Antimicrobial susceptibility pattern of Escherichia coli isolated from dairy calves with diarrhoea in Akaki Kality, Addis Ababa, Ethiopia

ABSTRACT Calf diarrhoea, the eighth most common cause of death in this age group, affects over 75% of newborn calves under three weeks old. Fifty calves with diarrhoea from the nine intensive dairy farms were assessed during the study period. Of these, 72% (n =36) were positive for E. coli There was a strong link between the prevalence of E. coli in calf diarrhoea and the age groups. Therefore, those under the age of one month were 1.3 more likely to be infected with E. coli than those between one and three months of age. The Antibiogram test revealed that E. coli isolated was highly responsive to the antibiotics tetracycline, chloramphenicol, sulfamethoxazole, streptomycin and doxycycline, but less susceptible to the drugs amoxicillin, ceftazidime, nitrofurantoin, and kanamycin and resistant to the antibiotics cefotaxime and vancomycin. Results from interviews and regular observations in the study area indicate that young calves were more impacted by diarrhoea, which accounted for about half of all health issues in calves. The E. coli recovery rate was significantly higher in calves that were one week old. These findings supported the hypothesis that the level of calf diarrhoea was excessive and would be detrimental to dairy output.


Introduction
According to the Animal Number Census (CSA 2021) Ethiopia has the largest population of livestock in Africa, with an estimated 70 million cattle, 42.9 million sheep, 52.5 million goats, 8.1 million camels and 57 million chickens.When compared to the annual growth rate of the human population, which is 2.5%, estimates that cattle will expand at a rate of 1.1% (ALM 2001).
Calf diarrhoea, the eighth most common cause of death in this age group, affects over 75% of newborn calves under three weeks old (Blowey 2016).Enterotoxigenic E. coli, Salmonella, Yersinia, Proteus, Rotavirus, Coronavirus, Cryptosporidium and Emeria species are the main causes of diarrhoea in calves (Foster and Smith 2009).
Calf scour is a clinical condition connected to a number of illnesses marked by diarrhoea rather than a single disease entity.Regardless of the source, altered gastrointestinal fluid absorption results in potentially fatal electrolyte imbalances.The scouring calf experiences fast dehydration, acidosis, electrolyte loss and fluid loss.Although infectious pathogens may first harm the gut, dehydration, acidosis and electrolyte loss are most often the causes of death from scour.Implementing efficient prevention and therapeutic approaches requires the identification of infectious organisms that cause scour (Sakha et al. 2014).It is crucial to determine the aetiological and predisposing factors associated with calf diarrhoea in order to develop preventative strategies and minimize losses during the first few months of life.This will boost productivity per livestock unit without increasing the number of cattle (Lorino et al. 2005).
Despite immunization campaigns and management strategies, E. coli-related diarrhoea is one of the most prevalent illnesses in young calves, necessitating treatment with antibiotics and fluid therapy (Gyles et al. 2008).E. coli is a member of the enterobacteriacea family that is gram negative, facultatively anaerobic, motile or non-motile and non-spore producing.Some strains have virulence genes, which increase an organism's capacity to infect both humans and animals with a range of intestinal illnesses and diarrhoeal disorders (Kaper et al. 2004).
The rise and spread of antimicrobial resistant E. coli has been noted as a major issue on a global scale (Schroeder et al. 2002).Antimicrobial therapy is a crucial technique in lowering the prevalence and signs of diarrhoea in cattle.However, there is widespread antimicrobial drug resistance that worries veterinarians (Blanco et al. 1998;Flament-Simon et al. 2020).According to several studies, the processing of calves, pigs and poultry is where antibiotic resistance and selection pressure are most prevalent (Prescott and Baggot 2000).
A survey published in 2005 by the World Organization for Animal Health (OIE) revealed that some countries have no regulations controlling the use of antimicrobial agents in livestock.However, the majority of the countries do have laws and regulations governing the approval and use of veterinary medicines (Valarcher et al. 2006).Without any doubt, the introduction and use of antimicrobial agents in veterinary medicine had and still has an enormous impact on the health and welfare of animal species.Antibiotic use in animals contributes to the selection and spread of resistant bacteria among animals, between herds and between countries (McEwen and Fedorka-Cray 2002).Therefore, the objectives of this study were to isolate and determine Antimicrobial susceptibility pattern of Escherichia coli isolated from dairy calves with diarrhoea in Akaki Kality, Addis Ababa, Ethiopia.

Study area
The study was conducted in the Akaki Kality sub-city, Addis Ababa, which is the dairy belt of the capital city.Akaki Kality, a subcity of Addis Ababa, is situated at an elevation of 2500 m above sea level.In May, the year's highest and lowest temperatures are attained.The main wet season lasts from June through September.Akaki Kality's relative humidity fluctuates between 40 and 50% when it's dry and between 70 and 80% when it's raining.The region receives 1800 mm of rain on average per year (NMSA 2003) (Figure 1).

Study animals
Dairy calves younger than one year of age from various farm types, including smallholder (fewer than five dairy cows), medium-sized (6-50 dairy cows) and large scale (more than 50 dairy cows), dairy farms were included in the study population.For this investigation, a total of 50 dairy calves were used.Each of the farms in this study has an intensive production system and keeps crossbred animals (Holstein x local breeds) (Lenta et al. 2000).All diarrhoeal calves during the sampling time were included in this study.A number of data points about potential risk factors for the occurrence of diarrhoea, including feed, management practices and the herd size of each animal in the chosen dairy farms were recorded.

Study design and sampling methodology
From November 2013 to April 2014 E.C, a case study was conducted on selected dairy farms in the Akaki Kality subcity.Calves with clinical cases of diarrhoea were purposively chosen for sample collection, and important clinical indicators like an elevated body temperature, depression, dehydration, a rough coat, weight loss, weakness, and soiling of the hindquarter and tail with diarrhoeal feces were taken into account (Lenta et al. 2000).About 10 gram of fecal contents was extracted from newly empty diarrhoeic feces or from the rectum of calves by direct stimulation after wearing disposable latex gloves and kept in a sterile universal bottle and were labelled.Finally, the fecal samples were transported using ice box in cold chain to Bacteriology laboratory, for microbiological analysis.The temperature of the feces was kept at 4°C till processing time.Each calf's age, breed and tag number were recorded along with the name of the farm, the time the sample was taken, the consistency and colour of the faeces.

Questionnaire survey
Dairy owners were interviewed to assess the general calf husbandry practice during sampling.In general, the questionnaire covers all farm practices related to calf health care, farm hygiene, farm health problems and types of diarrhoea that affect calves' growth.

Bacteriological examination
Every sample was grown on blood agar and kept under aerobic conditions for 24-48 h at 37°C.Pure colonies were further subcultured on nutritional agar and MacConkey agar for 24-48 h at 37°C after morphological analysis, hemolysis type and gram staining features.
The fundamental criterion for proceeding further with the isolation and identification of E. coli was the growth of a sample on MacConkey agar.In addition, the MacConkey agar colony characteristics were employed to divide a suspected E. coli strain into two categories: lactose fermenters and nonlactose fermenters.Eosin-Methylene Blue was used to subculture suspect E. coli in order to identify specific E. coli species based on their propensity to ferment lactose.The distinctive colonies on Eosin-Methylene Blue were identified by their blue-black to brown colour or green metallic sheen.For additional biochemical analyses, all isolated colonies were kept on nutritional media.

Biochemical tests
Gram staining was used to determine the purity of each isolate and oxidase, catalase and oxidation-fermentation tests were used for the initial biochemical evaluations.The indole, methyl red, voges-proskauer (VP) Test, citrate and urease tests were employed for the isolation and identification of bacterial isolates.

Antibiotic susceptibility test
According to (Lema et al. 2001), 11 discs containing chemotherapeutic drugs were used to perform an antibiogram on the different isolated groups of E. coli using the in vitro Kirby-Bauer disc diffusion technique on Mueller Hinton agar plates (amoxicillin, cefotaxime, ceftazidime, vancomycin, tetracycline, chloramphinicol, nitrofurantoin, sulfamethoxazole, streptomycin, doxacillin and kanamycin).
Four to five isolated colonies of the test organism were taken using a sterile inoculating loop, suspended in a test tube with 2 ml of sterile normal saline and the saline tube was vortexed to create a smooth suspension.The turbidity of the suspension was adjusted at 0.5 McFarland standard by adding more organism when the suspension was too light or diluting with sterile saline when the suspension was too heavy in which McFarland Densitometer was used as a standard measurement for checking the turbidity.A sterile swab was dipped in to the inoculum tube and was rotated against the side of the tube to remove excess fluid.The dried and well prepared Muller-Hinton agar plates were inoculated by streaking the swab three times over the entire agar surface and were rotated approximately 60° each time to ensure an even distribution of the inoculum.At last, the plates were rimmed with the swab in order to pick up any excess fluid and allowed to dry for 5 min.After the plates were dried, the antimicrobial disks were placed on the surface of the agar using sterile forceps and the disks were gently pressed with the forceps to ensure complete contact with the agar surface.Finally, once all disks were in place, the plates were incubated at 37°C for 18 h.Antibiotic disk test was repeated three times for one isolate.
After 18 h of incubation, the diameter of inhibition zone formed around each disk was measured using digital caliper based on the guidelines from the Clinical and Laboratory Standards Institute (Abdullah et al. 2013) and on the standardized table supplied by the manufacturer.

Data analysis
Microsoft Excel 2007 was used for data entry and validation, while SPSS software version 20 was used for processing in order to conduct the necessary statistical analysis.In order to determine the occurrence of E. coli among various variables, descriptive statistics were used.

Bacteriological examination of diarrhoeic calf feces
Out of 50 total samples examined for the isolation of E. coli, 36 (72%) samples were positive and 14 (28%) samples were negative.E. coli isolated from diarrhoeic calves' feces was subjected to a battery of physiochemical tests to determine its biochemical properties and the degree of variation in its reactivity pattern (Table 1).

Prevalence of E. coli isolates in different farms
Out of 21 diarrhoeic samples from large scale (14 of them), 18 from medium scale (14 of them) and 11 from small scale, eight of them were positive for E. coli (Table 2).

Types of diarrhoea associated with E. coli
Out of 50 samples examined, the occurrence of E. coli was higher in watery diarrhoea (30%) than in other types of diarrhoea (Table 3).

E. coli age-related risk
The prevalence of E. coli among calves of various age groups was identified in the current study.The highest E. coli prevalence were observed in calves less than 1 month old (54%), and reduced as the calves' ages increased (Table 4).

Antibiotic susceptibility test
All of the isolate were subjected to 11 antibiotic discs to determine the antibiotic sensitivity.The antibiotic sensitivity test revealed that the E. coli isolated from diarrhoeic were highly sensitive to sulfamethoxazole, streptomycine, doxacilline, tetracycline and chloramphinicol; less sensitive to amoxicilline, ceftazidime, nitrofurantoin and kanamycine; and the isolates were resistant to cefotaxime and vancomycine (Table 5).

Questionnaire results
All dairy farms grew their own replacement stock and used a stall-fed feeding regimen.Dairy farms in the Akaki Kality area primarily grew heifer calves; male calves were immediately sold after birth.Almost all of the farms, with the exception of a few smallholder dairy farms, had a calving facility (separate calving enclosure).None of the farms used navel therapy when calves were born.Everyone is aware of the colostrum's role in immunity, and most farms, with the exception of a few small-scale dairy farms, practice colostrum feeding immediately after delivery.All of the study farms gave whole milk to the calves twice a day, but none of the farms employed a specific starter feed; instead, some of the feed given to the cows was used for the calves.Hay and concentrate mixture are some of these.Different farms have different weaning ages and non-milk feed introduction ages (Table 6).
Calf diarrhoea (52%) was the most frequent calf health issue among the many illness categories identified, followed by pneumonia (20%) and lameness (10%).The consumption of colostrum and the cleanliness of the home were the two most significant risk factors that were found to affect the likelihood of calf health issues.According to the farm respondents, there were differences in the incidence of diarrhoea across different age groups of calves, with calves under a month old being more severely impacted than other age groups (Table 6).

Discussion
In the present investigation, a total of 50 fresh fecal samples were collected from calves suffering from diarrhoea, of which 36 samples were found positive for E. coli.They gave a positive reaction to lactose fermentation on MacConkey agar plate, metallic sheen colonies on Eosin-Methylene Blue (EMB) plates, and a deep blue colour on green coloured Simmon's citrate agar.Similar cultural characteristics were also corroborated by (Aggernesh 2010).This bacterium was also isolated from diarrhoeic calves by many other authors in different parts of the country (Demissie 2007;Dereje 2012).Our finding was higher than the reports of (Blanco et al. 1996).This may be due to the difference in climatic conditions, sample size, feed management, personal hygiene and age group.
E. coli is an intestinal commensal bacterial species, commonly isolated from fecal samples.Therefore, isolation of E. coli from feces may not be directly associated with the causation of diarrhoea.However, the finding that E. coli was predominately recovered from diarrhoeic calves in the present study may be an indication of its importance in calf diarrhoea in the study area.An in vitro antibiotic sensitivity assay of E. coli isolates to different antibiotics was carried out.A slight variation was noticed in the results of the sensitivity of isolates against the 11 different antibiotics used.The isolated E.coli was highly sensitive to tetracycline, chloramphinicol, sulfamethoxazole, streptomycin and doxacilline.E. coli was less sensitive to amoxicillin, ceftazidime, nitrofurantoin and kanamycin, and it was resistant to cefotaxime and vancomycine.These findings disagree with those reported by (Sharma et al. 2004), who recorded the highest percentage of antibiotic resistance with tetracycline, streptomycin, kanamycine, chloramphinicol and sulfamethoxazole in the treatment of E. coli in diarrhoeic calves in Spain.Sharma et al (Blanco et al. 1996) carried out antibiotic sensitivity tests on E. coli strains from diarrhoeic calves and found that E. coli strains were most sensitive to kanamycin, followed by streptomycin and doxycycline.Sharma et al., (Orden et al. 1999) studied the antibiotic resistance pattern of diarrhoeic calf E. coli isolates and found E. coli isolates showed resistance to tetracycline, streptomycin, chloramphinicol, nitrofurantoin and kanamycin.Orden et al (Blood et al. 1994) determined the in vitro activities of several cephalosporins and quinolones against E. coli isolated from dairy calves affected by neonatal diarrhoea.Ceftazidime and cefotaxime were highly effective against the E. coli isolates tested.This variation may be due to different factors such as timing of disc application; temperature of incubation; incubation time; size of plate; depth of agar medium; and spacing of the antibiotic discs; potency of the antibiotic discs; and composition of the medium.
In the present study, the observation of diarrhoea was more frequent in calves under 1 month old.Accordingly, age groups younger than one month old were at a high risk of being affected by diarrhoea.This could be due to a number of factors.Among the potential factors, the investigated age of the calf from calf factors, the time of first colostrum feeding and house hygiene from management factors were significantly associated with calf health problems.Incidentally, these risk factors, especially colostrum feeding and house hygiene, are amenable to intervention.Therefore, there is more room to improve the present scenario through management intervention.
From a questionnaire survey, all the farms assessed administer colostrum to new-born calves.However, the efficiency of colostrum intake and gut absorption may be affected by farm management practices.Colostrum is known to be efficiently absorbed from the gut of neonates within 24 h of birth (Godden 2008).Therefore, delays in administration could lead to a lack of colostrum derived from maternal antibodies to protect calves from enteric pathogens.This finding is compared well with the findings of (Blanco et al. 1996;Demissie 2007) also reported that calves aged less than one month were at great risk of diarrhoea, particularly during the first week of life and risk decreases with age.This is well explained by (Olson 2017), who reported that the structure of the bovine placenta impedes easy acquisition of immune globulins by unborn calves during pregnancy and therefore; calves are borne without circulating protective antibodies.So they are more susceptible to different pathogens.
From the questionnaire survey, calf diarrhoea was among the major problems in the study area of the nine farms.Calf diarrhoea was the leading cause of calf morbidity (52%), followed by pneumonia (20%) and lameness (10%).The finding of calf diarrhoea as the predominant calf health problem was in agreement with the findings of other studies (Lenta et al. 2000;Demissie 2007;Dereje 2012).Unlike reports by (Debnath et al. 1995;Sivula et al. 1996;Shiferaw et al. 2002), who reported pneumonia as a second important disease complex that affects calf health, there are studies which found pneumonia as the leading calf health problem (Wudu et al. 2008), and calf diarrhoea as a leading health problem in the present finding, which suggests the significance of poor hygienic handling of feeding utensils and calf house observed during the study.The relatively lower cases of pneumonia encountered in this study might be due to small calf herd size per farm.Stocking density has a strong correlation with environmental stress that exposes calves to respiratory problems.It was observed that a 50% decrease in stocking density increased the ventilation rate by 20 times, thereby decreasing the risk of pneumonia (Blowey 2016).
Even though the sampling method and level of analysis do not allow statistical comparison of calf health problems between farm types, calf diarrhoea was apparently higher in medium-sized and larger dairy farms than in smallholder dairy farms.This was in agreement with the report of (Ingle et al. 2013).All dairy farms of the dairy farmers had knowledge of the advantage of colostrums over ordinary milk, and most of them knew the optimum time to feed colostrums to calves.None of the farms practiced immediate isolation of sick calves.In most of the farms, diarrhoeic calves received recommended treatments by veterinarian.This was also in agreement with the findings of (Blanco et al. 1996).
The results of the current study's bacterium isolation, identification, biochemical tests, frequency distribution and antibiotic sensitivity indicate that the management system may play an important role in the development of calf diarrhoea and that alternative treatment approaches should be sought.

Conclussion
The isolated E. coli was highly sensitive to tetracycline, chloramphenicol, sulfamethoxazole, streptomycin and doxacilline, whereas was less sensitive to amoxicillin, ceftazidime, nitrofurantoin and kanamycin, and it was resistant to cefotaxime and vancomycin.In this study, there appears to be an association between the agent that has been investigated and diarrhoea in the first month of life.However, the poor detection rate, there after suggested that the cause of this age group must be sought elsewhere, possibly in nutritional and husbandry factors.Regarding the observational and questionnaire surveys, it revealed that separate calf pens were used in all of the farms, with the exception of some smallholder dairy farms, and that they were aware of colostrum feeding.In the present investigation, calf diarrhoea appears to be one of the major scenarios that reduce calf performance and that can be achieved through good management.Special emphasis should be given at the time of colostrum feeding, especially the times of colostrum administration to neonate calves.The hygiene of the calf house and isolation of sick calves should be considered an important factor.Highly sensitivity of E. coli was recorded against tetracycline, chloramphenicol, sulfamethoxazole, streptomycin and doxycycline therefore calves should be treated with such drugs.Further studies should be conducted to reduce antibiotic resistance in E. coli.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Consent for publication
I fully agree that this paper can be published in our journal.

Table 1 .
Secondary biochemical test results.

Table 2 .
Prevalenece of E. coli in the feces examined by farm types.

Table 3 .
Types of diarrhoea associated with E. coli.

Table 4 .
Age of diarrhoeic calves associated with E. coli.

Table 5 .
Antibiotic susceptibility pattern of the E. coli isolates from diarrhoeic calves.
Note:The above values are presented as Mean ± SD of triplicate experiments where:-++ = highly susceptible,+ = less susceptible, R = Resistant, SD = standard deviation.

Table 6 .
Questionnaire assessment of each farm.