Comparison of Different Commercial Serological Tests for the Detection of Toxoplasma gondii Antibodies in Serum of Naturally Exposed Pigs

Toxoplasma gondii is the aetiological agent of the zoonotic disease toxoplasmosis and transmitted among other ways by chemically and physically untreated, that is, raw pork to humans. The detection of Toxoplasma gondii is impossible by currently practiced meat inspection, but serological tests can be used to detect Toxoplasma gondii antibodies in pig herds and can consequently be helpful to identify potentially contaminated pork. Therefore, appropriate serological tests are required. In this study, serum samples of 1368 naturally exposed slaughter pigs from 73 Austrian farms were collected. Serum samples of at least 16 slaughter pigs per farm were tested. The prevalence of Toxoplasma gondii antibodies in serum was measured by a commercial available modified agglutination test (MAT) and compared to three different commercial available enzyme‐linked immunosorbent assays (ELISA). The MAT detected 6.5%, ELISA I 6.7%, ELISA II 4.8% and ELISA III 4.3% of the pigs as Toxoplasma gondii antibody positive. The agreement, according to the kappa coefficient (κ), was substantial between the MAT and ELISA I (κ = 0.62), II (κ = 0.64) and III (κ = 0.67). A better agreement was determined between ELISA I and II (κ = 0.715), ELISA I and III (κ = 0.747) and ELISA II and III (κ = 0.865). At least one pig per farm was detected Toxoplasma gondii antibody positive in 17 (23.3%) farms by the MAT, 26 (35.6%) farms by ELISA I, 16 (21.9%) farms by ELISA II and 11 (15.1%) farms by ELISA III. Pig farms with a high number of Toxoplasma gondii antibody‐positive pigs or high antibody titres were identified by all of the four used serological tests. Concerning the occurrence of Toxoplasma gondii antibodies in Austrian pig farms, a monitoring and surveillance programme would be reasonable to find high‐risk farms.     

Receiver-operating characteristic curves and likelihood ratios: improvements over traditional methods for the evaluation and application of veterinary clinical pathology tests

Receiver-operating characteristic (ROC) curves provide a cutoff-independent method for the evaluation of continuous or ordinal tests used in clinical pathology laboratories. The area under the curve is a useful overall measure of test accuracy and can be used to compare different tests (or different equipment) used by the same tester, as well as the accuracy of different diagnosticians that use the same test material. To date, ROC analysis has not been widely used in veterinary clinical pathology studies, although it should be considered a useful complement to estimates of sensitivity and specificity in test evaluation studies. In addition, calculation of likelihood ratios can potentially improve the clinical utility of such studies because likelihood ratios provide an indication of how the post-test probability changes as a function of the magnitude of the test results. For ordinal test results, likelihood ratios can be calculated on a category-specific basis from the empirical data or by using the slope of the line joining adjacent category limits on the ROC curve. For continuous test results, data need to be categorized into intervals for estimation of likelihood ratios, or they can be calculated as the slope (tangent) to the ROC curve at a unique test value. We use ROC analysis and calculate likelihood ratios to evaluate the performance of tests reported in 2 articles previously published in this journal.     

Emergency presentations of 4 dogs with suspected neurologic toxoplasmosis

Objective: To review the signalment, clinical signs, abnormal laboratory data, therapeutics, and response to therapy of dogs with clinical signs consistent with toxoplasmosis infection. Series summary: A retrospective review was performed on the records of 4 dogs presented to the Animal Emergency Center between January 1998 and February 2000 exhibiting neurologic signs and having elevated titers for Toxoplasma gondii. A tentative diagnosis of toxoplasmosis was based upon one of the following criteria: (1) a serial 4‐fold or greater change in serum T. gondii IgG titers; 2) serially decreasing serum T. gondii IgM titers with concurrent increasing serum T. gondii IgG titers; or 3) positive cerebrospinal fluid (CSF) T. gondii titers. In addition, inclusion of cases was limited to dogs that showed improvement of neurologic signs following treatment with antiprotozoal drugs. Trimethoprim–sulfamethoxazole treatment was associated with successful elimination of clinical signs in all of the dogs. Two of the dogs developed side effects potentially attributed to the trimethoprim–sulfamethoxazole (TMS), and antiprotozoal treatment was continued using clindamycin. Unique information presented: Toxoplasmosis is an important differential diagnosis in any dog that presents as an emergency with central or peripheral neurologic signs. Affected dogs need not be immunocompromised for clinical signs of toxoplasmosis to occur. Appropriate treatment with TMS or clindamycin can lead to resolution of clinical signs.     

Seroprevalence of <Emphasis Type="Italic">Toxoplasma gondii</Emphasis> antibodies from slaughter pigs in Chongqing,China

Toxoplasmosis is a disease caused by the protozoan Toxoplasma gondii which infects most genera of warm-blooded animals, including humans. The objective of this investigation is to evaluate the seroprevalence of toxoplasmosis in pigs in Chongqing Municipality, southwest China. Slaughterhouse pigs’ serum samples collected from six different regions in Chongqing were assayed for T. gondii antibodies by an indirect hemagglutination test. The average seroprevalence of T. gondii were found in 30.6% (278/908) in slaughter pigs, ranging from 21.6% to 40.9% among different sampling sites. The results indicated that toxoplasmosis in swine of Chongqing Municipality was relatively serious, and the pork may be an important source for human infection with T. gondii. Comprehensive measures are needed to strengthen further prevention and control of the disease in Chongqing.     

Serological Survey of Toxoplasma gondii Infection in the Domestic Goose (Anser domestica) in Southern China

In the present study, the antibodies to Toxoplasma gondii in 191 farm‐bred and 83 house‐bred geese (Anser domestica) were assessed for the prevalence of T. gondii infection in southern China with the modified agglutination test. Antibodies to T. gondii (MAT ≥ 1 : 5) were found in 27 (14.14%) of farm‐bred geese and 14 (16.87%) of house‐bred geese. Geese infected with T. gondii may be a source of T. gondii infection for humans and cats.     

The design of a Social Cost‐Benefit Analysis of preventive interventions for toxoplasmosis: An example of the One Health approach

Toxoplasma gondii infections cause a large disease burden in the Netherlands, with an estimated health loss of 1,900 Disability Adjusted Life Years and a cost‐of‐illness estimated at €44 million annually. Infections in humans occur via exposure to oocysts in the environment and after eating undercooked meat containing tissue cysts, leading to asymptomatic or mild symptoms, but potentially leading to the development of ocular toxoplasmosis. Infection in pregnant women can lead to stillbirth and disorders in newborns. At present, prevention is only targeted at pregnant women. Cat vaccination, freezing of meat destined for undercooked consumption and enhancing biosecurity in pig husbandries are possible interventions to prevent toxoplasmosis. As these interventions bear costs for sectors in society that differ from those profiting from the benefits, we perform a social cost‐benefit analysis (SCBA). In an SCBA, costs and benefits of societal domains affected by the interventions are identified, making explicit which stakeholder pays and who benefits. Using an epidemiological model, we consider transmission of T. gondii after vaccination of all owned cats or cats at livestock farms. To identify relevant high‐risk meat products that will be eaten undercooked, a quantitative microbial risk assessment model developed to attribute predicted T. gondii infections to specific meat products will be used. In addition, we evaluate serological monitoring of pigs at slaughter followed by an audit and tailor made advice for farmers in case positive results were found. The benefits will be modelled stochastically as reduction in DALYs and monetized in Euro's following reference prices for DALYs. If the balance of total costs and benefits is positive, this will lend support to implementation of these preventive interventions at the societal level. Ultimately, the SCBA will provide guidance to policy makers on the most optimal intervention measures to reduce the disease burden of T. gondii in the Netherlands.     

Toxoplasma gondii in edible fishes captured in the Mediterranean basin

The issue of whether market fish can be involved in the transmission of Toxoplasma gondii in the marine environment is highly debated since toxoplasmosis has been diagnosed frequently in cetaceans stranded along the Mediterranean coastlines in recent times. To support the hypothesis that fishes can harbour and effectively transmit the parasite to top‐of‐the‐food‐chain marine organisms and to human consumers of fishery products, a total of 1,293 fishes from 17 species obtained from wholesale and local fish markets were examined for T. gondii DNA. Real‐time PCR was performed in samples obtained by separately pooling intestines, gills and skin/muscles collected from each fish species. Thirty‐two out of 147 pooled samples from 12 different fish species were found contaminated with T. gondii DNA that was detected in 16 samples of skin/muscle and in 11 samples of both intestine and gills. Quantitative analysis of amplified DNA performed by both real‐time PCR and digital PCR (dPCR) confirmed that positive fish samples were contaminated with Toxoplasma genomic DNA to an extent of 6.10 × 10^?2 to 2.77 × 10⁴ copies/ml (quantitative PCR) and of 1 to 5.7 × 10⁴ copies/ml (dPCR). Fishes are not considered competent biological hosts for T. gondii; nonetheless, they can be contaminated with T. gondii oocysts flowing via freshwater run‐offs (untreated sewage discharges, soil flooding) into the marine environment, thus acting as mechanical carriers. Although the detection of viable and infective T. gondii oocysts was not the objective of this investigation, the results here reported suggest that fish species sold for human consumption can be accidentally involved in the transmission route of the parasite in the marine environment and that the risk of foodborne transmission of toxoplasmosis to fish consumers should be further investigated.     

Seroprevalence of Toxoplasma gondii infection in sheep and alpacas (Llama pacos) in Chile

Serum samples from 408 sheep from different regions of Chile and 447 alpacas (Llama pacos) from the north of the country were tested for Toxoplasma gondii antibodies. The indirect haemagglutination test (IHAT) was used in both species and the indirect immunofluorescence test (IIFT) was also used on the sheep samples in order to compare the performance of the tests in that species. In both tests, titers ≥1:16 were considered diagnostically significant. Sera from 49 sheep (12%) were positive to T. gondii antibodies by the IHAT. When using the IIFT, 114 sheep sera (28%) were positive. The different results obtained in sheep sera between the tests were significant (p<0.0001). No differences were observed between geographical locations or sex of the sampled sheep regarding serological detection of T. gondii antibodies in sheep. As expected, adult sheep showed higher T. gondii reactivity than young sheep (p=0.0008). The corrected prevalence of toxoplasmosis in alpaca was 16.3% (32 positive out of 447). The rather low prevalence in alpacas may be associated with their extensive management as well as the extreme climatic conditions of The Andes which apparently would not be favorable for the transmission of the parasite.     

Seroprevalence and risk factors of caprine toxoplasmosis in Minas Gerais, Brazil

The objective of this work was to carry out a study on caprine toxoplasmosis in the state of Minas Gerais, Brazil. To determine the prevalence of toxoplasmosis in goats in Minas Gerais, 767 sera from goats were tested by ELISA (enzyme-linked immunosorbent assay) and IFAT (indirect fluorescence antibody test). The prevalence of antibodies to Toxoplasma gondii was 43.0% and 46.0% by ELISA and IFAT, respectively. It was observed that 26.8% of the goats show low-avidity IgG to T. gondii. These results suggest the presence of animals in recent phase of toxoplasmosis in Minas Gerais. The risk factors for toxoplasmosis in goats were: age over 36 months (OR = 1.21; IC 95% 1.02–1.44), use of pen (OR = 1.83; IC 95%1.01–3.31) and pure breed animals (OR = 2.49; IC 95% 1.11–5.59).     

Determination of the More Adequate Modified Agglutination Test Cut‐off for Serodiagnosis of Toxoplasma gondii Infection in Sheep

Toxoplasmosis is one of the most common food borne disease world‐wide. Among food animals, sheep seems to having higher prevalence of Toxoplasma gondii infection. However, there is no consensus about the best cut‐off for serodiagnosis in sheep. To estimate the more adequate cut‐off value of Modified Agglutination Test (MAT) for serodiagnosis in sheep, a commercial ELISA kit was used as a golden standard. Evaluation of the optimal sensitivity and specificity was calculated using Youden’s J‐statistics. Values obtained were used to estimate the prevalence of sheep toxoplasmosis. One thousand four hundred and sixty seven blood samples were collected randomly from 160 farms from northern Portugal, representing approximately 10% of the ovine population from the region. All sera were tested for anti‐T. gondii antibodies using the MAT. One hundred nine sheep (7.4%) presented a MAT titer ≥1 : 80; 45 (3.0%) a MAT titer of 1 : 40; 97 (6.6%) a MAT titer of 1 : 20 and 1216 (83.0%) a MAT titer ≤1 : 20. The best Youden’s J‐statistic was obtained at 1 : 20 titer (0.752), with 86.15% of sensitivity and 89.09% of specificity with negative and positive predictive values of 90.32% and 84.48% respectively, suggesting that the 1 : 20 was the most appropriate cut‐off for serodiagnosis of toxoplasmosis in sheep. Assuming this cut‐off, the prevalence of toxoplasmosis in the studied population was 17.1% and 92 (57.5%) of the 160 studied flocks having one or more positive sheep. Those results indicate that toxoplasmosis in Portugal should be considered in the differential diagnosis of abortions in sheep and neurological signs in lambs. Furthermore, while Portugal produces ovine meat for internal consumption and for exportation, isolation of T. gondii from ovine meat and further characterization of the isolates will be needed to understand the risk that ovine toxoplasmosis may represent for human health.     

Toxoplasmosis Prevention and Testing in Pregnancy,Survey of Obstetrician–Gynaecologists

Toxoplasmosis in pregnant women can lead to congenital disease with severe neurological and ocular complications in the foetus. In 2006, we surveyed US obstetrician–gynaecologists to determine their knowledge and practices about toxoplasmosis prevention and testing. Questionnaires were mailed (four mailings) to a random sample of 1200 of the 33 354 members of the American College of Obstetricians and Gynecologists (ACOG). Of the 1200 surveyed, 502 (42%) responded. The respondents were similar to all ACOG members by gender, region of the country and practice type (P > 0.5), and age (respondents were slightly younger, mean 46 years versus 47 years). To prevent toxoplasmosis, most respondents indicated that they counsel pregnant women about cat litter (99.6%), but fewer counselled about eating undercooked meat (77.6%), handling raw meat (67.4%), gardening (65.4%) or washing fruits and vegetables (34.2%). Many (73.2%) respondents were not aware that some Toxoplasma IgM tests have had a high false positive rate, and most (91.2%) had not heard of the avidity test, which can help determine the timing of Toxoplasma gondii infection in relation to pregnancy. There is a need for more education about T. gondii serological testing, particularly the Toxoplasma avidity test. US obstetrician–gynaecologists are providing beneficial counselling to their patients, but could provide more information about undercooked meat and soil risks.     

Development and evaluation of an enzyme-linked immunosorbent assay based on recombinant TgSRS2 for serodiagnosis of Toxoplasma gondii infection in cats

An enzyme-linked immunosorbent assay (ELISA) based on recombinant SAG1-related sequence 2 of Toxoplasma gondii (rTgSRS2) was developed to detect toxoplasmosis in cats. The specificity and sensitivity of rTgSRS2 ELISA were confirmed using a series of serum samples from T. gondii-experimentally infected mice. A total of 76 field samples from cats were examined by the developed ELISA. The rTgSRS2 ELISA showed a good diagnostic performance characterized by high concordance (88.16) and kappa value (0.76) with latex agglutination test (LAT). The sensitivity and specificity of the test were 92.68% and 82.86%, respectively. These results suggest that the ELISA based on rTgSRS2 could be a useful tool for serodiagnosis of T. gondii infection in cats.     

Evaluation of a Direct Agglutination Test for Detection of Antibodies Against Toxoplasma gondii in Cat,Pig and Sheep Sera

The protozoan parasite Toxoplasma gondii is the causative agent of the zoonosis toxoplasmosis. In sheep and goats, it is one of the most prevalent causes of infectious abortion. Also in pregnant women, a primary infection can result in miscarriage. Humans acquire the infection either by ingestion of oocysts excreted by cats, the definitive host of the parasite, or by eating raw or undercooked meat from latently infected animals (Dubey & Beattie 1988). In Sweden, toxoplasmosis is a notifiable disease, and cases of clinical disease in humans as well as animals must be reported. In both veterinary and human medicine serological assays based on detecting the humoral antibody response of the host against the parasite are used as diagnostic tools. So far, solid phase assays, such as the indirect fluorescent antibody test (IFAT) and the enzyme-linked immunosorbent assay (ELISA), have been widely used to diagnose T. gondii infection in many species including cats, pigs and sheep (Dubey & Beattie 1988). However, both IFAT and ELISA require appropriate anti-species specific immunoglobulins (Ig) that must be carefully evaluated for each species prior to use. This makes these assays complicated and time consuming. Consequently, alternative, simpler methods that do not require specific antisera would be of great value. The direct agglutination test (DA), which is based on the principle that formalin-treated organisms agglutinate in the presence of specific IgG antibodies, is such an assay (Fulton & Turk 1959). The DA-test is widely used in human medicine as a screening test for T gondii infection but it has not yet been thoroughly evaluated for use in veterinary medicine (Uggla & Buxton 1990).     

Principles and practical application of the receiver-operating characteristic analysis for diagnostic tests

We review the principles and practical application of receiver-operating characteristic (ROC) analysis for diagnostic tests. ROC analysis can be used for diagnostic tests with outcomes measured on ordinal, interval or ratio scales. The dependence of the diagnostic sensitivity and specificity on the selected cut-off value must be considered for a full test evaluation and for test comparison. All possible combinations of sensitivity and specificity that can be achieved by changing the test's cut-off value can be summarised using a single parameter; the area under the ROC curve. The ROC technique can also be used to optimise cut-off values with regard to a given prevalence in the target population and cost ratio of false-positive and false-negative results. However, plots of optimisation parameters against the selected cut-off value provide a more-direct method for cut-off selection. Candidates for such optimisation parameters are linear combinations of sensitivity and specificity (with weights selected to reflect the decision-making situation), odds ratio, chance-corrected measures of association (e. g. kappa) and likelihood ratios. We discuss some recent developments in ROC analysis, including meta-analysis of diagnostic tests, correlated ROC curves (paired-sample design) and chance- and prevalence-corrected ROC curves.     

Prevalence of anti-<Emphasis Type="Italic">Toxoplasma gondii</Emphasis> antibodies in sport horses from Qazvin,Iran

In the present study, the prevalence of antibodies to Toxoplasma gondii in sport horses of Qazvin was examined using modified agglutination test (MAT). On 52 horse sera totally examined for anti-Toxoplasma antibodies, 37 horses (71.2%) were seropositive by MAT. Results of the present study showed a high rate of Toxoplasma infection in horses in Qazvin area. More comprehensive study on equine toxoplasmosis is recommended.     

Toxoplasma gondii Infections in Chickens (Gallus domesticus): Prevalence,Clinical Disease,Diagnosis and Public Health Significance

Chickens are considered one of the most important hosts in the epidemiology of Toxoplasma gondii infection because they are an efficient source of infection for cats that excrete the environmentally resistant oocysts and because humans may become infected with this parasite after eating undercooked infected chicken meat. The objective of this study is to review worldwide prevalence of T. gondii infection in chickens and to assess the role of infected chickens in the epidemiology of toxoplasmosis in humans. A very high prevalence of the parasite was found in chickens raised in backyards (up to 100%) and free‐range organic (30–50%) establishments.     

Seroprevalence of Toxoplasma gondii in mainland and sub-Antarctic New Zealand sea lion (Phocarctos hookeri) populations

AIMS: To investigate the seroprevalence of antibodies to Toxoplasma gondii in New Zealand sea lions (Phocarctos hookeri), as a potential contributor to reproductive failure.

METHODS: Archived sera were sourced from New Zealand sea lions from two recolonising mainland populations in the Otago Peninsula (n=15) and Stewart Island (n=12), as well as a declining population at Enderby Island (n=28) in the New Zealand sub-Antarctic. Sera were tested for antibodies to T. gondii using a commercially available ELISA (with samples considered positive if the sample to positive ratio was?>30%), and latex agglutination test (LAT; with titres ≥1:32 considered positive). Western blot analysis was used to validate the results of a subset of 14 samples.

RESULTS: Five samples from sea lions in mainland locations were confirmed positive for antibodies to T. gondii. Two adult females exhibited high LAT antibody titres (min 1:2048, max 1:4096) on both occasions when sampled 1 and 2 years apart, respectively. No animals from Enderby Island were seropositive.

CONCLUSIONS: Toxoplasma gondii infection is unlikely to be a major contributor to poor reproductive success in New Zealand sea lions. However, continued surveillance is pertinent to assess subclinical and clinical impacts of the parasite on these threatened populations. The commercial tests evaluated here, with further species-specific threshold refinement could provide a fast, inexpensive and reliable indicator of T. gondii exposure in New Zealand sea lions.     

Prevalence of anti-Toxoplasma gondii and anti-Neospora caninum antibodies in sheep from Mossoró, Rio Grande do Norte, Brazil

Toxoplasma gondii is an obligate intracellular parasite with a variety of hosts, responsible for reproductive problems and economic losses in sheep flocks. Neospora caninum was recently identified and its clinical presentation in sheep is similar to that of toxoplasmosis, which can cause repeated abortions, though less frequently in this species. In order to confirm the prevalence of these agents in the city of Mossoró, Rio Grande do Norte, Brazil, 409 serum samples from adult sheep (364 females and 45 males) were tested by the indirect immunofluorescence antibody test, using cut-off point at a dilution of 1:64 and 1:50 for T. gondii and N. caninum, respectively. From the 35 properties examined, 23 (65.7%) had at least one seropositive animal for T. gondii and six (17.1%) for N. caninum. The prevalence of seropositive animals for T. gondii was 20.7% and for N. caninum 1.8%. There was no association between the presence of the agent’s antibody and gender, reports of reproductive problems and presence of dogs and/or cats in the properties. T. gondii is well distributed and N. caninum has low prevalence in sheep and in the properties of the studied region.     

Seroprevalence of and risk factors for Toxoplasma gondii in sheep raised in the Jaboticabal microregion, São Paulo State, Brazil

Seroprevalence of and risk factors for toxoplasmosis in sheep from different properties in the Jaboticabal microregion, São Paulo State, Brazil were determined. Antibodies to Toxoplasma gondii were found in sera of 52.0% of 488 sheep tested by indirect fluorescent antibody test (IFAT ? 64). T. gondii seropositivity in sheep was significantly associated with gender of the sheep, pasturing system, contact with cats, and the use of mineral supplements and the type of feed.