Survey on Prevalence of Newcastle Disease Antibodies in Village Poultry at Live Birds Markets in Gombe, Nigeria

The study aimed to determine the seroprevalence of Newcastle disease (ND) amongst some village poultry species at poultry markets in Gombe, Nigeria. A total of 1200 (841 Village chickens, 320 Guinea Fowls and 39 Pigeons) sera samples were tested. Haemagglutination inhibition test revealed an overall ND virus (NDV) antibodies prevalent rate of 53.7% (644/1200). The seroprevalence of antibodies to ND was found to be higher in Village chickens 527/841 (62.7%) followed by Pigeons 19/39 (48.7%) and Guinea Fowls 98/320 (30.6%) respectively and were found to be seropositive with Geometric Mean Titres (GMT) of 1.9 to 5.9. There was no significant difference (p>0.05) in antibodies to ND seroprevalence rates among the different sampling locations. Species of poultry was found to be associated with ND seroprevalence (P ≤ 0.05) in this study. Newcastle disease antibodies was statistically significantly (P<0.0001) higher in Village chickens compared to Guinea Fowls at 95 CI (odd ratio=0.4887). However, there was no statistical significance (P=0.4106) difference of ND antibodies between Village chickens and Pigeons at 95% CI (odd ratio=0.7775) and also between pigeons and Guinea Fowls (P=0.1426 at 95% CI, odd ratio=1.591). These poultry species could play significant role in the epidemiology and transmission of the Newcastle disease to more the susceptible commercial exotic chickens or other immune deficient village poultry species especially where reared in close proximity. Therefore, free routine ND vaccination campaign should be launched in the study areas with more emphases targeting the Village poultry species in order to block the epidemic cycle of the virus. Moreover, village poultry farmers should be enlightened on the economic significance of the disease and the need to maintain strict biosecurity measures on their poultry farms.


Introduction
In developing countries, poultry production is being subjected to great pressure to meet the demand for animal protein required by the increasing human population, and also to have surplus for international trade [1]. Village poultry production, consist of edible domestic birds including chickens, ducks, guinea fowls, geese, pigeons, turkeys and quails among others which are mostly raised under the free range extensive husbandry systems especially in the sub-urban and rural areas [2][3][4]. In Africa, village poultry production system has influenced human civilization in several aspects which include economic, nutritional and socio-cultural aspects of livelihoods of poor rural households [5]. Village poultry products have ensured household food security as it supplies high quality animal protein (meat and egg) where used as food, petty cash derived from sales of poultry products, poverty alleviation and create jobs for rural dwellers [6,7]. Village poultry are also shared as gift among relatives and friends; they are also used as sacrifices during religion and cultural festivals [6][7][8]. Compared to a number of other livestock species, fewer social and religious taboos are related to the production, marketing and consumption of poultry products in developing countries of Africa including Nigeria [9]. In Nigeria, village poultry represents about 84% (115.8 million) of the 137.6 million poultry population while the commercial exotic poultry is 16% (21.7 million), thus, the village poultry industry is considered an important form of poultry production system [10][11][12][13].
Unfortunately, village poultry production is faced by a number of constraints which include predators, poor housing, management, inadequate feeding and most importantly, the menace of infectious diseases which have been identified as one of the major constraints to successful village poultry production in Nigeria [14][15][16][17]. High losses of village poultry due to diseases pose a serious threat to food security and livelihood of many rural families [15,18]. The most devastating disease of rural poultry is Newcastle disease (ND) [19][20][21][22][23]. Newcastle disease (ND) is an acute, highly infectious and highly pathogenic viral disease of domestic poultry and other species of birds regardless of variation in sex and age [24-29]. Newcastle disease virus (NDV) have previously been reported to exists in 10 serotypes of Avian Paramyxovirus (APMV); APMV-1 to APMV- 10 [30,31]. However, more recently, two distinct serotypes APMV-11 and APMV-12 of NDV have been isolated which makes it twelve (12)  The epizootiology and threat posed by ND to the poultry industry have earlier being attached mainly to the commercial exotic poultry which are reared under the intensive management system. Attention, however, were turned to the scavenging village poultry species when various strains and pathotypes of NDV were isolated from apparently healthy village chickens, ducks, guinea fowls, pigeons and other nondomesticated avian species [51][52][53][54][55][56][57][58].
Newcastle disease is usually transmitted from farm to farm mainly through introduction of live infected birds, selling and giving away sick birds, through inhalation of virus from contaminated air, ingestion of virus in contaminated feed and water [59]. Outbreaks of the disease may be associated with mortality of up to 100% [55, [60][61][62][63]. Newcastle disease is economically significant since it causes high morbidity and mortality, reduces egg production, deteriorates egg quality and impairs live performance [64,65]. It has been argued that Newcastle disease may represent a bigger drain on the world economy than any other animal viral disease since it also affects international trade [19,26,31,40,66] Due to the severe nature of ND and the related consequences, NDV is included in "LISTED" agents (reportable disease) by Office International des Epizooties (OIE) [67,68]. Notification is required by OIE of any outbreak of ND [69], when it meets certain criteria of virulence [40,43]. The first documented outbreak of ND in Nigeria occurred between December, 1952 and February 1953 in and around Ibadan [22]. The disease has since this time remained a notable problem in poultry production systems in the country [70] and has become endemic with annual epidemics recorded in highly susceptible flocks with pockets of outbreaks occurring in between the annual epidemic periods [22,63]. Outbreaks of Newcastle disease in Nigeria were reported to be more likely in farms that rear exotic birds mixed with village poultry species [11,[71][72][73]. Although, the village poultry are apparently more resistance to the disease but the fact that they serves as NDV carriers and reservoirs they tend to exert a high viral pressure on the exotic commercial flocks thus revealing the slightest failure in vaccination [74]. The disease was also reported to be more common during the cold dry harmattan period (November-March) [61,73,75,76). Newcastle disease has been reported endemic in many developing countries of Africa such as Kenya [77,78]; Cameroon [74,79]; Tanzania [80]; Ethiopia [81,82] and Egypt [83]. In Nigeria, several prevalence studies on Newcastle disease virus antibodies in village poultry have been conducted [21,22,50,53,56,58,[84][85][86][87][88]. Moreover, the prevalence of Newcastle disease have been categorically reported in most parts of the Northern Nigeria such as Sokoto State [59] [84,88]. Establishment of Newcastle disease status in Gombe is therefore of great importance. This present study therefore aimed at detecting antibodies to Newcastle disease virus infection from some village poultry species present for slaughter in major live birds markets that serves as main collection center of sales of village poultry species brought into Gombe metropolis from different parts of Gombe State and other neighboring States.

Study area
This study was carried out in Gombe, the capital city of Gombe State, and Northeastern Nigeria. Gombe township lies between Latitude 10° 08' N and 11° 24' E and longitude 11° 02' N and 11° 18'E of the Greenwich Meridian. The size of the town is 20,265 km 2 , with an estimated population of 261,536 inhabitants. Gombe town is between 400-450 feet, above sea level. The climatic and edaphic factors favor crop and livestock agriculture. The occupation of most of the inhabitants is agriculture which includes food and cash crop production; village poultry, cattle, sheep and goat rearing under the extensive and semi-intensive animal husbandry management systems. The annual rainfall ranges between 850-1000 mm, with two distinct seasons. The rainy season which starts from May to October and dry season, from November-April. Average daily temperatures are 34°C in April and 27°C in August. The relative humidity ranges from 70-80% in August and decreases to about 15-20% in December. The natural vegetation is typically that of the Sudano-Sahelian Savannah, which is composed of shrubs, herbs, grasses and sparsely distributed trees. Cereals such as ground nut, maize, guinea corn, millet and cowpea are predominantly grown in the area and provide enough fodder for the animals [95,96]. species (Village chickens, guinea fowls and pigeons) with no previous history of conventional ND vaccination. These tubes were labeled appropriately and kept in a slanting position at room temperature to allow blood samples to clot. The clotted blood samples were centrifuged at 1,500 rpm and the serum samples were harvested using disposable Pasteur pipettes. The separated serum samples were transferred into labeled cryotubes and stored at -20°C until tested.

Sample size determination
The number of poultry required for this study was determined using the formula given by Thrusfield [97] for simple random sampling. The size of sample was determining using 95% level of confidence, 50% expected prevalence since there was no previous work in this study area and 0.05% desired absolute precision. Therefore, a total of 841 village chickens, 320 guinea fowls and 39 pigeons which give overall total of 1200 sera that were sampled.

Source of Newcastle disease vaccine
The Newcastle disease vaccine (LaSota) that was used for the Haemagglutination Inhibition (HI) test was obtained from the Viral Research Department, National Veterinary Research Institute (NVRI), Vom, Plateau State, Nigeria. The Haemagglutination (HA) and Haemagglutination Inhibition (HI) test was carried out according to the FAO protocol [98].

Serology
Serum samples were tested for Newcastle disease virus specific antibodies using a modification of the Haemagglutination Inhibition (HI) test as previously described by Baba et al. [99]. ND-HI titer of log23 and above is generally accepted as positive for specific immunity [100].

Data analysis
Haemagglutination inhibition titres obtained were expressed as geometric mean titre (GMT) values according to the method described by Garner et al., using the formula X geo =antilog 10 {1/n (∑f i log 10 X i )} where n=number tested, X i =the reciprocal of dilution and f i =frequency. The data obtained from the study were stored and coded accordingly using Microsoft Excel-2007. And data collected were analyzed by the Statistical Package for Social Sciences (SPSS) version 17.0. Chi-square test was conducted to find out the association between the occurrence of ND antibodies and the species of village poultry. The prevalence was expressed in percentage. Significance was determined when p<0.05. Calculation of the lower and upper limits of the 95% confidence interval for a proportion was done according to the methods described by Newcombe [101].

Results
An overall prevalence rate of 644/1200 (53.7%) was observed among the different species of village poultry examined. Out of 841 village chickens sera samples tested 527 (62.7%) were seropositive for ND antibodies, while out of 320 guinea fowls and 39 pigeons sera tested, 98 (30.6%) and 19 (48.7%) were seropositive for ND antibodies respectively. The ND seroprevalence in the different poultry species sera surveyed was expressed in Geometric Mean Titre (GMT) values and these ranges from 1.9 to 5.9 (Table 1). Table 1 Prevalence and Distribution of Newcastle disease HI antibody titres in different types of poultry species in Gombe, Nigeria (HI antibody titre of ≥ 4log2 was considered protective). The results of seroprevalence of Newcastle disease antibodies in village chickens, guinea fowls and pigeons from different poultry markets/dressing slabs in Gombe revealed that no poultry markets/dressing slabs was found to be free of ND antibodies. There was no statistical significance (P>0.05) noted in the distribution of the ND HI antibodies in all the poultry markets sampled, but the seroprevalence among the different poultry species varied significantly (P<0.05). The highest prevalence rate among village chickens sera tested occurred in Gombe main market (72.2%) and Pantami market (70.9%), while the lowest prevalence rate occurred in Dukku park market (47.8%). However, the highest prevalence rate among guinea fowls sera tested occurred in Pantami market (45.8%), while the lowest prevalence rate occurred in Dukku park market (20.3%). Moreover, the highest prevalence rate among pigeons sera tested occurred in Gombe main market (71.4%), while the lowest prevalence rate occurred in Tudun wade market (21.4%) ( Table 2). The result of comparative analysis of seroprevalence of Newcastle disease antibodies among the different village poultry species in Gombe shows that ND seroprevalence was statistically significantly (P<0.0001) higher in Village chickens compared to guinea fowls at 95% CI (odd ratio=0.4887). However, there was no statistical significance (P=0.4106) difference of ND antibodies between village chickens and pigeons at 95% CI (odd ratio=0.7775) and also between pigeons and guinea fowls (P=0.1426 at 95% CI, odd ratio=1.591) ( Table 3). Key: N, number of sera sampled; n, number of HI/HA positive sera; 95% Confidence interval; P ≤ 0.05 was considered as significant.

Discussion
Newcastle disease (ND) is considered an endemic disease among backyard and commercial poultry industry in Nigeria. Despite vigorous vaccination trials and campaigns there were still reports of frequent outbreaks of the disease in the country among village and commercial poultry population [74,102]. However, seroprevalence survey still remains an important step towards immediate detection and effective control of Newcastle disease (ND) among poultry species in Nigeria where the disease is endemic. Haemagglutination Inhibition (HI) antibody titer between 0log 2 and 3log 2 is considered negative because they produce no antibody against the virus while HI antibody titer between 4log 2 and 8log 2 is considered positive for antibodies production against the virus based on OIE recommendation of 2000 [103,104]. The overall seroprevalence of Newcastle disease virus (NDV) antibodies in village poultry in this study was 53.7%, this findings tally with 55.5% previously reported by Lawal et al. [105] in a ten years retrospective study of ND cases reported and diagnosed in Veterinary clinics in Gombe metropolis. The occurrence of antibodies to ND in the tested sera of apparently healthy village poultry species with no previous history of conventional ND vaccination as observed in this study is an indication of natural infection by non-virulent strain of NDV which may not cause clinical disease but act like vaccine. However, infection may be due to non-virulent strain of ND which might have stimulated immune response of the seropositive birds. The prevalence rate is higher in village chickens (62.7%) compared to pigeon (48.7%) and guinea fowls (30.6%). This suggested that the village chickens are more susceptible to the Newcastle disease virus than other village poultry species in the study area and should be considered as an important factor in the epidemiology of the disease. The economic importance of ND to the village poultry farmers is not only due to the high mortality rate in infected birds but that such affected birds if survived could eventually become carriers and enhance the dissemination of the virus to other healthy susceptible flocks. Of interest in this study is the detection of NDV antibodies from the village poultry sampled in all the live birds markets in Gombe metropolis. This observation shows that ND naturally infected birds are brought to these markets from various localities for sale and are either bought for human consumption or replacement in poultry flocks. This could enhance the continuous circulation and transmission of NDV amongst susceptible birds in the study area. However, It is noteworthy that these village poultry species have no history of previous vaccinated against Newcastle disease, therefore, detection of ND antibodies is an indication of natural infection and since the poultry species sampled are adults birds the presence of maternal antibodies can be ruled out, because maternal antibodies disappear after the age of 3-4 weeks [21].
The seroprevalence of 53.7% NDV antibodies recorded in village chickens in this study was closer to those reported from some States which shares bounders with Gombe State, 56.3% was reported in Bauchi State [57] and 51.0% in Borno State [21]. However, it is lower than 74.3% previously reported in Maiduguri [19,20] but higher than 34.5% reported by Sule et al. [90] in Yobe State. In Adamawa State, Bobbo et al. [106] have reported the susceptibility of three phenotypes (Frizzle, Naked neck and Smooth feathered types) of Village chickens to ND which revealed that no ecotype of village in Northeastern Nigeria is definitely resistance to velogenic strain of the disease. Other studies conducted on village chickens at live bird markets in Nigeria by Ameji et al. [107], Chollom et al. [108], Jibril et al. [89] and Eze and Ike, showed 96%, 35.8%, 25.5% and 65.1% seroprevalence rates respectively. These observed regional differences in ND seroprevalence showed ecological area variation in NDV activity and may perhaps be a reflection of the impact of environment on the viability and spread of NDV and its epidemiology [50]. It has also been reported that most village chickens flocks in Nigeria are seldom routinely vaccinated against ND using the conventional vaccines [109]. Therefore, detection of antibodies to ND in apparently healthy chickens is indicative of natural infection by non-virulent or lentogenic strains of the virus that may not cause clinical diseases but acts like vaccine. However, the bird may be at the period of sampling be incubating the disease in a subclinical state [18,50,59,74].
The ND seroprevalent rate of 30.6% reported in guinea fowls from this study is relatively lower than 39.8% reported in Borno and Yobe State by Hassan et al. 15.0% and 13.4% reported in Jos, Plateau State by Ibu et al. [110] and Mai et al. respectively. The difference may be as a result of the sample size, the period of sample collections and diagnostic methods employed. Although fatal outbreaks of ND has been reported among guinea fowls in Nigeria despite the fact that these species of birds seem to be more resistant to ND than chickens [93,[111][112][113] have reported that Newcastle disease virus is enzootic among guinea fowls in Borno and Yobe States, Northeastern Nigeria.
However, Boakye et al. [114] recently reported 48.6% ND seroprevalence in guinea fowls in Ghana. In rural households of Northern Nigeria, it is customary to see village chickens and guinea fowls mingling and roaming around scavenging for food together, these events can create the chances of close contact for direct transmission of ND among the two species of birds.
The seroprevalence of 48.7% of NDV antibodies recorded in pigeons is higher than 44% reported by Sai'du et al. in domestic pigeons in Zaria, Kaduna State. Domestic pigeons and doves were reported to be more susceptible to ND virus than other members of the columbiformes [115]. Although, there were no reports of clinical outbreaks of ND in domesticated pigeons in Nigeria, there has only been serological evidence of ND infection in pigeons in Nigeria [116]. Pigeons are nevertheless infected with ND virus and may therefore serve as reservoirs of ND virus for the more susceptible poultry species [115,117]. Outbreak of ND had been reported from UK due to contamination of poultry feed with faeces of pigeon [118]. This report signifies that ND infected pigeons can play a significant role in transboundry transmission of the disease since they can fly several miles across regions. As far as our knowledge, this is the first time a higher prevalence rate of 48.7% is recorded in pigeons in Northeastern Nigeria. Although, HI antibody titre of ≥ 4log0032 was considered protective against NDV infection [21,119], some of the sera had reciprocal titres close to 4log2. Birds partially immuned can succumb to a velogenic NDV infection or maintain a subclinical disease during which active excretion of the virus may occur making them a possible source of infection for incontact birds [58,120,121].

Conclusion
The finding of this study revealed the presence of antibodies to ND in sera of apparently healthy village poultry species in Gombe, Nigeria. Newcastle disease antibodies were more frequent in the tested sera of Village chickens compared to sera of Pigeons and Guinea Fowls. Village chickens may have been more exposed to the virus than the other poultry species or probably their antibody levels have decreased below the threshold. Village poultry species could play a significant role in the epidemiology and transmission of the infection to the more susceptible commercial exotic chickens or immune deficient village poultry species especially where reared together or in close proximity. The disease may therefore be considered a threat to successful village poultry production system in the study area. Therefore, it is recommended that routine ND vaccination campaign should be launched in the study area. To effectively block the epidemic cycle of the virus and control this disease, more attention should be given to village poultry species. Village poultry farmers should be enlightened on the economic significance of the disease and the need to maintain strict biosecurity measures on their poultry farms. Also, researches on isolation and characterization of the spreading virus strains should be carried out in order to provide more information that could be used in formulating and planning for effective control of Newcastle disease in Gombe State.