Calf diarrhea remains the major health challenges in cattle herds and is the most common cause of morbidity and mortality in neonatal calves. A cross sectional study was conducted from November 2018 to October 2019 to determine the prevalence of E.coli infection in diarrheic calves, investigate potential risk factors of the infection and to determine the antimicrobial susceptibility pattern of E. coli isolates in dairy farms of Holota and its surrounding area, central Ethiopia. A total of 278 diarrheic calves less than three months of age were purposively selected for this study. Fecal samples collected directly from the rectum of calves were subjected to bacteriological culture and biochemical tests to isolate and identify E.coli. The overall prevalence of E. coli infection in diarrheic calves was found to be83.5% (232/278×100).The Odds of isolate rates of E.coli for age group 5-8 week was 5.67 times higher as compared to 9-12 week of age (95% CI of OR = 1.18–27.31,P= 0.031). The odds of E.coli isolate rates from diarrheic calves was lower in calves with mucoid diarrhea as compared to those with bloody diarrhea (OR=0.06; 95% CI, 0.01–0.54; P=0.012). The isolate rates of E.coli was 14.5 times higher in calves that received colostrum within6-12 hour than calves that received at their early life in less than 6 hrs (95% CI: 3.53-60.17,P=0.000). Similarly, the isolate rates of E.coli was 29.5 times higher in calves that received colostrum after 12 hours of life than calves that received colostrum within the first 6 hrs of life (95% CI: 6.77–128.9,P=0.000). The likelihood of E.coli isolation from diarrheic calves was 40.4 times higher in those fed colostrum by hand than those received colostrum via suckling (95% CI: 7.78–210;P= 0.000). E.coli isolates were highly susceptible to Gentamicin (81.82%), sulphamethoxazole (78.79%) and Ciprofloxacin (75.76%), and showed resistance against Streptomycin (81.82%), Tetracycline (60.61%), Ceftriaxone (60.60%) and Cefoxitin (54.55%). It was concluded that E.coli infection is highly prevalent in diarrheic calves < 3 months of age, the majority of which were highly susceptible to Gentamicin, Sulphamethoxazole and Ciprofloxacin antibiotics. Moreover, dairy farms should implement good calf management and hygienic practices including feeding of sufficient quantity of colostrum within the first 6 hrs of birth to prevent E.coli infection.

Hailu S (2020) Isolation, Identification and Antibiotic Susceptibility of E.coli from Diarrheic Calves in and Around Holeta Town, Central Ethiopia. J Vet Med Res 7(5): 1197.

NCD: Neonatal calves Diarrhea; CSA: Central statically agency; EAggEC: Enteroaggregative E. coli EAdEC: Enteroadherent E. coli; CLSI: Clinical and Laboratory Standards institute CSA: Central statical Agency; EIEC: Enteroinvasive E.coli; EPEC: Enteropathogenic E.coli ETEC: Enterotoxigenic E. coli; MR: Methyl red; NAHDIC: National Animal Health Diagnostic and Investigation Center; TSI: Triple Sugar Ironing E. coli: VP: Vogues Proskauer

Newly born calves represent an important source of animal production for either meat or breeding worldwide. Diarrhea is one of the very common disease syndromes in neonatal calves in different countries, causing huge economic and productivity losses to bovine industry worldwide [1]. Calf diarrhea results from multifactorial including Neonatal Calf Diarrhea [NCD] as well as non-infectious factors related to the animals management flaws_ inadequate nutrition, exposure to severe environment, insufficient attention to new born calves, or a combination of these [2]. Calves are at greatest risk of developing diarrhea within the first month of life and the incidence of diarrhea decreases with age [3].

Several enteropathogens are implicated in neonatal calf diarrhea, the relative prevalence of which varies geographically; the most prevalent infections causing calf diarrhea including viruses (coronavirus and rotavirus), protozoa (Cryptosporidium spp) and bacteria (Escherichia coli, Clostridium perfringens, Salmonella) [2,4]. In most cases the causes of neonatal calf diarrhea outbreaks are multifactorial and associated with more than one of these agents [5,1]. Among the bacteria, Escherichia coli is the most common primary agent causing calf diarrhea and results inhuge economic loss with high morbidity (75%) and mortality (60%) in the cattle industry worldwide [4,6-8]. Escherichia coli are motile Gram-negative bacilli that falls within the family Enterobacteriaceae and usually found as normal inhabitant in the gastrointestinal tract of animals and human. It colonizes the infant gastrointestinal tract and can cause severe gastrointestinal illness [9,10]. Many strains of the bacterium are harmless to the calf, but certain strains that acquire virulence genes can cause moderate to severe scours and even death [11].

The strains inducing gastro enteric disease are known as Diarrhegenic E.coli (DEC). DEC are subdivided seven pathotypes based on their virulence properties:- Enteropathogenic E.coli (EPEC), Enterotoxigenic E.coli (ETEC), Enterohemorrhagic (EHEC), Enteroinvasive E. coli (EIEC), Enteroaggregative E. coli (EAggEC), and Diffusely Adherent E. coli (DAEC). Among E. coli pathogroups, the most common cause of NCD is ETEC stains that produce the K99 (F5) adhesion antigen (E. coli K99+) and heatstable (STaor STb) and/or heat-labile (LT1 or LT2) enterotoxins. The fimbrial adhesion F5 (K99) promotes the adhesion of bacterial cells to glycoproteins on the epithelial surface of the jejunum and/or ileum, and bacterial enterotoxin also causes damage to the epithelial cells, resulting in fluid secretion and diarrhea [12].

Neonatal calf diarrhea associated with enterotoxigenicE. coli (ETEC) usually occurs within the first 72 hours of life [13]. Severity of E.coli symptoms varies from mild transient diarrhea to severe sustained diarrhea resulting in death; typical presentation is in calves less than 7 days of age and as early as 12 hrs of life, and affected calves usually die within 2-3 days. Death sometimes occurs before diarrhea is observed, but typically the calf has profuse, watery diarrhea. E. coli infections typically produce secretory diarrhea switching the intestinal epithelial cells from an absorptive to a secretory mode [14,15]. Calf diarrhea accounts for approximately 75% of the mortality of dairy calves under three weeks of age [16]. The incidence risks of diarrhea in calves < 30 days old reported by several studies varies between 15% and 20% [17]. Diarrhea is typically whitish to yellowish in color [18,1].

Antibiotics are commonly used for the treatment of diarrheic calves, however, the practice of under dosing, over dosing as well as indiscriminate usage of drugs are common and leading for development of antimicrobial resistance in Ethiopia [19]. Although understanding of the antibiotic sensitivity pattern is critical to prevent antimicrobial resistance, recent and detailed information on the prevalence and drug susceptibility profile of pathogenic E.coli from diarrheic calves is limited.

Calf diarrhea is one of the leading causes of calf morbidity and mortality with a consequent reduction of replacement stock in dairy farms worldwide [1]. Causes of calf diarrhea are multifactorial, of which, E.coli infections are in the frontage [2]. Understanding the magnitude of E.coli infection in diarrheic calves and identifying the involved strains and their antimicrobial sensitivity in a given area is essential for designing cost-effective management strategy [20]. Although studies on the prevalence and distribution of E.coli infection have been conducted in some parts of the country, the magnitude and contribution of E.coli infection to calf diarrhea in Holota and its surrounding area is not well known. Furthermore risk factors associated with E.coli infection and the status of antimicrobial sensitivity pattern of the isolates in the study area is not well studied. Therefore, this research work was carried out in and around Holeta with the following objectives

• To Estimate the prevalence of E.coli infection in diarrheic calves

• To isolate and identify E.coli from diarrheic calves

• To determine the antimicrobial susceptibility pattern of E.coli isolates

Study area

The study was conducted in Holeta town and its surrounding areas. The town is found in Walmara district, Finfine surrounding Oromia special zone, Oromia Regional state, Ethiopia. It is located at a distance of 44 kilo meters west of Addis Ababa at a latitude of 09° 03′ 00’′N and longitude of 38° 30’00’’ E. The altitude of the area is 2391 m.a.s.l and the annual mean temperature ranges between 11-22 °C. Walmara district is bordered by Addis Ababa to the East; Ejere district to the West, Sululta district to the North and Sebeta Ha was district to the south and its weather condition is classified as 39% woinadega and 61% Dega. The area has a short rainy season from March to April and a long rainy season from June to September [21]. There are numerous small and large-scale dairy farms embracing local, exotic and cross-breed cows that supply milk and milk products to the surrounding towns and urban areas. The livestock population of Walmara district is estimated at 188,221 cattle, 108,652 sheep, 15,420 goats 365,294 poultry, 8,062 horses, 1,406 donkeys, 229 mule and 1,853 traditional beehives, 870 transitional beehives [21] (Figure 1).

Study animals

From the twenty legally registered dairy farms, eighteen dairy farms which had calves less than three months of ages were communicated and selected for the study during November 2018 to October 2019. From each selected dairy farms and locally reared dairy cattle exotic, cross and local breed diarrheic calves up to 3 months of age were purposively chosen and incorporated in the study.

Study design

A cross-sectional study design was employed from November 2018 to October 2019 and selection of farms was done by the purposive sampling method from the list of farms existing in Holota and its surrounding areas. Purposive sampling method was used to select the study calves based on their diarrheic status. Clinical examination of calves was performed depending on clinical parameters (elevated temperature, depression, dehydration, reduced suckling reflex, rough hair coat, loss of weight, weakness and soiling of hind quarter and tail with pastywatery feces) and the animals were classified as diarrheic (sick) and non-diarrheic( healthy).

Sample Size determination

The sampled size for this study was estimated using the formula described by [22] with a desired absolute precision of 5 %, confidence level of 95% and expected prevalence of 89.9% [20].

 

where:

n = Required sample size

Pexp =expected prevalence;

d =desired absolute precision.

Accordingly, the calculated sample size for estimating prevalence in purposive sampling was 140. However, in order to make a prevalence estimate more precise, the sample size was inflated two times and was set to 278. Hence, a total of 278 calves were sampled for the study.

Sample collection and Laboratory processing

Sample collection: Fecal samples were collected aseptically from 278 calves from eighteen farms and kept separately in peptone water. The samples were collected directly from rectum by manual stimulation using disposable latex glove and samples were cooled on ice packs and transported to Holeta Agricultural research center, Department of animal health and National animal health Diagnostic and investigation center (NAHDIC) laboratory for isolation of E.coli. The samples were processed as soon as possible after collection/if not keeping in refrigeration at +4?C.

Bacterial Culture and isolation: All medium used were prepared according to the manufacturer instruction. Isolation of E.coli was conducted following standard procedures described in Quinn et al. [32]. Fecal samples were cultured on MacConkey agar which selectively grows the members of Enterobacteriaceae and incubated at 37 °C for 24-48hrs. Colonies showing characteristic lactose fermenting and having pink color colonies grouped under the species E.coli. The presence of growth on MacConkey agar was used as primary criteria to proceed for isolation and identification of E.coli.

Furthermore, the colony characteristics observed on MaCconkey agar was used to classify suspected bacteria isolated into two groups: lactose fermenters and non-lactose fermenters. Suspected E. coli colonies were presumptively identified by their actose fermenting character (pink colonies) were further subcultured on Eosin methyl blue (EMB) agar medium to selectively identify E. coli. The characteristic colonies of E. coli were identified on EMB based on their green metallic sheen or blue-black to brown color. All isolated colonies were preserved on nutrient agar for further biochemical tests. All isolates were stained by Gram stain to determine the cell morphology, gram reaction and purity of the isolates under the oil immersion objective (X100 magnification).

Biochemical tests Biochemical tests used for differentiation of Enterobacteriaciae use different enzymes. The isolated bacterial pathogens with specific cultural characteristics of E.coli maintained on nutrient agar used for further biochemical tests such as Methyl red (MR), Vogues Proskauer (VP) test, Indole test, citrateutilization test, catalase test and Triple Sugar Iron (TSI) test.

Anti-microbial susceptibility test of Escherichia coli

Antimicrobial susceptibility was done by using Kirby Bauer’s disc diffusion method according to performance standards of CLISI clinical and laboratory standards institute.

Briefly, after pre-inoculation of pure bacterial colony in Tryptone soy broth/or saline water and adjustment of turbidity to a 0.5 McFarland turbidity standard, bacterial suspensions are plated as a full lawn onto freshly prepared Mueller-Hinton agar plates using sterile swabs. The antimicrobial discs of streptomycin (10μg), Cefoxitin (30μg), Ceftriaxone (5μg), Nitrofurantoin(30μg), Ciprofloxacin (5μg), Gentamicin (10μg), sulphamethoxazole (100μg) and tetracycline (30μg),were selected to test susceptibility pattern of Escherichia coli. The disks were placed on the surface of the inoculated agar plates at about 2cm apart and the plates were incubated at 37 °C for 18 to 24 h. After incubation, the antibiotic Inhibition Zone Diameters (IZD) were measured in millimeter using a caliper and interpreted as susceptible, intermediate and resistant as described by the CLSI [23].

Questionnaire survey

A pretested questionnaire was administered to dairy farm owners to assess the general calf husbandry practices at their respective farms. The questionnaire includes all practices in dairy farms including calf health care, hygiene, health problems, colostrum feeding time and method as well as types of diarrhea that affect the growth of calves.

Data analysis

All collected data were coded and entered in to the MSExcel spread sheet and then transferred to the STATA software (STATA version 14) for analysis. Descriptive statistics was used to summarize the generated data. Prevalence of E.coli infection was calculated as the number of positive samples divided by the total number of samples examined and multiplied by 100. The univariate and multivariate logistic regression tests were used to calculate the odds ratio (or) of different risk factors. A p-value <0.05 was considered to be indicative of a statistical significant difference.

 

Prevalence of Escherichia coli infection

The overall prevalence of Escherichia coli infection in diarrheic calves less than 3 months of age in the study area was found to be 83.5% (232). A statistically significant difference (P=000) was observed among the three age groups being young calves less than 4weeksshowed higher odds of infection(OR=8.35; 95% CI, 3.18–21.86; P=0.000) as compared to other age groups. The prevalence of Escherichia coli infection was higher (85.26%) in the Exotic breed calves as compared to in the local breed (57.69%) and cross breed (78.57%).However no statistically significant variation (P>0.05) was observed in prevalence of the infection among the three breed groups (Table 1).

Out of the total calves raised in poor, moderate and good hygienic barns, 90.43%,82.61% and 73.24%, were positive for E. coli infection, respectively. The difference in the prevalence of E.coli among the three hygienic barn categories was also statistically significant (χ2 =9.47, P=0.009).

Risk factors

The univariable logistic regression analysis showed significant association between isolation rates of E. coli and age (P= 0.000), size of farm (P= 0.001), type of diarrhea (P=0.000), time of colostrum feeding (P= 0.000), method of colostrum feeding (P=0.000) and house hygiene and sanitation (P= 0.003), whereas, sex, breed and BCS have not shown significant association (Table 1).

Multivariate logistic regression analysis revealed that age, type of diarrhea, time of colostrum feeding and methods of colostrum feeding are independent predictors of fecal E.coli isolate rates from diarrheic calves (P≤0.05)(Table 1).

Antimicrobial susceptibility profile of E. coli isolates

The susceptibility of E. coli isolates (N=33) was tested to 8 commonly used antimicrobials. The results revealed that E.coli isolates were highly susceptible to Gentamicin (81.82%), sulfamethoxazole (78.79% and Ciprofloxacin (75.76%), and were resistant against streptomycin (81.82%), tetracycline (60.61%), Ceftriaxone (60.60%) and Cefoxitin (54.55%) (Table 2).

Results of the present study demonstrated the presence of E.coli in high proportion of the diarrheic calves. The prevalence of E.coli obtained in this study, 83.5%, is in agreement with previous findings of 89.9% prevalence in Ethiopia reported by Zelalem et al. [20], 86.7% in Iran by Pourtaghi et al. [24] and 84.3% in central highlands of Ethiopia by Lema et al. [25]. However, the current result is higher than the 76% prevalence report in Bangladesh by Paul et al. [26], 73.12% prevalence in USA by Foster and Smith [10], 70.7% in Ethiopia by Yeshiwas and Fentahun [27], 44% in Bangladesh by Masud et al. [28] and the 43.1% in Addis Ababa, Ethiopia by Dereje [29]. Such variation may be attributed to difference in climatic conditions, sample size, sampling strategies, feeding managements, hygienic measures, age of the sampled animals as well as farm size [1].

A significant association was observed between age of calves and E. coli Infection in this study which is in harmony with findings of Yeshiwas and Fentahun [27], Aggernesh [30], Temesgen [31] and Dereje [29] who stated that calves aged between 1-30days, particularly during the first week of life were at greater risk of diarrhea and that risk decreases with age. The reason for this association is related to the ability E. coli bacteria to colonize the sterile intestine at early stage of neonate and higher susceptibility of newborn calves than adults, especially if they received low amount of colostrum [32]. Moreover, Godden [33], Mellor and Stafford [34] stated that the structure of the bovine placenta can impede easy acquisition of immune globulins to the unborn calves during pregnancy and consequently, calves may be borne without circulating protective antibodies rendering them more susceptible to different pathogens during their early life.

The current study demonstrated that calf diarrhea was higher in medium and large sized dairy farms as compared to small sized dairy farms. This finding is in consistent with a study report by Yeshiwas and Fentahun [27]. A marked increase in population density commonly results in an increase in the incidence of infectious diseases and mortality [35,36]. Herd size by itself has not a biological effect on the calf health; rather, it may be a measurement of other factors like management and care for calves. The other possible reason for the association of herd size and calf morbidity could be that adequate time may elapse between successive births in small herd size farms, which will reduce the concentration of infectious agents in the calf-rearing environment [27,35]. The present study showed that E.coli isolate rate was significantly associated with type of diarrhea, higher proportion of the calves showed watery diarrhea as compared to mucoid, bloody or mixed types of diarrhea,which is in agreement with previous findings of Aggernesh [30], Dereje [29] and Yeshiwas and Fentahun [27].

The occurrence of E. coli was significantly associated with colostrum feeding time of the calves; the highest prevalence of E. coli was in calves that were fed colostrum after 12 hours of age due to low level of immunoglobulin. Calves fed with colostrum at less than 6 hour of age had lower odds of E.coli isolates than those fed colostrum after 12 hours of age. This resultis in agreement with reports of Razzaque et al. [37] and Yeshiwas and Fentahun [27]. Each hour of delay in colostrum ingestion in the first 12 hours of age increases the chance of calves becoming ill by 10%. Delaying colostrum intake is known to decrease intestinal immunoglobulin and fat-soluble vitamins absorption and there is no immunoglobulin absorption into the circulation after 24hours due to closure of calf’s gut [38,39]. The time between birth and the first feeding is the prime factor for the failure of passive transfer of colostral immunity. Several studies had reported that calf mortality is significantly higher in those that got colostrum late after birth than those that got colostrum soon after birth [27]. This indicates that administering the first colostrum with proven quality and sufficient amount of immunoglobulins as soon as birth is critical to ensure successful passive transfer and health of neonatal calves.

In the current study, the occurrence of E. coli infection was higher in calves that fedcolostrum by hand feeding method as compared to those fed by suckling method. Similarly, Yeshiwas and Fentahun [27] reported that failure of passive immunity transfer in bottle feeding is higher than in naturally suckled calves. This might be attributed to refusing and inability of calves to ingest enough quantity of colostrum during hand feeding. Furthermore, risk of contamination of the colostrum with environmental pathogens is very high. Contaminated colostrum is one of the earliest potential exposures to infectious agents that cause diarrhea and septicemia in newborn calves [39].

Results of this study revealed a significant statistically association between E.coli isolate rates and calf house hygiene and sanitation. Calves with poor house hygiene and sanitation were more likely to acquire E.coli infection than calves in good house hygiene practices. This finding is comparable with the reports of Bazeley [38] that reported housing types and poor hygiene as risk factors for calf diarrhea. Similarly Yeshiwas and Fentahun [27] indicated that the occurrence of E. coli was high in muddy or wet livestock floor. Fecal contamination is more likely to occur during feeding, watering and handling from poor hygienic and unsanitary house and also increase risk of dissemination of Escherichia coli to newborn calves. Dairy cattle housed in barns with flush-type manure removal systems were more likely to have E. coli than animals housed in barns where manure was removed by scraping [40].

The present study showed that 81.82% of the E.coli isolates were susceptible to Gentamicin followed by 78.79% susceptibility to sulphamethoxazole and 75.76% susceptibilityt o Ciprofloxacin. This high susceptibility might be due to uncommonly usage/ unavailability of these drugs for animal disease treatment in Ethiopia. On the other hand, Streptomycin, Tetreacycline and Ceftriaxone showed the highest rates of resistance with 81.82%, 60.61% and 60.60%, respectively. Comparable findings have been reported by Bagre et al. [41] from Burkina Faso, in which all E. coli isolates were 100% susceptible to gentamicin and 15% resistant to sulphamethoxazole. The antibiogram study done by Yeshiwas and Fentahun [27] revealed that the E. coli isolates were highly sensitive to tetracycline, sulphamethoxazole, chloramphenicol, streptomycin, oxacillin; less sensitive to amoxicillin, ceftazidime, nitrofurantoin, kanamycin and resistance to cefotaxime, vancomycin. The recorded antimicrobial resistance of E.coli to streptomycin, tetreacycline and ceftriaxone in the current study might be as a result of suboptimal, prolonged and misuse use of antimicrobials for prophylaxis and treatment of infection. Antimicrobial resistance emerges from the use of antimicrobials in animals and human, and the subsequent transfer of resistance genes and bacteria among animals, humans, animal products and the environment [42].

The current study revealed somewhat high prevalence of E. coli infection in diarrheic calves in dairy farms of Holota and its surrounding areas, Central highlands of Ethiopia. Age of calves, farm size, time and method of colostrum feeding, as well as house hygiene and sanitation were the most determinant factors that predispose calves to E.coli infection. Isolates of E.coliin the study area were highly susceptible to gentamicin, sulphamethoxazole and Ciprofloxacin and resistant to streptomycin, tetreacycline and ceftriaxone.

• Based on above findings, the following recommendations are suggested:Implementation of good calf management practice is highly advised particularly feeding ofsufficient amount of colostrum shortly after birth before the gut closure is completed. 

• Preventive approaches against E.coli infection directed towards management risk factors should best strengthened to minimize morbidity and mortality associated losses.

• Veterinarians should pay attention to E.coli resistant antibiotics while prescribing remedies for the treatment of colibacillosis in calves

• Further investigation on the prevalence of E.coli should be under taken in order to design cost effective control measures.

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