Crimean-Congo Hemorrhagic Fever Virus Infection in Domestic Ruminants in Van Province, a Non-endemic Region in Turkey

Crimean-Congo hemorrhagic fever (CCHF) infection is a tick-borne viral disease that causes mild fever and severe hemorrhagic fever with non-specific symptoms and sometimes progresses to fatal bleeding in humans but is asymptomatic in infected animals. The mortality rate in cases reported between countries varies between 5 and 50% in humans (Whitehouse, 2004; Tuncer et al., 2014).

The etiological agent known as Crimean-Congo hemorrhagic fever virus (CCHFV) has an enveloped, negative-sense single-stranded RNA genome in the Orthonairovirus genus, which belongs to the Nairovirdae family. The viral genome consists of three segments; small (S), medium (M) and large (L), which encode the viral nucleocapsid (N), envelope glycoproteins (GN and GC) and RNA-dependent RNA polymerase (RdRp), respectively (Carter et al., 2012; Garrison et al., 2020).

CCHFV is widely distributed in some countries in Africa, Asia, the Middle East, eastern Europe and more recently in south-western Europe ((Yylmaz et al., 2009); Chinikar et al., 2010; Mahzounieh et al., 2012; Tuncer et al., 2014; Ozan and Ozkul, 2020). Based on the prevalence of infectious diseases and the one health concept, when the disease is evaluated on the basis of countries; among the five categories, Turkey is in the first category with Iran, Iraq and Pakistan. In countries in this category, human cases are reported annually through surveillance systems and the infection is endemic (Spengler et al., 2018). 

The CCHF infection in humans was first detected clinically in Turkey in 2002 and a definitive diagnosis was made in 2003. More than 10,000 confirmed cases have been reported in Turkey since 2002, with a relatively low mortality rate (4.6%) compared to other countries. In recent years, the number of suspected CCHF-infected cases in humans and the incidence rate have decreased. About 2/3 of the cases were seen among rural farmers and housewives (Vatansever et al., 2007; (Ergonul et al.,  2009)). 

The main vectors and reservoirs of the CCHFV are ticks. The life cycle of the virus in nature is maintained between ticks-vertebrates-ticks because it is possible for the virus to be transmitted between ticks transovarially, transstadially and vertically. Livestock serve as a transient reservoir for CCHFV, but do not show clinical signs (Kasi et al., 2020). Ticks of the genus Hyalomma marginatum are accepted as the primary vectors of the CCHFV for numerous wild and domestic vertebrates, including cattle, sheep and goats. The prevalence of human cases has proven to be closely related to vector and reservoir distribution. Asymptomatic CCHF infection is common in many vertebrate species, both wild and domestic. As it is currently known, domestic and wild animals serve as indicator animals for the circulation of CCHFV in the environment (Nalca and Whitehouse, 2007; Ergonul, 2012; Schuster et al., 2016; Spengler et al., 2016).

For the control and prevention of infectious diseases, it is very important among the effective factors to reveal epidemiologically dynamic information about the reservoir and circulation of the agent. There is limited data about the origin and current status of CCHFV in animals in Turkey. The present study was designed to reveal the epidemiological dynamic information on CCHF infection in three ruminant species in the Van region, a non-endemic region in Turkey.

Sampling
 
The study was carried out in the province of Van, located in the Eastern Anatolia region of Turkey (Fig 1).



A total of 491 whole blood and 491 blood serum were collected from three ruminant species, including 276 cattle, 166 sheep and 49 goats (Table 1). Sampling was made from ruminants over six months old that were bred by the public in different districts of Van province, especially in the districts near the Iranian border, between May 2020 and October 2021. Serological and virological studies of the samples were carried out in the laboratory of the Department of Virology at Van Yuzuncu Yil University, Faculty of Veterinary Medicine, in Turkey.


 
Serological study
 
To determine the CCHFV-specific antibody, all 491 serum samples of animals taken into tubes were centrifuged at 2000 rpm and the sera transferred to the stock tubes were inactivated at 56°C for 30 minutes in order to inactivate indigenous complement and kept at -80°C until testing. Sera samples were tested with a commercial double-Ag ELISA-Ab kit (IDVet, France) reported and validated in the previous study using the same commercial test kit (Sas et al., 2018) to detect specific antibodies against the CCHFV nucleoprotein. The ELISA assay was performed according to the manufacturer’s instructions.
 
Virological study
 
Viral RNA isolation was performed on a total of 491 whole blood samples taken into tubes containing EDTA using a universal blood RNA extraction kit for purification of genomic RNA (EURx, Poland, Cat. no. E3598) as indicated in the manufacturer’s recommendation. To detect the presence of European lineage 1 CCHFV nucleic acids and viraemic animals, a one-step RT-PCR kit (Grisp, Portugal, Ref. GK64.0100) and S-segment partial sequence encoding primers were used in the conventional reverse transcription polymerase chain reaction (RT-PCR) assay as described previously (Burt et al., 1998). Briefly, sense and antisense primers (5’-TGGACACCTTCACAAACTC-3’ and 5’-GACAAATTCCCTGCACCA-3’) targeting the S segment of the viral genome to amplify a 536-bp-length PCR product were used in a RT-PCR assay.

CCHFV antibodies detection
 
All 491 serum samples, comprising 276 cattle, 166 sheep and 49 goats, were examined. Specific antibodies against CCHFV were detected in all three of the ruminant species tested. As a result of double-Ag ELISA, the seropositivity rate was determined in cattle, sheep and goats as 11.6% (32/276), 64.45% (107/166) and 81.63% (40/49), respectively. Considering all the animals included in the study, the seropositivity rate was found to be 36.45% (179/491) (Table 1).

CCHFV nucleic acid detection
 
All 491 whole blood samples were taken for a RT-PCR assay for CCHFV nucleic acid detection. As a result of the testing with specific primers based on the S gene, 1.22% (6/491) positivity was detected in the tested animals (Table 1). Agarose gel photographs of the obtained amplicons are shown in Fig 2.



Annual cases from CCHFV-endemic countries have been reported during the past few decades (Mardani and Namazee, 2017). During the past 8 years, cases have been decreasing in Turkey, but the incidence of CCHF remains the highest worldwide, with 371 confirmed cases reported in 2016 (Cosgun et al., 2017). With the spread of CCHFV to wide geographic regions, changes in climatic conditions and no approved effective and specific treatment or vaccine for diseases, the virus is considered a public health risk for many regions (Mertens et al., 2013). Although the serological data on the presence of the CCHF infection in humans in Turkey has been proven for a long time (Swanpoel, 1994), this disease was first reported clinically in 2002 in the province of Tokat (WHO, 2008). In the following years, the number of cases increased and more than 9,700 human cases were reported between 2002 and 2016 (Leblebicioglu et al., 2016). This disease has been seen as a health problem in Turkey for the last decade. It has been shown that the infection tends to spread in non-endemic regions other than those defined as endemic (Tuncer et al., 2014).

Hyalomma ticks and various animal species (domestic and wild vertebrates) are known as the natural vector and reservoir of the virus; however, the role of wild and domestic animals as asymptomatic reservoirs in the endemic transmission cycle of the virus has been demonstrated. In vertebrate species, the asymptomatic viraemia period can last up to 7-15 days (Hoogstraal, 1979; Ergönül, 2006). It has also been proven that the distribution and prevalence of human cases are closely related to vector and reservoir distribution (Ergonul, 2012). Serological and virological studies reveal that many wild and domestic animals, such as cattle, sheep and goats, are susceptible to the CCHF virus. These animals are thought to serve as a replicative host to the virus, in addition to developing an antibody response and transient viraemia to the CCHFV (Whitehouse, 2004) and isolation of the virus from livestock and small mammals has been reported in previous studies (Hoogstraal, 1979; Whitehouse, 2004; Ergönül, 2006). Until now, in the Van province, there has been one virological tick survey study and virus detection in tick species (Dinçeret_al2017), but no studies on infection in domestic and/or wild animals. The presence of antibodies against the CCHFV in animals is the best indicator of the presence of the virus in that region and the risk of disease in humans (Tuncer et al., 2014; Mertens et al., 2015; Spengler et al., 2016; Whitehouse, 2004).

A serological survey of a total of 491 blood serums in the present study revealed that 36.45% of the ruminant species tested were exposed to CCHFV infection. Goats had the highest seroprevalence rate (81.63%), followed by sheep (64.4%) and cattle (11.6%). Between ruminant species, the difference in seroprevalence values was significant. The statistical differences between the three ruminant species may be related to the susceptibility of the animal species, the distribution of vector ticks and pasture use. A possible reason for the higher prevalence of infection in ovine ruminants is thought to be continuous pasture throughout the season.

Similar studies (Hassanein et al., 1997; Mohamed et al., 2008; Telmadarraiy et al., 2009; Williams et al., 2000; Sas et al., 2017) reported that the prevalence of CCHFV antibodies in small ruminants was consistent with our serological results and confirmed that it was higher than in cattle. To compare the results, data similar to this study from Turkey; Tuncer et al. reported the seropositivity rate of CCHFV infection in domestic animals as 33.1% in their study conducted in the Marmara region of Turkey, which is considered non-endemic for CCHF (2014). In the endemic region, the seroprevalence of the virus has been reported as 17% in cattle, 37% in sheep (Kirbas et al., 2010), 79% in cattle (Vatansever et al., 2007), 66% in sheep and 85% in goats (Albayrak et al., 2012). In a recent study, the antibody rate in cattle was reported as 1.2% in three different provinces (Sevik, 2018). The evaluation of these results together shows that CCHF may have different epidemiological patterns in animals between endemic and non-endemic areas.

In different countries, different seroprevalence rates have been reported in domestic animals. In a study conducted in Bulgaria, a high seroprevalence rate (18.4%) was determined for the virus in ruminants in many regions where there were no human cases (Christova et al., 2018). Similar seroprevalence rates exist for Balkan countries: Greece has 25% (Papa et al., 2014), Kosovo has 18.4% (Fajs et al., 2014), the Republic of Macedonia has 14.6% (Mertens et al., 2015) and Albania has the lowest seroprevalence rate of 4.7% (Lugaj and Bërxhol, 2014). The CCHFV seroprevalence rate has been reported to be 57.7% in Niger (Mariner et al., 1995), 39.3% in Iran (Telmadarraiy et al., 2009) and 3.13% in Egypt (Mohamed et al., 2008). The seropositivity rate of 36.45% in the present study is compared with some previous studies (Vatansever et al., 2007; Kirbas et al., 2010; Albayrak et al., 2012; Tuncer et al., 2014; Sevik, 2018) in Turkey, indicating an intermediate prevalence of infection in the population tested. In addition, different seroprevalence rates between provinces in the same country show the effect of microclimate conditions on the epidemiology of the infection.

In the present study, using the RT-PCR technique for CCHFV nucleic acid detection, six out of 491 blood samples were found to be positive. In the 2017 tick study in the mentioned region (Dincer  et al., 2017), the presence of the virus was shown in 52.2% of the tick pools tested. According to the findings of this study, the identification of 1.5% (6/491) of the tested animals as viraemic indicates active virus circulation in the Van province, which is considered a non-endemic area. In Turkey, there are many studies on the transmission of infection from endemic to non-endemic regions in humans (Boluk et al., 2009; Ertugrul et al., 2009; Hekimoglu et al., 2012). In a study conducted in Erzurum, a neighboring province of Van, in 2014, the seroprevalence of infection in humans was found to be 1.3% (Yagci-Caglayik et al., 2014). In addition, in a study conducted in the Van province, the serological presence of CCHFV in humans was reported at 14.4% (Bayram et al., 2017). Despite this active virus circulation, the low incidence of human cases in the region may be due to unidentified rural cases without fatalities or a lack of sufficient data or studies.   

As a result, this study evaluating the prevalence of CCHFV in three ruminant species in the Van region shows that infection is widespread in cattle, sheep and goats, even in non-endemic areas. High seroprevalence rates and viraemic animals found in farm animals could lead to future endemics in rural areas, as well as an increased risk of infection for humans in close contact with infected animals. It is emphasized to conduct a risk assessment for public health and conduct similar studies in non-endemic regions and with wild animals that may contribute to the creation of epidemiological dynamic data and the determination of effective control measures in the region.

The authors thank Prof. Dr. Aykut Ozkul, who kindly shared the CCHFV RNA positive control for this study.

The current study was financially supported by the scientific research projects coordination unit of Van Yuzuncu Yil University with project ID: TYD-2020-8900. 

The study was approved by the Van Yuzuncu Yil University Animal Experiments Local Ethics Committee (Approval date: 29/08/2019; Decision no: 2019/08) and Ministry of Agriculture and Forestry of the Republic of Turkey (Approval date and no: 07/05/2019-1401211).

The authors declared that there is no conflict of interest.