A filarial parasite potentially associated with the health burden on domestic chickens in Japan

Abstract

Chickens in free-range environments are at risk of exposure to various pathogens, such as filarioids transmitted via hematophagous vectors. However, the study of filarioids in poultry has been largely neglected compared to the extensive studies focused on viruses, bacteria, and protozoa. Here, we performed histological and molecular investigations of the filarioids detected in domestic chickens from two different flocks in Hiroshima Prefecture, Japan. In the first case, adult worms were present in the pulmonary artery and right ventricle, and microfilariae were present in multiple organs of deceased chickens. In the second case, similar filarioids were detected in the organs and blood of one necropsied layer. Phylogenetic analysis using 18S rRNA gene fragments positioned the filarioid in the same clade as that of Onchocercidae sp., previously identified in a deceased chicken from Chiba Prefecture, Japan, that is located 500 km away from Hiroshima Prefecture. Based on 28S rRNA and mitochondrial COI gene fragments, the filarioid was positioned distinctly from previously reported genera of avian filarioids. These results suggest that the filarioids are potentially associated with the health burden on domestic chickens and belong to the genus Paronchocerca. Furthermore, we developed a nested PCR assay targeting mitochondrial COI and detected the parasite DNA from the biting midge Culicoides arakawae captured near the flock, suggesting that it serves as a vector. Our findings fill the knowledge gap regarding avian filarioids, laying the groundwork for future studies examining the epidemiology, life cycle, and species diversity of this neglected parasite group.

Keywords: Avian filariasis; Backyard poultry; Filarial nematode; Mitochondrial genome; Phylogenetic analysis.

Figure 1

Microscopic appearances of Onchocercidae sp. detected in Chabos (Japanese Bantams) and a Hy-line Brown layer. (A) Histological section of a microfilaria in a small pulmonary vein of a Chabo. Hematoxylin and eosin (H&E) stain. Scale bar = 10 µm. (B) Microfilaria in the myocardium of a Hy-Line Brown layer. H&E. Scale bar = 10 µm. (C) Adult worms in a right ventricle of a Chabo. Arrowheads indicate the endocardium. The inset presents the adult worm marked with the asterisk, where many microfilariae were observed. H&E. Scale bar = 50 µm. (D) Microfilaria retrieved from a blood sample of a Chabo. Methylene blue stain. Scale bar = 10 µm.

Figure 2

Bayesian phylogenetic trees constructed using (A) the nuclear 18S rRNA and (B) the nuclear 28S rRNA gene sequences of nematodes. Blue indicates that the parasite has avian hosts, orange indicates mammalian hosts. Black dots indicate nodes with posterior probability of > 0.75.

Figure 3

Schematic diagram of the Onchocercidae sp. mitochondrial genome. The positions of 12 protein-coding sequences (red), 2 ribosomal RNA genes (blue), and 22 transfer RNA genes (green) are illustrated.

Figure 4

Bayesian phylogenetic tree constructed using the mitochondrial COI gene sequences. Blue indicates that the parasite has avian hosts, orange indicates mammalian hosts, and green indicates reptilian hosts. Black dots indicate nodes with posterior probability of > 0.75.

Figure 5

Molecular detection of Onchocercidae sp. from (A) Culicoides arakawae and (B) blood of Chabos that were housed with the dead Chabos using the nested PCR assay. The PCR products were analyzed in 2.0% agarose gels, and representative images are presented. (A) Lane M, DNA ladder; lane PC, positive control; lane NC, negative control; lanes 1–13, DNA extracted from C. arakawae. (B) Lanes 1–7, DNA extracted from the blood samples of Chabos. The original gel images are presented in Supplementary Fig. S5.

Figure 6

Unrooted Bayesian phylogenetic tree illustrating the relationships among Wolbachia detected in terrestrial arthropods and filarial nematodes. The analysis employed 821 bp of partial 16S rDNA sequences. Nodes with posterior probabilities exceeding 0.75 are denoted by black dots. Tip labels display GenBank accession numbers and their host, with green indicating arthropod hosts and blue indicating filarial nematode hosts. Supergroup classifications, as determined by previous research, are given in capital letters in the coloured circles.