Toxoplasma gondii: level of carriage in sheep of Marrakech region (Mnabha)

Toxoplasma gondii is an ubiquitous parasite with a prevalence variable from country to country. In Morocco very few studies were devoted to this prevalence. To fill this gap we were interested to study the epidemiology of this parasite and to know the level of carriage by the different vectors which are the sources of contamination in humans. The study was done by directly detecting the cysts in the cerebral tissue of the 50 sheep killed and destined for consumption. The results of this preliminary study show that 30% of the cases carry the cysts of T. gondii. To confirm this result and verify the virulence, cerebral specimens were inoculated into mice. These findings are encouraging to complete this study with serological tests and to look for the parasite in cows and goats of this region.     

Comparison Between Elisa and Dye Test for Detection of Naturally Acquired Toxoplasma Gondii Antibodies in the Goat

Toxoplasma gondii IgG antibodies were measured in 212 goat sera, comparing the Sabin-Feldman dye-test and a three-layer sandwich enzyme-linked immunosorbent assay (ELISA). With 98 % concordance obtained between these 2 tests, the results are at the same paragon as for human sera. Accordingly, the ELISA sandwich procedure appears to be suitable for large-scale analysis of goat sera. The discordant 2 % were ELISA positive and dye-test negative. One possible explanation of the divergent titres is given using an immunized goat model.Key words: Toxoplasma gondii, goat, antibodies, dye-test, ELISA     

龙岩市部分地区牛、羊弓形虫病的血清学调查

目的：采集龙岩市部分地区牛、羊血清,检测福建省牛和羊弓形虫病的感染情况。方法：应用酶联免疫吸附试验检测牛和羊血清弓形虫IgG抗体。结果：49份牛血清弓形虫IgG抗体均呈阴性;35份羊血清中,8份弓形虫IgG抗体呈阳性,阳性率为22．86％（8／35）。结论：龙岩市部分地区羊弓形虫的感染率较高,应引起人们重视。     

Specific serum antibody responses following a Toxoplasma gondii and Trichinella spiralis co-infection in swine

The aim of this study was to examine the dynamics of parasite specific antibody development in Trichinella spiralis and Toxoplasma gondii co-infections in pigs and to compare these with antibody dynamics in T. spiralis and T. gondii single infections. In this experiment, fifty-four pigs were divided into five inoculated groups of ten animals, and one control group of four animals. Two groups were inoculated with a single dose of either T. gondii tissue cysts or T. spiralis muscle larvae, one group was inoculated simultaneously with both parasites and two groups were successively inoculated at an interval of four weeks. Specific IgG responses to the parasites were measured by ELISA. T. gondii burden was determined by MC-PCR carried out on heart muscle and T. spiralis burden by artificial digestion of diaphragm samples. Specific IgG responses to T. gondii and T. spiralis in single and simultaneously inoculated animals showed a respective T. gondii and T. spiralis inoculation effect but no significant interaction of these parasites to the development of specific antibodies with the serum dilutions used. Moreover, our data showed that the specific IgG response levels in groups of animals successively or simultaneously co-infected were independent of a respective previous or simultaneous infection with the other parasite. Additionally, no differences in parasite burden were found within groups inoculated with T. gondii and within groups inoculated with T. spiralis. Conclusively, for the infection doses tested in this experiment, the dynamics of specific antibody development does not differ between single and simultaneous or successive infection with T. gondii and T. spiralis. However, lower parasitic doses and other ratios of doses, like low-low, low-high and high-low of T. gondii and T. spiralis in co-infection, in combination with other time intervals between successive infections may have different outcomes and should therefore be studied in further detail.     

Prevalence of specific IgG-antibodies against Toxoplasma gondii in domestic turkeys determined by kinetic ELISA based on recombinant GRA7 and GRA8

The protozoan parasite Toxoplasma (T.) gondii is one of the most common zoonotic infectious agents worldwide. Besides its sexual reproduction in cats, T. gondii can also infect a wide spectrum of other warm-blooded animals. These include animals used for human consumption such as pigs or chickens. Nevertheless, the role of turkeys for the epidemiology of T. gondii infections has not been studied thoroughly. We have established a kinetic ELISA (KELA) for the detection of T. gondii-specific IgG antibodies in turkey serum samples. The test is based on the recombinant dense granule antigens GRA7 and GRA8. These proteins were used as an antigen mixture at a concentration of 0.13 μg per well. The overall sensitivity of the assay was between 92.6% and 100% and the specificity ranged from 78.1% to 100%, depending on the method used to calculate these parameters. Using this KELA we examined 1913 turkey serum samples from 14 turkey farms from different areas of Germany. From these sera, 387 produced a signal in the KELA, corresponding to a true seroprevalence of up to 20.2%. The seropositivity rate in individual fattening cycles at individual farms ranged from 0.0% to 77.1%, whereas the rates were highly variable within the individual farms and individual fattening cycles. Consequently, conditions of animal husbandry could not be associated with particular seroprevalence rates. Although seropositivity cannot be linked directly to infectious tissue cysts in the muscle tissue of commercially produced turkey meat, we state that there is a potential risk of being infected by consuming turkey meat products that were not heat treated.     

Seroprevalence of Toxoplasma gondii antibodies in clinically ill cats in the United States

OBJECTIVE: To determine regional seroprevalence estimates of Toxoplasma gondi-specific IgM and IgG in clinically ill cats throughout the United States. Sample Population-Sera from 12,628 clinically ill, client-owned cats. PROCEDURE: Toxoplasma gondii-specific IgM and IgG antibodies were detected by use of ELISAs. Sera from clinically ill cats previously submitted for T. gondii antibody testing were sequentially selected from our serum bank and the sample submission paperwork reviewed. The country was divided into 12 geographic regions. Overall prevalence as well as prevalence for each region, age group, season, sex (male vs female), and breed (domestic shorthair vs other) was calculated. Data were analyzed by logistic regression analysis. RESULTS: Overall, 31.6% of the cats were seropositive for T. gondii-specific IgM, IgG, or both. Percentage of cats seropositive for T. gondii antibodies ranged from 16.1% (southwestern United States) to 43.5% (northeastern United States). As age increased, odds of positive T. gondii antibody assay results (IgM alone, IgG alone, and any combination of IgM or IgG) increased. Males were more likely than females to be seropositive for T. gondii antibodies (IgG alone and any combination of IgM or IgG). Domestic shorthair cats were more likely than other breeds to be seropositive for T. gondii antibodies (IgM alone, IgG alone, and any combination of IgM or IgG). CONCLUSIONS AND CLINICAL RELEVANCE: Toxoplasma gondii-specific antibodies are common in serum samples of clinically ill cats from all regions of the United States. Seroprevalence increases as cats age and is higher in male and domestic shorthair cats, compared with females and other breeds.     

Biology and epidemiology of Toxoplasma gondii in man and animals

Toxoplasma gondii is a coccidian parasite which utilizes felids as definitive hosts, and which has an unusually wide intermediate host range. The parasite was initially described by Nicolle and Manceaux in 1908 from the rodent, Ctenodactylus gundi. Infection with T. gondii is one of the most common parasitic infections of man and other warm-blooded animals. It has been found worldwide from Alaska to Australia. Nearly one-third of humanity has been exposed to this parasite; serologic surveys indicate that T. gondii infections are common in wild carnivores, including pigs, bears, felids, fox, raccoons, and skunks. Clinical and subclinical toxoplasmosis has been reported from wild cervids, ungulates, marsupials, monkeys, and marine mammals. Southern sea otter populations have been severely impacted by Toxoplasma infections.     

Toxoplasma gondii infection in sheep from Haute-Vienne, France: seroprevalence and isolate genotyping by microsatellite analysis

Ingesting meat of free-range livestock, mainly sheep, is associated with human toxoplasmosis in European countries. Data on Toxoplasma gondii infection in French ovine livestock are relatively scarce. Sera from 164 lambs and 93 ewes slaughtered in Haute-Vienne district, France, were tested by a direct agglutination test. Antibodies to T. gondii were found in 36 (22.0%) lambs and in 61 (65.6%) ewes. In addition, to attempt parasite isolation for genotyping, hearts from 50 other ewes were obtained from a local slaughterhouse, and were screened by a direct agglutination test. T. gondii was isolated in 8 of 30 seropositive hearts bioassayed in mice. All isolates were type II by genetic characterization at five microsatellite loci (TUB2, TgM-A, W35, B17, B18). These results indicate that bovines slaughtered in France may be highly infected by T. gondii with a potential risk of parasite transmission to humans by consumption of undercooked meat. Multilocus microsatellite analysis shows the predominance of type II in sheep as previously described in humans.     

Seroprevalence and Toxoplasma gondii-IgG avidity in sheep from Lajes, Brazil

Sheep are important domestic animals in the Northeast region of Brazil due to their minimal rearing and maintenance costs, and to their production of both meat and milk. In animals, Toxoplasma gondii infection results in significant reproductive and economic losses. The epidemiology of toxoplasmosis in sheep in the Northeast of Brazil has been little studied; particularly in Rio Grande do Norte State. Sera from 102 sheep intended for consumption in Lajes were subjected to the Toxoplasma-ELISA test to detect anti-T. gondii specific IgG confirming a past infection. Of the total tested, 30 (29.41%) sera were positive for IgG with an increasing number of positive animals with advancing age. We used IgG avidity ELISA in 30 positive samples and observed that 6 (20%) had low avidity antibodies and 24 (80%) had high avidity antibodies. Epidemiological studies are required in order to identify sources of infection for these hosts as well as their impact on animal breeding in the region and risk of transmission to humans.     

A serological study on the prevalence of Toxoplasma gondii in sheep of Lithuania

In the present study the seroprevalence of the protozoan parasite Toxoplasma gondii infection in sheep was investigated in 6 regions of Lithuania. Blood samples were taken from 354 sheep and were tested using commercial ELISA method. The total seroprevalence of Toxoplasma gondii infection in sheep was 42.1%. Significant differences in seroprevalence were observed between age groups (P < or = 0.05). The results of this investigation suggest that the Toxoplasma gondii parasite is widely spread, and can be one of reasons of sheep abortion in Lithuania.     

Serodiagnosis of Toxoplasma gondii infection in cats by enzyme-linked immunosorbent assay using recombinant SAG1

The gene encoding surface antigen 1 (SAG1, P30) of Toxoplasma gondii (T. gondii) was cloned into the plasmid pGEX-4T-3 and subsequently expressed in Escherichia coli (E. coli) as a glutathione-S-transferase (GST) fusion protein. The recombinant SAG1 (rSAG1) was refolded using 8M urea solution followed by dialysis and thereafter evaluated in an enzyme-linked immunosorbent assay (ELISA) for serological diagnosis of toxoplasmosis. The test sera were adsorbed with GST to block non-specific reactivity to the GST-SAG1 fusion protein. The ELISA with rSAG1 was able to differentiate very clearly between sera from cats or mice experimentally infected with T. gondii and sera from normal cats or mice. The ELISA detected no cross-reactivity with sera from mice experimentally infected with the closely related parasite Neospora caninum (N. caninum). Some 193 cat sera were tested for antibodies to T. gondii, out of which 40 (20.7%) reacted positively by ELISA with the rSAG1 while another 79.3% cats reacted negative to the assay. Both positive and negative sera were confirmed by Western blot analysis. The results of ELISA were in agreement with those of a commercially available latex agglutination test (LAT) kit, although the former had higher titers than the latter.     

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

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

Role of endogenous transplacental transmission in toxoplasmosis in sheep

To investigate the potential role of endogenous transplacental transmission of Toxoplasma gondii, 31 seropositive ewes presumed to be persistently infected with the parasite and 15 seronegative ewes were mated and monitored throughout pregnancy and lambing. Antibody titres were determined in precolostral sera from the liveborn lambs and in thoracic fluid from the dead lambs. A PCR for the B1 gene of T gondii was applied to the placentas from all the ewes and to the brains of the stillborn lambs. Samples of brain, lung, liver, spleen and heart from the dead lambs were examined by histopathology. No evidence of toxoplasmosis was detected by histopathology or PCR in any of the samples, but low titres of antibody to T gondii were detected in two liveborn, healthy offspring of a seropositive ewe by the immunofluorescent antibody test (3.2 per cent of pregnancies and 4.1 per cent of lambs in the seropositive group). Antibody to specific antigens of T gondii was demonstrated in sera from these two lambs by Western blotting.     

Farmed wild boars exposed to Toxoplasma gondii and Trichinella spp

The meat of wild boar (Sus scrofa L.) can be a source of human infections with zoonotic parasites Toxoplasma gondii and Trichinella spp. We screened 197 wild boar sera collected at slaughter from 25 Finnish farms in 2007-2008 for serological evidence of infections with these parasites. Using a commercial direct agglutination test at a serum dilution of 1:40, T. gondii-specific IgG antibodies were detected in 65 (33.0%) samples, on 14 (56.0%) farms. Females, animals older than 24 months, animals of small herds, and animals originating from south-western parts of Finland were more often T. gondii-seropositive than were males, younger animals, animals of larger herds, and animals originating from the north and east, respectively. Four (2.0%) of the sera, originating from three (12.0%) farms, tested Trichinella-seropositive with an in-house ELISA and a conservative cut-off for seropositivity. One farm had both T. gondii- and Trichinella-seropositive animals. Taken together, an infection source had been present on 16 (64.0%) farms, and 69 (35.0%) of the 197 farmed wild boars intended for human consumption had specific serological evidence of exposure to a zoonotic parasite.     

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.     

CpG-oligodeoxynucleotides enhance porcine immunity to Toxoplasma gondii

Protection against a challenge infection with Toxoplasma gondii VEG strain oocysts was examined in pigs after vaccination with T. gondii RH strain tachyzoites with or without a porcine specific synthetic oligodeoxynucleotides (ODN) containing immunostimulatory CpG motifs. Six groups of pigs were immunized with incomplete Freund's adjuvant (IFA) and either vehicle, tachyzoites alone or in combination with three different doses of CpG ODN or with CpG ODN alone. Protection from challenge was significantly (P < 0.05) improved in pigs vaccinated using CpG ODN as an adjuvant with tachyzoites compared to all other groups. The CpG ODN tachyzoite-immunized pigs also had higher serum parasite specific IgG antibody, no clinical signs of disease, and 52% had no demonstrable tissue cysts after the challenge infection. These data indicate that CpG ODN is a potential safe and effective adjuvant for the T. gondii RH strain vaccine in pigs.     

Specificity of the carbon immunoassay (CIA) test for the diagnosis of Toxoplasma infections.

A rapid and low cost procedure, the carbon immunoassay (CIA) test, was evaluated for the diagnosis of Toxoplasma gondii infections. Using a closely related parasite (Besnoitia jellisoni) as antigen, and homologous or heterologous immune sera, it was demonstrated by light and electron microscopy that CIA is a very reliable and specific test. As it is neither expensive nor time-consuming, it can be recommended for general and routine laboratory use.     

屠宰羊弓形虫感染情况调查

为了保障羊肉制品的生物安全,对屠宰羊进行弓形虫感染情况调查以及对羊肉进行弓形虫检测。采用间接血凝试验,对677份屠宰羊的血清进行了弓形虫抗体检测。结果显示,弓形虫抗体阳性有47份,阳性率为6.9%。     

Application of an indirect carbon immunoassay (CIA) for the rapid diagnosis of antibody to Toxoplasma gondii in sheep

Carbon immunoassay (CIA), a novel indirect rapid test for Toxoplasma antibody in sheep, was compared with indirect fluorescent antibody assay (IFA). CIA relies on the adherence of carbon particles of India-ink to rabbit immunoglobulin G.l Carbon labelled anti-sheep rabbit IgG was used for the detection of sheep antibody when attached to tachyzoites of Toxoplasma gondii. The result was read in an ordinary light microscope and there was a clearcut difference between negative and positive reactions. Out of a total of 97 sheep sera tested, 15 sera were negative in both tests and 12 were negative in CIA but showed low positive titres in IFA. The remaining 70 sera were positive in both tests but the titres were usually about 3 dilution steps lower when investigated with CIA as compared to IFA.     

IFN-γ expression and infectivity of Toxoplasma infected tissues are associated with an antibody response against GRA7 in experimentally infected pigs

Toxoplasma gondii, an obligate intracellular parasite, can be transmitted to humans via the consumption of infected meat. However, there are currently no veterinary diagnostic tests available for the screening of animals at slaughter. In the current work, we investigated whether cytokine responses in the blood, and antibody responses against recombinant T. gondii GRA1, GRA7, MIC3 proteins and a chimeric antigen EC2 encoding MIC2-MIC3-SAG1, are associated with the infectivity of porcine tissues after experimental infection with T. gondii. Two weeks after experimental infection of conventional 5-week-old seronegative pigs, an IFN-γ response was detected in the blood, with a kinetic profile that followed the magnitude of the GRA7 antibody response. Antibody responses to GRA1, MIC3 and EC2 were very weak or absent up to 6 weeks post infection. Antibodies against GRA7 occurred in all infected animals and were associated with the presence of the parasite in tissues at euthanasia a few months later, as demonstrated by quantitative real-time PCR and isolation by bio-assay. Remarkably, although brain and heart tissue remained infectious, musculus gastrocnemius and musculus longissimus dorsi were found clear of infectious parasites 6 months after experimental infection. Seropositive response in a GRA7 ELISA indicates a Toxoplasma infection in pigs and is predictive of the presence of infectious cysts in pig heart and brain. This new ELISA is a promising tool to study the prevalence of Toxoplasma infection in pigs. Clearance of the infection in certain pig tissues suggests that the risk assessment of pig meat for human health needs further evaluation.