Comments to reviewer:

 
1. Methods need to be set up with a more consistent structure that is closely followed in the results
and discussion sections. Suggested structure:
a. Viruses, dose and challenge methods
b. Birds, strain, housing,diets,and isolation
c. Clinical sigs, production and egg quality
d. Autoposy, histoloogy, microscopy
e. Serology
f. Viral isolation??
g. PCR methods and developments
h. Statistical methods
This has now been updated as per reviewer’s suggestion
 
2. In the methods and results section I was confused about the role of the previous
experiment AECL no. 06/06 UNE 85 verses experiment 1 and 2 in the new project AECL 08/14
UNE.
The project AECL 08/14 was an extension of earlier project AECL no. 06/06 UNE 85
(which was regarding development of real time PCR assay). The samples collected for
histology, electron microscopy and egg quality parameters were studied during AECL
08/14. Experiment 1 was conducted exclusively with unvaccinated hens for K.
Chousalkar’s study (which was not funded earlier by AECL). Oviduct samples from K.
Chousalkar’s study (Experiment 1) were used to quantify the virus load by real time PCR.
This is also mentioned in the text. In Experiment 2, 15 unvaccinated challenged hens were
maintained throughout the experiment to perform studies on egg and egg shell quality
parameters (Please see table 2.1 and 2.2)
3. In particular, Experiment 1 in Project 08/14 UNE is not clearly reported! Is this data from AECL
no. 06/06 UNE 85 .
Please refer to the above answer. The details of experiments are given in tables 2.1 and
2.2.
4. If Experiment 1 and 2 are described in the methods these experiments should be reported in the
results sections under  distinct headings.
5. It appears in the results section that the experiment 2 is divided into two sections for the reporting
3.1 and 3.2.and I am not clear on experiment 1.
6. Perhaps this could be made clearer in the methods section.  Methods section could describe
experiments 1, 2a and 2b or experiments 1, 2,and 3, and these should be precisely reported
against in the results section. 
For all above three points please refer answer to question 2.
7. Another major point is that in the complex data from experiment 2 presented in tables(3.7 - 3.10,
3.15-3.18) it is difficult to interpret, particularly the vaccine impacts. Not all the treatments are
illustrated,   VK, VN, VT,  VA, VV are missing from the tables.
All the tables have now been converted in to graphs and respective results are included.
8. I appreciate the that the statistical effects of the vaccine treatments are listed, but the absolute
values are not listed for ( VK, VN, VT,  VA, VV)  and this makes it difficult to relate the text in the
results section to the data. Clearer links between the data in the tables and the text are required.
All the tables have now been converted in to graphs and respective results are included.

9. I suggest that distinct graphs be included that are derived from the data in the tables( 3.7-3.10,
3.15-3.18) to illustrate the main and most significant challenge, vaccine responses. This could
involve graphs presenting means and standard errors over time comparing challenge treatments
or vaccinations.
 All tables are converted to graphs.
10. I believe that the ISA Brown was used in the experimentation? If this is correct? it should be
clearly stated and some analysis of the albumen quality presented on an "individual bird" basis
both before and after challenge and vaccination. The data could be presented in frequency
distributions for albumen height or haugh units. This is important because of the practical issues
of albumen quality and watery whites.
Julie could you please comment on this ?
11. The use of the T and N1/88 viruses should be justified on the basis of prevalence or relative
pathogenicity to assist in ensuring that the experimental model is relevant to industry and that the
experimental model reflects the real situation "On Farm".  Another point worthwhile highlighting is
the adequacy of the "antibody response to vaccination” to assist in experimental standardisation
to industry practice.
This has now been mentioned in the text.
12. The methods also describe the collection of blood for haematocrit and electrolyte analysis, but
this data is not reported in the results section?
This parameter was not committed in the project hence deleted.