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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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Szabo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The Drosophila lymph gland, the larval hematopoietic organ comprised of prohemocytes and mature hemocytes, has been a valuable model for understanding mechanisms underlying hematopoiesis and immunity. Three types of mature hemocytes have been characterized in the lymph gland: plasmatocytes, lamellocytes, and crystal cells, which are analogous to vertebrate myeloid cells, yet molecular underpinnings of the lymph gland hemocytes have been less investigated. Here, we use single-cell RNA sequencing to comprehensively analyze heterogeneity of developing hemocytes in the lymph gland, and discover previously undescribed hemocyte types including adipohemocytes, stem-like prohemocytes, and intermediate prohemocytes. Additionally, we identify the developmental trajectory of hemocytes during normal development as well as the emergence of the lamellocyte lineage following active cellular immunity caused by wasp infestation. Finally, we establish similarities and differences between embryonically derived- and larval lymph gland hemocytes. Altogether, our study provides detailed insights into the hemocyte development and cellular immune responses at single-cell resolution.

We performed gross and histological examinations of the livers of sika deer (Cervus nippon yesoensis) in Hokkaido, Japan. Out of 1,381 deer slaughtered for venison production, thickening and dilation of the large intrahepatic bile ducts and Fasciola flukes in the duct lumens were detected in 621 deer (45.0%). Furthermore, 107 non-bile lesions (75 intrahepatic and 32 capsular lesions) were detected during gross examinations. Histologically, the bile duct lesions included chronic proliferative cholangitis, papillary hyperplasia, goblet cell and pyloric gland metaplasia, and periductal fibrosis. Many of the intrahepatic non-bile duct lesions (53/75, 71%) were considered to be Fasciola fluke migration-associated lesions, including two lesion types: necrosis, hemorrhage, and eosinophilic granuloma formation (29 lesions), and lymphoid tissue formation (24 lesions). Lymphoid tissue formation was considered to result from the persistent immune responses against dead Fasciola flukes. An epidermoid liver cyst was found incidentally, which has not been reported in the veterinary literature. In summary, this study demonstrated the predominance of fascioliasis-associated lesions in sika deer livers. The gross and histological lesions caused by Fasciola flukes in sika deer were similar to fascioliasis in other animals. Moreover, we described lymphoid tissue formation as a fascioliasis-associated lesion for the first time. The fact that bile duct lesions (45.0%) had a markedly higher prevalence than fascioliasis-associated parenchymal lesions (53/1,381, 3.8%) indicated that sika deer are a permissive host for fascioliasis. Our results provide information that will aid pathological examinations of sika deer.

We characterized the histologic effects of two stressors (heat and coccidial infection) alone or in combination on bursa of Fabricius, thymus, and spleen in broiler chickens. Four hundred and eighty Cobb500 male chicks at 14 days of age were randomly assigned to two treatments in a 2×2 factorial design, with 15 replicates per treatment and eight birds per replicate. The treatment factors were temperature (25 and 35 C) and a mixed culture of 2.5 × 10 5 sporulated Eimeria acervulina and Eimeria maxima oocysts (infection or no infection). Histologic lesion severity was scored in these tissues at different ages. At 21 and 28 days of age, bursal and thymic tissues from birds raised at 35 C exhibited significant increases in lymphoid depletion severity compared with those raised at 25 C. No significant differences were detected in the lymphoid depletion severity of birds infected with Eimeria when compared with uninfected birds. These results indicate that continuous exposure to heat stress-inducing temperatures results in lymphoid depletion of the bursa and thymus in broiler chickens, a potential histologic marker for the immunologic changes known to arise as a result of heat stress. Bursal and thymic atrophy are thought to contribute to immunologic changes that underlie the negative effects of heat stress on poultry production characteristics.

Visceral leishmaniasis (VL) is a disease caused by Leishmania infantum, which is transmitted by phlebotomine sandflies. Dogs are the main urban reservoir of this parasite and the disease presents similar characteristics in both humans and dogs. In this paper, we investigated the potential pathways involved in plasma cell replacement of normal cell populations in the spleen, with respect to disease severity in dogs from an endemic area for visceral leishmaniasis. To this end, canine spleen samples were grouped into three categories: TYPE1SC- (non-infected dogs or without active infection with organized white pulp), TYPE1SC+ (infected dogs with organized white pulp) or TYPE3SC+ (infected animals with disorganized white pulp). We analyzed the distribution of different plasma cell isotypes (IgA, IgG and IgM) in the spleen. The expression of cytokines and chemokines involved in plasma cell homing and survival were assessed by real time RT-PCR. Polyclonal B cell activation and hypergammaglobulinemia were also evaluated. The proportion of animals with moderate or intense plasmacytosis was higher in the TYPE3SC+ group than in the other groups (Fisher test, P<0.05). This was mainly due to a higher density of IgG+ plasma cells in the red pulp of this group. The albumin/globulin ratio was lower in the TYPE3SC+ animals than in the TYPE1SC- or TYPE1SC+ animals, which evidences VL-associated dysproteinemia. Interestingly, TYPE3SC+ animals showed increased expression of the BAFF and APRIL cytokines, as well as chemokine CXCL12. Aberrant expression of BAFF, APRIL and CXCL12, together with amplified extrafollicular B cell activation, lead to plasma cell homing and the extended survival of these cells in the splenic red pulp compartment. These changes in the distribution of immunocompetent cells in the spleen may contribute to the progression of VL, and impair the spleen's ability to protect against blood borne pathogens.

The Leishmania granuloma shares some, though not all, properties with that formed following mycobacterial infection. As a simplified, noncaseating granuloma composed of relatively few and largely mononuclear cell populations, it provides a tractable model system to investigate intra-granuloma cellular dynamics, immune regulation, and antimicrobial resistance. Here, the occurrence of granulomatous pathology across the spectrum of leishmaniasis, in humans and animal reservoir hosts, is first described. However, this review focuses on the process of hepatic granuloma formation as studied in rodent models of visceral leishmaniasis, starting from the initial infection of Kupffer cells to the involution of the granuloma after pathogen clearance. It describes how the application of intravital imaging and the use of computational modeling have changed some of our thoughts on granuloma function, and illustrates how host-directed therapies have been used to manipulate granuloma form and function for therapeutic benefit. Where appropriate, lessons that may be equally applicable across the spectrum of granulomatous diseases are highlighted.

Wolbachia, an endosymbiont of filarial nematode, is considered a promising target for treatment of lymphatic filariasis. Although functional characterization of the Wolbachia peptidoglycan assembly has not been fully explored, the Wolbachia genome provides evidence for coding all of the genes involved in lipid II biosynthesis, a part of peptidoglycan biosynthesis pathway. UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) is one of the lipid II biosynthesis pathway enzymes and it has inevitably been recognized as an antibiotic target. In view of the vital role of MurA in bacterial viability and survival, MurA ortholog from Wolbachia endosymbiont of Brugia malayi (wBm-MurA) was cloned, expressed and purified for further molecular characterization. The enzyme kinetics and inhibition studies were undertaken using fosfomycin. wBm-MurA was found to be expressed in all the major life stages of B. malayi and was immunolocalized in Wolbachia within the microfilariae and female adults by the confocal microscopy. Sequence analysis suggests that the amino acids crucial for enzymatic activity are conserved. The purified wBm-MurA was shown to possess the EPSP synthase (3-phosphoshikimate 1-carboxyvinyltransferase) like activity at a broad pH range with optimal activity at pH 7.5 and 37°C temperature. The apparent affinity constant (Km) for the substrate UDP-N-acetylglucosamine was found to be 0.03149 mM and for phosphoenolpyruvate 0.009198 mM. The relative enzymatic activity was inhibited ∼2 fold in presence of fosfomycin. Superimposition of the wBm-MurA homology model with the structural model of Haemophilus influenzae (Hi-MurA) suggests binding of fosfomycin at the same active site. The findings suggest wBm-MurA to be a putative antifilarial drug target for screening of novel compounds.

Organ-specific immunity is a feature of many infectious diseases, including visceral leishmaniasis caused by Leishmania donovani. Experimental visceral leishmaniasis in genetically susceptible mice is characterized by an acute, resolving infection in the liver and chronic infection in the spleen. CD4+ T cell responses are critical for the establishment and maintenance of hepatic immunity in this disease model, but their role in chronically infected spleens remains unclear. In this study, we show that dendritic cells are critical for CD4+ T cell activation and expansion in all tissue sites examined. We found that FTY720-mediated blockade of T cell trafficking early in infection prevented Ag-specific CD4+ T cells from appearing in lymph nodes, but not the spleen and liver, suggesting that early CD4+ T cell priming does not occur in liver-draining lymph nodes. Extended treatment with FTY720 over the first month of infection increased parasite burdens, although this associated with blockade of lymphocyte egress from secondary lymphoid tissue, as well as with more generalized splenic lymphopenia. Importantly, we demonstrate that CD4+ T cells are required for the establishment and maintenance of antiparasitic immunity in the liver, as well as for immune surveillance and suppression of parasite outgrowth in chronically infected spleens. Finally, although early CD4+ T cell priming appeared to occur most effectively in the spleen, we unexpectedly revealed that protective CD4+ T cell-mediated hepatic immunity could be generated in the complete absence of all secondary lymphoid tissues.

In vertebrate animals, pleural and peritoneal cavities are repositories of milky spots (MS), which constitute an organised coelom-associated lymphomyeloid tissue that is intensively activated by Schistosoma mansoni infection. This study compared the reactive patterns of peritoneal MS to pleural MS and concluded from histological analysis that they represent independent responsive compartments. Whole omentum, lungs and the entire mediastinum of 54 S. mansoni-infected mice were studied morphologically. The omental MS of infected animals were highly activated, modulating from myeloid-lymphocytic (60 days of infection) to lymphomyeloid (90 days of infection) and lymphocytic or lymphoplasmacytic (160 days of infection) types. The non-lymphoid component predominated in the acute phase of infection and was expressed by monocytopoietic, eosinopoietic and neutropoietic foci, with isolated megakaryocytes and small foci of late normoblasts and mast cells. Nevertheless, pleural or thoracic MS of infected mice were monotonous, consisting of small and medium lymphocytes with few mast and plasma cells and no myeloid component. Our data indicate that compartmentalisation of the MS response is dependent on the lymphatic vascularisation of each coelomic cavity, limiting the effects or consequences of any stimulating or aggressive agents, as is the case with S. mansoni infection.

Critical events of HIV-1 pathogenesis occur in lymphoid tissues where HIV-1 is typically accompanied by infections with other pathogens (HIV co-pathogens). Co-pathogens greatly affect the clinical course of the disease and the transmission of HIV. The apicomplexan parasite Toxoplasma gondii is a common HIV co-pathogen associated with AIDS development. Here, we examined the interaction of T. gondii and HIV in coinfected human lymphoid tissue ex vivo. Both pathogens readily replicate in ex vivo infected blocks of human tonsillar tissue. Surprisingly, we found that live T. gondii preferentially inhibits R5 HIV-1 replication in coinfected tissues. This effect is reproduced by treatment of the tissue blocks with recombinant C-18, a T. gondii-encoded cyclophilin that binds to CCR5. These ex vivo findings raise the possibility that, in addition to being a co-factor in HIV disease, T. gondii may influence the outcome of viral infection by preferentially suppressing R5 variants.

An antiserum against Eimeria tenella sporozoites was used to localize and isolate Ag-binding cells in intestinal cecal tonsils of parasite-infected chickens. Based on their tissue localization, ultrastructural features, and expression of surface markers, two subpopulations of cells were isolated, CD45(+) interdigitating dendritic cells (IDCs) and CD45(-) follicular dendritic cells (FDCs). IDCs expressed MHC class I, MHC class II, and selectin, induced the proliferation of allogeneic naïve CD4(+) T cells, and increased the secretion of IFN-gamma by autologous T cells. FDCs expressed surface IgG, IgM, ICAM-1, and VCAM-1, stimulated the proliferation of LPS-treated allogeneic B cells, and augmented the secretion of IgG by LPS-treated autologous B cells. Final cell yields were 6 x 10(5) to 8 x 10(5) cells per chicken with >95% purity. In summary, this combination of methods using Abs against E. tenella and CD45 made it possible for the first time to obtain a highly enriched IDCs and FDCs which are functionally active in chickens. This novel method will enable the detailed biochemical and immunological characterizations of avian dendritic cells and facilitate the investigation of their role in initiating immune response in normal and disease states.

A single inoculation of mice with the live, attenuated Toxoplasma gondii uracil auxotroph strain cps1-1 induces long-lasting immunity against lethal challenge with hypervirulent strain RH. The mechanism for this robust immunity in the absence of parasite replication has not been addressed. The mechanism of long-lasting immunity, the importance of route of immunization, cellular recruitment to the site of infection, and local and systemic inflammation were evaluated. Our results show that infection with cps1-1 elicits long-lasting CD8+ T cell- mediated immunity. We show that immunization with cps1-1-infected dendritic cells elicits long-lasting immunity. Intraperitoneal infection with cps1-1 induced a rapid influx of GR1+ neutrophils and two stages of GR1+CD68+ inflammatory monocyte infiltration into the site of inoculation. CD19+ B cells and CD3+ T cells steadily increase for 8 days after infection. CD8+ T cells were rapidly recruited to the site of infection and increased faster than CD4+ T cells. Surprisingly, cps1-1 infection induced high systemic levels of bioactive IL-12p70 and a very low level and transient systemic IFN-gamma. Furthermore, we show significant levels of these inflammatory cytokines were locally produced at the site of cps1-1 inoculation. These findings offer new insight into immunological mechanisms and local host responses to a non-replicating type I parasite infection associated with development of long-lasting immunity to Toxoplasma gondii.

Immunity in the gastrointestinal tract is important for resistance to many pathogens, but the memory T cells that mediate such immunity are poorly characterized. In this study, we show that following sterile cure of a primary infection with the gastrointestinal parasite Trichuris muris, memory CD4+ T cells persist in the draining mesenteric lymph node and protect mice against reinfection. The memory CD4+ T cells that developed were a heterogeneous population, consisting of both CD62L(high) central memory T cells (T(CM)) and CD62L(low) effector memory T cells (T(EM)) that were competent to produce the Th type 2 effector cytokine, IL-4. Unlike memory T cells that develop following exposure to several other pathogens, both CD4+ T(CM) and T(EM) populations persisted in the absence of chronic infection, and, critically, both populations were able to transfer protective immunity to naive recipients. CD62L(high)CD4+ T(CM) were not apparent early after infection, but emerged following clearance of primary infection, suggesting that they may be derived from CD4+ T(EM). Consistent with this theory, transfer of CD62L(low)CD4+ T(EM) into naive recipients resulted in the development of a population of protective CD62L(high)CD4+ T(CM). Taken together, these studies show that distinct subsets of memory CD4+ T cells develop after infection with Trichuris, persist in the GALT, and mediate protective immunity to rechallenge.

TLRs expressed by a variety of cells, including epithelial cells, B cells, and dendritic cells, are important initiators of the immune response following stimulation with various microbial products. Several of the TLRs require the adaptor protein, MyD88, which is an important mediator for the immune response following Toxoplasma gondii infection. Previously, TLR9-mediated innate immune responses were predominantly associated with ligation of unmethylated bacterial CpG DNA. In this study, we show that TLR9 is required for the Th1-type inflammatory response that ensues following oral infection with T. gondii. After oral infection with T. gondii, susceptible wild-type (WT; C57BL/6) but not TLR9(-/-) (B6 background) mice develop a Th1-dependent acute lethal ileitis; TLR9(-/-) mice have higher parasite burdens than control WT mice, consistent with depressed IFN-gamma-dependent parasite killing. A reduction in the total T cell and IFN-gamma-producing T cell frequencies was observed in the lamina propria of the TLR9(-/-) parasite-infected mice. TLR9 and type I IFN production was observed by cells from infected intestines in WT mice. TLR9 expression by dendritic cell populations is essential for their expansion in the mesenteric lymph nodes of infected mice. Infection of chimeric mice deleted of TLR9 in either the hemopoietic or nonhemopoietic compartments demonstrated that TLR9 expression by cells from both compartments is important for efficient T cell responses to oral infection. These observations demonstrate that TLR9 mediates the innate response to oral parasite infection and is involved in the development of an effective Th1-type immune response.

A 65-year old Korean man, living in Mokpo-city, Jeollanam-do, Republic of Korea, visited a local clinic complaining of right upper quadrant pain and indigestion. At colonoscopy, he was diagnosed as having a carcinoma of the ascending colon, and thus, a palliative right hemicolectomy was performed. Subsequently, an adult fluke of Gymnophalloides seoi was incidentally found in a surgical pathology specimen of the lymph node around the colon. The worm was found to have invaded gut lymphoid tissue, with characteristic morphologies of a large oral sucker, a small ventral sucker, and a ventral pit surrounded by strong muscle fibers. This is the first reported case of mucosal tissue invasion by G. seoi in the human intestinal tract.

Oral infection with the nematode parasite Heligmosomoides polygyrus H. polygyrus is entirely restricted to the small intestine. Although the evoked Th2 response has been extensively studied in secondary lymphoid organs, little is known about the systemic dissemination of Th2 cells or type 2 associated eosinophils and basophils. In this study we use bicistronic 4get IL-4 reporter mice to directly visualize the type 2 response to H. polygyrus infection. We observed that CD4(+)/GFP(+) Th2 cells spread systemically and found that these cells accumulated in nonlymphoid "hot spots" in the liver, the lung airways, and the peritoneal cavity. Interestingly, the total number of Th2 cells in the peritoneal cavity was comparable to those found in the draining mesenteric lymph node or the spleen. Peritoneal Th2 cells were distinguished by an exceptionally low apoptotic potential and high expression of the intestinal homing receptor alpha(4)beta(7) integrin. CD4(+)/GFP(+) Th2 cells from these peripheral sites were fully functional as indicated by rapid IL-4 production upon polyclonal or Ag-specific restimulation. Th2 cells persisted in the intestinal tissue and the peritoneal cavity of drug-cured mice for weeks. The presence of peripheral memory Th2 cells in the intestine might be crucial for immunity to recall infections. These findings have important implications for the design of vaccination strategies because it may be necessary to establish and maintain memory CD4(+) T cells at the potential future site of infection.

The semi-nested PCR was conducted for detection of Wuchereria bancrofti in patients' blood. The primers were designed based on the repetitive DNA sequences of the parasite. The results demonstrated that the semi-nested PCR could detect as little as 0.001 fg of parasite DNA. In addition, the primers showed no PCR amplification from human and other hemoparasites such as Brugia malayi, Plasmodium falciparum and P. vivax DNAs. This technique was used for detection of 18 W. bancrofti infected blood samples with a long-term storage, the data revealed that all samples were positive. The results obtained from this study clearly indicated that the semi-nested PCR is specific, sensitive, and suitable for detection of the disease carriers.

Cricket haemocytes were derived from either haemolymph or haemopoietic organs (lymph glands) of insects and introduced to a primary culture. Varied isolation protocols, tissue culture vessels, media compositions and cell densities were tested to determine the optimal conditions for in vitro maintenance of haemocytes, and for subsequent light and electron microscopic analysis of monolayers. Freshly prepared Mitsuhashi and Maramorosh (MM;Sigma, Steinheim, Germany) insect medium (420 mOsm), buffered with sodium bicarbonate (pH 7.2) and supplemented with 10 % FCS, was found to be most appropriate for haemocyte maintenance. All tested tissue culture vessels (FLEXiperm units, multiwell plates and Thermanox slides, with the exception of Melineux agar plates), were suitable for cell attachment and haemocyte monolayers formation. Viability of cultured cells was confirmed by LIVE/DEAD Viability/Cytotoxity Kit for Eukaryotic Cells. Free circulating haemocytes were cultivated up to 27 days and then degraded. Infection with the microsporidian Paranosema grylli or the coccidian Adelina grylli caused noticeable swelling of host lymph glands (haemopoietic tissue) and increase in the number of cells comprising the glands. The cells derived from haemopoietic tissue were maintained for maximum 5 days; thereafter multiplication of bacteria normally inhabiting cricket lymph glands destroyed monolayers and killed the cells. Microsporidian and coccidian invasive stages (spores and sporozoites, respectively) were isolated from infected tissues, resuspended in MM medium and added to haemocyte monolayers in ratios 1 zoite per haemocytes or 10 spores per 1 haemocyte. Actively moving zoites contacted and penetrated the cultured cells. Unlike coccidian zoites, microsporidian spores were phagocytized by haemocytes. Application of fluorescent LIVE/DEAD kit allowed to visualize internalized parasites inside host cells as clearly shaped dark areas. The present study has demonstrated that 1) cricket haemocytes from both circulating haemolymph and lymph glands can be short-term cultivated on tissue culture vessel surfaces which made possible their further light and electron microscopic analysis; 2) short-term haemocyte cultures may be employed to study host-parasite interactions, in particular, to follow the initial steps of parasite internalization inside host cell; 3) Fluorescent assay with Viability/Cytotoxity Kit for Eukaryotic Cells (Molecular Probes, Oregon) allows to observe penetration of these parasites into cultured cells.

To compare the pathogenesis of human genotype 1 (HuG1) and bovine genotype 2 (BoG2) Cryptosporidium parvum, neonatal gnotobiotic pigs were given 1-10 HuG1 or BoG2 oocysts. The prepatent and patent periods were significantly longer for HuG1 than for BoG2 C. parvum (prepatent, 8.6 vs. 5.6 days; patent, 16.6 vs. 10.3 days). BoG2-infected pigs developed significantly more severe disease than did HuG1-infected pigs. BoG2 parasites were seen microscopically throughout the intestines during the prepatent and patent periods. HuG1 parasites were only detected during the patent period in the ileum and colon but colonized the mucosal surface in significantly larger numbers than did BoG2. Moderate-to-severe villus/mucosal attenuation with lymphoid hyperplasia was seen throughout the intestines of BoG2-infected pigs, whereas lesions in HuG1-infected pigs were mild to moderate and restricted to the ileum and colon. These findings provide additional support for the hypothesis that human and bovine C. parvum genotypes may be separate species.

The immunomodulatory role of neutrophils during infection with Toxoplasma gondii was investigated. Monoclonal antibody-mediated depletion revealed that neutrophils are essential for survival during the first few days of infection. Moreover, neutrophil depletion was associated with a weaker type 1 immune response as measured by decreased levels of gamma interferon, interleukin-12 (IL-12) and tumor necrosis factor alpha. IL-10 was also decreased in depleted animals. Additionally, splenic populations of CD4(+) T cells, CD8(+) T cells, and NK1.1(+) cells were decreased in depleted mice. Neutrophil-depleted mice exhibited lesions of greater severity in tissues examined and a greater parasite burden as determined by histopathology and reverse transcription-PCR. We conclude that neutrophils are critical near the time of infection because they influence the character of the immune response and control tachyzoite replication.

The study aimed to characterize T cell responses in calves to a primary E. bovis infection. For this purpose, peripheral blood lymphocytes (PBL) were isolated from six infected calves and three controls during prepatency (Day 12 post infection (p.i.), patency (Day 25 p.i.) and postpatency (Day 35 p.i.). In addition, lymphocytes were isolated from various lymphatic organs (lnn. cervicales superficiales, lnn. jejunales craniales, lnn. jejunales caudales, lnn. caecales, lnn. colici, Peyer's patches (PP) and spleen) at necropsy (Day 35 p.i.). FACS analyses determined the proportions of CD4+-, CD8+-, CD2+-, and gammadelta+-T cells. Proliferative responses of the cells after stimulation with Concanavalin A (Con A) and an E. bovis-merozoite I antigen (EbAg) were measured. Furthermore, in situ hybridization experiments were performed for the detection of IL-2 and IL-4 mRNA in histological sections of lymphatic organs. Proportions of CD4+-, CD8+- and CD2+-expressing PBL were significantly increased 12 days p.i. in infected calves. While the proportions of CD4+- and CD8+-PBL declined until day 25 p.i. and finally reached control values, proportions of activated PBL (CD2+-T cells) remained at a high level throughout the observation period. Those of gammadelta+-PBL, in contrast, remained unaffected. The proportions of CD4+-, gammadelta+- and CD2+-T cells in lymphatic organs were significantly increased in comparison to uninfected controls, when determined 35 days p.i. Concerning the proportions of CD8+-T cells of the organs, however, there were no differences between the groups. PBL and cells from lymphatic organs except those from the PP showed strong proliferative response to the mitogen Con A, without a significant difference between the groups. Reactions to EbAg in contrast differed significantly between controls and E. bovis infected calves. Proliferation responses of PBL of infected animals were highest 12 days p.i.; subsequently they decreased and 35 days p.i. they were found within the ranges of controls. Lymphocytes isolated from lymphatic organs of infected animals reacted significantly stronger than lymphocytes from control animals, whereby most marked differences occured with cells from lymph nodes draining E. bovis infested parts of the intestine and from the spleen. These reactions were accompained by an increased transcription of the IL-2 gene but not of the IL-4 gene in gut associated lymphnodes of infected calves when compared with infected controls. The data suggest strong antigenic stimuli by developing first generation schizonts, and of predominant involvement of (CD4+) Th1 cells in the course of a primary E. bovis infection of calves.

Two aspects of host-parasite relationships that seem worthy of more attention are: (a) the distribution of parasites among host organs in the early course of infection, and (b) the dynamics of host lymphocyte tissue localization and recirculation during the course of infection. We have employed the derivatized aminostyrylpyridinium dye, [125I] I 2P-Di-6-ASP, to provide a relatively stable tag on both a parasite, Trypanosoma musculi, and on host mouse splenocytes, enriched B and T lymphocytes, and natural killer cells. The organ distribution of the parasites, splenocytes, and lymphocytes in recipient, host mice was tracked. Radiolabeled T. musculi localized primarily in the liver with lesser numbers in spleen, lungs, and kidneys. Per unit wet weight, the spleen accumulated parasites most efficiently. When T. musculi were inoculated intraperitoneally, most of them remained in the peritoneal space and the numbers that gained access to liver, lungs, and spleen were significantly smaller than in mice inoculated intravenously. The acquisition of parasites by the spleen (and lungs) of mice with an existing T. musculi infection was markedly inhibited. This was true also of syngeneic splenocytes and lymphocytes. In addition, lymphocytes from infected mice were significantly less likely to take residence in the spleens of normal recipient mice and were especially unlikely to localize in the spleens of infected recipients. These and other findings suggested that the inability of circulating lymphocytes to gain access to lymphoid tissues in infected mice, coupled with the poor ability of those tissues to sequester parasite antigens, could account for the known prolonged delay in the development of curative antibody response characteristic of T. musculi-infected mice. It is likely that the marked disruption of lymphoid tissue histoarchitecture that is typical of T. musculi infection contributes significantly to the failure of the tissues to sequester parasites and lymphocytes. Because lymphoid tissue disruption is seen in many parasitic infections, the findings reported here may have fairly broad relevance. In any case, the procedure described here for labeling parasites and lymphocytes should be of general utility for tracking their disposition in vivo.

Resistance of adult C57BL/6 mice to severe Cryptosporidium parvum infection is dependent on CD4+alpha beta+ TCR lymphocytes. In this study, we demonstrated that treatment with anti-IFN-gamma mAb extended oocyst excretion 18 days longer, and anti-IL-4 mAb extended oocyst excretion at least 11 days longer than isotype control mAb treatment. Analysis of the specific activity of anti-IFN-gamma mAb present in treated mouse sera suggested that IFN-gamma may have a limited role in the resolution phase of infection. Changes were also documented in numbers of CD4+alpha beta+IFN-gamma+ and CD4+alpha beta+IL-4+ lymphocytes in Peyer's patches and intraepithelium of adult C57BL/6 mice during resolution of C. parvum infection. Resistance to initial severe infection was associated with CD4+alpha beta+IFN-gamma+ lymphocytes, and eventual resolution of infection was associated with CD4+alpha beta+IL-4+ lymphocytes. Analysis of cytokine expression following in vitro stimulation with C. parvum Ags during resolution of infection demonstrated consistent increases in CD4+alpha beta+IL-4+ lymphocytes, but not CD4+alpha beta+IFN-gamma+ lymphocytes. The relevance of CD4+alpha beta+IL-4+ lymphocytes in protection against C. parvum was then evaluated in C57BL/6 IL-4 gene knockout mice (IL-4(-/-)). Adult IL-4(-/-) mice excreted oocysts in feces approximately 23 days longer than IL-4(+/+) mice. Further, anti-IFN-gamma mAb treatment increased the severity and the duration of infection in IL-4(-/-) mice compared with those in IL-4(+/+) mice. Together, the data demonstrated that IFN-gamma was important in the control of severity of infection, and either IFN-gamma or IL-4 accelerated termination of infection. However, neither IL-4 nor IFN-gamma was required for the final clearance of infection from the intestinal tract of adult mice.

Superior cervical ganglia (SCG) from neonatal mice were cultured with adult murine lymphoid tissue explants in Matrigel (Collaborative Biomedical, Bedford, MA). After 1 and 2 days in culture, many neurites grew toward thymus and spleen. Normal mesenteric lymph node (MLN) induced a smaller effect; however, activated MLN (isolated from mice 10 days after infection with Nippostrongylus brasiliensis; Nb-MLN-10d) caused significantly increased neurite outgrowth. To determine the roles of nerve growth factor (NGF) and cytokines in the promotion of neuritogenesis by lymphoid tissues, anti-NGF and various anti-cytokines were added to cocultures. Anti-NGF inhibited most of the neurite outgrowth toward thymus and spleen but only partially that toward Nb-MLN-10d. Anti-mouse IL-1 beta also significantly reduced the number of neurites growing toward thymus, spleen, and normal MLN. The number of neurites growing toward Nb-MLN-10d was significantly reduced by anti-IL-1 beta, anti-IL-3, anti-IL-6, or anti-GM-CSF. Exogenous IL-1 beta and IL-3 caused neurite outgrowth in single SCG cultures; and the IL-1 beta-, but not the IL-3-, mediated effect was completely blocked by anti-NGF. In one-day thymus/SCG cocultures, endogenous IL-1 was not detectable at concentrations sufficient to cause nerve growth; however, ample NGF was present in the thymic tissues and culture supernatants, but not in SCG. These data suggest that IL-1 mediates NGF production in lymphoid tissues, which in turn induces the growth of sympathetic nerves. Moreover, IL-3, IL-6, or GM-CSF produced during inflammation might also play important roles in the stimulation of nerve growth in vivo.

Coccidia-free rabbits were inoculated with different doses of a pure strain of Eimeria coecicola and samples of gut were taken at 80, 96, 112, 128, 144, and 160 h postinoculation. The use of a very low infective dose (2-20 oocysts) was sufficient to study the last merogony. The number of merozoites in meronts increased when the infective dose decreased. Only the first merogony of this coccidium in lymphocytes or M-cells of gut-associated lymphoid tissue (GALT) has previously been described. Three other generations of meronts are described herein. All these endogenous stages were observed in the epithelium of the vermiform appendix, sacculus rotundus, and Peyer's patches, especially at the bases of the domes. However, in heavily infected tissues the gamonts were seen throughout the epithelium of the GALT. The third- and fourth-generation meronts were of two types. As in other eimerian species of the rabbit, type A meronts produced thick polynucleated merozoites, whereas type B meronts gave rise to large numbers of thin merozoites with one nucleus. Microgamonts were polynucleated and less numerous than macrogamonts. Type A meronts were also polynucleated and less numerous at the end of the merogony. Therefore, types A and B could correspond to a sexual phenotype differentiation occuring during the two asexual phases of multiplication.

This study examined the relationship of IL-4, IL-10 and IFN-gamma with regard to the local granuloma (GR) and draining lymph node (LN) response to Schistosoma mansoni eggs. Synchronized GR were induced in naive and schistosome-infected mice at the vigorous (8 weeks) and late chronic (20 weeks) stages. In LN cultures, IL-10 and IFN production peaked on day 4 and was greatest for 8 week-infected mice. All GR cultures contained IFN, but compared with naive mice IL-10 production was accelerated at 8 weeks and abrogated at 20 weeks, consistent with expansion and abatement of Th2 activity. Cytokine neutralization was performed in egg-challenged, naive mice that were adoptively sensitized with lymphoid cells from 8 week-infected donors. GR size, GR macrophage tumour necrosis factor (TNF) production and egg antigen-elicited IL-2, IL-4, IL-5, IL-10 and IFN were examined on day 4 of GR formation. Anti-IFN augmented GR area by 40%, increased local IL-4 and IL-10, but decreased IFN and TNF production. In corresponding LN cultures, IFN decreased by about 50%, while IL-2, IL-4, IL-10 and IL-5 increased by nearly two-, four-, five- and six-fold, respectively. Anti-IL-10 did not affect GR size or GR cytokines, but abrogated GR area by 40%, along with a reduction in local IL-4 and TNF production. In LN, IL-4 depletion reduced IL-4 and IL-5 by 60-70% and increased IFN levels. These results support the notion of a cross-regulatory network in which IFN inhibits Th2 and IL-10 inhibits Th1 cells. IL-4 fosters Th2 cells differentiation in LN, but also performs a critical recruitment function in the eosinophil-rich schistosome egg-induced GR, whereas IFN contributes to enhanced GR macrophage function.

The purpose of this study was to begin to define the changes in the local lymphoid tissues that accompany Ostertagia ostertagi infection in naive and immunized calves. Abomasal lymph nodes were taken from calves beginning as early as 2 days post-infection. Phenotypic changes in the resulting lymphocyte populations were assessed by flow cytometry utilizing monoclonal antibodies specific for the cell surface determinates CD2, CD4, and CD8. Changes in antigen specificity were determined by limiting dilution analysis utilizing antigen derived from fourth-stage O. ostertagi. Primary infection of naive calves caused a rapid 30-40% decrease in the percentage of T cells in the abomasal lymph nodes. This decrease in T cell percentage was due to a decrease in cells bearing the CD4 marker, a marker usually associated with helper T cells. Immunized calves were able to maintain normal T cell percentages of 50-60% for the first 5 weeks of infection. Immunization greatly increased the total number of Ostertagia-specific T cells in the abomasal lymph nodes owing to a marked increase in the size of the lymph nodes. Challenge infection of naive and immunized calves caused an increase in the frequency of parasite-specific T cells in both groups, but the increase was more rapid in the previously immunized calves. Within 5 weeks of infection, Ostertagia-specific cells could not be detected in the abomasal lymph nodes. These results indicate that the critical time period for expansion and regulation of Ostertagia-specific T cells in infected calves is early in the infection at a time that coincides with larval development. In addition, previous exposure to parasite antigens appears to result in more rapid responses and in the maintenance of normal ratios of T cell subpopulations in the draining lymphoid tissues.

The invasive phase of Eimeria coecicola was studied during the first 80 h postinoculation (p.i.). Using a method that synchronized the life cycle, sporozoites were observed in the duodenum and the jejunum until 32 h p.i. They were seen first in the villous epithelial cells or in host cells resembling intraepithelial lymphocytes (IEL). Later they were observed in IEL in the lamina propria. After 48 h p.i., no coccidian stage was identifiable in the mucosa of the small intestine but sporozoites appeared in the lymphoid cells of lymphatic follicles of the gut-associated lymphoid tissue (vermiform appendix, sacculus rotundus, and Peyer's patches). The first merogony was observed 64 h p.i. in these lymphoid cells and in membranous epithelial cells (M-cells) but was never seen in the epithelium itself. Morphologically there were two types of meronts, depending on the host cell type, but in both cases the merozoites contained a refractile body and resembled sporozoites. The first meronts of the second generation were observed 80 h p.i. in the villous epithelial cells of the domes of the follicles of the gut-associated lymphoid tissue, where the further development of this Eimeria takes place. This pattern of invasion strongly suggests that sporozoites take an exclusively extraintestinal route to reach the target cells. Moreover, to our knowledge this is the first description of an eimerian merogony that does not take place in epithelial cells.

Eleven rabbits were infected with 10 embryonated eggs of Toxocara vitulorum per g body weight on Days 0, 35 and 72. Embryonated eggs and larvae were enumerated in feces on Days 1-3 after each infection. Two rabbits were killed and larvae were enumerated in small intestine, liver, lungs, skeletal muscles, heart, kidney, brain, eye, uterus, and mammary glands on Days 5, 15, 30, 65 and 101. Serum was obtained on Days 0, 5, 15, 30, 42, 50, 65, 78, 86 and 101 to perform enzyme-linked immunosorbent assays (ELISAs) and Western blots against an extract of embryonated eggs. Between 4 and 10% of the administered parasites, almost all embryonated eggs, were found in the feces after the first or second infection, but 32% (27% of them larvae) after the third. Yields of tissue parasites were 4.1% of the administered dose on Day 5, 2% on Day 15, and 0.8% on Day 30 of the first infection, 0.1% on Day 30 of the second infection, and 0.06% on Day 30 of the third. Larvae were found only in liver, lungs and muscle, including heart. Larva content declined steadily in liver and lungs from Day 5 to 30 of the first infection, was absent in the liver at Day 30 of the second, and in both organs at Day 30 of the third. Muscle larva content increased from Day 15 to 30 of the first infection, and persisted throughout the third infection. Production of IgM antibody was minimal, IgG and the sum of IgMGA antibodies increased slightly or moderately after the first and second infections, but dramatically after the third. Western blots revealed the first antigens (12) by Day 15 of the first infection. Their total number increased with time and number of infections, but some antigens disappeared, whereas new antigens appeared in the course of the observations. Four antigens (32,500-41,000 mol.wt.) may be related to protection. Comparison of the Western blot patterns of two rabbits showed differences in the antigens, recognizable for each rabbit.

Male jirds (Meriones unguiculatus) were inoculated subcutaneously with 100 Brugia pahangi L3 each at 2, 6, 10, and 15 wk of age to compare their susceptibility and pathologic reactivity to infection. Adult worm recoveries (mean +/- SD) ranged from 24.1 +/- 15.1 to 36.4 +/- 13.9 at 60 days postinfection. No significant difference in susceptibility was measured among the 4 age groups. Jirds infected at 2 wk of age had significantly fewer (alpha less than or equal to 0.025) testicular and intralymphatic worms than all other age groups. Numbers of intralymphatic thrombi were significantly lower (alpha less than or equal to 0.01) in jirds infected at 2 wk of age. Lymphatic lesion severity, expressed as the number of intralymphatic thrombi per intralymphatic worm, was similar between age groups. These data indicate no differences in susceptibility or lymphatic lesion formation following B. pahangi infection in 2-wk-old male jirds, despite altered adult worm location.

Studies were carried out to determine changes in lymphoid organs i.e. spleen, lymph node and bone marrow (BM) in progressive experimental visceral leishmaniasis. Mononuclear phagocytes in the BM were increased; spleens showed a hypercellularity coupled with a rise in parasite burden while secondary follicles with no apparent depletion of paracortex were seen in the lymph node. This enhanced proliferation of mononuclear phagocytes in the BM and probably their subsequent recruitment in the spleen could be induced in naive recipients by injecting nylon wool-purified lymph node cells derived from infected mice together with sonicated leishmanial antigen(s). Similar changes could also be induced in the BM of naive recipients by injecting serum of infected mice. In control experiments where both donor and recipients were uninfected such changes were not apparent. A working hypothesis is proposed to delineate the role of lymphoid organs in the pathogenesis of experimental visceral leishmaniasis.

Infected immunocompetent mice were studied prospectively in a well-described murine model system with the use of ultrastructural techniques to establish normal distribution of Giardia muris trophozoites, their relationships to intestinal mucosa, particularly Peyer's patches, and structural indications of the normal reaction of intestine and intestinal immune organs. Trophozoites colonized the proximal 25% of the intestine, adhered to microvilli of columnar cells near the bases of villi, wedged into furrows in the epithelial surface, or lodged in mucus within the unstirred layer. Density of trophozoite colonization of the jejunal epithelium correlated with stool cyst excretion. Over Peyer's patches, Giardia adhered to columnar cells and not to M-cells, which transport soluble antigens and particulate material from the lumen into the lymphoid system. Giardia entered intestinal lymphoid structures by incursions through defects in the lymphoid follicle epithelial barrier. During clearance of parasites, lymphocytes crossed the epithelium and attached to Giardia in the lumen. Giardia produced no apparent ultrastructural damage in normal mice but elicited a previously undescribed intraluminal cellular immune response during clearance by the host.

Schistosoma mansoni antigens play a crucial role in the induction of immunopathological processes and in modulating the host immune system. A polyclonal rabbit antiserum to an antigenic fraction of adult schistosoma worms was used to localize worm and egg antigens in tissue sections of infected mice. Most granuloma formations identified in paraffin sections of portal tracts and intestinal mucosa were vigorous with florid cellular composition, consisting of macrophages and epithelioid cells, surrounding a central nidus of S. mansoni egg. Schistosome pigments were demonstrated at the periphery of the granuloma, within sinusoidal Kupffer's cells and within macrophages of intestinal mucosa. Large amounts of schistosome antigen were found sequestered in the mesenteric lymph nodes and in the spleen. A strong reaction for locally synthesized IgG was found. These findings suggest that the schistosome antigen-antibody complex found in the host lymphoreticular system plays a major role in the immunopathological process.