Albanian j. agric. sci. 2014;(special edition)

Agricultural University of Tirana

(Open Access)

RESEARCH ARTICLE

Serological survey of Crimean-Congo hemorrhagic fever virus in cattle in Berat and Kolonje, Albania ARTA LUGAJ1 MYNYR KONI2 MARC MERTENS3 MARTIN H. GROSCHUP3 KRISTAQ BERXHOLI4 1

Department of Biology, “Ismail Qemali” University of Vlora, Albania

2

Department of Biology, The Faculty of Natyral Sciences, University of Tirana

3

Friedrich-Loeffler-Institut, Greifswald, Germany

4

Department of Veterinary Public Health, Agricultural University of Tirana, Tirana, Albania

Abstract Crimean–Congo hemorrhagic fever (CCHF) is a tick-borne disease caused by the arbovirus Crimean–Congo hemorrhagic fever virus (CCHFV), which is a member of the Nairovirus genus (family Bunyaviridae). The disease occurs sporadically throughout Africa, Asia, and Europe and results in an approximately 30% fatality rate. Numerous genera of ixodid ticks serve both as vector and reservoir for CCHFV; however, ticks in the genus Hyalomma are particularly important to the ecology of this virus. The aim of this study was to examine the distribution of CCHFV among the cattle in Berat and Kolonje regions in Albania. The data taken of this study indicates the presence of CCHFV in these regions. The serum samples were conserved at -20°C and tested with indirect ELISA at Friedrich-Loeffler Institute (FLI), Greifswald Germany. By this technique it was possible to identify CCHFV specific antibodies in serum samples. From these results in Berat-Terpan we had an indication about the presence of IgG antibodies in 2 blood samples, 3 serum samples were equivocal and 45 serum samples were negative from the total of 50 serum samples in cattle. While in Kolonje-Erseke the results show the presence of IgG antibodies in 4 blood samples from 54 seum samples in cattle. Respectively the prevalence in these two countries in Albania is 4% and 7.4%. These results can clearly proved the presence of CCHFV in livestock in Albania. Key words: CCHFV, Nairovirus, Bunyaviridae, Indirect ELISA, FLI

1. Introduction

where CCHFV is endemic. Furthermore, nosocomial CCHFV infections among caregivers are also reported [4, 16]. Like other tick borne zoonotic agents, CCHFV circulates in nature in an enzootic tick-vertebrate-tick cycle. Humans are being infected mainly through direct contact with blood or tissues from infected livestock or through tick bites. CCHFV is transmitted by ticks of the genus Hyalomma and in particular by Hyalomma marginatum marginatum. Ticks of the genus Hyalomma serve as vectors and reservoir of CCHFV and the geographic distribution of the disease coincide with the global distribution of Hyalomma ticks [5, 16, 18]. The virus is reported in over 30 countries in Africa (Democratic Republic of Congo, Uganda, Mauritania, Nigeria, S. Africa, Senegal, ect), Southeast Europe (Russia, Bulgaria, Kosovo, Turkey, Greece, ect), the Middle East (Iraq, Iran, Saudi Arabia, Oman) and Asia (China, Kazakistan, Tajikistan, Uzbekistan, Pakistan) [8 13]. In this regard, the geographical distribution of CCHFV is the greatest among all tick-borne viruses genus ticks in the ‘60s and transovarial and transstadial transmissions have been already

The members of the family Bunyaviridae constitute a diverse group of viruses that infects animals, plants, humans, and insects [3, 10, 11]. The viruses contain three RNA segments of negative sense, which in turn encode three structural proteins: the small (S) segment encodes the nucleocapsid protein (NP), the medium (M) segment which encodes a glycoprotein precursor, resulting in the two envelope glycoproteins Gn and Gc, and the large (L) segment which encodes the RNA-dependent RNA polymerase. Certain members of the Bunyaviridae also encode non-structural proteins [2, 3, 11, 13]. Crimean-Congo hemorrhagic fever virus (CCHFV) belongs to the genus Nairovirus of the family Bunyaviridae and is the causative agent of a severe hemorraghic fever disease resulting in a case fatality rate of around 30% in humans [10, 15]. At present, Crimean-Congo hemorrhagic fever is a public health problem in many regions of the world, e.g., Asia, Eastern Europe, Africa, and Russia. CrimeanCongo hemorrhagic fever outbreaks have been handle livestock such as sheep, goats, and ostriches, in areas

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propagated since viral isolates have been also found in field collected eggs and unfed immature stages of H. marginatum [17]. CCHFV has been also isolated in laboratory from other tick genera Rhipicephalus, Ornithoros, Boophilus, Dermacentor and Ixodes spp. Although, the virus persists in ticks, vertebrates are needed to provide blood meals for the ticks and a variety of livestock can become infected with the CCHFV. In fact, numerous domestic and wild vertebrates have been reported to present antibody response and/or viremia [16]. This livestock includes cattle, goats, sheep, horses, pigs, hares, ostriches, camels, donkeys, mice and domestic dogs. In contrast to human infections the livestock’s infections generally result in inapparent or subclinical disease [7, 14, 6, 12, 9, 18]. However, the infected livestock during the viremic phase is dangerous for the disease transmission in humans. Domestic ruminant animals such as cattle, sheep and goats will present viremia for

one week [1]. Although it has been shown that the majority of birds is resistant to infection [18] the potential role of migratory birds in the disease dissemination could not be ignored. 2. Material and methods 2.1 Sera from cattle. We collected serum samples from Berat and Kolonje areas where we had indication about the presence of CCHFV in humans. Blood was taken from the jugular vein by vakutanier and was left for two hours to coagulate. Then the serum samples were taken by centrifugation (15 minutes at 3000 rfm / rotate for minute) and were preserved in freezing at -20 degrees. In animal CCHF infections does not cause clinical signs. The data of serum samples are presented in table 1.

Table 1 Collected serum samples.

Region/Location (village, farm)

Number

50

Animal species CT-cattle Cattle

Date of sample Collection (Day/Month/Year) 15/04/2013

Gender M-male/ F-female F-female

Housing S-stable/ P-pasture P-pasture

Tick defense Measures D-defense/ND-no defense ND-no defense

Berat-Terpan Kolonje-Erseke

54

Cattle

16/05/2013

F-female

P-pasture

ND-no defense

Total

104

Cattle

F-female

P-pasture

ND-no defense

background reactions. Each serum samples was added to two wells without the N-protein. In case CCHFVspecific antibodies were in a serum sample, they bind to the N-protein. All unspecific antibodies were washed away. As a secondary antibody a peroxidase labelled bovine specific conjugate was added to each well. This conjugate formed antibody-antibody complexes with the CCHFV-specific antibodies of the serum sample. For the detection of this complex, a substrate for the peroxidase was added. The substrate changes color upon reaction with the enzyme and shows therewith, that CCHFVspecific antibodies are in the serum samples which have bound to the N-protein. The higher the concentration of the primary antibody present in the serum, the stronger the color change. A spectrometer was used to give quantitative values for color strength.

Indirect ELISA. IgG and IgM antibodies are detectable from about 7 days after onset of disease in humans. Specific IgM declines to undetectable levels by 4 months postinfection, but IgG remains detectable for at least 5 years. All collected sera were sent to FriedrichLoeffler-Institute (FLI) in Greifswald, Germany in November 2013. The indirect ELISA was used for the detection of IgG antibodies in the serum samples. Briefly, the following ELISA protocol was used. A recombinant Nucleocapsid (N-) protein of CCHFV was used as antigen. It was added half of the wells of a 96-well microtiter plate, were it adhere to the plastic through charge interactions. A solution of skim milk was used for blocking all free binding sides and to reduce

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Serological survey of Crimean-Congo hemorrhagic fever virus in cattle in Berat and Kolonje, Albania

country. From our results we have different values in different areas. We found the presence of infection (antibodies) in animals in areas where cases with hemorrhagic fever were observed more than 10 years ago, where since then has not been observed cases with hemorrhagic fever in humans. In Berat-Terpan the prevalence is 4% while in Kolonje-Erseke the prevalence is 7.4%. This indicates that the infection in these areas is still present.

3. Results and discussion A total of 104 serum samples were tested with indirect ELISA at FLI, Germany. Through this technique it was possible to identify the CCHFVspecific antibodies in sera of infected animals. The ELISA results presented in the table below indicates the presence of CCHFV in two different areas of our

Table 2 The results obtained from indirect ELISA. Region/ (village)

Total samples

Positive sample

Equivocal

Negative

Prevalence (%) of positive sample

Berat-Terpan

50

2

3

45

4%

Kolonje-Erseke

54

4

0

50

7.4%

TOTAL

104

6

3

95

5.7%

structural protein sequences. J Gen Virol 2000: 2161–2166.

We think that these results should be a signal especially for human service which should take strong observation in these areas and in equivocal cases should immediately take appropriate measures. 10.00% 8.00% 6.00% 4.00% 2.00% 0.00%

3. Bishop DHL. Biology and molecular biology of bunyaviruses: New York, NY: Plenum Press. 1996. 4. BurneyMI, Ghafoor A, Saleen M, Webb PA, Casals J. Nosocomial outbreak of viral hemorrhagic fever caused by Crimean hemorrhagic fever-Congo virus in Pakistan, January 1976. Am J Trop Med Hyg 1980: 941– 947.

7.4% 4% Berat-Terpan

Kolonje-Erseke

5. Charrel RN, Attoui H, Butenko M. Tick-borne virus diseases of human interest in Europe. ClinMicrobiol Infect 2004; 10:1040-55.

Prevalence (%) of positive samples Figure 1 Prevalence of CCHFV-specific

6. El-Azazy OM, Scrimgeour EM. Crimean-Congo hemorrhagic fever virus infection in the Western province of Saudi Arabia. Trans R Soc Trop Med Hyg 1997; 91:275-8.

antibodies in Berat and Kolonje, Albania.

We also emphasize that this infection is found mainly in animals, which are kept in outdoor leisure suburbs which creates very favorable conditions for infestation. As shown in Table no.1, no measures were performed to protect those animals from ticks.

7. Logan TM, Linthicum KJ, Bailey CL, Watts DM, Moulton JR. Experimental transmission of Crimean-Congo hemorrhagic fever virus by HyalommatruncatumKoch. Am J Trop Med Hyg 1989; 40:207-12.

4. References

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17. Watts DM, Ksiazek TG, Linthicum KJ, Hoogstraal H. Crimean-Congo hemorrhagic fever. In: Monath TP, ed. The arboviruses: epidemiology and ecology, volume 2, Boca Raton FL, USA: CRC Press, 1988:177-260.

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