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Open Access
APPLICATION OF POLYMERASE CHAIN REACTION FOR RAPID EVALUATION OF HYGIENIC STATUS OF MILK
(COLLEGE OF VETERINARY AND ANIMAL SCIENCES-MANNUTHY,THRISSUR, 2008) DEEPA MARY. J. J.; B. Sunil
Rapid assessment of the bacterial load and detection of pathogens in milk is of public health significance. Molecular detection of pathogenic microorganisms is based on DNA amplification of the target pathogens. Therefore efficient extraction of DNA directly from milk is a major step. DNA could be efficiently extracted directly from milk by a prior sample preparation so as to remove the fat and milk proteins. The phenol chloroform method of DNA extraction was modified to reduce the time require for the procedure. The use of lysozyme helped the release of DNA from lysed gram positive Staphylococcus aureus. The extracted DNA was used as template in PCR. PCR was carried out with already published primers. PCR was modified with the use of PCR buffer containing PCR facilitators (BSA and Tween 20) to overcome PCR inhibition. The standardized procedure was used to assess the bacterial load and to detect Escherichia coli and S. aureus directly from milk. To assess the bacterial load dilutions of milk were made uptolO"'". DNA was extracted from each dilution with which PCR was carried out with primers specific for Pseudomonas. Aerobic Plate Count was also done for the same samples and compared with PCR. It could be concluded that the approximate APC of the milk sample by PCR is next lower dilution to the dilution giving the PCR amplification. The total time taken for the analysis was approximately five hours. Extraction of DNA and PCR was done with primers for detection of E. coli from the same milk samples and compared with culture. Percentage of samples positive both by culture and PCR was 50 and negative by both methods were 30. Twenty percentage of the samples were positive by PCR and negative by culture. Extraction of DNA and PGR was done with primers for detection of S. aureus from the same milk samples and compared with culture. Percentage of samples positive both by culture and PGR was 60 and negative by both methods were 20. Twenty percentage of the samples were negative by PGR and positive by culture. Hence, protocol developed for detection of 5. aureus needs further refinement to take care of false negative results by PGR, probably due to the low number of organisms present in milk.
Open Access
IDENTIFICATION OF BACTERIAL CRITICAL POINTS AND ANTIBACTERIAL EFFECT OF LACTIC ACID ON BEEF CARCASS
(College of Veterinary and animal Science,Mannuthy, 1994) K. RAJEEV; M. Soman
During the process of slaughter and subsequent processing, the beef carcass is exposed to bacterial contamination. Bacterial load is one of the parameters for assessment of the sanitary conditions in slaughter operations. It is tedious and time consuming to evaluate bacterial load of carcass surface as a whole. Therefore assessment of bacterial load on certain points (critical points) in the carcass which are more frequently exposed to contaminants will help in quick assessment of sanitary standard. in the present study six critical points were selected on beef carcasses to evaluate the bacterial contamination. Carcasses from two slaughter houses differing in infrastructural facilities were used for this assessment and comparison. There was significant difference in the level of bacterial contamination on critical points of carcasses obtained from the two sources. Significant difference was noticed between points as well. Among the critical points, neck-lateral has shown highest level of contamination. This may be due to chances of exposure to contaminants during bleeding and flaying. The abdomen-medial was comparatively less contaminated. In spite of conscious precautions, carcasses invariably get contaminated. m order to minimise the bacterial load on carcass at the end of slaughter line, washing carcass with sanitizers is one of the methods adopted in meat trade. Lactic acid one per cent solution, when used as sanitizer for washing beef carcasses immediately after slaughter, has shown significant reduction in total viable count, conform count and faecal streptococcal count estimated 1 h after treatment. When hot lactic acid solution at 70°C was used for washing, significant reduction in the above counts in comparison to the first treatment was observed. This added benefit can be attributed to the enhanced temperature of the solution. it is concluded that one per cent lactic acid solution, preferably at 70oc, can be effectively used as a sanitizer on beef carcass surface for reduction of initial bacterial load and this helps in prolonging the storage life under the retail marketing condition.
Open Access
OCCURRENCE AND SURVIVABILITY OF YERSINIA IN PORK
(COLLEGE OF VETERINARY AND ANIMAL SCIENCES Mannuthy - Thrissur, 1994) SUNIL, B.; Prabhakaran, P.; SUNIL, B.
Investigation was carried out to find out the the presence and survivability of Yersinia in pork. One hundred and seventy one samples were collected from 39 animals. Three Isolates of Yersinia pceudotnberculosis were obtained using Yersinia isolation agar (Himedia). Two of the isolates were from the buccal cavity and tonsil of the same animal and the third from the tonsil of another animal. Even when the organism could not be isolated by direct plating, cold enrichment enabled isolation of the organism from the same sample. The organism could be recovered from in^oculated and stored (4°C) pork samples upto seven days. At -15»C storage, the organism could be recovered upto 30 days by direct plating. Direct plating failed to recover the organism on 45th day, but resuscitation technique was effective, which underlined the importance of resuscitation for isolation of the organism from frozen samples, on 60th day resuscitation also failed to recover the organism.
Open Access
EFFECT OF ELECTRICAL STIMULATION ON BEEF QUALITY
(COLLEGE OF VETERINARY AND ANIMAL SCIENCES Mannuthy - Thrissur, 1993) SUNIL, M.; Padmanabha Iyer, R.; SUNIL, M.
Application of eleotrical stimulation to pre-rigor muscle is considered as a method to prevent cold-shortening and to improve tenderness and consumer acceptability. The present study was conducted to evaluate the effect of electrical stimulation on changes in pK, Non-protein nitrogen (NPN) content, Total viable count (TVC) and organoleptic characters of beef stored at ambient and refrigeration temperatures, at specified intervals of time. Ten carcasses of adult cattle were subjected to the study. Electrical stimulation (ES) (alternating current at 110 V, 50 Hz and 20 pulses per second) was applied on left fore-quarter for 120 seconds. The right fore-qijarter was kept as control (C). Triceps brachii muscles ''\'ere co] lected from ES and C sides immediately after stiiriulat.i on and stored at ambient temperature for 12 h and at refrigeration temperature for 24 h. The rate of fall in pH in ES was highly significant than in C at all inte3r>^als except at 24 h. The fall in pH in C was faster at ambient temperature compared to that under refrigeration temperature upto 8 h. Highly significant increase in NPN was observed during storage in ES compared to C. TVC was found to increase on storage at both temperatures. But the increase was significantly lower in ES than in C at ambient temperature at 8 h. In refrigerated samples, at 12 h TVC in ES was lower than in C and the difference was significant (P<0.05). The organoleptic characters of samples were evaluated by a 5 member semi-trained taste panel. The flavour score for ES at ambient temperature, was significantly higher than for C at 8 h. The juiciness score was significantly higher in ES than in C at 0 and 8 h at ambient temperature and at 24 h at refrigeration temperature. Tenderness score at 8 h was significantly higher in ES than in C at ambient temperature. Under refrigeration temperature difference in tenderness score between C and ES samples was highly significant at 8 and 24 h. No significant difference was noticed in connective tissue score between C and ES samples. There was highly significant increase in overall acceptedjility for ES than for C at 8 h at ambient temperature and 24 h at refrigeration temperature.
Open Access
ASSESSMENT OF MICROBIAL QUALITY, ADULTERANTS AND PRESERVATIVES IN PASTEURIZED MILK
(College of Veterinary and Animal Sciences, Mannuthy., 2008) BHAGYALAKSHMI P. S.; Dr. E. Nanu
In the present study 200 pasteurized milk samples were collected from the retail shops of Thrissur and Palakkad districts. From Thrissur district, 20 samples each were collected from five brands viz., A, B, C, D and E, and an equal number of samples were obtained from the brands F, G, H, 1 and J of Palakkad district. All the samples were analyzed for the microbial quality by estimating various bacterial counts and yeast and mould count and also assessed the presence of certain pathogenic and spoilage bacteria. The milk samples were also tested to detect the presence of adulterants viz., starch and cane sugar and preservatives like carbonates and formaldehyde. The study revealed that the samples belonging to Thrissur district had an overall mean total viable count, coliform count, Escherichia coli count, psychrotrophic count, faecal streptococcal count and yeast and mould count of 5.08 ± 0.05, 2.89 ± 0.09, 0.53 ± 0.11, 5.30 ± 0.01, 3.40 ± 0.14 and 1.89 ± 0.08 logio cfu/ml, respectively. The corresponding count in the samples of Palakkad district was 5.24 ± 0.04, 3.01 ± 0.20, 0.78 ± 0.12, 4.99 ± 0.05, 3.20 ± 0.07 and 2.03 ± 0.09 logio cfu/ml. According to the total viable count limit prescribed by BIS (1992) 50 per cent samples from brand E were graded as satisfactory and the samples from all other brands were graded as unsatisfactory. The highest mean total viable count was seen in the samples of brand A (5.39 ± 0.04 logio cfu/ml). Of the samples collected from Thrissur district, the lowest count (4.51 ± 0.08 logic cfu/ml) was recorded from the samples of brand E. The samples collected from 1 brand of Palakkad district had the highest mean total viable count (5.35 ± 0.05 logic cfu/ml) and the lowest count (5.19 ± 0.09 logic cfu/ml) was observed in the samples of brand G. Ot the 100 samples collected from Thrissur district, the samples belonging to brand A had the highest mean colifonri count (3.40 ±0.17 logic cfu/ml). An equal number of samples collected from Palakkad district revealed that the highest mean count (4.39 ± 0.02 logio cfu/rnl) was observed in the samples belonging to brand I. According to the bacterial count limit prescribed by BIS (1992) 70 per cent of the samples from brand H were graded as satisfactory and the samples belonging to all other brands were graded as unsatisfactory. The overall mean coliform count of the samples belonging to various brands from Thrissur and Palakkad districts were at the level of two and three logio cfu/ml, respectively. The samples belonging to brand E of Thrissur and brand H of Palakkad had the lowest mean count. The samples collected from brand A of Thrissur district had the highest mean Escherichia coli count (0.92 ± 0.31 logio cfu/ml) and the lowest count (0.10 ±0.10 logic cfu/ml) was observed in the samples belonging to the brand E. Among the samples collected from Palakkad district, the highest mean count (1.54 ±0.11 logic cfu/ml) was observed in the samples of the brand I and the lowest count (0.43 ± 0.23 logic cfu/ml) was seen in the samples belonging to the brand H. The highest mean psychrotrophic count (5.39 ± 0.02 logic cfu/ml) was seen in the samples belonging to brand C of Thrissur district and the lowest count (5.29 ± 0.05 logic cfu/ml) was observed in the samples of the brand B. Among the samples from Palakkad district, the highest mean count (5.43 ± 0.01 logic cfu/ml) was seen in the samples of the brand 1 and the lowest count (4.63 ±0.10 logic cfu/ml) was observed in the samples of the brand ff. Of the samples collected from Thrissur district, the lowest mean faecal streptococcal count (2.95 ±0.17 logic cfu/ml) was seen in samples belonging to brand C and the highest count (3.78 ±0.16 logic cfu/ml) was observed in the samples of the brand B. Among the samples belonging to various brands of Palakkad district, the lowest mean count (2.88 ±0.12 logic cfu/ml) was seen in samples of the brand G and the highest count (3.51 ± 0.21 logic cfu/ml) was observed in the samples of the brand 1. Among the samples collected from the five brands of Thrissur district, the highest mean yeast and mould count (2.46 ±0.17 log lo cfu/ml) was seen in the samples of the brand A and the lowest count (1.35 ± 0.08 log lo cfu/ml) was observed in the samples of the brand E. Of the samples belonging to the five brands of Palakkad, the highest mean count was seen in the samples of the brand J (2.40 ± 0.24 logio cfu/ml) and the lowest count was observed in the samples of the brand F (1.66 ± 0.09 logio cfu/ml). A highly significant (P<0.01) difference was noticed among the mean total viable count, coliform count, faecal streptoccal count and yeast and mould count of various brands of pasteurized milk from Thrissur district. Similarly a highly significant (P<0.01) difference was noticed among the mean coliform count, Escherichia coli count and psychrotrophic count of the samples belong to the five brands of pasteurized milk from Palakkad district. Escherichia coli was isolated from 10 per cent of the samples belonging to Thrissur and the isolates consisted of serotype of 04 (1), rough strains (2) and untypable strains (7). The organism was isolated from 11 per cent of the samples collected from Palakkad. One of the isolates was serotyped as 04 and three isolates were serotyped as O60. One isolate fell in the class rough and six isolates were untypable. Two isolates each from Thrissur and Palakkad districts revealed Congo red binding charecteristics. Staphylococcus aiireus could not isolate from the samples obtained from Thrissur and Palakkad districts. Pseudomonas organism was isolated from four and six per cent of the samples from Thrissur and Palakkad. The isolates were identified as Pseudomonas aeniginosa, Pseudomonas flourescens, Pseudomonas cepacia and Pseudomonas putida. Bacillus cereus was isolated from three samples obtained from Thrissur district and two samples belonging to Palakkad district. None of the samples from Thrissur and Palakkad districts revealed the presence of the adulterants like starch and cane sugar and preservative like bicarbonates. But formaldehyde was detected from 19 per cent of the samples from Thrissur and 47 per cent of the samples from Palakkad.
Open Access
ASSESSMENT OF BACTERIAL QUALITY AND SHELF LIFE OF PASTEURIZED MILK
(College of Veterinary and Animal Sciences, Mannuthy., 2007) ASHA. K.; Dr. E. Nanu
In the present study raw and pasteurized milk samples were collected from two processing plants viz., DPi and DP: and pasteurized milk from retail shops. A total of 254 samples were analyzed for the bacterial quality by estimating various bacterial counts and also assessed the presence of certain bacteria of public health importance. The bacterial, physical and organoleptic qualities of pasteurized milk samples from two dairies stored under refrigeration (4 ± CC) were evaluated. Raw milk revealed an inferior bacterial quality with 50 per cent samples graded as fair (based on total viable count) and 85.7 per cent as poor quality (based on coliform count). The total viable count from both dairies was obtained at the level of 7 logio cfu/ml but coliform count was high in the samples obtained from DP| (3.34 ± 0.05 loglO cfu/ml). The psychrotrophic count and faecal streptococcal count in the samples belonging to both sources were at the level of 7 and 3 logio cfu/ml. respectively. Bacteria of public health significance like Escherichia coli. Staphylococcus aiireus and Pseudomonas was detected from a few samples. Pasteurization reduced the level of total viable count, coliform count, psychrotrophic count and faecal streptococcal count to a highly significant (P<0.01) level. Pasteurized milk under refrigeration (4 ± 1°C) showed an increase in total viable count and psychrotrophic count throughout the storage period with a difference of more than 3 log with that of fresh sample. However, coliform count. Escherichia coli count, and faecal streptococcal count of samples belonging to DP| initially showed increasing tendency up to si.\ days and thereafter the counts decreased. The increase in total viable count, coliform count, Escherichia coli count, psychrotrophic count and faecal streptococcal count between zero and 10''^ day from DP; was 4.8. 1.95. 2.08. 4.78 and 2.32 logm cfu/ml. respectively. The increase in the counts during storage may lead to the reduction in shelf life due to bacterial deterioration of milk. Isolates oi' Escherichia coli was obtained from [)P| on all days c.xcept eighth and lO"' day. A total of si.\ isolates were obtained from DIA- I'hc isolates belonged to 01 16 (3). 022. 046. 065 (2). 095 and the rest were rough varietv. Sluphvlococcus ctiircus was also isolated Irom two samples stoied on sixth da\' and three from the samples stored on zero, second and fourth da>. respectively (DPi). from DP:, three isolates were obtained from the samples stored on lO"' da\ and one from fresh samples. A total of 22.62 and 20.24 per cent Pseiuiomonus were isolated from DP| and DP:, respectively and the isolates were identified as I'seudomonus piUida. Psendomonus ucnipinosu and Pseudomonas Jloiirescens. Sensory and physico-chemical (COB test) analyses of refrigerated milk samples showed an overall reduction in the score of colour and appearance, llavour. odour and body as the storage period increased, fhe mean total scores Irom Di'i revealed that the samples were of excellent quality for up to .second day of storage, fhe sensory qualit\ of the samples stored on fourth day was good and then the quality of milk remained lair till eighth day and on 10''" day the quality became poor. In DP: samples had excellent quality for upto second day of storage, fhe sensory quality of the sample stored up to si.xth day was good and thereafter the quality ol milk remained as fair till the end of storage period. COB test of samples from DP| showed positive test on all samples stored on 10*'^ day. However, one sample stored on day six was COB test positive, fhe samples belonging to DP: showed that three samples stored on lO"' day and one sample stored on eighth day was COB positive. ■fhe bacterial prollle of the retail milk samples of the brands A. B. C. D. P and P was assessed and the samples belonging to the brand D had highest mean total viable count (5.94 ; 0.09 loglO efu/ml), psychrotrophic count (5.09 i 0.16 logm cfu/ml) and faecal streptococcal count (2.87 i 0.24 logi,, cfu/ml). Highest coliform count was seen in the samples of brand A (2.40 i 0.14 logiu ctu/ml) and Escherichia coli count (3.44 ± 0.72 logio cfu/ml) in samples of the brand C. Low counts especially total viable count (4.89 t 0.79 logio cfu/ml) and coliform count (1.19 i 0.42 logio cfu/ml) were seen in the samples of the brand F. Escherichia coli were detected from 20.8 per cent samples and the isolates consisted of the serotypes 046, 065, 095. 01 16, 0166 and 0171. Out of 15 isolates obtained six showed a positive congo red reaction indicating their property of invasiveness. Staphylococciis aureiis was isolated from only six samples (6.94 per cent). All retail milk samples were also tested for the isolation and identification of Pseiidomonas and the organism was isolated from 16 (22.22 per cent) samples. 1 he isolates were identified as Pseiidomonas pntida (7), Pseudomonas aeniginosa (6) and Pseiidomonas floiireseens (3). Polymerase Chain reaction was employed to identify and confirm the Eseherichia coli isolates obtained from the milk samples and a 366 bp product was obtained.
Open Access
DEVELOPMENT OF MONOSPECIFIC ANTI-BEEF SERA
(COLLEGE OF VETERINARY AND ANIMAL SCIENCES Mannuthy - Thrissur, 1995) THANGTHUAMA, R.; Prabhakaran, P.; THANGTHUAMA, R.
Agar gel immunodiffusion is a simple and reliable test for identifying the species origin of meat, povided the antisera to be used are monospecif ic. A study was undertaken to make Rabbit anti-cattle serum (RAGS) and Rabbit anti-buffalo serum (RABS) monospecific by absorption with the freeze dried sera of goat (GFD), buffalo (BFD), cattle (GFD) and a combination of GFD and GFD or, GFD and BFD. Though it was found that the RAGS was made monospecific by absorption with BFD, production of monospp- ;ific RABS through absorption with GFD or GFD, is more desirable. Absorption of RABS with GFD alone enabled to identify both beef and buffalo meat samples which can be further confirmed by RABS absorbed with GFD. RAGS absorbed with BFD and RABS absorbed with GFD could identify a level of 25 per cent or above adulteration with beef and buffalo beef respectively. Filter paper was found to be good carrier of beef and buffalo meat extract antigens and storing it for upto 30 days did not influence the test result with unabsorbed antisera. All the three eluants, NaCl, PBS and PBS-T were found to be equally useful for elution of the meat antigen from the dried filter paper.
Open Access
ASSESSMENT OF BACTERIOLOGICAL QUAUTY OF RAW MILK IN TRICHUR AND ITS PUBLIC HEALTH IMPORTANCE
(COLLEGE OF VETERINARY AND ANIMAL SCIENCE,MANNUTHY, 1995) ANJU RAGHUNATHRAO KAPRE; NANU, E
Open Access
BOVINE BRUCELLOSIS IN RELATION TO PUBLIC HEALTH
(COLLEGE OF VETERINARY AND ANIMAL SCIENCES Mannuthy - Thrissur, 1999) VINOD.., V. K; Nanu, E.; VINOD., V. K.
A serological survey was undertaken to assess the extent of brucellosis in bovines and bumans. Serum samples from 1233 bovines and 747 human were collected. These samples were screened by Rose Bengal Plate Test (RBPT) and Standard Tube Agglutination Test (STAT). The samples which showed a positive reaction, either by RBPT or STAT or both were subjected to Heat inactivation test (HIT), 2- Mercaptoethanol test (MET) and EDTA agglutination test (EAT). Of the 610 slaughtered male bovine serum samples screened, 41 (6.72 per cent) was found positive by RBPT and 37 (6.07 per cent) by STAT. HIT, MET and EAT detected positive reaction in 32 (5.25 per cent), 26 (4.26 per cent) and 27 (4.43 per cent) samples respectively. A total of 23 samples were positive by all the above tests. Among the 518 slaughtered female bovine serum samples, RBPT detected 41 (7.92 per cent) samples as positive while only 38 (7.34 per cent) samples were found positive by STAT. HIT, MET and EAT detected positive reaction in 31 (5.98 per cent), 26 (5.02 per cent) and 28 (5.4 per cent) samples respectively. Twenty four serum samples were found positive for the disease by all the above tests. None of the serological test employed could detect a statistically significant difference in the seroprevalence of disease between males and females. Of the serum samples collected from 105 farm fed cows, six (5.7 per cent) samples were found positive for brucellosis by both RBPT and STAT. The number of samples found positive by HIT, MET and EAT were three (2.86 per cent), one (0.95 per cent) and two (1.9 per cent) respectively. Only one sample revealed a positive reaction for the disease by all the above serological tests. Among the 406 human male serum samples collected, six (1.47 per cent) revealed an agglutination reaction positive for the disease by RBPT while only four (0.99 per cent) showed an agglutination litre positive for the disease by STAT. HIT, MET and EAT detected three (0.74 per cent) samples each as positive for the disease. Three samples revealed a positive reaction in all the above serological tests. Of the 327 human female serum samples screened, RBPT and STAT recorded a positive reaction in five (1.53 per cent) and four (1.22 per cent) samples, respectively. Three samples (0.92 per cent) each were found positive by HIT, and MET and EAT. Only three samples were found positive for all the serological tests used in this study. It was observed that serological tests employed in this study could not detect a statistically significant difference in the seroprevalence of disease between males and females. None of the serum samples collected from veterinary surgeons (10), animal attendants (two), and slaughter house workers (two) were positive for the disease. Of the serological tests employed in this study, RBPT detected the highest number of samples as positive followed by STAT, HIT, EAT and MET. It was also observed that, of the RBPT and STAT positive cases, HIT recorded maximum number of positive cases followed by EAT and MET. The reason for difference observed in the agglutination titre of the serum samples by the above tests were discussed.

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