The Molecular Detection of Cryptosporidium and Giardia in Sika Deer (Cervus Nippon Centralis)in Japan

Abstract

Fecal specimens (271 samples) from wild deer, Cervus nippon centralis, were collected from nine different areas in Japan; these samples were subjected to a real-time reverse transcription PCR for Cryptosporidium-and Giardia-specific 18S ribosomal RNA to investigate the prevalence of Cryptosporidium and Giardia infection. The incidence of Cryptosporidium and Giardia in the nine areas ranged from 0% to 20.0% and 0% to 3.4%, respectively. The prevalence of Cryptosporidium among male and female deer was 8.1% and 3.9%, respectively, while that of Giardia was 0.7% and 0.8%. Sequence analysis identified the Cryptosporidium deer genotype, Cryptosporidium bovis, Cryptosporidium ryanae and Cryptosporidium meleagridis from the sequence of Cryptosporidium-specific partial 18S ribosomal RNA and Giardia intestinalis assemblage A from the partial sequence of Giardia-specific 18S rRNA. The variation in regional prevalence indicates that Cryptosporidium infection depends on environmental factors, and that bovine Cryptosporidium was detected more frequently than cervine Cryptosporidium. These data suggest that wild deer might be a healthy carrier of bovine Cryptosporidium.

1. Introduction

Cryptosporidium spp. and Giardia spp. are protozoan parasites that infect a wide range of vertebrates, including humans. They cause different types of severe diarrhea with shedding of a small number of oocysts¹⁾. Infection is caused by the ingestion of viable oocysts via food or water contaminated with the feces of the infected host. Both Cryptosporidium and Giardia are chlorine resistant. This makes water sanitation difficult and results in sporadic outbreaks of water-borne disease in both developing and developed countries. Thus far, many wild animals, including ruminants and domestic animals, have been reported to be infected with several species of Cryptosporidium¹⁾. Among these, cattle are known to be typically infected by five different species: Cryptosporidium parvum, C. andersoni, C. bovis, C. ryanae, and C. ubiquitum²⁾. In contrast, among a large number of Giardia species and genotypes, only Giardia intestinalis assemblage A and assemblage E are known to infect cattle^3,4). Recently, the population of wild deer has markedly increased in Japan, which has led to severe damage to farming industries. To cope with these problems, regional governments are trying to control deer populations, and simultaneously utilize their meat. Cases of food-borne cryptosporidiosis and giardiasis have increased in Japan and abroad⁵⁾; however, there are few reports of epidemiological studies on pathogenic microbes in wild animals. Studies of Cryptosporidium have mostly focused on species that infect livestock such as cows and hogs. Thus, while there have been many epidemiological reports from foreign countries on Cryptosporidium and Giardia infections in wild animals⁶⁾, the distribution of these parasites in wild animals in Japan remains largely unknown. In the present study, we attempted to determine the distribution of Cryptosporidium and Giardia species in wild deer in Japan in order to facilitate the establishment of preventive measures against Cryptosporidium and Giardia infection.

2. Materials and Methods

2-1. Collection of Samples

Between October 2012 and June 2016, the sampling of sika deer (Cervus nippon centralis) fecal samples was carried out during the hunting season in nine prefectures in Japan. A total of 271 (Season: January, n = 30; February, n = 13; May, n = 35; April, n = 11; May, n = 16; June, n = 19; July, n = 9; August, n = 4; September, n = 11; October, n = 38; November, n = 25; December, n = 60; Sex: male, n = 135; female, n = 129; undetermined at sample collection, n = 7) deer fecal samples were collected. Individual fecal samples were taken directly from the rectum of each deer during evisceration. The samples were transported to the laboratory in an insulated cooler and stored at 4°C until DNA extraction.

2-2. DNA Extraction

DNA was extracted from each sample within 24 hours after arrival and stored at -20°C until analyzed. Feces were diluted in deionized water and filtered through gauze into centrifuge tubes. Ethyl acetate was then added at a ratio of 1:4 (v/v). The tubes were vigorously shaken and centrifuged at 1000 × g for 5 min at room temperature and the supernatants were decanted. The pellet was suspended again in deionized water and then centrifuged at 1000 × g for 3 min at room temperature to recover the pellet. The recovered pellet was resuspended in the extraction buffer to a volume of 200 µl which was immediately thereafter subjected to lysing and DNA extraction. Briefly, the samples were lysed by 5 cycles of freezing at -196°C and thawing at 85°C, and then DNA was extracted using the Qiagen DNA Stool Mini Kit (QIAGEN, Hilden, Germany). DNA was eluted in water and then stored at -20°C before assay.

2-3. Real-time PCR

The molecular characterization and estimation of parasite density of Cryptosporidium were performed using a Cycleave RT-PCR Cryptosporidium (18S rRNA) detection kit (Takara Bio, Shiga, Japan) and a Cycleave RT-PCR Giardia (18S rRNA) detection kit (Takara Bio) according to the manufacturer’s protocol.

2-4. Statistical Analysis

Prevalence with respect to sampling area and sex were statistically analyzed using Fisher’s exact test. The percentage of the parasites in each deer group were compared by the nonparametric Kruskal-Wallis test and Mann-Whitney U-test.

2-5. DNA Sequencing

All PCR-positive samples were directly sequenced targeting the partial sequence of the Cryptosporidium and Giardia 18S rRNA genes to determine the species. For Cryptosporidium, the forward primer (5ʹ-aagctcgtagttggatttctg-3ʹ) corresponded to nucleotides 601 to 621, and the reverse primer (5ʹ-taaggtgctgaaggagtaagg-3ʹ) corresponded to nucleotides 1015 to 1035 (GenBank accession number L16996)⁷⁾. For Giardia, the forward primer (5’-CATCCGGTCGATCCTGCC-3’) corresponded to nucleotides 1 to 18, and the reverse primer (5’-AGTCGAACCCTGATTCTCCGCCAGG-3’) corresponded to nucleotides 268 to 292 (GenBank accession number AF473852)^8,9). The sequence data were examined using an ABI 3730xl Genetic Analyzer/DNA Sequencer (Life Technologies, Tokyo, Japan) with a Big Dye Terminator V.3.1 cycle sequencing kit (Applied Biosystems, Massachusetts, USA). The 18S rRNA of Giardia was also sequenced by Takara Bio Inc. using a Cycleave RT-PCR Giardia kit.

2-6. DNA Sequencing and The Phylogenetic Analysis

Sequence assemblies were analyzed using the ATGC (Ver. 7) Sequence Assembly Software (GENETYX, Tokyo, Japan) and Molecular Evolutionary Genetics Analysis (MEGA version 6: http://www.megasoftware.net/) software programs¹⁰⁾. To identify Cryptosporidium and Giardia at the species level, each of the resulting sequences were compared to those of the Cryptosporidium and Giardia species genotypes submitted to GenBank (National Institutes of Health, Bethesda, MD, USA), using the BLAST software program (v.2.2.12; http://www.ncbi.nlm.nih.gov/BLAST/, National Center for Biotechnology Information, Bethesda, MD, USA), and the homology was determined. We used the neighbor joining method for the phylogenetic analysis of our sequencing results and compared the sequences to other genotypes deposited in the GenBank database.

3. Results

For Cryptosporidium, a total of 18 samples (6.6%) were confirmed to be Cryptosporidium-positive (Table 1). The percentage of Cryptosporidium positive samples by prefectures were as follows: Chiba (11.1%), Shizuoka (15.6%), Nagano (2.5%), Kyoto (13.8%), Miyazaki (20.0%), and Nagasaki (3.4%). The percentage by region were as follows: east (9.8%), west (4.4%), north (7.9%) and south (3.3%). The percentage by season were as follows: December to February (8.7%), March to May (3.2%), June to August (6.3%) and September to November (4.1%) (data not shown). The percentages by sex were 8.1% and 3.9%, respectively.

Table 1. The prevalence of Cryptosporidium and Giardia in Sika deer in Japan

		Cryptosporidium			Giardia			Cryptosporidium / Giardia
Location / Sex	No. of deer	No. PCR- positive for 18S-rRNA	%	P value (Fisher’s exact test*)	No. PCR- positive for 18S-rRNA	%	P value (Fisher’s exact test*)	P value (Fisher’s exact test*)
Prefecture
Chiba	27	3	11.1		0	0.0
Shizuoka	45	7	15.6		0	0.0
Nagano	40	1	2.5		0	0.0
Shiga	25	0	0.0		0	0.0
Mie	23	0	0.0		0	0.0
Kyoto	29	4	13.8		1	3.4
Nagasaki	58	2	3.4		1	1.7
Kumamoto	19	0	0.0		0	0.0
Miyazaki	5	1	20.0	0.055	0	0.0	0.817
Region
East	112	11	9.8		0	0.0
West	159	7	4.4	0.088	2	1.3	0.513
North	189	15	7.9		1	0.5
South	82	3	3.7	0.288	1	1.2	0.514
Total	271	18	6.6		2	0.7		0.0004
Sex
Male	135	11	8.1		1	0.7
Female	129	5	3.9		1	0.8
Total	264†	16	6.6	0.2	2	0.7	0.974

The prevalence of the Cryptosporidium and Giardia deer genotype according to area and sex. Cryptosporidium and Giardia were identified by a reverse transcription polymerase chain reaction targeting the partial sequence of 18S ribosomal RNA (18S rRNA).

*Fisher's exact test was conducted to analyze the statistical analyses according to area (Prefecture: Chiba, Shizuoka, Nagano, Shiga, Mie, Kyoto, Nagasaki, Kumamoto and Miyazaki. Region: Chiba, Shizuoka and Nagano for east; Shiga, Mie, Kyoto, Nagasaki, Kumamoto and Miyazaki for west; Chiba, Shizuoka, Nagano, Shiga, Mie and Kyoto for north; Nagasaki, Kumamoto and Miyazaki for south) and sex (male and female).

^†The sex was unknown for seven samples.

Two samples (0.7%) were confirmed to be Giardia-positive (Table 1). The percentage of positive samples of Giardia in the two prefectures were Kyoto (3.4%) and Nagasaki (1.7%) and by region, were west (1.3%), north (0.5%) and south (1.2%). The percent positive samples by season were as follows: December to February (1.0%) and September to November (1.4%) (data not shown); and that by sex were 0.7% and 0.8%, respectively.

There was a significant difference (P<0.01) between the total number of positive samples of Cryptosporidium and Giardia collected. There were no statistically significant differences among the sampling areas and seasons for either Cryptosporidium or Giardia.

The results of the partial sequence analysis of 18S ribosomal RNA of Cryptosporidium and Giardia are summarized in Table 2. Sample C1 and C2 from Chiba prefecture showed 100% identity with C. sp. deer genotype (Genbank accession number KR260681), C. ryanae (AY587166), and C. bovis (KU168249). With regard to the samples from Shizuoka, S1 showed 100% identity with C. ryanae (KT922235) and C. sp. genotype (KR260681); S2 showed 100% identity with C. ryanae (AY587166); S3, S4 and S5 showed 100% identity with C. ryanae (KT922235), C. bovis (KU168249) and C. sp. genotype (KR260681); and S6 and S7 were suggested to have 100% identity with C. ryanae (KT922235). In the samples from Kyoto, K1 showed 100% identity with C. ryanae (KT922235) and C. sp. genotype (KR260681); K2 showed 100% identity with C. sp. genotype (KT260681); and K3 and K4 showed 100% identity with C. ryanae (AY587166). M1 from Mie prefecture showed 100% identity with C. sp. genotype (KR260681) and N1 from Nagasaki showed 100% identity with C. meleagridis (AF112574).

Table 2. The identification of Cryptosporidium and Giardia species in Sika deer in Japan

Date of capture	Sampling area	Sample no.	Sex	Identity (>99% homology)
Cryptosporidium
2015 Jun.13	Chiba	C1	F	C. ryanae, C. bovis, C. sp. deer genotype
2016 Jun. 26		C2	M	C. ryanae, C. bovis, C. sp. deer genotype
2012 Oct. 16	Shizuoka	S1	M	C. ryanae, C. sp. deer genotype
2012 Oct. 16		S2	F	C. ryanae
2012 Oct. 18		S3	F	C. ryanae, C. sp. deer genotype, C. bovis
2012 Dec. 14		S4	M	C. ryanae, C. sp. deer genotype, C. bovis
2012 Dec. 14		S5	M	C. ryanae, C. sp. deer genotype, C. bovis
2013 May 28		S6	M	C. ryanae
2013 May 28		S7	M	C. ryanae
2015 Jun. 6	Kyoto	K1	M	C. ryanae, C. sp. deer genotype
2013 Dec. 11		K2	F	C. sp. deer genotype
2013 Dec. 13		K3	M	C. ryanae
2014 Jan. 9		K4	M	C. ryanae
2014 Jan. 11	Miyazaki	M1	Unknown	C. sp. deer genotype
2014 Dec. 18	Nagasaki		F	ND
2016 Jan. 21		N1	M	C. meleagridis
Giardia
2014 Jan. 16	Kyoto	Kyoto 29	F	G. intestinalis
2015 Nov. 27	Nagasaki	Nagasaki 28	M	G. intestinalis

The identification of Cryptosporidium and Giardia species and genotypes by the 18S rRNA sequence in wild deer samples

ND: not determined.

Both of the Giardia-positive samples from Kyoto and Mie were suggested to have 100% identity with G. intestinalis (Table 2).

The phylogenetic analysis placed all of the Cryptosporidium-positive samples, with the exception of the Nagasaki sample (C1-2, S1-7, K1-4, M1), in a single cluster with ruminant-derived Cryptosporidium species; N1 was clustered in bird-derived Cryptosporidium (Fig. 1). The phylogenetic analysis of Giardia revealed that the sequences of both samples from Kyoto and Nagasaki were clustered with Giardia intestinalis assemblage A (Fig. 2).

Fig. 1.

The phylogenetic relationships of the Cryptosporidium deer genotype obtained in this study to other available Cryptosporidium species and genotypes from the NCBI database as inferred by a neighbor-joining analysis, based on 18S ribosomal RNA (18S rRNA) sequences. A phylogenetic tree was constructed using the maximum-likelihood method with a maximum composite likelihood substitution model.

Samples C1-C2 from Chiba, S1-S4 from Shizuoka, K1-K4 from Kyoto, M1 from Mie, and N1 from Nagasaki correspond to Table 2.

Fig. 2.

Phylogenetic relationships of the Giardia deer genotype obtained in this study to other available Giardia species and genotypes from the NCBI database as inferred by neighbor-joining analysis, based on the 18S ribosomal RNA (18s rRNA) sequences. A phylogenetic tree was constructed using the maximum-likelihood method with a maximum composite likelihood substitution model.

Samples Contig Nagasaki 28 from Nagasaki and Contig Kyoto 29 from Kyoto correspond to Table 2.

4. Discussion

The results of this study demonstrate the difference in the prevalence of Cryptosporidium spp. and Giardia spp. in Cervus nippon centralis in Japan. This result suggests that Cryptosporidium had already spread widely throughout a large part of central Japan but that Giardia had not. The difference in the number of positive samples of Cryptosporidium and Giardia was statistically significant (P<0.01). The Cryptosporidium positive male deer was twice that of female deer (Table 1). This situation may be due to the males’ independent behavior and wide habitation range, which increases opportunities for exposure to pathogens. All of the deer examined in this study were asymptomatic. This may be due to the fact that the samples collected in this study were mostly obtained from adult deer of >2 years of age²⁾. The estimated number of Cryptosporidium and Giardia oocysts in each positive sample calculated by the manufacturer’s protocol was ≤4 oocysts per 1 g of feces (data not shown), which may be another reason of asymptomatic presentation. In this study, most of the samples were clustered in ruminant-derived Cryptosporidium (Fig. 1). Wild deer have recently caused significant feeding damage to grazing land in Japan; this represents a major problem for the country’s cattle industry and suggests that some cases of Cryptosporidium infection in deer result from contact with feces of cattle.

Zoonotic outbreaks of enterohaemorrhagic Escherichia coli (EHEC), Salmonella, hepatitis E, and Trichinella from the consumption of game meat have been reported in Japan; most cases were caused by eating raw meat¹²⁾. In the guidelines for processing game meat, information about pathogenic microbes refers only to those affecting farm animals, which are generally raised under relatively sanitary conditions. The detection of Cryptosporidium spp. and Giardia spp. in wild deer in the present study may indicate that the guidelines need to be updated to include preventive measures against Cryptosporidium and Giardia contamination that can be implemented during the slaughter of Sika deer.

The variable prevalence of these pathogens in different areas may suggest that there are some regional trends regarding frequency of contact between wild deer and cattle in Japan. A similar situation has been reported in China and Spain^6,11). A possible reason for the observed diversity in regional prevalence of Cryptosporidium and Giardia in deer feces is that some of the study areas were located close to cattle farms so wild deer may have frequent contact with cattle. On the other hand, since other study areas were located deep in the mountains, frequency of contact between deer and cattle may be low. In this study, Shiga, Mie, and Kumamoto are forest areas, which may be why no Cryptosporidium or Giardia were detected from deer feces.

In some areas, two or three different species were detected with >99% sequencing homology. This does not necessarily indicate coinfection; however, there are some reports on both mixed¹³⁾ and single infection^11,14) with Cryptosporidium species in cattle. These species are highly prevalent in cattle, indicating that C. bovis and C. ryanae may infect both cattle and deer, reflecting the results of the phylogenetic analysis (Fig. 1). The deer that harbored Cryptosporidium spp. were asymptomatic, suggesting that these healthy carrier deer may spread Cryptosporidium species to cattle or other farm animals.

Human infection with Cryptosporidium derived from deer has been reported in several countries¹⁵⁾, and the genotypes that caused sporadic cases in humans were also detected in Cervus nippon Temminck in China⁶⁾. In the present study, C. meleagridis was detected from deer in Nagasaki (Table 2). C. meleagridis has been reported in humans, but the most common host of C. meleagridis is turkey or parrots¹⁶⁾, so the current study may represent the first report from wild deer.

Two G. intestinalis assemblage A isolates were detected in the current study. This is consistent with the finding that among 12 species of Giardia, only G. intestinalis assemblage A has been reported to use wild animals as a host. Since G. intestinalis assemblage A is a human pathogenic species, it will be prudent to monitor the prevalence of G. intestinalis assemblage A to prevent human infection. In fact, the Giardia-positive deer in Nagasaki were caught just 10 km from an urban area.

In conclusion, we detected Cryptosporidium and Giardia in wild deer in Japan. These data could provide valuable information in relation to preparing Japanese wild game in a hygienic manner, and evidence to support the control of deer populations to protect water sources from contamination.

Acknowledgments

Our work was supported by a Grant-in-Aid for Young Scientists from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

Conflict of interest

The authors have no conflict of interest.

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