• Users Online: 279
  • Home
  • Print this page
  • Email this page
Home About us Editorial board Ahead of print Current issue Search Archives Submit article Instructions Subscribe Contacts Login 


 
 
Table of Contents
RESEARCH ARTICLE
Year : 2022  |  Volume : 59  |  Issue : 3  |  Page : 241-245

Molecular detection of Crimean-Congo Haemorrhagic Fever (CCHF) virus in hard ticks from South Khorasan, east of Iran


1 Department of Veterinary Medicine, Faculty of Veterinary Medicine, University of Zabol, Zabol, Iran
2 Department of Clinical Sciences, Faculty of Veterinary Medicine, University of Zabol, Zabol, Iran
3 Department of Food hygiene, Faculty of Veterinary Medicine, University of Zabol, Zabol, Iran
4 Cellular and Molecular Research Center, Iran University of Medical Sciences, Tehran, Iran
5 Department of Arboviruses and Viral Hemorrhagic Fevers (National Ref Lab), Pasteur Institute of Iran (IPI), Tehran, Iran

Date of Submission22-Oct-2020
Date of Acceptance18-Feb-2022
Date of Web Publication08-Dec-2022

Correspondence Address:
Mehdi Rasekh
Department of Clinical Sciences, Faculty of Veterinary Medicine, University of Zabol, Zabol
Iran
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0972-9062.342400

Rights and Permissions
  Abstract 

Background & objectives: Crimean-Congo Hemorrhagic Fever (CCHF) is a deadly viral infection reported from more than 30 countries. It is considered a zoonosis↱ and tick bites are the main route of transmission in nature. So far, the virus has been identified in 31 species of hard (Ixodidae) and soft (Argasidae) ticks. The aim of this study was to determine the rate of CCHF virus infection in hard ticks from South-Khorasan province, east of Iran.
Methods: In this study, 684 livestock including 302 sheep, 344 goats, 16 cows and 22 camels were sampled from Birjand, Qaen, Khusf, Darmian and Sarbisheh counties. Genus and species of the ticks were diagnosed under stereomicroscope according to valid morphological keys. Reverse transcription-polymerase chain reaction (RT-PCR) method was used to detect the CCHF virus genome based on S segment in 100 ticks.
Results: RT-PCR detected CCHF virus genome in 7 out of 100 ticks. Positive ticks belonged to Hyalomma and Rhipicephalus genera. CCHF virus infected species were Rhipicephalus sanguineus, Hyalomma detritium and Hyalomma asiaticum. All the infected ticks were isolated from goat and sheep and were from Birjand county.
Interpretation & conclusion: Our results suggest that Hyalomma and Rhipicephalus may be the main vectors of CCHF virus in the study area.

Keywords: Crimean-Congo hemorrhagic fever; Hard ticks; South Khorasan; RT-PCR


How to cite this article:
Jafari A, Rasekh M, Saadati D, Faghihi F, Fazlalipour M. Molecular detection of Crimean-Congo Haemorrhagic Fever (CCHF) virus in hard ticks from South Khorasan, east of Iran. J Vector Borne Dis 2022;59:241-5

How to cite this URL:
Jafari A, Rasekh M, Saadati D, Faghihi F, Fazlalipour M. Molecular detection of Crimean-Congo Haemorrhagic Fever (CCHF) virus in hard ticks from South Khorasan, east of Iran. J Vector Borne Dis [serial online] 2022 [cited 2023 Feb 2];59:241-5. Available from: http://www.jvbd.org//text.asp?2022/59/3/241/342400


  Introduction Top


Crimean Congo Haemorrhagic Fever (CCHF) is a deadly viral infection (mortality rate 3–30%) reported from more than 30 countries[1]. It is considered a zoonosis↱and transmission is possible through tick bites, contact with blood, secretions and carcasses of infected animals or humans. Crimean-Congo Haemorrhagic fever virus (CCHFV) is an RNA virus that belongs to the genus Nairovirus and the Bunyaviridae family. In addition to animal-to-human transmission, human-to-human transmission as well as nosocomial transmission are also possible for this disease[2],[3]. Like other tick-borne zoonotic agents, the virus follows the tick-vertebrate-tick cycle, and despite the lack of evidence of clinical disease in animals, it occurs in a wide range of domestic and wildlife animals as reservoirs including horses, donkeys, goats, cows, sheep, pigs, hedgehogs, hares, buffalos, zebras, and giraffes. Unlike animals, humans show clinical signs of the disease include high fever, chills, severe headache, dizziness and back and abdominal pain, nausea, vomiting, diarrhea, and cardiovascular alternations. In severe cases, bleeding, such as petechiae and ecchymosis, can lead to death[4],[5]. The main route of transmission in nature is by tick bites, and so far, the virus has been identified in 31 species of hard (Ixodidae) and soft (Argasidae) ticks. Soft ticks do not play an important role in the geographical distribution of the virus, because viral replication cannot occur in their adult or nymph stages. Sexual, transovarial and transstadial virus transmission have been reported in ticks[6],[7].

CCHF is endemic in many countries of Asia, Europe and Africa[8]. For the first time in 1970, Shumakov and colleagues identified antibodies against the CCHF virus in the sera of 45 sheep sent from Tehran to Moscow. Five years later, Saeedi et al., detected antibodies against the virus in human samples from the Caspian Sea and East Azerbaijan province. Since the discovery of the first human case in 1999, the disease has been reported more accurately and rapidly, including testing for the virus or its antibodies in human populations, reservoirs, and vectors. The disease has been reported from 27 provinces (out of 31 provinces) of the country which Sistan and Baluchistan, Isfahan, Fars, and Khuzestan being the most affected provinces[9],[10]. The aim of this study was to determine the rate of CCHFV infection in ticks collected from different livestock in South-Khorasan province, east of Iran.


  Material & Methods Top


Study area and sample collection

The survey carried out in Birjand, Qaen, Khusf, Darmian and Sarbisheh counties from South Khorasan province, east of Iran (32.8653°N 59.2164°E). In this study, 684 livestock including 302 sheep, 344 goats, 16 cows and 22 camels were sampled in 2019. Multistage random sampling method was used in one village randomly selected from each city and three to five livestock holding units were randomly selected from each village for collecting tick samples. Hard ticks were randomly collected during summer in 2019 using forceps and were placed into separate labelled vials. Information such as sampling area, host type, gender, age, owner name and animal code were obtained and written on prepared lists. Specimens were kept frozen at -20°C after each sampling interval. Then, all samples were transferred to the Department of Medical Entomology, School of Public Health, Tehran University of Medical Sciences along with the cold chain for species identification. Genus and species were diagnosed under stereomicroscope according to valid morphological keys[11]. For molecular detection of CCHF virus (CCHFV), identified ticks were sent to the Arboviruses and Viral Haemorrhagic Fever Laboratory (National Reference Laboratory) in the Pasteur Institute of Iran [Figure 1].
Figure 1: Sampling areas in Iran are marked with orange asterisk.

Click here to view


RNA extraction and molecular detection

Ticks were individually washed twice by PBS (PBS, pH 7.4) and crushed with a mortar and pestle in 200–300 μl of PBS. Total RNA was extracted using a RNeasy mini kit (QIAGEN, Cat No. 2215716) according to the manufacturer’s instructions. The extracted total RNA was stored at -70°C until use. Ticks were not pooled and the genetic material of each single tick was considered as a separate sample for molecular testing. Then a master mix was prepared with QIAGEN one-step RT-PCR kit (Cat No. 210212) as follows: 28 μl of RNase free water, 10 μl of buffer (5×), 2 μl of dNTP mixture, 2 μl of enzyme mixture containing reverse transcriptase and Taq DNA polymerase enzymes, 1 μl of RNase inhibitor. 1 μl of forward primer (5’ TGGACACCTTCACAAACTC-3’) and 1 μl of reverse primer (5’-GACAATTCCCTACACC-3’) then were added. Primers amplify a 536 bp fragment inside the S-segment of the CCHFV genome. PCR products were visualized by electrophoresis technique in 1.5% agarose gels[12],[13].

Data analysis

The Chi square test was used for data analysis. Also, 95% confidence interval for prevalence of tick infestation was calculated using binomial distribution. SPSS version 25 was used for statistical analysis. The significant level was considered P <0.05.

Ethical statement

The study only involved the separation of ticks from livestock with the consent of the owners. The experiments comply with the guidelines laid down by the Institutional Ethical Committee and in accordance with the country’s law and regulations.


  Results Top


Only 104 livestock were infested with ticks (15%) and a total of 269 ticks were caught from infested livestock. The average rate of tick infestation per livestock was 0.39%. The percentage of livestock infection with ticks in Sarbisheh, Birjand, Qaen, Khosf and Darmian were 11.66%, 12.82%, 6.66%, 34.34% and 15.55% respectively. Of the total ticks caught, 72 (26.8%) were isolated from camels, 11 (4%) from cattle, 89 (32.7%) from goats and 97 (36.4%) from sheep. Two genera and 6 species were identified: 155 (58%) Hyalomma and 114 (42%) Rhipicephalus. Six identified species were: 111 Rhipicephalus sanguineus (41.3%), 24 Hyalomma detritium (8.9%), 6 Hyalomma marginatum (2.2%), 9 Hyalomma anatolicum (3.3%), 5 Hyalomma asiaticum (0.9%), 90 Hyalomma dromedarii (33.5%) and 10 Hyalommasp. (3.7%). Eleven (4.1%) Hyalomma nymphs and 3 (1.1%) Rhipicephalus nymphs were also identified. The highest frequency of genus and species were related to the Hyalomma and Rhipicephalus sanguineus, respectively. As previously mentioned, after RNA extraction, RT-PCR steps were performed using a one-step Qiagen kit. After RT-PCR, the products were identified on gel electrophoresis. In this method, a sequence of 536 bp of the S fragment of CCHF virus was detected and amplified by two specific forward and reverse primers. The presence of the virus was confirmed in 7 of the 100 samples sent to the laboratory. The genera in which the presence of the virus was confirmed were: Rhipicephalus sanguineus (1 sample out of 27 tested samples), Hyalomma detritium (3 samples out of 11 tested samples), Hyalomma asiaticum (3 samples out of 5 tested samples). It should be noted that other submitted species were reported negative for the presence of the virus. In this study, out of 7 positive samples, 5 samples (72%) were female and 2 samples (28%) were male. All infected samples belonged to Birjand county. Samples sent from other counties were reported negative. Two out of the 7 positive samples were isolated from goats and 5 samples were isolated from sheep. No positive ticks were caught from camels and cows. Also, all 7 positive samples were taken from hosts less than one-year-old, male and in lowland areas. Statistical tests showed that there was a significant relationship between the presence of virus genome and species of tested ticks, height of capturing areas, age and sex of hosts (P <0.05), while this was not true for host type and tick sex (P>0.05) [Table 1] & [Figure 2].
Table 1: Characteristics of screened ticks with RT-PCR test

Click here to view
Figure 2: CCHFV RT-PCR results: PC; positive control, Tick specimens; Lane 1-7, EXN; Extraction negative control; NTC; Negative control.

Click here to view



  Discussion Top


Crimean-Congo haemorrhagic fever (CCHF) is a zoonotic disease and some species of hard ticks play an important role in transmission of the virus between different hosts[14]. Humans are sensitive hosts while domestic and wild animals are considered as asymptomatic reservoirs and ticks complete the transmission cycle by transmitting the virus from animal to human[15]. Surveillance systems, up-to-date data about the fauna of vectors and reservoirs and their contamination to CCHFV, awareness of people at risk and understanding the dynamics of viral transmission in nature is vital factors in controlling and prevention of the disease[16]. In the present study, CCHFV was detected in 7% of tested ticks. In previous studies, the rate of virus infection in different parts of Iran was reported to be between 0.2 and 33%[17]. Two genera and three species including Rhipicephalus sanguineus, Hyalomma detritium and Hyalomma asiaticum were positive for the presence of the virus and the most infected genus was Hyalomma. Telmadarraiy et al., reported that the predominant genus in Iran is Hyalomma in terms of infection, and the virus was also isolated from ticks of other genera such as Rhipicephalus which is in line with the result of the present study[18]. Sedaghat et al, screened ticks caught from livestock in Golestan province, Iran and came to the conclusion that the highest viral infection is observed in the genera of Hyalomma and Rhipicephalus[6]. They revealed that the presence of CCHFV is 5.3% in the ticks selected for screening. Many other reports also indicate Hyalomma as the main vector of CCHFV in Iran and other parts of the world[8],[12],[19],[20],[21]. On the other hand, it should be noted that there are differences between the species obtained in the present research and other studies, in terms of prevalence and viral infection, but these differences can be justified due to the climate diversity, livestock management, breeding methods and transfer, spatial-temporal overlap of ticks with livestock grazing and livestock sensitivity. In the present study, viral infections of Hyalomma and Rhipicephalus were 8.9% (6 out of 67 ticks) and 3.7% (1 out of 27 ticks), respectively. Previous studies have indicated the infections rate of Hyalomma and Rhipicephalus in Iran range from 0.2%–33% and 1.8%–55% respectively[17].

In a study conducted in 2013 on the ticks caught from one-humped camels in the east of Iran, Champour et al, sampled from three counties of South Khorasan province (Birjand, Boshrooyeh, Sarayan and Nehbandan). The infection rate of ticks caught in these three counties indicated the infection of South Khorasan province was 25%. The infection rate for Birjand was reported to be 35%. In the present study, the infection rates of Birjand city and South Khorasan province were determined to be 15% and 7%, respectively. The reason for this difference can be attributed to the specificity of the host to the one-humped camels, the smaller volume and different sampling area in Champour study[22].

In another study conducted by Telmadarraiy et al, it was concluded that the infection rate of livestock ticks to CCHFV in South Khorasan province was 6%, which is consistent with the present study to some extent[18]. In both studies that were conducted in South Khorasan, the main tick vector of CCHFV was Hyalomma, which is in line with the results of the present study. South Khorasan is neighbored to endemic provinces of CCHF such as Sistan and Baluchestan and Kerman, sharing border with Afghanistan which is also considered endemic and deals with illegal importation of animals across the border, which may clarify why it is a CCHF-positive area. Other important predisposing factors are popularity of animal husbandry and the high prevalence of tick vectors, including various species of Hyalomma in the region. The most abundant species of tick collected in this research was R. sanguineus followed by various species of Hyalomma. It is mentioned before that Hyalomma ticks are the main vectors of CCHFV, and other hard tick species also reported to be involved in CCHFV circulation[23],[25].

Tick-borne diseases are a significant threat to human health. CCHF epidemiology is very complex as its cycle depends on both invertebrate (different species of ticks) and vertebrate (human and animals) hosts. Many linked factors determine the emergence and spread of CCHFV. Environmental conditions, climate change, tick fauna and animal population are key factors affecting the epidemiology of CCHFV. People’s knowledge, health facilities as well as persistent monitoring of endemic areas in terms of human, animals and ticks will result in reduction of CCHF incidence. The standard test for CCHFV detection is viral isolation. However, it has some limitations to be performed in all conditions. For example, CCHFV isolation requires biosafety level 4 laboratories which are limited. On the other hand, PCR (RT-PCR and qRT-PCR) as well as ELISA techniques are available practicable methods for detecting CCHFV genome and specific IgM/IgG antibodies, respectively. The test showed high reliability in epidemiological studies, especially when performed together[26],[27]. Also, serological methods are very effective for screening human populations in endemic areas due to their acceptable sensitivity and low costs[27]. Finally, it is worth mentioning that amplicon sequencing of positive PCR products as well as serology testing of animals for CCHFV IgG will lead to more reliable results and is highly suggested. However, due to some limitations in the present study, these tests were not achieved.


  Conclusion Top


In conclusion, the presence of CCHFV was confirmed in 7 samples out of 100 hard ticks and all positive samples were collected from Birjand county. CCHFV positive ticks were caught from sheep and goats, which are the most common livestock kept by the people of South Khorasan. Our findings suggest that Hyalomma and Rhipicephalus ticks are the main vectors of CCHFV in South Khorasan province, Iran. Persistent surveillance of livestock and hard ticks for CCHFV along with serologic screening of high-risk people is recommended.

Conflict of interest: None


  Acknowledgements Top


This article is an excerpt from veterinary doctorate dissertation of first author at Zabol University. The project was funded cooperatively by Zabol University [Grant numbers: UOZ-GR-9618-159 and UOZ-GR-9618-141] and members of the research team. The authors express their gratitude to Dr. Zakkyeh Telmadarraiy and the personnel of the Department of Arboviruses and Viral Hemorrhagic Fevers (National Reference Laboratory), Pasteur Institute of Iran for their technical support.





 
  References Top

1.
Shafei E, Dayer MS, Telmadarraiy Z. Molecular epidemiology of Crimean-Congo hemorrhagic fever virus in ticks in northwest of Iran. J Entomol ZooI Stud 2016; 4(5): 150–4.  Back to cited text no. 1
    
2.
Mehravaran A, Moradi M, Telmadarraiy Z, Mostafavi E, Mo-radi AR, Khakifirouz S, et al. Molecular detection of Crimean-Congo haemorrhagic fever (CCHF) virus in ticks from southeastern Iran. Ticks Tick Borne Dis 2013; 4(1–2): 35–8.  Back to cited text no. 2
    
3.
Chinikar S, Ghiasi SM, Hewson R, Moradi M, Haeri A. Crime-an-Congo hemorrhagic fever in Iran and neighboring countries. J Clin Virol 2010; 47(2): 110–4.  Back to cited text no. 3
    
4.
Maltezou HC, Andonova L, Andraghetti R, Bouloy M, Ergonul O, Jongejan F, et al. Crimean-Congo hemorrhagic fever in Europe: current situation calls for preparedness. Eurosurveillance 2010; 15(10): 19504.  Back to cited text no. 4
    
5.
Bente DA, Forrester NL, Watts DM, McAuley AJ, Whitehouse CA, Bray M. Crimean-Congo hemorrhagic fever: history, epidemiology, pathogenesis, clinical syndrome and genetic diversity. Antiviral Res 2013; 100(1): 159–89.  Back to cited text no. 5
    
6.
Sedaghat MM, Sarani M, Chinikar S, Telmadarraiy Z, Moghaddam AS, Azam K, et al. Vector prevalence and detection of Crimean-Congo haemorrhagic fever virus in Golestan Province, Iran. J Vector Borne Dis 2017; 54(4): 353.  Back to cited text no. 6
    
7.
Tahmasebi F, Ghiasi SM, Mostafavi E, Moradi M, Piazak N, Mozafari A, et al. Molecular epidemiology of Crimean-Congo hemorrhagic fever virus genome isolated from ticks of Hamadan province of Iran. J Vector Borne Dis 2010; 47(4): 211–216.  Back to cited text no. 7
    
8.
Yaser SA, Sadegh C, Zakkyeh T, Hassan V, Maryam M, Ali OM, et al. Crimean–Congo hemorrhagic fever: a molecular survey on hard ticks (Ixodidae) in Yazd Province, Iran. Asian Pac J Trop Med 2011; 4(1): 61–3.  Back to cited text no. 8
    
9.
Keshtkar-Jahromi M, Sajadi MM, Ansari H, Mardani M, Hol-akouie-Naieni K. Crimean-Congo hemorrhagic fever in Iran. Antiviral Res 2013; 100(1): 20–8.  Back to cited text no. 9
    
10.
Chinikar S, Ghiasi SM, Moradi M, Goya MM, Shirzadi MR, Zeinali M, et al. Phylogenetic analysis in a recent controlled outbreak of Crimean-Congo haemorrhagic fever in the south of Iran, December 2008. Eurosurveillance 2010; 15(47): 19720.  Back to cited text no. 10
    
11.
Walker AR. Ticks of domestic animals in Africa: a guide to identification of species. Bioscience Reports Edinburgh 2003.  Back to cited text no. 11
    
12.
Farhadpour F, Telmadarraiy Z, Chinikar S, Akbarzadeh K, Moemenbellah-Fard MD, Faghihi F, et al. Molecular detection of Crimean–Congo haemorrhagic fever virus in ticks collected from infested livestock populations in a New Endemic Area, South of Iran. Trop Med Int Heal 2016; 27(3): 340–7.  Back to cited text no. 12
    
13.
Burt FJ, Swanepoel R. Molecular epidemiology of African and Asian Crimean-Congo haemorrhagic fever isolates. Epidemiol Infect 2005; 133(4): 659–66.  Back to cited text no. 13
    
14.
Telmadarraiy Z, Ghiasi SM, Moradi M, Vatandoost H, Eshraghian MR, Faghihi F, et al. A survey of Crimean-Congo haemorrhagic fever in livestock and ticks in Ardabil Province, Iran during 2004–2005. Scand J Infect Dis 2010; 42(2): 137–41.  Back to cited text no. 14
    
15.
Mustafa ML, Ayazi E, Mohareb E, Yingst S, Zayed A, Rossi CA, et al. Crimean-congo hemorrhagic fever, Afghanistan, 2009. Emerg Infect Dis 2011; 17(10): 1940.  Back to cited text no. 15
    
16.
Tekin S, Bursali A, Mutluay N, Keskin A, Dundar E. Crimean-Congo hemorrhagic fever virus in various ixodid tick species from a highly endemic area. Vet Parasitol 2012; 186(3–4): 546–52.  Back to cited text no. 16
    
17.
Saghafipour A, Mousazadeh-Mojarrad A, Arzamani N, Tel-madarraiy Z, Rajabzadeh R, Arzamani K. Molecular and seroepidemiological survey on Crimean-Congo Hemorrhagic Fever virus in Northeast of Iran. Med J Islam Repub Iran 2019; 33: 41.  Back to cited text no. 17
    
18.
Telmadarraiy Z, Chinikar S, Vatandoost H, Faghihi F, Hosseini-Chegeni A. Vectors of Crimean Congo hemorrhagic fever virus in Iran. J Arthropod Borne Dis 2015; 9(2): 137.  Back to cited text no. 18
    
19.
Sharifinia N, Rafinejad J, Hanafi Baali, Chinikar S, Piazak N, Baniardalani M, et al. Hard ticks (Ixodidae) and Crimean-Congo hemorrhagic fever virus in south west of Iran. Acta Med Iran 2015; 53(3): 177–81.  Back to cited text no. 19
    
20.
Gergova I, Kunchev M, Kamarinchev B. Crimean-congo hemorrhagic fever virus–tick survey in endemic areas in Bulgaria. J Med Virol 2012; 84(4): 608–14.  Back to cited text no. 20
    
21.
Kasi KK, von Arnim F, Schulz A, Rehman A, Chudhary A, Oneeb M, et al. Crimean-Congo haemorrhagic fever virus in ticks collected from livestock in Balochistan, Pakistan. Transbound Emerg Dis 2020; 67(4): 1543–1552.  Back to cited text no. 21
    
22.
Champour M, Chinikar S, Mohammadi G, Razmi G, Mostafavi E, Shah-Hosseini N, et al. Crimean-Congo hemorrhagic fever in the one-humped camel (Camelus dromedarius) in East and Northeast of Iran. J Arthropod Borne Dis 2016; 10(2):1 68.  Back to cited text no. 22
    
23.
Gargili A, Estrada-Peña A, Spengler JR, Lukashev A, Nuttall PA, Bente DA. The role of ticks in the maintenance and transmission of Crimean-Congo hemorrhagic fever virus: A review of published field and laboratory studies. Antiviral Res 2017; 144: 93–119.  Back to cited text no. 23
    
24.
Hosseini Z, Salehi Vaziri M, Ahmadnia S, Fakoorziba MR, Jalali T, Telmadarraiy Z, et al. Hard ticks infesting domestic ruminants, species composition and infection with Crimean-Congo hemorrhagic fever virus in a highland province, SW Iran. J Heal Sci Surveill Syst 2019; 7(2): 52–9.  Back to cited text no. 24
    
25.
Telmadarraiy Z, Moradi AR, Vatandoost H, Mostafavi E, Oshaghi MA, Zahirnia AH, et al. Crimean-Congo hemorrhagic fever: a seroepidemiological and molecular survey in Bahar, Hamadan province of Iran. Asian J Anim Vet Adv 2008; 3(5): 321–7.  Back to cited text no. 25
    
26.
Nasirian H. Crimean-Congo hemorrhagic fever (CCHF) seroprevalence: A systematic review and meta-analysis. Acta Trop 2019; 196: 102–20.  Back to cited text no. 26
    
27.
Vanhomwegen J, Alves MJ, Zupanc TA, Bino S, Chinikar S, Karlberg H, et al. Diagnostic assays for Crimean-Congo hemorrhagic fever. Emerg Infect Dis 2012; 18(12): 1958–65.  Back to cited text no. 27
    


    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1]



 

Top
 
  Search
 
    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Access Statistics
    Email Alert *
    Add to My List *
* Registration required (free)  

 
  In this article
Abstract
Introduction
Results
Discussion
Conclusion
Material & Methods
Acknowledgements
References
Article Figures
Article Tables

 Article Access Statistics
    Viewed1398    
    Printed8    
    Emailed0    
    PDF Downloaded61    
    Comments [Add]    

Recommend this journal