Factors affecting Somatic Cell Count in milk of dairy cows in Costa Rica

Somatic cells, such as bacteria fi ghting leukocytes, are present in cow’s milk as an immune response to combat intramammary infection. The primary objective of this study was to determine the effect of various factors on the somatic cell count, or SCC, within the milk of dairy cows. Factors studied in comparison to the SCC include the number of bacteria within the cows’ milk, the age of the cows, the number of Hypoderma lineatum lesions on the cows, and the cows’ diet and the sanitation practice at separate farms. Milk samples were collected from 13 cows from Farm 1, located in La Cruz, Costa Rica, 20 cows from Farm 2, located in La Cruz, Costa Rica, and 30 cows from Farm 3, located in Monteverde, Costa Rica. Farmers at each of these three farms fed their cows a slightly different diet, and carried out different procedures in relation to cleanliness during their milking process. The stage of lactation was kept constant to eliminate this confounding variable. Milk samples were observed for somatic cell and bacteria counts using a compound light microscope under 1000X magnifi cation using gel immersion. The fi ndings from this study indicate that somatic cells are signifi cantly correlated to bacteria in cow’s milk, while somatic cells do not signifi cantly correlate with the cow’s age or the number of H. lineatum lesions a cow has. This study also indicates that the sanitation practices and milking procedure that farmers use can affect their somatic cell counts, and that using a new and disposable cotton cloth or paper to wipe down the udders before milking may lead to lower somatic cell and bacteria counts than using the same wet rag to wipe down every cow. Also, treating the udders with a disinfectant before milking, instead of after, led to the lowest somatic cell and bacteria count. Research Article Factors affecting Somatic Cell Count in milk of dairy cows in Costa Rica Kyle E Kline1*, Sofía Flores2 and Frank Joyce3 1Department of Molecular Environmental Biology, University of California, Berkeley, USA 2Education Abroad Program, University of California, Monteverde, Costa Rica, USA 3University of California Education Abroad Program, Tropical Biology and Conservation Program, Costa Rica, USA Received: 18 December, 2017 Accepted: 03 January, 2018 Published: 04 January, 2018 *Corresponding author: Kyle E Kline, Department of Molecular Environmental Biology, University of California, Berkeley, USA, Tel: 6302008899; E-mail:


Introduction
In the last three decades, world milk production has increased by more than 50 percent, with developing countries broadening their share in global dairy production in more recent years [1]. Both the income of dairy operations and the health of the consumers depend on a healthy composition of the milk that is processed and sold. It is propitious that milk has low levels of deleterious compounds such as somatic cells or bacteria: high levels of somatic cells in milk can hinder the production of other dairy products, and high levels of bacteria in milk can pose health risks for consumers [2].
Regulatory standards are set forth worldwide to determine the healthy limits of somatic cell and bacteria that can be present in milk before it can be accepted from farms and processed.
These regulatory standards vary between countries, but in all locations, milk is tested in a laboratory after being collected from farms for a bacteria count and somatic cell count [3]. The bacteria count is a detection of microbial contamination, and is generally used to evaluate the sanitation of the dairymen's equipment and the overall health of the herd [4]. In the United States, the Grade A Pasteurized Milk Ordinance (PMO) requires bacteria to be less than 100,000 bacteria per mL for Grade A milk shipments. This would be indicated by fi nding a count of fi ve bacteria in 20 total fi elds of view from a milk sample on a microscope slide under 1000X magnifi cation. In Costa Rica, it must meet the standardized guidelines outlined by the Servicio Nacional de Sanidad y Calidad Agroalimentaria (SENASA) through the Ministry of Agriculture, which also requires the bacteria to be less than 100,000 bacteria per mL (indicated by a count of fi ve bacteria on a microscope slide). Somatic cell count, or SCC, is also an indicator of the quality of milk, and generally a lower SCC indicates better animal health [5]. In the United States, the current legal limit put forth by the PMO is 750,000 somatic cells per mL for Grade A milk, which would be indicated by fi nding a count of 37 somatic cells from 20 total fi elds of view on a microscope slide under 1000X magnifi cation [6]. In Costa Rica, there must be less than or equal to 400,000 somatic cells per mL according to SENASA, which would be indicated by fi nding a count of 20 somatic cells from a milk sample on a microscope slide under 1000X magnifi cation. Milk from an uninfected mammary gland usually contains less than 100,000 cells per mL, and a milk SCC of more than 200,000 cells per mL may suggest that an infl ammatory response has been elicited or that the cow is recovering from infection [7].
The somatic cells present in the milk of lactating cows consist of leukocytes, or white blood cells, from the cows immune system and secretory cells (shed from the udder tissue). Leukocytes play an important role in the body for mediating immune responses against infectious microorganisms [2]. Just as nutrients are transported from the bloodstream into the udder of lactating cows to be converted into milk, leukocytes will migrate from blood into the mammary gland through the teat canal in response to bacteria within the udder. Such infecting bacteria in the udder multiply and can cause intramammary infection, leading to tissue infl ammation and damage. Neutrophils are involved in repairing this damage and destroying bacteria in the udder tissue [7].
The difference in somatic cell counts between cows can result from differences in physiological factors and the functioning of each cow's immune system [8]. A compromised immune system can make an animal more susceptible to infection, and the effi cacy of an immune response is best measured by how quickly the immune system reacts to an infectious challenge.
One factor that can affect the health and immune system of dairy cows is parasitic fl ies such as H. lineatum. These organisms cause furuncular myiasis in cows, which is an infestation of a vertebrate host by fl y larvae that feed on living tissue, body fl uids, or ingested foods. Following copulation, an adult H. lineatum will capture a mosquito or a small blood-feeding fl y and transfer its larva. When this mosquito or small fl y feeds on the body of a cow, the larvae are transferred to the subcutaneous layer of the cow. The larvae then act as parasites by existing under the subcutaneous layer of the cow's skin. This produces myiasis, which appears as boil-like lesions or open wounds on the outer surface of the cow's body. These lesions can get scratched and infl amed, leading to a secondary infection by the infestation of bacteria. When the larvae develop, they fall to the ground, burrow, pupate, and then molt into an adult fl y. These fl ies can transfer infections to animals and can cause cows to be more susceptible to diseases. The objective of this study was to investigate factors affecting SCC in milk of cows in Costa Rica. Factors evaluated included the number of bacteria, age, the sanitation and milking practices of the farm, the diet of the cow, and the number of H. lineatum lesions on the cow. It was predicted that an increase in the number of bacteria within the milk, an increase in H. lineatum lesions on the cow, and less meticulous sanitation practices on the farm would correlate with an increase in SCC in the milk.

Materials and Methods
The fi rst step in the experiment was to obtain milk samples from cows with a range of ages, a range in the number of H. lineatum lesions on their body, and from farms that practice different feeding and milking practices. Milk was collected from 13 cows at one farm, 20 cows from a second farm, and 30 cows from a third farm, all of which were during the second (afternoon) milking session of the day. The stage of lactation was kept constant to eliminate this confounding variable.

Farm 1
The fi rst farm was in La Cruz, Costa Rica. The cows at this farm switched off feeding on three pastures throughout the day, each one having a different type of grass: one with Cynodon nlemfuensis, one with Brachiaria brizantha, and one with Pennisetum purpureum. The cows were also supplemented with a food concentrate and a mineral supplement.
For the milking procedure at this farm, the farmer brought each cow into a stable, rinsed the udders with water, and wiped the udders off with a wet, cotton cloth. He used this same wet cloth to wipe the udders of each cow before milking them. The farmer released several squirts of milk from the udders with his hand to eliminate the foremilk and check for any clotting before milking. He then disinfected the udders with a solution that is 1% iodine after the milking is completed. The farmer used this order of procedures to help collect the milk samples from each cow.
After each milk sample was obtained, a permanent marker was used to label each plastic bag with the number on the cow's ear tag. The labeled sample was secured and placed it in ice to keep the milk under 40 degrees Fahrenheit in order to eliminate bacterial growth [9]. During this time, the cow's entire external body was examined for H. lineatum lesions and this number, as well as the cow's age, was counted and recorded. This process was repeated for all 13 cows at this farm.

Farm 2
The second farm was in La Cruz, Costa Rica. The cows at this farm fed on Cynodon nlemfuensis and Saccharum offi cinarum, and were also supplemented with a food concentrate and a mineral supplement. For the milking procedure at this farm, the farmer fi rst cleaned the udders by dipping each one in a solution containing 1% iodine in order to disinfect them. He then wiped and dried the udders with a piece of newspaper. He uses a new piece of dry newspaper for each cow. The farmer then released several squirts of milk into a strip cup to release the foremilk and check for any clotting in the milk before beginning the milking process. The farmer used this order of procedures to help collect the milk samples from each cow. Each bag was labeled with the cow's ear tag number before placing it in ice to keep the samples under 40 degrees Fahrenheit. Each cow's entire body was then observed for H. lineatum lesions and this number and the cow's age was recorded next to the cow's ear tag number in a notebook. This process was repeated for all 20 cows at this farm.

Farm 3
The third farm was in Monteverde, Costa Rica. The cows at this farm were fed only Cynodon nlemfuensis grass, and were supplemented with a food concentrate and a mineral supplement. For the milking procedure at this farm, the farmer rinsed the udders with water from a hose, while wiping them off with his hand. He then wiped the udders off with a wet, cotton cloth, while also releasing a few initial squirts of the foremilk with this cloth. He used a new, wet cloth from a bucket of water for each cow. After the milking is complete, he disinfected the udders with a 1% iodine solution. The farmer used this order of procedures to help collect the milk samples from each cow.
Each plastic bag was labeled with the cow's ear tag number and placed it in ice to keep the sample under 40 degrees Fahrenheit. The cow's entire body was observed for H. lineatum lesions, and this number was recorded as well as the cow's age next to the cow's ear tag number in a notebook. This process was repeated for all 30 cows on this farm.

Counting somatic cells and bacteria
In order to decipher the somatic cell count and the bacteria cell count of the milk samples, the standard methodology practiced within the laboratory at the Monteverde Cheese Factory, located in Monteverde, Costa Rica, to evaluate the milk they receive from farms before the milk is processed was used.
Sterile Pasteur pipette was used to transfer ten microliters of milk from each plastic bag onto a glass microscope slide. Before transferring the milk with the Pasteur pipette, each plastic milk sample bag was shook to mix the milk thoroughly before testing it. Each slide contained four ten microliter drops, each coming from a different milk sample. The drops were labeled with the corresponding ear-tag number from the cow that the sample was taken from. Using the tip of the pipette, ten microliters of milk was spread on the slide into a thin-layered circle. The slides, which had four drops each, were placed under a light bulb for ten minutes to dry. Once the milk was dry, the slides were placed in a methylene blue dye for fi ve minutes in order to stain the somatic cells and bacteria. After fi ve minutes, the excess dye was drained by placing the slides on their sides on absorbent paper. Once the slides were free of excess dye, they were dipped in distilled water and placed them under a light bulb for fi ve minutes until they were completely dry.   This average corresponds to 76,000 total bacteria. The highest was a cow having a bacteria count of ten bacteria/ml, and the lowest was a cow having a bacteria count of zero bacteria/ml.       This is crucial for people in the dairy industry, because the production of maximum quantities of high quality milk is the most important goal of every dairy operation. An increase in somatic cells in cows' udders can lower a farmer's milk yield, and therefore income. Infl ammation and infection of the udders is considered to be the disease that has the greatest fi nancial impact on the dairy industry [11]. Economic losses of     mastitis in dairy cows include the cost to treat the infection, reduced milk yield, changes in milk composition, or premature culling, which is the separation of particular animals from the herd based on specifi c criteria [11]. Milk cannot be processed or sold when somatic cell and bacteria counts are higher than the legal limits, such as those previously stated that are put forth by the PMO in the United States and SENASA in Costa Rica. This means that if somatic cell and bacteria counts in the milk coming from a farm are higher than these regulated limits, the farmers will experience economic losses.

Somatic cells vs. Bacteria
Milk is produced in specialized cells in the alveoli of the cow's udders. Increased somatic cells lead to decreased milk yield because when many leukocytes move into the udders, they push out milk secreting cells, making the alveoli less productive in producing milk [5]. Infl ammation resulting from higher bacteria in the udders also leads to compromised secretion function and altered volume and composition of milk secreted. Accordingly, it is imperative for farmers to use practices that work to thwart intramammary infections in their cows, because such infections increase somatic cell counts in the milk, lower the total milk yield, and therefore decrease the farmer's income.

Somatic cells vs. Age
Although previous research has shown that when age increases, the SCC increases [12], data from this study indicated that an increase in a cow's age does not correlate to a higher number of somatic cells in the milk. The older cows did not display more bacteria in their milk, or more somatic cells fi ghting off those bacteria. These fi ndings show that the age of a cow does not necessarily affect the functioning of the cow's immune system, and older cows do not display a greater chance of contracting an intramammary infection.

Somatic cells vs. H. lineatum lesions
Although previous studies [13], have shown that cows with higher numbers of parasites, particularly ticks, have shown higher numbers of somatic cells in their milk, results from this study indicate that cows with higher numbers of H. lineatum lesions on their body did not correlate with higher a somatic cell count in their milk. Affected regions where the fl y larva exist in the subcutaneous layer of the cow's skin cause a lesion that can get scratched, irritated, and infl amed, making it more prone to secondary infections by bacteria. These fl ies can also transmit diseases to cows, and can secrete enzymes that have been suggested to negatively affect the cows' immune system.
Although such factors can have a potentially overwhelming effect on the cow's immune system, data from this study indicated that this effect is not large enough to increase the cow's chance of developing an intramammary infection.
Therefore, according to these results, cows with more H. inject insecticides or parasiticides into the back of cows, or use low-volume sprays applied to the entire surface of the cow's body [14].

Differences between the Farms
These results indicate differences in the numbers of H.
lineatum lesions, somatic cell counts, and bacteria counts between the three farms that were analyzed. These three farms had different feeding and milking practices. The fi rst farm showed a signifi cantly higher average number of somatic cells, bacteria, and H. lineatum lesions than the other two farms, but The main difference in the milking procedure was that at Farm 1, the farmer did not use a disposable cotton cloth or paper  article emphasizes the need to dry the teats completely using individual clean cloth or paper towel, and to avoid getting the rest of the udder wet [15]. The farmers at both Farm 1 and Farm 3 contradicted these recommendations by rinsing the udders completely with water before milking, and by using wet cloths to wipe the udders off instead of drying them before milking.
The farmer at Farm 1, with the highest average somatic cell and bacteria counts, also went against these recommendations by not using an individual clean cloth for each cow. The farmer at Farm 2 on the other hand, did not rinse the udders with water, and used a new piece of newspaper to completely dry the udders after disinfecting them before milking. This was the farm with the lowest somatic cell count on average. Farm 2 was also the only farm where the farmer used the iodine solution as a disinfectant before milking, as opposed to after milking such as at Farms 1 and 3. A study done on the effect of premilking teat preparation found similar results on the benefi ts of using a disinfectant before milking in reducing bacteria; this study demonstrated that the use of some disinfectant products for pre-milking teat preparation can have reduce the levels of staphylococcal and streptococcal pathogens on cow's udders [16].
Results from this study display how the milking procedure and sanitation practice that a farmer uses greatly determines the health of the cows, and the cows' susceptibility to infection.
These precautious cleanliness procedures may decrease the risk for an intramammary infection in the cows by decreasing harmful microorganisms, leading to lower bacteria in the udders, and therefore less somatic cells in the milk fi ghting off this bacteria in the udder tissue.