Molecular Investigation for Matrix Gene of Newcastle Disease Virus in Non-vaccinated Village Chickens in Central Rift Valley of Oromia, Ethiopia

Background Newcastle disease (ND) is a major infectious disease of poultry caused by a virulent 1. It is a major threat to the industry in many the world including Ethiopia. Newcastle Disease Virus (NDV) is an enveloped, non-segmented, single-stranded negative-sense RNA virus with a helical morphology whose genome has six open reading frames (ORF) which encode for the following proteins: nucleoprotein (NP), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin-neuraminidase (HN) and RNA-dependent RNA polymerase (L). The aim of this study was to detect matrix gene (M-gene), for NDV by molecular tools and identify its risk factors in non-vaccinated village chicken in Central Rift Valley of Oromia, Ethiopia. Methods A total of 84 pooled in five swab samples from 420 cloacal and tracheal chickens were sampled and RNA was extracted from the 84 pooled samples to carry out real-time quantitative polymerase chain reaction (qRT-PCR). A real-time reverse transcriptase polymerase chain reaction (rRT-PCR) along with the quantification was also done for 10 positive qRT-PCR samples that were having high concentration of viral load Ct. value. Results Out of the 84 pools of five swab samples tested for M-gene using qRT-PCR, 16.7% (14/84) samples were detected which included 13 positives and 1 negative for NDV. The prevalence of ND in males was found to be 16.10% and that in females was 14.67%. Although the overall ND prevalence was 15.48% (13/84), the highest score was recorded in Adama, 42.86% (6/14), and no positive case was detected in Bote and Bishoftu (p <0.05) while intermediate scores were obtained from Batu, Arsi-negelle and Shashemene. six open reading frames (ORF) which encode for the following proteins: nucleoprotein (NP), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin-neuraminidase (HN) and RNA-dependent RNA polymerase (L). ND is caused by Newcastle disease virus (NDV) which is a highly infectious agent capable of causing high mortality in non-vaccinated chickens. The sub clinical forms of ND in vaccinated and/or NDV exposure flocks have synergistic effect with other bacterial or viral infections

3 segmented, single-stranded negative-sense RNA virus with a helical morphology whose genome has six open reading frames (ORF) which encode for the following proteins: nucleoprotein (NP), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin-neuraminidase (HN) and RNA-dependent RNA polymerase (L). ND is caused by Newcastle disease virus (NDV) which is a highly infectious agent capable of causing high mortality in non-vaccinated chickens. The sub clinical forms of ND in vaccinated and/or NDV exposure flocks have synergistic effect with other bacterial or viral infections [3,4].
In Ethiopia, like other African countries, ND causes devastating losses to the poultry industry [5]. In backyard poultry, the disease is endemic but recurs as frequent epidemic outbreaks with high mortality and thus affects the livelihoods of poor rural households who depend on poultry for food and income [6]. Backyard poultry management favors the existence and spread of diverse NDV strains by allowing free interaction of different poultry species and wild birds as well as the frequent introduction of birds from markets [7]. In many countries, matrix gene and fusion gene based qRT-PCR assays are often used as standard methods for NDV screening and pathotyping directly from clinical samples [8].
The qRT-PCR assay is not only faster and less cumbersome than the conventional diagnostic techniques, but also provides equal or even greater sensitivity of virus detection than the gold standard virus isolation method [9]. The choice of matrix gene detection in NDV screening is due its highly conserved nature in the NDV genome where matrix gene assay is able to detect most of NDV isolates [10]. Despite the wide spread problem of ND in Ethiopia in general and Oromia in particular [7,11,12], there is limited information about the genetic profile of NDVs circulating in backyard poultry in Ethiopia. Therefore, the aim of this study was to detect the existence of Newcastle Disease virus by molecular tool in non-vaccinated village poultry in central rift valley of Oromia, Ethiopia.

Materials And Methods
The study was conducted in selected Central Rift Valley areas of East Shoa (Bishoftu, Adama, Bote, Batu) and West Arsi Zone (Shashemene and ArsiNegelle) of Oromia Regional State (Fig. 1). East Shoa Zone extends between 70'33'50" N -90'08'56" N and from 380'24'10" E -400' 05' 34" E. The temperature of the area ranges from 10 0 C in uplands to over 30 0 C in rift valley depressions with the 4 mean temperature of 20 o C. Since the large portion of the zone is located along the rift valley system, rainfall varies from 600 mm to 1000 mm with mean annual rainfall of 816 mm . The livestock   population of East Shoa zone is estimated to be 1,090,091 cattle, 319,598 sheep, 568,761 goats,   10644 horse, 7039 mules, 284, 583 donkey, 6818 camels, 14627 beehives [14].

Study Populations
The study populations were non-vaccinated village chickens from East Shoa and West Arsi Zones in Central Rift Valley of Oromia regional State, Ethiopia.

Study Animals
Study animals were non-vaccinated village chickens from randomly selected districts in East Shoa and West Arsi zones. Village chickens were sampled in respective of districts, sex and age. The age was determined based on history from the owners as young 3-6 months, adult 7-16 months and older more than 16 months [15].

Study Design
A cross-sectional study was conducted in non-vaccinated village chickens found in the study areas for the detection of Newcastle Disease during the period of January to May, 2019.

Sample Size Determination
Sample size was calculated based on the prevalence of 38.4%, as previously reported by [11], using 95% confidence level and 5% marginal error. A formula according to [16] was used to determine the required sample size as indicated below: Where; n = Sample size, Z = Statistic for a level of confidence, d 2 = Required absolute precision, and P exp = Expected prevalence. Therefore, at 95% confidence level, the sample size was: Hence, a total of 364 non-vaccinated village chickens were required to be sampled. However, 420 domestic birds were randomly selected at poultry markets and villages in the study areas to increase the precision of the study. To calculate the prevalence the following formula was used:

Sampling and Method of Data Collection
A total of 420 non-vaccinated and healthy live chickens were sampled using random sampling technique from poultry markets and villages by equal distribution in the study areas on consecutive market days. Tracheal and cloacal swab samples 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 (2 ml volume) containing 500 µl of freshly prepared viral transport media (VTM). During sampling, all the samples were labeled, placed in ice pack (4 °C) and transported to molecular laboratory of National Animal Health Diagnostic and Investigation Center (NAHDIC), Sebeta, Ethiopia and stored at -80 °C until processing. Sample collection and transportation were conducted according to the standard techniques recommended by OIE [17].

RNA extraction
Viral RNA extractions from tracheal and cloacal swabs were conducted using Qiagen®viral RNA Mini kit according to manufacturer's instruction (Qiagen, 2014) as follows: -Swab samples collected with 6 viral transport medium were centrifuged briefly at 8000 rpm for 1 minute in order to get cell free supernatant. Then, 140 µl of the supernatant were lysed by adding 560 µl of buffer AVL containing carrier RNA. After ten minutes' incubation at room temperature, the sample was mixed and 560 µl of 96% ethanol was added to filtrate and again mixed thoroughly. Samples containing 630 µl of lysis buffer and absolute alcohol were transferred for binding to mini spin column and centrifuged at 8000 rpm for 1minute and old collection tube was replaced by a new collection tube. A 500 µl wash buffer (AW1) was added and centrifuged at 8000 rpm for 1 minute and a new collection tube was used. Again, a 500 µl wash buffer (AW2) was added and centrifuged at full speed of 14,000 rpm for 3 minutes in order to remove any unwanted protein and others. Again, new collection tube was used and centrifuged at full speed of 14,000 rpm for 1 minute without adding anything. A new and sterile 1.5 µl of micro centrifuge tube was used followed by adding 60 µl of elution buffer (AVE) to mini spin column and incubated for 3 minutes at room temperature. Finally, the eluted RNA was kept at -80 °C for storage.

Real -Time Quantitative Polymerase chain reaction (qRT-PCR)
One step qRT-PCR for conserved region of M-gene assay was performed using an Applied Biosystems isolates and RNase free water as positive and negative controls, respectively were used. After placing tubes, the thermal cycler or PCR machine was programmed for reverse transcription (RNA to cDNA) step and incubated for 30 min at 50 °C, followed by 15 min at 94 °C. The cycling conditions consisted 35 cycles and each cycle were having 30 second for denaturation at 94 °C, 1 min for annealing at 55 °C, and extension at 68 °C for 1 min and additionally one cycle at 68 °C for 7 min final extension [18].

Gel electrophoresis
A 2 µl of loading dye and 8 µl of amplified PCR products were mixed up and loaded in to separate lane 8 (wells on the gel) and the gel was run for 45-60 minutes. The gel was placed under UV transilluminator or Gel doc machine that was connected to the computer with its respective software (Image Lab software version 5.0) for imaging. Finally, the presence or absence of band were observed and the image was captured.

Quantitative real-time PCR (qRT-PCR) based molecular detection of NDV in village chickens
The current findings in village chickens for pooled five swab samples obtained in the study area for M gene qRT-PCR test with their Ct. values are presented in Table 1  The present findings of both positive and negative controls ( Figure 2) in non-vaccinated village chickens swab samples pooled five based on qRT-PCR test for M gene of NDV was indicated ( Figure 3) when read from Applied Biosystems 7500 fast real time PCR thermo cycler.

Prevalence of ND in village chickens using qRT-PCR for M gene test by Age and Sex
The prevalence of ND based on M gene assay was higher in male (16.10%) than female (14.67%) chicken even though there was statistically no significance difference (p > 0.05) by sex ( Table 2). On the other hand, the prevalence of ND was higher in Adult (16.59%) but lower in old chicken (11.11%) as shown in Table 2.

Prevalence of ND using qRT-PCR for M gene test in village chickens in the study districts
The overall ND prevalence was 15.48% (13/84) in the study districts where the highest score was from Adama (42.86%) while none was detected in Bote and Bishoftu (Table 3). There was statistically no significance difference (p > 0.05) among the study districts. The current qRT-PCR test results also revealed a higher detection rate for M-gene from tracheal swab samples (TS), 21.43% (9/42), as compared to cloacal swab samples (CS), 9.52% (4/42), collected from village chicken from the study areas (Table 4).

rRT-PCR-based molecular detection of NDV
Ten samples were tested by rRT-PCR assay for the detection of M-gene. Each band represents the extracted RNA from ten samples (Figure 4). Detection of M gene in the present study by rRT-PCR ( Figure 4) is a demonstration of infection by the virus in these chickens.

Test agreement result between qRT-PCR and rRT-PCR detection technique
The kappa test agreement between qRT-PCR and rRT-PCR were having a value of 0.615 which indicates substantial result agreement between the two raters ( Table 5).  (Fig. 4) is a demonstration of infection by the virus in these chickens. The occurrence of positive result supports the use of the technique to identify NDV. Positive samples obtained in this study can further be subjected to pathotyping and phylogenetic studies which is an advantage that goes beyond 13 the previous serological study. Even though, the distribution of the disease differs among the study areas in Eastern Shoa Zone using qRT-PCR analysis with statistically significance difference (p < 0.05), [12] reported an overall prevalence of 26.7% by using rRT-PCR for fussion (F) gene assay from Adama and Bishoftu which is in a close resemblance with our current findings (21.42%) when it was analyzed only for Adama and Bishofitu. In addition, using a rRT-PCR for M gene study conducted by [11] in three selected districts (Batu, Bishoftu and TikurWuha) in rift valley areas revealed an overall prevalence of 8.4% which is less than the current finding and this could be due to the small area coverage. Even though the negative results from the qRT-PCR test in Bishoftu and Bote seem strange, a molecular and serological study conducted for NDV from Iran in non-vaccinated village chicken also showed negative result on qRT-PCR for M gene assay but positive reaction on HI test [19]. They concluded that the serological finding may imply a circulation of NDV antibody on the native chickens in the area but by molecular test no virus was detected since there was no occurrence of active viral infection.
The result of the present study (15.48%) was higher than the findings of other researches done by serological detection method in Eastern Shoa and Kersana Kondaltity district with an overall seroprevalence of 5.9% and 5.6%, respectively [7,20]. In another serological study conducted in selected areas in rift valley an overall sero-prevalence of 5.6% was reported [15] and 11.6% [11] from which one can discern out that the results under the present study, molecular tool, outweighs the results of the former studies by serological (ELISA) method. The overall discrepancy of the findings might be due to variations on management system that may serve as a stress factor that favor infection or the diagnostic tools used.
The present higher proportion (42.86%) of qRT-PCR positive result for NDV in chickens from Adama district might indicate chickens have been previously infected by the non-virulent strains or survived outbreaks of the virulent NDV strains [21]. Moreover, the detected high prevalence of ND in this study may mean that natural infection could have occurred since all the chickens were non-vaccinated.
However, clinical signs of ND were not observed on the examined chickens during sample collection.
Furthermore, the higher prevalence recorded in Adama district cannot be attributed to more chicken 14 had been sampled (70 chicken sampled) compared to other study districts (70 chicken sampled), because as the number of sample size increase the probability of having higher prevalence's also increase. The present study also revealed significant variation with almost closer agro-ecology between East Shoa and West Arsi Zones.
The findings in village chickens of the present study also found out to be higher than the results reported from North Eastern Coast and Amazon Biome of Brazil from 1022 cloacal/tracheal samples collected from domestic and wild birds tested by qRT-PCR targeting the M gene segment of APMV-1, 7 (0.7%) were positive [22]. These variations could have been attributed to seasonal differences during sampling periods, geographical location and type of birds employed for the studies.
In the present study a higher detection rate by qRT-PCR for M gene assay was obtained from pooled tracheal 21.43% (9/42) than cloacal 9.52% (4/42) swab samples with statistically insignificance difference (p > 0.05). This is in agreement with the finding of [7] who reported higher positivity of tracheal than cloacal pooled swab samples by rRT-PCR for F gene assay speculating the presence of viscerotropic velogenic virulent NDV in the study areas. On the other hand, Haque and his colleagues detected NDV from pooled oropharyngeal and cloacal swabs and suggested that the chickens are in the early or advanced stage of the disease since NDV is transmitted through aerosol and ingestion or the chickens are on the shedding stage of the infection [23].
The difference in the prevalence between adult, young and old age was statistically significant (p < 0.05), which agrees with the finding of [24] who stated that the young had a significantly lower ND prevalence than the adult. Furthermore, the result of the current study corroborates with the findings of [25]

Consent for publication
Not applicable

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on request.
analysis, and drafted the manuscript. RB and AA participated in the optimizing laboratory works. All authors read and approved the final manuscript.