Experimental infection of pigs with three dose levels of Trichuris suis

The objective of the study was to follow the course of Trichuris suis infection in pigs given infective eggs at low (400 eggs), medium (4,000 eggs) and high inoculation dose (40,000 eggs), respectively. Interestingly, despite a 100-fold difference in dose level no significant difference was found in either blood parameters, total faecal egg excretion, fecundity or worm burdens at necropsy 12 weeks post inoculation. The highest and lowest median faecal egg output was found in the medium and high dose group, respectively. With increasing dose level, worm size, establishment and prevalence of T. suis positive pigs decreased while worms were dislocated aborally. In addition there was a highly significant correlation between female worm burden and faecal egg excretion.     

Worm burden-dependent disruption of the porcine colon microbiota by Trichuris suis infection

Helminth infection in pigs serves as an excellent model for the study of the interaction between human malnutrition and parasitic infection and could have important implications in human health. We had observed that pigs infected with Trichuris suis for 21 days showed significant changes in the proximal colon microbiota. In this study, interactions between worm burden and severity of disruptions to the microbial composition and metabolic potentials in the porcine proximal colon microbiota were investigated using metagenomic tools. Pigs were infected by a single dose of T. suis eggs for 53 days. Among infected pigs, two cohorts were differentiated that either had adult worms or were worm-free. Infection resulted in a significant change in the abundance of approximately 13% of genera detected in the proximal colon microbiota regardless of worm status, suggesting a relatively persistent change over time in the microbiota due to the initial infection. A significant reduction in the abundance of Fibrobacter and Ruminococcus indicated a change in the fibrolytic capacity of the colon microbiota in T. suis infected pigs. In addition, ∼10% of identified KEGG pathways were affected by infection, including ABC transporters, peptidoglycan biosynthesis, and lipopolysaccharide biosynthesis as well as α-linolenic acid metabolism. Trichuris suis infection modulated host immunity to Campylobacter because there was a 3-fold increase in the relative abundance in the colon microbiota of infected pigs with worms compared to naïve controls, but a 3-fold reduction in worm-free infected pigs compared to controls. The level of pathology observed in infected pigs with worms compared to worm-free infected pigs may relate to the local host response because expression of several Th2-related genes were enhanced in infected pigs with worms versus those worm-free. Our findings provided insight into the dynamics of the proximal colon microbiota in pigs in response to T. suis infection.     

Host age influence on the intensity of experimental Trichuris suis infection in pigs

The impact of age-related resistance on the regulation of population dynamics of adult Trichuris suis was investigated in an experimental pig model. Helminth-na?ve pigs varying in age from five weeks to four years were infected with T. suis to determine susceptibility to infection. Sows had a significantly lower establishment of adult T. suis worms compared with weaner pigs. Adult worm populations were highly overdispersed in both sows and grower pigs contrasted by a more even distribution among weaner pigs. Sows had significantly lower worm fecundities compared to weaner and grower pigs; T. suis from grower pigs, in turn, had reduced fecundity compared to worms in weaner pigs. In conclusion, we provide the first controlled experimental evidence that age-related resistance to T. suis occurs in pigs.     

Schistosoma japonicum: treatment of different developmental stages in mice with long-acting praziquantel implants

This paper reports the effective treatment of Schistosoma japonicum in a mouse model with long-acting praziquantel (PZQ)-loaded poly(ε-caprolactone) implants. The implants yielded stable, high plasma PZQ concentrations ranging 100–1600 ng/mL during the 40-day investigation period. For assessment of efficacy, the implants were implanted into mice immediately after infection and at 1, 2, 3 and 4 weeks after infection to treat the schistosomes at different developmental stages. All the mice were sacrificed at 6 weeks after infection for worm and egg recovery, worm morphology examination, and histopathological analysis of implantation site tissues. The worm burdens, egg burdens, and numbers of miracidia hatched from the retrieved eggs for all the implant-treated groups (except groups T2-A, T4 and T5) were reduced by 100% when compared with the control group. From groups T2-A, T4 and T5, some schistosome debris was recovered. Eggs were found in only group T5 for which the time between infection and implantation was 4 weeks, which enabled the maturation of juvenile female schistosomes into adult ones that lay eggs. Histopathological observations of implantation tissue showed no evidence of granulomatous foreign-body or lymphoid cell aggregation, demonstrating good biocompatibility of the PZQ implants. These results demonstrate that the long-acting PZQ implants can kill schistosomes at any developmental stages and attenuate/avoid the associated liver damage.     

The pathogenic Th17 cell response to major schistosome egg antigen is sequentially dependent on IL-23 and IL-1β

CBA/J mice infected with the helminth Schistosoma mansoni develop severe CD4 T cell-mediated hepatic granulomatous inflammation against parasite eggs associated with a robust Th17 cell response. We investigated the requisites for Th17 cell development using novel CD4 T cells expressing a transgenic TCR specific for the major Sm-p40 egg Ag, which produce IL-17 when stimulated with live schistosome eggs. Neutralization of IL-23 or blockade of the IL-1 receptor, but not IL-6 neutralization, abrogated egg-induced IL-17 secretion by transgenic T cells, whereas exogenous IL-23 or IL-1β reconstituted their ability to produce IL-17 when stimulated by syngeneic IL-12p40-deficient dendritic cells. Kinetic analysis demonstrated that IL-17 production was initiated by IL-23 and amplified by IL-1β. Significantly, schistosome-infected IL-12p40-deficient or IL-1R antagonist-treated CBA/J mice developed markedly reduced hepatic immunopathology with a dampened egg Ag-specific IL-17 response. These results demonstrate that the IL-23-IL-1-IL-17 axis has a central role in the development of severe schistosome egg-induced immunopathology.     

Deletion of IL-4Ralpha on CD4 T cells renders BALB/c mice resistant to Leishmania major infection

Effector responses induced by polarized CD4+ T helper 2 (Th2) cells drive nonhealing responses in BALB/c mice infected with Leishmania major. Th2 cytokines IL-4 and IL-13 are known susceptibility factors for L. major infection in BALB/c mice and induce their biological functions through a common receptor, the IL-4 receptor alpha chain (IL-4Ralpha). IL-4Ralpha-deficient BALB/c mice, however, remain susceptible to L. major infection, indicating that IL-4/IL-13 may induce protective responses. Therefore, the roles of polarized Th2 CD4+ T cells and IL-4/IL-13 responsiveness of non-CD4+ T cells in inducing non-healer or healer responses have yet to be elucidated. CD4+ T cell-specific IL-4Ralpha (Lck(cre)IL-4Ralpha(-/lox)) deficient BALB/c mice were generated and characterized to elucidate the importance of IL-4Ralpha signaling during cutaneous leishmaniasis in the absence of IL-4-responsive CD4+ T cells. Efficient deletion was confirmed by loss of IL-4Ralpha expression on CD4+ T cells and impaired IL-4-induced CD4+ T cell proliferation and Th2 differentiation. CD8+, gammadelta+, and NK-T cells expressed residual IL-4Ralpha, and representative non-T cell populations maintained IL-4/IL-13 responsiveness. In contrast to IL-4Ralpha(-/lox) BALB/c mice, which developed ulcerating lesions following infection with L. major, Lck(cre)IL-4Ralpha(-/lox) mice were resistant and showed protection to rechallenge, similar to healer C57BL/6 mice. Resistance to L. major in Lck(cre)IL-4Ralpha(-/lox) mice correlated with reduced numbers of IL-10-secreting cells and early IL-12p35 mRNA induction, leading to increased delayed type hypersensitivity responses, interferon-gamma production, and elevated ratios of inducible nitric oxide synthase mRNA/parasite, similar to C57BL/6 mice. These data demonstrate that abrogation of IL-4 signaling in CD4+ T cells is required to transform non-healer BALB/c mice to a healer phenotype. Furthermore, a beneficial role for IL-4Ralpha signaling in L. major infection is revealed in which IL-4/IL-13-responsive non-CD4+ T cells induce protective responses.     

Nose-Rings and Transmission of Helminth Parasites in Outdoor Pigs

Five growing pigs experimentally infected with low doses of Oesophagostomum dentation, Ascaris suum, and Trichuris suis were turned out with 5 helminth-naïve pigs on each of 3 pastures in June 1996 (Group 1). On one pasture all pigs received nose-rings. After slaughter of Group 1 in October, pasture infectivity was monitored using helminth-naïve, unringed tracer pigs. In 1997, helminth-naïve young pigs were turned out on the contaminated pastures in May (Group 2) and again in August (Group 3). Again all pigs on one pasture received nose-rings. All pigs and pastures were followed parasitologically and reduction in grass cover was monitored. Based on the acquisition of infection by the naïve pigs in Group 1, the estimated minimal embryonation times for eggs deposited on pasture were 23–25 days for O. dentatum, 5–6 weeks for A. suum and 9–10 weeks for T. suis. Results from tracer pigs and grass/soil samples indicated that pasture infectivity was light both years. Free-living stages of O. dentatum did not survive the winter. The nose-rings reduced rooting considerably, resulting in three-fold more grass cover on the nose-ring pasture compared to the control pastures by the end of the experiment. Nevertheless, the nose-rings did not significantly influence parasite transmission.     

The consequences of short-term grazing of bioactive forages on established adult and incoming larvae populations of Teladorsagia circumcincta in lambs

The objective of this study was to investigate the consequences of short-term grazing on bioactive forages on (i) the viability and fecundity of established adult Teladorsagia circumcincta population and (ii) the establishment and development of incoming T. circumcincta infective larvae. Forty-eight, parasite naive, 3-month old, grazing lambs were artificially infected with 8000 infective larvae of T. circumcincta on day 1 of the experiment. On day 21 p.i., lambs were allocated to one of three bioactive forage grazing treatments; chicory (Cichorium intybus), sulla (Hedysarum coronarium), lotus (Lotus pedunculatus), and the control grass/clover (Lolium perenne/Trifolium repens) forage. On day 28 of the experiment a second dose of 8000 T. circumcincta infective larvae was administered to the lambs to investigate the effects of forages on the ability of infective larvae to establish within the host. All animals were slaughtered for worm recovery on day 35, while liveweight gain, feacal egg counts (FEC) and total worm egg output were monitored regularly throughout the experiment. Although FEC or total egg output were similar among the groups, adult worm burdens at slaughter were significantly affected (P<0.05) by forage treatment during the 2 week grazing period. Lambs grazing chicory had the lowest adult worm burdens and significantly lower numbers of male worms compared to those grazing on grass/clover (P<0.01), while the lambs grazing on sulla or lotus had similar adult populations to grass/clover fed animals. The results from the worm recoveries of the second dose (immature worm burdens) were affected by physiologically and/or immunologically mediated mechanisms, which reduced larval establishment in all treatments. Nevertheless, immature worm burdens at slaughter were similar between chicory, sulla and grass/clover group, while the immature worm recoveries from the lotus group were significantly higher (P<0.05) compared to those from lambs grazing grass/clover. Overall, the results of the present study support the view that chicory can be a promising candidate species in pasture management practices to control T. circumcincta burdens.     

Experimental Infection of Danish Landrace/Yorkshire Crossbred Pigs with Schistosoma japonicum from the People’s Republic of China

This study was undertaken to assess the suitability of Danish Landrace/Yorkshire (L/Y) crossbred pigs as experimental hosts of a Chinese mainland strain of Schistosoma japonicum. Pigs were exposed to 200, 500 or 1000 cercariae and parasite burdens were determined by perfusion after either 8 or 11 weeks. All pigs became infected with onset of faecal egg excretion 6 to 7 weeks following exposure to cercariae. The pattern of faecal egg excretion differed markedly among the individual animals. Gross hepatic pathological lesions of varying degrees were noted in all of the pigs. Schistosome worm recoveries ranged from 1.5–23.4% of the cercarial exposure dose. Most schistosome eggs recovered from the tissues, expressed as eggs/g tissue, were found in the rectum (91%), caecum (3.1%) and liver (5.1%). The results show that Danish L/Y pigs may serve as appropriate experimental final hosts of the Chinese mainland strain of S. japonicum.     

IFN-γ plays an essential role in the pathogenesis of gastric lymphoid follicles formation caused by Helicobacter suis infection

In this study, we aimed to assess the role of helper T cells in the development of gastric lymphoid follicles induced by Helicobacter suis infection. C57BL/6J mice were orally inoculated with H. suis. Six weeks after infection, gastric lymphoid follicles were observed in the gastric mucosa by hematoxylin and eosin staining, and the number of follicles was increased throughout the infection period. An immunohistological examination showed that the lymphoid follicles were composed of B cells, CD4-positive helper T cells, and dendritic cells (DC). It was also revealed that the mRNA expression level of interferon-γ (IFN-γ) in the gastric mucosa was significantly increased at 12 weeks after infection. No gastric lymphoid follicles were detected in IFN-γ-deficient mice that had been infected with H. suis at 12 weeks after infection, although the development of lymphoid follicles in IL-4-deficient mice infected with H. suis was similar to that seen in the wild-type mice. In conclusion, IFN-γ, a Th1 cytokine, is deeply involved in the pathogenesis of gastric lymphoid follicles induced by H. suis infection, and it is suggested that CD4-positive T cells and DC aid in the expansion of gastric lymphoid follicles.     

Reproductive potential of Echinococcus multilocularis in experimentally infected foxes, dogs, raccoon dogs and cats

A total of 15 red foxes, 15 raccoon dogs, 15 domestic dogs and 15 domestic cats were each infected with 20,000 protoscolices of Echinococcus multilocularis. At 35, 63, and 90 days post inoculation (dpi), five animals from each group were necropsied and the worm burdens determined. The highest worm burdens in foxes (mean of 16,792) and raccoon dogs (mean of 7930) were found at 35 dpi. These declined to a mean of just 331 worms in foxes and 3213 worms in raccoon dogs by day 63 with a further decline to 134 worms in foxes and 67 worms in raccoon dogs by day 90. In dogs, there was no significant difference between worm burdens recovered at days 35 (mean of 2466) and day 90 (mean of 1563), although reduced numbers were recovered on day 63 (mean of 899). In cats, worms were found in four animals 35 dpi (mean of 642), in three at 63 dpi (mean of 28) and in two at 90 dpi (mean of 57). Faecal egg counts were determined at 3 day intervals from 25 dpi. A mathematical model of egg excretion dynamics suggested that the mean biotic potential per infected animal was high in foxes (346,473 eggs); raccoon dogs (335,361 eggs) and dogs (279,910 eggs) but very low for cats (573 eggs). It also indicated that approximately 114, 42 and 27 eggs per worm were excreted in the faeces of dogs, raccoon dogs and foxes, respectively. The fecundity of worms in cats was low with an average of less than one egg per worm. The peak levels of coproantigen were detected earlier in foxes and raccoon dogs than in dogs. Eggs recovered from foxes, raccoon dogs and dogs resulted in massive infections in experimental mice. However, metacestodes did not develop from eggs originating from infected cats. It is concluded that foxes, raccoon dogs and dogs are good hosts of E. multilocularis. In contrast, the low worm establishment, the very few excreted eggs and the lack of infectivity of eggs strongly indicate that cats play an insignificant role in parasite transmission.     

Memory-type CD8+ T cells protect IL-2 receptor alpha-deficient mice from systemic infection with herpes simplex virus type 2

IL-2Ralpha-deficient (IL-2Ralpha(-/-)) mice exhibit an impaired activation-induced cell death for T cells and develop abnormal T cell activation with age. In our study, we found that IL-2Ralpha(-/-) mice at the age of 5 wk contained an increased number of CD44(+)CD69(-)CD8(+) T cells in lymph nodes, which expressed a high intensity of IL-2Rbeta and vigorously proliferated in response to a high dose of IL-15 or IL-2. The T cells produced a large amount of IFN-gamma in response to IL-15 plus IL-12 in a TCR-independent bystander manner. When IL-2Ralpha(-/-) mice were inoculated i.p. with HSV type 2 (HSV-2) 186 strain, they showed resistance to the infection accompanied by an increased level of serum IL-15. The depletion of CD8(+) T cells by in vivo administration of anti-CD8 mAb rendered IL-2Ralpha(-/-) mice susceptible to HSV-2-induced lethality. These results suggest that memory-type CD8(+) T cells play a novel role in the protection against HSV-2 infection in IL-2Ralpha(-/-) mice.     

Type 1 and type 2 CD8+ effector T cell subpopulations promote long-term tumor immunity and protection to progressively growing tumor

Cytolytic CD8+ effector cells fall into two subpopulations based on cytokine secretion. Type 1 CD8+ T cells (Tc1) secrete IFN-gamma, whereas type 2 CD8+ T cells (Tc2) secrete IL-4, IL-5, and IL-10. Using an OVA-transfected B16 lung metastases model, we assessed the therapeutic effects of adoptively transferred OVA-specific Tc1 and Tc2 subpopulations in mice bearing established pulmonary malignancy. Effector cell-treated mice exhibiting high (5 x 105) tumor burdens experienced significant (p < 0.05) delays in mortality compared with those of untreated control mice, whereas high proportions (70-90%) of mice receiving therapy with low (1 x 105) tumor burdens survived indefinitely. Long-term tumor immunity was evident by resistance to lethal tumor rechallenge, heightened levels of systemic OVA Ag-specific CTL responses ex vivo, and detection of long-lived TCR transgene-positive donor cells accompanied by an elevation in the total numbers of CD8+ CD44high activated and/or memory T cells at sites of tumor growth. Long-lasting protection by Tc2 and Tc1 effector cells were dependent, in part, on both the level of tumor burden and effector cell-derived IL-4, IL-5, and IFN-gamma, respectively. We conclude that Tc1 and Tc2 effector cells provide immunity by different mechanisms that subsequently potentiate host-derived antitumor responses.     

Murine immune responses to a novel schistosome egg antigen, SmEP25

Pathology in schistosomiasis consists of granuloma formation around parasite eggs. There is considerable variation in the severity of disease in individuals with schistosomiasis, which may result from differential responses to egg antigens. The egg-induced immunopathology is mediated by CD4+ T helper cells sensitised to egg antigens. In this study, cellular responses to a 25-kDa fraction of egg proteins identified a novel T-cell antigen, SmEP25. The native SmEP25 elicited significant proliferative responses as well as gamma interferon (IFN-gamma), IL-2, IL-4, and IL-5 secretion in CD4+ cells from 8.5-week infected CBA and C57BL/6 mice. In C57BL/6 mice, proliferative responses to SmEP25 were relatively stronger than those directed against the major egg antigen Sm-p40, whereas in CBA mice the reverse was found. SmEP25 elicited stronger Th2 type response than Sm-p40 in both mouse strains. By comparison, recombinant SmEP25 elicited a smaller, Th1-polarised response, with significant IFN-gamma, low levels of IL-5 and essentially no IL-4. B-cell responses to SmEP25 coincided with the start of parasite egg production and SmEP25 protein was restricted to parasite eggs. The systematic identification of T-cell-sensitising egg components will lead to a better understanding of the processes involved in granuloma formation.     

Down-regulation of IL-7Ralpha expression in human T cells via DNA methylation

IL-7 is critical for the development and survival of T cells. Recently, we found two subsets of human CD8+ T cells expressing IL-7Ralpha(high) and IL-7Ralpha(low) with different cell survival responses to IL-7. Although these CD8+ T cell subsets have differential IL-7Ralpha gene expression, the mechanism for this is unknown. DNA methylation is an important gene regulatory mechanism and is associated with the inactivation of gene expression. Thus, we investigated a role for DNA methylation in differentially regulating IL-7Ralpha gene expression in human CD8+ T cells and Jurkat T cells. IL-7Ralpha(high)CD8+ T cells had decreased methylation in the IL-7Ralpha gene promoter compared with IL-7Ralpha(low)CD8+ T cells and Jurkat T cells with low levels of IL-7Ralpha. Treating Jurkat T cells with 5-aza-2'-deoxycytidine, which reduced DNA methylation, increased IL-7Ralpha expression. Plus, the unmethylated IL-7Ralpha gene promoter construct had higher levels of promoter activity than the methylated one as measured by a luciferase reporter assay. These findings suggest that DNA methylation is involved in regulating IL-7Ralpha expression in T cells via affecting IL-7Ralpha gene promoter activity, and that the methylation of this gene promoter could be a potential target for modifying IL-7-mediated T cell development and survival.