Neospora Caninum DNA Research Articles

A holstein heifer infected with Neospora caninum NcUru3 congenitally transmits this strain to a viable offspring although infection does not protect her from aborting by a different N. caninum genotype in the subsequent gestation.

Neospora caninum is a leading cause of bovine abortion worldwide. Although the genetic diversity of this apicomplexan parasite has long been recognized, there is little information on whether infection with different genotypes results in different clinical outcomes or whether infection by a given genotype impairs protective immunity against abortion induced by different genotypes. Here, we provide evidence supporting that natural subclinical infection with isolate NcUru3 of N. caninum in a pregnant heifer did not provide protection against abortion caused by a different N. caninum genotype in the subsequent gestation. A Holstein heifer delivered a healthy calf congenitally infected with N. caninum. Specific anti-N. caninum IgG was detected by indirect ELISA in sera obtained from the dam at calving and the calf before ingestion of colostrum, indicating in utero exposure to the parasite in the latter. A N. caninum strain named NcUru3 was isolated and characterized by multilocus microsatellite typing from the brain of this neonate euthanized at 9 days of age. Sixty days after calving, the cow got pregnant, although she aborted spontaneously at ~6 months of gestation. Pathologic examination of the aborted fetus and placenta revealed typical lesions of neosporosis, including encephalitis, myocarditis, hepatitis, myositis, and placentitis. Neospora caninum DNA was amplified from the fetal brain, heart, kidney, and placenta, and multilocus microsatellite typing revealed a genotype that differed from isolate NcUru3 at the level of microsatellite marker 6A (MS6A). Serum obtained from the dam at the time of abortion had IgG that cross-recognized isolate NcUru3, as demonstrated by immunoblotting, indicating that the humoral immune response did not prevent the other genotype from infecting the fetus and inducing fetoplacental lesions and abortion. This is the first description of one same dam transmitting two N. caninum genotypes to her offspring in subsequent gestations.

A survey on Apicomplexa protozoa in sheep slaughtered for human consumption

Infections with the Apicomplexa Toxoplasma gondii, Neospora caninum, and Sarcocystis spp. are common causes of reproductive disorders in sheep. However, few epidemiological studies regarding co-infections with these three protozoa are reported in sheep in Italy. For this reason, this study aims to evaluate possible co-infections with T. gondii, N. caninum, and Sarcocystis spp. in sheep slaughtered for human consumption. From April to July 2019, individual blood, brain, heart, and diaphragm samples were collected from 138 sheep after slaughtering. The presence of IgG anti-Toxoplasma in serum samples was evaluated through ELISA. DNA of the three protozoa was investigated using specific PCRs. Co-infection with T. gondii, N. caninum and Sarcocystis spp. was found in 66.7% of the examined sheep. Antibodies against T. gondii were found in the 36.2% of serum samples. The presence of T. gondii DNA was detected in the 67.4%, 77.5%, and 21.7% of the brain, heart, and diaphragm samples, respectively. Neospora caninum DNA was found in 72.5% of the examined brain samples. Sarcocystis spp. DNA was detected in 92% and 52.2% of the heart and diaphragm samples, respectively. Sequence analysis of the Sarcocystis spp. revealed the sole presence of Sarcocystis tenella. The present study demonstrates that sheep have a high risk of infection with the three Apicomplexa investigated, suggesting the need to adopt adequate measures to prevent the spread of these parasitic infections considering their clinical and economic impact on ovine production. Furthermore, the possible role sheep play in the zoonotic transmission of toxoplasmosis to humans was highlighted.

Apicomplexan Protozoa Responsible for Reproductive Disorders: Occurrence of DNA in Blood and Milk of Donkeys (Equus asinus) and Minireview of the Related Literature.

Donkeys may be susceptible to many pathological agents and may act as carriers of pathogens for other animal species and humans. This study evaluated the occurrence of potentially abortifacient apicomplexan protozoa DNA in blood and milk samples collected at different time periods during lactation (1, 6, and 10 months) from 33 healthy dairy jennies. A total of 73 blood and 73 milk samples were used for DNA extraction and analysis. Blood specimens from 11/33 (33%) jennies scored positive for Theileria equi, while milk samples scored negative. Blood and milk of 3/33 jennies yielded DNA of Toxoplasma gondii at 6 months (n. 1) and 10 months (n. 2) after parturition. Neospora caninum DNA was found in four milk and in five blood samples only at one month after parturition. This study is the first report about the presence of N. caninum DNA in milk of naturally infected jennies. Moreover, the excretion of N. caninum DNA in some of these jennies at 30 days from the parturition may suggest a possible occurrence of an endogenous cycle, while the presence of T. gondii DNA in the milk collected at 6 and 10 months after parturition may be suggestive of a discontinuous excretion.

Molecular Identification of Neospora caninum Infection in Aborted Fetuses of Sheep, Cattle, and Goats in Mazandaran Province, Northern Iran.

Background:We aimed to identify Neospora caninum DNA in the brain samples of aborted fetuses of cattle, goats, and sheep in Mazandaran, northern Iran, using PCR.Methods:In total, 133 aborted fetuses (51 sheep, 78 cattle, and 4 goats) were randomly collected from different stages of gestation in various regions of Mazandaran, Iran, from Mar 2016 to May 2017. The DNA was extracted from all the brain samples using phenol chloroform isoamyl alcohol instructions. The Nc-5 gene was used for the detection of N. caninum DNA by nested-PCR assay.Results:The detection of N. caninum DNA was confirmed by the observation of a 227 bp band in 24 samples of 133 aborted fetuses (18.1%). The highest prevalence rate of N. caninum was detected in the cattle (20.5%) followed by the sheep (15.6%); however, no positive cases were reported in the goats. The highest and lowest prevalence rates of the infection were reported as 23.8% and 8.6% in Qaemshahr, and Behshahr, respectively. The prevalence rate of infection (32%) in the early gestational period was higher than those in the middle (15%) and late (3.8%) gestational periods.Conclusion:The obtained data of the present study indicated that N. caninum infection may partly be responsible for abortion and economic loss in livestock farming in Mazandaran Province.

Neospora caninum DNA distribution in tissues of gerbils as experimental models of chronic neosporosis

ABSTRACT Neospora caninum is the main etiologic agent of neosporosis in domestic animals and its pathogenesis comprises two characteristic phases: acute and chronic. Rodents are used as experimental models to mimic acute and chronic bovine neosporosis. In this study, we inoculated a total of 27 female gerbils, with different doses of N. caninum tachyzoites aiming to induce chronic disease. DNA was extracted from different organs of each animal after spontaneous death or euthanasia. Encephalic tissues were submitted to a highly sensitive real time PCR aiming to detect chronically infected animals. All the other samples were submitted to standard PCR. A total of 11 gerbils died due to acute neosporosis, as confirmed by N. caninum DNA detection in organs. 5x103 tachyzoites/mL of N. caninum was the dosage of antigen that can induce chronic infection in gerbils. In the encephalon sections of some animals that showed clinical signs of persistent infection, we found 70% positive for the anterior encephalon section, suggesting this area as preferential for cyst formation. Therefore, we determined the doses of tachyzoites that cause acute or chronic infection and detection of positive tissues, preferably, systemic organs during acute and encephalon in chronic phases.

Molecular and histopathological identification of ovine neosporosis (Neospora caninum) in aborted ewes in Iraq.

Aim:The objective of the present study was to detect Neospora caninum DNA in the placenta of sheep and evaluate the association of risk factors to polymerase chain reaction (PCR) positive and histopathological analysis of the placenta and fetal tissue samples of aborted fetuses.Materials and Methods:Fresh placenta from 51 aborted ewes was collected for PCR assay. Placental and fetal tissues of aborted fetuses, including brain, heart, liver, lung, and thymus, were collected for histopathological analysis, besides the risk factor data were obtained during the time of sampling.Results:From 51 placentas examined by PCR, 13.73% appeared positive to N. caninum DNA. The relationship between PCR positive and the risk factors revealed a significant difference (p<0.05) in age of the dam, fetal age, feed source, water source, and the presence of other animals at farm, whereas the type of birth, stillbirth, and size of flock showed insignificant difference (p>0.05). Histopathological investigation of placental and fetal tissues of positive samples showed tissue cyst-like structure, necrotic foci, and infiltration of mononuclear cells. Other lesions were thickening in chorionic plate in placenta, severe vacuolization and death of neurons, microgliosis, demyelination, edema, and proliferation of astrocytes in brain. In addition, fibrous and fat deposition with stenosis in the heart, parenchymal necrosis, severe atrophy, vacuolization and hyalinization of hepatocytes, megakaryocyte, portal fibrosis in the liver, and interlobular septal thickening in lung without obvious lesions is seen in the thymus tissue samples.Conclusion:This is a unique study that confirmed N. caninum DNA in the placenta of aborted ewes in Iraq using PCR assay. Histopathological analysis of some aborted fetuses organs could provide a more confirmatory and reliable data for a significant role of neosporosis in increasing the rate of abortion in sheep, while the clinical data of risk factors could be used to control the transmission of N. caninum infection.

Detection of Neospora Caninum DNA in Wild Birds from Italy.

The role of avian species in the Neospora caninum life cycle has not been completely elucidated, and epidemiological data from Europe are scant. The aim of the present report was to evaluate the presence of N. caninum DNA in the tissues of 302 birds belonging to different avian species, along with IFAT titers. Forty-two out of the 302 birds (13.9%) showed low serological titers (1/50 and 1/100) against N. caninum. These data, positive for 31 animals (10.3%), were corroborated by PCR. Twenty-two hearts and eighteen brains scored positive, while nine subjects resulted had parasite DNA both in their hearts and brains. Serological data showed significantly higher results in waterfowl in respect to non-waterfowl avian species. This finding indicates a higher exposure of water birds to the parasite. These avian species, in fact, which feed directly from soil and/or water, are prone to ingest oocysts excreted by final canid hosts. The present study adds information to the state of art of N. caninum epidemiology in Italy, even if more investigations using bio-assays are needed to allow for a serological/parasitological follow-up to evaluate the real impact of the avian species in maintaining the parasite in main reservoirs.

Neospora caninum DNA in feces of crab-eating fox (Cerdocyon thous – Linnaeus, 1776) from northeastern Brazil

Neospora caninum is an obligate intracellular tissue cyst-forming coccidian parasite and it was first described in dogs. Despite the relevance of wild canids in the transmission chain of N. caninum, there are few studies in Brazil. The aim of the present study was to detect N. caninum DNA in feces of free-range and captive crab-eating fox (Cerdocyon thous) from different states of northeastern Brazil. Fecal samples of eighteen crab-eating foxes (fifteen individually and three pools) were collected in sterile containers and were kept cool at −20 °C until further processing. All fecal samples were subjected to DNA extraction. A nested PCR targeting the ITS-1 gene was performed for N. caninum. All the positive bands were extracted from the gel and purified. Forward and reverse strands were sequenced and the nucleotide sequences obtained were compared with N. caninum sequences deposited in Genbank. The alignment was edited and phylogenetic reconstruction was based on the ITS1 gene sequences. Thirteen stool samples were PCR-positive for N. caninum DNA. Nine out of thirteen positive samples showed similarity between 99%–100% for N. caninum in relation to the sequence U25044.1 stored at GenBank. The crab-eating fox could have an important role in the sylvatic cycle of Neospora caninum in Brazil. Experimental infections studies involving these wild canids may confirm if the crab-eating foxes are definitive hosts of N. caninum.

Toxoplasma gondii and Neospora caninum infections in synanthropic rodents from Argentina.

Toxoplasma gondii and Neospora caninum are closely related coccidian parasites (phylum Apicomplexa). This is the first study from urban synanthropic rodent species that involved serological and molecular diagnosis of T. gondii and N. caninum infection, and genotyping of T. gondii in Argentina. A total of 127 rodent samples were trap captured: Mus musculus (n = 78), Rattus norvegicus (n = 26) and Rattus rattus (n = 23). Antibodies against T. gondii and N. caninum were detected by IFAT in 32.8% (40/122) and 0.8% (1/122) of rodent samples, respectively, demonstrating contact with these protozoans. Additionally, T. gondii DNA was detected in 3.3% (4/123) of rodent central nervous system samples and 2 samples were genotyped by multilocus nPCR-RFLP. Neospora caninum DNA was not detected by PCR. The 2 genotyped samples were type III allele for all markers except for SAG-1 (type I for Rat1Arg and type II/III for Rat2Arg) and were identified as #48 and #2 (likely) according to the allele combinations reported on Toxo DB (Toxo-DB). The results of the present study revealed a wide distribution of T. gondii and less for N. caninum, in synanthropic rats and mice in the studied area.

Neospora caninum DNA in Coyote Fecal Samples Collected in an Urban Environment.

Definitive hosts of Neospora caninum are species of canids, such as domestic dogs ( Canis lupus familiaris), coyotes ( Canis latrans) and foxes ( Vulpes vulpes), whereas ruminants, such as cattle ( Bos taurus), sheep ( Ovis aries), and deer (Cervidae) serve as intermediate hosts. We investigated the presence of N. caninum in feces of coyotes in Calgary, Alberta, Canada. One hundred coyote fecal samples were collected from five city parks. Following DNA extraction, the presence of N. caninum DNA was determined by using real-time PCR analysis with a primer and probe pair targeting the Nc5 gene. Ten of the 100 samples contained detectable amounts of N. caninum DNA. White-tailed deer ( Odocoileus virginianus) and mule deer ( Odocoileus hemionus) are common in Calgary's city parks, and although we did not examine them for the presence of N. caninum bradyzoites, the presence of N. caninum DNA in coyote fecal samples is indicative that a sylvatic host of N. caninum exists within this urban environment.

Microsatellite pattern analysis of Neospora caninum from a naturally infected goat fetus

Neospora caninum is an apicomplexan protozoan responsible for abortion in ruminants. The present study aimed to diagnose an abortion from an Anglo Nubian goat from a dairy herd located in Buenos Aires province, Argentina. The goat delivered a fetus of approximately 3 months gestation that was studied by indirect fluorescent antibody test (IFAT), Immunoblot (IB), histopathology (HP), immunohistochemistry (IHC), and molecular assays (PCR, sequencing and microsatellite genotyping). Interferon gamma knock-out mice were inoculated with a pool of tissues for isolation attempts. The mother had IFAT titers of 1:3200 and 1:400 for N. caninum and Toxoplasma gondii, respectively, as well as positive IB reactions, whereas the fetus was seronegative to both parasites by IFAT and IB. The fetus had severe multifocal necrotizing myocarditis and hepatitis, moderate interstitial pneumonia, and nephritis. Myocardium sample resulted positive by IHC, evidencing clusters of N. caninum tachyzoites within myocardiocytes associated with histopathological lesions. Neospora caninum-DNA was detected by PCR in heart, liver, lungs, kidney, and muscle from the fetus, and was negative for T. gondii by PCR. NC-5 and 18 S rRNA gene fragment sequences showed 100% identity with N. caninum. Inoculated mice bled 30 days post-inoculation resulted seronegative to N. caninum and T. gondii by IFAT, and showed no clinical signs. Multilocus-microsatellite genotyping revealed a genetic profile that differed from previously reported N. caninum genotypes, with unique MS21 and MS10 alleles. These findings indicate that N. caninum was efficiently transmitted from the mother to the fetus. We report the first case of direct detection of N. caninum in a goat fetus in Argentina and N. caninum microsatellite genotyping in naturally infected goat.

A novel loop-mediated isothermal amplification-based test for detecting Neospora caninum DNA

BackgroundNeospora caninum is a cyst-forming, coccidian parasite which is known to cause neurological disorders in dogs and abortion and neonatal mortality in cows and other livestock. This study reports the development of a loop-mediated isothermal amplification (LAMP) assay based on the Neospora caninum Nc-5 gene and compares its efficacy for detecting DNA to that of a semi-nested PCR test.ResultsSix primers were designed based on the Nc-5 repeat region of N. caninum. Specific LAMP primers led to successful amplification of N. caninum DNA at 63 °C in 30 min. The LAMP assay was highly specific (i.e. it did not reveal cross-reactivity with other parasite species) and had a low N. caninum plasmid DNA limit of detection (1 fg), which is ten times higher than that for the semi-nested PCR. LAMP applicability was evaluated using a set of naturally-infected samples (59 from canine faeces and five from bovine abortions). Thirty-nine percent (25/64) of the naturally-infected samples were positive for N. caninum DNA by LAMP and 36% (23/64) by semi-nested PCR. However, the LAMP assay is much faster to perform than semi-nested PCR and provides results in 30 min.ConclusionThe optimized reaction conditions described in this study resulted in a sensitive, specific and rapid technique for detecting N. caninum DNA. Considering the advantages of LAMP for detecting N. caninum DNA, further assays aimed at testing its usefulness on a wider range of field samples are recommended.

Foetal death in naive heifers inoculated with Neospora caninum isolate Nc-Spain7 at 110 days of pregnancy

Neospora caninum infection is a leading cause of abortion in cattle worldwide. The pathogenesis of bovine neosporosis, particularly during the second term of gestation when most abortions occur in naturally infected dams, is poorly understood. In the present study foetal death was observed in 3 of 6 experimentally infected dams at 110 days of gestation after 6 weeks of experimental period. All experimental heifers were febrile between 3 and 5 days post infection (dpi). Inoculated dams seroconverted by 3–4 weeks post-infection with higher mean antibody titres in aborting dams compared to non-aborting heifers, although not significantly (p > 0.05). Neospora caninum DNA was detected in all infected foetuses and placentas, and three infected foetuses also had N. caninum antibodies. The parasite burden was higher in the brain of dead/aborted foetuses than in live foetuses. Interestingly, high IFN-γ production was detected in foetal fluids of a dead foetus found upon euthanasia of its dam, while no IFN-γ was observed in amniotic, allantoic and/or foetal fluids in the three infected foetuses that were alive upon maternal euthanasia. The present study confirms that the infection of dams on gestation day 110 with 107 tachyzoites of the Nc-Spain7 isolate causes abortion. The fact that some infected dams aborted and some did not is relevant to the understanding of N. caninum pathogenesis of abortion in naturally infected cows.

Identification of latent neosporosis in sheep in Tehran, Iran by polymerase chain reaction using primers specific for the Nc-5 gene.

Little is known about latent infection and molecular characterisation of Neospora caninum in sheep (Ovis aries). In this study, 330 sheep samples (180 hearts and 150 brains) were analysed for N. caninum DNA by nested polymerase chain reaction (PCR) targeting the Nc-5 gene. Neospora caninum DNA was detected in 3.9% (13/330) of sheep samples. The parasite’s DNA was detected in 6.7% of heart samples (12/180) and 0.7% (1/150) of brain samples. No clinical signs were recorded from infected or uninfected animals. Sequencing of the genomic DNA revealed 96% – 99% similarity with each other and 95.15% – 100% similarity with N. caninum sequences deposited in GenBank. To our knowledge, this is the first report on the use of PCR to identify latent neosporosis in sheep in Iran. The results of this study have the potential to contribute to our understanding of the role of N. caninum-infected sheep in the epidemiology of neosporosis.

Endoparasite survey of free-swimming baleen whales (Balaenoptera musculus, B. physalus, B. borealis) and sperm whales (Physeter macrocephalus) using non/minimally invasive methods.

A number of parasitic diseases have gained importance as neozoan opportunistic infections in the marine environment. Here, we report on the gastrointestinal endoparasite fauna of three baleen whale species and one toothed whale: blue (Balaenoptera musculus), fin (Balaenoptera physalus), and sei whales (Balaenoptera borealis) and sperm whales (Physeter macrocephalus) from the Azores Islands, Portugal. In total, 17 individual whale fecal samples [n = 10 (B. physalus); n = 4 (P. macrocephalus); n = 2 (B. musculus); n = 1 (B. borealis)] were collected from free-swimming animals as part of ongoing studies on behavioral ecology. Furthermore, skin biopsies were collected from sperm whales (n = 5) using minimally invasive biopsy darting and tested for the presence of Toxoplasma gondii, Neospora caninum, and Besnoitia besnoiti DNA via PCR. Overall, more than ten taxa were detected in whale fecal samples. Within protozoan parasites, Entamoeba spp. occurred most frequently (64.7%), followed by Giardia spp. (17.6%) and Balantidium spp. (5.9%). The most prevalent metazoan parasites were Ascaridida indet. spp. (41.2%), followed by trematodes (17.7%), acanthocephalan spp., strongyles (11.8%), Diphyllobotrium spp. (5.9%), and spirurids (5.9%). Helminths were mainly found in sperm whales, while enteric protozoan parasites were exclusively detected in baleen whales, which might be related to dietary differences. No T. gondii, N. caninum, or B. besnoiti DNA was detected in any skin sample. This is the first record on Giardia and Balantidium infections in large baleen whales.

Impact of spontaneous Neospora caninum infection on pregnancy loss and subsequent pregnancy in grazing lactating dairy cows

The impact of spontaneous Neospora caninum infection on pregnancy loss and subsequent pregnancy in grazing lactating dairy cows was evaluated. Data from 1273 females (878 multiparous and 395 first-calving cows) from six preselected dairy herds were analyzed. Cows were classified as seropositive (SP) (prevalence, 24%; range, 11%–33%) or seronegative (SN) by indirect immunofluorescence detection of antibodies against N caninum. Seropostive cows (prevalence, 40.0%) presented higher (P < 0.001) incidence of abortion compared with SN cows (prevalence, 4.1%). Neospora caninum DNA was detected by real-time polymerase chain reaction in 44.4% of intact aborted fetuses from SP cows, whereas none was found in those aborted from SN cows. The average daily milk production adjusted to 305 days was lower (P < 0.001) in SP (22.5 ± 0.3 L/day) than in SN cows (24.8 ± 0.2 L/day). Furthermore, SP cows presented greater occurrence of retained placenta (17.1% vs. 6.0%; P < 0.001) and acute postpartum metritis (9.8% vs. 2.4%; P < 0.001). Despite similar pregnancy rates after first postpartum artificial insemination (27.6% vs. 31.8%; P = 0.40), cumulative pregnancy rates during 300 days in milk (94.7% vs. 98.5%; P = 0.005) were greater in SN cows. A reduced (P = 0.0001) Cox proportional hazard of pregnancy rate at 300 days in milk and a longer interval from parturition or abortion to conception (median, 111 vs. 101 days) were observed in SP compared with SN cows. Spontaneous N caninum infection is a significant contributing factor of pregnancy loss and occurrence of uterine disease (i.e., retained placenta and metritis), negatively affecting subsequent pregnancy in grazing lactating dairy cows.

Detection of Neospora caninum-DNA in brain tissues from pigeons in Changchun, Jilin (China)

Neospora caninum is an intracellular protozoan infecting many domestic and wild animals. The domestic chicken (Gallus domesticus) and the sparrow (Passer domesticus) are known as natural intermediate hosts of N. caninum, whereas the role of other birds such as pigeons is still unclear. In the present study, pigeon brain tissues collected in Jilin of China were screened by N. caninum specific-nested PCR to determine whether pigeons functioned as the natural intermediate hosts of N. caninum. The prevalences of N. caninum DNA and Toxoplasma gondii DNA among the brain samples were 30% (63/210) and 13.33% (28/210), respectively. One brain sample was co-infected with N. caninum and T. gondii in naturally infected pigeon. Of the 63 positive samples 42 could be assigned to the NC-PR genotype, 10 to the NC-1 genotypes and 5, 3 and 3 respectively to the each of the three new genotypes identified, indicating genetic polymorphism of N. caninum in pigeons in Jilin of China. The present study expanded the list of intermediate hosts of N. caninum to include pigeons which suggests that pigeons are involved in the transmission of the N. caninum.

Survey for the presence of Neospora caninum in frozen bull's semen samples by PCR assay

ObjectiveTo evaluate the occurrence of Neospora caninum (N. caninum) DNA using polymerase chain reaction (PCR) technique in frozen semen samples of Iranian bulls that were used for artificial insemination. MethodsIn this study, 57 frozen bull's semen samples were collected randomly from artificial insemination centers and genomic DNA was extracted. For detection of pNC-5 gene of N. caninum by oligonucleotide primers were amplified using PCR technique and 1% agarose gel electrophoresis used for visualization of amplified PCR products. ResultsThe results of this study present 6 of 57 (10.53%) as positive samples. This show high presence of N. caninum infection and display importance of frozen semen samples of bulls, which were used for artificial insemination, in the spread of bovine neosporosis in Iran. These results present PCR as suitable technique for fast detection of this protozoan parasite in semen specimens. ConclusionsAccording to these results control and eradication programs, for example prevent vaccinations, as reduction of economic losses caused by this protozoan infection in Iranian cattle seems to be necessary.

Detection of Neospora caninum-DNA in feces collected from dogs in Shenyang (China) and ITS1 phylogenetic analysis

Neospora caninum is an intracellular protozoan that infects many domestic and wild animals. Dog is known as a definitive host of N. caninum and involved in transmitting infections to intermediate hosts by shedding oocysts. To investigate the epidemiology of dog neosporosis in China, 212 dog feces specimens in Shenyang were screened by nested-PCR using Nc5 primers and confirmed by N. caninum ITS1 PCR. The positive rate of N. caninum DNA was 34.90% (74/212). There were no significant correlations in prevalence of Neospora infections between different ages and genders. N. caninum DNA positive samples were further examined by PCR using Hammondia heydorni-specific primers. 37 out of 74 N. caninum DNA positive samples were also H. heydorni DNA positive. Only Nc5 primers positive and H. heydorni primers negative samples were used for ITS1 gene sequence analysis. Sequencing results from the 37 N. caninum positive samples revealed that ITS1 gene has 96–100% similarity with N. caninum sequences deposited in Genbank. Also, the presence of a new genotype indicated genetic polymorphism of N. caninum in infected dog feces in Shenyang of China.

Red foxes (Vulpes vulpes) in Romania are carriers of Toxoplasma gondii but not Neospora caninum.

Brain samples from 182 red foxes (Vulpes vulpes) from Romania were examined using a standard PCR technique. Results provide evidence of Toxoplasma gondii (11 foxes=6.0%) and Neospora caninum (1 fox=0.5%) DNA in red foxes from Romania. No coinfections were found.