Multiple host feeding in Glossina palpalis gambiensis and Glossina tachinoides in southeast Mali.

Changes in agricultural practices and the resulting extinction of wildlife have led to the reduction or disappearance of savannah tsetse species. Riparian tsetse such as Glossina palpalis gambiensis Vanderplank 1949 and Glossina tachinoides Westwood 1850 (Diptera: Glossinidae) continue to persist in peridomestic sites, transmitting trypanosomiasis. At present, little is known about interspecies differences in feeding behaviour in these two species in southeast Mali, or of the phenomenon of multiple bloodmeals. To study these topics, 279 samples of G. p. gambiensis and G. tachinoides containing host DNA, caught in the Sikasso region between November 2008 and April 2009, were analysed by applying host species-specific primers and sequencing. Human accounted for > 66% of G. p. gambiensis bloodmeals, whereas G. tachinoides contained in equal parts DNA of human, cattle or both, showing a significantly higher proportion of multiple host use. Further, the trypanosome infection rate was found to be three-fold higher in G. tachinoides. Logistic regression analysis revealed double-feeding and infection to be independent of one another, but showed infection to be correlated with engorgement in G. p. gambiensis and female sex in G. tachinoides. Enhanced host-seeking activities paired with the high trypanosome infection rate found in G. tachinoides would indicate that this species has a higher vectorial capacity than G. p. gambiensis.

Polymerase chain reaction and DNA probe hybridization to assess the efficacy of diminazene treatment in Trypanosoma brucei-infected cattle.

Four of eight Ankole longhorn cattle experimentally infected with Trypanosoma brucei were treated with 7 mg/kg diminazene aceturate (Berenil, Hoechst AG, Germany) at day 71 postinfection. The trypanocidal activity was monitored using polymerase chain reaction (PCR) and DNA probe hybridization. When extracted parasite DNA (without host DNA) was used, as little as 1 fg per reaction, which is equivalent to about 1-10% of the DNA in a single trypanosome, produced a specific product that was visible as a 177-bp band in an agarose gel. In infected cattle, specific PCR products could be amplified at as early as 1 day postinfection. PCR signals remained positive during infection, except in one sample, although aparasitemic phases occurred. In cases where treatment resulted in a significant clinical improvement, PCR signals disappeared at 3-4 days after the administration of the drug. By contrast, in cattle that showed clinical signs of CNS involvement after treatment, although aparasitemic, and died before the termination of the experiment, specific products could be amplified on several occasions following treatment. The PCR signals generated after treatment could be further enhanced by subsequent slot-blot hybridization with a T. brucei-specific DNA probe. We conclude that PCR coupled with DNA probe hybridization provides a highly sensitive tool for the assessment of therapeutic efficiency and disease progression in trypanosome infections, especially in chronic infections when the level of parasitemia is low or when trypanosomes are sequestered at cryptic sites.

Investigation of skin samples from red foxes (Vulpes vulpes) in eastern Brandenburg (Germany) for the detection of Borrelia burgdorferi s. l.

During earlier investigations a high prevalence of Borrelia (B.) burgdorferi s. l. in unfed Ixodes (I.) ricinus ticks in the Federal State of Brandenburg has been demonstrated. In the present study skin samples were obtained from 100 red foxes (Vulpes vulpes) from the districts where the highest B. burgdorferi prevalences had previously been found (i.e. Uckermark, Barnim, Märkisch-Oderland, Oder-Spree). BSK- and MKP-medium including inhibitory substances were used for cultivation of spirochaetes. Non-motile spirochaete-like organisms were observed in 26% of the samples. Additionally, by subcultures it was not possible to obtain motile helical forms characteristic for B. burgdorferi. On tryptose agar, the bacteria which produced nonmotile forms appeared as corynebacterium-like-colonies. Investigations by electron microscopy showed that the immobile spiral forms were giant whips (flagellae) which belonged to the contaminant flora. These forms proved to be negative for B. burgdorferi s. l. by the use of a nested-PCR. In a further study, the same skin samples were investigated for the presence of B. burgdorferi s. l.-DNA using a nested-PCR. Seven out of 100 samples were positive.

Clinical follow-up examination after treatment of canine leishmaniasis.

In 16 dogs, the diagnosis of canine leishmaniasis could be detected by direct microscopic identification, by determination of the antibody titre or by PCR method (peripheral blood/bone marrow). On the basis of the clinical and laboratory diagnostic results 9 cases of the cutaneous type and 7 dogs of the combined cutaneous-visceral type (+ mono- or polyarthritis, hepatopathy and/or renal insufficiency as well as occular manifestation) have been classified. Therapy was: GLUCANTIME in 6 dogs, allopurinol in 3 dogs as single agent, combination-therapy GLUCANTIME and allopurinol in 7 dogs. During GLUCANTIME-treatment the following adverse reactions could be observed: general weakness, reduced food intake up to anorexia, vomiting and diarrhoea. Laboratory parameters showed sporadically leucopenia or pancreatitis. Adverse reactions during allopurinol therapy were: vomitus/diarrhoea or urine concrements. One dog with GLUCANTIME therapy, 2 dogs with allopurinol as well as 2 dogs with combination therapy are clinically symptom-free at the moment (peripheral blood and bone marrow: PCR negative). The remaining 11 patients showed a good to very good improvement of the clinical symptoms. However, since the peripheral blood respectively the bone marrow continue to be PCR positive, relapses have to be expected in these dogs.

PCR follow-up examination after treatment of canine leishmaniosis (CaL).

A study has been performed to investigate the usefulness of the polymerase chain reaction (PCR) for both the diagnosis and the follow-up after treatment of canine leishmaniosis (CaL). Blood samples (PBL) and/or bone marrow aspirates (BM) could be examined in a total of 18 confirmed cases of primary CaL. PBL was PCR-positive in 87%, whereas the BM was found to be positive in all cases (n=14) tested. PBL and BM from a total of 13 patients were submitted to PCR examinations after meglumine antimoniate (Glucantime) treatment. Only one dog showed a negative PCR after 2 treatment cycles (days 1-2: 50 mg/kg bw; days 3-10: 100 mg/kg bw). Examination of the PBL and BM after 15 months remained further PCR negative. All other dogs, from which four were pretreated with allopurinol up to 5 weeks, continued to be positive (92%) at least in the BM. Ten dogs could be monitored by means of the PCR after allopurinol treatment (2 x 10 mg/kg/bw/day/) either as a monodrug therapy in seven or as a successive combination with Glucantime in three cases. Two out of three dogs which showed good clinical improvement after daily administration for five weeks were likewise PCR negative in both the PBL and the BM. The four other dogs remained positive after the single therapy with allopurinol up to 5 weeks. A further three dogs were treated with allopurinol for 5 weeks, 6 and 20 month, respectively, after being clinically cured with Glucantime. Two of them were PCR negative in the PBL and BM after 5 weeks and 20 month, respectively. The results presented suggest that longterm treatment with allopurinol has some therapeutic benefit in CaL especially when administered following treatment with the pentavalent antimonial Glucantime.

[The detection of Toxoplasma gondii in abortion tissues of sheep using the polymerase chain reaction]. / Der Nachweis von Toxoplasma gondii in Abortgeweben vom Schaf mittels der Polymerase-Kettenreaktion.

A polymerase chain reaction was applied to detect Toxoplasma gondii DNA in placental and fetal tissue samples of 47 unselected ovine abortions of the lambing season 1990/91 (Baden-Württemberg, Rhineland, Hesse). For the amplification a 190 bp or 223 bp sequence of the B1-gene of T. gondii was selected as the target sequence. Both sequences were detected in five abortions. All positive results were immunohistochemically confirmed using the peroxidase antiperoxidase technique (PAP-staining). Thus, in Germany, too, T. gondii infection in sheep during pregnancy should be considered as a possible cause of abortions, particularly in case of abortions of unknown genesis.

Production of interferon by Theileria annulata- and T. parva-infected bovine lymphoid cell lines.

Theileria annulata and T. parva-infected lymphoblastoid cells were examined for their capacity to produce interferon (IFN). Supernatants of such cells were tested in biological assay for their antiviral activity. Only T. parva-infected cells of T-cell origin were capable of producing IFN-gamma. Supernatants of some but not all T. annulata-infected cells showed also antiviral activity, which was greatly reduced after exposure to a pH of 2. Northern-blot analysis of the cells using an IFN-gamma cDNA probe confirmed the results obtained for T. parva-infected cells in a biological assay. No IFN-gamma mRNA was detected in T. annulata-infected cells. The importance of IFN for the pathogenesis of theileriosis is discussed.

Application of the polymerase chain reaction in the diagnosis of pulmonary toxoplasmosis in immunocompromised patients.

Bronchoalveolar lavage (BAL) fluid from 47 immunocompromised patients (26 with AIDS and 21 patients on immunosuppressive therapy) was analysed for the presence of Toxoplasma gondii DNA by means of the polymerase chain reaction (PCR). Specific target DNA derived from the B1 and P30 gene of Toxoplasma gondii was detected in BAL fluids from three patients with AIDS (6.4%). Pneumonia as the presenting feature of disseminated toxoplasmosis was confirmed by both clinical findings and by detection of Toxoplasma gondii DNA in blood obtained from two patients. The findings indicate that PCR has potential value in the detection of Toxoplasma gondii as an etiologic agent of atypical pneumonia in immunocompromised patients.

In vitro proliferative and cytotoxic responses of PBL from Theileria annulata-immune cattle.

Peripheral blood lymphocytes were isolated from healthy calves and were subsequently infected with sporozoites of Theileria annulata in vitro. The infected cells were passaged for 50 times and thereafter inoculated into animals from which they were previously isolated. Within 4-5 days, schizont-containing cells were demonstrable in the lymph nodes of all animals. Few days later, merozoites were detected in erythrocytes. A slight decrease in the counts of lymphocytes and leucocytes was also found. After 2 months these animals and a group of uninfected calves were heavily infected by tick-infestation and showed severe symptoms of theileriosis with 60% schizont-containing cells in the lymph nodes and a parasitaemia of about 35%. Because of the severity of the infection, all control calves were treated with Halofuginone. In contrast, the initially immunized cattle (by inoculation of culture cells), survived the infection without chemotherapy. Less than 10% of their lymph node cells contained schizonts, whereas less than 1% of their erythrocytes were found to be infected with merozoites. In all immunized animals, specific cytotoxic PBL, with the capacity to lyse autologous but not allogeneic infected cells, were demonstrated. In addition, a population of PBL were found to be able to inhibit the growth of T.annulata-infected culture cells in vitro. However, in comparison to PBL of immune animals, PBL of acute infected calves were superior in their capacity to inhibit the proliferation of schizont-containing cells. In mixed lymphocyte reactions, T. annulata-infected cells could induce a more pronounced proliferative response in PBL from immune than in PBL of uninfected animals.

Studies on the sequence of variable antigen types in ponies infected with a clone of Trypanosoma evansi.

The sequential appearance of variable antigen types (VATs) of a clone of Trypanosoma evansi was studied in four ponies. Using luminol-dependent chemiluminescence, VAT populations which had been isolated from parasitemic peaks of single ponies, were tested for specificity with serum samples collected from other ponies. When antibody activity was demonstrated in a combination of trypanosomes and serum, it was concluded that a major VAT appeared in common. In the serum of all animals antibody activity was demonstrated to all VAT populations isolated from the other ponies during the first 4 weeks of infection, indicating that up to this moment in all four animals the same major VATs developed. The sequence of major VATs was very similar in all ponies. Several parasitemic waves consisted of more than one major VAT, and in another pony a certain major VAT developed either in the same or in a neighbouring wave of the parasitemia.

Variant specific opsonization of Trypanosoma evansi measured by luminol-dependent chemiluminescence.

Using luminol-dependent chemiluminescence (LCL), the specificity of antibodies to variable antigen type (VAT)-populations of Trypanosoma evansi was studied in four infected ponies. Trypanosomes of each wave of parasitemia were isolated and multiplied in irradiated mice. Their opsonization by serum collected during the infection was investigated with LCL and results for isolated VAT-populations are shown in the paper. Antibodies specific to each VAT-population were first found three days after the maximum of a parasitemic wave. There was no cross reactivity between different VAT-populations. LCL proved to be a rapid and automatic method for the demonstration of antibodies with specificity to variable antigen types of trypanosomes.