Humoral immune response and delayed-type hypersensitivity in rabbits infected with Trypanosoma equiperdum.

Trypanosoma equiperdum is the causative agent of dourine, a parasitic venereal disease of equids. In this work, rabbits were infected with T. equiperdum strain OVI; serological tests (complement fixation test, ELISA and immunoblotting), used for the diagnosis of dourine in horses, were applied to study rabbit humoral immune response and to characterise T. equiperdum antigen pattern recognised by antibodies from infected rabbits. Moreover a protein extract of T. equiperdum strain OVI was produced and tested in skin tests on infected rabbits to detect the cell-mediated response induced by T. equiperdum, in order to evaluate its use in the field diagnosis of dourine. Sera of infected rabbits recognized in immunoblotting Trypanosoma protein bands with molecular weight below 37 kDa, providing a serological response comparable with that already observed in dourine infected horses. Moreover the trypanosome protein extract was capable to produce in vivo delayed-type hypersensitivity (DHT Type IV) in rabbits and proved itself to be non-toxic and non-sensitizing.

Delayed-type hypersensitivity skin test against Neospora caninum in heifers with undetectable specific antibodies.

Delayed-type hypersensitivity (DTH) has been used in human and veterinary medicine as a skin testing for evaluating in vivo cell-mediated immune responses (CMIR). Whereas CMIR is a key process to control intracellular pathogens, its value at identifying cattle exposed to the abortigenic intracellular coccidian parasite Neospora caninum is unknown. In this work, we have evaluated a DTH skin testing in cattle exposed to N. caninum and still seronegative. Female calves were experimentally sensitized by subcutaneous (SC) inoculation with live tachyzoites of N. caninum (NC-Argentina LP1) in sterile phosphate-buffered saline (PBS) (group A; n: 8) whereas other calveswere mock-sensitized with PBS (group B; n: 6). Two DTH skin tests were performed by intradermal inoculation with a soluble lysate of N. caninum tachyzoites (NC-Argentina LP1) in the neck region at 60d and 960 d after sensitization. Skinfold thickness at the intradermal inoculation site was measured at 0, 24, 48 h post each DTH skin test and skin biopsies taken for microscopic evaluation. Specific N. caninum antibodies kinetics was evaluated all throughthe experiment. We found that whereas N. caninum specific antibodies remained below the ELISA cut-off, a distinctive skinfold thickness increase was detected in sensitized animals (group A) at the DTH skin test site, showing induration, swelling and inflammatory infiltration. Mock sensitized animals (group B) showed no skinfold thickness growth and lacked specific antibody response. Thus, N. caninum DTH skin testing could be a useful diagnostic tool for the detection of CMIR during N. caninum infection in non-humoral responders.

Delayed hypersensitivity reaction to mammalian galactose-α-1,3-galactose (α-Gal) after repeated tick bites in a patient from France.

The α-Gal syndrome is a tick-associated and emerging IgE-mediated hypersensitivity reaction directed against the carbohydrate Galα1-3Galß1-(3)4GlcNAc-R (α-Gal) epitope after red meat intake. Herein, we describe a clinical case of a 44-year-old French patient who suffered from recurrent anaphylactic reactions after mammalian meat consumption for five years before the final diagnosis of the α-Gal syndrome was established in 2018. The patient also reported multiple tick bites prior to symptom onset. This unique type of allergy has increasingly been reported across the world, but it is still unknown in many European countries. Therefore, the present clinical case should increase awareness among primary care practitioners and further improve the early diagnosis of the α-Gal syndrome in affected individuals.

Delayed type hypersensitivity induced by intradermal inoculation of a Neospora caninum tachyzoite antigen in previously exposed cattle.

The aim of this study was to evaluate delayed type hypersensitivity (DTH) induced by the intradermal inoculation of a Neospora caninum tachyzoite soluble lysate in cattle previously exposed with the protozoa. Four experimental groups were selected according to the prior exposure to N. caninum antigen. All cows were intradermally injected with a N. caninum tachyzoite soluble lysate and skinfold thickness growth at the inoculation sites was measured at 0, 24, 48, 72 and 96 h post inoculation (hpi). Additionally, specific antibodies and IFN-γ production were assessed. Cows experimentally infected with live N. caninum tachyzoites and cows naturally exposed to N. caninum developed skin reactions compatible with DTH between 24 and 96 hpi (p < 0.05). Moreover, cows inoculated with an experimental N. caninum vaccine and cows without evidence of exposure to N. caninum did not show a significant increase in skin thickness (p > 0.05). Furthermore, serological status of the animals was not modified due to the intradermal inoculation. The highest IFN-γ production was observed at 15 days after intradermal inoculation (p < 0.05). Therefore, these results suggest that cattle previously exposed to N. caninum develop a reaction compatible with DTH which could be useful as in vivo cell mediated immunity parameter for assessed bovine neosporosis.

Immunomodulatory activity of Buchholzia coriacea seed methanol extract on Trypanosoma brucei brucei infected mice.

CONTEXT: The seeds of Buchholzia coriacea Engler (Capparaceae) are used in Eastern Nigeria to treat feverish conditions, and to treat malaria and sleeping sickness that cause fever. OBJECTIVE: The current study assesses the immunomodulatory activity of Buchholzia coriacea seed extract on Trypanosoma brucei brucei infected mice. MATERIALS AND METHODS: Delayed hypersensitivity reaction, humoral antibody response and in-vivo leucocyte mobilization tests were assessed in three different experiments to determine the effect of the extract on immune response. Seventy-five (75) mice (25 mice per experiment) were used for the study and were each infected with 1.00 × 106 trypanosomes intra-peritoneally. Groups A, B and C were given 250, 500 and 1000 mg/kg of the extract, respectively, group D received 7.5 mg/kg body weight of levamisole and group E was the control. Sheep RBCs were used as antigen. RESULTS: The acute toxicity tests did not cause clinical signs or death within 24 h post treatment at all the doses tested. The extract inhibited delayed hypersensitivity reaction by 20.9 and 20.8% at 250 and 500 mg/kg, respectively, while at 1000 mg/kg, the paw size increased (-101.9%) when compared with the control. The extract elevated the antibody titre from 1.60 ± 0.40 for control to 8.00 ± 3.58 for 500 mg/kg group. The extract increased in total leucocytes counts. DISCUSSION AND CONCLUSION: The extract has a very wide safety margin and was able to improve immune response. The results of the present study showed that Buchholzia coriacea seed methanol extract possesses immunostimulatory activity on trypanosome-infected mice.

The development of a murine model for Forcipomyia taiwana (biting midge) allergy.

BACKGROUND: Forcipomyia taiwana (biting midge) allergy is the most prevalent biting insect allergy in Taiwan. An animal model corresponding to the human immuno-pathologic features of midge allergy is needed for investigating the mechanisms and therapies. This study successfully developed a murine model of Forcipomyia taiwana allergy. METHODS: BALB/c mice were sensitized intra-peritoneally with midge extract on days 0, 7, 14, 21 then intra-dermally on days 28, 31 and 35. Serum midge-specific IgE, IgG1, and IgG2a were measured every 14 days by indirect ELISA. The mice were challenged intradermally with midge extract at day 40 and then sacrificed. Proliferation and cytokine production of splenocytes after stimulation with midge extract were determined by MTT assay and ELISA, respectively. The cytokine mRNA expression in response to midge stimulation was analyzed by RT-PCR. RESULTS: Serum IgE, total IgG, and IgG1 antibody levels against midge extract were significantly higher in the midge-sensitized mice than in the control mice. After the two-step sensitization, all mice in the midge-sensitized group displayed immediate itch and plasma extravasation reactions in response to challenge with midge extract. Skin histology from midge-sensitized mice showed marked eosinophil and lymphocyte infiltrations similar to that observed in humans. Stimulation of murine splenocytes with midge extract elicited significant proliferation, IL-4, IL-10, IL-13 and IFN-γ protein production, and up-regulation of mRNA in a dose-dependent manner in the midge-sensitized group, but not in the control group. CONCLUSIONS: A murine model of midge bite allergy has been successfully developed using a two-step sensitization protocol. The sensitized mice have very similar clinical and immunologic reactions to challenge with midge proteins as the reactions of human to midge bites. This murine model may be a useful platform for future research and the development of treatment strategies for insect bite allergy.

Delayed-type hypersensitivity to sand fly saliva in humans from a leishmaniasis-endemic area of Mali is Th1-mediated and persists to midlife.

Immunity to sand fly saliva in rodents induces a T(H)1 delayed-type hypersensitivity (DTH) response conferring protection against leishmaniasis. The relevance of DTH to sand fly bites in humans living in a leishmaniasis-endemic area remains unknown. Here, we describe the duration and nature of DTH to sand fly saliva in humans from an endemic area of Mali. DTH was assessed at 24, 48, 72, and 96 hours post bite in volunteers exposed to colony-bred sand flies. Dermal biopsies were obtained 48 hours post bite; cytokines were quantified from peripheral blood mononuclear cells (PBMCs) stimulated with sand fly saliva in vitro. A DTH response to bites was observed in 75% of individuals aged 1-15 years, decreasing gradually to 48% by age 45, and dropping to 21% thereafter. Dermal biopsies were dominated by T lymphocytes and macrophages. Abundant expression of IFN-γ and absence of T(H)2 cytokines establishes the T(H)1 nature of this DTH response. PBMCs from 98% of individuals responded to sand fly saliva. Of these, 23% were polarized to a T(H)1 and 25% to a T(H)2 response. We demonstrate the durability and T(H)1 nature of DTH to sand fly bites in humans living in a cutaneous leishmaniasis-endemic area. A systemic T(H)2 response may explain why some individuals remain susceptible to disease.

Accelerated healing of cutaneous leishmaniasis in non-healing BALB/c mice using water soluble amphotericin B-polymethacrylic acid.

Cutaneous leishmaniasis (CL) is a neglected tropical disease that causes prominent skin scaring. No water soluble, non-toxic, short course and low cost treatment exists. We developed a new water soluble amphotericin B-polymethacrylic acid (AmB-PMA) using established and scalable chemistries. AmB-PMA was stable for 9 months during storage. In vitro, it was effective against Leishmania spp. promastigotes and amastigote infected macrophages. It was also less toxic and more effective than deoxycholate-AmB, and similar to liposomal AmB. Its in vivo activity was determined in both early and established CL lesion models of Leishmania major infection in genetically susceptible non-healing BALB/c mice. Intradermal AmB-PMA at a total dose of 18 mg of AmB/kg body weight led to rapid parasite killing and lesion healing. No toxicity was seen. No parasite relapse occurred after 80 days follow-up. Histological studies confirmed rapid parasite clearance from macrophages followed by accelerated fibroblast mediated tissue repair, regeneration and cure of the infection. Quantitative mRNA studies of the CL lesions showed that accelerated healing was associated with increased Tumour Necrosis Factor-α and Interferon-γ, and reduced Interleukin-10. These results suggest that a cost-effective AmB-PMA could be used to pharmacologically treat and immuno-therapeutically accelerate the healing of CL lesions.

[Morphogenesis of the structural changes of the internal organs in superinvasive opisthorchiasis].

The study of the model of superinvasive opisthorchiasis (SO) in Syrian hamsters and autopsy material obtained from patients with SO, it was shown that during the early period of pulmonary disease, the lungs underwent changes developing on the basis of delayed-type hypersensitivity reactions. In the chronic phase of SO, exudative and predominant productive reactions were revealed leading to the interstitial lung disease, fibrosing alveolitis. In the heart in SO, eosinophilic cell myocarditis was found with subsequent diffuse cardiosclerosis. In the testes, sclerotic processes with atrophy of glandulocytes (Leydig cells) and sustentocytes (Sertoli cells) were detected. The structural changes in the lungs, heart and testes were induced by parasite metabolites, that are deposited in tissues in the form of granules and conglomerates.

The p110 delta isoform of phosphatidylinositol 3-kinase controls the quality of secondary anti-Leishmania immunity by regulating expansion and effector function of memory T cell subsets.

We showed previously that mice with an inactivating knockin mutation in the p110delta isoform of PI3K (referred to as p110delta(D910A) mice) displayed enhanced primary resistance to Leishmania major despite mounting paradoxically impaired T cell responses. In this study, we show that p110delta(D910A) mice are impaired in their secondary (memory) anti-Leishmania responses in vitro and in vivo. Following secondary L. major challenge, p110delta(D910A) mice exhibited reduced delayed-type hypersensitivity response and weaker parasite control compared to wild-type mice. Using adoptive transfer experiments, we show that immune T cells from healed p110delta(D910A) mice were impaired in their proliferation and effector cytokine (IFN-gamma) responses upon L. major challenge. Interestingly, Leishmania-reactive T cells from healed p110delta(D910A) mice contain severalfold lower numbers of CD62L(lo) and CD62(hi) T cells than those from healed wild-type mice. The reduction in numbers of CD62L(lo) T cells in p110delta(D910A) mice is due to failure of their CD62L(hi) T cells to downregulate CD62L expression in response to L. major. Furthermore, although CD62L(lo) cells from p110delta(D910A) mice could home efficiently to lymphoid organs, their ability to exit these tissues and emigrate to cutaneous sites of infection was greatly impaired. Collectively, our data identify PI3K signaling as important events that control memory T cell subset differentiation, generation, effector function, and recruitment to cutaneous tissues and suggest that manipulating this pathway could provide means of enhancing desired memory T cell subset, response during vaccination, or both.

Comparison of liposome based antigen delivery systems for protection against Leishmania donovani.

Liposomes have been widely exploited as antigen delivery systems for a variety of diseases including leishmaniasis. These vesicles can be prepared in various ways which may affect the immunogenicity of the encapsulated antigens. In this study we compared the vaccine potentiality of three cationic formulations with Leishmania donovani promastigote membrane antigens (LAg) and the best vesicle was evaluated for long-term protection against experimental visceral leishmaniasis. We immunized mice with LAg encapsulated in multilamellar vesicles (MLV), dehydration-rehydration vesicles (DRV) and reverse-phase evaporation vesicles (REV) and challenged them with parasites ten days after vaccination. LAg in MLV or DRV induced almost complete protection, while LAg alone or entrapped in REV exhibited partial resistance. Protection observed with antigen incorporated MLV or DRV was predominantly Th1 as evidenced by elicitation of significantly high DTH, IgG2a antibodies and IFN-gamma. MLV encapsulated LAg demonstrated durable cell-mediated immunity and mice challenged ten weeks after vaccination could also resist experimental challenge strongly. Field trials of L. donovani vaccine were unsatisfactory mainly due to lack of an appropriate adjuvant. Cationic MLV when used as adjuvant with protein antigens induced sustained Th1 immunity. Adjuvant potential of cationic MLV can be utilized to design subunit vaccines.

Immunogenicity assay of the Leishmune vaccine against canine visceral leishmaniasis in Brazil.

Leishmune is the industrialized version of the FML-saponin vaccine which has been shown to develop 92-95% protection in vaccinated dogs and 76-80% vaccine efficacy against field canine visceral leishmaniasis (CVL) in Brazil. Leishmune has been proven to be safe and tolerable and a transmission-blocking vaccine which renders vaccinated dogs non-infectious to sand fly vectors. In the present investigation, 550 healthy seronegative dogs of endemic and epidemic areas of Brazil were monitored for Leishmune-induced immunogenicity during a 2-year trial. Another group of 588 untreated exposed dogs was also studied in parallel. Both groups were seronegative on day 0. The strong immunogenicity induced by Leishmune vaccine was demonstrated by the 98% of FML-seroconversion, increase in absorbencies, the 82.7% DTH positive reactions and increase in skin test size diameters, the average increase in CD8+ total lymphocytes population in blood (27.1%), expected for QS21 saponin-containing vaccine, the sustained proportions of CD4+ T cells, and the average increased proportions of CD21+ B lymphocytes (42.3%). The Leishmune-induced protection against CVL is demonstrated by the results: 98.8% asymptomatic dogs (at the end of first year) and 99% healthy survivors (at the end of the second year) among vaccinated dogs, compared to the 79.4% asymptomatic and 61% survivor dogs (p<0.001) monitored in the untreated exposed cohort. In spite of the low vaccine coverage, it was possible to detect a 66.1% (p<0.005) reduction in Belo Horizonte and an 80.2% (p<0.005) reduction in Araçatuba of the incidence of CVL among vaccinated dogs, when compared to the global incidence of CVL of each town, respectively. Our preliminary results support the potential use of Leishmune to prevent CVL epidemics.

Anti-Leishmania humoral and cellular immune responses in naturally infected symptomatic and asymptomatic dogs.

Canine infections with Leishmania infantum represent a considerable veterinary medical and public health problem. In this study, immunoglobulin G1 (IgG1) and IgG2 specific humoral responses were measured and compared with the delayed type hypersensitivity (DTH) cellular response to a leishmanin, in three groups of dogs clinically and serologically characterised as: (I) asymptomatic and direct agglutination test (DAT)-seronegative; (II) asymptomatic and DAT-seropositive; (III) DAT-seropositive and symptomatic. IgG2 was regarded as a marker of disease, since significantly higher levels of this subclass were recorded in the symptomatic dogs. In contrast, the IgG1 response could not be related to clinically relevant infection. A high correlation was observed between IgG2 level and DAT titre; the correlations between IgG1 and IgG2 levels, and between IgG1 level and DAT titre were lower. This may indicate that IgG2 is the main subclass in the specific humoral response which is detected by the DAT. A reduced IgG2 response, albeit not significantly different, was recorded among dogs with clear cellular immune responses detected by a DTH positive reaction. Furthermore, no correlations were observed between cellular response measured by DTH and humoral responses quantified by DAT titre or IgG1 and IgG2 levels. Combining serology and DTH skin test is a practical procedure to assess anti-Leishmania immune responses in dogs.

Cutaneous hypersensitivity induced in rabbits by extracts of the tick Amblyomma cajennense (Acari: Ixodidae).

The cutaneous hypersensitivity test was used to correlate host resistance to ticks and type of reactions elicited by Aomblyomma cajennense (Fabricius, 1787) tick extract in rabbits. Rabbits were divided into 3 groups of 2 animals each: naive, pre-infested and control. Cutaneous hypersensitivity was induced by intradermal inoculation of 25 microg extract in 0.03 ml of phosphate buffered saline (PBS) in rabbit ears. Control rabbits were inoculated with PBS only. The ear thickness was measured with a Mitutoyo device before and 10 min, 1, 2, 4, 18, 24, 48, 72 and 96 h post-inoculation (PI). Pre-infested rabbits showed an immediate type reaction within the 1st 10 min PI (60% increase in ear thickness) and a delayed reaction (18 h) (85% increase), whereas the naive rabbits showed only the immediate reaction within the 1st 4 h (60% increase). PBS induced only mild reactions. These results point out the crucial role of the cellular immune response of rabbits in the expression of resistance to A. cajennense.

Cercarial dermatitis caused by bird schistosomes comprises both immediate and late phase cutaneous hypersensitivity reactions.

Avian schistosomes are the primary causative agent of cercarial dermatitis in humans, but despite its worldwide occurrence, little is known of the immune mechanism of this disease. Using a murine model, hosts were exposed to primary (1x) and multiple (4x) infections of Trichobilharzia regenti via the pinna. Penetration of larvae into the skin evoked immediate edema, thickening of the exposure site, and an influx of leukocytes, including neutrophils, macrophages, CD4+ lymphocytes, and mast cells. A large proportion of the latter were in the process of degranulating. After 1x infection, inflammation was accompanied by the release of IL-1beta, IL-6, and IL-12p40. In contrast, in 4x reinfected animals the production of histamine, IL-4, and IL-10 was dramatically elevated within 1 h of infection. Analysis of Ag-stimulated lymphocytes from the skin-draining lymph nodes revealed that cells from 1x infected mice produced a mixed Th1/Th2 cytokine response, including abundant IFN-gamma, whereas cells from 4x reinfected mice were Th2 polarized, dominated by IL-4 and IL-5. Serum Abs confirmed this polarization, with elevated levels of IgG1 and IgE after multiple infections. Infection with radiolabeled cercariae revealed that almost 90% of larvae remained in the skin, and the majority died within 8 days after infection, although parasites were cleared more rapidly in 4x reinfected mice. Our results are the first demonstration that cercarial dermatitis, caused by bird schistosomes, is characterized by an early type I hypersensitivity reaction and a late phase of cutaneous inflammation, both associated with a polarized Th2-type acquired immune response.

Leishmania (Viannia) braziliensis-induced chronic granulomatous cutaneous lesions affecting the nasal mucosa in the rhesus monkey (Macaca mulatta) model.

The present studies on infections with Leishmania (Viannia) braziliensis in rhesus macaques were made to characterize the evolution of different parasite strains and the immune responses they elicited in this experimental host. A standardized inoculum of promastigotes was injected intradermally either above the eyelid or on the forearm of each monkey. Sixteen infected monkeys developed longstanding infections which lasted until the end of the observation period (33 months). The time required for lesion development was very variable, not only for the isolates showing molecular differences but also for individual animals in groups infected with the same parasite strain. The inocula produced lesions of variable severity, ranging from localized cutaneous leishmaniasis (CL) with a tendency to spontaneous healing to non-healing disease. One infected animal developed persistent metastatic skin and mucosal lesions. Anti-Leishmania antibodies and parasite-specific T-cell responses were induced by the experimental infections. As the granulomatous inflammatory response found at the lesions in L. (V.) braziliensis-infected M. mulatta was similar to that in patients with CL, this primate model could be useful for studying the pathophysiology and immunoregulatory events associated with disease evolution, as well as for the evaluation of new drugs or candidate vaccines.

A Leishmania infantum multi-component antigenic protein mixed with live BCG confers protection to dogs experimentally infected with L. infantum.

The capacity of a quimeric protein, formed by the genetic fusion of five antigenic determinants from four Leishmania proteins, formulated with BCG, to protect dogs against Leishmania infantum infection is described. The data showed that after i.v. administration of 500,000 parasites of the L. infantum M/CAN/ES/96/BCN150 strain, zymodeme MON-1, the animals became infected as suggested by the humoral response against the parasite antigens. All control unvaccinated dogs had parasites in the lymph nodes at day 150 post-infection. One of these unvaccinated infected dog was parasite negative at day 634 behaving, thus, as resistant. In contrast, only 50% of the immunized dogs had parasites in the lymph nodes at day 150 post-infection. Four of these dogs became parasite negative by day 634 post-infection. The control animals developed at various times during the follow-up period clinical symptoms associated with Leishmaniasis. The control diseased dogs developed also in the liver and spleen some of the abnormal histological features associated with natural visceral Leishmaniasis. The immunized dogs, however, were not only normal at the clinical but also at the anatomo-pathological level. A positive delayed type hypersensitivity (DTH) response was observed in nine of the immunized protected dogs. The data indicated that Q+BCG confers 90% protection against infection and at least 90% protection at the clinical level.

Canine infection and the possible role of dogs in the transmission of American tegumentary leishmaniosis in Salta, Argentina.

Some Leishmania species affect humans in two principal forms: visceral and cutaneous leishmaniosis (CL). Several studies have identified dogs as the main reservoirs of the visceral leishmaniosis (VL) caused by Leishmania infantum. The purpose of this work was to carry out a survey of the canine population associated with human cases of American tegumentary leishmaniosis (ATL), in order to establish the clinical, parasitological, serological and immunological characteristics of the canine disease, in an endemic region for both ATL and Chagas' disease in the province of Salta, in northwestern Argentina. Two hundred and eight dogs from the endemic area were examined and 41 (19.7%) of them presented lesions compatible with leishmaniosis. In order to investigate the presence of antibodies against Leishmania spp. and Trypanosoma cruzi, sera were screened by ELISA using two complex antigens from these parasites and, because of cross-reactions between them, a specific antigen for diagnosis of T. cruzi infection. Sixty-two (29.8%) of 208 dogs were positive for the complex antigen F45 from Leishmania and 50 (24%) were positive for the complex antigen F105 from T. cruzi. Nine dogs (4.3%) were positive for the specific Ag163B6-cruzipain suggesting that these dogs were truly infected with T. cruzi. Furthermore, three of these nine dogs presented Leishmania sp. in their skin lesions and therefore were considered as infected by both, T. cruzi and Leishmania parasites. The prevalence of Leishmania infection detected by lesions and/or positive serology was 27.4% (57/208). On the basis of previous observations regarding the clustered appearance of human ATL, the dog population was divided into two groups: zone A, dogs living within a 100 m radius from houses with human cases, and zone B, dogs living beyond this limit. The prevalence of ATL in dogs was significantly higher in zone A (34.6%) than in zone B (7.3%), suggesting a strong correlation between canine and human cases. The average time required for a parasitological diagnosis by microscopy was six times longer for dog samples than human ones, and the average number of parasites per 100 microscopic fields was 14-fold lower in canine samples. The high prevalence of Leishmania infection and the close association with human cases, demonstrated that dogs are a very susceptible host for Leishmania infection, but the scarcity of parasites in their lesions suggests that they may not be the main reservoir of the parasite in this endemic area.

Association between the tumor necrosis factor locus and the clinical outcome of Leishmania chagasi infection.

A periurban outbreak of visceral leishmaniasis (VL) caused by the protozoan Leishmania chagasi is ongoing outside Natal, northeast Brazil. Manifestations range from asymptomatic infection to disseminated visceral disease. Literature reports suggest that both genetic and environmental factors influence the outcome of infection. Due to the association of the tumor necrosis factor (TNF) locus with other infectious diseases, we examined whether polymorphic alleles at this locus are associated with the outcome of L. chagasi infection. Neighborhoods with ongoing transmission were identified through patients admitted to local hospitals. Altogether, 1,024 individuals from 183 families were classified with the following disease phenotypes: (i) symptomatic VL, (ii) asymptomatic infection (positive delayed-type hypersensitivity [DTH+]), or (iii) no evidence of infection (DTH-). Genotypes were determined at a microsatellite marker (MSM) upstream of the TNFB gene encoding TNF-beta and at a restriction fragment length polymorphism (RFLP) at position -307 in the promoter of the TNFA gene encoding TNF-alpha. Analyses showed that the distribution of TNFA RFLP alleles (TNF1 and TNF2) and the TNF MSM alleles (TNFa1 to TNFa15) differed between individuals with VL and those with DTH+ phenotypes. TNF1 was transmitted more frequently than expected from heterozygous parents to DTH+ offspring (P = 0.0006), and haplotypes containing TNF2 were associated with symptomatic VL (P = 0.0265, transmission disequilibrium test). Resting serum TNF-alpha levels were higher in TNF1/2 heterozygotes than in TNF1/1 homozygotes (P < 0.05). These data led us to hypothesize that an individual's genotype at the TNF locus may be associated with whether he or she develops asymptomatic or symptomatic disease after L. chagasi infection. The results preliminarily suggest that this may be the case, and follow-up with larger populations is needed for verification.

Hypersensitivity induced in dogs by nymphal extract of Amblyomma cajennense ticks (Acari:Ixodidae).

In general, hosts develop resistance to ticks after repeated infestations; nevertheless, several studies on naturally occurring host-tick interactions were unable to detect resistance of hosts to ticks even after repeated infestations. The purpose of this investigation was to study the type of cutaneous hypersensitivity to unfed nymphal extract of A. cajennense in dogs, which, unlike guinea pigs, do not develop resistance. A first, but no second, peak in skin reaction was observed, suggesting that cellular immunity is an important mechanism of resistance to ticks. This may partially explain why guinea pigs, but not dogs, develop resistance against ticks.