Infectious Bronchitis Vaccination Protocols for Laying Hens

A research was conducted to investigate the effects of vaccination protocols for Infectious Bronchitis (IB) on egg production, egg quality, and IB antibody titres of laying hens. Different initial vaccination (Control, VicS eye, VicS spray, VicS water, A3 eye, A3 spray, and A3 water) for IB were administered to day-old Isa Brown hens. Half the hens were revaccinated regularly during lay whereas the other hens were not vaccinated. Results showed that initial vaccination treatment had significant effects on hen day egg production and egg quality of egg weight, shell reflectivity, shell breaking strength, shell thickness, albumen height, Haugh Units, and IB antibody titre levels, but had no effect on percentage of shell and yolk colour. Egg weight and shell reflectivity were less favourable in the control hens. In contrast, shell breaking strength and shell thickness were highest for the group that initially received A3 vaccine in water. However, regular revaccination had some deleterious effects on egg production and egg quality. There were no significant effects of revaccination on IB antibody titres. It is concluded that there was little advantage in regularly revaccinating laying hens for IB virus, since they had received appropriate initial vaccination.


INTRODUCTION
Infectious bronchitis (IB) is a highly infectious and contagious respiratory virus disease which has been widespread and identifi ed all over the world. The infec-tion has a great economical eff ect on the layer industry, because it aff ects on egg production. Besides respiratory lesions, early exposure to IB virus causes extensive damage to wide range of various epithelial surfaces of the body both in layers (Cavanagh, 2003). Chousalkar et al. (2007) reported IB virus aff ected on Harderian gland, tracheal mucosa, kidney, and region of oviduct such as the magnum, tubular shell gland, and shell gland pouch of laying hens. It is associated with nephritis and respiratory distress in broilers and with the so-called "false layer syndrome" and drops in egg production in breeders and layers. It is therefore causing major economic problems in IB-vaccinated fl ocks (de Wit et al., 2011). Butcher & Miles (2001) mentioned that for laying hens IB not only aff ects egg production but also egg shell and internal egg quality.
The syndrome may cause partial or complete failure of the oviduct to develop, and in laying period, hens will not ovulate normally. Poor egg specifi c gravity, reduced shell thickness, misshapen eggs, pale shell pigmentation, watery albumen that leads to greatly reduced Haugh Unit (HU) values are a refl ection of dysfunctional or damaged oviducts (Butcher & Miles, 2001). Therefore, it is relevant to investigate the suitability of the current vaccines in protecting birds in the fi eld.
Since there is no appropriate drug therapy for controlling this disease, increasing the resistance of birds by vaccination is still the cornerstone of strategy for protecting chickens from IBV infection (Lister, 2001). Although no homologous vaccine was available until very recently, some success is reported in controlling its eff ects using currently available IB vaccines (de Wit et al., 2011). Various IB virus serotypes are used as commercial Connecticut, Holland, Arkansas, Florida and JMK. Both live and inactivated virus vaccines are used in broilers and for the initial vaccination of breeders and layers (Cavanagh & Gelb Jr., 2008).
The most common strains of IB virus vaccine used by the egg industry were Webster's VicS (Fort Dodge) and A3 (Fort Dodge). Some producers use only one strain (usually VicS) whereas others alternate the two strains during the rearing phase. Routes of vaccine administration vary, with coarse spray at the hatchery, and either coarse spray or in water vaccination for subsequent vaccinations, being the norm. Some producers do not revaccinate for IB once the birds have come into lay. However, increasingly, poultry veterinarians are recommending regular revaccination, usually every 8 weeks throughout lay. Results from a previous experiment, using Webster's VicS IB vaccine strain with ISA brown cockerels, indicated that vaccination at either day-old or two weeks of age, by eye drop, coarse spray or water vaccination, protected birds against the eff ects of exposure to T strain IB virus.
The objectives of this experiment were to determine the eff ects of diff erent initial vaccination protocols (diff erent virus strains of vaccine and routes of vaccine administration) for IB and regular revaccination every 8 weeks during lay, as compared with no revaccination beyond 14 weeks of age on hen day egg production, egg quality and IB antibody titres of ISA Brown laying hens.

Chickens and Management
Day-old-ISA Brown layers (623 birds) were transferred to isolation pens. The birds were reared according to standard commercial practice. Seven (7) isolation fl oor sheds (2 x 3 m 2 ) were used. After being reared in isolation pens for 15 weeks, all birds were transferred to two poultry isolation sheds equipped with 3-bird commercial-style cages. Each cage had one feeder and at least two accessible drinking nipples. The birds were supplied with feed and drinking water ad lib. A pre-layer diet (pullet grower with added calcium) was fed until birds had reached 5% production, after which birds received a standard commercial layer mash. Natural light was applied until birds had reached 5% lay or 18 weeks of age, after which birds received 14 hours light (6 am to 8 pm). The light phase was increased by 30 minutes per week. Clinical signs and mortality were recorded if observed and all mortalities autopsied.

Experimental Design and Measurements
There were seven initial experimental groups set up at day-old, each of 89 birds: control (no vaccination), VicS eye (VicS vaccine by eye drop at day-old), VicS spray (VicS by coarse spray at day-old), VicS water (VicS in water at day-old), A3 eye (A3 vaccine strain by eye drop at day-old), A3 spray (A3 by coarse spray at day-old), A3 water (A3 in water at day-old). The Control group remained unvaccinated. At 14 weeks of age, all birds (including the control birds which had not been vaccinated previously) were revaccinated with VicS vaccine strain by eye drop. At 15 weeks of age, all birds were transferred to the two poultry isolation sheds. One-half of the birds from each treatment group were allocated to each shed, 2 birds per cage (a total of 14 groups). The birds in one shed (seven groups) were revaccinated every 8 weeks with VicS vaccine strain by coarse spray, whereas the birds in the other shed (the other seven groups) were not revaccinated beyond 14 weeks of age. For all vaccinated groups, the vaccines used were Webster's VicS (Fort Dodge) and A 3 (Fort Dodge) strains of IB virus. For regular revaccination, VicS vaccine strain was used and administered by coarse spray. Revaccination of the regularly revaccinated groups was conducted at 22, 30, 38, 46, and 54 weeks of age.
Egg production, average egg weight and the external appearance of the eggs were recorded daily. Percentages of egg production were calculated as henday production. On the day of lay, 30 eggs from each treatment group were weighed using an O'haus Balance. Every 4 weeks, 21 eggs from each initial vaccination treatment group from each shed were collected for egg and eggshell quality measurements. Shell breaking strength (Newton), shell refl ectivity (%), egg weight (g), albumen height (mm), Haugh Units (HU), yolk colour (Roche score), percentage shell (shell weight/egg weight, %) (using TSS egg analyser) and shell thickness (µm) (using a digital micrometer) were measured. Measurements were conducted after all eggs were stabilized in the cool room for 24 hours and were determined at 20,24,28,32,36,40,44,48, 52 and 56 weeks of age. Heparinised blood samples were centrifuged in a Hearaeus Christ centrifuge at 3000 RPM for 10 minutes to separate the blood plasma, and the blood plasma was stored frozen at -20 o C for before analysis. IB antibody titres were measured in samples of plasma using a Flock Check Infectious Bronchitis Virus Antibody Test Kit (IDEXX Laboratories).

Statistical Analysis
Analysis of variance was used to test the eff ect of initial vaccination treatment and regular revaccination on each measured parameter. Fisher's protected LSD was utilized to separate means when signifi cant eff ects were observed. Statements of statistical signifi cance were based on P<0.05 unless otherwise indicated.

Hen-day Egg Production
Initial vaccination treatment had signifi cant eff ect on hen-day egg production from 20 to 56 weeks (Table  1). Production was slightly lower for the control and A3 course spray initial vaccination treatment groups. There was eff ect of regular revaccination on hen-day egg pro-duction between 20 and 56 weeks of age, with the egg production of regularly revaccinated birds being lower than those without revaccinated (Table 2 and Figure  1). Egg production during this period averaged 88.1 eggs/100 hens/day in the birds that were not revaccinated and 86.6 eggs/100 hens/day in the birds that were revaccinated regularly every 8 weeks from 22 weeks to 54 weeks. Some researchers reported that applying live IB vaccines in fl ocks approaching peak production may produce a 10% fall in egg production which usually remained depressed for about three weeks. Whatever live vaccine is used, the application is a very critical step. IBV virus is a sensitive virus that can be inactivated easily (Cavanagh & Gelb, 2008), which may result in inadequate effi cacy of the vaccination under fi eld conditions (Jackwood et al., 2009;de Wit et al., 2010).
The major goal of a vaccination program is to prevent disease and economic losses associated with infection by disease agents. The vaccine may be applied by eye drop or nasal drop, spray or drinking water routes and it is essential that a high percentage of the birds Group HD egg production (%) receive a required dose of the vaccine in the right tissue (Cook et al., 2008). Initial vaccination treatment of IB had a number of eff ects on the subsequent performance of the fl ock, during the laying phase. The treatment given to the control group, which was no vaccination until VicS vaccine strain was administered at 14 weeks of age, had a number negative eff ects on the birds during lay. Production was lower for the control birds at 20-56 weeks than for the other groups except the A3 coarse spray group.
shell thickness being 5 microns greater for the birds that were not revaccinated regularly ( Table 2). The birds that were revaccinated regularly during lay had thinner shells in every initial vaccination treatment except for VicS eye drop and A3 coarse spray group. A3 in water and not revaccinated was the thickest and A3 eye drop and revaccinated regularly the thinnest. There was signifi cant diff erence between initial vaccination treatment groups on albumen height (Tables 1), with albumen quality being highest for the control and lowest for the A3 water group. However, there was no signifi cant diff erence in albumen quality between birds that were regularly revaccinated during lay, and those that were not (Table 2). There was a signifi cant diff erence among initial vaccination treatment groups for Haugh Units (Table  1), with Haugh Units being highest for the control and lowest for the A3 water group. There was a signifi cant eff ect of regular revaccination for Haugh Units, being lower for those that were revaccinated regularly during lay than those that were not. The birds that were revaccinated regularly during lay had lower Haugh Units in every initial vaccination treatment except for VicS coarse spray and A3 in water groups. However, there was no signifi cant eff ect of initial vaccination treatment or regular revaccination during lay on percentage yolk colour. All yolk colour scores were at optimal levels, by egg commercial standards.
Initial vaccination at day-old with A3 strain IB virus resulted in both advantages and disadvantages during lay. Shell colour (as shell refl ectivity) was darker in the A3 groups than for the control and VicS groups. Shell thickness was highest in the A3 water group. Eff ects on shell breaking strength were variable, with the A3 water group having the highest shell breaking strength but the A3 eye group having the lowest. However, albumen quality was lowest for the A3 water group.
Regular revaccination during lay (at 22, 30, 38, 46, and 54 weeks of age) had no overall eff ect on shell refl ectivity (shell colour), albumen height, and yolk colour. However, in general, egg production, eggshell quality (shell breaking strength, percentage shell, shell thickness), and egg internal quality (Haugh Units) were lower in the birds that were revaccinated regularly during lay. The negative eff ects of regular revaccination are most likely the result of the response of the birds to exposure to IB virus, even though as a vaccine strain. Furthermore, the negative eff ects of regular revaccination on egg and eggshell quality may indicate a negative eff ect of IB vaccine on the oviduct agrees with Cavanagh (2003) and Choulsalkar et al. (2007). Roberts (2004) reviewed factors that aff ect egg shell quality and egg internal quality including: the strain and age of hen, production system, nutrition, induced moult, storage, stress, and diseases. Infectious bronchitis has been reported to cause egg shells to be paler in colour and sometimes wrinkled in appearance.
The results above suggest that in fl ocks, whose antibody levels are high, no increased protection may be achieved by revaccination during lay and an adverse eff ect upon egg production and quality can be induced

Egg and Egg Quality
Egg weight was lower for the initial control vaccination group (birds not vaccinated at all until 14 weeks of age) than for all other treatments (Table 1). Egg weight was higher overall in the birds that were revaccinated regularly than those without revaccinated beyond 14 weeks of age (Table 2). There were signifi cant diff erences among initial vaccination treatments on shell refl ectivity, with shell colour being lightest for the control and VicS groups ( Table 1). The groups vaccinated initially with A3 had generally darker shells. Shell colour was not aff ected by regular vaccination (Table 2). Initial vaccination treatment group had a signifi cant eff ect on shell breaking strength. There was a signifi cant eff ect of regular revaccination with breaking strength being slightly higher for the eggs produced by the birds that were not revaccinated regularly (Table 2). Animal received eyedrop vaccine and water had a lower shell breaking compared to that of the other groups.
There was a signifi cant eff ect of initial vaccination treatment on shell thickness (Table 1). Shell thickness was higher in the group that received A3 in water initially than for all other groups. There was a signifi cant eff ect of regular revaccination on shell thickness with by the vaccine itself. Poor egg specifi c gravity, reduced shell thickness, misshapen eggs, pale shell pigmentation, watery albumen that leads to greatly reduced Haugh Unit (HU) values are a refl ection of dysfunctional or damaged oviducts (Butcher & Miles, 2001). Although the use of live and killed vaccines which promote a wide spectrum of antibody is suggested, however, the use of live vaccines on farms where laying birds are present should be avoided. Revaccination with mild IB virus vaccine during lay needs to be justifi ed on the grounds that the optimum egg production is obtained in fl ocks where IB is not circulating or introduced, although the role of IBV in cases of egg production drop or poor eggshell quality is unknown (Ignjatovic et al., 2001).

IB Antibody Titre Levels
There were signifi cant eff ects of initial vaccination treatment on IB antibody titre levels that measured at 4, 6, and 16 weeks of age. IB antibody titre was higher for A3 eyedrop than all peaks and control, but there were no diff erent with A3 coarse and A3 water (Table  3). However, there were not signifi cant eff ects of revaccination on IB antibody titre levels until 49 weeks of measurements. The titre levels of all groups increased signifi cantly from 4 weeks to 16 weeks of age. The con-trol group of birds remained negative for IB antibodies until they were vaccinated with VicS at 14 weeks of age ( Figure 2).
Even though there was no diff erent of IB antibody titre level between revaccinated regularly hens and not revaccinated hens, the titre levels of all groups increased signifi cantly from 4 weeks to 16 weeks of age. The results indicated the increase in immune response following vaccination. The increase of antibody titres is an indication that the vaccination has been carried out carefully and correctly (Cook et al., 1999).
Since there is no appropriate drug therapy for controlling this disease, vaccination is still the main strategy for protecting chickens from IB virus infection (Lister, 2001). For the initial vaccination of breeders and layers, and also for broilers, both live and inactivated virus vaccines are applicable. However, at point of lay in breeders and layers inactivated oil-emulsion vaccines are used in used in poultry industry. Commercial mass application methods of IB vaccination include coarse spray, aerosol, and drinking water (Cavanagh & Gelb Jr., 2008). It appears that good immunity can be achieved when vaccine is administered via a range of diff erent routes, provided that each bird receives an adequate vaccine dose.  reported that protection provided by live and inactivated virus vaccination against challenge with the virulent nephropathogenic infectious bronchitis virus (NIBV) strain PA/Wolgemuth/98 was assessed. Even though, serum IBV enzyme-linked immunosorbent assay antibody geometric mean titers (GMTs) after vacwere low, ranging from 184 to 1354, and prior to NIBV challenge at 10 wk of age.
Live IB vaccines are generally recognized as providing good initial protection against infection, but in trying to provide immunity throughout the laying period, revaccination of hens during lay is recommended. Continual use of live vaccines complicates diagnosis since no simple diagnostic tool can diff erentiate in a fi eld from a vaccine strain (Ignjatovic & Sapats, 2000). Studies of molecular epizootiology reveal a strong probability that vaccination may lead to the spread of a vac-Tabel 3. IB antiboby titres in 4-16 weeks of age of hens treated with initial vaccination  cine virus, causing various disease manifestations and a confusing epizootiological situation in a poultry population (Farsang et al., 2002). Nephropathogenic infectious bronchitis (NIB) was diagnosed in 28 infectious bronchitis virus (IBV)-vaccinated commercial chicken fl ocks in Pennsylvania that show minimal respiratory disease signs in broilers, egg production and shell quality declined in layers . All vaccines such as VicS, A3, Steggles, Inghams, and others that have been used in poultry industry are considered to give good protection against several virulent, nephritis-inducing viruses of diff erent sub-types. However, their safety and CONCLUSION ing laying hens for IB virus, since they have received appropriate initial vaccination.