Study on the effect of koumiss on the intestinal microbiota of mice infected with Toxoplasma gondii.

Toxoplasma gondii is a worldwide food-borne parasite that can infect almost all warm-blooded animals, including humans. To date, there are no effective drugs to prevent or eradicate T. gondii infection. Recent studies have shown that probiotics could influence the relationship between the microbiota and parasites in the host. Koumiss has been used to treat many diseases based on its probiotic diversity. Therefore, we explored the effect of koumiss on T. gondii infection via its effect on the host intestinal microbiota. BALB/c mice were infected with T. gondii and treated with PBS, koumiss and mares' milk. Brain cysts were counted, and long-term changes in the microbiota and the effect of koumiss on gut microbiota were investigated with high-throughput sequencing technology. The results suggested that koumiss treatment significantly decreased the cyst counts in the brain (P < 0.05). Moreover, T. gondii infection changed the microbiota composition, and koumiss treatment increased the relative abundance of Lachnospiraceae and Akkermansia muciniphila, which were associated with preventing T. gondii infection. Moreover, koumiss could inhibit or ameliorate T. gondii infection by increasing the abundance of certain bacteria that control unique metabolic pathways. The study not only established a close interaction among the host, intracellular pathogens and intestinal microbiota but also provided a novel focus for drug development to prevent and eradicate T. gondii infection.

Parasites of veterinary importance from domestic animals in uMkhanyakude district of KwaZulu-Natal province.

This study investigated the occurrence and phylogenetic relationship of protozoan parasites and Ehrlichia infecting domestic animals from three municipalities in uMkhanyakude district of KwaZulu-Natal province, South Africa. A total of 208 blood samples collected from clinically healthy cattle, sheep, goats and dogs from uMkhanyakude district were examined by polymerase chain reaction (PCR) assays, using either genus or species-specific primers to determine the occurrence and phylogenetic relationship of various protozoan parasites and Ehrlichia of veterinary importance. A total of 5/109 (4.6%) cattle were PCR-positive for the presence of Toxoplasma gondii, 33/109 (30.3%) for Babesia bovis, 24/109 (22.02%) for Babesia bigemina and 20/109 (18.3%) for Trypanosoma sp., while 3/10 (30%) of sheep were PCR-positive for Theileria ovis and none of the goats were positive for any of the detected pathogens. The co-infection of 4/109 (3.7%) B. bovis and B. bigemina was detected in cattle. Only Ehrlichia canis was detected in dogs with infection rate of 20/48 (41.7%). Sequences of PCR-positive isolates (B. bovis, B. bigemina, E. canis, T. ovis and T. gondii) showed that they were closely related to their relevant species from various countries. These findings have expanded our knowledge about the prevalence and phylogenetic similarity between protozoan parasites and Ehrlichia isolates of South African origin. To date, this is the first study in South Africa to detect T. gondii infections from cattle blood using PCR.

Infection-Induced Intestinal Dysbiosis Is Mediated by Macrophage Activation and Nitrate Production.

Oral infection of C57BL/6J mice with Toxoplasma gondii results in a marked bacterial dysbiosis and the development of severe pathology in the distal small intestine that is dependent on CD4+ T cells and interferon gamma (IFN-γ). This dysbiosis and bacterial translocation contribute to the development of ileal pathology, but the factors that support the bloom of bacterial pathobionts are unclear. The use of microbial community profiling and shotgun metagenomics revealed that Toxoplasma infection induces a dysbiosis dominated by Enterobacteriaceae and an increased potential for nitrate respiration. In vivo experiments using bacterial metabolic mutants revealed that during this infection, host-derived nitrate supports the expansion of Enterobacteriaceae in the ileum via nitrate respiration. Additional experiments with infected mice indicate that the IFN-γ/STAT1/iNOS axis, while essential for parasite control, also supplies a pool of nitrate that serves as a source for anaerobic respiration and supports overgrowth of Enterobacteriaceae Together, these data reveal a trade-off in intestinal immunity after oral infection of C57BL/6J mice with T. gondii, in which inducible nitric oxide synthase (iNOS) is required for parasite control, while this host enzyme is responsible for specific modification of the composition of the microbiome that contributes to pathology.IMPORTANCEToxoplasma gondii is a protozoan parasite and a leading cause of foodborne illness. Infection is initiated when the parasite invades the intestinal epithelium, and in many host species, this leads to intense inflammation and a dramatic disruption of the normal microbial ecosystem that resides in the healthy gut (the so-called microbiome). One characteristic change in the microbiome during infection with Toxoplasma-as well as numerous other pathogens-is the overgrowth of Escherichia coli or similar bacteria and a breakdown of commensal containment leading to seeding of peripheral organs with gut bacteria and subsequent sepsis. Our findings provide one clear explanation for how this process is regulated, thereby improving our understanding of the relationship between parasite infection, inflammation, and disease. Furthermore, our results could serve as the basis for the development of novel therapeutics to reduce the potential for harmful bacteria to bloom in the gut during infection.

First report on seroprevalence and risk factors of Toxoplasma gondii infection in sheep and goats in North Lebanon.

INTRODUCTION: Toxoplasmosis is of dual importance in both public and veterinary health due to the respective risk of transplacental transmission in primo-infected pregnant women and economic losses caused by abortions in mammals. One of the main routes of Toxoplasma gondii transmission to humans is the consumption of raw or undercooked meats containing parasitic cysts. Here, we performed the first epidemiological study to determine the seroprevalence and the risk factors of toxoplasmosis in livestock in Lebanon. METHODOLOGY: Using a modified agglutination test with a cut-off of 1:40, we tested the positivity rate of Immunoglobulin G antibodies in the sera of 100 sheep and 80 goats collected from 18 different livestock farms located in North Lebanon between March and June 2018. RESULTS: Anti-Toxoplasma gondii IgG antibodies were detected in 42% of sheep and 34% of goats. Adults (> 1 year) were significantly more infected by T. gondii than the lambs (< 1 year) in both species (p < 0.05). CONCLUSIONS: These findings indicated that food animals are highly exposed to T. gondii in Lebanon and could be potentially a major risk factor of T. gondii infection to humans. Consequently, national prophylactic strategies should be implemented to control and to prevent T. gondii transmission between animals and humans.

Toxoplasma gondii, a Foodborne Pathogen in the Swine Production Chain from a European Perspective.

Toxoplasmosis is a foodborne zoonosis transmitted by Toxoplasma gondii, a cosmopolitan protozoan that infects humans through exposure to different parasite stages, in particular by ingestion of tissue cysts or tachyzoites contained in meat, primary offal (viscera), and meat-derived products or ingestion of environmental sporulated oocysts in contaminated food or water. The pig is an important species for infection: raw or undercooked pork consumption not subject to treatment able to inactivate the parasite represents a risk to consumers' health. Broadening knowledge of transmission ways and prevalence concerning this important pathogen in swine, together with a thorough acquaintance with hazard management are key elements to avoid T. gondii spreading within the swine production chain. This review aims to illustrate why toxoplasmosis should be regarded as a veterinary public health issue through a careful description of the parasite, routes of infection, and inactivation treatments, highlighting the main prevention lines from pig breeding to pork consumption.

Z-DNA Binding Protein Mediates Host Control of Toxoplasma gondii Infection.

Intrinsic to Toxoplasma gondii infection is the parasite-induced modulation of the host immune response, which ensures establishment of a chronic lifelong infection. This manipulation of the host immune response allows T. gondii to not only dampen the ability of the host to eliminate the parasite but also trigger parasite differentiation to the slow-growing, encysted bradyzoite form. We previously used RNA sequencing (RNA-seq) to profile the transcriptomes of mice and T. gondii during acute and chronic stages of infection. One of the most abundant host transcripts during acute and chronic infection was Z-DNA binding protein 1 (ZBP1). In this study, we determined that ZBP1 functions to control T. gondii growth. In activated macrophages isolated from ZBP1 deletion (ZBP1(-/-)) mice, T. gondii has an increased rate of replication and a decreased rate of degradation. We also identified a novel function for ZBP1 as a regulator of nitric oxide (NO) production in activated macrophages, even in the absence of T. gondii infection. Upon stimulation, T. gondii-infected ZBP1(-/-) macrophages display increased proinflammatory cytokines compared to wild-type macrophages under the same conditions. These in vitro phenotypes were recapitulated in vivo, with ZBP1(-/-) mice having increased susceptibility to oral challenge, higher cyst burdens during chronic infection, and elevated inflammatory cytokine responses. Taken together, these results highlight a role for ZBP1 in assisting host control of T. gondii infection.

Low-level sequence variation in Toxoplasma gondii calcium-dependent protein kinases among different genotypes.

The causative agent of toxoplasmosis, Toxoplasma gondii, can infect virtually all nucleated cell types of warm-blooded animals. In this study, we examined the sequence variation in calcium-dependent protein kinase 2 (CDPK2) genes among 13 T. gondii strains from different hosts and geographical locations. The results showed that the lengths of the complete CDPK2 DNA and cDNA sequences were 3671-3673 and 2136 bp, respectively, and the sequence variation was 0-0.9% among different T. gondii strains. Phylogenetic analysis based on the CDPK2 gene sequences revealed that T. gondii strains of the same genotypes were clustered in different clades. Further analysis of all the other T. gondii CDPK genes in genotype I (GT1), II (ME49), or III (VEG) strains indicated the T. gondii CDPK gene family is quite conserved, with sequence variation ranging from 0 to 1.40%. We concluded that CDPK2 as well as all the other CDPK genes in T. gondii cannot be used as proper markers for studying the variants of different T. gondii genotypes from different hosts and geographical locations, but their sequence conservation may be a useful feature promoting them as anti-T. gondii vaccine candidates in further studies.

A Toxoplasma gondii mutant highlights the importance of translational regulation in the apicoplast during animal infection.

Toxoplasma gondii is an obligate intracellular parasite of all warm-blooded animals. We previously described a forward genetic screen to identify T. gondii mutants defective in the establishment of a chronic infection. One of the mutants isolated was disrupted in the 3' untranslated region (3'UTR) of an orthologue of bacterial translation elongation factor G (EFG). The mutant does not have a growth defect in tissue culture. Genetic complementation of this mutant with the genomic locus of TgEFG restores virulence in an acute infection mouse model. Epitope tagged TgEFG localized to the apicoplast, via a non-canonical targeting signal, where it functions as an elongation factor for translation in the apicoplast. Comparisons of TgEFG expression constructs with wild-type or mutant 3'UTRs showed that a wild-type 3'UTR is necessary for translation of TgEFG. In tissue culture, the TgEFG transcript is equally abundant in wild-type and mutant parasites; however, during an animal infection, the TgEFG transcript is increased more than threefold in the mutant. These results highlight that in tissue culture, translation in the apicoplast can be diminished, but during an animal infection, translation in the apicoplast must be fully functional.

Transmission of toxoplasmosis (Toxoplasma gondii) by foods.

Protozoan foodborne diseases are generally underrecognized. Toxoplasma gondii is the causative agent of toxoplasmosis, one of the most prevalent parasitic infections to humans and domestic animals. The most likely source of T. gondii occurring through food is the consumption of raw or undercooked meat contaminated with tissue cysts. Sporulated T. gondii oocysts, from the feces of infected cats, present in the environment are a potential source of infection. The ingestion of water contaminated with oocysts and the eating of unwashed raw vegetables or fruits were identified as an important risk factor in most epidemiological studies. This review presents information and data to show the importance of T. gondii transmission by foods.

Systemic toxoplasmosis and Gram-negative sepsis in a southern chamois (Rupicapra pyrenaica) from the Pyrenees in northeast Spain.

A 6-year-old, male southern chamois (Rupicapra pyrenaica) had an absence of flight response and was captured by hand in the Catalan Pyrenees in northeast Spain. On clinical examination, the animal was in good body condition, and only atrophy of the right eye was observed. Blood samples were collected and hematologic analysis performed, but no alterations were observed. The animal was sent to a Wildlife Rescue Centre, where it developed chronic wasting and died after 32 days in captivity. At necropsy, the animal was cachectic and had edematous, mottled lungs. Histopathologic examination revealed systemic toxoplasmosis and acute Gram-negative septicemia. The protozoan organisms were identified as Toxoplasma gondii based on immunohistochemistry. An indirect fluorescent antibody test was performed, and the animal was positive with an antibody titer of 150.

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.

Vaccination against murine toxoplasmosis using recombinant Toxoplasma gondii SAG3 antigen alone or in combination with Quil A.

PURPOSE: Surface antigen 3 (SAG3) of Toxoplasma gondii is very similar in structure to the major surface antigen 1 (SAG1). Although numerous studies have supported the importance of SAG1 in protection against T. gondii infection, few reports exist on SAG3. MATERIALS AND METHODS: Glutathione-S-transferase (GST)-fused SAG3 of T. gondii (rSAG3) were immunized into BALB/c mice alone or in combination with Quil A (rSAG3/Quil A), and then evaluated the protective immunity in vivo and in vitro against murine toxoplasmosis. RESULTS: Immunization with rSAG3 or rSAG3/Quil A resulted in significantly more survival days and fewer brain cysts after challenge with T. gondii compared to an infected control group. Mice immunized with rSAG3 alone or in combination with Quil A produced significantly more specific IgG2a antibody, whereas specific IgG1 antibody titers did not increase. The percentage of CD8+ T cells, IFN-gamma mRNA expression, and nitric oxide production significantly increased in rSAG3- and rSAG3/Quil A-immunized mice. CONCLUSION: These results indicate that vaccination with Toxoplasma rSAG3 results in partial protective immunity against T. gondii infection through induction of a Th1-type immune response, and that protective immunity is accelerated by the modulating effects of Quil A.

Acute toxoplasmosis in three wild arctic foxes (Alopex lagopus) from Svalbard; one with co-infections of Salmonella Enteritidis PT1 and Yersinia pseudotuberculosis serotype 2b.

Acute disseminated toxoplasmosis was diagnosed in three wild arctic foxes (Alopex lagopus) that were found dead in the same locality on Svalbard (Norway). The animals included one adult female and two 4-months-old pups. The adult fox was severely jaundiced. Necropsy revealed multifocal, acute, necrotizing hepatitis, acute interstitial pneumonia, and scattered foci of brain gliosis, often associated with Toxoplasma tachyzoites. One pup also had Toxoplasma-associated meningitis. In addition, the latter animal was infected with Yersinia pseudotuberculosis serotype 2b and Salmonella Enteritidis phage type 1 (PT1), which may have contributed to the severity of the Toxoplasma infection in this animal. The diagnosis of toxoplasmosis was confirmed by positive immunohistochemistry and detection of anti-Toxoplasma gondii antibodies in serum of all foxes. The animals were negative for Neospora caninum, canine distemper virus, canine adenovirus, and rabies virus on immunolabelling of tissue sections and smears.

Immunological comparison of 124 isolates of Toxoplasma gondii.

We tested 124 isolates of Toxoplasma gondii, as determined morphologically and by their ability to elicit antibodies in the dye test with the RH strain of Toxoplasma in mice. They were compared for their capacity to immunize CF-1 mice against isolate T-1, and T-1 immune mice for their capacity to resist each of the 123 other isolates. Of the 125 isolates, 52 had been isolated in the continental USA, 33 in Central America, 15 in Europe, 9 in Hawaii, five in Japan, two in Taiwan, five in Australia, one in Indonesia, one in Tunisia, and one was of unknown origin. Complete cross-immunity was found. This suggests that only one immunotype of Toxoplasma is prevalent in the United States, and perhaps all over the earth. Vaccines are likely to immunize against most or all Toxoplasma isolates.

Differentiation between mouse-virulent and -avirulent strains of Toxoplasma gondii by a monoclonal antibody recognizing a 27-kilodalton antigen.

Using a murine monoclonal antibody, we were able to differentiate between mouse-virulent and -avirulent strains of Toxoplasma gondii. Monoclonal antibody TB6G5 was reactive with eight clinical mouse-avirulent isolates but not with mouse-virulent laboratory strains RH and BK. The antibody-reactive antigen was identified by indirect immunofluorescence and immunoblot as a 27-kDa cytoplasmic protein expressed by tachyzoites as well as by bradyzoites.

Prevention of abortion and neonatal death due to toxoplasmosis by vaccination of goats with the nonpathogenic coccidium hammondia hammondi.

Six does serologically negative to toxoplasma gondii were vaccinated with 1 million oocysts of the nonpathogenic coccidiym Hammondia hammondi 17 to 73 days before breeding. Two does were not inoculated with H hammondi. All does were mated with a T gondii-free buck. Between the 51st and 119th days of their pregnancies, 5 of the 6 Hammondia-vaccinated does and the 2 controls were each inoculated orally with 1,000 infective oocysts of the GT-1 strain of T gondii. Four of the 5 Toxoplasma-inoculated vaccinated does gave birth to 8 apparently healthy kids at the expected gestation period. The 5th vaccinated doe gave birth to 3 kids that died during delivery or were born dead. Toxoplasma gondii was isolated in mice inoculated with tissues of all kids born to vaccinated does. The vaccinated doe which was not inoculated with T gondii gave birth to 2 healthy noninfected kids. Of the 2 control pregnant does (not vaccinated, but inoculated with T gondii), 1 aborted due to toxoplasmosis 17 days after inoculation. The other control doe had 2 dead fetuses and retained fetal membranes in uterus when necropsied at the expected parturition time. the results indicate that it might be possible to develop a vaccine against toxoplasmic abortions in animals.

Characterization of Toxoplasma gondii isolates from an outbreak of toxoplasmosis in Atlanta, Georgia.

Toxoplasma gondii was isolated from tissue of 5 of 7 cats and of 4 of 4 field mice trapped in a riding stable in Atlanta where an outbreak of toxoplasmosis had occurred in persons. Dye-test antibodies were not found in serum of 4 cats and 4 field mice shown to be infected with T gondii by mouse inoculation. The pathogenicity and infectivity of the 9 isolates of T gondii from cats and mice were compared in mice with the same characteristics of an isolate from a person who acquired toxoplasmosis in association with the stable. Oocysts of all 10 isolates were more pathogenic than cysts of the same isolates. Epidemiologic implications of isolates from cats and mice are discussed.

Experimental infection of the guinea pig inner ear with Toxoplasma gondii.

Based on the question whether toxoplasma gondii is a potential pathogenetic factor in sudden deafness and vertigo (especially in the acquired form of Toxoplasmosis) we started a series of investigations, dealing with direct, local, hematogenous, and intracisternal infection of the guinea pig cochlea with toxoplasma gondii. Three of ten directly inoculated and one of five hematogenously infected guinea pigs showed a severe labyrinthitis in electron and light microscopy. Thus, we could demonstrate that toxoplasma gondii is a potent pathogenetic factor in acute inner ear disturbances in laboratory animals. Toxoplasmosis should always be considered in cases with sudden deafness and vertigo without obvious other cause. With a specific therapy the labyrinthine disturbances can apparently be successfully treated.

Experimental toxoplasmosis in elk (Cervus canadensis).

One cow elk (approx 4 months pregnant) and one calf elk were each inoculated intraruminally with 10(5) infective oocysts of Toxoplasma gondii. Both animals were seronegative when inoculated, but became seropositive to T gondii. Sabin-Feldman dye test antibody titers in relation to day postinoculation were as follows: 1:1024, 1:1024, and 1:2048 (days 28, 69, and 73) for the calf; 1:2048, 1:128, and 1:128 (days 34, 69, and 73) for the cow; and 1:2546 (73 days after inoculation of the cow) for the fetus. Indirect hemagglutination antibody titers were not detected in sera of the cow or her fetus and remained low (1:64 or lower) in calf sera. Both the cow and the calf remained asymptomatic until necropsy 73 days after they were inoculated. Toxoplasma was isolated in mice and cats after they were inoculated with brain, spinal cord, heart, diaphragm, liver, pancreas, cervical lymph nodes, and intestines of the infected calf; with brain, heart, diaphragm, and skeletal muscles of the infected cow; and with brain, heart, cervical lymph nodes, and spleen of the fetus.

Inhibition or killing of an intracellular pathogen by activated macrophages is abrogated by TLCK or aminophylline.

Agents that are known to inhibit certain metabolic pathways or cell functions were evaluated to determine their effects on the capacity of activated macrophages to inhibit or kill obligate intracellular pathogens. Tosyllysine chloromethyl ketone (TLCK) and aminophylline abrogate inhibition or killing of Toxoplasma gondii by activated macrophages.