RESULTS AND DISCUSSION
During this study a total of 127 ticks were collected. Of these, 47 were identified as Dermacentor sp . questing ticks from the environment and 80 were confirmed as Riphicephalus sp. engorged ticks, detached from adult goats and sheeps. All ticks were tested for detection of the six CCHFV genotypes by real time qRT-PCR. For this purpose, real time RT-qPCR based on SybrGreen with specific primer sets for the detection of all the six known CCHFV genotypes (Sas et al., 2018) in questing and engorged ticks were used. The detection of CCHFV consisted in the amplification of a 180 bp region from S segment that is coding for NP. CCHFV is classified as a risk group 4 pathogen, being considered to have bioterrorism potential due its aerosol infectivity. For regulatory reasons a positive control was not included in reaction. By using the negative control, we have not encountered any false-positive RT-PCR results due to contamination. In all collection sites from Tulcea county, the tested samples using real time qRT-PCR were identified as negative for the six genotypes of CCHFV. The results are listed in table 3. As no positive result was recorded, this defines a maximal prevalence <2.5% (p=0.05). We consider this result as robust because of the good sensitivity of the RT-qPCR used in this study. Two copies/µl were detected for genotypes II (DR Congo), IV (Afghanistan), V (Kosovo) and VI (Greece), while the limits of detection for genotypes I and III were 200 copies/µl respectively (Sas et al., 2018). During the same study the specificity was favorably evaluated using ten viruses from the order Bunyavirales as none of the viruses was detected.
The present study highlight the nil or at most low prevalence of CCHFV RNA in Dermacentor sp. and Rhipicephalus sp . questing ticks and engorged ticks from south-eastern Romania, in 2019 from the same region (Tulcea county) where the presence of CCHFV antibodies in animals has been detected previously (Ceianu et al., 2012; Răileanu et al., 2015).
Crimean Congo hemorrhagic fever has a widespread geographic distribution, the disease being reported in many regions of Africa, Middle East, Europe, and Asia, and outbreaks caused by CCHFV have been recorded in several countries (Chinikar et al., 2010). A detailed review about the epidemiology of CCHF in Asia, Europe and Africa was published in 1979 by Hoogstraal (Hoogstraal, 1979).
The Balkan Peninsula is an endemic region for the disease, where Turkey and Bulgaria are countries with the majority of the cases (Estrada-Pena et al., 2007; Honig et al., 2004; Papa, Pappa, Panayotova, Papadopoulou, & Christova, 2016; Papa et al., 2013; Zeller et al., 1997). In Bulgaria, country that is on the border with Romania, CCHFV has an endemic evolution (Gergova & Kamarinchev, 2013) which could suggest the circulation of the virus in Romania, possibly by ticks carried on migratory birds or through the international livestock trade. Migratory birds’ role in spreading Crimean-Congo hemorrhagic fever virus (CCHFV) through attached ticks was demonstrated in Turkey (Leblebicioglu et al., 2014). The geographic area of our present investigations comprises one of the most active bird migration pathways in southeastern Europe, for both spring and fall migrations.
In livestock the CCHFV does not cause clinical disease. However, domestic animals are very important for the epidemiology of the virus. Sheep have been recognized as CCHFV reservoirs in certain endemic regions, and have been epidemiologically linked to human cases on several occasions (Humolli, Dedushaj, Zupanac, & Mucaj, 2010; Mostafavi, Haghdoost, Khakifirouz, & Chinikar, 2013). Not only are they hosts for adult vectors, but they also may amplify the virus and infect other ticks during their short-lived viremia, and thus may introduce the virus into new areas via movement and importation of tick-infested and virus-infected livestock (Gale et al., 2010).
So far, the presence of the CCHFV in ticks from Romania was not confirmed and, consistently with our results, CCHFV human disease has not been reported to date. There are few serological studies regarding the circulation of CCHFV on Romanian territory. In 2012, Ceianu tested 471 sheep serum samples from different localities in Tulcea county and obtained a prevalence of 27.8% for IgG antibodies against CCHFV (Ceianu et al., 2012). However, a more recent study conducted by Raileanu in 2015, reported an overall prevalence of 74% IgG antibodies by testing 90 domestic ruminants (Răileanu et al., 2015). In the study conducted by Raileanu 74 Rhipicephalus sp . ticks, collected from the positive animals, were tested using rRT-PCR method obtaining negative results, which suggest the seroconversion process occurred before the ticks were attached and the transmission of the virus to vectors was not achieved (Răileanu et al., 2015). Many studies suggest that the viral RNA in attached ticks does not directly indicate transmission to host species, and vice versa: infected ticks have been found on seronegative animals and uninfected ticks on seropositive animals (Zeller et al., 1997).
The results of this study together with previous published data may suggest a silent tick-vertebrate-tick cycle of CCHFV in Romania. Clinical cases occur when a range of factors related to tick, vertebrate host, and human behavior align, increasing the level of viral circulation, the interactions between humans and sources of infection. Thus, detection of CCHFV antibodies in domestic animals has been important in providing initial evidence of circulating virus and localizing CCHFV and increased risk for human infection (Spengler, Bergeron, & Rollin, 2016).
There are many factors that can influence the circulation of CHHFV. For example, abiotic variation by season and region is reported in CCHFV evolution (Spengler et al., 2016). Longitudinal studies demonstrated considerable variation when repeated sampling was performed in the same location. These studies reported September as the optimum period for detecting antibodies with a notable decrease in seroprevalence in the winter–spring period (Gale et al., 2010). This could also partially explain our negative results, because we collected the ticks only once, in July 2019. Variation in seroprevalence is often associated with competent vector distribution, host preference of competent tick vectors, and tick load on a particular animal species. Hyalomma sp. ticks were not tested, which is a limit of our study. This study can therefore be extended by exploring different tick species, areas and seasons of sampling.