Toxoplasma gondii and Neospora caninum antibodies in dogs from Grenada, West Indies

Toxoplasma gondii and Neospora caninum are structurally similar parasites with many common hosts. The prevalence of antibodies to T. gondii and N. caninum was determined in sera from dogs in Grenada, West Indies. Using a modified agglutination test, antibodies to T. gondii were found in 52 (48.5%) of the 107 dogs, with titers of 1:25 in 17, 1:50 in 19, 1:100 in 7, 1:1,600 in 5, and 1:3,200 or higher in 4. Seroprevalence increased with age from 2.2% in dogs <6 mo old to 18.9% in dogs older than 2 yr, indicating postnatal transmission of T. gondii in this population of canines. There was no correlation between the health of the dogs and the seroprevalence or magnitude of the T. gondii titer. Antibodies to N. caninum were determined by the indirect immunofluorescent antibody test (IFAT). Two of the 107 dogs had N. caninum antibodies (IFAT titers 1:100 and 1:400); these dogs had T. gondii titers of 1:1,600 and 1:50, respectively. Results indicate that these 2 structurally similar protozoa are antigenically different.     

Prevalence of Neospora caninum infection in dogs from beef-cattle farms, dairy farms, and from urban areas of Argentina

Prevalence of anti-Neospora caninum antibodies was determined in sera of 320 dogs from Argentina using the indirect fluorescent antibody test (IFAT). Antibodies to N. caninum were found in 121 of 320 (37.8%) sera with titers of 1:50 (21 dogs), 1:100 (23 dogs), 1:200 (23 dogs), 1:400 (17 dogs), 1:800 (23 dogs), and > or = 1:1.600 (14 dogs). The seropositivity (IFAT, > or = 1:50) was higher in dogs from dairy (48% of 125) and beef (54.2% of 35) farms than in dogs from urban areas (26.2% of 160). Prevalence of anti-N. caninum antibodies was higher in dogs more than 12 mo of age (47.7%, 105 of 222) versus in 12-mo-old or younger dogs (12.7% of 86), suggesting postnatal exposure of N. caninum infection in dogs.     

Neospora caninum and Toxoplasma gondii antibodies in dogs from Durango City, Mexico

Toxoplasma gondii and Neospora caninum are structurally similar parasites, with many hosts in common. The prevalence of antibodies to T. gondii and N. caninum was determined in sera from dogs from Durango City, Mexico. Using a modified agglutination test, antibodies to T. gondii were found in 52 (51.5%) of the 101 dogs with titers of 1:25 in 27, 1:50 in 11, 1:100 in 5, 1:200 in 4, 1:400 in 2, 1:800 in 2, and 1:3,200 or higher in 1. Antibodies to N. caninum were determined by the indirect immunofluorescent antibody test (IFAT) and the Neospora sp. agglutination test (NAT). Two of the 101 dogs had N. caninum antibodies; these dogs did not have T. gondii antibodies, supporting the specificity of the tests used. The N. caninum antibody titers of the 2 dogs were: 1:400 by IFAT and 1:200 by NAT in 1, and 1:25 by NAT and IFAT in the other. Results indicate that these 2 structurally similar protozoans are antigenically different.     

Estimation of Canine Leishmania Infection Prevalence in Six Cities of the Algerian Littoral Zone Using a Bayesian Approach

A large-scale study on canine Leishmania infection (CanL) was conducted in six localities along a west-east transect in the Algerian littoral zone (Tlemcen, Mostaganem, Tipaza, Boumerdes, Bejaia, Jijel) and covering two sampling periods. In total 2,184 dogs were tested with an indirect fluorescent antibody test (IFAT) and a direct agglutination test (DAT). Combined multiple-testing and several statistical methods were compared to estimate the CanL true prevalence and tests characteristics (sensitivity and specificity). The Bayesian full model showed the best fit and yielded prevalence estimates between 11% (Mostaganem, first period) and 38% (Bejaia, second period). Sensitivity of IFAT varied (in function of locality) between 86% and 88% while its specificity varied between 65% and 87%. DAT was less sensitive than IFAT but showed a higher specificity (between 80% and 95% in function of locality or/and season). A general increasing trend of the CanL prevalence was noted from west to east. A concordance between the present results and the incidence of human cases of visceral leishmaniasis was observed, where also a maximum was recorded for Bejaia. The results of the present study highlight the dangers when using IFAT as a gold standard.     

Investigation of anti-Toxoplasma gondii antibodies in cats of the Ankara region of Turkey Using the Sabin-Feldman dye test and an indirect fluorescent antibody test

Blood samples from 99 cats from the Ankara province of Turkey were examined for the presence of anti-Toxoplasma gondii antibody with the use of both the Sabin-Feldman dye test (DT) and an indirect fluorescent antibody test (IFAT). Forty of the 99 sera (40.3%) were positive for antibodies against T. gondii with the DT, whereas the IFAT assay detected antibodies in 34 (34.3%). The study also evaluated 3 factors for their potential association with the presence of T. gondii antibody: age (<1 yr, 1-2 yr, and >2 yr), gender (female vs. male), and outdoor access (stray, owned with outdoor access, or indoor only). The DT detected antibodies in 3 cats under 1 yr of age, 22 cats between 1 and 2 yr, and 15 cats older than 2 yr, whereas the IFAT found 1, 18, and 15 cats positive for antibodies, respectively, in each of these categories. Of 61 female cats, 27 (44.2%) were positive by the DT; and of 38 male cats, 13 (34%) were positive by the DT. For the IFAT, 24 female cats (39.3%) and 10 male cats (26.3%) were positive. The percent seropositivity in indoor cats was 30.8% by the DT and 23.1% by the IFAT. In stray cats, the percent seropositivity was 52.8% by the DT and 41.7% by the IFAT. Antibody presence was significantly associated with age, but not with outdoor access.     

Seroprevalence of Neospora caninum antibodies in cattle and water buffaloes in India

Neospora caninum is now recognized as a major cause of abortion in cattle worldwide, but there is no report of N. caninum infection in cattle in India. Serum samples from 427 dairy cattle and 32 dairy water buffaloes from 7 organized dairy farms located in Punjab, India, were tested for N. caninum antibodies using a commercial monoclonal antibody-based competitive enzyme-linked immunosorbent assay (ELISA). Antibodies to N. caninum were found in 35 of 427 cattle from 6 of the 7 farms; 9.6% of cows, 5.1% of heifers, and 5.0% of calves were seropositive, suggesting postnatal transmission of N. caninum on the farm. Antibodies to N. caninum were found in 16 of 32 buffaloes tested from 2 dairy farms. In total, 64 cattle and 16 buffalo sera already tested by ELISA were also evaluated by an indirect fluorescent antibody test (IFAT) to verify ELISA results. Of the 64 cattle samples, 29 sera were negative by both tests and of the 35 ELISA-positive sera, 12 had IFAT titers of 1:100 or higher (1 had IFAT titer of 100, 2 had IFAT titer of 200, and 9 had IFAT titers of 400 or higher). Of the 16 buffalo sera positive by ELISA, 1 had an IFAT titer of 1:400. Thus, antibodies to N. caninum were demonstrated in cattle sera by 2 serologic methods. To our knowledge this is the first report of N. caninum infection in cattle and buffaloes in India.     

Serological diagnosis of neosporosis in a herd of dairy cattle in southern Brazil.

Neospora caninum-specific antibodies were detected in 60 of 172 (34.8%) dairy cattle by enzyme-linked immunosorbent assay (ELISA) in a herd from Parana State, Brazil. The seropositive animals included 47 of 126 (37.3%) adult cows, 7 of 29 (24%) heifers (1-2 yr), 4 of 15 (27%) heifers (5 mo-1 yr), and 2 precolostral samples. Data collected over a 9-yr follow-up period revealed that the proportion of pregnancies ending in abortion was 20% (31/154) among ELISA seropositive cows and 8% (15/193) among seronegative cows. The farm recorded 46 abortions, of which 31 (67.3%) were from seropositive cows. All sera positive by ELISA (n = 60) and sera from cows (n = 11) that were ELISA-negative but that had aborted were tested by the indirect fluorescent antibody test (IFAT) at dilutions from 1:25 to 1:200. All sera from ELISA-positive cows (n = 47) had an IFAT titer of 1:25:35 (74%) of these sera were also seropositive at a dilution of 1:200 (IFAT). Cows seropositive by ELISA had a 4-fold increased risk of having aborted at least once, compared to ELISA-seronegative cows. This association was statistically significant (P = 0.0016). The attributable fraction for this association indicated that approximately 76% of the risk for a cow having a history of abortion was attributable to seroconversion to N. caninum.     

First report of Neospora caninum infection in adult alpacas (Vicugna pacos) and llamas (Lama glama)

Neospora caninum is a cyst-forming coccidian that mainly affects bovines, although Neospora infection has also been described in other domestic and wild ruminant species. Serum samples from 78 alpacas (Vicugna pacos) and 73 llamas (Lama glama) at a unique dilution of 1:50 tested by indirect fluorescent antibody test (IFAT) were further analyzed serologically by IFAT and Western blot in both ruminant species to avoid cross-reactions with closely related coccidian parasites and to confirm the existence of N. caninum-specific antibodies. IFAT titers ranging between 1:50 and 1:800 were found. When using Western blot, N. caninum tachyzoite-specific immunodominant antigens with apparent molecular weights of 17-18, 34-35, 37, and 60-62 kDa were also recognized, although some sera with 1:50 IFAT titers proved not to have N. caninum-specific antibodies. As expected, higher IFAT titers were associated with higher anti-N. caninum reactivity in Western blot. This report documents for the first time the presence of N. caninum infection in adult alpacas and llamas from Peru.     

Canine and bovine Neospora caninum control sera examined for cross-reactivity using Neospora caninum and Neospora hughesi indirect fluorescent antibody tests

Neospora caninum is a well known protozoan parasite of domestic and wild animals. Neospora hughesi is a closely related protozoan with an unknown life cycle, host range, and infection prevalence. Many serologic surveys of N. caninum have been performed without consideration of potential cross-reactions with N. hughesi, which could confound results. The aim of this study was to investigate whether postexposure sera from animals experimentally infected with N. caninum exhibit significant reactivity differences when tested using N. caninum and N. hughesi Immunofluorescent Antibody Tests (IFAT). Pre- and postinfection serum samples from 10 dogs, 20 calves, and 17 cows were tested by dual IFATs. All pre-exposure samples for N. caninum tested seronegative for both organisms. All postexposure samples that were seropositive for N. caninum were also positive for N. hughesi, although N. hughesi antibody titers were usually 1 dilution lower (P < 0.02). Serologic surveys for N. caninum may be confounded by cross-reacting titers with N. hughesi, but true positive N. caninum antibody titers are greater than, or equal to, cross-reacting N. hughesi antibody titers.     

Seroprevalence of Toxoplasma gondii antibodies in cats and pigs from rural Western Amazon, Brazil

Antibodies to Toxoplasma gondii were assayed in sera of 63 cats and 80 pigs from 71 farms located at Rond?nia State, Western Amazon, Brazil, by the modified agglutination test (MAT) and the indirect immunofluorescent antibody test (IFAT). Antibodies (MAT > or = 1: 25) were found in 55 of 63 cats (87.3%) with titers of 1:25 in 2, 1:50 in 2, 1:100 in 7, 1:200 in 1, 1:400 in 2, 1:800 in 9, 1:1,600 in 6, and 1:3,200 or higher in 26 cats. By IFAT, antibodies were found in 55 cats (87.3%) with titers of 1:25 in 2, 1:50 in 1, 1:100 in 4, 1:200 in 4, 1: 400 in 1, 1:800 in 13, 1:1,600 in 12, and 1:3,200 or higher in 18 cats. In pig sera, by MAT, antibodies were found in 30 of 80 pigs (37.5%) with titers of 1:25 in 2, 1:50 in 3, 1:100 in 2, 1:200 in 8, 1:400 in 3, 1:800 in 5, 1:1,600 in 3, and 1:3,200 or higher in 4 pigs. By using the IFAT (titers > or = 1:64), antibodies were found in 35 (43.7%) pigs. The ingestion of undercooked tissues of infected pigs can be a source of T. gondii infection for humans and cats. However, the high seroprevalence of T. gondii in cats from the Amazon seems most likely to be indicative of high contamination of the environment by oocysts.     

Qualitative evaluation of selective tests for detection of Neospora hughesi antibodies in serum and cerebrospinal fluid of experimentally infected horses

Neospora hughesi is a newly recognized protozoan pathogen in horses that causes a myeloencephalitis similar to Sarcocystis neurona. There are no validated serologic tests using the gold standard sera that are currently available to detect specific N. hughesi antibodies and, thus, no tests available to detect antemortem exposure or estimate seroprevalence in the horse. The objectives of the present study were to establish a bank of gold standard equine sera through experimental infections with N. hughesi and to assess several serologic tests for the detection of related protozoan antibodies. Seven horses were inoculated with N. hughesi tachyzoites, and 7 horses received uninfected cell culture material. The horses were monitored, and blood and cerebrospinal fluid were collected repeatedly over a 4-mo period. With the sera, 4 different serologic techniques were evaluated. including a whole-parasite lysate enzyme-linked immunosorbent assay (ELISA), a recombinant protein ELISA, a modified direct agglutination test, and an indirect fluorescent antibody test. Qualitative and quantitative evaluation of the results showed that the N. hughesi indirect fluorescent antibody test (IFAT) consistently discriminated between experimentally infected and noninfected horses, using a cutoff of 1:640. Sera from 3 naturally infected horses had titers >1:640. Cerebrospinal fluid in all but I infected horse had very low N. hughesi IFAT titers (<1:160), starting at postinoculation day 30.     

Seroprevalence of Neospora caninum and Toxoplasma gondii antibodies in the South American opossum (Didelphis marsupialis) from the city of São Paulo, Brazil

Antibodies to Neospora caninum and Toxoplasma gondii were assayed in sera of 396 opossums (Didelphis marsupialis) from the city of S?o Paulo, Brazil. Antibodies to N. caninum were assayed using the indirect immunofluorescent antibody test (IFAT). Antibodies (IFAT, approximately 1:25) to N. caninum were found in 84 opossums (D. marsupialis) in titers of 1:25 in 46, 1:50 in 20, 1:100 in 17, and 1:400 in 1. Antibodies to T. gondii were assayed with the modified agglutination test (MAT) and the IFAT. Antibodies to T. gondii (MAT, approximately 1:25) were found in 82 (20.4%) of the 396 opossums, in titers of 1:25 in 24, 1:50 in 26, 1:100 in 18, 1:200 in 13, and 1:800 in 1. The IFAT antibodies to T. gondii were found in 148 of 396 opossums, in titers of 1:16 in 41, 1:32 in 23, 1:64 in 13, 1:128 in 6, 1:256 in 20, 1:512 in 17, 1:1,024 in 10, 1:2,048 in 10, 1:4,096 in 7, and 1:8,192 in 1. This is the first report of N. caninum and T. gondii infections in D. marsupialis.     

Comparison of serological methods and blood culture for detection of Trypanosoma cruzi infection in raccoons (Procyon lotor)

The indirect immunofluorescent antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA) were compared with blood culture for the detection of Trypanosoma cruzi infection in 83 raccoons (Procyon lotor) trapped in 4 counties of southeast Georgia. Both IFAT and ELISA detected 24 of 25 culture-positive samples (96% sensitivity). Cultures from 25 raccoons (30%) were positive for epimastigotes, whereas a total of 50 raccoons (60%) was seropositive by either the IFAT or ELISA. Forty-five of 83 serum samples (54%) were positive for anti-T. cruzi antibodies with the ELISA, and 47 were IFAT positive (57%). Forty-two of the 50 seropositive raccoons (84%) were seropositive by both tests. Endpoint titers of IFAT-positive samples were determined by testing doubling dilutions from 1:40 to 1:1280. High titers of 640 and 320 were observed for 4 raccoons trapped in 1 county (St. Catherines Island, Liberty County) and titers of 160 for 1-2 raccoons from each of the 4 counties sampled. IFAT titers and ELISA optical density values were positively correlated. Both serological tests have a high sensitivity and should be excellent tools for studying the prevalence of T. cruzi in wildlife populations.     

Neospora caninum infections in bovine foetuses and dairy cows with abortions in Argentina

Antibodies to Neospora caninum were measured in bovine foetuses, dairy cows and beef cows in Argentina using the IFAT, the N. caninum agglutination test, and the recombinant NCDG1 and NCDG2 ELISA. Serum antibodies (IFAT titre 1:80) were found in 20 of 82 (24.4%) dairy cow foetuses and one of 22 (4.5%) beef cow foetuses. Microscopic lesions suggestive of neosporosis were seen in brains of seven of eight foetuses with IFAT titres of 1:80. Antibodies (IFAT) were found in 122 of 189 (64.5%) dairy cows that aborted. Serum antibody titres (IFAT) of 189 dairy cows that aborted were: < 1:25 (67 cows), 1:25 (four cows), 1:50 (16 cows), 1:200 (seven cows), 1:> or = 800 (95 cows). Of the 87 sera with IFAT titres of < or = 1:50, 57 had no antibodies in 1:40 dilution and 30 had titres of 1:40 in the N. caninum agglutination test. Thus, sera from at least 56 dairy cows which had aborted were seronegative both in the N. caninum agglutination test and the IFAT. The distribution of positive and negative sera was similar when measured by ELISA, except that, depending on cut-off titre, the ELISA indicated a greater number of seropositive cows that were negative by the IFAT and N. caninum agglutination test. These results suggest that transplacental transmission of N. caninum in dairy cows in Argentina is frequent.     

Serological survey for antibodies to Encephalitozoon cuniculi in ownerless dogs from urban areas of Brazil and Colombia

There are 3 strains of Encephalitozoon cuniculi that occur in mammals. Strain III is associated with clinical disease in dogs, although some can be asymptomatic carriers and excrete spores in their urine. Several cases of human E. cuniculi infection caused by strain III have been observed in immunocompromised patients, indicating that E. cuniculi should be considered a zoonotic agent. Encephalitozoon cuniculi can cause fatal disease in maternally-infected or young dogs. Clinical signs in these animals included blindness, encephalitis, retarded growth rate, and nephritis. Encephalitozoon cuniculi has also been associated with primary renal failure in adult dogs. The present study used the direct agglutination test (DAT, cut-off 1:50) and the indirect fluorescent antibody test (IFAT, cut-off 1:10) to examine the prevalence of antibodies to E. cuniculi in dogs from Brazil and Colombia. Using the DAG, 31 (27.4%) of 113 dogs from Brazil and 47 (18.5%) of 254 dogs from Colombia were seropositive. Nine (14.3%) of 63 dogs from Brazil and 18 (35.3%) of the 51 dogs from Colombia were seropositive by indirect immunofluorescent antibody test. These results indicate that dogs from Brazil and Colombia are exposed to E. cuniculi.     

Serological differences in Neospora caninum-associated epidemic and endemic abortions.

A sandwich enzyme-linked immunosorbent assay (ELISA) for the sensitive and specific detection of bovine antibodies to Neospora caninum was developed and evaluated using sera from cattle experimentally infected with N. caninum, Toxoplasma gondii, Sarcocystis cruzi, Sarcocystis hominis, Sarcocystis hirsuta, Eimeria bovis, Cryptosporidium parvum, Babesia divergens, and field sera from naturally exposed animals. Field sera were classified using a gold standard that included the results from an indirect fluorescent antibody test (IFAT) and an immunoblot (IB). Based on these gold standard results, i.e., IFAT-IB results, an equal relative sensitivity and specificity of 94.2%(theta0) was reached when a cutoff of 0.034 (d0) was employed. The analysis of IFAT-IB-positive field sera showed that within groups of aborting and nonaborting dams, the animals from herds with endemic N. caninum-associated abortions had significantly higher ELISA indices than animals from herds with N. caninum-associated epidemic abortions. By contrast, IFAT-IB-positive aborting dams from herds with endemic N. caninum-associated abortions had significantly lower IFAT titers than IFAT-IB-positive aborting dams from herds with epidemic N. caninum-associated abortions. This is the first time that statistically significant serological differences between herds exhibiting epidemic and endemic N. caninum-associated abortions are described.     

Seroprevalence of Toxoplasma gondii antibodies in humans from rural Western Amazon, Brazil

Antibodies to Toxoplasma gondii were assayed in sera of 266 humans from 71 farms located at Rond?nia State, Western Amazon, Brazil, by the modified agglutination test (MAT) and the indirect immunofluorescent antibody test (IFAT). Antibodies were found in 195 humans (73.3%), with MAT titers of 1:25 in 11, 1:50 in 11, 1:100 in 16, 1:200 in 27, 1:400 in 38, 1:800 in 37, 1:1,600 in 22, and 1:3,200 or higher in 33. From the 71 farms visited, 69 had seropositive humans. Prevalence of anti-T. gondii antibodies increased with age of the people (P < 0.05), and no difference was observed in the occurrence by gender (P > 0.05). A sanitary questionnaire was applied in each farm, and statistical association between the serologic status and several variables were analyzed. Home-grown vegetable consumption and origin of drinking water (well or river) were the independent variables that displayed significant association (P = 0.002 and 0.02, respectively). Higher values of occurrence were found in people with consumption of home-grown vegetables (76.1%) and people that drink well water (75.4%) compared with people that did not consume this type of food (61.9%) and drink river water (55.2%). By IFAT (> or = 1:16), 194 of 266 (73%) humans were seropositive and there was a good correlation between MAT and IFAT.     

Seroprevalence of antibodies to Neospora caninum in dogs and raccoon dogs in Korea

Neospora caninum is an important cause of abortion in cattle, and dogs are its only known definitive host. Its seroprevalence among domestic urban and rural dogs and feral raccoon dogs (Nyctereutes procyonoides koreensis) in Korea was studied by indirect fluorescent antibody test (IFAT) and by the neospora agglutination test (NAT), respectively. Antibodies to N. caninum were found in 8.3% of urban dogs and in 21.6% of dogs at dairy farms. Antibody titers ranged from 1:50 to 1:400. Antibodies to N. caninum were found in six (23%) of 26 raccoon dogs. However, the potential role of raccoon dogs as a source of horizontal transmission of bovine neosporosis needs further investigation. The results of this study suggest that there is a close relationship between N. caninum infection among dairy farm dogs and cattle in Korea. This study reports for the first time upon the seroprevalence of N. caninum infection in raccoon dogs in Korea.     

Evaluation of ELISA for the Detection of Toxoplasma Antibodies in Swine Sera

Enzyme-linked immunosorbent assay (ELISA) is compared with the indirect fluorescent antibody test (IFAT), the indirect haemagglutination test (IHAT) and the latex agglutination (LA) test for the detection of toxoplasma antibodies in swine sera. The 100 swine sera examined represent ELISA values from > 0 to 154 EIU. The agreement was highest (0.67) between ELISA and IFAT with an ELISA cut-off value of 30 EIU, and between ELISA and the LA test with an ELISA cut-off value of 50 EIU (0.74). All sera giving < 10 EIU were negative in the other tests, and all those with > 70 EIU were positive in 1, 2 or all of the reference tests. In order to avoid false positive results with ELISA, all sera giving 10–70 EIU should be confirmed with a test which has a good specificity, e.g. IFAT. ELISA is a sensitive test and is highly suitable for the screening of large amounts of samples, but it may be too complicated for screening toxoplasma antibodies in the laboratories of abattoirs.     

Malaria seroepidemiology: comparison between indirect fluorescent antibody test and enzyme immunoassay using bloodspot eluates.

Blood sampling on filter paper is a current practice in malaria seroepidemiological studies by indirect fluorescent antibody test (IFAT). There is, however, scant comparative information about the use of bloodspot eluates for detection of malarial IgG antibodies simultaneously by IFAT and enzyme immunoassay (ELISA). Here we report data obtained by both serological methods done on 219 bloodspot eluate samples collected in a rural community in Brazilian Amazon Basin (Alto Paraíso, Ariquemes municipality) where malaria is endemic. Plasmodium falciparum and P. vivax thick smear antigens were used in the IFAT; a detergent-soluble P. falciparum antigen was prepared for ELISA. Substantial agreement of results (Kappa coefficient k = 0.686) was observed when P. falciparum antigen was used in both tests, and IFAT titers were found to be strongly correlated to ELISA antibody units (Spearman correlation coefficient rs = 0.818, p < 0.001). Only moderate agreement (k = 0.467) between IFAT with P. vivax antigen and ELISA with P. falciparum antigen was observed. Spearman correlation coefficient value between quantitative results (IFAT titers and ELISA antibody units) in this case was numerically lower (rs = 0.540, p < 0.0001). Our results suggest that, with P. falciparum antigen, both IFAT and ELISA performed on bloodspot eluates are equivalent for seroepidemiological purposes.