Evaluation of Neospora caninum and Toxoplasma gondii infections in alpaca (Vicugna pacos) and llama (Lama glama) aborted foetuses from Peru

The aim of this study was to investigate the participation of Neospora caninum and Toxoplasma gondii in abortion cases of Peruvian llamas and alpacas. Fifteen aborted foetuses were recovered from two main rearing areas of camelids in Peru (Central or South Andean region). Foetal histopathology was used to detect the presence of protozoal-associated lesions in target organs. N. caninum and T. gondii infections were confirmed by immunohistochemistry (IHC) combined with PCR and by PCR alone, respectively. The influence of the species (llama and alpaca), foetal age (first, second and third gestational periods) and geographical location (Central or South Andean region) of the foetuses was also studied. Thirteen of the samples (26%, 13/50) showed lesions suggestive of protozoal infection. N. caninum infection was detected by either IHC or specific PCR in 14 out of 50 foetuses (28%), of which 8 also showed protozoal-associated lesions. T. gondii DNA was not detected in any of the foetuses analysed. Protozoal infection was more frequent in the foetuses from the second gestational period (P<0.05, Fisher F-test). No significant association was observed between protozoal infection and species or geographical location (P>0.05, chi2 test). The results of the present study indicate that neosporosis should be included during the differential diagnosis of abortion in llamas and alpacas.     

Abortion epidemic in a dairy herd associated with horizontally transmitted Neospora caninum infection

A dairy herd experienced an abortion epidemic during which 43 per cent of the cows at risk aborted. Neospora caninum infection was demonstrated in four of six fetuses suitable for examination and the group of at-risk cows that aborted had significantly higher N. caninum antibody concentrations than the at-risk cows that delivered a live calf at term (P<0.001). The antibody concentrations in the cow herd were significantly higher than in the youngstock (P<0.001), and the concentrations in the youngstock increased significantly (P<0.001) with age. When seven months to a year old, the calves born at term to the at-risk cows had significantly higher (P=0.007) antibody concentrations than age-matched calves born before the epidemic. At the time of the epidemic, there was a significant increase in the antibody levels of the herd that was not consistent with vertical infection alone, indicating that there appeared to have been a sudden large increase in the incidence of horizontal postnatal transmission of N. caninum to the cow herd, or to the surviving offspring of the at-risk cows, or to both of these groups.     

Diagnostic significance of Neospora caninum DNA detected by PCR in cattle serum

A nested PCR that successfully detected Neospora caninum DNA in serum of cattle was used for investigation of selected abortion cases and in a study of healthy pregnant cows at an abattoir. N. caninum DNA was not detected in serum from antibody positive dams that aborted due to N. caninum, but was present in serum of some antibody negative dams that aborted due to other causes. N. caninum DNA was also found in the serum of about half of the animals that aborted of undetermined cause, but was not detected in cow sera from two beef cattle herds in Western Australia with no recent history of abortion. In the abattoir study of 79 dams and their foetuses N. caninum DNA was found in serum of 3 dams and in material from 11 foetuses. The majority of the cows and all foetuses were antibody negative. Our findings suggest that there is no obvious relationship between the presence or absence of N. caninum DNA in serum and the presence of antibodies to N. caninum in dams, the presence of N. caninum DNA in foetuses or abortion due to N. caninum. This is the first report of the detection of N. caninum DNA in serum of cattle rather than the white blood cell fraction. It indicates the presence of free tachyzoites and/or parasite DNA in circulation. The results suggest that persistent infection in the absence of antibodies is a possible outcome of N. caninum infection. Infection of foetuses in the absence of antibodies supports the possibility of persistent infection due to immunotolerance to an early in utero infection. It is therefore important to test for N. caninum DNA as well as antibodies for the detection of exposed and/or infected animals. However, the presence or absence of N. caninum antibodies or DNA did not support nor exclude N. caninum as the cause of abortion. Additional criteria are required for a positive diagnosis of abortion caused by N. caninum.     

Diagnosis and epidemiology of Neospora caninum-associated abortions in cattle]

Neospora caninum, a recently discovered protozoan parasite closely related to Toxoplasma gondii, has world-wide been recognized as an important cause of bovine abortion. N. caninum possesses a wide host range. The dog can be a definitive host for N. caninum. In cattle, the infection is transmitted transplacentally with high efficiency, while the majority of congenitally infected calves is clinically normal at birth and thereafter. Whether horizontal transmission occurs in cattle and whether this potential mode of transmission has epidemiological significance, remains to be elucidated. N. caninum-associated abortions can occur in epidemic or endemic form in a herd. The clinical symptoms of bovine neosporosis are confined to the occurrence of abortion, stillbirth and weak calves. Multifocal nonsuppurative encephalitis represents the most frequent pathohistological finding in N. caninum-associated abortions. The causative agent can be demonstrated by immunohistochemistry or polymerase chain reaction. Serological techniques can be used for indirect diagnosis. On the basis of the available diagnostic methods and the present knowledge about the epidemiology of the infection proposals are made regarding diagnosis, epidemiological assessment and prophylaxis of N. caninum-associated abortion problems in cattle herds.     

Seroprevalence of Neospora caninum infection in dairy and beef cattle in Paraguay

Seroprevalence of Neospora caninum in 879 beef and dairy cattle in different locations of Paraguay was determined by an ELISA. In the survey, 262 (29.8%) cattle were positive to N. caninum, and animals with anti-Neospora antibody titre were observed in all the locations tested in the country. Serum samples taken from a herd that exhibited persistent abortion had the highest percentage of animals being positive to the parasite (17/30, 56.7%). In the same herd, abortion was significantly more likely in animals with high anti-Neospora antibody titre. Immunoblot analysis demonstrated that the banding pattern from positive Paraguayan cattle was similar to that seen with the positive control sample. In conclusion, N. caninum infection is present among Paraguayan beef and dairy cattle, and it may be an important cause of bovine abortion in Paraguay.     

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.     

Herd-specific and age-specific seroprevalence of Neospora caninum in 14 British dairy herds.

All the cattle in 14 dairy herds in England were tested for Neospora caninum-specific antibodies with a commercial ELISA. Three of the herds had had sporadic abortions, eight had had endemic abortions and three had had epidemic abortions associated with N caninum before the study. Of 4295 cattle tested, 17.1 per cent were seropositive and the herd-specific prevalence ranged from 7.3 per cent to 44.8 per cent. No significant effect of either herd size (P = 0.988), endemic (P = 0.869) or epidemic (P = 0.138) patterns of abortion on herd-specific prevalence was found by using logistic regression analysis. There was no evidence in any herd of a significant increase in prevalence with age; the prevalence in seven-to 12-month-old cattle was not significantly different (P > 0.400) from the prevalence in older cattle, except that there was a significantly lower prevalence (P = 0.017) in 13-to 24-month-old cattle. The results of this study are consistent with vertical transmission being the major route of N caninum transmission in these herds.     

Recent advances in understanding the epidemiology of Neospora caninum in cattle

INTRODUCTION: Neospora caninum is the most frequently diagnosed cause of cattle abortion in New Zealand and is an important pathogen worldwide. The parasite has been found in aborted bovine foetuses and in puppies with neurological disease. Recent developments have provided new insights into the epidemiology of the disease that are reviewed in this article. TRANSMISSION: Transplacental infection is of major importance in the spread of N. caninum overseas. Most congenital infections result in the birth of apparently healthy calves. Seroepidemiological studies indicate introduced point-source infections are also likely, as do investigations of abortion outbreaks in cattle herds attributed to N. caninum. Horizontal transmission is an important determinant of the stability of infection prevalence in a herd. Two potential sources of horizontal transmission that have recently been reported are: via a canine definitive host and; cow-to-calf transmission through pooled colostrum or milk. Transmission parameters for the organism have been estimated using mathematical modeling, to explore the epidemiology and options for control of N. caninum infection in dairy cattle in the absence of precise epidemiological data. LIFE-CYCLE: Seroepidemiologic studies, combined with the knowledge that dogs can be a definitive host, provide evidence supporting a dog-cattle life-cycle. The importance of dogs in the epidemiology of bovine neosporosis is not yet clear. It is likely that N. caninum oocysts in dog faeces could serve as a source of infection for cattle and recent studies have shown that the canine and bovine isolatesflare the same organism. Infection of cattle orally exposed to oocysts has been demonstrated experimentally but not in nature. It is not known if other wild carnivores may serve as definitive hosts of N. caninum. CONCLUSION: Despite active research for over a decade, very little is known about the mechanisms of transmission of N. caninum. Vertical transmission is well documented but horizontal spread and the pathogenesis of abortion from this disease need further investigation. It is evident that, even with a very high probability of vertical transmission, some form of horizontal transmission is required for the disease to persist in a herd.     

First Confirmed Case of Neospora caninum-associated Abortion Outbreak in Portugal

In a dairy herd experiencing an abortion outbreak, 49% of the 119 cows were positive for Neospora caninum antibodies and the remaining 51% were seronegative. Using immunohistochemical analysis of foetal brain tissue, Neospora cysts and bradyzoite clusters were identified in two of the 15 aborted foetuses submitted for testing. Other agents that commonly cause abortions were not found. These results, coupled with neurological clinical symptoms in new-born calves, implicated N. caninum as the major cause of abortions in this dairy herd. This is the first report that associates N. caninum infection with bovine abortions in Portugal.     

Tunicamycins, a class of nucleoside antibiotics similar to corynetoxins of the Rathayibacter toxicus, increase susceptibility of mice to Neospora caninum

Neosporosis is the leading cause of abortion in cattle. Neospora caninum-associated abortion may exhibit both endemic and epidemic patterns. It was reported that the epidemic outbreaks took place in the form of "abortion storms" and were not significantly correlated with seasonal changes or consumption of any particular feeds; and thus, the mechanisms by which the epidemic "abortion storms" are triggered remain unclear. Annual ryegrass toxicity (ARGT) is a severe or fatal neurological disorder of livestock of Australia and South Africa. This disorder is caused by the ingestion of several plant genera (Lolium, Polypogon, and Agrostis) colonized by a nematode (Anguina sp.) and a bacterium, Rathayibacter toxicus. Corynetoxins (CTs) produced by R. toxicus contaminate plants which are in turn ingested by sheep and cattle, causing severe or often fatal hepatocerebral disorders in affected animals. We hypothesize that N. caninum-associated fetal deaths and abortions in cattle may be potentiated by pre-exposure of sublethal levels of CT-contaminated plants prior to N. caninum infection or reactivation of a latent infection. The exposure of sublethal CTs may not cause clinical diseases, but may increase susceptibility to pathogens such as N. caninum. Indeed, CT poisoning surviving pregnant sheep can have up to 10% abortions. The present study investigated whether animals pre-exposed to tunicamycins (TMs), a functional substitute for CTs, had a lowered resistance to sublethal experimental infection by N. caninum tachyzoites in mice. The results showed that sublethal doses of TMs or N. caninum alone did not cause significant deaths. Sublethal doses of N. caninum induced high mortality in TM-treated mice in a dose dependent manner. When mice were treated with a low dose of TMs (5 μg/mouse), as few as 2.5 × 10(6) tachyzoites were needed to induce more than 30% of mortality which is equivalent to the mortality rate caused by 40 × 10(6) tachyzoites. Spleen cells of mice treated with TMs had reduced (p<0.05) Neospora antigen-induced interferon-gamma (IFN-g) production and Con A-stimulated proliferation. The results suggest that CT contaminated plants, when exposed to animals under natural conditions, may contribute to lowered host resistance and increased N. caninum-associated fetal morbidity and mortality in affected animals.     

Occurrence of Neospora caninum and Toxoplasma gondii infections in ovine and caprine abortions

Neospora caninum and Toxoplasma gondii are closely related cyst-forming apicomplexan parasites identified as important causes of reproductive failure in cattle and small ruminants, respectively. Protozoan abortion in small ruminants is traditionally associated with T. gondii, but the importance of N. caninum remains uncertain. The aim of this study was to investigate the presence of N. caninum and T. gondii infections in abortion cases in small ruminants submitted for diagnosis. For this purpose, 74 ovine and 26 caprine aborted foetuses were recovered from different areas in Spain. Foetal histopathology was used to detect the presence of protozoal-associated lesions in brain. The presence of N. caninum and T. gondii was confirmed by PCR. Protozoal infection was detected in 17 out of 100 (17%) foetuses examined by at least one of the diagnostic techniques used. Lesions suggestive of protozoal infection were observed in 10.8% (8/74) and 15.4% (4/26) of the ovine and caprine abortions respectively. N. caninum and T. gondii infection was detected by PCR in 6.8% (5/74) and 5.4% (4/74) of sheep foetuses, respectively, of which five showed protozoal-associated lesions. N. caninum DNA was detected in 11.5% (3/26) of goat foetuses, of which two showed protozoal-associated lesions, whereas T. gondii DNA was detected in one goat foetus with no lesions. The simultaneous presence of N. caninum and T. gondii DNA was detected in one sheep foetus with severe lesions. This study demonstrates that N. caninum plays a significant role in abortion in small ruminants in the studied population. In addition, our results highlight the importance of differentiating between protozoa whenever characteristic lesions are observed.     

Evaluation by different diagnostic techniques of bovine abortion associated with Neospora caninum in Spain

Eighty foetuses from some of the main cattle-producing regions in Spain were analysed to investigate the participation of Neospora caninum in cases of bovine abortion. Diagnosis of the infection was determined by histopathological analysis complemented with immunohistochemistry, serology (IFAT and ELISA) and PCR tests. A total of 38.8% of the bovine foetuses analysed were considered to be infected by at least one of the diagnostic techniques used. Microscopic lesions consistent with Neospora infection in brain were identified in 31.3% of the samples, whereas only 10.7 and 15.3% were positive using serological and PCR analysis, respectively. Perfect agreement was shown between IFAT and ELISA, although there was little agreement among results of the other diagnostic techniques. Gestational age of aborted foetuses checked ranged from <3 to 9 months, with a mean of 5.9 months, and no difference in age was found between infected and non-infected foetuses (P>0.05). This study confirms the importance of N. caninum as a cause of abortion in Spain and underlines the need to use different diagnostic techniques to increase the chance to detect the infection in aborted foetuses.     

Neospora caninum infection does not affect the fertility of dairy cows in herds with high incidence of Neospora-associated abortions

This present study, was designed to establish whether Neospora caninum infection affects the fertility of high-producing dairy cows, and was based on yearly serological screening for neosporosis and on the confirmation of N. caninum infection in aborted foetuses. Assessment was made of 7518 artificial inseminations (AI) (applied to parous cows) performed in three herds. Of these inseminations, 2540 (33.8%) resulted in pregnancy; 34% of which corresponded to seronegative cows (2226 of 6556 AI performed in seronegative cows) and 32.6% to seropositive animals (314 of 962). Abortion occurred in 97 (30.1%) of the 314 pregnancies recorded in dams seropositive for N. caninum, while only 93 (4.2%) of the 2226 pregnancies in seronegative animals ended in abortion. Logistic regression analysis indicated no significant effects of N. caninum antibody titre, days in milk and milk production at insemination on fertility. Factors found to affect fertility were herd, season of insemination, lactation and insemination number, semen-providing bull and AI technician. Our results indicate that N. caninum infection does not affect the fertility of high-producing dairy cows.     

Seroprevalence of Neospora caninum in aborting dairy cattle in the Czech Republic

A serological survey for antibodies against Neospora caninum in aborting cattle was carried out in the Czech Republic. Serum samples from 463 aborting dairy cows originated from 137 farms from different parts of the Czech Republic were tested for presence of N. caninum antibodies by use of an enzyme-linked immunosorbent assay (ELISA) and an indirect fluorescent antibody test (IFAT). Antibodies (> or = 1:640) to N. caninum were found in 18 (3.9%) of 463 aborting cows. Farm prevalence in aborting cows was 12.4% (17/137). The antibody titres of cows were 1:200 (9 cows), 1:640 (7 cows), 1:1280 (3 cows), 1:2560 (3 cows), 1:5120 (3 cows), 1:10,240 (2 cows) and 1:20,480 (0 cow). A case-control study was conducted to estimate the association of N. caninum infection and abortion. For this 407 serum samples were collected from cows on five dairy farms with repeated occurrence of endemic and sporadic abortion of unidentified etiology. These samples were obtained from aborting cattle (n=44) and normally calving cattle (control group; n=363) and tested for N. caninum antibodies by an immunofluorescent antibody test (IFAT). Overall, 3.19% (13/407) of cows sampled had positive N. caninum fluorescence with a cut-off titre of 1:200. The prevalence of N. caninum was significantly higher (P<0.05) in the aborting group (13.64%; 95% confidence interval (CI): 5.2, 27.4) than in the control group (1.93%; 95% CI: 0.8, 3.9). A strong association between seropositivity and abortion was found, with seropositive cows being eight times more likely to abort than seronegative cows (odds ratio=8; 95% CI: 2.6, 25.1). This first report on the serological prevalence of N. caninum in cows in the Czech Republic verified a strong association between N. caninum infection and abortions in five dairy farms. Thus, the neosporosis should be considered in differential diagnosis of bovine abortion.     

Outbreak of abortion associated with Neospora caninum infection in a beef herd

Abortion outbreaks caused by infection with Neospora caninum in beef cattle have not been well documented. Neospora caninum infection was confirmed in 4 fetuses that were aborted by cattle in a 350-head beef herd; an additional 58 cattle aborted during the next 2.5 months. Overall, 44.4% (157/354) of the cattle in the herd did not become pregnant or experienced fetal loss during this period. Initially, 81.3% (282/347) of the herd was seropositive for antibodies to N caninum, and seropositive cows were 6.2 times as likely to not be pregnant as were seronegative cows. Other potential causes of abortion were not identified in this herd. Following the outbreak, few cattle in the herd or in a second exposed herd seroconverted, but high antibody titers persisted in most seropositive cattle through the end of the calving season. Evidence of cow-to-fetus transmission of the organism was detected in > 82% of seropositive cows.     

A longitudinal study of Neospora caninum infection on a dairy farm in New Zealand

A 600-cow New Zealand dairy herd experienced an abortion storm in 1997 and was monitored (blood sampling at about 3-month intervals) from May 1997 until January 1999. Abortion risk reached 9% in 1997 and was highest in heifers at 19%. The abortion risk decreased in 1998 to 3.2% (still somewhat higher than during the years prior to the outbreak). The serological reaction pattern for Neospora caninum showed an association with abortion risk only around the time of the 1997 outbreak when seropositive cows were 4.2 times more likely to abort than negative ones. Over the whole study period, only 27% of cows that were sampled on all nine visits always tested negative. Offspring from dams which had positive tests for Neospora caninum were 2.4 times more likely to abort than those from dams testing consistently negative. Controlling for age and breed, seropositive cows produced more milk than those that were consistently negative. Infection might have been present endemically within this herd prior to the epidemic, but in 1997 an additional factor appeared to have triggered the outbreak.     

First Portuguese isolate of Neospora caninum from an aborted fetus from a dairy herd with endemic neosporosis

Neospora caninum was isolated from the brain of an aborted 4-month-old fetus from a dairy cow herd with endemic neosporosis in Porto, Portugal. The fetal brain homogenate was inoculated interperitoneally first into outbred Swiss Webster mice given dexamethasone and then the peritoneal exudates from these mice was co-inoculated with mouse sarcoma cells in the peritoneal cavity of mice given dexamethasone. N. caninum tachyzoites were seen in peritoneal exudate of the second passage. Tachyzoites from the peritoneal exudate reacted positively with anti-N. caninum antibodies and not with anti-Toxoplasma gondii antibodies and contained N. caninum specific DNA. This Portuguese isolate of N. caninum has been successfully maintained in cell culture. The dam of the aborted fetus had an antibody titer of 1:10240 in the Neospora agglutination test (NAT). Antibodies to N. caninum were found in 76 of 106 cows from this herd in titers of 1:40 in 31, 1:80 in 22, > or =1:160 or more in 23 in the Neospora agglutination test. This is the first isolation of a viable N. caninum-like parasite from any host in Portugal.     

Serum antibody profile and reproductive performance during two consecutive pregnancies of cows naturally infected with Neospora caninum.

The objective of this study was to record how the antibody levels change over time during pregnancy in dairy cows naturally infected with the protozoan parasite Neospora caninum, and relate this to the reproductive performance. Eighteen cows with antibodies to N. caninum were serum sampled monthly during their first pregnancy and 13 of them were also followed for a second pregnancy. In all, five pregnancies ended in abortion and two in stillbirth. Antibodies to N. caninum in serum were demonstrated by immune stimulating complex enzyme-linked immunosorbent assay (iscom ELISA). The N. caninum antibody titres remained well above the 1:100 cut-off limit for the test used during 2 years in all cows. In the non-aborting cows, mean values of antibody titres to N. caninum rose 1.5-2.5 dilution steps to reach a plateau 4-5 months before parturition, and thereafter decreased from 2 months before parturition. These changes were statistically significant (p < 0.001). The same pattern was seen in the aborting cows. The consistent pattern of rise in antibody titres observed during both pregnancies in all cows indicated a reactivation rather than a reinfection of the parasite at mid-gestation.     

First documentation of a neospora-induced "abortion storm" (exogenous transplacental transmission of neospora caninum) in a Swiss dairy farm

In a Swiss dairy farm (canton of Geneva) consisting of 73 animals 8 abortions were observed within 2 weeks. Serological and molecular biological analyses (PCR) on aborting dams, and abortion materials, respectively, revealed that the protozoan parasite Neospora caninum was the causative agent. Besides the 8 aborting animals, 12 other non-aborting heifers were found to be serologically positive for this parasite. All positive sera were further tested in an avidity-ELISA to elucidate the recency of infection. All seropositive animals but one showed low avidities at the time the abortion storm started. This indicated at a recent N. caninum-infection within the herd. Thus, the animals most probably were exposed to N. caninum-oocysts (e.g. by dog feces-contaminated forage) and the resulting abortion storm was due to an exogenous (formerly known as "horizontal") parasite transmission into a naive herd. This is the first documented record of such an event in Switzerland.     

Molecular and immunodiagnostic studies of bovine neosporosis in Switzerland

Cyst-forming coccidia may cause significant losses in livestock, primarily due to abortion, loss of young animals and neuromuscular diseases. Rather recently, Neospora caninum has been recognized as one of the major protozoal abortion-inducing parasites in cattle. The present study addressed the performance of different diagnostic tools (in vitro-cultivation; histology; immunohistochemistry; serology; PCR) suitable for the direct or indirect detection of N. caninum. By PCR, Neospora-DNA was detected in 24 brains (29%) from 83 bovine abortion, many of these brains were simultaneously characterized by histopathological findings typical for a protozoal, cerebral parasitosis. The diagnostic methods were furthermore assessed using samples of different tissues and body fluids from three experimentally Neospora-infected pregnant cows and their foetuses. The diaplacental passage of N. caninum to the foetus was successful in two of the three cases. In these two cases, PCR was positive for different foetal organs and, additionally, for the abomasal and amniotic fluid. The successfully infected cows developed anti-Neospora serum antibodies between 10 and 17 days post infection, foetuses remained serologically negative in all cases. The results obtained in the present study demonstrated the usefulness of PCR, complemented by serology, for the specific diagnosis of bovine neosporosis. Such tests may prove suitable to perform epidemiological investigations. Taken together, our data indicated that prenatal neosporosis may be an important cause of infectious bovine abortion in Switzerland.