Ecology of the Crimean-Congo Hemorrhagic Fever Endemic Area in Albania

Introduction

C rimean-Congo hemorrhagic fever virus (CCHFV) belongs to the Nairovirus genus in the Bunyaviridae family. It is transmitted to humans either by bites of Ixodid ticks (mostly of the Hyalomma genus) or by contact with blood or tissues from CCHF patients or viremic livestock (Ergonul 2006). The life cycle of the virus in nature includes transovarial and transstadial transmission among ticks and a tick-vertebrate host cycle involving wild and domestic animals. Smaller wildlife species such as hares and hedgehogs, as well as ground-feeding birds, act as hosts for the immature stages of the tick vectors, while adult ticks prefer large mammals such as livestock and wild boar. Although CCHFV causes a severe disease in humans, there is no evidence of disease in its natural hosts. However, seroepidemiological surveys in endemic areas revealed high prevalence of anti-CCHFV antibodies in domestic animals, particularly in cattle and sheep. Avian species are not regarded as amplifying hosts for CCHFV, but migrating birds have been held responsible for the transport of transovarially infected larval ticks or infected nymphal ticks along avian migration routes. Like all other vector-borne diseases, the presence and persistence of zoonotic foci of infection depend on biological and ecological relationships between three very different kinds of organisms: virus, ticks, and vertebrates (Randolph and Rogers 2007).

The geographic distribution of CCHF extends from China to Russia, the Balkans, and various African countries. Sporadic cases, or even outbreaks, occur every year in the Balkan peninsula (Drosten et al. 2002, Papa et al. 2002b, 2004). Albania, a country with a population of 3.6 million, is located in southeastern Europe. Since the disease was first identified in 1986, cases have been reported here almost every year. There have been outbreaks, mostly among family members, and nosocomial transmission has also been reported (Papa et al. 2002a). The most endemic CCHF regions in Albania are two neighboring districts in the northeastern part of the country, Kukes and Has (Fig. 1). Kukes has an area of 930 km² and a population of 83,000; Has is a 374 km² area with a population of 20,000. The villages where CCHF cases were observed in former years are located at an altitude of 600 m above sea level, surrounded by forests, which are inhabited by dense wildlife. The area is covered with herbaceous vegetation, sea line oak, and alpine meadows. From 2001 to 2006, an average of five confirmed cases have been reported annually in the above districts, mainly in family clusters. Tick bite was reported in 17.3% of the cases.

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FIG. 1.

Map of Albania. Endemic CCHF regions, Kukes and Has, are in the northeast of the country. Dots represent CCHF cases in 2003 and 2004 (presented by Dr. S. Bino in the Joint Intercountry Workshop on CCHF Prevention and Control, Istanbul, 2006).

An outbreak was observed in Albania in 2003, and most cases were epidemiologically related to one fatal case observed in the end of May (Papa et al. 2006). The patient was a farmer and had close contact with animals that were heavily infested with ticks. Aim of the present study was to estimate the prevalence of CCHFV among ticks, animals, and birds in the endemic regions of Albania, as they are the main organisms involved in the virus life cycle.

Materials and Methods

Three hundred and thirty-eight ticks were collected during 2003–2005 from cattle grazing in Kukes and Has districts. Collection by flagging method was unsuccessful; therefore ticks were collected directly from the animals. After identification using standard keys, ticks were sent in dry ice to Aristotle University of Thessaloniki.

A total of 24 serum samples were collected from domestic animals (10 goats and 14 cattle). On 18 June 2003 sera were collected from 10 goats and 6 cattle from a farm in Vlahen village, Has (the place where the outbreak was observed), while on 18 October 2005 8 sera were collected from cattle in Kukes. On 18 October 2005 sera were also collected from 4 hares, 3–5 months old, from villages in Kukes. In addition, during July to October 2005, sera were collected from 42 birds in Kukes, at a biological station located 1400 m above sea level.

Ticks were grouped according to species, gender, location, and year of collection. Thirty-one pools were prepared and homogenized in a cell disrupter (Fast Prep 120, BIO 101, La Jolla, CA). Viral RNA was extracted using TRIzol LS Reagent (Invitrogen Life Technologies, Carlsbad, CA), and RT-nested PCR was performed (Schwarz et al. 1996). Cleaned PCR products were sequenced in an ABI PRISM^® 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA). Sequences were aligned with respective ones retrieved from GenBank Database using CLUSTAL W, genetic distances were calculated by the Neighbor-joining method, and a phylogenetic tree was constructed using the Phylip software (http://evolution.gs.washington.edu/phylip.html). In positive samples, a quantitative real time RT-PCR was applied (Papa et al. 2007).

All serum samples were tested for the presence of specific IgG antibodies to CCHFV by indirect immunofluorescence assay on spot-slides containing Vero E6 cells infected with the prototype Nigerian CCHFV strain (IbAr 10200) at an initial serum dilution of 1:32.

Results

All 338 ticks were adult Ixodidae ticks, with Rhipicephalus and Hyalomma spp. constituting 56.2% and 25.4% of the total number of ticks, respectively (Table 1). During June 2003 to June 2005, tick density was 14–15 ticks per animal. An increase in tick population was seen during June 2006 to June 2007, reaching 30 ticks per animal, with cows presenting the highest infestation rate.

One of the 31 pools, consisting of four female Hyalomma spp. ticks, was found positive for CCHFV. They were collected in July 2005 from cattle in Kukes. Sequencing of the PCR product revealed that the strain presented 99.2–100% nucleotide homology to CCHFV strains detected in patients living in the same area (Fig. 2). Actually, the sequences of the ticks collected from Kukes in 2005 were identical with those of respective ones from a CCHF human case in 2006 in the same region (case 252/06). Although extensive genetic diversity is seen among CCHFV strains from different geographic regions, the virus seems to be stable in the same region. All Albanian CCHFV strains cluster in the “Europe 1” clade, together with strains from Bulgaria, Kosovo, Russia, and Turkey and differ by approximately 24% from the “Europe 2” clade, which consists of the Greek strain AP92 (Fig. 2).

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FIG. 2.

Phylogenetic tree based on a 255-nt fragment from the sRNA segment showing the clustering of the Albanian strains obtained from infected ticks and human cases. Representative strains of clades Europe 1 and 2, Asia 1 and 2, Africa 1, 2, and 3 are included; Dugbe virus was used as outgroup. The numbers indicate percentage bootstrap replicates (of 100).

Quantitative real time RT-PCR showed that the ticks carried high viral load (6.8 × 10⁷ copies/mL of homogenate). For comparison, the highest viral load (3.3 × 10⁹ copies/mL of plasma) was observed in a primary fatal CCHF case in Albania, while in secondary cases the viral load was lower than 3 × 10⁴ copies/mL of plasma (Papa et al. 2007).

Among animals tested, high titers (1:512) of IgG antibodies to CCHFV were detected in 2/10 goats in Vlahen village, Has. The goat sera were collected shortly after the family outbreak in the village in 2003. The 25-year-old shepherd of that flock was a relative of the fatal case from which the outbreak was initiated, and presented fatigue, myalgia, and lower-back pain. He was not hospitalized but was found positive by serology to CCHFV. Neither the woman who died nor the shepherd referred any tick bite; however, it is likely to have gone unnoticed.

Avian species included three Alectoris graeca, six Columbia livia, five Columbia spp., five Corvus corone cornix, one Lanius senator, six Larus spp., one Paser montanus, five Passer domesticus, six Streptopelia decaocto, one Streptopelia turtur, one Sturnus vulgaris, one Sylvia spp., and one Turdus spp. All birds were CCHFV negative.

Discussion

Various ecological factors affect the distribution and abundance of tick vectors, which play a key role in CCHF epidemiology. Collection of appropriate ecological and environmental data is not easy, but it is the basis for understanding the disease epidemiology to further proceed in disease prediction and prevention. Although CCHFV has been isolated from at least 30 species of ticks, Hyalomma spp. ticks are considered as the main vector of the virus. Actually, the occurrence of CCHF coincides with the global distribution of Hyalomma spp. ticks (Hoogstraal 1979). CCHFV was first isolated in former Yugoslavia in 1976 from Hyalamma marginatum marginatum (CCHFV strains Ciflik 1 and 6) and Ixodes ricinus ticks (CCHFV strain Ciflik 11) (Gligic et al. 1977). In addition, 41/273 (15%) H. marginatum ticks collected in Kosovo at the time of the 2001 epidemic were found to be infected with CCHFV (Duh et al. 2006). In a survey in an endemic region in Turkey, 47% and 46% of ticks collected from sheep and cattle were Rhipicephalus bursa and H. marginatum marginatum, respectively, and four tick pools (three R. bursa and one H. marginatum marginatum) were CCHFV RNA positive (Tonbak et al. 2006). Till date there are no data concerning the vector of the virus in Albania. All primary Albanian CCHF patients referred either a tick bite or tending animals infested with ticks. One patient referred that he was bitten by ticks twice in a 1-month interval: 1 month before and, again, 2 weeks before the onset of the disease, suggesting that ticks are abundant in the region. This was also shown by the heavy infestation of domestic animals. The predominant tick in the endemic regions was Hyalomma spp. Identification of ticks was performed to the genus level. However, in earlier studies, all Hyalomma ticks collected in the same region were H. marginatum marginatum (Christova et al. 2003), suggesting that this species most probably serves as the principal vector of CCHFV in Albania, as in most parts of the Palaearctic region (Hoogstraal 1979). The study by Christova et al. (2003) also revealed that approximately half of H. marginatum marginatum ticks in the region were positive for Rickettsia spp., increasing their importance and medical implication in public health.

Concerning animals, it was found that goats of the affected family presented high titers of CCHFV antibodies. Although viremia in animals is very short (7–10 days), serosurveys on domestic animals can be useful for early warning system. Wildlife populations were not tested extensively; however, during the epidemic years 2001 and 2003, the number of hares (on which immature stages of Hyalomma spp. feed) in the endemic region increased very much. Similar observations were also reported in the most affected areas in Turkey, where both wild boars and hares have increased considerably (Vatansever et al. 2007). All birds in the study were found negative, as in most previous studies (Nalca and Whitehouse 2007).

In conclusion, this report gives a first insight into the ecology related to CCHFV life cycle in the endemic regions in Albania. The results could be helpful to public health authorities in assessing the risk for the disease in humans.