Avian colibadllosis is typically a systemic or localized disease occurring secondarily when host defences have been impaired or overwhelmed by virulent Escherichia coli strains and is characterized by a variety of diseases in poultry such as pericarditis, perihepatitis, airsacculitis, peritonitis, salpingitis, panopthalmitis, omphalitis, cellulitis, colisepticaemia, coligranuloma and swollen- head syndrome. It is among the most frequently reported diseases in surveys of poultry health or condemnations at processing causing up to 30% of mortality in birds. Colibacillosis is responsible for large economic losses in poultry rearing through low performances, weight loss, delay in onset of egg production and mortality as the organism affects multiple body system of birds¹,²-³. The infection is generally initiated or enhanced by predisposing agents, such as mycoplasmal, viral infections and environmental factors and also been reported to cause immunosuppression in poultry by causing depletion of lymphocytes in lymphoid organs⁴. Avian colibacillosis was found widely prevalent in all age group of chickens with higher prevalence rate in broiler chicken (87.5%) than layer (76.04%)⁴.

Although antibacterial agents have been used successfully to prevent this disease, restrictions on antibiotic usage in poultry production and increasing resistance of bacteria to antimicrobial agents have made difficult to control colibacillosis⁵. The high consumption of chicken meat also warrants great care in safeguarding the industry against threatening factors such as human food poisoning, food-borne illnesses etc.² The present study reports the pathology of colibacillosis, characterization and antimicrobial resistance of isolated E. coli associated with the disease in poultry population of Mizoram.

During the period of June, 2016 to May, 2017, outbreaks of colibacillosis were recorded in 18 different poultry farms located in different districts of Mizoram. Affected birds were disinclined to move and showed a characteristic posture with their chest and beak resting on the ground with closed eyelids with reduced food consumption, loss of weight, weakness, laboured breathing, and ruffled feathers. In few cases, well grown healthy broiler birds showed sudden death without exhibiting any clinical signs.

The disease was found to occur round the year with highest incidence observed during November to February (54.14%). Commercial broiler birds in the age group of 4–8 weeks were affected most (80.28%) followed by 1–3 weeks old (12.86%) and 21–52 weeks old (6.86%) adult layer birds. The percent morbidity and mortality rates were recorded as high as 62.98% and 24.37%, respectively.

Detailed post-mortem examination revealed mostly colisepticaemic form of the disease in broiler bird followed by yolk sac infection in young birds and salpingitis and egg peritonitis in layer birds. Fibrinous pericarditis and perihepatitis with severely congested lungs, spleen and liver was observed consistently in colisepticaemic form of the disease (Fig. 1). Young chicks in the age group of 1–2 weeks showed retained inflamed and necrotic yolk materials with congested lungs and liver (Fig. 2). Peritonitis with yolk material, impacted oviduct with onion layered texture of the inflammatory exudates and necrosedeggs were observed in layer birds.

Histopathological examination of liver reveals severe to moderate fibrino-perihepatitis. The capsule of the liver was thickened due to deposition of layers of fibrin with infiltration of mononuclear inflammatory cells, congestion and oedema (Fig. 3). In chronic cases, fibroplasia was observed, which has lead adherence of the fibrino-purulent layer firmly with the parenchyma. The hepatic parenchyma showed congestion and fibrinous exudates in sinusoidal spaces leading to disorientation of hepatic cords and dilation. The hepatocytes showed varying degree of degenerative changes. The mononuclear inflammatory cell infiltration was more evident in the portal area. Tissue sections from lungs showed severe fibrinous pneumonia. Severe congestion of parenchyma of parabronchi with infiltrating mononuclear cells and dilation of inter parabronchiolar septae due to oedema was observed. The lumen of the parabronchi, respiratory atria and air capillaries contained fibrinous exudates leading to reduction of air passages (Fig. 4). Severe fibrinous pericarditis was observed in the tissue sections from heart. The pericardium was thickened with layers of fibrin deposits, degenerative granulocytes, macrophages, clumps of bacteria and congestion. Myocardium beneath the epicardium shows congestion and infiltration of mononuclear inflammatory cells with separation of myofibres (Fig. 5). Severe to moderate nephritis was observed in kidney. Interstitium of kidney parenchyma showed infiltration of mononuclear inflammatory cells and congestion. Degeneration, desquamation and necrosis were observed in renal tubular epithelium. Glomeruli showed swollen and hypercellular glomerular tuft. Severity of congestion was more in the cortico- medullary junction of the kidney. The splenic parenchyma showed severe congestion particularly in red pulp and surrounding the lymphoid follicles (Fig. 6). Lymphoid depletion was observed in the germinal centres of the follicles with infiltrations by histiocytes. Diffuse depletion of lymphocytes was also observed in the parenchyma.

A total of 504 E. coli were isolated and characterized by conventional methods of bacteriology⁶, of which 440 isolates from heart blood and liver (82.38%) with colisepticaemic lesions as evidenced by pathological examination. All the isolates were further confirmed by detection of 16S RNA gene of E. coli using the PCR assay⁸ (Fig. 7).

The antimicrobial susceptibility test of all the 504 isolates revealed multidrug resistance against most of the antimicrobial agents (Fig.8). Highest level of resistance was observed against amoxycillin (87.23%) followed by nalidixic acid (80.75%), tetracycline (75.91%) and ampicillin (41.43%). Minimum resistance was observed against imipenem (l.22%) (Table 1).

A total of 204 (40.47%) out of 504 isolates were confirmed as ESBL producers by DDST screening method. An increase in zone of inhibition of ≥5mm in combination disks than the original antibiotic was considered as DDST positive.

Poultry production plays acrucial role in providing valuable proteins and poverty alleviation in hilly states like Mizoram. Despite great potential and opportunities, poultry production is threatened by many disease outbreaks and these diseases are the major constraints for developing the poultry industry¹⁰. The epidemiological, clinical and pathological findings in the present study have recorded colibacillosis as a major health concern for the poultry population of Mizoram, which is responsible for high mortality in birds throughout the year. Colibacillosis has been recorded by many researchers in different countries as the primary cause of morbidity, mortality and condemnation of carcasses resulting significant economic losses in poultry industry worldwide¹¹,¹²-¹³.

Antimicrobial agents are being used not only to control and prevent the diseases, especially like colibacillosis, but also as growth promoter in commercial poultry farm practices¹⁴. Therefore, the antibiotic selection pressure for resistance in E. coli in poultry is high. Consequently their faecal flora contains a relatively high proportion of resistant E. coli and resistance to existing antimicrobials is widespread, which is of concern to poultry veterinarians¹⁵,¹⁶-¹⁷. The antimicrobial susceptibility test of total 504 E. coli isolates from birds that died of colibacillosis revealed alarmingly very high multidrug resistance in the present study. Among the total 18 nos. of antibiotic tested, highest level of resistance was observed against Amoxicillin (87.23%) revealing as the least effective antibiotic against E. coli in Mizoram. Amoxicillin is most widely used antibiotic against infection in man and animals. However, because of indiscriminate use, misuse and abuse of such antibiotic, bacteria developed the highest level of resistance, which makes the drug a completely irrelevant option against E. coli. Earlier researchers¹⁸ reported 84.81% resistance against Amoxicillin from E. coli isolates of swine from Northeast India. Tetracycline is another broad spectrum antibiotic very commonly used in poultry and livestock population of NER, India for therapeutics use. Resistance to tetracycline was observed as most common and the most prevalent resistance phenotype in animal isolates (71.1%)¹⁹. Present study has recorded 75.91% resistance against tetracycline in E. coli isolates from colibacillosis affected birds. On the other hand, imipenem, a carbapenem P-lactam which is not in use for veterinary practices in any of the NER states of India was found to be the most sensitive antibiotic. This result is in corroboration with the findings of previous workers²⁰,²¹, who also reported no resistance against imipenem. However, two out of 504 isolates showed resistance against imipenem which is a matter of great concern for treating infections caused by Gram negative bacteria in poultry and livestock, drawing attention to the urgent need of control strategies for use of antibiotics.

With the availability of third and fourth generation of cephalosporins and without any restriction upon their use in veterinary and human health practice in this country, a high level of resistance against them are increasingly evidenced and at the same time, a decrease in level of resistance against older antimicrobials is also evident. Expanded spectrumcephalosporins are important therapeutic agents in veterinary and human medicine and are often used as first line agents for invasive Gram negative infections. Present study has recorded a total of 204 (40.47%) isolates out of total 504 E. coli as ESBLs producer. The prevalence of third generation cephalosporin resistance in E. coli in poultry suggests that these agents may be rapidly losing their efficacy as treatment option. Most of the ESBL producing organisms were found to be co-resistant to flouroquinolones, aminoglycosides and co-trimoxazoleas as similarly described by earlier workers²²,²³-²⁴. Several workers from India and abroad have also reported the prevalence of ESBLs producers varying from 6.6% to 91% from time to time²⁵,²⁶-²⁷.

The resistance pattern of E. coli isolates against all the antimicrobial agents are variable within the isolates obtained from different poultry farms of Mizoram. Maximum antibiotic resistance up to against 16 nos. of antibiotics out of 18 antibiotics used was recorded in one isolate from birds affected with severe colisepticaemia. This particular isolate was sensitive only against imipenem and gentamycin. A total of 299 (59.32%) out of 504 isolates exhibited resistance against more than 10 antibiotics.

Prevalence of multidrug resistance E.coli and also ESBL producer in poultry population of Mizoram is a matter of health concern not only for the poultry population of the state but also for the public health. The poultry producer and veterinarian should work closely, when antibiotic therapy is needed in a flock and both must continue to work towards ensuring a safe food supply for consumers.

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