Immune status of semi-scavenging Sonali chickens in Bangladesh against Newcastle disease

P K Biswas, G M N Uddin, H Barua, K Roy*, D Biswas**, A Ahad and N C Debnath***

Department of Microbiology, Chittagong Government Veterinary College, Pahartali, Chittagong-4202, Bangladesh
*Department of Pathology and Parasitology, Chittagong Government Veterinary College, Pahartali, Chittagong-4202, Bangladesh
**Dept. of Medicine and Surgery, Chittagong Government Veterinary College, Pahartali, Chittagong-4202, Bangladesh
***Principal, Chittagong Government Veterinary College, Pahartali, Chittagong-4202, Bangladesh
biswaspk2000@yahoo.com

Abstract

A cross-sectional survey was undertaken to assess immune status of 'Sonali' (♂ Rhode Island Red x ♀ Fayoumi) chickens against Newcastle disease (ND). The chickens were reared at Key rearers'  households by semi-scavenging system in six southern districts of Bangladesh under the 'Smallholder Livestock Development Project (SLDP-2)'. A Key rearer generally rears five Sonali (>2 months of age) and a few 'Deshi' (non descriptive) chickens. Randomly selected 250 Key rearers' households in each of two districts - 'Potuakhali' and 'Noakhali' were targeted to collect blood sera from Sonali chickens. At the beginning of this survey, the median number of Sonali chickens of these Key rearers' flocks was  6. To collect blood from the chickens, monetary incentive @ Taka (Taka is the currency of Bangladesh: 1US$=66 Taka) 30 per chicken was offered to the flock owners. The survey was started in autumn (September-November) 2003 and continued until the Rainy (June-August) 2004. In each season blood sera were collected only from chickens of the flocks, owner of which, permitted to do these because of financial benefits. In each seasonal sampling, one bird was sampled per permitted flock. Altogether, in four seasons, 471 chickens were screened taking blood sera. For doing Haemagglutination Inhibition (HI) test, 8 HAU (Haemagglutination Unit) of Newcastle disease virus (NDV) was used.

The mean log₂ HI titer to NDV in blood sera of the study population was 4.32 (±0.16). Overall, 58% Sonali chickens had HI titers ³log₂4; of which, 4.0% chickens were recorded with HI titers ³log₂10. Based on the temporal samples analyzed covering the entire study population 64, 47.4, 62.6 and 56.3% Sonali chickens had protective HI titers in the autumn (September-November), winter (December-February), summer (March-May) and the rainy season (June-August), respectively. In the two districts, an equal proportion of chickens had protective level of antibodies against ND in the autumn (P=0.68). On the contrary, a higher proportion of Sonali chickens in Potuakhali district had protective titer in the winter and summer (P<0.01). A higher proportion of chickens (P=0.01) reportedly vaccinated with only V₄ (a thermostable but lentogenic strain of NDV), had HI titers ³log₂4 than that of vaccinated with 'Baby Chick Ranikhet Disease Vaccine' (BCRDV) (prepared from 'F'strain of NDV by the Department of Livestock Services, Bangladesh). No significant difference was observed in the proportion of chickens reportedly immunized against ND by booster vaccination with RDV (Ranikhet Disease vaccine: M strain of NDV) having them primed either with BCRDV or V₄.

Key words: Newcastle disease, Semi-scavenging chickens, Survey, Vaccination, Vaccine

Introduction

In Bangladesh, Newcastle disease (ND), which is locally known as 'Ranikhet' disease caused by Newcastle diseases virus (NDV), produces epidemics every year, particularly in semi-scavenging and backyard chickens (Barman 2002; Chowdhury et al 1982).The disease was found as the most killer one of the semi-scavenging chickens (Biswas et al 2005) reared in 17 northern districts of Bangladesh (Ahamed 2002; Biswas et al 2005). Due to scarcity of prophylactics and infrastructural facilities, in developing countries of Africa and Asia, backyard chickens are more vulnerable to this disease than chickens raised in commercial flocks (Gueye 2004; Spradbrow 2004;  Spradbrow 1988) under improved bio-security. To render the birds immune against ND, the Smallholder Livestock Development Project-2 ( SLDP-2), which is financed by the Danish International Development Agency-DANIDA, has designed a vaccination schedule for 'Sonali' (♂ Rhode Island Red x ♀ Fayoumi) chickens being reared under semi-scavenging system in rural areas of six southern districts of Bangladesh (Ahmed 2002). In this endeavor three vaccines have been introduced: 'Baby Chick Ranikhet Disease Vaccine' (BCRDV) prepared by the Department of Livestock services (DLS), Bangladesh--is a lentogenic strain of Newcastle Disease Virus (NDV) ; 'Ranikhet Disease Vaccine' (RDV-Mukteswar' strain of NDV) and V4 (a lentogenic strain of NDV which was imported from Australia). The former two vaccines are heat-sensitive and to retain their immunogenic potency they totally depend on the proper cooling chain; while the later is regarded as a thermo-stable vaccine because it does not entirely dependent on the cooling system (Spradbrow 2004). The immunogenic effectiveness of the V₄ vaccine at village conditions in different countries has been reported (Alders 2000; Bell 2000; Tran Dinh Tu 2000).

The objectives of this study were to screen the level of immunity based on HI (Haemagglutination inhibition) titer against ND of semi-scavenging Sonali chickens reared in southern part of Bangladesh and to compare the effectiveness of the V4 vaccine in these chickens in rural conditions of the country.

Materials and methods

Study population

About 104,000 Key rearers, all of whom were economically poor village-women, were involved in rearing Sonali chickens (>2 months of age) in the sixth southern districts of Bangladesh under the SLDP-2 (Ahmed 2002). Of these Key rearers' flocks 250 were selected randomly in each of two districts - 'Potuakhali' and 'Noakhali'  - for this serological survey. At the beginning of the survey, the median number of Sonali chickens of these flocks was 6 (minimum 1, maximum 20). A key rearer is defined as a beneficiary under SLDP-2 who generally rears five Sonali and a few 'Deshi' (non-descriptive) chickens in her homestead based on semi-scavenging system. The Key rearers' list provided by the Non Government Organization (NGO) from a district was used as a sampling frame.

Determination of sample size

The minimum sample required for the investigation was calculated to be 100 with a level of precision 10% based on the formula 4pq/l² (where n= No. of total samples required, p= prevalence, q=1-p, l = level of precision). Because the prevalence of the immune chickens against ND in this semi-scavenging population was not known, a 50% prevalence was considered as a rule to estimate minimum sample size required for each season in this survey.

Collection and preservation of blood sera from chickens

Among the randomly selected 500 flocks, blood samples were collected only from Sonali chickens of the flocks, owner of which allowed to do these after having received financial incentive at the rate of Taka30 (Taka is the currency of Bangladesh) per chicken. Four sampling were done in four seasons started from the autumn (September-November) 2003 and ended up in the rainy (June-August) 2004.In every seasonal sampling, blood was taken from one bird per permitted flock. Four 'Poultry Health Workers' (PHWs), who had been employed for the research period, collected blood sera from Sonali chickens of the permitted flocks. After having separated a serum sample from blood clots, it was preserved at first chemically with merthiolate with a concentration of 1:10,000 in an eppendorf tube to prevent bacterial growth if accidental contamination occurred. After that, the collected sample was stored frozen in a refrigerator placed at the Non-Governmental Organization Office (NGO) in each of the two districts. Each serum sample was accompanied by a sample submission form completed by the PHW with vaccine and vaccination information by interviewing the respective Key rearer. All samples collected in two districts were transferred by the PHWs themselves to the Department of Microbiology, Chittagong Government Veterinary College, Chittagong, Bangladesh on monthly basis. After having the samples received at the Department of Microbiology, they were kept further frozen at -84⁰C until analyzed.

Conducting HI test to screen level of NDV antibodies in blood sera of Sonali chickens

HI test was performed in V- bottomed-microtitration plates. The test procedure was conducted as per the methodology described in the OIE Manual (2002). For preparation of 1% chicken red blood cells (RBCs) used in the test, blood was taken from three Deshi chickens that had not been primed with Newcastle disease vaccine. These collected RBCs were pooled in an equal volume of Alsever's solution. After that, collected RBCs were washed thrice in phosphate buffered saline (PBS) before using as a 1% (packed cell v/v) suspension. Serum was tested in two fold dilutions. For virus suspension 8 HAU of 'F' strain of NDV was used. For negative control, SPF chicken serum supplied from Bio-check, Holland was used. For positive control, pooled serum collected from 5 chickens at 21 days post vaccination with RDV was used. Four log₂HI titer to NDV was considered the protective threshold against ND; titer below this was categorized as non-protective (OIE 2002).

Data analysis

All data were entered in the computer program MS EXCEL (Microsoft Co.) for descriptive and inferential statistics and data summary. Univariate analysis was done to assess the immunity status of Sonali chickens against ND and to compare this variable in temporal and spatial samples a χ² test was applied.

Results and discussion

Of the randomly selected and targeted flocks we succeeded to collect blood sera from 139,114,115  and 103 flocks in the autumn (September-November), winter (December-February), summer (March-May) and rainy seasons (June-August), respectively. Results on HI titers to NDV in the study population are shown in Table 1. The mean HI titer in blood sera of the investigated chickens was log₂ 4.32 (± 0.16). Overall, 58% chickens had HI titers ³ log₂4, of which the highest proportion i.e. 18.5% chickens were recorded having HI titer log₂8. There were 19 chickens (4.1%) recognized having HI titer ≥ log₂10. In these 19 birds, only 2 were in Potuakhali district and the rest in Noakhali. Of the total number of chickens investigated, 35.7% had HI titers to NDV ≤ log₂1-2. A higher proportion of chickens in Potuakhali district had protective titer to NDV than that of Noakhali (P=0.00).

Overall, 64, 47.4, 62.6 and 56.3% Sonali chickens in southern part of Bangladesh had protective HI titer against ND in the autumn (September-November), winter (December-February), summer (March-May) and the rainy season (June-August), respectively. In the autumn an identical proportion of Sonali chickens had protective HI titer in both the districts (Table 2). Conversely, a higher proportion of chickens in Potuakhali district was immune against ND in the winter and summer (P<0.01).

Of total birds investigated, 243 were reported (by the beneficiaries) to have been vaccinated by any combinations of the three vaccines - BCRDV, V₄ and RDV; and the reportedly vaccinated birds comprised 51.6% (Table 3).

In the section of vaccinated birds, 74.1% were reported to have been immunized either with BCRDV or V₄; among which 19.7% with the so called thermostable vaccine (V₄). And 9.9% chickens were reported to have been vaccinated with the vaccine RDV without having been primed with any of the less virulent vaccine strains of NDV. However, of total chickens vaccinated, 16.1% were identified as given booster vaccination with RDV having been initially immunized either with BCRDV or V₄. All of the chickens that had been given boosted immunity with RDV had protective level of HI titer to NDV regardless whether BCRDV or V₄ vaccine was chosen for primary vaccination. All chickens, which had reportedly been vaccinated with only RDV, also had protective level of HI titer against ND. A higher proportion of Sonali chickens had protective HI titer when vaccinated with V4 than that when vaccinated with BCRDV (P=0.01).

A section of chickens, especially in Noakhali district, was recorded having HI titers ³log₂10, which is significantly higher than the mean titer observed in chickens being reared in the mother flocks of Sonali chickens given booster vaccination with RDV (Faruque 2003). In this study, a higher level of HI titers in semi-scavenging chickens might indicate natural infection, in agreement with Barman (2002) and Bouzari et al (2004) who reported that unvaccinated village chickens may possess NDV antibodies as a result of natural infection.

In field conditions, a higher proportion of Sonali chickens had a protective level of HI titer to NDV. In laboratory condition, both V₄ and BCRDV produced more or less similar HI titer in Sonali chicks after 14 days of booster vaccination (Kafi et al 2003). A better efficacy of V₄ vaccine found in the field condition can be explained on the basis that this vaccine might have retained its potency in the interruptions of the cooling system which likely happened in the field conditions of Potuakhali and Noakhali districts of Bangladesh.

We failed to collect blood sera from Sonali chickens of all the selected 500 Key rearers' flocks despite the monetary incentives provided for blood collection. Most of the Key rearers declined to co-operate fully with the UPHs for the purpose of blood collection fearing that their chickens could die or egg production might cease due to giving of blood or blood-letting out. We managed to collect blood sera of Sonali chickens from at least 103 flocks (range, 103-139) from a particular season. This minimum number of chickens was adequate for the estimated sample size (100) of a particular season for the entire study population. However, in respect to an individual district, the surveyed sample size was ≥ 49 (range, 49-70), which is less than the probabilistic sample size. This was one of the limitations of this survey. We did not overcome this due to non-cooperation from the Key rearers as stated above. However, international readers may have a greater interest on the results reported covering the entire population rather than a particular district.

The results showed that a substantial section of Sonali chickens reared in the southern part of Bangladesh under the SLDP-2 remained unprotected to ND. These susceptible chickens can easily be affected with NDV from deshi/backyard chickens or wild birds as the population is a contiguous one (Biswas 2005; Glaucia et al 2003; Schelling et al 1999). It depends on the virus loads existing  in the companion 'deshi' or even in wild birds in a locality.

Conclusions

• Despite the efforts of SLDP-2, about 48% of the semi-scavenging chickens of Key rearers in southern part of Bangladesh remained un-vaccinated. In addition, the mean HI titer to NDV of this chicken population was only slightly above the protective threshold. The findings of this serological survey should alert the stakeholders of SLDP-2 that ND vaccination approaches have to be intensified further in their area and that particular attention should be focused on Noakhali district.

• To elevate the level of immunity against Newcastle disease, all chickens have to be given booster vaccination with RDV having been primed either with BCRDV or V4.

• For the first time, V4 vaccine was assessed in field conditions of Bangladesh and the results showed that this thermo-stable vaccine could give better protection in rural chickens against ND than that of the commonly used thermo-labile vaccine (like BCRDV). To control ND in backyard chickens, this V4 vaccine could also be introduced in other developing countries where supply of electricity in the rural areas is inconsistent and/ or interrupting.

• Having suffered from ND, about 4% of semi-scavenging Sonali chickens in Bangladesh could survive showing the HI titer to NDV >log₂¹⁰.

Acknowledgements

The study was a part of a research project financed by the DANIDA through the SLDP-2. We are expressing our thanks and gratitude to this organization for supporting the research program.

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Received 20 April 2006; Accepted 22 April 2006; Published 13 June 2006

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