The control of ND in village chickens can make a vital contribution to the improvement of household food security and poverty reduction in Ethiopia. Intensive commercial poultry farmers in Ethiopia vaccinate chickens routinely, but village chicken farmers do not . In the current study, five cereal grain species, in 3 different forms, were evaluated for suitability and efficacy as a carrier for the NDI2 vaccine as a way forward for developing suitable vaccine delivery system for village chicken production system. The carrier grains have been shown to adsorb the virus from an aqueous suspension and release it in a viable form in the digestive tract of chickens . However, the virus adsorption and releasing capacity of cereal grains varies among grain species and forms of preparation . Grain based vaccine efficacy could be assessed via (i) monitoring sero-conversion, (ii) post vaccination challenge and (iii) survival rate as recommended by Spradbrow [13, 21, 22]. In this study, five different cereal grains were evaluated for their suitability and efficacy as vaccine carrier using the above mentioned three assessment methods.
Maternal HI antibody titer
At day old, chicks included in this study have HI antibody titer above log2
3. Such high maternal antibody titer in the baby chicks is deleterious to vaccination . Thus, we waited until it declined to log2
1.5 titer at 14 days to overcome the risk of its interference with the vaccine. In line with this, it has been well established that chicks from immunized parents possess high level of maternal antibody which protects the chicks against virulent virus and interferes with vaccine antigens [23, 24].
Monitoring serological response using HI antibody titer
An HI titer of ≥ log2
3 following vaccination has been considered protective against virulent ND virus. HI titers lower than log2
3 have been associated with lower levels of protection [13, 20]. The results of the current study showed that administration of 2nd and 3rd booster vaccination significantly and progressively increased HI antibody titer in all treatment groups except the naïve control. Furthermore, different grains induced different level of HI antibody titer. This implies the presence of inherent variation in virus carrying capacity of different grains. This is an opportunity to screen grains of different species and varieties. Interestingly, treating grains (either cracking or parboiling) increased their efficacy as vaccine carrier, evident by induction of higher HI antibody titer than that was induced by untreated form. Similar results have been reported in Nigeria by Olabode  as to the efficacy of treated grain particularly maize compared to untreated grain. Grains have been known to contain tannins, anthraquinone, cardiac glycosides and alkaloids. Some of these chemicals have been shown to have antiviral properties [22, 26]. The higher HI titer induced by treated grains than untreated ones could be due to the fact that cracking grains increase the surface area of the grains to adsorb the vaccine virus [10, 13, 14, 18, 22, 25, 26]. Likewise, parboiling might destroy the antiviral factors from the seed of the grains [22, 26]. Hence, both cracking and parboiling grains would induce better HI antibody titer via adsorbing and releasing live virus in the gut. However, inconsistent results were observed for sorghum in which cracked, untreated and parboiled sorghum induced HI titer of ≥ log2
3 in 20 %, 46.7 % and 60 % of chickens, respectively, after the 3rd booster vaccination.
This implies that repeated vaccination induces progressively higher HI titer that could correspond to high levels of protection. Moreover, different grain species as well as their preparations in different forms have induced different level of HI antibody titers. Treatment of grains (parboiling or cracking) renders the grains suitable for vaccine carrier for oral vaccination as evident from their corresponding improved antibody response. Higher HI antibody titer corresponded to higher protection level for most grain carriers in the order of parboiled, cracked and untreated grains. This, however, is not the case for sorghum particularly untreated form was found to be better than cracked form in HI titer and post challenge survival. Exceptionally, the parboiled and untreated sorghum induced higher HI titer than the cracked sorghum. Cracking in sorghum might have released anti-viral factors (tannins) and enzymes that probably inactivated the virus. To this end anthraquinone, alkaloids and cardiac glycosides were reported to be abundant in sorghum .
Despite low HI antibody titer induced by some grains (sorghum), unexpectedly higher survival % was observed following the challenge. This perhaps highlights the weak correlation between low HI titer and protection for sorghum and the difficulties of using serum antibodies to determine protection to respiratory pathogens [27, 28]. In addition to serum antibody, secretory antibody (IgA) at mucosal surfaces and cell mediated immunity are thought to play a role in resistance to challenge . In general, however, this study showed a negative correlation between mean HI titer and mortality % at the group level (Fig. 3).
Other than the treatment (grain type for vaccine delivery) that of the pen effect has been handled by grouping of the chickens by randomization. However, pens are not replicated due to shortage of space in the experimental house and finance to account for the pen effect on immune response. We believe that the variance among pens is generally less than the variance of chickens within pens due to treatment effect.
Protection assessment following artificial viral challenge
In the current study, intramuscular route of the breast muscle was used to challenge the chickens. Intramuscular route was preferred as it allows birds to receive equal doses of the challenge virus. Water, cracked maize and parboiled barley were found to be carriers of NDI2 vaccine than the others. They protected 100 % (15/15) of the chickens against virulent challenge with ND strain compared to 20 % protection in the naïve group (3/15). In agreement with this results of full protection (100 %) using the NDI2 vaccination via drinking water and parboiled barley was previously reported in Ethiopia . Another similar heat stable vaccine (NDV4) coated cracked maize has also been reported to induce high levels of protection in Nigeria  which is similar to our NDI2 results. The 80 % protection achieved using parboiled sorghum currently is, however, in disagreement with previous results report in Ethiopia , Nigeria  and Tanzania .
It is widely accepted that the recommended protection level of ND vaccination is 80 % . However, we have achieved 100 % protection using NDI2 in water, cracked maize and parboiled barley at on-station condition.
The survival rate for the chickens vaccinated with untreated and cracked millet is 60 %, but 73.3 % for the parboiled millet. Our current finding is comparable to the 70 % survival rate reported elsewhere among chicken vaccinated with NDI2 using parboiledand broken millet .
The chicken survival rate is 71.4 % for cracked wheat, 80 % for untreated wheat and 85.7 % for parboiled wheat group. The untreated barley as carrier for NDI2 in the current study had 60 % survival rate. This is far lower than the previous work in Ethiopia  and what was reported elsewhere with 100 % protection . This variation in serological response between and within grain forms could be explained by (i) differences in the contents of anti-viral factors in the grains, (ii) variability in vaccine carrying capacity among different grains, (iii) difference in agro-ecology and soil characteristics that can have different effect on physico- chemical characteristics of the grains. Oakeley  suggested that grains grown in different agro-ecology and on different soil characteristics tend to vary in their vaccine virus carrying capacity due to variation in the grains’ physico- chemical characteristics, especially their surface properties and chlorine content .
In general, parboiled grains, followed by cracked ones, induced higher serological response and protection level than intact (untreated) grains. Heating, soaking, washing and cracking grains might be useful in developing a successful vaccine carrier feed. Similar findings have been reported from other countries [17, 35]. Cracked maize and parboiled barley are found to be better vaccine carriers under Ethiopian context. Our promising finding on wheat was consistent with Spradbrow . Thus, cracked maize, parboiled barley or parboiled wheat should be the base for a large scale grain screening and oral based ND vaccination program. They can be used widely as carrier for oral NDI2 vaccination. However, it should be noted that the protection level of the grain based NDI2 vaccine varies under laboratory conditions i.e. > 90 % protection  and under real village conditions i.e. < 60 %  and with vaccine delivered by farmers . This signals the necessity of pilot field trial at village level to evaluate the results of the current on-station study at real village conditions.
Durability of the virus in the grains and at room temperature
In the current study, the vaccine virus coated on grains was highly immunogenic after 6 h of exposure to room temperature; hence it could be used to vaccinate village chicken against ND. We haven’t measured the upper limit of the time when it is still efficacious in order to enable central vaccine production and then distribution to rural villages. A very important condition for successful development and use of any chosen feed as vaccine carrier is the ability to allow firm binding or adherence of the coated vaccine virus without interfering with the survival of the vaccine virus. In this regard different results have been reported from different countries. According to Tu et al. , the NDI2 in grains has substantial infectivity and induction of immunity in chickens under laboratory conditionsafter storage for 17 days and in village conditions after storage for 21 days. Echeonwu et al.  reported that the virus coated feed without additive remained stable and immunogenic for 3 weeks (millet); 3.5 weeks (sorghum) and 5 weeks for maize at room temperature. Nassir et al.  recovered viable vaccine virus after 14 h at room temperature on parboiled barley.