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.

Detection and quantification of Histomonas meleagridis by real-time PCR targeting single copy genes

“…In particular, a panel of various PCR assays, which enabled fast detection of parasites' DNA, were developed (Bleyen et al, 2007;Grabensteiner and Hess, 2006;Hafez et al, 2005;Huber et al, 2005;Xu et al, 2014). All these methods were designed to target the small subunit of ribosomal RNA that has hampered the specificity of some assays due to strong homology of this region with other closely related protozoa (Bilic et al, 2014). Only recently, a quantitative real-time PCR method based on the 5.8S rRNA locus was developed to quantify the amount of histomonads in a sample (Landman et al, 2015).…”

“…exclusively gave negative results. Reported literature and our own experience showed that these parasites are often detected in chicken and turkey samples suspicious for H. meleagridis, and the presence of all, except Blastocystis sp., can cause false positive results in PCR assays based on the 18S rRNA or ITS1-5.8S rRNA-ITS2 regions (Lollis et al, 2011;Bilic et al, 2014;Nguyen et al, 2015).…”

“…Recently, the separation of H. meleagridis isolates into two genotypes was reported (Bilic et al, 2014). Therefore, several genetically different H. meleagridis isolates from both genotypes were tested with both qPCR assays.…”

“…Primer pairs HmRpb1-F/HmRpb1-R (Bilic et al, 2014) and HmFeHyd-F/HmFeHyd-R were used in conventional PCR for amplification of partial rpb1 and FeHYD genes, respectively. For real-time PCR based on the rpb1 gene primers, HmRpb1-rt-F/HmRpb1-rt-R and probe HmRpb1-rt-P were used, whereas the real-time PCR based on the FeHYD gene was performed with primer pair HmFeHyd-rt-F/HmFeHyd-rt-R and probe HmFeHyd-rt-P.…”

“…and Dientamoebidae sp. in the poultry gut besides H. meleagridis, which may hinder its specific detection with conventional diagnostic techniques (Bilic et al, 2014;Kemp and Reid, 1965;Lollis et al, 2011;Nguyen et al, 2015;Stenzel and Boreham, 1996). Earlier, the disease was diagnosed based on gross lesions and in vitro isolation of the parasite which proved to be a time consuming method (McDougald, 2005).…”