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.     

Prevalence of antibodies to Neospora caninum in dogs.

Serum samples from 1077 dogs suspected of having Neospora caninum infections from 35 states in the United States and 3 provinces in Canada were tested for N. caninum IgG antibodies by the indirect fluorescent antibody test. Antibodies to N. caninum were found in 75 of 1077 (7%) of the samples. Twenty of the positive dogs were females, 17 were males and the sex was not recorded on 38 dogs. Chi square analysis indicated no differences (P > 0.05) based on sex were present. Dogs from the Southeast and Midwest regions of the United States were more likely to be N. caninum antibody positive than were dogs from the Northeast or West regions.     

Prevalence of antibodies to Neospora caninum in dogs from Amazon,Brazil

Neospora caninum is an important cause of abortion in dairy cattle worldwide. Dogs are important in the epidemiology of this parasite because they are the only hosts known to excrete N. caninum oocysts. Antibodies to N. caninum were assayed in serum samples from 157 dogs from Monte Negro, Rond?nia, Amazon, Brazil using the indirect fluorescent antibody test. Antibodies to N. caninum were found in 13 (8.3%) of dogs in titers of 1:50 in 1, 1:100 in 2, 1:200 in 5, 1:800 in 1, 1:1600 in 2, and 1:3200 in 2 dogs. These data indicate that N. caninum infection is prevalent even in remote areas of the Amazon.     

Factors associated with infection by Neospora caninum in dogs in Brazil

From August 2006 to 2008, 411 dogs in northeastern Brazil were evaluated for seropositivity to Neospora caninum. The dogs were clinically examined, and their owners were interviewed about the conditions in which the animals were maintained in order to assess the factors associated with infection by this parasite. A serum sample was taken from each dog for serological examination in an indirect fluorescent antibody test for N. caninum. The Yates' Chi-square test or Fisher's exact test was used to select the variables for the multivariate logistic regression model. Seropositivity was detected in 9.26% of dogs. The seropositivity rates of dogs from different environments were 2.6% (4/156) in urban areas, 13.1% (28/214) in peri-urban areas, and 14.6% (6/41) in rural areas. Factors associated with seropositivity for N. caninum were the following: contact with other dogs, access to food outside the home and residing in the peri-urban or rural environments (p<0.05). Results of this study confirm that dogs in urban, rural and peri-urban areas of northeastern Brazil are exposed to N. caninum. Control measures to prevent infection of dogs in the studied region should be focused primarily on preventing access to potential sources of infection, which include environments with other dogs, bovines, and other small intermediate hosts, such as birds and rodents.     

新孢子虫和弓形虫ELISA及western blot检测方法的建立及应用

为建立牛新孢子虫和弓形虫的免疫学检测方法,并调查新疆部分地区牛新孢子虫和弓形虫的感染情况,本研究应用纯化的新孢子虫重组蛋白SRS2(NcSRS2)和弓形虫重组蛋白SAG2(TgSAG2)作为包被抗原,分别建立新孢子虫和弓形虫的ELISA和western blot血清学检测方法,并进行特异性和重复性试验,以其检测662份疑似样品,并与商品化试剂盒检测结果比较验证。特异性和重复性试验结果表明,建立的方法特异性强、重复性良好。采用建立的两种ELISA方法对662份临床样品的检测结果表明,新孢子虫和弓形虫的抗体阳性率分别为13.44%(89/662)和5.29%(35/662);与IDEXX试剂盒和永辉试剂盒的符合率分别为94.11%和95.92%。此外,western blot检测的新孢子虫和弓形虫抗体阳性率分别为5.14%(34/662)和3.17%(21/662);与建立的ELISA检测方法的符合率分别为91.69%和97.89%。本研究为分析奶牛流产的原因提供了一定的依据。     

Serologic prevalence of Sarcocystis neurona, Toxoplasma gondii, and Neospora caninum in horses in Brazil.

OBJECTIVE: To determine serologic prevalence of Sarcocystis neurona, Toxoplasma gondii, and Neospora caninum in horses in Brazil. DESIGN: Prevalence survey. ANIMALS: 101 Thoroughbreds in Brazil. PROCEDURE: Blood samples were obtained from horses and tested for serum antibodies against S neurona by use of an immunoblot procedure with culture-derived S neurona merozoites as antigen, and for serum antibodies against T gondii and N caninum by use of a modified agglutination test with formalin-preserved tachyzoites and mercaptoethanol. RESULTS: Antibodies against S neurona and T gondii were detected in 36 and 16 of 101 horses, respectively. Cross-reactivity between antibodies against T gondii and S neurona was not detected. Antibodies against N caninum were not detected in any samples. CONCLUSIONS AND CLINICAL RELEVANCE: The high prevalence of antibodies against S neurona detected in clinically normal horses emphasizes the importance of examining CSF for antibodies when establishing a diagnosis of equine protozoal myeloencephalitis.     

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.     

Seroprevalence of antibodies to Neospora caninum in urban and rural dogs in north-west Italy

Sera were collected from 490 dogs from north-west Italy. One hundred and eighty-eight dogs were urban, while 302 dogs were rural. Among the latter, 190 were shepherd dogs and 112 were cattle farm dogs. Sera were tested for the presence of antibodies against Neospora caninum using the Neospora agglutination test. Seroprevalence at 1/40, 1/80, 1/160 dilution titres was significantly higher in rural (36.4%, 19.5%, 9.9% respectively) than in urban dogs (20.2%, 10.6%, 4.8% respectively). Seroprevalence did not differ significantly in males and females. In shepherd dogs, prevalence increased according to dogs' age, thus suggesting a post-natal exposure by horizontal transmission. The observed higher seroprevalence in rural dogs suggests the importance of lifestyle and alimentary habits (i.e. aborted foetuses, placentas and small mammals) in the acquisition of N. caninum infection. Our results confirm that dogs are exposed to N. caninum and play an important role in the epidemiology of N. caninum.     

Occurrence of antibodies to Neospora caninum and Toxoplasma gondii in dogs from Pernambuco, Northeast Brazil

A serological survey was carried out to assess the occurrence of anti-Neospora caninum antibodies in dogs from the State of Pernambuco. A total of 625 serum samples of dogs (289 from Paulista, 168 from Amaraji and 168 from Garanhuns) were tested by an immunofluorescence antibody assay for the detection of anti-N. caninum antibodies. A total of 177 (28.3%; IC 95%, 24.9-32.1) samples were positive. The seropositivity rates found in Paulista, Amaraji and Garanhuns were 26% (IC 95%, 21-31.4), 26.2% (IC 95%, 19.7-33.5) and 34.5% (IC 95%, 27.4-42.2), respectively. Of the 177 serum samples positive to anti-N. caninum antibodies, 170 were additionally tested for the presence of anti-Toxoplasma gondii antibodies and out of these 57.6% (IC 95%, 49.8-65.2) were positive. The results indicate that dogs from Amaraji, Paulista and Garanhuns are exposed to both N. caninum and T. gondii infections. The presence of dogs infected by N. caninum in Pernambuco represents a potential risk factor for the occurrence of outbreaks of abortion in cattle and small ruminants in this state. This study is the largest serological survey on the presence of anti-N. caninum antibodies in dogs carried out in Brazil and reports for the first time the exposure to N. caninum and T. gondii in dogs from Pernambuco.     

Immune response to Neospora caninum native antigens formulated with immune stimulating complexes in calves

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.     

Clinical Sarcocystis neurona, Sarcocystis canis, Toxoplasma gondii, and Neospora caninum infections in dogs

Sarcocystis neurona, Sarcocystis canis, Toxoplasma gondii, and Neospora caninum are related apicomplexans that can cause systemic illness in many species of animals, including dogs. We investigated one breeder's 25 Basset Hounds for these infections. In addition, tissues from dogs and other non-canine hosts previously reported as S. canis infections were studied retrospectively. Schizonts resembling those of S. neurona, and recognized by polyclonal rabbit anti-S. neurona antibodies, were found in six of eight retrospective cases, as well as in two additional dogs (one Basset Hound, one Springer Spaniel) not previously reported. S. neurona schizonts were found in several tissues including the central nervous system, lungs, and kidneys. Fatal toxoplasmosis was diagnosed in an adult dog, and neosporosis was diagnosed in an adult and a pup related to the one diagnosed with S. neurona. No serological reactivity to S. neurona antibodies occurred when S. canis-like liver schizonts were retrospectively assayed from two dogs, a dolphin, a sea lion, a horse, a chinchilla, a black or either of two polar bears. Sequencing conserved (18S) and variable (ITS-1) portions of nuclear ribosomal DNA isolated from the schizont-laden liver of a polar bear distinguished it from all previously characterized species of Sarcocystis. We take this genetic signature as provisionally representative of S. canis, an assumption that should be tested with future sequencing of similar liver infections in other mammalian hosts. These findings further extend the uncharacteristically broad intermediate host range for S. neurona, which also causes a neurologic disease in cats, mink, raccoons, skunks, Pacific harbor seals, ponies, zebras, lynxes, and sea otters. Further work is necessary to delineate the causative agent(s) of other cases of canine sarcocystosis, and in particular to specify the attributes of S. canis, which corresponds morphologically to infections reported from wide range of terrestrial and marine mammals.     

Failure of dogs to shed oocysts after being fed bovine fetuses naturally infected by Neospora caninum

Neospora caninum is a protozoan that causes abortion in cattle. The dog has recently been identified as a definitive host for N. caninum. To verify if bovine fetuses can infect dogs, nine 2-4-month-old dogs were fed bovine fetuses naturally infected by N. caninum. None of the dogs excreted oocysts, seroconverted, had clinical signs or lesions compatible with N. caninum infection. Additional studies will be necessary to determine the natural mode of infection of dogs by N. caninum.     

奶牛新孢子虫重组蛋白NcSRS2t间接ELISA方法的建立及初步应用

以纯化的犬新孢子虫(Neospora caninum)重组蛋白 NcSRS2t为抗原包被酶标板,通过对间接ELISA 各反应条件的优化,确定了最佳反应条件.采用已建立的间接ELISA反应条件,对218份阴性血清的检测结果进行统计学分析,确定了间接ELISA的判定标准,即OD450nm值大于等于O.32为阳性,小于O.32为阴性.应用建立的间接ELISA方法对128份阴性血清和50份阳性血清(经IDEXX公司新孢子虫抗体检测试剂盒证实)进行检测,结果表明,闻接ELISA方法的特异性为93.0%,敏感性为90.0%.采用本试验建立的新孢子虫间接ELISA 诊断方法对黑龙江省克山、海林、双城、甘南、克东、富裕、牡丹江、大庆等8个县(市)奶牛场的540份奶牛血清进行了检测.结果表明,被检的540份奶牛血清样本中奶牛新孢子虫的抗体阳性率为13.33%.该间接ELISA 诊断方法的建立及初步应用为新孢子虫病诊断试剂盒的研制和新孢子虫病的预防研究奠定了基础.     

Protozoal encephalomyelitis of dogs involving Neospora caninum and Toxoplasma gondii in New Zealand

In a retrospective study of 15 cases of encephalomyelitis in dogs, three cases of Neospora caninum and two cases of Toxoplasma gondii infection were identified using immunohistochemical staining of central nervous system sections. All cases of neosporosis showed ataxia and progressive hind limb paralysis due to multifocal non-suppurative meningoencephalomyelitis which was most severe in the spinal cord and base of the brain stem. Neospora tissue cysts could not be distinguished morphologically from those of T. gondii using light microscopy, but electron microscopy confirmed their characteristic features. Although Neospora abortion in cattle has only recently been recognised in New Zealand, this study has shown that neosporosis has been present in dogs since at least 1972.     

The detection of canine autoantibodies to thyroid antigens by enzyme-linked immunosorbent assay, hemagglutination and indirect immunofluorescence.

Autoantibodies to thyroid antigens in serums from 34 clinically hypothyroid dogs were detected by various methods. Antibodies to thyroglobulin were detectable by enzyme-linked immunosorbent assay (ELISA) in 59%, by chromic chloride hemagglutination in 53% and by indirect immunofluorescence on formalin-fixed, paraffin-embedded, trypsin-digested thyroid tissue in 73% of samples. Antibody to thyroid microsomal antigen was detectable by ELISA in 29% of serums. Indirect immunofluorescence showed cytoplasmic fluorescence of thyroid follicular cells in several serums, however, this could not be confirmed as specific for microsomal antigen by absorption trials. Hemagglutination tests using commercially available tanned red cells coated with human antigens and indirect immunofluorescence assays on formalin-fixed tissue without trypsin digestion, on Bouin's fixed tissue, or on cryostat, methanol-fixed sections, were insensitive. Cryostat sections without methanol fixation were unsuitable due to tissue fragility. No method was recommended for routine diagnostic use. The ELISA test, because of its convenience, may be useful as a screening aid or adjunct to the diagnosis of thyroid disease.     

Pattern of recognition of Neospora caninum tachyzoite antigens by naturally infected pregnant cattle and aborted foetuses

Different aspects of Neospora tachyzoite antigen recognition by Neospora-infected heifers and cows and aborted foetuses were studied. The pattern of antigen recognition and the relationship between IFAT titres and number of Neospora antigens detected, were evaluated. In addition, the tachyzoite antigens involved in the humoral immune response developed against infection in normal cows and cows that aborted were also characterised throughout pregnancy. Comparison of tachyzoite antigen recognition was carried out in 13 thoracic and/or abdominal fluids from Neospora aborted foetuses and 33 sera from Neospora infected cows that had aborted. The kinetics of Neospora-antigen recognition was studied in Neospora-infected heifers and cows that had aborted foetuses (7) or not (14) during pregnancy.Based on the frequency and intensity of recognition, four IDAs-17-18, 34-35, 37 and 60-62kDa antigens-have been described. Moreover, a correlation was found between Western blot results and IFAT titres in both age groups. In relation to antigen recognition throughout pregnancy by seropositive cows that had aborted or not, the antibody fluctuations throughout pregnancy described in the literature could be due to differences in the intensity and frequency of recognition of particular antigens, especially the 17-18kDa antigen. We emphasize the important role that the 17-18kDa antigen could play in the serological diagnosis of Neospora infection in cattle as this was intensely detected in 100% of the animals.     

吉林省延边地区宠物犬与牧场家犬的新孢子虫病流行病学调查

为了调查宠物犬和牧场内家犬的犬新孢子虫病的感染情况,以新孢子虫Nc GRA2t为诊断抗原,应用间接酶联免疫吸附试验(ELISA)对采自吉林省延边地区部分宠物医院的23份宠物犬血清和牧场内家犬的4份血清进行了检测。结果,宠物犬的犬血清感染率为0%,牧场内家犬的感染率为75%。牧场内家犬的新孢子虫病感染率较高,牧场内饲养牛存在感染新孢子虫的风险。     

Incidence of Neospora caninum and other intestinal protozoan parasites in populations of Swiss dogs

The protozoan parasite Neospora caninum is one of the most important abortifacient organisms in cattle worldwide. The dog is known to act as definitive host although its potential role as infection source for bovines still remains unelucidated. The aim of the present study was to compile initial epidemiological data on the prevalence and incidence of N. caninum in Swiss dogs acting as definitive hosts. Thus, 249 Swiss dogs were investigated coproscopically in monthly intervals over a period of 1 year. A total of 3289 fecal samples was tested by the flotation technique. Among these, 202 were shown to contain Sarcocystis sp. (6.1%), 149 Cystoisospora sp. (=Isospora sp.; 4.5%) and 25 Hammondia/Neospora-like oocysts (HNlO) (0.7%). All but one sample containing HNlO were from different dogs; one dog shed HNlO at two subsequent time points. Calculation of the yearly incidence for HNlO resulted in the surprisingly high value of 9.2%. Farm dogs exhibited a higher incidence for HNlO than urban family dogs. Thirteen out of the 25 HNlO-samples showed sporulation after 5 days incubation at room temperature. HNlO were further differentiated by species-specific PCR. However, all HNlO-samples were negative for N. caninum, Hammondia heydorni and Toxoplasma gondii. One reason may be the low oocyst density found in most fecal samples, which did not permit us to carry out PCR under optimal conditions. Three out of the 25 HNlO-cases contained enough oocysts to allow further enrichment and purification by the flotation technique. Subsequently, twenty to fifty sporulated HNlO-oocysts were orally administered to Meriones unguiculatus. All gerbils were seronegative for N. caninum at 5 weeks p.i. A N. caninum-seroprevalence of 7.8% was determined by ELISA upon 1132 serum samples collected from dogs randomly selected by veterinarians among their clinical patients.