Seroepidemiology of Nine Zoonoses in Viljujsk,Republic of Sakha (Northeastern Siberia,Russian Federation)

Introduction

D uring the Soviet era, information about zoonoses in Eastern Siberia and especially in the Autonomous Republic of Yakutia (now the Republic of Sakha) was scarce and difficult to handle, as it was almost always written in Russian and often lacked an English abstract. Since the 1990s, however, a better knowledge of the status of these infections has suggested that the Yakut people may be steadily exposed to various zoonoses. Nevertheless, large gaps remain, especially concerning populations living in the remote parts of the country. In the meantime, the Republic of Sakha has developed its large natural reserves of oil, gas, coal, diamonds, gold, silver, tin, tungsten, and many other resources, along with the more traditional timber industry. As a consequence, an increasing number of Russian and foreign workers from the mining, oil, and forestry industries have penetrated into the Eastern Siberia ecosystem, potentially exposing themselves to zoonotic infections.

In 2004, the Mission Archéologique Française en Sibérie Orientale research group, a collaboration between Paul-Sabatier University in Toulouse, France, and Yakutsk State University, was formed for the purpose of studying the people of Eastern Siberia. Thanks to funding from the French National Research Agency, the focus of this collaboration was subsequently extended to include the diversity of modern Yakutian populations and human ecology, with a special concentration on the problem of zoonoses. To assess the possible presence and prevalence of nine major zoonotic infections in Yakutia, a seroepidemiology survey was therefore carried out in 2007 in Viljujsk city, which is located in the northern part of the Republic of Sakha at 63°45′19′′ N and 121°37′29′′ E. Geographically, Viljujsk area belongs to the Central Siberian Plateau. Superficial soils are glacial deposits, mainly sands of which the granulometry is about 1 mm. The permafrost layer, which is between 200 and 500 m thick, is found at about 2.0 m under the ground surface. The vegetal coverage is represented by black taiga (Siberian forest), in which the deciduous Dahurian larch (Larix gmelinii) is predominant. Birch trees (Betula humilis) and scrubs are found only around alas (ponds or small lakes), or along natural ditches (Alekseev et al. 2007). Viljujsk (elevation 107 m) is the administrative center of Viljujsky Ulus (district). Established in 1634 by Russian settlers as a post for fur trading, this city has 9776 inhabitants and is watered by the large Viljuy River (2650 km long), which is a tributary of the Lena River. The town displays the typical look of a remote Siberian settlement: private houses, which often are traditional isbas, are wooden-built, and the sandy unpaved streets are arranged according to a square grid. Due to the nature of the soil and the harsh climatic conditions, no sewage system exists, and neither piped water supply nor wells are available.

Subjects and Methods

Apparently healthy adult volunteers were recruited from administrative departments (Environmental Protection and Forests, Health and Social Security, Roads and Transportation, and Veterinary Services), among the medical and support staff of a retirement home, and acquaintances of one of the investigators (V.I.). Ninety subjects (63 women and 27 men) gave their informed oral consent to be enrolled in the study. After the subjects completed a questionnaire inquiring about age, sex, occupation (present or past), ethnicity, housing conditions, and food habits, a 10-mL venous blood sample was taken in a vacuum-glass vial, and stored at 4°C. In addition, 10 soil samples were randomly collected from unpaved streets in Viljujsk. In Toulouse, immunodiagnoses were carried out for Lyme borreliosis by indirect immunofluorescence assay using a commercial Borrelia burgdorferi reagent (Lyme-Spot™; bioMérieux, Marcy-L'Etoile, France), for alveolar echinococcosis (AE) by enzyme-linked immunosorbent assay (ELISA; Bordier Affinity Products, Crissier, Switzerland), for toxocariasis by Western blot (WB) using in-house-produced Toxocara canis larval excretory–secretory antigens (Magnaval et al. 1991), and for toxoplasmosis by indirect immunofluorescence assay detecting specific IgG (Toxo-Spot™; bioMérieux). Immunodiagnosis of both cystic echinococcosis (CE) and trichinellosis relied upon a similar in-house ELISA. Briefly, 96-well flat-bottomed titration plates (Nunc Maxisorb™; Life Technology, Cergy Pontoise, France) were sensitized with a commercial soluble extract of Echinococcus granulosus protoscoleces or Trichinella spiralis larvae (SR2B, Avrillé, France), and the antigen concentration was 5 μg/mL for CE and 6 μg/mL for trichinellosis. For both methods, anti-human immunoglobulin serum (anti IgG+A+M) conjugated with peroxidase (Bio-Rad, Hercules, CA) was used along with a kit for complementary reagents, for example, buffers and substrate (Enzygnost TMB™; Behring-Siemens, Marburg, Germany). For CE, the sensitivity rate of the assay was found to be 79% and the specificity rate 98%, and for trichinellosis, 89% and 96%, respectively. Moreover, for AE and CE, any ELISA result displaying an optical density (OD) greater than 0.150 (cut-off optical density value = 0.400) was checked by WB (LD Bio, Lyon, France). Soil samples were analyzed for helminth eggs or larvae by a flotation method (Ovassay™; San Diego, CA). In Marseilles, detection of IgG specific for tick-borne encephalitis and West Nile infection was achieved by ELISA according to the technical procedures from the WHO-Collaborating Reference and Research Center for Arboviruses at Institut Pasteur in Paris, and for Crimea-Congo hemorrhagic fever (CCHF) by ELISA using a recombinant antigen provided also by Institut Pasteur (Garcia et al. 2006). Statistical analysis of the data was performed using Epi-Info™ (CDC, Atlanta, GA).

Results

Sixty-six subjects belonged to the Yakut ethnics, eight were Evenk (another local ethnicity), four were Polish, three were Russian, three were Tadjik, and six were half-castes of Russian with Bashkir, Khakass, or Kazak. Table 1 displays demographic and occupational data. All subjects lived in modern wooden-built houses, or in isbas. All relied for drinking water catering on ice or snow melting during winter, thanks to the very low price for natural gas supplying, and on rainwater collected in tanks during the rest of the year. Eighty-eight subjects (97.7%) ate cooked meat of wild game regularly, mostly during spring and fall, and 86 (95.5%) had a culinary habit of consuming raw horse meat. The results of the serological investigations are shown in Table 2. For each zoonosis, no significant difference was found according to sex, classes of age, ethnicity, occupation, housing conditions, or food habits. When results for two tick-borne zoonoses (Lyme disease and CCHF) were combined, no significant correlation was detected with the above-cited variables.

Discussion

First of all, it should be underlined that the study population was essentially a convenience group rather than representative of the larger Viljujsk population, so these results must be cautiously taken into consideration.

The prevalence found for Lyme borreliosis (3.44%) was consistent with results from a survey carried out among 41 rural subjects from the same Viljujsk area (Storch et al. 2008). Using ELISA followed by WB, these authors found a 19.5% positivity rate for specific anti-Borrelia IgG. The significant difference from our result (Fisher's exact test, p = 0.004) may be explained by the rural recruitment in Storch et al.'s study, as well as the better sensitivity provided by the couple ELISA/WB. In this study, no subject tested positive for AE or CE. Since the sensitivity rate of WB is about 97% for the detection of infection with Echinococcus genus (Liance et al. 2000), the transmission of both zoonoses must occur at a low level in Northern Yakutia. However, no recent data were available in the literature on this topic and for this region. Given the semirural Viljujsk environment, the 4.44% seroprevalence rate for toxocariasis was surprisingly low. In fact, it was very similar to that observed in adult inhabitants of the downtown areas of large Westernized cities (reviewed by Magnaval et al. 2001). Moreover, although stray dogs were observed in the streets, none of the 10 soil samples were positive for helminth eggs or larvae. Environmental conditions would provide an explanation for this negative result: in Viljujsk, the temperature is over 0°C only from June to mid-October, the average annual temperature is −9.4°C, the average temperature in January is −47°C, and the average annual precipitation is 200 mm (according to Climats™; Meteo France database, Toulouse, France), conditions that would lead to an almost permanent absence of helminth propagules in the soil. No information concerning toxocariasis was available for Yakutia, but a survey carried out in Western Siberia reported a 1.8% rate, which supports the above-cited environmental hypothesis (Frolova et al. 1997).

Toxoplasmosis is a protozoan zoonosis that is either soil-transmitted (ingestion of oocysts) or food-borne (ingestion of bradyzoites along with raw or undercooked meat). The first route is certainly limited by the Siberian climate, but the Yakutian people have a habit of eating raw meat, as demonstrated by our survey. The combination of these two opposite transmission factors resulted in an 8.9% seroprevalence, lower than that observed, for example, in Western European countries, but similar to that (10.8%) reported in the United States (Jones et al. 2001). This finding appears to be consistent with data from a survey previously carried out in Central and Southern Yakutia (Egorov et al. 1997). According to a recent article (Ozeretskovskaya et al. 2005), the overall incidence of trichinellosis is declining in Russia, and most of the cases reported in Eastern parts of the Federation are due to the consumption of wild game (bear or badger). Such eating habits were found to be very prevalent among the studied subjects. As a result, 4.44% of the subjects had anti-Trichinella antibodies, but the nature of the involved Trichinella species (T. spiralis, T. britovi, or T. nativa) remains to be determined by further investigation.

Concerning the arboviral infections caused by flaviviruses, three patients displayed a concomitant positive result for tick-borne encephalitis and West Nile infection, but not for other tested zoonoses. Specific IgM antibodies against both viruses were not found, so no molecular detection of viral RNA was attempted. A seroneutralization assay for clarifying the specificity of the antibodies was not possible. Serodiagnosis for CCHF yielded 10 positive results (11.1%), which was an notable finding. Previously, it was believed that the northern border of CCHF distribution was Southern Russia, Central Asia, and Xinjiang in China (Lvov et al. 2004). The diagnostic assay used in this study has been previously validated (Garcia et al. 2006), and no cross reaction was observed with sera from subjects infected by other nairoviruses. However, possible interferences caused by as yet unknown viruses circulating in the region could not be excluded. Further studies, including the molecular detection of viral RNA in ticks, are therefore needed to confirm this epidemiological discovery. Moreover, it should be noted that Viljujsky Ulus was the primary focus of the so-called Viljusjsk encephalomyelitis outbreak, of which the cause remains unknown, despite several surveys investigating potential bacterial, parasitic, viral, and genetic etiologies (Vladimirtsev et al. 2007, Storch et al. 2008). CCHF is not known to induce any chronic central nervous system involvement (Cevik et al. 2008), so a case–control study of Viljusjsk encephalomyelitis patients, including the result of an extended laboratory diagnosis for CCHF as an outcome variable, would be highly desirable. Alternatively, the involvement of mosquito- or tick-borne flaviviruses should be more extensively investigated.

Undoubtedly, the harsh continental and sub-Arctic Yakutian climate exerts a steamroller effect on the diffusion of soil-transmitted zoonoses, namely, AE, CE, toxocariasis, and, partially, toxoplasmosis. The only similar observations available in the English literature are from a survey carried out in a Cree (Indian) community, close to Lake Mistassini, Canada (50°57′N, 73°42′W). Although more humid than Northeastern Siberia, the climate in this area may also be described as harsh, according to the annual temperature curves from the Climats database. The Canadian authors investigated serologically 50 volunteers for eight zoonoses, among them CE, toxoplasmosis, toxocariasis, and trichinellosis (Lévesque et al. 2007). The observed seroprevalence rates (CE: 0/50; toxocariasis: 2/50; toxoplasmosis: 5/50; trichinellosis: 0/50) did not differ significantly from the results of the present Siberian survey.