Volume 10, Issue 2: 53-57; March 25, 2020  
Misoon Esam ALMOBARAK1, Reem Rabie MOHAMMED SALIH2 and Haytham Hashim GIBREEL3  
1Faculty of Veterinary Medicine, University of Khartoum, Sudan  
2Head of Department of Clinical Medicine, Faculty of Veterinary Medicine, University of Khartoum, Sudan  
3Head of the Department of Silviculture, Faculty of Forestry, University of Khartoum, Sudan  
Supporting Information  
ABSTRACT: Dairy industry has recently grown as a very important economic national source of income. In Sudan,  
many dairy owners introduced foreign blood. This might result in a progeny of mixed blood cows with lowered  
resistance to endogenous and locally prevailing diseases such as mastitis. In this study 60 milk samples were  
obtained from Frisian cows in Elrudoan and Elmouileh Convention in Omdurman, Khartoum State, Sudan.  
Samples positive for bacterial growth were identified using the gram stain and various conventional biochemical  
tests. Hundred species of bacteria were isolated from 60 samples of milk. A total of 70 (70%) were gram  
positive, and 30 (30%) were gram negative bacteria. Among the total of the gram positive isolates, 40 (57.1%)  
were Staphylococcus spp., 18 (25.7%) were Bacillus spp., 6 (8.6%) Streptococcus spp., 4 (5.7%)  
Corynebacterium spp., and 2 (2.9%) were Actinomyces spp. and from gram negative isolates, 26 (86.7%) were  
Enterobacter spp. and 4 (13.3%) were E. coli. Antibiotic susceptibility tests to Tylosin and Enroflaxcin were  
performed for the isolated bacteria (Staphylococcus aureus, Staph. epidermidis, Enterobacter aerogenes and  
Enterobacter faecalis). The isolated bacteria were found to be highly sensitive to Tylosin and Enrofloxacin.  
Keywords: Tylosin, Enroflxacin, Bovine, Mastitis, In vitro  
Bovine Mastitis is a multi-etiological and complex disease, which is defined as inflammation of parenchyma of  
mammary glands. Mastitis is considered the main disease in dairy herds (Kaneen and Bandhard, 1990). The  
occurrence of disease is an outcome of interplay between three major factors: infectious agents, host resistance,  
and environmental factors (Gera and Guha, 2011). It is characterized by physical, chemical and, usually,  
bacteriological changes in milk and pathological changes in glandular tissues (Radostitis et al., 2000). Mastitis is a  
global problem as it adversely affects animal health, quality of milk and the economics of milk production, affecting  
every country, including developed ones and causes huge financial losses (Sharma et al., 2007). It is the most  
important disease in dairy cattle and more affect in economic, the most damaging (Ashish et al. 2000; Sharma et  
al. 2012; Elango et al. 2010; Mostert et al. 2004). Mastitis is caused by several species of common bacteria, fungi,  
mycoplasmas and algae (Batavani et al., 2007). Most mastitis is of bacterial origin, with just a few of species of  
bacteria accounting for most cases.  
Mastitis pathogens are categorized as contagious or environmental (Kivaria, 2006). Contagious pathogens live  
and multiply on and in the cow’s mammary gland and are spread from cow to cow, primarily during milking.  
Contagious pathogens include: Staphylococcus aureus, Streptococcus agalactiae, Mycoplasma spp. and  
Corynebacterium bovis (Radostitis et al., 2000). Environmental mastitis can be defined broadly as those intra-  
mammary infections (IMI) caused by pathogens whose primary reservoir is the environment in which the cow lives  
(Smith et al., 1985). The most frequently isolated environmental pathogens are Streptococci, other than S.  
agalactiae, commonly referred to as environmental streptococci usually S. uberis and S. disagalactiae and gram-  
negative bacteria such as Escherichia coli, Klebsiella spp. and Enterobacter spp. (Hogan et al., 1999).  
Tylosin is macrolide antibiotic produced from Streptomyces fradiae and related structurally to erythromycin  
(Plumb, 2002; Giguere, 2013). It is the first antimicrobial of the fluoroquinolones group available to veterinarians,  
they are bactericidal, their wide spectrum of antimicrobial activity includes various microorganisms such as gram  
positive, gram negative bacteria , mycoplasma, and chlamydiae (Pyorala et al., 1994). Enrofloxacin is alternative  
Citation: Almobarak ME, Mohammed Salih RR and Gibreel HH (2020). In vitro efficacy of Tylosin and Enrofloxacin in treatment of bovine mastitis causing bacteria in  
drug. The aim of this study to identify the most common causes of bacterial mastitis in cows in Omdurman locality  
and to measure the effectiveness of Tylosin and Enrofloxacin in the treatment of bacterial mastitis in vitro.  
Study area  
Study area is Omdurman city which located at the intersection of latitude 15 degrees 41 minutes north,  
longitude 32 degrees 37 minutes east, on the west bank of the Nile opposite the coupler with a tributary of the  
White Nile, and off both Khartoum and Khartoum North, which are linked by the bridge of the White Nile and  
Shambat bridge. The numbers of dairy cows were more in this locality than the other sites of Khartoum province.  
A total of 30 suspected cows were examined clinically: took the body temperature, pulse, heart rate,  
respiration, auscultation and palpation the last examination especially for mammary gland and supra mammary  
lymph nodes for presence of mastitis. Sixty milk samples from mastitic cows were collected. Mastitis was  
diagnosed when there were visible or palpable singes of udder, inflammatory changes in milk secretion, or through  
bacteriological examination of milk. During the study 60 milk sample were encountered from 30 cows suffering  
from clinical and subclinical mastitis. Milk sample were taken under from infected quarters only under aseptic  
condition for bacteriological studies. The fore milk was stripped off and about 5 ml of milk were drawn in sterile  
disposable bottle. All samples collected were immediately placed on ice in a thermo flask after collection.  
Isolation and identification of bacteria  
Culture. Milk samples were collected from mastitic cows were cultured in two media: Blood agar and  
MacConkeys agar. After culturing the plates were incubated for 24 hours at 37ºC. Purification was achieved by  
further subculturing on nutrient agar and incubated at 37ºC for 24 hours. After purification, a full loop from purified  
culture was taken and a smear was made and stained with Gram´s stain to differentiate between Grams positive  
and Grams negative bacteria and to see the shape of bacteria. Plates were examined for cultural characteristics  
and biochemical reactions according to standard keys (Barrow and Feltham, 2003). Staphylococci were studied in  
particular for haemolysis and coagulase production using human plasma. A positive coagulase test was judged as  
any degree of clotting from a loose clot suspended in plasma to a solid clot (Barrow and Feltham, 2003).  
Purification of cultures. Purification of culture was made by sub-culturing a part of a typical and well isolated  
colony on nutrient agar. This process was repeated twice. The resulting of growth was checked for purity by  
examining smears stained with Gram´s stain method.  
Identification of bacteria. The purified isolated bacteria were identified according to criteria outlined by Barrow  
and Feltham (2003) which included of: Reaction of Grams stain, shape of the bacterial colonies, presence or  
absence of spores, motility, the colonial characteristics on different media, haemolysis of blood agar and  
biochemical tests. All biochemical tests for identification of isolated bacteria were performed according to Barrow  
Antibiotic sensitivity test. Some of the bacteria that isolated through microbiological procedures were  
subjected to antimicrobial susceptibility test by disc diffusion method to identify the effectiveness of the Tylosin and  
Enrofloxacin. The sensitivity against Tylosin and Enrofloxacin were determined on Mueller Hinton agar as described  
by National Committee for Clinical Laboratory Standards (NCCLS, 2002). The results were obtained by measuring  
the diameter of the growth inhibition zone around the antibiotic disc for each isolated bacterial strain and recorded  
as sensitive, intermediate and resistant.  
Identification of isolates  
Out of the 100 isolates 70 (70%) were gram positive, and 30 (30%) were gram negative bacteria. Among the  
total of the gram positive isolates, 40 (57.1%) were Staphylococcu spp., Bacillus spp. were 18 (25.7%),  
Streptococcus spp. were 6 (8.6%), Corynebacterium spp. were 4 (5.7%), Actinomyces spp. were 2 (2.9%) and from  
gram negative isolates, Enterobacter spp. were 26 (86.7%) and E. coli were 4 (13.3%) (Figure, 1). The isolated  
Staphylococcus spp. in this study was divided into two groups: Coagulase positive included Staph. aureus and  
Staph. hyicus. Coagulase negative were Staph. epidermidis, Staph. chromogens, Staph. simulans and Staph.  
Citation: Almobarak ME, Mohammed Salih RR and Gibreel HH (2020). In vitro efficacy of Tylosin and Enrofloxacin in treatment of bovine mastitis causing bacteria in  
hominis. The total 70 isolates: 18 (25.7%) were found to be bacillus species. There were (6%) isolates of  
Streptococcus spp obtained in this study. Other isolates were: Corynebacterium spp., Actinomyces spp.,  
Enterobacter spp. and E. coli. Table 1 shows the quality control limits for antibiotics. The results of sensitivity tests  
by used Tylosin and Enrofloxacin are shown in Tables 2 and 3. They were affected in Staph. aureus, Staph.  
epidermidis, Enterobacter aerogenosa, Enterococcus faecalis.  
Table 1 - Quality control limits for antibiotics  
Antimicrobial agent  
Zone diameter in mm  
Staph. aureus  
S: sensitive; I: intermediate; R: resistant  
Table 2 - The efficacy of Tylosin against different types of bacteria.  
Isolated bacteria  
Zone of inhibition  
Staph. aureus  
Staph. epidermidis  
Enterobacter aerogenosa  
Enterococcus faecalis  
S: Sensitive; I: Intermediate  
Table 3 - The efficacy of Enrofloxacin against different types of bacteria  
Isolated bacteria  
Zone of inhibition  
34 mm  
Staph. aureus  
Staph. epidermidis  
Enterobacter aerogenosa  
26 mm  
30 mm  
25 mm  
Enterococcus faecalis  
S: sensitive  
Figure 1 - Total of different isolated bacteria from mastitic milk samples  
Citation: Almobarak ME, Mohammed Salih RR and Gibreel HH (2020). In vitro efficacy of Tylosin and Enrofloxacin in treatment of bovine mastitis causing bacteria in  
Staph hyicus Staph hominis  
Staph aureus  
chromogens epidermidis  
Figure 2 - Species of Staphylococci isolates  
Bovine mastitis is a common disease entity of dairy cows, accompanied by physical, chemical, pathological and  
bacteriological changes in milk and glandular tissue (Samad, 2008). It is a harmful disease affecting the dairy  
industry worldwide and is a matter of great concern for leading milk producing country because of the losses  
incurred due to high morbidity, discarded milk, treatment costs and reduced milk production, thus drawing in more  
attention towards its treatment and control (Mohanty et al., 2013).  
Apart from the economic losses, mastitis can have serious implications on public health. Mastitis which is  
mostly caused by the interaction of multiple pathogenic agents (primarily bacteria), can expose human beings to  
various organisms through infected milk, thus serving as a media for transmission of various zoonotic diseases like  
T.B, brucellosis, diphtheria, scarlet fever and Q fever (Mahantesh and Kaliwal, 2011).  
In fact, S. aureus was one of the most frequently isolated staphylococci, supporting the assertion that this  
microorganism numbers among the main mastitis pathogens in the Czech Republic (Rysanek et al., 2007). In this  
study the isolation of E. coli, Bacillus spp., and Enterobacter spp., might be attributed to poor or absence of hygiene.  
This suggestion was supported by the statement of Quinn et al. (2004) who mentioned that Bacillus cereus and E.  
coli were isolated from mastitic milk of bovine. These results collectively support our results in this study also the  
result is in agreement with Sudhan et al. (2005).  
The detection of Actinomyces bovis in the mastitic milk in this study was in agreement with Quinn et al. (2004)  
who mentioned that this bacterium among a rarely Gram- positive rod shaped causing bovine mastitis.  
In this study found the isolates were sensitive to tylosin and enrofloxacin sensitive, these antibiotics are used  
either for treatment of clinical cases to avoid the spreading of the causative agent, in prophylactic measures to  
eliminate the susceptibility of animals for prevention of new infection or growth promotion in weight gain for  
fattening programmes. This study is an agreement with Anon (2011), who reported that indicated for the treatment  
of local signs (inflammation, milk quality and yield) associated with per acute/acute mastitis in lactating dairy  
cattle. Also the isolates in this study were affected with Tylosin and this is an agreement with (Pyorala et al., 1994).  
In this study we have showed that the incidence of bovine mastitis is high in Omdurman locality. The most frequent  
isolated bacteria are Staphylococci. In addition, other opportunistic and environmental organisms were isolated  
from mastitic milk samples. Inadequate stall or pasture management e.g. dirty and wet bedding material or muddy  
areas as well as in proper milking procedures lead to an increased infection risk.  
1- In dairy farm hygienic procedure must be from Practice sustainable.  
2- Antimicrobial sensitivity testing should be practiced before treatment of mastitis with antibiotics.  
3- Farmers should be aware about what suitable antibiotics to be used for specific mastitis case.  
4- The usage of antibiotics in dairy farm should be under supervision of veterinarian to avoid missed used  
which leading to the development of antibiotic resistance bacteria.  
Citation: Almobarak ME, Mohammed Salih RR and Gibreel HH (2020). In vitro efficacy of Tylosin and Enrofloxacin in treatment of bovine mastitis causing bacteria in  
Corresponding author  
E-mail: reemat7@yahoo.com ORCİD: 0000-0001-6611-5562  
Authors’ contribution  
All authors contributed equally to this work.  
Availability of data  
The data can be available to the journal upon request.  
Consent to publish  
Not applicable  
Conflict of interest  
The authors declare they have no competing of interests.  
The authors would wish to acknowledge the Department of Microbilology, Faculty of Veterinary Medicine, University  
of Khartoum for their support through the whole process of developing this publication.  
Anon (2011). Special precautions for the disposal of unused veterinary medicinal product or waste materials. 1.  
Batavani RA, Asri S and Naebzadeh H (2007). The effect of sub-clinical mastitis on milk composition in dairy cows. Iranian Journal of  
Veterinary Research. 8 (3): 205211. DOI: https://dx.doi.org/10.22099/ijvr.2007.925  
Elango A, Doraisamy KA, Rajarajan G and Kumaresan G (2010). Bacteriology of sub-clinical mastitis and anti-biogram of isolates  
recovered from cross-bred cows. Indian Journal of Animal Research. 44 (4): 280284. Google Scholar  
Gera S and Guha A (2011). Assessment of acute phase proteins and nitric oxideas indicator of subclinical mastitis in Holstein × Haryana  
cattle. Indian Journal of Animal Sciences. 81 (10): 10291031. Google Scholar  
Giguere S (2013). Macrolides, azalides and ketolides. In: Antimicrobial Therapy in Veterinary Medicine, 4th ed. (Giguere SJF, Prescott JD,  
Baggot RD, Walker PM, Dowling Eds.) Blackwell Publishing, London. Wiley Online Library, 191-205. Google Scholar  
Hogan SJ, Gonzales RN, Harmon JR, Nickerson SC, Oliver SP, Pankey JW and Smith LK (1999). Laboratory Handbook on Bovine  
Mastitis.Published by National Mastitis Council, Inc., Verona, WI 53593, USA. Google Scholar  
Kaneene JB, Hurd HS. (1990). The national animal health monitoring system in Michigan. III. Cost estimates of selected dairy cattle  
diseases. Preventive Veterinary Medicine. 8(2-3):127-40. Google Scholar, DOI: https://doi.org/10.1016/0167-5877(90)90006-4  
Kivaria FM (2006). Epidemiological studies on bovine mastitis in smallholder dairy herds in the Dares Salaam Region, Tanzania. Doctoral  
thesis, Utrecht University. The Netherlands. Google Scholar | Direct Link  
Mahantesh MK and Basappa BK (2011). Prevalence and antimicrobial susceptibility of bacteria isolated from bovine mastitis. Adv. Appl.  
Sci. Res. 228 (6): 229-235. Direct Link  
Mostert BE, Banga C, Groeneveld E and Kanfer FHJ (2004). Breeding value estimation for somatic cell score in South African dairy cattle.  
South African Journal of Animal Science. 34 (2): 3234. Google Scholar, DOI: https://dx.doi.org/10.4314/sajas.v34i6.3823  
Mohanty NN, Das P, Pany SS, Sarangi LN, Ranabijuli S and Panda HK (2013) Isolation and antibiogram of Staphylococcus, Streptococcus  
and E. coli isolates from clinical and subclinical cases of bovine mastitis, Veterinary World 6(10): 739-743. Google Scholar, DOI:  
Plumb DC (2002). Veterinary Drug Handbook. Iowa State Press, Ames, IA. 631- 633.  
Pyorala S, jousimies-somer H and Mero M (1994). Clinical, bacteriological and therapeutic aspects of bovine mastitis caused by aerobic  
and anaerobic pathogens. British Veterinary Journal. 148(1): 54-62. DOI: https://doi.org/10.1016/0007-1935(92)90067-B  
Quinn PJ, Carter ME, Markey B and Carter GR (2004). Clinical Veterinary Microbiology. Mosby Publishing, London. 43 (55):327-344. ISBN  
0-7234-1711-3, Academia  
Radostits OM, Gay CC, Blood DC, Hinchcliff KW. (2000). A textbook of the diseases of cattle, sheep, pigs, goats and horses. Veterinary  
medicine. 9: 603-700. Google Scholar  
Rysanek D, Babak, V and Zouharova M (2007). Bulk tank milk somatic cell count and sources of raw milk contamination with mastitis  
pathogens. Veterinarni Medicina. 52 (6): 223230. Google Scholar  
Samad MA (2008). Animal Husbandry and Veterinary Science, volume II, LEP pub no.11, Bangladesh. Google Scholar  
Sharma N, Maiti SK and Sharma KK (2007). Prevalence, etiology and antiobiogram of micro-organisms associated with sub-clinical  
mastitis in buffaloes in Durg, Chhattisgrh State (India). International Journal of Dairy Science. 2 (2): 145151. Google Scholar, DOI:  
Sharma N, Rho GY, Hong YH, Lee TY, Hur TY and Jeong DK. (2012). Bovine mastitis: an Asian perspective. Asian Journal of Animal and  
Veterinary Advances. 7: 454476. Google Scholar  
Smith KL, Todhunter DA and Schoenberger PS (1985). Environmental mastitis: cause, prevalence, prevention. Journal of Dairy Science. 68  
Sudhan NA, Singh R, Singh M and Soodan JS (2005). Studies on prevalence, aetiology and diagnosis of sub clinical mastitis among cross  
bred cows. Indian Journal of Animal Research. 39(2): 127-130. Google Scholar | Direct Link  
Citation: Almobarak ME, Mohammed Salih RR and Gibreel HH (2020). In vitro efficacy of Tylosin and Enrofloxacin in treatment of bovine mastitis causing bacteria in  
Ashish A, Sisodia RS, Sharma RK, Misraulia KS and Garg UK (2000). Incidence of sub-clinical mastitis in cows of Malwa Region of Madhya  
Pradesh. Indian Journal of Dairy Science. 53 (4): 328331. Google Scholar  
Citation: Almobarak ME, Mohammed Salih RR and Gibreel HH (2020). In vitro efficacy of Tylosin and Enrofloxacin in treatment of bovine mastitis causing bacteria in