用重组蛋白 NcSAG1t作为ELISA诊断抗原进行改良绒山羊Neospora caninum的血清学诊断

N caninum；NcSAG1t；ELISA；改良绒山羊；抗体     

Development of latex agglutination test with recombinant NcSAG1 for the rapid detection of antibodies to Neospora caninum in cattle

Neospora caninum, an apicomplexan protozoan parasite, is recognized as a major cause of abortion in cattle. Surface antigen 1 of N. caninum (NcSAG1) is an important immunodominant candidate for the development of a diagnostic reagent for neosporosis. The present study describes the development and evaluation of a latex agglutination test (LAT) with recombinant NcSAG1 (rNcSAG1) for the detection of antibodies to N. caninum in cattle. The rNcSAG1 gene was cloned in pET-28a and protein was expressed in Escherichia coli BL21 (DE3). Carboxylated latex particles were coated with rNcSAG1 and the degree of agreement between LAT and a commercial enzyme-linked immunosorbent assay (iscomELISA) was evaluated by using of 164 serum samples. Twenty-two (13.4%) and 23 (14.0%) of samples were positive for antibodies to N. caninum by LAT and ELISA respectively. Eighteen of 23 ELISA-positive samples were positive according to the LAT and a substantial agreement (κ=0.77) was found between the results of LAT and ELISA. The results indicated that the LAT with rNcSAG1 would be a rapid, simple, relatively inexpensive and suitable diagnostic test for detection of specific antibodies in N. caninum infection under field conditions. Improvement in purification of rNcSAG1 can reduce probable false positive reactions and so increase the degree of agreement between the LAT and ELISA.     

青海省乌兰县牧羊犬犬新孢子虫病的血清学调查

用犬新孢子虫的重组蛋白GST-NcSAG1t作为ELISA诊断抗原,对青海省乌兰县的部分牧羊犬进行了犬新孢子虫病的血清学调查。经过对所收集的80份牧羊犬血清的检测,共检出犬新孢子虫病阳性血清25份,阳性率为31.25%。结果表明青海省乌兰地区的牧羊犬中存在犬新孢子虫感染。     

青海省海西地区山羊和绵羊犬新孢子虫病的血清学调查

用犬新孢子虫的重组蛋白GST-NcSAG1t作为ELISA诊断抗原,进行了青海省海西地区山羊和绵羊群中犬新孢子虫病的血清学调查。经过对所收集的120份绵羊血清和531份山羊血清抗体的检测,共检出绵羊阳性血清10份,山羊阳性血清36份,其阳性率分别为8.33%和6.78%。说明青海省海西地区的绵羊和山羊群中存在犬新孢子虫的感染。     

Seroepidemiology of Toxoplasma gondii and Neospora caninum in seals around Hokkaido, Japan

Serological analysis was performed to detect Toxoplasma gondii and Neospora caninum infection in seals in Hokkaido. Serum samples were collected from 322 Kuril harbor seals (Phoca vitulina stejnegeri) at Nosappu, Akkeshi and Erimo, from 46 spotted seals (P. largha) at Nosappu, Erimo, Yagishiri Island, Hamamasu and Syakotan, and from 4 ribbon seals (P. fasciata) and a bearded seal (Erignathus barbatus) at Nosappu between 1998 and 2006. Recombinant surface antigen of T. gondii (SAG2t) and N. caninum (NcSAG1t) were used as antigens for ELISA to detect antibodies. Antibodies against SAG2t were detected from 4% of 77 Kuril harbor seals at Nosappu in 2005. Antibodies against NcSAG1t were detected from 2% (1/66) in 2003, 5% (4/79) in 2004 and 10% (8/77) in 2005 of Kuril harbor seals and 11% of 9 spotted seals in 2004 sampled at Nosappu. Eight percent of 12 Kuril harbor seals from Akkeshi and 25% of 4 spotted seals from Erimo in 2005 also contained antibodies against NcSAG1t. These suggest sporadic infection of T. gondii and N. caninum in Kuril harbor seals and spotted seals in Hokkaido. Of the ELISA-positive seals, 2 seals having anti-SAG2t antibodies and 3 seals having anti-NcSAG1t antibodies in 2005 were judged to be juveniles that have no maternal antibodies. These suggest that the protozoan infections have occurred in recent years. Infection of terrestrial protozoa such as T. gondii and N. caninum in seals indicates that the sea environment has been contaminated with protozoa.     

Elaboration of a crude antigen ELISA for serodiagnosis of caprine neosporosis: validation of the test by detection of Neospora caninum-specific antibodies in goats from Sri Lanka

The protozoan parasite N. caninum is a major pathogen in cattle and dogs. However, clinical symptoms are occasionally described for other potential hosts. Natural abortion in goats due to N. caninum has been rarely reported and only little data is available on the seroprevalence of N. caninum in this species. In the present study, 486 goats from Sri Lanka were tested in a crude antigen ELISA for the presence of serum antibodies against N. caninum. Additionally, the sera were analysed by N. caninum-Western blot and indirect fluorescence antibody test (IFAT). In all three tests applied, only three sera (0.7%) were scored clearly positive for anti-N. caninum antibodies. The optimal correlation between ELISA, IFAT, and Western blot confirms the suitability of the ELISA for large-scale seroepidemiologic studies, not only in cattle but also in goats.     

Development of an indirect ELISA-NcSRS2 for detection of Neospora caninum antibodies in cattle

Neosporosis is of alarming economic concern in the cattle industry. The effectiveness of diagnostic tests for detecting specific antibodies against Neospora caninum is hampered by potential cross-reaction with other coccidia. Use of a single specific antigen might improve test specificity. An indirect enzyme-linked immunosorbent assay (ELISA) was developed using the truncated protein NcSRS2 expressed in Escherichia coli. The ELISA results were compared with those of the indirect fluorescence antibody test (IFAT). Receiver Operating Characteristic (ROC) and Tests in the Absence of a Gold Standard (TAGS) analysis revealed an assay having 96% specificity and 95% sensitivity when applied to 145 positive and 352 negative sera from two distinct cattle populations. Using OD ≤ 0.095 as the cut-off point, the assay's negative and positive predictive values ranged from 98.8% to 50.8% and from 58.8% to 99.1%, respectively, depending on neosporosis prevalence in a given area. The novel ELISA-NcSRS2 format described in the present report constitutes a specific and sensitive method for detecting N. caninum in cattle.     

新孢子虫dNcSRS2重组蛋白间接ELISA的建立及其应用

利用新孢子虫体外重组表面蛋白dNcSRS2蛋白作为包被抗原，对各项条件进行优化，确定判定标准，建立了检测新孢子虫血清抗体的间接ELISA方法。经对多例血清检测表明，所建立的诊断试剂盒重复性好、特异性强、灵敏度高，与进口的IFAT及两种商品化ELISA试剂盒的检测结果相比较，符合率均达到92％以上。应用建立的ELISA方法对236份奶牛血清的新孢子虫抗体进行检测，阳性率为22％。这是国内首次利用重组蛋白建立的诊断试剂盒，该方法的建立将为牛新孢子虫病的诊断与流行病学调查提供有效的技术手段。     

Performance characteristics and optimisation of cut-off values of two enzyme-linked immunosorbent assays for the detection of antibodies to Neospora caninum in the serum of cattle

AIM: To determine the performance characteristics of two enzyme-linked immunosorbent assays (ELISAs) manufactured by Institut Pourquier (IP) for the detection of antibodies against Neospora caninum in bovine sera. METHODS: Sera from 526 cattle were assayed in two ELISAs (IP) for the detection of anti-N. caninum antibodies. Results from a further ELISA (IDEXX) were used to provide the "gold standard"N. caninum infection status of the cattle and the ELISA results assessed by two-graph receiver operating characteristic (TG-ROC) analysis. RESULTS: TG-ROC analysis suggested changes to one of the IP ELISA protocols, arriving at a cut-off threshold that was different to the one recommended by the manufacturer. With that change, both of the ELISAs performed with high sensitivity and specificity (in excess of 98%) for bovine sera. CONCLUSIONS: The analysis of the two IP ELISAs when used on individual bovine sera demonstrated high sensitivity and specificity. TG-ROC analyses optimised the cut-off point suggested by the manufacturer for one of these commercial diagnostic assays and found agreement with the manufacturer's cut-off regarding the other assay. This will help with the accurate identification of infected animals and thereby contributing to the control of neosporosis.     

Reduced cerebral infection of Neospora caninum in BALB/c mice vaccinated with recombinant Brucella abortus RB51 strains expressing N. caninum SRS2 and GRA7 proteins

Neospora caninum, an obligate intracellular protozoan parasite, is the causative agent of bovine neosporosis, an important disease affecting the reproductive performance of cattle worldwide. Currently there is no effective vaccine available to prevent N. caninum infection in cattle. In this study, we examined the feasibility of developing a live, recombinant N. caninum vaccine using Brucella abortus vaccine strain RB51 as the expression and delivery vector. We generated two recombinant RB51 strains each expressing SRS2 (RB51/SRS2) or GRA7 (RB51/GRA7) antigens of N. caninum. BALB/c mice immunized by single intraperitoneal inoculation of the recombinant RB51 strains developed IgG antibodies specific to the respective N. caninum antigen. In vitro stimulation of splenocytes from the vaccinated mice with specific antigen resulted in the production of interferon-gamma, but not IL-5 or IL-10, suggesting the development of a Th1 type immune response. Upon challenge with N. caninum tachyzoites, mice vaccinated with strain RB51/SRS2, but not RB51/GRA7, showed significant resistance to cerebral infection when compared to the RB51 vaccinated mice, as determined by the tissue parasite load using a real-time quantitative TaqMan assay. Interestingly, mice vaccinated with either strain RB51 or RB51/GRA7 also contained significantly lower parasite burden in their brains compared to those inoculated with saline. Mice vaccinated with strain RB51/SRS2 or RB51/GRA7 were protected to the same extent as the strain RB51 vaccinated mice against challenge with B. abortus virulent strain 2308. These results suggest that a recombinant RB51 strain expressing an appropriate protective antigen(s), such as SRS2 of N. caninum, can confer protection against both neosporosis and brucellosis.     

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.     

Seroprevalence of Neospora caninum in free living and farmed red deer (Cervus elaphus) in Poland

Serum samples from 47 free living and 106 farmed red deer (Cervus elaphus) from the Mazurian Lake District in north-east Poland were investigated for the presence of antibodies to Neospora caninum. A modified Neospora iscom-ELISA was used for initial screening. All sera with optical density (OD) values exceeding 0.400 absorbance units were further investigated by Western blot analysis. Eighteen sera were positive in both tests. Six of these were from free living and 12 from farmed animals giving prevalence of 13 and 11%, respectively. This is the first report of N. caninum infection in farmed and free-living red deer living in the same region where neosporosis was confirmed in cattle and the first evidence of exposure to the parasite in red deer in Poland.     

Detection of specific antibodies anti-Neospora caninum in the fallow deer (Dama dama)

Sera from 335 farmed fallow deer (Dama dama) at the breeding station in Kosewo Górne in the Mazurian Lake District, North-East Poland, were investigated for the presence of antibodies against Neospora caninum. The distribution of age groups was as follow: >4 years - 154 animals; 2 years - 76 animals; 1 year - 105 animals. Ten sera with the optical density exceeding 0.159 absorbance units (i.e., cut-off value) in ELISA test were also analyzed by Western blot. Western blot analysis revealed seroreactivity against immunodominant N. caninum antigens of 37, 25, and 16kDa; however, in some sera additional bands were also visible. This is the first screening studies for antibodies against N. caninum in farmed fallow deer in Poland, in the region where neosporosis was confirmed in cattle and in farmed and free-ranging European red deer (Cervus elaphus).     

Immunological relationship between Neospora caninum and Besnoitia besnoiti

Neospora caninum is a coccidian parasite identified as a major cause of abortion in cattle. A combined infection of N. caninum with another taxonomically related parasite of cattle, Besnoitia besnoiti can occur in geographical areas endemic for both species. Both infections are routinely diagnosed serologically, and incorrect diagnosis could occur if immunological cross-reactivity exists between the two parasites. To investigate the possible degree of cross-reactivity, we compared results obtained with two serological techniques, immunofluorescent antibody test (IFA), and Western blot analysis on known positive and negative sera. The test sera were derived from naturally infected cattle and from experimentally infected Mongolian gerbils. In IFA of bovine sera, no cross-reactvity was detected at the commonly used serum dilution cutoffs of 1:200 for N. caninum and 1:256 for B. besnoiti. However, at 1:64 dilution of both cattle and gerbil sera, anti-N. caninum sera reacted with B. besnoiti antigen in some individual samples. Anti-B. besnoiti serum did not react with N. caninum antigen at any dilution. This low level one directional cross-reactivity was confirmed by Western blot analysis. B. besnoiti antigen showed two immunoreactive bands when probed with anti-N. caninum serum, while no bands appeared when N. caninum antigen was probed with B. besnoiti antiserum. Immunization and challenge experiments in the highly susceptible Mongolian gerbil (Meriones unguiculatus) showed essentially no cross-protection between N. caninum and B. besnoiti.     

Nationwide seroprevalence of Neospora caninum among dairy cattle in Japan

Serum samples from 2420 clinically healthy dairy cattle, randomly selected from stored sera in 18 districts of Japan, were tested for the presence of Neospora caninum antibodies using an indirect fluorescent antibody test (titer > or =1:200). Nationwide seroprevalence is estimated at 5.7% (139/2420). Seropositive cattle were detected in all surveyed districts despite the evidence of confirmed case reports of bovine neosporosis, showing that N. caninum is widely distributed throughout Japan. Age-specific seroprevalence did not increase with cattle age, suggesting that Neospora infection is likely to be transmitted vertically rather than horizontally in Japan. Considering that N. caninum seropositive cows are thought to be more likely to abort, substantial fetal losses may be induced by N. caninum infection in Japan. Devising strategies are needed to reduce the economic impact on the Japanese dairy industry. This is the first study to investigate the nationwide seroprevalence of N. caninum in cattle in Asia.     

Anti-neosporal IgG and IgE antibodies in canine neosporosis

Neospora caninum infection provokes neurological disorders, recurrent abortion and death in dogs and cattle. Dogs are both intermediate and definitive host of N. caninum. Thus, the development of sensitive and specific immunoassays to diagnose canine neosporosis is essential to control this disease. This work investigated serum anti-neosporal IgG and IgE antibodies in 140 dogs represented by 30 healthy animals (group I), 11 dogs showing acute N. caninum infection (group II), 50 urban dogs with serological evidence of canine neosporosis in indirect fluorescent antibody test (IFAT) (group III) and 49 urban dogs without clinical and laboratory evidences of neosporosis (group IV). Enzyme-linked immunosorbent assay (ELISA) and western immunoblotting, both using a soluble N. caninum tachyzoite antigen (SNA), investigated these two isotypes of antibodies, while a Urea-ELISA measured the avidity of the IgG antibodies. Anti-Toxoplasma gondii IgG antibodies were also investigated in the animals. Anti-neosporal IgG was found in all animals from groups II and III, whereas 32.7% (16/49) of dogs from group IV were reactive. IgG antibodies of low avidity were demonstrated in dogs from group II (median 35.3%), while animals from groups III and IV had IgG antibodies of high avidity (medians of 61.5% and 61.7% respectively). IgE antibodies were found in four (13.3%) and five (16.6%) dogs from groups III and IV respectively. Dogs presenting acute infection (group II) or chronic infection (group III) had IgG antibodies to several neosporal antigens, mainly of 29-30 and 35 kDa, while 13 of 16 dogs from group IV recognized antigens from 14 to 170 kDa. Antibodies to T. gondii were detected in 36 of 50 (72%) sera from group III and 25 of 49 (51%) sera from group IV. We concluded that IgG-ELISA and Urea-ELISA with SNA may substitute for IFAT in both laboratory routine and epidemiological studies of canine neosporosis.     

Characterization of Neospora caninum microneme protein 10 (NcMIC10) and its potential use as a diagnostic marker for neosporosis

Improvements in the serological diagnosis of neosporosis are needed to differentiate acute versus chronic Neospora caninum infections. In the present study, N. caninum microneme protein 10 (NcMIC10), similar to other microneme proteins, was shown to be released in a calcium-dependent manner. NcMIC10 may be discharged during active invasion of host cells by the parasite, and thus represent an excellent marker for the diagnosis of neosporosis. In order to test this hypothesis, recombinant NcMIC10 (rNcMIC10) was expressed in Escherichia coli, and polyclonal antibodies were generated against non-overlapping fragments of the protein. A capture ELISA was developed using these antibodies, and was found to be highly accurate and reproducible with a detection range of 10-10,000 pg/ml. The anti-rNcMIC10 antibodies used in this study did not cross-react with the Toxoplasma gondii antigens. NcMIC10 was detected by the ELISA in sera of 9 out of 10 goats (90%) experimentally infected with N. caninum tachyzoites. In general, goats infected with a lower dose (10(4)) of the parasite displayed a peak in NcMIC10 levels between weeks 4 and 5 post infection. Goats infected with a higher parasite dose (10(6)) displayed a more rapid increase in NcMIC10 levels. In most animals, NcMIC10 decreased to undetectable levels by week 6 post infection. This is the first circulating Neospora antigen-based assay which may complement the existing antibody-based assays for a rapid and cost-effective definitive diagnosis of neosporosis in livestock.     

Risk factors for canine neosporosis in farm and kennel dogs in southern Italy

Neosporosis by Neospora caninum causes losses to livestock production through abortion in cattle while, in dogs, it induces neuromuscolar disease. This study investigated neosporosis seroprevalence associated risk factors (including the seropositivity to leishmaniosis) in dogs of southern Italy, determined the prevalence of N. caninum oocyst shedding and examined the relationship between seroprevalence of neosporosis in farm dogs and cattle. Using an inhibition ELISA, 20.9% of 306 dogs had percent inhibition values >10 (indicative of exposure) and farm dogs had a significantly (p<0.001) higher seroprevalence than dogs in a rescue kennel. Whilst N. caninum seroprevalence was associated with increasing age in dogs (p< or =0.01) there was no association between seropositivity for N. caninum and for Leishmania infantum. Oocysts of N. caninum were not detected in faecal samples from 230 dogs including 160 farm dogs. The results indicated that neosporosis infection is common in southern Italy both in dogs and in cattle and that dogs at higher risk of exposure are free-ranging ones living in farms. The lack of correlation between canine seroprevalence for N. caninum and L. infantum assumes a particular significance in an endemic area for leishmaniosis.     

Evaluation of Toxoplasma gondii and Neospora caninum infections in sheep from Uberlândia, Minas Gerais State, Brazil, by different serological methods

Toxoplasmosis and neosporosis have been recognized as economically important diseases with considerable impact on the livestock industry. Considering the scarce information on the occurrence of Toxoplasma gondii and Neospora caninum infections in sheep from Uberlandia, Minas Gerais State, Brazil, this study aimed to investigate the frequency of antibodies against these parasites in sheep sera from this region by using different serological methods. A total of 155 sheep serum samples were analyzed by the indirect fluorescence antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA) for the detection of IgG against T. gondii and N. caninum. Seroreactivity by IFAT showed 80% of samples with titers between 512 and 2048 for T. gondii (cutoff ≥ 64) and 78% presenting titers between 50 and 200 for N. caninum (cutoff ≥ 50). Seroreactivity by ELISA showed 75% of samples with ELISA index (EI) between 2.0 and 3.0 for T. gondii (cutoff ≥ 1.3) and 54% presenting EI between 1.3 and 2.0 for N. caninum (cut off ≥ 1.3). Discordant results by both tests were analyzed by immunoblot, resulting in a total seropositivity of 61% for T. gondii and 23% for N. caninum, with 41% to T. gondii only, 3% to N. caninum only, and 20% to both parasites. There was a significant positive association between seropositivity to T. gondii and age over one year (P<0.001), but such association was not found for N. caninum infection. In conclusion, as T. gondii and N. caninum infections are simultaneously present in sheep flocks of this region, it should be emphasized the importance to carry out a regular monitoring of Toxoplasma infection due to its high prevalence, its zoonotic potential and induction of reproductive disorders leading to economic losses. For neosporosis, sheep farmers should be instructed about the presence of the parasite in the flock, its risk factors and potential abortifacient role in sheep. Differential flock management could be valuable tool to establish the association of serological positivity and reproductive disease induced by N. caninum in sheep.     

Evaluation of serological tests for the diagnosis of Neospora caninum infection in dogs: optimization of cut off titers and inhibition studies of cross-reactivity with Toxoplasma gondii

Diagnosis of Neospora caninum infection in dogs is based on serological assays such as the indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assays (ELISA). This study evaluated two serological tests (IFAT and ELISA) for the detection of IgG antibodies to N. caninum in 300 serum samples of dogs through the optimization of cut off titers by using the two-graph receiver-operating characteristic (TG-ROC) curve. In addition, the identification of major cross-reactive antigens with Toxoplasma gondii was investigated by inhibition ELISA and immunoblotting (IB) assays. IFAT and ELISA results showed 74% agreement, with a good negative concordance (P(neg)=0.83), but a poor positive concordance (P(pos)=0.42). The great majority (86%) of sera with positive concordant results (IFAT+/ELISA+) recognized at least two out of three N. caninum immunodominant antigens, particularly the 29-32 and 35-37 kDa bands. Optimization of cut off titers in IFAT and ELISA was performed considering the reactivity to at least two out of three N. caninum immunodominant antigens as infection markers, obtaining a titer of 50 for IFAT and 200 for ELISA. Seropositivity to N. caninum was significantly associated with T. gondii-seropositive samples, particularly in ELISA (55.4%). Inhibition ELISA curves for N. caninum showed a partial heterologous inhibition, indicating some degree of cross-reactivity between N. caninum and T. gondii antigens. Inhibition IB assays showed a moderate heterologous inhibition for N. caninum antigens above 45-50 kDa. These results indicate that ELISA should be used critically when crude tachyzoite antigen preparations are employed, due to possible cross-reactivity with other related parasites as T. gondii. Also, the cut off dilution of 1:50 in IFAT showed to be the most appropriated for N. caninum serology in dogs. Therefore, we suggest that N. caninum immunodominant antigens, specially the 17 and 29-32 kDa proteins, should be selected markers in serological assays for canine neosporosis.