Review on Diagnostic Methods of Infectious Coryza in Chickens

    

    

    Figure 3: Blood Tryptose Agar (BTA) plate cross streaked with Staphylococcus aureus feeder.
    

Predisposing factors: Intercurrent respiratory viral and bacterial infections are predisposing factors.Concurrent respiratory agents, including Mycoplasma synoviae, Mycoplasma gallisepticum, Pasteurella species and infectious bronchitis virus, as well as stress factors, can exacerbate disease [9,37].

Physicochemical Property

Avibacterium paragallinarum is a fastidious gram-negative bacterium in the Pasteurellaceae family [14]. It is of paramount importance in the poultry industry because of its worldwide distribution and may contribute to the formation of “respiratory disease complex” under field conditions, thus leading to more severe clinical signs and provokes acute inflammation of the upper respiratory tract and facilitates the growth of other bacterial and viral pathogens [37,48]. Given its slow growth rate and a need for specialized laboratory media and conditions, the organism is difficult to detect by culture, particularly from sites colonized by normal flora [9]. The minimal and maximal temperatures for the growth of the bacterium are 37 and 38°C. It is able to produce acid from maltose, mannitol, sorbitol and sucrose [12].

Serological Variations

Two classification systems applied to Av. paragallinarum are the “Page” and the “Kume” methods. Based on the page method, which employed the use of the plate agglutination method, three different serotypes termed A, B and C was detected. On the other hand, the Kume method is based on hemagglutination and haemagglutination inhibition tests. Accordingly, three different serogroups I, II and III consisting of seven serovars (HA-1 to HA-7) were detected. I.e. HA-1 to HA-3 belonging to serogroup I, HA-4 to HA-6 belonging to serogroup II and HA-7 belonging to serogroup III. Kume’s serogroups I, II and III correspond to Page’s serovars A, C and B respectively. This subsequently led towards the proposal to alter the Kume scheme nomenclature concluding with the nine currently recognized serovars [52].

Pathogenesis

The initial step in the pathogenesis of infectious coryza is adherence to and colonization of the nasal mucosa. An important common mechanism for bacterial pathogenesis is the capacity to adhere to the host cells, which in turn leads to colonization and, finally, to infection of the host cells. Gene hmtp210 of Av. paragallinarum encodes a 210-kDa outer-membrane protein that functions as an HA and has been identified as an important protective antigen.

It has also been proposed that the HA antigen plays a key role in the pathogenicity of Av. Paragallinarum and a trimeric auto transporter adhesin that besides conferring hemagglutination, enables cell adherence and biofilm formation activities [60].

The virulence factors of Av. paragallinarum are not clearly understood. There have been studies that looked at the role of capsule, hemagglutinin, hemocins, lipopolysaccharide, and iron acquisition proteins [54].

Clinical Signs

Infectious Coryza has a short incubation period that develops clinical signs within 1-2 days after inoculation in to the chickens. The most prominent Clinical signs of IC are an acute inflammation of the upper respiratory tract including involvement of nasal discharge and sinuses with a serous to mucoid nasal discharge, sneezing, facial edema and swelling of the face under eye, conjunctivitis, anorexia, and retarded growth in young poultry [9,14,27,33].

Exhibition of clinical signs can vary depending on age and breed, and the duration and severity of these signs can be affected by factors such as poor housing, parasitism, inadequate nutrition, and mixed infection from other infectious diseases such as fowlpox, infectious bronchitis, laryngotracheitis, Mycoplasma gallisepticum, and pasteurellosis [14]. In addition to this, there may be diarrhea, decrease in feed and water consumption and a reduction in egg production in layer chickens. Losses due to persistent mortality are culling where up to 5%. Affected flocks may suffer an egg drop of up to 10%, and to 100% in more serious situations [48].

Pathological Change

Gross Lesions: Grossly acute catarrhal inflammation of mucous membranes of the nasal passages and sinuses, frequently a catarrhal conjunctivitis, subcutaneous edema of the face and wattles are evident of the IC. In acute cases, lesions may be limited to the infraorbital sinuses and there is a copious semifluid exudate from the nostril synovitis [5,13].

Microscopic Lesions

Major histopathological lesions include inflammation of the respiratory mucosa with severe damage to the epithelium and infiltration of heterophils. Furthermore, severe hemorrhages in tissues of the nasal cavity as well as in sinus infraorbitalis [58]. Upon histological examination of the URT, there are usually lesions ranging from necrosis of the respiratory epithelium to marked hypoplasia and squamous metaplasia of the sinuses [1,61].

Diagnosis

The diagnosis can be based on a history of rapid disease spread, clinical symptoms, and pathological changes caused by IC. Molecular, serological and bacteriological methods have been used for the diagnosis and characterisation of Av. paragallinarum in chickens [9,30,48].

Rapid and accurate detection of respiratory IC has become a challenge because of the involvement of more than one agent with similar clinical signs and lesions, which complicates diagnostic decisions, as well as treatment and control strategies [31]. To overcome such difficulties associated with conventional diagnostic methods, alternative approaches such as multiplex Polymerase Chain Reaction (PCR) [33,39,53] have been proved to be useful for specific and sensitive identification of Av. paragallinarum. The test used to establish the serogroups and serovars is the Haemagglutination (HA) and Haemagglutination-Inhibition (HI) tests. There are currently molecular techniques available for the successful and rapid diagnosis of IC. One of these techniques is a species-specific PCR termed HPG2-PCR [34].

Direct Isolation

The diagnosis can be based on a history of rapid disease spread, clinical symptoms and pathological changes caused by IC. Avibacterium paragallinarum mainly isolated from sterile cotton swabs obtained from the infraorbital sinus, trachea and air sac. The swab is then streaked onto or inoculated into Blood Tryptose Agar (BTA) plates and these agar plates were cross streaked by Staphylococcus aureus (feeder culture). The plates were incubated at 37°C for 48 hrs in candle jar. After incubation, the plates were observed for growth. The dew drops colonies used for further biochemical investigation (Akhter et al., 2013) [57].

Molecular Diagnostic Techniques

A species-specific PCR test (HPG-1-PCR and HPG2-PCR) and 16S Ribosomal RNA Sequencing were employed for molecular identification of Av. paragallinarum. HPG2-PCR is sensitive and reliable method for species specific identification of Av. Paragallinarum [40]. Both these PCR’s are specific and sensitive and give positive results with NAD+ -dependent and NAD+ -independent isolates [19].

Serological Diagnostic Tests

For the understanding of prevalence of strains in a particular region, it is essential to characterize and classify organisms on serological level. Hence, Av. paragallinarum were further typed by using Page serotyping scheme [28]. Serological diagnosis of the disease has not been performed because the progress of this disease is rapid and antibodies are not likely to be induced in chickens infected with Av. paragallinarum, particularly with serovar C, even after the disease onset. HI tests are used for identification of Av. paragallinarum; however, the procedure is complicated, and the sensitivity is insufficient. For this, serological classification both conventional Hemagglutination- Inhibition (HI) test as well as modern multiplex PCR (Molecular serotyping) based protocols were followed and described below [29].

Hemagglutination-Inhibition (HI) Test

The Page serotyping scheme was initially developed by using plate or slide agglutination test to recognize the three serovars i.e. serovar A, B and C. However, the use of Hemagglutination-Inhibition (HI) technology has been shown to be a much better method for the identification of Page serovars of Avibacterium paragallinarum isolates around the world. A drawback of the Page scheme is that some isolates could not be typed due to non- agglutination. The maximum serum dilution completely inhibiting hemagglutination was considered as HI titer and the antisera specific to serovar with highest HI titer was considered as serovar of isolate [14]. The Haemagglutination (HA) and Haemagglutination-Inhibition (HI) test, detecting haemagglutinins andthe method used involved treating bacterial cells with potassium thiocyanate (KSCN), followed by sonication. The result was the detection of an additional antigen, together with haemagglutinins that was able to agglutinate fresh and gluteraldehyde-fixed chicken erythrocytes [47]. Three main forms of HI tests have been recently recognized: termed simple, extracted, and treated HI tests.

Simple HI test is based on whole bacterial cells of Page serovar Av. paragallinarum and fresh chicken erythrocytes. Although simple to perform, this HI test can detect antibodies only to serovar A. It has been widely used to detect antibodies in infected as well as vaccinated chickens [45].

Extracted HI test is based on KSCN-extracted and sonicated cells of Av. paragallinarum and glutaraldehyde-fixed chicken erythrocytes. This extracted HI test has been validated mainly by using Page serovar C organisms. The test is capable of detecting a serovar-specific antibody response in Page serovar C-vaccinated chickens. A major weakness of this assay is that the majority of chickens infected with serovar C remain seronegative [47]. Treated HI test is based on hyaluronidase-treated whole bacterial cells of Av. paragallinarum and formaldehyde-fixed chicken erythrocytes. The extracted HI test has not been widely used or evaluated. It has been used to detect antibodies to Page serovars A, B, and C in vaccinated chickens, with only serovar A- and C-vaccinated chickens yielding high titers. It has also been used to screen chicken sera in Indonesia for antibodies arising from infection with serovars A and C [14].

The Kume proposed a new serovar scheme based on the detection of Hemagglutinin (HA) in Av. paragallinarum into three serogroups (A, B, and C), which currently recognizes as nine serovars (A-1, A-2, A-3 (HA-1 to HA-3), A-4(HA-8), B-1 (HA-7), C-1, C-2, C-3 (HA-4 to HA-6), and C-4 (HA-9) However, HA/ HI is a time-consuming technique and it is difficult to serotype accurately to the serovar level, making a molecular technique the preferred alternative [46,47].

Molecular Serotyping by Using Multiplex PCR

The multiplex PCR is based on the amplification of a hypervariable region within the haemagglutinin gene of Av. paragallinarum A and C-serovars. PCR using primer sets around the hypervariable region amplified 0.8, 1.1 and 1.6 kbp fragments for serovars A, B and C, respectively [62]. This region encodes an outer-membrane protein, HMTp210, which serves as a major protective antigen of Av. paragallinarum. HMTp210 gene is an important gene that is responsible to express a 210 kDa outer membrane protein of Av. paragallinarum [43,44].

Based on DNA sequence homology, HMTp210 gene is divided into 3 regions. Out of these 3 regions, region 1 and region 3 are highly conserved between Av. paragallinarum serovars A and C, respectively. On the other hand, the homology of region 2 is around 50% between serovars A and C. Thus, region 2 seems to be a serovar-specific region in HMTp210, which is used for protection against serovars A and C. For these reasons, we focused on region 2 as the target to identification of the serovars of Av. paragallinarum using PCR [53,62]. A molecular technique is less time consuming, which is of benefit to the poultry industry. The homology of region 2 was more than 99.8% within each serovar: 99.9% within serovar A, 100% within serovar B and 99.8% within serovar C. In this protocol, region specific oligonucleotide primers were used to carry out multiplex PCR. The PCR product was further analyzed based on size of amplicon.

Deferential Diagnosis

Infectious Coryza should be differentiated from other common chicken diseases like chronic fowl cholera, Newcastle disease, infectious bronchitis, avian influenza, avian metapneumovirus (swollen head syndrome), mycoplasmosis and infectious laryngotracheitis [14].

Economic Importance

Infectious Coryza is a highly contagious respiratory disease of chickens resulting in high mortality, reduced egg production, and huge economic losses to the poultry industry worldwide [21,35]. The economic impact of the disease is mainly associated with a significant reduction (10%-40%) in egg production, especially on multi-age farms [18,40]. The infection is more commonly observed in intensive poultry farms with multiple age groups, including large-scale egg production and breeding complexes [14].

Treatment

Antibiotic treatment will reduce the severity of the course of the disease. Many drugs have bacteriostatic effect on the organism. Erythromycin, Tetracycline, Fluoroquinolones, Sulphadimetoxine, and Sulphamethazine can be used as treatment medication. Some antibiotics are not suitable for layers producing eggs for human consumption [1,17].

    

    

    Table 1: Location of primers for multiplex PCR [53].