Peter-Henning Clausen

Monitoring of putative vectors of bluetongue virus serotype 8, Germany.

To identify the vectors of bluetongue virus (BTV) in Germany, we monitored Culicoides spp. biting midges during April 2007–May 2008. Molecular characterization of batches of midges that tested positive for BTV suggests C. obsoletus sensu stricto as a relevant vector of bluetongue disease in central Europe.

Feeding patterns of biting midges of the Culicoides obsoletus and Culicoides pulicaris groups on selected farms in Brandenburg, Germany

Host feeding patterns of engorged sibling species of the Culicoides obsoletus and Culicoides pulicaris groups captured during three nights on two selected farms maintaining either cattle, sheep, horses, and pigs (Seedorf, Brandenburg) or cattle, sheep, moufflons, and red and fallow deer (Paulinenaue, Brandenburg) were determined by polymerase chain reaction amplification using conserved primers and sets of species-specific primers derived from vertebrates mitochondrial cytochrome b. Out of a total of 177 blood meals analysed, 115 (65%) tested positive for a blood meal from vertebrates. 63.5% (n = 73) of the cyt b positive specimens could be further assigned down to the species level. Cattle appeared to be the most attractive hosts for Palaearctic biting midges (79.5%, n = 58) even if other large vertebrates were kept in their immediate vicinity. If pigs or horses were additionally maintained on a farm, they were likewise attacked by biting midges but at a distinctly smaller rate than cattle (pigs 13.7%, horses 2.7%). In this study, game animals appear to be less attractive than cattle since only a few engorged midges had taken a blood meal from red deer (4.1%). None of the blood meals analysed tested positive for sheep. Preliminary results reveal that biting midges of the C. pulicaris and C. obsoletus groups can feed on a range of vertebrate hosts but with a distinct preference for cattle even if other livestock are maintained in adjacent areas.

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.

Characterisation and validation of farmers' knowledge and practice of cattle trypanosomosis management in the cotton zone of West Africa

We carried out a knowledge, attitude, practice (KAP) survey on how farmers (n=895) manage cattle trypanosomosis in Burkina Faso, Mali and Guinea. Most farmers (96%) recognised the common signs of trypanosomosis, 70% knew the role of tsetse flies in transmitting the disease and 96% had knowledge of drugs used for treatment. Farmers reported that trypanosomosis was the most important cattle disease and estimated that 25% of their herd fall sick each year and 18% of the sick animals die. Nearly all sick animals (90%) were treated with trypanocides and most treatments were administered by untrained farmers. Giving drugs was the strategy most used as primary means of protection (50% of farmers) followed by avoiding high risk areas (32% of farmers) and keeping trypanotolerant cattle (7% of farmers). Few farmers knew about communal tsetse control methods and those who did, rarely practiced them. Farmer diagnosis of trypanosomosis in cattle presented at clinics (n=113) was in most cases (84%) supported by laboratory tests. However, the signs that most farmers considered indicative of trypanosomosis (staring coat and emaciation) were poor predictors of trypanosomosis. We tested farmer knowledge of injection sites and trypanocide dilutions (n=423 cattle), and while few (15%) farmers gave under-dosages or over-dosage (2% of farmers), injection techniques were poor with injection-related side effects in 24% of cattle treated by farmers. Despite this, therapeutic outcomes were both objectively (clinical parameters) and subjectively (carer assessment) satisfactory in 89% of cattle treated by farmers. This study found that farmers play a major role in successfully managing trypanosomosis and recommends the recognition and support for community based treatment.

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.

Prevalence of helminths in horses in the state of Brandenburg, Germany

The objective of the study was to estimate the prevalence of helminths in the horse population of the state of Brandenburg, Germany. One hundred and twenty-six horse farms in the state were selected by randomised stratified sampling. In total, 1,407 horses across all farms were examined coproscopically. The experimental unit was the horse farm: a farm was considered infected when at least one horse on the farm investigated was positive for helminth eggs. Animal details such as age, breed and sex were collected for all study horses and analysed for risk of infection. Risk was defined as horses having an above-average shedding of strongyle eggs. The following prevalence on horse farm level were established: Cyathostominae (98.4%), ascarids (16.7%), tapeworms (14.3%), pinworms (8.7%) and strongyloides (4,0%). The large strongyle Strongylus vulgaris was identified on only one farm. Liver flukes and lungworms were not found. Age, breed and sex were identified as risk factors for high shedding of strongyle eggs of individual animals: odds ratios for higher shedding intensities were 4.18 for yearlings and 2.42 for fillies compared to adult animals, and 3.69 for heavy breeds and 4.94 for wild horses compared to thoroughbreds. Mares and stallions did shed more strongyle eggs than geldings. Knowledge about the helminth prevalence will allow the issuance of specific treatment recommendations. Furthermore, the information on risk factors of individual horses will facilitate targeting single animals for selective treatments.

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.

A field study to estimate the prevalence of Trypanosoma equiperdum in Mongolian horses

From May to July 2000, a cross-sectional study was conducted to estimate the prevalence of Trypanosoma equiperdum in the horse population of the central province (Tuv aimag) of Mongolia. On average, four herds were selected from each of the 29 aimag subdivisions (119 herds). From each herd, 10 horses were sampled in proportion to sex and age categories in the respective herds (1190 horses). Sera from 1122 horses were analysed for T. equiperdum antibodies using two serological assays, the complement fixation test (CFT) and the enzyme-linked immunosorbent assay (ELISA). The crude estimate of the CFT and the ELISA seroprevalence was 7.6 and 6.7%, respectively. Concordance between the CFT and ELISA results was high (96%). The highest number of CFT positive animals was detected in one herd in Möngönmorit (6/10), followed by herds in Bayandelger (5/10) and in Bayantsagaan (5/10). Poor body condition was significantly correlated with positive serological status in both CFT and ELISA. A history of abortion appeared to be a risk factor for both CFT and ELISA seropositivity. Blood samples of all horses belonging to herds with at least three (3/10) seropositive animals (CFT and/or ELISA) were analysed by light microscopy and by PCR using a Trypanosoma (Trypanozoon) brucei specific primer pair. No trypanosomes or any other haemoparasites could be detected in Giemsa stained thin blood smears. Eight out of the 130 samples (6.2%) analysed by PCR gave positive signals. Seven out of the eight PCR positive horses were also serologically positive. One PCR (and ELISA) positive stallion from Möngönmorit showed emaciation, scrotal and preputial oedema and an oedematous skin plaque. From the serological and DNA-based results it is concluded, that trypanosome infections occur in horses in the Tuv aimag of Mongolia. Since at present neither serological nor DNA-based tests allow a subspecies specific identification within the subgenus Trypanozoon, no definitive diagnosis can be given for T. equiperdum. Whether the examined herds are infected with T. equiperdum or with T. evansi, the causative agent of surra, remains an open question. However, based on the clinical findings, the negative parasitological results and the concentration of conspicuous seroprevalences in single herds, circumstantial evidence supports the existence of infections with the causative agent of dourine.