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

Muslimin Lucia1, Sri Rahayu1, DzulHaerah1, and Wahyuni1 Veterinary Medicine, Faculty of Medicine, Hasanuddin University, Makassar, South Sulawesi Corresponding author: [email protected]

Abstract This study aims to detect the presence of Staphylococcus aureus (S. aereus) and (S. agalactiae) in subclinical mastitis infection in dairy cows and buffalo in Enrekang (region in South Sulawesi). Subclinical mastitis was pre-examined using California Mastitis Test (CMT) reagent and 33 samples were positively detected. The positive samples were then isolated with culture test on the Baird Parker Agar media (BPA), identified with catalyst, Gram , test, and isolated in Mannitol Salt Agar (MSA) media. To distinguish the S. aureus from others Staphylococcus species, the sample was tested on blood agar media to observe the presence of . For S. agalactiae identification, culture methods, Gram staining as well as biochemical tests with catalyst, Christie Atkins Munch-Peterson (CAMP) test, eskulin bile test, glucose, maltose, sucrose, motility test, , urea and blood agar test were used. The result showed that 4 samples were positive of S. aureus characterized with grayish colonies observed on the BPA, positive result on gram staining (formed purple cocci bacteria chain), catalyst test, coagulase test and blood agar test. Glucose, maltose, lactose tests were also positive, while the catalyst test, eskulin bile test, indole, test showed negative results. The result also showed that S. agalactiae was deteccted in one milk sample while the others 28 samples were negatively detected for both bacteria.

Key words: S. aureus, S. agalactiae, dairy cattle, dairy buffaloes

information on the factors that can influence its growth in milk (21). Streptococcus agalactiae continues to be a 1. Introduction major cause of subclinical mastitis in dairy. Cattle and as cource of economic loss for the industry.Veterinarians are As one of the dairy products, milk is an animal protein often asked to provide information on herd level control sourse that is increasing demand in improving life quality. and eradication of Staph.agalactiae mastitis (14). Strep. Staphylococcus aureus is a gram-positive bacterium that Agalactiae have attracted most attention due to their colonizes a variety of animal species . S. aureus infections economic significance (10) According to (1). in animals are most commonly reported as a cause of Streptococcus agalactiae, one of Streptococci species that mastitis in dairy-producing animals (including cattle and infecting both terrestrial and aquatic animals. The goats) and “bumblefoot” in chickens (24). Staphylococcus organisms have been isolated from numerous fish species aureus is the key causative agent for mastitis and in natural disease outbreaks and showed to be pathogenic responsible for sub-clinical cases although it is also to several fish species responsible for different forms of the disease.( 19.) Using Mastitis is a common disease and is a major problem in bacteriological, biochemical and PCR-based identification the world of dairy farming because it cause huge schemes, 12 (25.53%) isolates were confirmed as S. economic losses to the dairy farm in the world (4) The aureus. All the isolates showed β-hemolysis on 5% sheep economic losses caused by mastitis, especially subclinical blood agar. S. aureus specific nuc 27gene (target size 279- mastitis, includes declining milk production and quality, bp) was amplified in the cases of all isolates (12). Animal increasing maintenance costs and treatment, and early S aureus always evolve from human strains, such that livestock culling. Expressed by ( 8), the decline in milk every human strain may be the ancestor of a novel animal- production due to mastitis reach about 15-20% of the total adapted strain. The zoonotic transfer of IMI- and milk- milk production, while according to ( 27), milk associated strains of Staph. aureus between cattle and production declined to about 30%. Mortality from humans seems to be very limited and different hosts are infections associated with S. aureus bacteraemia can not considered as a source for mutual, spontaneous range from as low as 2.5% to as high as 40% (28) infections (6). Staphylococcus aureus (S. aureus) mastitis is extremely According to ( 31) isolated S. agalactiae caused 83% difficult to control by treatment alone. To date, successful subclinical mastitis in Bogor (region in West Java), 82% control is gained only through prevention of new in Boyolali (region in Central Java), and 80% in Malang infections and culling of infected animals (32) S. aureus (region in East Java). S. agalactiae is an important cause organisms colonize teat ends and/or teat lesions. Spread of of mastitis, it was considered desirable to obtain additional infection can occur through milkers’ hands, washcloths,  Fermentation test on Mannitol Salt Agar media (MSA): teat cup liners, and flie acid condition were produced as the color changes from pink to medium yellow which indicates the presence of S. aureus  Coagulation Test: the case of plasmatic clotting 2. Materials and Methods showing the presences of S. aureus  Hemolysis Test (Test Pathogenicity): the pathogenic S. Sample was collected during the afternoon milking. aureus is characterized by the ability to lyse red blood Before the samples were taken, they first performed cells, with the presence of a transparent zone around the Mastitis Test using a reagent california mastitis test colonies on Blood Agar media.( 16) (CMT). A total of 2 ml of milk is placed on the paddle and added with 2 ml reagent CMT, and then horizontally and The ability of Staphylococcus aureus to adhere to the gently shaken for 10-15 seconds. The test result is extracellular matrix and plasma proteins deposited on considered negative if the mixture remain homogeneous, biomaterials is a significant factor in the pathogenesis of positive 1 if sedimentation is visible, positive 2 if mixture orthopaedic-device related infections. S. aureus possesses thickens rapidly and gel moves to the middle, and adhesion proteins on its surface, but it is not known how positive 3 if a lot of gel formed which causes the surface they interact with each other to form stable interactions turns convex . with the substrate (11), also has emerged as a significant public health problem both in human and veterinary 2.1. Streptococcus agalactiae Test medicine (13). Methicillin-resistant Staphylococcus aureus (MRSA) is a critically important human pathogen Milk samples which is positive with CMT were grown that is also an emerging concern in veterinary medicine in a Brain Heart Infusion Broth media (BHIB) and then and animal agriculture. It is present in a wide range of incubated for 18-24 hours at 37° C, and later with Nutrient animal species, including dogs, cats, rabbits, horses, cattle, Agar (NA) media. Colonies suspected contain pigs, poultry, and exotic species, both as a cause of Streptococcus sp. characterized by round, small, smooth, infection and in healthy carriers (29) convex, transparent, and 0.5-1 mm in diameter. Bacterial The prevalence of S. aureus can most likely be culture on the suspected Streptococcus sp., were then attributed to the wide distribution of the organism inside identified through several biochemical tests as follows: mammary glands and on the skin of teats and udders.  Gram staining: Streptococcus sp., were characterized by S. aureus adapts very well in the udder and establishes purple, round (cocci) and long chain chronic and subclinical infections. From there it is shed  test: S. agalactiae is negative if no bubbling into the milk, which serves as a source of infection for formation. healthy cows during the milking process. (3)  Christie, Atkins, Munch-Peterson test (CAMP Test): CAMP test was conducted on Media Blood Agar with S. aureus as a marker. Staphylococcus aureus is 3. Result and Discussion streaked in the middle of the media and then streak the sample suspected with S. Agalactiae across a plate Subclinical mastitis examination was performed using perpendicular to the S. Aureus streak. Cultures were CMT reagent on each of the udder quarters of cows and then incubated for 24 to 48 hours at 37 C. buffaloes.The test results of subclinical mastitis shown in Establishment of an arrow-like zone at the junction of 2 Table 1. organisms indicated the presence of S. agalactiae colonies. Table 1. Number of research samples and the results of subclinical mastitis with CMT test in Enrekang  Sugar Test: positive results for glucose, sucrose, and No Name No. of No. of No. of No. of maltose tests indicate the presence of S. agalactia. samples quarters quarters patients with  Bile Bacteria Test: S. agalactiae not change to black (per that tested CTM subclinical color, indicating a negative result. cow) CTM positive mastitis (tail) (quarters) (quarter)  Motility test, indol test, urease test: negative results indicate the absent of S. agalactiae 1. L 17 68 19 14 (82.35%)  Hemolysis test of S. agalactiae were performed on a (27.94%)

Blood . 2. P 11 44 14 9 (81.81%) (31.81%) 2.2. S. Aureus Test Number 28 112 33 23 (82.14%)

Staphylococci are gram positive, non-motile and non (29.46%) sporeforming bacteria. Pathogenic staphylococci are identified by their ability to produce coagulase thus clot A total of 33 quarters (29.46%) out of 112 tested udder the blood (11) quarters were positive subclinical mastitis using CMT test, Detection and differentiation of S. aureus were while number of subclinical mastitis cows were 23 conducted on Baird Parker Agar (BPA) media. S. aureus individuals (82.14%) out of 28 tested dairy cows. Based colonies on BPA characterized by round, slimy/smooth, on the results in Table 1, the incidence of subclinical convex with diameter 2 mm-3mm, grayish to black, with a mastitis in Enrekang is quite high at 82.14%. These results clear halo develops around the edge of the colony. are in accordance with some previous research which Further, the presence of S. aureus was confirmed by the reported that the incidence of subclinical mastitis is quite following tests: high. The incidence of subclinical mastitis in dairy cows in Indonesia is as high as 95-98%. While ( 31), mentioned negative results CAMP test B) 1) Staph.aureus. 2) Staph.agalactiae the incidence of subclinical mastitis in Indonesia is 85%. CAMP positive.3) Streptococcus pyogenesnegative CAMP test results performed on isolates originating 3.1 and Identification of from two samples: samples with the code S.07 (2) and Stapylococcus agalactiae S.09 (3). Only one colony formed a vertical line identified as S. aureus and 1 colony forming vertical crescent zone The 33 subclinical mastitis milk samples were tested towards S. aureus is the colonies suspected of S. positive using the CMT reagent. Total of 19 milk samples agalactiae. The identification phase was then followed were collected from the Lebang village and 14 from the with several biochemical tests, among others, eskulin bile Pinang village. Isolation and identification of bacteria test, sugars (glucose, maltose, sucrose) test, motility test, were performed by inoculating milk sample into medium and indole urea test. Based on (5), the results of S. brain heart infusion broth (BHIB). On the other hand, agalactiae biochemical test shows negative eskulin bile identification of bacteria was carried out followed by test, positive glucose test, positive maltose test, positive Gram staining, resulted in 11 colonies of Gram-positive sucrose test, negative motility test, negative indole test, was revealed, proven by a group of bacteria that looks and negative urease test. Results from the 11 isolates purple with a necklace of cocci-shaped colonies. matched to Bergeys manual showed that isolates from the Results of Gram staining sample confirming the grown samples with the code S.07 is S. agalactiae. The bacterial colonies was Streptoccous based on the cell biochemical results are shown in Table 2. morphology that showed purple color or Gram positive, and a chain of cocci shaped.The next identification stage Table 2. Results of biochemistry test Streptococcus agalactiae

was performed by catalase test on 11 samples which Sugars Test

obtained negative catalase results, characterized by the

formation of air bubbles on the colony which indicates Forms

Bile

Indol

Gram

Glu

Suk

Mal Urease

CAMP

Eskulin Motility these samples are belongs to a Streptococcus sp. group. Catalase The identification of the samples were then performed by CodeSample CAMP test to pre-identify whether they were S 04 Streptococcus + - - + + + - - - - S 05 Streptococcus + - + - + + - - - - Streptococcus Group B or S. agalactiae, which obtained S 07 Streptococcus + - - + + + - - - + 98-100% positive results. This positive CAMP test results S 09 Streptococcus + - + - + + - - - - showed perfect hemolysis zones forming an arrow-like or S 11 Streptococcus + - - + + + - - - - a crescent shape in the area close to S. aureus colonies S 14 Streptococcus + - - + + + - - - - S 16 Streptococcus + - + + + + - - - - (vertical line). From 11 samples tested, only one isolate S 21 Streptococcus + - + + + + - - - - showed positive result which was originated from sample S 23 Streptococcus + - - + + + - - - - S.07. S 29 Streptococcus + - + - + + - - - - Strain of S. agalactiae increases the hemolytic activity S30 Streptococcus + - + - + + - - - - on Staphylococcal ß- toxins formed as an arrow-like shape After the S. agalactiae identification test, colonies were in CAMP reaction. According to (23) Streptococcus cultivated in blood agar (BA) to examine the ability of Group B has CAMP factor, that is the extracellular protein bacteria in performing hemolysis on erythrocyte proven that produces a synergistic hemolysis on sheep blood agar by the occurring of transparent zone around the colony. with Staphylococcal ß-lysine (sphingomyelinase C) shared According to (25) on blood agar media S. agalactiae is by S. aureus. A complete hemolysis phenomenon of round, transparent, convex and form hemolysis area that is CAMP test will establish an arrow-head zone. ( 17), stated slightly larger than its own colonies (0.5-1 mm in that the sphingomyelinase initiates sphingomyelin into diameter). The morphology of the colony suspected S. ceramide which makes erythrocytes easily lysed by agalactiae is shown on sample S.07. Based on the results CAMP factor activity. Mammalian erythrocytes affect the of laboratory tests on 33 samples of dairy cows showed CAMP factors performance in different ways, depending only one sample (3.03%) was positive S. agalactiae, while on the sphingomielin content in the cell membrane. the other 32 samples were negative. Sphingomielin content in sheep blood is 51% while in the Two pathogenic bacterias that often cause subclinical blood of rabbits and humans are 26 and 19%. The greater mastitis are S. agalactiae (92%) and S. aureus (67%). the sphingomielin content, the clearer the positive reaction They are grouped into non environment pathogens formed the CAMP test. (obligate parasites) and environment pathogen. Bacteria S.

agalactiae, in this classification, is an obligate parasites (33). According to (7) S. agalactiae is highly infectious and easily transmitted from cows to other lactating cows. The main reservoir of the bacterial infection is glandules of mammae. Bacteria S. agalactiae can survive in its host in a high temperature, depending on its ability to resist phagocytosis. Isolates from S. agalactiae produces a polysaccharide capsule which function as an important virulence factor. The capsules block phagocytosis and act as a complement in the absence of antibody (20). Meanwhile, according to (30) S. agalactiae has a capsule composed by sialic acid and other carbohydrate compounds that form the structure of oligosaccharides. Fig 1. CAMP test results compared to literature. A= 1) Staph.aureus. 2) This capsule, as one of the S. agalactiae virulence factors, isolates showed positive results CAMP test 3) isolates demonstrated prevents phagocytosis, controls bacteria survive ability major test procedure which is commonly used after and prevents the bacteria killing process. coagulation test in identifying S. aureus. The high concentration of sodium chloride (NaCl) in MSA media 3.2. Isolation and Identification of S. aureus makes this media selective to S. aureus, since other bacteria not survive in this condition. Figure 3B shows Mastitis positive milk is further tested to determine culture results on MSA media, S. aureus colonies has whether there is any further S. aureus bacterium. Milk golden color and change the color from pink to golden samples were isolated then followed by identification test yellow. It caused by the S. aureus ability in producing including the observation of the colony characteristics, acid which modify the medium. catalase test, Gram staining, coagulase test, mannitol Other bacteria from Staphylococcus genus which grows fermentation test using media mannitol salt agar (MSA), on this medium is S. epidermidis, yet, this bacteria cannot and pathogenicity test in media blood agar (BA). Isolation fermenting mannitol and therefore the colony looks white is conducted in media Baird Parker agar (BPA), which is a and the media remains pink. The test results showed that specific medium for staphylococcus, due to the content of four (25%) out of 16 samples are positive Staphylococcus sodium pyruvate that stimulates staphylococcus growth. sp. proven by positive coagulase and capable in The characteristic of S. aureus colonies are round, fermenting mannitol on the MSA media. Whereas, the slippery, grassy/sticky, gray to black color, and convex. mastitis negative samples does not grow on mannitol salt Results of bacteria in media Baird Parker agar (BPA) . agar (MSA) media and negative coagulase. Identification test is then performed starting from Following the identification of S. aureus, the colonies catalase test to distinguish between Streptococcus sp. and were then streak onto blood agar (BA) aimed to observe Staphylococcus sp. Staphylococcus genus is characterized the ability of bacteria to hemolysis blood erythrocyte, thus by positive catalase result. Catalase enzyme functions as a catalyst which diffused hydrogen peroxide (H2O2) into the hemolysis zone around colonies will be visible (Figure H2 and O2, thus, when the bacteria colonies are mixed up 3C) with H2O2, the gas bubbles produced. Figure7 (A) shows Subclinical mastitis is caused by various types of positive catalase test result which is characterized by gas bacteria, for example S. aureus. Results of observations bubbles. Staphylococcus genus is Gram-positive bacteria, during milking process showed that there is still feces/dirt which morphologically cocci and clustered together like around the cows or the cage. Milking is performed using grapes (Figure 2C). simple tools such as a plastic bucket milk container but some breeders were used old plastic bucket which were used to paint the walls, and the milk collective 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 cmt reagent offer a high level of sensitivity and specificity. this is also supported by (26), which compares the diagnostic for the Fig 2. Streptococcus agalactiae hemolysis in Blood Agar (A), Katalase detection of subclinical mastitis, and showed the result of positive test (B) and the morphology of Staphylococcus sp purple and 100% sensitivity for somatic cell count and 96% for CMT clustered (C) reactions. Sensitivity is the ability of a reagent to show The identification results of catalase test and Gram positive results in cows suffering from subclinical staining from 33 positive mastitis milk samples, there are mastitis, while specificity is the ability of a reagent to 20 samples (60.6%) containing the bacteria indicate a negative result in cows suffering from Staphylococcus sp. and from the 2 negative samples subclinical mastitis. The CMT reagent contains detergent mastitis, there is one sample containing Staphylococcus or surfactant which is a composition wherein the bacteria. surfactant can be used to detect the increasing degree of Coagulase test is an important identification test. The somatic cells in mastitis milk. According to ( 34) different production of coagulase enzyme becomes pathogenic types of surfactants have different effects on milk which factor of S. aureus that differentiates them from other contains Aril CMT alkyl sulfonate (3%), sodium Staphylococcus. Coagulase enzyme produced by S. aureus hydroxide (1.5%) and Bromocresol purple. Aryl alkyl is able to clot blood plasma since it resembles sulfonate (3%) had a great sensitivity to the milk pH, prothrombin, which can converts fibrinogen to fibrin. while bromocresol purple is a color indicator to highlight Figure 3 shows no clots in the blood plasma indicating the reaction during observation. In the milk affected by negative coagulation. 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%), it will react with milk somatic cells, including leukocytes, resulting in the increasing of milk concentration to becomes more viscous (thick) and forming a gel. Laboratory test on 33 positive mastitis samples revealed that 60.6% has bacteria Staphylococcus sp. characterized with catalase positive. In the catalase test, most of the Fig 3. Positif coagulase (A) S. aureus culture Yellow colour on MSA (B) and hemolysis of S. aureus culture on BA bacteria producing enzyme catalase that can decompose H2O2 into H2O and O2. Hydrogen peroxide is toxic to Identification test is then followed by mannitol cells because this material is inactivating enzymes in the fermentation tests using MSA as culture medium. This is a cells. Hydrogen peroxide is formed during aerobic metabolism, so that microorganisms grow in aerobic E. Ryan, O. Salat, P. Zangerl, A. Steiner, H.U. Graber.2016. environment needs to decompose the toxic material. 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