Molecular phylogenetic analysis of Echinococcus multilocularis from horses raised in Canada or Japan, using mitochondrial cytochrome b gene-targeted PCR

Abstract

Alveolar echinococcosis is a zoonotic disease caused by a larval-stage Echinococcus multilocularis infection. Geographical haplotyping targeting the parasite's mitochondrial cytochrome b (cob) gene has been reported for isolates from definitive and intermediate hosts (wild canids and rodents); however, there are limited reports on strain typing for the dead-end host, the horse, which could act as a sentinel for E. multilocularis. Accordingly, we investigated the diversity of E. multilocularis in isolates obtained from slaughtered Japanese and Canadian horses originating from the Iburi and Hidaka regions in Hokkaido and from Alberta, respectively, with PCR and haplogroup analyses targeting cob gene sequences obtained. Seventy horses were diagnosed with alveolar echinococcosis based on histopathology and cob-gene PCR testing. The E. multilocularis detected in these horses was classified as either an Asian (for Hokkaido-raised horses) or a European (for Alberta-raised horses) haplogroup, based on the obtained cob-gene sequence analysis. In addition, haplotype network analysis revealed that E. multilocularis isolated from Hokkaido-raised horses is highly homologous to Kazakhstan isolates, and E. multilocularis isolated from Alberta-raised horses is highly homologous to Austrian isolates. The results of this study suggest that cob-gene-targeted PCR analysis could be useful for the geographical genetic characterization of E. multilocularis isolated from horses.

Keywords: Alveolar echinococcosis; Cob-gene; Horse; Meat inspection; Mitochondrial cytochrome b.

Graphical abstract

Fig. 1

Horses slaughtered in Japan (December 2020–November 2022): Origins and slaughterhouse locations. Map of Japan showing the four provinces included in this study: Hokkaido, Aomori Prefecture, Fukuoka Prefecture, and Kumamoto Prefecture. The nodules used in this study were collected from the slaughterhouses in Aomori Prefecture, Fukuoka Prefecture, and Kumamoto Prefecture. Most horses in Japan were born in the Iburi and Hidaka regions of Hokkaido, where alveolar echinococcosis is endemic. Additionally, there is a map of Canada showing Calgary, the province of Alberta that was included in this study. Before being imported to Japan, the horses originating in Canada were born and raised in Calgary, Alberta, where alveolar echinococcosis is endemic. This map was prepared using MapChart (https://www.mapchart.net/).

Fig. 2

Phylogenetic tree for E. multilocularis isolated from horses originating in Japan and Canada based on the partial sequences (694 bp) of cob genes. The neighbor-joining method, based on the Tamura-Nei model, was performed. Numbers next to branches indicate the percentage of replicate trees based on 1000 bootstrap replicates. Only bootstrap values ≥50% are shown. The scale bar indicates 0.02 nucleotide substitutions per nucleotide position. The isolates in this study had accession numbers LC764417 to LC764425. The isolates obtained from horses originating in Japan (LC764421 to 764,425) are surrounded by dashed red lines, and those from horses originating in Canada (LC764417 to 764,420) are surrounded by dashed blue lines. In the phylogenetic tree, “Em” is an abbreviation for Echinococcus multilocularis.

Fig. 3

Haplotype network based on the partial sequences (694 bp) of cob genes of E. multilocularis constructed using PopART version 1.7 software. This analysis shows nine haplotypes, including five haplotypes obtained from horses originating in Japan and four haplotypes obtained from horses originating in Canada. The nine haplotypes obtained in this study are highlighted in yellow. The most common strains isolated from horses originating in Japan and Canada were LC764421 (74.2%) and LC764417 (50.0%), respectively. Each colour represents a unique haplogroup (Asia, North America, Europe, etc.), and the circle size is proportional to the number of isolates contained. One vertical line on the branch indicates a single base mutation. Black circles represent hypothetical haplotypes. The accession numbers of samples used for reference are as follows: AB461398 (Kazakhstan), AB461399 (Japan: Hokkaido), AB461395 (Austria), AB461396 (France), AB461397 (Slovakia), KC550003 (Canada: coyote), MK843307 (Canada: patient), AB461400 (USA: Alaska, St. Lawrence Island), AB461401 (USA: Indiana), AB461402 (China: Inner Mongolia). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)