Multi-locus typing of Histomonas meleagridis isolates demonstrates the existence of two different genotypes

Abstract

Histomonas meleagridis is the aetiological agent of histomonosis or "blackhead disease". Histomonosis is of special importance today, because there is no effective treatment to prevent its occurrence with considerable losses for the poultry industry. Despite its importance only a few molecular studies have yet been performed to investigate the degree of genetic diversity between different isolates of this parasite. In the present study a collection of well defined samples, previously shown positive for the DNA of H. meleagridis, was used to investigate genetic relatedness of the parasite. Samples originated from 25 turkey flocks collected in France between 2007 and 2010. Additionally, diagnostic samples, collected at our Clinic in Vienna, from different European countries and Azerbaijan, during 2010 to 2013 were included in the analyses. For the analysis three different genetic loci were analyzed: 18S rRNA, α-actinin1 and rpb1 genes. To amplify partial sequences of α-actinin1 and rpb1 genes, primers specifically targeting H. meleagridis were designed. Following PCR, the sequences of 18S rRNA, α-actinin1 and rpb1 loci were analyzed. Phylogenetic analyses demonstrated separation of H. meleagridis isolates in two different clusters. The majority of isolates grouped within the cluster 1 and originated from different European countries. The cluster 2 was rare and predominantly found in samples originating from France. Considering that the genetic variability of clusters can be seen as two distinct genetic types we propose the term genotype instead of cluster.

Figure 1. Phylogenetic tree of H. meleagridis isolates based on partial 18S rRNA sequences (approx. 544bp).

For the analysis 47 sequences (sequence types) were used. The phylogenetic analysis was performed by applying separately maximum likelihood and neighbor-joining methods, with Trichonympha agilis as outgroup. The tree generated by maximum likelihood method is shown. Samples (N = 34) originating from this study are labelled bold. The robustness of the tree was determined by bootstrap re-sampling of the multiple–sequence alignments (1000 sets neighbor-joining/100 sets maximum likelihood). Values on the nodes are bootstrap support values indicated as percentages from neighbor-joining and maximum likelihood, respectively. Asterisks indicate nodes with bootstrap values below 50% or with a different topology. Branch lengths are proportional to sequence divergence and can be measured relative to the scale bar shown (bottom right). The scale represents nucleotide substitution per position.

Figure 2. Phylogenetic tree based on partial α-actinin1 sequences of H. meleagridis (approx. 1048bp).

The phylogenetic analysis was performed by applying separately maximum likelihood and neighbor-joining methods. The tree generated by maximum likelihood method is shown. Due to the low amount of analyzed sequences (N = 3) robustness of the tree by bootstrap re-sampling could not be determined. Branch lengths are proportional to sequence divergence and can be measured relative to the scale bar shown (bottom right). The scale represents nucleotide substitution per position.

Figure 3. Amino acid alignment of H. meleagridis Rpb1 protein with Rpb1 proteins of Trichomonadea and Tritrichomonadea representatives.

The alignment demonstrates a conserved insertion within region A of Rpb1 found only in Tritrichomonadea, present also in H. meleagridis Rpb1. The insertion is boxed, conserved amino acids are shown in bold, and gaps in the alignment are indicated by dashes.

Figure 4. Phylogenetic trees based on partial rpb1 sequences.

(A) only H. meleagridis sequence types (N = 4) were used and the tree is based on 1242bp sequences. (B) H. meleagridis rpb1 sequence types (N = 4) and sequences from different Tritrichomonadea (N = 4) and Trichomonadea (N = 5) available in the database were used; the tree is based on 759bp. Both analyses were performed with PHYLIP v3.68 software package applying separately neighbor-joining and maximum likelihood methods. Trees generated by maximum-likelihood method are shown. The robustness of the tree was determined by bootstrap re-sampling of the multiple–sequence alignments (1000 sets neighbor-joining/100 sets maximum likelihood). The values on the nodes are bootstrap support values indicated as percentages from maximum likelihood and neighbor-joining, respectively. Asterisks indicate nodes with bootstrap values below 50% or with a different topology. Branch lengths are proportional to sequence divergence and can be measured relative to the scale bar shown (bottom right). The scale represents nucleotide substitution per position.

Figure 5. Analysis of ITS1-5.8S rRNA sequences from different H. meleagridis clonal cultures demonstrates the presence of multiple sequence variants within a single clone.

(A) Phylogenetic tree based on the complete ITS1-5.8S rRNA-ITS2 region. Phylogenetic analysis of ITS1-5.8S rRNA-ITS2 region was performed using MegAlign application of Lasergene software (DNASTAR Inc.) by applying distance methods with default settings. Different clones are labelled with c and number, e.g. c1. (B) Sequence alignment of 5.8S rDNA region. Only sequence differences are shown, conserved nucleotides are designated as dots. Sequences originating from the same clonal culture are separated with lines.