 crimean - congo hemorrhagic fever virus belongs to the genus nairovirus in the bunyaviridae family , and is a human pathogen that can cause a severe , and often fatal , hemorrhagic fever . 
cchf virus is the most geographically widespread tick - borne virus of medical importance ( ergonul 2006 ) .
the disease is endemic in large areas of sub - saharan africa and the middle and far east , as well as in eastern europe .
a significant increase of cases in countries such as kosovo , albania , turkey , iran , and greece has recently been observed ( alavi - naini et al .
the cchfv genome has been isolated from at least 31 different tick species in the ixodidae ( hard ticks ) and argasidae ( soft ticks ) .
hyalomma spp ticks are considered the most important in the epidemiology of cchfv ; however , the virus has also been isolated from ticks of other genera ( i.e. , rhipicephalus , boophilus , dermacentor , haemaphysalis , and ixodes spp , ( saijo et al .
humans in high - risk occupations ( e.g. , slaughterhouse workers , shepherds , health care workers , and veterinarians ) are prone to cchf infection ( garcia et al .
this study was performed to ascertain the prevalence of cchfv in ticks present on the one - humped camel ( camelus dromedarius ) , and to estimate the prevalence of cchfv igg in the one - humped camel s sera in three provinces : khorassan - e - shomali , khorassane - razavi , and khorassan - e - jonoobi of iran .
the study was conducted in three provinces : khorassan - e - shomali , khorassan - e - razavi , and khorassan - e - jonoobi located at 5517 , 6115e and 3024 , 3817n in northeastern and east of iran ( fig .
1 ) . khorassan - e - shomali is a mountainous region , with temperate , cold weather .
khorassan - e - razavi is a semi - desert region that has mild weather .
khorassan - e - jonoobi is a semi - desert region that experiences arid conditions .
the average annual rainfall is approximately 300400 mm in the northern areas ( khorassan - e - shomali ) and 150 mm in the central and southern areas ( khorassan - e - razavi and khorassan - e - jonoobi ) .
there are approximately 25 million camels in the world , and nearly 150,000 one - humped camels are in iran , this is 0.6% of the world camel population , and 3.8% of the asian camel population ( fao 2011 ) . the majority of iran s camels are dromedaries , and they are scat - tered across the country s provinces . in the khorassan
provinces the camel population is 37,400 ( ministry of agriculture jihad , 2002 ) , but in the authors experience at present the actual number is several times greater , the majority of these are in khorassan - e - shomali and khorassan - e - jonoobi .
study area in khorassan - e - shomali , khorassan - e - razavi and khorassan - e - jonoobi are shown with asterisks amplification of the s segment of the cchfv genome using rt - pcr , in tick samples from the khorasan province .
( pc : positive control , nc : negative control , s1 - 10 : samples , s1 , s2 , s3 , s4 , s5 , s6 , s7 and s9 are negative , s8 and s10 are positive ) from may 2012 to january 2013 , eleven cities and towns were randomly selected from three provinces : khorassan - e - shomali , khorassan - e - razavi and khorassan - e - jonoobi as 
clusters ,  and at least 14 one - humped camels were sampled from each cluster . from each animal ,
two or three ticks were collected and placed in separate sterile tubes ; the tubes were labeled with the date of collection , animal number , sex , age , and area .
collected ticks were sent to the laboratory and identified under a stereo microscope using general identification keys ( hoogstraal 1979 , apanaskevich and filippova 2007 ) .
the samples were then pooled according to the area , sex and species of tick ( each pool routinely contained 18 ticks , but occasionally the number was greater ) and immediately sent to the arboviruses and viral hemorrhagic fevers laboratory ( national reference laboratory ) , pasteur institute of iran , and stored at 70 c until analysis . for the serum assay , 20 ml of blood collected from the jugular vein of each camel then the sample was labeled with the date of collection , animal number , sex , age , and area .
the samples were immediately sent to a laboratory and centrifuged at 5000 rpm for 10 min .
the sera were then separated and transferred into holding tubes and sent to the arboviruses and viral hemorrhagic fevers laboratory and stored at 70 c until analysis . according to previous studies in animals ,
tick infectious was 10% and with assumption 5% accuracy , 95% confidence interval and design effect equal 1.5 we need at least 207 ticks . with assumption 20% seropositivity in the studied animals , 8% accuracy , 95% degree of confidence , and design effect equal 1.5 , the sample size will be 144 , each cluster has at least 14 sera .
reverse transcriptase polymerase chain reaction : ticks were individually washed twice by pbs ( pbs , ph 7.4 ) and crushed with a mortar and pestle in 200300l of pbs .
the extracted viral rna was stored at 70 c until analysis . for the rt - pcr ,
a master mix was prepared as follows : 28 l of rnase free water , 10 l of buffer ( 5  conc ) , 2 l of dntp mixture , 2l of enzyme mixture containing reverse transcriptase and taq dna polymerase enzymes , 1l of primer f2 ( 5-tggacaccttcacaaactc-3 ) , 1l of primer r3 ( 5-gacaattccctacacc-3 ) , 1l of rnase inhibitor and 5l of extracted viral rna as template .
the f2 and r3 primers amplify a 536 bp fragment inside the s - segment of the cchfv genome , 536 bp fragment is rt - pcr target .
the thermal cycling program for the rt - pcr , included 30 min at 50 c for reverse transcription reaction ( cdna synthesis ) , 15 min at 95 c for activation of hot star taq dna polymerase and inactivation of reverse transcriptase , followed by 35 cycles of 95 c for 30 sec , 50 c for 30 sec , 72 c for 45 sec , and a final extension at 72 c for 5 min . for gel - based rt - pcr product analysis ,
5l of the pcr product was mixed with 1l loading buffer ( 6  conc ) .
then , the mixture was load in agarose gel 1.5% , and visualized with ethidium bromide ( chinikar et al .
, the elisa plates were coated overnight at 4 c with mouse hyperimmune ascitic fluid diluted at 1:1000 in 0.05% tween 20-pbs containing 5% skim milk as a saturating reagent .
the native or the recombinant antigen ( produced in our laboratory ) diluted in pbstm ( pbs containing 0.05% tween and 3% skim milk ) was added to the plates and the plates were incubated for 1h at 37 c and extensively washed .
serum samples diluted in pbstm were added , and the plates were incubated for 1h at 37 c .
after washing , the peroxidase - labeled anti - human or animal immunoglobulin diluted in pbstm was added to each weel and the plates were incubated for 1 h at 37 c .
the plates were then washed 3 times with pbs containing 0.5% tween ( pbst ) . finally , hydrogen peroxide and tetramethylbenzidine ( tmb ) were added and the plates were incubated for 15 min at room temperature .
the enzymatic reaction was stopped by the addition of sulphuric acid ( 4n ) and the plates read by elisa reader ( anathos 2020 ) at 450 and 620 nm . taken together , an igg - positive serum was considered as positive control and a negative serum taken as negative control in the igg elisa ( garcia et al .
descriptive statistics ( i.e. prevalence and percentages ) were used to summarize the quantitative variables .
the study was conducted in three provinces : khorassan - e - shomali , khorassan - e - razavi , and khorassan - e - jonoobi located at 5517 , 6115e and 3024 , 3817n in northeastern and east of iran ( fig .
1 ) . khorassan - e - shomali is a mountainous region , with temperate , cold weather .
khorassan - e - razavi is a semi - desert region that has mild weather .
khorassan - e - jonoobi is a semi - desert region that experiences arid conditions .
the average annual rainfall is approximately 300400 mm in the northern areas ( khorassan - e - shomali ) and 150 mm in the central and southern areas ( khorassan - e - razavi and khorassan - e - jonoobi ) .
there are approximately 25 million camels in the world , and nearly 150,000 one - humped camels are in iran , this is 0.6% of the world camel population , and 3.8% of the asian camel population ( fao 2011 ) . the majority of iran s camels are dromedaries , and they are scat - tered across the country s provinces . in the khorassan
provinces the camel population is 37,400 ( ministry of agriculture jihad , 2002 ) , but in the authors experience at present the actual number is several times greater , the majority of these are in khorassan - e - shomali and khorassan - e - jonoobi .
study area in khorassan - e - shomali , khorassan - e - razavi and khorassan - e - jonoobi are shown with asterisks amplification of the s segment of the cchfv genome using rt - pcr , in tick samples from the khorasan province .
( pc : positive control , nc : negative control , s1 - 10 : samples , s1 , s2 , s3 , s4 , s5 , s6 , s7 and s9 are negative , s8 and s10 are positive )
from may 2012 to january 2013 , eleven cities and towns were randomly selected from three provinces : khorassan - e - shomali , khorassan - e - razavi and khorassan - e - jonoobi as 
clusters ,  and at least 14 one - humped camels were sampled from each cluster . from each animal ,
two or three ticks were collected and placed in separate sterile tubes ; the tubes were labeled with the date of collection , animal number , sex , age , and area .
collected ticks were sent to the laboratory and identified under a stereo microscope using general identification keys ( hoogstraal 1979 , apanaskevich and filippova 2007 ) .
the samples were then pooled according to the area , sex and species of tick ( each pool routinely contained 18 ticks , but occasionally the number was greater ) and immediately sent to the arboviruses and viral hemorrhagic fevers laboratory ( national reference laboratory ) , pasteur institute of iran , and stored at 70 c until analysis . for the serum assay , 20 ml of blood collected from the jugular vein of each camel then the sample was labeled with the date of collection , animal number , sex , age , and area .
the samples were immediately sent to a laboratory and centrifuged at 5000 rpm for 10 min .
the sera were then separated and transferred into holding tubes and sent to the arboviruses and viral hemorrhagic fevers laboratory and stored at 70 c until analysis .
according to previous studies in animals , tick infectious was 10% and with assumption 5% accuracy , 95% confidence interval and design effect equal 1.5 we need at least 207 ticks .
with assumption 20% seropositivity in the studied animals , 8% accuracy , 95% degree of confidence , and design effect equal 1.5 , the sample size will be 144 , each cluster has at least 14 sera .
reverse transcriptase polymerase chain reaction : ticks were individually washed twice by pbs ( pbs , ph 7.4 ) and crushed with a mortar and pestle in 200300l of pbs .
the extracted viral rna was stored at 70 c until analysis . for the rt - pcr ,
a master mix was prepared as follows : 28 l of rnase free water , 10 l of buffer ( 5  conc ) , 2 l of dntp mixture , 2l of enzyme mixture containing reverse transcriptase and taq dna polymerase enzymes , 1l of primer f2 ( 5-tggacaccttcacaaactc-3 ) , 1l of primer r3 ( 5-gacaattccctacacc-3 ) , 1l of rnase inhibitor and 5l of extracted viral rna as template .
the f2 and r3 primers amplify a 536 bp fragment inside the s - segment of the cchfv genome , 536 bp fragment is rt - pcr target .
the thermal cycling program for the rt - pcr , included 30 min at 50 c for reverse transcription reaction ( cdna synthesis ) , 15 min at 95 c for activation of hot star taq dna polymerase and inactivation of reverse transcriptase , followed by 35 cycles of 95 c for 30 sec , 50 c for 30 sec , 72 c for 45 sec , and a final extension at 72 c for 5 min . for gel - based rt - pcr product analysis , 5l of the pcr product
then , the mixture was load in agarose gel 1.5% , and visualized with ethidium bromide ( chinikar et al .
for igg antibody detection , the elisa plates were coated overnight at 4 c with mouse hyperimmune ascitic fluid diluted at 1:1000 in 0.05% tween 20-pbs containing 5% skim milk as a saturating reagent .
the native or the recombinant antigen ( produced in our laboratory ) diluted in pbstm ( pbs containing 0.05% tween and 3% skim milk ) was added to the plates and the plates were incubated for 1h at 37 c and extensively washed .
serum samples diluted in pbstm were added , and the plates were incubated for 1h at 37 c .
after washing , the peroxidase - labeled anti - human or animal immunoglobulin diluted in pbstm was added to each weel and the plates were incubated for 1 h at 37 c .
the plates were then washed 3 times with pbs containing 0.5% tween ( pbst ) . finally , hydrogen peroxide and tetramethylbenzidine ( tmb ) were added and the plates were incubated for 15 min at room temperature .
the enzymatic reaction was stopped by the addition of sulphuric acid ( 4n ) and the plates read by elisa reader ( anathos 2020 ) at 450 and 620 nm . taken together , an igg - positive serum was considered as positive control and a negative serum taken as negative control in the igg elisa ( garcia et al .
descriptive statistics ( i.e. prevalence and percentages ) were used to summarize the quantitative variables .
a total of 200 one - humped multi - purpose camels ( rearing for milk , meat , riding and offspring ) were examined .
tick infestation was observed in 170 of them , and 480 ixodid ticks ( 133 females and 347 males ) were collected from different regions in the khorassan - e - shomali , khorassan - e - razavi and khorassan - e - jonoobi ( table 1 ) .
positivity rates , number , and sex of ticks collected from one - humped camels from khorassan - e - shomali , khorassan - e - razavi and khorassan - e - jonoobi in east and northeast iran in the current study , only four species of hyalomma genus were observed .
population frequency of h. dromedarii ( 90.7% ) was higher than others and h. asiaticum had the lowest frequency ( 0.4% ) .
hyalomma marginatum comprised about 2.9% and h. anatolicum accounted for 6% of total collected species .
h. dromedarii is the most dominant tick species of camel in the khorassan region and a one humped camel is a suitable host .
hyalomma dromedarii , h. anatolicum and h. marginatum were collected from all three provinces but in contrast , h. asiaticum only was collected from khorassan - e - razavi s one - humped camels ( table 2 ) . the sex , species and cchf - positive rate of ticks infesting one - humped camels the cchfv genome was found in 49 ( 10.2% ) of 480 ticks , and three ( 6% ) of 50 pools .
the cchfv genome was detected in two out of four tick species , and of these , 42 ( 85.7% ) belonged to h. dromedarii and 7 ( 14.3% ) belonged to h. anatolicum ( table 2 ) .
the viral genome was detected in tick samples from three cities in khorassan - e - jonoobi .
the positivity rates were as follows : boshroyeh , 25 out of 480 ( 51% ) , birjand , 17 out of 480 ( 34.7% ) , and nehbandan , 7 out of 480 ( 14.3% , table 1 ) .
all three provinces , and six out of eleven cities and towns , were igg - positive for cchfv .
nine ( 5.3% ) out of 170 camels were igg - positive , this means that the igg prevalence was 5.3% .
the positivity rates for the provinces varied , and were as follows : boshroyeh , 12.5% , kanimani , 7.14 % , birjand , 8% , nehbandan , 16.67% , chehl dokhtaran , 7.14% , and sabzevar , 6.66% .
the highest rate of igg - positive samples was found in nehbandan ( 16.67% , two out of 12 sera ) , khorassan - e - jonoobi .
eight out of the nine positive samples were collected from female camels ( table 3 ) .
igg antibody - positive sera collected from one - humped camels from khorassan - e - shomali , khorassan - e - razavi and khorassan - e - jonoobi
in this study , h. dromedarii was the most dominant species of tick on one - humped camels , and this is in agreement with the results obtained by salimabadi ( 2010 ) in iran and lawal et al .
crimean - congo hemorrhagic fever infection was detected in 49 ( 10.2% ) of 480 tick samples and this is a higher percentage than that previously reported by salim - abadi ( 3.79% ) in the yazd provinces of iran ( 2011 ) .
hyalomma ticks are the primary vectors for the transmission of cchfv throughout europe , asia , the middle east , and africa ( ergonul 2006 ) .
although hyalomma ticks are considered the most important vector and reservoir for the cchfv , the virus has also been reported in other tick genera ( tahmasebi et al . 2010 ) .
the cchfv genome was detected in two out of the four tick species that were collected ( h. dromedarii , 85.7% and h. anatolicum , 14.3% ) .
( 2011 ) , therefore , these results suggest that h. dromedarii and h. anatolicum act as vectors for cchfv in one - humped camels .
hyalomma dromedarii is distributed throughout the world wherever camels are present , and h. anatolicum is widely distributed throughout iran .
hyalomma anatolicum transmits at least five arboviruses , and is a significant vector of cchfv to humans ( nabian and rahbari 2008 ) .
( 2010 ) detected the cchfv genome in rhipicephalus bursa ( in one of the three ticks that were sampled ) , but we failed to discover r. bursa in our study . in the present study , we only found the cchfv genome in one - humped camel ticks in khorassan - e - jonoobi ( birjand , boshroyeh , and nehbandan ) .
this province has an unusual geographical location , as it borders afghanistan to the east , the sistan - va - baluchistan province of iran in the south , and the khorassan - e - razavi province of iran in the north .
since 2000 , the disease has infected 23 out of 31 provinces in iran : sistanva - baluchistan ( 283 confirmed human cases ) , isfahan ( 44 confirmed cases ) , fars ( 26 confirmed cases ) , tehran ( 17 confirmed cases ) , and khorasan ( 12 confirmed cases ) ( chinikar et al . 2012 ) .
notably , sistan - va - baluchistan province ( south of khorassan - e - jonoobi ) has not just had the highest number of cchfv cases , but cchf infections have been observed in this area since 2000 ( chinikar et al .
2012 ) , as it shares a border with two cchf - endemic countries : pakistan and afghanistan ( chinikar et al . 2010 ) .
the unusual location of khorassan - e - jonoobi , which is connected in the north , south , and east to heavily infected or endemic areas of cchf , may explain why it was the only cchfv - positive area found in the study .
we could not ascertain why all the reverse transcriptase polymerase chain reaction - positive ticks were male , but this may have been due to the presence of more males than females on the animals , and also in our samples ( 347 males vs 133 females ) . during the last decade ,
an increasing number of human cchfv infections have been reported in various regions of iran ( chinikar et al .
igg - positive serum samples collected from sheep , goats , cows , and humans have been frequently reported in different parts of the country ( saidi et al .
we found cchfv igg antibodies in one - humped camel sera in all three provinces studied , and these results , as well as those from a previous study by chinikar et al .
( 2012 ) , may indicate that cchf is endemic to these regions , or that it has spread from neighboring countries . in total , nine ( 5.3% ) out of 170 camels were igg positive .
( 1975 ) , who found that cchf samples were negative in all 157 camels that they had studied , from south and southeast iran .
( 2000 ) found that 17 ( 16% ) out of 109 camels sampled in oman were tested positive for the cchf igg antibody .
sistan - va - baluchistan has been the most cchfv - infected province in iran since 2000 , because , as mentioned previously , it shares a border with two cchf - endemic countries , pakistan and afghanistan ( izadi et al .
khorasan , which is connected to heavily infected or endemic areas of cchf , shares a large border area with neighboring countries , shares common pasture with herds from cchf - endemic areas , and experiences the illegal importation of animals across the border , which may explain why it is a cchf - positive area .
another important issue is the presence of disease reservoirs and vectors , such as hyalomma spp .
the cchfv genome has been isolated from at least 31 different tick species in ixodidae ( hard ticks ) and argasidae ( soft ticks ) ( saijo et al . 2002 , tahmasebi et al .
2010 ) . as champour ( 2013 ) noted , the main tick species that affect one - humped camels in this region are h. dromedarii , h. anatolicum , h. marginatum , and h. asiaticum .
hyalomma spp ticks are considered the most important species in the epidemiology of cchfv in camels in this area ( hoogstraal 1979 ) .
six out of the eleven cities and towns studied yielded one - humped camel serum that was igg - positive for cchf , and it has been shown that cchf is distributed across these three provinces .
we could not ascertain why almost all of the positive - igg sera samples were collected from female camels ( 8/9 ) , but this may have been due to the presence of more females than males in the samples ( 136 females vs 34 males ) , possibly because female camels remained in herds longer than did males at the time of sampling .
our results reveal a lower prevalence of seropositivity in one - humped camels ( 5.3% ) than in other domestic animals ( 30% ) in iran ( chinikar et al . 2009 ) . however , due to the fact that camels remain in herds for longer than do other animals ( champour et al .
2013 ) , and because camel pasture is very widely geographically distributed , this small percentage has a significant effect on the epidemiology of cchf .
the importance of camels in the epidemiology of cchf in russia and astrakhan oblast has been previously reported by kurbanov , berezin , and chumakov ( steele 1994 ) .
because cchf is a serious threat to iran , imported animals , particularly one - humped camels that carry a large number of ticks , should be inspected and treated carefully . continued surveillance and strictly enforced importation and quarantine practices will be required to prevent human exposure and the on - going dispersal of infected ticks and livestock in these regions .
the use of commercially available insect repellent , and the use of clothing impregnated with permethrin , can give some protection against tick bites ( telmadarraiy et al .
further surveys of human and animal populations , particularly of those animals imported from neighboring countries to these regions , are recommended , in order to provide a better understanding of the distribution and epidemiology of the virus in these provinces .
 background : this comprehensive study was conducted on multi - purpose one - humped camel ( camelus dromedarius ) sera and ticks to assess the epidemiological aspects of the crimean - congo hemorrhagic fever virus ( cchfv ) in northeast iran.methods:from may 2012 to january 2013 , eleven cities were randomly selected in the khorasan provinces of iran as  clusters ,  and at least 14 one - humped camels were sampled from each area . 
 reverse transcriptase polymerase chain reaction was used for the detection of the cchfv genome in ticks . 
 sera were analyzed using specific enzyme - linked immunosorbent assay tests.results:four hundred and eighty ixodid ticks were collected , and the genome of the cchfv was detected in 49 ( 10.2% ) out of 480 ticks . 
 the cchfv genome was detected in two out of four tick species , and in tick samples from three cities in khorassan - e - jonoobi . 
 all three provinces , and six out of eleven cities , were cchfv - specific igg - positive . in total , 
 nine ( 5.3% ) out of 170 one - humped camels were igg - positive . 
 the highest rate of igg - positive samples was found in nehbandan ( 16.67%).conclusion : continued surveillance and strictly enforced importation and quarantine practices should be implemented to prevent human exposure and the on - going dispersal of infected ticks and livestock in these regions . 
 it is recommended that acaricides be used to prevent cchf transmission to humans , and to reduce the tick population . in addition 
 , care should be taken by abattoirs workers and people who work with one - humped camels . 
Introduction
Materials and Methods
Study area
Sampling
Tick sample size calculation
Serum sample size calculation
Molecular detection
IgG-sandwich ELISA
Statistical analysis
Results
Discussion
Conclusion