Molecular analysis of selected paramphistome isolates from cattle in southern Africa

Abstract

Paramphistomes are parasites of domestic and wild ruminants, the effects of which in animal health remain underestimated. Very few studies in Africa have been done using molecular techniques to resolve situations associated with taxonomical groupings and epidemiology of these parasites. In this study, the genetic variability of nine representative paramphistome isolates collected from southern African countries, namely Botswana, South Africa, Zambia and Zimbabwe, was assessed using both morphological and internal transcribed spacer 2 (ITS2) rDNA sequence data. Morphological characterization and identification were carried out using median sagittal sections of the paramphistomes. DNA of the individual paramphistomes was isolated, the ITS2 rDNA was amplified, purified and sequenced. The sequences were submitted to GenBank, which assigned them the following accession numbers: KP639631, KP639630, KP639632, KP639633, KP639634, KP639635, KP639636, KP639637 and KP639638. These sequences were used for phylogenetic analysis using MEGA 6. Morphological characterization revealed three species of paramphistomes belonging to three different sub-families: one Stephanopharynx compactus isolate, a member of the Stephanopharyngidae sub-family; one Carmyerius dollfusi isolate, a member of the Gastrothylacidae sub-family; and seven Calicophoron microbothrium isolates belonging to the Paramphistomidae sub-family. ITS2 sequence analysis using BlastN results indicated that this is the first report of S. compactus (KP639630) and C. dollfusi (KP639636). Phylogenetic reconstruction of the paramphistome isolates revealed three separate clades representing the three species. However, the clade with all the C. microbothrium isolates was the only one that was supported by a higher bootstrap value of 92%, although there was no differentiation of the isolates according to geographical locations. The low divergence values on the ITS2 sequences of the C. microbothrium isolates indicate that ITS rDNA sequences can be used as a molecular tool to infer knowledge for resolving taxonomic groupings.

Loads of trematodes: discovering hidden diversity of paramphistomoids in Kenyan ruminants

“…1) by matching genetic sequences (<0•6% for ITS2 and <1•3% cox1) collected from cercariae and adults: (1) ITS2 sequences from cercariae from C. natalensis (GU735645) collected in Kenya grouped with sequences from adult worms we recovered from cattle (clade 2), provisionally identified as Carmyerius exporous (Dinnik and Dinnik, 1960). 2Cercariae (clade 4) we collected from C. natalensis matched adults collected in this study as well as two adults from Botswana (KP639636) and Kenya (GU735658) identified as Carmyerius dollfusi by Dube et al (2015). The latter species was synonymized with C. mancupatus (Sey, 1991), a species known to be transmitted by C. natalensis (Dinnik, 1965).…”

“…The phylogenetic trees were useful, however, in providing preliminary hypotheses for how the various clades were related to one another (see the paragraph below). Relative to other paramphistomoid molecular phylogenetic studies involving specimens from African ruminants and snails, we recovered five out of the six previously reported taxa from Kenya, Egypt and Tanzania noted by Lotfy et al (2010), three of the three identified taxa from Zimbabwe, Zambia and Botswana (Dube et al 2015) and one of the two identified taxa from Algeria (Titi et al 2014). The extent of overlap among specimens recovered from all four studies suggests that at least some of the species have broad distributions in Africa.…”

“…Previous studies have used the easily obtained ITS2 sequence as a molecular marker to distinguish among paramphistomoid species (Itagaki et al 2003 ; Rinaldi et al 2005 ; Goswami et al 2009 ; Lotfy et al 2010 ; Sanabria et al 2011 ; Ichikawa et al 2013 ; Shylla et al 2013 ; Ghatani et al 2014 ; Dube et al 2015 ). ITS2 is helpful for distinguishing paramphistomoid genera and differentiating more divergent species within a genus (Rinaldi et al 2005 ; Ghatani et al 2012 ).…”

“…The number of studies that use molecular techniques to provide assessments of the diversity of paramphistomoids have in general been limited, especially so for African species (Lotfy et al 2010 ; Mansour et al 2014 ; Sibula et al . 2014 ; Titi et al 2014 ; Dube et al 2015 ).…”

“…Given the inherent difficulties in identification, coupled with a growing list of studies from other digenean groups documenting the presence of cryptic species (Detwiler et al 2012;Herrmann et al 2014;McNamara et al 2014), paramphistomoids are ideal for studies attempting to meld traditional morphological identification with sequence data characterization provided by molecular approaches. The number of studies that use molecular techniques to provide assessments of the diversity of paramphistomoids have in general been limited, especially so for African species (Lotfy et al 2010;Mansour et al 2014;Sibula et al 2014;Titi et al 2014;Dube et al 2015).…”