Morbidity due to IBV infection can reach up to 100%. Mortality rate may range from 25 to 30% in young chicks but may increase to 80% as a result of factors that are host-associated (age, immune status), virus-associated (strain, pathogenicity, virulence, and tissue tropism), or environmental (cold and heat stresses, dust, and presence of ammonia). Secondary bacterial infections (E. coli) or coinfection with immunosuppressive viruses such as Marek's disease virus, infectious bursal disease virus (IBDV) [33, 47, 48], may worsen the outcomes of IBV infection. Generally, nephropathogenic IBV strain causes high mortality, compared with strains infecting only the respiratory or reproductive systems.

Ornithobacterium rhinotracheale (ORT) is gram negative, non-motile, rod shaped, non-sporulating bacteria. ORT is reported in many countries worldwide as being associated with respiratory signs and is isolated from a wide variety of hosts such as pheasant, pigeon, rook, duck, ostrich, goose, guinea fowl, turkey, chicken, red-legged partridge, and falcon; in particular, in chickens and turkeys it causes airsacculitis and pneumonia. The pathogenicity of the ORT depends on the route of inoculation, virulence of the strain, environmental factors, the immune status of the host, and the presence of the concurrent infection. ORT spreads horizontally through aerosols and drinking water with longer survival rates at lower temperature, which also explains its dissemination during winter months and concurrent infection with other respiratory diseases common during winter season. In China, 83% of serum collected from birds with respiratory manifestation were seropositive for ORT, and 15% of serum collected from apparent healthy birds were seropositive for ORT; five of six ORT strains recovered were associated with LPAIV H9N2 infection.

Furthermore, experimental preinfection with ORT and secondary infection with LPAIV H9N2 (A/chicken/Shandong/2011 (H9N2)) three days later induced the highest mortality rate with development of severe pneumonia and airsacculitis. On the other hand, a lower mortality was induced by coinfection and pre-infection with LPAIV H9N2 than in association with a secondary infection of ORT. Lower mortality rate was also recorded by Azizpour, et al. with coinfection of ORT and LPAIV H9N2 but with a different route of inoculation. Altogether, ORT as a preinfection, concurrent infection, or secondary infection is able to exacerbate the virulence of LPAIV H9N2 as compared to infection with LPAIV H9N2 alone. However, pre-infection with ORT and secondary infection with H9N2 induces a higher mortality rate with unique histopathological lesions represented by severe pulmonary fibrosis.

The articles discussed in this review recapitulate the adverse impacts of co/secondary viral and/or bacterial infections on AIVs infection in poultry, as well as the synergy between different pathogens (Table 1). Moreover, this review provides important insights into the variation in the rates of severe morbidity and/or mortality that subsequently occur in the case of co-infection or pre-infection with another bacterial or viral pathogen. Coinfection with AIVs and a bacterial pathogen can exacerbate the course of the viral or bacterial disease.

It has also been documented that AI-related bacterial and viral infections overall may account for up to a remarkable percent of reported cases under field conditions in different countries. In developing countries, where less biosafety measures are applied, this percentage is much higher, leading to severe economic losses in the poultry industry. This could also blur syndromic surveillance. We recommend the diagnosis of more pathogens during the inspection of an infected poultry flock that could be varied due to co-infection or pre-infection history. Moreover, the application of good biosafety and biosecurity measures is likely to reduce the severity of co-infection, and can restrict the widespread transmission of those bacterial and viral pathogens. In conclusion, this review may hopefully contribute to future knowledge regarding the diagnosis and control of avian disease among different poultry sectors.

Generally the short incubation period for IBV varies with infective dose and route of infection. For example, while infection via the tracheal route may take a course as short as 18 hours, ocular inoculation leads to an incubation period of 36 hours.

Infectious bronchitis virus (IBV) is, by definition, the coronavirus of the domestic fowl. Although it does indeed cause respiratory disease, it also replicates at many nonrespiratory epithelial surfaces, where it may cause pathology, for example, kidney and gonads [1, 2]. Strains of the virus vary in the extent to which they cause pathology in nonrespiratory organs. Replication at enteric surfaces is considered to not normally result in clinical disease, although it does result in faecal excretion of the virus. Infectious bronchitis (IB) is one of the most important diseases of chickens and continues to cause substantial economic losses to the industry. Infectious bronchitis is caused by IB virus (IBV), which is one of the primary agents of respiratory disease in chickens worldwide. All chickens are susceptible to IBV infection, and the respiratory signs include gasping, coughing, rales, and nasal discharge. Sick chicks usually huddle together and appear depressed. The severity of the symptoms in chickens is related to their age and immune status. Other signs of IB, such as wet droppings, are due to increased water consumption. The type of virus strain infecting a flock determines the pathogenesis of the disease, in other words, the degree and duration of lesions in different organs. The upper respiratory tract is the primary site of infection, but the virus can also replicate in the reproductive, renal, and digestive systems. The conventional diagnosis of the IBV is based on virus isolation in embryonated eggs, followed by immunological identification of isolates. Since two or three blind passages are often required for successful primary isolation of IBV, this procedure could be tedious and time consuming. Alternatively, IBV may be isolated by inoculation in chicken tracheal organ cultures. Furthermore, IBV may be detected directly in tissues of infected birds by means of immunohistochemistry [6, 7] or in situ hybridization. The reverse transcription-polymerase chain reaction (RT-PCR) has proved useful in the detection of several RNA viruses [9, 10]. Outbreaks of the disease can occur even in vaccinated flocks because there is little or no cross-protection between serotypes [2, 11]. The necessity of IB prevention in chicken regarding the nature of the virus with a high mutation rate in the S1 gene dictates the necessity to develop effective vaccines. The first step is to study the virus strains distributed in the geographical region and determine their antigenicity and pathogenicity in order to choose a suitable virus strain for vaccination. This virus was isolated from a flock suspected of IB suffering from severe respiratory distress and experiencing high mortality. The objective of the present study was to clarify some aspects of pathogenesis of the disease caused by IRFIBV32 (793/B serotype) in experimentally infected broilers. RT-PCR test was performed to detect the presence of the virus in body tissues and samples. The clinical signs, gross lesions, and antibody response of the affected chicks were also monitored.

Infectious bronchitis (IB) is an acute and highly contagious respiratory disease of chickens characterized by respiratory signs, and in young chickens by severe respiratory distress and a decrease in egg production in layers.1 The chicken was considered the only natural host of infectious bronchitis virus (IBV) but recently pheasants has been introduced as the other natural host for IBV.2 The disease is transmitted by the respiratory route, direct contact and indirectly through mechanical spread.3 The virus belongs to Coronaviridae, Order Nidovirales. The IBV and other avian coronaviruses of turkeys and pheasants are classified as group 3 coronaviruses.4 Its genome consists of about 27 kb and codes for four structural proteins: the spike (S) glycoprotein, the membrane (M) glycoprotein, the nucleocapsid (N) phosphoprotein, and the envelope (E) protein.5,6 The spike glycoprotein (S) is anchored in the viral envelope and is post-translationally cleaved into two proteins S1 and S2.7 The S protein is very diverse in terms of both nucleotide sequence and deduced primary protein structure, especially in the upstream part of S1.8 Three hypervariable regions (HVRs) have been identified in the S1 subunit.9-11 The S1 subunit induces neutralizing, serotype-specific, and haemagglutination-inhibiting antibodies.12-17 Amino acid changes in the spike (S) glycoprotein lead to the generation of genetic variants.18,19 The high frequency of new IBV variants is a distinguished characteristic of this virus among other coronaviruses.20 Many IBV serotypes have been described probably due to the frequent point mutations that occur in RNA viruses and also recombination events. Therefore, the characterization of virus isolates which exists in the field is very important.21 More than 50 serotypes of IBV have been identified and new variants continued to emerge despite the use of live attenuated and killed IBV vaccines.22-24

The usage of live attenuated vaccines is the most important preventive measure of the disease, but anti-genically different serotypes and newly emerged variants from field chicken flocks sometimes cause vaccine breaks.18,19 The IBV Massachusetts (Mass) type was first detected in Iran by Aghakhan et al.25 In 1998, a virus similar to the European 793/B type was isolated in Iran (Iran/793B/19/08).26 In recent years, new variants of IBV have been reported from different part of the country.27-29 The aim of this study was to provide information on the molecular characteristic and the phylogenetic relationship of prevalent IBV genotypes circulating in chicken flocks in Bushehr province, Iran.

Clinical signs observed including gasping, coughing or depression started to appear from three days post-challenge with APEC or a mixed APEC and IBV infection. Bacteriophage treatment delayed the onset of the clinical signs to 6 days post-challenge (dpc) and in addition markedly reduced their severity in both groups (Figure 3). Regarding IBV infection, clinical signs were observed from four-days post-challenge, with bacteriophage treatment leading to a reduction of their severity, but not delaying their onset (Figure 3).

Bacteriophage treatment was not associated with mortality in single APEC or mixed APEC and IBV infected groups. In contrast, birds challenged with APEC alone and mixed APEC and IBV infection without bacteriophage treatment showed a 16% and 29% mortality rate at 8 and 7 days post-infection respectively (Figure 4). Bacteriophage treatment in combination with single IBV infection did not reduce the mortality of 26% (Figure 4).

Bacteriophage treatment significantly reduced APEC shedding after single APEC or mixed APEC and IBV challenge, with a gradual decrease of bacterial loads in lung tissues over time. In contrast, a non-treated and challenged group showed a significantly higher APEC load with a gradual increase over time especially at 9 and 15 dpc (Figure 5). Interestingly, bacteriophage treatment significantly reduced IBV shedding in the mixed infected group but not in the IBV alone infected group comparing to the mixed infected group without bacteriophage treatment. The bacteriophage treated group infected with IBV showed relatively comparable results to the infected non-treated group. Groups with single IBV infection and mixed APEC and IBV infection with bacteriophage treatment showed a reduction, but not statistically significant, of IBV comparing to single IBV infection without bacteriophage treatment, with the reduction only becoming statistically significant at 15 dpc (Figure 6).

Infectious bronchitis (IB) is primarily a respiratory disease of chickens but with potential to cause more widespread infection in the urinary and reproductive tracts in chicken leading to significant production losses in commercial broiler and layer flocks worldwide. The causative infectious bronchitis virus (IBV) belongs to the family Coronaviridae. The disease is usually characterized by high morbidity and low mortality in mature birds, whereas in naive young birds (2–3 weeks of age), mortality up to 100% can be observed. Being an RNA virus with the ability to mutate and recombine, IBV persist as numerous serotypes and strains. The control of IB relies on vaccination. Vaccines are available for commonly occurring serotypes and strains but they are not necessarily antigenically similar to the wild-type viral strains circulating in poultry barns. Although, these vaccine strains may provide some degree of protection for some related strains known as protectotypes, the commonly available vaccines may not elicit protective immune responses in a flock if the field strains are antigenically very different from the vaccine strains. Owing to this reason, vaccination against IBV is not currently considered to be a very effective control method and other biosecurity measures are necessary to prevent the introduction of IBV into poultry production facilities.

IBV is known to replicate in the respiratory tract leading to changes in the muco-cilliary clearance mechanism, as such, expose the IBV infected birds to secondary bacterial infections. Additionally, IBV has tropisms for a variety of tissues. However, the mode of dissemination from the common route of entry, i.e. the respiratory route, to the rest of the body systems could potentially be due to the initial viremia. Once disseminated, IBV infects epithelial cells of the reproductive and urinary systems, particularly the oviduct and kidney depending on the infecting strain. Recently, it has been shown that a nephro-pathogenic strain of IBV (B1648) could replicate in peripheral blood monocytes leading to viremia. The infection of circulating monocytes could potentially disseminate IBV to the urinary tract, liver and spleen.

Macrophages play roles in innate immune responses, as well as in mounting adaptive immune responses by functioning as antigen presenting cells, as such they are critical in protecting animals from microbial infections. Although it is known that macrophage numbers are elevated in the respiratory tract in response to IBV infection, the role played by macrophages in IBV infection, particularly if they serve as a target cell for viral replication is not known. Macrophages have been implicated to play in an important role in the pathogenesis of some animal and human viruses including Marek’s disease virus in birds, feline corona virus in cats, and human immunodeficiency virus (HIV). It was also shown that coronaviruses such as severe acute respiratory syndrome (SARS)-coronavirus (CoV) can replicate within human macrophages thereby interfering with macrophage functions leading to severe pathology. However, a single report based on in vitro studies indicated that IBV, particularly nonpathogenic Beaudette and Massachusetts type 82822 strains do not replicate in avian macrophages.

Therefore, in this study we investigated the interaction of IBV with macrophages in lungs and trachea in vivo and macrophage cell cultures in vitro using two IBV strains, Connecticut A5968 (Conn A5968) and Massachusetts-type 41 (M41) which are known to induce clinical disease and pathological lesions in chickens. As implicated in some other viruses, we hypothesized that these two strains of IBV replicate within avian macrophages leading to productive replication and interfering with selected macrophage functions in the process.

The present study has revealed that the chickens infected with the three tested strains showed the specific respiratory signs with high clinical signs. These infected chicks were sero-converted at 14 dpi and confirmed the spread of the inoculated virus. In addition, the macroscopic lesions and histopathological examination revealed specific lesions of trachea and lung, without renal manifestations. The histopathology of respiratory organs demonstrated a high lesional score of IBV/TU strain which correlate with the clinical score of 140. Moreover, virus re-isolation was performed successfully for all the three tested strains. Results obtained with real time PCR in organs sampled from infected birds (trachea, lung and kidney) and from fluid allantoic in the first passage in SPF eggs, showed a significant abundance of the virus in the respiratory tract. In other words, the findings of molecular analysis of virus re-isolation are strongly correlated with the histopathological tests.

Finally, this current report justify that Italy 02 genotype isolated from different regions of Morocco is capable to induce a severe respiratory disease with a wide distribution of respiratory system. However, this genotype do not cause any kidney damage and without causing mortality.

In the infected birds, clinical signs observed include white, soft-shelled eggs, greenish diarrhoea, and respiratory distress (coughing, sneezing, and rales). As at the time of sampling, egg production had dropped by approximately 55%. Mortality was observed before the birds came into lay and continued during lay with a range of 1%–2%. At post-mortem, lesions observed include cloudy air sacs, frothy and congested lungs, whitish, cheesy materials on the serosal surface of the intestine, white nodules on the surface of the ventricles, proventriculus and intestines, and presence of ascitic fluid in the abdomen. These signs and lesions are consistent with findings from previous reports (Awad et al., 2014b; Ballal et al., 2005). Due to the similarities in clinical and pathological lesions presented by infections involving respiratory viruses, clinician that based their diagnosis on these non-pathognomonic signs may miss the aetiological agents. Though the mortality rate in the affected flock was low (1%–2%), it is consistent with the report by Awad et al. (2014a). Generally, single infections with IBV result in low mortality. However, exacerbation by concurrent infection with other pathogens of viral or bacterial origin have been reported (Jackwood, 2012).

As shown in Figure 1, the tissue homogenate was positive for IBV by RT-PCR and negative for AIV and NDV. Upon inoculation of ECEs with the tissue homogenate, no noticeable changes were observed in the embryos in the first few passages. However, at passage four, embryo death with characteristic IB lesions, including curling, dwarfing, and hemorrhages on the embryos (13 d of age) were conspicuously discernible (Fig. 2). Allantoic fluids harvested from the eggs of both dead and live embryos did not cause agglutination of chicken red blood cells in spot hemagglutination test (data not shown) and this confirms the absence of hemagglutinating agent. In this study, we have shown that IBV which is less described and often given less attention and not NDV or AIV was the causative agent of infection in the 54-wk-old laying birds showing respiratory signs and severe drop in egg production. Although ND was first suspected by the consulting clinician due to its enzootic status in Nigeria. In a limited study, the prevalence of IB was found to equal that of ND confirming the increasing important enzootic status of IB in Nigeria poultry (Shittu unpublished data). In this study, successful isolation of IBV in embryonating eggs was accomplished after four blind passages with the embryos developing lesions characteristic of IB such as stunting and dwarfing (Fig. 2). For IBV isolation, ECE and tracheal organ cultures (TOC) are substrates of choice although TOC has an edge over ECE in that stasis of the tracheal cilia could be observed in the former upon primary inoculation (OIE, 2008). In this study, ECE isolation technique was found to be equally useful.

According to the farm records, the birds were vaccinated with inactivated oil-emulsion vaccine which contained IBV antigen. However, this seemed not to have protected the birds against morbidity, mortality, and decreased egg production. It has been reported that chickens with low antibody level to IBV serotype could experience severe drop in egg production, whereas those with high antibody level are less affected in terms of egg quality and production possibly as a result of immune protection (Ballal et al., 2005). Available literature show that the use of inactivated IB vaccines alone does not confer adequate protection on the birds except where they are first primed with live attenuated IB vaccines during the early stages of production (Cook et al., 2012). In addition, IBV serotypes do not cross-protect (Jackwood et al., 2010), thus the vaccine must be designed based on circulating serotypes in the locality. In Plateau State, there are no available data on the circulating IBV strains. Although most vaccines being used on the field in Nigeria are predominatly Mass serotype, detection of other serotypes in this investigation is a further indication and support speculation that the vaccine strains being used differ from some of the serotypes in circulation. Furthermore, Ducatez et al. (2009) identified a novel IBV serotype “IBADAN” from southwestern Nigeria and no information exists on the ability of the vaccine strains in use to protect birds against this novel strain (de Wit et al., 2010). It is, however, not known if this serotype circulates in the northern part of the country.

Antibody prevalence and high GMT titre distribution of the three serotypes of IBV used in the study for Mass, Conn, and Ark are 490.5, 215.3, and 534.9, respectively, as shown in Tables 2 and 3. In all 32 serum samples tested, 100% seropositivity was also observed for Mass, Conn, and Ark serotypes. In addition, concurrent infections with Mass/Conn, Mass/Ark, Conn/Ark, and Mass/Conn/Ark serotypes were observed.

Interestingly, the HI results for the three serotypes (Mass, Conn, and Ark) tested in this study revealed 100% seropositivity (Table 2). This clearly shows that the three IBV serotypes are present in the farm and may be in circulation in Plateau State with the possibility of other hitherto unreported serotypes. As reported by Jackwood (2012), several serotypes and variants of IBV circulate around the world with some having specificity for a particular location, making them indigenous to those places. Such may include the newly described serotype by Ducatez et al. (2009) which we could not test for in our samples due to unavailability of strain specific diagnostic reagent. The Mass strain of IBV has been reported to be widespread across the globe possibly due to its use as a vaccine (de Wit et al., 2010). In Nigeria, breeder stocks are often vaccinated with live IBV vaccine using Mass-like strains at much younger ages (Ducatez et al., 2009). However, in this case, a trivalent killed-adjuvanted vaccine containing IBV was said to have been administered without prior priming with live IBV vaccine. As previously reported (Bijlenga et al., 2004; de Wit et al., 2010), antibody response to killed-adjuvanted IBV vaccine without priming the birds with live attenuated IB vaccine are usually poor. It can, therefore, be deduced that the high titre of antibodies to the three IBV serotypes detected in this study (Table 3) may not have emanated from vaccination, but could be a result of recent or continuous infection with circulating strains of the virus as also shown by molecular detection and virus isolation. To the best of authors’ knowledge, co-circulation of multiple serotypes of IBV as described here is the first documented report from Nigeria.

The virus isolate used in this study was IRFIBV32 (GenBank: HQ123359.1). It was obtained from Shiraz Veterinary University and was propagated two times in 9- to 11-day-old embryonated chicken eggs. The embryo lethal dose (ELD50) was calculated according to the Reed and Muench formula.

Poultry production in Algeria faces many zootechnical and health constraints, such as viral infections like avian infectious bronchitis (IB). The avian IB virus (IBV), a member of the Coronaviridae family (order Nidovirales and genus Coronavirus), frequently infects broilers and egg-laying hens and leads to severe economic losses to the poultry industry. Since its discovery in the 1930s, the IBV has been identified as the major cause of respiratory infections and poor zootechnical performances. Interestingly, it can also multiply in the renal tissue and cause nephritis, a phenomenon first described in the United States. More recently, IBV-associated nephritis has been accepted as the most pressing problem in broiler flocks in many countries.

The most effective method of protecting poultry from IBV infections is through live or killed vaccines. However, nephritis associated with infectious bronchitis has been observed in several vaccinated flocks, suggesting that the current vaccination strategies against IBV may not provide adequate protection. In fact, outbreaks of IB are frequently caused due to the strains serologically different from those used for vaccination. Since its discovery in 1931, a large number of serotypes or variants of IBV have emerged, and little or no cross-protection occurs between these serotypes. Therefore, it is crucial to track epidemic-causing serotypes in each geographic region or country and produce new vaccines to control IB.

In Algeria, poultry flocks have been vaccinated against IB with the Massachusetts (Mass) strain combined with the IB 4/91 United Kingdom variant strain or 793/B since the past few years. However, kidney damage with suspicion of IB has also been reported in recent years in spite of vaccination but has not been confirmed so far. This has led to the speculation of the possible emergence of variant strains against which conventional vaccines are not completely effective.

The aim of this study was to investigate the presence of IBV among Algerian broiler flocks and its possible involvement in broiler kidney damage. Clinically diseased broiler flocks were sampled and analyzed by the hemagglutination inhibition (HI) test and reverse transcriptase-polymerase chain reaction (RT-PCR) followed by phylogenic analysis.

Bacteria and parasites have been considered the primary etiological agents of gastroenteritis in commercial poultry. However, many viral infections have been associated with enteric diseases of chickens and turkeys, including coronavirus, reoviruses, rotaviruses, adenoviruses, enteroviruses, and the members of Family Astroviridae (Chicken Astrovirus-CAstV and Avian Nephritis Virus-ANV) [22–27]. Infections with the previously mentioned viruses are believed to be important in the pathogenesis of the economically important enteric disease, such as runting and stunting syndrome (RSS), which affects young chickens, mainly broiler chickens [6, 9, 28, 29] and turkeys. Recently, studies have included the enterotropic strains of infectious bronchitis virus (IBV) as a possible etiological agent of enteritis in chickens. IBV can grow in many cells of the gastrointestinal tract, and some Asian strains were described to cause lesions in the proventriculus. IBV is believed to only persist in the gastrointestinal tract of young chickens and in layers without clinical disease.

Different denominations or terms have been used to describe the enteric disease in poultry because the clinical signs are infrequent or occur independently of previous conditions, such as the presence of primary or secondary etiological agents, the immune and nutritional status of the host, and environmental conditions [3, 4, 15, 33]. According to Saif, the gastrointestinal tract (GIT) is the primary organ of the body that is exposed and a variety of injuries against it could result in inefficient utilization of nutrients during the early stages of development. Of the various signs described for enteric disease, diarrhea and lack of normal development are the most consistently reported symptoms.

The results obtained in this study demonstrate a high level of infection with one or more of the seven viruses investigated in the chickens with clinical symptoms (65.4%), as shown in Table 2. However, samples taken from chickens without symptoms (15.4%) were also positive for these viruses (Table 2), which demonstrates a similar prevalence between these two groups. This result indicates that chickens should be shedding the virus via the enteric tract without showing any clinical symptoms; therefore, these chickens are considered asymptomatic carriers or reservoir, representing a potential source of infection. Other studies showed lower levels of rotavirus infection (4.1%) in normal chickens, while higher frequencies were found in asymptomatic flocks with 30% rotavirus and 30% for CAstV from healthy flocks of turkeys. However, these studies did not survey a wide range of viruses. In an extensive survey of turkey flocks with enteric disease and healthy turkey flocks in the United States, Rotaviruses were detected slightly more frequently in healthy than in diseased flocks. Astroviruses were detected in the intestinal contents of poultry prior to the onset of clinical disease and gross pathologic changes. ANV-induced clinical disease presents as kidney lesions in young chickens but only presents as a subclinical persistent infection in mature chicken. These conditions reflect those viruses, other than reovirus, coronavirus, and chicken parvovirus, which have been identified in samples from flocks that appear healthy and may have different degrees of pathogenicity [22, 25]. In fact, there are indications that different serotypes and even strains within the same serotype can vary in their ability to produce illness and death. Moreover, several factors influence the susceptibility of chickens to enteroviruses, such as age, passive immunity level, simultaneous infection with other pathogens, and management failures, which cause stress [14, 36, 37].

Regardless of the symptoms, most of the samples (226 samples) representing 80.7% were positive for one or more of the viruses, which demonstrates the high prevalence of these viruses in Brazilian chicken flocks (Table 2). In almost half of these positive samples (115 samples or 41.1% of total), two or more viruses were detected simultaneously, which highlights the hypothesis of multicausal etiology for the enteric disease. The distribution of the seven viruses in these concomitance samples was demonstrated and is shown in Table 2.

The combinations were scattered proportionally according to the prevalence of each virus and did not present a pattern or a constant frequency. None particular combination of the viruses was observed with significant repeatability. Rarely, a single agent is the sole contributing factor to enteric disease; moreover, the presence of different combinations of viruses could result in varied disease presentations.

IBV was the most prevalent virus detected in the samples that contained a single virus (Table 2) and in all of the samples (120 samples/42.9%), as shown in Table 3. IBV is the infectious agent associated with infectious bronchitis of chickens and is responsible for outbreaks in many countries, including Brazil. Infectious bronchitis (IB) is characterized by respiratory, reproductive, and, sometimes, renal signs. However, some strains of this virus have been demonstrated to multiply in intestinal cells and were referred to as possible agents of diarrhea or may at least have some role in enteric diseases. Recently, we isolated variant strains of IBV from the intestinal contents of chickens with enteritis but without the typical respiratory, reproductive, or renal signs. In these cases, IBV was the only pathogen present, while all of the samples were negative for astrovirus, reovirus, and rotavirus. When one-day-old SPF chicks were inoculated with filtrated IBV variants, respiratory signs but not diarrheal or renal signs were observed (data not published). In addition, these samples were not screened at for other likely agents of enteritis, such as ANV or chicken parvovirus. However, the IBV strains that were detected in the intestinal contents may not play a role as direct pathogens in enteric disease.

Of the viruses investigated, ANV was the second most prevalent by absolute numbers and was detected in 83 samples (29.6%), as shown in Table 3. If IBV is not the primary causal agent, then ANV is the most prevalent pathogen causing enteric disease that was detected in this study. Originally regarded as a picornavirus, ANV was recently characterized as a new member of the Family Astroviridae in 2000 and has been detected in kidney samples from young chickens with growth deficiencies in Hungary. A recent study that was performed in the United States to detect the presence of several enteroviruses in chicken flocks demonstrated that ANV was the most prevalent virus followed by coronavirus, reovirus, CAstV, and rotavirus. The characteristic signs of avian nephritis that are caused by AVN vary from none (subclinical) to outbreaks characterized by diarrhea, growth retard, renal failure with tubulonephrosis, interstitial nephritis, uricosis, and death [37, 39].

Chicken astroviruses (CAstV) were the third and rotaviruses were the fourth most frequent agents detected in this study, with 21.1% and 17.1%, respectively (Table 3). Previously, a high frequency of CAstV was reported by Pantin-Jackwood et al., where 21 of the 34 (61.7%) analyzed samples were identified as positive for CAstV. Astroviruses cause or have been associated with acute gastroenteritis in humans, cattle, swine, sheep, cats, dogs, deer, mice, and turkeys, as well as with fatal hepatitis in ducks [26, 29, 40, 41]. However, the clinical importance of CAstV remains unclear. On the other hand, rotaviruses are frequently associated with enteric disease, but the economic significance of rotaviruses to the poultry industry has not yet been defined [10, 36, 42]. Rotaviruses were present in 46.5% of the samples and in chicken flocks from all regions of the United States that were tested during 2005 and 2006. Studies on the classification of serogroups by PAGE have indicated that group D rotaviruses are the most frequently reported group in United Kingdom flocks. Furthermore, group D rotavirus infection has recently been implicated as a contributing factor to the development of RSS in 5- to 14-day-old broilers in Germany. The A, F, and G groups have also been detected in broiler flocks [36, 43]. In Brazil, a study identified nine distinct electropherogroups using PAGE, but only three were similar to the A group profile of avian rotavirus. More recently, rotavirus was also detected in 45.3% of chicken and layer flocks in Brazil, and approximately 15% of these samples were identified as belonging to group A. In a previous study using the same RT-PCR test for the NSP4 gene, four different genotypes of rotaviruses were detected in samples from commercial turkeys, reflecting the great genetic variability of rotaviruses similar to that reported in humans and other mammals.

Parvoviruses are known to cause gastrointestinal disease in mammalian species and have been implicated as a cause of malabsorption syndrome in chickens and enteritis in turkeys [9, 44]. However, the role of chicken parvoviruses in disease has not yet been determined. Previous studies using electron microscopy have identified parvovirus-like particles in the samples of chickens with enteric disease, but it was not possible to confirm the presence of parvoviruses until the development of new molecular tools such as PCR. In this survey, 12.1% of the chickens were positive for chicken parvovirus, which is slightly more than those which were positive for reovirus and adenovirus and indicates that chicken parvovirus should be considered as an important etiological agent of enteric disease in chicken. In a nationwide survey in the United States, a high prevalence of chicken parvovirus (77%) was detected in 54 chicken samples.

Fowl Adenovirus subgroup I had a frequency 9.6% positive samples among the viruses investigated in this study (Table 3). The subgroup I and II adenoviruses are considered widely distributed in poultry and are commonly found in enteric samples [10, 47]. Although the association between adenovirus and disease is well established for subgroup II (turkey hemorrhagic enteritis and related viruses and subgroup III (egg drop syndrome)), the role of most subgroup I avian adenoviruses as pathogens is not well defined.

Avian reoviruses are frequently detected in the intestinal tracts of poultry with enteric disease and are widely distributed in poultry. Avian reoviruses were identified in this study of lowest frequency with only 7.9% (Table 3). Avian reoviruses were identified in 62.8% of the chicken flocks tested from all regions of the United States during 2005 and 2006. Avian reoviruses have been isolated from a variety of tissues in chickens that are affected by assorted disease conditions including viral arthritis/tenosynovitis, respiratory disease, immunosuppression, and enteric disease or malabsorption syndrome. Moreover, avian reoviruses have also been demonstrated to cause a synergistic effect and enhance the pathogenicity of other agents, such as chicken anemia virus, Escherichia coli, and infectious bursal disease virus. However, the severity of the effects depended on the strain of reovirus. A clear relationship is frequently reported between arthritis/tenosynovitis and reovirus infection. However, the role of avian reoviruses in enteric disease remains unclear, when we consider other primary pathogens associated with enteric problems in chickens [49, 50].

The association between viruses and age showed that the viruses could be detected at different stages in the broilers, breeders, and pullet/layer hens; for example, virus was detected from the first week of age to the last week, especially in broilers. An important finding was the detection of CAstV in one-day-old breeder chicks, which may constitute an indicator of vertical transmission. According to McNulty et al., in a longitudinal study carried out in young poultry flocks, the avian rotavirus is normally detected after two weeks of age because of the modulation of the passive maternal immunity. However, Tamehiro et al. reported samples that were positive for rotavirus in one-week-old broiler chickens. Furthermore, Pantin-Jackwood et al. demonstrated the presence of rotavirus in samples collected from poults before placement, in addition to the presence of astrovirus throughout their lifetime.

In conclusion, the primary viruses detected in this study were the IBV, ANV, CAstV, and rotavirus, followed by chicken parvovirus, adenovirus, and reovirus. However, the astroviruses (ANV and CAstV) should be regarded as the most important pathogens because coronavirus (IBV), although present in a higher percentage of samples, has not been demonstrated to cause pathogenicity in the enteric tract in our previous experiments (data not published). Detection of virus was high among chickens with and without clinical signs of disease, which demonstrates the high prevalence of these viruses in asymptomatic carriers and indicates that these carries may represent a potential source of infection. Moreover, the presence of CAstV in one-day-old breeder chicks that was detected in this study may suggest a vertical transmission. In addition, the injuries that these viruses cause can interfere with the absorption capacity of the intestinal tract during the first weeks of life of these birds, which enhances the negative impact on productivity for the rest of the production cycle. This is the first study to demonstrate the presence of these viruses in association with enteric disease in Brazil. Experimental challenges should be conducted to establish the role of these viruses and to analyze their impact on commercial chicken productivity.

Three IBV field strains (coded, IBV/MN, IBV/RA and IBV/TU), were isolated from trachea, lungs and kidney of broiler chickens suffering from the specific clinical signs of IB, and were previously characterized to belong to Italy02 serotype at laboratory of Biopharma between 2010- 2014. Phylogenetically, the three strains of Italy02 genotype branched and clustered with Spanish genotypes, are very closely related to Italy 497/02-1 (98.9 %); Spain/05/866 (98.9 %); and Spain/04/221 (97.4 %). The sequences of the three strains have a S1 gene nucleotide identity ranged between 96.9 % and 98.7 % when compared to each other.

These IBV strains were propagated by inoculation in 9- to 11-day-old SPF egg as described by Owen et al. For virus titration 6-fold dilutions were inoculated into the allantoic cavity of six SPF chickens embryos, and incubation during six days at temperature of 37 °C +/- 2 °C and relative humidity of 75 % +/- 10 %. The titer is calculated following the Reed and Muench method.

All birds were observed twice daily and clinical signs recorded in accordance with. Briefly, no signs were recorded as 0, mild signs included mild gasping, coughing or depression and were recorded as 1, and severe gasping, coughing or depression with ruffled feathers was considered as severe signs and recorded as 2. Birds with severe signs unable to move were recorded as 3 and humanely culled and samples collected. Birds which unexpectedly succumbed to disease were also recorded as 3 and samples collected.

Infectious bronchitis (IB) is a highly contagious viral disease of the upper respiratory and urogenital tract of chickens, which is caused by infectious bronchitis virus (IBV). The disease is prevalent in all countries with an intensive poultry industry, affecting the performance of both broilers and layers, thereby causing the considerable economic loss in poultry industry worldwide.1 The virus is the coronavirus of the domestic fowl that is mainly observed in chicken. It possesses a positive sense single–stranded RNA genome that ranges from 27 to 31 Kb.2 The number of IBV serotypes that exist throughout the world is unknown. More than 50 different serotypes have been listed and new IBV variants continue to emerge.3 It is now well documented that a considerable number of different serotypes with antigenic and pathogenic differences exist in poultry industry of different parts of the world.4

The D274 type was the most common type of IBV in several western European countries in the early and mid-1980s.5 IBV strains of the 4/91 type, which are also known as 793B, were first reported and characterized in Britain, 1991,6 and have been the dominant genotype in Europe.7 The Serological survey has revealed a high incidence of IBV infection of the 793/B type in layer and broiler chickens worldwide.8 Infectious bronchitis still causes serious problems in the Iraqi poultry industry due to the inability of the vaccines to protect the different genotypes. Due to the limited network of poultry diagnostic laboratories in Iraq, differential diagnosis is only made according to clinical signs and gross lesions.

The characterization of IBV has raised additional problems in terms of both epidemiology and control. Although IBV in the poultry farms in Iraq (with H120 and 4/91 strains) is presently controlled by both inactivated and live attenuated vaccines, the outbreaks of IB have still been observed on broiler farms.9,10 In Iraq, the first report of identification and genotyping of IBV isolates has been from Kurdistan-Iraq, which indicated the circulation of 793/B ( with the prevalence rate of 25 %) along with a new IBV variant (Sul/01/09) in vaccinated (Ma5 , or 4/91) broiler farms.10 So far, there has been no report on the prevalence rate of IBV genotypes in the south of Iraq.

The aim of the present study was to detect of three IBV genotypes including (Massachusetts; Mass), 793/B and D274 in the south of Iraq.

Avian infectious bronchitis virus (IBV) is a highly contagious pathogen of chickens that replicates primarily in the respiratory tract and also in some epithelial cells of the gut, kidney and oviduct. IBV is a virus member of genus Coronavirus, family Coronaviridae, order Nidovirales. The virus possesses a positive stranded RNA genome that encodes phosphorylated nucleocapsid protein (N), membrane glycoprotein (M), spike glycoprotein (S) and small membrane protein (E). The spike glycoprotein is post-translationally cleaved into two subunits, S1 and S2. The S1 protein forms the N-terminal portion of the peplomer and contains antigenic epitopes mainly within three HVRs. Neutralizing and serotype specific epitopes are associated within the defined HVRs.

Variation in S1 sequences, has been recently used for distinguishing between different IBV serotypes. Diversity in S1 probably results from mutation, recombination and strong positive selection in vivo. Antigenically different serotypes and newly emerged variants from field chicken flocks sometimes cause vaccine breaks. The generation of genetic variants is thought to be resulted from few amino acid changes in the spike (S) glycoprotein of IBV.

In Egypt, isolates related to Massachusetts, D3128, D274, D-08880, 4/91 and the novel genotype; Egypt/Beni-Suef/01 were isolated from different poultry farms. The commonly used IBV attenuated vaccine is H120 while the Mass 41 (M41) strain is commonly used in inactivated vaccines.

In the present study, Egypt/F/03 was isolated from 25-day-old broiler chickens in Fayoum Governorate, identified by Dot-ELISA, RT-PCR and sequenced to determine its serotype. Pathogenicity test to 1-day-old chickens and protection afforded by the commonly used H120 live attenuated vaccine were also performed.

Intra-assay repeatability experiments showed that the coefficient of variation (CV) was 5.09% for ILTV and 8.05% for IBV. Inter-assay repeatability experiments showed that the CV was 8.11% for ILTV and 9.48% for IBV. All CVs were less than 10%, indicating that the method has high repeatability. The results were shown in Tables 4 and 5.

Avian infectious bronchitis (IB) is an acute and highly contagious viral respiratory disease of chickens. IB occurs worldwide and causes substantial economic losses in the poultry industry. IB is caused by the infectious bronchitis virus (IBV) which belongs to the order Nidovirales, family Coronaviridae, genus Gammacoronavirus. IBV causes upper respiratory illness, nephritic syndrome, and a drop in egg production in layer chickens. The mortality rate can be high in young chickens, especially with other secondary complications such as viral and bacterial infections.

Currently, novel serotype and variant IBV have emerged due to rapid mutation rates, viral recombination, and host selection pressure, which have resulted in IB prevention becoming a global challenge. Vaccination is the best method to control the disease, and the current widely used commercial IBV vaccines are live-attenuated or inactivated viruses with adjuvants derived from classical or variant serotypes, however, live-attenuated IBV vaccine strains have some limitations, including genetic instability, reversion to virulence, and recombination between vaccine and wild-type viruses. IBV-inactivated vaccines also face some defects such as lack of ability to elicit strong cellular immune responses and lack of cross-immunoprotective effects. Furthermore, chicken flocks suffer co-infection with IBV and avian orthoavulavirus-1 (AOAV-1), which is a member of the genus Orthoavulavirus of the family Paramyxoviridae, often referred to as Newcastle disease virus (NDV), which hinders the prevention and control of these diseases. These two pathogens remain a vital threat to domestic poultry production. Thus, it would be advantageous to develop a next-generation vaccine that could serve as a bivalent vaccine against IBV and NDV challenge.

In recent years, epitope-based vaccines have been widely studied for their advantages, including safety, genetic stability, ease of production, and the ability to elicit immune responses against multi serotype pathogens.. Thus, they have been broadly applied for a range of viral, bacterial, and parasitic diseases. The most important factors for designing a multi-epitope vaccine are predicting and screening the functional neutralizing B cell epitopes and species-restricted T cell epitopes. IBV spike glycoprotein (S) is proteolytically cleaved into a 92-kDa S1 subunit and a 84-kDa S2 subunit, and these play an important role in viral infections. The S1 subunit possesses a receptor binding domain (RBD) that is primarily responsible for adsorption and entry into host cells. Epitopes of neutralizing antibodies are also found mainly on the S1 subunit. Therefore, the S1 protein has an indispensable effect on the protective immune response of IBV. Our previously constructed IBV S1 protein multi-epitope cassette includes sequences encoding neutralizing epitope domains (aa 24–61, aa 132–149, and aa 291–398) of IBV Australian T strain (Genbank AY775779.1) and BF2-restricted CTL epitopes aa 413–421 and aa 517–525 of the QX-like IBV SH1208 strain, which shares an ancestor with the SAIBK strain (Genbank DQ288927.1), and aa 45–52 and aa 413–421 of the Holte strain (Genbank L18988.1). The immune-protection experiment demonstrated that the recombinant DNA vaccine, consisting of this IBV S1 protein multi-epitope cassette, could induce a high level of immune responses and full protection of chickens against a lethal dose of IBV challenge. The results indicated that the multi-epitope-based vaccine provides a promising strategy for vaccine development against IBV infection.

The technology of the reverse genetic system (RGS) for NDV was established in Europe two decades ago, which has been successfully applied to research on pathogenicity, replication regulatory mechanisms, and vaccine vectors. Meanwhile, the LaSota strain has been used as a safe and effective live vaccine that protects against NDV challenge for several decades, because it can elicit both humoral and cell-mediated immune responses, and vaccine production is cost-effective.. Thus, it would be very attractive to rescue a rNDV LaSota strain expressing the immunogenic proteins or functional epitopes of other respiratory viruses, such as the S1 protein of IBV.

The aim of this study was to develop a live vaccine using recombinant NDV expressing previously identified IBV multi-epitope cassette. The protective efficacy of the recombinant NDV vaccine was evaluated by heterologous IBV and velogenic NDV challenge. The findings could provide an alternative and promising strategy for the development of a cost-effective and extensively immune-protective vaccine for the control of ND and IB.

Avian infectious bronchitis (IB) is caused by a virus in the Coronaviridae family, genera Gammacoronavirus. It is a highly contagious disease with a short incubation period. The Avian coronavirus was previously classified, and is most commonly referred to, as avian infectious bronchitis virus (IBV). The IBV is responsible for respiratory disease, which manifests in clinical symptoms such as sneezing and tracheal-bronchial rales that can lead to the development of more severe symptoms [2, 3]. Infected birds exhibit reduced performance, consequently leading to a reduction in weight gain and deterioration in egg quality and quantity. Secondary bacterial infections will also contribute to economic losses. Carcass condemnation due to the development of airsacculitis [4, 5] negatively impacts commercial sales of bird meat and eggs. Brazil was once the world’s largest exporter of poultry and currently the world’s third largest producer of bird meat. The consequences of IBV are a significant threat to Brazil’s poultry industry.

The IBV genome consists of a non-segmented positive-sense single-stranded RNA that is approximately 27.6 kb in length. It encodes non-structural (accessory proteins) and four structural proteins: the nucleocapsid protein (N), the spike protein (S), the envelope protein (E), and the matrix protein (M). The nucleocapsid protein, or N protein, consists of 409 amino acids. It has a molecular mass of approximately 50 kDa and directly binds with the viral genome to form the virion nucleocapsid [6, 7]. Its structure is highly conserved, with different strains of IBV sharing a high degree of identity (94–99%). The N protein is also known for its immunogenicity, inducing specific antibody and cytotoxic T-cells mediated responses [9, 10]. There is significant interest in the use of the IBV N protein as an important target for diagnosis since it possesses the antigenic characteristics required for the development of serological assays that can be applied to detect or quantify antibodies against the IBV.

The laboratory diagnosis of IB is dependent on direct and indirect techniques. The direct techniques are employed for viral isolation and genomic or phenotypic identification of the virus, while the indirect methods are used to detect specific antibodies. In addition to being applied for serodiagnosis, serological techniques can also be employed to evaluate the immune responses stimulated by vaccines. Commercial ELISA kits are typically used to indirectly diagnose IBV. These kits, however, are expensive when large number of samples require screening and they are not acessible for applications with the scale of the Brazilian poultry industry [13–15].

ELISA techniques currently available are designed to detect polyclonal antibodies that target the whole virion. The use of nucleoprotein as the antigen for diagnosis and evaluation of vaccine immune responses is an interesting target to explore since this protein plays a important role in IBV virus replication and the induction of a specific immune response in infected birds [16, 17]. The use of recombinant antigens in the design of a specific diagnostic technique facilitates the development of highly sensitive and specific assays that display a high antigen concentration and, thereby, reduce or eliminate background reactions. The use of recombinant antigens also represents a viable method of reducing immunoassay development costs. Easy production of antigens in expression systems leads to simple and efficient antigen development which can reduce the production costs associated with diagnosis. The aim of the current study was to evaluate the combined use of an ELISA and Western blot (WB) to detect antibodies against the nucleocapsid protein of IBV.

Infectious bronchitis (IB) is a serious and highly contagious disease of chickens all over the world. Avian infectious bronchitis virus (IBV) was first reported in the USA for replicating in the respiratory tract and some other epithelial cells of gut, kidney, and oviduct. Subsequently, some strains of IBV caused pathology in non-respiratory organs (such as kidney and gonads) were documented. The clinical disease and production problems frequently cause catastrophic economic losses to the poultry industry, accompanied by decreased production performance in breeder flocks, diminished egg production and poor egg quality in laying flocks. In China, IB has a more profound social impact for chicken industrial contributes to the rural economy. More importantly, there is accumulating evidence that nephropathogenic type IB has been more and more prevalent in China recently, but the strains isolated in earlier years mainly caused respiratory signs, which suggested that selecting and immunization with the appropriate vaccine strain is of great importance to control IB infection.

Some researchers reported that satisfactory cross protection could be provided by appropriate vaccine programs against genetically or antigenically unrelated IBVs. However, this symphysial vaccine manner was restricted by the diversity of the IBV strains. Since IBV strains were first isolated and identified in China in 1982, various live-attenuated and inactivated vaccines derived from respiratory-typed strains have been widely and extensively used in chicken farms to reduce the adverse effect of the IBV. However, the disease continues to emerge and cause serious production problems, even occurred in routinely vaccinated layer and breeder flocks in China, and the situation gets worse as time progressed. The most possible explanation for this phenomenon may be that the vaccine effectiveness is diminished by poor cross-protection against the circulating strains.

The spike tip glycoprotein (S1) of virus particle has direct relation to induce virus neutralizing antibody, and determines the cross-protection. Our previous research had confirmed that the predominant IBVs were nephropathogenic IBVs were mainly A2-like strains in China during 2008-2009. This study was to further investigate the prevalence of nephropathogenic IB under immune pressure with routine vaccine strains in China. Additionally, the effectiveness of vaccination program using the common field strains practically against IB was also verified.

All experimental procedures were approved by the Institutional Animal Care Committee of the National Administration of the Algerian Higher Education and Scientific Research (Ethical approval number: 98–11, Law of August 22, 1998) and were conducted according to the recommendations of the “Guide for the Care and Use of Laboratory Animals.”

The sensitivities of xMAP and ELISA were compared using 1:50 to 1:3200 ILTV-positive sera and 1:100 to 1:6400 IBV-positive sera. The results showed that xMAP detected ILTV-positive sera at 1:1600 and IBV-positive sera at 1:3200, while ELISA detected ILTV-positive sera at 1:400 and IBV-positive sera at 1:1600 (see Table 6).

To compare duplex xMAP assay with ELISA for ILTV/IBV detection, 90 chicken serum samples from a chicken farm in Huizhou, China were used and the results are shown in Table 7. For ILTV, xMAP detected 76 positive cases whereas ELISA detected 69 positive cases; among them, 67 samples were detected positive by xMAP assay and ELISA. All 90 samples were detected as positive for IBV by both methods.

Six 12-month-old purpose-bred female ferrets (Mustela putorius furo), seronegative for currently circulating influenza A (pH1N1 and H3N2) and B viruses and Aleutian disease virus, weighing 600–900 g., were obtained from a commercial breeder (Euroferret). Animals were housed and they received water and food ad libitum. The experiments were conducted in strict compliance with European guidelines (EU directive on animal testing 86/609/EEC) and Dutch legislation (Experiments on Animals Act, 1997). The protocol was approved by an independent animal experimental ethical review committee DCC in Driebergen, The Netherlands. Animal welfare was monitored on a daily basis. Virus inoculation of ferrets was performed under anesthesia with a mixture of ketamine/medetomidine (10 and 0.05 mg/kg resp.) antagonized by atipamezole (0.25 mg/kg). All animal handling (swabbing and weighing) was performed under light anesthesia using ketamine to minimize animal suffering. All experiments with ferrets were performed under animal biosafety level 3 conditions in class 3 isolator cages.

The ferrets were inoculated intratracheally with 106 median tissue culture infectious dose (TCID50) of Seal/H10N7 in a 3-ml volume. Clinical scores were assessed daily and the activity status was scored as follows: 0, alert and playful; 1, alert and playful only when stimulated; 2, alert but not playful when stimulated; 3, neither alert nor playful when stimulated. For diarrhea, sneezing, nasal discharge, inappetence and dyspnea we scored: 0, not present; 1, present. Dyspnea was characterized by open-mouth breathing with exaggerated abdominal movement. Body weight was monitored daily. Humane endpoints in case of severe disease prior to the experimental endpoint were set to 20% of weight loss over the course of the experiment, 15% weight loss in a day or activity score 3. Nasal and pharyngeal swabs were collected every day and were stored at -80°C in transport medium (Hank’s balanced salt solution containing 10% of glycerol, 200U/ml P, 200 mg/ml S, 100U/ml polymyxin B sulphate (Sigma Aldrich) and 250 mg/ml gentamycin [ICN, Netherlands]) until end-point titration in MDCK cells. At 3 and 7 dpi, three animals were euthanized by exsanguination. Necropsies were performed according to a standard protocol and the following samples were taken for virological, histopathological and immunohistochemical analyses: nasal turbinates, trachea, bronchus, lung, tracheo-bronchial lymph nodes, tonsils, heart, liver, spleen, kidneys, adrenal glands, pancreas, jejunum, olfactory bulb, cerebellum and cerebrum. Tissues for virological examination were homogenized in transport medium using the FastPrep system (MP Biomedicals) with 2 one-quarter-inch ceramic sphere balls, centrifuged at 1500 g for 10 min, aliquoted and stored at -80°C until end-point titration in MDCK cells. Tissues for histological examination were fixed in 10% neutral-buffered formalin, embedded in paraffin wax, sectioned at 4 μm, and stained with HE for light microscopical examination. For detection of influenza A virus antigen by immunohistochemistry, sequential slides of all tissues were stained with a primary antibody against the influenza A nucleoprotein as described previously. In each staining procedure, an isotype control was included as a negative control and a lung section from a cat infected experimentally with high pathogenic influenza virus (HPAI) H5N1 was used as positive control.

Infectious bronchitis (IB), also called avian infectious bronchitis, is a common, highly contagious, acute, and economically important viral disease of chickens caused by coronavirus infectious bronchitis virus (IBV). The virus is acquired following inhalation or direct contact with contaminated poultry, litter, equipment or other fomites. Vertical transmission of the virus within the embryo has never been reported, but virus may be present on the shell surface of hatching eggs via shedding from the oviduct or alimentary tract. Dozens of serotypes and genotypes of IBV have been detected, and many more will surely be reported in future. The highly transmissible nature of IB and the occurrence and emergence of multiple serotype of the virus have complicated control by vaccination (Saif et al. 2008). To monitor the existing different IBV serotypes in a geographical region, several tests including virus isolation, virus neutralization, hemagglutination inhibition, ELISA and RT-PCR have been employed (Haqshenas et al. 2005; Saif et al. 2008). The ELISA assay is a convenient method for monitoring of both the immune status and virus infection in chicken flocks. Several commercial ELISA kits for IBV specific antibodies detection are already available, which used inactivated virions as coating antigen (Zhang et al. 2005). PCR on reverse transcribed RNA is a potent technique for the detection of IBV. In comparison with classical detection methods, PCR-based techniques are both sensitive and fast (Zwaagstra et al. 1992). Samples for IBV isolation must be obtained as soon as clinical disease signs are evident. Tracheal swabs are preferred and are placed directly into cold media with antibiotics to suppress bacterial and fungal growth and preserve the viability of the virus (Swayne et al. 1998). In Iran, IB is one of the most important viral respiratory diseases of broiler chickens. However, only the Massachusetts vaccine strain is officially authorized. Despite the use of the IBV vaccine it is common to find IBV problems in vaccinated chickens, causing a tremendous economic impact (Nouri et al. 2003). Several serotypes of infectious bronchitis virus have been reported from different parts of Iran (Seyfi-Abad Shapouri et al. 2002; Nouri et al. 2003; Shoushtari et al. 2008). There is no report about the serotypes and molecular detection of IBV in Zabol in the southeast of Iran. The aim of this study was molecular detection of IBV and the IBV serotypes in Zabol.