Serological Evidence of Hepatitis E Virus Circulation Among Reindeer and Reindeer Herders

Abstract

Various deer species are infected with hepatitis E virus (HEV) and may be a source of zoonotic infection for humans. So far, HEV has not been isolated from reindeer and the role of this domesticated deer species in HEV transmission is unknown. We tested serum samples from 191 reindeer (Rangifer tarandus) and 86 adult reindeer herders from the circumpolar regions of Yakutia (Russian Federation) for anti-HEV and HEV RNA. Anti-HEV IgG prevalence was 12.0% (95% confidence interval [95% CI]: 8.1–17.5) in reindeer and 4.7% (95% CI: 1.5–11.7) in reindeer herders. The latter was similar to the positivity rate observed in adult residents of the city of Yakutsk, the capital of Yakutia, who do not have routine contact with reindeer (3.7% [19/519, 95% CI: 2.3–5.7]). No samples positive for HEV RNA were identified. The study provides evidence of HEV circulation in reindeer. Nevertheless, the low seroprevalence in reindeer herders indicate a low risk of zoonotic HEV infection.

Introduction

Hepatitis E virus (HEV), which belongs to the genus Hepevirus and family Hepeviridae, is the etiological agent of acute hepatitis. HEV, found in mammals, including humans, belongs to the species Orthohepevirus A and includes eight genotypes (HEV-1 to HEV-8). Genotypes HEV-1 and HEV-2 infect only humans, HEV-3 is found in humans, pigs, wild boar, deer, rabbits, rats, mongooses, cattle, and sheep. HEV-4 is identified in humans, pigs, and goats. HEV-5 and HEV-6 have been found so far only in wild boars. HEV-7 and HEV-8 have been found in one-humped and two-humped camels, respectively (Kenney, 2019). In addition, a unique case of HEV-7 infection has been detected in a recipient of liver transplantation who regularly consumed camel meat and milk (Lee et al. 2016).

Developing countries in tropical and subtropical zones are considered to be endemic for hepatitis E. The disease in these regions is caused mainly by HEV-1 and HEV-2, which are transmitted through the water-borne route and are associated with poor sanitation and poor public health infrastructure. Zoonotic HEV-3 and HEV-4 cause sporadic autochthonous cases in humans in industrial countries located in temperate climatic zones. The main reservoir of HEV-3 and HEV-4 are domestic pigs (Doceul et al. 2016). Data from seroprevalence studies indicate that HEV is widespread in industrial pig farms all over the world (Geng et al. 2010, Burri et al. 2014, Pisano et al. 2018, Tsachev et al. 2019). Therefore, veterinarians and other persons who work with swine may have increased risk of HEV acquisition (Drobeniuc et al. 2001, Kantala et al. 2017, Lassen et al. 2017). HEV is also prevalent in wild boars, posing the possible risk of infection for hunters (Ivanova et al. 2015, Zele et al. 2016, Jemersic et al. 2017, Strakova et al. 2018, Tsachev et al. 2020).

Besides pigs, HEV-3 was repeatedly identified in different deer species found in Europe (red deer, fallow deer, roe deer), while HEV-4 was found in species common in Asia—tufted deer, Sika deer (Zhang et al. 2008, Reuter et al. 2009, Boadella et al. 2010, Forgach et al. 2010, Neumann et al. 2016, Anheyer-Behmenburg et al. 2017, Weger et al. 2017). Moreover, cases of human infection with HEV have been described in patients who ate deer meat and deer liver (Tei et al. 2003). However, the contribution of deer to zoonotic transmission of HEV is believed to be significantly lower compared with domestic pigs due to consistent lower viral loads in tissues of infected deer (Anheyer-Behmenburg et al. 2017).

Antibodies to HEV (anti-HEV) are also found in the human population in territories where pig breeding is not common and wild boar are not found, such as among Canadian Inuits, whereas the frequency of detection of anamnestic antibodies is not lower than the rates recorded in countries where pigs are the main zoonotic reservoir of HEV (Minuk et al. 2007). Furthermore, anti-HEV has been identified in Alaska Native peoples, although less frequently when compared with non-Native persons (Miernyk et al. 2019). These data indicate the existence of an unidentified HEV reservoir in the northern territories. Reindeer (Rangifer tarandus) are the most likely source of zoonotic transmission of HEV in these regions.

Reindeer living in North America (Canada, USA [Alaska]) were tested for HEV, but so far only anti-HEV antibodies have been detected in this species of deer, not the virus itself (Weger et al. 2017). Until now, there have been no studies of HEV circulation among the Asian reindeer, which are the principal domesticated animal and source of meat for the indigenous population of the polar regions of Siberia. Also, there are no comparative data on the prevalence of anti-HEV among Siberian reindeer herders and among the populations of the same regions that do not have close contact with reindeer. The aim of this study was to determine the prevalence of markers of HEV infection (anti-HEV, HEV RNA) among reindeer and reindeer herders in the circumpolar regions of the Republic of Sakha (Yakutia), a large region in the northeast of the Russian Federation.

Materials and Methods

Samples

All human and animal samples tested in this study were collected from March 2018 to August 2019 and are shown in Fig. 1 with their indicated geographic origin. A total of 191 reindeer (R. tarandus) serum samples from two different regions of Yakutia (Anabarsky district, 156 samples, and Oymyakonsky district, 35 samples) were obtained and tested for anti-HEV IgG and HEV RNA. The sex was known for 145 out of 191 animals, which included 87 males and 58 females. The age was known for 123 animals: 36 animals were under 1 year (over 6 months), 36 animals were 2 years old, 18 were 3 years old, and 33 were over 3 years (4–8 years). Serum samples from 86 reindeer herders (24–80 years old, mean age 43.3 years, male:female ratio 1:1.1) from Olenyoksky Evenk National District were tested for anti-HEV IgM and IgG; antibody positive samples were tested for HEV RNA.

FIG. 1.

Sampling territories (shaded on the map) and the numbers of sampled sera from reindeer, reindeer herders, and the general population in Yakutia.

As a control group of persons who do not have routine contact with reindeer, serum samples from 519 adult residents of the city of Yakutsk, the capital of Yakutia, were tested for anti-HEV IgG. Subjects were males and females 20–84 years of age, apparently healthy, with no symptoms of acute disease (self-reported) and were permanent residents in the study region. The mean age in this cohort was 45.0 years, male:female ratio 1:1.3.

The climate in all studied regions of Yakutia is sharply continental characterized by long winter and short summer periods (mean annual temperature is −7.5°C, average annual precipitation about 200 mm/m²). The number of reindeer in Yakutia is about 201,000, which is 13% of the total reindeer population in the Russian Federation. Written informed consent was obtained from human participants. The investigation was conducted in accordance with the principles of the Declaration of Helsinki. Blood sampling from animals was carried out in a manner consistent with the humane treatment of animals. The study design was approved by the Ethics Committee of the Mechnikov Research Institute for Vaccines and Sera in Moscow, Russia (Approval 1, dated February 28, 2018) and by the Ethics Committee of the North-Eastern Federal University named after M.K. Ammosov, Yakutsk, Russian Federation (Approval #39 dated June 26, 2014). All sera samples were coded and aliquoted, and aliquots were stored at −70°C until testing.

HEV testing

Anti-HEV IgG and IgM were tested in human sera using commercial enzyme immunoassay (EIA) kits (DS-EIA-ANTI-HEV-G and DS-EIA-ANTI-HEV-M, Diagnostic Systems, Russia) according to the manufacturer's instructions. Both tests have 95.5% specificity and 95.0% sensitivity according to the manufacturer's specifications. Anti-HEV IgG in reindeer sera were tested using the same kit (DS-EIA-ANTI-HEV-G), but rabbit anti-deer IgG polyclonal antibody labeled with horseradish peroxidase (KPL, MA) was used instead of human-specific conjugate from the kit. The working dilution of deer-specific conjugate for ELISA was 1:100 in phosphate-buffered saline. The modified test was validated using the panel consisting of 94 HEV-negative human serum samples.

Its specificity was determined to be 100% (95% CI: 95.3–100). The sensitivity of modified test was not determined due to the lack of reindeer serum samples confirmed to be positive for anti-HEV in second independent assay. The reproducibility of modified test was assessed in testing of 8 anti-HEV-positive and 8 anti-HEV-negative reindeer serum samples in 10 independent runs. Coefficient of variation values for optical density (OD) values were 2.3% or lower.

HEV RNA was detected using RT-PCR with degenerate nested primers targeting the open reading frame 2 region (Meng et al. 1997). RNA was isolated from all deer serum samples and from anti-HEV-positive human serum samples using the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) and MagNA Pure Compact Nucleic Acid Isolation Kit I (Roche Applied Science, Mannheim, Germany).

Statistical analysis

Data analysis was performed using graphpad.com. Statistical analysis includes the calculation of a 95% confidence interval (95% CI) and assessing the significance of differences of mean values between groups using Fisher's exact test and the chi-squared test (significance threshold p < 0.05).

Results

Anti-HEV was detected in 23 out of 191 reindeer serum samples (12.0%, 95% CI: 8.1–17.5), including 17 out of 156 samples from Anabarsky district (10.9%, 95% CI: 6.8–18.9) and 6 out of 35 samples from Oymyakonsky district (17.1%, 95% CI: 7.7–33.1). All reindeer sera tested negative for HEV RNA. The age and sex were known for 145 out of 191 animals, including 16 out of 23 positive for anti-HEV. Reactive samples were identified in all age groups of reindeer. The mean cut-off index (COI) (the ratio of OD of the sample to cutoff OD) in reactive samples was 3.31 ± 1.39. The distribution of anti-HEV-positive samples by region, age, and sex among reindeer with known age and sex is shown in Table 1. No statistically significant differences in anti-HEV prevalence were found between age groups or by sex (p > 0.05).

Table 1.

The Prevalence of Anti-Hepatitis E Virus Among Reindeer with Known Age and Gender

	Age, years				Gender
	<1	2	3	>3	Males	Females
Number of tested samples	36	36	18	33	87	58
Number of anti-HEV positive samples (%)	3 (8.3)	6 (16.7)	1 (5.6)	6 (18.2)	8 (9.2)	8 (13.8)
95% CI	2.1–22.6	7.5–32.3	0.1–27.7	8.2–34.8	4.5–17.3	6.9–25.2
p ^a	0.294^b	1.000^b	0.398^b	—	0.420

Fisher's exact test.

When compared with age group “>3 years.”

95% CI, 95% confidence interval; HEV, hepatitis E virus.

The anti-HEV IgG detection rate in reindeer herders was 4.7% (4/86, 95% CI: 1.5–11.7). The mean COI in reactive serum samples was 5.53 ± 1.92. All four positive samples were obtained from individuals over 55 years (mean age 67.3 ± 14.2 years). No samples positive for anti-HEV IgM were identified in reindeer herders. All anti-HEV IgG-positive samples tested negative for HEV RNA.

In the control group, represented by conditionally healthy adult residents of Yakutsk, the prevalence of anti-HEV IgG was 3.7% (19/519, 95% CI: 2.3–5.7), which was not statistically different from the prevalence observed in reindeer herders (p > 0.05). The distribution of anti-HEV IgG-positive rates in different age groups of reindeer herders and residents of Yakutsk is shown in Table 2. All anti-HEV-positive cases in reindeer herders and the majority of such cases among residents of Yakutsk were concentrated in the older age groups (50 and older); differences between age groups within each cohort were statistically significant (p < 0.05, chi-squared test).

Table 2.

Age-Specific Anti-Hepatitis E Virus IgG Prevalence in Reindeer Herders and in Residents of Yakutsk City

Cohort	Age group	Number of tested samples	Anti-HEV IgG positive		Chi-squared	df	p^a
Cohort	Age group	Number of tested samples	N	%	Chi-squared	df	p^a
Reindeer herders	20–49 years	57	0	0.0 (95% CI: 0–7.6)	8.25	1	0.004
Reindeer herders	50 years and older	29	4	13.8 (95% CI: 4.9–31.2)	8.25	1	0.004
Residents of Yakutsk city	20–49 years	323	6	1.9 (95% CI: 0.8–4.1)	7.89	1	0.005
Residents of Yakutsk city	50 years and older	196	13	6.6 (95% CI: 3.8–11.1)	7.89	1	0.005

Chi-squared test.

df, degrees of freedom.

Two samples from residents of Yakutsk were positive both for anti-HEV IgG and anti-HEV IgM. Both were identified in the subgroup 50 years of age and older (2/196, 1.0%, 95% CI: 0.0–5.5). No HEV RNA was detected in anti-HEV-positive sera.

Discussion

This study presents data on the prevalence of HEV infection among the indigenous inhabitants of the polar regions of the Asian part of the Russian Federation, as well as among reindeer inhabiting this territory. Our results indicate the possibility of the exposure of reindeer herders to HEV. Nevertheless, the rate of detection of antibodies to HEV in reindeer herders did not differ from that in individuals from the control group, who did not have regular contact with reindeer. Anti-HEV prevalence data obtained in Yakutian reindeer herders (Evenk and Yakut ethnicities) are similar to those observed in the isolated Canadian Inuit community (Minuk et al. 2007).

Interestingly, anti-HEV prevalence in Alaska Native persons, reported by Miernyk et al. (2019), was much lower than in non-Native persons in that study and in comparison to our data on seroprevalence in reindeer herders. Seroprevalence data largely depend on the sensitivity of the test used. The limit of anti-HEV IgG detection for ELISA test used in our study was previously shown to be 1000 mIU/mL (Kodani et al. 2017).

It should be noted that all anti-HEV-positive cases among reindeer herders in this study were identified in elderly people, which may be attributed to exposure to HEV long ago. The limited circulation of HEV among reindeer herders is supported by the absence of cases of detection of anti-HEV IgM, as well as by the absence of IgG antibodies among younger individuals. In the control group, however, the anti-HEV IgG-positive cases were also concentrated among people over 50 years old. This pattern—an increase in the proportion of seropositive individuals in older age groups—is typical for most industrialized countries where zoonotic HEV-3 and HEV-4 circulate (Faber et al. 2012, Lagler et al. 2014, Mansuy et al. 2016).

The only type of activity of the surveyed reindeer herders is breeding and hunting of wild reindeer and dressing their skins. Their only meat-based dietary component is domesticated and wild reindeer. Thus, contact with wild or domestic pigs or pig meat products is excluded. Moreover, wild boars are not found in the polar regions of Yakutia, and due to climatic features, domesticated pig breeding is not developed throughout Yakutia.

Our data on anti-HEV detection among reindeer indicate that HEV circulates in these animals in Yakutia and, thus, may cause HEV infection in reindeer herders. Anti-HEV in reindeer was detected with a similar frequency in two distant regions of Yakutia, while positive findings were observed in all age groups, from deer under 1 year of age to animals 3–4 years of age. Anti-HEV seroprevalence data in Yakutian reindeer were much higher than those observed in free-ranging caribou in Canada (3.2%) (Weger et al. 2017).

It was previously suggested that deer are not a true reservoir of HEV, but are infected incidentally by sharing the same habitat as wild boar, which represent a source of spillover HEV infections for deer in areas where ranges of these species overlap (Pavio et al. 2017). Our data on serological evidence of HEV circulation among reindeer in a territory where wild and domestic pigs are absent suggest that reindeer might be a true reservoir of HEV.

Until now, HEV itself has not been isolated from reindeer, despite the numerous isolations of HEV-3 and HEV-4 from other species of deer living in warmer climates. A possible reason for this may be a significant genetic difference between reindeer HEV and known strains of Orthohepevirus A, which prevents its detection with the used primer sets. The sensitivity of the PCR used in our study was 125 IU/mL when detecting genotypes HEV-3 and HEV-4 (Diarrassouba et al. 2016); therefore, it is capable of detecting the RNA of these zoonotic genotypes in reindeer samples when present.

The second possible reason is that the search for viral RNA was carried out among animals that were too old. Among domestic pigs, most cases of HEV infection occur in piglets between 2 and 4 months of age, as they become susceptible to the virus once maternal antibodies have disappeared (Feng et al. 2011). Likewise, most cases of HEV infection in reindeer may also occur at an early age, and the viral sequences should be sought in fawns.

This study has several limitations. First, the analysis included a relatively small number of serum samples from reindeer and reindeer herders. Moreover, only one ELISA Kit was used for anti-HEV detection without a second confirmatory test. Finally, the absence of HEV RNA-positive samples made phylogenetic analysis impossible. Despite these limitations, this is a first seroprevalence study of HEV among reindeer and reindeer herders in the Russian Federation, which provides new data on the epidemiology of HEV infection in polar regions.

Conclusion

The prevalence of anti-HEV in reindeer and its detection in reindeer herders at a rate similar to that observed in a general population suggest that reindeer may be a true reservoir of HEV. Nevertheless, the low seroprevalence in reindeer herders indicates the low risk of zoonotic HEV infection.

Footnotes

Authors' Contributions

All authors have taken part in writing the article, reviewing it, and revising its intellectual and technical content. All authors assume responsibility and accountability for the results. All authors read and approved the final article.

Author Disclosure Statement

No conflicting financial interests exist.

Funding Information

This study was funded by grant from the Ministry of Education and Science of the Russian Federation (contract no. 075-15-2019-1481 from 15.08.2019, project ID RFMEF161319X0091).

References

Anheyer-Behmenburg

, Szabo

, Schotte

, Binder

, et al. Hepatitis E virus in wild Boars and spillover infection in red and Roe Deer, Germany, 2013–2015. Emerg Infect Dis, 2017; 23:130–133.

Boadella

, Casas

, Martín

, Vicente

, et al. Increasing contact with hepatitis E virus in red deer, Spain. Emerg Infect Dis, 2010; 16:1994–1996.

Burri

, Vial

, Ryser-Degiorgis

, Schwermer

, et al. Seroprevalence of hepatitis E virus in domestic pigs and wild boars in Switzerland. Zoonoses Public Health, 2014; 61:537–544.

Diarrassouba

, Potemkin

, Lopatukhina

, Isaieva

, et al. The standardization of diagnostics of hepatitis E [in Russian]. Clin Lab Diagn, 2016; 61: 299–303.

Doceul

, Bagdassarian

, Demange

, Pavio

. Zoonotic hepatitis E virus: Classification, animal reservoirs and transmission routes. Viruses, 2016; 8:270.

Drobeniuc

, Favorov

, Shapiro

, Bell

, et al. Hepatitis E virus antibody prevalence among persons who work with swine. J Infect Dis, 2001; 184:1594–1597.

Faber

, Wenzel

, Jilg

, Thamm

, et al. Hepatitis E virus seroprevalence among adults, Germany. Emerg Infect Dis, 2012; 18:1654–1657.

Feng

, Zhao

, Li

, Harrison

, et al. Infection dynamics of hepatitis E virus in naturally infected pigs in a Chinese farrow-to-finish farm. Infect Genet Evol, 2011; 11:1727–1731.

Forgach

, Nowotny

, Erdelyi

, Boncz

, et al. Detection of hepatitis E virus in samples of animal origin collected in Hungary. Vet Microbiol, 2010; 143:106–116.

10.

Geng

, Wang

, Zhao

, Yu

, et al. Serological prevalence of hepatitis E virus in domestic animals and diversity of genotype 4 hepatitis E virus in China. Vector Borne Zoonotic Dis, 2010; 10:765–770.

11.

Ivanova

, Tefanova

, Reshetnjak

, Kuznetsova

, et al. Hepatitis E virus in domestic pigs, wild boars, pig farm workers, and hunters in Estonia. Food Environ Virol, 2015; 7:403–412.

12.

Jemersic

, Keros

, Maltar

, Barbic

, et al. Differences in hepatitis E virus (HEV) presence in naturally infected seropositive domestic pigs and wild boars—An indication of wild boars having an important role in HEV epidemiology. Vet Arhiv, 2017; 87:651–663.

13.

Kantala

, Kinnunen

, Oristo

, Jokelainen

, et al. Hepatitis E virus antibodies in Finnish Veterinarians. Zoonoses Public Health, 2017; 64:232–238.

14.

Kenney

. The current host range of hepatitis E viruses. Viruses, 2019; 11:452.

15.

Kodani

, Kamili

, Tejada-Strop

, Poe

, et al. Variability in the performance characteristics of IgG anti-HEV assays and its impact on reliability of seroprevalence rates of hepatitis E. J Med Virol, 2017; 89:1055–1061.

16.

Lagler

, Poeppl

, Winkler

, Herkner

, et al. Hepatitis E virus seroprevalence in Austrian adults: A nationwide cross-sectional study among civilians and military professionals. PLoS One, 2014; 9:e87669.

17.

Lassen

, Janson

, Neare

, Tallo

, et al. Prevalence of antibodies against hepatitis E virus in veterinarians in Estonia. Vector Borne Zoonotic Dis, 2017; 17:773–776.

18.

Lee

, Tan

, Teo

, Lim

, et al. Chronic infection with camelid hepatitis E virus in a liver transplant recipient who regularly consumes camel meat and milk. Gastroenterology, 2016; 150:355–357.e3.

19.

Mansuy

, Gallian

, Dimeglio

, Saune

, et al. A nationwide survey of hepatitis E viral infection in French blood donors. Hepatology, 2016; 63:1145–1154.

20.

Meng

, Purcell

, Halbur

, Lehman

, et al. A novel virus in swine is closely related to the human hepatitis E virus. Proc Natl Acad Sci USA, 1997; 94:9860–9865.

21.

Miernyk

, Bruden

, Parkinson

, Hurlbart

, et al. Human seroprevalence to 11 zoonotic pathogens in the U.S. Arctic, Alaska. Vector Borne Zoonotic Dis, 2019; 19:563–575.

22.

Minuk

, Sun

, Uhanova

, et al. Serological evidence of hepatitis E virus infection in an indigenous North American population. Can J Gastroenterol, 2007; 21:439–442.

23.

Neumann

, Hackl

, Piepenschneider

, Vina-Rodriguez

, et al. Serologic and molecular survey of hepatitis E virus in German Deer Populations. J Wildl Dis, 2016; 52:106–113.

24.

Pavio

, Doceul

, Bagdassarian

, Johne

. Recent knowledge on hepatitis E virus in Suidae reservoirs and transmission routes to human. Vet Res, 2017; 48:78.

25.

Pisano

, Martinez-Wassaf

, Mirazo

, Fantilli

, et al. Hepatitis E virus in South America: The current scenario. Liver Int, 2018; 38:1536–1546.

26.

Reuter

, Fodor

, Forgach

, Katai

, et al. Characterization and zoonotic potential of endemic hepatitis E virus (HEV) strains in humans and animals in Hungary. J Clin Virol 2009; 44:277–281. Strakova P, Kubankova M, Vasickova P, Juricova Z, et al. Hepatitis E virus in archived sera from wild boars (Sus scrofa), Czech Republic. Transbound Emerg Dis, 2018; 65:1770–1774.

27.

Tei

, Kitajima

, Takahashi

, Mishiro

. Zoonotic transmission of hepatitis E virus from deer to human beings. Lancet, 2003; 362:371–373.

28.

Tsachev

, Baymakova

, Ciccozzi

, Pepovich

, et al. Seroprevalence of hepatitis E virus infection in pigs from Southern Bulgaria. Vector Borne Zoonotic Dis, 2019; 19:767–772.

29.

Tsachev

, Baymakova

, Pepovich

, Palova

, et al. High seroprevalence of hepatitis E virus infection among East Balkan swine (Sus scrofa) in Bulgaria: Preliminary results. Pathogens, 2020; 9:911.

30.

Weger

, Elkin

, Lindsay

, Bollinger

, et al. Hepatitis E virus seroprevalence in free-ranging Deer in Canada. Transbound Emerg Dis, 2017; 64:1008–1011.

31.

Zele

, Barry

, Hakze-van der Honing

, Vengust

, et al. Prevalence of anti-hepatitis E virus antibodies and first detection of hepatitis E virus in wild boar in Slovenia. Vector Borne Zoonotic Dis, 2016; 16:71–74.

32.

Zhang

, Shen

, Mou

, Gong

, et al. Hepatitis E virus infection among domestic animals in eastern China. Zoonoses Public Health, 2008; 55:291–298.