Response to adrenocorticotropic hormone or corticotrophin-releasing hormone and vasopressin in lactating cows fed an immunomodulatory supplement under thermoneutral or acute heat stress conditions.

Adrenal responsiveness was tested in nonpregnant, lactating Holstein dairy cows fed diets supplemented with OmniGen-AF (OG; Phibro Animal Health Corp., Teaneck, NJ), an immune modulator, and in nonsupplemented control (CON) cows following bolus infusions of a combination of corticotropin-releasing hormone (CRH; 0.3 µg/kg of BW) and arginine vasopressin (VP; 1.0 µg/kg of BW) or ACTH (0.1 IU/kg of BW) in 2 environments: thermoneutral [TN; temperature-humidity index (THI) <60] for 24 h/d and heat stress (HS; THI >68 for 17 h/d). Cows (506) were initially fed OG (n = 254) or CON (n = 252) diets for 44 d before selection of a subgroup of cows (n = 12; 6 OG, 6 CON) for the study. The 2 subgroups were balanced for parity, milk yield, and days in milk. All cows were transported to and housed in 2 environmentally controlled rooms at the University of Arizona Agricultural Research Complex (Tucson). Cows were given 3 d to acclimate to the rooms and then underwent 12 d of TN conditions and then 8 d of HS conditions for a total of 24 d on experiment. Cows were infused with CRH-VP on d 9 of TN and on d 1 of HS and with ACTH on d 10 of TN and on d 2 of HS. Hormone infusions took place at 1000 h (0 h) on each infusion day. Blood samples, taken in 30-min intervals, were first collected at 0800 h (-2 h) and were drawn until 1800 h (8 h). Before infusion, serum progesterone was elevated in OG cows compared with CON cows. Infusion of releasing factors (CRH-VP or ACTH) caused increases in serum cortisol and progesterone, but cortisol release was greater in CON cows than in OG cows during HS, whereas progesterone did not differ between the 2 treatments. Serum ACTH increased following infusion of releasing factors, but this increase was greater following CRH-VP infusion than ACTH infusion. Serum bovine corticosteroid-binding globulin also increased following infusion of releasing factors in both treatment groups, but this increase was greater during HS in cows fed OG. The free cortisol index (FCI) increased following CRH-VP and ACTH and was higher in HS than in TN for both OG and CON cows. However, the FCI response was blunted in OG cows compared with CON cows during HS. Heat stress enhanced the adrenal response to releasing factors. Additionally, the adrenal cortisol and FCI response to releasing factors was reduced during acute heat stress in cows fed OG. Collectively, these data suggest that OG supplementation reduced the adrenal responsiveness to factors regulating cortisol secretion during acute HS.

Immunological responses and evaluation of the protection in dairy cows vaccinated with staphylococcal surface proteins.

Bovine mastitis is a significant cause of economic losses in the dairy industry. Staphylococcus aureus is one of the most common contagious mastitis pathogens, whereas Staphylococcus chromogenes increasingly became a significant cause of subclinical mastitis in dairy cows. Current mastitis control measures are not effective on all mastitis pathogens. There is no effective vaccine to control Staphylococcal mastitis in dairy cows. The objective of this study was to evaluate the immune responses and protection in dairy cows vaccinated with S. aureus surface proteins (SASP) or S. chromogenes surface proteins (SCSP). We divided eighteen Holstein dairy cows randomly into three groups of 6 animals each. We vaccinated group 1 and 2 animals with SASP and SCSP with Emulsigen-D adjuvant, respectively. We injected control (group 3) animals with PBS (pH 7.2) in Emulsigen®-D. We vaccinated animals three times at 28 and 14 days before drying off, and at dry off. Two weeks after the third vaccination, we challenged each animal by dipping all teats in S. aureus culture suspension once daily for 14 consecutive days. We evaluated milk or mammary secretion and serum antibody titers during vaccination and challenge periods. We evaluated milk samples for the number of bacteria shedding and somatic cell counts (SCC). Out of six cows vaccinated with SASP, one cow was removed from the study due to injury, two were infected clinically, another two were infected subclinically, and the remaining cow was not infected. No SCSP vaccinated cows developed clinical or subclinical mastitis. Out of six control cows, two developed clinical mastitis whereas four were infected subclinically. The SCSP vaccine cross-protected against S. aureus mastitis and reduced number of S. aureus shedding in milk. We concluded that the SCSP is a promising vaccine to control Staphylococcal mastitis in dairy cows.

Short communication: Evaluation of bulk tank milk microbiological quality of nine dairy farms in Tennessee.

The purpose of this study was to evaluate the bulk tank milk (BTM) quality of 9 East Tennessee dairy farms and to determine its relationship with selected quality milk parameters. Bulk tank milk samples (n=1,141) were collected over a 42-mo period (June 2006 through November 2009) from farms, based on their preliminary incubation count (PIC) history. Parameters of BTM quality evaluated in this study included somatic cell count (SCC), standard plate count (SPC), PIC, laboratory pasteurization count (LPC), Staphylococcus spp. count, Streptococcus spp. count, and coliform count. Strong correlations between SPC and Streptococcus spp. counts (0.72) and between SPC and PIC (0.70) were found. However, moderate correlations were seen among other milk quality parameters. In addition, seasonal variations for some milk quality parameters were noted. For example, milk quality parameters such as SCC, SPC, LPC, and coliform count were significantly higher in summer, whereas Streptococcus spp. counts were significantly higher in winter. No seasonal variation in PIC or Staphylococcus spp. counts was observed. Summarizing, results from this investigation showed the importance of using several bacterial counts (SCC, SPC, PIC, LPC, Streptococcus spp. count, Staphylococcus spp. count, and coliform counts) as simultaneous indicators of milk quality.

Prevalence and persistence of coagulase-negative Staphylococcus species in three dairy research herds.

Coagulase-negative Staphylococcus species (CNS) were isolated from 11.3% (1407 of 12,412) of mammary quarter milk samples obtained from cows in three dairy research herds in 2005. Approximately 27% (383/1407) of CNS was identified to the species level. The species distribution among those CNS identified from all herds was Staphylococcus chromogenes (48%), Staphylococcus hyicus (26%), Staphylococcus epidermidis (10%), Staphylococcus simulans (7%), Staphylococcus warneri (2%), Staphylococcus hominis (2%), Staphylococcus saprophyticus (1%), Staphylococcus xylosus (1%), Staphylococcus haemolyticus (<1%), Staphylococcus sciuri (<1%), and Staphylococcus intermedius (<1%). Staphylococcuschromogenes was the predominant CNS isolated from all three herds; however, differences were seen in the prevalence of other CNS species. A total of 158 CNS (S. chromogenesn=66, S. hyicusn=38, S. epidermidisn=37, S. simulans n=10, and S. warneri n=7) were analyzed by pulsed-field gel electrophoresis (PFGE). The majority (33/41) of CNS isolated from the same mammary quarter on more than one occasion had the same PFGE pattern indicating persistence of the same infection over time. When all PFGE patterns for each CNS were analyzed, no common pulsotype was seen among the three herds indicating that CNS are quite diverse. Composite milk somatic cell count (SCC) data were obtained +/-14d of when CNS were isolated. Average milk SCC (5.32 log(10)/ml) for cows in which CNS was the only bacteria isolated was significantly higher than the average milk SCC (4.90 log(10)/ml) from cows with quarter milk samples that were bacteriologically negative.

Antimicrobial susceptibility of coagulase-negative Staphylococcus species isolated from bovine milk.

Coagulase-negative Staphylococcus (CNS) isolates (n=168) obtained from milk from heifers and dairy cows were screened for minimum inhibitory concentration (MIC) to antimicrobials used commonly for mastitis therapy. Of the 10 CNS species included in the study, the predominant species were Staphylococcus chromogenes (n=61), Staphylococcus epidermidis (n=37), Staphylococcus hyicus (n=37), and Staphylococcus simulans (n=16). The majority of CNS was susceptible to ampicillin, oxacillin, cephalothin, and ceftiofur. Erythromycin and pirlimycin were also very effective in vitro inhibitors of CNS. The only exception was observed with S. epidermidis. Of 37 S. epidermidis evaluated, 13 (35%) exhibited efflux-based resistance to erythromycin (> or =16 microg/ml) encoded by msrA and one isolate carried ermC encoding ribosomal methylase-based resistance to both erythromycin (> or =64 microg/ml) and pirlimycin (> or =64 microg/ml). A total of 17 S. epidermidis, 11 S. chromogenes, and one S. hyicus exhibited phenotypic resistance to ampicillin (> or =0.5 microg/ml). Constitutive beta-lactamase production was observed in all ampicillin resistant isolates except 4 S. epidermidis that exhibited inducible beta-lactamase production. Induced beta-lactamase production was also observed in 13 S. epidermidis that were phenotypically susceptible to the entire MIC panel. All isolates that produced beta-lactamase either constitutively or by induction carried blaZ. S. epidermidis (n=12, 32%) that were resistant to methicillin (oxacillin > or =0.5 microg/ml) carried low affinity penicillin-binding protein encoded by mecA. Most multi-drug resistant (MDR) S. epidermidis (> or =2 resistance genes) were resistant to ampicillin, erythromycin and methicillin. All except one MDR S. epidermidis had icaAB, which encodes for polysaccharide intercellular adhesion. Based on pulsed field gel electrophoresis, MDR S. epidermidis were closely related genotypically, and were isolated from different cows on the same farm suggesting clonal dissemination. Bovine S. epidermidis share antimicrobial resistance patterns and virulence determinants of strains observed in human infections. Studying CNS at the species level can provide valuable information about species-specific differences that can be vital data for effective mastitis therapy and management.

Discriminating between strains of Escherichia coli using pulsed-field gel electrophoresis and BOX-PCR.

In this study, we evaluated two biomolecular techniques for discriminating between strains of Escherichia coli isolated form a variety of sources. The DNA of 211 strains of E. coli collected from dairy farms, calves, feces, pigs, primates, humans, and food products was analyzed by pulsed-field gel electrophoresis (PFGE) and repetitive-element polymerase chain reaction using the BOXA1 primer (BOX-PCR). Objectives of the present study were to compare PFGE and BOX-PCR for discriminating among strains of E. coli and investigate their capability in clustering E. coli strains according to the origin of bacterial isolation. Our results showed that PFGE and BOX-PCR were both able to distinguish closely related strains of E. coli; however, PFGE was able to discriminate between isolates indistinguishable by BOX-PCR and interpretation of PFGE data was easier. BOX-PCR proved to have good discrimination power, was less expensive, and could be performed in a PCR thermocycler. Neither of the methods used were effective in clustering E. coli strains according to the source of the organism.

Genetic characterization of a diverse Escherichia coli O157:H7 population from a variety of farm environments.

Many of the current studies on the genetic diversity of Escherichia coli O157:H7 have focused on pathogenic clinical, veterinary, or food isolates. These studies did not explore the diversity of the larger population in the farm environment. Research on selected farm isolates address this wider diversity but have typically been limited to a specific geographic locale or farm type, thus giving limited insight into the greater diversity across geographic regions and varied environments. The objective of this study was to evaluate a diverse population of E. coli O157:H7 collected from a variety of locations and farm environments. Eighty-eight isolates were collected from four farm types (swine, dairy, beef, and poultry) across the southeastern and western United States. Eighteen farms were sampled every 3 months over a period of 24 months. Isolates were analyzed by ribotyping and pulsed field gel electrophoresis (PFGE). Real-time PCR was used to determine the presence or absence of key pathogenic genes (stx1, stx2, and eae). The data indicate a significant amount of genetic diversity, however, ribotype analysis revealed meaningful clusters within the larger population. These groupings were consistent with PFGE analysis. Most of these isolates were clustered by location (i.e. from the same state or region) or farm type. Of the isolates in these clusters, most did not contain pathogenic genes. Of notable interest is a single group in which the majority of isolates, collected from four of the five states sampled, contained at least one stx gene and the eae gene suggesting the existence of a specific pathogenic cluster. These data suggest that, while there is notable diversity within the broader E. coli O157:H7 population, pathogenic isolates may be limited to a subset of strains within the population.

Simultaneous detection of mastitis pathogens, Staphylococcus aureus, Streptococcus uberis, and Streptococcus agalactiae by multiplex real-time polymerase chain reaction.

The objective of this study was to develop a multiplex real-time polymerase chain reaction (PCR) method for simultaneous detection of Staphylococcus aureus, Streptococcus agalactiae, and Streptococcus uberis directly from milk. A genetic marker specific for Staph. aureus was used for primers and dual-labeled probe design. The target for Strep. agalactiae primers and dual-labeled probe was selected from the cfb gene encoding the Christie-Atkins-Munch-Petersen factor. The plasminogen activator gene was the target for primers and dual-labeled probe design for Strep. uberis. Quarter milk samples (n = 192) were analyzed by the multiplex real-time PCR assay and conventional microbiological methods. An additional 57 quarter milk samples were analyzed in a separate real-time PCR assay for Strep. agalactiae only. Using an overnight enrichment step, the real-time PCR technique correctly identified 96.4% of all quarter milk samples; 91.7% of Staph. aureus, 98.2% of Strep. agalactiae, and 100% of Strep. uberis. Results of conventional microbiological methods were used to determine the sensitivity and specificity of the multiplex real-time PCR procedure. The sensitivity of the procedure to correctly identify Staph. aureus, Strep. agalactiae, and Strep. uberis directly from milk was 95.5%, and the specificity was 99.6%. Results of this study indicate that the multiplex real-time PCR procedure has the potential to be a valuable diagnostic technique for simultaneous identification of Staph. aureus, Strep. agalactiae, and Strep. uberis directly from quarter milk samples.

Extended ceftiofur therapy for treatment of experimentally-induced Streptococcus uberis mastitis in lactating dairy cattle.

Streptococcus uberis is an important cause of mastitis in dairy cows throughout the world, particularly during the dry period, the period around calving, and during early lactation. Strategies for controlling Strep. uberis mastitis are poorly defined and are currently inadequate. Objectives of the present study were to evaluate efficacy of ceftiofur, a new broad-spectrum cephalosporin antibiotic, for treatment of experimentally induced Strep. uberis intramammary infections (IMI) in lactating dairy cows during early lactation and to determine whether extended therapy regimens enhanced efficacy of ceftiofur. Efficacy of extended ceftiofur intramammary therapy regimens was investigated in 37 mammary quarters of 23 dairy cows that developed clinical mastitis following experimental infection with Strep. uberis during early lactation. Cows that developed clinical mastitis during the challenge period were allocated randomly to 3 groups representing 3 different ceftiofur treatment regimens: 2-d (n = 7 mammary quarters), 5-d (n = 16 mammary quarters), and 8-d (n = 14 mammary quarters) treatment regimens. For all groups, 125 mg of ceftiofur hydrochloride was administered via intramammary infusion. A bacteriological cure was defined as an experimentally infected quarter that was treated and was bacteriologically negative for the presence of Strep. uberis at 7, 14, 21, and 28 d posttreatment. Percentage of Strep. uberis IMI eliminated was 43, 88, and 100% for the 2-, 5-, and 8-d ceftiofur treatment regimens, respectively. Both the 5- and 8-d ceftiofur extended therapy treatment regimens had significantly higher bacterial cure rates than the standard 2-d ceftiofur treatment regimen. The bacterial cure rate of the 8-d ceftiofur extended therapy group was marginally better (P = 0.052) than the 5-d ceftiofur extended therapy group. Results of this study indicate that ceftiofur therapy was effective for eliminating Strep. uberis experimental IMI, and 5- and 8-d extended ceftiofur therapy regimens were more effective than the standard 2-d treatment.

Influence of prepartum pirlimycin hydrochloride or penicillin-novobiocin therapy on mastitis in heifers during early lactation.

A study was conducted in 2 dairy research herds to determine whether prepartum therapy of heifer mammary glands with penicillin-novobiocin or pirlimycin hydrochloride was effective for reducing the percentage of heifers and mammary quarters infected with mastitis pathogens during early lactation. Almost 96% of Jersey heifers (67 of 70) and 71.3% of quarters (199 of 279) were infected 14 d before expected calving. Of the quarters infected at 14 d before expected parturition, 75% (54 of 72) were uninfected following treatment with penicillin-novobiocin; 87% (61 of 70) were uninfected following treatment with pirlimycin, and 56% (32 of 57) were uninfected in the untreated negative control group. The majority of intramammary infections in Jersey heifers were due to coagulase-negative staphylococci (61%), Streptococcus species, primarily Streptococcus uberis (19%), and Staphylococcus aureus (8%). Almost 73% of Holstein heifers (40 of 55) and 34.3% of mammary quarters (73 of 213) were infected 14 d before expected calving. Of the quarters infected at 14 d before expected parturition, 76% (19 of 25) were uninfected following treatment with penicillin-novobiocin; 59% (17 of 29) were uninfected following treatment with pirlimycin, and 26% (5 of 19) were uninfected in the untreated negative control group. The majority of intramammary infections in Holstein heifers were due to coagulase-negative staphylococci (44%) and Staph. aureus (30%). In both herds, the bacteriological cure rate was significantly higher in heifer mammary glands treated with penicillin-novobiocin or pirlimycin hydrochloride than in untreated controls. Prepartum therapy of heifer mammary glands with penicillin-novobiocin or pirlimycin hydrochloride significantly reduced the percentage of heifers and quarters infected with mastitis pathogens during early lactation.

Efficacy of extended ceftiofur intramammary therapy for treatment of subclinical mastitis in lactating dairy cows.

Little research has focused on treatment of cows with subclinical mastitis during lactation. Ceftiofur is a new broad-spectrum, third-generation cephalosporin antibiotic for veterinary use that inhibits bacterial cell wall synthesis by interfering with enzymes essential for peptidoglycan synthesis. Ceftiofur should be effective against a wide range of contagious and environmental mastitis pathogens. Objectives of the present study were to evaluate the efficacy of ceftiofur for treatment of subclinical mastitis in lactating dairy cows, and to determine if extended therapy regimens enhanced efficacy of ceftiofur. Holstein and Jersey dairy cows (n = 88) from 3 dairy research herds were used. Cows were enrolled in the study based on milk somatic cell counts >400,000/mL and isolation of the same mastitis pathogen in 2 samples obtained 1 wk apart. Cows with one or more intramammary infections (IMI) were blocked by parity and DIM and allocated randomly to 1 of 3 different ceftiofur treatment regimens: 2-d (n = 49 IMI), 5-d (n = 41 IMI), and 8-d (n = 38 IMI) treatment regimens. For all groups, 125 mg of ceftiofur hydrochloride was administered via intramammary infusion. Eighteen cows with 38 IMI were included as an untreated negative control group. A bacteriological cure was defined as a treated infected mammary quarter that was bacteriologically negative for the presence of previously identified bacteria at 14 and 28 d after the last treatment. Efficacy of ceftiofur therapy against all subclinical IMI was 38.8, 53.7, and 65.8% for the 2-, 5-, and 8-d ceftiofur treatment regimens, respectively. Four of 38 (10.5%) IMI in control cows were cured spontaneously without treatment. All 3 ceftiofur treatment regimens were significantly better than the negative control, and the 8-d extended ceftiofur treatment regimen treatment group was significantly better than the standard 2-d treatment group. Pathogen groups had significantly different cure rates from one another. The cure rate for the 8-d extended ceftiofur treatment regimen was 70% for Corynebacterium bovis, 86% for coagulase-negative Staphylococcus species, 36% for Staph. aureus, 80% for Streptococcus dysgalactiae ssp. dysgalactiae, and 67% for Strep. uberis.

Detection of Escherichia coli O157:H7 and Listeria monocytogenes in beef products by real-time polymerase chain reaction.

Rapid methods for the detection of Escherichia coli O157:H7 and Listeria monocytogenes in food products are important to the food industry and for public health. Conventional microbiological methods and newly developed molecular-based techniques such as polymerase chain reaction (PCR)-based methods are time consuming. In this study, a faster method based on utilization of a hybridization probe with real-time PCR, was developed and applied for detection of E. coli O157:H7 and L. monocytogenes from artificially contaminated raw ground beef and fully cooked beef hotdogs. Target genes for E. coli O157:H7 and L. monocytogenes were rfbE and hylA, respectively. An analysis of 169 bacterial strains showed that the chosen primers and probes were specific for detection of E. coli O157:H7 and L. monocytogenes by real-time PCR. The assay was positive for nine of 10. E. coli O157:H7 strains, and all L. monocytogenes (7/7) strains evaluated. Bacterial strains lacking these genes were not detected by these assays. Detection limits of real-time PCR assays ranged from 10(3) to 10(8) colony forming units (CFU)/ml for E. coli O157:H7 in modified tryptic soy broth and 10(4) to 10(8) CFU/mL for L. monocytogenes in Fraser Broth. Detection sensitivity ranged from 10(3) to 10(4) CFU/g of raw ground beef or hotdog without enrichment for E. coli and L. monocytogenes. Approximately 1.4-2.2 CFU/g of E. coli O157:H7 in raw ground beef were detected following an enrichment step of 4 h. Approximately 1.2-6.0 CFU/g of L. monocytogenes in beef hotdogs were detected following an enrichment step of 30 h. The real-time PCR assays for detection of E. coli O157:H7 and L. monocytogenes in raw ground beef and beef hotdogs were specific, sensitive and rapid.

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.

Prepartum antibiotic treatment of heifers: milk production, milk quality and economic benefit.

Prepartum intramammary antibiotic infusion of heifer mammary glands at 7 or 14 d before expected parturition is an effective procedure for eliminating many infections in heifers during late gestation and for reducing the prevalence of mastitis in heifers during early lactation and throughout lactation. Mastitis pathogens were isolated from 76% of samples obtained from untreated control quarters 7 d before expected calving, from 47% of samples obtained 3 d after calving, and from 29% of samples obtained 10 d postpartum. Mastitis pathogens were isolated from about 30% of control quarters through 240 d of lactation. A similar percentage of samples (70%) was positive for mastitis pathogens at C-7 before antibiotic treatment. However, only 8% of samples obtained at 3 d after calving and 4% of samples obtained at 10 d postpartum from quarters of antibiotic-treated heifers contained mastitis pathogens. Throughout the remainder of lactation, mastitis pathogens were isolated from an average of about 11% of quarters. The percentage of samples with mastitis pathogens was higher in untreated controls than in antibiotic-treated quarters at all sampling intervals during lactation. A similar response was observed in heifers that were treated with antibiotics at 14 d before expected parturition. Prepartum antibiotic-treated heifers produced significantly more milk than control heifers and had significantly lower somatic cell count scores than untreated control heifers. These observations are likely associated with or due to the lower prevalence of mastitis pathogen isolation in prepartum antibiotic-treated heifers throughout lactation. Prepartum antibiotic-treated heifers produced 531 kg more milk than heifers in the untreated control group. Multiplying this increase by a milk price of 0.407 dollars/kg yielded a 216.24 dollars per-heifer increase in gross revenue. The cost of treatment, including the cost of testing for antibiotic residues, was estimated at 15.60 dollars for a net revenue of 200.64 dollars per heifer. Prepartum antibiotic treatment to reduce the rate of mastitis in heifers during lactation was highly effective and economically beneficial.

Age-related changes in porcine corticosteroid-binding globulin (pCBG) as determined by an enzyme-linked immunosorbent assay.

The objectives of this study were to develop an assay for the direct measure of porcine corticosteroid-binding globulin (pCBG) and to confirm age-related changes in plasma pCBG concentration. Isolation and purification of pCBG from plasma was performed by affinity chromatography and HPLC-DEAE anion exchange techniques. Analysis by SDS-PAGE revealed two polypeptides (54 and 59 kDa) having similar amino acid homology (>50%) to previously reported sequences of seven mammalian species for the first 33 amino acids. Porcine CBG (20 ng/well) was immobilized to microtiter plates and standards or samples added along with rabbit antiserum developed against the purified pCBG. Goat anti-rabbit IgG-alkaline phosphatase conjugate was added followed by p-NPP substrate. The resultant color development was read at 405 nm. Intra- and interassay coefficients of variation (n=26) of a pooled sample were 10 and 15%, respectively. Age-related changes (P<0.001) in plasma pCBG concentration (n=203) from day 3 through 168 of age confirmed that, in the pig, changes seen in the percent distribution of cortisol among protein bound and free forms around day 28 of age are associated with an increase in CBG concentration.

Clinical, epidemiological and molecular characteristics of Streptococcus uberis infections in dairy herds.

A longitudinal observational study (18 months) was carried out in two Dutch dairy herds to explore clinical, epidemiological and molecular characteristics of Streptococcus uberis mastitis. Infections (n = 84) were detected in 70 quarters of 46 cows. Bacterial isolates were characterized at strain level by random amplified polymorphic DNA (RAPD) fingerprinting. Persistent infections were usually attributable to one strain, while recurrent infections could be caused by different strains. When multiple quarters of a cow were infected, infections were mostly caused by one strain. In each herd, multiple strains were identified yet one strain predominated. The majority of all infections were subclinical, and infections attributed to predominant strains were more chronic than infections attributed to other strains. Epidemiological and molecular data suggest infection from environmental sources with a variety of S. uberis strains as well as within-cow and between-cow transmission of a limited number of S. uberis strains, with possible transfer of bacteria via the milking machine.

Molecular characterization of Salmonella spp. isolated from bulk tank milk and cull dairy cow fecal samples.

The consumption of meat from cull dairy cows and of raw milk has been associated with foodborne salmonellosis. This survey was conducted to establish the prevalence of Salmonella in cull dairy cow fecal samples and bulk tank milk and to determine the proportion of Salmonella-positive dairy farms (n = 30) in east Tennessee. Food and Drug Administration bacteriological analytical protocols were generally used for Salmonella isolation. Primary enrichment was performed with lactose broth, and secondary enrichment was conducted with tetrathionate broth. Eosin methylene blue, hektoen enteric, xylose lysine desoxycholate, bismuth sulfite, and brilliant green (BG) were used as isolation agars. BG agars supplemented with individual antibiotics and/or sulfur compounds were also evaluated. Six of 268 (2.24%) bulk tank milk samples and 9 of 415 (2.17%) fecal samples from 7 of 30 (25.3%) dairy farms were Salmonella-positive. Most isolates (11 of 15) were obtained between September and December. Salmonella isolates were further characterized using polyvalent somatic O Salmonella antiserum, o-nitrophenyl-beta-D-galactopyranoside (ONPG), and Analytical Profile Index (API) 20E strips for Enterobacteriaceae. Serological evaluation of presumptive positive Salmonella isolates resulted in substantial numbers of false positives (41.2%). ONPG and API 20E tests enabled further biochemical distinction of the majority of Salmonella spp. from Salmonella Arizonae and closely related members of Enterobacteriaceae like Citrobacter youngae. Pulsed-field gel electrophoresis of SpeI-digested Salmonella DNA was used to subtype isolates. The isolates grouped into four clusters. The baseline information generated in this survey is being used to develop preharvest pathogen reduction programs on selected farms.

Prevalence and molecular characterization of Escherichia coli O157:H7 in bulk tank milk and fecal samples from cull cows: a 12-month survey of dairy farms in east Tennessee.

A study on the prevalence of Escherichia coli O157:H7 was conducted on 30 dairy farms in east Tennessee between May 2000 and April 2001. This pathogen was isolated from 8 of 30 (26.7%) dairy farms at various sampling times. A total of 415 fecal samples from cull dairy cows and 268 bulk tank milk samples were analyzed. Overall, 10 of 683 (1.46%) samples (2 of 268 [0.75%] milk samples and 8 of 415 [1.93%] fecal samples) tested positive for E. coli O157:H7. Food and Drug Administration Bacteriological Analytical Manual protocols were used for the conventional isolation and confirmation of E. coli O157:H7. Samples were shake cultured (150 rpm) at 42 degrees C for 24 h in tryptic soy broth containing 2 mg of novobiocin per liter. White colonies isolated on cefixime-tellurite sorbitol MacConkey agar plates were evaluated for fluorescence on sorbitol MacConkey agar supplemented with 0.025 g of methylumbelliferyl-beta-D-glucuronide per liter. Nonfluorescing white colonies were biochemically typed and serologically confirmed. Multiplex polymerase chain reaction profiles of E. coli O157:H7 isolates indicated the presence of common virulence factors (Shiga toxin, enterohemolysin, and intimin) of Shiga toxin-producing E. coli, suggesting the potential human pathogenicity of bacterial isolates. Pulsed-field gel electrophoresis profiles of SpeI and XbaI restriction enzyme-digested genomic DNA were used to establish relatedness among bacterial isolates. Data from this study indicate that both cull dairy cows and bulk tank milk pose a potential hazard with regard to human foodborne illness. It is therefore imperative to develop on-farm and preharvest pathogen reduction programs to control the carriage of E. coli O157:H7 pathogens.

Efficacy of a new premilking teat disinfectant containing a phenolic combination for the prevention of mastitis.

A teat disinfectant containing a phenolic combination was evaluated in a natural exposure study in two dairy research herds. Premilking teat disinfection was compared with a negative control using a split-udder experimental design. In both herds, premilking and postmilking teat disinfections with the phenolic combination were significantly more effective in preventing new intramammary infection (IMI) than was postmilking teat disinfection only. Clinical mastitis and new IMI by Streptococcus uberis, Streptococcus dysgalactiae, Gram-negative pathogens, and coagulase-negative Staphylococcus species were significantly lower in quarters of cows with teats predipped and postdipped than in quarters with teats postdipped only. No chapping or teat skin irritation was observed. Premilking teat disinfection with the phenolic combination in association with good udder preparation and postmilking teat disinfection can further reduce the occurrence of new IMI by numerous mastitis pathogens during lactation.

Evaluation of a postmilking teat disinfectant containing a phenolic combination for the prevention of mastitis in lactating dairy cows.

A trial was conducted for 12 months in a herd of 120 Holstein cows in order to determine the efficacy of a teat disinfectant, which contained a phenolic combination, for the prevention of bovine intramammary infections during lactation. Postmilking teat disinfection was compared to a negative control using a split-udder experimental design. The percentage of quarters newly infected by mastitis pathogens was 45% lower in mammary glands with teats that had been dipped in the experimental teat disinfectant after milking than it was in undipped controls. New infections caused by Streptococcus uberis, Staphylococcus aureus, coagulase-negative Staphylococcus species, and Corynebacterium bovis were significantly lower in mammary glands with teats that had been dipped in the experimental teat disinfectant than in undipped controls. No statistical differences in the incidence of clinical mastitis between treatment groups were observed. No irritation or chapping of teats dipped in the experimental teat disinfectant were observed. The results of this study suggest that the experimental teat disinfectant containing a phenolic combination is an effective postmilking teat disinfectant for use in the prevention of new intramammary infections by both contagious and environmental mastitis pathogens.