Isolation of Neospora caninum from dairy zero grazing cattle in Israel

First Israeli Neospora caninum isolates were obtained from brain tissues of aborted fetuses (NcIs491 and NcIs580) from dairy farms endemic for neosporosis and maintaining cattle on zero grazing. Tissues from different parts of the fetus brains were used to infect Vero cells. Tachyzoites of N. caninum were first observed in cultures from days 30 and 32 after infection. To confirm the identity of the isolated parasites, DNA extracts from brains and cultures were tested by PCR with specific primers based on the Nc5 gene. Specific fragments were amplified by PCR from infected cultures of both fetuses on day 25. Susceptible seronegative gerbils (Meriones tristrami) were inoculated intraperitoneally with 10(3) to 10(5) tenfold dilutions of subculture tachyzoites. The inoculated gerbils developed specific antibodies to N. caninum, with end-point serum dilution of 1:4096 in the IFA assay, whereas no neurological signs or deaths were seen during 4 months of observation.     

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

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

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

AIM: To isolate Neospora caninum from the brains of naturally infected cattle and use molecular techniques to characterise the isolates.

METHODS: Neospora caninum tachyzoites were isolated in Vero cell culture from the brains of a cow and two calves. The isolates were characterised using polymerase chain reaction (PCR) methods, DNA sequencing, an immunofluorescent anti-body test (IFAT), transmission electron microscopy (TEM), and immunohistochemistry (IHC). The brains of the three cattle were subjected to histopathological examination. A pathogenicity study was conducted in 120 BALB/c mice.

RESULTS: Neospora caninum tachyzoites were isolated from all three cases and first observed in vitro between 14 and 17 days post-inoculation. Parasites were sub-cultured and maintained in Vero cell culture for more than 6 months. PCR products were generated for all three isolates, using two different primers. Sequencing of the PCR products and a subsequent BLAST search identified the isolates as N. caninum. In addition, the isolates tested positive using IFAT and IHC, and ultrastructure revealed by TEM was characteristic of N. caninum. Histopathological examination revealed lesions characteristic of N. caninum in 1/3 brains. In the pathogenicity study using BALB/c mice, the mortality rate was 3–7%.

CONCLUSION: This was the first successful isolation of N. caninum in New Zealand confirmed using molecular characterisation tests.     

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

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

In vitro isolation of Neospora caninum from a stillborn calf in the UK.

Neospora caninum was isolated in Vero cell culture from the brain of a stillborn calf. This isolate (designated NC-LivB1) is the first to be obtained from cattle in the United Kingdom and was confirmed as N. caninum by immunofluorescence with specific antibodies and by internal transcribed spacer 1 (ITS1) sequence analysis. Differences were found between NC-LivB1, other bovine isolates and canine isolates of N. caninum and closely related protozoal parasites, using random amplified polymorphic DNA polymerase chain reaction (RAPD - PCR) techniques.     

Isolation of Neospora caninum from the brain of a naturally infected adult dairy cow

Neospora caninum was isolated from the brain of a 2-year-old dairy cow that had aborted confirmed N. caninum-infected fetuses on two occasions. The cow had an indirect fluorescent antibody titer of 1:1600 to N. caninum. The cow was killed 24 days after its second abortion and the brain was bioassayed for N. caninum in nude mice. Multifocal areas of perivascular cuffing and glial nodules were observed in the cerebrum and mesencephalon of the cow, but N. caninum was not identified in histological sections of the brain. All three nude mice inoculated with brain homogenate of the cow, developed emaciation and paralysis. Microscopical examination of the nude mice revealed systemic N. caninum infection with demonstrable tachyzoites in various organs. The parasites isolated from fresh mouse brain were transferred successfully into Vero cell cultures. PCR procedure on the purified tachyzoites obtained from the Vero cell cultures amplified the specific DNA sequence for N. caninum.     

Comparison of proteome and antigenic proteome between two Neospora caninum isolates

This study was conducted to explore the relationship between two isolates of Neospora caninum (N. caninum) (KBA-2 and VMDL-1) using proteomics. To achieve the goal, proteins of N. caninum tachyzoite lysates of KBA-2 and VMDL-1 were separated by two-dimensional gel electrophoresis (2-DE), stained with silver-nitrate and analyzed using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) to compare protein profiles. In addition, proteins separated by 2-DE were transferred to membranes, probed with bovine anti-N. caninum KBA-2 immunoglobulin G, and reactive proteins were visualized and compared between the two isolates. Most spots on 2-DE profiles and antigenic spots on 2-DE immunoblot profiles were located at similar locations in terms of isoelectric point and molecular weight. Proteins common to both isolates included the following: heat shock protein 70, subtilisin-like serine protease, nucleoside triphosphatase, heat shock protein 60, pyruvate kinase, tubulin alpha, tubulin beta, enolase, putative protein disulfide isomerase, actin, fructase-1,6-bisphosphatase, putative ribosomal protein S2, microneme protein Nc-P38, lactate dihydrogenase, fructose-1,6-bisphosphatase aldolase, serine threonine phosphatase 2C, 14-3-3 protein homologue, N. caninum dense granule-1 and NcGRA2. As a consequence, even though N. caninum KBA-2 and VMDL-1 isolates were isolated from geographically distinct locations there were significant homology in the proteome and antigenic proteome profiles. In addition, proteomic approach was verified as a useful tool for understanding of host immune response against different isolates of protozoa.     

Evaluation of the embryo transfer procedure proposed by the International Embryo Transfer Society as a method of controlling vertical transmission of Neospora caninum in cattle

OBJECTIVE: To evaluate efficacy of embryo transfer into seronegative recipients, using the procedure proposed by the International Embryo Transfer Society (IETS), for preventing vertical transmission of Neospora caninum in cattle. DESIGN: Prospective clinical trial. ANIMALS: 87 recipient cows and heifers and their embryo transfer calves from 22 donors originating from 9 dairy herds. PROCEDURE: Neospora caninum serologic status of donors and recipients was determined before collection and transfer of embryos. Viable embryos were washed and treated with trypsin. Recipients in experimental groups A (n = 50) and B (29) were seronegative and received embryos from seropositive and seronegative donors, respectively. Recipients in group C (n = 8) were seropositive and received embryos from seronegative or seropositive donors. Antibody titers against N caninum were determined monthly during pregnancy in recipients and in calf blood samples collected at birth. Tissues collected from stillborn calves and aborted fetuses were analyzed histologically and by immunohistochemical (IHC) methods. RESULTS: 76 calves and 11 fetuses and stillborn calves were examined. All calves from groups A and B were seronegative (n = 70) or lacked evidence of infection by use of tissue analysis (9). In group C, 5 of 6 calves were seropositive at birth, and IHC results were positive for 1 of 2 calves. Vertical transmission rate was significantly lower in groups A and B (0%) than in group C (75%). CONCLUSION AND CLINICAL RELEVANCE: Embryo transfer into seronegative recipients, using the procedure proposed by IETS, is an effective way to prevent vertical transmission of N caninum. Results provide support for pretransfer testing of all embryo transfer recipients.     

Neospora caninum in sheep: a herd case report

Neospora caninum was detected by means of PCR in the brain of 4 out of 20 aborted fetuses in a flock of 117 sheep exhibiting a persistent abortion problem, and N. caninum tissue cysts were furthermore found in encephalitic lesions in one of the PCR-positive fetuses. Toxoplasma gondii was detected as aborting agent in another 3 out of 20 fetuses. Antibodies to N. caninum (by indirect fluorescence antibody test (IFAT)) were found in 10.3% of 117 ewes and antibodies for T. gondii were found in 97.4% of 117 ewes. Other organisms associated with abortion were Chlamydia psittaci in three fetuses and Pasteurella multocida in one fetus. This is the first report of N. caninum associated abortion in naturally infected sheep.     

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.     

Rare detection of Neospora caninum in placentas from seropositive dams giving birth to full-term calves.

Neospora caninum is thought to be transmitted to cattle by dogs, the only known definitive host. Although aborted fetuses seem the most likely source of infective material for dogs, placentas from seropositive dams appear also as a potential source of infective material. The objective of the study was to evaluate the presence of N. caninum organisms in placentas of full-term calves born to seropositive cows. Sixteen placentas, 11 from Neospora-seropositive cows, were examined histologically and by immunohistochemistry and polymerase chain reaction (PCR) assay for the presence of N. caninum. Mild placentitis was observed in all placentas. Neospora caninum was not identified by immunohistochemistry, but placentas from 2 seropositive dams were positive for N. caninum by PCR. These results suggest that placentas of full-term calves from seropositive cows may be a potential source of N. caninum for dogs, but the incidence of this mode of transmission is likely to be low.     

Induced transplacental transmission of Neospora caninum in cattle.

Three Jersey cows were inoculated SC and IM with 26 million Neospora caninum tachyzoites at 129 (cow 1), 126 (cow 2), and 81 (cow 3) days after mating. Cows remained clinically normal for at least 1 month after inoculation of N caninum. Cow 1 was euthanatized 32 days after inoculation because of gangrenous mastitis. Cow 1 had a live fetus with no gross lesions; however, microscopic lesions were seen in the fetus and consisted of severe nonsuppurative necrotizing encephalitis of the cerebral white matter. Neospora caninum was identified in lesions by staining with anti-N caninum serum in an immunohistochemical test, by bioassays in mice, and by inoculation of bovine monocyte cultures with fetal tissue homogenate. Neither N caninum nor lesions were associated with infection with the protozoon identified in tissues of cow 1. Cows 2 and 3 aborted small autolysed fetuses 101 and 74 days, respectively, after inoculation with N caninum; the fetuses and attached placenta were unsuitable for laboratory investigations. Cows 2 and 3 remained clinically normal 4 months after abortion. Results of this study indicated that N caninum can be transmitted transplacentally in cattle.     

Seroepidemiology of neosporosis in dairy cattle in Uruguay

A seroepidemiological survey of Neospora caninum was done in a dairy farm in Uruguay employing indirect fluorescent antibody test (IFAT). Sera from 217 cattle (155 cows, 31 heifers and 31 calves) were tested for N. caninum antibodies in July 2000 and it was found that abortions were significantly associated with the seropositivity of the cows by chi(2)-test (P = 0.025). The cows with an IFAT titre of 1:3200 were at a significantly higher risk of abortion than seronegative cows (P = 0.0013). Neospora organisms were detected in the brains of two aborted foetuses by immunohistochemical examination. The infection of N. caninum was considered postnatal because of the even distribution of seropositive cows (60%), low seroprevalence (20%) in calves and no significant correlation in the serostatus between calves and their dams (P = 0.43). A fitted binomial generalised linear model revealed that there was an increasing risk of abortion with increasing IFAT titre and increasing age between 2 and 6 years.     

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.     

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.     

Survey of Neospora caninum infection by nested PCR in aborted fetuses from dairy farms in Aguascalientes, Mexico

The aim of this study was to determine by nested PCR probe the presence of Neospora caninum in fetal brain tissue from aborted dairy fetuses in Aguascalientes, a state in the central part of Mexico. Forty-four fetal brains from eight dairy farms were analyzed using single tube nested PCR probe with primers NF1, NS2, NR1 and SR1, with histopathology as the reference technique. From the 44 bovine fetuses studied, 35 (80%) were diagnosed as being infected by N. caninum using PCR probe and 20 (45%) were considered positive by histopathology, because they had lesions suggestive of bovine neosporosis. Fair agreement was observed (31%) between both probes. The age of aborting cows ranged from 1.6 to 2.9-year-old in 17 (49%) of the positive cases diagnosed by PCR. Gestational ages of aborted fetuses ranged from <3 to 9 months, with average of 5.6 months, and no statistical significant difference was observed when ages of infected and no infected fetuses were compared (P > 0.05). The frequency of N. caninum positive cases observed could be considered to be higher than other areas of Mexico and other countries.     

Neospora-like protozoan infection as a major cause of abortion in California dairy cattle

Ninety-five aborted bovine fetuses received from California dairies over a 4.5-year period had histologic lesions of focal encephalitis. Protozoa that reacted with Neospora caninum antiserum were detected in the brain of 88 of these fetuses and in the heart of 1 fetus. Sarcocystis spp schizonts were seen in the vascular endothelium of 1 fetus. It was concluded that a Neospora-like cyst-forming coccidian may be a major cause of abortion in California dairy cattle.     

First identification of Neospora caninum infection in aborted bovine foetuses in China

The first identification of Neospora caninum infection in the tissues of aborted bovine foetuses in China is reported. Aborted foetuses were collected from 16 dams, and 12 of the dams had high serum antibody titres to N. caninum determined using an ELISA test kit. The Nc-5 gene of N. caninum was amplified from DNA samples extracted from brains of four aborted foetuses using a Neospora-specific PCR assay, confirming N. caninum infection in the aborted foetuses. Histology and immunohistochemistry showed thick-walled (3 microm) tissue cyst in 25 microm diameter in the brain of one foetus. Non-suppurative encephalomyelitis, focal haemorrhage, hepatic lesions consisted of lymphocyte infiltration and haemorrhage were also found in the heart and lung of the foetus. Thus, we have confirmed for the first time the infection of N. caninum in aborted foetuses of cattle in the People's Republic of China.     

Neospora caninum infection associated with stillbirths in captive antelopes (Tragelaphus imberbis)

Neosporosis was diagnosed in full-term stillborn twin calves of captive antelopes (Tragelaphus imberbis) in a German zoo. In both calves a multifocal non-suppurative encephalitis was present and infection with Neospora (N.) caninum was confirmed by foetal serology and PCR. N. caninum-DNA was detected in brain, heart, lung, liver and spleen of a third full-term stillborn calf. Histopathological lesions were absent in this calf and antibodies against N. caninum were not detected in pleural fluid. Immunohistochemistry failed to demonstrate N. caninum antigen in all three calves.