Isolation , molecular identification and antibiogram profiles of Escherichia coli and Salmonella spp . from diarrhoeic cattle reared in selected areas of Bangladesh

The present research work was undertaken to find out the passive causes of occurrences of diarrhoea in terms of age, sex, season and location differences through isolation and identification of the E. coli and Salmonella spp. using cultural, biochemical and molecular from the field samples of the diarrhoeic cattle and to study the antibiogram profiles of the isolated bacterial species. Considering above purposes, a total of 57 rectal swab samples were collected from the diarrhoeic cattle of Mymensingh sadar, Trishal, Valuka, Natore sadar and Gomostapur, Chapai Nawabganj. Different types of cultural media like Nutrient agar, MacConkey`s (MC) agar, Eosin Methylene Blue (EMB) agar, Salmonella-Shigella (SS) agar, Xylose-Lysine-Deoxycholate (XLD) agar and Blood agar were used to isolate and to study the cultural properties of the E. coli and Salmonella spp. Finally Gram’s staining and different biochemical tests were performed to identify those two bacterial species. Out of 57 samples, 27 were positive for E. coli and 8 were positive for Salmonella spp. On the basis of information from cattle owners and clinical signs the prevalence of diarrhoea was recorded as 30.99% and the pvalue was calculated as 0.001 (p<0.01) which was noted as highly significant. The prevalence percentages of the E. coli and Salmonella spp. were differed depending on different epidemiological parameters like age, sex, season and location. Moreover, the molecular identifications were further confirmed by means of PCR assay using specific primers for E. coli and Salmonella spp. This was done targeting 16S rRNA gene where they were found to be positive showing amplification of 585 bp for E. coli and 574 bp for Salmonella spp. From the study of the antibiogram profiles, it was revealed that E. coli were susceptible to ciprofloxacin, gentamicin and norfloxacin but resistant to tetracycline, erythromycin, amoxicillin and streptomycin whereas Salmonella spp. were susceptible to ciprofloxacin, gentamicin, amoxicillin and streptomycin but resistant to azithromycin, tetracycline and erythromycin. The findings of this research work would certainly help to select the proper antibiotics against diarrhoea in cattle of Bangladesh and to overcome the multi-drug resistant problem of the bacteria.


Introduction
Diarrhoea is one of the major community health hazards both for man and animal which is caused by bacteria (such as Campylobacter, Escherichia coli, Salmonella and Clostridium), virus (Rotavirus, coronavirus, Bovine viral diarrhoea virus, along with newly emerging enteric viruses such as bovine torovirus and caliciviruses like bovine norovirus and Nebovirus), fungus, protozoa (Coccidia, Cryptosporidium), helminths, chemical agents, clay, sands, nutritional deficiency factors, indigestion, managemental factors, hepatic cirrhosis and other toxic factors.These factors act singly or in combination for the production of diarrhoea complex (Sharif et al., 2005).
The prevalence of each of the pathogen and disease incidence can vary by geographical location of the farms, farm management practices and herd size (Cho and Yoon, 2014).Livestock population in Bangladesh is currently estimated to comprise 25.7 million cattle, 0.83 million buffaloes, 14.8 million goats, 1.9 million sheep, 118.7 million chicken and 34.1 million ducks (Banglapedia, 2015).Among livestock population cattle are considered as the most important animals in livestock sub-sector.Various microbial diseases are common in cattle like anthrax, haemorrhagic septicaemia, brucellosis, tuberculosis, mastitis, diarrhoea etc. that can act as alarming side for cattle production.Among these diseases, diarrhoea is very common and may be fatal if proper treatment is not given to the affected cattle.Diarrhoea in cattle due to bacterial species were detected in home and abroad as colibacillosis, pasteurellosis, salmonellosis, campylobacteriosis and clostridial infections where serious losses of income generation activities are being occurred either in terms of morbidity and mortality of cattle (Fulton et al., 2000;Mailk et al., 2013;Cho and Yoon, 2014;Muktar et al., 2015).Out of these bacterial species colibacillosis and salmonellosis were being targeted as a primary step to reduce the diarrhoea in cattle population of Bangladesh.The etiological agents of diarrhoea due to colibacillosis and salmonellosis could be isolated and identified through cultural, biochemical, serological and molecular techniques.A course of antibiotic therapy is necessary to treat bacterial diarrhoea in cattle.Incomplete course of treatment and continuous indiscriminate use of antibacterial drugs against diarrhoeal infection of man and animal might have influenced to produce a new generation of virulent and resistant type of bacteria (Marshall et al., 1990;Izumiya et al., 2001).Different parameters including prevalence, isolation, identification, antibiotic sensitivity testing, epidemiological investigation, plasmid profiling of E. coli of different species were studied by Paul et al., 2010;Hossain et al., 2013;Hasan et al., 2015.Isolation, identification, serotypic characterization, pathogenicity testing, antibiogram study of Salmonella of different species were also studied by Sarker et al., 2009;Nesa et al., 2011;Islam et al., 2013;Ansari et al., 2014;Muktar et al., 2015.However, isolation, molecular identification and antimicrobial susceptibility profiles of E. coli and Salmonella spp.from diarrhoeic cattle were not systematically and properly focused by the previous research works in context of Bangladesh.So, the present study was undertakento survey the diarrhoeic cattle, to isolate and identify the E. coli and Salmonella spp. with their antibiogram profiles that will be helpful towards cattle diseases management and enhance dynamism of cattle farming which not only alleviate poverty but also boost up the national economy of Bangladesh.

Sampling areas and period
A total number of 57 rectal swab samples were aseptically collected using sterilized cotton buds from diarrhoeic cattle of the Mymensingh sadar (25), Trishal (8), Valuka (5), Natore sadar (9) and Gomostapur, Chapai Nawabganj (10)during the period of June 2015 to May 2016.After collection, samples were brought to the laboratory in the Department of Microbiology and Hygiene, Bangladesh Agricultural University (BAU), Mymensingh-2202, Bangladesh for the isolation, molecular identification and antibiogram profiles of the E. coli and Salmonella spp.

Isolation of E. coli and Salmonella spp.
The collected rectal swab samples were enriched into nutrient broth by incubation at 37ºC for 24 hours.To isolate and to study the cultural properties of the E. coli and Salmonella spp., enriched cultures were streaked onto different types of differential and selective culture media like MC agar, EMB agar, SS agar, XLD agar and blood agar according to the method described by Cowan, 1985 where all of the media were brought from the Indian company, HiMedia.

Identification of E. coli and Salmonella spp.by conventional methods
For identification of isolated E. coli and Salmonella spp., Gram's staining and biochemical tests were performed.Gram's staining was performed according to the method described by Merchant and Packer, 1967 where all of the reagents like crystal violet, Gram's iodine, safranin, acetone alcohol, immersion oil were brought from the German company, Merck.Different types of biochemical tests like sugar fermentation test, MR-VP reaction, indole reaction, catalase test were performed according to the methods described by Douglas et al., 1998 andOIE, 2000 where all of the reagents were brought from the German company, Merck.Motility test using MIU medium base was also performed according to the method described by Cowan, 1985 to differentiate motile bacteria from the non-motile one where MIU medium base was brought from the Indian company, HiMedia.

Preparation of DNA templates
Extraction of DNA from the E. coli and Salmonella spp. was carried out by conventional boiling and rapid cooling method (Medici et al., 2003).In brief, 200 μl deionized water was taken into an eppendorf tube, a pure bacterial colony from nutrient agar was mixed with the deionized water.The tube then transferred to boiling water and boiled for 10 minutes then immediately to the icebox for cold shock about 10 minutes and then centrifuged at 10,000 rpm for 10 minutes.Supernatant were collected and used as DNA template during PCR.

Identification of E. coli and Salmonella spp. by PCR assay
Details of the oligonucleotide primers used for the amplification of 16S rRNA gene of the E. coli and Salmonella spp.are summarized in Table 1.PCR reaction mixture (25μl) for E. coli was prepared using 12.5μlmaster mixture (Promega, USA), 10 pmol primer (Bioneer, South Korea) of each, 5μl DNA template and 5.5μl nuclease free water.PCR reaction mixture (20 μl) for Salmonella spp. was prepared using 10 μl master mixture (Promega, USA),10 pmol primer (Bioneer, South Korea) of each, 3 μl DNA template and 5 μl Nuclease free water.For the amplification of 16S rRNA gene of E. coli, the cycling conditions consisted of initial denaturation for 5 minutes at 95ºC, followed by 30 cycles of denaturation at 94ºC for 30 seconds, annealing at 58°C for 1 minute, extension at 72ºC for 1 minute and final extension at 72ºC for 10 minutes where the cycling conditions for the amplification of 16S rRNA gene of Salmonella spp.consisted of initial denaturation for 5 minutes at 94ºC, followed by 35 cycles of denaturation at 94ºC for 20 seconds, annealing at 50°C for 30 seconds, extension at 72ºC for 30 seconds and the final extension at 72ºC for 5 minutes.Amplification was performed in a thermal cycler (Eppendorf, Germany).The amplified products were electrophoresed into 1.5% agarose (Sigma-Aldrich, USA) gel at 100 volt visualized under Gel doc/UV trans-illuminator (BioRad).100 bp (for Salmonella spp.) and 1 kb (for E. coli) DNA size marker (Promega, USA) were used.

Statistical analysis
The experimental data were analysed by SPSS software (version 20) where the prevalence of diarrhoea based on questionnaire survey was calculated by ANOVA table method and the prevalence percentages of the isolated E. coli and Salmonella spp.based on different epidemiological parameters like age, sex, season and location were calculated by Chi-square (χ2) test method.

Prevalence of diarrhoea in cattle as per collected data from study areas
For this study, data were collected through a developed questionnaire from study areas of Bangladesh.Prevalence was recorded as 22.92% at farm level and 39.06% at household level.Overall prevalence was recorded as 30.99%.The p-value was calculated as 0.001 (p<0.01) which was noted as highly significant.The prevalence percentages of diarrhoea from different locations are shown in Table 2.

Isolation and cultural characterization of E. coli and Salmonella spp.
The growth of E. coli and Salmonella spp. was indicated by the presence of turbidity in the nutrient broth after overnight incubation at 37ºC.On nutrient agar, Salmonella spp.produced circular, smooth, opaque, translucent colonies and E. coli produced circular, smooth, colorless colonies.On EMB agar, E. coli produced greenishblack colonies with metallic sheen.On MC agar, E. coli produced circular raised, bright pink colored colonies while Salmonella spp.produced colorless, smooth, pale and transparent, raised colonies.On SS agar, E. coli produced slight pinkish colored colonies while Salmonella spp.produced pinhead or lentil sized, raised, round or circular, smooth, glistening, opaque, black, transparent or translucent colonies.On blood agar, E. coli produced colorless colony without hemolysis while Salmonella spp.produced white, round, raised colonies with no hemolysis.On XLD agar, Salmonella spp.produced blackish centered colonies while E. coli produced yellow colored colonies.

Identification of E. coli and Salmonella spp. by conventional methods
In Gram's staining, the isolated E. coli revealed Gram negative, pink colored, short plump rod shaped appearance arranged as single, paired or in short chain and Salmonella spp.revealed Gram negative, pink colored, very short plump rod shaped appearance arranged as single or paired.All of the isolates of E. coli and Salmonella spp.were found as positive to motility test.Fermentation of five basic sugars with the production of acid and gas indicated that the isolates were E. coli positive while Salmonella spp.fermented dextrose, maltose and mannitol with the production of acid and gas but did not ferment lactose and sucrose.E. coli were found as indole positive where Salmonella spp.were found as indole negative.Both organisms (E. coli and Salmonella spp.) were found as MR test positive, VP test negative and catalase test positive.

Identification of E. coli and Salmonella spp. by PCR assay
DNA extracted from all of the isolates (27 E. coli and 8 Salmonella spp.) were used in PCR assay.Polymerase chain reaction with the primers ECO-1 and ECO-2 (for E. coli) and Sal 16S rRNA (for Salmonella spp.) identified all of those isolates as positive for E. coli and Salmonella spp.showing amplification of 585 bp and 574 bp respectively as presented in Figures 1 and 2.

Prevalence of the isolated E. coli and Salmonella spp. at different epidemiological parameters (age, sex, season and location)
Prevalence of E. coli was 52.00% (n=13/25) in Mymensingh sadar, 12.50% (n=1/8) in Trishal, 20.00% (n=1/5) in Valuka, 33.33% (n=3/9) in Natore sadar and 90.00%(n=9/10) in Gomostapur, Chapai Nawabganj whereas prevalence of Salmonella spp. was 12.00% (n=3/25) in Mymensingh sadar, 37.50% (n=3/8) in Trishal, 40.00% (n=2/5) in Valuka and no samples were detected as positive for Salmonella spp. in Natore sadar and in Gomostapur, Chapai Nawabganj.The p-value was recorded as 0.009 (p<0.01) for E. coli and 0.047 (p<0.05) for Salmonella spp. that meant the results were highly significant for E. coli and significant for Salmonella spp.The prevalence of the isolated E. coli was 64.00% (n=16/25) in 1-12 month aged cattle and 34.37% (n=11/32) in above 12 month aged cattle.The p-value was calculated as 0.026 (p<0.05) that meant the results were significant.The prevalence percentages of E. coli were 26.31% (n=5/19) in male cattle and 57.89% (n=22/38) in female cattle.The p-value was recorded as 0.024 (p<0.05) that meant the results were significant.The prevalence percentages of E. coli were 56.75% (n=21/37) in summer season, 71.42% (n=5/7) in rainy season and 7.69% (n=1/13) in winter season.The p-value was calculated as 0.004 (p<0.01) that meant the results were highly significant.The prevalence of the isolated Salmonella spp. was found as 28.00% (n=7/25) in 1-12 month aged cattle and 3.12% (n=1/32) in above 12 month aged cattle.The p-value was recorded as 0.007 (p<0.01) that meant the results were highly significant.The prevalence of the isolated Salmonella spp. was found as 21.05% (n=8/38) in female cattle and there was no prevalence of Salmonella spp. in male cattle.The p-value was calculated as 0.038 (p<0.05) that meant the results were significant.The prevalence percentages of the isolated Salmonella spp.were found as 13.51% (n=5/37) in summer season, 42.85% (n=3/7) in rainy season and out of 13 samples no samples were detected as positive for Salmonella spp. in winter season.The p-value was recorded as 0.031 (p<0.05) that meant the results were significant.The prevalence percentages of the isolated E. coli and Salmonella spp. at different epidemiological parameters (age, sex, season and location) are shown in Tables 3 and 4.

Antibiogram profiles
E. coli were susceptible to ciprofloxacin, gentamicin and norfloxacin; intermediate to azithromycin but resistant to tetracycline, erythromycin, amoxicillin and streptomycin.Salmonella spp.were susceptible to ciprofloxacin, gentamicin, amoxicillin and streptomycin; intermediate to norfloxacin but resistant to azithromycin, tetracycline and erythromycin as shown in Table 5.

Discussion
The cultural properties of the isolated E. coli were the production of metallic sheen on the EMB agar and rose pink colored colony on the MC agar which agreed with the findings of Ali et al., 1998;Sharada et al., 1999.In this study the colonies of Salmonella spp. on SS agar were opaque, translucent with black centers which were similar to the findings of Cheesbrough, 1985;Hossain, 2002.In Gram's staining, the morphology of the isolated E. coli was exhibited as Gram negative, short plump rod arranged as single, paired or in short chain which was reported by Merchant and Packer, 1967;Buxton and Fraser, 1977;Freeman, 1985;Sharada et al., 1999 whereas the morphology of the isolated Salmonella spp. was Gram negative, very short plump rod arranged as single or paired and those properties of Salmonella spp.were supported by Freeman, 1985 andHossain, 2002.Motility test revealed that the isolated E. coli and Salmonella spp.were motile which was correlated with the results of Merchant and Packer, 1967;Buxton and Fraser, 1977.E. coli isolates were able to ferment the five basic sugars by producing both acid and gas which was supported by Beutin et al., 1997;Thomas, 1998 whereas isolated Salmonella spp.were able to ferment dextrose, maltose and mannitol with the production of both acid and gas but did not ferment lactose and sucrose and those characteristics of Salmonella spp.were satisfied the statement of Hossain, 2002;Han et al., 2011.The isolated E. coli were found MR and indole test positive but VP test negative which were reported by Merchant and Packer, 1967;Ali et al., 1998;Mishra et al., 2002.The isolated Salmonella spp.were found MR test positive but indole and VP test negative that satisfied the statement of Douglas et al., 1998;OIE, 2000.The results of prevalence of the isolated E. coli and Salmonella spp.calculated on the basis of age, sex, season and location were more or less similar with the findings of Baule et al., 1997;Khan et al., 2009.On the basis of information from the cattle owners and clinical signs, the prevalence of diarrhoea was recorded as 30.99% which showed more or less similar results with the findings of Gulliksen et al., 2009;Drillich et al., 2015.Molecular identifications of the isolated E. coli and Salmonella spp.were done by means of PCR assay that was similar to the findings of Ziemer and Steadham, 2003;Schippa et al., 2010;Adamu et al., 2014.In this study, 8 different antibiotics available in the market were used to study antibiogram profiles of the E. coli and Salmonella spp.isolated from diarrhoeic cattle by disc diffusion method and the results were supported by Akond et al., 2009;Wouafo et al., 2010;Hyeon et al., 2011;De et al., 2012;Jeyasanta et al., 2012;Jahan et al., 2013.Due to financial crisis and limited time following further studies like collection of more samples covering many areas and farms of Bangladesh to get more precise information, molecular detection of shiga toxin producing E. coli, molecular characterization of the isolated bacterial species by pulsed field gel electrophoresis (PFGE), 16S rRNA gene sequencing of the isolated E. coli and Salmonella spp., serotyping, multiplex PCR of the isolated Salmonella spp., pathogenicity study would not be possible.

Conclusions
The findings of this research work would certainly help the veterinary practitioners to select the proper antibiotics against diarrhoea in cattle of Bangladesh.If the prescribers prescribe suitable antibiotics against diarrhoea in cattle then it would be possible to overcome the multi-drug resistant problem of bacteria, otherwise successful antibiotic therapy against diarrhoea in cattle would not be possible.

Table 2 . Results of prevalence of diarrhoea in cattle as per collected data based on questionnaire survey.
**means 1% level of sig.(p<0.01)