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Serological evidence for tick-borne encephalitis, borreliosis, and human granulocytic anaplasmosis in Mongolia
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International Journal of Medical Microbiology 296 (2006) S1, 69–75 www.elsevier.de/ijmm
Serological evidence for tick-borne encephalitis, borreliosis, and human granulocytic anaplasmosis in Mongolia Gernot Waldera,, Erdenechimeg Lkhamsurenb, Abmed Shagdarc, Jantsandoo Bataac, Tsetsegsaikhan Batmunkhb, Dorothea Ortha, Franz X. Heinzd, Galina A. Danichovae, Maksim A. Khasnatinove, Reinhard Wu¨rznera, Manfred P. Diericha a
Department of Hygiene, Microbiology and Social Medicine, Innsbruck Medical University, Fritz-Pregl-Str. 3, A-6020 Innsbruck, Austria b Department of Microbiology and Immunology, National Medical University of Mongolia, Ulaanbaatar, Mongolia c National Center for Communicable Diseases, Ulaanbaatar, Mongolia d Institute for Virology, Medical University of Vienna, Vienna, Austria e Institute for Epidemiology and Microbiology, Irkutsk Medical University, Irkutsk, Russia
Abstract Five hundred and forty-five serum samples from donors from various parts of Mongolia were investigated for antibodies against the tick-borne encephalitis (TBE) virus, Borrelia burgdorferi, and Anaplasma phagocytophilum. Seroprevalence against TBE was 5.1% in the province of Selenge and 0.9% in Bulgan province, seroprevalence against B. burgdorferi was 1.9% in Selenge province and Bulgan province, 13.9% in Dornogov province, and 3.0% in Tov province and Ulaanbaatar. Seroprevalence against A. phagocytophilum was 2.3% in Selenge province, 5.6% in Bulgan province, 2.8% in Dornogov province, and 3.0% in Tov province and Ulaanbaatar. We conclude that all three pathogens are endemic in Mongolia. r 2006 Elsevier GmbH. All rights reserved. Keywords: TBE; Borreliosis; Human granulocytic anaplasmosis; Central Asia; Mongolia
Introduction Mongolia, a vast, but scarcely populated country in Central Asia with increasing economic importance (including tourism), is one of the very few countries in Eurasia where data on tick-borne diseases are not available yet (Ebright et al., 2003). There is, however, evidence which supports the suggestion that these diseases may well be endemic in this country.
Corresponding author. Tel.: +43 512 507 3411; fax: +43 512 578 745. E-mail address: [email protected] (G. Walder).
1438-4221/$ - see front matter r 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.ijmm.2006.01.031
According to local entomologists (postal communication from Batsukh Zayat, Institute of Veterinary Medicine of Mongolia), ixodid ticks are found in the northern and central parts of Mongolia. Ixodes (I.) persulcatus occurs in the provinces of Huvsgul, Selenge, and Hentii and I. crenulatus in Ovorkhangai, Tov, and Dornod province. Dermacentor and Haemaphysalis ticks are even more widespread, covering the provinces of Huvsgul, Selenge, Hentii, Ovorhangai, Tov, Dornod, Bayan-Olgii, Hovd, Uvs, Gov-Altai, Omnogov, Dundgov, Bayanhongor, and Dornogov. Epidemiologic data from neighboring Siberia and China provide further support for the occurrence of tick-borne encephalitis (TBE), borreliosis, and human
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granulocytic anaplasmosis (HGA) in Mongolia. Siberia, where two of the three genetically distinct strains of the TBE virus are abundant (Zlobin et al., 1992), accounts for more than 50% of all TBE cases in Russia (Su¨ss, 2003) and seroprevalences of up to 66% have been reported for ruminants in the neighboring region of Irkutsk (Titenko et al., 2002). Recently, the virus has also been isolated from ticks in north-eastern China (Cai et al., 1995), but not in close vicinity to Mongolia. Borrelia (B.) burgdorferi sensu lato has been found in 20–45% of I. persulcatus ticks in northern China (Zhang et al., 1991) by direct immunofluorescence. This agent is abundant in the two border provinces to Mongolia, Xinjiang (Wan et al., 1998) and Inner Mongolia, where it has been identified by culture and PCR (Takada et al., 2001). Seroprevalences vary from less than 4% (Zhang et al., 1997) to more than 20% (Zhang, 1989), depending on the location and the applied serologic methods. Studies on borreliosis in Siberia are mostly limited to the western part of this region, where annual morbidities of 2/100,000 inhabitants have been reported (Lesniak et al., 1995) and seroprevalence was 6–12% (Lesniak et al., 1994). A case of borreliosis was reported from the Baikal area in 1991 (Anan’eva et al., 1991). Information on Anaplasma (A.) phagocytophilum is still very limited. The agent has been detected in ticks and in patients from northern China by means of PCR (Zhao et al., 2002), and recently a clinical case was reported in the Far East of the Russian Federation (Sidel’nikov et al., 2003). Here we describe the demonstration of antibodies against TBE, B. burgdorferi s.l., and A. phagocytophilum in volunteer donors from Mongolia.
Materials and methods Patients Two hundred and twenty-nine serum samples were taken from volunteer donors during August 2003 in the First Hospital in Ulaanbaatar and in Khuder soum in the upper Chikoy valley in the Selenge province. All patients were questioned as to their permanent place of residence, previous travels abroad, tick bites, the geographic region where the tick bites had been acquired, and possible clinical symptoms following these tick bites. An additional 316 sera from female donors participating in a study on sexually transmitted diseases in pregnancy was supplied by the National Center of Infectious Diseases. These sera were rendered anonymously prior to investigation, so that only gender, age, and the province in which the patient permanently resided were recorded. Sampling was done by or in cooperation with the National Center for Communicable Diseases of Mongolia according to the local laws. All patients had given consent to their sera being tested for antibodies
against infectious diseases, although not all of them were available for questions regarding previous tick bites.
Serological investigation Samples were screened for IgG against TBE virus using commercially available ELISA (Enzygnost TBE, Dade Behring, Marburg, Germany; sensitivity 96.8% and specificity 99.5%, as stated by the manufacturer). Positive results were confirmed using a neutralization assay as described earlier (Holzmann et al., 1996). Samples yielding a neutralization titer of X1:10 were rated positive. A commercial ELISA (Enzygnost Borrelia, Dade Behring, Marburg, Germany; sensitivity 92.5%, as stated by the manufacturer; specificity 75%, as assessed in our laboratory, data not shown) was used to detect anti-B. burgdorferi s.l. IgG. Positive results were confirmed by means of a recombinant immunoblot (Mikrogen, Martinsried, Germany). Samples which had antibodies against at least one antigen of panel A (41; 41g; 41a; A) and against one of panel B (100, 39, C, 18) or at least two of panel C (100, 39, A, C, 18) were considered positive for IgG. Serum reactivity against A. phagocytophilum was examined making use of a commercially available immunofluorescence asssay (IFA) (MRL Diagnostics, Cypress, California, USA) based on HL-60 cells which had been infected with a human isolate of the HGE-1 strain. Samples were tested at a starting dilution of 1:64 in accordance with the manufacturer’s protocol yielding a specificity of 96% (Walder et al., 2003a) and a sensitivity of 99.4% (Bakken and Dumler, 2000). Convalescence serum of an Austrian HGA-patient was used as a positive control. A negative control serum was supplied by the manufacturer. Samples were rated positive when they showed a clear green fluorescence of A. phagocytophilum inclusion bodies at a cut-off titer of 1:64. Positive samples were confirmed by means of an immunoblot based on purified antigen of A. phagocytophilum (MarDx, Trinity Biotech, Bray, Ireland). Samples that were reactive against the 41 or the 43 kDa-protein were considered to be positive. Both tests combined yielded a specificity of 100% when tested with 120 well-defined negative sera from Tyrolean donors (data not shown).
Statistics Fisher’s exact test was used for the analysis of frequencies for 2 2 tables. For the analysis of frequencies in different geographic regions, a w2 test was applied. A p-value o0.05 was considered statistically significant.
Results Patients Altogether 545 donors (133 males, 412 females) participated in this study, 229 of whom returned a completed questionnaire. Of these, 34 patients (14.8%) recalled having a tick bite, which in nearly all cases were
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Fig. 1. Sample numbers, reported tick bites, and areas of sampling in Mongolia in 2003. International and province (aimag) boundaries are indicated by thick and thin black lines, respectively. Municipalities with province status and villages (soum) are indicated by circles and squares, respectively. The tick symbol marks provinces from where tick bites were reported. The number of samples is given in parentheses for each province.
Table 1.
Seroprevalences in human probands from different parts of Mongolia in 2003
Region
Number
TBE-positive
Bb-positive
Selenge Bulgan Dornogov Tov/Ulaanbaatar Rest
214 111 108 66 46
11 (5.1%) 1 (1%) 0 0 0
4 3 15 2 0
Male Female
133 412
7 (5%) 5 (1.2%)
(1.9%) (3%) (14%) (3%)
1 (1%) 21 (5.1%)
HGA-positive 5 6 3 2 2
(2.3%) (5%) (3%) (3%) (3%)
1 (1%) 12 (2.9%)
Bb: Borrelia burgdorferi s.l.; HGA: human granulocytic anaplasmosis; TBE: tick-borne encephalitis.
acquired in the province of permanent residency (30 bitten persons, 88%). Tick bites were reported in nine provinces as shown in Fig. 1. The frequencies of tick bites (as recalled by patients) did not differ significantly between male and female participants. Only two patients reported any symptoms after a tick bite: One female patient from the province of Bayankongor reported a febrile illness after having a tick bite in her home province. She was found seropositive for antibodies against A. phagocytophilum (IFA titer 1:256). A female patient who reported multiple tick bites acquired in the surrounding of Ulaanbaatar recalled a rash after a tick bite, but was seronegative for antibodies to all three agents tested. Another female patient recognized an erythema migrans (EM) depicted
in the questionnaire. She recalled a similar lesion on her back, which persisted for several weeks, approximately 3 years prior to questioning, but she did not recall having suffered from fever, headache, or any other general symptoms. This patient had no recollection of having a tick bite, but lived in Ulaanbaatar since birth and had spent a vast amount of her time conducting outdoor activities around the city. She was found to be seropositive for antibodies against B. burgdorferi.
Seroepidemiology The results of the seroepidemiologic survey are shown in Table 1.
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TBE virus Forty-nine sera were found positive by ELISA, 12 of which (24%) could be confirmed by means of the neutralization assay, yielding neutralization titers of up to 1:60. In detail, six donors had a titer of 1:10; three donors had a titer of 1:15, two donors had a titer of 1:30, and one donor was positive with a titer of 1:60. Seropositivities differed significantly (p ¼ 0:006) dependent on the area of tick exposure. Most seropositive donors were found to be resident in the northern part of the Selenge province around Khuder soum, where 10% of the tested subjects had neutralizing antibodies against TBE virus. Seroprevalence was significantly higher among the male participants included in this study. Eleven seropositive participants had returned the completed questionnaire. None of these patients recalled having a tick bite or in the case of a tick bite, no subsequent clinical symptoms were reported. However, all these patients had a history of tick exposure in the vicinity of their home. B. burgdorferi Twenty-four samples (41.9%) which yielded a positive result in the screening assay were confirmed by means of Western blot. Seropositivity rates differed significantly dependent on the area of tick exposure (p ¼ 0:001). The majority of seropositives was obtained from patients resident in Dornogov province, in the south of the country. Donors from this province participating in the study were almost exclusively females. This led to a significantly higher frequency of seropositive females than males (p ¼ 0:027). Without the data from Dornogov province, there would have been no significant difference between genders in seropositivity. Five out of 24 seropositive probands (21%) had returned a completed questionnaire. One of them reported having tick bites in his home region (Khyalgant soum/Bulgan province), but denied having suffered from any subsequent clinical symptoms. A. phagocytophilum In all, 18.2% of those samples found positive at a screening dilution of 1:64 yielded a positive result in the Western blot. Their endpoint titers were between 1:64 and 1:512. In detail, an endpoint titer of 1:64 was found in three samples, 1:128 in eight samples, 1:256 in six samples, and 1:512 in one sample, altogether 18 positive samples. No serum was found positive at 1:1024 or higher dilutions. Aside from the provinces described in detail in Table 1, one female patient from Bayankhongor province and one female patient from Zavkhan province were seropositive at titers of 1:256 and 1:64, respectively. No significant differences were found between genders or between probands from different parts of the country. The patient from Bayankhongor province was one of the four seropositive donors who
had returned a completed questionnaire (22%) and who recalled having acquired multiple tick bites in the vicinity of his home. This patient, however, was unaware of having acquired any tick bites while located in other parts of the country. Only one patient, a female from Selenge province, was found seropositive for two agents, for TBE virus and B. burgdorferi. However, she did not recall having any tick bites.
Discussion The data from neighboring areas, taken together with the fact that ticks are abundant throughout the country, supports the possibility that individuals are exposed to tick-borne pathogens in Mongolia. The aim of this study was to confirm this by demonstrating specific seroreactivity against the TBE virus, B. burgdorferi, and A. phagocytophilum in voluntary donors. In order to increase specificity and the positive predictive value of the applied test system, a two-step method was applied consisting of a screening assay followed by a second independent confirmatory assay. Sampling was limited by time constriction as well as logistic and linguistic difficulties. In order to get a sufficient number of sera and to include also sera from one of the southern provinces, anonymous samples from a separate seroepidemiologic study were included, despite the disadvantages of missing questionnaires and a significantly higher number of female than male participants in that part of the study. A disparity in regard to gender may bias the results. Previous seroepidemiologic studies on tick-borne diseases have been inconclusive on this point. Several studies failed to record a significant difference in seropositivity between men and women, while others exhibited more seropositive participants among males (Gustafson et al., 1993; Su¨ss, 2003; Walder et al., 2003b). When looking at areas in which a comparable number of men and women participated in our study, no significant difference in seroreactivity against B. burgdorferi or A. phagocytophilum could be detected between genders. It is noted, however, that in Selenge province significantly more men than women were found to have antibodies against the TBE virus. Thus, seroprevalence for TBE may be in fact higher in those provinces where predominantly female probands participated in the study. The positive results in seroepidemiologic surveys may be misleading if probands had traveled to countries where the pathogens of interest have been endemic. In the present study, only two out of 229 questioned participants had traveled to outside Mongolia and neither of them recalled having any tick bites in foreign countries. Thus, one can deduce that contact with the
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pathogen was acquired in the vast majority of cases within Mongolia. Isolated cases of imported infections amongst participants who failed to complete a questionnaire cannot be excluded. It is unlikely, however, that this has much influence on our results. It is noteworthy that the likelihood of being seropositive for TBE, borreliosis, or HGA was not higher in participants who recalled having tick bites when compared to those who denied previous tick bites. It is probable that many tick bites escaped attention because it can be assumed that most probands had a history of tick exposure due to excessive outdoor activities or their nomadic lifestyle. This is further supported by the fact that several participants who denied any previous tick bites, also reported ticks being frequent ectoparasites on their livestock. As the local nomads do not roam the whole country but usually stay in a certain well-described area in their province, it is possible to link geographical exposure and serologic findings. Neutralizing antibodies against TBE virus were only observed among probands resident in the northern parts of the country, where the main vector, I. persulcatus, is abundant. The observed seroprevalence is in good accordance with that reported from other endemic areas in Eurasia (Su¨ss, 2003), but is below that reported from north-eastern China (Cai et al., 1995). Neglecting one donor, who was not questioned, none of the seropositive probands had traveled abroad or had been vaccinated against TBE virus or any other arboviruses. Thus, the observed antibodies are indeed due to autochthonous infection acquired within Mongolia. Furthermore, it is probable that infection was acquired near the abode, where all seropositives and their livestock had a history of extensive exposure to ticks. The total absence of anamnestic tick bites among seropositives may be due to a lack of attention of the proband (thus possibly leading to longer attachment and an increased risk of infection), but could also point towards single cases of infection through consumption of raw milk. While our data presents convincing evidence for the occurrence of TBE in some provinces in northern Mongolia (Fig. 1), the (non-)occurrence of TBE virus in most other parts of the country, particularly in the other northern provinces, requires further investigation. It is notable that nearly 75% of those samples which yielded a positive result in the screening ELISA could not be confirmed by the neutralization assay, although a specificity of 99.5% was observed with European patients (Dade Behring, company information). Infections with the TBE virus usually lead to lifelong immunity, and cross-neutralization between the western subtype and isolates from the Irkutsk region was recently demonstrated (Hayasaka et al., 2001). Although neutralizing antibody titers tend to be lower
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when tested against heterologous strains, this should not cause a significant number of false-negative results. It is more likely that the high number of false-positive results was due to previous infection(s) with other flaviviruses that had given rise to cross-reactive, but not neutralizing antibodies. It is a well-known fact that antibodies against Japan B encephalitis, West Nile virus, or Dengue fever lead to false-positive results in ELISA, but usually do not affect the results of the neutralization assay. Efforts should be made to isolate and characterize native flaviviruses in order to gain a better understanding of their potential role as human pathogens and their influence on the human immune system. Up to that point, the exclusive use of serologic tests as established for use in Europe cannot be recommended for testing the seroreactivity against TBE virus in Mongolia or in patients returned from that country. Positive results for TBE virus using full-antigen tests such as ELISA or IFA should be confirmed by means of neutralization assays in order to ensure a correct diagnosis. Immunization against TBE may be considered beneficial for persons traveling to northern Mongolia in the summer months who plan to spend extended periods of time with outdoor activities. Vaccines based on the western genotype of TBE virus might also offer sufficient protection against strains from Central Asia (Hayasaka et al., 2001). Specific antibodies against B. burgdorferi were found throughout the country, with seroprevalences comparable to those reported for north-eastern China (1–4%) (Zhang et al., 1997). Unlike TBE, contact with the pathogen is not limited to the northern parts of the country which are covered by the boreal needle-leaf forest of the Taiga where I. persulcatus is abundant, but it also occurs in the steppe region where Ixodes ticks have not been observed by local entomologists, as yet. Similar to observations made in China, Haemaphysalis ticks could serve as vectors there (Takada et al., 2001). Positive donors who were available for questioning negated having traveled to foreign countries. The existence of a seropositive participant who recognized an EM on a picture provides further evidence that borreliosis does occur in Mongolia. This patient had never traveled abroad and in the 6-month period prior to the onset of symptoms, had not left the province of Tov. Borreliosis must be considered in patients in Mongolia who display appropriate clinical symptoms. Similar to the guidelines proposed for other endemic areas, a two-step procedure consisting of a screening assay and subsequent Western blotting should be applied. In routine diagnosis of European patients, approximately 60% of the positive results in ELISA have been confirmed by Western blot in our laboratory (unpublished data). The percentage of confirmed cases among Mongolian donors is lower. Contact with Gramnegative bacteria or other closely related pathogens are
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known causes for false-positive results in ELISA and may contribute to a lower percentage of confirmed cases. The existence of relapsing fever spirochetes in Mongolia is currently unclear. There are reports about relapsing fever from more western parts of Russia and Central Asia, but not from Mongolia or neighboring areas in northern China or Siberia. As the abundance of Ornithodoros ticks was not confirmed by local entomologists, the investigation for relapsing fever spirochetes was not an aim of our study. Antibodies against B. recurrentis or B. duttonii causing tick- and louse-borne relapsing fever, respectively, may yield positive results in whole-cell ELISAs coated with B. burgdorferi, but in recombinant Western blots cross-reactivity is rare (Magnarelli et al., 2002). Single sera of relapsing fever patients may react with flagellin or OspC, thus we have rated sera which were only reactive against flagellin or OspC as negative. Remarkable is the finding that six sera from Dornogov province were rated positive due to the presence of antibodies against flagellin and OspC, which could theoretically point towards cross-reactivity with relapsing fever borreliae. However, given that our knowledge on cross-reactivity between relapsing fever borreliae and B. burgdorferi s.l. is still limited, a final rating of this finding is not possible at the moment. To our best knowledge, this is the first time that human seroprevalences against A. phagocytophilum have been reported for Central Asia. Similar to borreliosis, contact with this pathogen was not limited to the inhabitants of the northern parts of the country but was also found in comparable percentages amongst donors resident in other regions. Positive donors who were available for questioning negated having traveled to foreign countries. One seropositive participant reported having a large number of tick bites acquired in the vicinity of her abode in Bayankhongor province, thus providing further support that HGA is endemic in Mongolia. HGA must therefore be considered in patients with appropriate clinical symptoms. Similar to the observations made in the United States, only a minority of samples which were found positive by means of IFA could be confirmed by Western blotting (Aguero-Rosenfeld et al., 2002). The reasons for this are currently poorly understood. Although cross-reactivity with members of the genus Ehrlichia has only been described when titers were extraordinarily high (Dumler et al., 1995), this is also probable with other members of the genus Anaplasma. Recent studies on Anaplasmataceae in ticks produced a large number of different, and partially still unclassified, species in neighboring parts of Asia (Wen et al., 2003). Thus, the abundance of additional pathogenic, and in particular immunostimulating, Anaplasmataceae in this region requires further investigation.
Conclusion TBE, borreliosis, and HGA are endemic in parts of Mongolia and must be considered in Mongolian patients and in travelers returning from these regions who suffer from febrile illnesses and have a history of tick exposure. Travelers, who visit northern Mongolia during the summer months and engage in extensive outdoor activities, should consider vaccination against TBE. Further studies are necessary in order to establish endemic areas, the ecological relationships, and the clinical impact of tick-borne diseases in Mongolia. This is particularly necessary in order to decide which parts of the native population may benefit from vaccination against TBE.
Acknowledgements The authors are indebted to Baxter Austria for support in logistics and transportation, to the laboratory staff at the First Hospital in Ulaanbaatar, and to Prof. Pfeiffer from the Department of Biostatistics, Innsbruck Medical University, for support in statistical calculations. The study was conducted during (but not as part of) a medical educational project by fabula medicine, Austria.
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International Journal of Medical Microbiology 296 (2006) S1, 69–75 www.elsevier.de/ijmm
Serological evidence for tick-borne encephalitis, borreliosis, and human granulocytic anaplasmosis in Mongolia Gernot Waldera,, Erdenechimeg Lkhamsurenb, Abmed Shagdarc, Jantsandoo Bataac, Tsetsegsaikhan Batmunkhb, Dorothea Ortha, Franz X. Heinzd, Galina A. Danichovae, Maksim A. Khasnatinove, Reinhard Wu¨rznera, Manfred P. Diericha a
Department of Hygiene, Microbiology and Social Medicine, Innsbruck Medical University, Fritz-Pregl-Str. 3, A-6020 Innsbruck, Austria b Department of Microbiology and Immunology, National Medical University of Mongolia, Ulaanbaatar, Mongolia c National Center for Communicable Diseases, Ulaanbaatar, Mongolia d Institute for Virology, Medical University of Vienna, Vienna, Austria e Institute for Epidemiology and Microbiology, Irkutsk Medical University, Irkutsk, Russia
Abstract Five hundred and forty-five serum samples from donors from various parts of Mongolia were investigated for antibodies against the tick-borne encephalitis (TBE) virus, Borrelia burgdorferi, and Anaplasma phagocytophilum. Seroprevalence against TBE was 5.1% in the province of Selenge and 0.9% in Bulgan province, seroprevalence against B. burgdorferi was 1.9% in Selenge province and Bulgan province, 13.9% in Dornogov province, and 3.0% in Tov province and Ulaanbaatar. Seroprevalence against A. phagocytophilum was 2.3% in Selenge province, 5.6% in Bulgan province, 2.8% in Dornogov province, and 3.0% in Tov province and Ulaanbaatar. We conclude that all three pathogens are endemic in Mongolia. r 2006 Elsevier GmbH. All rights reserved. Keywords: TBE; Borreliosis; Human granulocytic anaplasmosis; Central Asia; Mongolia
Introduction Mongolia, a vast, but scarcely populated country in Central Asia with increasing economic importance (including tourism), is one of the very few countries in Eurasia where data on tick-borne diseases are not available yet (Ebright et al., 2003). There is, however, evidence which supports the suggestion that these diseases may well be endemic in this country.
Corresponding author. Tel.: +43 512 507 3411; fax: +43 512 578 745. E-mail address: [email protected] (G. Walder).
1438-4221/$ - see front matter r 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.ijmm.2006.01.031
According to local entomologists (postal communication from Batsukh Zayat, Institute of Veterinary Medicine of Mongolia), ixodid ticks are found in the northern and central parts of Mongolia. Ixodes (I.) persulcatus occurs in the provinces of Huvsgul, Selenge, and Hentii and I. crenulatus in Ovorkhangai, Tov, and Dornod province. Dermacentor and Haemaphysalis ticks are even more widespread, covering the provinces of Huvsgul, Selenge, Hentii, Ovorhangai, Tov, Dornod, Bayan-Olgii, Hovd, Uvs, Gov-Altai, Omnogov, Dundgov, Bayanhongor, and Dornogov. Epidemiologic data from neighboring Siberia and China provide further support for the occurrence of tick-borne encephalitis (TBE), borreliosis, and human
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granulocytic anaplasmosis (HGA) in Mongolia. Siberia, where two of the three genetically distinct strains of the TBE virus are abundant (Zlobin et al., 1992), accounts for more than 50% of all TBE cases in Russia (Su¨ss, 2003) and seroprevalences of up to 66% have been reported for ruminants in the neighboring region of Irkutsk (Titenko et al., 2002). Recently, the virus has also been isolated from ticks in north-eastern China (Cai et al., 1995), but not in close vicinity to Mongolia. Borrelia (B.) burgdorferi sensu lato has been found in 20–45% of I. persulcatus ticks in northern China (Zhang et al., 1991) by direct immunofluorescence. This agent is abundant in the two border provinces to Mongolia, Xinjiang (Wan et al., 1998) and Inner Mongolia, where it has been identified by culture and PCR (Takada et al., 2001). Seroprevalences vary from less than 4% (Zhang et al., 1997) to more than 20% (Zhang, 1989), depending on the location and the applied serologic methods. Studies on borreliosis in Siberia are mostly limited to the western part of this region, where annual morbidities of 2/100,000 inhabitants have been reported (Lesniak et al., 1995) and seroprevalence was 6–12% (Lesniak et al., 1994). A case of borreliosis was reported from the Baikal area in 1991 (Anan’eva et al., 1991). Information on Anaplasma (A.) phagocytophilum is still very limited. The agent has been detected in ticks and in patients from northern China by means of PCR (Zhao et al., 2002), and recently a clinical case was reported in the Far East of the Russian Federation (Sidel’nikov et al., 2003). Here we describe the demonstration of antibodies against TBE, B. burgdorferi s.l., and A. phagocytophilum in volunteer donors from Mongolia.
Materials and methods Patients Two hundred and twenty-nine serum samples were taken from volunteer donors during August 2003 in the First Hospital in Ulaanbaatar and in Khuder soum in the upper Chikoy valley in the Selenge province. All patients were questioned as to their permanent place of residence, previous travels abroad, tick bites, the geographic region where the tick bites had been acquired, and possible clinical symptoms following these tick bites. An additional 316 sera from female donors participating in a study on sexually transmitted diseases in pregnancy was supplied by the National Center of Infectious Diseases. These sera were rendered anonymously prior to investigation, so that only gender, age, and the province in which the patient permanently resided were recorded. Sampling was done by or in cooperation with the National Center for Communicable Diseases of Mongolia according to the local laws. All patients had given consent to their sera being tested for antibodies
against infectious diseases, although not all of them were available for questions regarding previous tick bites.
Serological investigation Samples were screened for IgG against TBE virus using commercially available ELISA (Enzygnost TBE, Dade Behring, Marburg, Germany; sensitivity 96.8% and specificity 99.5%, as stated by the manufacturer). Positive results were confirmed using a neutralization assay as described earlier (Holzmann et al., 1996). Samples yielding a neutralization titer of X1:10 were rated positive. A commercial ELISA (Enzygnost Borrelia, Dade Behring, Marburg, Germany; sensitivity 92.5%, as stated by the manufacturer; specificity 75%, as assessed in our laboratory, data not shown) was used to detect anti-B. burgdorferi s.l. IgG. Positive results were confirmed by means of a recombinant immunoblot (Mikrogen, Martinsried, Germany). Samples which had antibodies against at least one antigen of panel A (41; 41g; 41a; A) and against one of panel B (100, 39, C, 18) or at least two of panel C (100, 39, A, C, 18) were considered positive for IgG. Serum reactivity against A. phagocytophilum was examined making use of a commercially available immunofluorescence asssay (IFA) (MRL Diagnostics, Cypress, California, USA) based on HL-60 cells which had been infected with a human isolate of the HGE-1 strain. Samples were tested at a starting dilution of 1:64 in accordance with the manufacturer’s protocol yielding a specificity of 96% (Walder et al., 2003a) and a sensitivity of 99.4% (Bakken and Dumler, 2000). Convalescence serum of an Austrian HGA-patient was used as a positive control. A negative control serum was supplied by the manufacturer. Samples were rated positive when they showed a clear green fluorescence of A. phagocytophilum inclusion bodies at a cut-off titer of 1:64. Positive samples were confirmed by means of an immunoblot based on purified antigen of A. phagocytophilum (MarDx, Trinity Biotech, Bray, Ireland). Samples that were reactive against the 41 or the 43 kDa-protein were considered to be positive. Both tests combined yielded a specificity of 100% when tested with 120 well-defined negative sera from Tyrolean donors (data not shown).
Statistics Fisher’s exact test was used for the analysis of frequencies for 2 2 tables. For the analysis of frequencies in different geographic regions, a w2 test was applied. A p-value o0.05 was considered statistically significant.
Results Patients Altogether 545 donors (133 males, 412 females) participated in this study, 229 of whom returned a completed questionnaire. Of these, 34 patients (14.8%) recalled having a tick bite, which in nearly all cases were
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Fig. 1. Sample numbers, reported tick bites, and areas of sampling in Mongolia in 2003. International and province (aimag) boundaries are indicated by thick and thin black lines, respectively. Municipalities with province status and villages (soum) are indicated by circles and squares, respectively. The tick symbol marks provinces from where tick bites were reported. The number of samples is given in parentheses for each province.
Table 1.
Seroprevalences in human probands from different parts of Mongolia in 2003
Region
Number
TBE-positive
Bb-positive
Selenge Bulgan Dornogov Tov/Ulaanbaatar Rest
214 111 108 66 46
11 (5.1%) 1 (1%) 0 0 0
4 3 15 2 0
Male Female
133 412
7 (5%) 5 (1.2%)
(1.9%) (3%) (14%) (3%)
1 (1%) 21 (5.1%)
HGA-positive 5 6 3 2 2
(2.3%) (5%) (3%) (3%) (3%)
1 (1%) 12 (2.9%)
Bb: Borrelia burgdorferi s.l.; HGA: human granulocytic anaplasmosis; TBE: tick-borne encephalitis.
acquired in the province of permanent residency (30 bitten persons, 88%). Tick bites were reported in nine provinces as shown in Fig. 1. The frequencies of tick bites (as recalled by patients) did not differ significantly between male and female participants. Only two patients reported any symptoms after a tick bite: One female patient from the province of Bayankongor reported a febrile illness after having a tick bite in her home province. She was found seropositive for antibodies against A. phagocytophilum (IFA titer 1:256). A female patient who reported multiple tick bites acquired in the surrounding of Ulaanbaatar recalled a rash after a tick bite, but was seronegative for antibodies to all three agents tested. Another female patient recognized an erythema migrans (EM) depicted
in the questionnaire. She recalled a similar lesion on her back, which persisted for several weeks, approximately 3 years prior to questioning, but she did not recall having suffered from fever, headache, or any other general symptoms. This patient had no recollection of having a tick bite, but lived in Ulaanbaatar since birth and had spent a vast amount of her time conducting outdoor activities around the city. She was found to be seropositive for antibodies against B. burgdorferi.
Seroepidemiology The results of the seroepidemiologic survey are shown in Table 1.
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TBE virus Forty-nine sera were found positive by ELISA, 12 of which (24%) could be confirmed by means of the neutralization assay, yielding neutralization titers of up to 1:60. In detail, six donors had a titer of 1:10; three donors had a titer of 1:15, two donors had a titer of 1:30, and one donor was positive with a titer of 1:60. Seropositivities differed significantly (p ¼ 0:006) dependent on the area of tick exposure. Most seropositive donors were found to be resident in the northern part of the Selenge province around Khuder soum, where 10% of the tested subjects had neutralizing antibodies against TBE virus. Seroprevalence was significantly higher among the male participants included in this study. Eleven seropositive participants had returned the completed questionnaire. None of these patients recalled having a tick bite or in the case of a tick bite, no subsequent clinical symptoms were reported. However, all these patients had a history of tick exposure in the vicinity of their home. B. burgdorferi Twenty-four samples (41.9%) which yielded a positive result in the screening assay were confirmed by means of Western blot. Seropositivity rates differed significantly dependent on the area of tick exposure (p ¼ 0:001). The majority of seropositives was obtained from patients resident in Dornogov province, in the south of the country. Donors from this province participating in the study were almost exclusively females. This led to a significantly higher frequency of seropositive females than males (p ¼ 0:027). Without the data from Dornogov province, there would have been no significant difference between genders in seropositivity. Five out of 24 seropositive probands (21%) had returned a completed questionnaire. One of them reported having tick bites in his home region (Khyalgant soum/Bulgan province), but denied having suffered from any subsequent clinical symptoms. A. phagocytophilum In all, 18.2% of those samples found positive at a screening dilution of 1:64 yielded a positive result in the Western blot. Their endpoint titers were between 1:64 and 1:512. In detail, an endpoint titer of 1:64 was found in three samples, 1:128 in eight samples, 1:256 in six samples, and 1:512 in one sample, altogether 18 positive samples. No serum was found positive at 1:1024 or higher dilutions. Aside from the provinces described in detail in Table 1, one female patient from Bayankhongor province and one female patient from Zavkhan province were seropositive at titers of 1:256 and 1:64, respectively. No significant differences were found between genders or between probands from different parts of the country. The patient from Bayankhongor province was one of the four seropositive donors who
had returned a completed questionnaire (22%) and who recalled having acquired multiple tick bites in the vicinity of his home. This patient, however, was unaware of having acquired any tick bites while located in other parts of the country. Only one patient, a female from Selenge province, was found seropositive for two agents, for TBE virus and B. burgdorferi. However, she did not recall having any tick bites.
Discussion The data from neighboring areas, taken together with the fact that ticks are abundant throughout the country, supports the possibility that individuals are exposed to tick-borne pathogens in Mongolia. The aim of this study was to confirm this by demonstrating specific seroreactivity against the TBE virus, B. burgdorferi, and A. phagocytophilum in voluntary donors. In order to increase specificity and the positive predictive value of the applied test system, a two-step method was applied consisting of a screening assay followed by a second independent confirmatory assay. Sampling was limited by time constriction as well as logistic and linguistic difficulties. In order to get a sufficient number of sera and to include also sera from one of the southern provinces, anonymous samples from a separate seroepidemiologic study were included, despite the disadvantages of missing questionnaires and a significantly higher number of female than male participants in that part of the study. A disparity in regard to gender may bias the results. Previous seroepidemiologic studies on tick-borne diseases have been inconclusive on this point. Several studies failed to record a significant difference in seropositivity between men and women, while others exhibited more seropositive participants among males (Gustafson et al., 1993; Su¨ss, 2003; Walder et al., 2003b). When looking at areas in which a comparable number of men and women participated in our study, no significant difference in seroreactivity against B. burgdorferi or A. phagocytophilum could be detected between genders. It is noted, however, that in Selenge province significantly more men than women were found to have antibodies against the TBE virus. Thus, seroprevalence for TBE may be in fact higher in those provinces where predominantly female probands participated in the study. The positive results in seroepidemiologic surveys may be misleading if probands had traveled to countries where the pathogens of interest have been endemic. In the present study, only two out of 229 questioned participants had traveled to outside Mongolia and neither of them recalled having any tick bites in foreign countries. Thus, one can deduce that contact with the
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pathogen was acquired in the vast majority of cases within Mongolia. Isolated cases of imported infections amongst participants who failed to complete a questionnaire cannot be excluded. It is unlikely, however, that this has much influence on our results. It is noteworthy that the likelihood of being seropositive for TBE, borreliosis, or HGA was not higher in participants who recalled having tick bites when compared to those who denied previous tick bites. It is probable that many tick bites escaped attention because it can be assumed that most probands had a history of tick exposure due to excessive outdoor activities or their nomadic lifestyle. This is further supported by the fact that several participants who denied any previous tick bites, also reported ticks being frequent ectoparasites on their livestock. As the local nomads do not roam the whole country but usually stay in a certain well-described area in their province, it is possible to link geographical exposure and serologic findings. Neutralizing antibodies against TBE virus were only observed among probands resident in the northern parts of the country, where the main vector, I. persulcatus, is abundant. The observed seroprevalence is in good accordance with that reported from other endemic areas in Eurasia (Su¨ss, 2003), but is below that reported from north-eastern China (Cai et al., 1995). Neglecting one donor, who was not questioned, none of the seropositive probands had traveled abroad or had been vaccinated against TBE virus or any other arboviruses. Thus, the observed antibodies are indeed due to autochthonous infection acquired within Mongolia. Furthermore, it is probable that infection was acquired near the abode, where all seropositives and their livestock had a history of extensive exposure to ticks. The total absence of anamnestic tick bites among seropositives may be due to a lack of attention of the proband (thus possibly leading to longer attachment and an increased risk of infection), but could also point towards single cases of infection through consumption of raw milk. While our data presents convincing evidence for the occurrence of TBE in some provinces in northern Mongolia (Fig. 1), the (non-)occurrence of TBE virus in most other parts of the country, particularly in the other northern provinces, requires further investigation. It is notable that nearly 75% of those samples which yielded a positive result in the screening ELISA could not be confirmed by the neutralization assay, although a specificity of 99.5% was observed with European patients (Dade Behring, company information). Infections with the TBE virus usually lead to lifelong immunity, and cross-neutralization between the western subtype and isolates from the Irkutsk region was recently demonstrated (Hayasaka et al., 2001). Although neutralizing antibody titers tend to be lower
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when tested against heterologous strains, this should not cause a significant number of false-negative results. It is more likely that the high number of false-positive results was due to previous infection(s) with other flaviviruses that had given rise to cross-reactive, but not neutralizing antibodies. It is a well-known fact that antibodies against Japan B encephalitis, West Nile virus, or Dengue fever lead to false-positive results in ELISA, but usually do not affect the results of the neutralization assay. Efforts should be made to isolate and characterize native flaviviruses in order to gain a better understanding of their potential role as human pathogens and their influence on the human immune system. Up to that point, the exclusive use of serologic tests as established for use in Europe cannot be recommended for testing the seroreactivity against TBE virus in Mongolia or in patients returned from that country. Positive results for TBE virus using full-antigen tests such as ELISA or IFA should be confirmed by means of neutralization assays in order to ensure a correct diagnosis. Immunization against TBE may be considered beneficial for persons traveling to northern Mongolia in the summer months who plan to spend extended periods of time with outdoor activities. Vaccines based on the western genotype of TBE virus might also offer sufficient protection against strains from Central Asia (Hayasaka et al., 2001). Specific antibodies against B. burgdorferi were found throughout the country, with seroprevalences comparable to those reported for north-eastern China (1–4%) (Zhang et al., 1997). Unlike TBE, contact with the pathogen is not limited to the northern parts of the country which are covered by the boreal needle-leaf forest of the Taiga where I. persulcatus is abundant, but it also occurs in the steppe region where Ixodes ticks have not been observed by local entomologists, as yet. Similar to observations made in China, Haemaphysalis ticks could serve as vectors there (Takada et al., 2001). Positive donors who were available for questioning negated having traveled to foreign countries. The existence of a seropositive participant who recognized an EM on a picture provides further evidence that borreliosis does occur in Mongolia. This patient had never traveled abroad and in the 6-month period prior to the onset of symptoms, had not left the province of Tov. Borreliosis must be considered in patients in Mongolia who display appropriate clinical symptoms. Similar to the guidelines proposed for other endemic areas, a two-step procedure consisting of a screening assay and subsequent Western blotting should be applied. In routine diagnosis of European patients, approximately 60% of the positive results in ELISA have been confirmed by Western blot in our laboratory (unpublished data). The percentage of confirmed cases among Mongolian donors is lower. Contact with Gramnegative bacteria or other closely related pathogens are
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known causes for false-positive results in ELISA and may contribute to a lower percentage of confirmed cases. The existence of relapsing fever spirochetes in Mongolia is currently unclear. There are reports about relapsing fever from more western parts of Russia and Central Asia, but not from Mongolia or neighboring areas in northern China or Siberia. As the abundance of Ornithodoros ticks was not confirmed by local entomologists, the investigation for relapsing fever spirochetes was not an aim of our study. Antibodies against B. recurrentis or B. duttonii causing tick- and louse-borne relapsing fever, respectively, may yield positive results in whole-cell ELISAs coated with B. burgdorferi, but in recombinant Western blots cross-reactivity is rare (Magnarelli et al., 2002). Single sera of relapsing fever patients may react with flagellin or OspC, thus we have rated sera which were only reactive against flagellin or OspC as negative. Remarkable is the finding that six sera from Dornogov province were rated positive due to the presence of antibodies against flagellin and OspC, which could theoretically point towards cross-reactivity with relapsing fever borreliae. However, given that our knowledge on cross-reactivity between relapsing fever borreliae and B. burgdorferi s.l. is still limited, a final rating of this finding is not possible at the moment. To our best knowledge, this is the first time that human seroprevalences against A. phagocytophilum have been reported for Central Asia. Similar to borreliosis, contact with this pathogen was not limited to the inhabitants of the northern parts of the country but was also found in comparable percentages amongst donors resident in other regions. Positive donors who were available for questioning negated having traveled to foreign countries. One seropositive participant reported having a large number of tick bites acquired in the vicinity of her abode in Bayankhongor province, thus providing further support that HGA is endemic in Mongolia. HGA must therefore be considered in patients with appropriate clinical symptoms. Similar to the observations made in the United States, only a minority of samples which were found positive by means of IFA could be confirmed by Western blotting (Aguero-Rosenfeld et al., 2002). The reasons for this are currently poorly understood. Although cross-reactivity with members of the genus Ehrlichia has only been described when titers were extraordinarily high (Dumler et al., 1995), this is also probable with other members of the genus Anaplasma. Recent studies on Anaplasmataceae in ticks produced a large number of different, and partially still unclassified, species in neighboring parts of Asia (Wen et al., 2003). Thus, the abundance of additional pathogenic, and in particular immunostimulating, Anaplasmataceae in this region requires further investigation.
Conclusion TBE, borreliosis, and HGA are endemic in parts of Mongolia and must be considered in Mongolian patients and in travelers returning from these regions who suffer from febrile illnesses and have a history of tick exposure. Travelers, who visit northern Mongolia during the summer months and engage in extensive outdoor activities, should consider vaccination against TBE. Further studies are necessary in order to establish endemic areas, the ecological relationships, and the clinical impact of tick-borne diseases in Mongolia. This is particularly necessary in order to decide which parts of the native population may benefit from vaccination against TBE.
Acknowledgements The authors are indebted to Baxter Austria for support in logistics and transportation, to the laboratory staff at the First Hospital in Ulaanbaatar, and to Prof. Pfeiffer from the Department of Biostatistics, Innsbruck Medical University, for support in statistical calculations. The study was conducted during (but not as part of) a medical educational project by fabula medicine, Austria.
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