Seroprevalence of Toxoplasma gondii in outdoor dogs and cats in Bangkok, Thailand.

The aim of this study was to estimate the seroprevalence and risk factors associated with Toxoplasma gondii exposure in dogs and cats from Bangkok, Thailand. Blood samples from 318 dogs and 321 cats were tested for T. gondii antibodies by modified agglutination test (cut-off 1:25). Additionally, 18 dogs and 20 cats were longitudinally sampled for T. gondii antibodies during the same study period, between June and July 2019. The overall seroprevalence in dogs and cats was 7.9% (25/318; 95% CI 4.9­10.8%) and 18.7% (95% CI 14.4­23.0%), respectively. For dogs, risk factors identified were being a mixed-breed animal and living totally outdoors, while increasing age was shown to be a risk factor for cats. Seroconversion was not detected and titres from positive animals remained constant over longitudinal study. The present study indicates that there is a prominent presence of T. gondii in urban and peri-urban areas of Bangkok, suggesting that outdoor dogs and cats should be considered as a possible risk factor for humans.

Zoonotic Enteric Nematodes and Dermatophytes in Cat Cafés: An Investigation in the Bangkok Metropolitan Area, Thailand.

Cat cafés have gained significant popularity worldwide, offering a unique interface between humans and cats. The present study aims to assess the prevalence of potentially zoonotic endoparasites and dermatophytes from cats living in cat cafés situated in the Bangkok metropolitan area in 2017-2018. Cat fecal samples were subjected to microscopic examination employing centrifugal flotation and centrifugal sedimentation techniques. The hair samples from every cat were cultured on a dermatophyte test medium and Sabouraud dextrose agar and subsequently confirmed by visualization of the typical colony and macroconidia morphology. Findings from 11 cat cafés indicated an 18.2% (2/11) prevalence of gastrointestinal parasites, including Toxocara spp., Ancylostoma spp., Physaloptera spp., and Eucoleus aerophilus. Dermatophytes were prevalent in 16.2% (32/198) of the total number of cats tested, with Microsporum canis being the sole species identified. Notably, the presence of dermatophyte was significantly correlated with the presence of skin lesions and the cats' origin. In summary, the findings of this study have provided evidence of potentially zoonotic endoparasites and dermatophytes in cats residing in cat cafés. Therefore, it is imperative to heighten awareness and encourage preventive measures among cat café owners and customers to halt the dissemination of these pathogens.

A first attempt at determining the antibody-specific pattern of Platynosomum fastosum crude antigen and identification of immunoreactive proteins for immunodiagnosis of feline platynosomiasis.

Background and Aim: Feline platynosomiasis, also known as lizard poisoning, is a feline hepatic disease caused by the parasitic trematode Platynosomum fastosum. Since this helminth resides in biliary ducts and gallbladder, the heavy infection can lead to failure of the hepatobiliary system and can be associated with cholangiocarcinoma. The primary diagnostic tool currently used is conventional fecal microscopy. However, low sensitivity of detection could occur in the case of light infection or biliary obstruction. This study aimed to determine the antibody-specific pattern of P. fastosum crude antigen and to identify immunoreactive proteins to develop the immunodiagnostic techniques. Materials and Methods: We investigated potential antigens specific to P. fastosum infection using western blotting. Forty-six samples of cat serum, including 16 P. fastosum-infected sera, eight healthy control sera, and 22 sera infected with other endoparasites were used. The sensitivity, specificity, positive predictive value, and negative predictive value of each band were calculated. Immunoreactive bands with high diagnostic values were further analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify the protein components. Results: Using immunoblotting, three proteins of 72 kDa, 53 kDa, and 13 kDa were found to be immunogenic. LC-MS/MS identified these proteins as a 70 kDa heat shock protein, a hypothetical protein (CRM22_002083) (adenosine triphosphate synthase subunit beta), and histone H2B, respectively. Conclusion: This study is the first to reveal three proteins that could be candidates for developing diagnostic tools for feline platynosomiasis.

Retrospective analysis of canine monocytic ehrlichiosis in Thailand with emphasis on hematological and ultrasonographic changes.

Background and Aim: Canine monocytic ehrlichiosis (CME) is a tropical endemic tick-borne disease that causes fatality or chronic infection involving many organs in dogs. This study aimed to examine the prevalence, risk factors, and hematological and ultrasonographic changes in the liver, gallbladder, kidneys, and spleen following CME infection. Materials and Methods: This retrospective study used 30,269 samples collected from dogs at the hematology section of the pathology unit of a university veterinary hospital and 35 samples collected from dogs at the diagnostic imaging unit. CME was determined using the buffy coat smear method. Data were analyzed using descriptive statistics and odds ratios. Results: The data revealed that the average yearly prevalence of CME was 1.32%. Risk factors contributing to CME infection were a tick on the body during physical examination, lack of ectoparasite control, and outdoor living. All 148 dogs with CME infection had low platelet counts. The percentages of CME-infected dogs with elevated serum alanine aminotransferase, alkaline phosphatase, and both enzymes above the normal range were 33.6%, 65.9%, and 29.8%, respectively. The rates for elevated serum levels of blood urea nitrogen, creatinine, and both compounds were 33.1%, 19.1%, and 17.3%, respectively. The most common ultrasonographic changes were liver abnormalities (hyperechogenicity or hypoechogenicity, hepatomegaly, and hypoechoic nodules), hyperechogenicity of the kidneys, and an enlarged spleen. These ultrasonographic changes were consistent with the hematology results, which showed a greater elevation of serum liver enzyme levels than renal enzymes. Conclusion: Ultrasonographic changes during CME infection and after treatment with doxycycline can help to monitor and identify persistent pathological changes in the target organs resulting from immune response to CME.

Antigenic components, identification, and characterization of whole worm extract of Platynosomum illiciens.

The antigenic components of adult Platynosomum illiciens were characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting using sera from cats naturally infected with P. illiciens, Dipylidium caninum, Toxocara cati and uninfected cat sera. The whole worm extract (WWE) of P. illiciens was fractionated by Sephadex G-200 gel filtration chromatography. The results showed that WWE fraction and F2 were highly antigenic as well as F1 and F3, which were moderately antigenic. For SDS-PAGE and immunoblotting, the antigenic molecules of WWE and all three fractions were mostly at molecular weights (MW) ranging from 11 to 150 kDa. Four antigenic proteins of 11, 18, 27 and 75 kDa detected in WWE and F1-F3 were found to give a reaction with sera from P. illiciens infected cats, and these proteins were also identified using liquid chromatography-mass spectrometry (LC-MS/MS). For immunolocalization observation, it was revealed that the P. illiciens antigen was present in high concentration in the cytoplasm of vitelline cells in the vitelline glands, the shell of the eggs and the eggs within the uterus, but not in other organs, i.e., tegument, muscle, parenchymal cells, testes and oral and ventral suckers of adult fluke. This finding indicates that these proteins may be potential antigen candidates for the immunodiagnosis of feline platynosomosis caused by P. illiciens.

Comparative performance of five recombinant and chimeric antigens in a time-resolved fluorescence immunoassay for detection of Toxoplasma gondii infection in cats.

Felids are definitive hosts of Toxoplasma gondii, being the only hosts that can spread the infection through oocyst shedding in their feces. The elevated presence of this parasite in the domestic cat (Felis catus), and its close contact with humans, make it necessary to obtain reliable diagnostic methods to detect positive animals as a public health measure. For this reason, in this study, the diagnostic performance of five different recombinant antigen-based techniques was assessed to diagnose T. gondii infection in cat blood plasma samples. Specifically, four T. gondii recombinant antigens (GRA7, truncated GRA7, SAG2, and truncated SAG2) and a chimeric antigen (SAG1-GRA8) were used. A time-resolved fluorescence immunoassay (TRFIA) was developed for each antigen, and the results of each of these techniques were compared with those obtained by a commercial enzyme-linked immunoassay (ELISA) and a modified agglutination test (MAT) as reference techniques. The TRFIA based on SAG1-GRA8 antigen showed better discrimination between seropositive and seronegative cats (p < 0.001), as well as a better area under the curve (0.95), sensitivity (93.6%), and specificity (89.5%) values for the optimal cut-off, versus the other TRFIAs. In addition, SAG1-GRA8 TRFIA showed substantial agreement (kappa value = 0.78) and a moderate significant correlation (Spearman's correlation: r = 0.62, p < 0.001) compared with the reference techniques. On the other hand, since plasma samples were obtained from 101 cats in Bangkok city and four of them were Neospora caninum seropositive by indirect immunofluorescence assay (IFAT), this is the first time that anti-N. caninum antibodies are detected in cats in Thailand. In conclusion, our study highlights that the TRFIA with TgSAG1-GRA8 antigen is an accurate and recommended diagnostic technique for detecting anti-T. gondii antibodies in cats.

Prevalence of endoparasitic and viral infections in client-owned cats in metropolitan Bangkok, Thailand, and the risk factors associated with feline hookworm infections.

The prevalence of enteric parasites in cats in metropolitan Bangkok has not been updated in over 13 years. The main objectives of this study include updating the prevalence of endoparasitism in client-owned cats, status of retroviral infections and determining the association between feline hookworm infection and possible risk factors. A total of 509 fecal samples were collected from client-owned cats in 2014-2015 and examined by a wet fecal mount technique. If additional sample remained, a PBS-ethyl acetate sedimentation was done (n = 229), and ZnSO4 centrifugal flotation was also performed if there was sufficient remaining sample (n = 105). At least one parasite was observed in 32.0% (163/509) of cats, with Ancylostoma being the most common intestinal parasite detected in 21.6% (110/509) of cats. Other parasitic infections detected by fecal examinations included Toxocara (6.9%; 35/509), Platynosomum (3.7%; 19/509), Cystoisospora (3.5%; 18/509), Taenia (2.9%; 15/509), Spirometra (1.6%; 8/509), Dipylidium (0.4%; 2/509), and Opisthorchis-like trematode (0.2%; 1/509). Examination for Giardia infection was conducted with the SNAP® Giardia Test, a coproantigen test, on a subset of the fecal samples (233/509) and revealed a positive result on 3.9% (9/233) of samples. Plasma samples were analyzed using the SNAP® Triple Test detecting antigens of Feline Leukemia Virus (FeLV) and Dirofilaria immitis while also detecting antibodies to Feline Immunodeficiency Virus (FIV). Antigens of FeLV and antibodies to FIV were found in 7.1% (19/269) and 5.2% (14/269) of cats, respectively. None of the cats were found to have circulating antigen of Dirofilaria immitis using this test. No association between retroviral and endoparasitic infections was found. From multivariable logistic regression examining associated factors, the ability of cats to access the outdoors (adjusted OR = 3.22, 95% CI; 1.42-7.87) and having tapeworm segments or adult helminths in feces (adjusted OR = 3.31, 95% CI; 1.34-8.21) were significantly associated with the finding of hookworm eggs in feces. This work presents the most up-to-date data on enteric feline parasite prevalence in the metropolitan Bangkok area from which fecal samples were directly collected from cats. Consequently, this study emphasizes that diagnosis of parasitic infections and the routine use of antiparasitic medications should be encouraged by veterinarians and to owners in order to reduce the reservoir of potentially zoonotic parasites.

Liver- and Spleen-Specific Immune Responses in Experimental Leishmania martiniquensis Infection in BALB/c Mice.

Leishmania martiniquensis is a neglected cause of an emerging leishmaniasis in many countries, including France, Germany, Switzerland, the United States of America, Myanmar, and Thailand, with different clinical manifestations ranging from asymptomatic, cutaneous (CL), visceral (VL), and atypically disseminated CL and VL. The persistence of parasites and the recurrence of the disease after treatment are challenges in controlling the disease. To explore efficient prophylaxis and therapy, this study aimed to investigate infection outcome and organ-specific immune responses after inoculation with L. martiniquensis (MHOM/TH/2011/PG; 5 x 106 promastigotes) in BALB/c mice via intravenous and intraperitoneal routes. A quantitative PCR technique, targeting L. martiniquensis ITS1, was primarily established to estimate the parasite burden. We found that the infection in the liver resolved; however, persistent infection was observed in the spleen. Histopathology with Leishmania-specific immunostaining revealed efficient hepatic granuloma formation, while splenic disorganization with parasitized macrophages at different locations was demonstrated. The mRNA expression of Th1 cytokines (IFN-γ, TNF-α, IL-12p40) and iNOS in the liver and spleen was upregulated. In addition, high expression of IL-10 was observed in the spleen in the chronic phase, revealing a significant moderate correlation with the parasite persistence [r(12) = 0.72, P = 0.009]. Further clarification of the mechanisms of persistent infection and experimental infection in immunosuppressed murine models are warranted.

Dendritic cells expressing plasmacytoid marker PDCA-1 are Trojan horses during Toxoplasma gondii infection.

Plasmacytoid dendritic cells (pDCs) play a key role in the innate immune response to viral infection, due largely to their ability to produce large quantities of type I IFNs. These cells are also notable for their ability to differentiate into conventional dendritic cells after appropriate stimulation. Here, we show that a splenic population of murine CD11c(+) cells expressing pDC markers Gr-1, B220, and PDCA-1 is preferentially parasitized after infection with the virulent RH strain of Toxoplasma gondii. Although these markers are closely associated with pDCs, the population we identified was unusual because the cells express CD11b and higher than expected levels of CD11c. By adoptive transfer of CD45.1-positive cells into CD45.2 congenic mice, we show that CD11c(+)Gr-1(+) cells migrate from the peritoneal cavity to the spleen. During infection, these cells accumulate in the marginal zone region. Recruitment of infected CD11c(+)Gr-1(+) cells to the spleen is partially dependent upon signaling through chemokine receptor CCR2. Intracellular cytokine staining demonstrates that infected, but not noninfected, splenic CD11c(+)Gr-1(+) dendritic cells are suppressed in their ability to respond to ex vivo TLR stimulation. We hypothesize that Toxoplasma exploits pDCs as Trojan horses, targeting them for early infection, suppressing their cytokine effector function, and using them for dissemination within the host.

TLR adaptor MyD88 is essential for pathogen control during oral toxoplasma gondii infection but not adaptive immunity induced by a vaccine strain of the parasite.

TLR adaptor MyD88 activation is important in host resistance to Toxoplasma gondii during i.p. infection, but the function of this signaling pathway during oral infection, in which mucosal immunity assumes a predominant role, has not been examined. In this study, we show that MyD88(-/-) mice fail to control the parasite and succumb within 2 wk of oral infection. Early during infection, T cell IFN-gamma production, recruitment of neutrophils and induction of p47 GTPase IGTP (Irgm3) in the intestinal mucosa were dependent upon functional MyD88. Unexpectedly, these responses were MyD88-independent later during acute infection. In particular, CD4(+) T cell IFN-gamma reached normal levels independently of MyD88, despite continued absence of IL-12 in these animals. The i.p. vaccination of MyD88(-/-) mice with an avirulent T. gondii uracil auxotroph elicited robust IFN-gamma responses and protective immunity to challenge with a high virulence T. gondii strain. Our results demonstrate that MyD88 is required to control Toxoplasma infection, but that the parasite can trigger adaptive immunity without the need for this TLR adaptor molecule.

Absence of mitogen-activated protein kinase family member c-Jun N-terminal kinase-2 enhances resistance to Toxoplasma gondii.

The function of mitogen-activated protein kinase (MAPK) family member c-Jun N-terminal kinase (JNK)-2 in resistance and pathology during infection has not been greatly studied. Here, we employed Jnk2(-/-) mice to investigate the role of JNK2 in resistance and immunity during oral infection with the protozoan pathogen Toxoplasma gondii. We found increased host resistance in the absence of JNK2 as determined by lower parasite burden and increased host survival. Lack of JNK2 also correlated with decreased neutrophil recruitment to the intestinal mucosa and less pathology in the small intestine. In the absence of JNK2, IL-12 production was slightly but significantly increased in restimulated splenocyte populations as well as in purified splenic dendritic cell cultures. These results provide evidence that expression of JNK2 plays a role in T. gondii-induced immunopathology, at the same time in promoting susceptibility to this parasitic pathogen.

Understanding the multiple functions of Gr-1(+) cell subpopulations during microbial infection.

The murine cell surface determinant Gr-1 is expressed at high level on neutrophils. Depletion of polymorphonuclear leukocytes with anti-Gr-1(+) monoclonal antibody results in increased susceptibility and dysregulated immunity to many microbial pathogens, a finding widely interpreted to indicate the importance of neutrophils during infection. Yet, in recent years it has become clear that additional cell types express the Gr-1 determinant, including dendritic cell and monocyte subpopulations. In this review, we evaluate current knowledge on the functional aspects of Gr-1(+) cell populations. We focus on infection with the opportunistic protozoan Toxoplasma gondii, a case where host survival depends on an intact Gr-1(+) cell population.

Phosphoinositide-3-kinase-dependent, MyD88-independent induction of CC-type chemokines characterizes the macrophage response to Toxoplasma gondii strains with high virulence.

Chemokines play an important role in inflammation and infection due to their ability to recruit cells of innate and adaptive immunity. Here we examined mouse macrophage chemokine responses during intracellular infections with high- and low-virulence Toxoplasma gondii strains. The high-virulence type I strain RH induced a large panel of CC-type chemokines, whereas responses elicited by strains PTG (type II) and M7741 (type III) were much weaker. Strikingly, the T. gondii-induced chemokine response occurred independently of signaling through the Toll-like receptor adaptor MyD88. Instead, production of chemokines during infection was heavily dependent upon phosphoinositide-3-kinase signaling pathways. Because infection with type I strains such as RH results in an uncontrolled proinflammatory cytokine response, we hypothesize that this virulence phenotype is a consequence of early strong induction of chemokines by type I, but not type II or III, Toxoplasma strains.

Oyster is an effective transmission vehicle for Cryptosporidium infection in human.

OBJECTIVE: To determine the ability of oysters to trap and maintain viable Cryptosporidium oocysts, and the feasibility of Cryptosporidium multiplication in oysters' organs. METHODS: Seventy oysters were raised in experimentally seeded natural seawater for up to 3 months, with weekly oocysts inoculations. Cryptosporidium oocysts, viable and non-viable, as well as other stages were detected using two immunofluorescence vital staining techniques (Sporo-Glo and Merifluor(®)) with confocal microscopy. Viability rate at various times after inoculations were calculated. RESULTS: Cryptosporidium oocysts were found most concentrated in oysters' digestive organs than in gill and water inside the oysters. Oocysts numbers were 857.33 at 24 h after inoculation and strikingly decreased to 243.00 and 126.67 oocysts at 72 h and 7 days, respectively. The oocysts in oyster were also less viable over time; 70%, 60% and 30% viable at 24 h, 72 h and 7 days after inoculation, respectively. At 77 days, the number of oocysts was very low and none was found at 84 days onwards. Although some oocysts were ruptured with released sporozoites, there was no evidence throughout the study of sporozoites multiplication to indicate that oyster is a biological host. Despite the significant reduction in oocysts number after 7 days of inoculation, the remained viable oocysts can still cause cryptosporidiosis. CONCLUSION: The findings confirm that Cryptosporidium parvum does not multiply in oyster, and is therefore not a biological host. Nevertheless, the results suggest that oyster can be an effective transmission vehicle for Cryptosporidium oocysts, especially within 24-72 h of contamination, with viable oocysts present at up to 7 days post infection. Unless consuming well-cooked oyster dishes, eating raw oyster remains a public health concern and at least 3 days of depuration in clean sea water prior to consumption is recommended.

Serological survey of Toxoplasma gondii, Dirofilaria immitis, Feline Immunodeficiency Virus (FIV) and Feline Leukemia Virus (FeLV) infections in pet cats in Bangkok and vicinities, Thailand.

The seroprevalence of Toxoplasma gondii, Dirofilaria immitis (heartworm), feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) infections was examined using serum or plasma samples from 746 pet cats collected between May and July 2009 from clinics and hospitals located in and around Bangkok, Thailand. The samples were tested for heartworm, FIV, and FeLV using a commercial ELISA. Of the 746 samples, 4.6% (34/746) were positive for heartworm antigen, 24.5% (183/746) had circulating FeLV antigen, and 20.1% (150/746) had antibodies against FIV. In addition, the first 348 submitted samples were tested for T. gondii antibodies using a modified agglutination test (MAT, cut off 1:25); 10.1% (35/348) were seropositive. Of the 348 cats sampled for all four pathogens, 11, 10, and 1 were positive for T. gondii antibodies and FIV antibodies, FeLV antigen, or D. immitis antigen, respectively. Of the 35 T. gondii-seropositive cats, 42.9% (15/35) were co-infected with at least one of the other three pathogens. The presence of antibodies to FIV was significantly associated with both age and gender, while FeLV antigen presence was only associated with age. In the case of FIV, males were twice as likely to be infected as females, and cats over 10 years of age were 13.5 times more likely to be infected than cats less than 1 year of age. FeLV antigen was more common in younger cats, with cats over 10 years of age being 10 times less likely to be FeLV positive than cats under 1 year of age. This is the first survey for these four pathogens affecting feline health in Thailand.

Mouse neutrophils require JNK2 MAPK for Toxoplasma gondii-induced IL-12p40 and CCL2/MCP-1 release.

The MAPK family member JNK/stress-activated MAPK (SAPK) is involved in extracellular stress and proinflammatory cytokine responses, including production of cytokines such as IL-12. The JNK1 and 2 isoforms are widely expressed, but JNK3 is largely restricted to tissues of the brain, testis, and heart. In this study, we focus on mouse neutrophils, a cell type in which JNK/SAPK expression and activity has been given little study. We used Western blot analysis to examine expression patterns of JNK/SAPK in wild-type and JNK2-/- polymorphonuclear leukocytes (PMN). Surprisingly, neutrophils displayed a major deficiency in JNK1 expression, in contrast to macrophages that expressed high levels of both JNK1 and JNK2 MAPK. JNK1 expression was steadily reduced during the neutrophil maturation in bone marrow. We used PMN infection with the protozoan parasite Toxoplasma gondii to determine whether neutrophil JNK2 was functional. The parasite induced rapid JNK2 phosphorylation and intracellular FACS staining demonstrated preferential activation in infected neutrophils. Use of JNK2-/- neutrophils revealed that this MAPK family member was required for PMN IL-12p40 and CCL2/MCP-1 production. The chemotactic response displayed a minor JNK2 dependence but phagocytosis and oxidative burst activity did not require this MAPK. These findings are important because they demonstrate 1) a previously unrecognized unusual JNK expression pattern in mouse neutrophils, 2) JNK2 in PMN is activated by Toxoplasma invasion, and 3) a requirement for JNK2 in PMN IL-12p40 and CCL2/MCP-1 production in response to a microbial pathogen.

Herpesvirus chemokine-binding glycoprotein G (gG) efficiently inhibits neutrophil chemotaxis in vitro and in vivo.

Glycoprotein G (gG) of alphaherpesviruses has been described to function as a viral chemokine-binding protein (vCKBP). More recently, mutant viruses devoid of gG have been shown to result in increased virulence, but it remained unclear whether the potential of gG to serve as a vCKBP is responsible for this observation. In this study, we used equine herpesvirus type 1 (EHV-1) as a model to study the pathophysiological importance of vCKBP activity. First, in vitro chemotaxis assays studying migration of immune cells, an important function of chemokines, were established. In such assays, supernatants of EHV-1-infected cells significantly inhibited IL-8-induced chemotaxis of equine neutrophils. Identification of gG as the responsible vCKBP was achieved by repeating similar experiments with supernatants from cells infected with a gG-negative mutant, which were unable to alter IL-8-induced equine neutrophil migration. Furthermore, rEHV-1 gG was able to significantly reduce neutrophil migration, establishing gG as a bona fide vCKBP. Second, and importantly, in vivo analyses in a murine model of EHV-1 infection showed that neutrophil migration in the target organ lung was significantly reduced in the presence of gG. In summary, we demonstrate for the first time that EHV-1 gG not only binds to chemokines but is also capable of inhibiting their chemotactic function both in vitro and in vivo, thereby contributing to viral pathogenesis and virulence.

Toxoplasma gondii inhibits toll-like receptor 4 ligand-induced mobilization of intracellular tumor necrosis factor alpha to the surface of mouse peritoneal neutrophils.

Neutrophils are well-known to rapidly respond to infection through chemotactic infiltration at sites of inflammation, followed by rapid release of microbicidal molecules, chemokines, and proinflammatory cytokines. For tumor necrosis factor alpha (TNF-alpha), we recently found that neutrophils contain intracellular pools of the cytokine and display the capacity to upregulate transcriptional activity of the gene during lipopolysaccharide (LPS) stimulation. We now show that triggering of mouse peritoneal neutrophils with Toll-like receptor 2 (TLR2), TLR4, and TLR9 ligands, but not ligands of TLR3, induces upregulation of surface membrane TNF-alpha. However, neutrophils infected with the protozoan Toxoplasma gondii displayed an inability to respond fully in terms of TLR ligand-induced increases in membrane TNF-alpha expression. Infected neutrophils failed to display decreased levels of intracellular TNF-alpha upon LPS exposure. In contrast to intermediate inhibitory effects in nontreated neutrophils, T. gondii induced a complete blockade in LPS-induced surface TNF-alpha expression in the presence of the protein synthesis inhibitor cycloheximide. Despite these inhibitory effects, the parasite did not affect LPS-induced upregulation of TNF-alpha gene transcription. Collectively, the results show that Toxoplasma prevents TLR ligand-triggered mobilization of TNF-alpha to the neutrophil surface, revealing a novel immunosuppressive activity of the parasite.

Cathepsin B homologue at the interface between a parasitic nematode and its intermediate host.

Parelaphostrongylus tenuis is a parasitic nematode that causes a debilitating neurologic disease in many North American cervids and domestic livestock species. We produced a PCR-based cDNA library from infective larvae (L3) in order to identify molecules that mediate parasitism. A dominant 1,250-bp amplicon encoded a homologue of cathepsin B cysteine proteases. The sequence incorporated a C29G substitution in the putative active site. Antibodies generated against a recombinant form detected the native protein (PtCPR-1) in Western blot assays of L3, but not adult worm, extracts. Immunohistochemical methods revealed that PtCPR-1 synthesis was restricted to larval stages within the snail intermediate host (Triodopsis sp.), beginning as early as 2 days postinfection (dpi) of snails. The protein was present in the intestine and luminal contents and was lost from larvae over time. Concurrent studies showed that larvae induced an immune response in snails beginning at 1 dpi. Layers of hemocytes encapsulated larvae immediately after infection, and granuloma-like structures formed around parasites in chronic infections. Loss of PtCPR-1 from L3 and its accumulation in host tissues coincided with degeneration of granuloma architecture 90 to 105 dpi. Fully developed L3 emerged from the snail at this time. Our data implicate PtCPR-1 in larval development and possibly in the emergence of P. tenuis from the intermediate host. Emerged L3 survived desiccation and cold stress, suggesting that they could remain infectious in the environment. Molecules promoting emergence would facilitate dispersal of L3 and increase the likelihood of transmission to definitive hosts.