Dieter Mehlitz

Standardised tests in mice and cattle for the detection of drug resistance in tsetse-transmitted trypanosomes of African domestic cattle

Resistance to the drugs used to control African animal trypanosomosis is increasingly recognised as a constraint to livestock production in sub-Saharan Africa. The most commonly used tests for detection of trypanocidal drug resistance are tests using mice or ruminants, but these suffer from lack of standardisation and hence it may be difficult to compare the results of different investigators. Tests in mice are less expensive than tests in ruminants, but while tests in mice they may be useful as a general guide to resistance in a geographic area they should not be extrapolated to cattle on an individual trypanosome level. Moreover, the commonly used protocols are too laborious for their application to large number of trypanosome isolates on an area-wide basis. This paper presents guidelines for standardised testing of trypanocidal drugs in vivo, and introduces a simplified single-dose test for use in mice, which is convenient for use in areas with limited laboratory facilities. The single-dose test is appropriate for characterisation of geographic areas in terms of trypanocidal drug resistance using large numbers of trypanosome isolates, for making comparisons between areas, and for monitoring changes in trypanocidal drug resistance over time. Multiple-dose tests may be used to determine the degree of resistance of individual stabilates to be determined precisely in mice are also described, but for logistical reasons these will rarely be conducted on more than a few stabilates, and testing of a larger number of stabilates in the single-dose test will generally provide more useful information. Finally, we describe tests in cattle that may be used to determine the efficacy of recommended curative doses of trypanocidal drugs for the treatment of infection with individual trypanosome isolates, including Trypanosoma vivax, which is rarely infective for mice.

Use of a PCR Assay for the Specific and Sensitive Detection of Trypanosoma Spp. in Naturally Infected Dairy Cattle in Peri-urban Kampala, Ugandaa

ABSTRACT: The objective of this study was to compare the sensitivity and specificity of the polymerase chain reaction (PCR) with the hematocrit centrifugation technique (HCT) and the mini‐anion‐exchange centrifugation technique (m‐AECT) for diagnosis of trypanosome infections in livestock. In a cross‐sectional study, 486 cattle from 50 randomly selected farms in Mukono County, Uganda were investigated in June 1994. The direct parasitological techniques were performed in the field, resulting in 45 (9.3%) animals positive by HCT and 78 (16%) positive by m‐AECT. The total prevalence (combined evidence of HCT and m‐AECT) was 18.9%, with 78.2%Trypanosoma brucei only, 10.9%T. vivax and 10.9% mixed (T. brucei/T. vivax) infections. Trypanosomes of the subgenus Nannomonas were not detected. DNA was prepared by lysis from 181 randomly selected blood samples and amplified by PCR using species‐specific oligonucleotide primers. Overall, the PCR gave positive results in 63 (34.8%) blood samples, with 76.2% positive only for T. brucei, 20.6% positive only for T. vivax and 3.2% positive for mixed (T. brucei/T. vivax) infections. The preliminary results from this study demonstrate that the detection rate of PCR is about two times higher than that of the direct parasitological techniques, suggesting a higher sensitivity. The higher proportion of T. vivax infections detected by PCR as compared to HCT/m‐AECT is likely to be due to false parasitological classifications which might occur under field conditions.

Multiple-drug resistant Trypanosoma congolense populations in village cattle of Metekel district, north-west Ethiopia.

Investigations were carried out to determine the prophylactic activity of isometamidium chloride in village populations of cattle naturally infected with trypanosomes in Metekel district, northwest Ethiopia. In a cross-sectional study in March 1997, 484 randomly selected cattle from four villages were examined for trypanosome infections by the dark ground/phase contrast buffy coat technique (BCT). The trypanosome prevalence was 17.2%. Trypanosoma congolense was the dominant species accounting for 47.6% of the overall infections. Fifty parasitaemic cattle from two villages were treated with isometainidium chloride (Trypamidium(R)) at a prophylactic dose of 1.0 mg/kg body weight (b.w.) and thereafter monitored on a monthly basis for parasitaemia. Trypanosomes were detected in six cattle within 1 month and in 18 cattle within 2 months of treatment. Twenty three percent (6/26) of cattle infected with T. congolense at the time of treatment were detected parasitaemic with this trypanosome species 1 month after treatment. Mice were infected with three T. congolense isolates obtained from cattle which were detected parasitaemic within one or 2 months after isometamidium treatment. The mice were subsequently treated with ranges of doses of isometamidium chloride or diminazene aceturate (Berenil(R)) and thereafter monitored for parasitaemia for a period of 60 days. Isometamidium chloride at doses of 0.5-4.0 mg/kg b.w. and diminazene aceturate at doses of 3.5-28.0 mg/kg b.w. failed to cure T. congolense infections in any of the animals. Three clones were derived from one of the isolates; each clone expressed high levels of resistance to both trypanocides when tested in mice. Based on these results it is concluded that the prophylactic activity of isometamidium is greatly reduced for some of the T. congolense populations present in the area, and in addition there is resistance to diminazene aceturate in this trypanosome species.

Enzyme polymorphism and the identity of Trypanosoma brucei gambiense.

Thirty-two isolates from man in known areas of Gambian trypanosomiasis, in the Sudan, Kenya, Zaire, Nigeria, Ivory Coast, Burkina Faso, Liberia and Senegal, were examined by isoenzyme electrophoresis of 11 enzymes. Comparisons were also made with our previously published results on 23 other stocks of similar origins, which had been examined in the same manner. All those stocks of low initial virulence to laboratory rodents, which thus conform to the accepted view of the behaviour of Trypanosoma brucei gambiense can be identified by characteristic combinations of enzyme patterns, especially certain aminotransferase markers. A limited study of superoxide dismutase polymorphism suggested a further marker of value. The isolates of high initial virulence to rodents, which are thus behaviourally akin to T. b. rhodesiense, did not share these characteristics. We conclude that there exists a homogeneous group of trypanosomes of wide dispersion throughout tropical Africa, characterized by certain isoenzyme combinations and low initial virulence to rodents, which corresponds to the classical concept of T. b. gambiense. The features of limited antigenic repertoire, high resistance to normal human serum and restriction fragment length polymorphisms of the genes for certain variant surface glycoproteins also appear to be characteristic of this group.

Field studies of drug-resistant cattle trypanosomes in Kénédougou Province, Burkina Faso.

Field studies were conducted to assess the occurrence of resistance to isometamidium chloride and diminazene aceturate in trypanosomes infecting cattle in Kénédougou Province of Burkina Faso. Forty-five of the 166 villages in Kénédougou were randomly sampled and visited to assess livestock numbers, trypanosomosis risk, and tsetse challenge. The proportion of cattle infections associated with drug-resistant trypanosomes was assessed in the nine villages with the highest trypanosome infection prevalence and one village with a confirmed history of drug-resistant infections. These studies showed that resistance to both isometamidium and diminazene was widespread. However, there was considerable variation between villages in drug-resistance parameters, with the proportion of treated cattle with trypanosome infections 3 months after isometamidium prophylaxis varying from 6.9 to 63.8% and the proportion of cattle having infections 2 weeks after treatment with diminazene varying from 0 to 36.8%. The demonstration of widespread resistance to both isometamidium and diminazene has important implications, as administration of trypanocides is the most commonly employed method to control trypanosomosis in this area.

Seasonal dynamics of biting midges (Diptera: Ceratopogonidae, Culicoides spp.) on dairy farms of Central Germany during the 2007/2008 epidemic of bluetongue

The unforeseen outbreak of bluetongue in north-western Europe in August 2006 raised the question, which Culicoides spp. were involved in the transmission of bluetongue virus (BTV). Based on the decision 2007/20/EU of December 2006, a large-scale entomological surveillance programme was initiated in the five affected EU member states including Germany. This paper reports on the entomological findings obtained from March/April 2007 to May 2008 at 15 sampling sites in the federal states of Lower Saxony (eastern region), Mecklenburg-Western Pomerania, Brandenburg and Saxony-Anhalt: The number of captured biting midges in one trap varied from none or few Culicoides during winter (December 2007 to April 2008) to up to more than 12,500 individuals during summer and autumn. Catches of the C. obsoletus group were consistently higher than those of the C. pulicaris group. C. imicola, the principal afro-asiatic vector of BTV, was not detected. High numbers of midges were caught inside the cattle sheds. Eleven pools of biting midges were RT-PCR-positive to BTV-8 including pools of non-engorged midges of the C. obsoletus and of the C. pulicaris groups. The first BTV-genome positive pool of midges was detected in August 2007; the remaining genome-positive pools were detected during October and November 2007.

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

Abstract 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.

Impact of biological factors on the interpretation of bovine trypanosomosis serology

A total of 457 cattle from dairy farms in Mukono County, Uganda, were investigated for Trypanosoma antibodies by ELISA. The objective of the study was to identify explanatory covariate factors for seropositivity among nine farm-specific and four animal-specific variables. We used logistic regression models for parasitological and serological outcome variables and then compared the adjusted odds ratios for explanatory factors between the models. Age is positively correlated with seropositivity but not with the detection of the parasite. Therefore, age group-specific cut-off values were established using mixture-distribution analysis. This procedure, as well as a mixture-distribution-derived cut-off value for the total sample, resulted in a greater relative efficiency of the ELISA as compared to conventional interpretation (cut-off value defined using non-exposed negative controls). The relevance of age and other biological factors for the serological status is briefly discussed.

An appraisal of current and new techniques intended to protect bulls against Culicoides and other haematophagous nematocera: the case of Schmergow, Brandenburg, Germany

The outbreak of bluetongue (BTV-8) in many parts of north-western Europe led to efforts to curb the spread of the disease, particularly in farms with valuable livestock, as on a stud bull farm in Schmergow, Brandenburg, Germany. In the abundance of the putative BT vectors, Palaearctic Culicoides species, several vector control methods were applied in the hope for a reduction of the target insect populations. Insecticide-impregnated ear tags and regular treatments at 6-week intervals of all bulls with deltamethrin pour on were expected to achieve the desired control of the biting midges. Additionally, insecticide-treated mosquito fences circumventing much of the pens were tried for the first time against Culicoides. Two suction black-light traps (BioGents® sentinel traps) helped to monitor the densities of Culicoides and other haematophagous nematocera during the trial period from July to December 2007. Despite all efforts, the densities of Culicoides were not distinctly reduced. Several thousand midges were repeatedly recorded during one-night catches. Examinations of midges and other haematophagous nematocera (Aedes and Anopheles species) revealed high percentages of successful feedings between 10% and 35% for Culicoides and more than 50% for Aedes and Anopheles species. Since all insects were caught inside the pens, the concept of endophily vs exophily or endophagy vs exophagy for some Culicoides species needs to be revised accordingly. Also, stabling of valuable livestock does not reduce the host–vector interface and, hence, the risk of transmission of BT.

Managing Tsetse Transmitted Trypanosomosis by Insecticide Treated Nets - an Affordable and Sustainable Method for Resource Poor Pig Farmers in Ghana

An outbreak of tsetse-transmitted trypanosomiasis resulted in more than 50% losses of domestic pigs in the Eastern Region of Ghana (source: Veterinary Services, Accra; April 2007). In a control trial from May 4th–October 10th 2007, the efficacy of insecticide-treated mosquito fences to control tsetse was assessed. Two villages were selected – one serving as control with 14 pigsties and one experimental village where 24 pigsties were protected with insecticide treated mosquito fences. The 100 cm high, 150denier polyester fences with 100 mg/m2 deltamethrin and a UV protector were attached to surrounding timber poles and planks. Bi-monthly monitoring of tsetse densities with 10 geo-referenced bi-conical traps per village showed a reduction of more than 90% in the protected village within two months. Further reductions exceeding 95% were recorded during subsequent months. The tsetse population in the control village was not affected, only displaying seasonal variations. Fifty pigs from each village were ear-tagged and given a single curative treatment with diminazene aceturate (3.5 mg/kg bw) after their blood samples had been taken. The initial trypanosome prevalence amounted to 76% and 72% of protected and control animals, respectively, and decreased to 16% in protected as opposed to 84% in control pigs three months after intervention. After six months 8% of the protected pigs were infected contrasting with 60% in the control group.