Cross-neutralization of the coagulant activity of Crotalus durissus terrificus venom from the northeast of Argentina by bivalent bothropic antivenom

Cross-neutralization of Crotalus durissus terrificus venom coagulant activity was tested using bivalent horse antivenom against Bothrops alternatus and Bothrops diporus venoms. Our in vitro and in vivo experiments showed that bothropic antivenom neutralizes the thrombin-like activity of crotalic snake venom and this cross-reaction was demonstrated by immunoassays either with whole venom or a purified thrombin-like enzyme. These results suggest common antigenic properties and, consequently, similar molecular structure among venom thrombin-like enzymes. Besides, they provide information that could be further used in the development of new antivenom formulations.

Systemic alterations induced by a Bothrops alternatus hemorrhagic metalloproteinase (baltergin) in mice.

Systemic alterations induced by a Bothrops alternatus hemorrhagin, named baltergin, a 55kDa fibrinogenolytic metalloproteinase isolated from venom of north-eastern Argentina specimens, were studied in mice. It caused macroscopic hemorrhagic spots in lungs which was injected intravenously with a minimum pulmonary hemorrhagic dose of 10microg. Histological observations of lungs showed mainly hemorrhagic areas, evidenced by the presence of erythrocytes in the alveolar spaces, congestion and increase of thickness of alveolar septum due to polymorphonuclear infiltrate and mononuclear cells. Neither macroscopic hemorrhage in other organs nor histological alterations in heart and cerebrum/cerebellum were observed at doses assayed. However, kidney and liver were mildly affected. Kidney examination revealed congestion, subcapsular hemorrhage with local capsule detachment, inflammatory infiltrate and degeneration of tubular cells. Congestion of blood vessels and hydropic degeneration of hepatocytes were observed in liver. Besides, baltergin was able to further hydrolyze type IV collagen. Although the enzyme showed to be less lethal than whole venom, it induced severe pulmonary bleeding and affected kinder and liver in minor grade. In conclusion, baltergin is able to alter the integrity of capillary vessels and simultaneously, to interfere on the hemostatic system. Thus, this metalloproteinase contribute markedly to systemic alterations characteristic of B. alternatus envenomations.

Proteolytic, edematogenic and myotoxic activities of a hemorrhagic metalloproteinase isolated from Bothrops alternatus venom.

A hemorrhagic metalloproteinase has been isolated from Bothrops alternatus venom from specimens that inhabit the north-east region of Argentina. The present study aimed at evaluating the proteolytic, hemorrhagic, edematogenic and myotoxic activities of the purified metalloproteinase, in order to consider its participation on the phatophysiology of the intoxication by Bothrops alternatus venom. The hemorrhagic metalloproteinase was isolated by a combination of DEAE-Cellulose chromatography and gel filtration on Sephadex G-75. The enzyme showed a molecular mass around 55k Da, it exhibited a hemorrhagic activity with a minimal hemorrhagic dose of 1.9 microg, almost two fold minor than the whole venom (3.6 microg). The enzyme showed a weak proteolytic activity on casein (18.72 U/mg enzyme), similar to the one exhibited by the whole venom (20 U/mg venom). Besides, the ability to degrade casein could be detected by SDS-PAGE; beta-casein was the fraction that showed the higher degradation, followed by alphas(1)-casein and kappa-casein degradation. The hemorrhagic metalloproteinase rapidly hydrolysed the A alpha-chain of fibrinogen, followed by B beta-chain degradation and leaving the gamma-chain unaffected. Proteolytic activities were inhibited by EDTA whereas they were not inhibited by benzamidine and PMSF. The metalloproteinase showed several polypeptides chains after autocatalytic processing, including a chain of 28k Da, it could be the processed disintegrin-like and cysteine-rich domains. The isolated enzyme exhibited myotoxic activity with high CK levels at 6h, due to local ischemia resulting of its hemorrhagic activity, and a significant edema-inducing effect (MED=1.3 microg), corroborated both results by the histological observations of samples of gastrocnemius muscle. These findings showed that this hemorrhagic metalloproteinase, possesses high edematogenic and myotoxic activities and, in despite of exhibiting a weak proteolytic activity, it is able to degrade fibrinogen. So, this enzyme would contribute markedly to the phatophysiology of the bothropic envenomation.

Effects of biotin supplementation on performance and claw lesions on a commercial dairy farm.

A controlled 14-mo field trial was conducted to evaluate the effect of biotin supplementation on hoof lesions, milk production, and reproductive performance in a commercial dairy herd. One hundred seventy cows were studied and supplemented with either 0 or 20 mg/d of biotin by computer feeder. All were housed in the same free-stall facility with the same environment, base diet, and management. The feet of 99 cows were trimmed three times at 6-mo intervals, and hoof health was evaluated. Milk production and fertility data were captured monthly by the Dairy Herd Improvement Association. At the final hoof trimming, sole hemorrhages were significantly higher in control (50%) vs. biotin-supplemented animals (24%). The incidents of cows affected with double soles, hoof wall grooves, and heel horn erosion did not differ between control and biotin-supplemented animals. Biotin supplementation of trimmed cows resulted in 878 kg more milk than control cows when compared with previous lactation yield (n = 46 biotin supplemented, n = 48 control cows). At the end of the study, for both trimmed and untrimmed animals, biotin supplemented cows (n = 81) produced 481 kg more milk and 25 kg more fat than the controls (n = 81). There was no interaction between biotin supplementation and hoof trimming on milk production. There were variations in the response of fertility to biotin between age groups. First lactation heifers fed supplemental biotin had significantly fewer days from calving to conception and required fewer inseminations per pregnancy than controls of the same parity.

Multiresistant Salmonella Typhimurium DT104 infections of humans and domestic animals in the Pacific Northwest of the United States.

Salmonella Typhimurium definitive type 104 with chromosomally encoded resistance to five or more antimicrobial drugs (R-type ACSSuT+) has been reported increasingly frequently as the cause of human and animal salmonellosis since 1990. Among animal isolates from the northwestern United States (NWUS), R-type ACSSuT+ Typhimurium isolates increased through the early 1990s to comprise 73% of Typhimurium isolates by 1995, but subsequently decreased to comprise only 30% of isolates during 1998. NWUS S. Typhimurium R-type ACSSuT+ were consistently (99%) phage typed as DT104 or the closely related DTu302. S. Typhimurium isolates from cattle with primary salmonellosis, randomly selected from a national repository, from NWUS were more likely to exhibit R-type ACSSuT+ (19/24, 79%) compared to isolates from other quadrants (17/71, 24%; P < 0.01). Human patients infected with R-type ACSSuT+ resided in postal zip code polygons of above average cattle farm density (P < 0.05), while patients infected with other R-types showed no similar tendency. Furthermore, humans infected with R-type ACSSuT+ Typhimurium were more likely to report direct contact with livestock (P < 0.01) than humans infected with other R-types.

Fecal Escherichia coli O157:H7 shedding patterns of orally inoculated calves.

To assess the duration of fecal shedding upon initial infection, the duration of shedding after subsequent re-infection and the effects of dietary restriction and antibiotic treatment on shedding recrudescence, four, one-week-old calves were orally inoculated on three separate occasions with 5x10(8) cfu of Escherichia coli O157:H7 strain 86-24 Nal-R. Fecal shedding was followed by serial culture three times weekly. Following the first inoculation, the calves shed E. coli O157:H7 in their feces for a mean of 30 days, with a range of 20 to 43 days. Following the second and third inoculations, the calves shed E. coli O157:H7 in their feces for 3-8 days. In each of the three inoculations, feed was withheld from the calves for 24 h after they had become fecal culture negative. Two calves resumed shedding, one for 1 day and the other for 4 days, after food was withheld after the third inoculation, but not in the first two inoculations. In the third inoculation, one calf resumed shedding for one day after treatment with oxytetracycline. No E. coli O157:H7 strain 86-24 Nal-R was found in the calves at necropsy. These calves did not exhibit persistent low-level shedding, and did not appear to be persistently colonized with E. coli O157:H7.

Regulation of the immunoglobulin G1 receptor: effect of prolactin on in vivo expression of the bovine mammary immunoglobulin G1 receptor.

Induction of colostrogenesis in non-pregnant cows was used to evaluate the relationship between prolactin (PRL) and mammary immunoglobulin G1 (IgG1) receptor expression. Six of eleven non-pregnant, non-lactating Holstein cattle responded to a standard lactation induction protocol by development of elevated IgG1 concentrations in mammary secretions. In order to increase the diversity in PRL concentrations, two of the six cattle were treated with bromocriptine, and two others were treated with recombinant bovine PRL. Serum alpha-lactalbumin, serum PRL and mammary secretion IgG1 concentrations were measured throughout the experiment. Biopsies of mammary tissue were collected after induction of lactation, and after treatments to alter serum PRL. Immunohistochemistry was used to evaluate IgG1 receptor expression. Administration of recombinant bovine (rbPRL) was associated with increased lactogenic activity, decreased secretion IgG1 concentrations, and decreased IgG1 receptor expression. Decreased serum PRL, due to bromocriptine, was associated with decreased lactogenic activity and maintenance of IgG1 receptor expression. Results of this experiment are consistent with an effect of PRL in decreasing the expression of the bovine mammary IgG1 receptor at the onset of lactogenesis.

Partitioning the mortality risk associated with inadequate passive transfer of colostral immunoglobulins in dairy calves.

This study developed a method to partition the risk of mortality in dairy calves in the 1st 16 weeks of life. Observed population mortality and the relative risk of mortality in each serum protein concentration stratum were used to determine the population baseline mortality rate and the mortality rate due to inadequate passive transfer of colostral immunoglobulin. A total of 3,479 calves were studied, 8.2% of which died before 16 weeks of age. The population baseline mortality rate was 5.0% and the mortality rate due to inadequate passive transfer was 3.2%. Thirty-nine percent of the observed mortality was attributed to inadequate passive transfer. This partitioning of risk between passive transfer-related and unrelated sources should prove useful in conducting investigations of calf mortality problems in dairy herds.

Use of serum protein concentration to predict mortality in mixed-source dairy replacement heifers.

The relationship between serum protein concentration in the 1st week of life and survival to 16 weeks of age was examined in 3,479 Holstein replacement heifers over a period of 10 years on a farm with endemic salmonellosis. Thirty-four percent of calves studied had serum protein concentrations < 5.0 g/dL and 60.5% of calves had serum protein concentrations < 5.5 g/dL. Cumulative mortality was 7.9%, indicating that calves with marginal passive transfer status can be reared successfully under conditions of endemic salmonellosis. Optimal survival was observed in calves with serum protein concentrations > 5.5 g/dL. Calves with serum protein concentrations of 5.0-5.4 g/dL had only a slightly increased relative risk (RR) of mortality (RR = 1.3) compared to calves with serum protein concentrations > 5.5 g/dL. The highest RR was experienced by calves with serum protein concentrations < 4 g/dL (RR = 4.6) and 4.0-4.4 g/dL (RR = 3.1). Calves with inadequate passive transfer (serum protein concentration < 5.0 g/dL), experienced increased mortality until at least 10 weeks of age, indicating that failure of passive transfer has an effect on calf health that extends into the juvenile period. Models in which serum protein concentration was treated with as a continuous variable or as a categorical variable failed to demonstrate any significant interaction between baseline mortality and the RR of mortality. This finding suggests that the RR derived in the present study should be applicable to farms with dramatically different baseline mortality rates.

Expression of immunoglobulin G1 receptors by bovine mammary epithelial cells and mammary leukocytes.

The objective of this study was to identify and evaluate expression of IgG1 receptors by different cell types in mammary tissue sections and digest-dispersed cells from the bovine mammary gland. An immunohistochemical system utilizing avidin-biotin-peroxidase complex demonstrated epithelial expression of IgG1 receptors in mammary tissue sections from cows producing colostrum but not from cows in lactation. Fluorescence flow cytometry demonstrated that cells dispersed in digests from both tissues producing colostrum and lactating tissues selectively bound IgG1. Fluorescence flow cytometry, using monoclonal antibodies to cell surface molecules, cytokeratin, and IgG1, revealed that leukocytes constituted the largest percentage of cells and were the predominant cell type binding IgG1 in mammary tissue digests. Although IgG1 binding to epithelial cells predominated in the gland during colostrum production in situ, digestion and filtration to produce single cell suspensions resulted in the loss of large numbers of epithelial cells. Studies of Ig binding of cells produced by enzymatic digestion must account for the types of cells surviving the digestion process.

Effect of prolactin on in vitro expression of the bovine mammary immunoglobulin G1 receptor.

Explants of mammary tissue from cows in late pregnancy were incubated for 72 h in serum-free, hormonally defined media to investigate the regulation of the bovine mammary IgG1 receptor. Treatments included incubation in basal medium alone, basal medium plus estradiol-17 beta, basal medium plus prolactin, or basal medium plus estradiol-17 beta and prolactin. alpha-Lactalbumin production was measured by radioimmunoassay in culture supernatants collected at 24, 48, and 72 h. Explants were examined immunohistochemically for expression of the IgG1 receptor at 24, 48, and 72 h. alpha-Lactalbumin concentrations increased, and IgG1 receptor expression decreased, by 72 h with explants cultured in the medium containing prolactin. Results suggest that, in addition to its positive lactogenic effect, prolactin decreases expression of the bovine mammary IgG1 receptor.

Prediction of serum IgG1 concentration in Holstein calves using serum gamma glutamyltransferase activity.

We examined the relationship between serum gamma glutamyltransferase (GGT) activity and serum gamma immunoglobulin G (IgG1) concentration in Holstein calves. Blood samples were collected from calves aged 1 to 3 days. A follow-up sample was obtained from each calf 2, 7, or 15 days after the initial sampling. Serum GGT activity and IgG1 concentration were measured. Regression models were used to predict IgG1 concentration as a function of age and serum GGT activity measured 2, 7, or 15 days later. Serum GGT activity and calf age at the time of the second sample were directly related to serum IgG1 concentration in the initial sample in calves aged 3 to 17 days (r = .54) and in calves aged 3 to 10 days (r = .63). Models were used to estimate the serum GGT activity equivalent to a serum IgG1 concentration of 1,000 mg/dL. One-day-old calves should have serum GGT activities > 200 IU/L. Four-day-old calves should have serum GGT activities > 100 IU/L. One-week-old calves should have serum GGT activities > 75 IU/L. Calves with serum GGT activities < 50 IU/L should be classified as having failure of passive transfer.

Sensitivity of bacteriologic culture for detection of Escherichia coli O157:H7 in bovine feces.

The sensitivities of several plating and broth enrichment methods for the detection of Escherichia coli O157:H7 in (i) bovine fecal samples directly inoculated with E. coli O157:H7, (ii) fecal samples from cattle in herds previously positive for E. coli O157:H7, and (iii) fecal samples from calves shedding E. coli O157:H7 after experimental oral inoculation were compared. Three enrichment protocols and three plating protocols were evaluated with directly inoculated fecal samples. All broth enrichment methods were superior to direct plating when they were combined with subsequent plating on sorbitol-MacConkey with cefixime and tellurite (SMACct). SMACct was the most sensitive plating medium, and the three alternative broth enrichment methods gave similar improvements in sensitivity. Of 351 fecal samples from known positive herds, 24 samples (6.8%) were positive by one or more methods. By the most sensitive plating method, cultures of 10-g samples were slightly more sensitive (19 of 351 [5.4%]) than cotton-tipped swab fecal samples (14 of 351 [4.0%]); however, this difference was not significant. For samples from calves orally inoculated with E. coli O157:H7, separation by immunomagnetic beads was slightly more sensitive (79%) than broth enrichment followed by plating at two dilutions (10(-3) and 10(-4)) (71%); however, this difference was not significant. The combination of overnight enrichment of swab fecal samples (0.1 g) and plating on SMACct at two dilutions (10(-3) and 10(-4)) appears to be a sensitive method for detection in large-scale studies involving hundreds of samples per week.

Survey of dairy managers in the Pacific Northwest identifying factors associated with teat chapping.

Telephone interviews were conducted to evaluate the association of teat chapping with combinations of premilking and postmilking teat disinfectants (predip and postdip), other management practices, and climatic conditions. Holstein dairies (n = 100) in the DHIA SCC program were randomly selected from three northwest regions. During nonwinter seasons, herd managers using an iodophor dip without skin conditioner both premilking and postmilking were 9.7 times more likely to report chapping than were those using iodophor with conditioner. The herd managers least likely to report chapping problems were those using conditioner in both the predip and postdip. Use of conditioner in postdip only was associated with intermediate frequency of reported chapping. No conditioner in either predip or postdip was associated with the greatest frequency of reported chapping. Herd managers using a different germicide in the predip and postdip reported more chapping than herd managers using the same germicide. The frequency of chapping was highest during winter in all regions, and the percentage of herd managers reporting chapping within a region was associated with the temperature extremes of the region. For seasons other than winter, characteristics of predip and postdip combinations and parlor exit or barn design were the management factors that best described an association with reported chapping.

Immunoglobulin G1 transfer into induced mammary secretions: the effect of dexamethasone.

The purpose of this study was to correlate the time of administration of the synthetic glucocorticoid dexamethasone with IgG1 concentrations in hormonally induced mammary secretions of nonpregnant cattle. Nonpregnant, nonlactating Holsteins were randomly assigned to three experimental groups of 8 cows each, and all were given .05 mg/kg of estrogen and .125 mg/kg of progesterone by subcutaneous injection every 12 h for 7 d. In addition, cows in groups 1 and 2 received dexamethasone (20 mg/d) on d 8 to 10 and on d 11 to 13, respectively. Group 3 cows were not given dexamethasone. The IgG1 concentrations in the mammary secretions of all three treatment groups rose steadily during d 1 to 7. The IgG1 concentrations in mammary secretions of groups 1 and 2 decreased sharply within 3 d of the first dexamethasone injection. The IgG1 concentrations in mammary secretions of group 3 also tended to peak and then decrease, but the decreases were generally later than those of cows receiving dexamethasone. The results indicate that the IgG1 transfer that occurs during induction of lactation is affected by glucocorticoid administration in a pattern similar to that reported during colostrum formation in pregnant cows.

Evaluation of the hydrometer for testing immunoglobulin G1 concentrations in Holstein colostrum.

Hydrometer measurement in globulin and IgG1 concentration measured by the radial immunodiffusion technique were compared for 915 samples of first milking colostrum from Holstein cows. Least squares analysis of the relationship between hydrometer measurement and IgG1 concentration was improved by log transformation of IgG1 concentration and resulted in a significant linear relationship between hydrometer measurement and log10 IgG1 concentration; r2 = .469. At 50 mg of globulin/ml of colostrum, the recommended hydrometer cutoff point for colostrum selection, the sensitivity of the hydrometer as a test of IgG1 concentration in Holstein colostrum was 26%, and the negative predictive value was 67%. The negative predictive value and sensitivity of the hydrometer as a test of IgG1 in Holstein colostrum was improved, and the cost of misclassification of colostrum was minimized, when the cutoff point for colostrum selection was increased above the recommended 50 mg/ml.

Coagulase-positive Staphylococcus intramammary infections in primiparous dairy cows.

Objectives were to determine the prevalence of coagulase-positive staphylococcal IMI in primiparous cows at first parturition, to contrast the differences in coagulase-positive staphylococcal IMI in primiparous cows at parturition in herds with high and low prevalences of coagulase-positive staphylococcal IMI in the lactating herd, and to determine the percentage of primiparous cows having persistent coagulase-positive staphylococcal IMI. Milk samples were collected aseptically from cows at the start and end of the study, at dry-off, and at parturition. Herds (n = 18) were split evenly into two categories: high (> 10%) or low (< 5%) prevalence of coagulase-positive staphylococcal IMI. At the start, the mean prevalence of coagulase-positive staphylococcal IMI in high prevalence herds was 30%, ranging from 13 to 65%, and in low prevalence herds was 2%, ranging from 0 to 5%. Overall the prevalence of coagulase-positive staphylococcal IMI in primiparous cows at parturition was 8.1% (67 of 828), ranging from 0 to 27%. Although primiparous cows from high prevalence herds had a higher prevalence of coagulase-positive staphylococcal IMI (9.2%; 40 of 436) at parturition than did primiparous cows from low herds (6.9%; 27 of 392), the difference was not significant. Of primiparous cows with coagulase-positive staphylococcal IMI at parturition, 43% had coagulase-positive staphylococcal IMI at least 2 mo after parturition. Primiparous cows with coagulase-positive staphylococcal IMI at parturition may represent significant reservoirs of infection to uninfected herdmates.

The importance of colostrum to the health of the neonatal calf.

Colostrum feeding is the means by which newborn calves acquire passive immunity to infectious agents. The ability of the newborn calf to absorb colostral antibodies is limited to the first few hours of life. In most cases, natural suckling from the dam is the best method for beef-breed calves to obtain optimum passive immune protection, but in some cases intervention and hand feeding are indicated. The details of when and how to intervene in the passive transfer process are discussed.