Infectious bronchitis virus in Jordanian chickens: Seroprevalence and detection

Abstract

Infectious bronchitis (IB) is one of the most important viral diseases of poultry; it causes major economic losses to the poultry industry. This study was conducted to investigate the prevalence of infectious bronchitis virus (IBV) in commercial chicken flocks in Jordan. Serum samples from 70 commercial chicken flocks (40 broilers, 18 layers, and 12 broiler breeders) free from respiratory disease were collected and screened for the presence of Massachusetts-41 (M-41), D274, and 4/91 strain antigens of IBV by using the hemagglutination inhibition (HI) test. In addition, 51 commercial chicken flocks (25 broilers, 15 layers, and 11 broiler breeders) suffering from respiratory disease were tested for the presence of IBV, using the reverse-transcription polymerase chain reaction. Overall, 92.9% of the flocks free from respiratory disease were seropositive for antibodies to the M-41 strain, whereas 90% and 61.4% of the flocks were seropositive for antibodies to the 4/91 and D274 strains, respectively. Infectious bronchitis virus nucleic acid was detected in 16 broiler (64%), 8 layer (53%), and 6 broiler breeder (54.54%) flocks affected with respiratory disease. This study clearly demonstrates that several strains of IBV are present in poultry flocks in Jordan. Future work should include the isolation and serotyping of IBV in the region, so that a suitable vaccination program, using the common field serotypes as vaccines, can be adopted to protect against IBV-caused disease. Furthermore, farmers need to be educated about the clinical signs of IB and the importance of IBV.

Introduction

Infectious bronchitis (IB) is an acute, highly contagious, viral respiratory disease of chickens, characterized by tracheal rales, coughing, and sneezing. In addition, it may affect the kidneys, and in laying flocks, it decreases egg production and impairs the quality of eggs produced (1). The etiologic agent of IB, infectious bronchitis virus (IBV), is in the genus Coronavirus of the family Coronaviridae. The disease is common worldwide and has significant economic consequences (1). Although effective vaccines are available and utilized routinely in commercial poultry production, the virus has the tendency to mutate frequently (2). More than 20 serotypes within the IBV are recognized worldwide (3). The Massachusetts (Mass)-type was first isolated in Europe in the 1940s (4). The D274-type was the most common serotype in several western European countries in the early and mid-1980s (5,6). The 4/91-type was first isolated in 1985 in France; later its type was identified in the United Kingdom (UK) (7).

In Jordan, the poultry industry comprises broiler breeders, layers, and broilers, with the total number of birds ranging from 30 to 35 million (8). Serious respiratory diseases of unknown etiology have been affecting chickens for a number of years, causing economical losses to farmers and poultry companies. The prevalence and severity of respiratory diseases in commercial chicken flocks has increased recently, due to the intensification of the poultry industry (8). Infectious bronchitis is of significant economic concern for poultry producers both in Jordan (9) and worldwide (10). Published information describing the prevalence of respiratory diseases in poultry in Jordan is limited. One report describes the presence of the Mass, Arkansas (Ark), Delaware variant-072 (DE-072), and JMK serotypes of IBV in poultry flocks in Jordan, based on the results from the hemagglutination inhibition (HI) test (9). The study reported herein was designed to investigate the prevalence of IBV variant strains in commercial chicken flocks by screening flocks serologically for the IBV strain M-41, as well as for the IBV strains D274 and 4/91, not previously documented in Jordan, and, in addition, by testing for the presence IBV in chicken flocks in the acute phase of respiratory diseases by using the reverse-transcription polymerase chain reaction (RT-PCR).

Materials and methods

Chicken flocks: history and samples

Seventy commercial chicken flocks (40 broilers, 18 layers, and 12 broiler breeders), located in the northern and southern areas of Jordan and recorded as being free from any apparent clinical disease, were selected for investigation. There are 450 chicken farms in these areas with an average of 15 000 chickens per farm. Most of these farms are broiler-type chicken farms, with a few broiler breeder and layer farms. Each farm selected for investigation had 1 flock, and these flocks were of varying ages, ranging from 28 to 41 d, 40 to 48 wk, and 30 to 36 wk for broilers, layers, and broiler breeders, respectively. The median distance between farms was 42 km, ranging from 0.5 km to 30 km. All flocks had been vaccinated against the M-41 strain, whereas none of the flocks had been vaccinated against the 4/91 and D274 strains, of IBV. All flocks were contacted through the appropriate veterinarians and all owners agreed to participate in this study. Twenty serum samples were collected randomly from each of the above flocks and tested serologically for antibodies to the M-41, D274, and 4/91 strains of IBV.

Additionally, from each of 25, 15, and 11 broiler, layer, and broiler breeder flocks affected with respiratory disease, respectively, from a different area of Jordan, 5 chickens in the acute phase of respiratory disease were submitted to the Provimi Jordan Laboratory as suspected IBV cases. These flocks had been vaccinated against the M-41 strain before 29 d of age. Broiler flocks were showing moderate to severe signs of respiratory disease, a decrease in feed intake, and deaths (25% to 35%); whereas, layer and breeder flocks were showing mild signs of respiratory disease, a decrease in feed intake, and a decrease in egg production (10% to 17%). The chickens were necropsied, and tracheas were collected and stored at −80°C until used for testing by RT-PCR.

Hemagglutination and hemagglutination inhibition test (HA/HI)

The HA/HI test was performed, as described by the Office international des épizooties (11), using reference antigens for the IBV serotypes M-41, D274, and 4/91 [Gezondheidsdienst voor Dieren B.V. (GD); Animal Health Service, Deventer, The Netherlands]. The HA titer of the IBV antigen was calculated as the average of 2 dilutions 1:2 and 1:3. The HI test was performed by using the above-titrated 3 reference antigens (serotypes), 3 positive control antisera against the above antigens, and a negative control serum [Gezondheidsdienst voor Dieren B.V. (GD)]. The maximum dilution of each serum sample causing inhibition of HA was used as the endpoint. The HI titer of each serum sample was expressed as a reciprocal of the serum dilution (top to bottom). The results of HI titer of all the sera thus obtained were subjected to statistical analysis.

Statistical analysis of the HI titers

The statistical analysis used in this study was the same as that previously described (12). Briefly, the geometric mean titer (GMT) was calculated by taking an average of the well numbers showing HI activity from all serum samples within 1 flock. This well number average was then cross-checked against GMT values given in the Brug’s table and reported as the GMT for a given flock against H9N2 antigen. All analyses were performed by using statistical analysis system (SAS) software (13).

RNA extraction

Tracheas from each flock were swabbed with sterile swabs (Heinz Herenz Medizin GmbH, Hamburg, Germany). Swabs were placed in phosphate buffered saline (PBS) (pH 7.2) and scraped against the side of the tube to facilitate removal of materials from the head of the swab. Extraction of RNA was performed on the material pooled from the swabs from each flock, using generic purification (Purescript RNA purification kit; Gentra systems, Minneapolis, Minnesota, USA) according to the manufacturer’s instructions.

Identification of IBV by RT-PCR

The IBV detection primers used in this study had been evaluated previously by Jackwood et al (14). The primers’ (Alpha DNA, Montreal, Quebec) sequences are as follows: IBV-LC3′ = 5′-ACA GAT TGC TTG CAA CCA C-3′ and IBV-LC5′ = 5′-ACT GGC AAT TTT TTC AGA-3′. The RT-PCR produces a 383-bp fragment (Figure 1) common to all IBV in positive samples and does not differentiate between different strains. One-step RT-PCR was performed by using amplification of target RNA (Access RT-PCR System kit, Promega Corporation, Madison, Wisconsin, USA), according to the manufacturer’s instructions. Briefly, a 50-μL reaction volume per sample was prepared by adding 10 μL of avian myeloblastosis virus/reverse transcriptase Thermus filiformis DNA polymerase 5× reaction buffer, 1 μL of deoxy nucleotide 5′-triphosphate mixture (10 mM of each deoxy nucleotide 5′-triphosphate), 1 μL (50 pmol/μL) of each downstream and upstream primer (Alpha DNA, Montreal, Quebec), 2 μL of 25 mM MgSO₄, 1 μL of avian myeloblastosis virus reverse transcriptase (5 U/μL), 1 μL of T. filiformis DNA polymerase (5 U/μL), 28 μL of nuclease-free water, and 5 μL of RNA template. The RT-PCR was carried out in a DNA thermo cycler (DNA Engine thermal cycler; Bio-Rad Laboratories, Mississauga, Ontario). Reverse transcriptase was carried out for 1 RT cycle at 45°C for 60 min, followed by 94°C for 5 min, then 40 PCR cycles at 94°C for 45 s, 55°C for 45 s, and 72°C for 90 s, with a final extension cycle at 72°C for 5 min. The PCR products were analyzed in 2% agarose gels and stained by ethidium bromide (Promega Corporation). Agarose gel electrophoresis was run for 60 min at 100 V, 400 Ma (Bio-Rad Laboratories) and then the gel was visualized under ultraviolet light (AlphaImager; Alpha Innotech, San Leandro, California, USA). The IBV strain (M-41) [Gezondheidsdienst voor Dieren B.V. (GD)] was used as a positive control for RNA extraction and RT-PCR. A negative control (nuclease-free water) was also used in each run.

Figure 1.

Electrophoresis (2% agarose gel) for infectious bronchitis virus (IBV) detection by the LC3/LC5 set of primers. Lane M = 100 bp – 1.5 kbp DNA ladder marker (Promega Corporation, Madison, Wisconsin, USA). Lane 1 = IBV positive control (positive; band at 383 bp). Lane 2 = Negative control for IBV (no band). Lanes 3–9 = Positive flocks (positive; band at 383 bp). Lanes 10–12 = Negative flocks (no band).

Results

The seroprevalence of various strains of IBV in commercial layer, broiler breeder, and broiler flocks free from respiratory disease is recorded in Table 1. The prevalence of the M-41 strain of IBV was about 87.5% in broiler flocks and 100% in layer and broiler breeder flocks, with an overall combined prevalence of 92.9% in all flocks tested (Table 1). The prevalence of the 4/91 strain of IBV was 100%, 77.8%, and 75% in broiler, layer, and broiler breeder flocks, respectively, with an overall combined prevalence of 90% in all flocks tested (Table 1). The prevalence of the D274 strain of IBV was 62.5%, 61.1%, and 58.3% in broiler, layer, and broiler breeder flocks, respectively, with an overall combined prevalence of 61.4% in all flocks tested (Table 1).

Table 1.

Seroprevalence of 3 strains of infectious bronchitis virus (IBV) in commercial chicken flocks free from respiratory disease^a

		IBV strains
		M-41		4/91		D274
Type of flock tested	Number of tested flocks	Number of positive flocks	HI titerb	Number of positive flocks	HI titerb	Number of positive flocks	HI titerb
Broiler	40	35 (87.5)c	70–160	40 (100)c	65–205	25 (62.5)c	55–165
Layer	18	18 (100)c	85–205	14 (77.8)c	55–175	11 (61.1)c	75–180
Broiler breeder	12	12 (100)c	75–215	9 (75)c	70–180	7 (58.3)c	75–190
Total		65 (92.9)d		63 (90)d		43 (61.4)d

^aThe age of broiler, layer, and broiler breeder flocks ranged from 28 to 41 d, 40 to 48 wk, and 30 to 36 wk, respectively. All flocks were vaccinated against the M-41 strain, whereas none of the flocks were vaccinated against the 4/91 and D274 strains of IBV. Twenty serum samples were collected randomly from each flock. Samples were tested for the presence of antibodies against the 3 different IBV strains, using the hemagglutination inhibition (HI) test.

^bValues in these columns represent the range of the HI titers calculated grand geometric mean of 800, 360, and 240 blood samples from broilers, layers, and broiler breeder, respectively.

^cValues in parentheses represent percentage of a given serotype in the corresponding flock type.

^dThis value indicates combined percentage in all 70 flocks tested.

Table 2 shows the RT-PCR results in commercial layer, broiler breeder, and broiler flocks affected by respiratory disease. Thirty of 51 cases (58.8%) tested by RT-PCR were positive for IBV (Table 2) (Figure 1). Sixteen (64%) broiler flocks, 8 (53%) layer flocks, and 6 (54.54%) broiler breeder flocks were positive by RT-PCR.

Table 2.

Reverse transcription polymerase chain reaction (RT-PCR) results for infectious bronchitis virus (IBV) in different commercial chicken flocks affected by respiratory disease that were tested^a

Type of bird	Number of tested flocks	Number of positive flocks (%)
Broiler	25	16 (64)
Layer	15	8 (53)
Broiler breeder	11	6 (54.54)
Total	51	30 (58.8)b

^aFive tracheas per flock were collected and tested for IBV, using the RT-PCR test.

^bThis value indicates combined percentage for all of 51 flocks tested.

Discussion

All of the tested respiratory disease-free flocks had antibodies against 1 or more strains of IBV. The most frequently found strain was M-41 (Table 1). As in many other parts of the world, Mass (M)-type strain vaccines are the only officially authorized vaccines against IBV, which is why the commercial chicken flocks in Jordan were vaccinated with the M-41 IBV strain and why the results obtained by Saad (9) indicate that M-41 is the most commonly detected IBV strain in Jordan. Table 1 shows that against the IBV M-41 strain, layer and breeder flocks exhibited (100%) seropositivity, whereas broiler flocks exhibited (87.5%) seropositivity. This was to be expected, because the flocks used in the current study had been vaccinated against the M-41 strain. Surprisingly, not all broiler flocks were seropositive for the M-41 strain. This may indicate a vaccination failure in these flocks; however, determining the cause of this failure was not the aim of this study. From our field experience, vaccination failure in Jordan frequently stems from the use of live vaccines in chlorinated drinking water, which inactivates the vaccine virus. Also, mishandling and the improper storage conditions of live vaccines may result in failure of the vaccine.

The results of this study (Table 1) clearly show that the 4/91 and D274 variant strains of IBV are also present in commercial respiratory disease-free chicken flocks in Jordan. It is interesting to note that against the 4/91 and D274 strains, broiler flocks exhibited 100% and 62.5% seropositivity, respectively, whereas layer and broiler breeder flocks exhibited 77.8% and 61.1% and 75% and 58.3% seropositivity, respectively. This may be due to the younger age and higher susceptibility of broiler flocks compared with layer and breeder flocks (1). The HI results in this study and the results of previous study (9) confirm that IBV is present in Jordan and suggest that there are several variant strains of IBV in Jordan.

In Jordan, poultry producers seek assistance for disease diagnosis through a limited network of poultry diagnostic laboratories. In most cases, the diagnosis of IBV infection is based on clinical signs and gross lesions only and, likewise, other respiratory agents are excluded as the cause of the respiratory disease, based on clinical signs and gross lesions only. In the case of acute respiratory infection, tracheal tissue will harbor a significant amount of the virus, so it is the preferred tissue for virus detection (15,16). All the flocks tested by RT-PCR had received IBV vaccine 29 d before the clinical signs of respiratory disease were observed. Therefore, the detection of IBV by RT-PCR indicates that these flocks had experienced field exposure to IBV and excludes the possibility that the detected IBV was due to vaccination (17). Based on the RT-PCR results (Table 2), IBV was detected in 30 (58.8%) of the chicken flocks tested, confirming that IB disease is endemic in Jordan.

The current study clearly demonstrates that there is a relatively high prevalence of IB disease in commercial chicken flocks in Jordan. By utilizing diagnostic techniques such as those in this study, it is possible to conduct a detailed epidemiological investigation of the full economic impact of this disease. Future work should include the isolation and serotyping of IBV in the region in order to adopt a suitable vaccination program, using the common field serotypes as vaccines, to protect against IBV caused disease.