牛新孢子虫NcSRS2基因腺病毒穿梭载体的构建及在293细胞中的表达

应用PCR技术扩增牛新孢子虫NcSRS2基因,纯化PCR产物后与克隆载体pMD18-T Simple Vector连接,将PCR、酶切鉴定及测序分析正确的pMD-18T-NcSRS2重组质粒进行EcoRⅠ和XbaⅠ双酶切,克隆至相同酶切回收后的腺病毒穿梭载体pCR259中,再将PCR、酶切鉴定正确的pCR259-NcSRS2重组质粒转染293细胞,应用IF-AT和Western-blotting技术检测重组质粒在293细胞中的表达情况。结果显示,扩增的牛新孢子虫NcSRS2基因长度为1 227bp,与GenBank中发表的NcSRS2（AF061249）核苷酸序列同源性为99%,构建的pCR259-NcSRS2重组质粒在293细胞中得到瞬时表达,表达蛋白的相对分子质量为43 000,具有较好的反应原性。本试验为新孢子虫病腺病毒载体疫苗的构建奠定了基础。     

犬新孢子虫NcSRS2基因原核表达质粒的构建

根据已发表的犬新孢子虫NcSRS2基因序列,设计1对含有EcoRⅠ和NotⅠ酶切位点的引物。以提取犬新孢子虫虫体基因组DNA为模板,应用PCR扩增获得NcSRS2 ORF基因片段,将此基因片段克隆到pMD18-T Simple载体上,用EcoRⅠ和NotⅠ双酶切该片段,回收得到含有两个酶切位点黏端的Nc-SRS2 ORF基因,将此基因片段克隆至相同酶切回收后的PGEM-4T-2原核表达载体中,获得重组质粒pGEX-NcSRS2,经PCR鉴定,限制性内切酶分析和克隆片段序列测定比较,证实了重组质粒的正确性。     

新孢子虫NcSRS2基因的亚克隆和表达

本研究根据NcSRS2基因序列设计合成一对引物,将上、下游引物分别引入EcoRI,XhoI酶切位点,用PCR技术从pGEX-NcSRS2重组质粒扩增截去N端疏水氨基酸序列NcSRS2的基因片段（以下称dNcSRS2）,插入到pGEX-6p-1质粒的多克隆位点,转化大肠杆菌BL21感受态细胞,于氨苄阳性LB培养平板上筛选阳性克隆,酶切及PCR鉴定;经IPTG诱导在E.coli中表达,用SDS-PAGE和免疫印迹分析表达产物并纯化.结果表明,新孢子虫dNcSRS2基因体外扩增产物与预期值相符,约1041bp;所构建pGEX-dNcSRS2重组质粒经双酶切与PCR鉴定,与预期结果一致;SDS-PAGE和免疫印迹显示,表达融合蛋白的分子量约为62.6 kD,表达效率为32.3%,该蛋白具有特异的免疫反应性,为新孢子虫病诊断试剂盒的研制和疫苗的研制奠定了基础.     

犬新孢子虫NcSRS2基因真核表达质粒的构建

根据犬新孢子虫NcSRS2基因序列，设计了1对含有Kozak序列、PstⅠ和XbaⅠ酶切位点的引物，以含有NcSRS2基因的质粒P43为模板，经PCR扩增获得NcSRS2 ORF基因片段，用PstⅠ和XbaⅠ双酶切该片段，回收得到含有以上2个酶切位点黏端的NcSR2 ORF基因，将此基因片段克隆至相同酶切回收后的pcDNA3．1（＋）真核表达载体中，获得重组质粒pcNCSRS2。经PCR鉴定、限制性内切酶分析和克隆片段序列测定、比较，证实了重组质粒的正确性。     

牛源犬新孢子虫NcSRS2-NcGRA7融合基因的克隆与生物信息学分析

为了解牛源犬新孢子虫NcSRS2-NcGRA7融合基因的生物学特性,本试验提取牛源犬新孢子虫基因组DNA,应用PCR技术扩增犬新孢子虫表面蛋白基因NcSRS2和致密颗粒蛋白基因NcGRA7,SOE-PCR技术拼接NcSRS2和NcGRA7基因,构建NcSRS2-NcGRA7融合基因重组克隆质粒,并进行PCR鉴定、双酶切鉴定及生物信息学分析。结果,NcSRS2基因扩增片段大小为1 061 bp,NcGRA7基因扩增片段大小为364 bp,NcSRS2-NcGRA7融合基因扩增片段大小为1 482 bp;获得重组克隆质粒pMD18-NcSRS2-NcGRA7经PCR鉴定、双酶切鉴定正确;测序分析表明,与Gen-Bank中已发表的美国株犬新孢子虫(AF061249、AF176649)核苷酸序列同源性为99%;经DNAman等软件分析,预测NcSRS2-NcGRA7融合蛋白抗原指数较高,融合蛋白二级结构以α-螺旋和β-折叠为主,三级结构中2种蛋白独立折叠,并借助Linker互相连接,功能互不影响。本试验为牛源犬新孢子虫NcSRS2-NcGRA7融合蛋白的免疫学研究奠定了基础。     

Confirmation that the dog is a definitive host for Neospora caninum.

Two mixed-breed littermate dogs were fed mouse brains containing tissue cysts of the NC-beef isolate of Neospora caninum. Both dogs excreted N. caninum oocysts in their feces. Dog 1 which was given methylprednisolone acetate (MPA) prior to ingesting tissue cysts, excreted oocysts on days 5 to 10 inclusive and on day 17 after ingesting tissue cysts. Dog 1 had a serum antibody titer of 1:200 in the indirect fluorescent antibody test (IFAT) 35 days after it was fed tissue cysts. Dog 2, which was not treated with MPA, excreted oocysts on Day 6 and Day 9 after ingesting tissue cysts. Antibodies to N. caninum were not found in a 1:25 dilution of serum on any examination period for Dog 2 during the study. Neospora caninum was not found in the tissues of either dog by histological or immunohistochemical means following necropsy 42 days after being fed tissue cysts. The identity of the oocysts excreted in the feces of the dogs was confirmed by mouse inoculation studies.     

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.     

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.     

Disseminated infection with Neospora caninum in a ten-year-old dog

Naturally-occurring neosporosis with multiple organ involvement was identified in a 10-year-old neutered male Basset hound dog. Clinical signs were first noticed 3 weeks prior to referral and consisted of crouched stance and mild pelvic limb ataxia. Dexamethasone administration had provided transient improvement. On presentation to the teaching hospital, clinical signs included depression, pelvic limb ataxia, inability to stand without assistance, and pain on deep palpation of the cervical and lumbar vertebral column. Lesions were found in the myocardium, liver, spleen, adrenal glands, brain, and spinal cord. Tachyzoites of Neospora caninum were found in the myocardium and adrenal glands. Organisms stained with anti-Neospora caninum, but not to anti-Toxoplasma gondii serum in an immunohistochemical test.     

Neospora caninum is a cause of perinatal mortality in axis deer (Axis axis)

Neospora caninum is a worldwide distributed protozoan that may cause neuromuscular disease in dogs and reproductive failure in domestic and wild ruminants. One axis fawn (Axis axis) and four neonates from the same deer herd died at a zoo in Argentina within a four-month period. The fawn presented with dilatation of the anal sphincter at birth and incontinence, developed weakness and ataxia and died at 14 days of age. At necropsy, a mega formation of the distal large intestine was observed. Microscopically, non-suppurative encephalitis, suppurative bronchopneumonia, fibrin necrotic enteritis and degenerative changes in the liver were observed in hematoxilin and eosin-stained tissue sections, and thick-walled N. caninum-like cysts were observed in fresh brain samples. Serologic studies for N. caninum revealed an IFAT titer of 1:6400 in the fawn and 1:25, 1:400, 1:3200 and 1:6400 in the neonates. N. caninum DNA was detected in brain samples from the fawn and from one neonate by PCR, and the parasite was isolated in vitro from the fawn’ brain after passage through gerbils (Meriones unguiculatus) and gamma-interferon knock-out mice. N. caninum DNA obtained from the fawn, neonate and isolated parasites showed the same microsatellite pattern. This suggests a common infection source for both animals.The diagnosis of N. caninum infection was confirmed, suggesting its association with perinatal mortality in captive axis deer. To the best of our knowledge, this is the first report of clinical disease associated to N. caninum infection in axis deer and of isolation of the parasite from this wild ruminant species.     

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.     

Diagnosis and treatment of Neospora caninum infection in a dog

Neospora caninum, a protozoan organism, caused extensor rigidity of the pelvic limbs in a 12-week-old dog. Diagnosis was based on results of muscle biopsy, neuroelectrodiagnostics, serotesting, and cell culture. Indirect fluorescent antibody (IFA) titer to N caninum was 1:800 at time of admission and 1:3,200 after 4 and 6 weeks. A reciprocal IFA titer of 50 to N caninum was also found in the CSF. Serotesting for T gondii was negative. Treatment with clindamycin followed by sulfadiazine and trimethoprim did not change the pelvic limb extensor rigidity, but other signs of minor neurologic dysfunction improved.     

Serodiagnosis of Neospora caninum infection in cattle using a recombinant tNcSRS2 protein-based ELISA

The truncated NcSRS2 gene (tNcSRS2) by removal of the N-terminal hydrophobic sequence was cloned into the pGEX-6p-1 plasmid and subsequently expressed as a glutathione-S-transferase (GST) fusion protein. The purified recombinant tNcSRS2 protein was specific to Neospora caninum and was used in an ELISA for the diagnosis of neosporosis. There was a good agreement between tNctSRS2-based ELISA and three commercially available diagnostic kits (IDEXX ELISA, HIPRA ELISA and VMRD IFAT). Three hundred dairy cattle serum samples from 9 regions were tested by our ELISA. The herd prevalence was 100% and the overall prevalence was 20.3%. There was a statistically significant difference in seroprevalence between regions (P<0.01) and an association between abortion history and N. caninum seropositivity. Our study showed that neosporosis in dairy cattle is widespread in China.     

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.     

Congenital Neospora caninum infection in a calf with spinal cord anomaly

Neospora caninum was identified in a calf with spinal cord anomaly in Australia. The calf was full-term and born dead. The caudal cervical and cranial thoracic segments of the spinal cord of the calf were asymmetric because of marked unilateral reduction of ventral gray matter and focal cavitation. Mild focal disseminated nonsuppurative encephalomyelitis was associated with N caninum tissue cysts. Tissue cysts were 16 to 35 microns X 10 to 27 microns, and the cyst walls were 1 to 3 microns thick. In an immunohistochemical test, the parasite stained with N caninum serum but not T gondii serum.     

Astroglial cells in primary culture: a valid model to study Neospora caninum infection in the CNS

The protozoan Neospora caninum has a veterinary importance because it causes abortion in cattle and neuromuscular alterations in dogs. We infected rat astrocytes, in vitro, with different concentrations of N. caninum. Astrocytes responded to infection by producing the pro-inflammatory cytokine TNF-alpha and the neurotoxic-free radical NO, 24 and 72 h post-infection. These data suggest that astrocytes, which are essential for brain function, are targets for the parasite and this represents a practical and valid model to study the effects of N. caninum on the CNS.     

Neospora caninum infection as a cause of reproductive failure in a sheep flock

Neospora caninum has been detected only sporadically in cases of ovine abortion, and it has therefore traditionally been considered as an unimportant parasite in small ruminants. This study was carried out with the aim of identifying the pathogen causing serious reproductive problems on a commercial sheep farm. Sera from all rams and ewes tested negative for antibodies against Border disease virus, Schmallenberg virus and Coxiella burnetii, and infections by these agents were therefore ruled out. Nevertheless, seropositivity to N. caninum and/or Toxoplasma gondii was detected, although the seroprevalence was higher in the case of N. caninum. The percentage of lambings and the number of lambs per dam were significantly lower in ewes that were seropositive to N. caninum while no effect on these parameters was detected in ewes that were seropositive to T. gondii. There was also no evidence of infection by T. gondii in the foetal/lamb tissues analyzed by PCR and/or immunohistopathological techniques. On the contrary, the DNA of N. caninum was detected in 13 out of 14 foetuses/lambs descendant from dams seropositive to this parasite. Characteristic lesions caused by N. caninum and/or its antigen were also detected. Genotyping of the N. caninum DNA revealed only two closely related microsatellite multilocus genotypes. The results clearly demonstrate that infection by N. caninum was the cause of the low reproductive performance of this sheep flock.