Presence of viral and bacterial organisms in milk and their association with somatic cell counts.

About 20 to 35% of milk samples from cows with intramammary infection or high somatic cell count (SCC) are negative on bacteriological culture analysis. However, little is known about SCC in milk of cows infected with viruses. In the first part of our study, we developed a real-time PCR assay for detection of bovine herpesvirus (BHV) 1, BHV2, and BHV4, and bovine viral diarrhea virus (BVDV) in composite quarter milk samples. A total of 1,479 lactating cows of 1,964 cows in the dairy herd were initially selected because these cows had complete SCC data for at least 3 consecutive test results, of which 139 lactating cows from different lactation age groups were selected randomly and studied extensively. Composite quarter milk samples were collected on 3 alternate days and examined for viruses, SCC, and bacteriological analysis. In total, 10, 28, and 0.7% of the composite quarter milk samples from cows were positive for BHV1, BHV2, and BHV4, respectively; BVDV was not detected in composite quarter milk samples. Bovine herpesvirus was not associated with a particular bacterial species. Our study results indicate that cows positive for BHV in composite quarter milk samples alone are less likely to have elevated SCC compared with cows with bacterial intramammary infection; BHV1, BHV2, and BHV4 are probably not major udder pathogens.

Epidemiology of Mycoplasma bovis in Pennsylvania veal calves.

The objective of the study was to estimate the prevalence and incidence of Mycoplasma bovis, a common cause of pneumonia, in veal calves. Using simple random sampling, 252 calves from 4 veal herds located in central Pennsylvania were selected and longitudinally followed for monthly collection of nasal swabs. Bronchial swabs and lung lesions were collected at the slaughterhouse. Nasal, bronchial, and lung lesion swabs were cultured for bacterial respiratory pathogens. Ninety lung lesions were identified, of which 41.1, 1.1, 1.1, 7.8, and 4.4% were culture positive for M. bovis alone, Pasteurella multocida alone, Mannheimia haemolytica alone, M. bovis and P. multocida co-infection, and M. bovis and M. haemolytica co-infection, respectively. The data indicate that potential interventions, such as therapeutics, vaccines, or management control measures, would be most effective before 50 d of age based upon the cumulative incidence of colonization.

Molecular epidemiological analysis of Mycoplasma bovis isolates from the Pennsylvania Animal Diagnostic Laboratory showing genetic diversity.

We have examined the genetic variability of Mycoplasma bovis strains submitted to the Pennsylvania Animal Diagnostics Laboratory, University Park (PA-ADL), between December 2007 and December 2008. Of 4,868 total samples submitted for Mycoplasma testing, 302 were determined to be culture positive. Mycoplasma bovis (63.6%), Mycoplasma californicum (7.3%), Mycoplasma bovirhinis (2.7%), Mycoplasma bovigenitalium (0.7%), Mycoplasma alkalescens (4.9%), Mycoplasma putrefaciens (0.3%), and Mycoplasma dispar (1.3%) and unidentified Mycoplasma sp. (19.2%) were identified using PCR. Mycoplasma bovis represented the largest portion of the positive samples submitted. Each of the 192 M. bovis isolates was examined for variations in the BglII and MfeI restriction sites of the DNA using amplified fragment length polymorphism fingerprinting and subsequently compared with the M. bovis type strain PG45 (ATCC 25523). Similarity between strains was calculated using the Dice similarity coefficient, which ranged from approximately 0.7 to 1.0. When clustering the isolates at greater than 95% similarity, it was determined that 11 distinct clusters were present. The results are consistent with the existence of at least 2 clonally distinct groups. No clear geographical, month of isolation, or source origination relationship was identified, indicating that a currently unclassified characteristic is responsible for the strain heterogeneity. These data indicate strong heterogeneity of M. bovis isolates submitted to PA-ADL. Additionally, multiple sites throughout Pennsylvania had isolates of separate clonal lineages present concomitantly, indicating the ability of multiple overlapping outbreaks to occur at a single location. Mycoplasma bovis represents the largest portion of Mycoplasma species isolated from PA-ADL samples. We propose that amplified fragment length polymorphism may serve as a valuable tool for molecular characterization of M. bovis strains from the United States.

Effect of heat treatment of bovine colostrum on bacterial counts, viscosity, and Immunoglobulin G concentration.

A study was conducted to identify the optimal temperature and time at which heat treatment of bovine colostrum would least change viscosity and IgG concentrations yet reduce bacterial count. First-milking colostrum with >50g of immunoglobulins/L (measured by colostrometer) was collected from 30 Holstein cows. Aliquots of colostrum were heated for 0, 30, 60, or 90min at 57, 60, or 63 degrees C in a water bath. Samples were examined for viscosity, IgG(1), and IgG(2) concentrations, standard plate count, coagulase-negative staphylococci, environmental streptococci, coliform, gram-negative noncoliform, Streptococcus agalactiae, and Staphylococcus aureus counts. All heat treatments reduced counts of all bacteria groups measured compared with untreated colostrum samples. Heat treatment at >or=60 degrees C denatured IgG(1) compared with untreated colostrum; however, colostral IgG(2) levels were not reduced when temperature was held at 60 degrees C for <60min. Viscosity was not affected when temperature was held at 60 degrees C for <60min. In this study, heat treatment of bovine colostrum at 60 degrees C for 30 or 60min reduced bacterial count, slightly reduced IgG concentration, and did not affect viscosity.

Biofilm in milking equipment on a dairy farm as a potential source of bulk tank milk contamination with Listeria monocytogenes.

The objective of this study was to assess the presence of a Listeria monocytogenes-containing biofilm in milking equipment as a potential source of bulk tank milk contamination on a dairy farm where milk contamination had been previously documented. Samples were collected from milking equipment and milking parlor premises on 4 occasions and analyzed for the presence of L. monocytogenes. Pulsed-field gel electrophoresis (PFGE) typing was conducted on L. monocytogenes isolates from the milking equipment, parlor and storage room floors, bulk tank milk, and in-line milk filters. Pieces from milk meters and rubber liners were obtained to visually assess the presence of a biofilm using scanning electron microscopy. A total of 6 (15%), 4 (25%), and 1 (6%) samples were culture-positive for L. monocytogenes in the first, second, and third sample collection, respectively. Two samples were L. monocytogenes hly PCR-positive but were culture-negative in the fourth sample collection. Combined AscI and ApaI restriction analysis yielded 6 PFGE types for 15 L. monocytogenes isolates obtained from milking equipment, parlor, bulk tank milk, and milk filters. A predominant and persistent PFGE type (PFGE type T) was observed among these L. monocytogenes isolates (9/15 isolates). Scanning electron microscopy of samples from the bottom cover of 2 milk meters showed the presence of individual and clusters of bacteria, mainly associated with surface scratches. The presence of a bacterial biofilm was observed on the bottom covers of the 2 milk meters. Prevention of the establishment of biofilms in milking equipment is a crucial step in fulfilling the requirement of safe, high-quality milk.

Risk factors for bacteriological quality of bulk tank milk in Prince Edward Island dairy herds. Part 1: overall risk factors.

The objective of this study was to determine on-farm risk factors for bacteriological quality of bulk tank milk. Bulk tank raw milk quality was evaluated on all Prince Edward Island dairy herds (n = 235) over a 2-yr period (March 2005 to March 2007). Biweekly total bacterial, preliminary incubation, laboratory pasteurization, and coliform counts were conducted using a Petrifilm culture system. For the assessment of risk factors, a case-control study was conducted from January 2006 to May 2007. Case and control herds were defined based on the last 6 analyses of bulk tank bacterial counts before on-farm evaluation. Cases were herds that had multiple elevated counts for any of the parameters measured. A total of 69 herds (39 cases and 30 control herds) were evaluated. Data collection included 1) observation and questionnaire on basic hygiene and farm management practices; 2) complete wash analysis of the milking equipment, monitoring the presence of bacterial films on equipment and evaluation of cooling system function; and 3) environmental and cow hygiene scoring. Data were analyzed using multivariable logistic regression. The results of the final model indicated that high alkalinity in the wash water and poor teat-end cleanliness were associated with high bacterial counts in bulk tank milk (odds ratios = 12 and 5.3, respectively). It was also observed that high water temperature of detergent wash and the use of a water softener were associated with low bacterial counts in bulk tank milk (odds ratios = 0.87 and 0.11, respectively). A significant association between udder hair clipping and teat-end cleanliness was also observed. In conclusion, this study highlights the importance of udder hygiene and milking system washing factors on hygienic quality of bulk tank milk.

Risk factors for bacteriological quality of bulk tank milk in Prince Edward Island dairy herds. Part 2: bacteria count-specific risk factors.

A case-control study was conducted to identify specific on-farm risk factors that influence bacteriological quality of bulk tank milk in Prince Edward Island dairy herds. Total aerobic (TAC), preliminary incubation (PIC), laboratory pasteurization (LPC), and coliform (CC) counts were used to assess the bacteriological quality of bulk tank milk. Four case-control groups were defined based on the last 6 results of each test before on farm evaluation. A herd was classified as a TAC, PIC, or CC case when the herd had at least 4 high TAC, PIC, or CC counts out of the last 6 analyses for each test, respectively. For the LPC case group, a herd was required to have at least 3 high results out of the last 6 analyses. Control groups had low counts in the last 6 analyses for each test in the corresponding case group (TAC, PIC, CC, and LPC). The results of the study showed that TAC and PIC were mainly associated with cow and stall hygiene: washing the teats with water, not using teat predip, and dirty teats were risk factors. The LPC and CC were related to equipment hygiene, with high counts being associated with low temperature of the cleaning solution, high water-hardness score, and high alkalinity of alkaline detergent wash. Based on the findings of this study it can be concluded that TAC, PIC, LPC, and CC counts are of considerable value in identifying practices that could influence milk quality.

Environmental sampling to predict fecal prevalence of Salmonella in an intensively monitored dairy herd.

Although dairy cattle are known reservoirs for salmonellae, cattle that are shedding this organism are often asymptomatic and difficult to identify. A dairy herd that was experiencing a sustained, subclinical outbreak of Salmonella enterica subsp. enterica Cerro was monitored for 2 years. Fecal samples from the lactating cows were collected every 6 to 8 weeks and tested for the presence of Salmonella. Fecal prevalence of Salmonella fluctuated throughout the observation period and ranged from 8 to 88%. Manure composites and water trough samples were collected along with the fecal samples, and bulk milk and milk filters were cultured for the presence of Salmonella on a weekly basis. Over 90% of the manure composites--representing high-animal-traffic areas-were positive at each sampling. Salmonella was detected in 11% of milk samples and in 66% of the milk filters. Results of weekly bulk milk quality testing (i.e., bulk tank somatic cell score, standard plate count, preliminary incubation count) were typically well within acceptable ranges. Milk quality variables had low correlations with herd Salmonella fecal prevalence. When observed over time, sampling period average prevalence of Salmonella in milk filters closely paralleled fecal prevalence of Salmonella in the herd. Based on results of this study, milk filters appear to be an effective method for monitoring shedding prevalence at the herd level. In-line filter testing is also a more sensitive measure of Salmonella, and perhaps other pathogens, in raw milk than testing the milk alone.

A survey of bacteriological quality and the occurrence of Salmonella in raw bovine colostrum.

In recent years, bovine colostrum has gained popularity as a human food because it is an excellent source of bioactive proteins, which have been claimed to inhibit viral and bacterial pathogens, improve gastrointestinal health, and enhance body condition. A study was conducted to determine bacteriological quality and occurrence of Salmonella in colostrum collected from dairy herds (n = 55) in Pennsylvania. Colostrum samples were analyzed for standard plate count, preliminary incubation count, laboratory pasteurization count, Staphylococcus aureus, Streptococcus agalactiae, coagulase negative staphylococci, streptococci, coliforms, and non-coliforms. A standardized polymerase chain reaction assay was used for detection of Salmonella in colostrum. Salmonella were detected in 8 of 55 (15%) of colostrum samples. Streptococcus agalactiae (1000 colony-forming units [CFU]/mL) was detected in one colostrum sample. The mean standard plate count (977,539 CFU/mL), preliminary incubation count (12,094,755 CFU/mL), laboratory pasteurization count (615 CFU/mL), Staphylococcus aureus (306 CFU/mL), coagulase negative staphylococci (164,963 CFU/mL), streptococci (256,722 CFU/mL), coliforms (323,372 CFU/mL), and non-coliforms (111,544 CFU/mL) counts in colostrum were considerably higher than raw bulk tank milk counts reported previously from Pennsylvania. Analysis revealed that farm size did not influence the bacteriological quality of colostrum. Collection, handling, and storage of colostrum need to be addressed to improve bacteriological quality of colostrum intended not only for feeding calves but also for human consumption.

A survey of bovine colostrum composition and colostrum management practices on Pennsylvania dairy farms.

Colostrum composition and management were surveyed via sample and data collection from 55 dairy farms in Pennsylvania. Colostrum samples were analyzed for fat, protein, lactose, total solids, ash, Ig, lactoferrin, water- and fat-soluble vitamins, and minerals. Mean percentages of fat, protein, and lactose in colostrum were 6.7, 14.9, and 2.5, respectively. Concentrations of IgG1, IgG2, IgA, IgM, and lactoferrin were 35.0, 6.0, 1.7, 4.3, and 0.8 mg/mL, respectively. Mean concentrations of fat-soluble vitamins, including retinol, tocopherol, and beta-carotene, were 4.9, 2.9, and 0.7 microg/g, respectively. Mean concentrations of water-soluble vitamins were 0.34, 0.90, 4.55, 0.60, 0.15, 0.21, and 0.04 microg/mL for niacin, thiamine, riboflavin, vitamin B12, pyridoxal, pyridoxamine, and pyridoxine, respectively. Mean concentrations (mg/kg) of selected minerals in colostrum were also determined (Ca 4,716; P 4,452; Mg 733; Na 1,058; K 2,845; Zn 38; Fe 5.3; Cu 0.3; S 2,595; and Mn 0.1). The findings of this study revealed that the mean concentrations of most nutrients in colostrum have increased when compared with values previously reported. Results also showed that management practices have improved over time, particularly with regard to colostrum storage and feeding. Additionally, we observed that herd size influenced colostrum management and quality. It can be inferred, based on these findings, that although improvements have been made with regard to colostrum management and quality, there is still a need to educate producers on issues related to storage and timely feeding of colostrum to increase passive transfer and decrease the rate of calf morbidity and mortality.

A survey of foodborne pathogens in bulk tank milk and raw milk consumption among farm families in pennsylvania.

A 2-part study was conducted to determine the risk of exposure to human pathogens from raw milk. The first part of the study focused on determining raw milk consumption habits of dairy producers. A total of 248 dairy producers from 16 counties in Pennsylvania were surveyed. Overall, 105 (42.3%) of the 248 dairy producers consumed raw milk and 170 (68.5%) of the 248 dairy producers were aware of foodborne pathogens in raw milk. Dairy producers who were not aware of foodborne pathogens in raw milk were 2-fold more likely to consume raw milk compared with dairy producers who were aware of foodborne pathogens. The majority of dairy producers who consumed raw milk indicated that taste (72%) and convenience (60%) were the primary factors for consuming raw milk. Dairy producers who resided on the dairy farm were nearly 3-fold more likely to consume raw milk compared with those who lived elsewhere. In the second part of the study, bulk tank milk from the 248 participating dairy herds was examined for foodborne pathogens. Campylobacter jejuni (2%), Shiga toxin-producing Escherichia coli (2.4%), Listeria monocytogenes (2.8%), Salmonella (6%), and Yersinia enterocolitica (1.2%) were detected in the milk samples. Salmonella isolates were identified as S. enterica serotype Typhimurium (n = 10) and S. enterica serotype Newport (n = 5). Of the 248 bulk tank milk samples, 32 (13%) contained > or = 1 species of bacterial pathogens. The findings of the study could assist in developing farm community-based educational programs on the risks of consuming raw milk.

Prevalence and Spatial Distribution of Salmonella Infections in the Pennsylvania Raccoon (Procyon lotor).

A study was conducted to determine the prevalence and spatial distribution of Salmonella infection in Pennsylvania raccoons (Procyon lotor), common wildlife mammals known to occupy overlapping habitats with humans and domestic food animals. The Pennsylvania Game Commission provided a total of 371 raccoon intestinal samples from trapped and road-killed raccoons collected between May and November 2011. Salmonella was isolated from the faeces of 56 (15.1%) of 371 raccoons in 35 (54%) of 65 counties across Pennsylvania. The five most frequently isolated serotypes were Newport (28.6%), Enteritidis (19.6%), Typhimurium (10.7%), Braenderup (8.9%) and Bareilly (7.1%). Pulsed-field gel electrophoresis (PFGE) analysis of the Salmonella isolates and subsequent comparison to the Pennsylvania Department of Health human Salmonella PFGE database revealed 16 different pulsetypes in Salmonella isolates recovered from raccoons that were indistinguishable from pulsetypes of Salmonella collected from clinically ill humans during the study period. The pulsetypes of seven raccoon Salmonella isolates matched those of 56 human Salmonella isolates by month and geographical region of sample collection. Results from Clustered Regularly Interspaced Short Palindromic Repeats and Multi-Virulence Locus Sequence Typing (CRISPR-MVLST) analysis corroborated the PFGE and serotyping data. The findings of this study show that several PFGE pulsetypes of Salmonella were shared between humans and raccoons in Pennsylvania, indicating that raccoons and humans might share the same source of Salmonella.

A survey on antibiotic usage in dairy herds in Pennsylvania.

A survey was conducted (July 2001 to June 2002) on antibiotic usage of 113 dairy herds from 13 counties in Pennsylvania. Fifty percent of dairy farms surveyed maintained antibiotic treatment records. Only 21% of dairy producers had written plans for treating sick animals. Thirty-two percent of dairy producers sought veterinarian advice before administering antibiotics and on most farms (93%), antibiotics were administered by the owner/manager or designated herdsman. Twenty-four percent of the dairy producers said they always completed the course of antibiotic treatment. Any extra-label use of antibiotics was administered only on the guidelines of a veterinarian on majority of the farms. Comprehensive records from 33 dairy farms indicated that antibiotic usage was largest for calves with enteritis (36%) followed by pneumonia in calves (25%) and foot rot in cattle (16%). Twenty-four antibiotics including beta-lactams, spectinomycin, florfenicol, and tetracyclines were used on these farms. Beta-lactam antibiotics were used mostly for dry cow therapy, clinical mastitis, and on some farms for pneumonia and metritis. On 18% of the dairy herds surveyed, ceftiofur was used in an extra-label manner to treat mastitis in lactating cattle. On 70% of farms, calves were fed medicated milk replacers containing oxytetracycline and neomycin. The results of this study suggest that antibiotics are used extensively on dairy herds for both therapeutic and prophylactic purposes. Beta-lactams and tetracyclines were the most widely used antibiotics. There is considerable variation in the management practices associated with antibiotic use on dairy farms. It is anticipated that the findings of this survey will permit developing new strategies for prudent use of antibiotics on dairy herds.

Detection of new and persistent Streptococcus uberis and Streptococcus dysgalactiae intramammary infections by polymerase chain reaction-based DNA fingerprinting.

Polymerase chain reaction-based DNA fingerprinting was used as a tool to differentiate new and persistent Streptococcus uberis and Streptococcus dysgalactiae intramammary infections (IMI) in dairy cows. The same subtype of S. uberis or S. dysgalactiae was detected from some infected mammary glands from one lactation to the next documenting the persistence of these infections. Conversely, some streptococci isolated from mammary glands during a lactation or from one lactation to the next were different subtypes suggesting that a new IMI occurred. These new streptococcal IMI would never have been detected using phenotypic methods of streptococcal identification. Results of this study indicate that PCR-based DNA fingerprinting can be used as an effective procedure to differentiate new and persistent S. uberis and S. dysgalactiae IMI in dairy cows. This technique will be useful in epidemiological investigations, and drug and vaccine efficacy studies when attempting to delineate new and persistent IMI.

Phylogenetic relationships of Staphylococcus aureus from bovine mastitis based on coagulase gene polymorphism.

Coagulase gene restriction fragment length polymorphism (RFLP) patterns were analyzed to determine the phylogenetic relationship among isolates of Staphylococcus aureus from the Czech Republic (n = 27), France (n = 48), Korea (n = 115) and the United States (n = 278). A total of 468 isolates of S. aureus were subtyped into 41 coagulase genotypes. Cluster analysis placed the 41 types into nine clusters. Eighteen API Staph profiles were determined for 102 S. aureus isolates representing 1 to 4 isolates of each coagulase type. The results of the study suggest that based on coagulase gene RFLP analysis, several genetic variants of S. aureus are prevalent. Comparison of coagulase and API Staph profiles indicated that the two identification system were independent of each other.

Comparative evaluation of Vitek gram-positive identification system and API Rapid Strep system for identification of Streptococcus species of bovine origin.

The Vitek Gram-positive identification system (GPI, Vitek Systems, Inc., Hazelwood, MO) and the API Rapid Strep system (Analytab Products, Plainview, NY) were evaluated for species identification of streptococci isolated from bovine mammary glands and compared to conventional biochemical methods. A total of 144 strains including Streptococcus uberis (60), S. dysgalactiae (32), S. agalactiae (15), S. bovis (15), Enterococcus faecium (10) and Ent. faecalis (12) were evaluated. All reference strains were identified correctly by both systems. Vitek GPI card system identified 94.4% of strains, including 95% of S. uberis, 93.8% of S. dygalactiae, 93.3% of S. agalactiae and S. bovis II, 90% of Ent. faecium and 100% of Ent. faecalis. Majority of strains were identified with a 90-99% level of confidence, with an average of 8 h needed for identification. The API Rapid Strep system identified 96.5% of strains correctly, including 95% of S. 96.9% of S. dysgalactiae, 93.3% of S. agalactiae, and 100% of S. bovis II, Ent. faecium, and Ent. faecalis. Majority of strains were identified with excellent level of identification. With the exception of S. uberis, most strains were identified at 4 h of incubation.

Identification and sub-typing of Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium by randomly amplified polymorphic DNA.

A commercially available kit consisting of twenty 10-mer random primers was evaluated to allow selection of a suitable primer that would permit identification and sub-typing of Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium by randomly amplified polymorphic DNA (RAPD). A primer OPE-20 (5'-AAC-GGT-GAC-C-3') was identified to be the most suitable primer when tested with four ATCC reference strains of M. paratuberculosis and eight well characterized field strains each of M. paratuberculosis and M. avium. Primer OPE-20 was further tested for its ability to identify and subtype 200 field isolates of M. paratuberculosis. The fingerprint patterns of M. paratuberculosis (n=212) consisted of five unique common fragments (620, 450, 310, 230, 180bp) and nine variable fragments resulting in six distinct genotypes. The DNA fingerprints of M. avium (n=8) consisted of a single common fragment of 620bp, and 15 variable fragments resulting in six different genotypes. The cattle, human and goat isolates of M. paratuberculosis were genetically similar, but a sheep isolate had a different RAPD profile as compared to RAPD profiles from other species. RAPD was observed to be a rapid, reproducible and reliable technique for identification and sub-typing of M. paratuberculosis.

Encapsulation of Streptococcus uberis: influence of storage and cultural conditions.

Streptococcus uberis (n = 100) isolated from bovine mammary secretions were assessed by India ink for expression of capsule. Organisms were evaluated under four conditions; (1) after primary culture on blood agar, (2) following 5 passages on blood agar, (3) after 5 passages in Trypticase Soy Broth (TSB), and (4) after storage in 10% skim milk. Strains from primary culture (44 of 100) were positive for an unstained halo (capsule) by the India ink method. Number of strains expressing capsule decreased greatly after passage and following storage. Freeze-etching followed by electron microscopy confirmed results of India ink preparations. Strains were also cultured in various media to determine influence of medium components on capsule expression. Todd-Hewitt medium supplemented with either serum or egg yolk enhanced the size of capsule expressed. Results of this study may aid researchers investigating the pathogenicity of S. uberis.

Detection of Salmonella enterica somatic groups C1 and E1 by PCR-enzyme-linked immunosorbent assay.

Objectives of this study were to develop a PCR-based enzyme-linked immunosorbent assay (PCR-ELISA) for identification of Salmonella enterica somatic groups C1 and E1 and to evaluate this procedure along with a PCR-ELISA procedure for S. enterica somatic groups B, C2, and D in a masked study. Primers were selected from the rfb gene cluster, which is responsible for biosynthesis of O antigens of Salmonella lipopolysaccharide. Previously serogrouped Salmonella isolates (n = 169) were used to determine the sensitivity and specificity of the PCR-ELISA procedure. DNA from all isolates was amplified using the PCR procedure for selected somatic groups and amplified products were visualized on agarose gels, as well as subjected to the ELISA procedure. The PCR-ELISA technique correctly identified 97% of somatic group C1 and 87% of somatic group E1. The sensitivity of this procedure to correctly identify S. enterica somatic group C1 was 97% and 88% for somatic group E1. The specificity was 98% for both somatic groups C1 and E1. The PCR-ELISA techniques correctly identified 93% of Salmonella isolates belonging to somatic groups B, C1, C2, D, and E1. The overall sensitivity of this procedure to correctly identify S. enterica somatic groups was 96% and the specificity was 98%. Ninety-one percent of somatic group D, 92% of somatic group B, and 97% of somatic group C2 were identified correctly with this procedure. Results of this study indicate that the PCR-ELISA procedure is a rapid and accurate method for serogrouping Salmonella isolates. Utilization of the PCR-ELISA procedure for Salmonella serogrouping would aide in identification, surveillance, prevention, and control of Salmonella.

Studies on thermostable antigens, production of species-specific antiadrenal sera and comparison of immunological techniques in meat speciation.

Heat-stable antigens (BE forms: resistant to heat and ethanol precipitation) of adrenal and muscle tissues of cattle, buffalo, sheep, goat and pig were prepared for use in detection of adulteration in meats. The physico-chemical characteristics of these antigens revealed that the antigens of adrenals had only one component corresponding to 'Troponin T'. Muscle antigens also contained a major troponin T component but were associated with low molecular weight fractions. Rabbit antiadrenal BE sera were developed and made species specific by immunoabsorption. The species-specific antisera were employed for identification of origin of fresh and cooked meats and their mixtures, using an immunodiffusion test-agar gel precipitation test (AGPT), counterimmunoelectrophoresis (CIEP), enzyme-linked immunosorbent assay (ELISA) and the unlabelled antibody peroxidase antiperoxidase (PAP) technique. The results indicated that absorbed antisera could successfully differentiate the fresh, cooked meats and the meat mixtures from the species under study. AGPT and CIEP were useful in identification of 5-10% addition, using water extracts of fresh meats and BE forms of cooked meats, whereas ELISA and PAP could detect adulteration down to the level of 1% when water extracts were used. Among the tests employed in the study, the PAP technique proved to be most sensitive. The antisera were also proved useful in identifying the species in canned meat products, milk, serum, plasma, semen, urine, organs, skin and spoilt flesh, employing AGPT and CIEP.