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. Out of the 84 pooled five samples tested by qRT-PCR for M gene, 14 (16.7%) samples were detected for NDV where 13 (15.5%) were identified as positive for NDV and 1 is having a Ct. value of 36.38 considered as negative for NDV in non-vaccinated village chickens.
Table 1: M gene qRT-PCR test Ct. values for NDV detection in non-vaccinated village chickens in Central Rift Valley of Oromia, Ethiopia.
Sample ID
|
Study areas
|
Swab type
|
Ct. value
|
A1
|
Adama
|
Cloaca
|
24.82
|
A2
|
Adama
|
Cloaca
|
26.42
|
A3
|
Adama
|
Trachea
|
30.82
|
A4
|
Adama
|
Trachea
|
33.87
|
A5
|
Adama
|
Trachea
|
27.20
|
A6
|
Adama
|
Trachea
|
31.23
|
A7
|
Adama
|
Trachea
|
36.38
|
AN1
|
Arsi Negelle
|
Cloaca
|
23.41
|
AN2
|
Arsi Negelle
|
Cloaca
|
34.85
|
AN3
|
Arsi Negelle
|
Trachea
|
28.78
|
BA1
|
Batu
|
Trachea
|
31.50
|
S1
|
Shashemene
|
Trachea
|
28.96
|
S2
|
Shashemene
|
Trachea
|
24.39
|
S3
|
Shashemene
|
Trachea
|
23.68
|
Mean
|
28.41
|
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.
Table 2: Prevalence of Newcastle disease in village chickens by Sex and Age in the study area.
Risk factors
|
Number examined
|
Number positive
|
Prevalence %
|
p-value
|
Sex
|
|
|
|
|
Male
|
236
|
38
|
16.1
|
> 0.05
|
Female
|
184
|
27
|
14.67
|
|
Age
|
|
|
|
|
Young
|
146
|
23
|
15.75
|
< 0.05
|
Adult
|
211
|
35
|
16.59
|
|
Old
|
63
|
7
|
11.11
|
|
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.
Table 3: Prevalence of Newcastle disease in village chickens in the study Districts.
Districts
|
No. examined in pool of five
|
No. Positive
|
Prevalence %
|
p-value
|
East Shoa Zone
|
|
|
|
|
Adama
|
14
|
6
|
42.86
|
|
Batu
|
14
|
1
|
7.14
|
< 0.05
|
Bishoftu
|
14
|
0
|
0
|
|
Bote
|
14
|
0
|
0
|
|
West Arsi Zone
|
|
|
|
|
Arsi Negelle
|
14
|
3
|
21.43
|
> 0.05
|
Shashemene
|
14
|
3
|
21.43
|
|
Total
|
84
|
13
|
15.48
|
|
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).
Table 4: qRT-PCR test result for M-gene detection rate from swab samples from study area.
Swab type
|
No examined
|
No positive
|
Detection rate %
|
p-value
|
Trachea swab
|
42
|
9
|
21.43
|
> 0.05
|
Cloacal swab
|
42
|
4
|
9.52
|
|
Total
|
84
|
13
|
15.47
|
|
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).
Table 5: Kappa test agreement between qRT-PCR and rRT- PCR of Newcastle disease in the study area.
qRT-PCR * rRT-PCR Cross tabulation
|
|
rRT-PCR
|
Total
|
Negative
|
Positive
|
qRT-PCR
|
Negative
|
Count
|
1
|
0
|
1
|
% within qRT-PCR
|
100.0%
|
0.0%
|
100.0%
|
% within qRT-PCR
|
50.0%
|
0.0%
|
10.0%
|
Positive
|
Count
|
1
|
8
|
9
|
% within qRT-PCR
|
11.1%
|
88.9%
|
100.0%
|
% within rRT-PCR
|
50.0%
|
100.0%
|
90.0%
|
Total
|
Count
|
2
|
8
|
10
|
% within qRT-PCR
|
20.0%
|
80.0%
|
100.0%
|
|
% within rRT-PCR
|
100.0%
|
100.0%
|
100.0%
|
|
Symmetric Measures
|
|
Value
|
Asymptotic Standard Errora
|
Approximate Tb
|
Approximate Significance
|
Measure of Agreement
|
Kappa
|
.615
|
.337
|
2.108
|
.035
|
N of Valid Cases
|
10
|
|
|
|