High prevalence of Toxoplasma gondii in Nigerian wild rats by molecular detection.

Toxoplasmosis has been reported in Nigeria using several diagnostic tools with high prevalence in humans and some food animals. Rodents have been recognised as vital intermediate hosts of Toxoplasma gondii. However, there is paucity of information on the occurrence of T. gondii in wild rats found in Nigeria. This study aimed at molecular detection of T. gondii in Zyzomys pedunculatus and to evaluate its involvement in the epidemiology of toxoplasmosis in Nigeria. A total of 84 rats were sampled across three states of the North Central Nigeria, and DNA was extracted from the brain, lungs, kidney and intestine of the rats for the detection of T. gondii DNA by nested PCR to amplify the multicopy B1 gene. Sixty-four of the 84 samples (76.2%) were positive for T. gondii out of which 5 samples were sequenced and had an identity score of between 97.73% and 99.35% with the reference B1 gene of T. gondii in GenBank. This study suggests Nigerian wild rats may be an important intermediate hosts of T. gondii and may play a role in the epidemiology and maintenance of T. gondii circulation in Nigeria.

Anthelmintic, antimycobacterial, antifungal, larvicidal and cytotoxic activities of acetone leaf extracts, fractions and isolated compounds from Ptaeroxylon obliquum (Rutaceae).

ETHNOPHARMACOLOGICAL RELEVANCE: Ptaeroxylon obliquum (Thunb.) Radlk, sneezewood, is a commonly used medicinal plant in South Africa for the treatment of parasitic infections in animals, tuberculosis (TB) and related symptoms, and other microbial infections. AIM OF THE STUDY: In this study anthelmintic, antifungal, antimycobacterial, larvicidal and cytotoxic activities of the acetone leaf extract of P. obliquum, solvent-solvent derived fractions of the extract and isolated compounds (obliquumol and a mixture of lupeol and ß-amyrin) were determined. MATERIALS AND METHODS: The in vitro anthelmintic activity was tested against Haemonchus contortus ova and larvae using the egg hatch inhibition and larval development assays. The antifungal activity was investigated using a serial microplate dilution method against Aspergillus fumigatus, Cryptococcus neoformans and Candida albicans. Antimycobacterial activity was evaluated by a tetrazolium violet-based broth microdilution method against pathogenic Mycobacterium tuberculosis and Mycobacterium bovis. The larvicidal activity was evaluated against Aedes aegypti. Cytotoxicity was determined using human liver (C3A) and Vero African green monkey kidney cell lines. RESULTS: Obliquumol (ptaeroxylinol acetate) was more effective than the crude acetone extract against H. contortus ova and larvae with an LC50 of 95 µg/mL against the larvae. Obliquumol had very good antifungal activity with minimal inhibitory concentrations (MIC) values from 2 to 16 µg/mL. Lupeol and ß-amyrin mixture also had good activity against C. neoformans and C. albicans with an MIC of 16 µg/mL against both fungi. The crude extract and fractions had average to weak antimycobacterial activity against the two pathogenic mycobacteria with MICs of 313-625 µg/mL for the extract and fractions tested. Obliquumol had good activity with an MIC of 63 µg/mL against pathogenic M. tuberculosis ATCC 27115. The acetone crude extract was toxic against Vero cells with an CC50 of 14.2 µg/mL. Obliquumol and the mixture of lupeol and ß-amyrin were not toxic against both Vero and C3A cells at the highest concentration tested of 200 µg/mL and had good selectivity index values against the opportunistic fungal pathogens. No tested samples had any larvicidal activity against A. aegypti at the concentrations tested. CONCLUSION: The activity noted against H. contortus may support the ethnoveterinary use of this plant against animal parasites in South Africa although aqueous extracts were not active. The activity of obliquumol against opportunistic fungal pathogens was also promising. The cytotoxicity of the acetone leaf extract, however, raises concerns about the traditional use of P. obliquum to treat a wide range of diseases although if an aqueous extract is used the toxicity concern may not be relevant.

Molecular occurrence of trypanosomes, erythrocyte and serum sialic acid concentrations of Muturu and Bunaji cattle in Benue State, Nigeria.

One hundred each, of Muturu and Bunaji cattle were screened, using polymerase chain reaction (PCR), for trypanosomes in Makurdi and Gboko Local Government Areas of Benue State, Nigeria. Erythrocyte surface sialic acid (ESSA) and free serum sialic acid (FSSA) concentrations were determined and compared in both breeds with the aim of providing baseline data for research and diagnostic purposes. Five per cent (5%) and 23% of the Muturu and Bunaji cattle, respectively, were positive for trypanosomes. The result at p=0.005 was significantly different, with p value of 0.0002 and odd ratio of 0.1762. The Trypanosoma species circulating in Benue State, as detected in the two breeds of cattle, were Trypanosoma congolense, T. vivax, T. brucei and T. evansi. This study, therefore, reports for the first time a natural infection of cattle with T. evansi and the use of a novel PCR in the diagnosis of trypanosome infections in cattle in Benue State, Nigeria. The determination of the ESSA and FSSA concentrations in Muturu cattle in Nigeria is also reported for the first time. The Muturu cattle have a significantly higher ESSA than the Bunaji cattle, this may be responsible for their relative trypanotolerance.

The antibacterial activity, antioxidant activity and selectivity index of leaf extracts of thirteen South African tree species used in ethnoveterinary medicine to treat helminth infections.

BACKGROUND: Diseases caused by bacteria remain a major challenge globally and particularly in sub-Saharan Africa. The plants used in this study have been used in South Africa to treat helminth infections in livestock and humans. In a previous study we found a correlation between antifungal and anthelmintic activity in some cases. In this study we examined other potential uses of these thirteen plant species by determining the antibacterial and antioxidant activity of the leaf acetone extracts.The antibacterial activity was determined by using a serial microdilution method against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Enterococcus faecalis. Bioautography was used to determine the number of antibacterial compounds. The antioxidant activity was determined using the ABTS and DPPH methods. RESULTS: Maesa lanceolata and Leucosidea sericea with an MIC of 0.02 mg/ml had excellent antibacterial activity against Enterococcus faecalis and Pseudomonas aeruginosa. There was a poor correlation between antioxidant activity and antibacterial activity with R2 = 0.143. This is because antibacterial activity is mainly related to non-polar compounds and antioxidant activity to polar compounds. Maesa lanceolata extracts had a low cytotoxicity with a selectivity index of 5.2, 2.6, 2.6 and 1.3 for P. aeruginosa, E. faecalis, E. coli and S. aureus respectively. Strychnos mitis extracts had a therapeutic index of 1.1 for E. coli. CONCLUSIONS: This study shows that plant extracts of some species used in ethnoveterinary medicine as anthelmintic may also have excellent antibacterial activity.

Occurrence of tick-transmitted pathogens in dogs in Jos, Plateau State, Nigeria.

BACKGROUND: Canine babesiosis caused by Babesia rossi, transmitted by Haemaphysalis elliptica in South Africa, has also been reported from Nigeria. Although H. leachi (sensu lato) is widespread in sub-Saharan Africa, published literature on the occurrence of canine babesiosis is meagre. It has been postulated that the genotype of Babesia rossi Erythrocyte Membrane Antigen 1 (BrEMA1) may be linked to virulence of the specific isolate. The primary objective of this study was to detect and characterise tick-borne pathogens in dogs presented to a veterinary hospital using molecular techniques. In B. rossi-positive specimens, we aimed to determine whether the BrEMA1 gene occurred and to compare genotypes with those found in other isolates. Lastly, we wished to identify the tick species that were recovered from the sampled dogs. METHODS: Blood specimens (n = 100) were collected during January to March 2010 from domestic dogs presented at an animal hospital in Jos, Plateau State, Nigeria. They were screened for the presence of Babesia/Theileria and Ehrlichia/Anaplasma genomic DNA using PCR and Reverse Line Blot (RLB) assays. Positive B. rossi specimens were tested for the presence of the BrEMA1gene using an RT-PCR. In addition, ticks were collected from dogs found to be infested during sampling. RESULTS: On RLB, 72 (72%) of the specimens were positive for one or more haemoparasites. Of the positive specimens, 38 (53%) were infected with B. rossi; 9 (13%) with Theileria sp. (sable); 5 (7%) with either Ehrlichia canis or Anaplasma sp. Omatjenne, respectively; 3 (4%) with Theileria equi; and 1 (1%) with B. vogeli and E. ruminantium, respectively. Co-infections were detected in 13 (18%) of the specimens. Results of RT-PCR screening for the BrEMA1 gene were negative. A total of 146 ticks belonging to 8 species were collected and identified: Rhipicephalus sanguineus 107 (73%), Haemaphysalis leachi (sensu stricto) 27 (18%), R. turanicus 3 (2%), and Amblyomma variegatum, H. elliptica, R. lunulatus, R. muhsamae and R. senegalensis 1 (1%), respectively. CONCLUSIONS: Up to 8 tick-borne pathogens possibly occur in the dog population at Jos, with B. rossi being the most prevalent. The absence of the BrEMA1 gene suggests that B. rossi occurring in that area may be less virulent than South African isolates.

Efficacy and toxicity of thirteen plant leaf acetone extracts used in ethnoveterinary medicine in South Africa on egg hatching and larval development of Haemonchus contortus.

BACKGROUND: Helminthiasis is a major limitation to the livestock industry in Africa. Haemonchus contortus is the singular most important helminth responsible for major economic losses in small ruminants. The high cost of anthelmintics to small farmers, resistance to available anthelmintics and residue problems in meat and milk consumed by humans further complicates matters. The use of plants and plant extracts as a possible source of new anthelmintics has received more interest in the last decade. Our aim was not to confirm the traditional use, but rather to determine activity of extracts.Based on our past experience acetone was used as extractant. Because it is cheaper and more reproducible to evaluate the activity of plant extracts, than doing animal studies, the activity of acetone leaf extracts of thirteen plant species used traditionally in ethnoveterinary medicine in South Africa were determined using the egg hatch assay and the larval development test. Cytotoxicity of these extracts was also evaluated using the MTT cellular assay. RESULTS: Extracts of three plant species i.e. Heteromorpha trifoliata, Maesa lanceolata and Leucosidea sericea had EC50 values of 0.62 mg/ml, 0.72 mg/ml and 1.08 mg/ml respectively for the egg hatch assay. Clausena anisata; (1.08 mg/ml) and Clerodendrum glabrum; (1.48 mg/ml) extracts were also active. In the larval development assay the H. trifoliata extract was the most effective with an EC50 of 0.64 mg/ml followed by L. sericea (1.27 mg/ml). The activities in the larval development test were generally lower in most plant species than the egg hatch assay. Based on the cytotoxicity results C. anisata was the least toxic with an LC50 of 0.17 mg/ml, while Cyathea dregei was the most toxic plant with an LC50 of 0.003 mg/ml. The C. anisata extract had the best selectivity index with a value of 0.10 and 0.08 for the two assays, followed by H. trifoliata and L. sericea with values of 0.07, 0.07 and 0.05, 0.04. The C. dregei extract had the worst selectivity index with a value of 0.00019 for both assays. CONCLUSION: The result of this study indicates which species should be further investigated in depth for isolation of compounds.

Some southern African plant species used to treat helminth infections in ethnoveterinary medicine have excellent antifungal activities.

BACKGROUND: Diseases caused by microorganisms and parasites remain a major challenge globally and particularly in sub-Saharan Africa to man and livestock. Resistance to available antimicrobials and the high cost or unavailability of antimicrobials complicates matters. Many rural people use plants to treat these infections. Because some anthelmintics e.g. benzimidazoles also have good antifungal activity we examined the antifungal activity of extracts of 13 plant species used in southern Africa to treat gastrointestinal helminth infections in livestock and in man. METHODS: Antifungal activity of acetone leaf extracts was determined by serial microdilution with tetrazolium violet as growth indicator against Aspergillus fumigatus, Cryptococcus neoformans and Candida albicans. These pathogens play an important role in opportunistic infections of immune compromised patients. Cytotoxicity was determined by MTT cellular assay. Therapeutic indices were calculated and selectivity for different pathogens determined. We proposed a method to calculate the relation between microbicidal and microbistatic activities. Total activities for different plant species were calculated. RESULTS: On the whole, all 13 extracts had good antifungal activities with MIC values as low as 0.02 mg/mL for extracts of Clausena anisata against Aspergillus fumigatus and 0.04 mg/mL for extracts of Zanthoxylum capense, Clerodendrum glabrum, and Milletia grandis, against A. fumigatus. Clausena anisata extracts had the lowest cytotoxicity (LC50) of 0.17 mg/mL, a reasonable therapeutic index (2.65) against A. fumigatus. It also had selective activity against A. fumigatus, an overall fungicidal activity of 98% and a total activity of 3395 mL/g against A. fumigatus. This means that 1 g of acetone leaf extract can be diluted to 3.4 litres and it would still inhibit the growth. Clerodendrum glabrum, Zanthoxylum capense and Milletia grandis extracts also yielded promising results. CONCLUSIONS: Some plant extracts used for treatment of parasitic infections also have good antifungal activity. Because it is much easier to isolate antifungal compounds by bioassay guided fractionation, this approach may facilitate the isolation of anthelmintic compounds from active plant extracts. The viability of this approach can be tested by isolating the antifungal compounds and then determining its anthelmintic activity. Some of these plant extracts may also be useful in combating fungal infections.