Heligmosomoides bakeri and Toxoplasma gondii co-infection leads to increased mortality associated with changes in immune resistance in the lymphoid compartment and disease pathology.

Co-infections are a common reality but understanding how the immune system responds in this context is complex and can be unpredictable. Heligmosomoides bakeri (parasitic roundworm, previously Heligmosomoides polygyrus) and Toxoplasma gondii (protozoan parasite) are well studied organisms that stimulate a characteristic Th2 and Th1 response, respectively. Several studies have demonstrated reduced inflammatory cytokine responses in animals co-infected with such organisms. However, while general cytokine signatures have been examined, the impact of the different cytokine producing lymphocytes on parasite control/clearance is not fully understood. We investigated five different lymphocyte populations (NK, NKT, γδ T, CD4+ T and CD8+ T cells), five organs (small intestine, Peyer's patches, mesenteric lymph nodes, spleen and liver), and 4 cytokines (IFN©, IL-4, IL-10 and IL-13) at two different time points (days 5 and 10 post T. gondii infection). We found that co-infected animals had significantly higher mortality than either single infection. This was accompanied by transient and local changes in parasite loads and cytokine profiles. Despite the early changes in lymphocyte and cytokine profiles, severe intestinal pathology in co-infected mice likely contributed to early mortality due to significant damage by both parasites in the small intestine. Our work demonstrates the importance of taking a broad view during infection research, studying multiple cell types, organs/tissues and time points to link and/or uncouple immunological from pathological findings. Our results provide insights into how co-infection with parasites stimulating different arms of the immune system can lead to drastic changes in infection dynamics.

Molecular Models of Toxoplasma gondii in Humans and Animals.

Toxoplasma gondii (T. gondii) is an obligate intracellular, zoonotic protozoan parasite of interest to physicians and veterinarians with its highly complex structure. It is known to infect about one-third of the world's population. Since it is a zoonotic disease, it is necessary to keep the animal population under control in order to prevent human exposure. Many studies have been conducted on the detection of T. gondii and it has been determined that there are three clonal groups consisting of types 1, 2, 3. Developments in molecular studies have led to changes in the taxonomy and new developments in parasitic diseases. It has helped in diagnosis, treatment, development of antiparasitic drugs and research on resistance. They also provided research on vaccine studies, genetic typing and phylogenetics of parasitic diseases. Conventional polymerase chain reaction (PCR), real-time PCR and genotyping studies conducted today increase our knowledge about T. gondii. Methods such as B1, SAG1, SAG2, GRA1, 529-bp repeat element, OWP genes and 18S rRNAs are mostly used in PCR, and methods such as MS, MLST, PCR-RFLP, RAPD-PCR and HRM are used in genotyping. Toxoplasmosis is a disease that is within the framework of the concept of one health and must attract attention, has not yet been eradicated in the world and needs joint studies for humans, animals and ecosystems to be eradicated. This can only be possible by establishing interdisciplinary groups, conducting surveys and training.

[Preliminary observation on the development and dynamic changes of chronic toxoplasmosis in mice].

OBJECTIVE: To investigate the development and dynamic changes of cysts in the brain of mice following infection with different forms of Toxoplasma gondii, so as to provide insights into for toxoplasmosis prevention and control. METHODS: ICR mice at ages of 6 to 8 weeks, each weighing 20 to 25 g, were intraperitoneally injected with tachyzoites of the T. gondii PRU strain at a dose of 1 × 105 tachyzoites per mouse, orally administered with cysts at a dose of 20 oocysts per mouse or oocysts at a dose of 200 oocysts per mouse for modeling chronic T. gondii infection in mice, and the clinical symptoms and survival of mice were observed post-infection. Mice were orally infected with T. gondii cysts at doses of 10 (low-dose group), 20 (medium-dose group), 40 cysts per mouse (high-dose group), and the effect of different doses of T. gondii infections on the number of cysts was examined in the mouse brain. Mice were orally administered with T. gondii cysts at a dose of 20 cysts per mouse, and grouped according to gender (female and male) and time points of infections (20, 30, 60, 90, 120, 150, 180 days post-infection), and the effects of gender and time points of infections on the number of cysts was examined in the mouse brain. In addition, mice were divided into the tachyzoite group (Group T), the first-generation cyst group (Group C1), the second-generation cyst group (Group C2), the third-generation cyst (Group C3) and the fourth-generation cyst group (Group C4). Mice in the Group T were intraperitoneally injected with T. gondii tachyzoites at a dose of 1 × 105 tachyzoites per mouse, and the cysts were collected from the mouse brain tissues 30 days post-infection, while mice in the Group C1 were orally infected with the collected cysts at a dose of 30 cysts per mouse. Continuous passage was performed by oral administration with cysts produced by the previous generation in mice, and the effect of continuous passage on the number of cysts was examined in the mouse brain. RESULTS: Following infection with T. gondii tachyzoites, cysts and oocysts in mice, obvious clinical symptoms were observed on days 6 to 13 and mice frequently died on days 7 to 12. The survival rates of mice were 67.0%, 87.0% and 53.0%, and the mean numbers of cysts were (516.0 ± 257.2), (1 203.0 ± 502.0) and (581.0 ± 183.1) in the mouse brain (F = 11.94, P < 0.01) on day 30 post-infection with T. gondii tachyzoites, cysts and oocysts, respectively, and the numbers of cysts in the brain tissues were significantly lower in mice infected with T. gondii tachyzoites and oocysts than in those infected with cysts (all P values < 0.01). The survival rates of mice were 87.0%, 87.0% and 60.0%, and the mean numbers of cysts were (953.0 ± 355.5), (1 084.0 ± 474.3) and (1 113.0 ± 546.0) in the mouse brain in the low-, medium- and high-dose groups on day 30 post-infection, respectively (F = 0.42, P > 0.05). The survival rates of male and female mice were 73.0% and 80.0%, and the mean numbers of cysts were (946.4 ± 411.4) and (932.1 ± 322.4) in the brain tissues of male and female mice, respectively (F = 1.63, P > 0.05). Following continuous passage, the mean numbers of cysts were (516.0 ± 257.2), (1 203.0 ± 502.0), (896.8 ± 332.3), (782.5 ± 423.9) and (829.2 ± 306.0) in the brain tissues of mice in the T, C1, C2, C3 and C4 groups, respectively (F = 4.82, P < 0.01), and the number of cysts was higher in the mouse brain in Group 1 than in Group T (P < 0.01). Following oral administration of 20 T. gondii cysts in mice, cysts were found in the moues brain for the first time on day 20 post-infection, and the number of cysts gradually increased over time, peaked on days 30 and 90 post-infection and then gradually decreased; however, the cysts were still found in the mouse brain on day 180 post-infection. CONCLUSIONS: There is a higher possibility of developing chronic T. gondii infection in mice following infection with cysts than with oocysts or tachyzoites and the most severe chronic infection is seen following infection with cysts. The number of cysts does not correlate with the severity of chronic T. gondii infection, and the number of cysts peaks in the mouse brain on days 30 and 90 post-infection.

Detection of Toxoplasma gondii infection in buffaloes (Bubalus bubalis) and cattle (Bos taurus) at the Tabriz abattoir, Iran.

BACKGROUND: Toxoplasma gondii is a widely prevalent zoonotic protozoan parasite in humans and warm-blooded animals worldwide. Infection of humans by this parasite can result in severe clinical symptoms, particularly in individuals with congenital toxoplasmosis or immunocompromised patients. Contamination mainly occurs through foodborne routes, especially the consumption of raw or undercooked meat from animals. OBJECTIVES: The aim of this study was to use PCR to detect T. gondii in tissues and organs of buffaloes and cattle slaughtered at Tabriz slaughterhouse, in Iran. METHODS: Fifty grams of heart, thigh, diaphragm and tongue from 50 buffaloes and 100 cattle slaughtered at the Tabriz industrial slaughterhouse were selected for sampling using a combination of convenience sampling. The samples were tested using a previously published PCR method. RESULTS: Out of the 150 animal samples, T. gondii was detected in 10 (6.7%, 95%CI: 3.2-11.9), including one buffalo (2%, 95%CI: 0.1-10.6) and nine cattle (9%, 95%CI: 4.2-16.4). There was no statistically significant difference in the rate of T. gondii infection among cattle based on age and sex (p > 0.05). CONCLUSIONS: The results indicated a potential risk of T. gondii transmission to humans through the consumption of infected meat. Therefore, appropriate and effective preventive measures should be taken to limit the transmission of this parasite to humans, and the consumption of raw and undercooked meat should be discouraged.

Early immune response to Toxoplasma gondii lineage III isolates of different virulence phenotype.

Introduction: Toxoplasma gondii is an intracellular parasite of importance to human and veterinary health. The structure and diversity of the genotype population of T. gondii varies considerably with respect to geography, but three lineages, type I, II and III, are distributed globally. Lineage III genotypes are the least well characterized in terms of biology, host immunity and virulence. Once a host is infected with T.gondii, innate immune mechanisms are engaged to reduce the parasite burden in tissues and create a pro-inflammatory environment in which the TH1 response develops to ensure survival. This study investigated the early cellular immune response of Swiss-Webster mice post intraperitoneal infection with 10 tachyzoites of four distinct non-clonal genotypes of lineage III and a local isolate of ToxoDB#1. The virulence phenotype, cumulative mortality (CM) and allele profiles of ROP5, ROP16, ROP18 and GRA15 were published previously. Methods: Parasite dissemination in different tissues was analyzed by real-time PCR and relative expression levels of IFNγ, IL12-p40, IL-10 and TBX21 in the cervical lymph nodes (CLN), brain and spleen were calculated using the ΔΔCt method. Stage conversion was determined by detection of the BAG1 transcript in the brain. Results: Tissue dissemination depends on the virulence phenotype but not CM, while the TBX21 and cytokine levels and kinetics correlate better with CM than virulence phenotype. The earliest detection of BAG1 was seven days post infection. Only infection with the genotype of high CM (69.4%) was associated with high T-bet levels in the CLN 24 h and high systemic IFNγ expression which was sustained over the first week, while infection with genotypes of lower CM (38.8%, 10.7% and 6.8%) is characterized by down-regulation and/or low systemic levels of IFNγ. The response intensity, as assessed by cytokine levels, to the genotype of high CM wanes over time, while it increases gradually to genotypes of lower CM. Discussion: The results point to the conclusion that the immune response is not correlated with the virulence phenotype and/or allele profile, but an early onset, intense pro-inflammatory response is characteristic of genotypes with high CM. Additionally, high IFNγ level in the brain may hamper stage conversion.

Altered landscape of total RNA, tRNA and sncRNA modifications in the liver and spleen of mice infected by Toxoplasma gondii.

BACKGROUND: Pathogens can impact host RNA modification machinery to establish a favorable cellular environment for their replication. In the present study, we investigated the effect of Toxoplasma gondii infection on host RNA modification profiles and explored how these modifications may influence the host-parasite interaction. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed the modification levels of ∼ 80 nt tRNA and 17-50 nt sncRNAs in mouse liver, spleen, and serum using liquid chromatography and tandem mass spectrometry analysis. The results revealed alterations in RNA modification profiles, particularly during acute infection. The liver exhibited more differentially abundant RNA modifications than the spleen. RNA modification levels in serum were mostly downregulated during acute infection compared to control mice. Correlations were detected between different RNA modifications in the liver and spleen during infection and between several RNA modifications and many cytokines. Alterations in RNA modifications affected tRNA stability and protein translation. CONCLUSIONS/SIGNIFICANCE: These findings provide new insight into the role of RNA modifications in mediating the murine host response to T. gondii infection.

Molecular detection of Toxoplasma gondii in domestic and wild guinea pigs (Cavia spp.) from the Marangani district in Cuzco, Peru.

Toxoplasmosis is a foodborne disease caused by the protozoan Toxoplasma gondii, and transmitted to humans by eating raw or undercooked meat, mainly. Poultry, beef, and pork are the main meats consumed in Peru; despite this, guinea pig meat is also widely consumed. For this reason, the objective of this study was to molecularly detect T. gondii in domestic and wild guinea pigs from the Marangani district in Cuzco, Peru, and identify some risk factors associated with this pathogen. DNA was extracted from the brain tissue samples of guinea pigs (30 domestic and 30 wild), and PCR protocols were used to amplify the internal transcribed spacer (ITS-1) region and a 529 bp fragment from the T. gondii genome. T. gondii DNA was detected in 14 (23.3%) guinea pigs. T. gondii frequency was 33.3% in domestic guinea pigs and 13.3% in wild guinea pigs. Our results demonstrated that guinea pigs represent an important source for T. gondii infection in human populations in this locality.

Differential transcriptome study on the damage of testicular tissues caused by chronic infection of T. gondii in mice.

BACKGROUND: Toxoplasma gondii is an intracellular protozoan parasite that is widely distributed in humans and warm-blooded animals. T. gondii chronic infections can cause toxoplasmic encephalopathy, adverse pregnancy, and male reproductive disorders. In male reproduction, the main function of the testis is to provide a stable place for spermatogenesis and immunological protection. The disorders affecting testis tissue encompass abnormalities in the germ cell cycle, spermatogenic retardation, or complete cessation of sperm development. However, the mechanisms of interaction between T. gondii and the reproductive system is unclear. The aims were to study the expression levels of genes related to spermatogenesis, following T. gondii infection, in mouse testicular tissue. METHODS: RNA-seq sequencing was carried out on mouse testicular tissues from mice infected or uninfected with the T. gondii type II Prugniaud (PRU) strain and validated in combination with real-time quantitative PCR and immunofluorescence assays. RESULTS: The results showed that there were 250 significant differentially expressed genes (DEGs) (P < 0.05, |log2fold change| â‰§ 1). Bioinformatics analysis showed that 101 DEGs were annotated to the 1696 gene ontology (GO) term. While there was a higher number of DEGs in the biological process classification as a whole, the GO enrichment revealed a significant presence of DEGs in the cellular component classification. The Arhgap18 and Syne1 genes undergo regulatory changes following T. gondii infection, and both were involved in shaping the cytoskeleton of the blood-testis barrier (BTB). The number of DEGs enriched in the MAPK signaling pathway, the ERK1/2 signaling pathway, and the JNK signaling pathway were significant. The PTGDS gene is located in the Arachidonic acid metabolism pathway, which plays an important role in the formation and maintenance of BTB in the testis. The expression of PTGDS is downregulated subsequent to T. gondii infection, potentially exerting deleterious effects on the integrity of the BTB and the spermatogenic microenvironment within the testes. CONCLUSIONS: Overall, our research provides in-depth insights into how chronic T. gondii infection might affect testicular tissue and potentially impact male fertility. These findings offer a new perspective on the impact of T. gondii infection on the male reproductive system.

The Toxoplasma oxygen-sensing protein, TgPhyA, is required for resistance to interferon gamma-mediated nutritional immunity in mice.

As Toxoplasma gondii disseminates through its host, the parasite must sense and adapt to its environment and scavenge nutrients. Oxygen (O2) is one such environmental factor and cytoplasmic prolyl 4-hydroxylases (PHDs) are evolutionarily conserved O2 cellular sensing proteins that regulate responses to changes in O2 availability. Toxoplasma expresses 2 PHDs. One of them, TgPHYa hydroxylates SKP1, a subunit of the SCF-E3 ubiquitin ligase complex. In vitro, TgPHYa is important for growth at low O2 levels. However, studies have yet to examine the role that TgPHYa or any other pathogen-encoded PHD plays in virulence and disease. Using a type II ME49 Toxoplasma TgPHYa knockout, we report that TgPHYa is important for Toxoplasma virulence and brain cyst formation in mice. We further find that while TgPHYa mutant parasites can establish an infection in the gut, they are unable to efficiently disseminate to peripheral tissues because the mutant parasites are unable to survive within recruited immune cells. Since this phenotype was abrogated in IFNγ knockout mice, we studied how TgPHYa mediates survival in IFNγ-treated cells. We find that TgPHYa is not required for release of parasite-encoded effectors into host cells that neutralize anti-parasitic processes induced by IFNγ. In contrast, we find that TgPHYa is required for the parasite to scavenge tryptophan, which is an amino acid whose levels are decreased after IFNγ up-regulates the tryptophan-catabolizing enzyme, indoleamine dioxygenase (IDO). We further find, relative to wild-type mice, that IDO knockout mice display increased morbidity when infected with TgPHYa knockout parasites. Together, these data identify the first parasite mechanism for evading IFNγ-induced nutritional immunity and highlight a novel role that oxygen-sensing proteins play in pathogen growth and virulence.

RPA-CRISPR/Cas12a-LFA combined with a digital visualization instrument to detect Toxoplasma gondii in stray dogs and cats in Zhejiang province, China.

Toxoplasma gondii, which causes toxoplasmosis, is prevalent in warm-blooded animals, such as cats, dogs, and humans. T. gondii causes economic losses to livestock production and represents a potential risk to public health. Dogs and cats are common hosts in the epidemiology of toxoplasmosis. The current molecular diagnostic tools for T. gondii infection require high technical skills, a laboratory environment, and complex instruments. Herein, we developed a recombinase polymerase amplification (RPA)-clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a (Cas12a) assay to detect T. gondii. The lowest limit of detection of the assay was 31 copies/µL for the T. gondii B1 gene. In addition, we established a visual RPA-CRISPR/Cas12a lateral flow band assay (RPA-CRISPR/Cas12a-LFA) combined with a digital visualization instrument, which minimized the problem of false-negative results for weakly positive samples and avoided misinterpretation of the results by the naked eye, making the LFA assay results more accurate. The assay established in this study could identify T. gondii within 55 min with high accuracy and sensitivity, without cross-reaction with other tested parasites. The developed assay was validated by establishing a mouse model of toxoplasmosis. Finally, the developed assay was used to investigate the prevalence of T. gondii in stray cats and dogs in Zhejiang province, Eastern China. The positive rates of T. gondii infection in stray cats and dogs were 8.0% and 4.0%, respectively. In conclusion, the RPA-CRISPR/Cas12a-LFA is rapid, sensitive, and accurate for the early diagnosis of T. gondii, showing promise for on-site surveillance. IMPORTANCE: Toxoplasma gondii is a virulent pathogen that puts millions of infected people at risk of chronic disease reactivation. Hosts of T. gondii are distributed worldwide, and cats and dogs are common hosts of T. gondii. Therefore, rapid diagnosis of early T. gondii infection and investigation of its prevalence in stray dogs and cats are essential. Here, we established a visual recombinase polymerase amplification-clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a-assay combined with a lateral flow band assay and a digital visualization instrument. Detailed analyses found that the assay could be used for the early diagnosis of T. gondii without false-negative results. Moreover, we detected the prevalence of T. gondii in stray cats and dogs in Zhejiang province, China. Our developed assay provides technical support for the early diagnosis of T. gondii and could be applied in prevalence surveys of T. gondii in stray dogs and cats.

Toxoplasma gondii infection in European brown hares (Lepus europaeus) in Brazil.

Toxoplasmosis is a worldwide zoonosis that affects warm-blooded animals, including humans. Wild animals can act as intermediate hosts of this pathogen; thus, this study aims to detect Toxoplasma gondii infection in invasive European brown hares in Brazil. For this, 72 wild European brown hares were captured from July 2020 to June 2022 in three Brazilian states: São Paulo, Paraná, and Rio Grande do Sul. The diagnostic of Toxoplasma gondii infection was performed by bioassay in mouse, histopathology in Hematoxylin-Eosin-stained tissue sections (brain, liver, lungs, kidneys, and small intestine), serology by IFAT, and molecular techniques by conventional PCR and qPCR. The combined prevalence of the different diagnostic methods was 51.4% (37/72, CI= 40.1 - 62.6 %), and there was no statistical difference between sexes, age range, or geographical region of the hosts. Mouse bioassay was the technique that detected more positive hares. To our knowledge, this is the first confirmation of Toxoplasma gondii infection in invasive European brown hares in Brazil. These animals act as reservoirs and potential infection source for carnivores and other wild and domestic animals, including humans, thus contributing to perpetuate the disease cycle in São Paulo, Paraná, and Rio Grande do Sul States. Research such as the present study is necessary to raise awareness about the role of animals in the disease cycle.

Seroprevalence and risk factors of Toxoplasma gondii infection among goats in Algeria.

Toxoplasmosis is one of the most common zoonotic parasitic diseases worldwide and is caused by Toxoplasma gondii. It is implicated in reproductive disorders in small ruminants. This study aims to determine, for the first time in Algeria, the seroprevalence and associated factors of T. gondii infection in goats. The study was conducted in four regions, Ghardaia, Laghouat and Djelfa, southern Algeria, and Jijel region, northern Algeria. A total of 92 blood samples were collected including 74 females and 18 males. All sera were tested using an enzyme-linked immunosorbent assay (ELISA) to detect the T. gondii antibodies. The presence of anti-T. gondii antibodies was detected in 35 out of 92 goats (38.04%) (95% CI: 31.64%-44.44%) and in all flocks (100%). Risk factors that have a significant influence on the seroprevalence of T. gondii infection are breed, regions, production system, presence of cats, clinics and abortion history. However, variables such as age and gender were note significantly associated with toxoplasma infection in goats. The highest seroprevalences of infection was observed in saanen (52.94%) (p<0.001) and cross-breed race (44%) (p<0.01) in comparison with other breeds. Regarding regions, Jijel and Laghouat were most infected with seroprevalences of 50% (p<0.001) and 40.91% (p<0.01), respectively. Animals in intensive production systems were most infected, showing a seroprevalence of 51.85%, in comparison with extensive (28.13%) and semi-intensive systems (36.36%) (p<0.001). The presence of cats in farms was significantly associated with high seroprevalence (44.64%) (p<0.001). The infection was more prevalent in previously aborted females (50%) than females that had never aborted (3.35%) (p<0.001)and animals that have diarrhoea or poor health (41.67%) were significantly more infected than healthy animals (37.50%) (p<0.01). Seroprevalence in males (38.89%) was very close to those in females (37.84%) (p>0.05). Age-related seroprevalence did not vary significantly (ranged from 36.37% to 40%) between the three age classes. These results indicate that goat toxoplasmosis is widespread in Algeria, and goats may represent a high risk of contamination for humans. This requires more attention during consumption of goat meat.

A combination of GRA3, GRA6 and GRA7 peptides offer a useful tool for serotyping type II and III Toxoplasma gondii infections in sheep and pigs.

The clinical consequences of toxoplasmosis are greatly dependent on the Toxoplasma gondii strain causing the infection. To better understand its epidemiology and design appropriate control strategies, it is important to determine the strain present in infected animals. Serotyping methods are based on the detection of antibodies that react against segments of antigenic proteins presenting strain-specific polymorphic variations, offering a cost-effective, sensitive, and non-invasive alternative to genotyping techniques. Herein, we evaluated the applicability of a panel of peptides previously characterized in mice and humans to serotype sheep and pigs. To this end, we used 51 serum samples from experimentally infected ewes (32 type II and 19 type III), 20 sheep samples from naturally infected sheep where the causative strain was genotyped (18 type II and 2 type III), and 40 serum samples from experimentally infected pigs (22 type II and 18 type III). Our ELISA test results showed that a combination of GRA peptide homologous pairs can discriminate infections caused by type II and III strains of T. gondii in sheep and pigs. Namely, the GRA3-I/III-43 vs. GRA3-II-43, GRA6-I/III-213 vs. GRA6-II-214 and GRA6-III-44 vs. GRA6-II-44 ratios showed a statistically significant predominance of the respective strain-type peptide in sheep, while in pigs, in addition to these three peptide pairs, GRA7-II-224 vs. GRA7-III-224 also showed promising results. Notably, the GRA6-44 pair, which was previously deemed inefficient in mice and humans, showed a high prediction capacity, especially in sheep. By contrast, GRA5-38 peptides failed to correctly predict the strain type in most sheep and pig samples, underpinning the notion that individual standardization is needed for each animal species. Finally, we recommend analyzing for each animal at least 2 samples taken at different time points to confirm the obtained results.

First report on prevalence, molecular characterization and phylogenetic study of Toxoplasma gondii infecting sheep of the Malakand Division of Pakistan.

BACKGROUND: Toxoplasma gondii is an apicomplexan protozoan parasite that infects one-third of the population of the world, including humans, animals, birds, and other vertebrates. The present investigation is the first molecular attempt in the Malakand Division of Pakistan to determine the epidemiology and phylogenetic study of Toxoplasma gondii infecting small ruminants. METHODOLOGY: A total of (N = 450) blood samples of sheep were randomly collected during the study period (December 2020 to November 2021), and DNA detection was done using PCR by amplifying ITS-1 genes. SPSS.20 and MEGA-11 software were used for statistical significance and phylogenetic analysis. RESULTS: The overall prevalence of T. gondii infection among sheep was 14.44 % (65/450). A high infection rate was found in more than five-year-olds at 18.33 % (11/60). Sequencing and BLAST analysis of PCR-positive samples confirmed the presence of T. gondii. Randomly, three isolates were sequenced and submitted to GenBank under accession numbers (PP028089-PP028091), respectively. The BLAST analysis of the obtained sequences based on the ITS-1 gene showed 99 % similarities with reported genotypes found in goats of Malakand, Pakistan (PP028089) and dogs of Brazil (MF766454). The study concludes that T. gondii is notably prevalent among the sheep population in the region, emphasizing the significant role of risk factors in disease transmission across animals and potentially to humans. Further research, zoonotic potential analysis, and targeted control measures are warranted to address and manage this parasitic infection effectively.

Seroprevalence and risk factors of Toxoplasma gondii in goats (Capra hircus) in the state of Espírito Santo, Southeast Brazil.

Toxoplasma gondii is described as a potential cause of abortion in goats and as a threat to public health. To estimate the prevalence of goats infected by T. gondii, in different cities in the Espírito Santo State, and to identify possible risk factors for infection a serological study was conducted. A total of 146 goat serum samples from the cities of Cariacica, Serra and Vila Velha were analyzed. The presence of IgG Class Immunoglobulins was serologically evaluated by Immunofluorescence antibody test (IFAT) and by Enzyme-linked Immunosorbent Assay (ELISA). The seroprevalence of anti-T. gondii was 46.6% (68/146) in both techniques and the same samples got the same results in both techniques. Among the analyzed sera, 70.6% (48/68) exhibited high-avidity IgG antibodies, and 29.4% (20/68) exhibited low-avidity IgG antibodies, suggesting that the infection was chronic in the infected animals. Female sex, age group over two years old, water from the public supply system, storage of food and supplies in an open and unprotected place, and the presence of a domestic cat on the property were identified as risk factors for T. gondii infection in goats. The state of Espirito Santo has a high frequency of infected goats, and this is the first research on caprine toxoplasmosis seroepidemiology in that region.

Molecular prevalence, phylogeny and hematological impact of Toxoplasma gondii and Plasmodium spp. in common quails from Punjab, Pakistan.

This study investigates the molecular prevalence and phylogenetic characteristics of two prominent blood-borne pathogens, Toxoplasma gondii (T. gondii) and Plasmodium spp., in common quails (Coturnix coturnix) sampled from both wild (N = 236) and farmed (N = 197) populations across four districts (Layyah, Dera Ghazi Khan, Lahore, and Multan) in Punjab, Pakistan, during the hunting seasons from 2021 to 2023. Additionally, the impact of these pathogens on the complete blood count (CBC) of the hosts is examined. Out of 433 quails tested, 25 (5.8%) exhibited amplification of the internal transcribed spacer (ITS-1) gene for T. gondii, while 15 (3.5%) showed amplification of the Cytochrome b gene for Plasmodium spp. A risk factor analysis indicated that the prevalence of both pathogens was not confined to specific sampling sites or bird sexes (P > 0.05). District-wise analysis highlighted that hens were more susceptible to both T. gondii and Plasmodium spp. infections than cocks. Wild quails exhibited a higher susceptibility to T. gondii compared to farmed birds. Significant CBC variations were recorded in infected birds as compared to uninfected ones. BLAST analysis of generated sequences has confirmed the identity of recovered PCR amplicons as T. gondii and Plasmodium relictum. Phylogenetic analysis revealed that Pakistani isolates clustered with those reported from various countries globally. This study provides the first documentation of T. gondii and Plasmodium sp. infections in Pakistani quails, underscoring the need for detailed investigations across different regions to enhance our understanding of infection rates and the zoonotic potential of these parasites.

Correlations between the degree of infection by wild strain of Toxoplasma gondii in vitro and porcine hematological parameters.

The apicomplexa Toxoplasma gondii is capable of actively proliferating in numerous types of nucleated cells, and therefore has a high potential for dissemination and resistance. Thus, the present work aimed to correlate the inoculum concentrations and amount of post-infection parasites with porcine hematological parameters (including biochemistry) through in vitro culture. Porcine blood was incubated with different concentrations of parasites (1.2 × 107, 6/3/1.5 × 106 cells/mL), then the concentrations of red blood cells (RBC) and their morphology, total and differential leukocytes, and free peptides were evaluated. In addition, eight different blood samples analyzed before inoculation, where subsequent multivariate analysis was applied to correlate different variables with trophozoite concentration. The results showed no significant variation (p < 0.05) in the relative levels of free peptides, or the relative percentage of RBC at all the parasite concentrations tested. However, the normalized percentages of leukocytes and neutrophils showed a significant reduction, while those of lymphocytes, eosinophils and monocytes showed the opposite behavior. Semi-automatic processing of images exhibited significant microcytosis and hypochromia. The multivariate analysis revealed a positive correlation between the amount number of protozoa (AP) and the variables: "Red cells" and "Neutrophils", an indifference between the AP and the content of free peptides, and the concentration of monocytes in the samples; and a negative correlation for AP and the percentages of lymphocytes and eosinophils. Our results suggest that specific changes in hematological parameters may be associated with different degrees of parasitemia, demanding a thorough diagnostic process and adequate treatment.

The Development of Toxoplasma gondii Recombinant Trivalent Chimeric Proteins as an Alternative to Toxoplasma Lysate Antigen (TLA) in Enzyme-Linked Immunosorbent Assay (ELISA) for the Detection of Immunoglobulin G (IgG) in Small Ruminants.

This study presents an evaluation of seventeen newly produced recombinant trivalent chimeric proteins (containing the same immunodominant fragment of SAG1 and SAG2 of Toxoplasma gondii antigens, and an additional immunodominant fragment of one of the parasite antigens, such as AMA1, GRA1, GRA2, GRA5, GRA6, GRA7, GRA9, LDH2, MAG1, MIC1, MIC3, P35, and ROP1) as a potential alternative to the whole-cell tachyzoite lysate (TLA) used in the detection of infection in small ruminants. These recombinant proteins, obtained by genetic engineering and molecular biology methods, were tested for their reactivity with specific anti-Toxoplasma IgG antibodies contained in serum samples of small ruminants (192 samples of sheep serum and 95 samples of goat serum) using an enzyme-linked immunosorbent assay (ELISA). The reactivity of six recombinant trivalent chimeric proteins (SAG1-SAG2-GRA5, SAG1-SAG2-GRA9, SAG1-SAG2-MIC1, SAG1-SAG2-MIC3, SAG1-SAG2-P35, and SAG1-SAG2-ROP1) with IgG antibodies generated during T. gondii invasion was comparable to the sensitivity of TLA-based IgG ELISA (100%). The obtained results show a strong correlation with the results obtained for TLA. This suggests that these protein preparations may be a potential alternative to TLA used in commercial tests and could be used to develop a cheaper test for the detection of parasite infection in small ruminants.

Seroprevalence and risk factors of Toxoplasma gondii infection in goats from South Punjab Province, Pakistan.

Layyah District in South Punjab Province of Pakistan offers the most intensive caprine economy in the country; its Indus riverine and desert environment makes the area peculiar and worthy of specific investigations. This study aimed to investigate the prevalence of anti-Toxoplasma gondii (T. gondii) IgG-antibody in goats in serum samples and the potential risk factors. The prevalence of T. gondii infection was estimated using a two-stage sample design. All caprine farms in the study area were stratified by size, and from these 110 were randomly selected. Twelve goats (>1-year-old) were selected from each farm and a total of 1320 serum samples were collected and tested by ELISA. A questionnaire on the conditions and management practices of each farm was administered to 110 farmers. Four hundred and sixteen out of 1320 sera samples (31.5%) were found positive and 89% of the flock had at least one seropositive goat. The proportion of seropositive goats tested within each flock ranged from 8.3% to 83.3%. with several factors contributing to this heterogeneity. Goat age played a significant role in the presence of cats. Significant interactions were related to goat farms having floor of dirt and kitten presence. Moreover, age class, abortion history and water source supply were modulated by owner education levels. This is the first study to determine the prevalence of anti-T. gondii antibodies in goats sera in Layyah district and the largest carried out so far in Pakistan. The remarkable presence of T. gondii among goats in areas where goat farming plays a significant economic role may pose a production threat to the small-stock industry, as well as to public health and food safety. Therefore, investigations to identify high-risk goat populations are highly recommended in order to facilitate the implementation of local control strategies.

Molecular detection of Toxoplasma gondii and Neospora caninum in seabirds collected along the coast of Santa Catarina, Brazil.

Toxoplasma gondii and Neospora caninum are two closely related protozoans that infect a wide range of animals, including birds. However, the occurrence of N. caninum and T. gondii in seabirds is unknown. Therefore, this study aimed to determine the presence of T. gondii and N. caninum DNA in tissue samples of seabirds. Tissue samples of the pectoral muscles, heart, and brain were collected from 47 birds along the coastline of Santa Catarina State, SC, Brazil. The DNA was extracted from the tissues and screened using nested-PCR (nPCR) targeting internal transcribed spacer 1 (ITS1). T. gondii DNA was detected in tissues from seven seabirds (7/47, 14.8%), kelp gull (Larus dominicanus) (5/21), and Manx shearwater (Puffinus puffinus) (2/8). N. caninum DNA was detected in tissues of nine seabirds (9/47, 19.1%), the kelp gull (L. dominicanus) (4/21), Manx shearwater (P. puffinus) (2/8), neotropic cormorant (Phalacrocorax brasilianus) (1/4), brown booby (Sula leucogaster) (1/5), and white-chinned petrel (Procellaria aequinoctialis) (1/1); however, no co-infection was observed. In conclusion, this study showed the circulation of N. caninum and T. gondii in seabirds along the coastline of Santa Catarina State. Further studies are required to clarify the role of these birds in the epidemiology of neosporosis and toxoplasmosis.