Infection dynamics following experimental challenge of pigs orally dosed with different stages of two archetypal genotypes of Toxoplasma gondii.

Toxoplasma gondii is a food-borne zoonotic parasite widespread in a variety of hosts, including humans. With a majority of infections in Europe estimated to be meat-borne, pork, as one of the most consumed meats worldwide, represents a potential risk for consumers. Therefore, we aimed to investigate the progress of T. gondii infection and tissue tropism in experimentally infected pigs, using different T. gondii isolates and infectious stages, i.e. tissue cysts or oocysts. Twenty-four pigs were allocated to treatment in four groups of six, with each group inoculated orally with an estimated low dose of either 400 oocysts or 10 tissue cysts of two European T. gondii isolates, a type II and a type III isolate. The majority of pigs seroconverted two weeks post-inoculation. Pigs infected with the type III isolate had significantly higher levels of anti-T. gondii antibodies compared to those infected with the type II isolate. Histopathological exams revealed reactive hyperplasia of the lymphatic tissue of all pigs. Additionally, a selected set of nine tissues was collected during necropsy at 50 dpi from each of the remaining 22 pigs for T. gondii DNA detection by quantitative real-time PCR. A positive result was obtained in 29.8 % (59/139) of tested tissues. The brain was identified as the most frequently positive tissue in 63.6 % (14/22) of the animals. In contrast, liver samples tested negative in all animals. The highest mean parasite load, calculated by interpolating the average Cq values on the standard curve made of ten-fold serial dilutions of the genomic DNA, corresponding to 100 to 104 tachyzoites/µL, was observed in shoulder musculature with an estimated concentration of 84.4 [0.0-442.5] parasites per gram of tissue. The study highlights the variability in clinical signs and tissue distribution of T. gondii in pigs based on the combination of parasite stages and strains, with type III isolates, particularly oocysts, causing a stronger antibody response and higher tissue parasite burden. These findings suggest the need for further investigation of type III isolates to better understand their potential risks to humans.

Optimization of the most widely used serological tests for a harmonized diagnosis of Toxoplasma gondii infection in domestic pigs.

The intake of Toxoplasma gondii tissue cysts through raw or undercooked pork meat is one of the main infection sources for humans. Thus, surveillance is recommended to control and prevent infection in domestic pigs. However, the lack of comparative studies hampers the updating of their performance and the comparison of seroprevalence data. Therefore, the aim of this study was to develop and validate three in-house tests and accomplish a comparative analysis of the most widely used serological tests employed in pigs. A panel of sera from pigs experimentally infected with either oocysts or tissue cysts from type II and III isolates (n = 158) was used to develop and validate a tachyzoite-based Western blot assay. Then, this technique was used as a reference to develop and preliminary validate a lyophilized tachyzoite-based enzyme-linked immunosorbent assay and an immunofluorescence antibody test. Next, a comparative study of the three in-house tests and three widely used commercial ELISAs (IDScreen®, PrioCHECK™ and Pigtype®) was accomplished with the abovementioned sera together with an additional serum panel of pigs experimentally infected with oocysts from the type II isolate (n = 44) and a panel of naturally infected pigs (n = 244). The results obtained by the majority of the tests were regarded as reference, and data analyses included TG-ROC calculations and agreement tests. Finally, the kinetics of anti-T. gondii IgGs from experimentally infected pigs was analyzed. Excellent sensitivity (Se) and specificity (Sp) values (≥ 93%) and moderate to near perfect agreement (k = 0.63-0.91) were observed using sera from experimental infections without requiring further readjustment, except for PrioCHECK (100% Se, 73% Sp). However, the Se of IDScreen® (87%) and TgSALUVET WB (71%) and the Sp of PrioCHECK (72%) were slightly or notably reduced when sera from naturally infected animals were analyzed, which also influenced the kappa values (k = 0.30-0.91). Cutoff readjustments increased the Se and Sp values to equal to or above 97% for all tests, except for TgSALUVET WB, which can be used as a reference for initial validation of tests, but it is not recommended for routine diagnosis. Seroconversion was recorded from two weeks post-infection by most of the tests, with significantly higher IgG levels in sera from pigs infected with the T. gondii type III vs. type II isolate. Again, differences regarding the test employed were observed. Differences in the diagnostic performance among tests evidenced the need to harmonize serological techniques to obtain comparable and reliable results.

Inactivation of Toxoplasma gondii in dry sausage and processed pork, and quantification of the pathogen in pig tissues prior to production.

Toxoplasma gondii is an important zoonotic foodborne parasite. Meat of infected animals appears to be a major source of infection in Europe. Pork is the most consumed meat in France, with dry sausages well represented. The risk of transmission via consumption of processed pork products is largely unknown, mainly since processing will affect viability but may not entirely inactivate all T. gondii parasites. We investigated the presence and concentration of T. gondii DNA in the shoulder, breast, ham, and heart of pigs orally inoculated with 1000 oocysts (n = 3) or tissue cysts (n = 3) and naturally infected pigs (n = 2), by means of magnetic capture qPCR (MC-qPCR). Muscle tissues of experimentally infected pigs were further used to evaluate the impact of manufacturing processes of dry sausages, including different concentrations of nitrates (0, 60, 120, 200 ppm), nitrites (0, 60, 120 ppm), and NaCl (0, 20, 26 g/kg), ripening (2 days at 16-24 °C) and drying (up to 30 days at 13 °C), by a combination of mouse bioassay, qPCR and MC-qPCR. DNA of T. gondii was detected in all eight pigs, including in 41.7% (10/24) of muscle samples (shoulder, breast and ham) and 87.5% (7/8) of hearts by MC-qPCR. The number of parasites per gram of tissue was estimated to be the lowest in the hams (arithmetic mean (M) = 1, standard deviation (SD) = 2) and the highest in the hearts (M = 147, SD = 233). However, the T. gondii burden estimates varied on the individual animal level, the tissue tested and the parasitic stage used for the experimental infection (oocysts or tissue cysts). Of dry sausages and processed pork, 94.4% (51/54) were positive for T. gondii by MC-qPCR or qPCR, with the mean T. gondii burden estimate equivalent to 31 parasites per gram (SD = 93). Only the untreated processed pork sample collected on the day of production was positive by mouse bioassay. The results suggest an uneven distribution of T. gondii in the tissues examined, and possibly an absence or a concentration below the detection limit in some of them. Moreover, the processing of dry sausages and processed pork with NaCl, nitrates, and nitrites has an impact on the viability of T. gondii from the first day of production. Results are valuable input for future risk assessments aiming to estimate the relative contribution of different sources of T. gondii human infections.

Anatomical distribution of Toxoplasma gondii in naturally and experimentally infected lambs.

Consumption of raw or undercooked meat containing Toxoplasma gondii tissue cysts is one of the main sources of infection for humans worldwide. Among the various species intended for human consumption, sheep appear to be a high risk for human infection. The present study focused on the detailed anatomical distribution of Toxoplasma gondii in naturally and experimentally infected lambs using fresh and frozen samples of various pieces of meat, from a public health perspective. The first objective was to rank the edible parts intended for human consumption according to the detectable parasite burden by real-time PCR targeting the 529-bp repeated element. The second objective was to evaluate the impact of freezing by comparing the detection efficiency of the quantitative PCR between fresh and frozen tissues, as imports of lamb carcasses/cuts may arrive frozen or chilled. The highest estimated parasite loads were observed in skeletal muscles, and more particularly in edible portions such as quadriceps femoris muscle, intercostal muscles, deltoid muscle and diaphragm, with a significant difference in detectable parasite burden between fresh and frozen samples (p < 0.0001) or natural and experimental infection (p < 0.0001). Thoracic and pelvic limbs (3278-1048 parasites/g muscle) were ranked at the top of the list. Toxoplasma gondii DNA was detected in all the edible parts of lamb studied. These results suggest that lamb meat represents a risk for consumers. Further investigations are needed in order to confirm these differences in larger numbers of animals and in different breeds.

Title: Distribution anatomique de Toxoplasma gondii chez des agneaux infectés naturellement et expérimentalement. Abstract: La consommation de viande crue ou insuffisamment cuite contenant des kystes tissulaires de Toxoplasma gondii est l'une des principales sources d'infection pour l'homme dans le monde. Parmi les différentes espèces destinées à la consommation humaine, le mouton apparaît à haut risque d'infection humaine. La présente étude s'est concentrée sur une distribution anatomique détaillée de Toxoplasma gondii chez des agneaux infectés naturellement et expérimentalement à l'aide d'échantillons frais et congelés de divers morceaux de viande, du point de vue de la santé publique. Classer les parties comestibles destinées à la consommation humaine, selon la charge parasitaire détectable par une PCR en temps réel ciblant l'élément répété de 529 pb était un premier objectif. Un second objectif était d'évaluer l'impact de la congélation en comparant l'efficacité de détection de la PCR quantitative entre les tissus frais et congelés, car les importations de carcasses/coupes d'agneau peuvent arriver congelées ou réfrigérées. Les charges parasitaires estimées les plus élevées ont été observées dans les muscles squelettiques et plus particulièrement dans les parties comestibles telles que le quadriceps fémoral, les muscles intercostaux, le deltoïde et le diaphragme avec une différence significative de charge parasitaire détectable entre les échantillons frais et congelés (p < 0,0001) ou l'infection naturelle et expérimentale (p < 0,0001). Les membres thoraciques et pelviens (3278 à 1048 parasites/g de muscle) ont été classés en tête de liste. L'ADN de T. gondii a été détecté dans toutes les parties comestibles étudiées de l'agneau. Ces résultats suggèrent que l'agneau représente un risque pour les consommateurs. Des investigations supplémentaires doivent être effectuées afin de confirmer les différences mentionnées ci-dessus chez plus d'animaux et dans différentes races.

Burden and regional distribution of Toxoplasma gondii cysts in the brain of COBB 500 broiler chickens following chronic infection with 76K strain.

Toxoplasmosis is a worldwide zoonosis caused by the obligate intracellular apicomplexan parasite Toxoplasma gondii (T. gondii). Chickens are ground-feeders and represent, especially if free-range, important intermediate hosts in the epidemiology of toxoplasmosis and are used as sentinels of environmental contamination with T. gondii oocysts. Until now, little is known about the burden and regional distribution of T. gondii cysts in the chicken brain. It was therefore the aim of this study to investigate the abundance and specific distribution of T. gondii cysts within the chicken brain following chronic infection with a type II strain (76 K) of T. gondii. A total of 29 chickens were included in the study and divided into control group (n = 9) and two different infection groups, a low dose (n = 10) and a high dose (n = 10) group, which were orally inoculated with 1500 or 150,000 T. gondii oocysts per animal, respectively. Seroconversion was detected in the majority of chickens of the high dose group, but not in the animals of the low dose and the control group. Moreover, T. gondii DNA was detected most frequently in the brain and more frequently in the heart than in liver, spleen, thigh and pectoral muscle using qPCR analysis. The number of T. gondii cysts, quantified in the chicken brain using histological analysis, seems to be considerably lower as compared to studies in rodents, which might explain why T. gondii infected chickens very rarely, if at all, develop neurological deficits. Similar to observations in mice, in which no lateralisation for T. gondii cysts was reported, T. gondii cysts were distributed nearly equally between the left and right chicken brain hemispheres. When different brain regions (fore-, mid- and hindbrain) were compared, all T. gondii cysts were located in the forebrain with the overwhelming majority of these cysts being present in the telencephalic pallium and subpallium. More studies including different strains and higher doses of T. gondii are needed in order to precisely evaluate its cyst burden and regional distribution in the chicken brain. Together, our findings provide insights into the course of T. gondii infection in chickens and are important to understand the differences of chronic T. gondii infection in the chicken and mammalian brain.

Experimental infection with Toxoplasma gondii in broiler chickens (Gallus domesticus): seroconversion, tissue cyst distribution, and prophylaxis.

Toxoplasma gondii is a widespread zoonotic protozoan that infects most species of mammals and birds, including poultry. This study aimed to investigate the course of T. gondii infection and the efficacy of diclazuril and Artemisia annua in preventing infection in experimentally infected chickens. Seventy-five 1-month-old chickens, female and male, were randomly divided into five groups (n = 15 each) as follows: (1) uninfected untreated (negative control, NC); (2) infected with T. gondii genotype II/III isolated from a wild cat (group WC); (3) infected with T. gondii genotype II isolated from a domestic cat (group DC); (4) infected with T. gondii domestic cat strain and treated with the anticoccidial diclazuril (group DC-D); and (5) infected with T. gondii domestic cat strain and treated with the medicinal plant Artemisia annua (group DC-A). Clinical signs, body temperature, mortality rate, weight gain, feed conversion ratio, hematological parameters, and the presence of T. gondii-specific IgY antibodies were recorded in all groups. Five chickens per group were euthanized 28 days post-infection (p.i.) and their brains, hearts, and breast muscle tested for T. gondii by mouse bioassay and polymerase chain reaction (PCR). No clinical signs related to the experimental infection were observed throughout the study period. T. gondii-specific antibodies were detected by day 28 p.i., but not in all infected chickens. Overall, T. gondii DNA was detected (bioassay or tissue digests) in all infected and untreated chickens (10/10), while viable parasite (bioassay) was isolated from 7 out of 10 chickens. The parasite was most frequently identified in the brain (7/10). There were no differences in the T. gondii strains regarding clinical infection and the rate of T. gondii detection in tissues. However, higher antibody titers were obtained in chickens infected with T. gondii WC strain (1:192) comparing with T. gondii DC strain (1:48). A. annua reduced replication of the parasite in 3 out of 5 chickens, while diclazuril did not. In conclusion, broiler chickens were resistant to clinical toxoplasmosis, irrespective of the strain (domestic or wild cat strain). The herb A. annua presented prophylactic efficacy by reduced parasite replication. However, further studies are required aiming at the efficacy of diclazuril and A. annua for the prevention of T. gondii infection in chickens using quantitative analysis methods.

Evaluation of immunogenicity and protection of the Mic1-3 knockout Toxoplasma gondii live attenuated strain in the feline host.

Toxoplasmosis is a zoonotic disease caused by the parasite Toxoplasma gondii. Up to a third of the global human population is estimated to carry a T. gondii infection, which can result in severe complications in immunocompromised individuals and pregnant women. Humans and animals can become infected by ingesting either tissue cysts containing T. gondii bradyzoites, from raw or undercooked meat, or sporulated oocysts from environmental sources. T. gondii oocysts are released in the faeces of cats and other felids, which are the parasite's definitive hosts, leading to environmental contamination. Therefore, vaccination of the feline host against T. gondii is an interesting strategy to interrupt the parasitic life cycle and subsequently limit contamination of intermediate hosts. With this goal in mind, we tested in cats, an attenuated live strain of T. gondii deleted for the Mic1 and Mic3 genes (Mic1-3KO) that was previously shown to be an efficient vaccine candidate in mouse and sheep models. Subcutaneous or oral vaccination routes induced a high specific antibody titer in the cat sera, indicating that the Mic1-3KO strain is immunogenic for cats. To assess protection induced by the vaccine candidate strain, we followed oocysts shedding by vaccinated cats, after oral challenge with a T. gondii wild-type strain. Surprisingly, a high antibody titer did not prevent cats from shedding oocysts from the challenge strain, regardless of the vaccination route. Our results show that the Mic1-3KO vaccine candidate is immunogenic in the feline host, is well tolerated and safe, but does not confer protection against oocysts shedding after natural infection with wild type T. gondii. This result highlights the particular relationship between T. gondii and its unique definitive host, which indicates the need for further investigations to improve vaccination strategies to limit environmental and livestock contaminations.

Toxoplasma gondii in water buffaloes (Bubalus bubalis) from Romania: what is the importance for public health?

The purpose of our study was to evaluate the prevalence of Toxoplasma gondii infection in autochthonous Carpathian buffaloes from northwestern Romania by serology, PCR techniques, and mouse bioassay. Agreement between MAT and ELISA, correlation between indirect and direct detection methods, and risk factors were evaluated. The apparent overall seroprevalence of T. gondii was 8.1% by MAT and 6.6% by ELISA. The agreement between ELISA and MAT was fair. The apparent seroprevalence was significantly higher in adult buffaloes (12.5%) compared to calves (0.0%) and juveniles (1.9%) by MAT. Most of the positive adult buffaloes detected by MAT had antibodies at a low sera dilution and the highest dilution was 1:768 in a juvenile female (30 months). No viable T. gondii was detected by mouse bioassay, as no T. gondii cyst or DNA was found in the brain of mice and they did not seroconvert. However, T. gondii DNA was detected in two buffaloes: in a 30-month-old male buffalo by qPCR on the diaphragm digest and in a 252-month-old female buffalo by RE nPCR on the mesenteric lymph node. Both animals were negative in MAT and ELISA. The total prevalence of T. gondii by direct detection methods was 2.7%. There was no correlation between indirect and direct detection methods. Since no viable T. gondii was detected in buffaloes, the risk of human infection from buffalo meat is minimal. Buffaloes' biological response to a T. gondii infection appears to be very similar to the response of cattle.

The first isolation and molecular characterization of Toxoplasma gondii from horses in Serbia.

BACKGROUND: Consumption of undercooked or insufficiently cured meat is a major risk factor for human infection with Toxoplasma gondii. Although horsemeat is typically consumed rare or undercooked, information on the risk of T. gondii from infected horse meat to humans is scarce. Here, we present the results of a study to determine the presence of T. gondii infection in slaughter horses in Serbia, and to attempt to isolate viable parasites. METHODS: The study included horses from all regions of Serbia slaughtered at two abattoirs between June 2013 and June 2015. Blood sera were tested for the presence of specific IgG T. gondii antibodies by the modified agglutination test (MAT), and samples of trypsin-digested heart tissue were bioassayed in mice. Cyst-positive mouse brain homogenates were subjected to DNA extraction and T. gondii strains were genotyped using 15 microsatellite markers (MS). RESULTS: A total of 105 slaughter horses were sampled. At the 1:6 cut-off 48.6% of the examined horses were seropositive, with the highest titre being 1:400. Viable parasites were isolated from two grade type mares; both parasite isolates (RS-Eq39 and RS-Eq40) were T. gondii type III, and both displayed an increased lethality for mice with successive passages. These are the first cases of isolation of T. gondii from horses in Serbia. When compared with a worldwide collection of 61 type III and type III-like strains, isolate RS-Eq39 showed a combination of MS lengths similar to a strain isolated from a duck in Iran, and isolate RS-Eq40 was identical in all markers to three strains isolated from a goat from Gabon, a sheep from France and a pig from Portugal. Interestingly, the source horses were one seronegative and one weakly seropositive. CONCLUSIONS: The isolation of viable T. gondii parasites from slaughter horses points to horsemeat as a potential source of human infection, but the fact that viable parasites were isolated from horses with only a serological trace of T. gondii infection presents further evidence that serology may not be adequate to assess the risk of toxoplasmosis from horsemeat consumption. Presence of T. gondii type III in Serbia sheds more light into the potential origin of this archetypal lineage in Europe.

Toxoplasma gondii, Dirofilaria immitis, feline immunodeficiency virus (FIV), and feline leukemia virus (FeLV) infections in stray and pet cats (Felis catus) in northwest China: co-infections and risk factors.

This study was conducted to estimate the prevalence of Toxoplasma gondii, Dirofilaria immitis, feline immunodeficiency virus (FIV), and feline leukemia virus (FeLV) infections among stray and pet cats in Lanzhou, northwest China, and to identify the influence of age, gender, and regions on seropositivity. T. gondii antibodies were examined in cat sera by the modified agglutination test (MAT). The circulating antigens of D. immitis and FeLV and specific antibodies to FIV were examined using kits commercially available. The overall prevalence of T. gondii, FIV, FeLV, and D. immitis was 19.34, 9.12, 11.33, and 3.04 %, respectively. For the genetic characterization of T. gondii genotypes in cats, genomic DNA was extracted from the seropositive cats and the T. gondii B1 gene was amplified using a semi-nested PCR. DNA samples giving positive B1 amplification were then genotyped using multilocus PCR-RFLP. Two T. gondii genotypes (ToxoDB#9 and ToxoDB#1) were identified. Results of the multivariate logistic regression analysis showed that older cats are more likely to be seropositive than juveniles for T. gondii, FIV, FeLV, and D. immitis. This is the first report of T. gondii genotypes in cats in northwest China. Moreover, the present study is the first study of retrovirus and D. immitis seroprevalence in cats in China. The results revealed that T. gondii, FIV, and FeLV infections are common in stray and pet cats in northwest China.

Molecular characterization of Taenia multiceps isolates from Gansu Province, China by sequencing of mitochondrial cytochrome C oxidase subunit 1.

A total of 16 Taenia multiceps isolates collected from naturally infected sheep or goats in Gansu Province, China were characterized by sequences of mitochondrial cytochrome c oxidase subunit 1 (cox1) gene. The complete cox1 gene was amplified for individual T. multiceps isolates by PCR, ligated to pMD18T vector, and sequenced. Sequence analysis indicated that out of 16 T. multiceps isolates 10 unique cox1 gene sequences of 1,623 bp were obtained with sequence variation of 0.12-0.68%. The results showed that the cox1 gene sequences were highly conserved among the examined T. multiceps isolates. However, they were quite different from those of the other Taenia species. Phylogenetic analysis based on complete cox1 gene sequences revealed that T. multiceps isolates were composed of 3 genotypes and distinguished from the other Taenia species.

Identification of Trichinella spiralis early antigens at the pre-adult and adult stages.

Three expression cDNA libraries from Trichinella spiralis worms 14 h, 20 h and 48 h post-infection (p.i.) were screened with serum from pigs experimentally infected with 20,000 T. spiralis muscle larvae. Twenty-nine positive clones were isolated from the 14 h p.i. cDNA library, corresponding to 8 different genes. A putative excretory-secretory protein similar to that of T. pseudospiralis was identified. Three clones corresponded to a T. spiralis serine proteinase inhibitor known to be involved in diverse functions such as blood coagulation and modulation of inflammation. Screening of the 20 h p.i. cDNA library selected 167 positive clones representing 12 different sequences. The clone with the highest redundancy encoded a small polypeptide having no sequence identity with any known proteins from Trichinella or other organisms. Fourteen clones displayed sequence identity with the heat shock protein (HSP) 70. HSPs are produced as an adaptive response of the parasite to the hostile environment encountered in the host intestine but their mechanism of action is not yet well defined. From the 48 h p.i. T. spiralis cDNA library, 91 positive clones were identified representing 7 distinct sequences. Most of the positive clones showed high similarity with a member of a putative T. spiralis serine protease family. This result is consistent with a possible major role for serine proteases during invasive stages of Trichinella infection and host-parasite interactions.

Cloning and analysis of a cDNA encoding a putative serine protease comprising two trypsin-like domains of Trichinella spiralis.

The cDNA encoding a putative serine protease, TsSerP, was cloned by degenerative polymerase chain reaction and screening of the cDNA library from Trichinella spiralis adult-newborn larvae stage. Sequence analysis revealed the presence of two trypsin-like serine protease domains flanking a hydrophilic domain, with the catalytic triad residue histidine in the alpha domain substituted by an arginine residue. Southern blots indicated that this was a single copy gene in the parasite genome. Northern blots demonstrated a single 2.3-kb transcript during the muscle larvae and adult stages of T. spiralis. The recombinant protein from the TsSerP beta domain (betaSerP) was produced but not recognised by T. spiralis-infected swine serum. An anti-betaSerP polyclonal serum detected a 69-kDa polypeptide in the soluble antigens of T. spiralis muscle larvae. Immunolocalisation analysis located TsSerP on the inner layer of the cuticle and oesophagus of the parasite, suggesting a potential role in its moulting and/or digestive functions.