Relationship between Somatic Cell Count and Different Microbial and Chemical Quality Parameters of Bulk Tank Milk

| The objective of this study was to investigate bulk tank somatic cell count and associated risk factors influencing the hygienic quality and chemical composition of raw milk; additionally, to assess the impact of somatic cell count (SCC/ml) on the microbial and chemical profiles of Bulk Tank Milk (BTM). A total of 214 BTM samples were collected from different dairy farms in Alexandria, Menofia and El-Dakahlia Governorates, Egypt. Samples were collected during the period from January 2019 to September 2020. The mean values of SCC/ml, Aerobic Plate Count APC (cfu/ml), Coliform count (MPN/ml), Staphylococcus aureus count and Bacillus cereus count (cfu/ml) were 6.85×10 5 ±0.33×10 5 , 7.09×10 6 ±2.76×10 6 , 2.77×10 4 ±0.63×10 4 , 1.27×10 3 ±0.25×10 3 and 3.32×10 3 ±0.13×10 3 , respectively. Identification of the isolated strains reveals the presence of S. aureus ; coagulase negative staphylococci (CNS); St. uberis, E. coli and B. cereus with incidence of 19.16%, 62.15%, 21.50%, 32.24% and 10.28% of the examined BTM samples, respectively. The correlation between SCC and different bacteriological and chemical parameters was estimated using Pearson’s correlation coefficient ( r ). Most BTM samples tested for milk quality parameters were below the acceptable limits suggested by the legislations; however, food poisoning bacteria ( E. coli, B. cereus and S. aureus ) were isolated. Thus, there is a need to improve milking hygiene, proper herd and udder health management to improve quality and safety of raw milk.

in lactose, fat and casein contents in milk in consequent of SCC elevation (Lindmark-Månsson et al., 2006). The elevation of SCC in BTM is an indicator of both raw milk quality and the prevalence of subclinical mastitis in dairy herds. Therefore, most countries set a critical limit for SCC, above which milk cannot be collected from the farm, also some milk processing plants and/or marketing cooperatives adopted more stringent quality standards to the farms and offer bonus payments as a real financial incentive to produce milk of low SCC (Olechnowicz and Jaśkowski, 2012;Alhussien and Dang, 2018).
Milk is a nutritious product and is a good medium for the growth of a wide diversity of microorganisms. The consumption of contaminated milk may lead to foodborne diseases. The most accused pathogens in outbreaks of foodborne illnesses due to consumption of raw milk and its products were S. aureus, E. coli especially Shiga toxinproducing one, Salmonella species, Klebsiella species, Proteus species, Yersinia enterocolitica, Campylobacter jejuni, Pseudomonas aeruginosa and Listeria monocytogenes from different countries (Sarkar, 2016).
Escherichia coli is a member of coliform group and considered as an indicator organism reflecting the hygienic quality of food and indicates direct or indirect fecal contamination. Additionally, E. coli is responsible for serious fatalities and milk borne disease outbreaks around the world (Ahmed and Fahim, 2018).
Staphylococci are normal commensals of the skin and mucosa of both healthy human and animals. They are an important agent of bovine mastitis, leading to economic losses in dairy farms. S. aureus is considered as a major public health problem, it produces many extracellular proteins and toxins which probably contribute to the virulence of this organism. Staphylococcal food poisoning is very common, the symptoms subside within 24-48 h, but the disease may remain for 7-10 days (Mohamed et al., 2016;Fisher and Paterson, 2020).
Coagulase negative staphylococci (CNS) have appealed increasing interest as they have been isolated from mastitis in dairy animals, they adapted to survive in the bovine environment or called opportunistic pathogens, they are potential zoonotic pathogens, and they have the capability to produce enterotoxins in food (Torky and Abu Tabeikh, 2016).
Bacillus cereus is an important foodborne pathogen, which causes two distinct types of food poisoning; diarrheal and emetic type caused by two different types of toxins. Centers for Disease Control and Prevention (CDC) reported domestically acquired foodborne illness caused by B. cereus in the US, with estimated number of 63,400 cases annually (Naas et al., 2018). In addition, B. cereus has been identified as an occasional cause of bovine mastitis in New Zealand (Cressey et al., 2016).
Streptococci are considered as main human and animal pathogens include more than 40 subspecies and several groups. They continue to be a major cause of subclinical mastitis in dairy cattle, causing economic losses for the dairy industry especially St. agalactiae, St. uberis and St. dysgalactia. Streptococcal infections in human are associated with scarlet fever, bacterial endocarditis, rheumatic fever, sore throat, tonsillitis, and pneumonia (Youssef and Mohamed, 2015).
The guidelines to evaluate the BTM quality vary between studies on SCC and their effect on other milk constituents (Bi et al., 2016;Macedo et al., 2018) to the prevalence of foodborne pathogens (Youssf and Mohamed 2015; Kupradit et al., 2020); Additionally, the quality of BTM is determined through different bacterial parameters as enumeration of total aerobic bacteria, coliform and S. aureus in relation to SCC (Moheebi-Fani et al., 2016;Naing et al., 2019). Therefore, the present study was conducted to evaluate the quality and safety of BTM through determination of SCC with its impact on the hygienic and chemical profiles, and the assessment of microbiological parameters (Aerobic Bacterial count, Coliform, E. coli, total staphylococci, S. aureus, CNS, B. cereus and Streptococci); in addition to the correlation between SCC, milk constituents and microbial parameters. The degree of acceptability of the examined bulk tank milk vs. the Egyptian and European standards was evaluated.

CollECtioN of Btm SamplES
A total of 214 BTM samples were collected from different dairy farms in Alexandria, Menofia and El-Dakahlia Governorates, Egypt. Samples were collected during the period from January 2019 to September 2020 under complete aseptic conditions as described by Jayarao and Wolfgang (2003). Collected samples were transferred to the laboratory in insulating icebox to be immediately examined for the following: dEtErmiNatioN of SomatiC CEll CouNt/ml (SCC/ ml) The technique described by Zecconi et al. (2002) was adopted using Nucleo Counter ® SCC-100 TM (Denmark, Chemometic 0613-019-061uol).

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StatiStiCal aNalySiS
The obtained data were analyzed using SPSS software statistics version 20. Pearson's correlation coefficient (r) was used to evaluate correlation between milk hygiene indicators and variables of milk composition. Significance was set at P-value < 0.05.

BulK taNK SomatiC CEll CouNt (SCC/ml)
The mean SCC in the examined BTM samples was 6.85×10 5 cells/ml, Table 1, (41.59%) of the samples have SCC < 500 X 10 3 , (24.77%) of the samples have SCC within the range 500-750 x10 3 / ml and (33.64%) of the samples have SCC within the range 750 X 10 3 ->10 6 ( Table 2). According to the requirements of the Egyptian Specifications of raw milk (SCC not >750x10 3 ) and the European specifications (SCC not >400x10 3 ); 66.36% and 31.78% of the examined samples complied to both standards, respectively, (Table 4).

BaCtEriologiCal paramEtErS
Aerobic mesophilic microbial count and coliform count were recorded in all examined BTM samples with mean values of 7.09×10 6 cfu/ml and 2.77×10 4 MPN/ml, respectively. Staphylococcus aureus and B. cereus pathogens were detected in the examined BTM samples with mean count of 1.27×10 3 and 3.32×10 3 cfu/ml, respectively, Table  3.

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CorrElatioNS BEtwEEN SCC aNd miCroBial paramEtErS
The obtained results show statistically significant positive correlations between SCC and microbial parameters (Total aerobic bacteria, Coliform, S. aureus and B. cereus count), positive correlations between different bacterial groups (Total aerobic bacteria, Coliform and S. aureus), Table 3.

ChEmiCal aNalySiS of ExamiNEd SamplES aNd itS CorrElatioN to SCC
The mean values of Fat, protein, S.N.F, Lactose, Ash, Chlorine % and pH of the examined bulk tank milk samples were 4.33±0.07, 3.17±0.01, 8.37±0.01, 4.64±0.01, 0.67±0.02, 0.094±0.002 and 6.32±0.01, respectively. Significant weak positive correlations were detected between SCC and fat %, and between SCC and ash % (Table 6). Somatic Cell count of BTM is used to determine the quality of the produced milk and to differentiate between normal and abnormal milk (Ruegg and Pantoja, 2013).

Advances in Animal and Veterinary Sciences
October 2021 | Volume 9 | Issue 10 | Page 1664 Most of the examined BTM show high SCC (> 500 X 10 3 cells/ml), which parallel to the results recorded by Gillespie et al. (2012), while higher results were recorded by Macedo et al. (2018) and Olatoye et al. (2018). Lower SCC were recorded by Moheebi-Fani et al. (2016).
The accepted limit of SCC/ml of bulk tank milk vary in different countries as from <400 X 10 3 in European Union, Australia, New Zealand and Canada, to <1,000,000 cells/ mL in Brazil (Ruegg and Pantoja, 2013). Our study revealed that 66.36% and 31.78% of the examined BTM samples complied to the requirements of the Egyptian (SCC not > 750x10 3 ) and the European specifications (SCC not > 400x10 3 ), respectively.
The high SCC of the examined BTM samples may be attributed to the stage of lactation, prevalence of subclinical mastitis and/or seasonal variation (Ruegg and Pantoja, 2013). The increased milk SCC may lead to reduction in milk yield, short shelf life of liquid milk, higher levels of proteolytic and lipolytic enzymes, altering the manufacturing properties of milk as reducing cheese yield and affecting the shelf life of dairy products (Olechnowicz and Jaśkowski, 2012).
The increased competitions between dairy companies encourage farms to produce high quality raw milk, as the dairy companies impose system of financial penalty if the SCC of bulk milk rises above a certain threshold and also offer 'bonus payments' if the SSC is under 200,000 or 250,000/ml. Other companies do not rely on SCC only but also set quality standards based on the bacterial counts of BTM (Blowey and Edmondson, 2010).
The Aerobic Plate Count (APC) is considered an indicator of the general hygienic condition in the dairy farm and the health state of the udder (Olatoye et al., 2018). The obtained results of APC of the examined BTM were nearly similar to that recorded by Olatoye et al., 2018, lower counts were recorded by (Elmoslemany et al., 2016), while higher APC was recorded by (Naing et al., 2019;Kupradit et al., 2020). Aerobic Plate Count less than 5,000 cfu/mL of BTM is considered as an index of proper hygiene. The high APC of the examined BTM samples considered a bad indication revealed low hygiene and poor quality milk. Therefore, many processors provide incentive programs to encourage dairy farmers to produce milk with lower APC and SCC than the required regulatory limits (Olatoye et al., 2018).
Milk secreted from a healthy udder contains only a very few bacteria of about 500 to 1,000 per milliliter. The main sources of milk contamination include infected udder and/or teats, animal skin, faecal soiling of the udder, contaminated milking and storage equipment, and water used for cleaning (Pandey and Voskuil, 2011). The wide diversity of sources of contamination of raw milk diminishes the accuracy of APC in identifying the contamination sources. Therefore, using APC to evaluate the hygienic quality of milk is recommended to be accompanied by counting of specific groups of microorganisms such as S. aureus, coliform and B. cereus (Macedo et al., 2018).
The coliform group of bacteria comprises all aerobic and facultative anaerobic, gram-negative, non-spore forming rods able to ferment lactose with production of acid and gas at 32˚C within 48 hr (Hogan and Smith, 2003). Our results showed contamination of all examined BTM with coliforms, which parallel to the results obtained by Kunda et al., 2015, Comparatively higher results were recorded by (Ombarak and Elbagory, 2015) and (Naing et al., 2019); while, lower results were recorded by (Olatoye et al., 2018).
Coliforms are commonly found in the feces of cows and widely distributed in the farm environment. Therefore, coliform count reflects the hygienic and sanitation practices tracked on the farm, high coliform count of BTM indicates fecal contamination and unhygienic milk production practices. Consequently, it can result in milk spoilage and severe human diseases (Macedo et al., 2018). Likewise, contamination of milk with E. coli often reflects fecal contamination; it is the known causative agent of diarrhea and other foodborne-related illnesses through the ingestion of contaminated foodstuffs (Mohamed et al., 2017).
Our study revealed the contamination of examined BTM samples with E. coli with percentage of 32.24 %, which were analogous to the results documented by Bi et al. (2016), while, higher incidences were given by (Kupradit et al., 2020;El-Leboudy et al., 2014) failed to detect E. coli in BTM samples. E. coli has a zoonotic importance, as it is responsible for serious fatalities and milk borne disease outbreaks worldwide, and it is a major mastitis pathogen in dairy animals. The Antimicrobial resistance among some commensal E. coli strains isolated from cattle can be associated with the presence of virulence factors which is of a major public health risk with the introduction of these bacteria to the food chain (Fahim et al., 2019).
Staphylococci are normal inhabitants of skin and mucosa of healthy human and animals, the organisms are important agent of bovine mastitis, leading to economic damage to dairy farms. Staphylococcus aureus is regarded as a zoonotic pathogen implicated in both clinical medicine and food safety, it was isolated from animal body surfaces, hands of the milking operators, as well as from several dairy utensils, especially teat cups (Azevedo et al., 2016;Fisher and Paterson, 2020). The analyzed BTM contained S. aureus with incidence of 19.16%, that is nearly similar to the incidence reported by Moheebi-Fani et al. (2016),
Staphylococcus aureus is a major problem of public health causing food borne outbreaks. It produces many extracellular proteins and toxins (Mohamed et al., 2016). The produced enterotoxins are resistant to inactivation by gastrointestinal proteases and show thermal stability, making their elimination difficult to achieve, (Schelin et al., 2011). Staphylococcus aureus food borne illness can be caused by ingestion of as little as 20 ng of enterotoxin that obtained from S. aureus count of 10 5 cfu/ml (Hassan et al., 2015). Therefore, determination of S. aureus count is critical for risk management and surveillance in the field of food safety.
It is clear from the obtained results that 17.76 % and 12.15 % of the examined BTM contained S. aureus count > 100 and > 500 cfu/ml, respectively, and disagree with the Egyptian and European standards. Nearly similar S. aureus counts were recorded by Kupradit et al. (2020), lower count was reported by El-Leboudy et al. (2014), while higher counts were reported by Ombarak and Elbagory (2015).
Coagulase Negative Staphylococci (CNS) were detected in the examined BTM samples with high incidence, which may represent food safety risk. Coagulase Negative Staphylococci contaminate bulk milk through milking of mastitis animals or from the surrounding environment (NMC, 2016). Coagulase Negative Staphylococci are potential zoonotic pathogens, as a relatively high percentage of CNS strains possess enterotoxin genes making them able to produce enterotoxins in food (Oliveira et al., 2011). On the other hand, CNS are considered emerging mastitis pathogens, with a small SCC contribution for bulk milk (Azevedo et al., 2016).
Bacillus cereus is ubiquitous and found in soils, water, dust, plants, animals and humans. It was isolated from contaminated foods of both plant and animal origin. Contamination of animal teats with farm soil or faeces and milking equipment are the most important routes for introducing the organism into raw milk (Kupradit et al., 2020). Our findings recorded that 10.28% of the examined BTM samples were contaminated with high count of B. cereus that is not accepted by the Egyptian (< 1 cfu/ml) and European (< 10 cfu/ml) specifications. Our results were comparable to the results obtained by Kupradit et al. (2020). Higher incidences of B. cereus were given by Kassa et al. (2017).
Presence of B. cereus in raw milk considered as a potential threat due to its ability to form thermoduric endospores that are able to survive pasteurization, to grow and survive at refrigeration temperature and to produce toxins; in addition, it has the capacity to grow over a broad pH range of 4.9 to 9.3 (Cressey et al., 2016). In most B. cereus outbreaks, the number of the organism associated with diarrhea ranged from 10 5 to 10 8 cfu/ml of food (Chitov et al., 2008). This indicate that the count of B. cereus obtained in the present study was not high enough to cause illnesses.
Streptococci are important zoonotic pathogens, they are main causes of subclinical mastitis in dairy cattle and a source of economic losses for the dairy industry especially St. agalactiae, St. uberis and St. dysgalactiae. In human, streptococcal infections are associated with scarlet fever, sore-throat, tonsillitis, bacterial endocarditis, rheumatic fever and pneumonia (Youssef and Mohamed, 2015).
The current study revealed high incidence of streptococcal contamination of BTM, with the isolation of St. uberis from 21.50% of BTM samples, which was comparable to Asfour et al. (2016).
Contamination of BTM with environmental streptococci indicates poor hygiene during milking and improper cleaning and sanitation of equipment, in addition, the infected cows are a major source of St. uberis in milk. Therefore, control of streptococcal mastitis, specifically that caused by St. uberis and St. agalactiae, is recommended to improve the microbial quality of raw BTM (NMC, 2016).

CorrElatioN BEtwEEN SCC aNd diffErENt miCroBial paramEtErS
Data obtained from this study show statistically significant positive correlations between SCC and APC, coliform, S. aureus and B. cereus count (p< 0.01); which approved by Pantoja et al. (2011), NMC (2016) and Olatoye et al. (2018). On contrary to our results, Moheebi-Fani et al. (2016) found no correlation between SCC and other bacterial parameters.
Considering the correlation between different bacteria; significant positive correlation was recorded between APC, coliform and S. aureus count (p< 0.05), which is in accordance with (Zadoks et al., 2004;Elmoslemany et al., 2016;Moheebi-Fani et al., 2016); while (Macedo et al., 2018;Olatoye et al., 2018) recorded weak correlation between APC and coliform and suggested that single quality parameters could not predict the others.

ChEmiCal paramEtErS
Results concerning the chemical analysis declared that all BTM samples agreed with the Egyptian specifications of raw cow milk, concerning fat % (3%) and SNF % (8.25%). Our findings declared statistically significant positive correlations between SCC and fat % and ash % ((p<0.05), and no correlations were detected between SCC and other
El-Tahawy and EL-Far (2010) observed a rise in plasmin levels in milk with high SCC which increases the breakdown of proteins, milk fat and other solids in milk, leading to a decrease in the levels of milk constituents. Jatawa et al. (2011) explained the increase in fat concentration in infected cows as not to be the result of increased synthesis, but due to a larger decrease in milk and lactose synthesis in comparative to fat.
Food quality assurance programs focus on producing milk with low somatic cell and bacterial count, resulting in better quality products with longer shelf life (Olech Nowicz and Jaśkowski, 2012).

CONCLUSIONS AND RECOMMENDATIONS
The obtained results revealed the relation between SCC and the microbial and chemical parameters of bulk tank milk of the examined dairy farms. High SCC and APC in most of examined farms indicate bad hygiene that was confirmed by the high coliform count, but SCC/ml has a weak remarkable effect on the milk constituents. Most samples tested for milk quality parameters were below the acceptable limits suggested by legislations. The study showed high incidence of food poisoning bacteria (E. coli, B. cereus and S. aureus) which represent serious hazards to human health. Consequently, there is a need for improving the quality and ensuring the safety of bulk tank raw milk which can be achieved through application of Good Manufacturing Practices.

NOVELTy STATEMENT
This study provided an innovative data about the quality of bulk tank milk and declared the association between somatic cell count and the chemical constituents, microbiological profile and quality of bulk tank milk.