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1.
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.  相似文献   

2.
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.  相似文献   

3.
Neosporosis is a major cause of abortion in cattle over the world. One of the methods of preventing vertical transmission within the herd is to avoid breeding replacement heifers from infected dams. Another procedure suggested and recommended by the International Embryo Transfer Society (IETS) is embryo transfer (ET) from infected dams into uninfected recipients. Oocytes and embryos taken from seropositive cows were examined for the presence of Neospora caninum DNA. A modified PCR protocol using Np21 and Np6 primers was applied to detect parasite DNA in the samples. The expected 328 bp product was not obtained in oocytes and/or embryos collected from seropositive dams. The results confirmed that transfer of the embryos from seropositive donors into seronegative recipients is an appropriate method to eliminate vertical transmission of neosporosis in a herd. The present study demonstrated that oocytes and embryos are not exposed to N. caninum in the uterine cavity of seropositive dams.  相似文献   

4.
To test the different sensitivity of rodents of the subfamily Murinae and Gerbillinae, Wagner's gerbils (Gerbillus dasyurus), Tristram's jirds (Meriones tristrami) and multimammate rats (Mastomys natalensis) were inoculated with Neospora caninum tachyzoites. Clinical signs of neosporosis appeared in all inoculated animals. Histopathological examination confirmed the presence of tachyzoites in brains, lungs, skeletal muscle, myocardium, liver, in serosa of stomach and intestines, and in vesicular accessory genital glands. An examination of brains by PCR revealed presence of N. caninum DNA in all experimentally N. caninum infected rodents. The susceptibility of Wagner's gerbils and Tristram's jirds further proved the high sensitivity of gerbiline rodents to the N. caninum infection. The finding of N. caninum tachyzoites in the vesicular accessory genital glands of the infected gerbils suggests the usefulness of the rodent model for demonstration of N. caninum in the male reproductive system. Moreover, the multimammate rat was a susceptible experimental host to be the first immunocompetent rodent of the subfamily Murinae.  相似文献   

5.
为了了解鸡传染性贫血病病毒（CIAV）和J亚群禽白血病病毒（ALV-J）对AA肉种鸡鸡胚和1日龄雏鸡的感染情况,试验直接从山东省3个不同AA肉种鸡场的鸡胚和1日龄雏鸡的肝、脾、肾、胸腺、骨髓、法氏囊等组织（器官）提取DNA,进行了PCR扩增及PCR产物的克隆和序列测定。结果显示,被检的3个肉种鸡场的鸡胚和1日龄雏鸡体内均有这2种病毒核酸的检出,其中CIAV的阳性率是20.42%,ALV-J的阳性率是15.83%,二者共感染的阳性率是6.25%;在阳性检出率中弱雏〉死胚〉健康雏〉正常胚。病毒核酸在各感染组织（器官）的含量也有所差异,CIAV以脾的含量最多,ALV-J以肾的含量最多。对肝进行细菌分离鉴定时发现,3个鸡场还存在大肠杆菌、沙门氏菌和霉形体的混合感染。结果提示,在AA肉种鸡鸡胚和雏鸡体内存在CIAV、ALV-J的感染和二者的共感染以及继发性大肠杆菌、沙门氏菌和霉形体的混合感染。  相似文献   

6.
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.  相似文献   

7.
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。  相似文献   

8.
Neospora caninum is one of the most important causes of abortion in dairy cattle worldwide. The distribution of N. caninum in tissues of adult cattle is unknown and the parasite has not been demonstrated histologically in tissues of cows. In the present study the distribution of N. caninum in different tissues of adult cattle was evaluated by bioassays in dogs. Seventeen dogs (2-3 month-old) were fed different tissues of 4 naturally exposed adult cattle (indirect fluorescent antibody test N. caninum titer ≥ 400): 5 were fed with masseter; 5 with heart, 3 with liver, 4 with brain, and 3 pups were used as non-infected control. Two dogs fed masseter, 2 fed heart, 1 fed liver, and 3 fed brain shed oocysts, and all dogs presented no seroconvertion to N. caninum during the observation period of 4 weeks. The oocysts were confirmed as N. caninum based on the detection of N. caninum-specific DNA by PCR and sequencing. The results indicate that dogs can be infected by N. caninum with different tissues of infected cattle.  相似文献   

9.
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.  相似文献   

10.
In September 2004, a neonatal sea otter pup was found alive on the beach in northern Monterey Bay, CA. Efforts to locate the mother were unsuccessful. Due to a poor prognosis for successful rehabilitation, the pup was euthanized. Postmortem examination revealed emaciation, systemic lymphadenopathy and a malformation of the left cerebral temporal lobe. On histopathology, free tachyzoites and tissue cysts compatible with Toxoplasma gondii were observed in the brain, heart, thymus, liver, lymph nodes and peri-umbilical adipose. The presence of T. gondii within host tissues was associated with lymphoplasmacytic inflammation and tissue necrosis. Immunofluorescent antibody tests using postmortem serum were positive for anti-T. gondii IgM and IgG (at 1:320 and 1:1280 serum dilution, respectively), but were negative for IgG directed against Sarcocystis neurona and Neospora caninum (<1:40 each). Brain immunohistochemistry revealed positive staining for tachyzoites and tissue cysts using antiserum raised to T. gondii, but not S. neurona or N. caninum. T. gondii parasite DNA was obtained from extracts of brain and muscle by PCR amplification using the diagnostic B1 locus. Restriction enzyme digestion followed by gel electrophoresis and DNA sequencing confirmed the presence of Type X T. gondii, the strain identified in the majority of southern sea otter infections.  相似文献   

11.
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.  相似文献   

12.
The aim of this study was to evaluate whether Neospora caninum tachyzoites (Nc-1) inoculated by the conjunctival route in pregnant cows were able to generate infection in their fetuses. Group 1 contained 2 naturally infected cows; group 2 contained two cows inoculated intravenously with 2.5 × 108 tachyzoites, group 3 contained two cows inoculated with 2.5 × 108 tachyzoites by the conjunctival route, and group 4 contained two uninfected control cows. The four inoculated cows from groups 2 and 3 were challenged at 23 weeks of gestation. An indirect fluorescent antibody test (IFAT), recombinant NcGRA7-based ELISA, ELISA for IgG subisotypes and Western blot analysis were assessed to characterize the humoral immune response in dams. Sera from their fetuses were tested also using Western blot analysis. Routine microscopic evaluation of H&E stained fetal tissues was made and any fetal tissues and placentas with lesions compatible with Neospora-infection were processed by immunohistochemistry (IHC). DNA extraction from fresh and formalin-fixed, paraffin-embedded fetal tissues were tested by nested PCR. All dams from groups 1, 2 and 3 were seropositive by IFAT, rNcGRA7-based-ELISA and Western blot. IgG1/IgG2 ratios were ≤1 at weeks 27 and 29 of gestation. Only fetuses from groups 1 and 2 developed N. caninum specific antibodies by Western blot. Histopathological lesions compatible with those caused by N. caninum were observed in fetuses from groups 1 and 2. N. caninum cysts and tachyzoites were observed by IHC on fetal tissues from groups 1 and 2. Only fetal samples from group 2 were positive by PCR. Further work is needed not only to characterize the cellular immune response but also to clarify the consequences on the dam after conjunctival inoculation of N. caninum tachyzoites. This study shows that N. caninum tachyzoites inoculated by the conjunctival route were not vertically transmitted in pregnant cows.  相似文献   

13.
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.  相似文献   

14.
Diagnosis of Neospora caninum associated bovine abortion is often made by histological examination of fetal tissues, although this procedure is not very sensitive. One hundred and forty-four undiagnosed bovine abortion cases were evaluated for N. caninum associated bovine abortion using a revised diagnostic protocol. As a result, 12 (8.3%) of these previously undiagnosed bovine abortion cases were definitively diagnosed as N. caninum positive. The 12 new positive cases included 7 that had exhibited histological lesions, and 5 that had not exhibited histological lesions when examined prior to this study. None of the 12 cases had been immunohistochemically (IHC) stained prior to this study. Also, IHC staining implemented during this research revealed tachyzoites without associated histological lesions in at least 1 tissue from 69 (47.9%) of 144 aborted bovine fetuses, and positive IHC staining of cytoplasmic N. caninum antigen in macrophages in at least 1 tissue from 44 (30.6%) of 144 aborted bovine fetuses. These results demonstrate the necessity for more aggressive evaluation of bovine fetuses for neosporosis.  相似文献   

15.
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.  相似文献   

16.
不同新城疫病毒Ｆ４８Ｅ８、Ｎ－７９、ＬａＳｏｔａ和分离株Ｘ以１∶１００００稀释接种９日龄ＳＰＦ鸡胚,每胚０．２ｍｌ,结果发现,Ｆ４８Ｅ８和分离株Ｘ毒力较强,可在５０小时左右致死鸡胚,ＬａＳｏｔａ和Ｎ－７９较弱。Ｆ４８Ｅ８和Ｘ株接种鸡胚的肌肉、肝脏、脑和尿囊液均有大量病毒存在,而Ｎ－７９和ＬａＳｏｔａ只在尿囊液中有大量病毒存在,胚体病毒极少。  相似文献   

17.
Eighteen outbreaks of inclusion body hepatitis (IBH) were identified in broiler chickens in Baghdad in 1977 and 1978. The disease was seen mainly in 4-to-6-week-old broiler chickens. The mortality rate did not exceed 1% in any outbreak investigated. The most common gross findings were stellate or punctiform hemorrhagic areas in markedly fatty livers. Noticed in many cases were enlarged and pale kidneys, hemorrhagic lesions in the skeletal muscles, and pale fattened bone marrow. Histological examination of tissues revealed fat droplets and intranuclear inclusion bodies in degenerated liver cells. Eosinophilic inclusion bodies were seen in all cases. Only five cases had basophilic inclusions along with the eosinophilic ones. The etiologic agent was isolated on chorioallantoic membrane (CAM) of 12-day-old embryonated chicken eggs. The disease was produced experimentally in 4-week-old chicks using infected CAM suspension.  相似文献   

18.
The aim of this study was to compare the immune responses to live Neospora caninum tachyzoites and N. caninum native antigens formulated with immune stimulating complexes matrix (ISCOM-matrix) in calves. Fifteen calves were used in this study: 3 were intravenously inoculated with 1 × 10(8) live tachyzoites (Group A), 3 were inoculated twice with N. caninum native antigens formulated with ISCOMs (Group B); 3 with N. caninum native antigens in phosphate-buffered saline (PBS) (Group C); 3 received ISCOM-matrix (ISCOMs without antigen) (Group D) and 3 were negative controls receiving PBS (Group E). The last four groups were inoculated subcutaneously. The specific total IgG and its subtypes were analyzed by an indirect enzyme-linked immunosorbent assays (ELISAs) and by Western blot. IFN-γ levels in plasma was quantified using a commercial kit. All calves were challenged intravenously with 1 × 10(8) live tachyzoites at week 11 after receiving the first dose. Parasitemia was assessed in plasma samples by semi-nested PCR. Neospora-specific antibodies were detected in animals from Groups A and B in the week 2 after inoculation. The ELISA OD values were higher in Group B compared with Group A from weeks 6 to 11 (P<0.05). Analysis of the subisotype specific antibodies in experimentally infected calves revealed a predominant IgG(2) response; however, a predominant IgG(1) response was observed in animals inoculated with N. caninum native antigens formulated with ISCOM-matrix. Control calves remained seronegative until challenge infection. The pattern of bands by Western blot was similar when testing sera from animals in Groups A and B. The levels of IFN-γ production after respective immunization schedules were similar between Groups A and B. Neospora-DNA was detected in plasma samples shortly after intravenous challenge in calves from all groups including those receiving the experimental vaccine formulation. The duration of the parasitemia was similar in all groups.  相似文献   

19.
从哈尔滨市某肉鸡养殖场疑似传染性支气管炎的病死鸡中分离到1株肾型IBV,并对其进行鸡胚矮小化、血凝性、电镜下特征、新城疫干扰试验、致病性等生物学鉴定和N基因的RT-PCR鉴定。结果表明,该病毒分离株在鸡胚上传至第四代(F)4开始出现死亡或侏儒胚;病毒不凝集鸡红细胞;透射电镜下可见多呈球形、直径约80～120nm的病毒粒子,具有冠状病毒的典型形态特点;该病毒可干扰新城疫LaSota株在鸡胚中的增殖;将分离毒第4代尿囊液接种于6日龄雏鸡,7d后开始出现死亡,死亡率高达67%(6/9),病死鸡剖解后可见肾脏明显肿大、苍白,具有传染性支气管炎的典型病变;分离毒第5代尿囊液经N基因特异性RT-PCR获得大小约438bp的目的片断。初步确定所分离病毒为肾型IBV。  相似文献   

20.
Immunohistochemical diagnosis of Neospora caninum in tissue sections   总被引：10，自引：0，他引：10  
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.  相似文献   

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