Prevalence of Newcastle disease virus in village indigenous chickens in varied agro-ecological zones in Kenya

L W Njagi, P N Nyaga, P G Mbuthia, L C Bebora, J N Michieka, J K Kibe and U M Minga*

Department of Veterinary Pathology, Microbiology and Parasitology, P.O. Box 29053 - 00625, Kangemi, Kenya
* Open University of Tanzania, P.O. Box 23409, Dar es salaam, Tanzania
njagiluc@mail.uonbi.ac.ke

Abstract

It was hypothesized that the agro-ecological zone in which village indigenous chickens were farmed influenced the level of diseases occurrence.  One hundred and forty four apparently healthy chickens (71 from lower highland 1, a cold zone and 73 from lower midland 5, a hot zone) were randomly sampled. Oro–pharyngeal and cloacal swabs were collected from each bird and processed for virus isolation in 10-12 day old embryonated chicken eggs. In addition, blood, without anticoagulant was obtained from each bird through wing venipuncture. Haemagglutination inhibition assay was performed for all sera samples.

Prevalence of Newcastle disease (NDV) virus was significantly higher (17.8%) in the dry hot zone (lower midland 5) compared to the cool wet zone (lower highland 1) at 9.9% showing evidence for climate as a risk factor in the occurrence of NDV in village chicken. Female birds had higher mean Newcastle disease viral titers than their male counterparts. All Newcastle disease virus isolates recovered were from healthy appearing birds and were all velogenic. Sero-prevalence was significantly highest (p<0.05) in adult birds (10%) while growers had 5.1% and chicks 2.9%. Apparently healthy-appearing birds were reported to be reservoirs of velogenic Newcastle disease virus strains that could initiate endemicity NDV cycles in the village setting.

Key words: cool and dry zones, healthy-appearing chickens, reservoirs, velogenic Newcastle disease virus

Introduction

Many reports and studies (Bell et al 1990; McBride et al 1991) suggest a continuous presence of Newcastle disease virus (NDV) in village poultry populations. Some of the risk factors that have been associated with the maintenance of NDV include: carrier chickens, village poultry population dynamics, other poultry species, wild birds and heterogeneity of NDV (Awan et al 1994). Although clinically diseased chickens are the most important hosts for NDV, latently infected birds and survivors of natural infection, which still harbour the agent, may also act as reservoirs. Village chickens may be exposed naturally to virulent virus shed from recovered birds, vaccinated birds having various levels of antibodies in their blood, non – susceptible species carrying virulent virus or susceptible birds yielding virulent virus, which may have evolved from passages in birds of mesogenic viruses (Westbury et al 1984; De Leeuw et al 2003). It was hypothesized that the agro - ecological zone in which village indigenous chickens were farmed influenced the level of diseases occurrence.  

The aim of this study was therefore to determine the prevalence of NDV and NDV antibodies in naturally exposed non – vaccinated multi-age, village indigenous chickens, in varying climatic and ecological zones in Kenya.

Materials and methods 

Sources of chickens

The farms from which the chickens were purchased were located in two varied zones in terms of climate and ecology namely: lower midland 5 (LM5) in Mbeere district and lower highland 1(LH1) in Embu district. The flock size was variable and all the birds were on free – range system in both agro – ecological zones.

The lower highland zone 1 (LH1) is a tea – dairy cattle zone at an altitude of 2070 metres above sea level. It has a long to very long cropping season followed by a medium one, with an average rainfall of 1080mm. The first rains start in mid March while the second, starts in mid October. Predominant crops grown in this zone are tea, peas, cabbages, lettuce, carrots, kales, potatoes, maize, beans, pyrethrum and plums. The grassland and forage include Kikuyu grass, napier grass and clover legume (Jaetzold and Schmidt 1983). This is a cold climatic zone.

The lower midland 5 zone (LM5), also referred to as livestock – millet zone, covers the central belt of Mbeere district extending to Mwea plains on the south west, at an altitude of 760 metres above sea level; has an average rainfall of 180mm per year. It has two short cropping seasons. The first rains starts at end of March while the second, end of October. The following crops are grown: bulrush and foxtail millet, green grams, cowpeas, chickpeas, bambarra groundnuts, dwarf sunflower, moth beans and maize. The vegetation in this zone includes mixed short grass savannah with buffel grass, horsetail grass and saltbush (Jaetzold and Schmidt 1983). This is a hot climatic zone.

Experimental design

One hundred and forty four apparently healthy chickens (71 from LH1 and 73 from LM5), consisting 59 growers (31 females, 28 males); 35 chicks (15 females, 20 males; and 50 adults (26 females, 24 males) were randomly sampled. There were equal numbers of females and males (72 each). The chicks were less than 2 months old; growers were between 2 to 8 months old; and adults, above 8 months of age. All the birds were transported in cages to Kabete, University of Nairobi campus for sampling. Cloacal and oro - pharyngeal swabs were processed for NDV  isolation as described below while the serum samples were tested for NDV specific antibody by haemagglutination inhibition (HI) test. The isolated viruses were then tested for pathogenicity using mean death time (MDT) and intracerebral pathogenicity index (ICPI) tests.  

Collection and processing of swabs and blood

Swabs were taken from oro–pharynx and cloaca using sterile cotton – tipped applicator swabs. The swabs were placed in 2 ml viral transport medium comprising minimum essential medium, with penicillin and streptomycin at a concentration of 5000 units / ml and 2.5 mg/ml amphotericin B and transported in a cool box to the laboratory. The swabs were expressed, centrifuged at 3500 rpm for 10 minutes and the supernatant transferred to a sterile bijoux bottle. All the samples were stored at -20^°C until virus isolation was done.

In addition, blood from the brachial vein was collected into universal bottles, without anticoagulant. Serum samples were separated from respective clotted blood samples by centrifugation at 500 rpm for 15 minutes, and then heated at 56^°C for 30 minutes to inactivate non-specific hemagglutination inhibitors. The serum samples were then decanted, aliquoted into screw capped vials and kept frozen at -20^°C until HI tests were performed.

Virus isolation

Virus was recovered from the tissues and swabs by culture in primary chicken embryo fibroblasts and inoculation in embryonated chicken eggs as previously described (Kumanan and Venkatesan 1994; Nyaga et al 1985).

Serology

Presence of NDV antibody was detected by hemagglutination inhibition test as described by OIE (2000). 

Pathogenicity tests

Mean death time and intracerebral pathogenicity index were carried out as described in OIE (2000).

Statistical analysis

Data on HI titres and virus recovery were log transformed before being analysed using SAS software (SAS Institute Inc., Cary, NC, USA, 2002 -2003).   The Waller – Duncan K – ratio test and Ryan – Einot – Gabriel – Welsch multiple range test (Steel and Torrie 1980) were used to analyse the antibody responses. The titres were compared across the various age groups, between sexes and agro -ecological zones.

Results 

Prevalence of Newcastle disease antibodies and virus in the Lower highland 1 (wet - humid) and Lower midland 5 (dry - hot) agro – ecological zones

The prevalence of NDV was higher in the Lower midland 5 (17.8%) as compared to Lower highland 1 (LH1) which had a prevalence of 9.9 % (P < 0.05). Lower midland 5 (LM5) had a NDV seroprevalence of 8.2% while LH1 had 4.2% (Figure 1).

Prevalence of Newcastle disease antibodies and virus in different age groups and sexes of village indigenous chickens

Figure 2 shows that prevalence of Newcastle disease virus was highest in female birds (22.2%) of all the age groups, while males had a prevalence rate of 5.6%. Similarly, the seroprevalence was higher in females than males of the same age groups: 6.9% and 5.6% for females and males, respectively. In this study, the difference in NDV prevalence between the males and females was statistically significant (P < 0.05) while the seroprevalence was not significant (P > 0.05).

Among the three age groups, the growers had the highest NDV prevalence of 25.4%, while chicks and adults had an NDV prevalence of 8.6% and 4%, respectively. In respect to antibody prevalence, adult birds had the highest NDV seroprevalence of 10% followed by growers (5.1%) and lastly chicks with 2.9%. Newcastle disease virus prevalence between the three age groups was statistically significant (P < 0.05) (figure 3).

Regarding different sexes, the grower females group had higher NDV prevalence of 38.7% compared to that of their male counterparts (10.7%). Female chicks had an NDV prevalence of 13.3% while males had 5%, and adult females had 7.7% while no virus was isolated from adult males. In respect to antibody prevalence, adult females had the highest NDV seroprevalence of 11.5% and their male counterparts 8.3%. Grower females and males had a seroprevalence of 6.5% and 3.6% respectively. Lastly, male chicks had a seroprevalence of 5% while all female chicks were seronegative (figure 4).

Effect of age, sex and climate on Newcastle disease virus titers in village indigenous chickens

Chickens from lower highland 1 had higher mean titres of antibodies as compared to those from Lower midland 5. Female birds had the highest geometric mean titres of both NDV and antibodies in the two agro - ecological zones. The NDV titres were highest in adults, followed by growers and lowest in chicks while antibody levels were highest in the growers, followed by chicks and lowest in adults (Table 1). 

Age and sex of the birds did not affect the mean titres of NDV and antibody in various chicken groups (P > 0.05).

Pathogenicity of recovered Newcastle disease virus isolates

The mean death time (MDT) of 12 isolates tested ranged between 48 and 56 hours. The lowest intracerebral pathogenicity index (ICPI) was 1.5 while the highest was 1.8. Majority of the isolates had an ICPI of 1.7. All the 12 isolates tested were from female birds only (Table 2).

All were velogenic strains. Thus all the isolates were velogenic by both mean death time and intra-cerebral pathogenicity tests despite being from apparently healthy looking birds.

Discussion 

The prevalence of Newcastle disease virus was higher in LM5 than the LH1 and the difference was statistically significant. The fact that NDV prevalence rate was higher in LM5 than LH1 may be attributed to the diversity in the climate, management practices, including confinement, mode of disposal of poultry waste and carcasses and recovery rates of chicken from disease outbreaks all which form circumstances favouring the maintenance of the virus in the village poultry populations. These findings agree with the responses from farmers on the occurrence of Newcastle disease (ND) in the two agro – ecological zones whereby the farmers reported occurrence of more outbreaks of the disease in LM5 compared to LH1. Previous studies have associated ND with change of seasons, some reportedly associated with the start of the wet season (Thitisak et al 1988; Jintana 1987). Cold weather has been associated with ND outbreaks in some countries (Dao and Pham 1985), while in others it is hot weather (Bell et al 1990) and others with both cold and hot seasons (Nyaga et al 1985).

The seroprevalence differed across the two agro - ecological zones, with LM5 having a higher seroprevalence than the LH1 although the difference was not statistically significant (P > 0.05). Yongolo (1996) reported a sero prevalence varying from 25 % to 81.5% in Tanzania, which also had variation in different months and localities, but the data had not been aggregated to reflect occurrence in specific agro – ecological zones. Some of the risk factors identified in this study and the free-range management system may be responsible in creating uninterrupted cycles of infections whereby the virus passes from one chicken to another throughout the year. The different climatic environments and the management practices further enhance the differences in disease outbreaks for the different agro-eological zones

Female chickens had higher mean NDV titres compared to the male birds. Hens and pullets may therefore be playing a significant role in the carriage and maintenance of the virus in these rural poultry populations. Previous studies by Kutubuddin (1973) indicated that male birds were more affected by NDV than female birds. However, in this study the females had higher viral load than male birds in all age groups. Huchzermeyer (1993) noted that brooding hens and hens with chicks that were kept segregated could also escape infection. Chickens that may have survived previous ND outbreaks produce chicks, which become susceptible to ND after the maternal antibodies have waned (Huchzermeyer 1993). The actual cause of this apparent sex related differences in NDV carriage is not yet understood.

All the NDV isolates recovered were velogenic by both MDT and ICPI tests. It is not clear why apparently healthy birds, which had no antibodies to the virus, would harbour velogenic NDV strains and show no signs of disease. Perhaps they were protected through other non – antibody mediated protective mechanisms, e.g. cell mediated immunity from prior exposure to virus. This would be the case assuming that antibodies from the previous outbreak had declined to zero levels as was the case demonstrated in these birds. On the other hand, the birds could have been incubating the disease and would have shown clinical disease if they were sampled several days later. However, the two hypotheses may not have been the underlying explanation and therefore, this phenomenon requires further investigation. Although virulent NDV isolates have been recovered from healthy looking birds previously (Awan et al 1994), it was not recorded whether these birds had high antibody levels. For the one isolate in our study that was recovered from a healthy looking bird with high antibody titre, one can explain why the bird still looked healthy. In this case, though infected, it can be taken that the birds had been protected from clinical disease by the antibodies. This is what normally occurs when vaccinated birds are challenged by virulent virus in an epidemic. The birds with protective levels of antibodies get infected but they overcome this infection by neutralizing the virus and infection does not progress into full disease (Alexander 1997).

Conclusions

• The present study indicates that NDV occurrence in Kenya is higher in village chickens that are kept in warm (LM5) than cold climates (LH1).

• Further, the study demonstrates for the first time that in Kenya, factors such as climate, ecology, age and sex of birds influence the viral carriage in the village birds.

• It is still not clear why healthy looking birds yield virulent NDV and this requires further investigation.
   

Acknowledgments 

The authors would like to thank the poultry farmers in Embu and Mbeere districts and Ann Munene, Mary Mutune and Rose Nyawira for technical assistance; and Mr. Crispin Matere for data analysis. Prof. John E. Olsen is acknowledged for his constructive criticism. This study was funded by the Danish International Development Agency (DANIDA) through the ENRECA project ‘Productivity and health of smallholder livestock in Eastern Africa.’

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Received 10 February 2010; Accepted 22 March 2010; Published 1 May 2010

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