Clinicopathological evaluation of Escherichia coli infection in broiler chickens of the north eastern plain zone of Uttar Pradesh

Introduction

The poultry industry is the fastest growing sector in animal agriculture and is crucial for providing food security, nutritional supplement, job creation and economic development. India is currently among the top producing countries of poultry meat where commercial broiler production is a major contributor to the livestock economy. Productivity and profitability of poultry industry is greatly hindered by the occurrence of infectious diseases, whereby bacterial diseases are among the major sources of economic losses through higher mortality rate, poor growth performance, inefficient feed conversion ratio and condemnation of carcasses (Hofstad, 1975; Gross, 1991) [7,6] . Escherichia coli bacteria are one of the important pathogens associated with disease in commercial poultry. Though E. coli is a normal constituent of intestinal flora of healthy avian and mammalian animals, some of the strains of the bacterium have the capacity to cause diseases to poultry. These strains are collectively referred to as avian pathogenic Escherichia coli (APEC) because of their ability to develop localized and systemic infections. The disease complex called avian colibacillosis is characterized by airsacculitis, pericarditis, perihepatitis, omphalitis, cellulitis, salpingitis and septicemia, causing immense economic losses to the poultry industry (Jawetz et al., 1984; Gross, 1991; Dho-Moulin and Fairbrother, 1999) [8,6,4] . Escherichia coli is a Gram-negative, facultative anaerobe, motile, sporeless, lactosefermenting bacillus that belongs to the bacterial family Enterobacteriaceae. Traditional bacteriological methods based on colony appearance, Gram staining, motility, and biochemical tests are the standard tools used in laboratories to diagnose E. coli infections. They are accurate, cost-effective, and commonly used to identify pathogenic E. coli strains in birds (Croxen et al., 2013) [2] . Avian colibacillosis is usually associated with stress conditions, lack of good biosecurity, improper ventilation, overcrowding, and simultaneous respiratory infections that lead to the susceptibility of birds to opportunistic E. coli infection (Gross, 1991; Dho-Moulin and Fairbrother, 1999) [6,4] . Despite the number of reports about the occurrence of E. coli infection in broiler chickens from various parts of India, there is no sufficient information

Isolation of Escherichia coli

Each of the tissue samples used in the study was mixed with 10 ml of sterile normal saline. Approximately 5ml of the tissue sample was then inoculated in nutrient broth and was incubated at 37 °C for 24 hours. The enriched culture obtained from nutrient broth was streaked onto MacConkey agar and was incubated at 37 °C for 24 hours. Lactose fermenters were sub cultured into new medium and maintained on nutrient agar slants at 4 °C.

Identification and Characterization of E. coli E. coli was identified on the basis of colony morphology, Gram staining, motility, and biochemical properties as described by Cruickshank et al., (1975) [3] and Edwards and Ewing, (1972) [5] .

Statistical Analysis

Prevalence of Escherichia coli infections was calculated in percentages in each district. The outcomes of culturing and biochemical identification were presented descriptively.

Results

Isolation and Identification of Escherichia coli

A total of 677 samples from naturally dead broilers were used to conduct bacteriological investigations. Out of these, 327 (48.30%) isolates were confirmed to be Escherichia coli. Cultural prevalence of Escherichia coli isolates varied between districts. The maximum isolation rate was found in Basti (53.49%, 199/372), then in Bahraich (42.85%, 93/217) and Siddharth Nagar.

Culture and Morphology

The colonies grown on MacConkey agar had the appearance of being smooth, round, elevated and bright pink colonies that fermented lactose. The colonies grown on EMB agar had a metallic green sheen, which helped to confirm the identification of E. coli. Cultures grown in nutrient broth resulted in uniform turbidity. Gram staining indicated Gramnegative, short rods in single or pair formation.

Biochemical Characterization of Escherichia coli

The isolates showed positive results for indole test, methyl red, TSI test, nitrate reduction, glucose, lactose and mannitol fermentation tests. Most of the isolates were negative for Voges-Proskauer test, citrate test, urease test and hydrogen sulphide. Variable results were shown by sucrose, dulcitol, adonitol and salicin fermentation tests (Table 2).

Discussion

This study revealed the high incidence of E. coli infections in broiler chicken birds belonging to the North Eastern Plain Zone of Uttar Pradesh, which is an indication that colibacillosis remains to be one of the prominent bacterial diseases in commercial poultry production. The high incidence of E. coli infection observed in the Basti district might be due to differences in management, stocking density, hygiene, and biosecurity among others. Similar findings were made by Gross (1991) [6] , Dho-Moulin and Fairbrother (1999) [4] , where E. coli was found to be one of the prevalent bacteria causing economic losses in poultry production.

Pink colored lactose fermenter colonies in the MacConkey agar medium and metallic green colonies in the Eosin Methylene Blue agar medium obtained in the current study agree with the cultural characteristics of E. coli described by Edwards and Ewing (1972) [5] and Cruickshank et al. (1975) [3] . Similarly, Gram staining showed Gram negative rods in the samples. The biochemical characterization of the isolates, which included positive reactions for indole, methyl red, Triple sugar Iron medium, nitrate reduction, glucose, lactose, and mannitol fermentation, along with negative reactions for Voges-Proskauer, hydrogen sulfide production, citrate utilization, and urease test, was consistent with the standard biochemical properties of Escherichia coli reported by Croxen et al. (2013) [2] . Any deviations from the standard reactions with respect to citrate utilization, urease reaction, and carbohydrate fermentation could be due to strains differences. The results obtained from this study provide further evidence regarding the prevalence of Escherichia coli in broiler flocks of the current study area and highlight the significance of regular bacteriological survey, biosecurity, and better farm management practices to prevent and control avian colibacillosis.

Conclusion

From the above study, it has been found that the bacterium Escherichia coli is commonly found in broiler chickens within the North Eastern Plain Zone of Uttar Pradesh. Cultural, morphological, and biochemical techniques were successfully used to isolate and identify the bacteria. It is important to emphasize the need for regular monitoring of diseases to reduce the economic loss from avian colibacillosis in the poultry industry.

References

1. Barnes HJ, Nolan LK, Vaillancourt JP. Colibacillosis. In: Saif YM, Fadly AM, Glisson JR, McDougald LR, Nolan LK, Swayne DE, et al., editors. Diseases of Poultry. 12th ed. Ames (IA): Blackwell Publishing; 2008. p. 691-732.
2. Croxen MA, Law RJ, Scholz R, Keeney KM, Wlodarska M, Finlay BB. Recent advances in understanding enteric pathogenic Escherichia coli. Clin Microbiol Rev. 2013;26(4):822-880.
3. Cruickshank R, Duguid JP, Marmion BP, Swain RHA. Medical Microbiology. 12th ed. Edinburgh: Churchill Livingstone; 1975.
4. Dho-Moulin M, Fairbrother JM. Avian pathogenic Escherichia coli (APEC). Vet Res. 1999;30(2-3):299316.
5. Edwards PR, Ewing WH. Identification of Enterobacteriaceae. 3rd ed. Minneapolis (MN): Burgess Publishing Company; 1972.
6. Gross WB. Colibacillosis. In: Calnek BW, Barnes HJ, Beard CW, Reid WM, Yoder HW Jr, editors. Diseases of Poultry. 9th ed. Ames (IA): Iowa State University Press; 1991.
7. Hofstad MS. Diseases of Poultry. 7th ed. Ames (IA): Iowa State University Press; 1975.
8. Jawetz E, Melnick JL, Adelberg EA. Review of Medical Microbiology. 16th ed. California: Lange Medical Publications; 1984.