ABSTRACT
The prevalence, morphometry and molecular characterisation of Fasciola species from slaughtered cattle and sheep, and snails from Maiduguri, Gombe and Jos were investigated. Prevalence of Fasciola spp. was studied by determination of eggs of the parasite in both faeces and bile, while morphometric description was done using standard keys and descriptions. For molecular characterisation, the first internal transcribed spacer (ITS-1) of nuclear ribosomal DNA (rDNA), 28S rDNA and NADH dehydrogenase subunit 4 (NAD4) respectively were amplified from individual Fasciola isolated from bile duct by polymerase chain reaction (PCR). All collected snails were subjected to morphological identification using standard keys. The DNA of Fasciola spp. was similarly characterised in Lymnaea natalensis by the use of 28S rDNA, while the Lymnaea (Radix) natalensis was characterised by the use of 18S rDNA. Representative amplicons of both Fasciola spp and Lymnaea natalensis were sequenced, and NCBI databases were used for sequence homology analysis using BLAST and ClustalW programs, while phylogenetic analysis was done in ApE and Molecular Evolutionary Genetics Analysis (MEGA). Combined location prevalence rate was 27.52% for cattle and 11.97% for sheep. For cattle, sex, age or breed had no significant (p≥0.05) impact on prevalence rate, while for sheep, only age had an impact, as more adult than young sheep were infected (12.62% compared to 2.63%). Jos, had a significantly (p<0.05) higher prevalence of 35.43% followed by Gombe (26.99%), while Maiduguri had the lowest (19.63%). In cattle, there was a negative association between the number of positive animals and egg per gram of faeces and bile (EPG), with Maiduguri having a mean EPG of 65.85±13.2 followed by Gombe 45.48± 10.8 and Jos the least (14.4± 1.34). For sheep, Jos had significantly (p<0.0.010) higher prevalence rate (24.35%) than both Maiduguri (6.16%) and Gombe (5.52%). Actual mean EPGs were 19.71, 36.34 and 14.47 for the respective locations. For both cattle and sheep, mean EPG were significantly (p<0.05) higher by the bile sedimentation method than by faecal analysis. For cattle, values were 41.12±5.8 versus 15.72± 3.8, while for sheep values were 50.88±15 versus 8.36±1.9 for bile and faeces respectively. Month of sampling had a significant (p≤0.05) influence on infection rate with most animals infected in January – February, being the months with highest infections. Morphological differences were observed in linear measurements and ratios. Three useful morphological parameters (BL, CL, CW) and one ratio (BW/BL) showed significant (p<0.05) variations among samples from the locations, and may therefore be relevant for phenotypic differentiation of species. The molecular identification using ITS-1, 28S rDNA and NAD4 and the sequencing revealed the presence of both F.hepatica and F.gigantica in the study areas. Analaysis of the overall genetic sequence data showed that 64.7% of the sequences of Fasciola isolates were F. gigantica, while 35.3% were F. hepatica. Of the F.hepatica isolates, 66.6% were from Jos. The phylogenetic tree constructed based upon the ITS-1 sequences revealed a close relationship (95-98%) with isolates of F. gigantica from Bukina Faso and South Africa, while the F. hepatica isolate had 84% identity with that from Iran. The 18S rRNA of Lymnaea natalensis was identified molecularly at 450bp. The sequences had between 99-100% similaritiesamong themselves and 98-100% with other deposited reference Lymnaea natalensis sequences. All our Lymnaea (Radix) natalensis sequences formed a clade different from the clade formed by other reference sequences. Lymnaea (Radix) natalensis sequences from Nigeria seem to clade separately from deposited sequences from GenBank. Conclusively, the prevalence of Fasciola spp indicates a high morbidity in the sampled animals. The study had shown that, a well defined relationship exists between egg counts in bile and faeces of cattle, but not sheep in this study.
This study also confirmed the presence of F.gigantica and strongly suggests the existence of F.hepatica for the first time, to the best of our knowledge, using molecular tools. The findings ofthe two species of Fasciola (F. hepatica and F.gigantica) may have implications for livestock and human infections and vaccine types to be developed in the control of fasciolosis in the study locations and Nigeria.The findings also confirmed the existence of Lymnaea (Radix) natalensis and its role as intermediate host of Fasciola spp in the study areas. In addition, experimental infection of different breeds of cattle and sheep with Fasciola spp, complete sequence of the ITS-1, 28S rDNA, NAD4 of Fasciola spp and 18S rDNA of Lymnaea (Radix) natalensis and investigation of snails such as Melanoides tuberculata and Biomphalaria pfeifferei as potential intermediate hosts of Fasciola spp are recommended for further studies.
