Abstract: This study aimed to identify staphylococcal mastitis milk in Peranakan Etawa (PE) or Etawa crossbred goats in Indonesia. Total of 93 milk samples used in this study, there were 49 (52.7%) subclinical and 3 (3.2%) clinical mastitis samples. From 52 subclinical and clinical mastitis samples could be isolated 20 (38.5%) staphylococci. Phenotypic identification were performed based on the Gram staining, Mannitol Salt Agar (MSA), catalase, Voges-Proskauer (VP) and coagulase tests. Molecular identification were determined using the Polymerase Chain Reaction (PCR) to detect several genes such as 23S rRNA, nuc and coa genes and confirmed with DNA sequencing. Based on phenotypic and genotypic identification, could be identified 10 Coagulase Positive Staphylococci (CPS) indicated Staphylococcus aureus and 10 Coagulase Negative Staphylococci (CNS) isolates, including Staphylococcus pasteuri (3 isolates), Staphylococcus xylosus (5 isolates) and Staphylococcus haemolyticus (2 isolates). From 3 clinical mastitis of PE goats in our study could be isolated three different staphylococci i.e., S. aureus, S. pasteuri and S. xylosus. This finding indicated that both CPS and CNS have important role as causative pathogen of mastitis in PE goats.
INTRODUCTION
In Indonesia, smallholders keep goats as an important component of farming activities. Small ruminant production plays an important role as an income generating activity, particularly for the smallholders, whilst being a source of animal protein to support the national program. Peranakan Etawa (PE) goat is descended originally from crossings between the Kacang goat (indigenous breed of goat in Indonesia) with Etawa (Jamnapari India) goat. Intra mammary infections among small ruminants raised in Indonesia are not well documented in spite of research overseas report that described the susceptibility of cows and goats to Staphylococcal infections.
Mastitis in goats is commonly found and caused economic losses due to decrease in milk production (Bleu et al., 2006). Staphylococcus aureus is one of bacteria caused clinical mastitis or subclinical mastitis in small ruminants as well PE goats (Contreras et al., 2007; Purnomo et al., 2006). Staphylococcus aureus is also an important food-borne pathogen because of its ability to produce a wide range of extracellular toxin proteins and virulence factors that contribute to the pathogenicity of the organism (Greig et al., 2007; Salasia et al., 2011).
Staphylococcus aureus is characterized as a Coagulase Positive Staphylococci (CPS) and known as the main pathogen of mastitis infections in dairy animals. Mostly researcher focused on this pathogen as an important mastitis agent. However, misidentification of this bacteria with the coagulase negative staphylococci are commonly occurred, raising the mastitis diagnostic problem.
Coagulase Negative Staphylococci (CNS) cause mainly subclinical mastitis and also clinical mastitis in cows (Bochniarz et al., 2013; Taponen et al., 2006). Clinical mastitis due to CNS are characterised by mild symptoms but associated with increased Somatic Cell Count (SCC) (Taponen et al., 2007) and decreased milk production, which results in economic losses (Bochniarz et al., 2013). The infection of CNS in cows, sheep and goats hasmarkedly increased (Contreras et al., 2007). Coagulase-negative staphylococci have also reported as predominant pathogens in human infections (Banerjee et al., 1991). It is important to accurately identify the CNS isolates to further define the epidemiology of these microorganisms.
This study aimed to identify coagulase-positive and negative staphylococci as the causative agents of subclinical and clinical mastitis cases in PE goats. Both phenotypic and genotypic characteristics Staphylococcus strains can be used as the basis of epidemiological studies and mastitis control in goats.
MATERIALS AND METHODS
Bacterial isolates: Samples were obtained from 3 PE goats with clinical mastitis (3 milk samples) and 37 PE goats with subclinical mastitis (60 milk samples) in the region of Yogyakarta, Central Java Indonesia, 30 milk samples from 17 PE goats with subclinical mastitis in the region of Riau Indonesia. Staphylococcus aureus strain BY7 isolated from milk cow described in previous research (Salasia et al., 2011) was used as a control strain and non-staphylococcal isolate used as a negative control. A clinical examination of each udder was conducted in order to detect the goats with signs of clinical mastitis. Each teat was thoroughly washed and then disinfected. The first few streams of foremilk were discarded and the middle stream of each milk sample was aseptically collected into sterile containers. Samples collected from apparently normal halves of the udder were subjected to California Mastitis Test (CMT).
The strains were identified as S. aureus and coagulase-negative staphylococci by their properties in culture, Gram staining and biochemical tests. The biochemical tests included ones that measured Mannitol Salt Agar (MSA), catalase, coagulase and Voges-Proskauer (VP) as described previously (Harrigan, 1998; Salasia et al., 2004). The MSA test was observed based on the change of phenol red in the media by bacteria to yellow colour, indicating mannitol fermentation. The catalase test was done by placing a drop of hydrogen peroxide on a microscope slide. A small amount of bacterial isolate was added to hydrogen peroxide, bubbles of oxygen were observed for catalase-positive. The coagulase test was performed by cultivation of the bacteria in the tube coagulase test (Bactident-Coagulase; Merck, Germany). The presence of coagulation was observed at 6 and 24 h. The VP positive test was observed by the change of VP medium colour to pink.
Molecular identification: Furthermore, the strains were identified by Polymerase Chain Reaction (PCR) amplification of the 23S rRNA gene (Cremonesi et al., 2005; Straub et al., 1999), thermonuclease nuc gene (Brakstad et al., 1992; Cremonesi et al., 2005) and coagulase (coa) gene (Cremonesi et al., 2005; Hookey et al., 1998). The reaction mixture (25 μL) contained 1 μL primer 1 (20 pmol), 1 μL primer 2 (20 pmol; Invitrogen, USA), 12.5 μL PCR mix containing Taq DNA polymerase, MgCl2 and dNTPs (KAPA Biosystem, USA), 2 μL of DNA template and 8.5 μL distilled water. The DNA of the isolates was prepared with the QIAamp DNA mini kit (Qiagen, Germany) as described by the manufacturer. The amplification of the genes was carried out with a thermal cycler (Mastercycler, Eppendorf, Germany). The oligonucleotide primers and the thermal cycler programs are shown in Table 1.
The PCR products were separated by gel electrophoresis in a 1.5% (w/v) agarose gel (Roth, Germany) in 0.5×TBE buffer (containing a mixture of Tris base, boric acid and EDTA). A 100 bp DNA ladder (Geneaid, Taiwan) was used as a size marker. The resulting bands were visualized using FloroSafe (1St Base, Singapore) staining under UV transillumination.
DNA isolation and purification: DNA sequences of four different species were isolated from milk PE goats (Table 2). A QIAmp DNA mini kit (Qiagen, Germany) was used to purify the DNA from S. aureus according to the manufacturer’s protocol. The bacterial strains were cultivated on blood agar base (Oxoid, Germany) containing 5% defibrinated sheep blood for 24 h at 37°C.
| Table 1: | Oligonucleotide primers and PCR programs used for amplifying the gene encoding staphylococcal 23S rRNA, nuc and coa genes |
| *1: 30 cycles 94°C for 5 min, 94°C for 60 sec, 56°C for 60 sec, 68°C for 60 sec, 72°C for 7 min, 2: 37 cycles 94°C for 5 min, 94°C for 40 sec, 64°C for 60 sec, 72°C for 75 sec, 72°C for 5 min, 3: 37 cycles 94°C for 5 min, 94°C for 1 min, 55°C for 30 sec, 72°C for 30 sec, 72°C for 5 min, 4: 30 cycles 94°C for 5 min, 94°C for 1 min, 58°C for 1 min, 72°C for 1 min and 72°C for 5 min | |
| Table 2: | DNA sequences of bacterial isolates from milk PE goats in Indonesia |
| PE: Peranakan Etawa | |
A total of 5~10 S. aureus colonies were suspended with 180 μL TE buffer (10 mM Tris-HCl and 1 mM EDTA [pH 8]) containing 5 μL lysostaphin (1.8 U μL–1; Sigma, USA) in 2 mL microfuge tubes. The suspension was incubated for 1 h at 37°C and 25 μL of proteinase K (14.8 mg mL–1; Sigma, USA) and 200 μL of AL buffer (containing reagents AL1 and AL2; Qiagen, Germany) were then added. The suspensions were incubated for 30 min at 56°C and then for 10 min at 95°C before being spun at 6,000×g for a few seconds. A total of 420 μL ethanol was added to each sample and placed in a spin QIAmp column. After centrifugation at 6,000×g for 1 min, the spin columns were placed in a clean collection tube a nd the sample was washed twice with 500 μL of AW buffer (Qiagen, Germany). After the second wash and a centrifugation at 6,000×g for 3 min, the QIAamp spin columns were placed in a clean 2 mL microfuge tube and the DNA was eluted twice with 200 and 100 μL of AE buffer (Qiagen, Germany). DNA was stored at -20°C.
DNA Sequencing: The PCR products of 23S rRNA that were amplified with the oligonucleotide primers described by Straub et al. (1999) and Cremonesi et al. (2005) were purified and sequenced (1st BASE, Singapore) before alignment. A nucleotide sequence homology search was performed through the National Center for Biotechnology Information (NCBI) BLAST network service according to the algorithm of Altschul et al. (1990).
RESULTS
A total of 93 milk samples were collected from 57 goats in different dairy goat farms in Indonesia for microbial examinations are shown in Table 3. From total of 93 milk PE samples used in this study, there were 49 (52.7%) subclinical and 3 (3.2%) clinical mastitis samples. From 52 subclinical and clinical mastitis samples could be isolated 20 (38.5%) staphylococci. According to the results of cultural and biochemical properties, along with amplification of the 23S rRNA and sequencing for species specific, all 20 isolates could be identified 10 Coagulase Positive Staphylococci (CPS) indicated Staphylococcus aureus and 10 Coagulase Negative Staphylococci (CNS) isolates, including Staphylococcus pasteuri (3 isolates), Staphylococcus xylosus (5 isolates) and Staphylococcus haemolyticus (2 isolates). From 3 clinical mastitis of PE goats in our study could be isolated three different staphylococci i.e., S. aureus, S. pasteuri and S. xylosus.
| Table 3: | Samples for microbiological examination |
| CMT: California mastitis test | |
| Table 4: | Phenotypic and genotypic characteristic of Staphylococcus aureus and Coagulase Negative Staphylococci (CNS) isolates from milk PE goats in Indonesia |
| 1: Yogyakarta, Central Java, 2: Pakanbaru, Riau, sc: Subclinical mastitis, c: Clinical mastitis, CMT: California mastitis test, MSA: Mannitol salt agar, VP: Voges proskauer, 1Cremonesi et al. (2005), 2Straub et al. (1999), 3Brakstad et al. (1992), 4Hookey et al. (1998) | |
All 20 cultures investigated were Gram positive, positive for catalase, fermented mannitol in MSA and positive for Voges-Proskauer with an exception for 2 isolates of S. pasteuri and S. xylosus. All 10 S. aureus were positive for coagulase, whereas S. pasteuri, S. xylosus and S. haemolyticus isolates were negative for coagulase.
The PCR results of the 23S rRNA gene amplified with the primers designed by Cremonesi et al. (2005), all S. aureus could be observed amplicon with the size of about 499 and 1250 bp (Fig. 1a) with the primers described by Straub et al. (1999). All CNS isolates revealed single amplicon 23S rRNA gene with a size of about 499 bp (Fig. 1b). After sequencing the PCR product of 23S rRNA of both amplicons 1250 and 499 bp, revealed that sequence of 1250 bp were 99-100% identical with S. aureus, whereas the sequence of 499 bp turned out to be identical to S. pasteuri (99%), S. xylosus (96%) and S. haemolyticus (97%).
The PCR amplification of the nuc gene revealed that all S. aureus isolates had a single amplicon approximately 400 bp in size (Fig. 2a) with the primers designed by Cremonesi et al. (2005) and 279 bp (Fig. 2b) with the primers according to Brakstad et al. (1992). The PCR products of the coa gene of all S. aureus amplified with the primers of Cremonesi et al. (2005) showed a single amplicon with the size of 204 bp (Fig. 3a), whereas amplification with the primers described by Hookey et al. (1998) revealed five different PCR products of 430, 600, 700, 800 and 840 bp (Fig. 3b).
| Fig. 1(a-b): | PCR-amplified 23S rRNA genes of selected, (a) Staphylococcus aureus isolates and (b) CNS isolates from PE goats, lane M: 100 bp molecular weight marker, lane A, a: Staphylococcus aureus control strain, lane B, b: A negative isolate, lane C-F: Amplicon of 1250 bp, lane c-f: Amplicon of 499 bp |
| Fig. 2(a-b): | PCR-amplified nuc genes of selected, (a) Staphylococcus aureus isolates and (b) CNS isolates from PE goats, lane M: 100 bp molecular weight marker, lane A, a: Staphylococcus aureus control strain, lane B, b: A negative isolate, lane C-F: Amplicon of 279 bp, lane c-f: Amplicon of 400 bp |
| Fig. 3(a-b): | PCR-amplified coa genes of selected, (a) Staphylococcus aureus isolates and (b) CNS isolates from PE goats, lane M: 100 bp molecular weight marker, lane A, a: Staphylococcus aureus control strain (204, 850 bp), lane B, b: A negative isolate, lane C-F: Amplicon of 204 bp, lane c: Amplicon of 680 bp, lane d-f: Amplicon of 600 bp |
All CNS isolates could not be detected the coa gene. The pheno and genotypic characteristic of Staphylococcus aureus and Coagulase Negative Staphylococci (CNS) are shown in Table 4.
DISCUSSION
Our finding showed that based on the phenotypic identification, S. aureus and CNS could be differentiated through the ability to coagulate plasma. Based on the genotypic identification, S. aureus could be differentiated with CNS as follows: S. aureus could be identified based on 23S rRNA, nuc and coa genes for both amplicon resulted by references showed in the Table 1. Staphylococcus pasteuri, S. xylosus isolates could be distinguished only by 499 bp 23S rRNA and 400 bp nuc genes, whereas S. haemolyticus only by 499 bp 23S rRNA. This findings have been confirmed by sequencing based on the PCR products of 23S rRNA with the oligonucleotide primers as described by Brakstad et al. (1992) and Cremonesi et al. (2005). Identification to distinguish CNS and S. aureus should be determined by genotypic identification, using specific primers to detect gene 23S rRNA, nuclease gene (nuc gene) and gene encoding of coagulase (coa gene). The difference in the amplicon size of the 3 end of the coa gene can be explained in polymorphisms of this gene by the typical 81 bp tandem short sequence repeats (Goh et al., 1992). The existence of 23S rRNA, nuc and coa gene can be used as a marker specific species between Staphylococcus spp. (Brakstad et al., 1992; Cremonesi et al., 2005; Hookey et al., 1998; Salasia et al., 2004; Straub et al., 1999).
In the present study most of S. aureus and CNS isolated from the milk of PE goats with subclinical mastitis (85%) and only 3 isolates (4.8%) from clinical mastitis. Mastitis can cause a serious economic burden for the dairy industry as well as for small ruminant holders in Indonesia. Recently reported that more than 50 staphylococcus species and subspecies have been characterized to cause staphylococcal mastitis (El-Jakee et al., 2013). Coagulase-negative staphylococci can cause clinical mastitis, nonetheless, they primarily induce poorly noticeable subclinical conditions. The incidence of CNS intramammary infections of the udder in cows, sheep and goats has markedly increased (Contreras et al., 2007). In the majority of cases, CNS cause subclinical mastitis (Taponen et al., 2007; Waller et al., 2011). However, they can also cause clinical mastitis, characterised by mild symptoms but associated with increased Somatic Cell Count (SCC) (Taponen et al., 2007) and decreased milk production, which results in economic losses. The CNS have become the most common bacterial pathogens isolated from milk samples in many countries causing bovine intramammary infections (Czerw et al., 2007; El-Jakee et al., 2013) and could be described as emerging mastitis pathogens (Pyorala and Taponen, 2009).
It was of interest that from 3 clinical mastitis of PE goats in our study could be isolated three different staphylococci i.e., S. aureus, S. pasteuri and S. xylosus. S. aureus is world wide known as pathogen that can cause clinical and subclinical staphylococcal mastitis in ruminants and small ruminants. Staphylococcus aureus is also an important food-borne pathogen because of its ability to produce a wide range of extracellular toxin proteins and virulence factors that contribute to the pathogenicity of the organism (Salasia et al., 2011). Staphylococcus aureus was detected in goat and sheep bulk-tank milk samples (Muehlherr et al., 2003) with contain of various Staphylococcal Enterotoxin (SE) genes (Scherrer et al., 2004).
Staphylococcus pasteuri was firstly recognized as a new coagulase-negative species within the genus Staphylococcus in 1993 by Chesneau et al. (1993). Staphylococcus pasteuri is emerging as an agent of nosocomial infections and a blood derivatives contaminant, its role in causing human disease mostly remains controversial (Savini et al., 2009). The information of Staphylococcus pasteuri and its role in causing mastitis in goat is still not widely known.
Staphylococcus xylosus is part of coagulase-negative staphylococci that commonly found on skin of mammals, goat milk and cheese (Meugnier et al., 1996). Staphylococcus xylosus have been isolated from nosocomial infections and described as multi-resistant to diverse antibiotics (Barriere et al., 2001). It was mentioned that this bacteria is the causing agent of urinary tract infections patients (Al-Mathkhury et al., 2008). The present study showed that besides S. aureus, S. xylosus also dominated of CNS species isolated from the milk of PE goats with mastitis. This finding correlates with the results published by Malinowski et al. (2006) and Bochniarz et al. (2013), that S. xylosus was also dominating of CNS isolated from clinical and subclinical mastitis cows.
Staphylococcus haemolyticus is also a pathogen frequently isolated from dairy cows and small ruminants. However, it always appears in only a few animals and not as a major pathogen. Leitner et al. (2009) reported an outbreak in a dairy goat herd of approximately 25.6% isolates were identified as S. haemolyticus.
CONCLUSION
Considering the fact that beside of S. aureus, coagulase-negative staphylococci are a part of important causative agent of clinical and subclinical mastitis, special caution should be paid to milk sampling and identification of culture results. The identification of Staphylococcus spp. phenotypically need to be supported by the identification of the molecular approach in order to facilitate the determination of specific species of pathogen that causes clinical and subclinical mastitis in goats. Data obtained in this study could be used to consider the identification of S. aureus and coagulase-negative staphylococci.
ACKNOWLEDGMENTS
This work was supported by Directorate General of Higher Education (DGHE), through the PMDSU Study Grant Program of 2014 with grant number LPPM-UGM/ 2316/LT/2014. We would like to thank Rahmi Febriyanti, DVM, M.Sc. for the kind help in collecting milk PE goats samples in Pakanbaru Riau.