Localized mammary gland changes in milk composition and venous blood metabolite concentrations result from sterile subclinical mastitis.

Subclinical mastitis reduces milk yield and elicits undesirable changes in milk composition, but the mechanisms resulting in reduced milk production in affected mammary glands are incompletely understood. This study investigated the effects of sterile inflammation on mammary gland metabolism by assessing changes in milk and venous blood composition. Mid-lactation primiparous Holstein cows (n = 4) had udder halves randomly allocated to treatments; quarters of 1 udder half were infused with 2 billion cfu of formalin fixed Staphylococcus aureus (FX-STAPH) and quarters of the opposite udder half infused with saline (SAL). Blood samples were collected from the right and left subcutaneous abdominal veins in 2.6 h intervals until 40 h post challenge and analyzed for blood gas and metabolite concentrations. Milk from FX-STAPH udder halves had significantly increased SCS by first milking at 8 h post-challenge. By 16 h post-challenge, FX-STAPH udder halves had increased concentrations of protein and lactate and lower lactose concentrations than SAL udder halves. Milk fat concentrations, milk yields, energy corrected milk yields, and the ferric reducing antioxidant power of milk were not significantly different between SAL and FX-STAPH udder halves. Venous blood of FX-STAPH halves had marginally greater concentrations of saturated O2, partial pressures of O2, and glucose concentrations than SAL halves. Conversely, total and partial pressures of CO2 did not differ between udder half treatments suggesting a shift in local metabolite utilization in FX-STAPH udder halves. These results indicate that changes in milk composition resulting from mastitis are accompanied by changes in some key blood metabolite concentrations. The shift in venous blood metabolite concentrations, along with the marked increase in milk lactate, suggests that local mammary tissue and/or recruited and immune cells alters metabolite usage in mammary tissues. Future studies are needed to quantify the uptake of key milk precursors during mastitis.

Genomic Characterization of Mycoplasma arginini Isolated from a Housefly on a Dairy Farm and Comparison with Isolates from Bovine Milk and Lung Tissue.

Mycoplasma mastitis can be highly contagious, unresponsive to treatment, and cause severe economic problems in affected herds. Notable routes of Mycoplasma spp. transmissions are contaminated milking equipment and animal contact through respiratory secretions. Only a few studies report the environment as a possible source of infection. Our group studied the presence of pathogens in houseflies (Musca domestica) in a New York State dairy in the United States. Among others, a Mycoplasma spp. was found in the gut of a housefly captured in the sick pen and identified as M. arginini. Here, we characterized its genome and investigated its relatedness with eight isolates from milk, one isolate from lung tissue collected in the same dairy, and five other dairies in New York State. We applied whole-genome sequencing and phylogenetic analysis based on the sequences of the 16S rRNA gene and 76 conserved proteins. We also assessed an in silico virulence profile by considering a panel of 94 putative virulence genes. As a result of the genome analysis, the housefly M. arginini isolate was highly similar to the milk isolates; interestingly, the similarity was highest with M. arginini isolated from milk on the same dairy farm where the housefly was captured. The housefly and milk M. arginini isolates possessed 54 of the 94 pathogenicity genes considered. Our data support the hypothesis that houseflies are carriers of Mycoplasma spp. and can be considered within the possible roots of environmental transmission of infection in dairy cows. Nevertheless, M. arginini pathogenicity will need to be investigated with dedicated studies. IMPORTANCE It is critical to control the spread of bovine mastitis caused by Mycoplasma spp., as this disease can be highly contagious and have a severe economic impact on affected dairies. A better understanding of possible transmission routes is crucial for infection control and prevention. Based on our data, the composite milk isolates are genetically similar to the housefly isolate. This provides evidence that the same Mycoplasma species found in milk and associated with mastitis can also be isolated from houseflies captured in the dairy environment.

Leukocytes, microRNA, and complement activity in raw, heat-treated, and frozen colostrum and their dynamics as colostrum transitions to mature milk in dairy cows.

The objectives of this study were to evaluate the abundance and viability of leukocytes, the abundance of microRNA, and the activity of the complement pathway in (1) colostrum following heat-treatment or freezing, and (2) colostrum, transition milk, and mature milk. In experiment 1, composite colostrum samples were harvested from individual cows (n = 14) on a commercial dairy farm in NY and split into 3 aliquots using single-use colostrum bags. One aliquot was immediately cooled on ice following harvest (RAW) and stored at 4°C overnight, one was heat-treated for 60 min at 60°C (HT) before being cooled on ice and stored at 4°C overnight, and one was frozen at -20°C overnight (FR). The following morning, all samples were warmed to 40°C before further processing. In experiment 2, cows were sampled in a longitudinal study where composite samples were collected from colostrum (first milking, n = 23), transition milk (3 to 4 d postpartum, n = 13), and mature milk (6 to 7 d postpartum, n = 13). In both experiments colostrum was harvested from the first milking within 8 h of calving and samples were processed within 14 h of collection. Colostral leukocytes were isolated before viability was determined by trypan blue exclusion and manual differential cell counts were performed. Extracellular vesicles were isolated from whey by ultracentrifugation to isolate and quantify microRNA. Activity of the alternative complement pathway was determined in casein-depleted whey by semi-solid phase hemolysis assay. Somatic cell counts were determined for all raw samples. Macrophages and neutrophils made up the greatest proportion of leukocytes in colostrum followed by lymphocytes. Lymphocyte proportion increased as colostrum transitioned to mature milk, but overall somatic cell numbers declined concurrently. Viable cells were not isolated from HT or FR samples. Abundance of microRNA isolated from transition and mature milk was decreased compared with colostrum, did not differ between HT and RAW, but was increased in FR compared with RAW. Alternative complement pathway activity was decreased in HT, but not FR compared with RAW, and was not measurable in transition or mature milk. Postharvest heat-treatment and freezing of colostrum eliminated viable colostral leukocytes and affected microRNA abundance and complement activity. Leukocyte proportions, microRNA abundance, and complement activity changed as colostrum transitioned to mature milk. Although there were clear changes in the colostral components under study in relation to treatment and transition to mature milk, the biological significance of the described treatment effects and temporal changes were not investigated here.

Eucalcemia during lipopolysaccharide challenge in postpartum dairy cows: I. Clinical, inflammatory, and metabolic response.

Hypocalcemia induced by immune activation is a conserved response across mammalian species; however, administration of Ca is discouraged in other species as it is associated with increased morbidity and mortality. Early postpartum cows experience a decrease in circulating Ca concentration following acute inflammation. Corrective Ca therapy during the transition period, particularly in dairy cows experiencing acute disease, is common practice. However, the effect of Ca administration on the inflammatory response during acute immune activation is unknown. Our objective was to compare the clinical, inflammatory, and metabolic response to an intravenous (IV) lipopolysaccharide (LPS) challenge between postpartum cows infused, or not, with IV Ca to maintain eucalcemia. Cows (n = 14, 8 ± 1 d in milk) were enrolled in a matched-pair randomized controlled design to receive IV Ca (IVCa) or sterile 0.9% NaCl (CTRL) during an IV LPS challenge (0.040 or 0.045 µg of LPS/kg of body weight over 1 h). Ionized Ca (iCa) was monitored cow-side, and IV Ca infusion was adjusted in a eucalcemic clamp for 12 h following the start of LPS infusion. Cows were monitored during the 24 h following challenge and serial blood samples were collected to quantify concentrations of glucose, ß-hydroxybutyrate, nonesterified fatty acids, urea nitrogen, cytokines, acute-phase proteins, and cortisol. Blood iCa concentration decreased to 0.87 ± 0.03 mM in CTRL during challenge, and by design, iCa concentration was maintained within 3% of baseline in IVCa. Body temperature, heart rate, and respiratory rate were monitored for 24 h following the start of challenge and did not differ between groups. A treatment × time interaction was identified such that serum cortisol concentrations increased in both groups at 2 h but decreased to a greater extent at 6 h in IVCa compared with CTRL. Rumination time (min/h) over the first 12 h following challenge was greater in IVCa, but total rumination time in the 24 h following challenge did not differ from CTRL. Serum glucose and nonesterified fatty acid concentrations decreased, and ß-hydroxybutyrate and urea nitrogen concentrations increased over time, but did not differ between groups. Acute leukopenia occurred in both groups at 4 h before leukocytosis was observed at 24 h with total white blood cell counts returning to baseline within 72 h. Plasma concentrations of tumor necrosis factor (TNF) and interleukin-10 (IL-10) increased within 1 h following the start of challenge and did not differ between groups. Serum haptoglobin and serum amyloid A concentrations increased within the 24 h following challenge and were elevated through 72 h but did not differ between groups. Eucalcemia during the acute systemic inflammatory response did not alter the TNF or IL-10 cytokine response, or the acute-phase protein SAA and haptoglobin response in this LPS challenge model; however, eucalcemia was associated with a more rapid decline in cortisol response and greater rumination time in the first 12 h following challenge. We did not find evidence that eucalcemia exacerbated the inflammatory response in early postpartum cows, but Ca administration may alter the clinical response to acute systemic inflammation.

Prospective cohort study of the relationship between milking machine liner slip, milking performance, and cow characteristics.

We conducted a prospective cohort study to investigate the associations of machine milking liner slip with (1) milking performance and (2) cow characteristics. Parlor data including milk flow characteristics and data on the occurrence of milking machine liner slips from a 4,000-cow dairy with a thrice-daily milking schedule were obtained with electronic on-farm milk meters over a 2-mo period. We analyzed data from a total of 686,330 milking observations. A multivariable general linear mixed model revealed no association between liner slip and milking unit on time. Least squares means (95% confidence intervals, 95% CI) were 237 (235-238) s for milking observations with and without a liner slip. We observed statistically significant differences in average milk flow rate; however, these were biologically irrelevant. Least squares means were 3.40 (3.37-3.42) kg/min for a milking observation with and 3.42 (3.40-3.44) kg/min without a liner slip. A multivariable generalized linear mixed model showed an association between liner slip and cow characteristics. Compared with late-lactation cows, the odds ratios (OR, 95% CI) of occurrence of a liner slip were 2.03 (1.59-2.59) in early lactation cows and 1.26 (0.97-1.64) in cows from 101 to 200 days in milk. Presence of a nonlactating quarter increased the odds of liner slip occurrence [OR, 95% CI: 10.35 (8.02-13.35)]. Bimodal milking observations had higher odds of occurrence of a liner slip compared with milking observations with a unimodal milk flow curve [OR, 95% CI: 1.05 (1.005-1.09)]. A 1-kg increase in 2-min milk yield increased the odds of a liner slip [OR, 95% CI: 1.26 (1.25-1.28)]. We conclude that, in the study cohort presented herein, the negative effect of liner slips on milking performance can be diminished. The identified cow characteristics could offer unique opportunities to identify and manage cows at increased risk of liner slips.

Pathogens associated with houseflies from different areas within a New York State dairy.

Houseflies (Musca domestica) are nonbiting muscoids of importance because they can be mechanical vectors of many kinds of pathogens such as bacteria, protozoa, viruses, and helminth eggs. This study aimed to evaluate the bacterial communities associated with houseflies captured in 3 different areas on a dairy farm located in New York State. Variations in the bacterial community were also evaluated based on the flies' sex and external or internal location where the bacteria were isolated. A total of 101 flies were collected: 27 flies from the sick pen, 42 from calf hutches, and 32 from the milking parlor. A total of 485 organisms were isolated, 233 (48.0%) from 53 female flies and 252 (52.0%) from 48 male flies. Most (74%) bacteria were found in the internal parts of the flies, with only 26% isolated from the external surfaces. The number of isolates detected per fly ranged between 1 and 11. A total of 392 bacteria were identified at the species level. We isolated 26 species reported to be bovine contagious or environmental mastitis pathogens. Within the group of organisms considered contagious, we isolated Staphylococcus aureus and Mycoplasma arginini. This was the first time that a Mycoplasma species was isolated from houseflies. We identified 5 organisms considered foodborne pathogens that affect human health: Salmonella spp., Escherichia coli, Staph. aureus, Bacillus cereus, and Bacillus subtilis. Four of the organisms isolated in this study were also linked with milk spoilage, including Pseudomonas aeruginosa, Bacillus cereus, Bacillus licheniformis, and Paenibacillus lactis. This study confirmed that houseflies carry a high bacterial diversity, including organisms associated with animal infections, organisms that could be a concern for public health, or organisms that could negatively affect milk quality.

Risk factors for delayed milk ejection in Holstein dairy cows milked 3 times per day.

Delayed milk ejection, manifested most often as bimodal milk flow, occurs when the cisternal milk fraction is removed before the alveolar milk reaches the gland cistern. It is thought to be a consequence of not meeting cows' physiological needs, due to insufficient premilking teat stimulation, inadequate timing of milking unit attachment, or both. It has been associated with decreased milking efficiency, reduced milk yield, and impaired teat and udder health. Traditionally, portable electronic milk meters have been used to assess the presence of delayed milk ejection in dairy cows. By contrast, incremental milk flow rates from on-farm milk meters and their suitability as a measure to assess delayed milk ejection have not been studied by rigorous methods. The objectives were (1) to describe a protocol for identification of cows with chronically delayed milk ejection (CDME) and (2) to investigate risk factors for CDME using incremental milk flow rates obtained from automated on-farm milk meters. In a retrospective case control study, milk flow data from a 4,300-cow dairy with a thrice-daily milking schedule were obtained over a 1-wk period. Incremental milk flow rates (0-15 s, 15-30 s, 30-60 s, and 60-120 s) were used to identify cows with delayed milk ejection. Cases of CDME were defined as presence of delayed milk ejection at all 21 milking observations. Cows that had no delayed milk ejection at any of the same 21 milking observations were included as controls. A total of 171 cases and 393 controls were included in the study based on these criteria. A logistic regression model was used to evaluate associations of the following risk factors with CDME: parity (1, 2, ≥3), stage of lactation (<100, 101-200, >200 DIM), presence of a nonlactating quarter, milk somatic cell count, average daily milk production, and health and management events. Parity and CDME were associated such that compared with cows in their third or greater lactation, the odds (95% confidence intervals, 95% CI) of CDME were 1.27 (0.71-2.25) for cows in their first and 4.77 (2.47-9.22) for animals in their second lactation. The odds of CDME increased with increasing stage of lactation, with an odds ratio of 0.20 (0.11-0.36) for early and 0.28 (0.15-0.52) for mid-lactation animals, respectively, compared with late lactation cows. A 1-kg increase in average daily milk production was associated with decreased odds of CDME [odds ratio (95% CI): 0.89 (0.87-0.92)]. A lameness event during the study period increased the odds of CDME [odds ratio (95% CI): 8.04 (1.20-53.83)], as did a vaccination event 1 wk before the study period [odds ratio (95% CI): 4.07 (0.99-16.71)]. This study confirmed associations between CDME and previously reported risk factors and identified several previously less rigorously investigated health and management events that could be associated with CDME. Incremental milk flow rates from individual cows serve as an automated tool to evaluate milk flow dynamics. This information could be used to improve individual premilking udder preparation to meet the animal's physiological requirements, improve teat and udder health, and enhance parlor efficiency.

Lipopolysaccharide challenge following intravenous amino acid infusion in postpartum dairy cows: I. Production, metabolic, and hormonal responses.

Postpartum cows experience a nadir in energy and AA deficit early postpartum. At the same time, cows are challenged with inflammatory stimuli and often show heightened immune responsiveness, further increasing their metabolic needs during this critical time. This study investigated the response to a systemic inflammatory stimulus after a 4-d intravenous (IV) AA infusion designed to ameliorate the estimated metabolizable protein (MP) deficit in postpartum cows. Our objectives were to (1) describe the production and metabolic responses to early postpartum IV AA infusion, (2) determine the metabolic and hormonal responses to an acute IV lipopolysaccharide (LPS) challenge in early postpartum cows, and (3) compare these metabolic and hormonal responses between IV AA treated and control cows. Cows (n = 14, 4 ± 1 d in milk) were continuously IV infused for 4 d in a matched-pair randomized controlled design and received IV AA (IVAA) or 0.9% NaCl (CTRL). Treatment with IV AA consisted of 1 g/kg of BW per day of combined essential AA (EAA) and nonessential AA (NEAA). After infusion ended, cows were challenged IV with LPS (0.0625 µg/kg of BW over 1 h), and serial blood samples were collected to quantify AA, metabolite, and hormone concentrations. Amino acid infusion increased plasma EAA and NEAA concentrations and ameliorated the estimated MP deficit but not the metabolizable energy deficit in IVAA cows. Patterns of dry matter intake during infusion were different between groups. Milk yield and milk protein content and yield were unaffected, but IV AA was associated with increased milk fat content and yield of both de novo and preformed fatty acids. Before LPS infusion, plasma EAA and NEAA concentrations were greater in IVAA compared with CTRL. During LPS challenge, plasma AA concentrations decreased to a greater degree in IVAA than CTRL. Glucagon concentrations were greater and glucose concentrations lower in IVAA during challenge; however, previous AA infusion did not affect the time-dependent changes in concentrations of energy metabolites or glucoregulatory hormones. Plasma urea nitrogen concentration increased in both treatments following challenge, although the temporal pattern depended on treatment. Effects of AA infusion on milk fat response were pronounced and likely due to a combination of increased lipolysis and de novo milk fat synthesis. Despite differences in circulating concentrations of nutrients and hormones before challenge, metabolic responses to systemic inflammation did not differ between the 2 treatments. We conclude that AA infusion changed metabolic status and milk fat but did not appear to alter the metabolic response to subsequent systemic inflammation.

Lipopolysaccharide challenge following intravenous amino acid infusion in postpartum dairy cows: II. Clinical and inflammatory responses.

Amino acids (AA) are integral nutrients for a functioning immune system. Postpartum cows experience AA deficits early postpartum that may influence the response to immune activation. This study investigated the clinical and inflammatory responses to a systemic inflammatory stimulus after a 4-d intravenous (IV) AA infusion with a mix of essential and nonessential AA designed to ameliorate the estimated metabolizable protein deficit in early postpartum cows. Our objectives were (1) to describe the clinical and inflammatory response to an acute IV lipopolysaccharide (LPS) challenge in early postpartum cows, and (2) to compare these clinical and inflammatory responses between IV AA-treated and control cows. Cows (n = 14, 4 ± 1 d in milk) were continuously infused IV for 4 d in a matched-pair randomized controlled design and received 0.9% NaCl (CTRL) or IV AA (IVAA) to supply 1 g/kg of BW per day of combined essential and nonessential AA. After infusion ended, cows were challenged with IV LPS (0.0625 µg/kg of BW over 1 h), and serial blood samples were collected for complete blood cell counts and to quantify plasma cytokines and acute-phase proteins. Body temperature, heart rate, and respiratory rate were monitored for 24 h during challenge. During challenge, maximum body temperature was greater in IVAA (41.3 ± 0.20°C) than in CTRL (40.6 ± 0.19°C). In both groups, respiratory rate increased during the first 2 h following challenge, whereas heart rate first decreased over the first 2 h and then increased to reach a maximum at 4 h. Acute leucopenia occurred within 1 h of challenge in both groups before leukocytosis was observed at 24 h, with white blood cell counts returning to baseline values within 72 h. Plasma haptoglobin and serum amyloid A concentrations increased 3-fold and 4-fold in both groups and peaked at 48 and 24 h following challenge, respectively. Plasma concentrations of TNF-α and IL-10 increased within 1 h and peaked at 2 h following the start of challenge. Plasma IL-10 concentrations increased to a greater extent in CTRL compared with IVAA during challenge. Despite differences in IL-10 concentration, previous AA infusion did not alter the acute-phase protein response to LPS challenge. We conclude that AA infusion before systemic inflammatory challenge decreased the anti-inflammatory response but did not alter concentrations of other systemic markers of inflammation.

Short communication: Comparative performance of 3 on-farm culture systems for detection of mastitis pathogens interpreted by trained and untrained observers.

On-farm culture (OFC) systems facilitate pathogen-based mastitis management and can facilitate antimicrobial stewardship on dairy farms. Interpretation of the results, however, may present a challenge for those with limited microbiology experience. Here, we compared results of 3 OFC systems interpreted by trained and untrained observers against results of a standard laboratory reference method (aerobic culture and mass spectrometry). Milk samples (280 quarter and 60 composite) were selected from submissions for routine diagnostic testing to Quality Milk Production Services (Cornell University, Ithaca, NY) between August 2017 and January 2018. Samples were cultured simultaneously using the standard laboratory reference method and 3 commercially available OFC systems that varied in detail of pathogen identification (provided in parentheses) as follows: (1) Minnesota Easy Culture System II Bi-plate (University of Minnesota Laboratory for Udder Health, St. Paul; gram-positive, gram-negative), (2) Minnesota Easy Culture System II Tri-plate (gram-positive, gram-negative, some genus level), and (3) FERA Diagnostics and Biologicals AccuMast plate (Ithaca, NY; genus level, some species level). After 18 to 24 h of incubation, OFC plates were interpreted by 1 trained observer (>10 yr of experience in milk microbiology) and 6 untrained observers with no previous milk microbiology training, using only the manufacturers' instructions for guidance. Strength of agreement (κ) between observer groups and the reference method was determined for the available outcomes of each system. Interpreted by the trained observer, agreement was moderate for identifying gram-positive organisms (Bi-plate, κ = 0.56) and substantial for Streptococcus spp. (Tri-plate, κ = 0.64, AccuMast κ = 0.61). Interpretation by untrained observers resulted in fair agreement (κ = 0.29-0.37) for these organisms. Moderate agreement (κ = 0.43-0.59) was found across all 3 OFC for the identification of gram-negative organisms (Bi-plate), non-aureus staphylococci (Tri-plate and AccuMast), Lactococcus spp., and Enterococcus spp. (AccuMast) when interpreted by the trained observer, and fair to moderate agreement was found (κ = 0.31-0.53) among untrained observers. Across all 3 OFC, agreement was almost perfect (κ = 0.80-0.89) for Staphylococcus aureus for the trained observer, and moderate to substantial (κ = 0.56-0.61) for untrained observers. We concluded that all 3 OFC appeared suitable to support pathogen-based mastitis management when operated by trained observers. Training beyond the instruction manual is a prerequisite to make OFC systems useful for pathogen-based mastitis management.

Mycoplasma species isolated from bovine milk collected from US dairy herds between 2016 and 2019.

Determining the species of mycoplasma isolated from culture-positive milk samples is important for understanding the clinical significance of their detection. Between August 2016 and December 2019, 214,518 milk samples from 2,757 dairy herds were submitted to Quality Milk Production Services (QMPS) at Cornell University for mycoplasma culture. From these samples, 3,728 collected from 204 herds were culture positive. Based on the request of herd managers, owners, or veterinarians, 889 isolates from 98 herds were subjected to molecular identification by PCR and amplicon sequencing. The largest proportion of the identified isolates were from New York State (78.1%), while the others came from the eastern United States (17.8%), Texas (2.0%), and New Mexico (2.1%). As expected, Mycoplasma spp. were the most common (855 isolates, 96.2%) and Acholeplasma spp. accounted for the remainder (34 isolates, 3.8%). Mycoplasma bovis was the most prevalent Mycoplasma species (75.1%), followed by M. bovigenitalium (6.5%), M. canadense (5.9%), M. alkalescens (5%), M. arginini (1.7%), M. californicum (0.1%), and M. primatum (0.1%). A portion of the isolates were confirmed as Mycoplasma spp. other than M. bovis but were not identified at the species level (16 isolates, 1.8%) because further information was not requested by the manager, owner, or veterinarian. Mycoplasma bovis was the only species identified in 59 of the 98 herds. However, more than 1 Mycoplasma sp. was identified in 29 herds, suggesting that herd infection with 2 or more mycoplasmas is not uncommon. Moreover, a Mycoplasma sp. other than M. bovis was the only species identified in 8 herds. From the subset of 889 mycoplasma culture-positive isolates from 98 herds, we determined that over a third of the herds had either more than 1 Mycoplasma sp. or a Mycoplasma sp. other than M. bovis detected in their milk samples. In conclusion, we observed that M. bovis is the most common pathogenic Mycoplasma species found in mastitic milk, but other Mycoplasma species are not uncommon. Our results suggest that it is critical to test milk samples for mycoplasmas using diagnostic tests able to identify both the genus and the species.

Heat treatment of bovine colostrum: II. Effects on calf serum immunoglobulin, insulin, and IGF-I concentrations, and the serum proteome.

In-depth analysis of colostrum components has identified hundreds of proteins, but data are sparse regarding their systemic uptake in the newborn calf. Moreover, heat treatment may influence these colostral components and their absorption. Our objectives were to describe the serum proteome of newborn calves before and after colostrum feeding and the possible effects of colostral heat treatment. Newborn Holstein heifer calves (n = 22) were randomized within pair and fed heat-treated (n = 11; 60°C, 60 min) or raw (n = 11) colostrum at 8.5% of birth body weight by esophageal feeder within 1 h of birth. After the single colostrum feeding, calves were not fed until after the 8-h time point, when milk was offered free-choice. Blood samples were taken immediately before feeding (0 h), as well as 4, 8, and 24 h after feeding. Whole blood packed cell volume (%), serum Brix percentage, and plasma glucose concentrations were determined for all time points. Plasma insulin and insulin-like growth factor-I concentrations were determined by radioimmunoassay for selected time points. Serum IgA and IgG were measured by radial immunodiffusion at 24 h. The serum proteome was analyzed using nano-scale reverse-phase chromatography and tandem mass spectrometry (nano LC-MS/MS) in 0- and 8-h samples. For proteomics analysis, ratios of results for 8-h to 0-h samples were analyzed with false discovery rate adjustment. For all other outcomes, repeated-measures ANOVA was performed with the fixed effects of group, time, and their interaction, and random effect of pair. Serum Brix percentage and glucose concentrations increased over time and were independent of colostrum treatment. Serum IgG and IgA concentrations at 24 h did not differ between groups. Nano LC-MS/MS identified a total of 663 unique proteins in serum, of which 261 increased in abundance, whereas 67 decreased in abundance after feeding in both groups. Among serum proteins that increased in abundance and that were previously identified in colostrum, many belonged to those involved in immune response, coagulation, the classical complement pathway, or the antimicrobial peptide class of cathelicidins. Serum proteins that decreased in abundance and that were identified in colostrum belonged to the alternative complement pathway and the membrane attack complex. Thirty-eight proteins differed in calves that were fed heat-treated colostrum compared with those fed raw colostrum. Decreased abundances in calves fed heat-treated colostrum included several enzymes involved in glycolysis or glycogenolysis, whereas the incretin gastric inhibitory polypeptide and serum insulin were increased in this group. Our findings point to important innate immune defense pathways associated with colostrum ingestion in newborn calves. Furthermore, calves fed heat-treated colostrum showed differences in serum proteins and enzymes associated with carbohydrate metabolism.

Blood perfusion of teat tissue in dairy cows: Changes associated with pre-milking stimulation and machine milking.

Mechanical forces during machine milking of dairy cows evoke circulatory impairment of the teat tissue that may affect the teats' defense mechanisms against mastitis pathogens. Ample research describes dimensional changes of different teat traits after machine milking, whereas reports that describe changes in blood circulation of dairy cows' teats are limited. Therefore, the objectives of this study were to (1) describe changes in teat blood circulation that occur after pre-milking teat stimulation and machine milking and (2) study the effect of 2 different milking liners on machine milking-induced changes in teat blood flow. In a randomized trial, Holstein dairy cows were stratified by parity, stage of lactation, and average daily milk yield during the previous week, and allocated to 1 of 2 treatment groups. Treatment consisted of 1 milking observation with either a round or multisided concave milking liner. Teat scans were taken of the left front and the right hind teats using power Doppler ultrasonography. Imaging occurred before pre-milking udder preparation (T1), after completion of pre-milking udder preparation but before milking-unit attachment (T2), and immediately after unit detachment (T3). Perfusion intensity measurements from teat scans were performed with a commercially available software program. Data from 109 cows were analyzed. A general linear mixed model showed differences in perfusion intensity between time points. Least squares means (95% confidence intervals) for T1, T2, and T3, respectively, were 0.035% (0.026-0.047), 0.124% (0.093-0.164), and 0.095% (0.073-0.124). Conversely, no statistically significant differences between treatment groups were observed. We conclude that teat blood circulation is subjected to several influences, including inherent circulatory regulation mechanisms, as well as extrinsic factors such as machine milking. Future research is warranted to decipher the magnitude of their influence and to further our understanding of how these changes relate to the susceptibility to intramammary infection and milking performance.

Case study: Evaluating quarter and composite milk sampling for detection of subclinical intramammary infections in dairy cattle.

Our objective was to evaluate a 200,000 cells/mL somatic cell count (SCC) cut-point on both the quarter and composite level to determine its effectiveness at identifying subclinical mastitis infections in one commercial dairy herd in Central New York. Milk samples from 107 Holstein cows were used for analysis. All cows were eligible for enrollment provided they had 4 working udder quarters, were >14 and <365 d in milk, and had no clinical mastitis event or treatment with intramammary antibiotics ≤14 d prior to d of sampling. A total of 428 quarter and 107 composite samples from 34 primiparous and 73 multiparous animals were analyzed for total SCC and aerobic culture. Performance of SCC for identification of subclinically infected animals was evaluated against the gold standard aerobic culture. Sensitivities for a 200,000 cells/mL cut-point on both the quarter and composite basis were 28.6%, and specificities were 91.5% and 87.3% on the quarter and composite basis, respectively. Receiver operating characteristic (ROC) curves determined on a quarter basis found the cut-point that optimized the sensitivity and specificity of a positive culture was 32,000 cells/mL, with a sensitivity of 76.2%, a specificity of 62.4%, and an area under the curve (AUC) of 0.73. The ROC curve cut-point that optimized the sensitivity and specificity for the composite samples was 75,000 cells/mL, with a sensitivity of 57.1%, a specificity of 78.9%, and an AUC of 0.67. A large proportion of culture positive primiparous cows (38%) had low SCC (median of 101,000 cells/mL on the quarter and 80,000 cells/mL on the composite level), and therefore, when multiparous cows were examined separately, the cut-point that optimized sensitivity and specificity on the quarter basis increased to 645,000 cells/mL with a corresponding sensitivity of 34.8%, specificity of 97.5%, and AUC of 0.65. On the composite basis, the cut-point based on multiparous cows only was 152,000 cells/mL, with a corresponding sensitivity of 60.0%, and specificity of 82.0%, and an AUC of 0.65. Our data indicate that the 200,000 cells/mL cut-point was inefficient in identifying subclinically infected animals, regardless of whether quarter or composite sampling was used. The low prevalence of subclinical infections as well as the large proportion of minor pathogens, especially in primiparous animals, contributed to this inefficiency. This case study provides evidence that, with continued improvement upon mastitis control and reduction in major mastitis pathogens, blanket cut-points may no longer provide the same diagnostic usefulness as they once did.

Nutrient-sensing kinase signaling in bovine immune cells is altered during the postpartum nutrient deficit: A possible role in transition cow inflammatory response.

Transition dairy cows experience a nutrient deficit, particularly in the immediate postpartum period. At the same time, the inflammatory balance is altered and cows exhibit an immune response primed for inflammatory response rather than tolerance. The mechanistic link that might be underlying the immunological effects due to the lack in nutrients is not fully understood. Studies in other species demonstrate an orchestrating role of nutrient-sensing kinases in the determination of immune phenotypes and immune cell proliferation and differentiation. Our primary objective was to investigate changes in energy storage and signaling through the protein kinase B (AKT)/mechanistic target of rapamycin complex 1 (mTOR) pathway in bovine immune cells in the transition period, as well as the association with cytokine expression profiles. A secondary objective was to test if supplementation with branched-chain amino acids alone or in combination with oral propylene glycol had any effect on the measured parameters. To assess cellular energy storage, glycogen concentration was measured by an enzymatic-fluorometric method in peripheral blood mononuclear cells (PBMC) of multiparous Holstein cows (n = 72) at 3 time points in the transition period (21 d before, 7 and 28 d after calving). At the same time points, phosphorylation of proteins in the AKT/mTOR pathway was assessed by immunoblotting in PBMC from 60 animals. Whole-blood leukocyte cytokine gene expression of IL12B, IL6, IL1B, TNF, and IL10 was measured in samples from 50 animals by reverse-transcription quantitative PCR with and without stimulation of samples with 10 ng/mL of lipopolysaccharide. Compared with glycogen concentration of prepartum PBMC, glycogen concentration decreased by 37% on d 7 postpartum. The activation of AKT/mTOR in bovine PBMC postpartum was reduced compared with prepartum values. Results of reverse-transcription quantitative PCR showed an increase in cytokine gene expression postpartum compared with prepartum values. Supplementation with branched-chain amino acids alone or in combination with oral propylene glycol did not alter glycogen storage, AKT/mTOR activity, or inflammatory balance as assessed by the measured parameters in this study. We conclude that the nutrient deficit of the immediate postpartum period is sensed by bovine immune cells, and that it affects their energy storage as well as cellular signaling pathways postpartum. Temporal associations with changes in cytokine gene expression are intriguing and warrant further investigation of the role of this pathway as a possible link between metabolism and immune phenotype postpartum.

Intramammary immunization with ultraviolet-killed Escherichia coli shows partial protection against late gestation intramammary challenge with a homologous strain.

The objective of this study was to evaluate the efficacy of intramammary immunization with UV-killed Escherichia coli ECC-Z on prevention of intramammary colonization after a challenge with a dose of the homologous E. coli ECC-Z live bacteria. A total of 10 cows were included in a study to evaluate the efficacy of intramammary immunization. All 10 cows received an intramammary immunization of 100 cfu of UV-killed E. coli ECC-Z bacteria into one hind quarter at the time of dry off. Approximately 2wk before the anticipated calving date, both hind quarters of all cows were challenged with 100 cfu of live E. coli ECC-Z bacteria. Five of the cows were vaccinated parenterally with a commercial J5 bacterin, and 5 cows served as controls with no parenteral vaccination. The cows were then followed over time and infection risk, clinical scores, somatic cell count, and milk production were observed over time. The results of these 10 cows showed partial protection of intramammary immunization on the outcome of a subsequent homologous intramammary challenge. Immunization resulted in a lower probability of infection, a lower bacteria count, lower somatic cell counts and milk conductivity, a lower clinical mastitis score, and increased milk production compared with unimmunized control quarters. Once the analysis was corrected for immunization, parenteral J5 vaccination had no significant effect on any of the measured parameters. These results provide the first evidence that intramammary immunization may improve the outcome of an intramammary E. coli infection in late gestation and onset of mastitis immediately following parturition. Unlike systemic vaccination, which generally does not reduce the intramammary infection risk, the intramammary immunization did show a 5-times reduced odds of an established intramammary infection after challenge. Cytokine profiles indicated a local return of proinflammatory response after challenge as the data showed a more pronounced increase in in IFN-γ with a subsequent negative feedback due to a spike in the level of IL-10 in immunized quarters relative to nonimmunized quarters. Although these results are preliminary and obtained on only 10 cows, the results provide insight into the biological benefits of triggering mucosal immunity in the mammary gland.

Host-response patterns of intramammary infections in dairy cows.

Many different bacterial species have the ability to cause an infection of the bovine mammary gland and the host response to these infections is what we recognize as mastitis. In this review we evaluate the pathogen specific response to the three main bacterial species causing bovine mastitis: Escherichia coli, Streptococcus uberis and Staphylococcus aureus. In this paper we will review the bacterial growth patterns, host immune response and clinical response that results from the intramammary infections. Clear differences in bacterial growth pattern are shown between bacterial species. The dominant pattern in E. coli infections is a short duration high bacteria count infection, in S. aureus this is more commonly a persistent infection with relative low bacteria counts and in S. uberis a long duration high bacteria count infection is often observed. The host immune response differs significantly depending on the invading bacterial species. The underlying reasons for the differences and the resulting host response are described. Finally we discuss the clinical response pattern for each of the three bacterial species. The largest contrast is between E. coli and S. aureus where a larger proportion of E. coli infections cause potentially severe clinical symptoms, whereas the majority of S. aureus infections go clinically unnoticed. The relevance of fully understanding the bovine host response to intramammary infection is discussed, some major gaps in our knowledge are highlighted and directions for future research are indicated.

Ex vivo phagocytic overall performance of neutrophilic granulocytes and the relation to plasma insulin-like growth factor-I concentrations in dairy cows during the transition period.

Impaired function of polymorphonuclear neutrophilic leukocyte (PMNL) during the peripartal period is a major reason for increased susceptibility of dairy cows to infections in this critical interval. Factors dysregulating PMNL function are widely unknown. Insulin-like growth factor (IGF-I) enhanced PMNL functions in vitro. The objective of this study was to investigate the influence of IGF-I and, additionally, ß-hydroxybutyrate and nonesterified fatty acid concentrations on phagocytic activity (PA, percentage of viable PMNL) and phagocytic capacity (PC, mean fluorescence intensity of phagocytic PMNL) assessed by flow cytometry. Antepartum (i.e., wk -3, -2, -1; before calving), plasma concentrations of IGF-I were high (80-110 ng/mL) without significant differences between primiparous and pluriparous cows (n=18 and n=41, respectively). Concentrations of IGF-I declined toward the week of calving (wk 1). Postpartum (i.e., wk 2, 3, and 4; after calving), IGF-I remained lower than before parturition, with concentrations higher in primiparous compared with those of pluriparous cows. The PA was constant in primiparous cows throughout the study period. Conversely, PMNL of pluriparous cows had a significantly increased and higher PA in wk 2 and 3 postpartum compared with that of primiparous cows. The PC decreased significantly only in primiparous cows the week of calving, whereas the number of PMNL in primiparous cows exceeded that of pluriparous cows significantly. The phagocytic power (PP, a product of PA by PC), but not the phagocytic overall performance (POP, a product of PA, PC, and PMNL number), differed between primiparous and pluriparous cows in wk 3 postpartum. No significant differences in POP were found, except in wk 4 after calving between the primi- and pluriparous cows. In both groups, POP increased in the week of calving (wk 1). In contrast to ß-hydroxybutyrate, which was weakly positive correlated with PA and PP in pluriparous cows in the transition period (wk -3 antepartum to wk 4 postpartum), pluriparous animals had weak negative correlations of PMNL number, PA, PP, POP, and IGF-I concentration in this period. In primiparous animals, only PP and PC were weakly negatively correlated with IGF-I in the transition period. Increased plasma IGF-I concentrations were not associated with enhanced phagocytosis function of bovine blood PMNL ex vivo and, thus, can not be regarded as a suitable predictor for this function.