Evaluation of an in-house TgSAG1 (P30) IgG ELISA for diagnosis of naturally acquired Toxoplasma gondii infection in pigs

Toxoplasma gondii is an apicomplexan protozoan parasite which is able to infect a large variety of warm-blooded animals. Raw or undercooked pork has been regarded as an important source of infection for humans. The aim of this study was to evaluate an in-house enzyme-linked immunosorbent assay to diagnose natural T. gondii infection in swine using native affinity chromatography-purified T. gondii surface protein-1 (TgSAG1-ELISA) as antigen, comparing its performance to that of indirect fluorescent antibody test (IFAT) and immunoblotting (IB). To obtain a panel of sera showing the evolution of the antibody response in the time course 12 pigs were experimentally inoculated intravenously (iv) with tachyzoites of the T. gondii strains RH (clonal type I), ME49 (clonal type II) and NED (clonal type III) and serologically monitored for a period of 11 weeks. Both IFAT and ELISA showed a similar time course of antibody response to T. gondii; but by IFAT this response was characterized by rapidly rising titers with peaks at two weeks post inoculation (wpi), while the ELISA indices increased slowly and reached a maximum in most animals at five wpi. Three-hundred randomly selected sera from a total of 602 pigs of different ages derived from outdoor and indoor farms from Argentina were analyzed. Serum samples testing either positive or negative by both IFAT and IB were considered as "relative standards of comparison" (RSC). Sensitivity and specificity of TgSAG1-ELISA were obtained by a Receiver Operating Characteristics (ROC) analysis and statistical agreement among serological tests was evaluated. Antibodies to T. gondii were detected in 160 of 300 sera (53.3%) by IB, in 133 of 300 (44.3%) by IFAT and in 123 of 300 sera (41%) by TgSAG1-ELISA. One hundred and eleven sera tested positive and 118 sera tested negative by both IFAT and IB (RSC); 103 of 111 positive RSC sera tested positive by TgSAG1-ELISA, and 116 of 118 negative RSC sera tested negative by TgSAG1-ELISA. Agreement observed between RSC and TgSAG1-ELISA was almost perfect (κ=0.9124, p≥0.05) and between IFAT and IB was moderate (κ=0.53, p≥0.05). Relative sensitivity and specificity of the TgSAG1-ELISA using a cut-off index of 0.204 were of 92.8% and 98.3%, respectively. ROC analysis revealed that TgSAG1-ELISA was highly accurate (AUC=0.983) relative to the RSC. According to the results in this study, the ELISA based on affinity purified T. gondii surface antigen TgSAG1 was useful for the specific and sensitive detection of antibodies to this protozoan parasite in naturally infected pigs.     

Detection of specific antibodies to Neospora caninum and Toxoplasma gondii in naturally infected alpacas (Lama pacos), llamas (Lama glama) and vicunas (Lama vicugna) from Peru and Germany

Sera of an experimentally Neospora caninum infected llama and a non-infected control llama were used to establish an immunoblot, an ELISA and an IFAT to detect antibodies against N. caninum tachyzoites. Subsequently, serum samples collected from a total of 871 South American Camelids (SAC: Lama glama, Lama pacos, Lama vicugna) of two farms in Peru and from 32 SAC of a farm in central Germany were examined for antibodies against N. caninum and Toxoplasma gondii. Based on the recognition of specific bands in the immunoblot, sera of SAC from Peru were differentiated into N. caninum-positive (n = 18) and T. gondii-positive (n = 30) samples and into samples negative or inconclusive for both parasites. Using the immunoblot results as the reference, a modified version of the p38-ELISA and the IFAT were evaluated for detecting N. caninum antibodies in SAC sera. Applying a cut-off as determined by two graph-receiver operating characteristic analysis both, the ELISA and the IFAT, exhibited a sensitivity and specificity of about 95% in the SAC sera from Peru. Serological testing confirmed that SAC may become infected with N. caninum under field conditions in Peru. In addition to alpacas and llamas also 114 wild living vicunas had been examined for antibodies against N. caninum. However, only the alpacas and llamas but no vicunas were found N. caninum-positive. In contrast, T. gondii-seropositive animals were detected in all three SAC species. The lack of N. caninum-seropositive vicunas indicates that in the study area in Peru wild canids might not serve as definitive hosts of N. caninum while for T. gondii a life cycle including wild felids is likely. On the German farm no N. caninum- but only T. gondii-seropositive SAC (n = 14) were detected. The seroprevalence of T. gondii infection was significantly higher in adult SAC (alpacas in Peru, llamas in Germany) than in crias (i.e. < 12 months old foals) indicating that the predominant route of infection is post natal. Since the present study was restricted to a few farms, the seroprevalences determined are not representative. However, our results confirm natural infections with N. caninum and T. gondii in SAC. Whether these infections are linked to any disease, e.g. reproductive losses, has to be clarified in further studies.     

Comparative serological diagnosis of toxoplasmosis in horses using locally isolated Toxoplasma gondii

A total of 420 serum samples collected from horses of different ages, sexes and breeds, located at some horse farms in Egypt, were used for serological studies. A crude antigen of the locally isolated Toxoplasma gondii tachyzoites from horse tissues (LA) was used for the detection of T. gondii antibodies in horses. It showed good diagnostic efficiency (38.1%) by Enzyme Linked Immunosorbent Assay (ELISA). To increase this efficiency, an affinity purification process was performed. Two fractions were obtained from LA by CNBr-Sepharose 4B affinity column chromatography named; unbound (LAunb) and bound (LAb). LAb showed the highest diagnostic potency (51.7%), while LAunb showed the lowest value (31.7%) using ELISA. The electrophoretic profile of LA (12 bands), LAb (6 bands) and LAunb (6 bands) showed molecular weights ranged from 25.1 to 184.3kDa. The immunoreactive bands of each of the three antigens were identified with infected horse sera by immunoblot assay. Four immunogenic bands of 155.8, 115.1, 83.2 and 66.2kDa were identified in LAb and probably were responsible for the highest diagnostic potency. Examination of horse sera by Indirect Fluorescence Antibody Test (IFAT) at a dilution of 1: 64 and Modified Agglutination Test (MAT) at a dilution of 1: 25 revealed that 170 (40.5%) and 202 (48.1%) had antibodies against T. gondii, respectively. The current research introduces crude and purified fractions (bound and unbound) obtained from the locally isolated tachyzoites (equine origin), which are utilized globally for the first time in detection of T. gondii antibodies in horses. Furthermore, this study recommended utilization of the bound fraction in diagnosis of toxoplasmosis using indirect ELISA which proved better diagnostic potency compared with IFAT and MAT.     

广东清远鹅弓形虫病血清学调查

本调查采用MAT方法对来自广东省清远市12个规模化养鹅场的520份血清进行了弓形虫病血清学调查。结果发现,所调查的清远市8个肉鹅场中,有2个场弓形虫血清阳性率均为2.22%,其余6个场未发现阳性样品;4个种鹅场均有弓形虫血清阳性样品,阳性率分别为:2.5%、5%、7.5%、2.5%。种鹅的弓形虫感染率(4.38%)明显高于肉鹅(0.56%)。结果表明,清远市规模化养鹅场的鹅弓形虫血清阳性率较低,种鹅弓形虫血清阳性率高于肉鹅,差异显著(P<0.05),种鹅可能成为弓形虫的传播源。     

Serological survey and first finding of Neospora caninum in Taiwan, and the detection of its antibodies in various body fluids of cattle

A serological survey for antibodies against Neospora caninum in cattle, goats and farm dogs in Taiwan was carried out. Sera of 613 cattle from 25 dairy farms, 24 goats from six goat farms and 13 dogs from six dairy cattle farms were tested for antibodies against N. caninum using indirect fluorescence antibody test (IFAT). The same sera were also tested for antibodies against Toxoplasma gondii using latex agglutination test. Of the 613 cattle sera, 44.9% (275/613) were found to have antibodies against N. caninum. Among these 275 positive cattle, 77 also possessed antibodies against T. gondii. Nevertheless, 92 cattle which were negative for N. caninum showed antibodies against T. gondii. Of the 24 goat sera tested, none was found to be positive for N. caninum but 50% (12/24) were positive for T. gondii. Of the 13 farm dogs tested, three were found to possess antibodies against N. caninum, two of which tested negative for T. gondii antibodies. Besides sera, antibodies to N. caninum in cattle could be observed in the milk, vaginal secretion and saliva. However, the order of higher frequency of antibodies detection is in sera, milk, vaginal secretion and saliva. This is the first demonstration of the presence of antibodies to N. caninum in vaginal secretion and saliva of cattle. A 50microm cyst was observed in the brain of one of the 13 prednisolone-treated SPF ICR mice which had been peritoneally inoculated 4 months earlier with the brain homogenate of a serologically N. caninum positive but T. gondii negative cattle. Thus, we have confirmed for the first time the presence of N. caninum in Taiwan and also observed that it is widespread among dairy cattle and farm dogs.     

Unexpected oocyst shedding by cats fed Toxoplasma gondii tachyzoites: in vivo stage conversion and strain variation

Tachyzoites, bradyzoites (in tissue cysts), and sporozoites (in oocysts) are the three infectious stages of Toxoplasma gondii. The prepatent period (time to shedding of oocysts after primary infection) varies with the stage of T. gondii ingested by the cat. The prepatent period (pp) after ingesting bradyzoites is short (3-10 days) while it is long (18 days or longer) after ingesting oocysts or tachyzoites, irrespective of the dose. The conversion of bradyzoites to tachyzoites and tachyzoites to bradyzoites is biologically important in the life cycle of T. gondii. In the present paper, the pp was used to study in vivo conversion of tachyzoites to bradyzoites using two isolates, VEG and TgCkAr23. T. gondii organisms were obtained from the peritoneal exudates (pex) of mice inoculated intraperitoneally (i.p.) with these isolates and administered to cats orally by pouring in the mouth or by a stomach tube. In total, 94 of 151 cats shed oocysts after ingesting pex. The pp after ingesting pex was short (5-10 days) in 50 cats, intermediate (11-17) in 30 cats, and long (18 or higher) in 14 cats. The strain of T. gondii (VEG, TgCKAr23) or the stage (bradyzoite, tachyzoite, and sporozoite) used to initiate infection in mice did not affect the results. In addition, six of eight cats fed mice infected 1-4 days earlier shed oocysts with a short pp; the mice had been inoculated i.p. with bradyzoites of the VEG strain and their whole carcasses were fed to cats 1, 2, 3, or 4 days post-infection. Results indicate that bradyzoites may be formed in the peritoneal cavities of mice inoculated intraperitoneally with T. gondii and some bradyzoites might give rise directly to bradyzoites without converting to tachyzoites.     

Comparison of Dot-ELISA, ELISA and IFAT for the detection of IgG antibodies to Sarcocystis muris in experimentally infected and immunized mice

The dot enzyme-linked immunosorbent assay (Dot-ELISA) was used for the detection of IgG antibodies to Sarcocystis muris and compared with the enzyme-linked immunosorbent assay (ELISA) and the indirect fluorescent antibody test (IFAT). In experimentally infected mice, first positive reactions occurred in the Dot-ELISA between 18 and 32 days after infection (dpi), in the ELISA between 18 and 49 dpi, and in the IFAT between 11 and 25 dpi. Maximum titers were 1:40 960 in the Dot-ELISA, 1:1280 in the ELISA and 1:2560 in the IFAT. High titers persisted until the end of the examination period (182 dpi) in all 3 tests. In immunized mice, all 3 tests detected antibodies 7 days after the first injection of protein antigen. The highest titers of 1:5120 and 1:10 240 were recorded in the Dot-ELISA after 35 days; titers of 1:1280 and 1:2560 were observed in the ELISA, and titers of 1:160 and 1:320 in the IFAT after 42 days. No false-positive reactions occurred in the Dot-ELISA and in the IFAT when 177 sera from non-infected mice were examined, but 1% (2/177) of the sera reacted positively in the ELISA. Sixty-three percent (94/150) of sera from mice infected experimentally with Toxoplasma gondii showed slight positive reactions up to 1:40 in the Dot-ELISA; 9% (13/150) of the sera reacted positively up to 1:40 in the IFAT and 4% (6/150) up to 1:20 in the ELISA. The Dot-ELISA appears to be a good alternative to the ELISA and the IFAT in the serodiagnosis of sarcosporidiosis and should be further evaluated for the serodiagnosis of other parasitic diseases.     

Detection of IgG antibodies to Neospora caninum and Toxoplasma gondii in dogs examined in a veterinary hospital from Brazil.

A total of 163 dogs with neuromuscular, respiratory and/or gastrointestinal disorders, was admitted at the Veterinary Hospital, Federal University of Uberlandia, Brazil, and submitted to serology for Toxoplasma gondii and Neospora caninum. Assays for T. gondii included indirect haemagglutination (IHA), indirect fluorescent antibody (IFAT-Tg), immunoenzymatic (ELISA), and immunoblotting (IB-Tg). Assays for N. caninum included IFAT-Nc and immunoprecipitation (IP-Nc). Based on concordant results by three serological tests (IHA, IFAT-Tg and ELISA) for T. gondii, and divergent results further confirmed by IB-Tg for reactivity to TgSAG1, the 163 sera were divided into two groups: 59 (36%) Tg-seropositive samples and 104 (64%) Tg-seronegative samples. Antibodies to Neospora were detected in 11 (6.7%) out of 163 analyzed dog sera, with 5 (3.1%) samples reactive to both parasites (Tg+/Nc+), and 6 (3.7%) reactive only to Neospora (Tg-/Nc+). Antibodies only to T. gondii were found in 54 (33%) samples. Among the 11 Neospora-positive sera analyzed by IB-Tg, the five sera Tg+/Nc+ showed strong reactivity to Toxoplasma antigens, especially to TgSAG1 (p30). No reactivity was observed to TgSAG1 in the six samples Tg-/Nc+. By IP-Nc, two highly immunodominant antigens (29 and 35kDa proteins) were recognized by all 11 IFAT-Nc positive sera. Our results suggest that the infection by N. caninum can be concomitantly present in dogs from this area, although less common, and therefore should be considered in the differential clinical diagnosis with T. gondii in dogs presenting neuromuscular, respiratory and/or gastrointestinal disorders.     

Serological investigation of aborted sheep and pigs for infection by Neospora caninum

Serology for Neospora caninum was undertaken using direct ELISAs on sera from 660 aborted sheep and 454 breeding sows, which had aborted or were considered infertile. All ovine sera were further tested by indirect fluorescent antibody test (IFAT) for N. caninum, and a latex agglutination test (LAT) for Toxoplasma gondii was performed on 423 of the samples, including all those positive by ELISA. ELISA-positive porcine sera were tested by IFAT and an inhibition ELISA for antibodies to N. caninum and by LAT for T. gondii. Only 3 (0.45%) of the ovine sera were seropositive for N. caninum by both ELISA and IFAT whereas although 40 porcine sera were seropositive by ELISA all were negative by IFAT. The results suggest that environmental exposure to N. caninum occurs rarely in sheep and pigs.     

Soil contamination of Toxoplasma gondii oocysts in pig farms in central China

Toxoplasmosis in pigs is a large threat to pig industry as well as pork consumers. Most pigs become infected by ingestion of oocysts from contaminated environment (soil, water and feed) or infected animal tissues postnatally. In the present study, field studies were conducted to evaluate the relationship between soil contamination status of Toxoplasma gondii oocysts and T. gondii infection in pigs in 12 pig farms with different density of cats in central China. The presence of T. gondii oocysts in soil were determined by PCR and loop-mediated isothermal amplification (LAMP). T. gondii DNA was found in 11 farms with different cat density excepting one farm exposed to low cat density. Twenty (21.1%) and 36 (37.9%) of 95 soil samples were T. gondii positive by PCR and LAMP, respectively (0.01相似文献   

Cross-sectional survey in pig breeding farms in Hesse, Germany: seroprevalence and risk factors of infections with Toxoplasma gondii, Sarcocystis spp. and Neospora caninum in sows

A cross-sectional survey was performed to estimate the prevalences of antibodies to Toxoplasma gondii (ELISA, IFAT), Sarcocystis spp. (ELISA, using S. miescheriana as antigen) and Neospora caninum (ELISA, immunoblotting) in sows from breeding farms in southern Hesse, Germany. A total of 2041 plasma samples of sows from 94 randomly selected farms was examined. Data on farm profiles, husbandry management and sows were collected by a questionnaire and exploratively analysed. For T. gondii the ELISA results agreed well with the results obtained by IFAT (kappa=0.71). Antibodies to T. gondii were detected by ELISA in 19% of the sows. Sixty-nine percent of the farms had at least one seropositive sow, and a within-farm seroprevalence of >or=50% was observed in 14% of all farms. The prevalence of anti-T. gondii antibodies was positively correlated with the age of sows. The within-herd seroprevalence was significantly higher in farms with reproductive disorders than in those without such problems. On the farm level, the farm type 'piglet production' (versus 'pedigree breeding' or 'farrow-to-finish') was the only risk factor associated with the presence of T. gondii-seropositive sows. Antibodies to Sarcocystis spp. were found in 29% of the sows. Seventy-two percent of the farms harboured at least one seropositive sow, and a within-farm seroprevalence of >or=50% was detected in 23% of all farms. The seroprevalence increased significantly with the age of sows. On the farm level, only the farm type 'piglet production' (versus 'pedigree breeding') and the replacement of sows by purchasing (versus raising on the own farm) were identified as risk factors for seropositivity. Antibodies to N. caninum were detected in one sow using both the screening ELISA and the confirmatory immunoblotting technique. This may indicate the first natural N. caninum infection in pigs.     

Isolation and molecular characterization of Toxoplasma gondii from chickens and ducks from Egypt

The prevalence of Toxoplasma gondii in free range chickens is a good indicator of the prevalence of T. gondii oocysts in the environment because chickens feed from the ground. In the present study, prevalence of T. gondii in 121 free range chickens (Gallus domesticus) and 19 ducks (Anas sp.) from a rural area surrounding Giza, Egypt was assessed. Blood, heart, and brain from each animal were examined for T. gondii infection. Antibodies to T. gondii, assayed with the modified agglutination test (MAT), were found in 49 (40.4%) chickens in titers of 1:5 in 11, 1:10 in four, 1:20 in four, 1:40 in eight, 1:80 in 10, and 1:160 or more in 12 chickens. Antibodies were found in three ducks each with a titer of 1:80. Hearts and brains of seropositive (MAT > or = 1:5) chickens and ducks were bioassayed in mice. Additionally, hearts and brains of seronegative (MAT<1:5) animals were bioassayed in T. gondii-free cats. T. gondii was isolated from 19 of 49 seropositive chickens (one with a titer of 1:5, two with a titer of 1:20, one with a titer of 1:40, five with a titer of 1:80, three with a titer of 1:160, and seven with a titer of > or = 1:360). One cat fed tissues pooled from 15 seronegative chickens shed T. gondii oocysts, while two cats fed tissues of 34 seronegative chickens did not shed oocysts. T. gondii was isolated from one of the seropositive ducks by bioassay in mice. The two cats fed tissues from 16 seronegative ducks did not shed oocysts. Genotyping of 20 chicken isolates of T. gondii using the SAG 2 locus indicated that 17 isolates were type III and three were type II. The duck isolate of T. gondii was type III. The mice inoculated with tissue stages of all 21 isolates of T. gondii from chickens and ducks remained asymptomatic, indicating that phenotypically they were not type I because type I strains are lethal for mice. Infections with mixed genotypes were not found.     

重庆部分地区鹅副黏病毒的分离鉴定及血清学调查

从重庆部分地区规模化鹅场收集以肝、脾肿大瘀血,肠黏膜出血、坏死为主要病理变化的病死鹅,无菌取其肝脏、脾脏组织,进行病毒的分离培养、血清学试验和动物回归试验,分离鉴定出一株鹅副黏病毒;并做了鸡、鸭、鹅的鹅副黏病毒和鸡新城疫病毒HI抗体检测,以了解该地区鸡、鸭、鹅群中副黏病毒的流行情况,结果表明,鸡的阳性率分别为70.1%、83.9%,鸭分别为1.7%、6.8%,鹅分别为61.2%、55.4%。证实鹅副黏病毒在这些地区感染比较严重,同时与鸡新城疫病毒存在交叉免疫原性。应引起当地养鹅专业户的高度重视。     

Serologic immunoreactivity to Neospora caninum antigens in dogs determined by indirect immunofluorescence, western blotting and dot-ELISA

Neospora caninum, is a coccidian protozoan known as a major cause of bovine abortion and canine neuropathies. The aim of the present study was to develop a reliable and quick test to detect antibodies to N. caninum in dog sera. Sixty-five serum samples from dogs, including 35 positive and 30 negative for N. caninum antibodies were used for standardization of the test. In parallel, immunoreactivity of the sera to Toxoplasma gondii antigens was investigated using a passive agglutination test. A dot-ELISA test, using soluble extract of N. caninum tachyzoites on nitrocellulose ester membranes, was developed and standardized. SDS-PAGE and complementary analysis of reactivity by Western blotting were used for the characterization of the immunoreactive fractions of all tested sera. The sensitivity and specificity of the dot-ELISA were 94 and 73%, respectively, compared to IFAT at a cut-off of 1:50, and 87 and 100% compared to IFAT at a cut-off of 1:25. Among the sera that tested positively for both IFAT and dot-ELISA, only 8.6% were reactive to T. gondii. The most immunoreactive fractions in Western blots were the 14-, 33-, 42- and 55 kDa bands, with percentages of 42, 60, 42 and 37%, respectively. The 60 kDa band showed a non-specific reaction in 43% of neosporosis-negative animals by both dot-ELISA and IFAT. These results indicate that the dot-ELISA using N. caninum antigen present good sensitivity and specificity, and might be used as a screening test to detect antibodies to N. caninum in dogs.     

Serodiagnosis of postnatally and prenatally induced toxoplasmosis in sheep

Nineteen pregnant (45 to 90 days of gestation) and 9 nonpregnant ewes were inoculated orally with 1,000 or 10,000 oocysts of Toxoplasma gondii. Pregnant ewes were euthanatized at days 14 (2 ewes), 21 (1 ewe), 23 (1 ewe), 28 (2 ewes), 35 to 42 (6 ewes), and 49 to 62 (6 ewes), and antibody titers in fetal and maternal sera were assayed, using the modified agglutination, latex agglutination, indirect hemagglutination, and dye tests. Although all ewes developed antibody titers of greater than or equal to 1,024 within 28 days after inoculation, fetuses were seronegative up to 28 days, using the modified agglutination test. Toxoplasma gondii antibodies were found in fetuses, using the modified agglutination and dye tests 35 days after ewes were inoculated. Latex agglutination and indirect hemagglutination tests were insensitive for detection of T gondii antibodies in ovine fetal sera. Toxoplasma gondii antibody titers in nonpregnant ewes were similar to those in pregnant ewes. Passively acquired T gondii antibodies from the colostrum decreased from 1,024 to less than 16 between 49 and 56 days of age in 1 lamb and between 62 and 106 days in its twin.     

Protective immunity to toxoplasmosis in pigs vaccinated with a nonpersistent strain of Toxoplasma gondii

The RH strain of Toxoplasma gondii is highly virulent; 1 infective organism is uniformly lethal for mice. Three pigs inoculated SC with 10(3) tachyzoites of the RH strain developed fever, but otherwise remained normal, and T gondii was not demonstrated in their tissues by bioassay into mice. To determine whether vaccination with the RH strain could induce protective immunity to oral challenge with T gondii oocysts, 12 pigs were divided into 3 groups (A, B, C) of 4 pigs each. Pigs in groups A and B were inoculated IM with 10(6) tachyzoites of the RH strain and 4 pigs in group C served as uninoculated controls. Except for fever, the pigs remained clinically normal after inoculation with the RH strain and T gondii was not found by bioassay in mice of tissues from 4 pigs euthanatized 64 days after inoculation. Pigs in groups B and C were challenge-inoculated orally with 10(4) (4 pigs) or 10(5) (4 pigs) T gondii oocysts 72 days after vaccination with the RH strain. The previously uninoculated pigs developed fever, anorexia, and diarrhea from 3 to 8 days after the oocyst challenge. One of the 2 pigs given 10(5) oocysts became moribund because of toxoplasmosis and was euthanatized 9 days after inoculation. Pigs vaccinated with the RH strain remained free of clinical signs after challenge with oocysts. Results of the bioassays indicated that fewer tissue cysts developed in the RH strain-vaccinated pigs than in the previously uninoculated control pigs.     

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.     

Seroprevalence of Toxoplasma gondii in sows from slaughterhouses and in pigs from an indoor and an outdoor farm in Argentina

The objective of the present study was to determine the prevalence of Toxoplasma gondii antibodies from slaughter sows and from pigs raised at an indoor and an outdoor swine farm. Serum samples were obtained from 230 slaughter sows belonging to 83 farms distributed in 5 provinces. Blood samples were collected monthly from pigs of different ages from an intensive management indoor farm (farm 1). A cross-sectional study was carried-out from an outdoor farm (farm 2). All sera were tested for T. gondii antibodies by the modified agglutination test (MAT), using formalin-fixed tachyzoites as antigen. An antibody titer > or =1:25 was considered positive. Antibodies to T. gondii were detected in 87 (37.8%) of 230 sows sera. Distribution among provinces was: 37.1% from Santa Fe, 62.8% from Buenos Aires, 3.3% from San Luis, 58.7% from La Pampa and 24% from Córdoba. Four of 88 (4.5%) serum samples from farm 1 had antibodies to T. gondii and none of the negative pigs seroconverted. However, 45 of 112 samples from farm 2 were positive (40.2%) with the following distribution: sows 100%; nursery 40%; growers 13.8% and fatteners 20%. It is concluded that the prevalence of T.gondii antibodies among sows seems to be quite variable. T. gondii prevalence was related to the facilities and management of the farm.     

Establishment of a competitive ELISA (cELISA) system for the detection of influenza A virus nucleoprotein antibodies and its application to field sera from different species

A recombinant baculovirus (RBV) encoding the nucleoprotein (NP) of avian influenza virus (AIV) was generated and the appropriate protein was expressed in Sf9 cells. Purified recombinant NP and the NP-specific monoclonal antibody HB65 were used to establish a competitive ELISA (cELISA) system for the detection of NP-specific antibodies in sera of ducks, geese and wild birds. Tests to evaluate this method were carried out using sera of ducks experimentally infected with AIV, pre-immune duck and chicken sera, and poultry field sera, which tested negative in the haemagglutination inhibition (HI) assay, and field sera of several poultry species experimentally infected with other viruses. The evaluation of the test demonstrated a high sensitivity and specificity of this method. Tests carried out using field sera of duck and goose flocks revealed widely corresponding results obtained by HI assay and cELISA indicating that this test is applicable for flock diagnosis. Differing results were obtained for individual samples. It can be assumed that for the most part this was because of a better recognition of the conserved NP antigen by serum antibodies, although some results remained unclear.     

Toxoplasma gondii infection in adult llamas (Lama glama) and vicunas (Vicugnavicugna) in the Peruvian Andean region

The present study was designed to investigate Toxoplasma gondii infection in adult llamas (Lama glama) and vicunas (Vicugna vicugna) in the Peruvian Andean region, for which to date no information has been available. Serum samples from 43 llamas (L. glama) and 200 vicunas were tested by IFAT detecting titres of 1:50 or higher in 55.8% (33.9-70.9%) and 5.5% (2.8-9.6%), respectively. IFAT titres ranged from 1:50 to 1:6400. In order to avoid cross reactions with closely related coccidian parasites and to confirm the existence of T. gondii specific antibodies, IFAT positive sera from both ruminant species were also analysed by western blot. T. gondii specific antigens were recognised by IFAT positive sera, although different IFAT cut-off points could be selected for llamas (1:200) and vicunas (1:50) meaning seroprevalence of 44.2% (29.1-60.1%) and 5.5% (2.8-9.6%), respectively. Based on the frequency and intensity of tachyzoite antigen recognition, at least three immunodominant antigens with apparent molecular weights of 22-24, 30, and 38-40 kDa were detected, together with other minor protein fractions located in the 18-73 kDa range. This study documents for the first time the presence of T. gondii infection and reports the target T. gondii antigens in adult llamas and vicunas in Peru.