细胞因子在犬新孢子虫感染中的作用

犬新孢子虫是一种专一性细胞内寄生虫，它的感染导致了宿主Th细胞的分化。一方面，刺激宿主产生Th1型细胞因子反应，这些细胞因子包括IL-12、IFN-7和INF-α。Th1型细胞因子反应同时还激活了产生氧自由基（FOR）、一氧化氮（NO）及其代谢物，这些细胞因子对犬新孢子虫均具有致命的杀伤力。另一方面，引发宿主产生IL-4、IL-5和IL-10等Th2型细胞因子，Th2型细胞因子主要在宿主妊娠过程中发挥作用。     

Intrauterine Neospora caninum inoculation of heifers

Here, we studied the potential of Neospora caninum tachyzoites to infect heifers when administered in utero by artificial insemination via contaminated semen. Eighteen primiparous cyclic heifers were hormonally synchronized and artificially inseminated. Nine of them, which were inseminated with semen containing 10(7) live N. caninum NC-1 isolate-tachyzoites, reacted with seroconversion and a specific IFN-gamma response. Moreover, N. caninum DNA was demonstrated by a nested-PCR in the blood of all nine heifers and in brain, lungs, liver and uterine horn of several of them. In contrast, nine heifers inseminated with tachyzoite-free semen developed no antibody or IFN-gamma responses, and no parasite DNA was detected in blood or organs. At necropsy, viable embryos were detected in one and six of the infected and non-infected heifers, respectively. No specific Neospora DNA was detected in any of the embryos. This study provides evidence that intrauterine inoculation via contaminated semen cause N. caninum infection in cattle.     

Experimental studies on the transmission of Neospora caninum between cattle

Three studies were conducted to investigate the transmission of Neospora caninum between cattle by the oral route. In the first study, six calves were dosed with 10(7)N caninum tachyzoites (NC LivB1) in colostrum and/or milk replacer on four occasions. In the second study, two calves and two cows were fed placental tissues from N caninum -infected cows, and, in the third study, seven uninfected calves were fostered onto N caninum -infected dams. In the first study, all six calves developed antibody responses and five calves developed antigen-specific lymphoproliferation responses, including two calves initially challenged at 1 week of age. No evidence of N caninum infection was found in the brain or heart of these calves by histology or polymerase chain reaction (PCR). In the second and third studies, there was no evidence of N caninum infection in any of the calves and cows. The results confirm that calves up to 1 week of age can be experimentally infected via the oral route, but suggest that this is not an important natural route of transmission for N caninum between cattle.     

新孢子虫与宿主关系研究进展

新孢子虫是一种传播范围极广的寄生性原虫,是导致母畜流产、死胎,新生胎儿运动或神经障碍的主要病原之一。论文详细介绍了新孢子虫表面抗原、致密颗粒抗原及棒状体抗原等侵入相关抗原,并对新孢子虫黏附、侵入、繁殖、释放的过程和相关侵入机制等方面进行了阐述,同时探讨了虫体侵入宿主后增强机体细胞免疫、体液免疫反应状况及所引起的相关Th1/Th2型免疫应答,阐明了宿主妊娠期间抵抗新孢子虫由强至弱的动态变化及相关性,并对未来犬新孢子虫的相关研究做了展望。     

Repeated transplacental transmission of Neospora caninum in dogs

Four litters of German Shorthaired Pointers from one owner developed a toxoplasmosis-like illness. According to the records, 29 of 39 dogs had hind limb paralysis. Six dogs from 2 litters were necropsied and had generalized encephalomyelitis. Tachyzoites and tissue cysts of Neospora caninum were found in the brain and spinal cord of each dog. Lesions were found in the eyes, extraocular muscles, or both in all of the dogs, and N caninum was detected microscopically in the eyes (retina and choroid in 1 dog), extraocular muscles, or both in 5 of the 6 dogs. Ocular lesions consisted of focal retinitis, choroiditis, mild nonspecific iridocyclitis, and myositis of extraocular muscles. Organisms stained with anti-N caninum serum, but not with anti-Toxoplasma gondii serum in an immunohistochemical test, except in 1 dog. In one dog, aged thick-walled N caninum tissue cysts reacted mildly with anti-T gondii serum.     

Detection of Neospora caninum in semen of bulls

In cattle, transplacental infection is the main route of Neospora caninum transmission, but postnatal transmission by the oral uptake of sporozoite-containing oocysts shed by dogs may also be possible. Other routes of horizontal transmission, such as the venereal route, have not been investigated. In this study, we evaluated the presence of N. caninum DNA by a nested-PCR in fresh non-extended semen and frozen extended semen straws of five Holstein-Friesian bulls with naturally-acquired neosporosis. The infection status was assessed by an immunofluorescent antibody test (IFAT) and confirmed by immunoblotting (IB). Because of inhibitory components of semen, a protocol was developed to purify N. caninum DNA from bovine semen. Sporadically, N. caninum DNA was detected in non-extended fresh semen samples and frozen extended semen straws of the five seropositive bulls. In all positive samples, specific DNA was consistently found in the cell fraction of semen and not in seminal plasma. The parasite mean load in positive fresh semen samples determined by a real-time PCR was low oscillating between 1 and 2.8 parasites/ml of semen (maximum parasite load detected in one sample was 7.5 parasites/ml of semen). In parallel, another three similar but uninfected bulls acted as controls and no N. caninum DNA was amplified in any of their fresh and straw semen samples assayed. Whether venereal transmission plays a role in the spread of bovine neosporosis needs to be determined.     

奶牛新孢子虫病的血清学诊断

1984年,犬新孢子虫(Neospora caninum)第一次在挪威的一条狗体内被发现并被报道[1].目前,犬新孢子虫病已经是世界范围内的一种牛和狗的严重疾病[2].欧洲、美洲、澳大利亚、新西兰等国家都有发生,我国周边的日本、韩国等国家和我国台湾地区都有报道.刘群等[3]对北京地区的30头有流产史的奶牛进行调查,结果表明血清中N.caninum的抗体阳性率为26.67%.目前新孢子虫病的研究已成为国外兽医寄生虫学界的热点问题,研究报道较多,其检测试剂已经商品化.     

奶牛新孢子虫病血清流行病学调查

本研究用酶联免疫吸附试验（ELISA）对采集自北京地区的94份奶牛血清（随机采集）和河北地区的55份奶牛血清（有流产史奶牛），进行Neospora caninum血清抗体检测。结果发现，北京地区随机采集的奶牛血清N．caninum抗体阳性率为18．1％（17／94），河北地区有流产史的奶牛N．caninum血清抗体阳性率为23．6％（13／55）。采用牛奶记录体系（DHI）对北京地区17头N．caninum血清抗体阳性牛进行了日产奶量、乳中蛋白率和乳脂率的测定，并与同群牛中134头阴性牛比较。结果表明，N．caninum血清抗体阳性牛日产奶量比阴性牛降低9．7％，乳中蛋白率和乳脂率分别降低20％和15．4％。初步证明N．caninum血清抗体阳性奶牛产奶量降低及奶品质的下降。对不同N．caninum抗体滴度阳性牛的泌乳期主要生产性能比较发现，其生产性能的变化与抗体滴度无明显相关性。     

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.     

Evidence of Neospora caninum DNA in wild rodents

Seventy-five house mice (Mus musculus), 103 rats (Rattus norvegicus) and 55 field mice (Apodemus sylvaticus) from North-West Italy were PCR analysed for Neospora caninum infection. Brain, kidney and muscle tissues collected from the above mentioned animals were tested by PCR using Np6 and Np21 primers. The brain tissue from 2 house mice and 2 rats, the kidney from 4 rats, 1 house mouse and 1 field mouse and muscle from 10 rats, 8 house mice and 1 field mouse were tested positive for N. caninum. Sequencing showed a 96-97% identity of PCR products with N. caninum NC1 sequence. Our findings support previous report on house mouse and rat, and for the first time, provides the evidence of the infection also in field mice. Based on our data, it could be hypothesized that mice can act as a reservoir of N. caninum, and they can play a role in maintaining/spreading N. caninum infection also in the sylvatic cycle. The possibility that dogs could be infected by eating infected house mice suggests new opportunities for N. caninum prophylaxis and control.     

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.     

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.     

Immunohistochemical diagnosis of Neospora caninum in tissue sections

An avidin-biotin-peroxidase complex immunoperoxidase staining method was developed to detect Neospora caninum in formalin-fixed, paraffin-embedded tissue sections. Specific antiserum to N caninum was made in rabbits and used to probe tissues from dogs naturally and experimentally infected with N caninum. The test detected tachyzoites and bradyzoites of N caninum. A reaction was not observed to Toxoplasma gondii, Hammondia hammondi, Sarcocystis cruzi, S capricanis, S tenella, Besnoitia jellisoni, Caryospora bigenetica, Hepatazoon canis, Atoxoplasma sp, or the organism causing canine dermal coccidiosis. When antiserum made in rabbits to T gondii was used in the test, reaction to N caninum was not observed.     

Neospora caninum: oocyst challenge of pregnant cows

Three pregnant cows were each orally challenged at 10 weeks of gestation with 600 sporulated oocysts of Neospora caninum. The number of oocysts was limited by those available. In concurrent bioassays, one oocyst per os infected each of two gerbils. Challenged cattle developed Neospora-specific antibody, cell proliferation and gamma-interferon responses. N. caninum specific PCR demonstrated persisting infection in the brains of cows 4 months after calving. Abortion was not induced and there was no evidence of transplacental infection in the healthy calves born at full-term. This experiment suggests that the dose threshold for induction of abortion exceeds 600 oocysts.     

Seroprevalence of Neospora caninum in non-carnivorous wildlife from Spain

Serum samples from 1034 non-carnivorous wildlife from Spain were tested for antibodies to Neospora caninum by competitive screening enzyme linked immunosorbent assay (ELISA) and confirmed by an indirect fluorescent antibody test (IFAT). High agreement was observed between results in both techniques (kappa value higher than 0.9). Prevalences of N. caninum antibodies positive by both techniques were 11.8% of 237 red deer (Cervus elaphus), 7.7% of 13 barbary sheep (Ammotragus lervia), 6.1% of 33 roe deer (Capreolus capreolus) and 0.3% of 298 wild boar (Sus scrofa). In one of 53 hares (Lepus granatensis), antibodies were found in the ELISA but could not be confirmed by IFAT due to lack of sample. Antibodies to N. caninum were not found in any of 251 wild rabbits (Oryctolagus cuniculus), 79 fallow deer (Dama dama), 27 mouflon (Ovis ammon), 40 chamois (Rupicapra pyrenaica) and three Spanish ibex (Capra pyrenaica). Statistically significant differences were observed between N. caninum seroprevalence in red deer and management of hunting estates (open versus fenced) with higher prevalence in fenced estates, and among sampling sites. Seroprevalence was particularly high in some areas (MO estate in South-Central Spain or some estates of Catalonia, North-East Spain), while no contact with N. caninum was observed in others. Results indicate that in certain areas of Spain, N. caninum is present in wildlife, especially in red deer. These results have important implications in both sylvatic cycles and may influence the prevalence of infection in cattle farms in those areas. To our knowledge, this is the first report of antibodies to N. caninum in wildlife from Spain and the first report of N. caninum antibodies in barbary sheep and wild boar.     

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.     

Seroprevalence of antibodies to Neospora caninum in Belgian dogs

Sera from 300 dogs from Ghent and Antwerp were tested for antibodies to Neospora caninurn using an Indirect fluorescent antibody test. Overall, 11 per cent (995 to 13 per cent; confidence interval of 95 per cent) of dogs were seropositive, at titres of 1:50 to 1:800. No sex or breed differences were detected, but there was an Increase In seropositivity with age.     

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。