Isolation, Identication and Antimicrobial Susceptibility Tests of E. Coli and Salmonella from Diarrheic Calves in and Around Sebeta Town

Calves are important assets for replacement of cows for the future dairy and beef herd sustainability. However, calf hood diseases have a signicant nancial impact on dairies resulting from treatment costs, genetic loss, and impaired future performance. The purpose of the present research is for isolation and identication of Salmonella and E. coli infection in diarrheic calves, assessing risk factors for occurrence of calf diarrhea and examining antimicrobial susceptibility of isolated bacteria’s. A cross- sectional study on the isolation, identication and antimicrobial susceptibility of E. coli and Salmonella was carried out on calves up to two months of age from November, 2018 to March, 2019 in and around Sebeta town, Ethiopia. The detection of E. coli and Salmonella was done by conventional bacteriological method and biolog (semiautomatic machine). The data was analyzed by Stata 12.


Introduction
Livestock is an integral part of the agricultural production system in some countries and plays an important role in national economy as well as in socio-economic development of millions of rural household. Ethiopia basically constitutes an agrarian society; the socio-economic activities of about 85% of the population are based on farming and animal husbandry. Livestock plays a critical role for the majority of the Ethiopian population. Domestic animals are mainly used as drought animals, source of milk, meat, hide and skin and as pack animals. Apart from this they also serve as a means of risk diversion and accumulation of wealth among the rural community [1].
Dairy production is a critical issue in Ethiopian livestock -based society where livestock and its products are important sources of food and income. The substantial demand-supply variance in milk and milk products for the major urban centers in Ethiopia is a great opportunity for the development and ourishing of peri-urban dairy farms. Peri-urban and urban dairies are intensive production systems, which keep high-grade cows and have improved management practices. This is usually associated with increased susceptibility to disease, poor survival rate and poor reproductive. The country has large potential for dairy development mainly due to large livestock population, the favorable climate for improved high yield breed, and relatively diseases-free environment [2] Newly born calves are important source of animal production for either meat or breeding worldwide. Calves are important assets for replacement of cows for the future dairy and beef herd sustainability. It is needed to rear healthy calves. In the calf, losses have been as high as 50% of calf crop because of poor management and low adaptation of exotic dairy breed to tropical environment [3]. Calf hood diseases have, therefore, a signi cant nancial impact on dairies resulting from treatment costs, genetic loss, and impaired future performance [4]. Diarrhea is a leading cause of economic losses to most countries [5]. Its etiology is complex involving management, environmental, nutritional, physiological variations and variety of pathogens including bacteria, viruses, protozoa and intestinal parasites are described as important agents causing diarrhea (either separately or in combination) in calf [6].
Calf diarrhea caused by bacterial infection has a bad effect on the dairy industry all over the world when calves are reared intensively. It involves signi cant economic loss for labor and capital, calf mortality, loss in calf value and veterinary costs [7; 8]. Among these bacteria E.coli and salmonella are believed to be the major microbial causes of calf diarrhea [9; 10]. According to Lorenz [11], calf morbidity and mortality by diarrhea have short-term and long-term detrimental effects on performance of a dairy farm.
They impair both growth rate and replacement capacity of the herd. The economic losses occur not only from mortality but also from treatment costs and time spent on care as well as subsequent chronic ill, thrift and impaired growth performance [12]. In order to increase the productivity per livestock unit without increasing livestock numbers and to devise preventive measures as well as to reduce losses during the initial months of life it is important to identify the etiological agents involved in calf diarrhea [13; 14]. Loading [MathJax]/jax/output/CommonHTML/jax.js Current treatment regimens for the treatment of neonatal calf diarrhea center on antimicrobial therapy and uid therapy. Antimicrobial agents are considered popular to ght diarrhea in calves. Nevertheless, their wide spectrum of activity, the emergence of microbial tolerance of different antimicrobial agents has become a well-known phenomenon, which represents a major concern. Resistance to antimicrobial agents was frequently occurred in Salmonella species and E. coli particularly in pre-weaned dairy calves [16]. Antibiotic resistance to bacteria is a serious and growing phenomenon and has emerged as one of the preeminent public Health concerns of this century. The choice of which antibiotic is likely to be most effective requires knowledge of potential resistance. The practice of under dosing, over dosing as well as indiscriminate usage of drugs are not uncommon in Ethiopia, As a result, bacterial strains are being developed which are multidrug resistant and new types of antibiotics are required for the prevention and control of diseases.
Discontinuation or incomplete course of treatment and continuous indiscriminate uses of antibacterial drugs against diarrheal infection of man and animal creates a potential health risk to animals and humans in terms of drug residues and the development of resistant bacterial strains. Although routine laboratory isolation and drug sensitivity testing are expensive and impractical, the periodical check of the pattern of the drug sensitivity of organisms is more important. It is, therefore, important that sensitivity of different bacteria isolated from diarrheic calves needs to be studied from time to time in order to formulate appropriate therapeutic measures [17]. In order to increase the productivity per livestock unit without increasing livestock numbers, it is important to identify the etiological and predisposing factors involved in calf diarrhea in order to devise preventive measures and reduce losses during the initial months of life [18; 13].
Dairy farming is a growing livestock production system in Ethiopia. It is primary source of income for urban and peri-urban poor communities. Because of better availability of milk market, most of the dairy farms are concentrated in urban and peri-urban areas of the country. They also increase the use of exotic dairy cattle and their crosses in order to enhance milk production. In Ethiopia in general calf morbidity and mortality due to calf diarrhea is a challenging problem for dairy producers. However, there is no coordinated surveillance and assessment for prevalence and risk factors inducing this problem and there is also little information available regarding to this problem. Accordingly, the objectives of this research were to isolate and identify of salmonella and E. coli infection in diarrheic calves, to assess risk factors for occurrence of calf diarrhea and to examine antimicrobial susceptibility of isolated bacteria's in and around Sebeta town.

Description of study area
The study was conducted on diarrheic calves between November 2018 to March 2019 in and around Sebeta town, located at suburb of Addis Ababa in Oromia special zone surrounding Fin nne of Oromia region. Sebeta is situated at latitude of 8º5440"N and longitudeof38º3717"E and an elevation 2356 Loading [MathJax]/jax/output/CommonHTML/jax.js meters (7730 feet) above sea level. The average temperature is 17.4ºC and the town receives annual rainfall of 1650 mm, the monthly precipitation is being 150 mm are mostly wet and below 30 mm mostly dry. The farming system of the study area is intensive, semi-intensive and extensive. However this study was conducted on the intensive farming system.

Study population
Study animals were local and crossbred calves managed under intensive management system in and around Sebeta town. The farms and house hold where were visited during the study period to enable us to observe the health status of the calves. During each visit the calf's management, housing and sanitation situations were also observed. The sick animals were treated after the clinical investigation and after the necessary samples were collected. Age, sex, body condition, manure removal, breed of each animal were recorded during sample collection.

Study design
A cross sectional study was conducted in dairy farms (small, medium sized and large) in and around Sebeta from November 2018 to march 2019 and a purposive sampling methods used to collect samples from calves that showed major clinical sign of diarrhea (elevated temperature, depression, dehydration, reduced suckling re ex, rough hair coat, loss of weight, weakness and soiling of hind quarter and tail with diarrheic feces [19].

Fecal sample collection, storage and transportation
From November 2018 to march 2019 a total of 129 diarrheic samples were collected purposively from the rectum of the calves less than two month old with surgical gloves and placed in sterile dry screw-cup universal bottles and transported to the Microbiology Laboratory of NAHDIC in an ice box within 3-4 hrs of collection from farms and small holder which found in and around Sebeta town. The samples were collected from calves showed clinical sign of diarrhea, but had not been treated with antibiotics before. At the time of sampling the name of the farm, date of sampling, consistency of feces, age, breed, body condition, tag number; and feeding management practices as well as past history of diarrhea were recorded for each calf on a recording format. A calf was considered as diarrheic if feces were semi-uid (loose) to uid with or without mucus and/or blood. In the laboratory, isolation and identi cation of Salmonella and E. coli were conducted using conventional methods as described by [40].

Isolation and identi cation Salmonella
The fecal samples for isolation of salmonella were done brie y as follows: 1 g of feces was pre-enriched in 9 ml of buffered peptone water (BPW) and incubated overnight at 37 °C. From the overnight culture 100 µl pre-enriched suspension was added into 10 ml of Rappaport-Vassiliadis enrichment Broth (RVS) (Oxoid, USA) and incubated at 42 °C for 24 hr. cultures showing characteristic growth on RVS broth was streaked on to Xylose Lysine deoxychlorate (XLD) (Oxoid, USA) selective media and the plates were incubated at 37 °C for 24 to 48 h. Then the colony was examined for the presence of typical salmonella Loading [MathJax]/jax/output/CommonHTML/jax.js colonies. Super imposed on the red colonies is the production of H2S so red colonies is with a black center in Xylose lysine deoxychlorate (XLD) medium can likely be salmonella, which was subjected to successive biochemical tests after cultured pure colonies on nutrient agar. After suspected colonies were selected and cultured on nutrient agar media, biochemical tests: TSI test, citrate utilization test, Urease test, Indole test, methyl red test and Vogues Proskauer test were conducted. For con rmation and for sub species identi cation of isolated bacteria, Biolog (semiautomatic machine) was utilized.

Isolation and identi cation of E. coli
To detect E. coli, samples were inoculated on MacConkey's agar and Eosin Methylene Blue (EMB) agar used for the purpose of observing growth of E. coli and incubated at 37ºC overnight. The presence of growth on MacConkey's agar was used as primary criteria to proceed for isolation and identi cation of E. coli. Furthermore the colony characteristics observed on MacConkey's agar was used to classify suspected bacteria isolated into two groups: lactose fermented and non-lactose fermented. Suspected E. coli colonies were presumptively identi ed their lactose fermenting character (pink colonies), were con rmed as E. coli according to [20; 21; 22]. Lactose positive colonies sub cultured on Eosin methyl blue (EMB) agar medium to identify selectively E. coli. Colonies producing metallic sheen or blue-black to brown color on EMB agar were considered as having E. coli. In addition, various biochemical tests were done for the con rmation of E. coli [22]. All isolated colonies were sub-cultured on nutrient agar to obtain pure colony for further biochemical tests:-TSI test, citrate utilization test, Urease test, Indole test, methyl red test and Vogues Proskauer test were conducted. For con rmation, Biolog (semiautomatic machine) 65 con rmed E. coli isolate from each positive sample was stored at Brain heart infusion slant agar until drug susceptibility testing.

Antimicrobial susceptibility test
The antimicrobial susceptibility test was performed following the standard agar disk diffusion method according to [41] using commercial antimicrobial disks. The selection criteria of the antibiotics depended on the regular use of the antimicrobials in the ruminants, potential public health importance and recommended from the guideline of antimicrobial susceptibility testing. Pure colonies incubated for 6 hours in Tryptone Soya Broth (Oxid, England) will be made to have a turbidity of 0.5 macFarland standards and inoculated on already prepared Muller-Hinton agar (Bacton Dickinson company and Cockeysville USA). Antibiotic discs were then placed and incubated for 24 hours at 37oC. The results will be classi ed as resistance, intermediate and susceptible after the zone of inhibition of bacterial growth is appreciated, hence a standardized table is given by the manufacturer for this classi cation [41] The following disks were used: Gentamicin (10 µg), Ampicillin (10 µg), Chloramphenicol (30 µg), sulphamethoxazole trimethoprim (30 µg), polymixin B and Tetracycline (30 µg).

Data management and analysis
The data generated from the study was arranged, coded and entered to Excel spread sheet (Microsoft® o ce excels, 2007). Prevalence of Salmonella and E.coli were calculated as a percentage of Salmonella Loading [MathJax]/jax/output/CommonHTML/jax.js and E.coli culture-positive samples among the total number of samples examined. Chi-square test was done by STATA version 12 to study association between Salmonella and E.coli isolates and risk factors. The signi cance level was set at 0.05 and 95% con dence level.

Result S And Discussions
Laboratory examination of 129 fecal samples from diarrheic calves during the study period was done to isolate and identify Salmonella and E. coli associated with calf diarrhea. The overall occurrence of E.coli and Salmonella from isolated samples were 65 (50.3%) samples revealed the presence of E. coli and 8 (6.2%) of isolates were identi ed as Salmonella, showed in Table 1. Table 1 The overall occurrence of E. coli and salmonella isolated from diarrheic calf in and around sebeta town. The culturally and biochemically identi ed Salmonella isolates were further tested for con rmation and sub species identi cation by using Biolog (semiautomatic machine). A total of 8 Salmonella isolates, consisting of two different subspecies were identi ed. Of the sub species identi ed during study were S. enterica subspecies enterica and S. enterica subspecies thyphimurium. S. enteric subspecies enterica constitutes 75% (6 isolates) and S. enterica subspecies thyphimurium constitutes 25% (2 isolates).
The distribution of E. coli and salmonella among calves of different age groups, sex, farm size and times of colostrum feeding was determined. In this regard, the isolation rates of E. coli decreased with increasing age of calves and when the times of colostrum feeding, the highest being in calves of < 2 week old and as the time of colostrum feeding exceed 6 hours. There was statistically signi cant difference between the age groups of E. coli and salmonella isolation rate. The difference in isolation rates of E. coli in different age groups and times of colostrum feeding were statistically signi cant (p < 0.05) which is showed in the following Tables 2,3

Antimicrobial susceptibility tests result
Antimicrobial study of the isolates indicated that out of 65 isolates of E. coli, all of the isolates were highly susceptible to sulphamethoxazole trimethprim, chloramphenicol, gentamicin and ampicillin. Most of the isolates were highly resistant to tetracycline and followed by polymixin B, Ampicillin and Chloramphenicol, 29.23% 6.1%, 1.54% and 1.54% respectively. None of the isolates showed resistance for sulphamethoxazole trimethprim and gentamicin. From 8 isolates of salmonella all showed 100% susceptibility for sulphamethoxazole trimethprim and polymixin B, 87% to Ampicillin, chloramphenicol and tetracycline as well as 75% to gentamicin. Only one isolates showed mono drug (tetracycline) resistance.

Discussion
The present study was made to isolate and identify salmonella and E. coli in diarrheic calves, to assess the association of different risk factors and also to detect the antimicrobial susceptibility of those Loading [MathJax]/jax/output/CommonHTML/jax.js isolated bacteria's. Accordingly, the detection of E. coli in this study was 50.3%, which is higher than the reports of [23; 24; 25; 29; 39 and 9] who has reported 44%, 43.1%, 23%, 25%, 49% and 38.6%; respectively from different countries and lower than [26; 27; 28; 31 and 30] who has reported 75%, 76%, 70.7%, 76%, and 60% from different localities. The detection of salmonella in this study was 6.2% which is lower than the ndings of [32] 13%, [33] 9.61% and [9] 21.9%; and higher than the ndings of [34]) 4%, [25] 5%. This prevalence difference may be due to the difference in sex, age and farm size of our study and others. As a result this study revealed that calf diarrhea was apparently higher in large sized dairy farms (61.24%) than medium (27.13%) and small dairy farms (13.18%) though it was not statistically signi cant (x2 = 2.15, P = 0.34).
Similarly, diarrhea was highly associated with age of calves (E. coli, x2 = 9.249, p = 0.024 and salmonella (x2 = 8.161, p = 0.043). Calves in age group of 2 to 4 weeks were least susceptible while that of age group 0-2 weeks are highly susceptible and 5-8weekswere more resistant. The result can be associated with many factors, at younger age; delay in rst colostrums feeding was associated with higher risk of being affected with diarrhea. The nding that delayed colostrums intake (latter than 6 hours of age) associated with high risk of being affected with diarrhea reach agreement with other reports found that each hour of delay in colostrums ingestion in the rst 12 hours of age increased the chance of a calf becoming ill by 10%. 61% of colostral immunoglobulin containing 80 mg/ml of 43 IgG is absorbed in six hours and decreases sharply thereafter [35].
This indicates that the rst six hours are the period in which maximum absorption of colostral immunoglobulin takes place. Therefore, delays in administration could lead to lack of colostrums originated from maternal antibodies to protect calves from enteric pathogens [36; 37].
Another risk factor supposed with the occurrence of diarrhea associated with E. coli and salmonella was feeding colostrum. It was signi cantly associated with the occurrence of diarrhea E. coli, (x2 = 7.510, p = 0.023) and salmonella( x2 = 6.678, p = 0.035). Sex of calves was also supposed to be risk factor associated with the prevalence of E. coli in diarrheic calves. As a result this study revealed that calf diarrhea was apparently higher in male calves (35/65) than females calves (30/65) (p = 0.001). This may due to the male calf in dairy farms were not as much as needed in the production system and the producer mainly weaned the female (heifer) one for replacements.
The result also shows that the calves with severe diarrhea had highest percentage positivity to bacterial scours. This a rms the virulent ability of the organism to destroy the mucosa of the intestinal lining which affects the absorption and secretion ability of the gastro intestinal system and result in profuse watery foul smelling diarrhea. Management system of the farm also played a role in bacterial scours.
Moreover, in this study diarrhea was highly associated with housing and E. coli as well as salmonella but it was not signi cant (p = 0.095). The intensive system of management had high percentage positivity to the disease while that with an extensive system of management had low percentage of positivity. This probably is due to free cycling of these bacteria between animals in a limited host environment once the Loading [MathJax]/jax/output/CommonHTML/jax.js pathogen gets access to the farm in animals kept indoors. The fact that the use of processed feed is more common in animals kept indoors than those kept outdoors might also suggest the possibility of indoor kept animals being infected with bacteria from contaminated animal feed. It can be inferred that calves reared in extensive management have low risk of infection and are more resistant while those reared in intensive are exposed to causes of infection and less resistant to infection. Reasons for this could be poor hygiene in intensive, feeding animals with contaminated concentrates, hay and forages animal to make choice of what to eat could also be a contributing factor [11]. The earlier study has also shown livestock waste generated by animals consuming a diet chie y composed of grass were less likely to harbor Salmonella spp. [38]. Therefore, satisfactory and essential attention should be given to management of calf by ensuring that the environment where calving takes place is disinfected properly from bacteria from previous calving, colostrums is taken by calf in the rst few hours of life, in case of di cultly in feeding, and calf should be hand fed with feeders. Overcrowding of calves and lack of proper ventilation of the pen is also a risk factor and should be prevented as much as possible. Calf kept in housed pens should be housed individually in clean pens. Farms practicing intensive system of farming should pay ultimate attention to hygiene since they are at greater risk of the infection. I acknowledged limiting ourselves to isolation of bacterial pathogens in calves diarrheic cases in the course of this study due to the facts that other etiological agents such as viral, parasitic, fungal and even mycotic agents are not as common as bacterial pathogens, also approach to management of this condition require the use of an appropriate and most sensitive antibacterial agent.
In this study the highly sensitivity of E. coli to chloramphenicol and Sulfamethoxazole reach agreement with the reports of [28] and [30] and Sherwood and Snodgrass (1983) who found that all the isolates were sensitivity to chloramphenicol. These ndings were in agreement with the results of Guerra et al. (2006) who stated that most of the microorganisms isolated from calves suffering from diarrhea were highly susceptible to chloramphenicol. The resistance percentage (29.23%) of tetracycline in this study was in contrary with reported by [39] and [29] reported more than 80% and Hossain et al. (2012), documented 100% sensitivity to tetracycline.

Conclusion And Recommendations
The present study con rms the calf diarrhea was mainly caused by E. coli and sometimes there is involvement of pathogenic Salmonella species by isolating and identi ying and of E. coli and Salmonella from diarrheic calves in in the study area. This has showed that calf diarrhea was found to be high and could affect dairy production; and the detection of higher E. coli (50.3%) and lower Salmonella (6.2%) from diarrheic calves may be an indication for their importance in calves as causative agent for diarrhea in the study area. For the higher detection of E. coli from diarrheic calves in the study area sex, age and time of feeding colostrums were found to be incredibly important risk factors. With regarding to antimicrobial susceptibility most isolates were resistant to atleast one commonly used antimicrobials. This seeks attention from veterinary practitioners in order not to use antimicrobials simply without de nitive diagnosis of the case. Although high percentage of E. coli were detected from diarrheic calves in and around Sebeta town, isolation of E. coli from feces may not be directly associated with causation of diarrhea. Since methods required for identifying pathogenic strains of E. coli are not established. Based on recent ndings, we are highly recommending improved calf management practice, early colostrum feeding, and rational usage of antimicrobials.

Declarations
Ethics approval and consent to participate Permission on ethical approval was obtained from the College of Veterinary Medicine Samara University. Fecal Samples were collected following prior explanation of the objectives of the study to the dairy farm owners and sampled based on their consent.

Consent for publication
Not applicable.

Availability of data and material
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.