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OPEN Molecular detection of pathogenic Escherichia coli strains and their antibiogram associated with risk factors from diarrheic calves in Jimma Ethiopia Destaw Asfaw Ali1*, Tesfaye Sisay Tesema2 & Yosef Deneke Belachew3

Diarrheagenic Escherichia coli are a number of pathogenic E. coli strains that cause diarrheal infection both in animal and human hosts due to their virulence factors. A cross sectional study was conducted between November, 2016 and April, 2017 to isolate and molecularly detect pathogenic E. coli from diarrheic calves to determine the pathogenic strains, antibiogram and associated risk factors in Jimma town. Purposive sampling technique was used to collect 112 fecal samples from diarrheic calves. Conventional culture and biochemicalARTICLE methods were conducted to isolate E. coli isolates. Molecular method was followed to identify virulence factors of pathogenic E. coli strains. Antimicrobial sensitivity patterns of the isolates were tested using the Kirby–Bauer disk difusion method. A structured questionnaire was also used to collect information from dairy farms and socio- demographic data. The overall isolation rate of E. coli in calves was 51.8% (58/112) (95% CI 42.0–61.0). The occurrence of the bacterium difered signifcantly by age, colostrum feeding time, amount of milk given per time and navel treatment (P < 0.05). Multivariable analysis revealed that the odds of being infected was signifcantly highest in calves which fed 1–1.5 L amount of milk per a time (OR 5.38, 95% CI 1.66–17.45, P = 0.005). The overall virulence genes detection rate was 53.5% (95% CI 40.0–67.0). Eleven (19.6%) of eaeA, 6 (10.7%) of Stx1 and 13 (23.2%) of Stx2 genes were detected from calves isolates. Except ciprofoxacillin, all isolates were resistant to at least one drug. Multi drug resistance was recorded in 68.0% (38/56) of calves isolates. Neomycin, 83.3% (25/30), followed by amoxicillin, 53.3% (16/30) were the highest resisted virulence genes. The study demonstrated considerable isolation rate, multiple antimicrobial resistant isolates and high resistant virulent genes in diarrheic calves. It also indicated that the potential importance of calves as source of pathogenic E. coli strains and resistant genes for human diarrhea infection. Improving the hygienic practice of farms and wise use of antimicrobials could help to reduce the occurrence of pathogenic E. coli in farms. Hence, further studies are needed to describe all virulent factors and serotypes associated with the emergence of drug resistant pathogenic E. coli strains in calves.

Abbreviations AMGPT Amount of milk given per time AOR Adjusted odds ratio CI Confdence interval CF Calving facilities RETRACTEDCLSI Clinical Laboratory and Standard Institute COR Crude odds ratio CSA Central Statistical Agency CSAASS Central Statistical Agency Agricultural Sample Survey DACA​ Drug Administration and Control Authority

1College of Veterinary Medicine and Animal Sciences, University of Gondar, Gondar, Ethiopia. 2Institute of Biotechnology, Addis Ababa University, Addis Ababa, Ethiopia. 3College of Agriculture and Veterinary Medicine, Jimma University, Jimma, Ethiopia. *email: [email protected]

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dNTP Deoxyribo nucleotide triphosphate EIAR Ethiopian Institute of Agricultural Research EVT EnteroVero toxin FCFT F irst colostrum feeding time IgG Immunoglobulin G ISO International Organizations for Standardization OIE Ofce International des Epizooties OPEDJZ Ofce of Planning and Economic Development for Jimma Zone P Probability PCR Polymerase chain reaction SPSS Sofware Package for Social Science USDA United State Department of Agriculture χ2 Chi-square

Background Cattle production plays an important role in the economy and livelihood of farmers and pastoralists worldwide. Despite the large livestock population of Ethiopia, the economic benefts remain marginal due to prevailing diseases, poor nutrition, poor animal production systems, reproductive inefciency, management constraints and general lack of veterinary c­ are1. Te future of any dairy and beef production depends on the successful rais- ing of calves and heifers for replacement. Te health and management of replacement animals are important components of total herd proftability. Diarrhea is one of the very common disease syndrome in neonatal calves in diferent countries and this can have severe impacts both economically and in terms of animal w­ elfare2. Calf diarrhea is a multifactorial disease which, despite decades of research in the topic, remains the most common cause of calf ­mortality3. Tere is multitude of interaction in the none infectious causes like gaps in management of animals, inadequate nutrition, exposure to severe environment, insufcient attention to the new borns, and the infectious diseases. According to C­ ho4, 80% of diarrheic calves tested were positive for at least one of the target enteric pathogens suggesting that infectious factor is still a major cause of calf ­diarrhea5. Te prevalence, antibiogram and epidemiological features of E. coli as the causative agent of diarrhea vary from region to region around the world, and even between and within countries in the same geographical a­ rea6. Neonatal diarrhea has been commonly attributed to multiple enteric pathogens of bacteria, protozoa and ­viruses7,8. Although co-infection considerably worsens ARTICLEthe disease, sometimes single infection is recorded and many of the cases are predominantly related to bacterial ­pathogens9,10. Among the bacterial pathogens, diarrhea in calves due to E. coli remains common devastating disease all over the world, particularly in calves less than 3 months of a­ ge11,12. Although E. coli is the predominant member of the animal and human intestinal microfora, a number of strains have developed the ability to cause diseases particularly of the gastrointestinal in animal and human ­hosts13. Diarrheagenic strains of E. coli are categorized based on distinct epidemiological and clinical features, specifc virulence determinants and association with certain s­ erotypes14,15. New strains of E. coli arise all the time from the natural biological process of genetic variability and hence monitoring the levels of E. coli contamination is important. Te development of highly applicable nucleic acid based diagnostic analysis on the basis of culture media isolation and biochemical reactions enabled the detection of diferent pathotypes of diarrheagenic E. coli. Among these, PCR is a commonly used molecular method that gives rapid and reliable detection of pathogenic E. coli strains that could show the prevalence and distribution of these ­pathogens16,17. Te advent of advanced molecular studies showed that animal to human disease transmission is by cross infection in experimental models, animal handling and contaminated food consumption. Te direct and indirect economic impacts of neonate animals is one of the most common and devastating conditions encountered in the animal agriculture ­industry18. Te high frequency and persistence of calf diarrhea in farms has gained the interest of many researches. In central and south eastern part of Ethiopia, limited researches indicate calf mortality rate and the supposed infectious agents. A few studies show mortality in the frst 6 months of calf hood ranging from 7 to 25%18,19. E. coli isolates in calf diarrhea accounts 37% Gebregiorgis and ­Tessema20 in Kombolcha District, 50.9% ­Yakob21 in Arsi zone and 69.5% ­Yimer22 in North Shewa zone of Ethiopia. In south western part of the country, particularly in Jimma town and its surroundings, the information on cause of diarrhea in calves and its associated factors is minimal. Understanding the population structure of pathogenic E. coli is important since it impacts the efectiveness of molecular epidemiological studies. Molecular detection of the supposed pathogenic E. coli strains isolated from calves in Ethiopia is limited. Tis hinders implementation of efective control and preventive measures. In addition, most pathogenic bacteria that are commonly involved in causing infection to animals and humans RETRACTEDhave shown considerable degree of resistance to commonly used antimicrobials in this ­country23. Uncontrolled application of antimicrobials is rooted from lack of particular pathogenic strain identifcation from a host at particular place that leads to an increase in the rate of antimicrobial resistance which has signifcant efect for the problem to persist. Terefore, this study was carried out to isolate and molecularly detect virulence genes of pathogenic E. coli strains, identifes their antibiogram and associated risk factors from diarrheic calves in dairy farms in Jimma town.

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Category Samples Variables Category Number examined Number positive proportion (%) proportion (%) χ2 P value 1–2 44 33 75.0 29.5 Age(week) 3–8 39 16 41.0 14.3 16.31 0.001 9–16 29 9 31.1 8.0 Female 89 49 55.0 43.8 Sex 1.86 0.177 Male 23 9 39.1 8.0 Cross 94 48 51.1 42.9 Breed 0.398 0.390 Local 18 10 55.6 8.9 > 6 h 20 16 80.0 14.3 FCFT 5.26 0.027 < 6 h 92 42 45.7 37.5 Same barn 66 37 56.1 33.0 CF 1.18 0.279 Separated 46 21 45.7 18.8 1–1.5 L 49 37 75.5 33.1 AMGPT 1.5–2.5 L 33 9 27.3 8.0 20.66 0.001 Unknown 30 12 40.0 10.7 No 54 35 64.8 31.3 Navel treatment Yes 58 23 39.7 20.5 7.09 0.008 Each total 112 51.8

Table 1. Over all occurrence of E. coli isolates with diferent factors in diarrheic calves. FCFT frst colostrum feeding time, AMGPT amount of milk given per time, CF calving facility; χ2 Chi square, P probability.

Univariable Multivariable Risk factors Category No. tested Sample positive (%) COR(CI) P AOR(CI) P 1–2 44 33 (29.5) 6.66 (2.35–18.89) 0.001 4.029 (1.22–13.27) 0.022 Age (weeks) 3–8 39 16 (14.3) ARTICLE1.55 (0.56–4.26) 0.399 0.968 (0.28–3.31) 0.954 9–16 29 9 (8.0) 1.00 1.00 Female 89 49 (43.8) 1.91 (0.75–4.86) 0.177 2.55 (0.79–82.13) 0.116 Sex Male 23 9 (8.0) 1.00 1.00 Cross 94 48 (42.9) 0.64 (0.23–1.78) 0.390 – – Breed Local 18 10 (8.9) 100 – > 6 h 20 16 (14.3) 3.41 (1.15–10.19) 0.027 3.73 (1.01–13.76) 0.048 FCFT < 6 h 92 42 (37.5) 1.00 1.00 Te same barn 66 37 (33.0) 1.51 (0.72–3.24) 0.279 – – CF Separated pen 46 21 (18.8) 1.00 – 1–1.5 L 49 37 (33.1) 4.63 (1.74–12.30) 0.002 5.38 (1.66–17.45) 0.005 AMGPT 1.5–2.5 L 33 9 (8.0) 0.56 (0.19–1.62) 0.287 0.73 (0.20–2.84) 0.631 Unknown 30 12 (10.7) 1.00 1.00 No 54 35 (31.3) 2.80 (1.30–6.04) 0.008 1.93 (0.75–4.94) 0.173 Navel treatment Yes 58 23 (20.5) 1.00 1.00

Table 2. Risk factors associated with E. coli isolates in calves by univariable and multivariable logistic regression. COR crude odd ratio, AOR adjusted odd ratio, CF calving facility, AMGPT amount of milk given per time, CI confdence interva, P probability, 1.00 rreference group.

Results Overall isolation of Escherichia coli in calf diarrhea. Escherichia coli was recovered in 58 (51.8%) out of the 112 diarrheic calves that showed calf diarrhea. In the present study, the occurrence of diarrhea due to E. RETRACTEDcoli difered signifcantly by age, colostrum feeding time, amount of milk given per time and navel treatment. A higher occurrence of diarrhea due to E. coli was detected in calves of 1–2 weeks old (29.5%), in calves frst colos- trum fed less than 6 h (37.5%), calves that fed 1–1.5 L of milk per a time (33.1%) and in not navel treated calves during birth (31.3%). Its occurrence did not difer by sex, breed and calving facility (Table 1).

Univariable and multivariable logistic regression analysis of risk factors associated with calf diarrhea. From univariable logistic regression analysis, independent variables such as age, frst colostrum feeding time, amount of milk given per time and navel treatment were signifcantly associated with E. coli iso- lates (P < 0.05). On the other hand sex, breed and calving facility were not signifcantly associated with E. coli

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Sample source Genes Isolates examined Positive isolates (%) Diarrheic samples (%) χ2 P (95% CI) Calves eaeA 56 11 (19.6) 11 (9.8) 11.86 0.001 (0.000–0.052) stx1 56 6 (10.7) 6 (5.4) 5.82 0.016 (0.00–0.113) stx2 56 13 (23.2) 13 (11.6) 14.67 0.001 (0.000–0.052) Total 30 (53.5) 30 (26.8)

Table 3. PCR detection rate of virulence genes in E. coli isolates in diarrheic calves and children. χ2 Chi square, P Probability, CI confdence interval.

isolates as shown in Table 2. Te variables with P value < 0.25 in univariable logistic regression analysis were taken to multivariable logistic regression analysis to control confounders. Followed, age, sex, time of frst colos- trum feeding, amount of milk given per time and navel treatment were entered to multivariable logistic analysis. Multivariable logistic regression analysis was carried out to observe the independent efects of each risk factors in relation to the occurrence of E. coli isolates in diarrheic calves. Accordingly, variables such as age (P = 0.022), frst colostrum feeding time (P = 0.048) and amount of milk given per time (P = 0.005) were identifed as the signifcant independent predictors for occurrence of E. coli isolates in calf diarrhea. Te log odds ratio indicates that calves within the age category between 1 and 2 weeks (AOR = 4.029,9 5% CI 1.22–13.27, P 0.022) are more susceptible to diarrhea due to E. coli compared to other age categories of diarrheic calves. Calves that were fed frst colostrum in more than 6 h (AOR = 3.730, 95% CI 1.01–13.77, P = 0.048) were more susceptible than those fed before 6 h. Te occurrence of the bacteria in calf diarrhea was also more likely in calves also signifcantly associated with thethat fed 1–1.5 litter amount of milk given per time (AOR = 5.38, 95% CI 1.66–17.45, P = 0.005) than those fed more. None of the variables found to be collinear and there was insignifcance diference between the observed and the predicted values with Chi-square = 3.610, P = 0.890 which was ftted well with the data.

Description of dairy farms and owners based on questionnaire and observation. A total of 35 questions comprised fve areas of interest namely farm characteristics, calving and care of the newborn, calf housing, calf feeding, weaning and calf disease in pre-weaned calves were administered. Owners of 54 farms used for sample collection were interviewed. Te farms harbor cross Holstein Friesian 94 (83.9%) and local 18 (16.1%) breeds of calves. Seventeen (31.5%) of the farmsARTICLE in the study were intensively managed with an average number of 8 calves per herd and most of smallholder farms had 2 calves per herd. More than three-forth of the dairy farmers had knowledge of the advantage of colostrum feeding. Ninety two (82.1%) out of 112 diarrheic caves fed frst colostrum within less than 6 h and 46 (41.1%) of calves were kept in separated calving pen that were ofen not cleaned and disinfected regularly. Navel treatment during birth of calves was practiced in 58 (51.8%) of the visited farms. Te practice of providing bedding for calves was limited in large dairy farms and none in small holders. All study farms fed whole milk for calves two times daily by bucket feeding with the exception of few small holder farms that allowed calves to suckle their dams. Special starter feed was not used in any of the farms rather straw, hay and concentrates that were given to cows were used for calves. Forty six (85.2%) of farms weaned calves at 3 months of age whereas 8 (14.8%) of them weaned at 4 months. In general, the weaning age was lower for male calves, mostly under 3 months. In all large dairy farms, there were vet personnel employed to deal with health aspects of the farms. Smallholder dairy farms call private veterinary practitioners whenever their animals face health problems. From farm managers or owners that mentioned calf health problems, majority of them complained that diarrheal and respiratory tract diseases were the most frequent diseases encountered in calves.

Polymerase chain reaction based detection of virulence genes. Of the total 112 fecal samples from diarrheic calves 58 isolates were presumed to be E. coli positive by biochemical tests. Fify six isolates, two of the isolates were not recovered during DNA extraction, were then subjected to virulence genes specifc PCR assays. Te overall detection rate of the three genes tested from positive isolates in calves was 30 (53.5%). Eleven (19.6%) of eaeA gene, 6 (10.7%) of stx1 gene and 13 (23.2%) of stx2 gene positive isolates were detected by PCR from diarrheic calves (Table 3) and illustrated by documented jell as in Fig. 1. All the virulence genes involved were signifcantly associated with diarrhea due to E. coli (P < 0.05).

Antimicrobial resistance profles of Escherichia coli. Mono drug resistance. Te antimicrobial sus- ceptibility features of 56 isolates from diarrheic calves are given in Table 4. Te highest sensitivity of ciprofoxa- cillin and norfoxacillin were recorded in 98.2% of the isolates. Norfoxacillin and oxytetracycllin for all isolates RETRACTEDdid not show intermediate resistance while another tested drugs was recorded as intermediate resistance in one or more of the tested isolates. Te highest resistance isolates was recorded for neomycin (76.8%) followed by amoxicillin (48.2%). Resistance to chloramphenicol and norfoxacillin were observed in 1.8% of the isolates from calves isolates.

Multi drug resistance. Te multi-drug resistance features of the E. coli isolates are shown in Table 5. Of the tested 56 isolates from calves, 38 (68.0%) were resistant to two or more (up to eight) antimicrobials. Twenty three multi drug resistance profles were observed and the number of isolates resistant to two drugs were higher

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ARTICLE

Figure 1. (A–C) Amplifcation of virulence genes in E. coli isolates from diarrheic calves and the images are a product of time averaged data. M marker, N negative control, P positive control; Numbers = 1–11 representative sample numbers; (A) eaeA gene with 450 bp; (B) stx2 gene with 350 bp; (C) stx1 gene with 110 bp.

Antibiotics Susceptible (%) Intermediate (%) Resistant (%) Amoxacillin 26 (46.4) 3 (5.4) 27 (48.2) Chloramphenicol 50 (89.3) 5 (8.4) 1 (1.8) Ciprofoxacillin 55 (98.2) 1 (1.8) – Cefoxitin 41 (73.2) 10 (17.9) 5 (8.9) Gentamycin 53 (94.6) 1 (1.8) 2 (3.6) Neomycin 4 (7.1) 9 (16.1) 43 (76.8) RETRACTEDNorfoxacillin 55 (98.2) – 1 (1.8) Oxytetracycliine 41 (73.2) – 15 (26.8) Streptomycin 44 (78.6) 7 (12.5) 5 (8.9) Sulfonamides 30 (53.6) 4 (7.1) 22 (39.3) Trimethoprim 35 (62.5) 1 (1.8) 20 (35.7) Total (%) 434 (70.45) 41 (6.65) 141 (22.9)

Table 4. Number (%) of E. coli isolates (56) resistant to antimicrobials.

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Isolates from calves (n = 56) Number of antimicrobials Resistance pattern (no. of isolates) No. of isolates (%) Two NEO, AMC (7); W, AMC (3); AMC, S3 (1); W, S3 (2); NEO, S3 (1); NEO, OT (1) 15 (26.8) NEO, AMC, S3 (1); W, AMC, S3 (1); NEO, W, AMC (2); NEO, OT, S3 (2); NEO, AMC, Tree 8 (14.3) OT (1); S, W, S3 (1) NEO, AMC, OT, CXT (1); NEO, W, AMC, S3 (2); NEO, AMC, CXT, S3 (1); NEO, Four 9 (16.1) AMC, OT, S3 (2); NEO, W, OT, S3 (3) Five W, AMC, OT, CXT, S3 (1); NEO, W, AMC, OT, S3 (1) 2 (3.6) Six NEO, S, W, AMC, OT, S3 (1); GEN, NEO, S, W, CXT, S3 (1) 2 (3.6) Seven GEN, NEO, W, AMC, OT, CHL, S3 (1) 1 (1.8) Eight NEO, S, W, AMC, OT, CXT, NOR, S3 (1) 1 (1.8) Total 38 (67.8)

Table 5. Multiple antimicrobial resistance profles of E. coli isolates. AMC Amoxacillin, CHL Chloramphenicol, CPR Ciprofoxacillin, CXT Cefoxitin, GEN Gentamycin, NEO Neomycin, NOR Norfoxacillin, OT Oxytetracycliine, S Streptomycin, S3 Sulfonamides, W Trimethoprim.

Resistant level of Pathogenic strain harboring virulence factor genes (%) Antimicrobials eaeA (n = 11) stx1 (n = 6) stx2 (n = 13) All (n = 30) Amoxacillin 5 (45.5) 1 (16.6) 10 (76.9) 16 (53.3) Chloramphenicol – – – – Ciprofoxacillin – – – – Cefoxitin – – 3 (23.1) 3 (10) Gentamycin – – – – Neomycin 9 (81.8) 4 (66.6) 12 (92.3) 25 (83.3) Norfoxacillin – – 1 (7.7.) 1 (3.3) Oxytetracycliine 3 (27.3) 1 (16.6) 3 (23.1) 7ARTICLE (23.3) Streptomycin 2 (18.2) – 1 (7.7) 3 (10) Sulfonamides 3 (27.3) 2 (33.3) 5 (38.5) 10 (33.3) Trimethoprim 3 (27.3) 2 (33.3) 3 (23.1) 8 (26.7)

Table 6. Antimicrobial resistance profles of pathogenic E. coli strains genes (n = 30). n number.

followed by four drugs resistant isolates. Te NEO, AMC (7/38) phenotype occurred more frequently followed by the NEO, W, OT, S3 (3/38) phenotype from calves isolates.

Virulence genes resistance. Te antimicrobial resistance profle of the pathogenic 30 E. coli strain genes is shown in Table 6. Te highest resistance to the strains of sampled calves was recorded for neomycin 25 (83.3%) followed by amoxicillin 16 (53.3%) whereas resistance to norfoxacillin 1 (3.3%) showed least resistance. Out of 30 calves isolated of E .coli genes, eaeA gene 9 (81.8%), stx1 gene 4 (66.6%) and stx2 gene 12 (92.3%) encoded isolates showed highest resistant for neomycine. On the other hand, streptomycine 2 (18.2%) for eaeA gene, amoxicillin and oxytetracycllin 1 (16.6%) for stx1 gene and norfoxacillin and streptomycin for stx2 gene showed all 1 (7.7%) least resistant from calves isolates. Chloramphenicol, ciprofoxacillin and gentamycin in diarrheic calves isolates did not show resistance to the identifed virulent genes of E. coli. Discussion Tis study was conducted to assess the overall isolation rate, virulence factors of pathogenic strains and anti- biogram profle of E. coli isolated from diarrheic calves in Jimma town. Te importance of detecting pathogenic E. coli from diarrheic calves has great signifcance. Calf morbidity and mortality result in great economic losses as calves are replacement stocks in a cattle production system and diarrheic calves are also potential source of RETRACTEDthe bacterium for human infection. In this study, the overall isolation rate of E. coli in calves was 51.8% and signifcantly difered by age, time of frst colostrum feeding, amount of milk given per time and navel treatment during birth. Furthermore, pathogenic E. coli strains were detected molecularly and antimicrobial susceptibility patterns of the isolates were also investigated.

Overall isolation of Escherichia coli in calf diarrhea. Te overall isolation rate of E. coli in diarrheic calves was 51.8%. Tis isolation rate was in agreement with the fndings of Y­ akob21 in Arsi Zone (50.9%), Ghada et al.24 in Egypt (50%) and Hossain et al.25 in Bngladish (49%) from diarrheic calves. Te result obtained was lower than the fndings of ­Dawit26 in Addis Ababa and Debre Zeit (64%), ­Yimer22 in North Shewa (69.5%) and Sunday et al.27 in Nigeria (63.2%). In contrast, this fnding was higher than the reports of Abdisa and M­ inda28

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in Holeta (12.5%), Gebregiorgis and ­Tessema20 in Kombolcha (36.8%) and Bekele et al.19 in Hawasa (37%). Te variation in isolation rates could be due to diference in sample size, age of the calves, geography, management of the farms and the isolation methods used. In a study that looked at equal 50 dairy farms in Ohio and Norway, E. coli was not found in any of the Norwegian farms but was found in 4 of the Ohio farms indicated that its preva- lence may be afected by geography and management ­variations6.

Risk factors associated with calf diarrhea. Te occurrence of E. coli causing calf diarrhea was associated with many risk factors that were found to be signifcantly associated but some were not. E. coli in diarrheic calves was isolated from all age groups examined, but the odds of being infected was high among age category of 1–2 weeks. Te association between the age level and diarrhea was being curvilinear as observed e­ lsewhere20,22,29. Tis could be related to the beginning of stress due to environmental exposure and infection pressure when the immune system of the calves is still de­ veloping30. Neonatal calves under 1 week of age are particularly susceptible because of incomplete establishment of the normal fora of the intestine, the presence of naive immune system and access of receptors for the adhesions of E. coli on the frst week of life of the calves. Te isolation rates of the bacterium in the subsequent age groups were shown to decrease as supported by the li­ terature31. Calving management and care of the newborn are important for the calves’ health. In the present study, the occurrence of E. coli in diarrheic calves due to the time of frst colostrum feeding was considered and signifcantly difered (P < 0.05). Te isolation rate of E. coli isolates was signifcantly higher in calves that fed frst colostrum in more than 6 h than those fed before 6 h early. Tis is in accordance with study performed in North Showa in which the isolation rate of E. coli was 100% out of 23 diarrheic calves that fed frst colostrum in more than 6 ­h22. Klein et al.32 in Austria also indicated that the importance of early colostrum management concerning the diar- rheal infection in neonates. Matte et al.33 found that 61% of colostral immunoglobulin containing 80 mg/ml of IgG is absorbed in 6 h and decreases sharply thereafer. By 24 h, the gut can absorb only 11% of what it originally could have absorbed at birth and digestive enzymes break down and digest all of the a­ ntibodies34. Tis indicates that the frst 6 h are the period in which maximum absorption of colostral immunoglobulin takes place. For adequate passive transfer of immunoglobulin, in addition to time, quantity and quality of the immunoglobulin fed to the calf plays an important r­ ole32. Te occurrence of E. coli in diarrheic calves was also signifcantly associated with the amount of milk given to the calves per time (P < 0.05). Tus, diarrheic calves that fed less (1–1.5 litter) milk per time were highly infected with E. coli than those fed more milk. Te fnding is in line with the idea feeding sufcient amounts of milk as per the calf body weight has more beneft on health, growth and performance later in life than reduced milk f­eeding35. Furthermore, the isolation rate of the bacterium wasARTICLE signifcantly higher in calves which did not get navel treatment during birth. Tis could be as the contaminated umbilicus provides the bacterium with an easy route of entry to the neonatal calf’s bloodstream and body. When a calf has septicemia due to E. coli, as the bacterium or its toxins presented in blood, the infection disseminates and damages many diferent organs and develops diseases including diarrhea as documented by ­Walter36. However, in the multivariable logistic regression analy- sis, the association of navel treatment was not signifcant (P > 0.05). In the present study sex, breed and calving facility of calves were not signifcantly associated with the occurrence of E. coli in calf diarrhea which is similar with the fnding of ­Yimer22 and Klein et al.32.

Detection of virulence genes of Escherichia coli. Detailed studies of the virulence factors produced by E. coli strains in farm animals are needed. In this study, 30 (53.5%) pathogenic E. coli strains out of 56 isolates in diarrheic calves were positive for at least one of the virulence factors involved and signifcantly associated (P < 0.05). Eleven (19.6%), 6 (10.7%) and 13 (23.2%) positive strains for eaeA, stx1 and stx2 genes, respectively were found positive. Te occurrence of E. coli eaeA virulent genes in this study was signifcantly associated with E. coli infection in calf diarrhea. Te result obtained is comparable with the fndings of Hur et al.37 in Korea (17%) and Andrade et al.38 in Brazil (24.3%) eaeA gene. Reports of Tan et al.39 in Vietnam (9.8%) and Islam et al.29 in Bangladish (12.5%) showed lower isolation rate of the same gene isolated from diarrheic calves. In contrary, higher prevalence of eaeA gene was reported by Dastmalchi and ­Ayremlou40 in Iran (26.9%) and 100% eaeA gene prevalence in I­raq41 of the same gene from diarrheic calves. Shiga toxin producing E. coli strains from diarrheic calves isolates were 33.9% (10.7% of stx1 gene and 23.2% of stx2 gene). Te presence of stx1 gene (10.7%) found in this study was signifcantly associated with calf diarrhea due to E. coli. Te occurrence of stx1 gene coincided with the report of ­Tahamtan42 (2010) in Iran (10.3%) calf isolates. Te current fnding was diferent from lower report of Askari et al.43 in Iran (5%); and higher reports of Yahya et al.44 in Turkey (42.8%), Tan et al.39 in Vietnam (46%) and Andrade et al.38 in Brazil (82.8%). Te present fnding on stx2 gene (23.2%) was also signifcantly associated with calf diarrhea due to E. coli in calves. Te result was in agreement with the report of Orden et al.45 for stx2 gene in Iran (20.9%) diarrheic caves; however, it was higher than that of Andrade et al.38 report from Brazil (4%). In contrast, it is lower than the reports of Yahya et al.44 from Turkey (71.4%) and Tan et al.39 from Vietnam (73%). RETRACTEDIn this study, higher frequency of stx2 (23.2%) genes than stx1 gene (10.7%) was observed which is in agree- ment with Dastmalchi and ­Ayremlou40 who reported stx2 (30%) and stx1 (10%) in diarrheic calves in Iran. How- ever, it is contrary to the observations of Srivani et al.46 who reported predominance of stx1 (16.04%) over stx2 (12.64%) in diarrheic calves in India. Te relative occurrence of STEC virulence factors changed as calves aged with stx1 positive isolates replaced by stx2 positive ­isolates47. Te diferences of the virulent genes were likely due to geographical as per LeJeune et al.6, season of sample taken as Fernandez et al.48, sample size or number of experimented isolates, age of samples and the calves and diferences in detection ­methods47. High rates of pathogenic E. coli strain colonization found in cattle in many countries indicate that cattle are important reservoir

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of diferent E. coli ­pathotypes49. Te current study suggests that calves could serve as important reservoir for pathogenic E. coli strains or human infection in Ethiopia. Te virulence factors found from the present study indicated that diarrhea in calves due to E. coli could be ascribed mainly to Enteropathogenic E. coli (EPEC) consisted eaeA genes and Enterohaemorrhagic E. coli (EHEC) of shiga toxin producing E. coli pathotypes which was supported by Alikhani et al.50. Te present investigation of the virulent genes was restricted to individual genes due to resource limitation. However, E. coli strains in others previously done studies harbored the eaeA, stx1 and stx2 genes were found alone as well as in combination­ 51. Some groups and strains of E. coli could share similar virulence traits and there are many over- laps in the mechanisms of pathogenesis for various pathotypes. Te virulence genes carried by these pathogenic E. coli groups are contained within mobile genetic elements and can be transferred between strains to create “emerging” strains as documented by Lucia et al.52. Te relatively high occurrence of the stx2 gene comparing to stx1 gene in this study suggests that E. coli carrying a combination of the eaeA and stx2 genes is more common than the combination of eaeA and stx1 genes. Te virulence factors eaeA, stx1 and stx2 genes were indicated as important diarrhea causative pathogens of the neonates and other extra enteric severe human illnesses like haemolytic uraemic s­ yndrome53. However, the present study could not screen all the remained virulence genes of the pathotypes and serotype characterization.

Antimicrobial resistance profles of Escherichia coli isolates. Antimicrobial resistance levels have markedly increased over the years that could be due to indiscriminate and widespread uses of antimicrobials both in the veterinary and public health ­practices54. Te highest sensitivity for ciprofoxacillin and norfoxacillin were recorded in 98.2% of isolates from diarrheic calves. Tis fnding was consistence with high susceptibility fnding of E. coli isolates to norfoxacillin (98%) as Ewa et al.55. reported in Poland, and ciprofoxacillin (98.8%) and norfoxacillin (100%) found in ­Bangladish56. Te high sensitivity to the mentioned drugs might be attrib- uted to recent development of the drugs and their seldom use in treatment of enteric ­infections57,58. Except ciprofoxacillin, all isolates were resistant to at least a single antimicrobial agent. Neomycin (76.8%) was the antimicrobial that presented the higher frequency of resistance among E. coli isolates, followed by amoxacillin (48.2%). Resistance to neomycin in calves isolates was in agreement with Ewa et al.55 who reported 76% resistance in Poland dairy farms. Te present neomycin resistance level was higher than the report of Rigobelo et al.59 in Brazil (26%). Te resistance level of the calves isolates to amoxacillin in this study was higher than the resistance repots of ­Yimer22 who noted 39.7% resistant isolates from diarrheic calves. Contrarily, higher resistance (100%) of E. coli isolates to amoxacillin was found by Taye et al.60 in Haromaya and Ewa et al.55 in Netherland dairy farms. Resistance to sulfonamide (39.3%) in calves isolates, wasARTICLE one of the most common resistance profles identifed among the current isolates. A report in USA indicated that sulfonamides (93.1%) was resisted by E. coli isolates and the resistance genes were commonly associated with mobile genetic e­ lements61. Tat report also documented that sulfonamides resistant E. coli isolates were also co-transferred to tetracycline and streptomycin. Moderate rate of resistance to oxytetracycline (26.8%) in calves isolates were obtained in this study. Te fnd- ing was closer to the 33.3% report of Taye et al.60 in Haromaya of the same isolates. Although 100% sensitivity of tetracycline was reported by Hossain et al.25 in Bangaladish, the resistance of the isolates to tetracycline was found higher elsewhere as ­Yimer22 found 74% in North Shewa, 61.4% in Bangladish by Islam et al.29 and 100% resistance in Indian report of Malik et al.12 from diarrheic calves. Escherichia coli isolated from calves also showed moderate 8.9% resistance level to streptomycin. In contrast, Yimer­ 22 found that the resistance of streptomycin to E. coli isolates was 74% in North Shewa and 29.7% resist- ance was recorded by Tadesse et al.61 in USA calves isolates. Te variation of the resistance level could be due to methods of susceptibility used with diferent break points and frequency of use of the drug in the study areas. Although tetracycline and streptomycin had moderate resistance in this study, they are routine chemoprophylaxis used among livestock in Ethiopia. Tey are readily available in diferent dosage forms and in combination with other antibiotics and vitamins. Te resistance of E. coli isolates to the relatively cheaper and commonly available antimicrobials is disturbing as the resistance causes more expensive therapies and longer duration of ­sickness62. Tis study showed the presence of multidrug resistant E. coli in diarrheic calves. A considerably higher proportion of the isolates 38 (68%) out of 56 were resistant to two or more of the antimicrobials. Twenty three multi drug resistance profles were observed. Multi drug resistance was considered when an isolate is resistant simultaneously to two or more ­drugs63. Te number of isolates resistant to two drugs was higher followed by four drugs resistant isolates. In general, as the number of drugs got higher the number of resistant isolates decreased that indicates combined use of antimicrobials may be useful for efective treatment. Diferent multidrug resist- ance profle for which E. coli isolates were resistant in this study were reported earlier from ­Ethiopia22,64, in other parts of A­ frican58,65 and European ­countries38,42 from diferent food animals, food products and humans. Te increasing development of multidrug resistant bacteria is signaling a serious alarm from treatment point of view or the possible transfer of resistance genes to other related ­pathogens66. In the present study, isolates were also assessed for the presence of resistance among the virulent genes. RETRACTEDTe highest resistance in isolates from calves was recorded for neomycin 25 (83.3%) followed by amoxicillin 16 (53.3%). Isolates possessing the virulence factors eaeA gene 9(81.8%), stx1 gene 4 (66.6%) and stx2 gene 12 (92.3%) encoded isolates showed highest resistant for neomycine. Resistance to norfoxacillin 1 (3.3%) was the least in isolates carrying all the three virulence genes; while chloramphenicol, ciprofoxacillin and gentamycin resistance was not shown in calves isolates possessing all the three virulent genes. Neomycin resistance of the strains of E. coli virulence genes in this study approached to the 100% neomycine resistance E. coli fnding of Anshu et al.67 in Indian isolated from animal and human hosts. Contrarily, this resist- ance fnding is higher than the 39% neomycin resistant stx encoded E. coli strains isolated from cattle and human in ­Germany68. Te variation could be due to the method of sensitivity test used and the frequency of the drugs

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used in the areas as well as diference in involved molecular mechanisms and transfer of antibiotic resistance genes among isolates from diferent l­ocalities69. In general, the present study indicated that resistance of stx2 positive E. coli predominated over the resistance profle of stx1 and eaeA positive E. coli from the diarrheic calves isolates as supported by previous ­report70. High frequency of antimicrobial resistance among pathogenic E. coli strains isolated from calves was observed. Tis could indicate drug resistance E. coli isolates from farm animals as potential reservoirs for resistance genes in human as supported by Call et al.71. Antimicrobial resistance profle of non specifed E. coli isolates which were not encoded with the virulence factor genes involved were also considered and considerable resistance isolates were found. Commensally existing E. coli could be a potential reservoir for resistance genes in farm animals as the resistance genes could be transferred between bacteria, environments and food p­ roducts72. Resistance development also related to exchange of resistance factors between related bacteria that could disseminate mul- tiple antimicrobial resistance genes in E. coli isolates­ 73. Terefore, identifying these commensally reservoirs and mechanisms of persistence could be a key to reducing the load of resistant pathogenic strains. Conclusion and recommendations Te study was the frst conducted to determine pathogenic E. coli strains and their antibiogram, as well as asso- ciated risk factors from diarrheic calves in Jimma town. Te isolation rate of E. coli from diarrheic calves was found to be high and signifcant proportion of pathogenic E. coli strains were identifed. Calf and managemental factors were found to be independent predictors for occurrence of E. coli associated diarrhea in calves. Higher rate of stx2 positive E. coli strains were detected than stx1 and eaeA genes. Considerable rate of mono and multidrug resistance to commonly prescribed antimicrobials was observed in calves isolates. Te overall high isolation rate of E. coli from calves indicates that the infection is widely distributed in the area. Calf diarrhea can cause economic losses in livestock production in the area. Diarrheic calves could be also potential reservoirs of pathogenic strains and resistant genes carrying E. coli strains to humans. Tis study has limitations as all the virulence genes and serotypes were not described because of time and resource limitation. As a recommendation, attention should be paid on proper dairy farm management practices and care of the newborns to protect calf health. eaeA of EPEC and STEC of EHEC pathotypes are prevalent in diarrheic calves and could be considered in designing control and preventive measures. Proper antibiotic prescription in veterinary and human practices and continuous monitoring of the resistance patterns in bacterial pathogens, in general, and in E. coli, in par- ticular, is mandatory to guide appropriate antimicrobial therapy. Further studies are needed to describe all the virulence genes and serotypes of pathogenic E. coli strains in calves that favor the emergence of drug resistant isolates and developing preventive measure. ARTICLE Methods Description of the study area. Te study was conducted between November, 2016 and April, 2017 on dairy farms found in Jimma town, Oromia Regional State, Ethiopia. Jimma town covers 19, 305.5 ­km2, which is the capital of Jimma zone and is 352 km Southwest of Addis Ababa, capital city of Ethiopia. Te zone bor- dered in Northwest by Illubabor, in East by Shewa Zone, in West by Wellega, and in South by Southern Nations and Nationalities People’s Regional State. In general, the zone lies at 7° 40′ 43″ N latitude and of 36° 50′ 18″ E longitude. Te elevation of the town is 1704 m above sea level while in the zone the altitude varies from 1000 to 3360 m above sea level with maximum and minimum temperatures in range of 25–30 °C and 7–12 °C, respectively. Te area has chromic nitosol, combisol and fuvisol soil ­types74 having sub-humid tropical climate with average annual rainfall ranging from 1200 to 2800 mm. Approximately, 70% of the total annual rainfall is received during the main rainy season, which lasts from June to S­ eptember75. According to the 2007 Population and Housing Census of Ethiopia, the total population of the Jimma zone was 2,642,114, from these Jimma town populations accounts 177,900, with 49.7% and 49.3% females and males, respectively­ 76 Among this, 2,204,225 (88.66%) is the rural population which directly depends on agricultural activities for domestic use and exchange of commodities with the urban residents. Te predominant economic activities involve mixed farming, which broadly includes cultivation of cereal crops, cash crops including primar- ily cofee and production of livestock. According to the Central Statistical Agency Agricultural Sample Survey 2011 of Ethiopia, Jimma zone has an estimated 2,317,678 heads of cattle, 824,485 sheep, 310,642 goats, 97,716 horses, 72,667 donkeys, 23,638 mules, 1,804,739 poultry and 479,703 beehives p­ opulation77. Te present study was conducted on dairy farms found in Jimma town. Te map of the town is shown in Fig. 2.

Study subjects. Animals that included in this study were local and cross breeds of dairy calves of both sexes up to 4 months of age that were clinically afected with diarrhea and exhibited signs of systemic disease (poor appetite, fever, sunken eye, dehydration, reduced suckle refex, and defecate pasty watery feces). In the study area there are few relatively large dairy farms and many market oriented smallholder dairy farms. During the RETRACTEDstudy, 54 out of the total 74 dairy farms, 3 institutional and 71 composed of large and smallholder dairy farms registered at Jimma town Bureau of Urban Agriculture Development were included. Te majority of market oriented small scale dairy farms were organized under dairy cooperatives in their respective localities and the farms kept local and crosses of Holstein breed of calves. Ages of diarrheic calves were categorized into three groups: 1–2 weeks, 3–8 weeks and 9–16 weeks of age based on post-natal silent stress response coupled with lack of immunocompetence, pre-weaning and post weaning strategies in which calves are ofen susceptible to enteric disease 78,79.

Inclusion and exclusion criteria. Diarrheic calves aged less than or equal to 4 months at dairy farms in Jimma town and owners willing to provide sample from their claves were included as the population of this

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Figure 2. Map of the study area. (Generated by Destaw A. Ali using QGIS 2.18 sofware. https://​qgis.​org/​en/​ site/​forusers/​ ​visualchan​ ​gelog​218/​index.​html.

study. Whereas, calves aged above 4 months, that were on antibiotic therapy for 2 weeks and those whose owners did not agree to allow samples taken were excluded from this study.

Study design and sampling methodology. Study type, type of sampling and source of samples. Cross sectional study type was conducted in dairy farms. SelectionARTICLE of farms were done purposively based on the avail- ability of clinical case (diarrheic calves) in the farms and based on willingness of the owners of the calves. Te health status of each calf was evaluated by clinical examination. Calves free from diarrhea were classifed as healthy whereas sick calves that show abnormal stool consistency and/or signs of dehydration, sunken eye, di- arrhea and weakness were classifed as diarrheic. Description of fecal amount, type, consistency, color, smell and mixtures (presence of blood or particles of undigested food, blood clots or pieces of intestinal tissue) were recorded. In addition, farm management practices were assessed. Te pre-tested and structured questionnaire surveys were distributed and collected on owners during the time of sample collection in the study period to assess relevant information of the farm management system. Te questionnaires were developed in accordance with the objectives of the study and designed in a simple manner to get accurate information from the dairy farm owners.

Sample size determination. Non-probability purposive sampling was used for the selection of farms as well for calf sample. Te sample size was determined based on availability of clinical case (diarrheic calves) and on will- ingness of the owners in the farms. Based on that 112 diarrheic calves were included in the study.

Sample collection procedure. Fecal samples were collected directly from rectum of non treated diarrheic calves preferably soon afer onset of diarrhea. Te samples were collected from all diarrheic calves aged from newborn to 4 months of age present on the farm at the time of the visit and on emergency calls from the farm owners. Sufcient amount (25–50 g) of fecal samples were collected directly from the rectum using gloved hands and transferred to 50 ml sterile wide mouth screw caped universal bottles. Sterile swabs were used to scratch inside the rectum of the calves whenever there were insufcient amount of feces is obtained from diarrheic calves. Te bottles were clearly labeled with information on date of sampling, age, sex and breed of the calves. Te samples were transported under cold conditions in ice box to Microbiology Laboratory of School of Veterinary Medicine, College of Agriculture and Veterinary Medicine of Jimma University.

Study methodology. Cultural procedures for isolation of Escherichia coli. Isolation and identifcation of RETRACTEDE. coli were conducted following standard procedures described in Quinn et al.80 and the technique recom- mended by the International Organization for Standardization ISO-1665481. Upon arrival at the laboratory, im- mediately or afer overnight storage in refrigerator at 4 °C and thawing at room temperature, the samples were manually homogenized by using vortex mixer for approximately 30 s. Twenty fve gram of fecal sample was stirred in to 225 ml of sterile bufered peptone water (Himedia, India), or at 1:9 ratio whenever there is little amount of sample present, in a sterile fask. Te pre-enriched samples were homogenized for two minutes in the fask and were incubated aerobically at 37 °C for 24 h. All the media used for the study were prepared following the instructions of the manufacturers.

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PCR condition (35 cycles) Nucleotide sequence Target Gene Denaturing Annealing Extension Product size (bp) References 5′-AAACAG​ ​GTG​AAA​ EAEF CTG​TTG​CC-3′ eaeA 95 °C, 60 s 55 °C,60 s 72 °C,60 s 450 86 5′-CTC​TGC​AGATTA​ ​ACC​ EAER TCT​GC-3′ 5′-ATC​AGT​CGT​CAC​TCA​ EVSF CTG​GT-3′ stx1 95 °C, 60 s 55 °C,60 s 72 °C,60 s 110 87 5′-CTG​CTG​TCA​CAG​TGA​ EVCR CAA​A-3′ 5′-CAA​CAC​TGG​ATG​ATC​ EVTF TCA​G-3′ stx2 95 °C, 60 s 55 °C,60 s 72 °C,60 s 350 87 5′-CCC​CCT​CAA​CTG​CTA​ EVTR ATA​-3′

Table 7. Primer gene sequence and PCR conditions. F Forward, R Reverse, eae efacing and attaching, ev Verocytotoxin, stx shiga toxin, bp base pair.

Pre-enriched broth of 0.1 ml (a loop full) sample dilution was inoculated aseptically onto sterile MacConkey agar (Himedia, India) and incubated at 37 °C overnight. From each plate isolates of lactose fermenting colonies were inoculated on Eosin Methylene Blue (EMB) agar medium (Himedia, India). Preliminary characterization and colonies showing characteristic metallic sheen on EMB agar were then picked up and considered as presump- tive E. coli. Te isolated characteristic colonies of E. coli were stored in nutrient broth for further identifcation by biochemical tests and other studies. All the isolates were stained by Gram stain to determine the cell morphology and purity of the ­isolates82.

Biochemical characterization for identifcation of Escherichia coli. Escherichia coli isolates were identifed preliminarily by using indole, methyl red, Voges Proskauer and citrate utilization (IMViC) biochemi- cal tests. In these tests, indole production from tryptophan (indole test), production of strong acid causing red color in methyl red indicator (methyl red test), productionARTICLE of acetoine (Voges Proskauer test) and use of citrate as the only carbon source (citrate test) were conducted. Te isolates which exhibited IMViC pattern (++−−) were presumed as E. coli isolates. Ten afer, the presumed isolates that kept cold in ice packed box were transported to Institute of Biotechnology, Addis Ababa University for PCR based detection of the possible pathogenic E. coli pathotypes and to observe their antimicrobial resistance profle.

Polymerase chain reaction. DNA extraction. Escherichia coli isolates were grown in nutrient broth at 37 °C overnight. exactly 1.5 ml of the culture was pipetted and spun by centrifugation at 13,000 rpm for 10 min in autoclaved eppendorf tube. Te bacterial pellet was lysed by boiling in 50 µl of nucleus free water in a water bath at 95 °C for ten minutes. Te lysate was centrifuged again as before and an aliquot of supernatant was trans- ferred to another autoclaved eppendorf tube and then 3 µl of the extracted DNA was used directly as template for PCR ­amplifcation83. Afer extraction of the target DNA, E. coli isolates were subjected to PCR for the presence of virulent genes. According to the component optimization of the compatible enzyme used, three types of PCR assays were performed. Te frst two PCR assay were carried out independently to detect the presence of eaeA and stx1 genes in a 22 µl master mix reaction volume. Te mix contained 12 µl of nucleus free water, 1 µl of 0.5 µmol of each primer ( EAEF, EAER and EVSF, EVCR), 2.5 µl of PCR bufer with 2 µl of 1.5 mmol Mg­ Cl2, 2 µl of solution S, 1 µl of 0.35 mmol of dNTPs (dATP, dCTP, dGTP, dTTP), 0.5 µl of 1U Taq polymerase enzyme (Solis Biodine), and 3 µl of template DNA. Te third PCR assay was performed similarly in a 22 µl master mix reaction volume to detect stx2 gene with a diferent source of Taq polymerase enzyme, the PCR bufer and MgC­ l2 used (Himedia, India). Te reaction volume contained 14 µl of nucleus free water, 1 µl of 0.5 µmol of each primer (EVTF, EVTR), 2.5 µl of PCR bufer with 2 µl of 1.5 mmol MgC­ l2, 1 µl of 0.35 mmol of each dNTPs (dATP, dCTP, dGTP, dTTP), 0.5 µl of 1U Taq polymerase enzyme (Himedia, India), and 3 µl of template DNA. Te reaction mixtures of both assays were amplifed with 35 cycles, each consisting of 3 min initial denatura- tion at 95 °C, 60 s denaturation at 95 °C, 60 s annealinig at 55 °C and 60 s elongation at 72 °C in thermal cycler (TC-412; Version 34.11)84. For all the PCR reactions, additional extension step of 10 min at 72 °C were performed. RETRACTEDNegative control (PCR grade water in place of the template) and known pooled positive E. coli genes as positive control for each primer involved were also placed along with the samples.

Detection of PCR products / Agarose gel electrophoresis. Amplifed PCR products (expected 110-450 bp) were analyzed by gel electrophoresis at 120 V for 45 min in 2% agarose (Conda, cat.8010.11) made in 1 × tris acetate bufer (EDTA) containing ethidium bromide (0.5 µg ­ml−1) using a marker DNA ladder of 100 ­bp85. Te prod- ucts were visualized on ultraviolet illuminator and imaged with gel documentation system (BIO-RAD). Details of primer gene sequences and the diferent reaction temperatures that were carried out in the PCR assays are indicated in Table 7.

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Antimicrobial susceptibility testing. Te antimicrobial susceptibility testing of the E. coli isolates were performed using panel of commonly used antimicrobials using Kirby–Bauer disk difusion test according to the Clinical and Laboratory Standard Institute guideline (CLSI) (M100-S25)88. From each isolate, four to fve well isolated colonies recovered and grown on EMB agar were aseptically transferred into test tubes containing 5 ml of nutrient broth. Te broth cultures were incubated at 37 °C for 24 h and the turbidity of the broth suspension were adjusted by normal 0.9% saline solution with visual comparison of 0.5 McFarland turbidity standards (Remel, USA). Sterile cotton swab was dipped into the suspension, rotated several times, pressing frmly on the inside wall of the tube above the fuid level to remove excess inoculums and swabbed uniformly over the surface of Muller Hinton agar plate. Te plates were held at room temperature for 10 min to allow drying. Ten eleven antibiotic discs with known concentration of antimicrobials were placed on the cultured Muller Hinton agar plate of appropriate distance of each disc with famed forceps, inverted and incubated with agar side up for 16–18 h at 37 °C. Following incubation, the diameters of zone of inhibitions were measured with a ruler to the nearest millimeter. Interpretation of the results depended on categorization of isolates into susceptible, interme- diate or resistant according to CLSI guide­ lines36.

Questionnaire survey. Informed consent was obtained from the owners and the permission was granted by the owners to perform experiment on their calves. Pre-tested structured questionnaire was administered to dairy farm owners or farm managers during the time of sample collection in the study period to assess relevant information on calf husbandry practices and the general farm management system. Te questionnaires were developed in accordance with the objectives of the study and designed in a simple manner to get accurate infor- mation from the dairy farm owners. Te questionnaires included practices in the farm which can have impact on the proper rearing of calves associated with risk factors responsible for calf diarrhea. Tese include age of calf, farm size, colostrum feeding, general health care, animal housing, hygiene and sanitation of farms, occurrence of calf diarrhea, disease preventive and control measures practiced in the farms.

Data management and statistical analysis. All the data obtained from questionnaires and labora- tory describing the conditions of the study suggestive of E. coli infection on calves along with the risk factors were fltered, coded and entered in to Microsof Excels pread sheet 2007. Te collected data were computed by using SPSS version 20.0 sofware (SPSS INC. Chicago, IL) for appropriate statistical analysis. Descriptive analy- sis was used to describe the study population in relation to risk factors. Te point prevalence was calculated as the number of infected individuals divided by the number of individuals sampled times 100. Te associations between occurrence of E. coli isolates and the risk factors,ARTICLE as well the presence of the diferent virulent genes detected by PCR and diarrheal infection due to positive E. coli were analyzed using person’s χ2 test. Variables with P value < 0.25, for controlling the possible efect of confounders, in univariable logistic regression were ft- ted into multivariable logistic regression model to observe the strength of the association between risk factors and the outcome. Te suitability of the model was checked by multicollinearity diagnosis among independent variables by contingency coefcient. Te goodness of ft of the model with the data was assessed by Hosmer and Lemeshow test. Afer selecting the fnal model of multivariable logistic regression, the beta (β) coefcients of each independent variable were observed to estimate odds ratio (OR) which is used for assessing strength of association. Efects were reported as statistically signifcant as P value was less than 0.05.

Ethics approval and consent to participate. Te incorporated work was ethically cleared from the Institutional Review Board (HIRPG/248/07) of Jimma University Institute of Health Sciences. All methods were carried out in accordance with relevant guidelines and regulations, on involving humans and animals in the study. Informed consent was obtained from the farm owners and the permission was granted by the owners to perform experiment on their calves. No fees were requested. Data availability Te data sets used during the current study are available from the corresponding author on reasonable request.

Received: 22 January 2021; Accepted: 22 June 2021

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Specifcally the AAU TR project entitled “Biotechnologi- cal Approaches to Harness Microbe-Host-Environment interactions for Sustainable Development of Health and Agricultural Productivity; Ref. No. VPRTT/LT-030/08/15”. We very grateful to the Institution and the

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Universities. All participants of the study such as farm owners, technical assistances and grant sources are duly acknowledged. Author contributions D.A.A. was prior responsible for collecting the samples, processing all the laboratory works and writing up the paper work and shaping of this manuscript to be submitted. Y.D.B was closely monitoring and guiding the work in addition to shaping the paper and managing part of the aforded costs. T.S.T. was chief responsible for guiding the entire work, managing all the resources and shaping of this manuscript to be submitted.

Competing of interests Te authors declare no competing interests. Additional information Supplementary Information Te online version contains supplementary material available at https://doi.​ ​org/​ 10.​1038/​s41598-​021-​93688-6. Correspondence and requests for materials should be addressed to D.A.A. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afliations. Open Access Tis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. Te images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creativeco​ mmons.​ ​org/​licenses/​ ​by/4.​0/. © Te Author(s) 2021 ARTICLE

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