2.1 Measurement of chicken embryos weight from 6th do 20th day of development.
Every day from the 6th to the 20th day of the incubation period, 28 ECEs from a total of 420 were used in the project. All ECEs were opened and embryos were weighed. Each egg’s weight ranged from 55g to 60g, both in the test and control groups beside the day of experimentation. Embryos inoculated with HAdV-D36 showed significant differences in weight between test and control groups (Figure 1). The greatest differences between the infected and control groups were observed for inoculation into the yolk. In this group, weight differences between infected and control embryos up to day 12 of development varied between 0.14g and 1.09g. In older embryos, the greatest deviations were noted on days 15 (3.51g) and 18 (5.41g).
In the case of infection into the allantoic cavity, the differences were not as significant, ranging between 0.1g and 3.33g (day 20 of development). On days 6,10,17 and 19 the weight of control ECEs was even higher than that of infected ECEs.
Detailed data of the embryos infected in the yolk sack and in the allantoic cavity are presented in the Table 1.
Table 1. Mean body weights with standard deviation of embryos used in tests.
The weight of the tested embryos was given in grams
|
Yolk- sack inoculation
|
Allantoic cavity inoculation
|
Day
|
Tested group
|
Control group
|
Tested group
|
Control group
|
6
|
0.38 ± 0.12
|
0.15 ± 0.04
|
0.24 ± 0.10
|
0.34 ± 0.02
|
7
|
0.55 ± 0.17
|
0.57 ± 0.01
|
0.47 ± 0.14
|
0.40 ± 0.11
|
8
|
1.23 ± 0.38
|
0.63 ± 0.08
|
1.47 ± 0.51
|
1.16 ± 0.26
|
9
|
1.93 ± 0.39
|
1.50 ± 0.38
|
2.15 ± 0.21
|
2.03 ± 0.43
|
10
|
2.28 ± 0.43
|
1.93 ± 0.13
|
2.26 ± 0.45
|
2.51 ± 0.47
|
11
|
3.06 ± 0.65
|
2.39 ± 0.23
|
3.82 ± 0.90
|
3.78 ± 0.33
|
12
|
4.41 ± 1.32
|
3.12 ± 0.54
|
3.78 ± 1.00
|
4.58 ± 0.15
|
13
|
7.33 ± 2.04
|
6.13 ± 0.83
|
7.30 ± 1.44
|
5.52 ± 0.12
|
14
|
9.64 ± 2.37
|
8.84 ± 2.78
|
10.86 ± 0.46
|
10.86 ± 2.00
|
15
|
14.48 ± 2.25
|
10.97 ± 2.12
|
15.93 ± 0.53
|
13.20 ± 1.60
|
16
|
17.12 ± 4.29
|
16.12 ± 1.51
|
17.87 ± 0.33
|
16.82 ± 0.62
|
17
|
20.91 ± 0.65
|
18.62 ± 2.91
|
21.66 ± 0.50
|
22.63 ± 3.62
|
18
|
26.04 ± 1.13
|
20.63 ± 2.65
|
24.08 ± 0.39
|
23.52 ± 3.62
|
19
|
27.59 ± 2.01
|
26.45 ± 2.04
|
26.19 ± 0.19
|
27.66 ± 0.98
|
20
|
29.13 ± 2.32
|
27.91 ± 1.83
|
34.51 ± 0.30
|
31.18 ± 0.18
|
2.2 Histopathological examinations
Whole embryos (up to 10 days) and livers taken from infected embryos were subjected to histopathological examination in order to observe the changes that caused HAdV-D36 infection in the tissues of the examined organism.
Very clear changes in embryonic development were observable as early as 48 hours after HAdV-D36 infection. In general, accelerated proliferation of whole-body cells was observed. Mesenchyme of the dermis in 7-day-old embryos after infection with the virus, was in a significantly more advanced stage of development compared to control. This early connective tissue contained more collagen fibres and was more optically dense then in the control group. Both the liver and the kidney were characterised by accelerated growth and increased angiogenesis.
On day 8, embryos infected with HAdV-D36 had cartilage and intervertebral muscles as well as the liver and kidneys that were all much more advanced in development, than those observed in the control group.
After 4 days of observation of infected embryos, it was possible to conclude that their development was about 2 days more advanced than the development of the control group.
On the 9th day of development, in infected ECEs, it was possible to notice the initial stages of cartilage tissue remodelling, the formation of joints in the limbs or the initial phases of tissue organisation in the eye. The liver and kidneys were still enlarged and more advanced than in the control group. No increased number of adipocytes in the chicken body was observed.
5 days after infection, the initial phases of respiratory development were observed in embryos, including clearly forming lungs, which is a unique picture in birds at this stage of development because the lungs usually begin to develop in the later days of the embryo's life.
On the 13th day of embryo development, in embryos infected with HAdV-D36, a glandular period in developing lungs was noted as well as the first drops of fat appearing in the liver were observed. Until the 20th day of embryo development, in infected embryos, we observe a constant increased growth of all organs, with particular emphasis on the liver and kidneys, which are characterised by a significantly increased size every day, however, on day 15, the first inflammatory changes in the liver appear, which in the following days progress giving a picture of extensive limocytic-macrophage infiltrates both in the liver parenchyma and in the vicinity of blood vessels.
On day 20, we observe a pronounced inflammation of the liver with the initial stages of a very pronounced liver lipidosis. Compared to the infected group, the embryos in the control group developed at a much slower rate. The amount of fat in the cells and tissues remained at a normal level, in some cases, in the control, could also be observed steatosis of some organs, however, the absence of any accompanying cytopathic effects in the tissues allows the determination their individual characteristics, not resulting from viral infection (histopathological pictures are presented as a Figure 2, 3 and 4).
Histopathological analyses are consistent with the measurements of the weight of the examined embryos, however, thanks to the conducted research, we can conclude that the increased mass of the examined embryos does not result from an increase in the number of fat cells in the tissues of infected embryos, but from increased cell division in all tissues of the body, which resulted in an increase in the number of cells in the tissues and thus all organs presented accelerated development and increased size. The observed changes appear to occur through a previously unknown mechanism induced by HAdV-D36 infection.
2.3 Real Time PCR
The real time PCR showed that the highest Cq (quantification cycle) concentration of HAdV-D36 genetic material can be found on day 20 and the lowest on day 17 for both tested groups (Figure 5 ). Despite the low Cq value, the viral DNA was present in each of tested samples from infected embryos.
2.4 Statistical analysis
By comparing the results of individual days and the average weight gains obtained from a group of 7 chickens on each day, a correlation plot was prepared (Figure 6). It was found that the polynomial relationship of second degree is the best to describe the situation. It was observed that between days 12 and 13 (the area marked in the figure with a grey, dashed line) (Figure 7), there was a change in weight gain in all study groups (greater gains than before are observed after day 12). For the purposes of further analyses, a change in the angle of inclination of the results in relation to the axis was noticed and two correlation analyses were carried out: the first one only from day 6 to 12 and the second one covering days from the 13th to the 20th. The results are presented in the 7th figure (Figure 7).
On days 6 to 12, all the obtained linear correlations were statistically significant (p <0.001), regression coefficients showed a very strong positive correlation, and it was noticed that the coefficient b in the group infected in the allantoic cavity and the group infected in the yolk sac were almost equal (0.6437 and 0.6483). In the case of the control group inoculated in the yolk sack, a much lower value of b (0.4936) was obtained, and in the control, group inoculated into allantoic cavity, this value was higher (0.7429). Detailed results are presented in the table (Figure 8). Then, comparisons were made after day 12 (days 13 to 19). From the 13th day onwards, we obtained in all cases much greater slope coefficients (b) than for the cases up to day 12, which indicates greater mean weight gains compared to the period preceding day 12. The same tendency can be observed as in the previous days, i.e., for the experimental groups infected in the allantoic cavity and infected in the yolk sack, we obtained similar slope parameters of the lines (3.7137 and 3.3257 respectively), with a stronger increase in mass in the group infected in the allantoic cavity. This tendency is also maintained in the control groups, i.e., we expected a greater weight gain in the allantoic cavity control group as compared to the yolk sack group.