In this study, for H. contortus worms isolated from the abomasa of suspected infected ruminants, mtDNA COI genes were utilized to determine the presence and the extent of genetic variations of Haemonchus spp. populations among four major domestic ruminants (sheep, goats, camels, and cattle) in Egypt. Sequence alignment of each worm isolated from same animal species showed the same nucleotide sequence even from different localities in Egypt so each worm sequence was represented in one sequence of sheep, goats, camels, and cattle. The population structure was elucidated by comparing the sequences from Egypt with the sequences of Haemonchus isolates from other countries published in GenBank.
An approximately 709-bp length of a partial COI gene from different ruminants was successfully amplified, and a band specific to Haemonchus spp. was obtained in all reactions using COI-specific primers (Fig. 1).
The specificity of PCR products was proved by sequencing of DNA amplicon. The resulted nucleotide sequences were edited utilizing MEGA 6.0 program producing a 262-bp length of target size that corresponded to nucleotide position 288 to 547 of the entire mitochondrial genome of Haemonchus worms and submitted in GenBank with accession numbers (KT826575, KT826574, KT826573, and KT826572) from sheep, goats, cattle, and camels, respectively. Analysis of the nucleotide sequence of PCR amplicons found that they belonged to the COI gene of H. contortus in sheep, goat, and cattle isolates but belonged to that of H. longistipes in the camel isolate.
The sequences of the local Egyptian Haemonchus spp. from four different ruminants were compared over an alignment length of 240 bp. The G + C contents of COI sequences of sheep, goat, cattle, and camel isolates were 32%, 31.2%, 35.1%, and 32.8%, respectively. The COI fragments for four isolates studied identical in 76.3%; the sequences coincided for 200 out of 262 nucleotides. Nine basic regions of interspecific homology (positions 27–34, 42–58, 84–98, 102–115, 132–139, 150–157, 183–191, 221–232, and 239) can be distinguished. Sequence difference between isolates occurred at 60 positions, comprising 56 substitutions (point mutations) and four deletions/insertions. Single base deletion was reported for positions 11 and 235, in sheep isolate, and only for position 11 in camel isolate.
The percent of identity and diversion between the Egyptian Haemonchus isolates from different hosts was reported. The highest identity percent was 93.5% between sheep and goat isolate with divergence percent of 4.4%. The lowest identity percent was 80.2% between sheep and camel isolates with divergence percent of 21.9%. The identity percent of sheep isolate were 93.5, 88.9, and 80.2% with divergence percent of 4.4, 9.6 and 21.9% with goat, cattle, and camel isolates, respectively. The identity percent of goat isolate were 93.5 and 81.7% with divergence percent of 6.1 and 19.8% with cattle and camel isolates, respectively. The identity percent of cattle isolate was 83.6% with divergence of 17.3% with camel isolate.
The phylogenetic analysis in Fig. 2 showed a phenogram depicting genetic similarity of Haemonchus COI sequences based from pairwise sequence comparisons. H. longistipes isolated from camels was the genetically most distinct taxa. The phylogenetic tree showed clustering of sheep and goat H. contortus isolates which differ well from cattle H. contortus isolates.
The sequences were first aligned using Clustal W (1.82) program, and the phylogenetic analyses were conducted using PHYLIP package. Nucleotide substitutions are shown underneath the tree.
In order to further comprehend the population structure, a correlation of the partial genomic sequences (240 bp) of Haemonchus COI gene resulted from different hosts in Egypt with sequences of ten reference genotype sequences retrieved from GenBank from other countries and genus (Fig. 3). Egyptian sequences from sheep, goats, and cattle showed little variations among all published sequences which ranged from seven to ten substitutions (Fig. 3). On the other hand, Egyptian sequence from camels demonstrated great variation with others including 30 substitutions. The percent of identity and diversion between the Egyptian isolates and reference strains from the GenBank data was reported. Our isolates showed highest identities with Haemonchus isolated from Pakistan involving accession numbers KJ724402 (94.9% identity and 4% divergence with sheep isolate), KJ724377 (98.3% identity and 1.7% divergence with goat isolate), and KJ 724399 (96.2% identity and 2.6% divergence with cattle isolate), while camels isolate had the highest identity with H. longistipes isolated from Pakistan (KJ724419 with 97.4% identity and 2.6% divergence).
The phylogenetic analysis of aligned COI sequences of these Egyptian isolates from different hosts and countries (Fig. 3) demonstrated discrete clusters and grouping that revealed close ancestral and relative genetic origin among those retrieved from GenBank. The Neighbor-Joining (NJ) dendrogram generated with 26 replicates (Fig. 3) revealed four main clades. The first clade consisted of H. contortus while the second clade included H. placei and Trichostrongylus axei. The other two clades consist of H. longistipes isolates and Cooperia oncosphora.