Partial prediction of the duration and the clinical status of Staphylococcus aureus bovine intramammary infections based on the phenotypic and genotypic analysis of isolates.

Staphylococcus aureus is a bacterial pathogen causing bovine intramammary infections (IMIs) often leading to chronic clinical or subclinical mastitis. Predicting the outcome of S. aureus IMIs (duration and clinical vs subclinical) based on the characterization of isolates would help to make better case management decisions. For this purpose, 583 S. aureus isolates from series of quarter milk samples were characterized by genotypic tests (detection of virulence genes seg, tst, lukM), epidemiological typing (spa type) and by a phenotypic test (biofilm production). VNTR typing (variable number of tandem repeats) was used to establish persistence of the same S. aureus strain in each series of sequential isolates. This allowed to associate each strain to a clinical/subclinical status and to validate the duration of infection. We found differences in the distribution of spa types between the strains from clinical and subclinical cases. Prevalence of lukM was also higher in strains from clinical cases than in strains from subclinical cases. A Kaplan-Meier analysis was then used to determine factors influencing the duration of the infection. Considering a multivariable model of the logistic regression, time to elimination was shorter with the strains of the subclinical lactation series compared to the clinical series (series with at least one clinical case). Strains from the spa type t359 and t529 were less likely to persist compared to those of spa type t13410. In sum, strain characterization including determination of the spa type helps to predict duration of infection and the clinical or subclinical outcome of S. aureus IMIs.

Innovative dairy cow management to improve resistance to metabolic and infectious diseases during the transition period.

The incidence of metabolic and infectious diseases varies greatly during the lactation cycle. Most new cases of clinical mastitis appear at the beginning of lactation, and the incidence increases with the level of milk production. In addition to mastitis, many other infectious diseases become clinically apparent during the first 2weeks of lactation. During this time, cows are in a negative energy balance and must mobilize body reserves to balance the deficit between food energy intake and energy required for milk production. The relationships between energy deficit and metabolic diseases, such as ketosis and hepatic lipidosis, are well known. Furthermore, cows in energy deficit have a weakened immune system and are therefore more susceptible to infections. There is now good evidence that the increase in circulating non-esterified fatty acids impairs immune cell functions. Therefore, management approaches that reduce the negative energy balance and the increase in non-esterified fatty acids at the beginning of lactation are likely to improve resistance to infection. Improving the nutrient supply through periparturient nutritional management has been the subject of considerable research. However, another way to reduce the imbalance between nutrient supply and demand is to temporarily decrease the latter. In this review, we examine how management strategies such as conjugated linoleic acid feeding, prepartum milking, or limiting postpartum milk production could be used to reduce metabolic perturbations and immunosuppression during the transition period. At this stage, it appears that reducing the amount of milk harvested postpartum by means of partial milking in the first days after calving is the most promising approach to reduce metabolic stress and immunosuppression without compromising the productivity of high-yielding dairy cows.

Genotypic and phenotypic characterization of Staphylococcus aureus causing persistent and nonpersistent subclinical bovine intramammary infections during lactation or the dry period.

Staphylococcus aureus is a significant pathogen frequently causing persistent intramammary infections (IMI) in dairy cows. We compared some genotypic and phenotypic characteristics of 285 strains collected from quarter milk samples from cows with persistent and nonpersistent subclinical IMI across Canada. Variable number of tandem repeats typing was used to infer the persistence of the same S. aureus strain in 3 consecutive quarter milk samples collected at intervals of 3 wk during lactation or before and after dry-off. All first isolates of the series were used as the representative strains from persistent IMI and were compared with nonpersistent strains for the presence of genes seg, sen, sec, and tst as well as by spa typing. Biofilm production in vitro and hld-RNAIII expression levels were also quantified. The gene seg was associated with a reduction in the likelihood of the bacteria to cause a persistent IMI during lactation. Strains persisting through the dry period produced significantly more biofilm in vitro than strains that do not persist after calving. Also, we showed that strains expressing more hld were more likely to be nonpersistent during either lactation or through the dry period. Three spa types were predominant (t529, t267, and a novel type: t13401). In the strains studied, the spa type tbl 2645 was the most frequent, and 97.0% of the strains of this spa type carried both sen and seg. Strains from the spa type tbl 2645 were less likely to cause a persistent IMI in the dry period. Most (86.7%) of the strains of the novel spa type (t13401) were negative for seg, sen, or both and produced significantly more biofilm in vitro than tbl 2645 and t267. The present study expanded our current knowledge on the genotypic and phenotypic traits of S. aureus strains recovered from persistent and nonpersistent IMI in Canada.

Experimental treatment of Staphylococcus aureus bovine intramammary infection using a guanine riboswitch ligand analog.

Staphylococcus aureus is a leading cause of intramammary infections (IMI). We recently demonstrated that Staph. aureus strains express the gene guaA during bovine IMI. This gene codes for a guanosine monophosphate synthetase and its expression is regulated by a guanine riboswitch. The guanine analog 2,5,6-triaminopyrimidine-4-one (PC1) is a ligand of the guanine riboswitch. Interactions between PC1 and its target result in inhibition of guanosine monophosphate synthesis and subsequent death of the bacterium. The present study describes the investigational use of PC1 for therapy of Staph. aureus IMI in lactating cows. The in vitro minimal inhibitory concentration of PC1 ranged from 0.5 to 4 µg/mL for a variety of Staph. aureus and Staphylococcus epidermidis strains and required a reducing agent for stability and full potency. A safety assessment study was performed, whereby the healthy quarters of 4 cows were infused with increasing doses of PC1 (0, 150, 250, and 500 mg). Over the 44 h following infusions, no obvious adverse effect was observed. Ten Holstein multiparous cows in mid lactation were then experimentally infused into 3 of the quarters with approximately 50 cfu of Staph. aureus strain SHY97-3906 and infection was allowed to progress for 2 wk before starting PC1 treatment. Bacterial counts reached then about 10(3) to 10(4) cfu/mL of milk. Infected quarters were treated with 1 of 3 doses of PC1 (0, 250, or 500 mg) after each morning and evening milking for 7d (i.e., 14 intramammary infusions of PC1). During the treatment period, milk from PC1-treated quarters showed a significant reduction in bacterial concentrations. However, this reduction of Staph. aureus count in milk was not maintained during the 4 wk following the end of the treatment and only 15% of the PC1-treated quarters underwent bacteriological cure. The somatic cell count and the quarter milk production were not affected by treatments. Although bacterial clearance was not achieved following treatment with PC1, these results demonstrate that the Staph. aureus guanine riboswitch represents a relevant and promising drug target for a novel class of antibiotics for the animal food industry.

Effect of postcalving serum nonesterified fatty acids concentration on the functionality of bovine immune cells.

The periparturient period is marked by metabolic, hormonal, and immunological changes, which have an effect on the incidence of infectious and metabolic diseases. In a previous study, a slower increase in milk production was induced by milking cows once daily during the first week of lactation, leading to an improvement in levels of several metabolites, including nonesterified fatty acids (NEFA) and ß-hydroxybutyrate (BHBA). The aim was to determine the influence of serum collected on d 2, 5, and 61 postpartum from cows milked once or twice daily on immune cell functions and to determine which of the constituents were responsible for these effects. Peripheral blood mononuclear cells (PBMC) and polymorphonuclear leukocytes were collected from healthy midlactation cows and their immune functions (i.e., proliferation and interferon-γ production and chemotaxis, phagocytosis, and oxidative burst, respectively), were evaluated in presence of serum, NEFA, and BHBA. Proliferation of PBMC was greater with d-61 (65.1±1.6%) serum than with d-2 (37.3±2.4%) or d-5 (48.4±1.6%) serum and greater with d-2 and -5 serum from cows milked once (42.2±3.7 and 54.0±2.5) compared with cows milked twice daily (32.4±3.0 and 42.9±2.1). Proliferation was inversely correlated with the concentration of NEFA and BHBA in the serum (r=-0.86). Adding NEFA to d-61 serum to reach the level present in d-5 serum decreased proliferation to the level observed with d-5 serum. No effect of BHBA addition was observed. The release of interferon-γ by PBMC was lower in d-5 serum (766±63 pg/mL) than in d-61 serum (1,187±90 pg/mL) and by NEFA. Milking frequency did not affect chemotaxis, phagocytosis, or oxidative burst of polymorphonuclear leukocytes. Phagocytosis decreased over time in serum from d 2 to 61. Similarly, oxidative burst was greater with d-5 serum (12.7×10(8) ± 1.6×10(8) relative light units) than with d-61 serum (9.0×10(8) ± 1.6×10(8) relative light units). The NEFA had a negative effect on oxidative burst, but BHBA did not. In conclusion, several immune cell functions appear affected by the NEFA concentration. Therefore, strategies that prevent increases in blood NEFA during the transition period may limit postpartum immunosuppression.

Shelf life of pork from five different quality classes.

A total of 117 loins were selected on the cutting line at 24h post-mortem to study the long term shelf life (35 days, 4 degrees C) of vacuum packaged pork from five different quality classes (PSE: pale, soft, exudative; PFN: pale, firm, non-exudative; RSE: red, soft, exudative; RFN: red, firm, non-exudative; and DFD: dark, firm, dry). The microbial load at 0 d was not significantly different (P>0.05) among the pork quality classes, indicating that the initial microflora was influenced by the dressing conditions at the plant, not by the meat quality class. But after 35 d of storage, total aerobic mesophilic and presumptive lactic acid bacteria counts were higher (P<0.05) in DFD pork due to its higher ultimate pH. RSE was the second quality class most susceptible to spoilage, whereas PFN, RFN and PSE pork had similar microbial loads. Further research is needed to elucidate the causes of the shorter shelf life in RSE pork.

Impact of postpartum milking frequency on the immune system and the blood metabolite concentration of dairy cows.

The transition from pregnancy to lactation is marked by metabolic, hormonal, and immunological changes that have an impact on the incidence of infectious and metabolic diseases. The aim of this study was to evaluate the effect on immune function and blood metabolite concentration of limiting milk production in early lactation to reduce negative energy balance. Twenty-two multiparous Holstein cows were milked either once a day (1x) or twice a day (2x) for the first week postpartum. All cows were milked twice daily for the rest of lactation. Blood concentrations of nonesterified fatty acids (NEFA), beta-hydroxybutyric acid (BHBA), calcium, bilirubin, urea, phosphorus, glucose, leptin, stanniocalcin-1, and 17beta-estradiol were determined in samples collected from 5 wk before scheduled calving to 5 wk after calving. Polymorphonuclear leukocytes (PMNL) were isolated from blood to conduct assays for chemotaxis, phagocytosis, and respiratory burst. Peripheral blood mononuclear cells (PBMC) were isolated to evaluate lymphocyte proliferation and cytokine production (tumor necrosis factor-alpha, IL-4, and interferon-gamma). Cows milked 1x produced 31% less milk than cows milked 2x during the first week of lactation. Over the following 13 wk of lactation, the milk production of cows milked 1x during the first week was 8.1% lower than for cows milked 2x. However, because the percentages of fat and protein were greater in the milk from 1x cows, the yields of milk components and energy-corrected milk were similar. Calving induced an increase in the concentrations of NEFA, BHBA, urea, and bilirubin. The increases in levels of NEFA and BHBA were greater in cows milked 2x than in cows milked 1x. During the same period, the serum glucose concentration decreased but remained greater in cows milked 1x. Serum calcium on d 4 and serum phosphorus on d 4 and 5 were greater in cows milked 1x. The differences between the 2 groups persisted beyond treatment until postpartum d 24 for NEFA and glucose and until postpartum d 14 for BHBA. After calving, the concentrations of leptin and stanniocalcin-1 decreased. During the first week postpartum, the decrease of leptin was less marked in cows milked 1x. The immune functions of PBMC and PMNL isolated from experimental cows and incubated using a standard medium did not show clear-cut peripartum immunosuppression. These variables were not significantly affected by the treatments, with the exception of interferon-gamma secretion, which was greater on d 5 and 14 in cows milked 1x. In conclusion, limiting milk production in early lactation had positive effects on metabolite concentration, but larger studies are necessary to establish if this could reduce disease incidence.

Serum levels of stanniocalcin-1 in Holstein heifers and cows.

Stanniocalcin-1 is a hormone that possesses both paracrine and endocrine functions and has recently been identified in mammals. While paracrine functions have been determined for several organs, the role of circulating stanniocalcin-1 in cattle is still unclear but, observations in mice and cows suggest that stanniocalcin-1 plays a role in both gestation and lactation. The changes in serum stanniocalcin-1 content in different physiological states have never been evaluated in ruminants. We measured stanniocalcin-1 levels in sera from cattle ranging in age from post-weaned calves to 17-month-old heifers and in sera from cows during lactation and pregnancy. Our results indicate that the blood concentration of stanniocalcin-1 is increased by pregnancy, but not by lactation. The highest levels of stanniocalcin-1 were found in young calves and during the non-lactating period preceding calving. This suggests that stanniocalcin-1 is important for gestation and the preparation of the mammary gland for lactation.

Efficacy of a lactoferrin-penicillin combination to treat {beta}-lactam-resistant Staphylococcus aureus mastitis.

The efficacy of intramammary (IM) treatments containing penicillin G (PG) alone or a combination of PG and bovine lactoferrin (bLF) was evaluated using a model of experimentally induced chronic bovine mastitis caused by a clinical isolate of Staphylococcus aureus highly resistant to beta-lactam antibiotics. First, we confirmed that this strain could cause mastitis and infection could not be cured with PG alone. In a second trial, chronic mastitis was induced in 19 late-lactating cows by injecting a low dose of Staph. aureus through the teat canal of all quarters. After 15 d, cows with stable infections in their 4 quarters had their mammary quarters randomly assigned, within cow, to 1 of 4 IM treatments as follows: 1) citrate buffer, 2) 100,000 IU of PG, (3) 1 g of bLF, or 4) 1 g of bLF + 100,000 IU of PG. Treatments were repeated twice a day for 5 d. A third trial was undertaken to investigate the effect of an extended therapy on chronic mastitis acquired in a previous lactation. One month before dry-off, 20 gravid cows regrouped by dates of calving were infected in their 4 quarters. Once infections were established, cows were dried off abruptly. After calving, aseptic milk samples were collected separately from all quarters for 4 wk to monitor infection. Mammary quarters from enrolled cows were then randomly assigned, within cow, to 1 of 2 treatments as follows: 1) 100,000 IU of PG or 2) 250 mg of bLF + 100,000 IU of PG. Treatments were administered IM twice a day for 7 d. For all trials, milk samples were taken to monitor bacterial concentration and somatic cell count. Bacteriological cure rate was determined using milk samples taken 3 and 4 wk after initiation of treatments. For the second trial, cure rate was null for control quarters, 11.1% for bLF, 9.1% for PG, and 45.5% for the bLF + PG combination. For cows infected in their previous lactation, cure rate was higher for the bLF + PG combination (33.3%) compared with PG alone (12.5%). In conclusion, bLF added to PG is an effective combination (i.e., 3- to 5-times higher cure rate) for the treatment of stable Staph. aureu infections highly resistant to beta-lactam antibiotics.