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PubMed2001

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.

Agglutination Tests↗
PubMed2001

Leishmania infantum and Neospora caninum simultaneous skin infection in a young dog in Italy.

Leishmania infantum, the agent of canine leishmaniasis in Mediterranean countries, and Neospora caninum, a recently recognized protozoal pathogen in dogs, were diagnosed in a 9-month-old Argentine Dogo dog. Both skin lesions and neurological signs were present. Histopathology of cutaneous lesions revealed a suppurative, diffuse dermatitis with numerous intracellular protozoa. Serology was positive for both L. infantum (1:640) and N. caninum (1:800). Double-label immunohistochemical staining of skin samples with hyperimmune serum from L. infantum-infected dogs was positive for protozoa within macrophages, while the polyclonal antibody specific for N. caninum showed positive reactions for protozoa in endothelial cells and fibroblasts. Transmission electron microscopy (TEM) confirmed the infection with both protozoa. This is, to the authors' knowledge, the first case of simultaneous infection with L. infantum and N. caninum in a dog. It is possible that the immunosuppressive effects of Leishmania infection or long-term steroid therapy may have been a contributing factor to the development of N. caninum in this dog.

Animals↗
PubMed2001

A Swiss case-control study to assess Neospora caninum-associated bovine abortions by PCR, histopathology and serology.

Neospora caninum is one of the most frequent infectious organisms causing abortion in cattle worldwide. The present case-control study was designed to assess the importance of bovine neosporosis for causing abortion in Swiss cattle and to identify selected risk factors. Infection was primarily diagnosed by a N. caninum-specific PCR and serology, complemented with histopathology and immunohistochemistry. A total of 113 case and 113 corresponding control-farms were studied for 1.5 year. During this time period, 242 abortions were reported and referred for bacteriological, virological, parasitological and pathohistological examinations. N. caninum was detected by PCR in the brains of 21% of all aborted fetuses. Microscopic lesions indicative for cerebral protozoa infection were detected in 84% of PCR-positive fetal brains. Bovine viral diarrhea virus (BVDV) was demonstrated in 7% of the cases, and bacterial infections were detected in 4% of the abortions. One or more N. caninum-abortions occurred in 20% of the herds (41 case-farms and 3 control-farms). Serological examination of aborting mother cows revealed a significantly higher percentage of N. caninum-seropositive animals (44%) in comparison to the prevalence in a randomly selected population (12%). However, in eight cases (4% of all investigated abortions) seronegative cows aborted N. caninum PCR-positive fetuses, and in 50 cases the fetus remained negative although the respective mother cow was N. caninum-seropositive. Repetitive serological investigations (at a 3-12 months interval) of 3551 cows from case- and control-farms showed a decrease of the overall N. caninum-seroprevalence from 17 to 12%. Ninety out of 3008 seronegative animals were converted to N. caninum-seropositivity. Conversely, 212 out of 543 initially seropositive animals became seronegative for their second serum sample. The obtained data underlined the importance of N. caninum as a causative agent for abortion in Swiss cattle. Furthermore, PCR was confirmed to be a valuable diagnostic tool for the primary diagnosis of N. caninum in aborted fetuses. On the other hand, the value of serology appears to be hampered by the temporal instability of N. caninum antibody concentrations in adult cattle, including especially seronegativity of some individual animals. Thus, seronegativity in a mother cow or heifer does not exclude N. caninum-associated abortions.

Abortion, Veterinary↗
PubMed2002

In vitro antiprotozoal effects of artemisinin on Neospora caninum.

Neospora caninum is an intracellular apicomplexan parasite that infects a wide range of mammals and has been associated with abortion in cattle worldwide. Artemisinin is an effective antimalarial compound derived from a traditional Chinese herbal remedy, qinghao or Artemisia annua L. In the study reported, the cultured host cells (vero cells or mouse peritoneal macrophages) infected with N. caninum tachyzoites were incubated with alpha-MEM (minimal essential medium) 10%HS supplemented with various concentration or artemisinin (20, 10, 1, 0.1 and 0.01 microg/ml) to examine the efficacy of artemisinin against N. caninum tachyzoites intracellular multiplication. In long-term studies, at 20 or 10 microg/ml for 11 days, artemisinin reduced N. caninum and completely eliminated all microscopic foci of N. caninum. At 1 microg/ml for 14 days, artemisinin reduced N. caninum and completely achieved elimination of all microscopic foci of N. caninum. There was no apparent toxicity to host cells in long-term studies. In short-term studies, at > or = 0.1microg/ml, artemisinin reduced N. caninum tachyzoites intracellular multiplication, significantly (P < 0.05) and appeared to depend on the artemisinin concentrations. Pretreatment of host cells or N. caninum tachyzoites with artemisinin had no effect on N. caninum tachyzoites intracellular multiplication. These results demonstrate that artemisinin inhibited N. caninum tachyzoites intracellular multiplication.

Animals↗
PubMed2002

Point source exposure of cattle to Neospora caninum consistent with periods of common housing and feeding and related to the introduction of a dog.

Eight dairy herds with evidence of post-natal transmission of Neospora caninum were used to test the hypothesis of a point source exposure by a retrospective analysis of the housing and feeding of infected age-groups. The first N. caninum-associated abortion or birth of N. caninum-seropositive offspring from the post-natally infected age-group was considered as the first indication of the infection. In seven of the eight dairy herds, a point source exposure to N. caninum of the infected age-groups was found during a limited period of common housing and feeding. In all herds studied, the analysis indicated that the cattle had been infected shortly before the first abortions occurred. In all, except one herd, the post-natal infection was more directly related to housing than to feeding. Therefore, it appeared that the feed was contaminated in the feeding alley. In one herd, the total mixed ration was found to be the probable path of infection. In all farms studied, a new dog (young, adult dog or litter) had been introduced within a period of 1.5 years prior to the first indication of N. caninum infection in the cattle. As there was evidence in all herds of vertical transmission of neosporosis for years, it is hypothesized that the newly introduced dog was infected with N. caninum by materials from already infected cattle and subsequently transmitted the infection to other cattle by shedding of oocysts.

Abortion, Veterinary↗
PubMed2002

Natural transmission routes of Neospora caninum between farm dogs and cattle.

Twelve dairy herds with evidence of post-natal infection with Neospora caninum were compared with 21 control herds with no evidence of post-natal infection. On the former farms, dogs consumed placenta or licked uterine discharge in 75 and 67% of the farms, respectively, while on control farms these activities occurred in 38 and 24% of the farms, respectively. On all control farms and all but three post-natally infected farms the dogs were fed colostrum or milk. Defecation of dogs on the feeding alley was observed in 92% of the post-natally infected farms and in 24% of the control farms. The same trend was observed for defecation of dogs in grass silage, in 75% of the post-natally infected farms and in 19% of the control farms; and in corn silage, in 50% of the post-natally infected farms and in 10% of the control farms. Consumption of placenta, material of aborted foetuses or uterine discharge in combination with defecation on the feeding alley, storage of grass or corn silage was observed in 19% of the control farms and in 75% of the post-natally infected farms. This study supports the hypothesis that farm dogs may become infected by foetal fluids or placental material of infected cattle, and may subsequently cause a post-natal infection of cattle in the herd by shedding oocysts.

Animal Feed↗
PubMed2002

Immunological relationship between Neospora caninum and Besnoitia besnoiti.

Neospora caninum is a coccidian parasite identified as a major cause of abortion in cattle. A combined infection of N. caninum with another taxonomically related parasite of cattle, Besnoitia besnoiti can occur in geographical areas endemic for both species. Both infections are routinely diagnosed serologically, and incorrect diagnosis could occur if immunological cross-reactivity exists between the two parasites. To investigate the possible degree of cross-reactivity, we compared results obtained with two serological techniques, immunofluorescent antibody test (IFA), and Western blot analysis on known positive and negative sera. The test sera were derived from naturally infected cattle and from experimentally infected Mongolian gerbils. In IFA of bovine sera, no cross-reactvity was detected at the commonly used serum dilution cutoffs of 1:200 for N. caninum and 1:256 for B. besnoiti. However, at 1:64 dilution of both cattle and gerbil sera, anti-N. caninum sera reacted with B. besnoiti antigen in some individual samples. Anti-B. besnoiti serum did not react with N. caninum antigen at any dilution. This low level one directional cross-reactivity was confirmed by Western blot analysis. B. besnoiti antigen showed two immunoreactive bands when probed with anti-N. caninum serum, while no bands appeared when N. caninum antigen was probed with B. besnoiti antiserum. Immunization and challenge experiments in the highly susceptible Mongolian gerbil (Meriones unguiculatus) showed essentially no cross-protection between N. caninum and B. besnoiti.

Animals↗
PubMed2002

Seroprevalence of antibodies against Neospora caninum in diagnostic equine serum samples and their possible association with fetal loss.

A case-control study of the association between the presence of serum antibodies against Neospora spp. and fetal loss was performed on serum samples submitted to a veterinary diagnostic laboratory in northwestern United States. Control sera were randomly selected from those submitted from healthy horses for routine equine infectious anemia testing required for regulatory health certification. Case sera were randomly selected from those submitted from aborting mares for diagnostic workup. Based on a 1:50 or greater titer on the indirect fluorescent antibody test, 8% of the 160 control sera and 13% of the 140 case sera were titer positive. The association odds ratio of 1.67 fell short of statistical significance (p=0.124).

Abortion, Veterinary↗
PubMed2002

Serological survey of Neospora caninum infection in dairy cattle herds in Aguascalientes, Mexico.

A serological survey for antibody activity to Neospora caninum was carried out in Aguascalientes, a state in the central part of Mexico. One-hundred and eighty-seven serum samples from 13 dairy herds were tested by the ELISA test. The herd prevalence was 100% and the overall prevalence was 59% (n=110). Seventy-six of 97 seropositive cows had previous records of abortion. There was a statistically significant difference between the groups (P<0.05). However, the odds ratio was 1.4, suggesting an association between abortion and seropositivity. Neosporosis in dairy cattle appears to be widespread in Mexico, warranting more epidemiological studies to determine the distribution of the causative protozoan.

Abortion, Veterinary↗
PubMed2002

p38-avidity-ELISA: examination of herds experiencing epidemic or endemic Neospora caninum-associated bovine abortion.

An enzyme-linked immunosorbent assay (ELISA) was established to measure the avidity of bovine IgG directed against p38, a surface antigen (NCSRS2) of Neospora caninum tachyzoites. In the sera of dams intravenously infected with N. caninum NC-1, the p38-specific avidity increased from initially below 40% up to avidity indices between 50 and 80% after days 23 and 91 p.i. The p38-avidity-ELISA was used to examine various herds that had experienced endemic and epidemic N. caninum-associated bovine abortions. In herds with endemic abortion, generally high avidity indices of N. caninum-specific IgG were detected. This finding and the observation of an association between the seropositivity of dams and that of their offspring suggested a predominantly vertical transmission of the parasite among the animals of these herds, thus indicating chronic infection of these cattle. In contrast, for herds experiencing epidemic abortion: (i) an association regarding seropositivity of dams and their daughters could not be shown and (ii) a generally low avidity of p38-specific IgG in the sera from aborting dams was determined. This indicates recent postnatal transmission of N. caninum in these herds. A linear regression model explaining the avidity of p38-specific IgG was significantly influenced by the time span between sampling and the occurrence of the first abortion of the epidemic in herds with an N. caninum-associated abortion storm. Another factor significantly contributing to the model was the proportion of dams at risk that had aborted in the herds (i.e. the severity of the abortion epidemic). A possible explanation for this observation is that herds experiencing heavy abortion are sampled earlier after the onset of the epidemic than others that have a less severe abortion storm.

Abortion, Veterinary↗
PubMed2002

Serological survey of Neospora caninum and Leishmania infantum co-infection in dogs.

A seroprevalence survey and risk analysis of Neospora caninum and Leishmania infantum was conducted in dogs from an area of the Campania region of southern Italy, in order to investigate the co-infection of these two protozoa. Blood samples were collected from 1058 asymptomatic dogs over a 18 months period. Serum samples were tested for antibodies to N. caninum and to L. infantum using the indirect fluorescent antibody test. Epidemiological data (breed, age, sex, and utilization) were collected and statistically analysed in relation to N. caninum and to L. infantum seropositivity and antibody titres. Out of the 1058 sera samples tested, 68 (6.4%) were found to have antibodies to N. caninum, and 222 (21.0%) to have antibodies to L. infantum. The co-presence of antibodies to N. caninum and to L. infantum was found in 46 (4.3%) dogs. Thus, 67.6% of the dogs positive for N. caninum also had antibodies to L. infantum. The major risk factor for N. caninum seropositivity was the presence of antibodies to L. infantum, and the major risk factor for L. infantum seropositivity was the presence of antibodies to N. caninum. In addition, high N. caninum seroprevalence was closely correlated to Boxer breed, and high L. infantum seroprevalence was correlated to masculine gender and Setter and Pit bull breeds. Low L. infantum seroprevalence was closely correlated to Yorkshire breed. The findings of this survey indicate that in the Campania region of southern Italy the co-presence of antibodies to N. caninum and to L. infantum is very common in dogs, and that infection by one protozoan seems to enhance the susceptibility to the other one. This is probably due to the immunological status of the tested dogs.

Animals↗
PubMed2002

Seroepidemiology of beef and dairy herds and fetal study of Neospora caninum in Argentina.

The purpose of the present work was to study the epidemiology of Neospora caninum in beef and dairy herds in the Humid Pampas of Argentina. The seroprevalence of N. caninum was evaluated in 2414 serum samples of cows from beef and dairy farms. An indirect fluorescent antibody test (IFAT) was used to determine specific antibodies. The sera was screened at a dilution >or=1:200 and >or=1:600 in cows with reproductive disease antecedents and without them, respectively. Cows without history of reproductive diseases from nine beef and fifteen dairy farms were grouped according to the percentage (> or or <or= to 50%) of seropositive dogs. The analysis of 966 serum samples from aborted cows, demonstrated positive 18.9% (41/216) and 43.1% (323/750) from beef and dairy herds, respectively. Microscopic lesions compatible with N. caninum were observed in 43 of 188 (22.8%) fetuses and/or placentas evaluated. The protozoan was identified in 29 of 43 (67.4%) aborted specimens, being the largest number of positive results in dairy fetuses. The results obtained demonstrate a high association between neosporosis and dairy herds, however, our data also reveals that N. caninum is an important risk factor for reproductive losses in the extensively farmed beef cattle in the Humid Pampas of Argentina.

Aborted Fetus↗
PubMed2002

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.

Animals↗
PubMed2002

Neospora caninum NcSRS2 is a transmembrane protein that contains a glycosylphosphatidylinositol anchor in insect cells.

We investigated the terminal location of NcSRS2, a surface antigen of Neospora caninum that has potential use for diagnosis, and demonstrated its importance as a vaccine component against neosporosis, in an insect-baculovirus expression system. To examine the role of the hydrophobic C-terminal tail in NcSRS2, four types of recombinant baculoviruses were constructed. Immunoblotting and N-terminal amino acid analysis revealed cleavage of a 6 kDa of the N-terminal signal peptide in the mature NcSRS2 protein. The recombinant NcSRS2 (rNcSRS2) lacking 25, and 62 amino acids from the termination codon were detected in supernatants from recombinant virus-infected cells, but not in recombinants with truncated 147 amino acids from the termination codon, and intact NcSRS2 gene (401 amino acids). By flow cytometric and confocal laser scanning microscopic analyses, the truncation of the hydrophobic C-terminal tail in NcSRS2 was shown to result in the reduction of protein expression on the cell surface relative to intact rNcSRS2. Except for the recombinant lacking the 147 C-terminal residues, three other rNcSRS2 were detected in the supernatants after treatment with phosphatidylinositol-specific phospholipase C. Our results demonstrate that the N. caninum NcSRS2 is a transmembrane protein that contains a glycosylphosphatidylinositol-anchor molecule in insect cells, and that the hydrophobic C-terminal domain is an essential component for GPI-membrane attachment. We have likewise shown the usefulness of the insect-recombinant baculovirus system in the expression of rNcSRS2.

Animals↗
PubMed2002

A high rate of seroconversion for Neospora caninum in a dairy herd without an obvious increased incidence of abortions.

The purpose of this paper is to investigate the high rate of seroconversion for Neospora caninum in a dairy herd. Forty-five of 95 (47%) of the seronegative animals seroconverted within a period of 6 months. Seropositive animals were not equally distributed among age-groups. A high seroprevalence age-group of 8-30 months was housed together during a period of 4 months, indicating a point source infection within this period. A lack of association between the serological status of daughters and mothers also indicated horizontal transmission of the infection. Low avidity indices in the seroconverted animals confirmed a recent infection in this herd. However, there was no increased abortion rate in connection with the seroconversion, which indicates that mass seroconversions in dairy herds may remain unnoticed. There was circumstantial evidence that the farm dog played a role in the transmission of the infection to the cattle. However, also evidence was found for an unexplained ongoing horizontal transmission after the initial point source exposure.

Abortion, Veterinary↗
PubMed2002

Use of bulk milk for detection of Neospora caninum infection in dairy herds in Thailand.

The relationship between the level of Neospora caninum antibodies in bulk milk and the seroprevalence in lactating cows was investigated. Bulk milk was also used to estimate the prevalence of N. caninum infection in dairy herds in the northeast and north Thailand. Bulk milk and individual serum from all lactating cows in 11 herds as well as 220 bulk milk samples from nine milk collection centres were analysed for presence of N. caninum antibodies using an iscom ELISA. In the 11 herds the bulk milk absorbances ranged between 0.04 and 0.89 and the seroprevalences varied between 0 and 46%. Five herds had milk absorbances below 0.20, among those were the two herds housing only seronegative lactating cows. In the remaining three herds with such low bulk milk absorbances one or two cows (5-14%) were seropositive. Six of the investigated herds had bulk milk absorbances above 0.20. In the two herds with the highest bulk milk absorbances more than 30% of the cows were seropositive. Using an absorbance of 0.20 to discriminate between negative and positive herds, 102 (46%) of 220 bulk milk samples were judged positive. There was no significant difference in mean bulk milk absorbance between the milk collection centres within each region. However, the proportion of herds with bulk milk absorbances > or =0.50 in the north was statistically (P < 0.01) higher than that in the northeast. It was concluded that bulk milk antibody testing can be used to identify N. caninum-infected herds and that N. caninum is a common infection in dairy herds in Thailand.

Animals↗
PubMed2003

Evaluation of a single serological screening of dairy herds for Neospora caninum antibodies.

Twenty-one dairy herds with a history of Neospora caninum-associated abortions were used for a longitudinal serological study. A total of 1,676 animals were blood sampled 3 times and used to evaluate a single serological screening for N. caninum antibodies. The results of the first serological screening were compared with the results based on three consecutive samples, whereby two or more positive or negative test results per animal were considered to determine its serological status as positive or negative, respectively. In both test regimes 95.3% of the animals had the same interpretation, of which 33.9% were seropositive, and 61.3% seronegative. Relative sensitivity of one-time sampling compared to three consecutive samplings was 94.7%, while relative specificity was 95.6%. Relative specificity differed between herds. Predictive values positive and negative of one-time sampling were 92.4 and 97%, respectively. The agreement between one-time sampling and three consecutive samplings, kappa, was 0.90. For evaluation of discrepant results age distribution and pedigree data were used to provide clues regarding likelihood of transmission. Age clustering of seropositive animals was interpreted to indicate a point source infection. Daughter-mother relationships were used for the interpretation of congenital infections. The proportion of congenital infection decreased with increasing parity of the mother. Seropositive heifers had 80% congenitally infected offspring, while in older cows 66% of the offspring was congenitally infected, possibly due an increased immunity to transplacental infection with age. It is concluded that a single serological screening of a whole herd in connection with an analysis of age distribution and pedigree data is a rapid and valid method to interpret the serologic status of individual animals and to study the mode of transmission of N. caninum.

Aging↗
PubMed2003

Reactivity against Sarcocystis neurona and Neospora by serum antibodies in healthy French horses from two farms with previous equine protozoal myeloencephalitis-like cases.

Sarcocystis neurona is considered a leading cause of equine protozoal myeloencephalitis (EPM), a common infectious neurological disease in horses in the Americas. EPM-like cases associated with S. neurona peptide reactive antibodies in Western blots were recently described in Normandy, France. In this report, antibodies reacting with S. neurona merozoites were detected using an agglutination assay at titers ranging from 50 to 500 in sera from 18/50 healthy horses from two farms with a previous EPM-like case. Higher values were found in older animals. Four out of six horses which traveled or stayed in the US exhibited titers over 50, a higher figure than in the group which did not travel out of France or stayed in an other European country. No correlation was found between anti-S. neurona and anti-Neospora sp. antibody titers. Data prompt further study of significance of anti-S. neurona antibodies in clinically healthy or diseased European horses, and identification of putative immunizing parasite(s) and their host(s).

Animals↗