Wide variation of heterozygotic genotypes of recent fasciolid hybrids from livestock in Bangladesh assessed by rDNA internal transcribed spacer region sequencing and cloning

Abstract

Fascioliasis causes high economic losses in livestock and underlies public health problems in rural areas, mainly of low-income countries. The increasing animal infection rates in Bangladesh were assessed, by focusing on host species, different parts of the country, and rDNA sequences. Fasciolid flukes were collected from buffaloes, cattle, goats and sheep from many localities to assess prevalences and intensities of infection. The nuclear rDNA internal transcribed spacer (ITS) region including ITS-1 and ITS-2 spacers was analyzed by direct sequencing and cloning, given the detection of intermediate phenotypic forms in Bangladesh. The 35.4% prevalence in goats and 55.5% in buffaloes are the highest recorded in these animals in Bangladesh. In cattle (29.3%) and sheep (26.8%) prevalences are also high for these species. These prevalences are very high when compared to lowlands at similar latitudes in neighboring India. The high prevalences and intensities appear in western Bangladesh where cross-border importation of animals from India occur. The combined haplotype CH3A of Fasciola gigantica widely found in all livestock species throughout Bangladesh fits its historical connections with the western Grand Trunk Road and the eastern Tea-Horse Road. The "pure" F. hepatica sequences only in clones from specimens showing heterozygotic positions indicate recent hybridization events with local "pure" F. gigantica, since concerted evolution did not yet have sufficient time to homogenize the rDNA operon. The detection of up to six different sequences coexisting in the cloned specimens evidences crossbreeding between hybrid parents, indicating repeated, superimposed and rapidly evolving hybridization events. The high proportion of hybrids highlights an increasing animal infection trend and human infection risk, and the need for control measures, mainly concerning goats in household farming management. ITS-1 and ITS-2 markers prove to be useful for detecting recent hybrid fasciolids. The introduction of a Fasciola species with imported livestock into a highly prevalent area of the other Fasciola species may lead to a high nucleotide variation in the species-differing positions in the extremely conserved fasciolid spacers. Results suggest that, in ancient times, frequent crossbreeding inside the same Fasciola species gave rise to the very peculiar characteristics of the present-day nuclear genome of both fasciolids.

Keywords: Animal importation; Bangladesh; Crossbreeding hybrids; Fasciola gigantica and F. hepatica; Heterozygotic sequence complexity; ITS-1 and ITS-2; Livestock; Nuclear rDNA internal transcribed spacer region; Prevalences and intensities; Sequencing and cloning.

Graphical abstract

Fig. 1

Geographic map of Bangladesh and neighboring countries, showing zones where human infection by fasciolid flukes have been reported. h.i.r. = human infection reported (h.i.r. labels noted in zones of reports except in Myanmar). Background from composed satellite map of Asia orthographic projection by NASA (full resolution of 1887 × 1962 pixels; public domain) via Wikimedia Commons.

Fig. 2

Geographic map of Bangladesh, showing the sites where the hosts of the liver flukes analyzed were collected. Coordinates and altitudes of each site are indicated in Table 1. 1) Chakaria; 2) Bhola Sadar; 3) Rangamati Sadar; 4) Sylhet Sadar; 5) Patnitola; 6) Shapahar; 7) Naogaon Sadar; 8) Ishwardi; 9) Ishwarganj; 10) Lalmonirhat Sadar; 11) Mymensingh Sadar; 12) Dhunat; 13) Shailkupa; 14) Jhenaidah Sadar. Yellow dots = rural localities where only rDNA ITS region sequences of the combined haplotype Fg-CH3A of “pure” F. gigantica were found; red dots = rural localities where heterozygotic sequences of the rDNA ITS region were found; pink dots = localities surrounding Mymensingh city where heterozygotic sequences of the rDNA ITS region were also found; pumpkin lines = routes from the West followed by F. gigantica along the Grand Trunk Road; green lines = routes from the North and the East followed by F. gigantica along the Tea-Horse Road (for more details about the Grand Trunk Road and the Tea-Horse Road, see [8]). Background from composed satellite map of Asia orthographic projection by NASA (full resolution of 1887 × 1962 pixels; public domain) via Wikimedia Commons. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)