Protection of chickens vaccinated with combinations of commercial live infectious bronchitis vaccines containing Massachusetts, Dutch and QX-like serotypes against challenge with virulent infectious bronchitis viruses 793B and IS/1494/06 Israel variant 2

ABSTRACT

Infectious bronchitis virus (IBV) is a coronavirus which affects chickens of all ages. IBV mainly causes respiratory disease but can also result in reduced weight gain, reduced egg production, increased frequency of abnormal eggs and increased rates of mortality. Vaccination is the most important way to control the disease. Nevertheless, novel strains of infectious bronchitis (IB) continue to emerge in the field. In order to respond promptly, combinations of existing IB vaccines are frequently tested to see whether they can provide cross-protection. The efficacy of a combination of vaccines based on Massachusetts, Dutch and QX-like IB strains against emerging IB Israel variant 2 and IB 793B strains was assessed by means of four challenge studies. At least 80% of the birds vaccinated with IB H120 (Mass type) combined with IB D274 (Dutch type) followed by a QX-like IB vaccine booster or vaccinated with a combination of IB H120, IB D274 and QX-like IB were protected against a challenge with IB 793B. In addition, IB 1263 (Mass type) boosted by QX-like IB showed an 85% protection following challenge with IB 793B. A combination of IB H120 and IB D274 boosted by QX-like IB vaccine conferred 70% protection whilst H120 and IB D274 combination on its own showed 61.1% protection against Israel variant 2 challenge. IB 1263 boosted by a QX-like IB vaccine showed 50% protection against IB Israel variant 2. Therefore, it can be concluded that a combination of the IB H120, IB D274 and QX-like IB confers broad protection against different non-related virulent IB strains.

KEYWORDS:

• IB 793B
• IB Israel variant 2
• IB H120
• IB D274
• cross-protection
• antibodies
• QX-like IB vaccine

Introduction

Infectious bronchitis virus (IBV) is a coronavirus that affects chickens of all ages though younger birds show more severe signs of disease and higher mortality especially when a co-infection with a secondary bacterial pathogen(s) is present. IBV mainly causes respiratory disease and nephritis in chickens but can also result in reduced weight gain, reduced egg production, increased frequency of abnormal eggs and increased rates of mortality (Cavanagh & Gelb, 2008). IBV can genetically mutate and recombine, resulting in antigenic shifts and drifts which present a constant challenge to existing vaccination programmes (Cook et al., 2012). Variants such as D207 (D274) and D212 (D1466) (Davelaar et al., 1984), 793B (4/91) (Gough et al., 1992), D388 (QX) (Worthington et al., 2008) and Israel variants 1 and 2 (Callison et al., 2001) are examples of pathogenic variants that have evolved over the last decades. Hence, multiple serotypes of the virus have been identified worldwide and for the most part do not cross-protect (Jackwood et al., 2012).

After its emergence, the 793B virus was further characterized by Adzhar et al. (1997). In Europe, 793B like IBVs are prominent and have been detected in more than 30% clinical samples collected from commercial chickens at the beginning of this century (Worthington et al., 2008). The virus mainly causes respiratory disease but, in some outbreaks, it has been associated with unusual pathology for infectious bronchitis (IB), including bilateral myopathy and mortality in mature birds (Gough et al., 1992).

In recent years new vaccines such as the ones based on IB strain 4/91 (Cook et al., 2001) or IB L1148 (QX-like) (Geerligs et al., 2011) have been developed from pathogenic IB strains. However, new vaccines cannot be developed in prompt response against every emerging variant. As an alternative, the ability of some vaccine combinations to cross-protect against IBV strains of different serotypes has been assessed. Different cross-protection studies have been described (Wang et al., 1996; Cavanagh et al., 1997; Lim et al., 2012) where cross-protection can range from very poor to moderate.

Israel variant 2 IB viruses such as the IS/1494/06 isolate are known to be nephropathogenic (Susan et al., 2010) and can also affect the respiratory system. Persistence of problems associated with the IS/1494/06 has been reported despite the use of the H120 vaccine in some countries in the Middle East (Selim et al., 2013). The IS/1494/06 has been identified circulating in Libia (Awad et al., 2014) and in Turkey (Kahya et al., 2013).

Although vaccines which offer homologous protection against the circulating field strains are preferred, in the field birds can be exposed to different IB variant strains at the same time. Therefore, it is very important to know if certain vaccine combinations can confer sufficient cross-protection to protect the birds against both the homologous strains covered by the vaccine combination used and other heterologous strains that may be prevalent.

Currently, vaccines based on a QX-like IB strain are on the market in the EU. The QX IBV is one of the newest variants which was first isolated in China (Wang et al., 1998). Since then, QX-like viruses have spread worldwide and are present in European countries such as Germany, Holland, Belgium, Italy, France and UK (Monne et al., 2008; Worthington et al., 2008), as well as others. The QX-like variants cause the false layer syndrome, respiratory and kidney disease resulting in increased mortality and loss of performance (Worthington et al., 2008; de Wit et al., 2011).

In the studies described in this paper, we investigated whether this QX-like vaccine (Geerligs et al., 2011) in combination with vaccines containing Massachusetts (Mass)-type vaccine viruses, that is, IB H120 and IBMM, and a Dutch strain IB D274 confer protection against an IB 793B and Israel variant 2 (IS/1494/06) challenge viruses.

Materials and methods

Birds

In studies 1, 2 and 4, chickens were hatched from specific pathogen-free (SPF) eggs from Valo BioMedia España in Salamanca (Lohmann Tierzucht). For study 3, chickens used were hatched from SPF eggs from Animal Health Service, GD, Deventer, the Netherlands. The chickens were housed by treatment group in isolators. Environmental temperatures inside the isolators, starting at 30°C and decreasing progressively until 20–24°C, were monitored daily during the studies. Commercial compound feed suitable for the age of bird and tap water were supplied ad libitum.

In all the studies, birds were maintained and experiments performed in accordance with the Animal Health and Welfare regulations; procedures were approved by the local Animal Welfare committee before the study started.

Vaccines and challenge virus

Different commercial vaccines were used in the studies: a vaccine containing strain IBMM 1263 marketed under the name Poulvac^®IBMM; a vaccine containing strains IB H120 and IB D274 marketed under the name Poulvac^® IB Primer and a vaccine containing an attenuated QX-like IB strain, marketed under the name Poulvac^® IB QX. For all the vaccines mentioned above, the batch release titre was used to calculate the dilution needed in phosphate-buffered saline (PBS) in order to have a minimal titre per dose of 10^3.0 embryo Infective Dose 50% (EID₅₀) per strain. In cases where two vaccines were administered at the same time, the volume of PBS used in the dilutions was calculated in order to have the same minimal titre in both vaccines.

For the challenge, IB strain 793B from the University of Liverpool UK (Adzhar et al., 1997) and IB Israel variant 2 (IS/1494/06) isolated from an IBV commercial outbreak in Egypt were used (Susan et al., 2010).

Vaccines and challenge viruses were kept in an ice bath until use. In all cases, the time between vaccines and challenge viruses preparations and administrations did not exceed 2 hours.

Experimental design

The design of all studies was based on the European Pharmacopoeia (EP) for live avian infectious bronchitis vaccines (04/2013:0442). The design used in the studies is summarized in Table 1.

Table 1. Experimental design, summary of ciliostasis test scores and percentage of protected birds.

Study	Vaccination regime		Challenge		Ciliostasis score^a	% protection^b
	Day 0	Day 14	Day	Strain
1	IB H120 + IB D274 and QX-like IB	None	21	IB 793B	1.50	83.3^c
	None	None	21	IB 793B	8.40	3.6^c
2	IBMM	QX-like IB	35	IB 793B	1.00	85^d
	IB H120 + IB D274	QX-like IB	35	IB 793B	0.85	85^e
	None	None	35	IB 793B	4.32	42^d,e
3	IBMM	QX-like IB	35	Israel variant 2	5.00	50^f,g
	IB H120 + IB D274	QX-like IB	35	Israel variant 2	3.00	70^f,h
	Placebo	Placebo	35	Israel variant 2	10.00	0^g,h
4	IB H120 + IB D274	None	21	Israel variant 2	3.20	61.1^i
	None	None	21	Israel variant 2	10.00	0^i

^aMean ciliostasis score/bird for the 20 tracheas examined in each group; maximum possible score 10.00 (no protection).

^b% protected birds from total challenged. A chicken was considered protected if not fewer than 9 out of 10 tracheal rings/bird showed normal ciliary activity.

^cP < 0.05.

^dP < 0.05.

^eP < 0.05.

^fP = 0.167.

^gP < 0.05.

^hP < 0.05.

ⁱP < 0.05.

In study 1, a group vaccinated at one day of age with a combination of the vaccines containing IB H120, IB D274 and QX-like IB was included.

In study 2, two vaccine groups were included; one was vaccinated with a vaccine containing IBMM 1263, and the second was vaccinated with a combination of IB H120 and IB D274 at one day of age; both groups were followed by QX-like IB vaccine at 14 days of age.

In study 3, two vaccine groups were included; one was vaccinated with IBMM 1263 at one day of age, and the second was vaccinated with IB H120 and IB D274 at one day of age; both groups were vaccinated with QX-like IB at 14 days of age.

In study 4, a group vaccinated at one day of age with IB H120 and IB D274 was included.

In all the studies, a non-vaccinated challenged group was included. Each group, either vaccinated or control, consisted of 20 chickens. Chickens were individually identified with a unique wing tag number.

In studies 1 and 2, chickens were challenged with IB 793B strain; and in studies 3 and 4, chickens were challenged with Israel variant 2 strain. In all cases, challenge was done at three weeks after the last vaccination; at 21 days of age in studies 1 and 4, and at 35 days of age in studies 2 and 3 where a booster at 14 days of age was administered.

After challenge with IB 793B, the chickens in study 1 were mixed and randomly distributed in three isolators. In study 2, the chickens remained in the isolators after challenge. In study 3, following challenge with Israel variant 2, the isolators were connected so that the chickens were all allowed to mix. In study 4, after challenge each group was randomly divided into two with each sub-group housed in an independent isolator. In all the studies, after challenge the chickens were observed for the presence of clinical signs until five days post-challenge, when chickens were euthanized by intravenous inoculation of 20% sodium-pentobarbital. The tracheas were collected, cut into 10 rings per chicken and the rings were examined microscopically for ciliary movement.

During the studies blood samples were collected on the days just before vaccination, just before challenge, and at five days post-challenge (except in study 3) and tested for antibody titres against IB.

Vaccination

The lyophilized vaccines were reconstituted and further diluted in distilled water according to the instructions of the manufacturer. The diluted vaccines were used within the 2 hours in-use shelf life defined by the manufacturer for the diluted product. In the studies in which the chickens were challenged with IB 793B (studies 1 and 2) and in one of the studies where chickens were challenged with Israel variant 2 (study 4), the vaccines were administered by coarse spray using a sprayer in accordance with the instructions of the manufacturer. In the remaining study, in which the chickens were challenged with the IB Israel variant 2 virus (study 3), the chickens were vaccinated by eye/nose drop with each bird receiving a total of 0.1 ml of the diluted vaccine (0.05 ml in the eye and 0.05 ml in the nostril).

Challenge

Three weeks after last vaccination chickens were challenged with IB 793B or IB Israel variant 2. Before the study started, challenge strain stocks were titrated in eggs to calculate the EID₅₀. IB 793B strain was administered at a dose of 10^5.0 EID₅₀ and IB Israel variant 2 was administered at a dose of 10^4.0 EID₅₀. The challenge viruses were diluted in distilled water and the diluted viruses were kept chilled in cold water until use. The challenge viruses were administered by eye/nose drop in a volume of 0.1 ml per dose.

Ciliostasis test

The ciliary activity of the tracheal explants was examined five days after the challenge in all studies. Tracheal samples were collected after euthanasia and transverse sections were made, three sections from the upper part, four sections from the middle and three sections from the lower part. The tracheal explants were placed in PBS at 37°C in Petri dishes and examined by low-magnification microscopy for ciliary activity not later than 2 hours after collection. For a given tracheal section, ciliary activity was considered as normal when at least 50% of the internal ring showed vigorous ciliary movement. Each section with normal ciliary activity received a score of 0; if the section had a ciliary activity of the internal ring less than 50%, it received a score of 1. A total score (between 0 and 10) per chicken was then calculated. A chicken was considered not affected if more than 9 out of 10 rings showed normal ciliary activity (total score ≤1).

Serology

As day-old chickens are too young to keep alive after bleeding, at least three chickens from the same flock and hatch were bled and euthanized just before the first vaccination. In the studies with a second vaccination at two weeks of age, blood samples of all chickens were taken just before re-vaccination. Blood samples were also collected from all chickens (except for study 4 where 50% of the birds were bled) prior to challenge and five days after challenge. No samples were collected after the challenge from study 3. All sera samples were analysed by enzyme-linked immunosorbent assay (ELISA; FlockChek Infectious Bronchitis Virus Antibody Test Kit by IDEXX Europe B.V., Hoofddorp, the Netherlands) to determine antibody titres against IBV.

Statistical analysis

The significance level for the ciliostasis test (percentage of protected birds) and the serological response (percentage of seropositive birds) was set at 0.05. For all four studies, the total ciliostasis score for each bird was determined and the mean ciliostasis score for each treatment group was then calculated. The percentages of protected birds per treatment group were calculated and compared between the vaccinated and non-vaccinated groups. A pairwise treatment test was used for the analysis in study 1 and the Fishers exact test in study 2.

In studies 3 and 4, the Pearson’s chi-square and Fisher’s exact tests were used in the analysis of data. In cases where the expected frequencies in one or several cells of the contingency tables were less than 5, Fisher’s exact test was deemed more appropriate, otherwise conclusions are based on Pearson’s chi-square tests. One-sided tests were chosen if vaccinated groups were compared with non-vaccinated groups whilst two-sided tests were considered more appropriate when two different vaccinations were compared. Pearson’s test was also used to compare the results of studies 3 and 4.

Serology

The percentage of seropositive birds for each treatment group in each study was calculated.

Results

Clinical observations and ciliostasis test

No clinical signs such as coughing, sneezing or conjunctivitis were observed by visual examination, in any groups throughout the different studies.

Table 1 summarizes both the ciliostasis score and percentage of protected chickens per group for the four studies. The maximum ciliostasis score is 10 and the minimum is 0. The results show that there were clear differences in ciliostasis scores between vaccinated and non-vaccinated birds after IB 793B challenge. The results of study 1 complied with the requirements for efficacy of the EP, that is, at least 80% of the non-vaccinated controls should not be protected, whereas in the vaccinated group at least 80% should be protected. The differences between the vaccinated and non-vaccinated birds were statistically significant (P < 0.05).

The results of study 2 also showed protection of at least 80% in the vaccinated groups, although in the non-vaccinated controls the level of non-protection was below 80%. However, differences between vaccinated groups and non-vaccinated groups in study 2 were statistically significant (P < 0.05). There were statistically significant differences in protection between the two vaccinated groups and non-vaccinated group after the challenge with IB Israel variant 2 (P < 0.05) in study 3. The protection generated by the combination IB H120 and IB D274 administered at day of age and QX-like IB at 14 days was 70%, whilst the combination IB 1263 at day of age and QX-like IB at 14 days of age was less protective, that is, 50%. This difference in percentage protection between the two vaccinated groups was not statistically significant (P = 0.167). In study 4, there were statistically significant differences between the vaccinated group and the non-vaccinated group following challenge with the Israel 2 variant (P < 0.05). The protection generated following vaccination with IB H120 and IB D274 on day one of age was 61.1%.

Serology

All sera taken from one-day-old chickens were negative for antibodies against IBV. There was a measurable serological response following completion of the vaccination regime used for each study. At challenge, 15% of vaccinated chickens from study 1, 40–50% from study 2, 58.3–65.2% from study 3 and 60% from study 4 had seroconverted. Except for study 1, the differences in the percentage of seropositive birds between vaccinated and control birds at the time of challenge were significant.

Control birds were seronegative throughout the studies.

Discussion

Improvement in the protection against challenge with different serotypes of IB viruses is frequently observed after using a combined vaccination programme which incorporates two IB vaccines which are antigenically different (Cook et al., 1999). The most common vaccines used are the ones based on the Massachusetts type viruses, such as IB H120 or IBMM, or bivalent vaccines such as H120 + D274 or IBMM + Arkansas.

In all studies performed, ciliostasis tests showed that vaccinated birds had a significantly higher percentage of protection when compared with the non-vaccinated birds. This indicated that protection was achieved in the vaccinated birds probably due to local protection of the upper respiratory tract induced by the live vaccines. The ciliostasis test was selected to evaluate the cross-protection because it is the reference test prescribed in the EP. This is a well-known test that gives a good indication of protection.

The results of two of the studies described showed that vaccination consisting of IB H120 + D274 or IB 1263 used at day old and boosted by QX-like vaccine 14 days later or a combination of H120 + D274 + QX-like at day old, clearly generated protection against IB 793B challenge. Only a small numerical difference was found between vaccination with IB H120 + IB D274 and QX-like IB at one day old and vaccination with IB H120 and IB D274 at one day old and QX-like IB at 14 days, and then challenge with IB 793B (83% vs. 85%). As previously described (Cook et al., 1999), broader cross-protection was shown when antigenically distinct live attenuated vaccines were administered two weeks apart than when the vaccines were combined on the same day.

Challenge with the IS/1494/06 Israel variant 2 after vaccination with IB 1263 (a Mass-type vaccine) followed by a QX-like vaccine 14 days later conferred not more than 50% protection to the vaccinated birds. However, boosting with a QX-like vaccine 14 days after initial vaccination with IB H120 and IB D274 increased the percentage of protection to 70%. Challenge with Israel variant 2 14 days after vaccinating with IB H120 + IB D274 conferred 61% protection. A lower protection (25%) following challenge with an Israel variant 2 (IS/585/98) was previously observed in a study following vaccination with an H120 vaccine strain (Gelb et al., 2005). A protection of 30–40% was observed after challenge with an Israel variant 2 (D1344/2/4/10 EG) following vaccination with a vaccine containing an H120 strain boosted 11 days later with a vaccine closely related to the 793B serotype (Tatár-Kis et al., 2012). The results obtained in our studies indicate that combining H120 with D274 vaccines might increase the protection levels.

The EP requirements for product authorization in the European Union specify that at least 80% of the non-vaccinated challenged chickens should not be protected, whereas in the vaccinated groups at least 80% should be protected. The results of study 1 are fully compliant with the EP, whereas results for study 2 do not meet the EP requirement for challenge as the percentage non-protection of the controls was lower than the requirements, possibly due to a virus diluted to a too low titre. Results for studies 3 and 4 also do not fully meet the EP requirements as protection in vaccinated groups was lower than 80%.

The reason that cross-protection occurs has never been fully explained. A cell-mediated immunity (CMI) response after initial vaccination with the H120 strain was observed (Timms & Bracewell, 1981) and that response was observed to be cross-reactive (Dhinakar Raj & Jones, 1997). It was shown that the H strain had a rare ability to cross-protect against serotypes which are heterologous making it potentially effective in the control of a broader range of serotypes (Bijlenga et al., 2004). This cross-protection could be due to the virus genome remaining unchanged whilst new variants have emerged, which make it more relevant to think in terms of protectotypes rather than serotypes (Zanaty et al., 2013).

ELISA was used for the detection of antibodies against IBV. ELISA has been shown to be a reliable and sensitive method to monitor vaccination schedules and for the rapid detection of initial increase of antibodies against IB (Ghadakchi et al., 2005). The results in this study show that the percentage of seropositive birds post vaccination using the same vaccination scheme was variable for the different studies at the time of challenge. The percentage of seropositive birds was 40% and 65.2% in the two groups where H120 + D274 vaccine combination was used initially followed by a QX-like vaccine booster. The percentage of protected birds was 50% and 58.3% after using IB 1263 at day old followed by a QX-like vaccine booster 14 days later. The percentage of protected birds at challenge after vaccination with IB H120 + IB D274 was 60%. Vaccinating with H120 + D274 + QX-like at day old showed the lowest percentage of protected birds at the time of challenge at 15%. The differences in the percentage of seropositive birds between the vaccinated and control groups at the time of challenge in studies 2, 3 and 4 were significant. Although low to medium humoral response is expected following the administration of live IBV vaccines (Terregino et al., 2008), antibodies against IBV do not always correlate with protection (Raggi & Lee, 1965; Cavanagh, 2003) and the results from the studies presented in this paper support this observation. It has to be considered also that live vaccines stimulate local, systemic and CMI to IBV (Bijlenga et al., 2004).

SPF chicks were used in the four studies presented in this paper. In the field, it is likely that chicks will have maternally derived antibodies (MDAs). The presence of MDAs has been shown to have no adverse effect on the efficacy of live IBV vaccines which are administered to one-day-old chicks (Davelaar & Kouwenhoven, 1977).

Based on the results obtained, a vaccination schedule using an initial combination of H120 + D274 vaccine followed by a QX-like vaccine two weeks later appears to confer more protection (70%) than an IB 1263 boosted by a QX-like vaccine (50%) following challenge with an Israel 2 variant IB strain. Therefore, in areas where the IS/1494/06 Israel variant 2 strain of IBV is prevalent, the statistical significant differences observed once compared with the control group indicate that H120 + D274 vaccine combined with QX-like could aid in the protection of birds in the field. Concerning the results from 793B IB challenge studies, the same level of protection (85%) was shown between birds vaccinated with IB H120 + D274 vaccine combination boosted by a QX-like vaccine 14 days later and those where an IB 1263 vaccine is initially used followed by a QX-like vaccine 14 days later. The protection level following an initial vaccination with IB H120 + D274 and a QX-like vaccine was similar (83%). Therefore, any of these vaccination approaches could be considered in areas where the 793B IB variant is prevalent. The importance of identifying the IB strains circulating and prevalent in a particular area is essential as only then can the appropriate vaccination regimes be used. The data presented in this article and the data obtained from the literature clearly demonstrate that the use of certain combinations of IB vaccines can result in protection against a virulent IB strain, which is genetically different from the vaccine strains. This knowledge is very important as it shows that the development of novel vaccines based on newly emerging strains is not always necessary. Cross-protection studies can be performed relatively quickly, and if the results are adequate, a short-term solution can be available. For longer term, the need for the development of novel vaccine strains should be evaluated.

Acknowledgements

Studies 1, 2 and 4 were performed at Olot, Zoetis VMRD facilities and study 3 was performed at GD Animal Health in Deventer the Netherlands under the supervision of Dr G.J. Boelm.

Disclosure statement

No potential conflict of interest was reported by the authors.