Received date: August 24, 2016; Accepted date: September 19, 2016; Published date: September 19, 2016
Citation: Miressa Y, Cherinet Y, Bsrat A, Tadesse F (2016) Investigation of Newcastle Disease Virus Using Reverse Transcription Polymerase Chain Reaction in Selected Districts of Eastern Shewa, Ethiopia. J Vet Sci Technol 7:384. doi: 10.4172/2157-7579.1000384
Copyright: © 2016 Miressa Y, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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In Ethiopia Newcastle disease virus is the most important cause of loss in village-dwelling as well as commercially raised chickens. The disease occurs almost at any time of a year throughout the country including East Shewa Zone. A cross sectional study was therefore conducted from December 2014 to May 2015 to determine the prevalence of Newcastle disease in market and village chicken in selected districts of East Shewa zone of Ethiopia. Molecular diagnostic technique, Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) was employed to detect the viruses during the study. A total of 300 swab samples was collected using simple random sampling technique in Adama and Bishoftu Districts. An overall prevalence of 26.7% (40/300) was recorded during this study from the collected swab samples using real time PCR. The result of this study indicated that village chicken flock are endemically infected with Newcastle disease virus which could pose a threat to commercial poultry farms. Attention should therefore be given for regular monitoring of Newcastle disease virus in village chickens and wild birds and measures to prevent this infection should be taken.
East Shewa; Ethiopia; Newcastle disease; RT-PCR
Poultry play an important economic, nutritional and sociocultural role in the livelihoods of poor rural households in developing countries, including Ethiopia. The total poultry population in Ethiopia is estimated at 56.87 million, 95.86% of which are village chickens . In developing countries, chickens are most commonly owned by rural families. Many of these families have scarce resources and many may be headed by women. Increasing the productivity of their chickens would make a significant contribution towards increasing their food security and their ability to have secure livelihoods .
Despite this fact the contribution of poultry production to farm household and national income is not proportional to their population numbers due to various constraints like low inputs of feeding, poor management, the presence of diseases of various natures and lack of appropriate selection and breeding practices [3-5]. Poultry production is hampered by wide arrays of constraints among which infectious diseases including Newcastle disease (ND) . ND is one of the major problems in village chickens in most parts of Ethiopia and the disease has become endemic in poultry population and recurs every year inflicting heavy losses [7-9].
ND induces up to 100% mortality as well as a decrease in egg production and poor egg quality. It is also highly contagious, septicemic, fatal and destructive disease which attacks chiefly chickens and turkeys usually in an acute, sometimes in sub-acute or even chronic form. Occasionally human being and even wild birds may be also infected with the virus . It is present in endemic form with frequent out break indifferent parts of the country and it remains as a constant threat to the backyard poultry . It disease occurs almost at any time of the year and widely distributed throughout the country for marked economic losses across the country despite the fact that, this has not been substantiated with laboratory investigations. Therefore, the objective of this study was to determine the prevalence of ND in Village chicken in selected Eastern Shewa zone of Oromia region, Ethiopia.
Study design and area
A cross sectional study was conducted in December 2014 to May 2015 in selected districts of East Shewa namely Bishoftu and Adama Zones of Oromia Regional State which are located south east of Addis Ababa at 45 and 99 km. The total area coverage of the zone is 11,607 km2 while the altitude of zone is ranging from 1000-3100 meter above sea levels. The mean annual rainfall of the zone is 500-1200 mm and the daily mean maximum and minimum temperatures were 30°C-42°C and 10°C-28°C respectively .
The population of this study was all backyard unvaccinated chickens in the two selected Districts. The study participants included all unvaccinated backyard chickens managed under traditional free ranging system randomly selected from the live bird market and village chicken population located within 10 kms area coverage from migratory birds` movement area.
Sample size determination
The Sample size was determined according to the formula given by Thrusfield with a 50% expected prevalence (considering no previous study was undertaken), 95% confidence level and 5% precision, sample size was calculated to be 384 . However due to logistic reasons 300 samples were collected using simple random sampling technique.
The samples (tracheal and cloacal) were collected from live chickens by inserting sterile cotton tipped swab into the trachea gently swabbed its wall and cloacal swab deeply into the vent and gently swabbing the wall of the vent. Both the tracheal and cloacal swab samples were placed in sterile separate cryovial containing 2 ml of freshly prepared viral transport media (VTM). After sampling, the specimens were labeled, placed in ice pack and transported to the molecular laboratory at National Animal Health Diagnostic and Investigation Center (NAHDIC), Sebeta, Ethiopia. Sample collection and transportation were conducted according to the standard techniques recommended by OIE .
RNA extraction: Viral RNA extraction from 300 tracheal and cloacal swabs were conducted using QIAamp viral RNA extraction kit according to manufacturer’s instruction. The sample was centrifuged briefly in order to get cell free supernatant. The supernatant was lysed by adding 560 μl of prepared buffer AVL containing carrier RNA in to 1.5 micro centrifuge tube and 140 μl of sample was added to the buffer AVL carrier RNA in the micro centrifuge tube. The solutions were then mixed by pulse-vortexing and incubated at room temperature (15- 25°C) for 10 minutes. The tube was then briefly centrifuged to remove drops from inside the lid. Then equal 560 μl of 96% ethanol was added to filtrate and mixed thoroughly and washed with 500 μl washing 1 and 2 (500 μl buffer AW 1 and 500 μl AW 2), any unwanted protein and DNA were removed. Then, 60 μl of elute solution (AVE) was added to collect the RNA. Finally, the eluted RNA was kept at-20°C (Qiagen, Inc., Gaithersburg, MD, USA).
Real-Time Polymerase Chain Reaction (RT-PCR): After RNA was extracted the Master Mix preparations were carried out in Ice bath. The reaction mixes were prepared by pipetting all the components of the Master Mixture in to 2 ml PCR tube according to the kit protocol. The reaction components were template RNA, primer solutions, dNTP Mix, and 5x QIAGEN One Step RT-PCR buffer, RNase-free water, probe and enzyme mix. Total RNA was extracted by scraping cells with RLT buffer and isolated according to manufacturers’ instructions using the RNeasy Mini Kit (QIAGEN). The control that we used were NDV vaccine strain La Sotaa and RNase free water as positive and negative controls, respectively. Then Applied Bio-systems 7500 Fast Real- Time PCR thermo cycler were used for amplification of the extracted RNA. A primer probe combination from a conserved region of the M gene APMVI F M+4100 5’-AGT GAT GTG CTC GGA CCT TC-3’, APMV-I R M- 4220 5’-CCT GAG GAG AGG CATTTG CTA-3’and Probe APMV-1M+4169 5’-FAM TTCTCT AGC AGT GGG ACA GCC TGC TAMRA -3’ was used to amplify all NDV isolates (Qiagen, Inc., Gaithersburg, MD, USA).
Data management and analysis
All the data collected were entered in Microsoft excel spread sheet and coded appropriately. The coded data was transferred in to SPSS version 20 software for analysis descriptive statics was used.
Out of 300 tracheal and cloacal swabs samples, 40 (26.7%) were found positive for ND using RT-PCR analysis with a distribution of 16.7% and 10% in Adama and Bishofitu respectively (Table 1).
The current study indicated that ND was observed as endemic disease in the study districts with the overall prevalence of 26.7% using RT-PCR. The highest prevalence in this finding was obtained from Adama Districts (16.7%) followed by Bishoftu (10%). The result has found in accordance with the previous report in Eastern Shewa who reported a prevalence of 24.2% and 14.2% in dry and wet seasons, respectively . However, the finding was lower than previous report in Debre Berhan (28.7%) and Sebeta/ Nazaret areas (38.33%) . ND prevalence of 6% and 5.9% in the wet and dry seasons respectively findings had reported from Eastern Shewa zone . On the other hand, higher report (43.68%) was also recorded in central Ethiopia among local scavenging chickens kept under traditional management system . Furthermore, the finding of the current study found higher than previous study result in southern Ethiopia of 19.78%  and in rift valley area of Ethiopia (11%) .
The overall variation of the findings might be due to variation on management system that may serve as a stress factor and favor infection. Poor sanitary conditions, continuous exposure of chickens to range conditions and wild birds, nutritional deficiencies, absence of vaccination in traditionally managed chickens, and contact of chickens of one village with those in other villages may facilitate the spread of NCD .
Furthermore, the free range management system of poultry in the study areas might also allow the uninterrupted circulation of infection among the poultry flocks. Chicken could also prone to acquire infection from wild birds and local open markets where huge numbers of chicken are gathered which might also serve as continuous foci of infection .
The current study indicated that ND is endemic in village chicken of Eastern Shewa of Oromia region. Furthermore, important information has been provided by the study on epidemiological distribution of the disease in the area which alerts to look existence of the disease in other parts of the country and then to development effective prevention and control measures. This may indicate importance of implementing surveillances and bio-security practices in live poultry markets.
Based on the above concluding remarks the following recommendations were forwarded. Further studies should be conducted to understand and identify the circulating strains and pathos-types of ND virus with their dynamics in the study area. Strategic control and prevention measures should be implemented to reduce the impact of this disease on poultry production in the area. Extension work should be conducted to create awareness in flock owners about the transmission and prevention measure of the disease.
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