Disparate HI titre dynamics following Newcastle disease vaccination to six local chicken ecotypes of Tanzania

P L M Msoffe, U M Minga**, M M A Mtambo and P S Gwakisa*

Department of Veterinary Medicine and Public Health, P.O. Box 3021, Morogoro, Tanzania
*Department of Veterinary Microbiology and Parasitology, P. O. Box 3019,Morogoro, Tanzania
**Open University of Tanzania, P. O. Box 23409, Dar es salaam, Tanzania
makengamsoffe@yahoo.co.uk

Abstract

A study was conducted to evaluate the HI dynamics of six local chicken ecotypes of Tanzania to Newcastle disease vaccine. The ecotypes were locally known as Ching'wekwe, Mbeya, Morogoro-medium, Pemba, Tanga and Unguja. Parents were vaccinated and HI titres measured after two weeks on hens. Three weeks later, eggs from vaccinated hens and later chicks hatching from the eggs were also tested for HI titres profiles. Some chicks were monitored until their HI titres were down to zero and then were vaccinated against Newcastle disease. HI titres were monitored weekly for 28 days; 70 days later, the chicks were re-vaccinated and titres were observed for a further 28 days.

The HI titres in chicks were higher than those of hens and eggs. Tanga ecotype showed early protective immunity while Morogoro-medium and Mbeya ecotypes showed persistently higher responses. There is an indication from these results that local chicken ecotypes have divergent responses towards Newcastle disease vaccine and that these findings can be useful during planning and execution of ND control programmes. Further studies are required to establish the factors that are associated with these variations.

Key words: haemagglutination, local chicken ecotypes, Newcastle disease, vaccination

Introduction

The free-range local chicken (FRLC) industry is characterized by low production, high morbidity and high losses due to diseases and predation (Kitalyi 1998). Diseases are reported to be the major hindrance with Newcastle disease (ND) the leading cause of mortalities in the FRLC (Melewas 1989; Minga et al 1989; Awan et al 1994). Other diseases such as Gumboro, coccidiosis, fowl pox, fowl typhoid, fowl cholera, infectious coryza, chronic respiratory disease (CRD) and both internal and external parasites have also been reported (Melewas 1989; Yongolo 1996). However, the local chickens survive amidst all these constraints with virtually no or very little input from the owners (Crawford 1984). This has led to the assumption that local chickens are naturally resistant to diseases (Kulube 1990; Chrysostome et al 1995).

However, there are very limited research findings supporting this assumption. In one study, five local chicken ecotypes in Tanzania were described and one ecotype was shown to have some resistance to experimental fowl typhoid (Msoffe et al 2002). Nevertheless, the study did not involve all the possible ecotypes found in the region and the sample size was small. In another study, the Nigerian local chickens were found to be more susceptible to Gumboro disease than the exotic chickens (Okoye and Aba-Adulugba 1998). Currently there is no report that indicates resistance of chickens to ND.

Disease resistance is a trait often controlled by multiple genes as well as interactions between several factors (Hartmann 1997). Testing of disease resistance potential can be direct, by infecting the host with the virulent pathogens (Okoye and Aba-Adulugba 1998; Permin and Ranvig 2001; Mdegela et al 2002). From the infection studies response of the host towards the pathogen (ranging from effect on production to mortality) is evaluated. However, some pathogens of high virulence such as the ND virus will cause severe disease and mortalities in the host precluding chances for efficient observation as well as raising hosts' welfare issues. Indirect measurements of the immune response parameters such as antibody production, cellular dynamics and complement activation following challenge with non-pathogenic immunogenic substances, vaccines or low virulence pathogens is hence a relevant option (Prescot et al 1982; Max et al 1997; Msoffe et al 2001).

This study was undertaken to test the disease resistance potential in the local chicken ecotypes of Tanzania using ND vaccine.

Materials and methods

Specimens

This experiment utilized sera from hens and chicks as well as yolk materials totalling 87 for each group. These specimens were collected from six local chicken ecotypes namely Mbeya (N=14), Morogoro-medium (16), Ching'wekwe (16), Pemba (15), Tanga (12) and Unguja (14). The description of the chickens was done on our earlier publication (Msoffe et al 2004). In addition a total of 90 sera (15 from each ecotype) from chicks whose HI status was followed down to zero (naïve) after hatching from ND vaccinated dams were also included.

Experimental design

The hens from the parental stock (Msoffe et al 2004) were vaccinated against Newcastle disease (La Sota strain, Hipra Laborotorios, SA, Spain) by eye drop method. A fortnight later sera were prepared separately for each chicken ecotype. Eggs laid on the third week after vaccination were collected separately for each ecotype and some were incubated while others were stored in the refrigerator for determination of HI titres from the yolk. Chicks (same numbers as hens) from each ecotype hatching from eggs of vaccinated hens were sacrificed within the first three days of their life and sera were prepared separately for each ecotype. The haemagglutination inhibition (HI) test was then conducted on sera and egg yolk of local chicken ecotypes that were inoculated with a live Newcastle disease vaccine.

HI titres determination

HI titres for sera obtained from hens and chicks were determined following a procedure described by Allan and Gough (1974). The standard antigen used in HI was locally prepared in the virology laboratory Sokoine University of Agriculture. From the eggs, the yolk material was carefully prepared by breaking each egg into a separate petri dish, removing the albumen and incising the yolk membrane. One millilitre of yolk material was taken and diluted into 9ml PBS (making a 10% yolk solution). HI procedure similar to that performed in the sera was conducted.

For the chicks whose HI titres were monitored down to zero after hatching (four weeks were required to achieve this), 15 chicks from each ecotype were then vaccinated using live ND La Sota vaccine. The sero-conversion following vaccination was monitored by HI test at weekly intervals for four weeks (28 days) then on day 63 after immunisation. On day 70 the chicks were re-immunized with the ND vaccine and the HI titres were monitored for a further 28 days. The rationale for re-vaccination was because HI titres in some individuals were falling below the acceptable protective levels.

Statistical analysis

HI titres data were analysed using Statistix ® statistical package where descriptive statistics, analysis of variance (ANOVA) and comparisons of means (LSD) were conducted at 5% level of significance.

Results

HI titres in hens, eggs and chicks from the local chicken ecotypes

Table I shows the overall mean HI titres as measured in hens, eggs and chicks. It is seen that the mean HI titres in chicks were significantly higher than those in hens and eggs (P<0.05). However, the range of the three datasets was not different.

Figure 1 shows the mean HI titres (expressed as Log₂10) in the three categories separately for each local chicken ecotype. It was observed that Pemba ecotype hens had the lowest mean (5.4), which differed significantly from that of Tanga ecotype. Tanga ecotype had the highest mean HI titre (7.2) that was significantly different from Ching'wekwe, Mbeya, Morogoro-medium and Unguja ecotypes with HI titres means of 5.6, 6.2, 5.9, and 5.6 respectively (P<0.05).

Mean HI titres in eggs revealed Morogoro-medium and Unguja ecotypes as having the lowest values that were significantly different from Ching'wekwe, Mbeya and Tanga ecotypes. In chicks, the mean HI titres were the lowest in the Ching'wekwe and highest in the Pemba ecotypes and these two had HI titre means that were significantly different from each other (P<0.05). The mean HI titre for Morogoro-medium and Tanga ecotypes were similar but significantly different from that of Pemba ecotype.

Dynamics of HI titres in naïve local chicken ecotypes immunised with ND vaccine

The overall HI mean titres for the local chicken ecotypes following two consecutive immunizations with ND vaccine are shown in Figure 2 and 3. It was observed that there was a two-fold increase in the overall mean HI titre from day 7 to day 14 after immunization. There was only a slight increase between day 14 and day 28 after immunisation. The mean HI titres decreased from 5.6 to 3.7 between day 28 and day 63. After the second vaccination, the mean HI titre rise seemed slower than in the first vaccination and by day 28 after re-immunisation there was already a decreasing trend. Comparison of the overall HI titre means shows that in the first immunisation, the day seven value was significantly lower than other values. The mean HI titre for day 28 was significantly higher than mean day 7, day 14 and day 63 but not day 21. Following the second immunisation on day 70, the mean HI titres for day 1 were significantly lower (P<0.05) compared to other days. The mean HI titres on days 14, 21 and 28 were statistically similar.

Viewing each ecotype separately (Figure 2) it was seen that seven days after the first immunisation, Morogoro-medium ecotype had not yet sero-converted, whilst Tanga ecotype had a mean HI titre above 3 (with a range between 2 - 7). In the subsequent days (days 14, 21, 28 and 63) the Mbeya ecotype showed a high and consistent mean HI titre. There was a difference on the peaking of the mean HI titres in the different local chicken ecotypes. Pemba, Tanga and Unguja ecotypes showed their peak mean HI titres on day 21 while the other ecotypes peaked on day 28 after immunisation. Except for Tanga ecotype, all other ecotypes had a mean value at day 63 that was above the day 7 HI mean.

On re-immunization (Figure 3), most chicken ecotypes showed an increase in the mean HI titres up to day 21, followed by a decrease on day 28 after re-immunization. Deviations from this trend were shown by the Mbeya ecotype whose mean remained constant from day 14 onwards and Morogoro-medium ecotype whose mean peaked on day 21 after immunisation. The existence of wide ranges within the dataset indicates the presence of chickens with very low HI titres and some with very high titres. Comparing the means for the re-immunisation indicated significant variation on the mean HI titres between the ecotypes except on the day 1 values. Tanga ecotype showed consistently lower mean HI titres for the whole experimental period. This lower mean HI titre was significantly different from that of Ching'wekwe ecotype (throughout the experiment), Morogoro-medium and Pemba ecotypes (on day 7 and 14), and the rest of the ecotypes (on day 7). The Ching'wekwe ecotype showed a high mean HI titre throughout the experimental period. However, the high mean HI titres were only significantly different to (apart from that of Tanga ecotype) the Unguja ecotype on days 14 and 21 after re-immunisation. Other ecotypes showed means that were not significantly different from each other.

Discussion

HI titres in hens, eggs and chicks

The overall mean HI titres for the three categories showed that the titres were significantly higher in chicks compared to either hens or eggs. The comparable HI titre means between eggs and hens provides the possibility of using eggs to test the flock immunity instead of the invasive blood/serum method. This is particularly useful in free-range chickens where eggs would be easier to acquire than the chickens themselves. Although there were significant differences in mean HI titres between the different categories of the local chickens ecotypes, chicks had high titres across all the ecotypes. The differences between ecotypes with regard to antibody responses to ND vaccine are consistent with earlier reports by Gwakisa et al (1994). In that study, divergent immune responses were observed between four local chicken ecotypes of Tanzania. However, the criteria for the description of ecotype was different in the two studies.

The results of the current work showed that the mean HI titres for chicks across all ecotypes were above 6Log_2. This indicated that regardless of the ecotype, chicks hatched from eggs of recently vaccinated hens had sufficient protective maternal antibodies. This observation is in agreement with an earlier hypothesis that day old chicks from immunised parents possessed high level of maternal antibodies to prevent a haematological spread of the virulent virus during the first seven days of life (Kouwenhoven 1993). Allan and Gough, (1974), have reported that HI titres of 3log₂ were protective hence the current experiment has shown that vaccination of free-range hens would offer protection to the offsprings. The maternally derived protection waned gradually and was down to zero in four weeks after hatch. This is consistent with previous studies that reported maternally derived antibodies for up to five weeks (Awan et al 1994).

HI titres in immunised chicks

The results on the overall responses of the chicks whose HI status was zero at the time of the immunization showed that chicks were barely protected at day seven after immunisation. This observation is based on protection afforded by HI titres of 3log₂ (Allan and Gough 1974), although birds with HI titres below 3log₂ may also be protected probably by cell mediated or mucosal immunity (Beard and Hanson 1984). However, the range of 1 to 7log₂ was wide enough to suspect between ecotype variations. Results from day 14 to 28 and the subsequent immunization on day 70 showed that the chickens had protective HI titres.

When the local chicken ecotypes were compared on their response to ND vaccine, significant differences in mean HI titres were observed. It was clear that only a few individuals responded seven days after immunisation and in one ecotype (Morogoro-medium) there was no response at all. The mean and the range for the responding ecotypes indicated that some chickens had already attained the protective HI titre within the first week and could hence be protected. For instance mean HI titre for Mbeya ecotype was 3.3 (ranging from 2 to 7) while that of Morogoro-medium ecotype was zero. Early response to immunization would be of great advantage when it comes to survival during ND outbreaks. In two separate studies Mtambo et al (1999) and Waihenya et al (2002), reported that high levels of HI antibodies contributed towards the recovery of the infected birds. Early responders to immunisation will most probably attain high HI titres before the disease outbreak when strategic immunisation is applied. Bearing in mind that the most prevalent local chicken ecotype in Tanzania is the Morogoro-medium, this information would be invaluable in strategic mass vaccination campaigns for the free-range chickens in the country.

It was shown in the current study that two months after immunization although the mean HI titres indicated protective levels, but the range indicated that some chickens within each ecotype were no longer protected. This may be an important fact in explaining the few ND deaths that occurs in free-range local chickens vaccinated at three months intervals (Unpublished observations). The lack of significant differences between ecotypes was an indication that the waning of the HI antibodies was common to all ecotypes. The slow response during re-vaccination may be due to the fact that some chickens were re-vaccinated while still having high HI titres from the first vaccination. Earlier studies have shown differences in opinion on whether susceptibility to ND was genetically controlled. While Higgins and Shortridge (1988), were of the opinion that there were no host genetic influences, Ratanasethakul (1989), believed the native chickens to be more resistant. Nevertheless, recent observations suggest that limited evidence exists to support genetic resistance to ND in chickens (Schat and Davies 2000). However, from our results the possibility of selection for increased antibody responses to live or killed ND vaccines seems to exist.

The current study is increasingly supportive of selection for high antibody responses to vaccine as well as for early responses. It has been seen in earlier studies that the response of the local chickens to sheep red blood cells could be divided into high and low responders (Msoffe et al 2001). The existence of birds with high and low, early and late response to ND vaccine in each ecotype will provide the best starting point. It would be of great interest to study the response of the high/low responders or the early/late responders to infection with the virulent virus after immunisation. It would also be of interest to find genetic markers that are associated with the named categories of chickens.

Acknowledgements

This study was supported by DANIDA through an ENRECA programme "Improvement of health and productivity of rural chickens in Africa". The authors are grateful for the support. Prof. John E Olsen and Dr. M G S Yongolo are acknowledged for their constructive criticisms. Mr. Maulid Mdaki is acknowledged for technical assistance.

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