Differential somatic cell count in milk before, during, and after lipopolysaccharide- and lipoteichoic-acid-induced mastitis in dairy cows.

Intramammary infections induce the initiation of the inflammatory response, resulting in an increase in somatic cell count (SCC) in milk. The SCC includes several different types of cells but does not differentiate between them. On the contrary, the new differential somatic cell count (DSCC) parameter allows for the differentiation between 2 groups of cells: polymorphonuclear neutrophils (PMN) and lymphocytes versus macrophages. Therefore, the aim of this paper was to describe the changes of both DSCC and SCC during mastitis induced by cell wall components from typical mastitis-causing pathogens [lipopolysaccharide (LPS), Escherichia coli; lipoteichoic acid (LTA), Staphylococcus aureus] known to trigger different severities of mastitis. In addition, the effect the glucocorticoid prednisolone (PRED), which is known to attenuate the immune response in the mammary gland, was investigated. Twenty dairy cows were equally divided into 5 groups and treated with LPS, LTA, LPS+PRED, LTA+PRED, or a saline control. Milk samples were taken at the following time points: baseline (d -3, -2, and -1), right before treatment (d 0), 5 h after treatment (d 0.2), early cure phase (d 1 and 2), and late cure phase (d 3, 4, 5, 6, 7, and 14) and analyzed for DSCC and SCC. Mean DSCC values increased significantly from <60% at baseline and right before treatment to >81% 5 h after treatment and the early cure phase in all groups, except for the groups control and LTA+PRED. This increase clearly reflects a shift in cell populations to predominantly PMN. The SCC increased significantly following the stimulation, too, as expected. Interestingly, we observed cases where SCC increased moderately only whereas DSCC showed an evident increase, meaning that the shift in cell populations occurred even at low SCC levels. The PRED clearly lowered the cell migration in group LTA+PRED. This is the first ever study investigating DSCC during induced mastitis under controlled conditions. The combination of DSCC and SCC could be employed for the earlier detection of mastitis by revealing the shift in cell population independent from the SCC level. Furthermore, combining DSCC and SCC information could help to determine the stage of mastitis because we observed high DSCC and SCC results in the early stage of mastitis but evidently lower DSCC and high SCC in the cure phase. Hence, our results offer the first fundamental insights on how mastitis monitoring could be improved in the frame of dairy herd improvement programs.

Supraphysiological oxytocin increases the transfer of immunoglobulins and other blood components to milk during lipopolysaccharide- and lipoteichoic acid-induced mastitis in dairy cows.

Bacterial mastitis causes pathogen-dependent changes of the blood-milk barrier, and these changes can influence the differential transfer of blood components to milk. It is well known that gram-negative pathogens such as Escherichia coli can cause a greater activation of the immune system and thus a more comprehensive transfer of blood components including IgG than gram-positive pathogens such as Staphylococcus aureus. Supraphysiological doses of oxytocin (OT) have been shown to increase the permeability of the blood-milk barrier; however, the effect of OT during experimentally induced mastitis has not been investigated. Therefore, the objective of this study was to examine if intravenous administration of OT during lipopolysaccharide (LPS)- or lipoteichoic acid (LTA)-induced mastitis could influence the transfer of blood components to milk. The hypothesis was that OT could induce a greater transfer of blood components during mastitis. Twenty-seven dairy cows were injected via the teat canal with LPS, LTA, or a saline control followed by an intravenous injection of OT 2h following intramammary challenge. Milk samples were collected every half hour and analyzed for somatic cell count (SCC), IgG, lactate dehydrogenase (LDH), and serum albumin (SA). Due to the chosen dosage of LPS and LTA, there was no difference in SCC between quarters challenged with only LPS or LTA. Quarters challenged with LPS and OT had a higher SCC and a greater transfer of IgG, LDH, and SA compared with quarters challenged with only LPS. Quarters challenged with LTA and OT had a greater transfer of IgG, LDH, and SA, whereas the SCC increase did not differ from quarters only treated with LTA. In quarters treated only with OT, SCC, LDH, and SA increased, but no difference was observed in IgG concentration from untreated control quarters. In conclusion, there are pathogen-specific changes in the blood-milk barrier and OT can induce a greater transfer of blood components to milk in both LPS- and LTA-induced mastitis. Oxytocin could have implications for use as a mastitis therapy, as there was an increased transfer of IgG into the milk.

Differential glucocorticoid-induced closure of the blood-milk barrier during lipopolysaccharide- and lipoteichoic acid-induced mastitis in dairy cows.

Bacteria invading the mammary gland can cause pathogen-dependent differences in the permeability of the blood-milk barrier leading to the differential paracellular transfer of blood and milk components. Glucocorticoids such as prednisolone (PRED) are known to increase the integrity of the blood-milk barrier and quickly restore the decreased milk quality associated with mastitis. The objective of this study was to examine the effect of intramammary PRED on the differential permeability of the blood-milk barrier during mastitis induced by lipopolysaccharide (LPS) from Escherichia coli or lipoteichoic acid (LTA) from Staphylococcus aureus. Thirty-one dairy cows, divided into 6 groups, were injected via a teat canal with LPS, LTA, LPS and PRED, LTA and PRED, saline (control), or PRED. Milk and blood samples were collected 0 to 8h after challenge and analyzed for somatic cell count, IgG, serum albumin, and lactate dehydrogenase in milk, or α-lactalbumin in plasma. Somatic cell count was similarly elevated in LPS- and LTA-challenged quarters and was reduced to control quarter levels only in LTA-challenged quarters with PRED administration. Lactate dehydrogenase activity was highly elevated in LPS quarters and only slightly elevated in LTA quarters, but decreased to control quarter levels with PRED administration. For serum albumin and IgG, only LPS quarters showed an elevation in concentration and PRED treatment reduced the concentration to control quarter level. We found no differences in α-lactalbumin concentrations in plasma in PRED-treated cows compared with cows that only received LPS or LTA. In conclusion, the pathogen-specific appearance of blood constituents in milk during mastitis demonstrates a differential activation of the blood-milk barrier that, in turn, can be manipulated by intramammary glucocorticoids. The results show that the administration of PRED during mastitis increases the blood-milk barrier integrity but has implications in reducing the transfer of IgG that specifically occurs during E. coli mastitis. In addition, it can also reduce the number of migrating immune cells dependent on the mastitis-inducing pathogen. Potential effects of PRED on the cure of naturally occurring mastitis have to be taken into consideration.

Blood-derived proteins in milk at start of lactation: Indicators of active or passive transfer.

Colostrum has a different composition compared with milk in established lactation. This difference is in part due to the partially open blood-milk barrier, which, when closed, is designed to prevent the interdiffusion of blood and milk components. In the first days of lactation, α-lactalbumin (α-LA), a milk protein, is typically present in blood and several blood-derived proteins are also present in milk, such as IgG1, IgG2, serum albumin (SA), and lactate dehydrogenase (LDH). With the exception of IgG1, which is known to be transferred by active transcellular transport, the other proteins are thought to pass paracellularly through the temporarily open barrier. Along with an exchange of blood and milk components, somatic cell count (SCC) is typically high in colostrum. The decline of these proteins and SCC can be used as indicators to determine transcellular or paracellular transport. Two hypotheses were tested. The first hypothesis was that the decline curve for a protein or SCC would be the same as IgG1, indicating transcellular transport, or the decline curve would be different than IgG1, indicating paracellular transport. The second hypothesis was that the decline curves of SCC and all proteins that are thought to have paracellular transport would be the same. Ten Holstein cows were milked at 4 h after parturition, the next 5 consecutive milkings, and the afternoon milking on d 5, 8, 10, and 14 of lactation for a total of 10 milking time points, and sequential jugular blood samples were also taken. Blood and milk samples were analyzed for the concentrations of LDH, SA, IgG1, IgG2, and α-LA and milk samples were measured for SCC. Protein concentration and SCC curves were generated from all 10 time points and were evaluated using the tau time constant model to determine the rate of decline of the slope of each protein. When examining the first hypothesis, the concentration of IgG1 declined significantly faster in the milk than the proteins IgG2 and LDH, but declined at the same rate as SA. Immunoglobulin G1 also declined significantly faster than SCC and α-LA in plasma. The second hypothesis showed that IgG2, LDH, and SA in milk were declining at the same rate, but were declining significantly faster than SCC and α-LA in plasma. These results indicate that only active transcellular transport of IgG1 occurred, with a sharp decline at parturition, compared with IgG2, SA, LDH, α-LA, and SCC, which are likely following paracellular transport.

Changes in milk L-lactate, lactate dehydrogenase, serum albumin, and IgG during milk ejection and their association with somatic cell count.

In both conventional and automatic milking systems (AMS), sensitive and reliable mastitis detection is important for profitable milk production. Mastitis detection parameters must be able to detect mastitis when the somatic cell count (SCC) is only slightly elevated. Owing to the pre-milking teat cleaning process in AMS, sampling cannot take place before the occurrence of alveolar milk ejection and importantly, this can affect the ability of parameters to detect mastitis. The aim of the present study was to examine the effect of alveolar milk ejection on L-lactate, lactate dehydrogenase (LDH), serum albumin (SA) and immunoglobulin G (IgG) compared with SCC, a commonly used indicator of mastitis. In this experiment, milk samples were collected every 20 s from one quarter during a 120-s manual teat stimulation in ten cows. Samples were analysed for SCC, L-lactate, LDH, SA and IgG. Quarters were grouped by low (<5.0 log10 cells/ml), mid (5.0-5.7 log10 cells/ml), and high (>5.7 log10 cells/ml) SCC using the sample at t=0 s. Neither L-lactate nor LDH could statistically differentiate between low and mid-SCC quarters, but there were a significant difference in levels between the high-SCC quarters and low and mid-SCC quarters. SA could not differentiate between the low and mid-SCC quarters, but the SA levels for the high SCC quarters remained statistically different compared with low and mid-SCC quarters throughout the experiment. IgG could statistically differentiate between low and mid-SCC, although the high-SCC quarters were not statistically different from the mid-SCC quarters after 60 s. In the high-SCC quarters, a decrease was shown in all parameters during milk ejection, after t=60 s. In conclusion, alveolar milk ejection reduces the effectiveness of detection parameters when compared with SCC. With the exception of IgG, the ability of other tested parameters was not satisfactory to differentiate between quarters with low to mid-SCC levels.

Effect of intramammary administration of prednisolone on the blood-milk barrier during the immune response of the mammary gland to lipopolysaccharide.

OBJECTIVE: To investigate effects of intramammary administration of prednisolone on the immune response of mammary glands in cows. ANIMALS: 5 lactating Red Holsteins. PROCEDURES: Cows received a different intramammary infusion in each mammary gland (10 mg of prednisolone, 100 µg of lipopolysaccharide [LPS], 100 µg of LPS and 10 mg of prednisolone, or saline [0.9% NaCl] solution). Milk samples were collected before (time 0) and 3, 6, 9, 12, 24, and 36 hours after treatment. Somatic cell count (SCC), lactate dehydrogenase (LDH) activity, and concentrations of serum albumin (SA) and tumor necrosis factor (TNF)-α in milk and mRNA expression of TNF-α, interleukin (IL)-8, and IL-1ß in milk somatic cells were analyzed. RESULTS: Saline solution or prednisolone did not change SCC, LDH activity, and SA and TNF-α concentrations in milk and mRNA expression of TNF-α, IL-1ß, and IL-8 in milk somatic cells. The SCC and TNF-α concentration in milk increased similarly in glands infused with LPS, independent of prednisolone administration. However, the increase of LDH activity and SA concentration in milk after LPS infusion was diminished by prednisolone administration. The mRNA expression of TNF-α, IL-8, and IL-1ß in milk somatic cells increased after LPS infusion and was unaffected by prednisolone. CONCLUSIONS AND CLINICAL RELEVANCE: Intramammary administration of prednisolone did not induce an immune response and did not change mRNA expression of TNF-α, IL-8, and L-1ß during the response to intramammary administration of LPS. However, prednisolone reduced disruption of the blood-milk barrier. This could influence the severity and cure rate of mastitis.

Development of an anti-Salmonella phage cocktail with increased host range.

Salmonella shedding in many livestock species can increase significantly after transport and lairage. Preprocessing increases in shedding can amplify the amount of Salmonella that enters the processing facility and the likelihood of end-product contamination. We previously produced an anti-Salmonella phage cocktail that reduced colonization in swine when the pigs were exposed to an environment heavily contaminated with Salmonella, similar to what might be seen in a transport trailer or processing facility holding pen. The aim of this study was to increase the efficacy of the phage treatment by (1) expanding the host-range of the cocktail and (2) developing a more cost-effective microencapsulation technique. We collected samples from wastewater treatment facilities and isolated 20 distinct phages belonging to either the Siphoviridae or Myoviridae families. From this library we identified 10 phages that together lysed a mixed culture of Salmonella enterica Typhimurium, Enteriditis, and Kentucky--three serovars commonly associated with meat and poultry products. The phages were microencapsulated using two sodium-alginate-based methods that only reduced the cocktail titer by 1.0-1.5 logs (premicroencapsulation: 10.4 log(10) PFU/mL; postmicroencapsulation method one: 9.2 log(10) PFU/mL; postmicroencapsulation method two: 8.9 log(10) PFU/mL). Microencapsulated phages remained stable at both 4°C and 22°C for up to 14 days with no appreciable drop in titer (mean titer: 8.9 log(10) PFU/mL). These data indicate that phage cocktails with wider host ranges are possible and a cost-effective microencapsulation process can protect the phages over an extended period, making simultaneous treatment of large numbers of animals with feed- or water-based delivery possible.

Contamination rates and antimicrobial resistance in bacteria isolated from "grass-fed" labeled beef products.

Grass-fed and organic beef products make up a growing share of the beef market in the United States. While processing, animal handling, and farm management play large roles in determining the safety of final beef products, grass-fed beef products are often marketed as safer alternatives to grain-finished beef products based on the potential effects of all-forage diets on host microbiota. We conducted a series of experiments examining bacterial contamination rates in 50 beef products labeled as "grass-fed" versus 50 conventionally raised retail beef products. Coliform concentrations did not differ between conventional and grass-fed beef (conventional: 2.6 log(10) CFU/mL rinsate; grass-fed: 2.7 log(10) CFU/mL rinsate). The percentages of Escherichia coli positive samples did not differ between the two groups (44% vs. 44%). Enterococcus spp. were frequently isolated from both grass-fed beef products (44%) and conventional beef products (62%; p = 0.07). No Salmonella or E. coli O157:H7 isolates were recovered from any of the meat samples. Enterococcus spp. isolates from conventional beef were more frequently resistant to daptomycin and linezolid (p < 0.05). Resistance to some antimicrobials (e.g., chloramphenicol, erythromycin, flavomycin, penicillin, and tetracyline) was high in Enterococcus spp. isolated from both conventional and grass-fed beef. There were no differences in the percentages of antimicrobial resistant E. coli isolates between the two groups. Taken together, these data indicate that there are no clear food safety advantages to grass-fed beef products over conventional beef products.

Phage therapy to reduce preprocessing Salmonella infections in market-weight swine.

Contamination of meat products with food-borne pathogens usually results from the carcass coming in contact with the feces of an infected animal during processing. In the case of Salmonella, pigs can become colonized with the organism during transport and lairage from contaminated trailers and holding pens, resulting in increased pathogen shedding just prior to processing. Increased shedding, in turn, amplifies the likelihood of carcass contamination by magnifying the amount of bacteria that enters the processing facility. We conducted a series of experiments to test whether phage therapy could limit Salmonella infections at this crucial period. In a preliminary experiment done with small pigs (3 to 4 weeks old; 30 to 40 lb), administration of an anti-Salmonella phage cocktail at the time of inoculation with Salmonella enterica serovar Typhimurium reduced Salmonella colonization by 99.0 to 99.9% (2- to 3-log reduction) in the tonsils, ileum, and cecum. To test the efficacy of phage therapy in a production-like setting, we inoculated four market-weight pigs (in three replicates) with Salmonella enterica serovar Typhimurium and allowed the challenged pigs to contaminate a holding pen for 48 h. Sixteen naïve pigs were randomly split into two groups which received either the anti-Salmonella phage cocktail or a mock treatment. Both groups of pigs were comingled with the challenged pigs in the contaminated pen. Treatment with the anti-Salmonella phage cocktail significantly reduced cecal Salmonella concentrations (95%; P<0.05) while also reducing (numerically) ileal Salmonella concentrations (90%; P=0.06). Additional in vitro studies showed that the phage cocktail was also lytic against several non-Typhimurium serovars.