Epidemiological Survey, Molecular Detection and Risk Factors of Camel ORF Virus in Arero District, Ethiopia

Background: While camels (dromedaries) were traditionally believed to be resistant to most livestock diseases, research has demonstrated that they are susceptible to a large number of infectious agents. Based on the clinical appearance of typical lesions, Camel contagious ecthyma (CCE), caused by a Orf virus, is thought to be one of the most common viral diseases of camelids in Ethiopia. However, the epidemiology of the disease has not been formally described and the causative agent has never been molecularly confirmed. Methods: a cross-sectional study was conducted from November 2013 to April 2014 in Arero district of Borena Zone, to assess morbidity and mortality rates of the of disease consistent with CCE, isolate and molecularly identify the causative agents and to find out the potential risk factors. Molecular technique, namely, PCR based on B2L gene-specific primers of ORFV was used for the confirmatory diagnosis of CCE virus from the skin lesions. Results: Majority (86.8%) of the respondents indicated occurrence of CCE outbreaks in their herds in the past one year (a year preceding the start of the study). The overall morbidity and mortality rates attributed to CCE was 43.6% (95 % CI: 41.2%–46%) and 6.3% (95 % CI: 5.2 –7.6%) respectively. Confirmatory diagnosis of the suspected Orf virus isolates using conventional PCR techniques generated the expected amplification product of 1200bp for one of the samples. No product was amplified from the DNA samples of the negative control. This study showed that young camels (calves) had higher odds of becoming affected by CCE than adults [OR=3.44 (95 % CI: 2.29 –4.09); (p<0.05)]. The disease had marked seasonality with most of the cases occurring during rainy season. Acacia trees significantly contribute to virus dissemination through damaging the lips of browsing camels. Conclusions: This study confirms the presence and importance of CCE in Ethiopia and establishes the basis for further research.


Background
The one-humped camel (Camelus dromedarius) is a key livestock species that is uniquely adapted to harsh environments [1]. Dromedaries provide a reliable source of livelihood especially for some of the most food insecure pastoral and nomadic communities. In addition to providing milk, meat and local transportation, they are the source of cash [1, 2, &3] for households.
East Africa is home to a large number of dromedaries [4]. Ethiopia possesses over 1 million dromedaries [5], mainly found in the Afar, Somali and Borana areas [6]. The Borana pastoralists, who traditionally depend on cattle husbandry for milk production, have increasingly turned their attention to camel milk in recent years. As a result there is an increased demand for camel production in the area [7], however regional disease knowledge is limited [8 & 9].
Camel contagious ecthyma (CCE), also known as Orf in camels is caused by a pox virus of the genus parapoxvirus (PPV) of the family Poxviridae [10]. CCE is clinically recognized by the appearance of papules, vesicles, pustules and rapidly growing scabs confined to the lips and muzzle of the affected animals [11][12][13][14]. Infected animals are weak, fail to thrive, and are more disposed to other bacterial infections [ 13& 14]. In addition to its economic impact (from morbidity and mortality), CCE has important zoonotic implications [15]. Field observations have indicated that CCE as one of the major viral infections causing economic losses and potential public health threat to camel herders in many parts of Ethiopia including Borena pastoral area. Molecular technique, namely, PCR based on B2L gene-specific primers of ORFV was extensively used for the confirmatory diagnosis of contagious ecthyma in infected animals [16]. Therefore, the aim of this study was to quantify morbid and mortality caused by CCE, isolate and molecularly identify ORF virus and to find out the potential risk factors of CCE in Arero district, Ethiopia.

Description of the study area
The study was conducted in Arero district of Borana zone, Oromia regional state of Ethiopia.
Arero district was selected on the basis of its camel population, and easy access from a major road. The district has an area of 10,890 Km 2 . Its altitude ranges from 750-1700 meters above sea level. The annual average temperature and rainfall is 19 o C and 716 mm respectively.
Animal husbandry in the area is characterized by extensive pastoral productions system with seasonal migration. Camels and cattle are the key livestock species in the area. As aridity is gradually increasing and drought is a recurrent phenomenon in the area, the principal stock is shifting from cattle to camels [17]. Our study was specifically carried out in three pastoral associations (Haro-Dimtu, Kaarra-Gumaata and Silala) where there are large numbers of camel populations.

Study Methods and sample size determination
The study employed cross-sectional study design (November 2013 to April 2014). A total of 129 volunteer camel owners were interviewed to explore data relevant to the study objectives such as herd size of the respondents, herding experiences, occurrence and seasonality of CCE outbreak in the past one year (a year preceding the start of the study), number of animals affected and died due to CCE and common plant browse that are potentially associated with CCE occurrences. Herders' ability to identify CCE infection from other diseases with similar clinical signs and symptoms was cross-checked by enquiring about the clinical signs of the diseases. The questionnaire data were collected only after confirming that the respondent had described the disease correctly.

Sample collection and sampling procedures
The protocol for field studies and collection of animal samples was carried out in accordance the area was disinfected with 70% alcohol and the scabs were aseptically collected using a scalpel blade. The collected samples were put in a labeled sterile universal tube containing phosphate buffered saline (PBS) at pH 7.2 and supplemented by antifungal and antibiotics to prevent further contamination. Samples with PBS were instantly transferred into a cold box and transported to the National Veterinary Institute (NVI, Ethiopia) for laboratory analysis.

Virus isolation on cell culture
Tissue homogenates (10 % w/v) were prepared using sterile PBS and centrifuged at high speed using a refrigerated centrifuge. Then, 2% Glasgow Minimum Essential Medium (GMEM), calf serum and PBS at 37 o C were kept warm in water bath. The laminar flow hood was disinfected with alcohol and the UV light. The media was discarded from the cellular monolayer and 0.5ml of sample suspension was inoculated into each 25cm 2 tissue clutter flask. Inoculated flasks were then incubated at 37 0 c for one hour to adsorb on to the cell and the suspension was subsequently discarded for monolayer formation. After washing with PBS suspension, 2% GMEM media was added. Loosely closing the stopper, cells and virus containing flask was incubated at 37 0 C. Samples were considered negative when no cytopathic effect (CPE) was observed following three blind passages [18].

Polymerase chain reaction (PCR)
Confirmatory diagnosis of the cell culture positive samples was attempted using conventional PCR techniques with primers that amplify the B2L gene. This gene helps to distinguish ORF virus from other orthopoxviruses [19]. DNA was extracted using the DNeasy Tissue Kit (Qiagen, Germany) based on the manufacturer's instructions. Amplification was carried out in a final volume of 50ml containing 5ml of 10 × PCR buffer, 1.5ml of MgCl2 (50mM), 1µl of dNTP (10mM), 1ml of forward primer, 1µl of reverse primer, 5ml of DNA template, 0.5µl of Taq DNA polymerase and 35µl of nuclease-free water.
Amplification was performed by using primer pair 5`CGCAGACGTGGCTCAGTACGT-3à nd 5`TGAGCTGGTTGCCGCTGTCCT-3'. The reaction conditions were first denaturation for five minutes at 94°C followed by 29 cycles of one minute at 94°C, followed by one minute at 45°C, and finally 2.5 minutes at 72°C. A final extension step at 72°C for 10 minutes completed the amplification. Gel electrophoresis was prepared and ethidium bromide (loading dye) was added after forming a comb. The sample was subjected to electrophoresis for 1hour and 20 minutes, and subsequently observed under UV light.

Data collection and analysis
Data related to the location of the herds/specific PAs, seasonal occurrence of CCE, the age groups of camels diseased and died of CCE, and the types of camel browses potentially predisposing to CCE were collected through the questionnaires. Laboratory results abstained from each tested sample were also recorded. The collected data were entered and managed in Microsoft excel sheet.
Data were analyzed using the Statistical Packages for Social Sciences (SPSS of version 17).
Descriptive (proportion) and inferential (logistic regression model) statistics were used to analyze the data. Potential risk factors associated with occurrence of CCE were assessed using logistic regression model and odds ratio (OR) were used to determine the strength of association between the risk factors (independent variables) and disease (dependent variable).
In all the analyses, confidence levels at 95 % and a p < 0.05 were used for statistical significance test.

Results of the questionnaire survey
Herd profile in relation to camel CCE and herding years of the respondents was described in Table 1. Herd size was used to evaluate the relative contribution of camels to pastoralists in the study area, and herding year was used to estimate their practices in camel husbandry.
Average herd size of the respondents was 14 camels (range one to 29) and average herding year was 37 (range one to 75). A total of 129 herds (1,679 camels) were investigated in the present study. Majority (86.8%) of the respondents indicated occurrence of CCE outbreaks in their herds in the past one year. The overall morbidity and mortality rates attributed to CCE was 43.6% (95 % CI: 41.2%-46%) and 6.3% (95 % CI: 5.2 -7.6%) respectively.
Age wise morbidity and mortality rate of the disease was presented in Table 2. The results, therefore, showed that young animals (below two years of age) had higher odds of becoming affected by CCE than adults (OR=3.44; 95%CI: 2.3-6.4) and the difference was statistically significant (p < 0.05). The disease had marked seasonality with most of the cases occurring during rainy season. Acacia trees significantly contributed to virus dissemination in the study area through damaging the lips of browsing camels.

PCR result
The sequence of the PPVB2L gene, a homologue of the major enveloped antigen, p37K of the vaccinia virus, has been used as a molecular target for virus recognition, diagnosis of infection and for identification of species within the genus Parapoxvirus [20]. In the present study, the extracted DNA was amplified and only 1 out of 10 tested samples were found The overall morbidity and mortality rate rates of CCE in the present study were comparable with the reports from Southwest Iran [22] and Mongolia [23]; but lower when compared to the findings of Khalafalla of Sudan [24]. The variation could be related to the age structure of animals included in the studies, difference in the husbandry and health management system in different countries.
During our field clinical investigation, we noticed that majority of the animals showing clinical signs of suspected pox virus infection were camel calves. The morbidity and the mortality rates attributed to CCE are also higher in this age group than in adults. Our findings concur with the reports of different researchers [25,26]. Severity of the disease in young animals might be due to lack of prior exposure to infecting pathogens or due to absence of fully developed immune system.
The present study season was evaluated for potential risk factors for occurrence of CCD; rainy season being associated with the disease. This finding concurs with previous reports [25][26][27][28]. The increase in incidence of CCE in the rainy season could be associated with the abrasion of the lips due to browsing thorny Acacia trees locally named 'Ammarressa' and and poor primer binding due to variation in DNA sequence at the binding sites.

Conclusions
The present study confirmed the existence of CCE in Arero district of Borena zone.

Ethics approval and consent to participate
Ethical clearance was obtained from Jimma University, College of Agriculture and Veterinary Medicine. Written and oral consent was obtained from camel owners before questionnaire survey and sample collection.

Consent for publication
Not applicable

Availability of data and material
The dataset analyzed during the current study is available from the corresponding author on reasonable request.

Competing interest
The authors would like to declare that they have no any financial and personal relationships with other people or organizations that could inappropriately influence (bias) their work.

Funding
The present study received financial support from Jimma University, Vice president for research and publication office (VRPO).

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. Tables2.pdf Questionnare.pdf