Eight raw milk samples collected from different areas of Dhaka city contained MRSA (Nusrat et al., 2015). To obtain a rough estimate of herd udder health, the milk somatic cell count (BMSCC) can be measured (Wilson et al., 1997). It became a good source of income for landless, smallholders and marginal farmers (Saadullah et al., 2002).
Thus, it became a reliable test to identify pathogenic microorganisms from BM samples (Koskinen et al., 2009).
Somatic cell count
MALDI-TOF is used to analyze DNA, proteins, peptides and sugars, as well as large organic molecules and other macromolecules. Mass spectra are produced which are analyzed with dedicated software and assimilated with profiles stored in enriched databases. MALDI-TOF is based on biochemical tests which are cost-effective, faster and more accurate procedures for the identification of bacterial species in microbiological laboratories (Bahr et al., 1992; Sandrin et al., 2013).
Furthermore, not only organisms, but also antibiotic susceptibility of bacteria can be predicted by MALDI-TOF.
Sources of Staphylococcus aureus in bulk milk
Effect of Staphylococcus aureus on humans
Emergence of MRSA
MRSA in animals and personnel working with animals
Contamination of raw milk with MRSA can be transmitted to farmers as well as people who consume raw milk or can cause an initial contamination in the production chain of raw milk products (Oliver et al., 2009). It was reported by Kluytmans et al., (1995) that 5 out of 21 patients died from the first foodborne outbreak of MRSA.
Somatic cell count in bulk milk
- Poor udder health
- Management
- Relationship between bulk tank somatic cell count and mastitis prevalence
- Effects of high BMSCC 1. Milk production
- Milk composition and quality
- Somatic cell count and monitoring udder health
- Guidelines for monitoring BMSCC and other data 1. Entry into the milking herd
- New infections
- Cure
- Culling
In multiparous cows, the risk of developing CM increases with increasing parity (Steeneveld et al., 2008). Significant association was obtained between the occurrence of CM and increased level of SCC (Barkema et al. 1998). Acceptable threshold level of SCC must be set as well as feasible time for the result must be tracked (Lam et al., 2011).
Cows with negative economic value as well as chronic mastitis should be considered for culling (Schukken et al., 2003).
Bulk milk analysis in Bangladesh
17 the hygiene score, the evaluation of slaughtered animals that have chronic infection and the evaluation of the herd environment (Schukken et al., 2003) should be critically evaluated. Cows that had high SCC on the previous test day and were treated in lactation should have at least a 50% chance of having a low SCC on the current test day (Sol et al., 1994). Recent cases of clinical mastitis data including cow ID, lactation number, current days in milk, calving date, mastitis date and treatment notes can be recorded to monitor the rate of recovery from bacterial infection ( Sol et al., 1994; Sol, 2002).
Knowledge gaps are hygienic standard maintained in the farm, TBC for pasteurized as well as raw milk, prevalence S.
Chapter-3: Materials and Methods
- Experimental design
- Sample collection, preservation and transportation
- Bulk milk somatic cell count
- Bacteriological examination of bulk milk
- Preservation of isolates
- Matrix associated laser desorption/ ionization- time of flight
- Minimum inhibitory concentration
- Statistical analysis
- Descriptive statistics
- Risk factors analysis
The number of TES in BM samples was assessed using modified Edward's base (Himedia® Ltd., Mumbai, India. Isolates were screened for hemolysis, catalase production and coagulase production (NMC, 2017; Jayarao et al., 2004).milk samples were thoroughly mixed by gently inverting the milk bottle 20 to 25 times (Marshall, 1992).
Colony count at the final countable dilution × dilution factor/mL milk = CFU/mL original culture. Bulk milk samples were diluted to 10-6 and 20μL of each dilution was plated on Mannitol salt agar (HIMEDIA® Ltd., Mumbai, India) and incubated at 37°C for 48 hours. Bulk milk samples were diluted to 10-6 and 20μL of each dilution was plated on modified Edward's base (Himedia® . Ltd Mumbai, India) and incubated at 37°C for 48 hours.
Bulk milk samples were diluted to 10-6 and 20μL of each dilution was plated on MacConkey's agar (HIMEDIA® Ltd., Mumbai, India) and incubated at 37°C for 48 hours. Then 2-3 colonies were transferred to Brain Heart Infusion broth (BHB) (Oxoid, Basingstoke, UK) and incubated at. Staphylococcus aureus isolates were transferred to the National Veterinary Institute (SVA), Uppsala, Sweden using Copan Transsystem® (Capon, Brescia, Italy) transport media tube for species confirmation using Matrix-Associated Laser Desorption/Ionization- Time of Flight (MALDI-TOF) Biotyper 3.0 (Bruker Daltonics GmbH, Bremen, Germany) for validation (score > 1.8) of phenotypic identification accuracy.
Results were presented as total number of holdings and percentage and 95% confidence interval (CI). These factors included farm size, year of establishment, educational qualifications, farmer's age, number of employees, number of dairy cows, stable system, source of animals, refreshed water per frequency per day, drainage score, milking system, calf suction, number of milkers, environmental temperature and humidity.
Chapter-4: Results
- Dairy cattle herd characteristics
- Assessment of somatic cell count and bacterial contamination in bulk milk
- Correlation among the variables
- Prevalence of pathogens isolated from bulk milk
- Minimum inhibitory concentration determination of selected S. aureus
- Univariate analysis
- Multivariable linear regression model for bulk milk somatic cell count
Mean, min-max and quartile estimates of BMSCC and bacterial contaminants (TBC, TSC, TESC and TCC) in bulk milk samples from 24 dairy herds in Chattogram, Bangladesh. BMSCC= Bulk milk somatic cell count; TBC=Total bacterial count; TSC= Total staphylococcal count; TESC=Total environmental streptococcal count; TCC=Total coliform count. Summary statistics (mean, min-max and quartile estimates) of somatic cell count and milk contamination (TBC, TSC, TESC and TCC) in bulk milk samples from 24 dairy herds in Chattogram, Bangladesh, using Log10-transformed values.
Prevalence of Staphylococcus aureus and MRSA in bulk milk from 24 dairy farms in Chattogram, Bangladesh. Resistance1 (numbers in brackets) and distribution of MIC for Staphylococcus aureus (n=4) from 24 dairy farms in Chattogram, Bangladesh. 1Resistance percentage was calculated as the number of isolates identified as resistant according to the cutoff divided by the total number of isolates tested for a specific type of bacteria tested (S. aureus/NAS); **White boxes indicate the range of dilutions tested for each substance.
In the MIC plate, there was a designed well for Trim-sulfa containing combination of 0.25 µg/mL trimethoprim and 4.75 µg/mL Sulfamethoxazole. When no cutoff values were available, bacteria were not categorized as susceptible or resistant. Univariable associations between the farm-level factors and the mean number of somatic milk cells of lactating cows in Chattogram during 6 months (n=24 farms).
According to the final model: own livestock as replacement (p=0.09) compared to both purchased and own livestock, dry floor (p=0.06) compared to watery floor and weekly or more frequent cleaning (p=0.1) compared with 1-4 The number of cleanings per day was associated with a lower BMSCC level (Table 4.7). Results of multivariable linear regression between the farm level factors and the average bulk milk cell count of lactating cows over the 6-month period (n=24 farms).
Chapter-5: Discussion
- Farm characteristics
- Somatic cell count and bacterial contamination in bulk milk Bulk milk somatic cell count
- Correlation with bulk milk somatic cell count with different bacterial counts
- Prevalence of S. aureus and MRSA Staphylococcus aureus
- Risk factors associated with bulk milk s omatic cell count
- Limitation of the study
The lowest TBC mean ranged from 400 CFU/mL indicating good quality milk, and the highest mean was 1,890,567 CFU/mL milk in our study. According to the Standards Institute of Sri Lanka 1983, the expected standard is less than 30,000 CFU/ml (Gunasena and Siriwardhana, 2021). Internationally, acceptable limits for coliforms in raw milk are less than 100 CFU/mL milk (Mubarack et al., 2010; Salman and Hamad, 2011).
In this study, the lowest TCC was found to be 33 CFU/mL, while the highest value was 1,500 CFU/mL milk, which is inconsistent with the results from neighboring countries. A positive significant correlation was found between TBC and TSC (r=0.65), which was also reported in several studies (Jayarao et al., 2004; Pyz-Łukasik et al., 2015). In developing countries like Bangladesh, more than 70% of the infectious bacteria have been termed multidrug-resistant strains (Jilani et al., 2008; Dutta et al., 2013).
Eight samples of raw milk collected from different areas of Dhaka city contained MRSA (58.8%) (Nusrat et al., 2015). The result of this study contrasts with other studies that mentioned that larger farm size was a risk factor for increased BMSCC (Elmoslemany et al., 2010; Jayarao et al., 2004). 34 In this study, dry floor condition compared to wet floor conditions was significantly related to lower level of BMSCC, which is in agreement with Santman-Berends et al. 2016), who found an increased incidence of CM in herds where floor cleaning is performed once a day compared to cleaning 4 times a day.
Higher BMSCC was associated with animals purchased from other farms compared to replacement from own stock, which is consistent with Schukken et al., (2011). Own stock animals are more concordant in preventing infection due to strong herd immunity (Schukken et al., 2011).
Chapter- 6: Recommendations and Future Directions
Recommendations
Future directions
Further studies on the isolation of zoonotic bacteria from bulk milk and their route of transmission together with implications for public health should be considered.
Prevalence and zoonotic significance of methicillin-resistant Staphylococcus aureus in raw milk and some dairy products at Ismailia city, Egypt. Characterization of Staphylococcus aureus from milk and dairy products sold in some local markets of. High production of LukMF'in Staphylococcus aureus field strains is associated with clinical bovine mastitis.
Molecular typing of Staphylococcus aureus isolated from bovine mastitic milk based on toxin genes and coagulase gene polymorphisms. Methicillin (oxacillin)-resistant Staphylococcus aureus strains isolated from large food animals and their potential transmission to humans. Biochemical profiles, restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD) and multilocus variable number tandem repeat analysis (MLVA) for typing of Staphylococcus aureus isolated from dairy products.
Management practices associated with bulk milk prevalence of Staphylococcus aureus on Canadian dairy farms. Methicillin-resistant Staphylococcus aureus (MRSA): An emerging veterinary and zoonotic pathogen of public health concern and some studies in Malaysia. Phenotypic and genotypic characteristics of Staphylococcus aureus isolates from raw goat and sheep milk samples.
Appendix-I
Baseline Information
Appendix-Ⅱ
Appendix-Ⅲ
- Catalase test
- Coagulase test
- Horse plasma collection
- Tube coagulase test
- Indole test
- Oxidase test
- Methyl red test
- Voges-Proskauer (VP) test
- Preservation of stock culture
- Fifty percent sterile buffered glycerin was made by mixing 50 parts of pure glycerin and 50 parts of buffered glycerol saline
- Pure culture of isolated bacteria from blood agar was incubated overnight in Brain heart infusion broth
- Then the bacterial culture was mixed with 50% sterile buffered glycerin in 1.5 ml Eppendorf tubes (700µl broth culture and 300µl glycerol)
- Then preserved at -80°C for long term use
60 A piece of filter paper was placed in a clean petri dish and 2 drops of oxidase reagent was added to the filter paper. A colony of test organisms was removed using a wire loop (not an oxidized wire loop) and rubbed onto treated filter paper. Using a light inoculum, tubes of MR-VP media were inoculated with 24-hour pure cultures of the test organisms.
After incubation, 2.5 ml of culture was transferred into a new sterile culture tube and 5 drops of the methyl red reagent was added to the tube. Red color on the surface of the medium due to high acid production and a decrease in the pH of the culture medium was indicated as a positive test. Fifty percent sterile buffered glycerin was made by mixing 50 parts pure glycerin and 50 parts buffered glycerin salt.
The bacterial culture was then mixed with 50% sterile buffered glycerin in 1.5 ml Eppendorf tubes (700 µl broth culture and 300 µl glycerol).
Appendix-Ⅳ
Minimum Inhibitory Concentration (MIC)
- Preparation of inoculum
- Inoculation and incubation
