This study presents evidence of many highly divergent novel chaphamaparvoviruses in the bile of free-range laying chickens showing gross pathology consistent with SLD. Strikingly, this study reports for the first time 14 novel GaChPVs circulating in laying chickens that show from 64.1–87.4% similarities to other parvoviruses isolated globally. All novel genomes of GaChPV sequence derived from free-range laying chickens contain all the major structural and functional genes, and NS1 genes exhibit from 45.29–88.26% amino acid identities compared to other parvoviruses. Except for the NS1 gene of GaChPV-2, all other NS1 genes of GaChPVs sequenced in this study are significantly divergent and clearly represent separate species, as demonstrated by the low degree of amino acid identities (between 45.29% and 77.63%) to the closest parvovirus genes. Following the recent nomenclature of considering species of parvovirus 18, we propose the 14 GaChPVs found to be novel parvoviruses under the genus Chaphamaparvovirus, family Parvoviridae and subfamily Hamaparvovirinae.
Although this is an interesting finding and opens a lot of opportunities for further investigation, some aspects of this case are difficult to explain fully without conducting ethically debatable virus-transmission experiments. The direct or indirect effect of these parvoviruses on the livers of laying chickens is unknown. However, a recent finding in another Galliformes species – pheasants (Phasianus colchicus) – shows several outbreaks of hepatitis with high mortality, which were caused by a novel chaphamaparvovirus. The aetiology of hepatitis outbreaks in pheasants was confirmed by pathology, sequencing and in-situ hybridisation 23; pathologically, extensive areas of severe diffuse degeneration and hepatic necrosis with sparse infiltration of mononuclear inflammatory cells and heterophils were observed in liver tissue, with degenerative hepatocytes presenting large amphophilic to acidophilic intranuclear inclusion bodies. Additionally, parvovirus strains from the Anseriform dependoparvovirus 1 species within the Dependoparvovirus genus, are also capable of causing acute disease in young geese and ducks, with characteristic lesions (including hepatitis) and the presence of similar intranuclear inclusion bodies 24. In contrast, the etiological agent of SLD-causing bacterium, C. hepaticus, is isolated and characterised from the bile of laying chickens, and the organism produces histological changes in liver tissue 25,26 very similar to hepatitis in pheasants (Phasianus colchicus) 23. However, authors Phung, et al. 25 reported that infection with C. hepaticus may not be sufficient to induce disease; some other predisposing factors may also be required 25; this claim is supported by various previous research efforts 26. For example, due to the absence of bacteria in necrotic hepatic foci, it has been suggested that a bacterial toxin, such as a member of the cytolethal distending toxin (CDT) group commonly found in C. jejuni, could be associated with liver pathology 27. In contrast, a study by Petrovska, et al. 28 reported that CDT genes were not detected in their studies of C. hepaticus. Though the presence of toxin-induced pathology cannot be discounted, the influence of such entities in C. hepaticus pathology remains inconclusive. It is likely possible that there are other pathogens, including viruses that might contribute to the development of liver pathology, which require further investigation. The pathogenic effects of parvoviruses detected in this study on the liver is largely unknown; however, considering that several outbreaks of hepatitis in pheasants 23, geese and ducks 24 were caused by parvoviruses, the long-term consequences in susceptible populations and their pathology remain to be determined.
Our phylogenetic analysis provides strong evidence of multiple circulating chaphamaparvovirus lineages in the free-range chicken farm in Australia (Fig. 3) and supports that the newly sequenced GaChPVs are representative species of the genus Chaphamaparvovirus. The detected GaChPVs are divergent from the viruses belonging to other genera of the subfamily. Thus, our results suggest that there are multiple lineages, including a unique lineage of chaphamaparvoviruses that has emerged during the evolution of parvoviruses in free-range chickens in Australia. Interestingly, the GaChPVs detected in this study were shown to be highly divergent genetically from chaphamaparvoviruses that infect other galliform species, such as chickens, turkeys and peafowl 20,21. In the absence of any parvovirus sequence in Australian chickens, it is difficult to elucidate the origin of the GaChPVs from this case alone. It is likely that these viruses are circulating in free-range and/or captive chickens in Australia but have not been detected yet. In support of our findings, two recent studies on peafowl 20 and pheasants 23, in which the authors discovered novel chaphamaparvoviruses in tissues, showed that case fatalities were linked to parvoviruses; however, many viruses designated as Chaphamaparvovirus were identified from faecal samples 21,29−31, swabs 32 or asymptomatic tissues 33.
Interestingly, CTChPV-2 is basal to many known avian chaphamaparvoviruses, including five GaChPVs detected in this study (lineage-VII, Fig. 3), which suggests that all the avian chaphamaparvoviruses clustered in this lineage may have evolved from the ancestral duck species that gave rise to CTChPV-2. The duck species (chestnut teal, Anas castanea) from which CTChPV-2 was isolated, is one of the few species of Australian ducks 19 and is found in southwestern and southeastern Australia, Rockhampton, Queensland to Ceduna, South Australia, and is most common in New South Wales, Victoria and Tasmania. However, it is not documented whether free-range chickens intimately share any ecological niche with any known duck species. Free-range chickens are usually reared to roam and forage outdoors for at least eight hours a day, an activity that has increased in the last decades. Such husbandry systems present more biosecurity challenges than conventional poultry farms, implying a higher risk of the introduction and transmission of pathogens. A possible scenario for transmission could be the ingestion of wild duck-contaminated food within their pasture since parvovirus is likely to persist in the environment for a time, facilitating exposure to susceptible animals 34. It is well known that wild birds harbour a number of bacterial, fungal, viral and parasitic diseases, which can be transmitted to captive and/or free-range poultry; the potential for diseases to transfer from wildlife to poultry may increase when poultry have access to the outdoors 35.