New supporting data to guide the use of evident toxicity in acute oral toxicity studies (OECD TG 420).

Currently there are three test guidelines (TG) for acute oral toxicity studies of substances or mixtures from the Organisation for Economic Co-operation and Development (OECD). TG 423 and TG 425 use lethality as an endpoint, while TG 420 replaces death with 'evident toxicity', defined as clear signs that exposure to a higher dose would result in death. However, the perceived subjectivity of 'evident toxicity' may be preventing wider use of TG 420. To address this, the UK National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) and the European Partnership for Alternative Approaches to Animal Testing (EPAA) collaborated to provide recommendations on the recognition of 'evident toxicity'. Historical data from acute oral toxicity studies were analysed for clinical signs at the lower dose that could have predicted death at the higher dose. Several signs including ataxia, laboured respiration, and eyes partially closed, alone or in combination, are highly predictive. Others such as lethargy, decreased respiration, and loose faeces have lower but still appreciable positive predictive value (PPV). The data has been used to develop recommendations to promote use of TG 420 and thus reduce the suffering and numbers of animals used in acute oral toxicity studies.

Prevalence of bovine trypanosomosis and assessment of trypanocidal drug resistance in tsetse infested and non-tsetse infested areas of Northwest Ethiopia.

The Northwestern region of Ethiopia is affected by both tsetse and non-tsetse transmitted trypanosomosis with a significant impact on livestock productivity. The control of trypanosomosis in Ethiopia relies on either curative or prophylactic treatment of animals with diminazene aceturate (DA) or isometamidium chloride (ISM). In the present work; questionnaire survey, cross-sectional and experimental studies were carried out to; a) assess the utilization of trypanocidal drugs; b) determine the prevalence of bovine trypanosomosis and; c) assess the drug resistant problems respectively in Tsetse and non-tsetse infested areas on NW Ethiopia. A total of 100 respondents were included for the survey and the questionnaires focused on the drug utilization practices for the control of Trypanosomosis. Blood from cattle 640 (324 cattle tested in 2011, 316 cattle tested in 2012) and 795 (390 cattle tested in 2011, 405 cattle tested in 2012) were examined from tsetse infested and non-tsetse infested areas respectively using the buffy coat technique and thin blood smear for the detection of trypanosomes and measurement of packed cell volume (PCV). For the assessment of trypanocidal drug resistance three isolates, one from tsetse (TT) and two from non-tsetse (NT) areas were used on thirty six trypanosome naïve calves. The experimental animals were divided randomly into six groups of six animals (TT-ETBS2-DA, TT-ETBS2-ISM, NT-ETBD2-DA, NT-ETBD2-ISM, NT-ETBD3-DA and NT-ETBD3-ISM), which were infected with T. vivax isolated from a tsetse-infested or non-tsetse infested area with 2 × 106 trypanosomes from donor animals, and in each case treated with higher dose of DA or ISM. The results of the questionnaire survey showed trypanosomosis was a significant animal health constraint for 84% and 100% of the farmers questioned in non-tsetse and tsetse infested areas of Northwest Ethiopia respectively. Responses on trypanocidal drug utilization practices indicated that risk factors for the development of drug resistance are common and treatment failures are frequently seen. Accordingly, the majority of farmers in tsetse infested area get trypanocides from drug stores and unauthorized sources whereas those from non-tsetse area get from veterinary clinics. Moreover, treatment administration is mainly by animal health personnel and treatment frequency is a maximum of three times/year/animal in non-tsetse area whereas it is administered mainly by the farmers more than seven times/year/animal in tsetse infested area. The prevalence of trypanosomosis varied from 17.59% in 2011 to 25.0% in 2012 in tsetse infested areas with a significant (P = 0.023) difference. Similarly, in non-tsetse infested area the prevalence was varied from 3.85% in 2011 to 5.93% in 2012 without significant rise. Trypanosoma congolense (75%) was the most prevalent followed by T. vivax (20.58%) and mixed infections (4.41%) in tsetse infested area while in non-tsetse infested area only T. vivax was detected. The overall mean PCV in parasitaemic animals (20 ± 2.3 SD) was significantly (P < 0.001) lower than that of aparasitaemic animals (27 ± 4.3 SD). The assessment of trypanocidal drug resistance tests revealed one isolate of non-tsetse infested area against DA in group NT-ETBD2-DA is resistant to the higher dose used with 3 relapsing animals (50% relapses) in the group. Another two relapses were detected one against ISM for the isolate from tsetse infested area (TT-ETBS2-ISM) and one against DA for another isolate (NT-ETBD3-DA) from the non-tsetse area. In conclusion, trypanosomosis is widely prevalent in both study areas causing significant reduction in the mean PCV values. Farmers' trypanocidal utilization practices appear to pose risks of drug resistance problems. The in vivo drug resistance tests indicated the presence of resistant parasites with the higher dose against DA for NT-ETBD2 isolate and suspected resistance problems were detected against ISM and DA for TT-ETBS2 and NT-ETBD3 isolates respectively. Therefore, trypanosomosis is a major constraint in Northwest Ethiopia and drug resistance is a threat in the control of trypanosomosis in both study areas.

Development and evaluation of an ITS1 "Touchdown" PCR for assessment of drug efficacy against animal African trypanosomosis.

Animal African trypanosomoses (AAT) are caused by flagellated protozoa of the Trypanosoma genus and contribute to considerable losses in animal production in Africa, Latin America and South East Asia. Trypanosoma congolense is considered the economically most important species. Drug resistant T. congolense strains present a threat to the control of AAT and have triggered research into discovery of novel trypanocides. In vivo assessment of trypanocidal efficacy relies on monitoring of treated animals with microscopic parasite detection methods. Since these methods have poor sensitivity, follow-up for up to 100 days after treatment is recommended to increase the chance of detecting recurrent parasitaemia waves. Molecular techniques are more amendable to high throughput processing and are generally more sensitive than microscopic detection, thus bearing the potential of shortening the 100-day follow up period. The study presents a "Touchdown" PCR targeting the internal transcribed spacer 1 of the ribosomal DNA (ITS1 TD PCR) that enables detection and discrimination of different Trypanosoma taxa in a single run due to variations in PCR product sizes. The assay achieves analytical sensitivity of 10 parasites per ml of blood for detection of T. congolense savannah type and T. brucei, and 100 parasites per ml of blood for detection of T. vivax in infected mouse blood. The ITS1 TD PCR was evaluated on cattle experimentally infected with T. congolense during an investigational new veterinary trypanocide drug efficacy study. ITS1 TD PCR demonstrated comparable performance to microscopy in verifying trypanocide treatment success, in which parasite DNA became undetectable in cured animals within two days post-treatment. ITS1 TD PCR detected parasite recrudescence three days earlier than microscopy and had a higher positivity rate than microscopy (84.85% versus 57.58%) in 66 specimens of relapsing animals collected after treatments. Therefore, ITS1 TD PCR provides a useful tool in assessment of drug efficacy against T. congolense infection in cattle. As the assay bears the potential for detection of mixed infections, it may be applicable for drug efficacy studies and diagnostic discrimination of T. vivax and T. congolense against other pathogenic trypanosomes, including T. brucei, T. evansi and T. equiperdum.