Echinococcus multilocularis Surveillance Using Copro-DNA and Egg Examination of Shelter Dogs from an Endemic Area in Hokkaido,Japan

Abstract

Surveillance of Echinococcus multilocularis infection among 156 shelter dogs was conducted in an urban area (Sapporo city) in Hokkaido, where the parasite is endemic in Japan using copro-DNA and fecal egg examination from September 2013 to April 2017. Echinococcus infection was detected in three dogs (1.9%), including one dog that excreted eggs. The results suggested that free-roaming or stray dogs in urban area may be infected by capturing wild voles containing parasitic cysts and could be a source of human infection. Dog-to-human transmission is a significant concern, and the risk of such transmission is present even in urban areas in Hokkaido. We recommend deworming within 1 month (e.g., before egg excretion) of capture for free-roaming or stray dogs in Echinococcus-endemic area to prevent potential human infection.

Introduction

Hokkaido is the only island in Japan where Echinococcus multilocularis is highly endemic among wild animals (fox–vole cycle). The prevalence of the tapeworm in Hokkaido has been estimated as 1.0% of stray dogs by necropsy examination (data reported by the Hokkaido government) and 0.4% of companion dogs by coprological examination (Nonaka et al. 2009). The risk of dog-to-human transmission by eggs excreted from infected dogs is a significant concern, and prevention against such transmission is necessary.

Administration of the deworming agent praziquantel is effective for eliminating the worms in dogs (Sakamoto et al. 1976) and would contribute to preventing E. multilocularis transmission. However, such measure has not been widely applied, and one of the reasons may be lack of information concerning the recent prevalence of E. multilocularis in urban dogs in Hokkaido.

We focused on sheltered dogs in a local government animal shelter, where dogs are sheltered when captured in the wild or dropped off by their owners. Especially for stray dogs, survival conditions are unclear, for example, the dog may have captured wild voles containing E. multilocularis cysts. To evaluate the importance of preventive deworming on free-roaming or stray dogs in an urban area, we determined the prevalence of E. multilocularis infection using copro-DNA and fecal egg examination in shelter dogs in an urban city in a highly endemic area in Japan.

Materials and Methods

Fecal collection

From September 2013 to April 2017, stool samples (deposit feces) were collected from 156 dogs sheltered in an animal center of an urban city in Hokkaido, Japan; 126 dogs were strays and 30 were dropped off by their owners. Feces were heated for inactivation of Echinococcus eggs at 70°C for 12 h and then stored at −20°C until use.

After fecal sampling, dogs were administered deworming agent praziquantel (5 mg/kg body weight) by the attending veterinarian, or returning/new owners were instructed to have their dogs receive veterinary examination.

Fecal examination for E. multilocularis infection

Copro-DNA examination specific for E. multilocularis was performed on 1 g of feces as previously described (Irie et al. 2017). For polymerase chain reaction (PCR)-positive samples, DNA was extracted from a subsample of feces two more times for confirmation, followed by sequencing analysis of the cytochrome c oxidase subunit 1 (COI) gene (Nonaka et al. 2008).

Fecal egg examination was performed using 2 g of feces by the Army Medical School III (AMS III) method (Hunter et al. 1948). For species identification, eggs were collected from a fecal subsample of egg-positive dogs using nylon mesh and egg DNA was analyzed. Briefly, approximately 1 g of feces was mixed with distilled water and passed through a wire mesh (pore size: 150 μm). The filtrate was then filtered through large (pore size: 80 μm) and small (pore size: 20 μm) nylon mesh. Materials trapped on the small mesh were washed off into water, and eggs were collected under a stereomicroscope. Egg-DNA was then extracted using the same method for copro-DNA extraction and PCR was performed for E. multilocularis U1 small nuclear RNA (U1 snRNA) (Bretagne et al. 1993) and COI genes, followed by sequencing analysis. PCR for the 12S ribosomal RNA (12S rRNA) gene was also performed, followed by restriction fragment length polymorphism (RFLP) analysis (Yagi et al. 1999).

Results

Three of 156 dogs (1.9%; 2 stray dogs and 1 dropped-off dog) were positive for E. multilocularis copro-DNA (Table 1). The sequences obtained (243 bp) were completely identical to COI sequences of E. multilocularis detected in Hokkaido (GenBank Sequence ID: AB461416), China (Sequence ID: KY446505), and Russia (Sequence ID: AB688126), and a 1-bp difference was observed compared with sequences obtained in France (Sequence ID: AB461413) and Austria (Sequence ID: AB461412).

Table 1.

Number Of Echinococcus Multilocularis-Positive Dogs by Copro-Dna and Egg Examination

	No. of dogs
			Egg positive (%)
Sheltered situation	Examined	DNA positive (%)	Em	Tc	Tl	Tv	M	E
Strayed	126	2 (1.6)	1^a (0.8)	1 (0.8)	2 (1.6)	4 (3.2)	5 (4.0)	1 (0.8)
Dropped-off	30	1 (3.3)	0	0	0	0	0	1 (3.3)
Total	156	3 (1.9)	1 (0.6)	1 (0.6)	2 (1.3)	4 (2.6)	5 (3.2)	2 (1.3)

The dog also positive in copro-DNA examination.

Em, Echinococcus multilocularis; Tc, Toxocara canis; Tl, Toxascaris leonina; Tv, Trichuris vulpis; M, Metagonimus spp.; E, Echinostoma spp.

Taeniid eggs (11 eggs/g feces) were detected by the AMS III method in one of copro-DNA-positive stray dog (0.6%). The COI gene sequence from egg-DNA was completely identical to COI sequences obtained from copro-DNA. The U1 snRNA gene sequence (337 bp) was 99% (336/337) identical to the registered sequence (GenBank Sequence ID: M73768; only one sequence was available in GenBank). RFLP analysis of the 12S rRNA gene identified E. multilocularis (373–165/103/105 bp).

In fecal egg examination, eggs of Toxocara canis, Toxascaris leonina, Trichuris vulpis, Metagonimus spp., and Echinostoma spp. were also found in feces of surveyed dogs (Table 1).

Discussion

In this study, 1.9% of shelter dogs had E. multilocularis by copro-DNA examination. The prevalence was slightly higher than that reported for companion dogs (Nonaka et al. 2009), which may suggest that surveyed dogs, especially for stray dogs, had a greater chance to capture infected intermediate hosts (i.e., wild vole). Our results explain the presence of an infectious source to the definitive host (e.g., dog) and demonstrated that the parasite life cycle could be maintained even in urban areas in Hokkaido, Japan.

One dropped-off dog was positive for E. multilocularis copro-DNA, thus, it was likely infected while kept as a companion dog. Companion dogs allowed to roam freely, even for only a few hours, may catch an infected vole outside of their owners' eyesight (Morishima et al. 2006). For the dog that excreted E. multilocularis eggs, it was unclear how long it had been a stray before being sheltered. However, in this dog, infection before becoming a stray could not be completely denied because egg excretion occurs ∼1 month from ingestion of an infected vole (Matsumoto and Yagi 2008).

Dogs infected with the tapeworm are mostly asymptomatic (Deplazes and Eckert 2001), thus, it is difficult to determine infection by only a normal health checkup. Therefore, the risk of transmission from infected dogs to humans by shedding of parasitic eggs remains a significant concern. To prevent excretion and spreading of eggs, deworming agent praziquantel (5–10 mg/kg body weight) (Sakamoto et al. 1976) should be administered to free-roaming and stray dogs for as long as 1 month (i.e., before egg excretion). The importance of such measure on free-roaming dogs should be imparted to dog owners and local veterinarians in E. multilocularis-endemic areas. Additionally, preventive deworming for shelter dogs is expected to be accepted in animal shelters to prevent infection in workers because they handle many dogs, which have various histories and some dogs might shed tapeworms, as explained by this study.

Fecal egg examination detected infection by several parasites in surveyed dogs, including Toxocara, which is a known zoonotic parasite. Surveillance on dog parasites conducted in our study will be useful to evaluate risk of parasite transmission, including E. multilocularis, from dog to human.

In this study, prevalence of E. multilocularis infection in shelter dogs in an endemic area in Japan was determined by copro-DNA and egg examination. Dog-to-human transmission is a significant concern, and the risk of such transmission is present even in urban areas. Allowing dogs to freely roam in E. multilocularis-endemic area should be avoided to prevent human infection. Additionally, prompt preventive deworming before egg excretion is recommended for dogs that accidentally run away.

Footnotes

Acknowledgments

The authors thank the staff of Sapporo Animal Welfare and Management Center, Hokkaido, Japan, for their assistance with fecal sampling of shelter dogs. This research was supported by the Research Program on Emerging and Re-emerging Infectious Diseases from Japan Agency for Medical Research and Development, AMED (17fk0108315h0003) and a research grant from the Akiyama Life Science Foundation (2016 Grants-in-Aid).

Author Disclosure Statement

No competing financial interests exist.

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