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.

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.

Experimental transmission of Leishmania major to vervet monkeys (Cercopithecus aethiops) by bites of Phlebotomus duboscqi (Diptera: Psychodidae).

Experimental transmission of Leishmania major to vervet monkeys (Cercopithecus aethiops) was accomplished by bites of Phlebotomus duboscqi sandflies. Three-day-old, laboratory-reared P. duboscqi were fed on leishmanial lesions on hamsters infected with L. major. The flies were re-fed on monkeys 10 d after infection. Five adult male vervet monkeys were used in concurrent transmission trials. Two of the monkeys received subcutaneous inoculations with stationary-phase promastigotes (2 x 10(6) promastigotes in 0.1 ml of medium) on the base of the tail. Putatively infected P. duboscqi were allowed to feed on the remaining 3 monkeys at sites on the base of the tail and on the right eyebrow. Challenges by sandfly bites resulted in multiple leishmanial lesions at all bite sites and, consequently, more lesion area than was produced by needle challenges. Post-feeding dissection of sandflies indicated that multiple lesions could be caused by bites of a single fly, and that probing alone, without imbibing blood, was sufficient for transmission. These first experimental transmissions of L. major to vervets by bites of P. duboscqi demonstrate that sandfly challenge is an efficient alternative to needle challenge, making available a unique Leishmania-sandfly-non-human primate model for use in vaccine development.

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.

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.

IFN-gamma and delayed-type hypersensitivity are associated with cutaneous leishmaniasis in vervet monkeys following secondary rechallenge with Leishmania major.

IFN-gamma levels and delayed-type hypersensitivity (DTH) responses were evaluated in vervet monkeys, following secondary infection with Leishmania major (L. major). The animals had previously been vaccinated with leishmanial antigen, exposed to a primary infection and allowed to self-cure. Supernatants of peripheral blood mononuclear cell (PBMC) cultures, stimulated with either L. major antigen or Concanavalin A (Con A), were examined for the presence of IFN-gamma in a double sandwich enzyme-linked immunosorbent assay (ELISA). Significant levels of IFN-gamma were detected during active disease and following self-cure in both antigen and Con A supernatants. Higher levels of IFN-gamma were, however, present during active disease as compared with after self-cure. Positive and strong DTH responses were elicited in all experimental animals, following intradermal injection of fixed promastigotes (5 x 10(7)/animal) before rechallenge, during active infection and following self-cure. Again, strongest DTH responses were obtained during active infection as compared with the other sampling points. There was a correlation between IFN-gamma levels and DTH responses. It was concluded that IFN-gamma secretion and positive DTH responses are associated with secondary L. major infection and represent specific immunological correlates of protection in this disease model.

Generation of bovine intraspecies hybridomas with initial suppressed growth.

Bovine B-cell hybrids were generated by fusing pokeweed mitogen (PWM) activated normal bovine peripheral blood lymphocytes (PBL) with an aminopterin-sensitive bovine B-cell line BL20. The fusion partner was derived by irradiation and growth in medium containing increasing concentrations of 6-thioguanine (6TG). Resultant cells were designated ATS/BL20. Polyethylene glycol-induced (PEG) fusion was used and hybrids were selected in hypoxanthine-aminopterin-thymidine (HAT) medium. Hybrid cell growth was noticed after 4 to 6 weeks of fusion following a period of quiescence. Hybrid formation was confirmed by selection in HAT medium, expression of cytoplasmic IgM (cIgM) and surface antigens and karyotype analysis.

Selected phenotypic and cloning properties of a bovine lymphoblastoid cell line, BL20.

A bovine lymphoblastoid cell line, BL20, was shown to express a BoLA antigen and surface IgM, implying that it was probably of B-cell origin. At low density (less than 10(5)/ml), the cells failed to grow. Inclusion of growth factor-containing supernatants from concanavalin A or pokeweed mitogen (PWM)-activated bovine lymphocytes or from thymus fibroblast-like cells did not improve cloning of the cells. Feeder cells also did not enhance cloning of the cells except for bovine thymus fibroblast-like cell monolayer.

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 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.

In vitro secretion of bovine immunoglobulins during pokeweed mitogen or pokeweed mitogen and antigen activation of lymphocytes.

Activation of bovine peripheral blood mononuclear cells (PBM) towards immunoglobulin (Ig) synthesis and secretion was examined in vitro using pokeweed mitogen (PWM) or PWM plus sheep red blood cells (SRBC). Bovine PBM were composed of 7-30% B cells, 25-56% T cells and 1-4% macrophages. Cell density and PWM concentrations were critical parameters for obtaining reproducible maximum lymphocyte proliferation and polyclonal B cell activation. Inclusion of aminopterin, a folic acid antagonist, reduced cellular proliferation and viability but had no apparent effect on the time of appearance of peak proliferation. Co-culture of PBM with SRBC and PWM generated anti-SRBC specific antibodies.

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.

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.

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.

Antibodies to Leishmania tropica promastigotes during infection in mice of various genotypes.

After intradermal injection of 10(7) promastigotes of a particular isolate of the intramacrophage protozoan parasite, Leishmania tropica, the development of disease (cutaneous lesions) is much more severe in BALB/c than in three other mouse strains, C57BL/6,C3H/He and CBA/H. Using fixed promastigotes and 125I-labelled protein A in a solid-phase radioimmunoassay (RIA), titres of antibody were shown to increase up to about day 50 of infection. However, titres were not markedly different in BALB/c mice compared with the other three resistant strains, although antibody levels were highest in sera from the diseased BALB/c mice at late time points. Using isotype-specific antisera in the RIA, and sera from the day 50 time point, the isotype distribution of anti-promastigote antibodies was not noticeably different in sera from mice of the four genotypes with IgG1 and IgG2a (+/- IgG2b) antibodies predominating. It is concluded that differences in susceptibility to disease in this murine model of cutaneous leishmaniasis do not correlate with any decrease or increase in any particular antibody response to the promastigote.