey the seroprevalence of antibodies against T. gondii and N. caninum and their co-infection rate in dogs in Korea. In total, sera from 553 domestic dogs were collected from different breeds, sexes, and ages of dogs from nine provinces across the country of Korea during 2006 and 2007. The presence of antibodies against T. gondii and N. caninum was analyzed using the latex agglutination test (LAT) with a cut-off value of 1:32, and the indirect fluorescent antibody test (IFAT) using a serum titer of 1:100. In the total dog population, 71 (12.8%) dogs were positive for anti-T. gondii antibodies and only 20 (3.6%) were positive for anti-N. caninum antibodies. Relatively higher seropositive frequencies of antibodies against T. gondii (20.1%) and N. caninum (4.9%) were detected in the dog population from the Gyeonggi. A higher proportion of animals seropositive for anti-T. gondii antibodies was found in stray dog populations as compared to household dog populations: 18.5% (59/319) vs 5.1% (12/234), respectively. The Chi-square tests revealed significant differences in the seropositive frequencies of antibodies against T. gondii between stray and household dogs in the total population (p<0.0001), and in dogs from the Gyeonggi (p<0.01). No significant differences were observed for the presence of antibodies against T. gondii or N. caninum when compared across the sex or age (p>0.05). The first serological survey on antibodies against both T. gondii and N. caninum parasites across the entire country showed that co-infection was not common in these canine populations with a seropositive level of 0.72%. The significantly higher positive frequency of T. gondii antibodies in stray dogs in both, Gyeonggi and in the total dog populations suggests that further investigation on the seroprevalence of parasites should focus on stray dogs.     

Clinical neosporosis in three dogs in Shahrekord, Iran

Three crossbreed male dogs were referred from the veterinary administration of Chaharmahal-Bakhtiari province in Iran with clinical signs of acute hind limbs ataxia and rigid hyperextension. Serum samples and cerebrospinal fluid were collected for serologic investigations. Evaluated samples had no detectable antibodies to Toxoplasma gondii, but all three dogs had high titers of IgG antibodies to Neospora caninum; two cases were sero-positive with titers of 1:3,200 and one with a titer of 1:6,400. Evaluated cerebrospinal fluid samples showed detectable anti-N. caninum IgG antibodies. The dogs were treated with clindamycin hydrochloride, and recovery was seen within 2 weeks.     

Gerbils (Meriones unguiculatus) are highly susceptible to oral infection with Neospora caninum oocysts

Laboratory-reared gerbils (Meriones unguiculatus) were found to be highly susceptible to oral infection with Neospora caninum (NC-Liv strain) oocysts. Gerbils fed ∼1000 oocysts became sick or died at 6–13 days post feeding of oocysts (PFO). N. caninum was isolated in cell culture and from γ-interferon-knockout mice inoculated with homogenates of mesenteric lymph nodes of gerbils examined as early as 1 day PFO. Numerous N. caninum tachyzoites were found in ulcerative lesions in the intestines of gerbils examined at 7–9 days PFO. In a gerbil fed 10 oocysts, N. caninum tachyzoites were found in lesions in the brain. Gerbils fed 10 oocysts developed antibodies to N. caninum by 18 days PFO as determined by the Neospora agglutination test (titers ≥1:500). All gerbils remained negative for antibodies to Toxoplasma gondii as determined by the Toxoplasma agglutination test. Received: 10 September 1999 / Accepted: 10 September 1999     

Serologic study of anti-Neospora caninum antibodies in household dogs and dogs living in dairy and beef cattle farms in Tehran, Iran

A few studies have been done on the seroepidemiology of anti-Neospora caninum antibodies in dairy and beef cattle farms in Iran, which suggested the presence of N. caninum in these areas, but there is no published information directed on the presence or epidemiology of this organism in the dogs in Iran. To investigate anti-N. caninum antibodies in household dogs and dogs living in cattle farms, 100 blood samples were collected: 50 from dogs living in dairy and beef cattle farms and 50 from household dogs. Serum samples were screened for detection of anti-N. caninum IgG antibodies using indirect fluorescent antibody test (IFAT; ≥50). Antibodies were seen in 10 (20%) of 50 household dogs and in 23 (46%) of 50 farm dogs. There were significant statistical differences in seropositivity between these two groups (P = 0.005). The IFAT antibody titers were as follows: 1:50 in seven dogs, 1:100 in eight dogs, 1:200 in six dogs, 1:400 in seven dogs, 1:800 in three dogs, 1:1,600 in one dog, and 1:12,800 in one dog. There were no significant differences in seropositivity between males and females. The positive results were increasing with age, and positive results were significantly different in the age group of older than 2 years compared to the dogs of age group under 1 year (P = 0.000) and 1–2 years (P = 0.007). The results confirm the exposure of household and farm dogs to N. caninum in Tehran and the higher rate of exposure for the dogs of dairy and cattle farms around Tehran.     

Evaluation of bovine abortion associated with <Emphasis Type="Italic">Neospora caninum</Emphasis> by different diagnostic techniques in Mashhad,Iran

Twelve aborted foetuses (gestational ranged from 4–9 months) and dams from dairies cattle farms in (Mashhad) Iran were analysed to investigate the participation of Neospora caninum in abortion. Diagnosis of the infection was determined by histopathology, serology (indirect fluorescent antibody test [IFAT]) and semi-nested polymerase chain reaction (PCR). A total 33% of bovine foetuses were considered to be infected by PCR technique. Microscopic lesions consistent with N. caninum infection in foetal brains were observed in 25% of the samples, whereas 33% were positive using IFAT (with a cut-off titre of 1:20). This study confirms the importance of N. caninum as an important cause of abortion in Iran.     

Transplacental transmission in cattle: is Toxoplasma gondii less potent than Neospora caninum?

We compared the transplacental-transmission ability of Toxoplasma gondii and Neospora caninum in cattle. One uninfected pregnant heifer served as control, while three were inoculated with N. caninum K9WA strain and four with T. gondii RH strain at their midgestational period. Both infected groups showed clinical signs and antibodies either to N. caninum or T. gondii, while the control animal was normal. Two (50%) Toxoplasma dams aborted on days 6 and 11 postinoculation. T. gondii tachyzoites were found in various organs of those dams that had abortions but not in their fetuses. Two Neospora dams did not abort but gave birth to subclinically infected calves. The remaining two Toxoplasma dams and one from Neospora group became recumbent. Those two dams and their fetuses showed disseminated Toxoplasma DNA, but no Neospora DNA was found. Our findings suggest that maternal toxoplasmosis could be a cause of abortion and congenital toxoplasmosis in cattle, especially when they are infected by virulent strains.     

Epidemiology of neosporosis in dairy cattle in Galicia (NW Spain)

This comprehensive study of neosporosis in dairy cattle in Galicia (NW Spain) included: (1) a comparative study of three serological techniques for detection of Neospora caninum antibodies (direct agglutination, enzyme-linked immunosorbent assay and indirect immunofluorescence); (2) a cross-sectional serological survey in which 276 herds and 5,196 animals were tested; (3) a study of N. caninum antibody dynamics; (4) the isolation of viable tachyzoites of N. caninum. Data were analysed to determine the risk factors associated with the infection. A total of 219 herds (79.3%) and 816 heads of cattle (15.7%) were found to be seropositive. Seropositivity was higher on farms with dogs than on farms without dogs, and there was a negative correlation between the size of the herds and seroprevalence. Co-infection with Toxoplasma gondii increased the risk of seropositivity. Cows infected with N. caninum were 5.3 times more likely to abort than non-infected cows. The dynamics study showed an increase in anti-N. caninum antibody titres during the third trimester of pregnancy. Viable tachyzoites were isolated from brain samples. These results indicate that the economic impact of N. caninum is high in Galicia, and therefore, the inclusion of control measures for neosporosis in the official control health programmes is strongly recommended.     

Transplacental transmission of Toxoplasma gondii in reinfected pregnant female canines

Twelve pregnant female canines, naturally infected with Toxoplasma gondii, were reinfected with T. gondii: three (GI) received tachyzoites subcutaneously (1.0 × 107), three (GII) were orally inoculated with oocysts (1.5 × 104), and six (GIII) were kept as a nonreinfected control group. All the reinfected female canines (GI and GII) miscarried or presented fetal death, while only one GIII female presented a stillborn in a litter of four pups (P < 0.01). Fever, lymphoadenopathy, miscarriage, and fetal death were the main clinical alterations observed. The highest serological titers detected through the indirect fluorescence antibody test (IFAT) were 1,024 (GI) and 4,096 (GII). In group III, the titers ranged between 64 and 256. By bioassays in mice, T. gondii was isolated in 17 organs of the reinfected adult canines, in 11 of the control group, and in 20 of the neonates. Positive immunostaining of cysts and/or tachyzoites were observed in 26 canine tissues (14 from GI and GII and ten from GIII). The agent was detected by immunohistochemistry in the encephalon of a neonate and in the spinal cord of a stillborn, thus, confirming that T. gondii infected canine fetuses, provoking miscarriages, even in bitches that presented primoinfection.     

Seroprevalence of Neospora caninum in cats from the Czech Republic

Sera of 414 cats coming from different parts of the Czech Republic were tested for N. caninum antibodies. Sera samples were collected during years 2002–2011. N. caninum antibodies were detected by a commercial competitive-inhibition enzyme-linked immunosorbent assay (cELISA) with cut off ≥30% inhibition. Samples positive in cELISA were confirmed by an indirect fluorescence antibody test (IFAT); titre ≥50 was considered positive. In total, 137 (33%) cats reacted positively in cELISA; N. caninum antibodies in IFAT were detected in 16 (3.86%) cats with titres 50 and 100. In 6 cats, positive for N. caninum antibodies, T. gondii antibodies were also detected by IFAT. It is the first report of N. caninum antibodies in domestic cats from the Czech Republic and third report in Europe.     

Investigation of Neospora caninum, Hammondia sp., and Toxoplasma gondii in tissues from slaughtered beef cattle in Bahia, Brazil

Neospora caninum, Hammondia sp., and Toxoplasma gondii are parasites with morphological and genetic similarities. N. caninum and T. gondii are important abortive agents of cattle and sheep, respectively, and may infect numerous animal species. Hammondia sp. is not known to induce disease in animals, but may cause confusion in the identification of closely related coccidia. The aim of this study was to investigate infection rates caused by N. caninum, Hammondia sp., and T. gondii in beef cattle using a nested PCR for Toxoplasmatinae rDNA, followed by sequencing of the PCR products. Antibodies to N. caninum and T. gondii were also investigated in the tested animals. Brains and hearts were obtained from 100 beef cattle in a slaughterhouse in Bahia. Seven samples from brain tested positive for Toxoplasmatinae DNA. No positive reactions were found in heart tissues. After sequencing of the PCR products from all positive tissues, five sequences matched with N. caninum and two matched with T. gondii. Antibodies to N. caninum and T. gondii were found in 20% and 26% of the animals, respectively. The confirmation of N. caninum and the absence of Hammondia heydorni in the tested animals is suggestive that cattle are not efficient intermediate hosts of H. heydorni; however further studies need to be performed using a greater variety of tissues and a higher sample size. The detection of T. gondii DNA in bovine tissues reinforces the potential risk of transmission of this parasite to humans and other animals through the consumption of bovine meat.     

Occurrence of antibodies anti-Neospora caninum, anti-Toxoplasma gondii, and anti-Leishmania chagasi in serum of dogs from Pará State, Amazon, Brazil

A cross-sectional study was conducted to determine the occurrence of anti-Toxoplasma gondii, anti-Neospora caninum, and anti- Leishmania chagasi antibodies in dogs of the state of Pará, Brazil. For this purpose, 129 blood samples were collected from dogs of different ages and gender. Samples of 72 dogs were collected from 39 rural properties from 19 municipalities, and 57 samples were from stray dogs, collected after captivity by the Center of Zoonosis Control from the municipality of Santarém. The sera were analyzed for anti-T. gondii and anti-N. caninum antibodies by indirect fluorescent antibody tests with cutoff values of 1:16 and 1:50, respectively. For the presence of L. chagasi antibodies, enzyme-linked immunosorbent assay was used and positive results were confirmed by immunochromatographic method using the recombinant antigen K39. Of the total of 129 dogs, 90 (69.8%) were positive for T. gondii, 16 (12.4%) for N. caninum, and 30 (23.3%) for L. chagasi. Antibodies for all three parasites were found simultaneously in seven dogs (5.4%), mostly in urban dogs (six of seven). No association was observed related to gender and location (urban or rural) of dogs and occurrence of N. caninum and T. gondii antibodies although, regarding L. chagasi, higher prevalence was found in females (P < 0.02) and in dogs from urban location (P < 0.001). From the 39 farms, in 30 (76.9%) at least one dog was positive for T. gondii or N. caninum or both. Higher occurrence of Leishmania antibodies was observed in N. caninum-negative dogs (P < 0.05).     

Immunoanalysis of three litters born to a Doberman bitch infected with <Emphasis Type="Italic">Neospora caninum</Emphasis>

Neospora caninum is a tissue cyst-forming coccidium that may cause neuromuscular disorders in dogs. Infected bitches can transmit the parasite to their pups in utero. Vertical transmission may occur after primary infection during pregnancy and in subsequent pregnancies. The reason why only a few pups develop clinical neosporosis is unknown. We obtained sera from a Doberman bitch and its offspring delivered in three litters. The bitch had a titer of 1:640 in an indirect fluorescent antibody test (IFAT). At least three pups of litter A, one pup of litter B, and two pups of litter C were also seropositive for N. caninum. However, clinical neosporosis developed only in one pup of litter C, which had the highest IFAT titer (1:5,120) of all dogs examined. Western blots carried out after one-dimensional and two-dimensional separation of N. caninum tachyzoites revealed that the largest number of antigens was recognized by sera derived from the bitch. The lowest number of antigens was recognized by serum from the pup with clinical neosporosis. However, this pup uniquely recognized a major antigen with a molecular weight of about 17,000. The information collected in this study adds to our knowledge on why some pups develop clinical neosporosis and others do not.     

Prevalence of antibodies to Neospora caninum in stray dogs of Urmia, Iran

To investigate anti-Neospora caninum antibodies in stray dogs living in Urmia city, 135 blood samples were collected. Serum samples were screened for detection of anti-N. caninum IgG antibodies using indirect fluorescent antibody test (IFAT; ≥50). Antibodies were seen in 36 (27%) of 135 dogs. The IFAT antibody titers were as follows: 1:50 in 16 dogs, 1:100 in ten dogs, 1:200 in six dogs, 1:400 in one dog, 1:800 in two dogs, and 1:1,600 in one dog. There were no significant differences in seroprevalence of Neospora infection between different genders (p > 0.05). The seropositive results were increased with age and the differences were statistically significant (p < 0.05). The results confirm the presence and exposure of stray dogs to N. caninum in Urmia city and the importance of this protozoan as a cause of disease in dogs of the region.     

The chloramphenicol acetyltransferase vector as a tool for stable tagging of Neospora caninum

Neospora caninum is an obligate intracellular Apicomplexa, a phylum where one of the current methods for functional studies relies on molecular genetic tools. For Toxoplasma gondii, the first method described, in 1993, was based on resistance against chloramphenicol. As in T. gondii, we developed a vector constituted of the chloramphenicol acetyltransferase gene (CAT) flanked by the N. caninum dihydrofolate reductase-thymidylate synthase (DHFR-TS) 5′ coding sequence flanking region. Five weeks after transfection and under the selection of chloramphenicol the expression of CAT increased compared to the wild type and the resistance was retained for more than one year. Between the stop codon of CAT and the 3′ UTR of DHFR, a Lac-Z gene controlled by the N. caninum tubulin 5′ coding sequence flanking region was ligated, resulting in a vector with a reporter gene (Ncdhfr-CAT/NcTub-tetO/Lac-Z). The stability was maintained through an episomal pattern for 14 months when the tachyzoites succumbed, which was an unexpected phenomenon compared to T. gondii. Stable parasites expressing the Lac-Z gene allowed the detection of tachyzoites after invasion by enzymatic reaction (CPRG) and were visualised macro- and microscopically by X-Gal precipitation and fluorescence. This work developed the first vector for stable expression of proteins based on chloramphenicol resistance and controlled exclusively by N. caninum promoters.     

Neospora caninum: Is it really different from Hammondia heydorni or is it a strain of Toxoplasmagondii ? An opinion

The published data concerning Toxoplasma gondii, Hammondia hammondi, H. heydorni and Neospora caninum on one side and between T. gondii on the other were neglected by most authors. As conclusion we are convinced that there are only two valid species: Isospora (Toxoplasma) gondii and Hammondia heydorni. The first includes as a strain H. hammondi and the latter N. caninum. In any case there is absolutely no reason (with respect to general Zoological nomenclature) to create new genera! Received: 20 October 1999 / Accepted: 22 October 1999     

Serological Evidence of Human Infection with the Protozoan Neospora caninum

Neospora caninum is a protozoan parasite that is closely related to Toxoplasma gondii. Dogs are a definitive host. Prior to its discovery in 1988, N. caninum infection in animals was often mistakenly diagnosed as toxoplasmosis. Neosporosis in animals is characterized by encephalitis, abortion, and other conditions that clinically and pathologically resemble toxoplasmosis. The potential of N. caninum to infect humans is unknown. Therefore, evidence of human exposure to this parasite was sought by screening for antibodies in blood donors by indirect fluorescent antibody (IFA) tests and immunoblotting. Of 1,029 samples screened, 69 (6.7%) had titers of 1:100 by IFA testing. Fifty of the 69 (72%) sera that were positive for N. caninum were also negative for a closely related protozoan pathogen of humans, T. gondii. Immunoblot analysis confirmed the specificity of the positive sera for N. caninum antigens, with several sera recognizing multiple Neospora antigens with molecular masses similar to those of antigens recognized by monkey anti-N. caninum serum. An immunodominant antigen of approximately 35 kDa was observed with 12 sera. These data provide evidence of human exposure to N. caninum, although the antibody titers in healthy donors were low. The significance of human exposure to, and possible infection with, this parasite is unknown and warrants further study.     

Characterization of a Swedish bovine isolate of Neospora caninum

The brain of a stillborn calf, seropositive to Neospora caninum and born to a seropositive cow, was homogenized and cultured on Vero cells, where growth of Neospora-like tachyzoites was detected after 8 weeks. The ultrastructural features of the new isolate (Nc-SweB1) corresponded to those of previously published Neospora isolates. In indirect immunofluorescence tests, antigens on Nc-SweB1 tachyzoites were recognized by antibodies raised to a canine N. caninum isolate (Nc-1) but not by antibodies to Toxoplasma gondii, Sarcocystis cruzi, S. tenella, Eimeria alabamensis, Babesia divergens, or B. motasi. Immunoblot analyses revealed no major antigenic difference between Nc-SweB1 and Nc-1, whereas several differences were seen between Nc-SweB1 and protozoa related to N. caninum. The sequences of 16S-like rRNA and the internal transcribed spacer 1 of Nc-SweB1 revealed complete homology with corresponding sequences of two canine N. caninum isolates. Thus, no dissimilarity between Nc-SweB1 and the canine isolates was found, confirming that Nc-SweB1 is N. caninum and suggesting that Neospora-like organisms isolated from cattle are indeed N. caninum.     

Prevalence of <Emphasis Type="Italic">Toxoplasma gondii</Emphasis> indirect fluorescent antibodies in naturally- and experimentally-infected chickens (<Emphasis Type="Italic">Gallus domesticus</Emphasis>) in Thailand

Toxoplasma gondii infections in free-range (FR) chickens (Gallus domesticus) are potential public health risks. Antibodies for T. gondii were found in 194 out of 303 serum samples (64.03%) from FR chickens in Thailand tested by the indirect fluorescent antibody test (IFAT, 1:16). To verify the validity of serologic data in this survey, sera from chickens experimentally infected with the RH strain of T. gondii were tested by the IFAT. Antibodies against T. gondii were detected as early as 7 days p.i., peaked at 2 weeks, and then declined by 10 weeks p.i.     

Seroepidemiology of Toxoplasma gondii, Neospora caninum, and Leishmania spp. infections and risk factors for cats from Brazil

The seroprevalence of infection by Toxoplasma gondii, Neospora caninum, and Leishmania spp. was detected through an indirect immunofluorescence in 70 cats from the Andradina Municipality, S?o Paulo State, Brazil. Anti-T. gondii antibodies (titer >64) were detected in 15.7% (11/70) of animals, whereas positivity for N. caninum (titer 16) was not observed in any animal. Of the cats from urban and rural areas, 10.4% (5/48) and 27.2% (6/22) were positive for T. gondii, respectively. Breed, age, food, and contact with animals of other species were significant for considering the positivity for T. gondii (P ≤ 0.0001). Cats having access to streets (17.1%, 11/64), cats cohabiting with rats (19.6%, 10/51), and cats feeding on homemade food and raw milk (27.2%, 6/22) were positive for T. gondii. In addition, 4.2% (3/70) of the cats were positive for Leishmania spp. by ELISA technique and negative by IFAT without coinfection with T. gondii and Leishmania spp. There was no serological positivity against feline immunodeficiency virus or feline leukemia virus. In conclusion, T. gondii infection in part of the feline population from Andradina is not linked to immunosuppressions or coinfections but probably to postnatal infection in association with the type of diet and presence of rats.