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International Journal of Dairy Science

Year: 2020 | Volume: 15 | Issue: 1 | Page No.: 38-47
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ABSTRACT

Background and Objective: The hygienic measures of the farm especially during milking and the storage condition are all key agents in the count and kind of pathogen contamination present in the bulk milk, so the influence of some hygienic measures (bedding, silage, equipment swabs, worker hand swabs and SCM cow’s milk samples) of the farm on the prevalence of subclinical mastitis (SCM) pathogens was evaluated in this study. Material and Methods: Swabs (workers hands and milking equipment) silage, bedding, SCM samples were collected from dairy farm located in Fayoum district, Egypt and the different types of SCM pathogens were isolated. Bulk milk tank and water samples were subjected to count (total colony and coliforms), respectively. The significant (p<0.05) relationship between hygienic condition of environmental samples and prevalence of different subclinical mastitis pathogens was calculated. Results: In this study, 51 BTM (bulk tank milk) samples were examined through the 4 seasons of the year for the TCC, the highest mean value was 4.8×104±3.6×103 CFU mL–1 in the spring. The results of coliforms count of the examined 24 water samples from trough showed that, the highest mean value 8.3×10±3.45 MPN/100 mL was observed at the winter. Conclusion: There was a significant relation between the hygienic condition of the farm and microorganisms causing subclinical mastitis which could enter the food chain and accidently mixed with bulk tank milk which may induce health hazards to human, so adequate sanitary measures should be taken by improving the personnel, cow, equipment and farm hygiene. Producers should well know the influence of each hygienic measure on a load of microbes in raw milk and how they can control to prevent the occurrence of clinical form of mastitis.
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INTRODUCTION

Specific cells in mammary gland are responsible for milk synthesis (sterile in udder). After this phase, the microbial contamination may occur from three main sources, interior or exterior of the udder and during handling, storage of the milk1. Bovine mastitis is highly significant disease of dairy cows from an economic, social and public health point of view2. It is an inflammation of the mammary gland of differ etiology. The disease can sitting in a clinical and subclinical form. In SCM, there is no detectable change in the udder and there are no remarkable abnormalities in the milk but the production of milk reduced3. However, mastitis is caused by multiple bacterial species; it is the result of contest between the pathogen and defense mechanisms of host. Abundant environmental causes favor the bacterial invasion of the mammary gland as; incorrect milking procedures, unhygienic condition of milking machines, inadequate sanitary level of the farm and cows4. Mastitis is caused by a broad spectrum of microbes and, assorted into contagious pathogens are those for which udders of infected animals serve as the main reservoir. They spread from cow to cow, primarily during milking and tend to result in sub-clinical infections with flare-ups of clinical form. Contagious microbes include: Staphylococcus aureus and Streptococcus agalactiae5. Environmental mastitis can be defined as those Intra-Mammary Infections caused by pathogens whose primary barrage is the environment. Pathogens of environment include E. coli and Klebsiella spp., infection of these microbes was characterized by short duration6. An administrative ability of the dairy farm is a key and critical points for mastitis control or for prevention the existing infections7. Therefore, this study was carried out for monitoring the influence of some of hygienic measures of the farm on the prevalence of SCM and its causing microorganisms that had a highly potential hazard on the milk consumers.

MATERIALS AND METHODS

Study area: Private dairy farm located in Fayoum district, Egypt was chosen for this study all over 4 seasons during the period of December, 2016 to November, 2017. The farm was depending on automatic Afi milking machine system where the cows were milked 3 times daily and the milk was collected automatically in large milk tanks. The water source was running water supply from municipal pipes, while the cows obtained their daily water needs from common water trough.

Collection and preparation of samples: A total of 284 different environmental samples were obtained from bedding, silage, swabs from worker hands, milking equipment in some selected points in the farm in addition water samples from troughs and pipes (24 each) and fifty one samples of Bulk Tank Milk (BTM) were subjected to bacteriological analysis for detection of influence of some hygienic measures on the prevalence of subclinical mastitis (SCM) applied on the farm.

Hands of workers and milking equipment swabs: The swabs were removed from the sterile wrapping and moisten the tip by immersing it in a tube containing sterile buffered peptone water (Oxoid, CM1049). The swabs were pressed against the wall of the tube to remove excess liquid. Moistened sterile swabs were rubbed onto the palm of both hands, area between fingers, finger tips and streaked an estimated area of 10 cm2 on the surface of the equipment to be investigated as teat cups, pipelines as well as collecting tanks.

Each swab was inserted in the tubes (labeled with date) and transported to the laboratory immediately in an ice box. The tubes containing swabs were mixed and homogenized using shaker (Fisher scientific vortex mixer)8.

Silage and bedding samples: Fifty grams of bedding were collected from the area in which the udder of recumbent cow was resting and 50 g of silage also were collected from different areas were placed into sterilized bags, labeled, kept in an ice box and transported to the laboratory immediately. The bedding and silage samples were mixed with two hundred ml of Peptone Water (Oxoid, CM0009) in 2-chamber filter bags and stomached (Seward 400 scientific lab blender) for 1 min9.

SCM cow’s milk samples: The 444 SCM cow’s milk samples were collected from 695 lactating Holstein Friesian cows according to Radostits et al.10 and prepared for bacterial investigation according to Carter and Cole11, where the percentage of SCM in the farm was found to be at cows and quarter level 63.88 and 41.18%, respectively by California mastitis test (CMT) according to Schalm et al.12.

The initial suspension of all prepared samples was incubated aerobically at 37°C (24-48 h) for aerobic bacterial growth. A loopful from the initial suspension was sub-cultured on 10% sheep blood agar (Oxoid, CM0055), MacConkey agar (Oxoid, CM0115), Pseudomonas specific agar media (Oxoid, CM0559), Bacillus specific agar media (Oxoid, CM0617), Edwards media (Oxoid, CM0027), EMB agar media (Oxoid, CM0069), Baird parker agar media (Oxoid, CM1127+SR0054) and Mannitol salt agar plates (Oxoid, CM0085) and incubated aerobically at 37°C (24-48 h) for isolation of some microorganisms. The suspected colonies were isolated on nutrient slope agar (Lab M, LAB008) and incubated at 37°C/24-48 h for further identification. The bacterial isolates were identified by the Vitek 2 compact system according to BioMerieux13.

The significant (p<0.05) relationship between hygienic condition of environmental samples and prevalence of different subclinical mastitis pathogens was calculated using Chi-square value and also p-value was calculated between the mean values of TCC of examined BTM samples and MPN for water samples (SPSS software; version 25).

Total colony count of BTM samples: Sixty milliliters of bulk tank milk samples were collected after proper turn on the sterile agitator from the top of the bulk tank using clean sanitized dipper. Samples were immediately labeled and transported to the laboratory in an ice box. Tenth fold serial dilutions were prepared for the collected samples and the total colony count was performed within 24 h using standard plate count agar medium (Oxoid, CM0463)14.

Coliforms count in water samples: A water sample of 100 mL was collected from water troughs in the yard or pipes of the examined dairy farm in sterile screw capped bottles of 150 mL capacity by following strict aseptic procedures. Collected samples were identified and kept in an ice box and transported immediately to the laboratory. Five tubes were prepared for three serial dilutions of a water sample and incubated at 37°C for 24-48 h. The MPN index is determined by comparing the pattern of positive results (the number of tubes showing growth at each dilution) with standard statistical tables. The tabulated value is reported as MPN/100 ml of sample15.

RESULTS

Bacterial species isolated from environmental samples: Table 1 reveals that the most often microorganisms isolated from the bedding samples (n = 38) were Escherichia coli 29 (76.30%), Enterococcus species 18 (47.40%), Staphylococcus aureus 16 (42.10%), Pseudomonas aeruginosa 11 (28.94%), Proteus species 3 (7.90%), while Enterobacter aeruginosa, Serratia and Klebseilla species were isolated in a percentage of 5.30% for each.

Bacterial species isolated from SCM cow’s milk samples: A total of 444 SCM cow’s milk samples were subjected to bacteriological investigation. The data obtained showed that the most isolated microorganisms from the samples were Staphylococcus aureus, Enterococcus species, Escherichia coli and Streptococcus agalactiae in a percentage of 66.66, 51.80, 47.29 and 23.87, respectively. While S. chromogenes, Klebsiella oxytoca, Bacillus subtilis, Pseudomonas aeruginosa and Bacillus cereus were found in the examined samples in a percentage of 17.34, 13.73, 10.13, 7.20 and 6.08, respectively.

Table 1:
Prevalence of bacterial species isolated from different environmental samples and SCM cow’s milk samples
Image for - Influence of Some Hygienic Measures on the Prevalence of Subclinical Mastitis in a Dairy Farm
n: Number of examined samples, Number: Number of isolated microorganisms (Bedding = 83, silage = 49, equipment = 42, worker hand = 56, SCM milk = 1153)

Relation between hygienic conditions of environmental samples and SCM pathogens: There is a significant (p<0.05) relation between hygiene of environmental samples and prevalence of (Enterococcus species, Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli ) with a chi-square value 41.63, 8.58, 32.68 and 101.19, respectively (Table 2).

Results of total colony count (CFU mL–1) of the examined BTM samples (n = 51): In the present study, 51 BTM samples were examined through the four seasons of the year for the TCC, the data presented in the Table 3 revealed that the highest mean value of the TCC of the BTM was observed in the spring (4.8×104±3.6×103 CFU mL–1), followed by summer, winter and autumn seasons with a mean values 4.4×104±3.4×103, 4.0×104±3.1×103 and 2.7×104±2.3×103 CFU mL–1, respectively and absence of significant difference between all seasons of year (p>0.05).

Table 2:
Relationship between hygienic conditions of environmental samples and prevalence of the different SCM pathogens
Image for - Influence of Some Hygienic Measures on the Prevalence of Subclinical Mastitis in a Dairy Farm
*Significance difference (p<0.05)

Table 3:
Statistical analytical results of total colony count (CFU mL–1) of the examined bulk tank milk samples (n = 51)
Image for - Influence of Some Hygienic Measures on the Prevalence of Subclinical Mastitis in a Dairy Farm
n: Total number of examined samples, aNon-significant difference (p>0.05)

Results of coliforms count (MPN/100 mL) of the examined water samples (n = 48): The data presented in Table 4 demonstrate that the mean value of coliforms count of the examined 24 water samples from pipes is <3 MPN/100 mL all over the year, while results of coliforms count of the examined water samples from trough showed that the highest mean value (8.3×10±3.45 MPN/100 mL with a percentage of 83.3) was observed at the winter, followed by summer and autumn with a mean values of (4.3×10±1.79 and 4.1×10±1.71 MPN/100 mL with a percentage of 83.3 and 50.0), respectively.

DISCUSSION

Milk is a perfect medium for the propagation of various microorganisms, particularly bacterial pathogens. The SCM is the most prevalent disease that affects milk hygiene in dairy animals16. Choosing the environment in which lactating cows will spend most of their time has great effect on the productivity, health, reproduction of cows as well as milk quality and farm profitability. Dirty housing, unsuitable hygiene and the incorrect cow preparation for milking are all prerequisite factors that can potentially lead to SCM17. The degree of cleanliness of the milking system significantly influences the TCC and coliforms count. Environmental contaminants from bedding, manure and feeds as well as the less efficient cleaning and the absence of sanitizers support the growth of variety of microbes18.

The most often microorganism could be isolated from the environmental samples and SCM cow’s milk samples was Staphylococcus aureus in a percentage of 42.10, 52.60, 28.90, 30.50 and 66.66 from bedding, silage, equipment swabs, worker hand swabs and SCM cow’s milk samples, respectively, while Escherichia coli was 76.30, 26.30, 7.80, 6.80 and 47.29%, respectively. In this study, the highest mean value of TCC of BTM was observed in the spring, while the Coliforms count of the examined trough water samples was 8.3×10±3.45 MPN/100 mL observed in the winter.

The results in Table 1 are nearly similar to data recorded by Edberg et al.18, Hogan et al.19 and Kristula et al.20 for Klebseilla and Enterobacter species, while the Staphylococcus aureus incidence was nearly similar to those reported by Hassan21 and Saied22, higher incidence were recorded by Nahed et al.23, however, lower incidence reported by Elbably et al.24 and Mcauley et al.25.

Table 4:
Statistical analytical results of coliforms count (MPN/100 mL) of the examined water samples (n = 48)
Image for - Influence of Some Hygienic Measures on the Prevalence of Subclinical Mastitis in a Dairy Farm
n: Total number of examined samples, a,bSignificant difference (p<0.05)

In addition, the microorganisms which isolated from 38 samples of silage were S. aureus, Escherichia coli, Enterococcus species and Bacillus cereus in a percentage of 52.60, 26.30, 21.10 and 10.50, respectively. While Pseudomonas aeruginosa, Enterobacter aeruginosa and Citrobacter species were isolated in a percentage of 5.30 for each, However, Enterobacter cloaca complex was detected in a percentage of 2.60.

Staphylococcus aureus, Escherichia coli, Enterococcus species and Pseudomonas aeruginosa, were isolated from the 90 equipment swab samples in a percentage of 28.90, 7.80, 6.70 and 3.30, respectively. These results are nearly similar to those reported by Nahed et al.23, higher incidence were recorded by Saied22.

One hundred eighteen samples of worker hand’s swabs were positively for Staphylococcus aureus, Escherichia coli, Enterococcus species and Enterobacter aeruginosa in a percentage of 30.50, 8.45, 6.80 and 1.70, respectively. These data are nearly similar to results reported by Elbably et al.24, higher incidence was recorded by Saied22, Nahed et al.23 and Mohamed et al.26, however, lower incidence was reported by Lee et al.27, Parmar et al.28 and Zeinhom and Abdel-Latef29. Escherichia, Klebsiella and Enterobacter species are considered to be an indicative of fecal contamination30.

Bacteria involved in bovine mastitis are classified as either contagious or environmental pathogens based on their epidemiological association with the disease. The SCM is a multietiologic disease, many microorganisms is implicated as causes. Contagious pathogens are those organisms transmitted from animal to animal where the primary reservoir harboring the pathogens is the cow. The SCM dominant contagious pathogens are S. aureus and Streptococcus agalactiae. Environmental pathogens are transmitted during milking from the environment serving as the primary source of these organisms. The main pathogens in this set are Coliforms as Escherichia coli, Klebsiella and Enterobacter species, these organisms are usually found in the animal environment and can be contracted by udder31,32.

If Staphylococcus aureus persists in mammary gland, the milk comes into contact with teats of infected animals, the infection becomes contagious33. The results were showed in (Table 1) reveal that the nearly similar percentage of the isolated S. aureus from the SCM cow’s milk samples was obtained by Michael et al.34, Alemu et al.35 and Zenebe et al.36. The presence of S. aureus may be due to lack of effective udder washing, post milking teat as well as improper disinfection in the milking area37.

Enterococcus deserves widely distributed and makes up a large proportion of the saprophytes related with the gastrointestinal tracts of humans and animals38,39. The nearly similar percentage of isolation of the Enterococcus species was reported by Giraffa40, Domig et al.41 and Scheidegger et al.42.

Streptococcus agalactiae is considered one of the major economic losses in dairy farms, that don’t apply strict sanitary measures. It breaks the natural udder barriers, enters the teat canal and ascends in the milk through the quarter. In later phase, the acini become filled with scars resulting in quarter fibrosis43. The percentage of Streptococcus agalactiae in this study was found to be in accordance with that reported by Alemu et al.35 and Biressaw and Deme44. On the other hand the higher prevalence was recorded by Abdel-Rady and Sayed45, Ramirez et al.46 and Momtaz et al.47, however, the lower prevalence was reported by Gao et al.48, Mpatswenumugabo et al.49 and Mulate et al.50.

Coagulase negative staphylococci (CNS) have become the main SCM pathogens in several countries and its prevalence may be a consequence of poor hygienic practices in farm51. These results are supported by the data obtained from previous researches52-54.

Coliforms cause up to 25% of cows mastitis in proper managed animals55. The high percentage of SCM caused by Coliforms indicates to unsanitary production56. According to Mellenberger et al.57, Coliforms may multiply in polluted water, contaminated bedding and manure. Coliforms invade the udder through the teat sphincter when teat-ends come into contact with an environment that is contaminated with these microorganisms58.

The relatively near percentage of the isolated E. coli in this study is obtained by Ahmed et al.59 and Barbour et al.60, while lower incidence was obtained by Biressaw and Deme44, Darbaz et al.54 and Mia et al.61.

Klebsiella mastitis has become a trouble in sand bedding farms, this may be attributed to fecal shedding of Klebsiella by healthy animals62. Incidence of Klebsiella oxytoca in this study is agreed to results that reported by Chander et al.63 and Saidi et al.64. While the higher prevalence recorded by Oluchi65 and Ali and Ali66, but lower findings were observed by Shahid et al.67, Kateete et al.68 and Ahmed et al.69. Mouth ingestion of Klebsiella spp. could be due to the presence of the organism in and/or on crops, or due to fecal contamination of water and the fecal shedding of Klebsiella lead to contamination of dairy animals and their environment70. Klebsiella mastitis causes a considerable decrease in milk production, with average losses of 7.6 kg/day after infection71. Thus, prevention of exposure is the main strategy for control of this type of mastitis20.

Pseudomonas aeruginosa subclinical mastitis has increased in large dairy farms, causing significant problems. The origin of SCM due to P. aeruginosa has been traced to contaminated water sources. Contamination of the parlor floor by fore-milking also facilitated its transmission72. The low prevalence of P. aeruginosa species in this study could be attributed to the intermittent shading nature of this organism from the udder to milk. The same prevalence of P. aeruginosa in this study is in agreement with the results reported by Ayano et al.73.

Sewage, water, soil and food are mostly sources of Citrobacter amalonaticus. It produces enterotoxins acting on intestinal mucosa74. The incidence of Citrobacter species in this study was nearly similar to that isolated from mastitic cow’s milk by Kateete et al.68 and Turutoglu et al.75, while the higher incidence was reported by Oluchi65.

Bacillus cereus mastitis has a lower incidence than Staphylococcus spp. and Streptococcus spp.61. Bacillus species are environmental and also found among normal flora of the teat skin. So, Bacillus spp. can infect mammary gland when animals have incoming to pasture76. The prevalence of Bacillus in this study was nearly similar to that reported by Mulate et al.50 and Abera et al.77. While the higher incidence reported by Mpatswenumugabo et al.49, Darbaz et al.54 and Vasquez-Garcia et al.78.

In this study, a Chi square test of independence was performed to examine the relation between hygienic conditions of environmental samples and prevalence of the different SCM pathogens. There was a significant (p<0.05) relation between hygiene state of (bedding, silage, equipment swabs, worker hand swabs) and all investigated SCM pathogens expect Enterobacter aeruginosa and Enterobacter cloaca complex. The results presented in Table 2 are nearly similar to the data that recorded by Abera et al.77, DeVries et al.79, Nwankwo et al.80, Azevedo et al.81, Suleiman et al. 82 and Sanotharan et al.83.

Low bacterial count milk production involves multiple factors related to cow health and udder hygiene, hygiene of the milking environment in which the animals are lived. Contamination of BTM occurs by different types of microorganism84,85. Collected milk from clean, healthy animals has TCC value (<1×103) CFU mL–1. Higher count suggest that contaminating bacteria are entering the milk from a variety of possible sources and poor cleaning and sanitizing during milking. Counts of 10×103 CFU mL–1 or lower should be achievable by most dairy farms86.

The obtained results (Table 3) are nearly similar to those reported by Hassan21, Saied22, Axelsson87, Al-Tahiri88, Perkins et al.89, Mhone et al.90 and El-Leboudy et al.91. The cooler condition, the more bacteria are found within the Bulk Tank Milk because of an increase in bacteria from the animal directly. The difference in count of bacteria between the seasons was larger, with increasing total milk production; the effect of season on bacterial count in milk was the least89,92,93. Coliforms count of water is necessary to determine sanitary quality. According to the Egyptian Standard94 the water should be free from coliforms, while Oshiro95 recorded that for animal consumption (young calves), coliforms should be <1/100 mL. For adult animals, coliforms should be <10/100 mL.

This result (Table 4) is similar to data reported by Rice and Johnson96. significant difference (p<0.05) could be obtained between the mean values of coliforms count in (spring-summer, spring-autumn and spring-winter) season, but the higher coliforms count in the winter may be due to that the water troughs were near feed bunks, or they were exposed to improper cleaning and sanitizing. This data is nearly similar to those reported by LeJeune et al.97, while higher mean value recorded by Sanderson et al.30. Cattle may contaminate the troughs with fecal material as well as extraneous matter (dust, feed or bedding) may also contaminate the trough98.

CONCLUSION

From this present study, there is a significant association between the hygienic condition of the farm environment and some of SCM pathogens (S. aureus, B. cereus, E. coli and pseudomonas aeruginosa). This significant association indicates the efficiency degree (level) of hygienic measures which applied to environment of the farm. Lack of maintenance of strict hygiene and good sanitary environment may be a contributory factor in the cause of SCM. To achieve high raw milk quality, producers should be aware of the all previous factors that influence contamination of raw milk and how they can be controlled.

SIGNIFICANCE STATEMENT

This study discover the strong relationship between some of inadequate hygienic measures which applied on this dairy farm and the prevalence of SCM pathogens, that can be beneficial for the farm to pay more attention during applying of these measures. This study will help the researcher to uncover the critical areas of zoonotic pathogens of SCM and the demand for more investigation concerning the SCM.

REFERENCES

  1. Muhammad, K., I. Altaf, A. Hanif, A.A. Anjum and M.Y. Tipu, 2009. Monitoring of hygienic status of raw milk marketed in Lahore city, Pakistan. J. Anim. Plant Sci., 19: 74-77.
    Direct Link
  2. Firth, C.L., C. Laubichler, C. Schleicher, K. Fuchs and A. Kasbohrer et al., 2019. Relationship between the probability of veterinary-diagnosed bovine mastitis occurring and farm management risk factors on small dairy farms in Austria. J. Dairy Sci., 102: 4452-4463.
    CrossRefDirect Link
  3. De Vliegher, S., I. Ohnstad and S. Piepers, 2018. Management and prevention of mastitis: A multifactorial approach with a focus on milking, bedding and data-management. J. Integr. Agric., 17: 1214-1233.
    CrossRefDirect Link
  4. Cvetnic, L., M. Samardzija, B. Habrun, G. Kompes and M. Benic, 2016. Microbiological monitoring of mastitis pathogens in the control of udder health in dairy cows. Slovenian Vet. Res., 53: 131-140.
    Direct Link
  5. Youssif, N.H., N.M. Hafiz, M.A. Halawa and H.M. Aziz, 2019. Genes conferring antimicrobial resistance in cattle with subclinical mastitis. Bulg. J. Vet. Med., (In Press).
    CrossRefDirect Link
  6. Abebe, R., H. Hatiya, M. Abera, B. Megersa and K. Asmare, 2016. Bovine mastitis: Prevalence, risk factors and isolation of Staphylococcus aureus in dairy herds at Hawassa milk shed, South Ethiopia. BMC Vet. Res., Vol. 12.
    CrossRefDirect Link
  7. Risvanli, A., I. Seker, N. Saat, B. Karagulle, A. Koseman and E. Kaygusuzoglu, 2017. The management practices and microbiological quality of a dairy farm with low bulk tank milk somatic cell count. Pak. Vet. J., 37: 175-179.
    Direct Link
  8. ISO., 2004. Microbiology of food and animal feeding stuffs—Horizontal methods for sampling techniques from surfaces using contact plates and swabs. ISO 18593:2004, International Standard Organization, Geneva, Switzerland.
  9. Clegg, F.G., S.N. Chiejina, A.L. Duncan, R.N. Kay and C. Wray, 1983. Outbreaks of Salmonella newport infection in dairy herds and their relationship to management and contamination of the environment. Vet. Rec., 112: 580-584.
    CrossRefPubMedDirect Link
  10. Radostits, O.M., C. Gay, K.W. Hinchcliff and P.D. Constable, 2007. Veterinary Medicine: A Textbook of the Diseases of Cattle, Horses, Sheep, Pigs and Goats. 10th Edn., Saunders Ltd., London, UK., ISBN-13: 9780702039911, pp: 1576-1580.
  11. Carter, G.R. and J.R. Cole Jr., 2012. Diagnostic Procedure in Veterinary Bacteriology and Mycology. 5th Edn., Academic Press, New York, USA., ISBN-13: 9780323138185, Pages: 620.
  12. Schalm, D.W., E.J. Carroll and N.C. Jain, 1971. Bovine Mastitis. Lea and Febiger, Philadelphia, USA., ISBN-13: 9780812103328, pp: 20-158.
  13. BioMerieux, 2013. VITEK® 2: Technology product information manual. BioMerieux Inc., France.
  14. APHA., 2004. Standard Methods for the Examination of Dairy Products. 17th Edn., American Public Health Association Inc., Washington, DC., USA., ISBN-13: 978-0875530024, Pages: 570.
    Direct Link
  15. ISO., 2012. Water quality—Enumeration of Escherichia coli and coliform bacteria—Part 2: Most probable number method. ISO 9308-2:2012, International Standard Organization, Geneva, Switzerland.
  16. Gonzalo, C., 2018. Milk hygiene in small ruminants: A review. Spanish J. Agric. Res., Vol. 15, No. 4.
    CrossRefDirect Link
  17. Bewley, J.M., L.M. Robertson and E.A. Eckelkamp, 2017. A 100-year review: Lactating dairy cattle housing management. J. Dairy Sci., 100: 10418-10431.
    CrossRefDirect Link
  18. Edberg, S.C., E.W. Rice, R.J. Karlin and M.J. Allen, 2000. Escherichia coli: The best biological drinking water indicator for public health protection. J. Applied Microbiol., 88: 106S-116S.
    CrossRefDirect Link
  19. Hogan, J.S., S.L. Wolf and C.S. Petersson-Wolfe, 2007. Bacterial counts in organic materials used as free-stall bedding following treatment with a commercial conditioner. J. Dairy Sci., 90: 1058-1062.
    CrossRefDirect Link
  20. Kristula, M.A., Z. Dou, J.D. Toth, B.I. Smith, N. Harvey and M. Sabo, 2008. Evaluation of free-stall mattress bedding treatments to reduce mastitis bacterial growth. J. Dairy Sci., 91: 1885-1892.
    CrossRefDirect Link
  21. Hassan, A.M., 2008. Prevalence of environmental mastitis in dairy farms. M.Sc. Thesis, Suez Canal University, Egypt.
  22. Saied, M.S., 2016. Prevalence and antimicrobial resistance assessment of some pathogens in bulk tank milk and dairy farm environment in Egypt. Ph.D. Thesis, Cairo University, Egypt.
  23. Nahed, M., K. Dalia, K. Ahlam and A. Abeer, 2013. A biosecurity measures application with proper treatment to overcome the risk factors that limit effective control of subclinical mastitis in dairy buffalo farms-a field study. Nat. Sci., 11: 140-151.
    Direct Link
  24. Elbably, M.A., H.H. Emeash and N.M. Asmaa, 2013. Risk factors associated with mastitis occurrence in dairy herds in Benisuef, Egypt. World's Vet. J., 3: 5-10.
    Direct Link
  25. Mcauley, C.M., K. McMillan, S.C. Moore, N. Fegan and E.M. Fox, 2014. Prevalence and characterization of foodborne pathogens from Australian dairy farm environments. J. Dairy Sci., 97: 7402-7412.
    CrossRefDirect Link
  26. Mohamed, A.A., M.M. Nasser, L.E. Mowafy, A.A. Magda and A.A. Heba, 2004. Tracing some sources of infection of some zoonotic diseases due to some bacteria among family Enterobacteriacae. Zagazig Vet. J., 32: 1-14.
  27. Lee, S.H.I., B.L.C. Mangolin, J.L. Goncalves, D.V. Neeff, M.P. Silva, A.G. Cruz and C.A.F. Oliveira, 2014. Biofilm-producing ability of Staphylococcus aureus isolates from Brazilian dairy farms. J. Dairy Sci., 97: 1812-1816.
    CrossRefDirect Link
  28. Parmar, B.C., M.N. Brahmbhatt, A.J. Dhami and J.B. Nayak, 2014. Isolation, characterization and antibiotics sensitivity pattern of Staphylococcus aureus from man, animal and environment. Scholars J. Agric. Vet. Sci., 1: 173-179.
    Direct Link
  29. Zeinhom, M.M. and G.K. Abdel-Latef, 2014. Public health risk of some milk borne pathogens. Beni-Suef Univ. J. Basic Applied Sci., 3: 209-215.
    CrossRefDirect Link
  30. Sanderson, M.W., J.M. Sargeant, D.G. Renter, D.D. Griffin and R.A. Smith, 2005. Factors associated with the presence of coliforms in the feed and water of feedlot cattle. Applied Environ. Microbiol., 71: 6026-6032.
    CrossRefPubMedDirect Link
  31. Spakova, T., J. Elecko, M. Vasil, J. Legath, P. Pristas and P. Javorsky, 2012. Limited genetic diversity of Aerococcus viridans strains isolated from clinical and subclinical cases of bovine mastitis in Slovakia. Polish J. Vet. Sci., 15: 329-335.
    CrossRefDirect Link
  32. Zeryehun, T. and G. Abera, 2017. Prevalence and bacterial isolates of mastitis in dairy farms in selected districts of Eastern Harrarghe Zone, Eastern Ethiopia. J. Vet. Med., Vol. 2017.
    CrossRefDirect Link
  33. Rofaida, M.E., 2010. Isolation and identification of the bacteria associated with bovine mastitis and detection of their specific antibodies in milk and sera. M.Sc. Thesis, Department of Microbiology, Faculty of Veterinary Medicine, University of Khartoum, Sudan.
  34. Michael, L.G., B. Deressa, F. Begna and A. Mekuria, 2013. Study on prevalence of bovine mastitis in lactating cows and associated risk factors in and around Areka town, Southern of Ethiopia. Afr. J. Microbiol. Res., 7: 5051-5056.
    CrossRefDirect Link
  35. Alemu, G., G. Almaw and M. Abera, 2014. Incidence rate of Staphylococcus aureus and Streptococcus agalactiae in subclinical mastitis at smallholder dairy cattle farms in Hawassa, Ethiopia. Afr. J. Microbiol. Res., 8: 252-256.
    CrossRefDirect Link
  36. Zenebe, N., T. Habtamu and B. Endale, 2014. Study on bovine mastitis and associated risk factors in Adigrat, Northern Ethiopia. Afr. J. Microbiol. Res., 8: 327-331.
    CrossRefDirect Link
  37. Vakkamaki, J., S. Taponen, A.M. Heikkila and S. Pyorala, 2017. Bacteriological etiology and treatment of mastitis in Finnish dairy herds. Acta Vet. Scand., Vol. 59.
    CrossRefDirect Link
  38. Linden, P.K., 2007. Optimizing therapy for Vancomycin-Resistant Enterococci (VRE). Semin. Respir. Crit. Care Med., 28: 632-645.
    CrossRefDirect Link
  39. Hamzah, A.M. and H.K. Kadim, 2018. Isolation and identification of Enterococcus faecalis from cow milk samples and vaginal swab from human. J. Entomol. Zool. Sci., 6: 218-222.
    Direct Link
  40. Giraffa, G., 2002. Enterococci from foods. FEMS Microbiol. Rev., 26: 163-171.
    CrossRefPubMedDirect Link
  41. Domig, K.J., H.K. Mayer and W. Kneifel, 2003. Methods used for the isolation, enumeration, characterisation and identification of Enterococcus spp.: 1. Media for isolation and enumeration. Int. J. Food Microbiol., 88: 147-164.
    CrossRefDirect Link
  42. Scheidegger, E.M.D., S.A.P. Fracalanzza, L.M. Teixeira and P. Cardarelli-Leite, 2009. RFLP analysis of a PCR-amplified fragment of the 16S rRNA gene as a tool to identify Enterococcus strains. Memorias Inst. Oswaldo Cruz, 104: 1003-1008.
    CrossRefDirect Link
  43. Lucia, M., S. Rahayu, D. Haerah and D. Wahyuni, 2017. Detection of Staphylococcus aureus and Streptococcus agalactiae: Subclinical mastitis causes in dairy cow and dairy buffalo (Bubalus bubalis). Am. J. Biomed. Res., 5: 8-13.
    Direct Link
  44. Biressaw, S. and T. Deme, 2015. Prevalence of bovine mastitis and determinant of risk factors in Lemu Bilbilo district, Arsi zone. Global J. Vet. Med. Res., 3: 80-85.
    Direct Link
  45. Abdel-Rady, A. and M. Sayed, 2009. Epidemiological studies on subclinical mastitis in dairy cows in Assiut Governorate. Vet. World, 2: 373-380.
    Direct Link
  46. Ramirez, N.F., G. Keefe, I. Dohoo, J. Sanchez and O. Arroyave et al., 2014. Herd- and cow-level risk factors associated with subclinical mastitis in dairy farms from the High Plains of the Northern Antioquia, Colombia. J. Dairy Sci., 97: 4141-4150.
    CrossRefDirect Link
  47. Momtaz, H., R. Soleimani and A. Jazayeri, 2017. Prevalence of virulence factors and antimicrobial resistance of Streptococcus agalactiae and Streptococcus uberis in ruminant sub-clinical mastitic milk in Iran. Int. J. Med. Lab., 4: 34-47.
    Direct Link
  48. Gao, J., H.W. Barkema, L. Zhang, G. Liu and Z. Deng et al., 2017. Incidence of clinical mastitis and distribution of pathogens on large Chinese dairy farms. J. Dairy Sci., 100: 4797-4806.
    CrossRefDirect Link
  49. Mpatswenumugabo, J.P., L.C. Bebora, G.C. Gitao, V.A. Mobegi, B. Iraguha, O. Kamana and B. Shumbusho, 2017. Prevalence of subclinical mastitis and distribution of pathogens in dairy farms of Rubavu and Nyabihu districts, Rwanda. J. Vet. Med., Vol. 2017.
    CrossRefDirect Link
  50. Mulate, B., S. Abegaz and S. Nazir, 2017. Antibiogram of bacterial pathogens isolated from subclinical mastitis in Kombolcha, South Wollo, Ethiopia. Anim. Health Prod., 65: 37-47.
    Direct Link
  51. De Visscher, A., S. Piepers, F. Haesebrouck, K. Supre and S. De Vliegher, 2017. Coagulase-negative Staphylococcus species in bulk milk: Prevalence, distribution and associated subgroup- and species-specific risk factors. J. Dairy Sci., 100: 629-642.
    CrossRefDirect Link
  52. Oliveira, C.S.F., H. Hogeveen, A.M. Botelho, P.V. Maia, S.G. Coelho and J.P.A. Haddad, 2015. Cow-specific risk factors for clinical mastitis in Brazilian dairy cattle. Prev. Vet. Med., 121: 297-305.
    CrossRefDirect Link
  53. Bhat, A.M., J.S. Soodan, R. Singh, I.A. Dhobi, T. Hussain, M.Y. Dar and M. Mir, 2017. Incidence of bovine clinical mastitis in Jammu region and antibiogram of isolated pathogens. Vet. World, 10: 984-989.
    CrossRefDirect Link
  54. Darbaz, I., A. Bastan and S. Salar, 2018. Investigation of udder health and milk quality parameters of dairy farms in Northern Cyprus. Part I: SCC and bacteriologic examination. Ankara Univ. Vet. Fak. Derg., 65: 145-154.
    Direct Link
  55. Schukken, Y., M. Chuff, P. Moroni, A. Gurjar, C. Santisteban, F. Welcome and R. Zadoks, 2012. The “Other” Gram-negative bacteria in mastitis: Klebsiella, Serratia and more. Vet. Clin. North Am.: Food Anim. Pract., 28: 239-256.
    CrossRefDirect Link
  56. Bradley, A.J., K.A. Leach, J.E. Breen, L.E. Green and M.J. Green, 2007. Survey of the incidence and aetiology of mastitis on dairy farms in England and Wales. Vet. Rec., 160: 253-257.
    CrossRefPubMedDirect Link
  57. Mellenberger, R. and C.J. Roth, 2009. California mastitis test: Fact sheet. Department of Animal Sciences, Michigan State University and Department of Dairy Science, University of Wisconsin-Madison, USA.
  58. Ssajjakambwe, P., G. Bahizi, C. Setumba, S. Kisaka and P. Vudriko et al., 2017. Milk hygiene in rural Southwestern Uganda: Prevalence of mastitis and antimicrobial resistance profiles of bacterial contaminants of milk and milk products. Vet. Med. Int., Vol. 2017.
    CrossRefDirect Link
  59. Ahmed, W.M., S.I. Abd El-Moez and G.M. Nabil, 2008. Observations on sub-clinical mastitis in buffalo-cows with emphasis on measuring of milk electrical resistance for its early detection. Global Vet., 2: 41-45.
    Direct Link
  60. Barbour, E.K., T.J. Kassabian, H. Shaib, Z. Kassaify and A. Iyer et al., 2015. The significance of Escherichia coli-induced mastitis in cows associated with the presence of virulence genes and wide range-resistance to twenty antimicrobials. Int. J. Applied Res. Vet. Med., 13: 51-63.
    Direct Link
  61. Mia, M.T., M.K. Hossain, N.A. Rumi, M.S. Rahman, M.S. Mahmud and M. Das, 2016. Detection of bacterial species from clinical mastitis in dairy cows at Nilphamari district and their antibiogram studies. Asian J. Med. Biol. Res., 2: 656-663.
    CrossRefDirect Link
  62. Munoz, M.A. and R.N. Zadoks, 2007. Patterns of fecal shedding of Klebsiella by dairy cows. J. Dairy Sci., 90: 1220-1224.
    CrossRefDirect Link
  63. Chander, Y., M.A. Ramakrishnan, N. Jindal, K. Hanson and S.M. Goyal, 2011. Differentiation of Klebsiella pneumoniae and K. oxytoca by multiplex polymerase chain reaction. Int. J. Applied Res. Vet. Med., 9: 138-142.
    Direct Link
  64. Saidi, R., D. Khelef and R. Kaidi, 2014. Antibiotic susceptibility of enterobacteriaceae species isolated from mastitic milk in Algeria. Asian Pac. J. Reprod., 3: 311-316.
    CrossRefDirect Link
  65. Oluchi, U.S., 2016. A study on raw milk for the isolation of coliforms ason herd life in two New York State dairy herds. Prev. Vet. Med., 71: 105-125.
  66. Ali, M.A.A. and Z.A.A.R. Ali, 2017. Antibiotic resistance of Raouitella ornithinolytica strains isolatd from some cow's and buffalo's milk samples causing subclinical mastitis in Misan city. Transylvanian Review, Vol. 25, No. 22.
    Direct Link
  67. Shahid, M., N. Sabir, I. Ahmed, R.W. Khan, M. Irshad, M. Rizwan and S. Ahmed, 2011. Diagnosis of subclinical mastitis in bovine using conventional methods and electronic detector. ARPN J. Agric. Biol. Sci., 6: 18-22.
    Direct Link
  68. Kateete, D.P., U. Kabugo, H. Baluku, L. Nyakarahuka and S. Kyobe et al., 2013. Prevalence and antimicrobial susceptibility patterns of bacteria from milkmen and cows with clinical mastitis in and around Kampala, Uganda. PLoS ONE, Vol. 8, No. 5.
    CrossRefDirect Link
  69. Ahmed, H.F., H.A.E. Asfour, A.M.K. Sobeih, S.F. Darwish, M. Abu El-Magd, S.A. Darwish and H.I.A. El Saka, 2016. Phenotypic and molecular identification of Klebsiella and Salmonella species isolated from subclinical mastitis milk of Egyptian buffalo. Global Vet., 16: 500-507.
    Direct Link
  70. Zadoks, R.N., H.M. Griffiths, M.A. Munoz, C. Ahlstrom, G.J. Bennett, E. Thomas and Y.H. Schukken, 2011. Sources of Klebsiella and Raoultella species on dairy farms: Be careful where you walk. J. Dairy Sci., 94: 1045-1051.
    CrossRefDirect Link
  71. Grohn, Y.T., D.J. Wilson, R.N. Gonzalez, J.A. Hertl, H. Schulte, G. Bennett and Y.H. Schukken, 2004. Effect of pathogen-specific clinical mastitis on milk yield in dairy cows. J. Dairy Sci., 87: 3358-3374.
    CrossRefPubMedDirect Link
  72. Kawai, K., Y. Shinozuka, I. Uchida, K. Hirose and T. Mitamura et al., 2017. Control of Pseudomonas mastitis on a large dairy farm by using slightly acidic electrolyzed water. Anim. Sci. J., 88: 1601-1605.
    CrossRefDirect Link
  73. Ayano, A.A., F. Hiriko, A.M. Simyalew and A. Yohannes, 2013. Prevalence of subclinical mastitis in lactating cows in selected commercial dairy farms of Holeta district. J. Vet. Med. Anim. Health, 5: 67-72.
    Direct Link
  74. Suwansrinon, K., H. Wilde, V. Sitprija and R. Hanvesakul, 2005. Enteric fever-like illness caused by infection with Citrobacter amalonaticus. J. Med. Assoc. Thailand, 88: 837-840.
    PubMedDirect Link
  75. Turutoglu, H., S. Mudul and F. Pehlivanoglu, 2002. Antibiotic susceptibility and β-lactamase prevalence for Staphylococci isolated from bovine mastitic milk samples. Acta Vet., 52: 337-344.
    CrossRefDirect Link
  76. Swartz, T. and C. Petersson-Wolfe, 2016. Bacillus spp.: A practical summary for controlling mastitis. Publication DASC-62P, Virginia Cooperative Extension, Virginia State University, Petersburg, VA., USA.
  77. Abera, M., T. Habte, K. Aragaw, K. Asmare and D. Sheferaw, 2012. Major causes of mastitis and associated risk factors in smallholder dairy farms in and around Hawassa, Southern Ethiopia. Trop. Animal Health Prod., 44: 1175-1179.
    CrossRefDirect Link
  78. Vasquez-Garcia, A., T.D.S. Silva, S.R.D. Almeida-Queiroz, S.H. Godoy, A.M. Fernandes, R.L. Sousa and R. Franzolin, 2017. Species identification and antimicrobial susceptibility profile of bacteria causing subclinical mastitis in buffalo. Pesquisa Vet. Brasil., 374: 447-452.
    CrossRefDirect Link
  79. DeVries, T.J., M.G. Aarnoudse, H.W. Barkema, K.E. Leslie and M.A.G. von Keyserlingk, 2012. Associations of dairy cow behavior, barn hygiene, cow hygiene and risk of elevated somatic cell count. J. Dairy Sci., 95: 5730-5739.
    CrossRefDirect Link
  80. Nwankwo, I.U., N. Amaechi and W.A. Adiele, 2015. Microbial evaluation of raw milk from dairy farms in Udi L.G.A Enugu State, Nigeria. IOSR J. Agric. Vet. Sci., 8: 60-65.
    Direct Link
  81. Azevedo, C., D. Pacheco, L. Soares, R. Romao and M. Moitoso et al., 2016. Prevalence of contagious and environmental mastitis-causing bacteria in bulk tank milk and its relationships with milking practices of dairy cattle herds in Sao Miguel Island (Azores). Trop. Anim. Health Prod., 48: 451-459.
    CrossRefDirect Link
  82. Suleiman, T.S., E.D. Karimuribo and R.H. Mdegela, 2018. Prevalence of bovine subclinical mastitis and antibiotic susceptibility patterns of major mastitis pathogens isolated in Unguja Island of Zanzibar, Tanzania. Trop. Anim. Health Prod., 50: 259-266.
    CrossRefDirect Link
  83. Sanotharan, N., M. Pagthinathan and M.S.M. Nafees, 2016. Prevalence of bovine subclinical mastitis and its association with bacteria and risk factors in milking cows of Batticaloa district in Sri Lanka. Int. J. Scient. Res. Innov. Technol., 3: 137-150.
    Direct Link
  84. Murphy, S.C. and K.J. Boor, 2000. Trouble-shooting sources and causes of high bacteria counts in raw milk. Dairy Food Environ. Sanit., 20: 606-611.
    Direct Link
  85. Elmoslemany, A.M., G.P. Keefe, I.R. Dohoo and B.M. Jayarao, 2009. Risk factors for bacteriological quality of bulk tank milk in prince edward island dairy herds. Part 2: Bacteria count-specific risk factors. J. Dairy Sci., 92: 2644-2652.
    CrossRefDirect Link
  86. Tassew, A. and E. Seifu, 2011. Microbial quality of raw cow's milk collected from farmers and dairy cooperatives in Bahir Dar Zuria and Mecha district, Ethiopia. Agric. Biol. J. North Am., 2: 29-33.
    Direct Link
  87. Axelsson, A., 2004. A comparison between bulk tank milk samples and individual milk samples from dairy herds in Uruguay. Degree Project 2004:28, Faculty of Veterinary Medicine, Swedish University of Agricultural Sciences, Uppsala, Sweden. https://stud.epsilon.slu.se/11172/1/axelsson_a_170926.pdf.
  88. Al-Tahiri, R., 2005. A comparison on microbial conditions between traditional dairy products sold in Karak and same products produced by modern dairies. Pak. J. Nutr., 4: 345-348.
    CrossRefDirect Link
  89. Perkins, N.R., D.F. Kelton, K.J. Hand, G. MacNaughton, O. Berke and K.E. Leslie, 2009. An analysis of the relationship between bulk tank milk quality and wash water quality on dairy farms in Ontario, Canada. J. Dairy Sci., 92: 3714-3722.
    CrossRefDirect Link
  90. Mhone, T.A., G. Matope and P.T. Saidi, 2011. Aerobic bacterial, coliform, Escherichia coli and Staphylococcus aureus counts of raw and processed milk from selected smallholder dairy farms of Zimbabwe. Int. J. Food Microbiol., 151: 223-228.
    CrossRefPubMedDirect Link
  91. El-Leboudy, A.A., A.A. Amer and S. Abd El-Mohsen, 2014. Detection of some pathogenic organisms from dairy farm milk. Alex. J. Vet. Sci., 44: 111-118.
    Direct Link
  92. Zurbrigg, K., D. Kelton, N. Anderson and S. Millman, 2005. Stall dimensions and the prevalence of lameness, injury and cleanliness on 317 tie-stall dairy farms in Ontario. Can. Vet. J., 46: 902-909.
    PubMedDirect Link
  93. Tomazi, T., J.L. Goncalves, J.R. Barreiro, M.A. Arcari and M.V. dos Santos, 2015. Bovine subclinical intramammary infection caused by coagulase-negative staphylococci increases somatic cell count but has no effect on milk yield or composition. J. Dairy Sci., 98: 3071-3078.
    CrossRefDirect Link
  94. EOS., 2005. Decree No. 1589/2005 for standards of natural purified drinking bottled water. Egyptian Organization for Standardization and Quality Control (EOS), Cairo, Egypt.
  95. Oshiro, R.K., 2002. Method 1604: Total coliforms and Escherichia coli in water by membrane filtration using a simultaneous detection technique (MI medium). EPA 821-R-02-024, September 2002, U.S. Environmental Protection Agency (EPA), Washington, DC., USA.
  96. Rice, E.W. and C.H. Johnson, 2000. Survival of Escherichia coli O157:H7 in dairy cattle drinking water. J. Dairy Sci., 83: 2021-2023.
    CrossRefDirect Link
  97. LeJeune, J.T., T.E. Besser, N.L. Merrill, D.H. Rice and D.D. Hancock, 2001. Livestock drinking water microbiology and the factors influencing the quality of drinking water offered to cattle. J. Dairy Sci., 84: 1856-1862.
    CrossRefDirect Link
  98. Bartram, J., J. Cotruvo, M. Exner, C. Fricker and A. Glasmacher, 2003. Heterotropic Plate Counts and Drinking-water Safety: The Significance of HPCs for Water Quality and Human Health IWA Publishing, London, UK., ISBN-13: 978-9241562263, Pages: 256.
    Direct Link

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