Molecular detection of Ehrlichia ruminantium in engorged ablyomma variegatum and cattle in Ogun State, Nigeria.

Early diagnosis of Ehrlichia ruminantium in cattle is a recipe for effective control of heartwater in ruminants. Hence, we assessed the presence of E. ruminantium in the blood of cattle and the engorged Amblyomma variegatum by nested PCR. The electrophoresed PCR products obtained after primary and secondary amplifications revealed amplicon sizes of about350 bp and 280 bp respectively, which corresponded with the partial region of pSC20 gene amplified. Sequences obtained had 95-99% homology with those sequences available in GenBank. The prevalence of the E. ruminantium in ticks (50%; 126/252) was significantly (p < 0.05) higher than that in cattle blood 23.55% (61/259). The prevalence was significantly (p < 0.05) higher in ticks from adult cattle 51.47% (133/259) than those from the young cattle 44.86% (116/259) and in tick from females 54.55% (141/259) than in ticks from the males 41.38% (107/259). Alignment of autochthonous sequences revealed that the three sequences were polymorphic with two sequences showing similar nucleotides deletion at points 87-91 and 107-108. The phylogenetic trees inferred by ML showed topologies with two autochthonous sequences, one each from cattle blood and tick, clustering together in one clade and the other clustering within those sequences from South Africa and Zimbabwe in another clade. In conclusion, this study revealed a higher prevalence of E. ruminantium in engorged A. variegatum than in the blood of infected cattle. Hence, it is suggested that the amplification that targets the pCS20 gene in engorged ticks may be more suitable to determine the E. ruminantium carrier status of cattle.

Multi-locus genotyping and phylogenetic analyses of Giardia intestinalis isolates from indigenous goats in Ogun State, Nigeria.

Giardiasis is a common gastrointestinal disease of humans and various animal species worldwide. In this study, 302 stool samples were collected from West African Dwarf and Sokoto Red breeds of goats in Ogun State, Nigeria, and screened for Giardia intestinalis coproantigens using enzyme-linked immunosorbent assay (ELISA). The genotypes of G. intestinalis in faecal samples collected from 152 goats raised on selected farms were identified by polymerase chain reaction (PCR) amplification and sequence analyses of the small subunit ribosomal RNA (ssu rRNA), glutamate dehydrogenase (gdh), triosephosphate isomerase (tpi) and ß-giardin (bg) genes. Based on ELISA, an overall prevalence of 45.7% was recorded with the infection rates in pre-weaned (60.2%) and post-weaned goat kids (51.5%) being significantly (p < 0.05) higher than in adults (28.2%). Giardia intestinalis DNA was amplified in 31.6% and 29.6% of goat faeces at the ssu rRNA and gdh loci respectively. These were genotyped at the ssu rRNA locus as assemblages B (n = 13) and E (n = 36). Similar results were observed at the gdh locus except that eight isolates contained assemblage E mixed with either assemblage A or B. Additionally, sub-assemblages BI (n = 7) and BIII (n = 2) were identified with up to four single nucleotide polymorphisms (SNPs) occurring in these isolates. Multilocus genotypes (MLG) of all assemblage E isolates were identified using the ssu rRNA and gdh loci while MLG of all isolates containing assemblage B and mixed assemblages were determined after further typing at the tpi and bg loci. Forty-two MLG isolates were identified and these comprised 32, 8 and 2 (sub)-assemblage E, BI and BIII respectively. All isolates with mixed assemblages at the gdh locus were consequently designated as assemblage E by MLG. The assemblage E isolates from goats were genetically related to isolates from cattle, sheep and goats while the assemblage B isolates were related to isolates of human, pig and lemur origin. This suggests that G. intestinalis isolated from goats bred in Ogun State, Nigeria may be capable of cross-species transmission and may be of zoonotic importance.

Prevalence and multilocus genotypes of Giardia duodenalis infecting pigs in Ogun state, Nigeria.

Giardia duodenalis is an intestinal flagellated protozoan parasite that is infectious to humans and a wide range of animals worldwide. While varying prevalence rates have been reported in pigs worldwide, there are currently no published reports on the genotypes of Giardia infecting pigs in any African country. The present study is on the prevalence and genotypes of G. duodenalis in 209 pigs raised on four farms in Ogun State Nigeria. Using an enzyme-linked immunosorbent assay (ELISA) kit, Giardia duodenalis coproantigens were detected on all farms and in 25.4% (53/209) of pigs sampled. However, there was no significant influence (p > 0.05) of age, sex and stool consistencies of the pigs on the distribution of the infection. Genotyping of Giardia duodenalis in all ELISA-positive samples, achieved by the amplification of the small subunit ribosomal RNA (ssu rRNA), glutamate dehydrogenase (gdh), triosephosphate isomerase (tpi) and beta giardin (bg) genes, identified 14 and 37 assemblage B and E isolates respectively while mixed infection by both assemblages was recorded in two isolates. Novel nucleotide substitutions were identified in four assemblage B isolates at the ssu rRNA locus. Genetic diversity was observed among the assemblage B isolates after multiple alignment analyses of the gdh, tpi and bg sequences whereby sub-assemblages BII (n = 2), BIII (n = 9) and BIV (n = 3) were identified. The assemblage B isolates from pigs in this study were phylogenetically related to isolates from humans, marmoset and cattle while the assemblage E isolates were related to isolates from sheep, goats and cattle. These findings suggest that pigs in southwest Nigeria predominantly harbour G. duodenalis isolates that could be infectious to other animal species and to a lesser extent, isolates that may be of zoonotic importance.

Detection and molecular characterisation of Ehrlichia canis in naturally infected dogs in South West Nigeria.

Canine ehrlichiosis is an important tick-borne rickettsial disease mainly caused by Ehrlichia canis. This study aimed to detect and characterise E. canis in dogs in Abeokuta, Nigeria by microscopy and nested PCR. Blood samples were collected from 205 dogs, thin smears were made, field-stained, and DNA was extracted from the blood samples. A partial region of the 16S rRNA gene was amplified by polymerase chain reaction (PCR) and sequenced unidirectionally. Ehrlichial morulae were detected in three dogs (1.5%). The PCR test revealed that 47 dogs (22.9%) were positive for E. canis. The lengths of the sequences obtained range from 374 bp to 376 bp with an average G-C content of 37% and 98-99% homology with the reference sequences in GenBank. The aligned autochthonous sequences were less polymorphic. The phylogenetic analysis separated sequences reported previously in Nigeria from the autochthonous sequences. The present work shows that the strain of E. canis detected in the study area is genetically different from those reported in the northern part of Nigeria and more closely related to sequences from Brazil and India.

Multilocus genotyping of Giardia duodenalis infecting rabbits in Ogun State, Nigeria.

Giardiasis is a cosmopolitan gastrointestinal protozoal parasite that infects humans and various animals worldwide. To assess the zoonotic transmission potential of Giardia, molecular characterization is required. We are unaware of any report on the genotypes of Giardia infecting rabbits in Nigeria. Molecular detection and genotyping of Giardia duodenalis were conducted in a herd of adult Chinchilla rabbits (Oryctolagus cuniculus) managed on the Teaching and Research farm of the Federal University of Agriculture, Abeokuta located in a southwestern state of Nigeria by analysis of the small-subunit ribosomal RNA (ssu rRNA), glutamate dehydrogenase (gdh), triosephosphate isomerase (tpi) and beta-giardin (bg) genes. An overall prevalence of 72.3% (60/83) was recorded in the rabbits with no statistically significant (p > .05) influence of sex on the distribution of the infection in the herd. All the 19 isolates amplified at the four genetic loci were identified as G. duodenalis assemblage BIV by multiple alignment analysis of their consensus sequences. Novel nucleotide substitutions were identified in two isolates at the ssu rRNA locus. Phylogenetic analysis revealed that all ssu rRNA genotypes were closely related to G. duodenalis assemblage B of cattle and human origin. Findings of this study suggest that the rabbits harbour potentially zoonotic assemblage BIV that portends a high risk to students and staff of the University who are in regular contact with the animals.