Introduction
Newcastle disease (ND) is a major threat to the poultry production industry throughout developing countries. The Newcastle disease viruses (NDVs) infecting industrialized and indigenous poultry in Iran are velogenic strains and responsible for the frequent outbreaks of ND in poultry farms even in vaccinated flocks causing serious economic losses in the commercial and indigenous poultry ( Habibi et al., 2015 ). With this endemic nature of ND either in commercial or village chicken and little possibility of enforcing efficient biosecurity measures to prevent the spread from village to commercial poultry, vaccination remains the only alternative strategy for controlling and minimizing the effect of the disease ( Alexander, 2001 ). The eradication of the virus is not feasible; therefore, vaccination programs should be continual and sustainable. Despite the plethora of vaccines and aggressive vaccination programs being practiced by the poultry industry all over the world to control the disease, ND has defied all logic and continues to rear its ugly head in both epidemic and endemic forms causing monumental economic losses in the industry. None of the usual commercial ND vaccines will partially protect chickens against the disease if the vaccines reach the chickens in a potent form. However, there are frequent outbreaks of the disease even in vaccinated flocks. Many commercial vaccines are thermolabile and sometimes extremely thermolabile. Cold chains are too expensive to maintain and develop. Heat-resistant vaccines enable distributors and users to reduce the problems associated with the inadequacy of cold chains in the field. While evaluating vaccines for their ability to protect from ND, it may be useful to include their ability to decrease virus shedding in vaccinated birds following exposure to wild field virus, which potentially reduces the spread of NDV, especially in an outbreak setting. A thermostable ND vaccine strain I-2 has been initially recommended for use in developing countries for the protection of village chickens against ND ( Bensink and Spradbrow, 1999 ). However, the present study aimed to establish the efficiency of the thermostable variant of strain I-2 as the thermostable vaccine in broiler chickens locally produced at the Avian Diseases Research Center of Shiraz University in Iran. Three vaccination strategies were used to obtain the following results: 1) the efficacy of the I-2 vaccine in broiler chickens challenged with highly virulent virus under laboratory conditions. 2) The horizontal transmission of the I-2 vaccine virus. 3) The transmission of the disease
Material and Methods
Experimental design. A total of 180 commercial day-old broiler chicks (Cobb 500) of mixed gender were purchased from a local hatchery in Fars province, Iran. All the experimental measures were conducted in the controlled laboratory condition of the Avian Diseases Research Center of Shiraz University. A total of 150 four-week-old chicks, which had been tested negative for ND antibody, were randomly allocated to experimental groups to test whether the locally produced I-2 NDV would stimulate chickens to produce a protective haemagglutination inhibition (HI) antibody titer or not. During the study, water and food were provided ad libitum. All birds were fed on a standard commercial diet based on corn and soybean meal. Strict sanitation practices were maintained in the unit before and during the course of the experiment. Sixty chicks identified with individual wing tags were randomly placed into rooms 1, 2, 3, and 4 (15 chicks in each room). Rooms 5, 6, and 7 were kept as the positive control, vaccine control, and negative control groups, respectively (Table 1). Birds in rooms 1, 2, and 6 were vaccinated by means of drinking water, and the birds in rooms 3 and 4 were vaccinated with food containing vaccines. The chickens (in rooms 1, 2, 3, 4, and 6) were vaccinated on day 28. The chickens in rooms 2 and 4 were revaccinated 1 week later (day 35).
| Room | Group | Birds (n) | Challenge (day 42) | |
|---|---|---|---|---|
| 1 | 1 | Vaccinateda via drinking water | 15 | + |
| 2 | In contactb with vaccinated chickens of group 1 | 8 | + | |
| 3 | In contact with chickens of group 1 and 2 after challenge | 7 | - | |
| 2 | 4 | Vaccinated twicec via drinking water | 15 | + |
| 5 | In contact with vaccinated chickens of group 4 | 8 | + | |
| 6 | In contact with chickens of groups 4 and 5 after challenge | 7 | - | |
| 3 | 7 | Vaccinated via food containing vaccine | 15 | + |
| 8 | In contact with vaccinated chickens of group 7 | 8 | + | |
| 9 | In contact with chickens of groups 7 and 8 after challenge | 7 | - | |
| 4 | 10 | Vaccinated twice via food containing vaccine | 15 | + |
| 11 | In contact with vaccinated chickens of group 10 | 8 | + | |
| 12 | In contact with chickens of groups 10 and 11 after challenge | 7 | - | |
| 5 | 13 | Positive control | 10 | + |
| 6 | 14 | Vaccine control (vaccinated via drinking water) | 10 | - |
| 7 | 15 | Negative control | 10 | - |
| aPrimary vaccination was performed on day 28. bIn-contact chickens were not vaccinated and were seronegative to Newcastle disease virus. c Secondary vaccination was performed on day 35. | ||||
Transmission study. To evaluate the horizontal transmission of the I-2 vaccine virus, 2 h after the termination of vaccination, eight unvaccinated chickens were kept in contact with the vaccinated chickens of the rooms 1, 2, 3, and 4 throughout the experimental period. To assess the transmission of the disease, 2 h after the challenge (day 42) of the vaccinated and in-contact chickens, seven sensitive chickens (seronegative to ND virus) were added to the rooms 1, 2, 3, and 4.
I-2 strain. The strain I-2 of NDV was propagated in 9-day-old embryonated fowl eggs from the vaccine master seed produced and supplied by the Department of Veterinary Pathology of the University of Queensland, Australia ( Spradbrow and Copland, 1996 ). The vaccine harvested from embryonated eggs was reported with a titer of 1010.2 EID50/ml.
Coating carrier food. The wheat grain was cracked to small sizes to ease swallowing by the birds. Then, it was sieved for the removal of dust content. To remove the effects of some inactivating and inhibitory substances on the wheat grain (e.g., acids and lectins), it was autoclaved at 121°C for 20 min, and 0.3 kg of cracked wheat was mixed with 30 mL of blended 10% glycerol before mixing with the vaccine. Thirty doses of vaccine (each dose equal to 106.5 EID50/bird) from the allantoic fluid of the strain I-2 virus were reconstituted in 30 ml of phosphate-buffered saline and thoroughly mixed with a wheat carrier. After mixing, the coated food vaccine was spread on trays and kept at room temperature to be dried for 3 h. The virus content of the dried coated wheat was estimated to be 105.5 EID50/g.
Vaccination. A 10 g of the coated food vaccine (105.5 EID50/g), equal to a dose for one chicken, was presented to each bird. For vaccination via drinking water, a 40 dose vial of NDV I2 strain was reconstituted in 1 liter of distilled water, and each bird was given 25 ml of the reconstituted vaccine. Each dose contained 10 6.5 EID50/ml ND I-2 virus.
Challenge trail. A very virulent field isolate of the velogenic NDV was selected as the challenge virus previously characterized and registered in GeneBank by the name of JF820294.1. The embryo infective dose (EID50) was calculated according to the Reed and Muench ( Habibi et al., 2015 ) formula and reported as 108.3 EID50/ml. The intravenous pathogenicity index (IVPI) of the challenge virus was 2.46, and the mean death time of chicken embryos was 59 h. Birds were intranasally challenged with 0.05 ml allantoic fluid containing 104.3 EID50/ml at day 42.
Serology. Sera samples for subjecting to the HI test were collected to determine the antibody titers of NDV according to the method of Allan and Gough ( Allan and Gough, 1976 ). The ND HI antigen was manufactured by Razi Vaccine and Serum Research Institute in Iran. The haemagglutination (HA) titer was diluted to contain 4 HA units for the use in the HI test.
Clinical observation and statistical analysis. The birds were daily monitored after the challenge for clinical signs, and on days 5, 10, and 17 after the challenge, two chickens from each group were humanely euthanatized by cervical dislocation and examined for the presence of gross lesions. Morbidity, mortality, and protection rates for each group were calculated in the study. The data of the mean titers of the immune response to the NDV were analyzed using one-way analysis of variance by SPSS software (version 16.0).
Significant differences among different groups were identified at 5% by Duncan’s multiple range test.
Results
The HI titer of maternal antibody in one-day-old chicks was log2 5.9. Before vaccination, all chickens were seronegative to the NDV antibody (HI Geometric Mean Titer [GMT] log2<1). The HI antibody response to different vaccination protocols is shown in Table 2. On day 42 (14 days after the primary vaccination and/or on the day of the challenge), 100% of the chickens in the vaccinated groups and even of in-contact chickens had a HI titer above log2 3. The birds of group 4 (which were vaccinated via drinking water and revaccinated 1 week later) had the highest HI antibody titer with the mean titer of log2 4.8 among vaccinated groups (P<0.05). In addition, 17 days after the challenge, the highest HI antibody mean titers were reported for groups 4 and 10 (Table 2; P<0.05). All the vaccinated and in-contact birds appeared healthy after the challenge and did not show any clinical signs of ND until the end of the experiment (17 days following the challenge). Furthermore, the sensitive birds added to rooms 1, 2, 3, and 4 to evaluate the transmission of the disease did not show any signs of the disease; however, they serologically became positive for NDV (Table 2). In this study, 100% protection and no gross lesion were observed in necropsy among the groups of rooms 1, 2, 3, and 4 that were humanely euthanized. On the other hand, 100% of positive control birds showed clinical signs, including dyspnea, nasal discharge, anorexia, green diarrhea, tremor, as well as torticollis, and finally, all of them died up to day 11. In the necropsy of the birds in the positive control group, petechial hemorrhages in the proventriculus, cecal tonsils, hyperemia, and exudates in the trachea and lungs were observed.
| Room | Group | 1 week after primary vaccination | 2 weeks after primary vaccinationa | Challenge (day 42) | 17 days after challenge | Protection rate (%)b |
|---|---|---|---|---|---|---|
| Drinking water containing vaccine (1x) | 1 | 1.9±0.8 | 4.2±0.8 | + | 6.75±0.8 | 100 |
| 2 | 1.3±0.5 | 4±0.5 | + | 6.75±1.1 | 100 | |
| 3 | 0.75±0.5 | 0.5±0.5 | - | 5±1.8 | 100 | |
| Drinking water containing vaccine (2x) | 4 | 2±1.0 | 4.8±1.0 | + | 7.66±1.0 | 100 |
| 5 | 1.2±0.8 | 4.4±0.8 | + | 6.5±1.2 | 100 | |
| 6 | 0.75±0.5 | 0.5±0.5 | - | 4.8±2.0 | 100 | |
| Food containing vaccine (1x) | 7 | 1.6±0.7 | 3.5±0.7 | + | 6.75±0.8 | 100 |
| 8 | 1.1±0.7 | 3±0.7 | + | 6±0.9 | 100 | |
| 9 | 0.75±0.5 | 0.5±0.5 | - | 5.5±1.3 | 100 | |
| Food (2x) | 10 | 1.5±0.5 | 4±0.5 | + | 7.33±1.2 | 100 |
| 11 | 1.3±0.8 | 3.25±0.8 | + | 6.25±1.6 | 100 | |
| 12 | 0.75±0.5 | 0.5±0.5 | - | 5±1.9 | 100 | |
| Positive control | 13 | 0.75±0.5 | 0.5±0.5 | + | -c | |
| Vaccine control | 14 | 1.3±0.7 | 4.4±0.5 | - | 4.75±0.5 | - |
| Negative control | 15 | 0.75±0.5 | 0.5±0.5 | - | 0.5±0.6 | - |
| aSecondary vaccination in groups 4 and 10 was performed 1 week after the primary vaccination. bNo clinical signs and death were considered 100% protection. cAll birds died up to day 11 after the challenge. | ||||||
Discussion
Vaccination as a means of protecting birds against ND is routinely practiced around the world. Despite the extensive use of vaccines, the outbreaks of ND have still been recorded in Asia and Middle East that might be due to the genetic or antigenic variation in vaccine and circulating viruses, inadequacy of vaccines, or no correct application of vaccines ( Shabbir et al., 2013 ).
In the present study, it was tried to investigate, under controlled laboratory conditions, the efficacy of thermostable ND virus strain I-2 in broiler chickens. This experiment provided the information on antibody responses and adequate protection in broiler chickens, where the birds vaccinated twice via drinking water with a one-week interval produced higher HI titer values (Table 2). However, all groups showed 100% protection against overt clinical signs and mortality upon the challenge with highly virulent NDV (IVPI 2.46). The production of higher HI antibody titer by means of drinking water vaccination than that by food coated vaccine was also previously shown in several studies ( Habibi et al., 2015 ; Abdi et al., 2016 ; Lawal et al., 2016 ). It was indicated that the direct oral drench vaccine demonstrated a more uniform response and consequently higher GMT values in comparison to the maize coated vaccine.
Although the lower limit of the recommended protective antibody level measured by HI test for ND vaccines in intensively reared commercial chickens is 4 based on log2 ( Sarcheshmei et al., 2016 ), it has been reported that HI antibody titer of log2 3 was considered to be adequate for food-based vaccines administered orally to scavenging chickens ( Echeonwu et al., 2007 ). The resistance of the chickens even with the HI antibody titer of log2 ≤ 4 challenged with the velogenic ND virus in the present study indicated that other immune system components could possibly contribute to the immunity against ND. This finding suggested a mechanism for reducing the amount of virus shedding in addition to the antibodies measured in HI assays. Several studies with orally administered ND vaccines showed that there was an increased number of secreting immunoglobulin A (IgA) referred to as secretory antibodies on the mucosal surfaces of the avian intestine, bronchi, and oviduct ( Jayawardane and Spradbrow, 1995 ). The measurement of mucosal IgA and cellular immune responses in future studies with these recombinants may provide direct evidence of the aforementioned finding. Due to close contact between birds when they are housed together, the lateral transmission of the live vaccine virus is possible ( van Boven et al., 2008 ). The obtained results of the present experimental study demonstrated that nonvaccinated birds in the same room (housed together with vaccinated birds) did not show any mortality following the challenge with a very virulent field strain. The horizontal spread of thermostable live NDV can result in the production of protective HI antibody, which was also previously reported ( Habibi et al., 2015 ). It has been shown that the vaccination of susceptible birds against ND and avian influenza protects birds against more serious consequences of the disease; however, virus replication and shedding may still occur, albeit at a reduced level ( Costa-Hurtado et al., 2015 ). In the present study, susceptible in-contact birds exposed to challenged birds became seropositive and did not show any clinical signs. This finding implies the inability of the I-2 strain virus for the full protection against viral replication and possible shedding of challenge virus but reduced viral shedding to induce disease in susceptible birds. The reduction of viral shedding from vaccinated birds infected with NDV could potentially minimize the impact of an outbreak and help to prevent the spread of the disease. Based on the previous recommendations regarding the vaccination of village chickens using food-based vaccines, the multiple administration of the vaccine is needed ( Spradbrow and Samuel, 1991 ; Wambura et al., 2000 ; Habibi et al., 2015 ). In the present study on broiler chickens, even the primary dose of food-based I-2 strain virus has been protective. The results of this study showed that wheat coated with ND I-2 strain was able to protect susceptible broiler chickens against the challenge with a very pathogenic field isolate of NDV (JF820294.1) in comparison to the control group. Previous studies showed that using parboiled sorghum and parboiled sorghum coated with gum has not been protective in local and commercial chickens ( Lawal et al., 2016 ), and uneven results from previous studies have been obtained in this regard ( Habibi et al., 2015 ). Antiviral factors constituent in the seed or introduced as preservatives have been reported to be responsible for insufficient protection in previous studies ( Echeonwu et al., 2007 ).
The basic principles for ND prevention are similar to those reported for other diseases, including good management practice and biosecurity or good hygiene in conjunction with vaccination. In the present experimental study, it was shown that the administration of I-2 vaccines via drinking water and/or coated on the wheat grain was safe and immunogenic and provoked the production of protective antibody response following the vaccination of broiler chickens. The successful implementation of field trials depends on good laboratory-field communications. Pretrial planning and extension are crucial to ensure farmer cooperation in these trials and any future vaccination programs.
Ethics
All animal studies were approved by the Ethics Committee of Shiraz University and performed in accordance with the ethical standards set in the 1964 Declaration of Helsinki and its later amendments.
Authors’ Contribution
Study concept and design: H Nili and K Asasi
Acquisition of data: H Habibi and S Firouzi
Analysis and interpretation of data: N Mosleh
Drafting of the manuscript: H Habibi and S Firouzi
Critical revision of the manuscript for important intellectual content: H Nili, H Habibi and S Firouzi
Statistical analysis: N Mosleh
Administrative, technical, and material support: H Habibi and S Firouzi
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