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.

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.

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.

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.

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.

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.

Gabexate mesilate (Foy) as an anticoagulant in extracorporeal circulation in dogs and sheep.

Gabexate mesilate (GM), a synthetic serine protease inhibitor with a short half-life time of approx. 80 sec, was applied as the exclusive anticoagulant in small scale extracorporeal circulation in narcotized dogs and sheep. The animals underwent a veno-venous bypass. As the blood was drawn out of the femoral vein, GM was infused immediately into the extracorporeal system. GM was greatly reduced because of its short half-life time before the blood reentered the animal via a brachial vein. In order to control the coagulatory state of the blood, the Activated Clotting Time (ACT), Partial Thromboplastine Time (PTT) and Recalcification Time (RT) were measured in regular intervals. In addition, a screen pressure test device was used to monitor on line during ECC the tendency of blood clotting. It was shown that the blood of the extracorporeal system was sufficiently anticoagulated by an infusion of 0.03 mg/ml/min GM. During two hours ECC, simultaneously measured ACT-values of the animals only differed up to 10 percent from the native values. Pilot studies have shown that the results of these model investigations could be successfully transferred to extracorporeal circulation with membrane oxygenators and pumps as they are clinically used.

Subtype specific ELISA for the detection of antibodies against influenza A H1N1 and H3N2 in swine.

A subtype specific ELISA using purified hemagglutinin (HA) from influenza A H1N1 and H3N2 was developed to detect antibodies present in swine previously exposed to either H1N1 or H3N2 influenza viruses. The HA was extracted using the detergent octylglucoside followed by ion exchange chromatography. All HA preparations were free of contaminating nucleoprotein and matrix protein contamination. Monospecific swine anti-H1N1 and swine anti-H3N2 sera were used to demonstrate the subtype specificity of the assay. Monospecific rabbit anti-H1N1 or H3N2 was used to sterically block crossreacting determinants and thus enhance assay specificity. A linear relationship between single dilution point ELISA and the hemagglutination inhibition (HI) test was established. This enabled the accurate estimation of HI titer from ELISA. Further refinement of this ELISA based HI estimation system could allow it to replace the current HI procedures in instances where identification at the subtype level of specificity is acceptable. The substantial specificity requirements associated with the detection of strain specific antibody would still necessitate the use of the HI procedure.

Class specific antibody response to influenza A H1N1 infection in swine.

Early detection of swine influenza A outbreaks is essential to understand the true cause and effect relationship that exists between this disease and other serious respiratory or herd health problems. Enzyme-linked immunosorbent assays (ELISAs) for the early detection of H1N1 subtype specific serum IgM, IgG and secretory IgA were compared to direct virus detection in in embryonated eggs. Elevated levels of H1 hemagglutinin (HA) specific IgM and IgG were detected as early as 3 days post experimental infection with a field strain of swine influenza A (H1N1). Influenza specific IgA in nasal mucous samples was detected on day 4 post infection (PI). This compared favorably with egg inoculation methods which detected virus 2-4 days PI. Identification of elevated H1 HA specific IgM in test herds could signify a recent influenza outbreak. Alternatively, ELISA analysis of nasal mucous samples for H1 HA specific IgA could provide a noninvasive method of obtaining similar information on the influenza specific immune status of the herd.

A dot immunobinding assay (dot-ELISA) for the rapid serodiagnosis of Salmonella enteritidis infection in chickens.

A dot immunobinding assay (DIA) was developed for the detection of antibodies to Salmonella enteritidis. Western blot analysis of outer membrane proteins from SE identified 2 polypeptides of molecular masses 43 and 46 kD that were specific for S. enteritidis. These 2 polypeptides were utilized as antigens in the DIA. The DIA was tested on sera from chickens experimentally infected with S. enteritidis. Results of the DIA were compared with that of conventional microagglutination and serum plate tests. The DIA was a highly specific and sensitive test that can be useful for screening birds to determine if they are infected with S. enteritidis. Its simplicity, reliability, reproducibility, and speed in interpreting the assay results makes it a useful screening test for flock monitoring.

ELISA method for detection of influenza A infection in swine.

An antigen-capture enzyme-linked immunosorbent assay (ELISA) was developed to monitor virus shedding associated with experimental infection with a field strain of swine influenza in pigs. The assay consisted of a monoclonal anti-nucleoprotein capture antibody and a biotinylated rabbit anti-influenza A (H1N1) sandwich antibody. The antigen-capture system was capable of detecting as little as 1 ng/ml purified virus. The ELISA system surpassed egg cultivation procedures in the detection of low levels of shedding virus. Egg cultivation procedures indicated that most viral shedding had ceased by day 10 postinfection. In contrast, antigen-capture ELISA still showed an ongoing presence of viral antigen. A virus-capture ELISA, using this capture-sandwich antibody system, is equivalent in sensitivity to conventional egg inoculation procedures for the detection of the early phases of virus shedding. The automative potential of an ELISA-based system coupled with a substantially reduced assay time requirement give this virus-capture ELISA a distinct advantage over other cell culture or egg-based diagnostic techniques.

Effects of intrauterine challenge with Leptospira interrogans serovar hardjo on fertility in cattle.

The purpose of this study was to determine the effects of Leptospira interrogans serovar hardio on fertility in cattle. Twenty seronegative mature dairy cows were assigned to two groups. Group I (challenged cows) was bred by a seronegative bull followed by intrauterine infusion (within 30 minutes) of Leptospira interrogans serovar hardjo. Group II was bred by the same bull followed by intrauterine infusion of 5 ml of sterile culture medium. Blood samples were collected at two-day intervals to monitor serum antibody titers. Daily blood cultures for 10 days and weekly urine cultures for five weeks were performed to monitor the animals for leptospiremia and leptospiuria. Cows were slaughtered 35 days post-breeding, and their reproductive tracts were examined. All animals remained clinically normal following intrauterine challenge. There was no difference in pregnancy rates (Group I, 7/10; Group II, 6/10). All embryos, reproductive tracts, and kidneys appeared normal. A microscopic agglutination test (MA) showed that 4 of 10 challenged cows developed serum antibody titers between 8 and 20 days after challenge. However, on the basis of the hamster passive protection test, all challenged cows had serum antibodies present. All blood and urine cultures were negative through the experimental period, as were the final kidney and uterine cultures. In a second experiment, six seronegative cows were infused with killed microorganisms immediately after insemination. Results of a microscopic agglutination test and a hamster passive protection test indicated that these cows did not develop humoral antibodies against serovar hardjo. These results indicated that intrauterine inoculation of Leptospira interrogans serovar hardjo (hamster-adapted strain) following breeding did not affect pregnancy rates despite an intrauterine challenge which caused the development of humoral antibodies.

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.