BackgroundConventional Newcastle disease (ND) vaccination strategies in village chicken production settings is impractical due to shortage of cold-chain, unsuitability of vaccine administration routes and demanding trained personnel and hence affected its adoption. Results from earlier works elsewhere showed that the heat stable vaccines such as NDI 2 are thought to be promising for village chickens. This study investigated the suitability and efficacy of Ethiopian cereal grains as carriers for the orally administrated NDI 2 vaccine in chickens. ResultsOf the 15 treatment groups, drinking water, cracked maize and parboiled barley induced significantly higher HI antibody titer than the other carrier grains and naive control.
The higher mean HI titer of chickens in drinking-water, cracked maize and parboiled barley group resulted in 100% survival rate. In general, there was an inverse relationship between chicken mortality (%) and mean HI titer. Chickens with higher HI antibody titers had better survival rate to the challenge experiment. Booster vaccination at age of day 35 and 105 induced progressively higher HI antibodies titers in all treatment groups. BackgroundEthiopia has several diverse indigenous chicken ecotypes. Their diversity is revealed in genetic diversity , morphology and production performance. Ethiopia has greater than 42.9 million chickens, with the majority (95%) kept in village scavenging systems.
Chickens in scavenging production systems in rural settings exist with little human input and are constrained by feed, management and disease problems. Newcastle disease (ND) is a devastating disease of both commercial farms and village chickens.In village chickens, different ND virus strains and velogenic pathotypes have been identified in clinically affected and apparently healthy chickens. Therefore, village chickens may serve as a reservoir to disseminate ND virus to the nearby commercial poultry farms. The control of ND in village chickens, therefore, not only reduces the impact of the disease within the village but may also prevent spread to nearby poultry commercial farms. Historically, annual ND outbreaks with high mortality are thought to have deterred the potential of owners to rear village chicken. Effective ND control could, therefore, improve rural farmers’ confidence in the profitability of village chickens production, and in turn play a role in the rural poverty reduction strategy. Vaccination is an effective control strategy against ND.However, the adoption of conventional vaccine strategies in a village production setting faces many challenges.
Such challenges include the dependency on cold-chain, large dose preparation per vial and vaccine administration methods (i.e. Eye drop and aerosol) developed for a commercial setting. For village chickens, heat stable, non-pathogenic ND strains (I2 and V4) have been identified as an innovate alternatives to traditional vaccines.
Heat stable vaccines, such as NDI 2, are cheaper to produce, do not rely on a cold-chain and can be easily administered with feed grain or water without catching individual bird, and are thought to be suitable and fit for village chickens. The reports from other countries indicated that NDI 2 vaccine retains potency in the absence of a cold chain, for eight weeks when stored in a cool, dark condition, or at 28 °C in a freeze-dried form. A standard dose of 10 6EID 50/bird can protect birds of all age categories when administered via eye-drop, drinking water, certain feeds or injection. Water-based oral delivery system of NDI 2 vaccine appeared to be constrained by variability in water mineral composition (hard water + high chlorine) at different locations.
Hence, carrier grains are preferred to water. Carrier grains adsorb the virus from an aqueous suspension and release it in a viable form in the digestive tract of chickens. Further treatment of grains particularly parboiling could probably remove some deleterious substances from the grain that hampers viability of the I2 virus, thus preserves NDI 2 ,. Oral administration of the vaccine, using a common chicken feed as a carrier, is well suited to village chicken production systems.Feed cultivated in different regions within the same country or different countries have unique characteristic and differ in the capacity to be utilized as vaccine carriers.
Therefore, vaccine carriers need to be validated for each local environment. Preliminary work has been undertaken to explore the most suitable carrier grain for the NDI 2 vaccine in Ethiopia. Although this study led to promising protection , only two carrier grains (parboiled barley and sorghum) were investigated despite the large variety of cereal and pulse crops in Ethiopia. Therefore, the current experimental study aimed to determine the suitability and efficacy of five different Ethiopian grains as a carrier for the heat stable NDI 2 vaccine for oral immunization of rural chickens. Experimental managementA total of 400 fertilized Bovans brown chicken eggs were obtained from Genesis Farms PLC and hatched at National Veterinary Institute mini-hatchery room.
Eggs were cleaned, fumigated and incubated. Finally, 300 chicks were hatched and collected at the 21st and 22nd day of incubation. Of these, the experimental study utilized 225 chicks.The 225 experimental chicks were brooded together until 14 days in a pen with infrared bulbs for heating and teff straw for bedding.
On the 14th day the chicks were randomly split into the 15 treatment groups as described under study design below (Fig. ). The chickens were fed on purchased starter commercial ration for 2 months, grower ration the next 3–5 months, and layer ration from 5 months onwards. Water was given ad libitum.
Antibiotic (oxytetracycline), minerals and vitamins mix in a sachet (i.e. Vytlet) was purchased and supplied for 3 days after each bleeding (Fig. ). Chickens showing signs of disease (suspected infectious coryza and coccidiosis) were given 20% oxytetracycline and amprolium. Mortality was recorded daily. Study design of the experimental NDI2 vaccine trial in Bovans brown chickens. Vaccine carrier groups (experimental units) were listed from No. 1 -15 in this figure.
Sera collection was performed across age in days (indicator, blue color) to get 1ml sera per chick at each sampling day from wing vein until day 119. However, for chickens of barely and cracked maize group the last sampling was made on day 126, not on day 119. Vaccination was given at a rate of 107 EID50 NDV I 2 per dose per chick three times (indicator, black color). The value of vaccination by each grain carrier was measured by monitoring antibody production post vaccination in the regularly collected sera and by survival rate of the chickens post artificial inoculation of a virulent local Alemaya strain ND virus at a rate of 0.5 × 106.5 EID50 per chick of all experimental units intramuscularly at breast muscle at day129 (indicator, red color). Vaccine and coating of grains with the vaccineVials of freeze dried NDI 2 vaccine (300 doses per vial) were purchased from National Veterinary Institute (NVI) located in Bishoftu. Vials were reconstituted in 150 ml of clean, sterile, non-chlorinated distilled water (manufacturer’s instruction). About 48.5 ml of clean, non-chlorinated water was added to the grain first to wet it.
Subsequently, for each treatment group ( n = 15 chickens) 7.5 ml of vaccine suspension (0.5 ml per chick) was mixed with 150 g of carrier grain (10 g per chick) to deliver one dose (10 7 EID 50) as described by Wambura et al. The final suspension of vaccine was stored at room temperature for 6 h. Prior to vaccination, feed was withheld for 7 h, after which the chickens were given the grains coated with the vaccine.
Commercial feed was not provided until the chickens consumed the coated grains completely. Haemagglutination inhibition (HI) assayHI assay was conducted within serology laboratory of NVI.
Serum prepared from sequential blood collections (Fig. ) was heat inactivated at 56 °C for 30 min and stored at −20 °C. The level of ND virus antibodies in serum samples were determined using the HI test as described by OIE. The HI test has 98% specificity and 69–98% sensitivity. HI titration was made to determine the right HI concentration via 2-fold serial dilution of 25 μl sera in 25 μl PBS followed by 25 μl loading of viral antigen per well. Then, after 30 min 25 μl of 1% RBC per well was loaded and kept for 45 min to determine the end point of haemagglutination.
The antibody level for each serum sample was expressed as a log to the base two and recorded. For convenience, the titer was recorded as just the log index. For example, the titer of log 2 2 was recorded as two. The geometric mean titers (GM) were calculated. In this study we used the published cut off value for the protective HI antibody titer (HI titer ≥ log 2 3 i.e.
5^0 Equals
GM ≥ 3) for ND vaccination in chickens ,. Experimental challenge by virulent ND virusAll the vaccinated and non-vaccinated chickens were challenged by lethal dose (0.5 × 10 6.5 ELD 50 based on viral titration).
Local virulent ND Alamya strain was obtained from NVI and inoculated via intramuscular route into the breast muscle. The Alamya strain has a mean embryonic death time of 51.1 h, an intracerebral pathogenicity index of 1.84 and an intravenous pathogenicity index of 2.51. The challenge time was at the age of 129 which was 3 weeks after the 3rd vaccination.Post challenge, the chickens were examined daily for 4 weeks until the age of 160 day for clinical signs and death due to ND. Statistical analysisThe mean value and standard deviation (SD) of HI antibody titers were determined and classified according to treatment groups. The post vaccination mean HI antibody titers were compared by General Linear model of SPSS version 15.
Where the HI results were significant, least square difference (LSD) was used to compare antibody response via pair-wise treatment comparisons. Proportion of chickens with HI antibody titer ≥ log 2 3 between treatment groups was used as a cut-off value to compare and decide the protective level ,. Serology (HI) was to predict level of protection. However, real level of protection was evaluated using challenge experiment. Subsequently, the time to death among treatment groups was compared using Kaplan-Meier survival curve with log rank test used to assess equality of survival distribution among the groups. Chickens were censored in Kaplan-Meier survival analysis whose death was not related to events of interest (i.e.
Mixvibes Cross V1 5 0 Cracked Wheat Oil
Death due to any other causes than ND challenge). Significant differences set at 5% alpha and at 95% confidence interval. The relationship between chicken mortality (%) and% of chicken with HI titer above cut-off value were compared to the mean HI titer and statistically tested by Pearson correlation. Geometric mean (log2 2) ± SE of HI antibody titer of chicken vaccinated by coating on 13 different types of grains in comparison to vaccination via water (positive control) and negative control. Red arrows labeled with V1, V2 and V3 on top indicated the day of vaccination. Maternal antibody at day old was high but reduced at day 14. One time vaccination using different carrier grains induced HI titer differently.
Second and third round vaccination (booster) upgraded the induction of HI titer significantly ( p. Vaccine carrier typePost vaccination GM ± SD HI antibody titer (log2) chickens vaccinated by different methods at different ages in days ( N = 15)Day 28Day 49Day 77Day 105Day 119Day 126Naive. Ano = represented sera not collected. Figures outside the brackets indicated number of chickens in each group having HI titer above cut off value whereas figures in brackets indicated percentagesFollowing the 1st vaccination, HI titer of ≥ log 2 3 was detected in 80% of chickens vaccinated via conventional (water) and cracked maize. It was 66.7% in the parboiled barley treatment group and 50% in the parboiled wheat, cracked wheat, untreated wheat and parboiled millet treatment groups. The percentage of chickens’ with HI titer ≥ log 2 3 was less in the cracked sorghum and untreated sorghum coated treatment groups (Table ).
After the first booster vaccination, there was an increase in the proportion of chickens with HI titer above ( log 2 3) in comparison to the first vaccination. The highest percentage was observed in chicken vaccinated via conventional method (100%), followed by cracked maize (86.7%), parboiled barley (75%) and parboiled wheat (73%) in that order, as shown in Table. The cracked sorghum and untreated sorghum coated with vaccine had lowest titer but they scored better than their titer induced during the first vaccination. After the 2nd booster vaccination, all (100%) of the vaccinated chickens via conventional (water) and cracked maize had HI titer ≥ log 2 3. In the naive control chicken, none (0%) of the chickens had HI titer ≥ log 2 3 throughout the study period as shown in Table. Monitoring the survival rate of chickens following artificial challengeThe mean HI titer and mortality (%) were inversely correlated as shown on Fig. ( r = −0.938, p.
N † represented number of chickens present in each group at age of 129 day (onset of the challenge). Figures outside the brackets indicated number of chickens in each group whereas figures in brackets indicated percentages.
A Chickens in the treatment groups listed up to parboiled millet had significantly ( p. Survival analysisThe overall differences in mortality (%) were statistically significant (Log-rank = 77.35, D.F. DiscussionThe 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 NDI 2 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 ,. 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 titerAt day old, chicks included in this study have HI antibody titer above log 2 3. Such high maternal antibody titer in the baby chicks is deleterious to vaccination. Thus, we waited until it declined to log 2 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 ,. Durability of the virus in the grains and at room temperatureIn 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 NDI 2 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. This study was financed by the World Bank and CIDA as channeled via Ethiopian Institute of Agricultural Research under National Agricultural Research Fund Project. Ethiopian Ministry of Science and Technology also co-funded. Addis Ababa University College of Veterinary Medicine and Agriculture hosted the implementation of the study activities within its premises. The virulent Alemaya (local) strain of ND virus for the challenge experiment was granted by National Veterinary Institute. Professor Rob Christley and Dr Stacey Lynch of Liverpool University commented this manuscript.