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Detection of Staphylococcus Aureus and Streptococcus Agalactiae: Subclinical Mastitis Causes in Dairy Cow and Dairy Buffalo (Bubalus Bubalis)

Muslimin Lucia*, Sri Rahayu, Dzul Haerah, Dan Wahyuni

Veterinary Medicine, Faculty of Medicine, Hasanuddin University, Makassar, South Sulawesi

Abstract: This study aims to detect the presence of Staphylococcus aureus (S. aureus) and Streptococcus agalactiae (S. agalactiae) in subclinical mastitis infections in dairy cows and buffalos in Enrekang (in South Sulawesi, Indonesia). Subclinical mastitis was pre-examined using the California Mastitis Test (CMT) reagent, and 33 samples were detected as positive. The positive samples were then isolated with a culture test on the Baird Parker Agar media (BPA), identified with a catalyst, Gram staining, and a coagulase test, and isolated in Mannitol Salt Agar (MSA) media. To distinguish the S. aureus from others Staphylococcus species, the sample was then tested on blood agar media, in order to observe the presence of hemolysis. For the identification of S. agalactiae, culture methods and Gram staining were used, as well as biochemical tests using a catalyst, the Christie Atkins Munch-Peterson (CAMP) test, an eskulin bile test, glucose, maltose, sucrose, motility tests, an indole test, and finally urea and blood agar tests. The results showed that four samples were positive for S. aureus, characterized by grayish colonies observed on the BPA, while there were positive results in the gram staining (forming a bacteria chain of purple cocci), in the catalyst test, the coagulase test and the blood agar test. The glucose, maltose, and lactose tests were also positive, while the catalyst test, the eskulin bile test, the indole, and the urease test all showed negative results. The results also showed that S. agalactiae was detected in one milk sample, while the other 28 samples detected none of the two bacteria.

Keywords: S. aureus, S. agalactiae, dairy cattle, dairy buffaloes

Citation: Muslimin Lucia, Sri Rahayu, Dzul Haerah, Dan Wahyuni, Detection of Staphylococcus Aureus and Streptococcus Agalactiae: Subclinical Mastitis Causes in Dairy Cow and Dairy Buffalo (Bubalus Bubalis). American Journal of Biomedical Research, 5 (1). 8-13. January 2017. doi:10.12691/ajbr-5-1-2

1. Introduction

As one of the dairy products, milk is an animal protein source, demand for which has been increasing, in terms of improving life quality. Staphylococcus aureus is a gram-positive bacterium that colonizes a variety of animal species. S. aureus infections in animals are most commonly reported as a cause of mastitis in dairy-producing animals (including cattle and goats) and “bumblefoot” in chickens [25]. Staphylococcus aureus is the key causative agent for mastitis and is responsible for sub-clinical cases, although it is also responsible for different forms of the disease [21]. Using bacteriological, biochemical, and PCR-based identification schemes, twelve (25.53%) isolates were confirmed as S. aureus. All the isolates showed β-hemolysis on 5% sheep blood agar. The S. aureus specific nuc 27gene (target size 279-bp) was amplified in the cases of all isolates [13]. Animal S. aureus always evolves from human strains, such that every human strain may be the ancestor of a novel animal-adapted strain. The zoonotic transfer of IMI- and milk-associated strains of S. aureus between cattle and humans seems to be very limited, and different hosts are not considered as a source for mutual, spontaneous infections [6].

According to [31], isolated S. agalactiae causes 83% of subclinical mastitis in Bogor (West Java, Indonesia), 82% in Boyolali (Central Java, Indonesia), and 80% in Malang (East Java, Indonesia). S. agalactiae is an important cause of mastitis; it was considered desirable in this study to obtain additional information on the factors that can influence its growth in milk [22]. Streptococcus agalactiae continues to be a major cause of subclinical mastitis in dairy cattle and is a source of economic loss for the industry. Veterinarians are often asked to provide information on herd level control, regarding the eradication of S. agalactiae mastitis [15]. S. agalactiae has attracted most attention, due to its economic significance [10]. According to [1], Streptococcus agalactiae, one of the Streptococci species, infects both terrestrial and aquatic animals. The organisms have been isolated from numerous fish species in natural disease outbreaks and showed to be pathogenic to several fish species.

Mastitis is a common disease and is a major problem in the world of dairy farming, as it causes huge economic aquatic animals. These organisms have been isolated from numerous fish species in natural disease outbreaks, and have been shown to be pathogenic for several fish species. losses to dairy farms worldwide [4] The economic losses caused by mastitis, especially subclinical mastitis, include declining milk production and quality, increased maintenance costs and treatment, and early livestock culling. As shown in [8], the decline in milk production due to mastitis reaches about 15-20% of the total milk production, while according to [28], milk production has declined to about 30%. Mortality from infections associated with S. aureus bacteremia can range from as low as 2.5%, to as high as 40% [29]. Staphylococcus aureus (S. aureus) mastitis is extremely difficult to control by treatment alone. To date, successful control has been achieved only through the prevention of new infections and culling infected animals [32]. S. aureus organisms tend to colonize teat ends and/or teat lesions. The infection’s spread can occur through milkers’ hands, through washcloths, teat cup liners, and flies.

2. Materials and Methods

The sample used in this study included 28 female lactating dairy cows from the Cendana District in Enrekang, Indonesia, whereas CMT was applied to all the nipples (112 nipples) during afternoon milking. Before the samples were taken, they were assessed via a Mastitis Test, using a reagent California mastitis test (CMT). A total of two ml of milk were placed on the paddle, adding two ml reagent CMT, then horizontally and gently shaking this for ten-fifteen seconds. The test result is considered negative if the mixture remains homogeneous, positive one if sedimentation is visible, positive two if the mixture thickens rapidly and the gel moves to the middle, and positive three if a lot of gel forms, causing the surface to turn convex.

2.1. Streptococcus Agalactiae Test

Milk samples positive with CMT were grown in a Brain Heart Infusion Broth medium (BHIB), incubated for 18-24 hours at 37°C, and later placed in a Nutrient Agar (NA) Media. Colonies suspected to contain Streptococcus sp are characterized by round shapes that are small, smooth, convex, and transparent, and that are 0.5-1 mm in diameter. The bacterial culture on the suspected Streptococcus sp. was then identified through several biochemical tests, as follows:

• Gram staining: Streptococcus sp. is characterized by a purple color, round cocci and a long chain.

• Catalase test: S. agalactiae is negative if there is no bubbling formation.

• Christie, Atkins, Munch-Peterson Test (CAMP Test): the CAMP Test was conducted on the Media Blood Agar, with S. aureus as a marker. Staphylococcus aureus was streaked in the middle of the media, then streaking the sample suspected with S. agalactiae across a plate, perpendicular to the S. aureus streak. Cultures were then incubated for 24 to 48 hours, at 37 C. The establishment of an arrow-like zone at the junction of two organisms indicated the presence of S. agalactiae colonies.

• Sugar Test: positive results for glucose, sucrose, and maltose tests indicate the presence of S. agalactia.

• Bile Bacteria Test: S. agalactiae that does not change to a black color, indicating a negative result.

• Motility test, indole test, urease test: negative results indicate the absence of S. agalactiae.

• A hemolysis test of S. agalactiae was performed on a Blood Agar Plate.

2.2. The S. aureus Test

Staphylococci are gram positive, non-motile and non spor-forming bacteria. Pathogenic staphylococci are identified by the ability to produce coagulase, hence, clotting the blood [12].

The detection and differentiation of S. aureus were conducted on Baird Parker Agar (BPA) medium. S. aureus colonies on BPA are characterized by their round shape; they are slimy/smooth, convex with a diameter of two mm-three mm, have a grayish to black color, and a clear halo that develops around the edge of the colony. Further, the presence of S. aureus was confirmed by the following tests:

• Fermentation test on Mannitol Salt Agar medium (MSA): the acid condition, as the color changes from pink to medium yellow, indicates the presence of S. aureus.

• Coagulation Test: Plasmatic clotting, showing the presences of S. aureus.

• Hemolysis Test (Test Pathogenicity): the pathogenic S. aureus is characterized by the ability to lyse red blood cells, with the presence of a transparent zone around the colonies on Blood Agar Media [17].

Staphylococcus aureus’s ability to adhere the extracellular matrix and plasma proteins deposited on biomaterials is a significant factor in the pathogenesis of orthopedic-device related infections. S. aureus possesses adhesion proteins on its surface, but the way they interact with each other to form stable interactions with the substrate is not known [12]. This emerged as a significant public health problem, both in human and veterinary medicine [14]. Methicillin-resistant Staphylococcus aureus (MRSA) is an important human pathogen, also raising emerging concerns in veterinary medicine and animal agriculture. Its presence in a wide range of animal species, including dogs, cats, rabbits, horses, cattle, pigs, poultry, and exotic species, has caused infection and unhealthy carriers [11].

The prevalence of S. aureus can most likely be attributed to the wide distribution of the organism inside mammary glands, as well as on the skin of teats and udders. S. aureus adapts very well in the udder and can establish chronic and subclinical infections. From there, these are then shed into the milk, serving as a source of infection for healthy cows, during the milking process. [3]

3. Results and Discussion

An examination for subclinical mastitis was performed using a CMT reagent on each of the udder quarters of cows and buffaloes. The test results of subclinical mastitis are shown in Table 1.

Table 1. Number of research samples and the results of subclinical mastitis with the CMT test in Enrekang, Indonesia

A total of 33 quarters (29.46%), out of 112 tested udder quarters, were positive for subclinical mastitis, using the CMT test, while the number of cows with subclinical mastitis were 23 (82.14%), out of the 28 dairy cows that were tested. Based on the results in Table 1, the incidence of subclinical mastitis in Enrekang is quite high, at 82.14%. These results are in accordance with some previous research, reporting that the incidence of subclinical mastitis is quite high. The incidence of subclinical mastitis in dairy cows in Indonesia is as high as 95-98%, while [31] mentioned that the incidence of subclinical mastitis in Indonesia is 85%.

3.1. Isolation and Identification of Staphylococcus agalactiae

The 33 subclinical mastitis milk samples tested positively, using the CMT reagent. A total of nineteen milk samples were collected from Lebang village, and fourteen from Pinang village (both villages are in Enrekang, South Sulawesi, Indonesia). Isolation and identification of the bacteria were performed by inoculating the milk samples into a medium brain heart infusion broth (BHIB). Additionally, the identification of bacteria using Gram staining resulted in eleven colonies that were Gram-positive, proven by a group of bacteria looking purple, with a necklace of cocci-shaped colonies.

The results of the Gram staining sample confirmed that the grown bacterial colonies were Streptococcus, based on the cell morphology showing a purple color or being Gram positive, and a chain being cocci shaped. The next identification stage was performed using the catalase test on eleven samples, which obtained negative catalase results. The formation of air bubbles on the colony indicates that these samples belong to a Streptococcus sp. group. The identification of the samples was then performed by a CAMP test, in order to pre-identify whether they involved Streptococcus Group B, or S. agalactiae. The test indicated 98-100% positive results. This positive CAMP test results showed perfect hemolysis zones, forming an arrow-like or crescent shape, in the area close to S. aureus colonies (vertical line). From eleven samples tested, only one isolate showed a positive result, originating from sample S.07.

The strain of S. agalactiae increases the hemolytic activity on Staphylococcal ß- toxins that formed as an arrow-like shape in the CAMP reaction. According to [23], Streptococcus Group B has a CAMP factor, that is, the extracellular protein producing a synergistic hemolysis on sheep blood agar with Staphylococcal ß-lysine (sphingomyelinase C), shared by S. aureus. A complete hemolysis phenomenon of the CAMP test will establish an arrow-head zone. [18] stated that the sphingomyelinase initiates sphingomyelin into ceramide, making erythrocytes easily lysed by CAMP factor activity. Mammalian erythrocytes affect the CAMP factors’ performance in different ways, depending on the sphingomyelin content in the cell membrane. The content of sphingomyelin in sheep blood is 51%, while in the blood of rabbits and humans it is 26 and 19%, respectively. The greater the sphingomyelin content, the clearer a positive reaction was formed by the CAMP test.

Figure 1. CAMP test results compared to literature. A= 1) S. aureus. 2) isolates showed positive results CAMP test 3) isolates demonstrated negative results CAMP test B) 1) S. aureus. 2) S. agalactiae CAMP positive.3) Streptococcus pyogenes negative

The CAMP test results were performed on isolates, originating from two samples: samples with the code S.07 (2), and those with the code S.09 (3). Only one colony formed a vertical line identified as S. aureus, while one colony formed a vertical crescent zone towards S. aureus, which involved the colonies suspected of S. agalactiae. The identification phase was then followed by several biochemical tests; amongst others: the eskulin bile test, the sugars (glucose, maltose, sucrose) test, the motility test, and the indole urea test. Based on [5], the results of the S. agalactiae biochemical test shows a negative eskulin bile test, a positive glucose test, a positive maltose test, a positive sucrose test, a negative motility test, a negative indole test, and a negative urease test. Results from the eleven isolates matched to the Bergeys manual showed that isolates from the samples with the code S.07 involved S. agalactiae. The biochemical results are shown in Table 2.

Table 2. Results of the biochemistry test Streptococcus agalactiae

After the S. agalactiae identification test, colonies were cultivated in blood agar (BA), in order to examine the ability of bacteria in performing hemolysis on erythrocyte, proven by the occurrence of a transparent zone around the colony. According to [26], on blood agar media S. agalactiae is round, transparent, convex, and forms a hemolysis area that is slightly larger than its own colonies (0.5-1 mm in diameter). The morphology of the colony suspected that S. agalactiae is shown on sample S.07. Based on the results of laboratory tests on 33 samples of dairy cows, it was shown that only one sample (3.03%) was positive for S. agalactiae, while the other 32 samples were negative.

Two pathogenic bacteria, that often cause subclinical mastitis, are S. agalactiae (92%) and S. aureus (67%). They are grouped into non-environment pathogens (obligate parasites) and environment pathogens. The Bacterium S. agalactiae, in this classification, is an obligate parasite [32]. According to [7], S. agalactiae is highly infectious, and can be easily transmitted from cows to other lactating cows. The main reservoir of the bacterial infection involves glandules of mammals. Bacteria S. agalactiae can survive in its host in a high temperature, depending on its ability to resist phagocytosis. Isolates from S. agalactiae produce a polysaccharide capsule, which functions as an important virulence factor. The capsules block phagocytosis and act as a complement in the absence of antibodies [21]. Meanwhile, according to [30], S. agalactiae has a capsule, composed by sialic acid and other carbohydrate compounds, that forms the structure of oligosaccharides. This capsule, as one of the S. agalactiae virulence factors, prevents phagocytosis, controls the ability of bacteria to survive, and prevents the bacteria killing process.

3.2. Isolation and Identification of S. aureus

Mastitis positive milk was further tested, to determine whether there was any further S. aureus bacterium. Milk samples were isolated, then followed by an identification test, including observing the colony characteristics, using a catalase test, Gram staining, a coagulase test, a mannitol fermentation test using media mannitol salt agar (MSA), and a pathogenicity test in media blood agar (BA). Isolation was conducted in media Baird Parker agar (BPA), which is a specific medium for staphylococcus, due to the content of sodium pyruvate that stimulates the growth of staphylococcus. The characteristics of S. aureus colonies are that they are round, slippery, grassy/sticky, of a gray to black color, and convex.

An identification test was then performed, starting from a catalase test to distinguish between Streptococcus sp. and Staphylococcus sp. The staphylococcus genus is characterized by a positive catalase result. Catalase enzyme functions as a catalyst, which diffused hydrogen peroxide (H2O2) into H2 and O2; thus, when the bacteria colonies are mixed up with H2O2, gas bubbles are produced. Figure 2A shows a positive catalase test result, which is characterized by gas bubbles. The staphylococcus genus involves Gram-positive bacteria, which morphologically are cocci and are clustered together like grapes (Figure 2C).

Figure 2. Streptococcus agalactiae hemolysis in Blood Agar (A) Catalase positive test (B) and the morphology of Staphylococcus sp purple and clustered (C)

The identification results of the catalase test and of Gram staining, based on 33 positive mastitis milk samples, showed that there were twenty samples (60.6%) containing Staphylococcus sp. bacteria, and from the two samples that were negative for mastitis, there was one sample containing Staphylococcus bacteria.

The coagulase test is an important identification test. The production of the coagulase enzyme becomes the pathogenic factor of S. aureus, differentiating it from other types of Staphylococci. The coagulase enzyme produced by S. aureus is able to clot blood plasma, since it resembles prothrombin, which can convert fibrinogen to fibrin. Figure 3 shows no clots in the blood plasma, indicating negative coagulation.

Figure 3. Positive coagulase (A) S. aureus culture yellow color on MSA (B) and hemolysis of S. aureus culture on BA

An identification test was then followed by mannitol fermentation tests, using MSA as a culture medium. This is a major test procedure, which is commonly used after the coagulation test in the context of identifying S. aureus. The high concentration of sodium chloride (NaCl) in MSA media makes this media selective to S. aureus, since other bacteria do not survive in this condition. Figure 3B shows culture results on MSA media; S. aureus colonies have a golden color and their color changes, from pink to golden yellow. This is caused by the S. aureus’ ability to produce acid, which modifies the medium.

Other bacteria from the Staphylococcus genus, which grow in this medium, include S. epidermidis; yet, this bacterium cannot ferment mannitol. Therefore, the colony looks white and the media remains pink. The test results showed that four (25%) out of sixteen samples were positive for Staphylococcus sp., proven by the positive coagulase, and were capable of fermenting mannitol on the MSA media. Whereas, the mastitis negative samples did not grow on mannitol salt agar (MSA) media and negative coagulase.

Following the identification of S. aureus, the colonies were then streaked onto blood agar (BA), in order to observe the bacteria’s ability to induce hemolysis blood erythrocyte, to visualize the hemolysis zone around colonies (Figure 3C).

Various types of bacteria cause subclinical mastitis, for example, S. aureus. Results of observations during the milking process have shown that there feces/dirt remains around the cows and/or the cage. Milking is performed using simple tools, such as a plastic bucket milk container, but some breeders used old plastic buckets, which had previously been used to paint the walls, while the milk collecting containers were not closed during milking. Additionally, the farmers’ habit of not washing the nipples (dipping) before and after milking may increase the risk of mastitis.

Examination of subclinical mastitis using a CMT reagent offers a high level of sensitivity and specificity. This is also supported by [27], which compares the diagnosis for the detection of subclinical mastitis, and showed the result of 100% sensitivity for somatic cell count and 96% for CMT reactions. Sensitivity test, which assess the ability of a reagent, showed positive results in cows suffering from subclinical mastitis, while Specificity test indicates a negative result in cows suffering from subclinical mastitis. The CMT reagent contains a detergent or surfactant, which is a composition wherein the surfactant can be used to detect the increasing degree of somatic cells in mastitis milk. According to [33], different types of surfactants have different effects on milk containing Aril CMT alkyl sulfonate (3%), sodium hydroxide (1.5%) and Bromocresol purple. Aryl alkyl sulfonate (3%) was shown to have great sensitivity in terms of milk pH, while bromocresol purple is a color indicator highlighting the reaction during observation. In the milk affected by mastitis, there will be an increasing number of leukocytes; therefore, the pH will be more alkaline. If an active substance, such as alkyl sulfonate Aril, is added (3%), this will react with milk somatic cells, including leukocytes, resulting in the increase of milk concentration to becomes more viscous (thick), forming a gel. The laboratory test on 33 positive mastitis samples revealed that 60.6% had bacteria Staphylococcus sp., characterized as catalase positive. In the catalase test, most of the bacteria producing enzyme catalase that can decompose H2O2 into H2O and O2. Hydrogen peroxide is toxic to cells, because this material inactivated enzymes in the cells. Hydrogen peroxide forms during aerobic metabolism, so that microorganisms grow in an aerobic environment, needing to decompose the toxic material. Catalase is one of the enzymes that uses micro-organisms to decompose hydrogen peroxide. This means that H2O2 is not decomposed by catalase-negative bacteria, thus not producing oxygen. Catalase-negative bacteria do not contain the catalase enzyme, which decomposes H2O2, including the genus of Streptococcu22s. [19] suggests that mastitis caused by Staphylococcus can reach 70% within a farm. The inhibitory titer was progressively increased, when five to 30 μg of ammonium thiocyanate per ml of milk were added to pasteurized milk, and the diluent [9]. [16], found the bacteria S. aureus in 145 (48.33%) of their 300 mastitis milk samples. Based on previous research conducted by [2], it can be concluded that the incidence of mastitis caused by S. aureus on a farm is quite high. S. aureus is a contagious bacterium, which can be isolated from the skin surface of humans or animals. Its role as a cause of mastitis in cows is related to environmental sanitation and milking hygiene. The observation of environmental conditions showed that some cages do not have proper sanitation, whereas sewer and fesses containers are too close to the water or to feed storage areas. In such conditions, the possibility of S. aureus strains infection not derived from humans and animals is very high. In accordance with [23], besides being able to be isolated from the skin, S. aureus can also be isolated from the equipment in cages, from the cage environment and from milking tools. Generally, strains of S. aureus found in milk were not derived from cows or human contamination. Based on the results of 28 samples of milk from dairy buffaloes spread in Enrekang, no milk samples were found positive for S. aureus and S. agalactiae.

4. Conclusion

Based on the milk samples from 33 buffaloes (Bubalus bubalis), which were positive for subclinical mastitis in Enrekang, one milk sample (3.03%) was positive for S. agalactiae, while four samples (12.12%) were positive for S. aureus, whereas 28 milk samples from the dairy cows were negative for both S. agalactiae and S. aureus bacteria.

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