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Isolation, Identification and Antibiogram of Bacterial Pathogens from Bovine Subclinical Mastitis in Asella, Ethiopia

Mahendra Pal Dereje Lemu Tsion Bilata
Vol 7(8), 62-70
DOI- http://dx.doi.org/10.5455/ijlr.20170524115206

Mastitis, a disease complex of multiple etiologies, results into considerable financial losses to dairy industry worldwide. A cross sectional study was carried out between July 2013 to August 2013 in Asella town and its surrounding areas in order to determine the prevalence of subclinical mastitis and conducting antibiotic sensitivity test on the isolated pathogens. The study populations comprised of lactating crossbred dairy cows that were found in different age groups, parities and lactation stages. The milk samples were aseptically collected from a total of 780 quarters of teats from 195 apparently healthy cows. The collected milk samples were screened by using Californian Mastitis Test (CMT) and 393 (50.3 %) were found to be positive at quarter level and 100 (51.2%) at cow level. The prevalence of blind teats accounted 7 (0.89%). Only the results of the strong positive (CMT Score > 4) milk samples from the quarters were cultured on the media of blood and Mac Conkey agar plates, and 126/265 (47.5%) yielded microbial growths. The bacterial identification and isolation was done on the basis of colony morphology, microscopic examination of Gram-stained smears, catalase production and biochemical tests. The commonly recovered organisms were Staphylococcus aureus, CNS (Coagulase negative Staphylococcus), Streptococcus agalactae, Streptococcus uberis, Streptococcus intermidius, Proteus spp., Escherichia coli, Corynebacterium bovis, Corynebacterium pyogenes, Micrococcus spp. and Bacillus spp. Among the isolated pathogens, S. aureus, Corynebacterium bovis, Corynebacterium pyogenes and E. coli were the most prevalent that accounted 15.9%, 11.9%, 12.7% and 11.1%, respectively. The results of antibiotic sensitivity test indicated that kanamycin (85.3%), norfloxacilline (61.3%), cloxacillin (56.8%), nitrofurantoin (53.1%) and cloxacilin (56.8%) were found to be sensitive against the total isolates. Substantial resistance of the isolated strains were recorded against cefotaxime, penicillin G, amoxicillin, ampicillin and vancomycin, which accounted 97.9%, 96.4%, 80.4%, 80.2% and 78%, respectively. Staphylococcus aureus revealed resistance against penicillin G, amoxacillin and tetracycline, which accounted 97.9%, 96.4% and 80.4%, respectively and Escherichia coli showed resistance against vancomycin, ampicillin and penicillin G, which indicated 83%, 74% and 65%, respectively. Coagulase Negative Staphylococcus aureus (CNS) were found resistance to penicillin G. Subclinical mastitis is a major disease in the worldwide with economic and public health importance. The routine inspections of subclinical mastitis by using CMT and SCC are imperative and cost effective methods for the diagnosis of mastitis in dairy cows. The isolation of pathogens and conducting of antimicrobial susceptibility tests are essential in order to achieve effective treatment and controlling of the disease and also preventing the development of the resistant bacterial strains. Since misuse of antimicrobials leads to the presence of residues in foods of animal origin, there is a need for stakeholders in the livestock industry and public health sector to intervene urgently on the impacts of drug residues with regards to animal and human health.


Keywords : Antibiotic Sensitivity Test California Mastitis Test Dairy Cows Microorganisms Public Health Subclinical Mastitis

Introduction

Mastitis, an inflammation of mammary gland, is one of the most important production disease of dairy animals such as cows, buffaloes, goats, sheep, and camels (Pal and Jadhav, 2013; Pal, 2015).The disease is caused by various pathogens, which include bacteria, fungus, actinomycetes, viruses, prototheca and mycoplasma (Gobu and Pal, 2005; Quinn et al., 2010; Pal and Jadhav, 2013; Pal, 2015). It affects all breeds of dairy cows, but high yielding, exotic and cross breed cows are highly susceptible (Gobu and Pal, 2005; Pal and Jadhav, 2013). Approximately, 140 species of microorganisms have been identified as etiological agents of bovine mastitis. Milk of the infected animal is of inferior in quality, low in quantity; and may pose public health concern because of suspecting several zoonotic pathogens, which can cause serious illness in humans, especially, when the milk is consumed raw ( Pal,2007; Shamila-Syuhada et al., 2016). In this context, Pal (2007) has listed many zoonotic diseases such as brucellosis, tuberculosis, campylobacteriosis, listeriosis, cornybacteriosis, yersiniosis, collibacillosis, salmonellosis, staphyloccosis, streptococcosis, coxillosis, toxoplasmosis, anthrax, Haver Hill fever, poliomyelitis, and infectious hepatitis, which are transmitted through the consumption of raw milk. Subclinical mastitis has been reported in many countries including Ethiopia (Ayano et al.,2013; Abrahmsen et al.,2014; Dieser et al., 2014; Amanu et al., 2016; Claudia et al.,2016;Tassew et al., 2016). Certain measures such as application of good sanitary and hygienic practices, adequate housing with proper sanitation, regular screening for early detection and treatment, regular bacteriological monitoring to adapt the prophylactic treatment plan and follow up chronic cases, culling of older cows with repeated attacks and prompt treatment of teat or udder injuries are required to control mastitis in dairy animals (Gobu and Pal, 2005).

Globally, the use of antibiotics to treating and preventing udder infections in cows were the major component of controlling plan in many countries (Jamali et al., 2014). Nevertheless, the prolonged use of these drugs may lead to the emerging of the resistant bacterial strains. Nowadays, antimicrobial resistance (AMR) bacterial strains and the resulting failure of therapies in human medicine are increasing a threat to effective treatment of bacterial infections. These microorganisms represent a serious problem in the treatment of infectious diseases including mastitis. Such antimicrobial resistant organisms can pose serious health related problems to animals as well as human beings (Jamali et al., 2015; Shamila-Syuhoda et al., 2016).

Several studies have been conducted in order to determine the antimicrobial sensitivity tests on isolated microorganisms from bovine mastitis (Bedada and Hiko, 2011; Abebe et al., 2013; Amunu et al.,2016; Pirzada et al., 2016; Shamila-Syuhada et al., 2016; Tassew et al., 2016). These investigators have reported the essentiality of antimicrobial susceptibility test in order to suggest suitable antibiotic treatments of dairy animals. Hence, the current study is also attempted to determine the prevalence of subclinical mastitis by using by using California Mastitis Test (CMT), isolating and identifying the major bacterial pathogens and conducting antibiotic sensitivity test on the isolated pathogens.

Materials and Methods

Study Area and Population

Asella is located about 175 km south east of Addis Ababa at 6º59′ to8°49’N latitudes and 38º41′ to 40°44’E longitudes. The altitude of the area ranges from 2600-3000 m.a.s.l. It is characterized by mid subtropical weather with minimum and maxim temperature ranging from 8.4°C-22.6°C and the relative humidity ranging from 43% to 60%. The average rainfall is 2000 mm. The area has a bimodal rainfall occurring from March to April (short rainy season) and July to October (long rainy season). The study animals included lactating crossbred dairy cows, which were found in different age groups, parities and lactation stages. The selected cows were reared under intensive management system.

Study Design

The cross sectional study was carried out between July 2013 to August 2013 in order to determine the prevalence of subclinical mastitis and to conduct antimicrobial sensitivity test on the isolated pathogens in the study area. Purposive sampling method was used to select the cows according to the willingness of the owner. The sampling unit was each udder quarter, with apparently normal milk secretions.

Milk Sample Collection

The milk samples were aseptically collected from cows not treated early with either intra mammary or systematic antimicrobials agents. The sterile collection tube was used and the first stream of milk from each quarter was discarded. Later, the milk samples were kept in cold chain and transported to the laboratory.

Detection of Mastitis

The collected milk samples were further investigated by California Mastitis Test (CMT). This test was carried out as a screening test in order to diagnose the presence of subclinical mastitis in the collected samples according to the standard procedure of (Quinn et al., 2010).

Isolation of Bacteria

Milk samples with the results of CMT ≥4 were examined for presence of bacteria. The samples were subjected to culture on 5% sheep blood agar and Mac Conkey agar (MAC) plates. The plates were incubated under aerobic conditions at 37 °C for 18 h – 24 h. The bacterial Identification and isolation was done on the basis of colony morphology, microscopic examination of Gram-stained smears, catalase production and biochemical properties according to the (Quinn et al., 2010).

Antibiotic Sensitivity Test

The isolated microorganisms were further tested for antimicrobial susceptibility test according to Kirby-Bauer disc diffusion method on Mueller-Hinton agar plates according to the procedures described by Quinn and others (2010). The susceptibility of bacterial isolates was tested to kanamycin, norfloxacilline, cloxacillin, nitrofurantoin, cefotaxime, penicillin G, amoxicillin, ampicillin, vancomycin, tetracycline, and gentamycin discs according to the guidelines of National Committee for Clinical Laboratory Standards (NCCLS, 1990). In the study areas, several antibiotics such as lactoclox, penstrip, penicillin, streptomycin, oxytetracycline and sulpha drugs have been extensively used.

Data Management and Analysis

The prevalence of sub-clinical mastitis was calculated as the number of sub-clinical cow-cases with at least one quarter affected per 100 cows. The quarter-sub-clinical mastitis prevalence was defined as the number of quarters infected per 100 quarters. The collected data was coded and entered to MS Excel spread sheet. The data was analyzed by descriptive statistics with the help of SPSS version 22.

Results and Discussion

A total of 780 quarter samples of 195 apparently healthy lactating dairy cows were examined by using CMT. California Mastitis Test is an indirect cow-side test that is widely used as on-farm screening test in order to estimate somatic cell count (SSC) from the individual cow and quarter. California Mastitis Test is subjectively graded using a five-point scale where each score represents an approximate SCC range (Schalm and Noorlander,1957).The details of scoring system and somatic cell count range is summarized in Table 1.

Table 1: California Mastitis Test (CMT) scoring system with interpretation and somatic cell count (SCC) range

CMT Score Interpretation SCC per ml
1 Negative 0-200 000
2 Trace 150 000- 500000
3 Weak positive (1+) 400 000 – 1500 000
4 Distinct positive (2+) 800 000 -5 000 000
5 Strong positive (3+) >5 000 000

The prevalence of subclinical mastitis in the study area was found to be 100/195 (51.2%) in cow level and 393/780 (50.3%) in quarters level (Table 2). The prevalence of blind teats accounted 7 (0.89%). The results of our findings in this study indicated that subclinical mastitis is the most dominant disease of the dairy farms in the study area. The prevalence of sub-clinical mastitis in quarter level was higher when compared to the central region of Algeria, which accounted 28.57% (Saidi et al., 2013) and was lower than the prevalence reported by Kifle and others (2008) in North Shoa Zone, Ethiopia, which was 89.5% in cow and 63.1% in quarter level, respectively. In Asella town of Ethiopia, almost similar prevalence of 55.8% at cow level was registered by Bedada and Hiko (2011).

Table 2: Subclinical mastitis at individual cow and quarter level

Types No. of Sample CMT positive Prevalence
Cows 195 100 51.20%
Quarters 780 393 50.30%

From the total of 393 affected quarters , only 265 quarter samples, that have the CMT Score + and +3 (Table 3) were inoculated on microbial media for the presence of bacteria.

Table 3: Results of California Mastitis Test (CMT) score of the affected quarters of dairy cows

CMT Score No. of CMT Positive Quarters
3+ 126
2+ 170
1+ 90
Trace 7
Total 393

Out of the cultured milk samples, only 47.5% (126/265) of yielded microbial growth (Table 4). In this study, isolation of Staphylococcus aureus, Coagulase negative Staphylococcus (CNS), Streptococcus agalactae, Streptococcus uberis, Streptococcus intermidius, Proteus spp., Escherichia coli, Corynebacterium bovis, Corynebacterium pyogenes, Micrococcus spp. andBacillus spp. were incriminated as causes of mastitis in the study areaThe most prevalent isolated pathogens were Staphylococcus aureus, Corynebacterium bovis, Corynebacterium pyogenes and Escherichia coli, which accounted (15.9%), (11.9%), (12.7%) and (11.1%), respectively.

Table 4: Types of bacterial isolates from the cultured milk samples

No. Isolated Bacteria Frequency Prevalence (%)
1 Bacillus cerues 12 -9.5
2 CNS(Coagulase negative Staphylococcus) 15 -11.9
3 Corynebacterium bovis 15 -11.9
4 Corynebacterium pyogenes 16 -12.7
5 Escherichia coli 14 -11.1
6 Micrococcus spp 10 -7.9
7 Proteus Spp 5 -4
8 Staphylococcus aureus 20 -15.9
9 Str.agalactae 3 -2.4
10 Streptococcus intermidius 9 -7.1
11 Streptococcus uberis 7 -5.6
Total 126 100

These findings are partially similar to Chandrasekaran and co-investigators (2014), which indicated that Staphylococcus aureus and Escherichia coli as the most common causes of contagious and environmental clinical mastitis. It was also reported that, Staphylococcus aureuswas the major pathogen recovered from the milk samples positive to CMT (Mahantesh et al.,2011; Zenebe et al., 2014).

The results of our study on antibiotic sensitivity test indicated that kanamycin (85.3%), norfloxacilline (61.3%), cloxacillin (56.8%), nitrofurantoin (53.1%) and cloxacillin (56.8%) were found to be the most sensitive drugs against all the total isolates. Substantial antibiotic resistance of the microorganisms was shown against cefotaxime, penicillin G, amoxicillin, ampicillin and vancomycin, which accounted 97.9%, 96.4%, 80.4%, 80.2% and 78%, respectively.This study is in agreement with the findings of Jamali and others (2015). Among the isolates, the highest antibacterial resistance against S. aureus was observed to penicillin G, amoxacillin and tetracycline, which was 97.9%, 96.4% and 80.4%, respectively. This finding is also in agreement with other results reported by several workers (Sharma et al., 2015; Amanu et al., 2016; Shamila-Syuhada et al., 2016; Tassew et al., 2016). Staphylococcus aureus are common causes of bacteraemia in humans. Among the different strains involved, the sources of major concern are methicillin-resistant S. aureus (MRSA) and vancomycin resistant S. aureus (VRSA).In most countries, the widespread and continuous use of penicillin and tetracycline leads to increase in resistance of antimicrobial strains towards these agents (Jamali et al., 2015). This problem has become a major challenge now to the dairy industry in many countries of the world.

Escherichia coli were found to be resistant to vancomycin, ampicillin and penicillin G, which accounted 83%, 74% and 65%, respectively. Similar results were reported by Badada and Hiko (2011), which indicated the resistant of E. coli to penicillin and streptomycin. Our observation goes parallel with findings of Chandrasekaran and others (2014) who recorded E. coli resistance to penicillin G. In this study, CNS was found to be resistant to penicillin G. This finding is also in agreement with the observation of previous investigators (Bedada and Hiko, 2011; Claudia, 2016) who reported that the isolates were resistant to penicillin. The remaining microbial strains were susceptible for chloramphenicol and gentamycin. This finding is similar to the documented reports of the Asella Regional Veterinary Laboratory (ARVL) operational areas (Personal observation).

One of the most important reasons for the failure of mastitis treatment in dairy farms is assumed to be indiscriminate use of these antibiotic drugs. For example, penicillin-streptomycin combination, and tetracycline are heavily used for the treatment of bacterial infections in all animal species in Ethiopia. The using of the wrong drugs, reducing the dosage of the drugs or length of the treatment, decreased activity of the drug and without conducting antibiotic sensitivity test on the causative organisms, leads to increasing resistant strains of the microorganisms. The failure of these practices will result in the development of resistance strains of microorganisms.

Conclusions

The results of our investigation revealed that subclinical mastitis is a dominant disease in dairy cattle farms in the study area with a prevalence of 51.2% in cow and 50.3% in quarter level. The major isolated pathogens were S. aureus, C. bovis, and C. pyogenes, which accounted 15.9%, 11.9%, 12.7%, respectively. Staphylococcus aureus was resistance to many drugs while E. coliand CNS were both resistant to penicillin G. The resistance of staphylococci to antibiotics is a major obstacle to the successful treatment of mastitis in dairy cattle. The indiscriminate and ill-advised administration of antibiotics and irrational treatment of mastitis with different antibiotics have chances for the development of multiple drug resistance, which in turn has an impact on public health. Hence, subclinical mastitis is considered to be one of the most important diseases that threaten public health and the dairy industry. It is imperative to screen the affected animal from the herd for timely treatment or culling. The awareness of risk factors and mastitis causing pathogens are essential to control the disease in developing countries. Also, preventive measures are important to minimize antimicrobial usage. This could help further for the prevention of antibiotic resistance strains and provision of good quality milk to the consumers.

Acknowledgements

The authors wish to thank the staff of Tiyo Livestock Development and Fishery office and Tiyo Animal Health for their cooperation and help during the study period. The help of Anubha in computer work is also appreciated.

References

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