A novel herpesvirus in the sanctuary chimpanzees on Ngamba Island in Uganda.

BACKGROUND: Recent studies in non-human primates have led to the discovery of novel primate herpesviruses. In order to get more information on herpesvirus infections in apes, we studied wild born captive chimpanzees. METHODS: Chimpanzees of the Ngamba island sanctuary, Uganda, were analyzed with pan-herpes polymerase chain reaction (PCR) targeting the herpesvirus DNA polymerase gene and the glycoprotein B gene. The obtained sequences were connected by long-distance PCR, and analyzed phylogenetically. RESULTS: Twenty-one of 40 individuals were infected with members of the Gammaherpesvirinae, two of them with a novel member of this subfamily. Phylogenetically, the novel virus fell into a clade of primate rhadinoviruses and the Kaposi sarcoma herpesvirus (human herpesvirus 8), representing a third distinct rhadinovirus in chimpanzees. CONCLUSION: Non-human primates harbor several herpesviruses many of which are still unknown. This has implications to management of primates in sanctuaries requiring continuous updates on the management protocols to deal with potential occupational pathogens.

Distinct immunity in patients with visceral leishmaniasis from that in subclinically infected and drug-cured people: implications for the mechanism underlying drug cure.

Significant levels of IgG3 and IgG4 and high levels of IgG1 leishmania-specific antibody differentiated the immune states in 10 patients with visceral leishmaniasis from those of virtually all 20 drug-cured and 18 subclinically infected subjects, whereas the level of IgG2 antibody was nondiscriminating. The most extreme "subclinically infected" outlier subsequently developed disease. Overall, the immune states in subclinically infected and drug-cured persons were mutually indistinguishable but were readily distinguished from those of patients. These findings may have implications for the immunologic mechanism underlying drug cure in visceral leishmaniasis.

Circulating TNF-alpha, TGF-beta, and IL-10 in tuberculosis patients and healthy contacts.

Levels of tumour necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta, and interleukin (IL)-10 in plasma of pulmonary tuberculosis (TB) patients and healthy contacts and plasma and pleural fluid of patients with tuberculous pleuritis were examined by enzyme immunoassay. Plasma TNF-alpha and IL-10 were elevated to significant levels in healthy contacts. High levels of TGF-beta and IL-10 were also detected in plasma from TB patients and healthy contacts. Pleural fluid contained all three cytokines with the level of IL-10 being highest followed by TGF-beta and TNF-alpha. Plasma of tuberculous pleuritis patients also had detectable levels of the three cytokines. Increased levels of TNF-alpha in plasma of contacts and to some extent pleural fluid of pleuritis patients, is perhaps to limit the infection, while elevated IL-10 in plasma of TB patients and contacts and pleural fluid would perhaps modulate excess proinflammation. Elevated TGF-beta in TB patients suggests its role in the immunopathogenesis.

Vervet monkeys vaccinated with killed Leishmania major parasites and interleukin-12 develop a type 1 immune response but are not protected against challenge infection.

Leishmania major is a protozoan parasite that causes chronic cutaneous lesions that often leave disfiguring scars. Infections in mice have demonstrated that leishmanial vaccines that include interleukin-12 (IL-12) as an adjuvant are able to induce protective immunity. In this study, we assessed the safety, immunopotency, and adjuvant potential of two doses of IL-12 when used with a killed L. major vaccine in vervet monkeys. The induction of cell-mediated immunity following vaccination was determined by measuring delayed-type hypersensitivity, in vitro lymphocyte proliferation, and gamma interferon (IFN-gamma) production. Protection was assessed by challenging the animals with L. major parasites and monitoring the course of infection. At low doses of IL-12 (10 microg), a small increase in the parameters of cell-mediated immunity was observed, relative to those in animals that received antigen without IL-12. However, none of these animals were protected against a challenge infection. At higher doses of IL-12 (30 microg), a substantial increase in Leishmania-specific immune responses was observed, and monkeys immunized with antigen and IL-12 exhibited an IFN-gamma response that was as great as that in animals that had resolved a primary infection and were immune. Nevertheless, despite the presence of correlates of protection, the disease course was only slightly altered, and protection was low compared to that in self-cured monkeys. These data suggest that protection against leishmaniasis may require more than the activation of Leishmania-specific IFN-gamma-producing T cells, which has important implications for designing a vaccine against leishmaniasis.

The African Green Monkey model for cutaneous and visceral leishmaniasis.

Non-human primates are valuable models for biomedical research because of their similarities to human anatomy, immunology and physiology. Leishmaniasis, a disease caused by protozoan parasites, has a worldwide distribution and results in high morbidity and mortality. Availability of a non-human primate model of leishmaniasis would facilitate the study of different aspects of this disease and would accelerate the development of vaccines and new drugs. In this article, some interesting features of the vervet monkey (African Green monkey) model of human cutaneous and visceral leishmaniasis are discussed.

T-cell recognition of Mycobacterium tuberculosis culture filtrate fractions in tuberculosis patients and their household contacts.

We examined the immune responses of patients with active pulmonary tuberculosis (TB) and their healthy household contacts to short-term culture filtrate (ST-CF) of Mycobacterium tuberculosis or molecular mass fractions derived from it. Our goal was to identify fractions strongly recognized by donors and differences among the donor groups of possible relevance for vaccine development. The study population consisted of 65 human immunodeficiency virus-negative donors from the Hossana Regional Hospital, Hossana, Ethiopia. Peripheral blood leukocytes from the donors were stimulated with different antigens and immune responses were determined. Household contacts produced significantly higher levels of gamma interferon (IFN-gamma) than the TB patients in response to antigens present in ST-CF and the 10 narrow-molecular-mass fractions. A similar difference in leukocyte proliferative responses to the antigens between the two groups was also found. In general, while all fractions stimulated immune responses, the highest activity was seen with the low-molecular-mass fractions, which include well-defined TB antigens such as ESAT-6. Leukocytes from contacts of TB patients with severe disease produced higher levels of antigen-specific IFN-gamma than those from contacts of patients with minimal disease. Both groups of contacts exhibited higher cell-mediated responses than the patients themselves. The enhanced immune response of healthy contacts, especially those of patients with severe disease, to secreted mycobacterial antigens is suggestive of an early stage of infection by M. tuberculosis, which could in time result in overt disease or containment of the infection. This possibility is currently being investigated by follow-up studies of the household contacts.

Human T cell responses to the ESAT-6 antigen from Mycobacterium tuberculosis.

Human T cell responses to ESAT-6 and eight synthetic overlapping peptides were investigated in tuberculosis (TB) patients and control subjects from regions of high and low endemicity for TB. ESAT-6 was recognized by 65% of all tuberculin purified protein derivative-responsive TB patients, whereas only 2 of 29 bacille Calmette-Guérin-vaccinated Danish healthy donors recognized this molecule. In Ethiopia, a high frequency (58%) of healthy contacts of TB patients recognized ESAT-6. All of the peptides were recognized by some donors, indicating that the molecule holds multiple epitopes. Danish and Ethiopian patients differed in the fine specificity of their peptide responses. Recognition of the C-terminal region (aa 72-95) was predominant in Danish patients, whereas recognition of aa 42-75 was predominant in Ethiopia. The relationship of these differences to the distribution of HLA types in the two populations is discussed. This study demonstrates that ESAT-6 is frequently recognized during early infection and holds potential as a component of a future TB-specific diagnostic reagent.

The presence of Cryptosporidium oocysts in stools of clinically diarrhoeic and normal nonhuman primates in Kenya.

A total of 114 nonhuman primates comprising 51 vervet monkeys (Cercopithecus aethiops) and 63 olive baboons (Papio anubis) were examined for Cryptosporidium oocysts using the modified Kinyoun's acid-fast staining technique. About 51.7% (59/114) of all the specimens examined, representing 78.4% (40/51) of the vervet monkeys and 30.1% (19/63) of the olive baboons were positive. Bright red, refractile Cryptosporidium oocysts were observed in the stained faecal smears against a blue background. Up to 4/6 (66.7%) of the diarrhoeic vervets and 2/3 (66.7%) baboons, respectively, were positive while the rest were negative. To the best of our knowledge, this report is the first on cryptosporidiosis in old world nonhuman primates in Kenya and probably the first report of the infection in olive baboons. Given the high frequency of oocysts in diarrhoeal specimens, the parasite may have been associated with clinical diarrhoea in the sampled animals. Cryptosporidium, which has been reported in humans in Kenya, is also suspected to occur in livestock. Its isolation from clinically ill, normal colony-borne and newly caught feral nonhuman primates has significant implications for both public health and animal agriculture in Kenya.

Heterologous protection by Leishmania donovani for Leishmania major infections in the vervet monkey model of the disease.

The study was aimed at analyzing immunological cross-reactivity between Leishmania major and Leishmania donovani and possible cross-protection between the two parasite species in the vervet monkey model of the disease. Nine vervet monkeys (Cercopithecus aethiops) from the institute animal colony were sued in the study. Five of the animals had been previously infected with L. donovani but had remained asymptomatic while the other four animals were naive and comprised the control group. Immunological responses to both L. major and L. donovani antigens in the five animals with prior exposure to L. donovani were examined before challenge. High antibody titers to the two antigens were demonstrated in an enzyme-linked immunosorbent assay, but the antibody titers to L. donovani were significantly higher than those to L. major (P < 0.005). Positive in vitro peripheral blood leucocyte (PBL) proliferation to L. major and L. donovani antigens was also demonstrated, but there was no significant difference in the response to the two antigens (P > 0.1). High and varying levels of interferon gamma (IFN-gamma) were secreted in PBL from the five vervet monkeys when stimulated with L. major antigen, but vervet monkey 1296 secreted marginal levels of IFN-gamma. When the animals were challenged intradermally with 1 x 10(5) virulent L. major promastigotes mixed with sandfly vector salivary gland lysate all four vervet monkeys in the control group developed nodules of varying sizes at the inoculation sites that eventually ulcerated. However, nodule formation and ulceration occurred at different times among these animals. The other five animals (animals with prior exposure to L. donovani) did not pick up the infection at all, but one animal from this group, vervet monkey 1296, developed a transient lesion that healed within 9 weeks, the same animal that had been shown to secrete low levels of IFN-gamma. The results demonstrate high cross-reactivity between L. donovani and L. major and that L. donovani protects against L. major infections. This finding is important for vaccine development studies against leishmaniasis.

Vaccination of vervet monkeys against cutaneous leishmaniosis using recombinant Leishmania 'major surface glycoprotein' (gp63).

Vervet monkeys (Cercopithicus aethiops) were shown to give a positive delayed-type hypersensitivity (DTH) reaction to gp63, a major surface glycoprotein of Leishmania parasites, and also produce antibodies to the molecule following a triple vaccination with a total dose of 150 micrograms of recombinant gp63 mixed with Bacille Calmette Guerin (BCG). However, peripheral blood leucocytes (PBL) from these animals neither proliferated nor produced any interferon-gamma (IFN-gamma) following in vitro stimulation with the antigen. Analysis of lymphocyte subsets following vaccination did not reveal any striking phenotypic alteration of cellular sub-populations in PBL. When vaccinated animals were rechallenged, via the needle, with virulent Leishmania major promastigotes containing salivary gland extracts from vector sandflies, only partial protection was achieved. We concluded from these studies that rgp63 produced in Escherichia coli is a safe vaccine molecule which gives only partial protection following vaccination in the vervet monkey host. The molecule requires further improvement for vaccine and/or immunodiagnosis application.

The chemotactic effect of Phlebotomus duboscqi (Diptera: Psychodidae) salivary gland lysates to murine monocytes.

The possibility that salivary gland lysates of Phlebotomus duboscqi are able to attract vertebrate monocytes was investigated. In vitro studies showed that salivary gland lysates of P. duboscqi, the vector of Leishmania major in Kenya, are chemotactic to mouse peritoneal monocytes. This attraction of monocytes by vector salivary gland lysates may form part of the mechanisms through which sandfly saliva ensures successful parasitization of macrophages in a susceptible host by Leishmania parasites.

Visceral leishmaniasis in vervet monkeys: immunological responses during asymptomatic infections.

Nine vervet monkeys (Cercopithecus aethiops) were infected intradermally with 8 x 10(7) virulent L. donovani promastigotes. Four animals developed clinical visceral leishmaniasis and died over a period of 18 months. The remaining five animals have remained asymptomatic for a period of 3 years now. Attempts to isolate parasites from spleen and liver through biopsies were fruitless. Immunological responses of these subclinically infected animals were examined. Enzyme-linked immunosorbent assay (ELISA) and western blot analyses demonstrated Leishmania specific antibodies in these animals, but the antibody titres were low. When proliferation of peripheral blood monocytes (PBMC) to Concanavalin A (Con A) of these animals was compared with control 'disease free animals' there were no significant differences in response. However, L. donovani antigen (fixed promastigotes) specific proliferation was demonstrated in the five subclinically infected animals. High and varying levels of interferon gamma (IFN-gamma) were secreted in PBMC cultures from the five vervet monkeys when stimulated with either Con A or L. donovani antigens. In control animals, IFN-gamma was only detected when PBMC were stimulated with Con A. Marked delayed-type hypersensitivity (DTH) responses were demonstrated in the five subclinically infected animals 48 h after injection with formalin fixed promastgotes. It was concluded that the visceral Leishmania disease spectrum due to L. donovani observed in humans could be induced in vervet monkeys and that L. donovani asymptomatic/cryptic infected animals have competent humoral and cellular responses to homologous parasites.

Trypanosomatid cysteine protease activity may be enhanced by a kininogen-like moiety from host serum.

African trypanosomes contain cysteine proteases (trypanopains) the activity of which can be measured by in vitro digestion of fibrinogen, after electrophoresis in fibrinogen-containing SDS/polyacrylamide gels. When assessed by this procedure, trypanopain from Trypanosoma brucei (trypanopain-Tb) is estimated to have a molecular mass of 28 kDa. However, two additional bands of trypanopain activity (87 kDa and 105 kDa) are observed if serum is added to the trypanopain before electrophoresis. Formation of the 87 and 105 kDa bands is frequently accompanied by a reduction in the intensity of the 28 kDa activity which suggests that the extra bands are complexes of the 28 kDa trypanopain-Tb and a molecule from rat serum called rat trypanopain moledulator (rTM). The rTM-induced activation of cysteine proteases is not restricted to T. brucei as it is also observed with proteases from other protozoan parasites such as bloodstream forms of Trypanosoma congolense and the mammalian-infective in vitro-derived promastigote forms of Leishmania donovani and Leishmania major. The physical properties of rTM resemble those of the kininogen family of cysteine protease inhibitors. rTM is an acidic (pI 4.7) heat-stable 68 kDa glycoprotein with 15 kDa protease-susceptible domains. This resemblance between rTM and kininogens was confirmed by the positive, albeit weak, immunoreactivity between anti-(human low-molecular-mass kininogen) antibody and rTM as well as anti-rTM antibody and human low-molecular-mass kininogen. Furthermore, commercial preparations of human-low-molecular-mass kininogen and chicken egg white cystatin mimicked rTM by forming extra bands of proteolytic activity in the presence of trypanopain-Tb. In some instances, low-molecular-mass kininogen was also observed to increase the rate of hydrolysis of 7-(benzyloxycarbonyl-phenylalanyl-arginyl-amido)-4- methylcoumarin by live T. brucei. Although this effect was rather erratic, in no instance was significant inhibition observed when this putative cysteine protease inhibitor was used under these conditions. The activation of parasite cysteine proteases by commonly accepted cysteine protease inhibitors is unexpected and may have important pathological repercussions.

Cell-mediated responses of immunized vervet monkeys to defined Leishmania T-cell epitopes.

A population of vervet monkeys was immunized with killed parasites and infected with Leishmania major promastigotes either by needle or by infected-fly bite. The responses of recovered monkeys to mitogens, killed parasites, and molecularly defined T-cell epitopes were then compared with those of control animals. Peripheral blood mononuclear cells (PBMC) from both naive and recovered animals proliferated strongly in response to both B- and T-cell mitogens, although the responses of the recovered animals were less strong than those of the naive animals. Cells from recovered vervets, but not those from naive vervets, also proliferated in response to parasite antigens and synthetic T-cell epitopes. Likewise, cells from recovered animals released gamma interferon and either interleukin 2 (IL-2) or IL-4 into culture media in response to both of the above-mentioned antigens, whereas cells from control animals did not. The fact that no IL-5 could be measured following parasite antigen or synthetic T-cell epitope stimulation of PBMC suggested that cells proliferating in response to these molecules belonged to the Th1 subset. Phenotypic analysis of the PBMC showed a marked increase in T-cell but not B-cell populations in recovered animals. Among this population was an increased number of CD45R0+ memory cells. The data from this study are in keeping with the earlier finding that vervet monkeys provide an excellent model system for leishmaniasis. Further, these data support the contention that synthetic T-cell epitopes are prime candidates for molecularly defined Leishmania vaccines.

Comparative susceptibility of African green monkeys (Cercopithecus aethiops) to experimental infection with Leishmania leishmania donovani and Leishmania leishmania infantum.

The leishmaniases are global health problems that affect both humans and animals. The availability of nonhuman primate models is desirable for such important areas as testing candidate vaccines and newly developed chemo- and immunotherapeutic agents. Visceral leishmaniasis was experimentally induced in African green monkeys (Cercopithecus aethiops) by intravenously inoculating 10(7) amastigotes/kg of body weight of either Leishmania leishmania donovani of human origin (group 1) or L. l. infantum of canine origin (group 2). The infected monkeys were monitored for 12 weeks. The monkeys developed persistent infections, became emaciated, and lost between 9 and 22% of their body weights. Splenomegaly developed by 6 to 10 weeks postinfection. All infected monkeys developed normocytic, normochromic anemia (3.5 to 3.8 x 10(6)/microliters), leukopenia (3,000 to 3,700/microliters), and neutropenia of varying severity. Hyperproteinemia with hyperglobulinemia (5.22 to 6.12 g/dl) was present in all monkeys to various degrees. Antibody responses gradually increased to peak values at 2 weeks postinfection in the L. l. donovani group and by 6 weeks postinfection in the L. l. infantum group. Lymphocyte blastogenesis proliferation responses were mildly decreased in all infected monkeys at 10 to 12 weeks postinfection. Parasite numbers were consistently higher in the livers than in spleens, and parasites were present in smears or cultures of the liver, spleen, bone marrow, and lymph nodes. Contrasting data between the two groups included 20-fold-higher parasite numbers in the livers (3.23 to 9.48 x 10(9)) and 39-fold-higher parasite numbers in the spleens (6.7 x 10(8) to 2.69 x 10(9)) of group 1. Granulomatous inflammatory reactions of various severity and intensity were observed in the liver, spleen, lymph nodes, thymus, and bone marrow of all infected monkeys. Within the granulomatous inflammatory reactions, clusters of macrophages, often containing amastigotes, were present. The morphologic changes in the bone marrow suggested a myelophthisic disease and those in lymph nodes and spleen suggested a B-cell proliferation. The clinicopathologic changes, mild suppression of cell-mediated immunity, and high antibody response in all infected monkeys indicated that African green monkeys can be a useful laboratory model for studying the clinicopathologic and immunopathologic changes induced by both L. l. donovani and L. l. infantum.

Reactivity of some monoclonal antibodies specific for human lymphocytes with vervet monkey peripheral blood mononuclear cells.

Reactivities of some monoclonal antibodies to human lymphocyte surface antigens were tested on vervet monkey peripheral blood mononuclear cells (PBMC), using flow cytometry and immunoperoxidase techniques. A number of antibodies were identified which reacted with similar populations of lymphocytes from vervet monkeys. These included antibodies that define T cells, suppressor/cytotoxic cell subset, pan B cells, monocytes and MHC class I and II. A number of anti-CD4 markers examined were unsuitable for use in the vervet system.