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.     

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.     

不同药物体外抑制新孢子虫的比较

应用青蒿素、桦褐孔菌多糖、黄芩苷及乙胺嘧啶等药物对Vero细胞中培养的犬新孢子虫进行了体外抑制试验。结果显示,青蒿素、桦褐孔菌多糖、黄芩苷、乙胺嘧啶均有抑制虫体的效果,尤其青蒿素在高浓度试验组中抑虫效果最为明显,质量浓度为20 mg/L时的抑制率为95.17%,Vero细胞形态较为完整,虫体组织包囊体积最小、数量均最少;桦褐孔菌多糖(25 mg/L)、黄芩苷(20 mg/L)和乙胺嘧啶(1.25 mg/L)高质量浓度时的抑虫效果相近。结果表明,青蒿素、桦褐孔菌多糖、黄芩苷和乙胺嘧啶均可以作为抗新孢子虫的潜在药物,其中桦褐孔菌多糖为天然药物提取物,在抗新孢子虫药物中更具有研究价值。     

Incidence and vertical transmission rate of Neospora caninum in sheep

The infection by Neospora caninum in sheep can lead to abortion and the birth of weak and debilitated lambs. The aim of this study was to assess the incidence of natural infection by Neospora caninum and the vertical transmission rate among sheep. A flock of 50 sheep was monitored for serum antibody titres against N. caninum and seroconversion over a period of six months using an indirect ELISA technique. The offspring of the herd was also investigated regarding anti-N. caninum antibodies to determine the vertical transmission rate through the indirect immunofluorescence technique. The initial and final prevalences of infection by N. caninum were 26.0% (13/50) and 72.0% (36/50), respectively, and the incidence of infection by N. caninum in the present study was 62.2% (23/37). The vertical transmission rate found was 15.4% (2/13). A high incidence of infection by N. caninum in sheep was observed, and this is the first report assessing the incidence of N. caninum among naturally infected sheep.     

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.     

Influence of culture medium pH on internalization, growth and phenotypic plasticity of Neospora caninum

Neospora caninum, a strictly intracellular protozoan, is a major leading cause of parasite-induced abortion in cattle. A widely held view of N. caninum infection is that both cellular proliferation and stage interconversion (tachyzoite-bradyzoite transformation) are triggered, perhaps even modulated by, changes in cultural conditions. This study tested the hypothesis that exposure of N. caninum tachyzoites to different pH culture media affects the parasite's entry, proliferation and cyst formation in cultured cells. The endocytic pathway for N. caninum entry into the K562 cell line was found to be mediated by low pH of culture medium. Internalization of N. caninum by host cells was significantly increased in acidic and alkaline culture medium compared to cells maintained in neutral medium as revealed by transmission electron microscopy. Parasite proliferation within Vero cells was assessed by plaque formation assay and was found to be highest when pH level was optimum, paralleled by a decrease in the number of cysts. In contrast, parasite encystation increased when the pH level was alkaline or acidic, as evaluated by indirect immunofluorescence and immunocytochemical analyses. Acidic pH regardless of state of host cell infection suppressed the rate of host cell division. These findings suggest that culture medium pH has a determinable effect on the host cell-N. caninum interaction and support the hypothesis that pH of culture medium influence the entry, growth, and phenotypic plasticity of N. caninum in mammalian cells.     

Neospora caninum: in vivo and in vitro treatment with artemisone

Neosporosis caused by Neospora caninum has global economic, clinical, and epidemiological impacts, mainly in the cattle industry. Currently, there is no useful drug for treatment of neosporosis. This publication is the first to describe the significant benefits that artemisone has on Neospora infections both in vitro and in vivo. Artemisone is a new semi-synthetic 10-alkylamino artemisinin that is superior to other artemisinin derivatives in terms of its significantly higher antimalarial activity, its tolerance in vivo, lack of detectable neurotoxic potential, improved in vivo pharmacokinetics and metabolic stability. Low micromolar concentrations of artemisone inhibited in vitro Neospora development. Prophylactic and post-infection treatment profoundly reduced the number of infected cells and parasites per cell. In the in vivo gerbil model, a non-toxic dose prevented typical cerebral symptoms, in most animals. There were no signs of clinical symptoms and brain PCR was negative. Most treated gerbils produced high specific antibody titer and were protected against a challenge. Overall, artemisone could be considered as a future drug for neosporosis.     

Association of serologic status for Neospora caninum and postweaning feed efficiency in beef steers.

OBJECTIVE: To determine the effects of serologic status for Neospora caninum on short-term weight gain, feed intake, and feed efficiency (feed intake/gain). DESIGN: Longitudinal observational study. ANIMALS: 34 weaned mixed-breed beef steers. PROCEDURE: Serologic status for N. caninum was determined for each steer on days 0 (weaning), 88, 116, 144, 172, and 200, using an agglutination test. Individual steer body weight was measured on days 0, 88, 116, 144, 172, 200, and 242 (slaughter). Daily feed intake was monitored from days 116 through 242. Serologic status was matched to animal performance for the period immediately following serum sample collection. A mixed mode, using repeated-measures with an unstructured covariance matrix, was used in the analysis. Breed, age, and pen effects were controlled for in the analysis. RESULTS: A reduction in average daily gain for the period following a positive serologic result was detected for the entire trial (6 measurements/steer). This may have been attributed to a significant impairment in feed efficiency rather than to an impairment in feed intake. Changes in serologic status in individual steers over time were common; additionally, the effects of serologic status on steer performance were also transitory. CONCLUSIONS AND CLINICAL RELEVANCE: Significant reductions in short-term weight gain and feed efficiency were associated with the presence of antibodies against N. caninum in postweaning beef steers.     

Neospora caninum serostatus and culling of Holstein cattle

OBJECTIVE: To determine whether time until culling or risk of culling was associated with Neospora caninum serostatus among Holstein cattle in dairy herds in Ontario. DESIGN: Retrospective cohort study. ANIMALS: 3,412 cows in 56 herds. PROCEDURE: Blood samples were collected, and serum was tested for antibodies against N. caninum. Information on cows that were culled was collected during the 1- to 2-year period that producers were unaware of serostatus of individual cows in their herds. RESULTS: Herd prevalence of N. caninum-seropositive cows ranged from 0 to 68.3% (median, 7.0%). During the time of the study, 184 of 359 (51.3%) N. caninum-seropositive cows were culled, compared with 1,388 of 3,053 (45.5%) seronegative cows. Mean time from blood sample collection to culling for seronegative cows (289 days; 95% confidence interval, 280 to 299 days) was not significantly different from mean time for seropositive cows (296 days; 95% confidence interval, 269 to 323 days). Survival analysis indicated that N. caninum serostatus was not associated with time to culling or risk of culling. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that N. caninum serostatus of Holstein cows in Ontario was not significantly associated with either time to culling or risk of culling. Thus, N. caninum serostatus alone should not be used to determine whether cows should be culled.     

吉林株犬新孢子虫的分离与鉴定

将疑似感染犬新孢子虫而流产的牛胎儿脑组织接种于Vero细胞进行体外培养,同时感染长爪沙鼠进行动物试验.提取牛流产胎儿脑组织DNA、沙鼠脑组织DNA、细胞培养虫体DNA,用犬新孢子虫特异性引物进行PCR扩增.结果显示,PCR扩增出了1条约为681 bp的目的条带;测序结果表明,扩增出的特异性目的基因序列与GenBank中发表的犬新孢子虫NcSAG1基因(AF113004)核苷酸序列的同源性为99.4%.结果表明,在吉林省牛流产胎儿脑组织中分离出犬新孢子虫.     

抗弓形虫和新孢子虫药物体外筛选方法的研究进展

弓形虫和新孢子虫是2种在形态结构及生物学特征方面非常相似的细胞内寄生性原虫,二者引起的疾病对人和动物健康造成了严重危害。开发和筛选高效、低毒的抗弓形虫和新孢子虫药物一直是抗原虫药物研究领域的重要方向之一。药物筛选包括体外筛选和体内筛选,体外筛选的结果是进行体内筛选的依据和基础。对抗弓形虫和新孢子虫药物的体外筛选方法研究进展进行综述,以期为开发更加高效的抗弓形虫和新孢子虫药物体外筛选技术提供参考。     

新孢子虫对小鼠致病力及在小鼠体内动态分布的研究

小鼠是研究新孢子虫的动物模型。通过小鼠毒力试验,检测3株新孢子虫(Nc-1,Nc-LIV和Nc-BJ)对BALB/c小鼠的致病力差异;同时研究了低剂量Nc-LIV株(1×10~6)感染后,新孢子虫在小鼠的心、肝、脾、肺、肾、脑和后肢肌肉中的分布情况及其在血清中抗体的动态变化规律。结果显示,Nc-LIV对BALB/c小鼠的毒力最强,Nc-BJ其次,Nc-1最弱。小鼠血清中新孢子虫抗体在感染后4d出现至第32天达到峰值。感染后第8天在脑组织中可检测到新孢子虫特异性基因Nc-5,在第32天达到峰值后下降;其他组织中则分布很少。通过观察新孢子虫Nc-LIV在小鼠体内各器官组织动态分布的规律,为进一步建立小鼠感染新孢子虫的动物模型,研究新孢子虫病奠定了基础。     

新孢子虫SRS2基因的克隆及原核表达

为构建表达新孢子虫重组蛋白SRS2原核表达系统,本研究以新孢子虫基因组为模板PCR扩增SRS2蛋白部分基因,获得大小约为998bp的目的片段,并克隆到大肠杆菌PET-32a载体中,IPTG诱导重组大肠杆菌,通过SDS—PAGE和免疫印迹分析表达产物并鉴定其生物学活性。经SDS—PAGE分析,表达的重组SRS2蛋白分子量约为54kD,免疫印迹结果表明,表达的重组蛋白与牛抗新孢子虫阳性血清反应形成一条特异性蛋白条带。本实验为进一步研究诊断和治疗新孢子虫病奠定了基础。     

Neospora caninum serostatus and milk production of Holstein cattle

OBJECTIVE: To determine whether Neospora caninum serostatus was associated with milk production among Holstein cattle in Ontario. DESIGN: Case-control study and cross-sectional observational study. ANIMALS: 3,702 Holstein cows in 83 herds (case-control study) and 3,162 Holstein cows in 57 herds. PROCEDURE: Herds in the case-control study were grouped on the basis of N. caninum abortion status. Herds in the observational study were considered representative of Ontario dairy herds. The N. caninum serostatus of individual cows was determined with a kinetic ELISA. Milk production was modeled to compare seropositive with seronegative animals while controlling for parity, days since parturition, and herd clustering. RESULTS: In the case-control study, 305-day milk production of seropositive cows was significantly less than milk production of seronegative cows in herds with abortions attributable to N. caninum infection and in herds with abortions attributable to pathogens other than N. caninum, but not in herds without abortion problems. In the observational study, 305-day milk production for seropositive cows was not significantly different from milk production of seronegative cows. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that the association between N. caninum serostatus and milk production in Ontario Holstein dairy cattle may depend on abortion status of the herd. In herds with abortion problems, regardless of cause, N. caninum-seropositive cattle produced less milk, whereas in herds without abortion problems, N. caninum-seropositive cattle produced the same amount of milk as seronegative cattle.     

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.     

新孢子虫二温式PCR检测方法的建立及应用

根据新孢子虫Nc2基因保守区设计特异性引物,应用均匀设计法优化反应中Taq DNA聚合酶、引物浓度和退火温度等,建立新孢子虫二温式PCR检测方法。结果显示,扩增条带与预期目的片段大小(266bp)相符,未扩增出弓形虫等虫种的阳性DNA片段;扩增片段经克隆、测序发现与引物基因序列的同源性为100%;最低检出量为32.5fg/μL,是三步法PCR的10-1倍,但循环时间缩短了38min;重复3次对10-4、10-5、10-6和10-7稀释的阳性质粒进行二温扩增,10-7稀释均未出现扩增条带。同时对156份牛全血DNA进行检测,检出率为10.90%(17/156),与《新孢子虫病检疫技术规范》(SN/T 3499—2013)的检出符合率为83.33%(30/36),二者检测结果差异性不显著(P0.05)。由此可见,建立的二温式PCR检测方法可用于临床诊断和日常监测新孢子虫,为监控"带虫宿主"提供技术支撑。     

牛源犬新孢子虫NcGRA7基因的克隆及原核表达分析

为了解牛源犬新孢子虫NcGRA7基因的生物学特性,本实验采用PCR技术扩增牛源犬新孢子虫NcGRA7基因,并连接至pMD-18T载体中,构建重组质粒pMD 18-NcGRA7,经PCR、酶切鉴定及测序分析,将NcGRA7基因连接到原核表达载体pGEX-4T-1中,构建重组表达质粒pGEX-NcGRA7,转化大肠杆菌BL21,筛选阳性克隆,将PCR和酶切鉴定正确的重组质粒进行IPTG诱导表达,SDS-PAGE和westem blot分析表达产物.结果表明,扩增的NcGRA7基因片段大小为701 bp,编码233个氨基酸,与GenBank中登录的NcGRA7(AF176649)核苷酸序列同源性为98.7％;western blot分析表达蛋白的分子量为52 ku,具有较好的反应原性.本实验为犬新孢子虫NcGRA7基因疫苗研究奠定了基础.     

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.     

Prevalence and transmission of Neospora caninum within infected Swedish dairy herds

In this long-term study, the changes in within-herd Neospora caninum seroprevalences and the relative importance of horizontal and vertical parasite transmission in Swedish dairy herds were investigated. The within-herd prevalences varied between 5.8% and 65.0% when the herds were first sampled. Comparing the prevalences of year 2000 with those found 2-3 years later, nine out of 14 investigated herds had a similar or lower within-herd prevalence while five herds had an increased portion of seropositive individuals. Three herds were free or almost free from infection at their last sampling. Changes in prevalence were not related to the level of prevalence that each herd had when first sampled. Antibody avidity measurements revealed that the majority of seropositive individuals in all herds were chronically infected. Dam-calf pair investigations and inspection of genealogic trees supplemented with antibody test results showed that vertical transmission was by far the dominant transmission route in all herds. Taken together, these results indicate that the prevalence of N. caninum infection in Swedish dairy cattle can be successfully reduced if actions to eliminate infection in a herd are combined with preventive measures to avoid re-infection.