Chronic respiratory disease (CRD) is a bacterial infection caused by one of the group of organisms known as pleuro pneumonia like organism (PPLO), but the particular organism directly associated with CRD is Mycoplasma gallisepticum (MG), sometimes with secondary complications (Ley, 2008). Chronic respiratory disease has been reported worldwide causing heavy economic losses in large commercial operations. The infection may be in-apparent or result in varying degrees of respiratory distress with slight to marked rales, difficulty in breathing and cough or sneezing. Morbidity is high and mortality low in uncomplicated cases (Bahatti et al., 2013).

Diagnosis of avian mycoplasmosis can be made on the basis of characteristic gross and histological lesions (Uddin et al., 2010), on serological assays to detect antibody production and/or on isolation and identification of the organism (OIE, 2008). Detection of Mycoplasma by polymerase chain reaction (PCR) has high specificity and sensitivity, which can amplify even very small amount of nucleotides that cannot be easily detected by other methods (Ramadass et al., 2006 and Saritha et al., 2010). The prevalence and pathology of Chronic Respiratory Disease (CRD) caused by Mycoplasma gallisepticum in broilers were studied.

Materials and Methods

Samples were collected from twenty medium sized organised broiler flocks comprising between 5000-30000 birds and situated in a radius of approximately 50 miles.

Collection of Sample

Sixty pooled swab samples from nostrils, choanal cleft, trachea and air sacs were collected at necropsy from broiler flocks which were suspected for avian mycoplasmosis and were subjected to PCR for the amplification of 16S rRNA of 185 bp as per, OIE Terrestrial manual, 2008, guidelines (OIE, 2008). All the birds found dead during the study period were examined for lesions indicative of mycoplasmosis. Tissue samples including the trachea and lungs were collected from thirty-six broilers suspected for CRD based on the gross lesions.

Pathological Studies

Gross Pathology

Broiler carcasses (n = 339) were examined for the presence of gross respiratory lesions indicating CRD during post mortem.  Overall occurrence of chronic respiratory disease was evaluated as proportion of bird necropsy. Chronic respiratory disease was suspected in birds with characteristic gross lesions as described by Ley (2008). The lesions comprised sinusitis, catarrhal exudate in nasal passages, catarrhal exudate adherent to the tracheal wall with congestion and haemorrhages, bronchitis with accumulation of caseous material, congestion of lungs and foamy, cloudy air sacculitis with cheesy caseous exudates deposited over air sacs. Swabs from nostrils, trachea, choanal cleft and air sacs were collected from the birds suspected for CRD.

Histopathology           

Sections of all trachea and lung tissues collected from broiler carcasses suspected for CRD based on gross lesions were processed for microscopic study (Gridley, 1960). Special staining such as Mayer’s Mucicarmine staining and Masson’s Trichrome staining were carried out in tissue samples for demonstration of mucin and connective tissue proliferation, respectively.

Molecular Diagnosis of Mycoplasma gallisepticum

DNA was extracted from swab samples (three- five pooled) suspended in 1 ml of PCR grade PBS. The suspension was centrifuged for 30 minutes at 14,000 g at 4°C. The supernatant was then carefully removed with pipette and the pellet was suspended in 25 µl of PCR-grade water. The tube along with the contents was boiled for 10 minutes and then placed over ice for 10 minutes and then centrifuged at 14,000 g for 5 minutes. The supernatant thus obtained containing DNA, was used in PCR. The oligo primer (Table 1) specific to 16S rRNA of MG (OIE, 2008) was synthesized by Integrated DNA Technology (IDT) Inc. and utilized in the present study. Oligo primer supplied in freeze dried order form were reconstituted in Milli-Q water to the volume equivalent to the mass of primer (Mass/µg) and further diluted in Milli-Q water to give a final concentration of 10 pmol/µl.

Table 1: Detail of primer used in PCR

The PCR amplification of 16S rRNA gene was carried out in a final reaction volume of 25 µl. The PCR master mix for sixty samples was prepared and aliquot of 24 µl were added in each PCR tube. 1 µl genomic DNA was added in each tube, to make the final volume of 25 µl. Components of PCR reaction mixture are Ultra-pure water (9.5 µl), PCR master mix (12.5 µl), F primer (1 µl), R primer (1 µl) and genomic DNA (1 µl). For every reaction, a positive control from genomic DNA of live vaccine Nobilis MG 6/85 (Merck Sharp & Dohme. Inc.) and a negative control were used to cross check any contamination of foreign DNA in reaction component.

The PCR tube was kept in a pre-programmed thermocycler (Veriti- 96 well thermocycler, ABI, USA) and thus standardized reaction programme including denaturation (94ºC for 30 seconds), annealing (57º C for 30 seconds), initial extension (72º C for 60 seconds) and final extension (72ºC for 5 minutes) was carried out. PCR amplification was confirmed by running 10 µl of PCR product mixed with 1 µl of 6X gel loading dye from each tube on 2% agarose gel at a constant voltage, 70-80 V for 60 minute in 0.5 X TAE buffer. Ethidium bromide was incorporated in 100ml of agarose gel at the rate of 5 µl of 1% solution. The amplified product was visualized as a single compact fluorescent band of expected size under UV light and documented by gel documentation system (Syngene, Gene genius bio imaging).

Statistical Analysis

Statistical analysis for association between gross CRD lesions and molecular detection of MG by PCR was determined by Fischer’s exact test (Mcdonald, 2014). IBM SPSS-20 (IBM Corp.) statistical software was used for statistical test. The P value less than 0.05 (P<0.05) was considered statistically significant for the test.

Results

Gross Lesions

Total 36 birds (10.61%) had lesions strongly suggestive for chronic respiratory disease. The gross lesions like sinusitis, exudate in nasal passages, caseous exudate adherent to the tracheal wall with congestion and haemorrhages (Fig. 1), airsacculitis with cheesy caseous deposits over air sacs (Fig. 2), congestion of lungs were characteristically observed in these birds (Table 2).

Table 2: Summary of organ wise distribution of gross lesions

Microscopic Lesions

Sections of trachea and lung collected from the cases affected with CRD based on gross lesions were studied for microscopic changes. The percent distribution of microscopic lesions in trachea of affected broilers were hyperaemia (72.22%) (Fig. 3A), surface epithelial destruction (58.33%), infiltration of leukocytes in lamina propria (50.00%), mucous gland hypertrophy (44.44%) (Fig. 3B), haemorrhage (33.33%), mucosal thickening (27.77%) and squamous cell metaplasia (13.88%). The lesions in trachea were further classified as acute catarrhal tracheitis (41.66%), acute haemorrhagic tracheitis (33.33%), chronic catarrhal tracheitis (13.88%) and fibrinous exudative tracheitis (08.33%), as per the microscopic observation.

Fig. 3:  Microscopic section of trachea with mucous gland hypertrophy and leukocytic infiltration, H&E X 400 (A); Hypertrophied mucous gland with excess mucin deposition (arrow) over mucosal epithelium of trachea, Mayer’s mucicarmine X 400 (B).

Fig. 4:  Microscopic section of MG positive broiler lung with oedema and thickened interstitium, H&E X200 (A); Septal thickening (arrow). Masson’s trichrome X100 (B); Thickened arteriole, congested capillaries and areas of necrosis. H&E X200 (C).

The overall findings noticed in lungs of broilers affected with CRD were congestion (91.66%), haemorrhage (83.33%), infiltration of cells (66.66%), oedema (33.33%) (Fig. 4A), septal thickening (33.33%), secondary bronchi mucosal hyperplasia (27.77%), necrosis and denudation (22.22%) and respiratory atrial muscle thickening (13.88%). Microscopically, depending upon the extent of involvement, the lesion was categorized as mild, moderate and severe congestion (Fig. 4C). The occurrence of mild, moderate and severe congestion was 12.12%, 42.42% and 45.45%, respectively.

The occurrence of pneumonia was broadly classified as bronchopneumonia and bronchointerstitial pneumonia which accounts for 69.50% and 30.50%, respectively. Bronchopneumonia was further classified as haemorrhagic, catarrhal, necrotic and sero-fibrinous with 22.22%, 19.44%, 16.66% and 11.11% distribution, respectively. Bronchointerstitial pneumonia was observed in 30.50% cases. Special staining of microscopic section of lung with Masson’s trichrome staining revealed thickened interbronchial septa (Fig.4B).

Prevalence of Mycoplasma gallisepticum by PCR

Of sixty samples, 8 samples were found positive for MG. In positive samples the PCR amplified product of 185bp of 16S rRNA of Mycoplasma gallisepticum was clearly visible (Fig. 5). The overall prevalence of MG was determined as 13.33% in broilers by PCR. Amongst these, 75% of birds found positive for MG were less than three weeks of age and 25% of samples positive for MG were from birds of seven to eight weeks of age.

Fig. 5: PCR amplification of 185 bp of 16S rRNA for identification of MG in broiler.

L02, L04, L08 : Positive sample

M                     : 100bp Ladder

L13                  : Negative control

L14                  : Positive control (Nobilis MG 6/85 vaccine)

The average body weight of chicks at 2 weeks of age and infected with MG was determined to be 201.66 g, whereas average body weight of chicks at 3 weeks of age and infected with MG was 341.66 g. The body weight of chicks at 2 and 3 weeks was much less than the expected average ideal body weight. Association between gross CRD lesions and molecular detection of MG by PCR were determined by Fisher’s exact test. The results are shown in Table 3. The significant value, P= 0.017, indicates the positive association between gross CRD lesions and molecular detection of MG.

Table 3: Association between gross CRD lesions and molecular detection of MG by PCR

^*Significant at P<0.05; MG- Mycoplasma gallisepticum

Discussion

The overall prevalence of chronic respiratory disease based on gross lesions was found to be 10.61%. Earlier workers determined the prevalence of CRD based on gross lesions as 11.50%, 9.87%, 12.84%, 11.81% and 11.58% respectively (Yunus et al., 2008; Uddin et al., 2010; Razia et al., 2012; Sultana et al., 2012 and Rajkumar et al., 2017). This mild variation in percent prevalence may be observed due to the geographical variation, higher prevalence of infection in a study area and various secondary factors that contributes to the increased virulence of organism to produce clinical disease.

In the current study the significant lesions observed in birds suspected for CRD were similar to the earlier findings (Islam et al., 2011). Haemorrhages in trachea were similar to the findings of Brar et al. (2017) and the thickened mucosa and infiltration of cells were in accordance with Stipkovits et al. (2012). Hypertrophy of mucous gland, mucosal thickening, infiltration of leukocytes and surface epithelial destruction were in accordance with histopathological findings in trachea (Thilagavathi et al., 2016). Presence of congestion, haemorrhage and cell infiltration in lungs were in accordance to many authors’ findings of CRD in poultry (Islam et al., 2011; Brar et al., 2017 and Thilagavathi et al., 2017). In our study maximum of severe type of congestion (45.45%) was observed, which was in discordance with other study where moderate type of congestion (42.86%) was most prevalent in birds showing respiratory lesions (Itoo et al., 2014).

In another study the bronchopneumonia was observed in MG infection at 2.00% of the cases (Ibrahim et al., 2015); the percent occurrence of bronchopneumonia in our current study was 69.50%. The occurrence of catarrhal and fibrinous type of bronchopneumonia in birds affected with MG has been reported (Yilmaz and Timurkaan, 2011).  Timurkaan et al. (2008) found the most prevalent type of pneumonia observed in broilers were catarrhal type of bronchopneumonia. In our current study we found the percent occurrence of catarrhal type of bronchopneumonia as 19.44%. Secondary bronchial hyperplasia was noticed in 27.77% of birds affected with CRD. This finding was also in agreement with earlier reports where bronchial hyperplasia in birds affected with CRD has been reported (Sivaseelan et al., 2013 and Thilagavathi et al., 2017).

In the current study the molecular diagnosis of MG was found to be 13.33%. In accordance with our findings Rajkumar et al. (2018) determined the prevalence of MG by PCR as 11.65%. A higher prevalence of 18.46% and 27.00% respectively was reported by Reddy (2014) and Tomar et al. (2017). The wide variation of MG prevalence and detection rates in different studies might be due to the sample size, sample type, detection techniques, rate of infection, biosafety and biosecurity in the respective study area (Rajkumar et al., 2018). Sample in the present study were largely based on organised farms where strict biosecurity measures are practised. Good biosecurity management practices followed in farms may play a major role in control of organisms which in turn can be manifested as low detection of organisms from sampling. The lack of identification may occur due to low antigen concentrations in the samples, which is common for chronic infections like chronic respiratory disease (Lockaby et al., 1998). The stage of infection may also influence the detection of organism as tracheal or choanal number of organisms in chronically infected birds may be so low that Mycoplasma gallisepticum organism may not be detected by sampling. In other words acute infections generally have higher DNA copies in samples than chronic infections, as in chronic infection attenuation of organism due to immune response takes place (Hossam et al., 2016).

MG affects younger birds more severely than mature birds. In the present study 75.00% of birds found positive for Mycoplasma gallisepticum were less than three weeks of age and 25% of samples positive for Mycoplasma gallisepticum were from birds of seven to eight weeks of age. This finding was in accordance with the study where the prevalence of MG was found high in birds of 8 to 21 days of age (Haque et al., 2015). This higher prevalence of Mycoplasma gallisepticum at broiler chicks may be due to vertical transmission in commercial breeder poultry flocks of India, which results in spread of MG infection from breeder hens to chicks (Tomar et al., 2017). Yoder (1972), found development of resistance in MG infected adult birds. This development of immunity against infection may also decrease the detection of organism in adult birds. Thus, higher infection in the young chickens might be due to the lesser immunity level and it can be speculated that, until and unless parent flocks are not free from mycoplasmosis, the disease cannot be controlled from commercial flocks.

Antibiotics such as tetracyclines, tylosin, fluroquinolones, aminoglucosides, tiamulin were reported to be effective against MG infection in birds. MG are resistant to antibiotics which acts by inhibiting cell wall like penicillin. Constant monitoring of breeder flocks and acquisition of birds free from MG plays a vital role in prevention of the disease. Cleaning and disinfection must be carried out regularly using disinfectants containing phenol, cresylic acid, Hypochlorite and glutaraldehyde.

Conclusion

To conclude a significant prevalence of CRD was observed in the study area. Mycoplasma gallisepticum inducing destruction of cilia and tracheal epithelial cells could lead to immune modulations and superimposed infections. Strict biosafety measures are to be applied to reduce economic losses in the poultry industry.

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