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.     

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.     

In vitro isolation and characterization of bovine Neospora caninum in Korea

The brains of nine aborted bovine fetuses and two newborn calves born from dams suspected to be infected with Neospora caninum were homogenized and inoculated into Vero cells. All fetuses and calves were from cows determined as seropositive to N. caninum by an IFA test. Sera and thoracic fluids of all fetuses and calves also revealed high antibody titer to N. caninum by IFAT ranging from 1:800 to 1:3200. N. caninum was isolated from the brains of one aborted fetus and one newborn calf when the brain homogenates were grown continuously in Vero cell culture. N. caninum tachyzoites, giemsa-positive, were first observed on Days 45 and 56 postinoculation in the newborn calf and the aborted fetus, respectively. The isolates (KBA-1 and KBA-2) were morphologically and ultrastructurally similar to previously published Neospora isolates. The isolated parasites were confirmed as N. caninum by means of the antigenic reactivities, immunostaining, PCR and southern blotting, and electron microscopy.     

牛源犬新孢子虫孕鼠感染模型的建立

为建立牛源犬新孢子虫孕鼠感染模型,深入研究牛源犬新孢子虫对孕鼠的致病作用,本试验以雌性BALB/c小鼠为试验动物,分离Vero细胞中培养的牛源犬新孢子虫速殖子,分不同剂量组腹腔接种雌性BALB/c小鼠后,与雄性BALB/c小鼠合笼,每天观察小鼠临床症状和发病情况,观察主要脏器组织的病理变化,应用PCR方法检测孕鼠脑、肝脏、脾脏等脏器组织及胎盘中犬新孢子虫Nc5基因,并测定孕鼠胎盘湿重和胎盘系数。结果显示,感染模型小鼠的最佳攻虫剂量为10⁵个虫体;感染犬新孢子虫孕鼠先后出现精神不振、共济失调等临床症状,并有不同程度死亡;病理学观察模型小鼠脑、肝脏、脾脏等脏器组织出现充血、出血、肿大等病理变化;在模型小鼠脑、肝脏、脾脏等脏器组织及胎盘中检测到犬新孢子虫Nc5基因;随攻虫天数的增加,模型小鼠胎盘重量和胎鼠重量均不断增加,胎盘系数逐步降低,在第12、14、16天时,模型组与对照组相比,胎盘重量和胎盘系数均差异显著（P < 0.05）。本试验成功建立了牛源犬新孢子虫孕鼠感染模型,为犬新孢子虫致病机制研究奠定了基础。     

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.     

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.     

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.     

Neospora caninum infected the alimentary tract of nude mice and was transmitted to other mice by intraperitoneal inoculation with the intestinal contents

Neospora caninum (BT-2 strain) that originated from the brain of a Holstein calf was serially passaged through 10 generations of BALB/c nude mice by intraperitoneal inoculation. Histological examination of the mice revealed that numerous clusters of tachyzoites appeared in the pancreas, stomach and small intestine as well as in the central nervous system (CNS) and skeletal muscles. Intestinal contents of the infected mice were inoculated intraperitoneally into uninfected nude mice and 3 of the 17 inoculated mice showed clinical signs at post inoculation days 3 to 10. The present experiments demonstrated a proliferation of N. caninum tachyzoites in the mucosa of the alimentary tract and pancreas of the nude mice and the intestinal contents of the mice were infective to other nude mice.     

Isolation of Neospora caninum from a calf in Malaysia

In order to attempt isolate the protozoan parasite Neospora caninum, an N. caninum seropositive pregnant Sahiwal Friesian cross heifer from a large-scale dairy farm in Malaysia was kept for observation until parturition at the Veterinary Research Institute, Ipoh. The heifer gave birth to a female calf that was weak, underweight and unable to rise. Precolostral serum from the calf had an N. caninum indirect fluorescent antibody test titre of 1:3200. It died 12 h after birth and necropsy was performed. Brain homogenate from the calf was inoculated into 10 BALB/c mice that were kept for 3 months after which brain tissue from the mice was inoculated onto 24 h fresh monolayer Vero cell lines. The cell cultures were examined daily until growth of intracellular protozoa was observed. DNA of the organisms from the cell cultures was analyzed by PCR and DNA sequencing. DNA fragments of the expected size were amplified from the isolate using N. caninum-specific primers, and sequence analysis of ITS1 clearly identified the isolate as N. caninum. This is the first successful isolation of N. caninum from a bovine in Malaysia, and the isolate is designated Nc-MalB1.     

Use of a serum-free medium to produce in vitro Neospora caninum and Toxoplasma gondii tachyzoites on Vero cells

Neospora caninum and Toxoplasma gondii are cyst-forming coccidian parasites of human and veterinary clinical relevance. In vitro cultivation of the protozoans using Vero cells is usually performed in order to produce antigenic materials. Quantitative and qualitative comparisons of Vero cells grown in RPMI medium supplemented either with foetal calf serum (FCS), horse serum (HS) or a specific serum-free additive (DefCell) were performed. A serum-free cell culture system used to propagate N. caninum (NC-1 isolate) and T. gondii tachyzoites (Rh stain) were compared with the other two cell culture systems. FCS supplemented media was found to be more effective than the others in promoting Vero cells and N. caninum tachyzoites. However, it was found unable to support adequate T. gondii tachyzoite proliferation. Vero cells, T. gondii and N. caninum tachyzoite production gave similar growth patterns with either HS or DefCell supplemented media. Defcell was considered as a good alternative to supplement culture medium.     

Confirmed clinical Neospora caninum infection in a boxer puppy from Argentina

Generalized neosporosis was diagnosed in a 2-month-old boxer puppy. The dog had a history of progressive paralysis and muscle atrophy, followed by cervical weakness, stiff jaws and dysphagia. The dog had a 1:12,800 antibody titer for Neospora caninum and was negative for antibodies to Toxoplasma gondii by the indirect fluorescent antibody test (IFAT). After euthanasia a complete necropsy was carried out. The puppy had a megaesophagus. Microscopically, tachyzoites and tissue cysts were observed in histologic brain sections. Severe myositis was observed in esophagus and striated muscle sections and several groups of tachyzoites were associated with these lesions. Immunohistochemically, parasites in the brain and striated muscle reacted to anti-N. caninum antiserum. Western blot analysis allowed the identification of three major and four minor antigens of N. caninum tachyzoites corresponding to 30, 37, 45-kDa and 28, 29, 43, 47 and 67-kDa bands, respectively. Cerebral homogenate of the dog was inoculated into four Mongolian gerbils (Meriones unguiculatus). Forty-nine days after inoculation, all the gerbils had positive IFAT titers to N. caninum (1:200, 1:400, 1:100 and 1:400). Genomic DNA was isolated from the brain, lung and striated muscle from the puppy and from the brain of one of the inoculated gerbils. The N. caninum specific primer pair Np 6/21 produced 328 bp amplicons on electrophoretic gels. This is the first confirmed clinical case of generalized canine neosporosis in Argentina.     

Neosporosis in Beagle dogs: clinical signs, diagnosis, treatment, isolation and genetic characterization of Neospora caninum

Clinical neosporosis was diagnosed in a litter of five pups born to a Beagle bitch from Virginia, USA. Four of the pups developed limb weakness starting at 4 weeks of age. The dogs were suspected to have neosporosis based on clinical signs and empirically treated with Clindamycin (75 mg, oral, twice daily, total 150 mg) starting at 9 weeks of age and the dosage was doubled at 13 weeks of age. Antibodies to Neospora caninum were detected in sera of the dam and pups when first tested serologically at the age of 4 months. The owner donated the pup with the worst clinical signs and the dam for research; both dogs were euthanized. Viable N. caninum was isolated in gamma interferon gene knock out (KO) mice and in cell culture from the pup killed at 137 days of age. Tissue cysts, but no tachyzoites, were found in histological sections of brain and muscles. The isolate was also identified as N. caninum by PCR and sequence analysis and designated NC-9. N. caninum was neither isolated by bioassay in KO mice nor found in histological sections of tissues of the bitch. Clinical signs in the remaining three pups improved considerably after a 6-month treatment with Clindamycin; N. caninum antibody titers were still persistent in these pups at 23 months of age. Results indicate that medication with Clindamycin can improve clinical condition but not eliminate N. caninum infection.     

Finding of Neospora caninum in the wild brown rat (Rattus norvegicus)

Nine rats (16.4%) out of 55 (Rattus norvegicus) from cattle farms were seropositive to Neospora caninum. Two of the seropositive rats were also PCR positive but all were negative by immunohistochemistry and PAS staining. The brains of all the captured rats were homogenized and initially inoculated intraperitoneally into nude mice or into SPF ICR mice, which had been immunosuppressed with prednisolone. One mouse that was inoculated with brain material from a seropositive rat became infected with N. caninum, as demonstrated by the presence of a tissue cyst in the brain and confirmed by immunohistochemistry and PCR. This is the first finding of N. caninum in naturally infected farm rats. The findings show that natural N. caninum infection occurs in wild brown rats and thus rats may serve as a reservoir for the protozoan on the cattle farm.     

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.     

Monoclonal antibodies preventing invasion of Neospora caninum tachyzoites into host cells

Twelve monoclonal antibodies (MAbs) against Neospora caninum tachyzoites were produced to specify the antigens related to the invasion of tachyzoites into host cells. In the assay to evaluate the inhibition activity, all these MAbs prevented the cultured Vero cells from the invading by the tachyzoites. These MAbs recognized approximately a 73 kDa antigen in Western blot analysis. Immunofluorescence assay and immune electron microscopy revealed that this 73 kDa antigen is a part of the surface antigens of N. caninum tachyzoite, and that the tachyzoite antigen identified plays an important role for invasion of host cells.     

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.     

Neospora hughesi: experimental infections in mice, gerbils, and dogs

Neospora hughesi is a recently described cause of equine protozoal myeloencephalitis (EPM). A rodent model for pathogenicity would facilitate development of therapies to be used in horses. In the present study, we examined the susceptibility of BALB/c gamma-interferon gene knockout (gamma-INFKO), BALB/c, CD-1, and C57BL/6 strains of mice and gerbils to infection with tachyzoites of the Nh-A1 strain of N. hughesi isolated from a horse from AL, USA. Only the gamma-IFNKO mice developed severe clinical disease following infection with N. hughesi and died 19-25 days after infection and exhibited severe cardiac lesions. In contrast, experimental infection of gamma-INFKO mice with tachyzoites of the NC-1 or NC-Liverpool strains of Neospora caninum resulted in deaths 8-10 days after infection. The most severe lesions were in the livers, spleens, and lungs of these mice. Gerbils inoculated with N. hughesi did not develop clinical disease, had few microscopic lesions, but did seroconvert. Two dogs fed the brains of mice, shown to contain N. hughesi tissue stages by cell culture and gamma-IFNKO mouse bioassay, did not shed N. caninum-like oocysts over a 23 days observation period. The marked difference in pathogenicity between the two species of Neospora in gamma-IFNKO mice, and lack of oocyst excretion by dogs fed N. hughesi infected mice provide additional evidence that the species distinction between N. caninum and N. hughesi is valid.     

IgG avidity pattern in cattle after ingestion of Neospora caninum oocysts

The avidity (functional affinity) of specific antibodies are being used to estimate duration of bovine Neospora caninum infection. Here, we report for the first time the avidity pattern in cattle orally inoculated with N. caninum oocysts. In all, 16 pregnant cows and 7 calves were administered N. caninum oocysts. In the cows, the avidity increased during the early course of infection. In all but one, the avidity was < or = 35 during the first 6 weeks after infection and no cow had an avidity value >50 until week 9. The calves were sampled either week 6 (n = 3) or week 9 (n = 9) after infection, and by then had avidities between 2 and 17. The results are in agreement with results from previous investigations of naturally infected cattle, and calves that were experimentally infected with tachyzoites. They further validate the ability of the N. caninum iscom avidity ELISA to accurately assess the duration of infection.     

Isolation of Neospora caninum from the brain of a naturally infected dog, and production of encysted bradyzoites in gerbils

Neospora caninum was isolated from the brain of an adult dog in Brazil. Cerebral tissue from the dog was inoculated into Mongolian gerbils. Gerbils were euthanized 3-4 months later and bradyzoite-containing tissue cysts were observed in their brains. N. caninum (designated NC-Bahia) was isolated in cell culture after inoculation with tissue cysts from the gerbils. The identity of the parasite was confirmed by immunohistochemical examination and polymerase chain reaction (PCR). Gerbils may be a useful alternative to immunosuppressed mice for isolation of N. caninum and for production of encysted bradyzoites.     

Gray wolf (Canis lupus) is a natural definitive host for Neospora caninum

The gray wolf (Canis lupus) was found to be a new natural definitive host for Neospora caninum. Neospora-like oocysts were found microscopically in the feces of three of 73 wolves from Minnesota examined at necropsy. N. caninum-specific DNA was amplified from the oocysts of all three wolves. Oocysts from one wolf were infective for the gamma interferon gene knock out (KO) mice. Viable N. caninum (designated NcWolfUS1) was isolated in cell cultures seeded with tissue homogenate from the infected mouse. Typical thick walled tissue cysts were found in outbred mice inoculated with the parasite from the KO mouse. Tissue stages in mice stained positively with N. caninum-specific polyclonal antibodies. Our observation suggests that wolves may be an important link in the sylvatic cycle of N. caninum.