Clinical manifestation and aetiology of a genital associated disease in Olive baboons (Papio hamadryas anubis) at Lake Manyara National Park, Tanzania
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The aim of the study was to investigate a genitally associated disease and to describe its clinical manifestation and aetiology in baboons at Lake Manyara National Park in the United Republic of Tanzania.Lake Manyara National Park is located in the northern part of the country, 160 km northwest of the Mt. Kilimanjaro. It is among the smallest protected areas, but belongs to the extended ecosystem of the Serengeti, Ngorongoro Conservation Area, Lake Manyara and Tarangire National Park. The area is famous for its baboon population, the highest in Africa with an estimated number of 2,000 2,500 individuals inhabiting an area of roughly 110 km2. First sightings of baboons with severe genital ulceration were reported in 1994. This PhD study made use of 63 immobilised and sampled Olive baboons (P. h. anubis), 39 of which displayed outward signs of genital ulceration and 24 of those looking healthy. Animals with genital lesions were grouped into initial , moderate or severe affected. Immobilisation and sampling followed a standardised protocol. A combination of ketamine (10 mg/kg bw) and xylazine in a dose-range of 0.1 0.2 mg/kg bw was used as anaesthetic. Baboons were continuously monitored using a Nelcor OxiMax N-65 pulse-oximeter, starting at the time of discovery until fully recovered. The field site was left not before the animal regained consciousness. Photo documentation of genital lesions and the sampling procedures were conducted under general anaesthesia. Baboons were sampled for blood, oral and genital swaps and skin tissue biopsies. In addition, a vaginal exfoliative cytology was performed in female baboons. Sample storage and preservation followed the principle of prevention of loss through cooling-chain breakdown and protection of sample quality under extreme tropical conditions.In the laboratory tissue samples were prepared for histological and molecular biological analysis. Formalin preserved tissue was embedded into paraffin and cut into 4-µm thick layers. All slides specimens were stained with Hematoxyline-Eosin, Periodic-Acid-Shiff and Warthin-Starry-silver, according to the manufacturer s protocol. In some cases an additional Giemsa staining was performed. The light-microscopic examination was done twice, with stepwise increasing magnification from 100x to 1,000x.Immunohistochemistry for the detection of Treponema pallidum made use of rabbit-polyclonal antibodies diluted 1:500. Furthermore primary mouse-anti-human B-cell CD20 and rabbit-anti-human T-cell CD3 antibodies were used in dilutions of 1:300 and 1:50, respectively, to differentiate B- and T-cells semi-quantitative. Biotinylated secondary antibodies, streptavidin and diaminobenzidin were applied as chromagen according to the supplier´s instructions.Fluorescence-in-situ-hybridisation was used in baboons with and without genital ulceration and of different stages of clinical infections. The technique utilised the probe EUB 338 to illustrate a broad range of prokaryote structures in-situ in paraffinised tissue slides.DNA-extraction from RNA-Later conserved tissue samples was achieved by usage of a commercially available extraction kit. A broad screening for bacteria with 16S rRNA PCR was followed by PCR assays for specific pathogens. Products were cloned and sequenced. Sequences were compared with those of existing prokaryotes to the public GenBank database. In addition, all DNA-extracts were tested for the presence of T. pallidum (the bacterium that causes yaws, syphilis and bejel), Klebsiella granulomatis (the bacterium that causes donovanosis), Haemophilus ducreyi (the bacterium that causes soft chancre) and Herpesvirus papio 2 (baboon genital herpes), all known to cause genital ulceration in primates. Furthermore 26 baboons were tested for the presence of any other herpesviruses with pan-herpes consensus PCR. Products were gel-extracted and sequenced.A real-time PCR was used for the quantitative detection of T. pallidum. Primers were set to include a 67 bp sequence of a highly conserved part of the DNA polymerase I gene.Phylogenetic analysis made use of six different polymorphic regions previously determined to aid in differentiation between the T. pallidum subspecies. An alignment was used to create phylogenies constructed in Phylogenetic Analysis Using Parsimony (PAUP) software. Polymorphisms, which fell within regions in which no signature of recombination was detected, were concatenated and aligned in ClustalX version 1.83. Recombination was ruled out using genomic searches for possible donor regions at highly polymorphic sites, utilising Recombination Detection Analysis from Sequence Alignments Software, and by analysis of dN/dS ratios to detect recombination events. All excluded polymorphisms were located in hypervariable regions of the tp92 gene. Phylogenetic trees were constructed in PAUP using Treponema paraluiscuniculi, the agent that causes rabbit syphilis, as an outgroup. Maximum Parsimony and Maximum Likelihood methods were applied, with 1,000 replicates used to obtain bootstrap support and starting trees were obtained through random, step-wise addition.The group size of observed baboon troops varied from 8 to 250 individuals. Observation of genital ulcerated animals was sometimes difficult due to dense vegetation or limited access to the residence. Baboons went down 4 5 minutes after injection of the anaesthetic. The induction time (time from injection until full anaesthesised) was not significantly influenced by the dose of xylazine and the antagonisation of xylazine in a dosage range of 0.1 2.0 mg/kg bw, in single injected animals, had no significant effect on the recovery time (time from injection until fully recovered).The macro- and microscopic findings of infected baboons displayed a uniform character. The chronic-active ulcerative disease was accompanied by a massive destruction of the genitalia. Progressive scarification of the tissue lead to a vagina and anus that are permanently ajar in females, while males displayed a substantial to complete loss of the penis through autoamputation and mutilation of the external reproductive organ. The age of the baboons was not significantly correlated with the extend of genital ulceration.According to the characteristic picture of the vaginal exfoliative cytology it was possible to assign genital ulcerated female baboons, which did not show any or reduced physiological sexual swelling of the ano-genital area, to the different sexual cycle stages.The histology showed a chronic-active inflammatory-cell composition, most often condensed around the dermal blood vessels, resulting in superficial and deep perivascular dermatitis. Skin lesions were characterised by irregular epidermal proliferation of different extent with acanthosis, acantholysis and exocytosis. In severe cases the overlaying epidermis was ulcerated and a chronic granulomatous reaction ensued. The morphologic illustration of the spirochete in tissue samples was possible in 16 cases by immunohistochemistry. Significantly more often in clinically affected than clinically non-affected baboons. The pathogene was mainly distributed in the centre of the lesion, showing a clear tropism for the epidermis-dermis region (epitheliotropic pattern) and displaying a vasculotropic pattern. A single organism was characteristic spiral-like shaped and about 6.0 15.0 µm in lengh. Warthin-Starry-silver and fluorescence-in-situ-hybridisation were not able to depict treponemes. In all cases, more T- than B-cells were present among the lymphocytic inflammatory-cell population.16S rRNA PCR found a broad, but not meaningful, spectrum of bacteria. Thrirty-eight baboons tested positive for T. pallidum by PCR and/ or quantitative real-time PCR. Baboons with positive PCR result showed significantly more often clinical signs than those that were negative in PCR. In addition Olive baboons with histological signs of an infection were significantly more often positive in T. pallidum PCR than those without histological alterations. Baboons of moderate clinical infection stage had the highest number of T. pallidum gene copies, followed by those of severe and clinically initial stage. Clinically non-affected animals had the lowest number of T. pallidum copies. Particularily worth mentioning is also that 30 % of the animals without genital ulceration were PCR positive for T. pallidum.Molecular biological tests for K. granulomatis and H. ducreyi were negative in all samples. Only one baboon out of 57 (63 individuals: in three baboons DNA extraction failed, two were not sampled for skin tissue and in one animal only RNA was extracted) was positive for Herpesvirus papio 2. In addition a variety of different herpesviruses were found in 23 of 26 tested individuals. Sequence analysis of the herpesvirus genomes revealed a high prevalence of lymphocrypto- and Baboon cytomegaloviruses.This study is the first detailed description of macroscopic, histological and molecular biological findings of a genital associated disease in baboons, caused by T. pallidum. In comparison to previously published information regarding polymorphic sites useful in distinguishing the subspecies and epidemiologically it is interesting that the Lake Manyara National Park simian strain is genetically most closely related to non-venereal human T. pallidum strains (ssp. pertenue), raising the question if the potential for predominat genital associated ulceration is present in treponemes that fall outside the monophyletic ssp. pallidum clade, too. Results gained from this study indicate that the prevalence of the disease in reality is much higher than previously estimated by field observations. Though treponemal infections in wild nonhuman primates are not a new phenomenon, they never have been considered to cause genital associated diseases. Further research is needed to understand the disease s transmission dynamics, as well as its temporal and spatial distribution, especially in the context of proven zoonotic transmission of non-venereal Fribourg-Blanc simian strains of West Africa.Verknüpfung zu Publikationen oder weiteren Datensätzen
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Giessen : VVB Laufersweiler