Drug-resistant trypanosome isolates populations in dogs in Enugu North Senatorial Zone, Southeastern Nigeria.

African animal trypanosomosis is an important wasting and endemic protozoan disease causing morbidities and mortalities in animals in the sub-Saharan Africa. Currently, chemotherapy is the widely used method of African animal trypanosomosis control, especially in dogs in the sub-Saharan Africa. However, their efficacy is threatened by the emergence of drug-resistant trypanosomes owing to their extensive use and misuse over several decades amongst other factors. Thus, this study focused on the trypanocidal sensitivity and characterization of Trypanosoma species isolated from dogs in Enugu North Senatorial Zone (ENSZ), Southeastern Nigeria. Trypanosoma brucei (n = 44) and T. congolense (n = 4) isolated from naturally infected dogs in ENSZ, Southeastern Nigeria, between January and August 2016 were subjected to single dose test to assess their sensitivity to diminazene aceturate (DA) and isometamidium chloride (ISM). Subsequently, DA and multidrug-resistant isolates were further subjected to DA multi-dose test and CD50 was determined and was used to characterize the drug-resistant trypanosomes. Clones were derived from a randomly selected multidrug-resistant isolate and their sensitivity also assessed. 100% and 83.3% of T. congolense and T. brucei respectively were resistant to the trypanocides. Amongst the drug-resistant isolates, 50%, 16.7%, and 33.3% were resistant to DA, ISM, and both trypanocides respectively with CD50 ranging between 11 and 32.34 mg/kg. Drug-resistant trypanosomes were characterized into highly resistant (CD50 = 11-24.99 mg/kg) and very highly resistant (CD50 = > 25 mg/kg) trypanosome isolates. Clones also expressed high levels of resistance to both DA and ISM with CD50 values between 35.58 and 38.85 mg/kg. Trypanocidal resistance was, thus, confirmed and appears to be widespread in dogs in ENSZ, Southeastern Nigeria. The adoption of an integrated trypanosomosis control strategy in ENSZ is most desirous.

Prevalence and Molecular Identification of Trypanosomes in Dogs in Enugu North Senatorial Zone, South East Nigeria.

PURPOSE: Dogs are of immense social, psychological and economic importance in Nigeria and are severely affected by African trypanosomosis. However, the prevalence of canine African trypanosomosis (CAT) in Nigeria is underreported and the identification of the parasites relies mostly on basic morphological characteristics under the microscope, which could be misleading. The present study was carried out to determine the prevalence and characterize trypanosomes isolated from dogs in South east Nigeria. METHODS: A cross-sectional survey was carried out to determine the prevalence and molecular identification of trypanosomes in dogs in Enugu North Senatorial Zone (ENSZ), South east Nigeria. Dogs (n = 450) were randomly sampled, their blood collected and some characteristics such as sex, breed, sampling location, season and age duly noted. The blood samples were screened for trypanosomosis using standard trypanosome detection techniques. Trypanosome-positive blood samples were spotted on FTA® cards for molecular identification using nested Tubulin-PCR, ITS-PCR, TgsGP-PCR, and DNA sequencing. Some hematological parameters of the dogs such as packed cell volume (PCV), total leucocyte count (TLC), red blood cell count (RBC) were also determined. RESULTS: Of the 450 dogs sampled, 51 dogs were positive for trypanosomes with a prevalence rate of 11.3% (95% CI = 0.087-0.146). Trypanosoma brucei was the predominant trypanosome species infecting dogs in the study area. T. congolense, T. evansi, and T. vivax were also identified. The prevalence of canine trypanosomosis was significantly associated with season (χ2 = 13.821, df = 1, P = 0.0001) and the sampling location (χ2 = 6.900, df = 2, P = 0.032) while sex, breed, and age were not. The PCV and RBC of the infected dogs were significantly lower (p < 0.0001) than those of the uninfected dogs. CONCLUSIONS: CAT due to T. brucei is very prevalent in Enugu North Senatorial Zone, South east Nigeria and is associated with hematological changes. Our study also detected T. vivax in dogs in South east Nigeria which appears to be the first report of T. vivax in a dog in Nigeria.

Azithromycin and Diminazene Aceturate Combination Therapy in Experimental Multidrug-resistant Trypanosoma brucei brucei Infection in Albino Rats.

Azithromycin and diminazene aceturate combination therapy in experimental multidrug-resistant Trypanosoma brucei brucei infection in albino rats was evaluated. A total of forty-five female albino rats were used. These rats were randomly assigned to nine groups of five rats each. Group 1 was the uninfected-untreated group while groups 2 - 6 were infected with 1 × 106 trypanosomes suspended in 0.3 ml of normal saline intraperitoneally. Following infection and parasitaemia, group 2 was untreated while group 3 was treated once with 7 mg/kg diminazene aceturate. Groups 4 - 6 were treated with 10, 20 and 30 mg/kg azithromycin respectively for 7 days. Groups 7 - 9 were treated with combination of 7 mg/kg diminazene aceturate (DA) once and 10, 20 and 30 mg/kg azithromycin (AZT) respectively for 7 days. Level of parasitaemia, haematological indices (packed cell volume, total erythrocyte count, total leukocyte count, haemoglobin concentration, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration), survivability, body weight and rectal temperature were used to assess the effectiveness of the combination therapy. A significant reduction in parasitaemia levels was observed in the DA-treated group and AZT-treated group 6 while clearance of parasitaemia was observed in the DA-AZT treated groups 7 - 9 for periods between 1 and 5 days post treatment. The haematological indices and survivability of the DA-AZT treated groups were better than the DA-treated group despite the relapse recorded in those groups. One rat each in the DA-AZT combination groups survived till the end of the experiment. In conclusion, the DA-AZT combination treatment can be used as a possible adjunct to DA in the treatment of multidrug-resistant T. brucei brucei. The combination also enhanced survivability and decreased the effect of the disease in rats.

Identification of Trypanosoma brucei gambiense in naturally infected dogs in Nigeria.

BACKGROUND: Animal trypanosomosis is endemic in Nigeria, while the human disease caused by Trypanosoma brucei gambiense is rarely reported nowadays after efforts to bring it under control in the 20th century. The University of Nigeria Veterinary Teaching Hospital (UNVTH) is a reference centre located within the Nsukka area and serves Enugu and neighboring states, Benue, Kogi, Anambra and Delta. Among dogs presented to the UNVTH with canine trypanosomosis, T. brucei is frequently reported as the causative agent. However, this is by morphological identification under the microscope, which does not allow distinction of human-infective (T. b. gambiense) and non-human-infective (T. b. brucei) subspecies. Here, we used subspecies-specific PCR tests to distinguish T. b. gambiense and T. b. brucei. METHODS: Blood samples were collected on FTA cards from 19 dogs presenting with clinical signs of trypanosomosis at the UNVTH from January 2017 to December 2018. All dogs had a patent parasitaemia. DNA was extracted from the FTA cards using Chelex 100 resin and used as template for PCR. RESULTS: All infections were initially identified as belonging to subgenus Trypanozoon using a generic PCR test based on the internal transcribed spacer 1 (ITS1) of the ribosomal RNA locus and a PCR test specific for the 177 bp satellite DNA of subgenus Trypanozoon. None of the samples were positive using a specific PCR test for T. evansi Type A kinetoplast DNA minicircles. Further PCR tests specific for T. b. gambiense based on the TgsGP and AnTat 11.17 genes revealed that two of the dogs harboured T. b. gambiense. In addition to trypanosomes of subgenus Trypanozoon, T. congolense savannah was identified in one dog using a species-specific PCR test for this taxon. CONCLUSIONS: Nineteen dogs presenting with canine African trypanosomosis at UNVTH were infected with trypanosomes of the T. brucei group and in two cases the trypanosomes were further identified to subspecies T. b. gambiense using specific PCR tests. Thus T. b. gambiense is one of the parasites responsible for canine African trypanosomosis in the Nsukka area of Nigeria and represents a serious danger to human health.

Effect of diminazene aceturate, levamisole and vitamin C combination therapy in rats experimentally infected with Trypanosoma brucei brucei.

OBJECTIVE: To investigate the effect of diminazene aceturate (DA) alone or in combination with either levamisole and/or Vitamin C in albino rats experimentally infected with Trypanosoma brucei brucei. METHODS: Thirty adult male albino rats, randomly assigned into 6 groups (A-F) of 5 rats each were used. They were either infected with 1×10(6) trypanosomes intraperitoneally (groups A-E) or uninfected (group F). The different groups were treated respectively as follows: group A-with 3.5 mg/kg DA; group B-3.5 mg/kg DA and 7.5 mg/kg levamisole; group C-3.5 mg/kg DA and 100 mg/kg vitamin C; and group D-3.5 mg/kg DA and 7.5 mg/kg levamisole and 100 mg/kg vitamin C. Group E was left untreated. Parameters assessed include: rectal temperature, body weight changes, packed cell volume (PCV), Haemoglobin concentration (Hb), total leucocyte count (TLC) differential leucocyte count (DLC), parasitaemia, clinical signs and survivability. RESULTS: Average pre-patent period of 5 days was recorded. Parasites in the blood were cleared in all treated groups (A-D) within 48 hours post treatment (PT). Untreated rats in group E died between 25 and 32 days post infection (PI). Relapse was not recorded in all the treated groups (A-D). The initial reduction in PCV, Hb, TLC and increases in rectal temperature following infection were reversed by the treatments. The rats that received drug combinations (groups B, C and D) showed faster and higher recovery rates than the uninfected control and group A. CONCLUSIONS: Levamisole and/or Vitamin C combination with DA were more effective in the treatment of rats infected with Trypanosoma brucei brucei.