Effects of different infectious Bursal disease vaccination regimes on biochemical and haematological parameters of indigenous chicken in Kenya

M O Bwana, L W Njagi, P N Nyaga, P G Mbuthia, L C Bebora, M W Wahome, W U Mutinda¹ and P M Kitala²

Department of Veterinary Pathology, Microbiology and Parasitology, University of Nairobi, PO Box 29053-00625, Kangemi, Nairobi, Kenya
bwanamoses@gmail.com
¹ Department of Animal Science, Pwani University, PO Box 195-80108, Kilifi Kenya
² Department of Veterinary Public Health, Pharmacology and Toxicology, University of Nairobi, PO Box 29053-00625, Kangemi, Nairobi, Kenya

Abstract

The effects of four infectious bursal disease (IBD) vaccination regimes on glucose, plasma protein, differential white blood cell counts and heterophil to lymphocyte (H/L) ratios in specific antibody negative (SAN) indigenous chicken were evaluated. A total of twenty SAN indigenous chicks were divided into five groups of four chicks each, under different IBD vaccination regimes. Group 1 received Jovac D78^®alone, Group 2 received K1 (local inactivated vaccine), Group 3 received combined D78^® and K1 on the same day while Group 4 birds received D78^® and K1 30 days apart and Group 5 was control. Birds were bled and data collected weekly for seven weeks. Data were analysed using GraphPad Prism Version 7.01.

Group 3 and Group 4 had the highest initial mean weekly total plasma protein levels (3.75g/dL) two weeks after primary IBD vaccination. There were significant differences (p˂0.05) in plasma protein levels between Groups 1 and 3 and the control and between groups 1 and 2. Significant (p˂0.05) correlations between mean weekly plasma protein and glucose levels (r=0.39) were observed in Group 3. There were significant (p˂0.05) increases in mean weekly relative counts of lymphocytes 14 days after primary vaccination in groups 1, 3 and 4. Heterophil to Lymphocyte (H/L) ratios decreased with age in all the groups and had a negative correlation (r=-0.39) with plasma protein levels in groups 1 and 4. These preliminary results indicate that use of the imported D78^® IBD vaccine alone; and in combination with the caandidate local K1vaccine in a prime-boost program gives the best outcomes in the biochemical and hematologic parameters associated with immunity in indigenous chickens in Kenya.

Key words: H/L ratios, plasma Protein levels, prime-boost program

Introduction

Infectious bursal (Gumboro) disease is one of the most important contagious viral diseases of young chicken in the global poultry industry (Gardin et al 2009). The disease is caused by an Avibirnavirus of the Birnaviridae family of viruses with a bi-segmented ds-RNA genome designated the Infectious Bursal Disease (IBD) virus (Sara et al 2014).  The virus is highly stable and resistant to many physical and chemical agents and prone to extreme antigenic variability with increased virulence (Jackwood and Sommer-Wagner, 2011). Chicken are more susceptible at 3 to 6 weeks of age and get infected via the faecal-oral route in a direct mode of transmission since the virus is shed in high amounts in faeces from 48 hours up to 2 weeks post infection (El-mahdy et al 2013). The IBD virus, like most poultry viruses, has no specifically known treatment and vaccination remains its main prevention and control strategy (Gardin et al 2009).

The disease is immunosuppressive in nature and causes high economic losses in indigenous chicken compared to other categories of chicken in Kenya (Mutinda et al 2013). This is further complicated by lack of a standard vaccination regime for indigenous village chickens against the disease coupled with a poor evaluation and validation of the available imported vaccines which hampers real prevention and control of the disease in Kenya. Development of a new poultry vaccination program from scratch is often a time consuming and costly process hence not tenable during emergencies and in resource-poor settings of the developing world (Gardin et al 2015). This can however be overcome by the much faster specialized screening of existing standardised IBD vaccination programs for other chicken breeds under different combinations to reveal the best program that may work against the disease in indigenous village chicken (Parker and Sjaak, 2014).  Screening for a vaccination program is a three step process that involves an initial laboratory evaluation of the effects of the existing programs on key immunity and fitness markers in the host of interest such as total plasma proteins, glucose levels, white blood cell counts and Heterophil to lymphocyte ratios (Ojiezeh et al 2014), followed by testing the efficacy of the programs and a final field trial of the identified vaccination program (Sarachai et al 2010).

The primary aim of this study was therefore to evaluate the effects of four IBD vaccination regimes on glucose concentration, plasma proteins and differential white blood cell counts in indigenous chickens with the secondary aim of providing preliminary data for the establishment of a standard vaccination regime.

Materials and Methods

Study Birds and Site

Day-old SAN indigenous chicks were obtained from a local experimental birds hatchery which did not vaccinate their chicken against IBD and had no history of outbreaks. The birds were reared in deep litter brooders for 7 days and transferred to individual cages at the department of Veterinary Pathology, Microbiology and Parasitology of the University of Nairobi. Birds were kept under standard conditions and feed and water were provided ad libitum.

Study Vaccines

The imported live attenuated IBD vaccine Jovac D78^® (10^3.0 TCID50 IBDV strain D78) was procured from local agrovet shops. Local inactivated K1 (10^3.9 EID₅₀/ml) was prepared from four samples of local isolates of IBDV (designated E7, E9, E19 and E42) collected from field outbreaks across the country and proven as good vaccine candidates based on their virulence and pathogenicity profiles in a previous study by Mutinda et al (2015a). The local IBDV isolates were propagated in four weeks old SAN indigenous chicken whose bursae of Fabricius were collected 72h after inoculation and homogenised to make 20% antibiotic treated (1000 IU/ml, streptomycin-penicillin) bursal suspensions as described by Mutinda et al (2015b). The bursal homogenate virus suspension were pooled  and the virus titre (10^3.90 EID₅₀/ml) determined by the Spearman-Kaeber method (Dougherty, 1964) after inoculation of white leghorn eggs, was used for antigen production. The rest of the pooled bursal homogenate virus suspension was used to prepare the inactivated local IBD vaccine (K1) as described by Habib et al (2006a). Briefly, 10μl of 40% formalin was added to 1990 μl of the local IBD virus suspension, to make a final concentration of 0.2%, incubated for 48 hours to inactivate the virus and clarified by filtration through 0. 22μm pore filters (Habib et al 2006a). 

Experimental Design and Vaccination Protocols

The birds (n=20) were randomly allocated into five groups (Group 1, Group 2, Group 3, Group 4 and Group 5) each consisting four wing-tagged birds. They were put under four different IBD vaccination regimes as follows:

• Group 1 birds received Jovac D78^® at 14 days of age and were boosted 14 days later.

• Group 2 birds received K1 (local killed vaccine) at 14 days of age and were boosted 14 days later.

• Group 3 birds received a combination of D78^® and K1 on the same day (14 days of age) and were boosted 14 days later with the same combination.

• Group 4 birds received K1 30 days (44 days of age) after receiving D78^® on days 14 and 21 of age respectively.

• Group 5 birds acted as control and received no vaccines.

Jovac D78^® was given via drinking water while K1 (10^3.9EID₅₀/ml) was administered intramuscularly at 0.3 ml/bird (Habib et al 2006b). Birds were bled (from 14 days of age) on days 0, 7, 14, 21, 28, 35, 42 and 49 post vaccination. No pre-vaccination treatment (deworming) was done to the birds since they were from a clean flock free from worms. The trial lasted from 25^th February to 16^th August 2016.

Measured Parameters

Blood was collected from the wing vein into labeled Hawksley’s heparinised capillary tubes (3tubes/bird), centrifuged at 2500 rpm for 5 minutes and total plasma protein was determined using a refractometer (Rwuaan et al 2009; Bahman et al 2011).  Thin blood smears (two/bird) were also made immediately after blood collection and air dried for determination of differential cell counts  and morphology;  they were fixed with absolute methanol for 1 minute,  stained with Wright- Giemsa, washed and air dried before examination at  x100 oil emulsion light microscope  (Campbell and Ellis 2007). At least 100 white cells were examined and categorized as heterophils, lymphocytes, eosinophils, monocytes, or basophils on the basis of staining characteristics and morphology in 5 microscope fields and expressed as a percentage of the total number of leucocytes counted in the five fields (Fildman et al 2000). Heterophil to Lymphocyte (H/L) ratios were determined from the differential counts data as described by Krams et al (2012). Blood glucose was measured immediately during blood sampling using a portable digital glucometer (SD Biosensor, CodeFree, Yeongtong-gu, republic of Korea) used for routine monitoring of human blood glucose levels (Gould et al 2016; Borin et al 2012).

Data Analysis

Data were analysed using GraphPad Prism Version 7.01 and differences between and variations among means were compared by Student’s t-tests and ANOVA respectively. Correlation analysis was computed for the three variables and p≤0.05 was taken to be significant.

Results

Effect of the Vaccination Regimes on Plasma Glucose levels in Indigenous Chicken

Group 1 birds showed the highest mean weekly glucose concentrations (21.95 mmol/L) while group 2 birds recorded the lowest (17.08 mmol/L) compared to the control group (19.2 mmol/L) 7 days post-primary IBD vaccination before stabilizing at levels ˃18 mmol/L in week 3 (Figure 1). There were no significant difference and variations (p˃0.05) in mean weekly glucose levels between and among the vaccine groups and control group.

Figure 1: Weekly mean glucose levels of indigenous chicken under different vaccination programs

Effects of the Regimes on Total Plasma Proteins in Indigenous Chicken

Total plasma protein level showed increases in weeks two and six respectively among most of the vaccine groups and declines in weeks 3 to 5 in all the groups except group 2 which showed a slight increase between weeks three and four (Figure 2). Group 3 and Group 4 had the highest initial mean weekly total plasma protein levels (3.75g/dL) two weeks after primary IBD vaccination, followed by Group 1 (3.9g/dL) in week six and Group 4 (3.9g/dL) in week 7, 3 and 4 weeks post-booster IBD vaccinations respectively. There were highly significant variations (p˂0.0001) among the mean weekly total protein levels of the groups and significant differences (p˂0.05)  in mean weekly total protein levels between the vaccine groups 1 and control (p=0.0001), 3 and control (p=0.0001), & 4 and control (p=0.0012) with the most significant differences seen between Groups 1 & 3 and the control. There were also significant differences (p˂0.05) between Groups 1 & 2 (p=0.0023), and 3 & 2 (p=0.0208) respectively with the most significant differences seen between groups 1 & 2.

Figure 2: Weekly mean plasma protein levels in indigenous chicken under different vaccination programs

Correlation of the Effects of the Vaccination Regimes on Total Plasma Protein Levels  

There were significant positive correlations (p˂0.05) between vaccination regime and mean weekly total plasma protein levels of Group 1 & 4 (p=0.0184, r =0.41) and Group 3 and 4 (p= 0.0002, r =0.61, Figure 3) with the closest seen between groups 3 & 4. There were also significant correlations (p˂0.05) between mean weekly plasma protein and glucose levels (r= 0.39) among Group 3 birds.

Figure 3: Relationship between the effects of the vaccination regimes of group 3 and group 4 on total plasma proteins in indigenous chicken.

Effects of the Regimes on Differential White Cell Counts

Lymphocytes: There was a general increase in mean relative counts of lymphocytes between days 0 and 14 post-vaccination in all the groups (Table 1). There were significant (p˂0.05) increases in the mean relative counts of lymphocytes on day 14 after vaccination in groups 1, 3 and 4 with the highest initial increases in groups 1 and 4 on day 14 (Table 1). High relative lymphocyte counts were associated with high total plasma protein level in groups 1, 3 and 4 on day 14 (week 2, figure 2); groups 1 and 3 on day 42 (week 6) and in groups 1 and 4 on day 49 (week 7, figure 2).  

Heterophils: In contrast to relative lymphocyte counts (Table 1), there was a general decrease in mean relative counts of Heterophils between days 0 and 14 post-primary IBD vaccination in all the groups with a slight increase on day 7 (Table 2). There were significant (p˂0.05) increases in mean relative heterophil counts on days 14 and 28 post-vaccination in group 1 (p=0.0101). Low relative heterophil counts (Table 2) were associated with high plasma protein levels in groups 1 and 4 on day 14 (week 2, figure 2).

Monocytes, Basophils and Eosinophils: There were increases in the mean relative counts of Monocytes on day 14 after primary IBD vaccination in all the vaccine groups except the control (group 5) which had a general decline in counts. The highest initial post-vaccination increase in Monocyte counts was seen in groups 1, 3 and 4 on day 14 (Table 3). There were significant differences in the mean relative counts of Monocytes on days 0 and 14 after vaccination within group 3 (p=0.0372) and on day 28 between groups 3 and 4 (p=0.0319).

There were no significant differences (p˃0.05) detected in basophil and eosinophil cell counts between the groups. Mean relative Basophil and Eosinophil counts were less than 4 % in all the groups, in the entire period, corresponding to normal physiological levels (0-4%).

Heterophil to Lymphocyte Ratios (H/L): There was a general decrease in H/L ratios with age in all the groups (Table 4). Group 4 birds had the lowest pre-vaccination (day 0) H/L ratio which corresponded with high relative lymphocyte counts, glucose and plasma protein levels on day 14 (week 2; Tables 2 & 4; figures 1 & 2). Group 3 had the highest pre-vaccination (Day 0) H/L ratio which corresponded with low relative lymphocyte cell counts on day 14 (Tables 1 & 4). There were slight increases in H/L ratios post-primary IBD vaccination in group 4 and control (group 5) birds on day 7 (Table 4).There were no significant differences in mean weekly H/L ratios between the groups but there was a significant difference (p=0.0301) within group 3 on days 0 and 42. There was a significant negative correlation between H/L ratio and total plasma proteins in group 1 (r =-0.39, p=0.026) and group 4 (r = -0.39, p=0.026).

Discussion

The costs of mounting humoral, innate and cell-mediated immune responses to a vaccine virus involves lymphocyte proliferation and diversification which require substantial energy and nutrients hence groups receiving the live IBD vaccine (Groups 1, 3 and 4) which replicates in the birds expressed the highest glucose levels 7 days post-vaccination while that receiving the inactivated IBD vaccine (Group 2) expressed the lowest levels within the same time period (Vatsalya  and Kashmiri 2011; Rauw 2012). These findings are supported by Talebi, (2006) who also observed high glucose levels (20.7 Mmol/L) 7 days post-vaccination and an average of 17.05 Mmol/L in broilers vaccinated with IBD (D78^®), infectious Bronchitis and Newcastle Disease vaccines.

The increases in total plasma protein levels observed from week 6 (42 days post vaccination) among most of the vaccinated groups are similar to the findings of Talebi (2006) and Rwuaan et al (2009) who had increases from day 42 (our week six) when birds were vaccinated with IBD (D78^®), infectious Bronchitis and Newcastle Disease vaccines; and corresponds to the longer lasting secondary immune response in the form of immunoglobulins after primary exposure to the vaccine antigen (Angela 2011). The early initial increases of plasma proteins (weeks 2 and 3 post-vaccination) seen in birds receiving the live and a combination of the live and inactivated IBD vaccines (groups2, 3 and 4) correspond to the ages when chicken are most susceptible to IBD hence may indicate primary (early) immune responses in the form of immunoglobulins elicited by their respective vaccination regimes (El-mahdy et al 2013). Live intermediate plus IBD vaccines are much faster in stimulating immune responses and elicit a more rapid onset of immunity often seen within 3 days post-vaccination as is alluded to by the high plasma protein levels in Group 1 birds receiving the live D78^® IBD vaccine (Gardin et al 2015). Combinations of a live and an inactivated IBD vaccine in prime-boost program is theorised to give the best protective immune responses since the booster doses are thought to outlive the maternal antibodies barrier to vaccine response (Gardin et al 2015); which is confirmed by the high plasma protein levels seen in group 3 birds receiving a combination of the D78 and K1 IBD vaccines. The close correlation (r=0.61) in plasma protein levels between group 3 and 4 birds suggest an almost similar effect of their vaccination regimes on total plasma protein levels which is further validated by the fact that they both had a combination of the imported Jovac D78^® and the local killed (K1) IBD vaccines (Jackwood et al 1999). The positive correlation (r= 0.39) between mean weekly plasma protein and glucose levels among groups 3 birds suggest an almost linear relationship between the variables as a result of the combination of the D78^® and K1 IBD vaccines in their vaccination regimes (Vatsalya & Kashmiri 2011; Rauw 2012).

Lymphocyte numbers are often positively correlated with the levels of complement and antibody diversity hence protective immunity (Matson et al. 2005). The general increase in mean relative counts of lymphocytes between days 0 and 14 post-vaccination in all the groups are contrary to the findings of Hassan and Ghada (2016) who observed a decrease in lymphocyte counts when broilers were vaccinated with a live IBD vaccine. These findings are however similar to those of Gottstein et al (2015) who found gradual increases in all groups but with decreases on days 14 and 21 when hybrid chicks were vaccinated with an HVT FC Mareks Disease vaccine which indicates a successful antigen presentation and stimulation of the acquired immune system. The regimes of groups 1, 3 and 4 stimulated a high degree of lymphocyte proliferation after the first and second vaccinations which correlated with high plasma protein levels in the form of immunoglobulins and Cytokines, which in turn correspond to immune protection against IBD after the first two weeks of life in indigenous chicken (Ali et al 1997; Liu et al 2010). Heterophils are highly active phagocytic cells which generally arrive first at a site of inflammation due to antigen challenge (Angela 2011). The general decrease in mean relative counts of Heterophils between days 0 and 14 post-vaccination in all the groups is similar to the observations of Hassan and Ghada (2016) who observed an average heterophil count of 45% and Gottstein et al (2015) who found declines in all groups but with slight increases on days 14 and 21 when broilers and hybrid chicks were vaccinated with a live IBD and Mareks Disease vaccines, respectively. An intense heterophil proliferation often follows increased heterophil functional efficiency as a result of the presence of pro-inflammatory cytokine/chemokine (IL-1β) produced after the first recognition of conserved molecular motifs (dsRNA) unique to a particular antigen; as was confirmed in group 1 after the second vaccination (day 28)with a live D78^® IBD vaccine (Swaggerty et al 2006). The association of low relative heterophil counts with high plasma protein levels in groups 1 and 4 on day 14 could be due to the relative increase in lymphocyte counts after a successful antigen presentation to the acquired immune system (Matson et al 2005).

Monocytes are blood cells which become macrophages in tissues and are important in antigen presentation, phagocytosis, cytokine production, antiviral and cytotoxic activities (Angela 2011). Increases in the mean relative counts of Monocytes seen on day 14 after IBD vaccination indicate activation of the innate immune system which modulates the quality, strength and persistence of the adaptive immune responses (Angela 2011). Group 3 birds receiving a combination of D78^® and K1 on the same day simulated a better monocyte proliferation after the first vaccination due to the two modes of vaccine antigen exposure; Intramuscularly (K1) stimulating macrophages in tissues and Orally (D78^®), stimulating other antigen presenting cells in the gut associated lymphoid tissues (Angela 2011). Basophils in blood and mast cells in tissues release histamine and are important in the development of allergies while Eosinophils are phagocytic cells that are more important in resistance to parasites (Angela 2011).

H/L ratios can be used both as a determinant of the magnitude of the humoral immune response in terms of antibodies to the vaccine antigens and also as an index of acute and chronic stress in indigenous chicken (Krams et al 2012). The observed decrease in H/L ratio with age is similar to the finding of Ojiezeh et al (2014); they found a similar decline when broilers and local chicken were vaccinated with Newcastle Disease vaccines in a prime-boost regime. High pre-vaccination H/L ratios seen in group 3 could be due to increased blood corticosterone levels due to stress which supresses lymphocyte proliferation hence antibody production while low pre-vaccination H/L ratios seen in group 4 birds could indicate no stress hence stimulation of lymphocyte proliferation and antibody production (Shini et al 2010; Krams et al 2012). The slight increase in H/L ratios post-vaccination in group 4 and Control (group 5) birds on day 7 (Table 5) could be associated with the stress of handling during vaccination or a mild opportunistic infection (Zulkifli et al 2000; Shini 2003). The negative correlation between H/L ratios and total plasma protein in groups 1 and 4 birds suggests an inverse relationship between the two variables (Rauw 2012).

Conclusion

• The results of this study indicate that use of the imported D78^® vaccine alone (group 1) and in combination with the experimental local K1 vaccine in a prime-boost program, as demonstrated by group 3 and 4 birds, gives the best outcomes in biochemical and hematologic parameters associated with immunity in indigenous chickens in Kenya.

  
  

• To establish a standard vaccination regime, further humoral immune response, efficacy, optimal time of vaccination and field trials data are needed.

Acknowledgement

This work was funded under the RUFORUM Graduate Research Grant -128 awarded to the University of Nairobi. The corresponding author also expresses his gratitude to the Department of Veterinary Pathology, Microbiology and Parasitology staff; Mary, Lydia, Nyaga and Jack, who assisted in the study.

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Received 13 February 2017; Accepted 16 February 2017; Published 1 May 2017

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