Evaluation of the antigen relatedness and efficacy of a single vaccination with different infectious bronchitis virus strains against a challenge with Malaysian variant and QX-like IBV strains

Infectious bronchitis (IB) is an acute, contagious disease affecting the respiratory, renal, and reproductive systems of chickens, causing a decrease in carcass weight, egg production, and death of the chickens, as well as significant economic losses worldwide [1]. The etiology of the disease is an infectious bronchitis virus (IBV), a highly prevalent coronavirus of chickens. IBVs primarily infect the respiratory tract of chickens, but some strains can cause lesions in the kidneys, oviduct, and other epithelial tissues [2].

In Malaysia, an IB case was first detected in 1967 [3]. Between 1967 and 1977, outbreaks of IB were due primarily to the Mass strains [3]. In the 1990's, a new IBV Malaysian variant strain MH5365/95 was detected, causing kidney disease, resulting in 20 to 30% mortality in the infected chickens [4]. In 2004, a new IBV variant was detected circulating in poultry farms and was reported as Malaysian IBV strain V9/04 [5]. Recently, a study carried out between 2014 and 2015 indicated the presence of Malaysian IBV QX-like and a local variant (MV) causing respiratory and kidney disease in broiler and layer chickens in several states in Malaysia [6]. A subsequent study indicated that the IBV QX-like is the predominant strain (47%) followed by the MV (13%) affecting poultry farms in Malaysia [7].

Besides biosecurity, the best strategy to control IB is by vaccination. On the other hand, the control of IB via vaccination is problematic because of the emergence of new variant IBV strains that are antigenic distinct. Moreover, vaccination with certain strains of IB failed to induce protection [8]. In most cases, poor protection of IB-vaccinated flocks is associated with the inability of the vaccine to provide adequate protection against the circulating field IBV.

In studies of chickens vaccinated with the Mass vaccine strain, the most commonly used IB vaccine could not provide adequate protection against the field IBV Belgian B1648 strain [9], Holland D1466, South Africa 890/80, Brazil 50/96 [10], Iranian QX-like [11], and Thai QX-like [12]. In another study, chickens vaccinated with the variant IB 4/91 vaccine showed poor protection against local IBV Holland D1466, South Africa 890/80, and Honduras 22/97 strains [10]. The main reason for the failure of the IB vaccine to provide protection is due primarily to antigenic differences at the S1 protein of the vaccine and the field strains of the viruses [13]. Furthermore, the diminished similarity of S1 protein can reduce the degree of cross-protection against homologous and heterologous IBV [14].

A previous study confirmed the high amino acid variation of the S1 protein of the Malaysian QX-like and MV compared to several IBV strains [6]. The Malaysian QX-like and MV showed high amino acid differences in the S1 protein against Mass H120 (EU822341; 29%–30% variations), 793/B (KP118884; 21%–27% variations), D274 (X15832; 22%–29% variations), Italy-02 (AJ457137; 21%–27% variations), KM91 (JQ977698; 17%–23% variations), K2 (JQ920378; 17%–21% variations), and Malaysian MH5365/95 (EU086600; 11%–26% variations). The variations in the S1 protein may influence the cross-protection ability of IBV vaccine strains against the Malaysian QX-like and MV. Cavanagh [15] reported that differences as little as 5% in the S1 sequences could cause poor cross-protection against an IB challenge.

For effective vaccination, the live attenuated vaccines against IB are given to day-old chicks at a hatchery or later in the farm by either drinking water or spray. Boosters with inactivated vaccines are required for laying hens [16, 17, 18]. Although vaccination against IB is used widely, vaccination with 1 serotype does not ensure complete protection against different IB strains [10]. Therefore, this study examined the antigenic relatedness between different IB vaccine strains and the Malaysian QX-like and MV strains as well as the efficacy of different IB vaccine strains in conferring protection against the Malaysian QX-like and MV in specific-pathogen-free (SPF) chickens.

Currently, both Mass and variant IBV vaccine strains have been used to control IB in commercial poultry farms in Malaysia. On the other hand, limited studies have been carried out to determine the antigenic relatedness and efficacy of Mass and non-Mass (variant) vaccine strains in conferring protection against the Malaysian field IBV strains. In addition to the vaccine strains, studies have indicated that 2 distinct strains, the MV and QX-like, are the prevalent field IBV affecting commercial poultry farms in Malaysia [6, 7]. Based on genotype classification analysis [24], the Malaysian QX-like is grouped with the QX-like viruses from other countries as genotype GI-19. In contrast, the MV strain is a unique strain closely related to a previously isolated nephropathogenic strain, MH5365/95 (data not shown). In this study, the efficacy of single inoculation with the Mass strain (H120) or with different variant strains (4/91 or K2) was evaluated in SPF chickens following a challenge with MV (IBS37A/2014) and QX-like (IBS130/2015) at 21 dpv.

An antigenic relatedness study indicated that the MV strain IBS037A/2014 has a minor subtype difference with the H120 and 4/91 strains but antigenically different with IBS130/2015. On the other hand, the Malaysian QX-like IBS130/2015 has no antigenic relatedness with IBS037A/2014, H120, and 4/91 strains but has a minor subtype difference with the K2 strain. This result is based on serologic r-value analysis that provides quantitative information on the antigenic relatedness of different virus strains using a mathematical calculation to predict the variation in the antigenic composition of the strains against the specific immune serum used [22]. An analysis of the serologic antigenic differences of IBVs is important because different serotypes of IBVs generally do not cross-protect, which is the main reason for the failure of the IB vaccine to provide protection [13]. The antigenically different IBVs usually induce poor cross-protective immunity. On the other hand, some IBV strains can provide cross-protection against other serotypes [10]. In many studies, these IBV strains are identified as protectotype strains [11, 25, 26, 27].

Following a single IB vaccination, the detected ELISA antibody titers were not consistent at different days post-vaccination. On the other hand, all the vaccinated groups elicited antibody profiles at 21 dpv. The actual reasons of the different patterns in antibody titers in the vaccinated groups are unclear, but it could be influenced by various factors, such as host immune response [28], vaccine strain used [29], type of vaccination application method [30], and other local variables, such as temperature and feed quality [31]. Hence, the antibody titers alone are not a good indicator of a successful IB vaccination because the antibody titer does not always correlate with protection. The results of this study are supported by Roh et al. [32], who demonstrated a poor induction of antibody level detected by the Mass-type of ELISA antigen following vaccination with a live attenuated Ark-type IBV vaccine.

To evaluate the protection induced by the vaccine strains following the challenge with MV and QX-like IB, a few parameters, such as the clinical sign scores, macroscopic lesions examination, and ciliary activity, were measured. The same parameters were also used in a previous study to evaluate the protection of vaccinated and challenged chickens against homologous and heterologous IBV strains [8]. On the other hand, other studies reported that clinical signs usually vary and are challenging to quantify. Moreover, it is difficult to differentiate between vaccination or challenge groups [33]. In terms of practicality, direct observations, such as macroscopic lesions examination and ciliary activity estimation, are the most important parameters for a protection evaluation in many studies [8, 33]. In addition, an indirect examination based on a VN assay is also helpful in differentiating the IBV serotypes [34].

The clinical signs and lesions in the trachea and kidneys, ranging from mild to severe, were observed in chickens following a challenge with IBS037A/2014 and IBS130/2015. Generally, the clinical signs and lesions were far more severe in the unvaccinated chickens than the vaccinated chickens. Depression, lacrimation, coughing, and ruffled feathers are the common signs observed. Sometimes gasping, sneezing, and the presence of nasal exudates also can be detected. Furthermore, the tissue samples collected from these birds tested positive to IBV based on RT-PCR. These findings were in line with other studies with a single vaccination using IB vaccine virus strains, such as H120 and CR88 challenged with TN20/00 [25], H120, Ma5 and 793/B challenged with M41 and QX-like strains [26], and H120 and Conn IB vaccines challenged with Thai QX-like [12]. On the other hand, less severe clinical signs and lesions were recorded when the chickens were given the Mass-type vaccine and booster with variant IB vaccine strains [27, 35]. Protection studies indicated that the homologous IBV vaccine strains, either Mass-type or variant strains, usually induce better protection against an IBV challenge [10]. On the other hand, an assessment of cross-protection of some IBV vaccine combinations against circulating IBV strains of different serotypes could be an alternative approach to IB control [27]. A combination of the Mass-type (H120) and 4/91 vaccine strains, to some extent, could provide adequate protection against the heterologous IBV field variant strain [35]. These could be explained by the combination of different IBV vaccine strains broadening the protection by increasing the cellular and local immune responses [18]. This has also been postulated as the mechanism behind some of the protectotype IB strains in inducing a robust immunity against challenges with different serotypes of IBV.

The unvaccinated challenged groups demonstrated clinical signs and trachea lesions, as well as increased mucin secretion and swollen kidneys with urate deposition, as reported in a previous study [36]. All the vaccinated groups challenged with IBS037A/2014 demonstrated mild to moderate trachea and kidney damage between 3 and 7 dpc. On the other hand, the vaccinated chickens challenged with IBS130/2015 demonstrated mild to moderate tracheal lesions, whereas moderate kidney damage was observed in all the vaccinated groups between 5 and 7 dpc. Unlike an evaluation of the clinical signs and lesions, studies have shown that a tracheal ciliary activity examination is an efficient method to assess the protection against an IBV challenge [8, 10, 37]. The ciliary activity examination for assessing the damage to the chicken caused by IBV is fast and easy to perform [30]. By estimating the score or percentage of the surface of the tracheal epithelium that is showing ciliary movement, the protection can be assessed in IBV post-challenge birds [8]. The recommended time for assessing a protection post-challenged assessment is between 4 and 7 dpc [37]. Therefore, most vaccine efficacy studies assess protection from 5 to 7 dpc [38]. In this study, the tracheal ciliary activity of the vaccinated groups challenged with the Malaysian QX-like IBS130/2015 (48% to 52%) is slightly lower than the vaccinated groups challenged with the MV strain IBS037A/2014 (56% to 59%). On the other hand, the percentage mean ciliary activity failed to confer protection against the respective challenged viruses, which is probably due to the antigenic variations between the tested vaccines and challenge virus strains.

Several studies have shown that some IBV vaccine strains can provide protection after a single vaccination [12, 26]. Therefore, in this study, the birds were vaccinated once with a single heterologous IB vaccine strain to evaluate the efficacy of the vaccine against challenge virus of Malaysian QX-like IBS130/2015 and MV IBS037A/2014. The results from single-dose heterologous vaccination obtained showed that the vaccines used could not provide adequate protection against the challenge viruses. Currently, limited information is available on the common neutralizing epitopes found in different IBV serotypes, including protectotype strains that can confer protection. A previous study reported that a few changes in the S1 amino acid composition might produce a new IBV serotype, even though the remaining virus genome remained unchanged, indicating the importance of the S1 gene in determining the IBV serotype [14]. S1 also reported where the IBV antigenic determinants were located [2]. Therefore, if many antigens can be shared, this might suggest that a combination of currently available vaccines should provide robust protection against challenge IBVs belonging to different serotypes [10].

Several studies using IB booster vaccinations showed better protection against field IBV than a single vaccination alone [8, 10]. For example, chickens primed with Mass M41 at 1-day-old, followed by a booster vaccination with IB-Var2 in 14-day-olds, showed better protection against field IBV Middle East IBV variant 2 (80%–100% protection) [26]. In another study, chickens primed with the Mass vaccine at 1-day-old followed by a booster vaccination with IBV 793/B showed effective protection against field M41 (90%–96% protection) and 793/B (93% protection) [27]. Chickens primed with Mass H120 at 1-day-old, followed by a booster vaccination with IB QX-like vaccine strain 1148 or 4/91, showed adequate protection against Thai QX-like (100% protection) [39]. Hence, improved protection was observed in the combination of 2 different serotypes IB vaccines, which was observed in the combined vaccination [10]. Moreover, another study showed that the sequential vaccination of different IBV vaccines could increase the broadness of the reacting antibody titers [40], which was proven by evaluating the cross-reactivity of sera collected from the prime and prime-boosted vaccinated chickens [38]. Therefore, this may explain the possibility of combined immunity between Mass and variant IBV strains against MV and QX-like. On the other hand, combination vaccinations of Mass and variant vaccine strains are not applied widely in Malaysia. This type of vaccination may be used in large commercial farms, but most small scale farmers usually prefer single vaccination due to the limitation of costs and human resources. Hence, vaccination with a Mass strain followed by a variant strain may be the answer to providing effective protection against a challenge with the MV and QX-like IB viruses.

In conclusion, although the Malaysian variant IBS037A/2014 and the H120 and 4/91 IB vaccine strains are grouped under 1 subtype, and the Malaysian QX-like strain IBS130/2015 and the K2 vaccine strain belong to another subtype group, single vaccination with the above vaccine cannot confer protection in SPF chickens.