Involvement of the α7-nicotinic acetylcholine receptors in the anti-inflammatory action of the thymulin-related peptide (PAT).

BACKGROUND AND PURPOSE: Peptide analog of thymulin (PAT) has been shown to have anti-hyperalgesic and anti-inflammatory properties in animal models of inflammation. Recent reports suggest that the peripheral cholinergic system has an anti-inflammatory role mediated by α7-nicotinic acetylcholine receptor (α7-nAChR). Our aim is to investigate whether the action of PAT is mediated, via the cholinergic pathway. EXPERIMENTAL APPROACH: The anti-hyperalgesic and anti-inflammatory action of PAT was assessed in rat models of inflammatory nociceptive hyperactivity (carrageenan and endotoxin) and in a mice air-pouch model for localized inflammation, respectively; the possible attenuation of PAT's effects by pretreatment with the α7-nAchR specific antagonist methyllycaconitine citrate (MLA) was also investigated. In another series of experiments, using two electrode recordings, the effect of PAT on the α7-nAChRs, expressed in Xenopus Oocytes, was also determined. KEY RESULTS: Administration of PAT reversed inflammatory nociceptive hyperactivity and cold and tactile hyperactivity in rats. This effect was partially or totally prevented by MLA, as assessed by different behavioral pain tests. Treatment with PAT also reduced the alteration of cytokines and NGF levels by carrageenan injection in the mouse air pouch model; this effect was partially antagonized by MLA. Electrophysiological recording demonstrated that PAT significantly potentiated the α7-nAchR expressed in Xenopus Oocytes. These effects were not observed when a control peptide, with a reverse sequence (rPAT), was utilized. CONCLUSIONS AND IMPLICATIONS: The behavioral and electrophysiological observations described in this report demonstrate that PAT mediates, at least partially, its anti-inflammatory action by potentiating the α7-nAChR. These results indicate that PAT has a potential for new therapeutic applications as anti-inflammatory and analgesic agent.

Heat-treated colostrum and reduced morbidity in preweaned dairy calves: results of a randomized trial and examination of mechanisms of effectiveness.

A randomized controlled clinical trial was conducted using 1,071 newborn calves from 6 commercial dairy farms in Minnesota and Wisconsin, with the primary objective being to describe the effects of feeding heat-treated colostrum on serum immunoglobulin G concentration and health in the preweaning period. A secondary objective was to complete a path analysis to identify intermediate factors that may explain how feeding heat-treated colostrum reduced the risk for illness. On each farm, colostrum was collected each day, pooled, and divided into 2 aliquots; then, one aliquot was heat-treated in a commercial batch pasteurizer at 60°C for 60 min. Samples of fresh and heat-treated colostrum were collected for standard microbial culture (total plate count and total coliform count, cfu/mL) and for measurement of immunoglobulin G concentrations (mg/mL). Newborn calves were removed from the dam, generally within 30 to 60 min of birth, and systematically assigned to be fed 3.8L of either fresh (FR, n=518) or heat-treated colostrum (HT, n=553) within 2h of birth. Venous blood samples were collected from calves between 1 and 7d of age for measurement of serum IgG concentrations (mg/mL). All treatment and mortality events were recorded by farm staff between birth and weaning. Regression models found that serum IgG concentrations were significantly higher in calves fed HT colostrum (18.0 ± 1.5 mg/mL) compared with calves fed FR colostrum (15.4 ± 1.5 mg/ml). Survival analysis using Cox proportional hazards regression indicated a significant increase in risk for a treatment event (any cause) in calves fed FR colostrum (36.5%, hazard ratio=1.25) compared with calves fed HT colostrum (30.9%). In addition, we observed a significant increase in risk for treatment for scours in calves fed FR colostrum (20.7%, hazard ratio=1.32) compared with calves fed HT colostrum (16.5%). Path analysis suggested that calves fed HT colostrum were at lower risk for illness because the heat-treatment process caused a significant reduction in colostrum total coliform count, which was associated with a reduced risk for illness as a function of improved serum IgG concentrations.

Heat treatment of colostrum on commercial dairy farms decreases colostrum microbial counts while maintaining colostrum immunoglobulin G concentrations.

This study was conducted on 6 commercial dairy farms in Minnesota and Wisconsin to describe the effect of heat treatment (at 60°C for 60 min) on colostrum, on colostrum bacteria counts, and immunoglobulin G concentrations. First-milking colostrum was collected each day, pooled, divided into 2 aliquots, and then 1 aliquot was heat treated in a commercial batch pasteurizer at 60°C for 60 min. Frozen samples of pre- and post- heat-treated colostrum were submitted for standard microbial culture (total plate count and total coliform count, cfu/mL) and testing for immunoglobulin G concentrations (mg/mL). Data were analyzed from 266 unique batches of colostrum. Linear regression showed that heat treatment decreased colostrum total plate counts (-2.25 log(10)) and coliform counts (-2.49 log(10)), but, overall, did not affect colostrum IgG concentration. Though higher-quality batches of colostrum did experience a greater magnitude of loss of IgG as a result of heat treatment as compared with lower- or intermediate-quality batches of colostrum, the colostral IgG concentrations in these batches remained high overall, and within acceptable limits for feeding. This study demonstrates that batch heat treatment of colostrum at 60°C for 60 min can be successfully conducted on commercial dairy farms by farm staff to decrease colostrum microbial counts while maintaining colostrum IgG concentrations.

The selective treatment of clinical mastitis based on on-farm culture results: I. Effects on antibiotic use, milk withholding time, and short-term clinical and bacteriological outcomes.

The objective of this multi-state, multi-herd clinical trial was to evaluate the efficacy of using an on-farm culture system to guide strategic treatment decisions in cows with clinical mastitis. The study was conducted in 8 commercial dairy farms ranging in size from 144 to 1,795 cows from Minnesota, Wisconsin, and Ontario, Canada. A total of 422 cows affected with mild or moderate clinical mastitis in 449 quarters were randomly assigned to either (1) a positive-control treatment program or (2) an on-farm, culture-based treatment program. Quarter cases assigned to the positive-control group received immediate on-label intramammary treatment with cephapirin sodium. Quarters assigned to the culture-based treatment program were cultured on-farm and treated with cephapirin sodium after 18 to 24h of incubation if they had gram-positive growth or a mixed infection. Quarters with gram-negative or no growth did not receive intramammary therapy. The proportion of quarter cases assigned to positive-control and culture-based treatments that received intramammary antibiotic therapy because of study assignment was 100 and 44%, respectively; the proportion of cases that received secondary antibiotic therapy was 36 and 19%, respectively; and the proportion of cases that received intramammary antibiotic therapy because of study assignment or secondary therapy was 100 and 51%, respectively. A tendency existed for a decrease in the number of days in which milk was discarded from cows assigned to the culture-based treatment program versus cows assigned to the positive-control group (5.9 vs. 5.2 d). No statistically significant differences existed between cases assigned to the positive-control and cases assigned to the culture-based treatment program in days to clinical cure (2.7 vs. 3.2 d), bacteriological cure risk within 21 d of enrollment (71 vs. 60%), new intramammary infection risk within 21 d of enrollment (50 vs. 50%), and treatment failure risk (presence of infection, secondary treatment, clinical mastitis recurrence, or removal from herd within 21 d after enrollment; 81 vs. 78%). In summary, the use of an on-farm culture system to guide the strategic treatment of clinical mastitis reduced intramammary antibiotic use by half and tended to decrease milk withholding time by 1 d, without significant differences in days to clinical cure, bacteriological cure risk, new intramammary infection risk, and treatment failure risk within 21 d after the clinical mastitis event.

The selective treatment of clinical mastitis based on on-farm culture results: II. Effects on lactation performance, including clinical mastitis recurrence, somatic cell count, milk production, and cow survival.

The objective of this multi-state, multi-herd clinical trial was to report on the efficacy of using an on-farm culture system to guide strategic treatment decisions in cows with clinical mastitis. The study was conducted in 8 commercial dairy farms ranging in size from 144 to 1,795 cows from Minnesota, Wisconsin, and Ontario, Canada. A total of 422 cows affected with mild or moderate clinical mastitis in 449 quarters were randomly assigned to either (1) a positive-control treatment program or (2) an on-farm culture-based treatment program. Quarter cases assigned to the positive-control group received immediate on-label intramammary treatment with cephapirin sodium. Quarters assigned to the culture-based treatment program were not treated until the results of on-farm culture were determined after 18 to 24h of incubation. Quarters in the culture-based treatment program that had gram-positive growth or a mixed infection were treated according to label instruction using intramammary cephapirin sodium. Quarters assigned to the culture-based treatment program that had gram-negative or no-growth did not receive intramammary therapy. It was already reported in a companion paper that the selective treatment of clinical mastitis based on on-farm culture results decreases antibiotic use by half and tends to decrease milk withholding time without affecting short-term clinical and bacteriological outcomes. The present article reports on long-term outcomes of the aforementioned study. No statistically significant differences existed between cases assigned to the positive-control program and cases assigned to the culture-based treatment program in risk and days for recurrence of clinical mastitis in the same quarter (35% and 78 d vs. 43% and 82 d), linear somatic cell count (4.2 vs. 4.4), daily milk production (30.0 vs. 30.7 kg), and risk and days for culling or death events (28% and 160 d vs. 32% and 137 d) for the rest of the lactation after enrollment of the clinical mastitis case. In summary, the selective treatment of clinical mastitis based on on-farm culture resulted in no differences in long-term outcomes, such as recurrence of clinical mastitis in the same quarter, somatic cell count, milk production, and cow survival for the rest of the lactation after clinical mastitis.

Relationship between in vitro susceptibility test results and treatment outcomes for gram-positive mastitis pathogens following treatment with cephapirin sodium.

The selection of antimicrobial agents for the treatment of mastitis has often been based on results of in vitro susceptibility testing. However, the results of in vitro susceptibility tests have been shown to be poor predictors of treatment outcomes. The objective of this study was to determine if an association existed between results of antimicrobial susceptibility tests and outcomes of mastitis caused by gram-positive pathogens recovered from quarters that received treatment with cephapirin sodium. Mastitis pathogens were obtained from a multi-site clinical trial that evaluated the benefits of using an on-farm culturing system. Target pathogens (n = 187) comprised coagulase-negative staphylocci (65%), Streptococcus spp. (14%), other pathogens (12%), and Staphylococcus aureus (11%), which were recovered from quarters that received treatment using cephapirin sodium. The antimicrobial susceptibility profile to cephapirin was determined using the broth micro-dilution technique. The overall bacteriological cure rate achieved by cephapirin treatment was 82%. Bacteriological outcomes (cure or treatment failure) were not associated with pathogen type. A recurrent case of mastitis was observed in 10 quarters classified as cures and 3 quarters classified as treatment failures. Recurrence of mastitis was not associated with bacteriological outcomes or susceptibility test results. In vitro susceptibility to cephapirin was exhibited by 94.8 and 91.2% of pathogens recovered from quarters classified as cures and treatment failures, respectively. Bacteriological outcomes of mastitis treated using cephapirin were not associated with in vitro susceptibility test results or in vitro minimum inhibitory concentration values. In this population, there was an 82% probability of treatment success when the isolate was susceptible but only a 27% probability of treatment failure when the isolate was resistant. Based on this research, results of in vitro susceptibility tests should not be used as the primary guide for treatment decisions regarding intramammary cephapirin sodium.

Onset and duration of protective immunity against clinical disease and renal carriage in dogs provided by a bi-valent inactivated leptospirosis vaccine.

Protection against clinical disease and prevention of the renal carrier state remain the key objectives of vaccination against leptospirosis in the dog. In the present paper, groups of dogs were vaccinated twice with a commercial bacterin (EURICAN L) containing Leptospira interrogans serovars icterohaemorrhagiae and canicola and challenged with heterologous representatives of both serovars at 2 weeks (onset of immunity) or 14 months (duration of immunity) after the second vaccination. Control dogs were not vaccinated against leptospirosis and kept with the vaccinated dogs. The challenges, irrespective of the serovar, reliably produced clinical signs consistent with Leptospira infection in the control pups with up to 60% mortality. As expected clinical disease in the adult controls was less severe, but we were able to induce morbidity and mortality as well. Under these extreme challenge conditions, clinical signs in the vaccinated dogs were rare, and when observed, mild and transient in nature. Following experimental infection, 100% of the control pups and 83% of the adult controls became renal carriers. Despite the heavy challenges, none of the 18 vaccinated puppies (onset of immunity studies) and only 2 out of the 16 vaccinated adult dogs (duration of immunity studies) developed a renal carrier state. These results show that a primary course of two doses of EURICAN L provided quick onset and long-term protection against both clinical leptospirosis and the renal carrier stage. This vaccine should provide veterinarians with a powerful tool to prevent clinical disease in dogs and zoonotic transmission of leptospirosis to humans.

Molecular subtyping of mastitis-associated Klebsiella pneumoniae isolates shows high levels of diversity within and between dairy herds.

Despite advances in controlling mastitis (inflammation of the mammary gland), udder infections caused by Klebsiella pneumoniae continue to affect dairy cattle. Mastitis caused by K. pneumoniae responds poorly to antibiotic treatment, and as a consequence, infections tend to be severe and long lasting. We sought to determine whether a nonrandom distribution of specific genotypes of K. pneumoniae was associated with mastitis from 6 dairy herds located in 4 different states. A total of 635 isolates were obtained and fingerprinted by repetitive DNA sequence PCR. Significant genetic diversity was observed in 4 of the 6 dairy herds analyzed, and a total of 49 genotypic variants were identified. Within a herd, Simpson's diversity indices were 91.0, 94.1, 91.7, 88.6, 53.3, and 64.3% for dairies A, B, C, D, E, and F, respectively. The association between matrices of genetic similarity and matrices of temporal distance was negative in all the dairies analyzed. Four dairies had a high incidence of K. pneumoniae mastitis during the winter. The majority of genotypes were unique to herds of origin, and only 5 genotypes were detected in more than 2 dairies. Genotype 1 (arbitrary designation) occurred most frequently across dairies and was found in 25.2% of all mastitis cases and among 22.8% of reinfected and culled cows in dairy A. Specific genotypes also tended to be associated with a specific bedding type and dairy location. Analysis of molecular variance showed that 18% of the genetic diversity was due to variation among herds within states, and 82% of the genetic diversity was accounted for by variation of genotypes within herds. The data support the idea that mastitis is caused by a diverse group of K. pneumoniae genotypes and thus has major implications for the diagnosis, prevention, and treatment of udder infections in dairy cows.

Ability of organic and inorganic bedding materials to promote growth of environmental bacteria.

The major objective of this study was to contrast the ability of 4 commonly utilized bedding materials to promote growth of environmental bacteria under controlled conditions. A second objective was to describe the relationship between bacterial growth and specific biochemical or nutritional properties of these bedding materials. Unused samples of clean sand (CS; n = 20), recycled sand (RS; n = 21), digested manure solids (DS; n = 15), and shavings (SH; n = 15) were collected from bedding storage areas on 49 commercial Minnesota and Wisconsin dairy farms. Sterilized bedding samples were inoculated with Klebsiella pneumoniae and Enterococcus faecium then incubated, in triplicate, for 72 h at 37 degrees C. Subsamples were collected after 0, 24, 48, and 72 h of incubation for culture and enumeration of bacteria. Subsamples of bedding were also tested for pH, total C content (%), and total N content (%). If bacterial growth occurred, peak levels were typically achieved within 24 h. Digested manure solids promoted the greatest amounts of growth of K. pneumoniae, followed by RS and then SH, whereas CS promoted the least. There would seem to be a tradeoff in selecting SH as a bedding material, because it supported moderate growth of K. pneumoniae but caused a rapid decline in the numbers of E. faecium. However, RS, CS, and DS each only supported relatively small amounts of growth of E. faecium, so the benefit of SH relative to other bedding materials is limited. High bedding pH may partially explain why some bedding materials supported growth of E. faecium (e.g., DS and RS). Both high bedding pH (e.g., as for DS or RS) and high total C (%) content (e.g., as for DS and SH) may partially explain why some bedding materials supported growth of K. pneumoniae.

Genetic diversity of mastitis-associated Klebsiella pneumoniae in dairy cows.

The objectives of this study were to determine the level of genetic diversity of Klebsiella pneumoniae isolated from clinical mastitis cases and to define genotypes most commonly associated with the disease. Individual quarter milk samples were collected from a single privately owned dairy herd over a 2-yr period and submitted to the Laboratory for Udder Health, Minnesota Veterinary Diagnostic Laboratory, University of Minnesota, for bacteriological culture. Eighty-four K. pneumoniae isolates were obtained and fingerprinted by repetitive DNA sequence PCR, 43 by pulsed-field gel electrophoresis (PFGE), and 29 by multilocus sequence typing (MLST). Significant genetic diversity was observed among the isolates regardless of the fingerprinting method used. Simpson's diversity index was 93.5, 96.1, and 97.0% when analyzed by repetitive DNA sequence PCR (n = 84), pulse field gel electrophoresis (n = 43), and MLST (n = 29), respectively. In some cases more than 1 genotype was obtained from a single milk sample originating from an individual quarter. The majority of infections were observed during the winter and accounted for 69.0% of K. pneumoniae mastitis cases. There was a negative correlation between a matrix of fingerprints similarity and a matrix of temporal distances. The MLST results revealed 5 new and novel allelic types, which have not been previously reported in the MLST database. Three isolates shared MLST types with human clinical isolates, raising the possibility that some K. pneumoniae isolates, of bovine origin, may be capable of causing disease in humans. There were 21 genotypes present within the herd, and there was no evidence for nonrandom distribution of genotypes uniquely associated with mastitis. We have shown, using 3 distinct genotyping methods, that K. pneumoniae isolated from clinical mastitis within a single dairy herd is caused by a genetically diverse population and that multiple genotypes can be isolated from a mastitic quarter. The data suggest that mastitis can be caused by a variety of K. pneumoniae genotypes. Diverse genotypes may have different levels of invasiveness and virulence and may originate from various sources within the dairy.

Passive transfer of immunoglobulin G and preweaning health in Holstein calves fed a commercial colostrum replacer.

The objective of this study was to describe passive transfer of IgG and preweaning health in newborn calves fed a commercially available plasma-derived colostrum replacement (CR) product or maternal colostrum (MC). Twelve commercial Holstein dairy farms enrolled singleton newborn heifer calves to be fed fresh MC (n = 239 calves) or one dose of CR containing 125 g of Ig (n = 218 calves) as the first colostrum feeding. For 7 of these farms that routinely provided a second feeding of 1.9 L of MC to their calves 8 to 12 h after the first colostrum feeding, calves assigned to the CR treatment group were offered a second feeding consisting of 1.9 L of commercial milk replacer supplemented with one dose of a commercially available plasma-derived colostrum supplement, containing 45 g of Ig per dose, 8 to 12 h after the first colostrum feeding. A blood sample was collected from all calves between 1 to 8 d of age for serum IgG and total protein (TP) determination, and records of all treatment and mortality events were collected until weaning. Serum IgG and TP concentrations were significantly higher in calves fed MC (IgG = 14.8 +/- 7.0 mg/mL; TP = 5.5 +/- 0.7 g/dL) compared with calves fed CR (IgG = 5.8 +/- 3.2 mg/mL; TP = 4.6 +/- 0.5 g/dL). The proportion of calves with failure of passive transfer (serum IgG <10.0 mg/mL) was 28.0 and 93.1% in the MC and CR treatment groups, respectively. Though a trend was present, the proportion of calves treated for illness was not statistically different for calves fed MC (51.9%) vs. CR (59.6%). Total number of days treated per calf (MC = 1.7; CR = 2.0), treatment costs per calf (MC = $10.84; CR = $11.88), and proportion of calves dying (MC = 10.0%; CR = 12.4%) was not different between the 2 colostrum treatment groups. The mean serum total protein concentration predictive of successful passive transfer (serum IgG = 10 mg/mL) was 5.0 g/dL in calves fed MC or CR. Long-term follow-up of these calves (to maturity) is ongoing to describe the effects of feeding CR on longevity, productivity, risk for Johne's disease, and economics.

Heat-treatment of bovine colostrum. II: effects of heating duration on pathogen viability and immunoglobulin G.

Batches (30-L) of first-milking bovine colostrum, inoculated with Mycoplasma bovis (10(8) cfu/mL), Listeria monocytogenes (10(6) cfu/mL), Escherichia coli O157:H7 (10(6) cfu/mL), Salmonella enteritidis (10(6) cfu/mL), and Mycobacterium avium subsp. paratuberculosis (Map; 10(3) cfu/mL), were heat-treated at 60 degrees C for 120 min in a commercial on-farm batch pasteurizer system. Duplicate 50-mL subsamples of colostrum were collected at 15-min intervals throughout the heat-treatment process for the purpose of bacterial culture and for measurement of IgG concentration (mg/mL) and antibody activity [log2(bovine viral diarrhea virus type 1 serum neutralization titer)]. Four replicate batches of colostrum were run for each of the 5 pathogens studied. There was no effect of heating moderate- to high-quality colostrum at 60 degrees C for at least 120 min on mean IgG concentration (pre = 60.5 mg/mL; post = 59.1 mg/mL). Similarly, there was no effect of heat-treatment on the mean log2 bovine viral diarrhea virus type 1 serum neutralization titer (pre = 12.3; post = 12.0). Viable M. bovis, L. monocytogenes, E. coli O157:H7, and S. enteritidis added to colostrum could not be detected after the colostrum was heat-treated at 60 degrees C for 30 min. Average bacteria counts showed that Map was not detected when batches were heated at 60 degrees C for 60 min. Although the authors believe that heat-treating colostrum at 60 degrees C for 60 min should be sufficient to eliminate Map from colostrum in most situations, further research is needed to determine whether these findings may be replicated, given that variability was observed in Map culture results.

Heat treatment of bovine colostrum. I: effects of temperature on viscosity and immunoglobulin G level.

The objective of this study was to identify the critical temperature, at or below which heat-treatment of bovine colostrum would produce no significant changes in viscosity, IgG concentration, or Ig activity. Results of preliminary work, using a Rapid Visco Analyzer (RVA) to heat 50-mL aliquots from 6 unique batches of bovine colostrum at 59, 60, 61, 62, and 63 degrees C, suggested that colostrum could be heated to 60 degrees C for up to 120 min without changing viscosity or IgG concentration. This finding was confirmed by heating 50-mL aliquots from 30 unique batches of colostrum in an RVA for 120 min at 60 and 63 degrees C. Heating colostrum to 63 degrees C resulted in an estimated 34% decrease in IgG concentration and 33% increase in viscosity. However, there was no difference in IgG concentration between preheat-treated (73.4 +/- 26.5 mg/mL) and post-heat-treated (74.5 +/- 24.3 mg/mL) samples after heating colostrum to 60 degrees C in an RVA for 120 min. Similarly, viscosity was unaffected after heating colostrum to 60 degrees C in an RVA for 120 min. High quality colostrum (> or =73.0 mg/mL) suffered greater losses of IgG and greater viscosity changes when heated to 63 degrees C than did moderate quality colostrum (<73.0 mg/mL). However, the effects of colostrum quality were minor if high quality colostrum was only heated to 60 degrees C. The results of a bovine viral diarrhea serum neutralization assay suggested that antibody activity was unchanged after heating colostrum to either 60 or 63 degrees C. However, these results were interpreted as being inconclusive due to a high proportion of missing results because of the congealing of many samples after heat treatment. The results of this study indicate that 50-mL volumes of bovine colostrum can be heat treated at 60 degrees C for up to 120 min in an RVA without affecting IgG concentration or viscosity.

Preventing bacterial contamination and proliferation during the harvest, storage, and feeding of fresh bovine colostrum.

The objectives of this study were to identify control points for bacterial contamination of bovine colostrum during the harvesting and feeding processes, and to describe the effects of refrigeration and use of potassium sorbate preservative on bacteria counts in stored fresh colostrum. For objective 1, first-milking colostrum samples were collected aseptically directly from the mammary glands of 39 cows, from the milking bucket, and from the esophageal feeder tube. For objective 2, 15-mL aliquots of colostrum were collected from the milking bucket and allocated to 1 of 4 treatment groups: 1) refrigeration, 2) ambient temperature, 3) refrigeration with potassium sorbate preservative, and 4) ambient temperature with potassium sorbate preservative. Subsamples from each treatment group were collected after 24, 48, and 96 h of storage. All samples underwent bacteriological culture for total plate count and coliform count. Bacteria counts were generally low or zero in colostrum collected directly from the gland [mean (SD) log10 cfu/mL(udder) = 1.44 (1.45)]. However, significant bacterial contamination occurred during the harvest process [mean (SD) log10 cfu/mL(bucket) = 4.99 (1.95)]. No additional bacterial contamination occurred between the bucket and the esophageal feeder tube. Storing colostrum at warm ambient temperatures resulted in the most rapid increase in bacteria counts, followed by intermediate rates of growth in nonpreserved refrigerated samples or preserved samples stored at ambient temperature. The most effective treatment studied was the use of potassium sorbate preservative in refrigerated samples, for which total plate count and total coliform counts dropped significantly and then remained constant during the 96-h storage period.

The distribution of Mycobacterium avium ssp. paratuberculosis in the environment surrounding Minnesota dairy farms.

The objective of this study was to characterize the distribution of Mycobacterium paratuberculosis (Map) in the environment of infected and uninfected Minnesota dairy farms. Eighty herds known to be infected from Minnesota's Johne's Disease Control Program (JDCP) and 28 herds known to be uninfected from Minnesota Voluntary Johne's Disease Herd Status Program (VJDHSP) were sampled. Fecal samples from up to 100 cows in each herd were cultured in pools of 5 cows. Two environmental samples were obtained from each farm from various locations. All samples were tested using bacterial culture for Map. Eighty percent of the JDCP herds had at least one positive pool. Environmental samples were cultured positive in 78% of the JDCP herds. Two (7%) of the VJDHSP herds had one positive pool, and one herd had one positive environmental sample. Environmental samples were cultured positive in cow alleyways (77% of the herds), manure storage (68%), calving area (21%), sick cow pen (18%), water runoff (6%), and postweaned calves areas (3%). There was an association between maximum level of colonies per tube from cow alleyways and manure storage and fecal pool prevalence. Herds with both areas cultured negative were estimated to have 0.3 to 4% fecal pool prevalence. Herds with both areas having a heavy load of bacteria were estimated to have 53 to 73% fecal pool prevalence. The study results indicate that targeted sampling of cow alleyways and manure storage areas appears to be an alternative strategy for herd screening and Johne's infection status assessment and for estimating herd fecal prevalence.

Serological diagnosis of brucellosis caused by Brucella canis.

Blood serum samples from 2,328 dogs were tested to detect antibodies against Brucella canis with the agar gel immunodiffusion (AGID) and 2-mercaptoethanol slide agglutination test (ME-SAT) using Brucella ovis as the antigen. All blood serum samples were also evaluated for antibodies against Brucella abortus and Brucella melitensis using the Rose Bengal test. Twentyfive (1.07%) of the sera evaluated were considered positive with AGID test. Only 4 (16%) of these blood serum samples were positive when evaluated with ME-SAT. The 25 AGID positive samples and 25 AGID negative serum samples were also examined by: the complement fixation test (CFT) using B. ovis hot saline extract (HSE) as the antigen, indirect enzyme linked immunosorbent assay (ELISA) and immunoblotting (IB) using B. canis and B. ovis HSE antigens. Two positive canine sera from culture positive dogs and the serum of an experimentally RM6/66 B. canis-infected rabbit were employed as positive controls and one serum from a known uninfected dog as a negative control. ELISA with B. canis antigen gave 9 (18%) positive results (6 AGID-positive and 3 AGID-negative sera). ELISA performed with B. ovis antigen detected 15 (30%) positive samples (10 AGID-positive, 5 AGID-negative and 8 B. canis ELISA positive sera). IB analysis of known positive controls sera employing B. canis antigen detected bands with molecular weights of 94-80, 64-50, 35, 32-30, 28, 23, 20-18, 15-12 kDa. The same sera tested with B. ovis antigen revealed bands of 35, 32-30, 25, 23, 20-18, 15-12 kDa. No bands were observed with the negative control serum and the 50 canine tested sera.

Effectiveness of an internal teat seal in the prevention of new intramammary infections during the dry and early-lactation periods in dairy cows when used with a dry cow intramammary antibiotic.

The objectives of this study were to determine the effect of infusion with an internal teat seal at dry off, when used as an adjunct to long-acting antibiotic infusion at dry off, on the risk for acquiring a new intramammary infection (IMI) during the dry period, prevalence of IMI and linear score (LS) after calving, and risk for experiencing a clinical mastitis event between dry off and 60 DIM. A total of 437 cows from 2 dairy herds, with no clinical mastitis and 4 functional quarters, were enrolled at dry off. Prior to the final milking, all quarters were sampled for bacteriological culture and SCC analysis. After milking, all 4 quarters were infused with a commercially available long-acting dry cow antibiotic. Two contralateral quarters were then infused with an internal teat seal (Orbeseal, Pfizer Animal Health, New York). Following calving the teat seal was stripped out at first milking. Duplicate milk samples were collected between 1 to 3 DIM and again between 6 to 8 DIM for culture and SCC analysis. Quarters treated with Orbeseal had significantly lower prevalence of IMI at 1 to 3 DIM (tx = 22.8%, control = 29.1%), had significantly fewer quarters that acquired a new IMI between dry off and 1 to 3 DIM (tx = 20.2%, control = 25.4%), and had significantly fewer quarters affected by a clinical mastitis event between dry off and 60 DIM (tx = 5.9%, control = 8.0%). Multivariable analysis showed a significant effect of treatment, with treated quarters being 30% less likely to develop a new IMI between dry off and 1 to 3 DIM, 31% less likely to have an IMI present at 1 to 3 DIM, 33% less likely to experience a clinical mastitis event between dry off and 60 DIM, and having significantly lower linear score measures at 1 to 3 DIM and 6 to 8 DIM, compared with control quarters.

Field validation of a milk-line sampling device for monitoring milk component data.

The objective of this study was to investigate the ability of a milk-line sampling device to obtain a representative sample under field conditions by comparing the milk component composition between milk-line and bulk-tank samples, for milk harvested from the same group of cows at the same milking. A total of 42 paired milk-line and bulk-tank samples were collected at separate milking events from 21 different dairy herds. Samples were analyzed for milk fat (%), true protein (%), and milk urea nitrogen (mg/dl). Concordance correlation coefficients showed a very high level of agreement between the two sample types, with values ranging between 0.82 and 0.95 for the components measured. ANOVA showed that milk component data derived from milk-line samples were neither statistically nor numerically different from milk component data derived from bulk-tank samples. The strategy of monitoring milk component data through milk line sampling should provide dairy producers with inexpensive, timely, and accurate information that will help to improve ongoing nutritional monitoring programs for individual groups of cows within the dairy herd.

Field validation of a milk line sampling device for monitoring milk quality and udder health.

The objective of this study was to investigate the ability of a milk line sampling device to obtain a representative sample by comparing SCC and bacterial culture results between milk line and bulk tank samples for milk harvested from the same group of cows at the same milking. A total of 42 paired milk line and bulk tank samples were collected at separate milking events from 21 different herds. Concordance correlation coefficients showed a high level of agreement between the two sample types, with values ranging between 0.74 and 0.99 for all parameters and bacterial species measured. ANOVA showed that SCC and bacterial culture results for Streptococcus agalactiae, Staphylococcus aureus, Streptococcus non-agalactiae, Coliforms, and coagulase-negative staphylococci were neither numerically or statistically different between milk line and bulk tank samples. KAPPA analysis showed that overall agreement beyond chance between milk line and bulk tank samples in determining whether a herd was positive or negative for either Strep. agalactiae or Staph. aureus were 100 and 75%, respectively. While further research is needed to fully assess the utility of this tool for the purpose of bacterial culture, the results of this study suggest that the strategy of milk line sampling is a very promising monitoring tool. This sampling strategy should provide producers with inexpensive and timely information that will help to improve programs for monitoring milk quality and udder health in commercial dairy herds.