Seroprevalence report on tick-borne encephalitis virus and Crimean-Congo hemorrhagic fever virus among Malaysian’s farm workers

We have shown the seropositivity for TBEV among a small group of farm workers in Peninsular Malaysia. Although our initial data showed that the prevalence of IgG against TBEV was 36.5 %, after testing the positive sera we found that only five (4.2 %) samples could be linked to the presence of TBEV. It was not surprising that there was cross reactivity between TBEV, WNV and DENV because they are all flaviviruses that share a complex antigenic relationship [17]. Our findings contradict previous studies by Thayan et al. [16] in which they stated that none of 600 tested samples of Malaysian patients with encephalitis carried TBEV antibodies. The difference is probably due to their selection of subjects who were not from a high-risk group and therefore did not meet the study requirements. To study a tick-borne disease, it is recommended to select subjects at high risk of tick bites, which is how most humans become infected [18]. Another reason for the difference between our findings and Thayan et al.’s could be the type of ELISA kit: the type of kits used in the two studies had different sensitivity and specificity. The TBEV IgG ELISA kit (Abnova® Corporation, Taiwan) that we used has a range of relative avidity index value of between 64 and 99 % in high avidity serum samples, and between 6 and 36 % in low avidity serum samples. Moreover, LGTV could be another reason for the detected seropositivity in our study [19]. LGTV is a Malaysian counterpart of TBEV with more than 80 % genome homology. Therefore, even using a virus neutralisation test, there could still be the chance of false positivity in the TBEV IgG ELISA results. Our findings show that 70.97 % of identified seropositive participants mentioned tick-bite experience and LGTV is also a tick virus, although without any clinical symptoms in humans. Our data showed that only five (4.2 %) sera were reactive against TBEV without cross reactivity with other tested flaviviruses, but there was uncertainty about the accuracy of the positivity of those samples because a virus neutralisation test was not performed. We were not able to do a virus neutralisation test using TBEV because this virus was not available in our laboratory and it is not an endemic virus in Malaysia. This was clearly one of the limitations of the study. We therefore strongly suggest considering a virus neutralisation test by collaborating with other certified laboratories when conducting future studies. CCHFV is claimed to be the most endemic tick-bornevirus with the widest geographical range compared with other tick-borne viruses [7]. Our data suggest that it is still not a threat to Malaysian farm workers. However, because there is evidence of sudden epidemics in nonendemic areas of different countries such as India, it is crucial to watch out for this infection. Although CCHFV is considered a potential occupational hazard among agricultural workers, a study by Sargianou et al. [20] showed a similar result of low seroprevalence (3.4 %) in Achaia, where citizens are almost entirely dependent on livestock farming. A serological survey conducted in Madagascar also showed very low occurrence of CCHFV infection in only 16 of 1995 tested workers [21]. However, higher prevalence data of 28.5 % have been reported in Iran due to CCHFV infection among livestock handlers and farmers [22]. A possible explanation for the low seropositive outcome for TBEV and the seronegative outcome of CCHFV between different studies and our study might relate to acaricides and rotational grazing systems. Seven of the eight farms in this study used acaricides and had a rotational grazing system. Acaricides are pesticides that kill ticks and mice. Rotational grazing combined with acaricide usage is a management practice to disrupt the parasitic life cycle and reduce the tick population on farms [23]. By keeping the pasture host-free to break down the natural life cycle of ticks, the system reduces the chances of tick survival by tick starvation [24]. According to Horst and Seifert, the larvae and nymph of ticks can be starved by keeping livestock away from a pasture for about 6 or 7 months. To eradicate adult ticks, the pasture must be host-free for about 14 or 15 months. The practice is most effective during hot and dry seasons [24]. However, in an area with strong winds, rotational grazing might be less efficient be- cause tick larvae may become wind-borne and carried away to neighbouring paddocks [24]. Tick management reduces the probability of infection among livestock and humans, leading to less positive results in seroprevalence studies. Methods of laboratory diagnosis are also closely related to outcomes obtained. The molecular method is useful for the early detection of a virus, while the serological method can only detect the presence of antibodies after the second week of infection. Detection of IgG only reveals previous virus infection and does not identify people who are currently infected [25]. There is a possibility that viruses are present in serum samples at an early phase of infection when antibodies are not detected by ELISA, and the analysis may therefore result in a false negative reading [26]. The outcome of this study suggested that seropositivity of TBEV did not increase with age and gender, as reported in another study in Lithuania [27]. Although the difference did not reach statistical significance, participants within the older age group (51–60 years) showed the highest seropositivity rate. This outcome was consistent with Mangarov et al.’s study in which they stated that the incidence and severity of TBEV were highest in 307 people over 50 years old [18]. This finding is most likely related to urbanisation, because it is common for younger citizens with a better educational background to migrate to urban areas [28]. Youngsters tend to move away from villages because they have a better opportunity to make a living in a working environment that is more convenient and less demanding than farming. This leads to the isolation of older people and youngsters with a lower educational background in rural areas, where farming is one of their main sources of income. The greater experience of older people means that they handle livestock more frequently than younger farmers. Since ticks feed from the blood of large mammals, frequent handling also increases the risk of a tick bite. Besides, antibodies accumulate and remain in the circulation for longer periods of time, which may lead to a higher titer in older individuals [29]. Certain groups of workers, such as farmers, veterinarians, shepherds and slaughterhouse staff, are considered at risk of zoonotic infections including TBEV and CCHFV because of the nature of their jobs [11]. Healthcare personnel are also at high risk because of exposure to infected patients’ blood and body fluids. This might be an alternative pathway for viral transmission, consistent with some investigations that have proven that infection by tick-borne viruses is more prevalent among high-risk than low-risk groups [22]. Nabeth et al. [30] suggested that direct contact with viraemic animals’ blood is the primary mode of animal-to-human transmission in high-risk groups. However, one study conducted in Iran suggested that CCHFV was not highly prevalent in high-risk professions [31]. As mentioned previously, there is no outbreak or reported cases of TBEV or CCHFV in Malaysia. This is in contrast to neighbouring countries, for example China, where the TBEV-Fe is endemic in certain parts of the country including Jilin Province, Inner Mongolia Province, Heilongjiang Province, Xinjiang Uygur Autonomous region and Yunan Province, and also Tibet [32]. In Japan, the first case of TBEV was reported in Hokkaido Prefecture. A phylogenetic study by Suzuki [5] showed that TBEV had been transmitted between Russia and Japan at least three times, most probably due to transmission of infected ticks by migratory birds travelling across the sea. Although there are no reported and confirmed cases of TBEV and CCHFV in Malaysia, there is no assurance that Malaysia will always stay a TBEV- and CCHFV- free country because many other factors may contribute to the viruses’ transmission, such as climate change and adverse environmental conditions including temperature and humidity [16].