IV. RESULTS. unorganized sectors located in and around Bangalore (Table 1). The milk samples were

Transcription

1 RESULTS

2 IV. RESULTS The present study was carried out with an objective to isolate predominant mastitis pathogens viz., S. aureus, Streptococcus and E. coli prevailing specially in cases of subclinical bovine mastitis, to develop PCR for their identification and to standardize multiplex-pcr to detect these pathogens directly from the bovine milk samples. The results obtained during the period of study are documented under the following sub headings. 4.1 Screening of the milk samples A total of 246 bovine milk samples were collected from four organized and three unorganized sectors located in and around Bangalore (Table 1). The milk samples were collected from all lactating animals irrespective of the age and the lactation. These samples were subjected to EC test and SCC for screening for subclinical bovine mastitis in the herds. Based on the results of SCC, milk samples were grouped into four different groups viz., 0-1 lakh, 1-2 lakh, 2-5 lakh and >5 lakh cells /ml. Of these, 78 milk samples revealed a SCC value of < 1 lakh cells / ml; 19 revealed 1-2 lakh cells / ml; 38 revealed 2-5 lakh cells / ml and 111 samples revealed SCC >5 lakh cells / ml. A preliminary evaluation of SCC of 246 milk samples indicated SCM at 45 per cent since, 111 out of 246 samples were positive for SCM, if the conventional criteria of SCC >5 lakh cells / ml was considered to declare positivity.

3 87 Whereas, EC categorized these samples into two broad groups, 92 (38 per cent) samples revealed an EC value of < 6.5 ms/cm and remaining 150 (62 per cent) revealed EC 6.5 ms/cm. A total of 186 samples which included those milk samples positive for SCM by either of the two criteria and randomly selected samples from different SCM negative groups were subjected for isolation and characterization by conventional microbiological methods. Farm wise SCC and EC values are shown in Table 10. Table 10. Farm wise SCC value and EC value FARM SCC 0-1lakh cells/ml SCC 1-2 lakh cells/ml SCC 2-5 lakh cells/ml SCC >5 lakh cells/ml EC<6.5 ms/cm EC>6.5 ms/cm A Nil Nil B C D Unorganized sector A Unorganized sector B Unorganized sector C Isolation and biochemical characterization A total of 186 milk samples were subjected for isolation of bacteria following standard methods. The biochemical profiles of all the bacterial isolates were examined as per William et al. (1991), Barrow and Feltham (1993), FDA bacterialogical analytical manual manual (2001) and Collee et al. (2008). The isolated bacteria were identified up to genus level based on colony characteristics of individual primary isolates, hemolytic patterns on blood agar, growth on Mannitol salt agar, MacConkey agar and EMB agar, Gram staining. Further, these

4 88 isolates were subcultured on plain BHI agar plates and then subjected to primary tests such as catalase, using pure young cultures and were preserved at 4 C on nutrient agar slants. All the isolates were subjected to the cultural and biochemical tests such as Esculin hydrolysis test, Sodium hippurate hydrolysis test, Voges Proskauer, Citrate test, Urease test, Indole test, PYR test, Catalase test, Coagulase test, Thermonuclease test, M.R test and Sugar fermentation tests such as Glucose, Mannitol, Ribose, Sorbitol, Raffinose and Sucrose as per standard procedures described by Collee et al. (1989) to identify and group them up to genus or species level. A total of 323 bacterial isolates were recovered from 186 milk samples which included Staphylococci {S. aureus (95) and CoNS (95)} followed by E. coli (48) and Streptococci (85) isolates. Details of the test results are furnished in Tables 11, 12 and 13. Farmwise distributions of these isolates are depicted in Table 14. Distribution of these isolates according to SCC is depicted in Table Statistical analysis The distribution of isolates according to SCC was subjected for statistical analysis and analysed by using Graph pad prism software version -5. There was no significant difference between the distributions of the organisms with respect to different groups of SCC (Fig. 1). Distribution of the organisms was also not significant between organised farm and unorganised sector (Fig.2). It was found that there was a significant difference (P<0.0003) in the distribution of coagulase negative staphylococcus between the groups, whereas distribution of other organisms did not show any significant difference.

5 89 Interestingly in the organized farms, the occurrence of CoNS was significant (P<0.004) and there was no significant difference in the occurrence of other bacteria, whereas in unorganized farms, there was a random distribution of organisms (Fig.2). There was no significant difference in the distribution of S. aureus and CoNS in milk samples from sub clinically affected animals of both organized and unorganized farms (Fig.3). 4.4 Gram staining Gram staining of all the 85 Streptococcal isolates from 24 hr old pure broth culture revealed Gram positive cocci arranged in chains and Staphylococcus aureus (95 isolates) arranged in clusters, and E. coli isolates (48 isolates) as Gram negative bacilli. 4.5 Identification of isolates by polymerase chain reaction Genus specific PCR for Streptococcus and Staphylococcus Primers targeting tuf gene encoding Tu elongation factor were designed and employed for the genus specific confirmation of the Streptococcus and Staphylococcus isolates. All the 85 Streptococcal isolates yielded 110 bp amplicon of tuf gene (Fig.4) whereas, Staphylococcal isolates revealed 235 bp amplicons of the tuf gene (Fig.5). The amplification was seen in the reference cultures which were used as positive controls. S. aureus and E. coli were used as negative controls for amplification of Streptococcal isolates, while reference Streptococcal strains and E. coli were used as negative controls

6 90 for amplification of Staphylococcal isolates. No amplification was seen in negative controls and no template control (NTC) Species specific PCR for Streptococcus A house keeping gene 16S rrna gene was targeted for the designing of species specific primers for the identification of the 85 Streptococcal isolates confirmed at the genus level. Out of 85 Streptococcal isolates, seven isolates and reference S. agalactiae (AD1) yielded 329 bp amplicon of 16S rrna gene confirming them as S. agalactiae (Fig.6), none of the isolates yielded 549 bp amplicon which otherwise would confirm them as S. dysgalactiae and none of the isolates yielded 854 bp amplicon which otherwise would confirm them to be S. uberis. However, the reference cultures yielded the desirable amplicons (Fig.7 and Fig.8). 4.6 Amplification of virulence genes Surface immunogenic protein (sip) gene specific PCR for S. agalactiae The sip gene encoding a potent immunogenic surface protein was targeted to design the primers specific to the sip gene of S. agalactiae. The sip specific PCR was carried out using designed primer pairs on all the 85 isolates. Out of 85 isolates, only seven yielded specific amplicon of 266 bp confirming the presence of sip gene in all the seven isolates and reference (Fig.9).

7 Plasminogen activator (paua) gene specific PCR for S. uberis The pau gene encoding a plasminogen activator was targeted to design the primers specific to the paua gene of S. uberis. The paua gene specific PCR was carried out using designed primers on all the 85 isolates. Out of 85 isolates, none of the isolates yielded specific amplicon of 439 bp. However, reference isolates (AD2 and AD6) yielded a specific amplicon of 439 bp (Fig.10). 4.7 Identification of Staphylococcus aureus The staphylococcal isolates were differentiated as Coagulase positive Staphylococcus (CoPS) (S. aureus based on catalase, coagulase and thermonuclease test, VP test and Urease test) and Coagulase negative staphylococci (CoNS). Primers were designed targeting nuc gene and soda gene specifically to identify S. aureus. All the 95 S. aureus and reference strain amplified specific amplicons of 181 bp confirming the specificity of nuc gene present in all the isolates (Fig.11), whereas when these isolates were screened by soda gene, there was specific amplification of 159 bp amplicon in 87 isolates (Fig.12). 4.8 Identification of Escherichia coli by amplification of alr gene Out of 52 isolates suspected as E.coli by growth on EMB agar as metallic sheen colonies, 48 isolates were amplified by alr gene based PCR (Fig.13). The biochemical tests (IMViC) also confirmed 48 isolates as E. coli.

8 Multiplex PCR to identify E.coli When 52 isolates suspected as E. coli were screened by multiplex PCR targeting the primers described earlier, only 48 were specific to E. coli, which was confirmed both by biochemical tests and alr gene amplification (Fig.14) TraT gene specific PCR for E. coli TraT gene, serum survival gene was targeted to design the primer specific for E. coli. The TraT gene specific PCR was carried out using designed primers on all the 48 isolates. Out of 48 isolates, 40 isolates yielded specific amplicon of 313 bp (Fig.15). 4.9 Nucleotide sequencing of the reference isolates The PCR product of reference isolate of genus specific tuf gene, species specific 16S rrna amplicons, sip and paua, nuc, alr gene were sequenced at M/s Chromous biotech, Bangalore, and the sequences were subjected for BLAST analysis with NCBI nucleotide sequence data library (Fig. 29 to Fig.36), which confirmed the specificity of the designed primers. The amplicons product of unidentified species of Streptococcal isolates were also sequenced and subjected for nucleotide BLAST analysis and identified these isolates belonging to Streptococcus bovis equinus complex (Fig.37) Two tube Multiplex PCR to identify isolates from the milk samples DNA was extracted from the milk samples processed for isolation and two tube multiplex PCR was carried out. Initial standardization was carried out using reference strains (Fig.16).

9 93 In all, the two tube multiplex could detect seven sip gene specific amplification indicating S. agalactiae, 95 nuc gene specific amplification of S. aureus and 44 alr gene specific amplification of E. coli from the respective milk DNA samples. None of the DNA extracted from the milk samples processed for isolation yielded either S. uberis or S. dysgalactiae (Fig.17) Sensitivity and Detection limit The sensitivity of PCR with DNA extracted from spiked normal saline and milk revealed 1cfu/mL for S. agalactiae, S. dysgalactiae and S. aureus, where as it was 10 3 cfu / ml in normal saline and 10 4 cfu / ml in milk for E. coli and S. uberis (Fig.18 to Fig. 22). Detection limit of PCR for S. agalactiae was 22 pg /µl (Fig.23), S. dysgalactiae was 77 pg / µl (Fig.24), S. aureus was 7 pg / µl (Fig.25), S. uberis was 25.5 pg / µl (Fig.26) and E. coli was 140 pg / µl (Fig.27) Screening of bulk milk samples A total of 147 bulk milk samples were collected from four milk chilling centres in Bangalore district. Each sample represented milk pooled and procured from one Milk producers cooperative society (MPCS). The bulk milk samples were initially screened for SCC and EC. The results are represented in Table 16. A preliminary evaluation of SCC of 147 bulk milk samples indicated SCM at 78 per cent since, 115 out of 147 samples were positive for SCM, if the conventional criteria of SCC >5 lakh cells / ml is considered to declare positivity.

10 94 Whereas, EC categorized these samples into two broad groups, 55 (37 per cent ) samples revealed a EC value of < 6.5 ms/cm and remaining 92 (63 per cent) revealed EC 6.5 ms/cm. Chilling centre wise SCC and EC values are shown in Table 16. The result of screening of these 147 bulk milk samples by two tube m-pcr is presented in Table 17. Of the 147 bulk milk samples, 66 were negative for all the five bacterial species screened, 30 samples were positive for more than one species (S. aureus and E. coli). The species most frequently present in bulk milk was S. aureus, being found in 35 (43 per cent) samples followed by E. coli in 34 (42 per cent) samples. S. dysgalactiae and S. uberis were detected in 7 (9 per cent) and 5 (6 per cent) samples respectively (Fig.28).

11 Table 11. Biochemical profile of the Streptococcal isolates Isolate Esculin VP Sod hip PYR Glucose Man Rib Sor Raff Suc Tentative species identification S. uberis /S.parauberis S. dysgalactiae sub sp dysgalactiae S. dysgalactiae sub sp dysgalactiae/ S. pyogenes/ S. equi/s. zooepidemicus/s. phocea/ S. dedifis Others Others S. dysgalactiae sub sp dysgalactiae/ S. pyogenes/ S. equi/s. zooepidemicus/s. phocea/ S. dedifis S. uberis /S. parauberis S. uberis /S.parauberis Others Others S. uberis /S. parauberis S. uberis /S. parauberis S. uberis /S. parauberis S. agalactiae S. uberis /S. parauberis 95

12 S. uberis /S. parauberis S. agalactiae S. uberis /S. parauberis S. uberis /S. parauberis S. agalactiae S. uberis /S. parauberis Others S. anginosus / S. constallatus / S. pleurinimalium/ S. dysgalactiae sub equsimilis/s. canis/s. urinalis S. dysgalactiae sub sp dysgalactiae/ S.pyogenes/ S. equi/s. zooepidemicus/ S. phocea/ S. dedifis S. anginosus /S. constallatus/ S. pleurinimalium/ S. dysgalactiae sub equsimilis/s. canis/s. urinalis S. agalactiae S. anginosus /S. constallatus/ S. pleurinimalium/ S. dysgalactiae sub equsimilis/s. canis/s. urinalis S. anginosus /S. constallatus/ S. pleurinimalium/ S. dysgalactiae sub equsimilis/s. canis/s. urinalis S. anginosus /S. constallatus/ S. pleurinimalium/ S. dysgalactiae sub equsimilis/s. canis/s. urinalis S. agalactiae 96

13 Others S. dysgalactiae sub sp dysgalactiae/ S. pyogenase/ S. equi/s. zooepidemicus/ S. phocea/ S. dedifis S. dysgalactiae sub sp dysgalactiae/ S. pyogenase/ S. equi/s. zooepidemicus/ S. phocea/ S. dedifis S. dysgalactiae sub sp dysgalactiae/ S. pyogenase/ S. equi/s. zooepidemicus/ S. phocea/ S. dedifis Others S. anginosus /S. constallatus/ S. pleurinimalium/ S. dysgalactiae sub equsimilis/s. canis/s. urinalis S. anginosus /S. constallatus/ S. pleurinimalium/ S. dysgalactiae sub equsimilis/s. canis/s. urinalis S. anginosus /S. constallatus/ S. pleurinimalium/ S. dysgalactiae sub equsimilis/s. canis/s. urinalis Others S. agalactiae 97

14 Isolate Esculin VP Sod hip PYR Glucose Man Rib Ara Sor Suc Raf Interpretation S. anginosus/s. constallatus/ S. pleurinimalium/ S. dysgalactiae sub equsimilis/s. canis/s. urinalis S. adjacent/s. uberis /S. parauberis S. sanguis/ others 44 + W S. anginosu/s.constallatus/s. pleurinimalium/ S. dysgalactiae sub equsimilis/s. canis/s. urinalis S. anginosus /S. constallatus/ S. pleurinimalium/ S. dysgalactiae sub equsimilis/s. canis/s. urinalis S. pyogenes/s. equii. Spp. equis/s. porcinus 47 - W S. adjacens 48 + W S. pyogenes/s. equii. Spp. equis/s. porcinus 49 + W S. oralis/ S. mitis/ S. adjacens S. defectives/others S. sanguis/s. mutens/ S. constellatus/ S. porcinus S. sanguis/s. mutens/ S. constellatus/ S. porcinus 53 + W S. equii. Spp. equis/s. pyogenes 54 + W S. equii. Spp. equis/s. pyogenes 55 + W S. sanguis/s. agalactiae 56 + W S. agalactiae/s. pyogenes/ S. defectivus S. sanguis/s. mutens/ S. constellatus/ S. porcinus 98

15 S. equii. spp. zooepidermicus S. acidominimus 60 - W S. equii. spp. equis/s. agalactiae/s. pyogenes/ S. defectivus S. agalactiae/ S. mitis/ S. adjaceans S. porcinus/ S. mutans S. pyogenes/ S. equii. spp. zooepidermicus S. equii. spp. equi/ S. agalactiae/s. pyogenes/ S. defectivus S. equii. spp. equis/ S. adjacent/s.uberis /S.parauberis 66 + W S. pneumonia/ S. fecalis/ S. pyogenes S. sanguis/s. agalactiae S. agalactiae/s. pyogenes/ S. defectivus S. oralis/ S. mitis/ S. adjacens 70 + W S. equii. spp. equis/ S. adjacent/s. uberis / S. parauberis S. pyogenes/s. equii. spp. equis/s. porcinus S. pneumonia/ S. fecalis/ S. pyogenes S. pyogene/s. agalactiae/ S. mitis/ S. adjaceans S. equii. spp. equis/s. porcinus S. pyogenes/s. equii. spp. equis/s. porcinus 99

16 76 - W S. equii. spp. equis/s. porcinus 77 - W S. equines/s. agalactiae/s. pyogenes/ S. defectivus S. sangui/s. bovis/s. mutans/s. equii. spp. zooepidermicus S. pyogenes/s. acidominimus S. pyogenes/s. acidominimus 81 + W S. sanguis/s. bovis/s. mutans/ S. equii. spp. zooepidermicus S. equii. spp. equis/s. oralis/ S. mitis/ S. adjacens 83 - W S. pyogenes /S. defective S. sanguis/s. bovis/s. mutans/s. equii. spp. zooepidermicus S. pyogenes/s. equii. spp. equis/s. porcinus W+ : Weak positive; V: Variable

17 Table 12. Biochemical profiles and PCR results of S. aureus isolates Isolate No. Catalase Coagulase Thermonuclease Urease Mannitol PCR based confirmation at genus level tuf gene W W PCR based confirmation at species level nuc gene 101 1

18 W W W

19

20 W+ : Weak positive

21 105 Table 13. Biochemical profile of Escherichia coli isolates Isolate no. Indole MR VP Citrate Urease Interpretation E. coli E. coli E. coli E. coli E. coli E. coli E. coli E. coli E. coli E. coli E. coli E. coli E. coli E. coli E. coli E. coli E. coli E. coli E. coli E. coli E. coli E. coli E. coli E. coli E. coli E. coli E. coli E. coli E. coli

22 E. coli E. coli E. coli E. coli E. coli Salmonella? Citrobacter? E. coli W+ + - Salmonella? Yersinia? E. coli E. coli E. coli E. coli E. coli E. coli E. coli E. coli E. coli Enterobacter? E. coli E. coli Citrobacter? E. coli 54 - W Yersinia? W+: weak positive

23 107 Table 14. Farm/Unorganized sector wise distribution of Streptococci, S. aureus, CoNS and E. coli Farm Streptococcus species Staphylococcus aureus CoNS E. coli A B C D Unorganized A Unorganized B Unorganized C Total Table 15. Distribution of isolates according to SCC SCC Farm 0-1 lakh cells /ml 1-2 lakh cells / ml 2-5 lakh cells /ml >5 lakh cells / ml Strepto Sau EC CoNS Strepto Sau EC CoNS Strepto Sau EC CoNS Strepto Sau EC CoNS Organised farm A nil nil nil nil nil nil nil nil 1 nil nil nil Organised farm B nil nil nil nil nil nil nil nil Nil nil nil nil Organised farm C 1 nil nil nil nil nil Organised farm D nil nil nil 6 Unorganized A nil 5 Nil Unorganized B nil nil Unorganized C nil nil

24 108 Distribution of organisms versus somatic cell count Somatic cell count lakh cells/ml 1-2 lakh cells/ml 2-5 lakh cells/ml >5 lakh cells/ml 0 Strepto S. aureus E. coli CoNS Isolates classified based on somatic cell count Fig.1 Distribution of organisms in different groups of SCC

25 109 Organised Farm lakh cells/ml 1-2 lakh cells/ml 2-5 lakh cells/ml >5 lakh cells/ml 20 0 Streptococcus sp. S. aureus E. coli CoNS Unorganised farm lakh cells/ml 1-2 lakh cells/ml 2-5 lakh cells/ml >5 lakh cells/ml 10 0 Streptococcus sp. S. aureus E. coli CoNS Fig.2 Distribution of organisms in organized and unorganized farms according to groups of SCC

26 110 Distribution of S. aureus and CoNS in organized and unorganized farms with SCC more than 5 lakh cells. 60 S. aureus CoNS Organized Unorganized Distribution of S. aureus and CoNS in more than 5 lakh SCC 60 Organized farms Unorganized sectors S. aureus CoNS Fig.3 Distribution of S. aureus versus CoNS in organized and unorganized farms

27 111 Table 16. SCC and EC values of bulk milk samples Sl. No. Chilling centre Total no of sample SCC>5lakh cells/ml SCC <5lakh cells /ml EC>6.5 Ms/cm EC<6.5 Ms/cm 1 A B nil C D Table 17. Major mastitis pathogen in bulk milk samples by Multiplex PCR Organisms Sl. No. Chilling centre Total no. of sample S. aureus S. agalactiae Streptococcus S. dysgalactiae S. uberis E. coli Total no. of isolates 1 A nil nil B 36 nil nil 1 nil C nil nil nil D 36 8 nil 6 nil 9 23 TOTAL : 81

28 112 Fig.4 PCR amplification of 110 bp tuf gene of Streptococcus species isolated from subclinical bovine mastitis cases Lane M : 100 bp DNA ladder Lane 1-3 : Positive control (reference strains AD1, AD3, AD6) Lane 4-9 and 10-16: PCR amplified 110bp product of Streptococcus isolates Lane 17-19: Negative controls (S. aureus, E. coli, NTC) Fig.5 PCR amplification of 235 bp tuf gene of Staphylococcus species isolated from subclinical bovine mastitis cases Lane M : 100 bp DNA ladder Lane1 : Positive control Lane 2-18 and 21-24: PCR amplified 235 bp product of Staphylococcus isolates Lane 19 and 20: Non template control

29 113 Fig.6 PCR amplification of 329 bp 16S rrna gene of S. agalactiae isolated from subclinical bovine mastitis cases Lane M : 100 bp DNA ladder Lane 1 : Positive control (reference strains AD1) Lane 2-8 : PCR amplified 329 bp product of S. agalactiae isolates Lane 9 : Non template control Lane : Negative controls (S. aureus, E. coli, NTC) Fig.7 PCR amplification of 549 bp 16S rrna gene of S. dysgalactiae isolated from bovine mastitis case Lane 1: 100 bp DNA ladder Lane 2: S. dysgalactiae AD3 Ref. (Positive control)

30 114 Fig.8 PCR amplification of 854 bp 16S rrna gene of S. uberis isolated from subclinical bovine mastitis cases Lane M: 100 bp DNA ladder Lane 1 : S. uberis AD2: Reference (Positive control) Lane 2 : S. uberis AD6 : Reference (Positive control) Fig.9 PCR amplification of 266 bp sip gene of S. agalactiae isolated from subclinical bovine mastitis cases Lane M : 100 bp DNA ladder Lane 1 : Positive control (AD1) Lane 2-8 : PCR amplified 266 bp product of S. agalactiae isolates Lane 9 : Non template control Lane : Negative controls (S. dysgalactiae, S. uberis, S. aureus, E. coli)

31 115 Fig.10 PCR amplification of S. uberis pau A (439 bp) gene Lane M: 100 bp DNA ladder Lane 1 : PCR amplified 439 bp products of pau A gene (AD2) Lane 2 : PCR amplified 439 bp products of pau A gene (AD6) Lane 3 : Negative control S. aureus Lane 4 : Negative control E. coli Fig.11 PCR amplification of 181bp nuc gene of S. aureus isolated from subclinical bovine mastitis cases Lane M : 100 bp DNA ladder Lane1 : Positive control S. aureus (SAU-3) Lane 2-6, : PCR amplified181bp product of S. aureus isolates Lane 7-12 : Negative controls (S. agalactiae, S. dysgalactiae, S. uberis, E. coli, NTC)

32 116 Fig.12 PCR amplification of 159 bp soda gene of S. aureus isolated from subclinical bovine mastitis cases Lane M : 100 bp DNA ladder Lane1 : Positive control S. aureus (SAU-3) Lane 2-9 & : PCR amplified159 bp product of S. aureus isolates Lane : Negative controls (S. agalactiae, S. dysgalactiae, S. uberis, E. coli, NTC) Fig.13 PCR amplification of 366 bp alr gene of E. coli isolated from subclinical bovine mastitis cases Lane M : 100 bp DNA ladder Lane 1 : Positive control (EC11) Lane 2-7, and 20-24: PCR amplified 366 bp product of E. coli isolates Lane 8-12 : Negative controls (S. aureus, S. agalactiae, S. dysgalactiae, S. uberis, NTC)

33 117 Fig.14 PCR amplification of E. coli isolated from subclinical bovine mastitis cases by E. coli multiplex PCR Fig.15 PCR amplification of 313 bp TraT gene of E. coli isolated from subclinical bovine mastitis cases Lane M Lane 1 Lane 2-5 Lane 6-8 : 100 bp DNA ladder : Positive control (EC-11) : PCR amplified 313 bp product of E. coli isolates : Negative controls (S. aureus, AD1, NTC)

34 118 Fig.16 Standardization of two tube multiplex PCR Lane M : 100 bp DNA ladder Lane1and 2 : Amplification of sip and pau A Lane 3-4 : Amplification of nuc, alr and 16S rrna dys Lane 5 : Non template control Fig.17 Screening of milk samples processed for isolation by PCR 181 bp : S. aureus 366 bp : E. coli 549 bp : S. dysgalactiae

35 119 Fig.18 Sensitivity of PCR for the detection of S. agalactiae (AD1) spiked to normal saline and milk CFU Lane M : 100 bp DNA ladder Lane 1-9 : Spiked to normal saline from neat and CFU Lane : Spiked to milk CFU and neat Fig.19 Sensitivity of PCR for the detection of S. dysgalactiae (AD3) spiked to normal saline and milk CFU Lane M : 100 bp DNA ladder Lane 1-9 : Spiked to normal saline from CFU and neat Lane : Spiked to milk CFU and neat

36 120 Fig.20 Sensitivity of PCR for the detection of S. aureus (SAU3) spiked to normal saline and milk CFU Lane M : 100 bp DNA ladder Lane1-8 and 10 : Spiked to normal saline from CFU and neat Lane and 19 : Spiked to milk from CFU and neat Fig.21 Sensitivity of PCR for the detection of S. uberis (AD6) spiked to normal saline and milk CFU Lane M : 100 bp DNA ladder Lane 1-9 : Spiked to normal saline from CFU and neat Lane : Spiked to milk CFU and neat

37 121 Fig.22 Sensitivity of PCR for the detection of E. coli (EC11) spiked to normal saline and milk CFU Lane M : 100 bp DNA ladder Lane1-9 : Spiked to normal saline from CFU and neat Lane : Spiked to milk CFU and neat Fig.23 Detection limit of S. agalactiae (AD1) Lane M : 100 bp DNA ladder Lane 1 : 2200 ng / µl Lane 2 : 220 ng / µl Lane 3 : 22 ng / µl Lane 4 : 2.2 ng / µl Lane 5 : 220 pg / µl Lane 6 : 22 pg / µl

38 122 Fig.24 Detection limit of S. dysgalactiae (AD3) Lane M : 100 bp DNA ladder Lane 1 : 770 ng / µl Lane 2 : 77 ng / µl Lane 3 : 7.7 ng / µl Lane 4 : 770 pg / µl Lane 5 : 77 pg / µl Fig.25 Detection limit of S. aureus (SAU-3) Lane M : 100 bp DNA ladder Lane 1 : 7000 ng / µl Lane 2 : 700 ng / µl Lane 3 : 70 ng / µl Lane 4 : 7 ng / µl Lane 5 : 700 pg / µl Lane 6 : 70 pg / µl Lane 7 : 7 pg / µl

39 123 Fig.26 Detection limit of S. uberis (AD6) Lane M : 100 bp DNA ladder Lane 1 : 2550 ng / µl Lane 2 : 255 ng / µl Lane 3 : 25.5 ng / µl Lane 4 : 2.55 ng / µl Lane 5 : 255 pg / µl Lane 6 : 25.5 pg / µl Fig.27 Detection limit of E. coli Lane M : 100 bp DNA ladder Lane 1 : 1400 ng / µl Lane 2 : 140 ng / µl Lane 3 : 14 ng / µl Lane 4 : 1.4 ng / µl Lane 5 : 140 pg / µl

40 124 Fig.28 Screening of bulk milk samples by PCR 181 bp : S. aureus 266 bp : S. agalactiae 366 bp : E. coli 439 bp : S. uberis 549 bp : S. dysgalctiae Fig.29 NCBI BLAST report for Streptococcal tuf gene amplification product Streptococcus agalactiae ATCC elongation factor Tu (tuf) gene, partial cds Length=761 Score = 204 bits (96), Expect = 8e-50 Identities = 96/96 (100%), Gaps = 0/96 (0%) Strand=Plus/Plus Query 1 TCCAGAACCAGAACGTGATACTGACAAACCTTTACTTCTTCCAGTTGAAGATGTATTCTC 60 Sbjct 270 TCCAGAACCAGAACGTGATACTGACAAACCTTTACTTCTTCCAGTTGAAGATGTATTCTC 329 Query 61 AATCACTGGACGTGGTACAGTTGCTTCAGGACGTAT 96 Sbjct 330 AATCACTGGACGTGGTACAGTTGCTTCAGGACGTAT 365

41 125 Fig.30 NCBI BLAST report for the sequence of Streptococcus agalactiae 16S rrna gene amplification product Streptococcus agalactiae clone WWD_7B_MC51 16S ribosomal RNA gene, partial sequence Length=274 Score = 479 bits (259), Expect = 4e-132 Identities = 269/273 (98%), Gaps = 4/273 (1%) Strand=Plus/Plus Query 3 AGAGT-ATTAACACATGTTAGTTATTTAAAAGGAGCAATTGCTTCACTGTGAGATGGACC 61 Sbjct 175 AGAGTAATTAACACATGTTAGTTATTTAAAAGGAGCAATTGCTTCACTGTGAGATGGA-C 233 Query 62 CTGCGTTGTATTAGCTAGTTGGTGAGGTAAAGGCTCACCAAGGCGACGATACATAGCCGA 121 Sbjct 234 CTGCGTTGTATTAGCTAGTTGGTGAGGTAAAGGCTCACCAAGGCGACGATACATAGCCGA 293 Query 122 CCTGAGAGGGTGATCGGCCAACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGC 181 Sbjct 294 CCTGAGAGGGTGATCGGCCA-CACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGC 352 Query 182 AGCAGTAGGGAATCTTCGGCAATGGACGGAAGTCTGACCCGAGCAACGCCGCGTGAGTGA 241 Sbjct 353 AGCAGTAGGGAATCTTCGGCAATGGACGGAAGTCTGACC-GAGCAACGCCGCGTGAGTGA 411 Query 242 AGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTA 274 Sbjct 412 AGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTA 444

42 126 Fig.31 NCBI BLAST report for the sequence of Streptococcus dysgalactiae 16S rrna gene amplification product ( S. dysgalactiae AD3) Streptococcus dysgalactiae strain ATCC S ribosomal RNA gene, partial sequence Length=1471 Score = 891 bits (482), Expect = 0.0 Identities = 504/513 (98%), Gaps = 7/513 (1%) Strand=Plus/Plus Query 6 GGA-GGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTCCCGAGCGTTGGTCCGGA 64 Sbjct 481 GGACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTCCCGAGCGTT-GTCCGGA 539 Query 65 TTTATTGGGCGTAAAGCGAGCGCAGGCGGTTCTTTAAGTCTGAAGTTAAAGGCAGTGGCT 124 Sbjct 540 TTTATTGGGCGTAAAGCGAGCGCAGGCGGTTCTTTAAGTCTGAAGTTAAAGGCAGTGGCT 599 Query 125 CAACCACTGGTACGCTTTGGAAACTGGAGAACTTGAGTGCAGAAGGGGAGAGTGGAATTC 184 Sbjct 600 CAACCACT-GTACGCTTTGGAAACTGGAGAACTTGAGTGCAGAAGGGGAGAGTGGAATTC 658 Query 185 CATGTGTAGCGGTGAAATGCGTAGATATATGGAGGAACACCGGTGGCGAAAGCGGCTCTC 244 Sbjct 659 CATGTGTAGCGGTGAAATGCGTAGATATATGGAGGAACACCGGTGGCGAAAGCGGCTCTC 718 Query 245 TGGTCTGTAACTGACGCTGAGGCTCGAAAGCGTGGGGAGCAAACCAGGATTAGATACCCT 304 Sbjct 719 TGGTCTGTAACTGACGCTGAGGCTCGAAAGCGTGGGGAGCAAAC-AGGATTAGATACCCT 777 Query 305 GGTAGTCCACGCCGTAAACGATGAGTGCTAGGTGTTAGGCCCTTTCCGGGGCTTAGTGCC 364 Sbjct 778 GGTAGTCCACGCCGTAAACGATGAGTGCTAGGTGTTAGGCCCTTTCCGGGGCTTAGTGCC 837 Query 365 GGAAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAG 424 Sbjct 838 GGA-GCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAG 896 Query 425 GAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAG 484 Sbjct 897 GAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAG 956 Query 485 AACCTTACCAGAGTGGGTGACATCCTCCTGACC 517 Sbjct 957 AACCTTACCAG-GTCT-TGACATCCTCCTGACC 987

43 127 Fig.32 NCBI BLAST report for the sequence of Streptococcus uberis 16S rrna gene amplification product (S. uberis AD6) Streptococcus uberis gene for 16S rrna, strain:hn1 Length=1501 Score = 1240 bits (671), Expect = 0.0 Identities = 679/682 (99%), Gaps = 3/682 (0%) Strand=Plus/Plus Query 1 CTGCGTTGTATTAGCTAGTTGGTAAGGTAACGGCTTACCAAGGCGACGATACATAGCCGG 60 Sbjct 213 CTGCGTTGTATTAGCTAGTTGGTAAGGTAACGGCTTACCAAGGCGACGATACATAGCC-G 271 Query 61 ACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGC 120 Sbjct 272 ACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGC 331 Query 121 AGCAGTAGGGAATCTTCGGCAATGGGGGGAACCCTGACCGAGCAACGCCGCGTGAGTGAA 180 Sbjct 332 AGCAGTAGGGAATCTTCGGCAATGGGGGGAACCCTGACCGAGCAACGCCGCGTGAGTGAA 391 Query 181 GAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGAGAAGAACGGTAATGGGAGTGGAAAAT 240 Sbjct 392 GAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGAGAAGAACGGTAATGGGAGTGGAAAAT 451 Query 241 CCATTACGTGACGGTAACTAACCAGAAAGGGACGGCTAACTACGTGCCAGCAGCCGCGGT 300 Sbjct 452 CCATTACGTGACGGTAACTAACCAGAAAGGGACGGCTAACTACGTGCCAGCAGCCGCGGT 511 Query 301 AATACGTAGGTCCCGAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTT 360 Sbjct 512 AATACGTAGGTCCCGAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTT 571 Query 361 GATAAGTCTGGAAGTTAAAGGCTGTGGCTTAACCATAGTTCGCTTTGGAAACTGTCAAAC 420 Sbjct 572 GATAAGTCTG-AAGTTAAAGGCTGTGGCTTAACCATAGTTCGCTTTGGAAACTGTCAAAC 630 Query 421 TTGAGTGCAGAAGGGGAGAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGATATATGG 480 Sbjct 631 TTGAGTGCAGAAGGGGAGAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGATATATGG 690 Query 481 AGGAACACCGGTGGCGAAAGCGGCTCTCTGGTCTGTAACTGACGCCTGAGGCTCGAAAGC 540 Sbjct 691 AGGAACACCGGTGGCGAAAGCGGCTCTCTGGTCTGTAACTGACGC-TGAGGCTCGAAAGC 749 Query 541 GTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAGG 600 Sbjct 750 GTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAGG 809 Query 601 TGTTAGGCCCTTTCCGGGGCTTAGTGCCGGAGCTAACGCATTAAGCACTCCGCCTGGGGA 660 Sbjct 810 TGTTAGGCCCTTTCCGGGGCTTAGTGCCGGAGCTAACGCATTAAGCACTCCGCCTGGGGA 869 Query 661 GTACGACCGCAAGGTTGAAACT 682 Sbjct 870 GTACGACCGCAAGGTTGAAACT 891

44 128 Fig.33 NCBI BLAST report for the sequence of Streptococcus agalactiae surface immunogenic protein (sip) gene amplification product Streptococcus agalactiae strain GB01068 surface immunogenic protein (sip) gene, sip-8.1 allele, complete cds Length=1305 Score = 361 bits (195), Expect = 1e-96 Identities = 212/219 (96%), Gaps = 6/219 (2%) Strand=Plus/Plus Query 1 CGC-AGTGTTC-AGCACAAG-AACAGATACGACGTTGGACAGCACGTACAGTTTCAGTAG 57 Sbjct 60 CGCAAGTGTTCAAGCACAAGAAACAGATACGACG-TGGACAGCACGTACTGTTTCAG-AG 117 Query 58 GTAAAGGCTGATTTGGTAAAGCAAGACAATAAATCATCATATACTGTGAAATATGGTGAT 117 Sbjct 118 GTAAAGGCTGATTTGGTAAAGCAAGACAATAAATCATCATATACTGTGAAATATGGTGAT 177 Query 118 ACACTAAGCGTTATTTCAGAAGCAAATGTCAATTGATATGAATGTCTTAGCAAAAATTAA 177 Sbjct 178 ACACTAAGCGTTATTTCAGAAGCAA-TGTCAATTGATATGAATGTCTTAGCAAAAATTAA 236 Query 178 TAACATTGCAGATATCAATCTTATTTATCCTGAGACAAC 216 Sbjct 237 TAACATTGCAGATATCAATCTTATTTATCCTGAGACAAC 275

45 129 Fig.34 NCBI BLAST report for the sequence of Streptococcus uberis plasminogen activator A (pau) gene amplification product (S. uberis AD6) Streptococcus uberis strain QMP B5-144 plasminogen activator A (paua) gene, paua-9 allele, complete cds Length=861 Score = 706 bits (382), Expect = 0.0 Identities = 399/406 (98%), Gaps = 5/406 (1%)Strand=Plus/Plus Query 6 TGATT-CGACTACTACGCTAGATATATTGATCCCGATGAAAATAAAATAACATTTGCCAT 64 Sbjct 87 TGATTCCGACTACTACGCTAGATATATTGATCCCGATGAAAATAAAATAACATTTGCCAT 146 Query 65 AAATGGTTGATGGTTTTGTCGAAGGTAGTAATCAAGAAATCCTTATTAGAGGAATTCATC 124 Sbjct 147 AAAT-GTTGATGGTTTTGTCGAAGGTAGTAATCAAGAAATCCTTATTAGAGGAATTCATC 205 Query 125 ATGTTTTAACAGATCAAAACCAAAAGATTGTTACAAAGGCCGAGTTGTTAGACGCTATTA 184 Sbjct 206 ATGTTTTAACAGATCAAAACCAAAAGATTGTTACAAAGGCCGAGTTGTTAGACGCTATTA 265 Query 185 GACATCAAATGGTTCTTCTACAATTGGATTATTCCTATGAACCTAGTCGACTTTGCGCCT 244 Sbjct 266 GACATCAAATGGTTCTTCTACAATTGGATTATTCCTATGAAC-TAGTCGACTTTGCGCCT 324 Query 245 GATGCACAATTATTAACACGAGATCGACGGCTTTTATTTGCCAATCGAAATTTTGAGGAA 304 Sbjct 325 GATGCACAATTATTAACACGAGATCGACGGCTTTTATTTGCCAATCGAAATTTTGAGGAA 384 Query 305 ATCCGTATCACTTGAAGATACTATTCAAGAATACCTTTTAAAAGGGCATGTTATTCTCAG 364 Sbjct 385 -TCCGTATCACTTGAAGATACTATTCAAGAATACCTTTTAAAAGGGCATGTTATTCTCAG 443 Query 365 AAAACGGGTTGAAGAACCCTATCACTCATCCTACTAACACTGCTAA 410 Sbjct 444 AAAACGGGTTGAAGAACC-TATCACTCATCCTACTGAGACTGCTAA 488

46 130 Fig.35 NCBI BLAST report for the sequence of Staphylococcus aureus (nuc) gene amplification product (SAU 3) Staphylococcus aureus strain R1 thermostable nuclease (nuc) gene,partial cds Length=655 Score = 329 bits (178), Expect = 3e-87 Identities = 181/182 (99%), Gaps = 1/182 (1%), Strand=Plus/Plus Query 1 GTGCTGGCATATGTATGGCAATTGTTTCAATATTACTTATAGGGATGGCTATCAGTAATG 60 Sbjct 20 GTGCTGGCATATGTATGGCAATTGTTTCAATATTACTTATAGGGATGGCTATCAGTAATG 79 Query 61 TTTCGAAAGGGCAATACGCAAAGAGGTTTT-CTTTTTCGCTACTAGTTGCTTAGTGTTAA 119 Sbjct 80 TTTCGAAAGGGCAATACGCAAAGAGGTTTTTCTTTTTCGCTACTAGTTGCTTAGTGTTAA 139 Query 120 CTTTAGTTGTAGTTTCAAGTCTAAGTAGCTCAGCAAATGCATCACAAACAGATAACGGCG 179 Sbjct 140 CTTTAGTTGTAGTTTCAAGTCTAAGTAGCTCAGCAAATGCATCACAAACAGATAACGGCG 199 Query 180 TA 181 Sbjct 200 TA 201 Fig.36 NCBI BLAST report for the sequence of Escherichia coli alanine racemase E. coli complete genome gene amplification product (EC- 3) Score = 544 bits (294), Expect = 3e-151 Identities = 301/304 (99%), Gaps = 1/304 (0%) Strand=Plus/Minus Query 37 CGTCTGGGCGTAAGGCCGGAACAGGCTGAGGCGTTTTATCATCGCCTGACCCAGTGCAAA 96 Sbjct CGTCTGGGCGTAAGGCCGGAACAGGCTGAGGCGTTTTATCATCGCCTGACCCAGTGCAAA Query 97 AACGTTCGTCAGCCGGTGAATATCGTCAGCCATTTTGCGCGCGCGGATGAACCAAAATGT 156 Sbjct AACGTTCGTCAGCCGGTGAATATCGTCAGCCATTTTGCGCGCGCGGATGAACCAAAATGT Query 157 GGCGCAACCGAGAAACAACTCGCTATCTTTAATACCTTTTGCGAAGGCAAACCTGGTCAA 216 Sbjct GGCGCAACCGAGAAACAACTCGCTATCTTTAATACCTTTTGCGAAGGCAAACCTGGTCAA Query 217 CGTTCCATTGCCGCATCGGGTGGCATTCTGCTGTGGCCACAGTCGCATTTTGACTGGGTG 276 Sbjct CGTTCCATTGCCGCATCGGGTGGCATTCTGCTGTGGCCACAGTCGCATTTTGACTGGGTG Query 277 CGCCCGGGCATCATTCTTTATGGCGTCTCGCCGCTGGAAGATCGCTCCACCGGGGGGCGA 336 Sbjct CGCCCGGGCATCATTCTTTATGGCGTCTCGCCGCTGGAAGATCGCTCCACCGGTGC-CGA Query 337 TTTT 340 Sbjct TTTT

47 131 Fig.37 NCBI BLAST report for the sequence of unidentified streptococcus by biochemical test Accession GU Description Streptococcus equinus strain AC1 16S ribosomal RNA gene, partial sequence Max score Total score Query coverage E value Max ident % % FJ EU EU EU Streptococcus sp. G3 16S ribosomal RNA gene, partial sequence Streptococcus infantarius subsp. infantarius strain GMRS2 16S ribosomal RNA gene, partial sequence Streptococcus equinus strain KLDS S ribosomal RNA gene, partial sequence Streptococcus lutetiensis strain S ribosomal RNA gene, partial sequence % % % % % % % % AM Uncultured Streptococcus sp. partial 16S rrna gene, clone 302C10(oral) % % EU AJ AB AB AB AB Streptococcus bovis isolate 53 16S ribosomal RNA gene, partial sequence Streptococcus sp. R S rrna gene, strain R Streptococcus bovis gene for 16S rrna, partial sequence, strain:dst 1607 Streptococcus sp. Je2 gene for 16S rrna, partial sequence Streptococcus pasteurianus gene for 16S ribosomal RNA, partial sequence Streptococcus gallolyticus gene for 16S rrna, partial sequence, strain: Tokushima % % % % % % % % % % % %