A postbiotic from Aspergillus oryzae attenuates the impact of heat stress in ectothermic and endothermic organisms.

Heat stress is detrimental to food-producing animals and animal productivity remains suboptimal despite the use of heat abatement strategies during summer. Global warming and the increase of frequency and intensity of heatwaves are likely to continue and, thus, exacerbate the problem of heat stress. Heat stress leads to the impairment of physiological and cellular functions of ectothermic and endothermic animals. Therefore, it is critical to conceive ways of protecting animals against the pathological effects of heat stress. In experiments with endothermic animals highly sensitive to heat (Bos taurus), we have previously reported that heat-induced systemic inflammation can be ameliorated in part by nutritional interventions. The experiments conducted in this report described molecular and physiological adaptations to heat stress using Drosophila melanogaster and dairy cow models. In this report, we expand previous work by first demonstrating that the addition of a postbiotic from Aspergillus oryzae (AO) into the culture medium of ectothermic animals (Drosophila melanogaster) improved survival to heat stress from 30 to 58%. This response was associated with downregulation of genes involved in the modulation of oxidative stress and immunity, most notably metallothionein B, C, and D. In line with these results, we subsequently showed that the supplementation with the AO postbiotic to lactating dairy cows experiencing heat stress decreased plasma concentrations of serum amyloid A and lipopolysaccharide-binding protein, and the expression of interleukin-6 in white blood cells. These alterations were paralleled by increased synthesis of energy-corrected milk and milk components, suggesting enhanced nutrient partitioning to lactogenesis and increased metabolic efficiency. In summary, this work provides evidence that a postbiotic from AO enhances thermal tolerance likely through a mechanism that entails reduced inflammation.

Effects of acute lying and sleep deprivation on metabolic and inflammatory responses of lactating dairy cows.

Dairy cows that are restricted from lying down have a reduced ability to sleep. In other species, sleep loss is a key risk factor for disease, mediated by changes in metabolic and inflammatory responses. The cumulative effect of lying and sleep deprivation on cow health is unknown. The objective was to determine the effects of lying and sleep deprivation on metabolic and inflammatory responses of dairy cows. Data were collected from 8 multiparous and 4 primiparous lactating cows (199 ± 44 d in milk, 77 ± 30 d pregnant; mean ± standard deviation) enrolled in a study using a crossover design. Each cow was exposed to 2 treatments meant to induce sleep loss: (1) human disturbance (imposed by researchers making noise or physical contact when the cow's posture suggested sleep) and (2) lying deprivation (imposed by a wooden grid placed on the pen floor). Cows experienced a 24-h baseline period (d -1) followed by a 24-h treatment period (d 0), with a 12-d washout period between treatments. Baseline and treatment periods were imposed from 2100 to 2059 h. Cows were housed in individual pens during the acclimation period (d -3 and -2), d -1, and d 0. Nonesterified fatty acid and glucose concentrations were measured at 0300, 0900, 1500, and 2059 h on d -1 and 0. Proinflammatory cytokine mRNA [tumor necrosis factor (TNF), interleukin-1B (IL1B), and interleukin-6 (IL6)] abundance in whole-blood leukocytes, both nonstimulated and stimulated with lipopolysaccharide, were assessed at 2059 h on d -1 (end of baseline) and d 0 (end of treatment). Nonesterified fatty acids and glucose varied by time of day but were not affected by treatment or day. The abundances of TNF and IL1B from both stimulated and nonstimulated cells were higher following 24 h of lying deprivation (d 0) compared with baseline (d -1). Abundance of IL6 was increased in nonstimulated cells after lying deprivation compared with baseline. In contrast, human disturbance for 24 h did not alter TNF, IL1B, or IL6 abundance relative to baseline levels. These results suggest that a short period of lying deprivation generally increases inflammatory responses but not metabolic responses.

Comparing dairy farm milk yield and components, somatic cell score, and reproductive performance among United States regions using summer to winter ratios.

Heat stress abatement is a challenge for dairy producers in the United States, especially in the southern states. Thus, managing heat stress is critical to maintain dairy cow performance in the summer. The ability to employ a metric to measure heat stress and evaluate abatement strategies may benefit dairy producers by providing meaningful feedback on the effectiveness of current and future management strategies with the goal of improving heat stress management. Therefore, this study aimed to explore the use of the summer to winter performance ratio metric to quantify and compare farm performance variables among regions of the United States. Monthly performance data recorded by the Dairy Herd Improvement Association from 2007 to 2016, for all US Dairy Herd Improvement Association herds processing records through Dairy Records Management Systems (Raleigh, NC), were obtained. Season dates were based on the astronomical definition of the Northern Hemisphere with summer as June 21 to September 21 and winter as December 21 to March 19. States were grouped into regions based on climate zone classification. Performance records included a total of 16,573 herds [Northeast (n = 7,955), Midwest (n = 6,555), Northern Plains (n = 305), Southeast (n = 1,370), and Southern Plains (n = 388) regions]. Herd test day performance variables energy-corrected milk, somatic cell score, milk fat and protein percentage, conception rate, heat detection rate, and pregnancy rate in summer and winter were used to calculate summer to winter ratios for each region. The MIXED procedure of SAS 9.4 (SAS Institute Inc., Cary, NC) was used to compare test day performance variables. The effects of year, mean days in milk, mean 150-d milk, mean herd size, and number of milkings per day were included as covariates in the models. Dairy cattle performance in all climate regions was negatively affected by summer heat stress, but some regions greater than others. A difference was also observed among regions when comparing summer to winter ratios for each performance parameter. This indicates that summer performance varies by climate region identified by the summer to winter ratio and demonstrates usefulness of the metric to monitor degree of heat stress based on performance.

Short communication: Evaluation of an automated method for assessing white blood cell concentrations in Holstein dairy cows.

Assessing white blood cell (WBC) differentials is one way to assess cow health and well-being. The objective of this study was to determine the agreement between WBC populations identified by manual evaluation via microscopy and an automated approach. Data were collected from mid-to-late lactation dairy cows sampled at 6-h intervals starting at 2100 over a 48 period (n = 192). The agreement between the two methods was calculated using a regression model in SAS (v9.4, SAS Institute Inc., Cary, NC) to construct limits of agreement. Data were analyzed by comparing the mean response of the methods for each sample against the difference between the methods The maximum allowable difference (MAD) was set at ±10% for each response variable. Agreement between methods was evident for neutrophils and lymphocytes, but not for monocytes and eosinophils. Agreement for these factors was established from an insignificant P-value, a low R2 value, and concentration of the data within the MAD. This data indicates that the automated method is appropriate for analysis of neutrophil and lymphocyte concentrations. However, accuracy needs to be improved for analysis of monocytes and eosinophils if differentiation of all WBC populations is necessary.

Quantification of bovine oxylipids during intramammary Streptococcus uberis infection.

Streptococcus uberis mastitis results in severe mammary tissue damage in dairy cows due to uncontrolled inflammation. Oxylipids are potent lipid mediators that orchestrate pathogen-induced inflammatory responses, however, changes in oxylipid biosynthesis during S. uberis mastitis are unknown. Thus, the current objective was to determine how oxylipid concentrations change in milk and mammary tissues during different stages of S. uberis mastitis. Increased arachidonic acid and linoleic acid-derived oxylipids were significantly increased in S. uberis-infected bovine mammary tissue. Linoleic acid metabolites, hydroxyoctadecadienoic acid (HODE) and oxooctadecadienoic acid, predominated in tissue and milk. Furthermore, in vitro exposure of bovine mammary endothelial cells to 13-hydroperoxyoctadecadienoic acid, upstream metabolite of HODE, significantly increased cyclooxygenase-2 expression, but 13-HODE exposure had no effect. The findings in the current study indicate lipidomic profiling may explain some of the dynamics of inflammation during bacterial challenge, however continued research is necessary to determine sample compartments which best reflect disease pathogenesis.

Association between interleukin-8 receptor-α (CXCR1) polymorphism and disease incidence, production, reproduction, and survival in Holstein cows.

The objective was to evaluate the association between the single nucleotide polymorphism at position +735 in the interleukin-8 receptor-α (CXCR1) gene (CXCR1c.735) and disease incidence, milk production, reproductive performance, and survival in Holstein cows. Three-hundred fifty Holstein cows were enrolled. No association was found between CXCR1c.735 genotype and retained fetal membranes, metritis, or endometritis. Incidence rate of clinical mastitis was associated with CXCR1c.735 genotype; cows with genotypes CC and GC had a decreased incidence rate of clinical mastitis compared with GG cows. Milk yield was associated with CXCR1c.735 genotype; cows with genotype GC had greater milk yield than GG cows. Hazard of pregnancy was not associated with CXCR1c.735 genotype. Cows that had clinical mastitis had decreased hazard of pregnancy, and cows that had endometritis tended to have a decreased hazard of pregnancy. Hazard of death or culling was not associated with CXCR1c.735 genotype. Multiparous cows and cows that had mastitis had increased hazard of death or culling. In contrast to what we expected, cows with the genotype GG had an increased incidence rate of clinical mastitis and decreased milk yield.

Differential calcium signaling in dairy cows with specific CXCR1 genotypes potentially related to interleukin-8 receptor functionality.

Neutrophil migration and activation are critical components of innate immunity and are mediated by a variety of inflammatory mediators, which include interleukin-8 (IL-8) and epithelial-derived neutrophil activating peptide-78 (ENA-78). Limited knowledge on the expression of receptors for these inflammatory mediators (CXCR1 and CXCR2) in bovine, in addition to the association of a polymorphism (G-->C) in position +777 of the CXCR1 gene with impaired neutrophil function, prompted evaluation of CXCR1 and CXCR2 mRNA and protein expression, ligand binding affinity, and intracellular receptor signaling in neutrophils from cows with different CXCR1 genotypes. Initial observations revealed that overall IL-8 receptor numbers appeared to be lower in cows with a CC genotype compared to cows with a GG genotype. However, in the presence of SB225002, a CXCR2 inhibitor, CXCR1 affinity was about fivefold lower in cows with a CC genotype and may have resulted in an underestimation of receptor numbers in cows with this genotype. In addition, intracellular calcium ([Ca++]i) release was lower in cows with a CC genotype when cells were stimulated with IL-8 but not ENA-78. Furthermore, when neutrophils were stimulated with an optimal dose of IL-8 in the presence of SB225002, [Ca++]i release was lower in cows with a CC genotype, suggesting differential CXCR1 signaling among genotypes. These findings offer knowledge of the role that each of these receptors plays in the inflammatory response in the bovine and provide insight into the potential mechanisms that may be affected in neutrophils of cows with different CXCR1 genotypes.

Association of a bovine CXCR2 gene polymorphism with neutrophil survival and killing ability.

Recent research in our lab has demonstrated a significant association between the incidence of subclinical mastitis and specific polymorphisms of the CXCR2 gene in Holstein dairy cows. This gene encodes a receptor for interleukin-8 (IL-8), a key regulator of neutrophil migration, killing and survival. Because of the importance of this gene in neutrophil function, we hypothesized that differences in neutrophil killing and survival may exist among the CXCR2 genotypes and potentially contribute to the observed variation in intramammary infections. To test this hypothesis, neutrophils were isolated from cows representing each CXCR2 +777 genotype (GG, GC or CC) and tested for suppression of apoptosis, reactive oxygen species (ROS) generation, glutathione levels, and bactericidal activity. A significant increase in survival was observed in neutrophils from cows with a CC genotype when compared to those with a GG genotype in response to IL-8, but not dexamethasone. In contrast, a significant reduction in neutrophil ROS generation in response to phorbol-13-myristate-12 acetate (PMA) was observed in cows with a CC genotype when compared to those with a GG genotype. However, no differences in bactericidal activity or glutathione levels were observed among genotypes. The functional activity of neutrophils from cows heterozygous for this polymorphism was intermediate between those with homozygous genotypes for those assays where differences were observed among homozygous genotypes. In summary, our results suggest that neutrophils from Holstein cows with different CXCR2 genotypes vary in their ability to suppress apoptosis and produce ROS. These differences have the potential to influence overall neutrophil function and may partially explain the variation observed with respect to mastitis in vivo. These results provide a foundation for future research aimed at better understanding the basic differences between dairy cows genetically more or less susceptible to mastitis and has the potential to provide novel preventive and therapeutic measures against inflammatory diseases such as mastitis.

Impaired neutrophil migration associated with specific bovine CXCR2 genotypes.

Bovine mastitis continues to be the most detrimental factor for profitable dairying. Recent research conducted within our laboratory has identified a genetic marker in the CXCR2 gene associated with mastitis susceptibility. The objective of the present study was to evaluate the migratory ability of neutrophils from cows with different CXCR2 +777 genotypes. Neutrophils isolated from peripheral blood of 30 Holstein cows were tested for in vitro migration and adhesion molecule expression. Cows with the CC or GC genotype at CXCR2 +777 showed significantly lower neutrophil migration to recombinant human interleukin-8 (rhIL-8) than cows with the GG genotype (P < 0.05). Cows with the CC genotype at CXCR2 +777 also showed decreased neutrophil migration to zymosan-activated serum compared to these same cows (P < 0.05). Decreased upregulation of CD18 expression was observed after stimulation with rhIL-8 in cows expressing the CXCR2 +777 CC genotype compared to cows expressing the GG genotype (P < 0.05). A similar trend was observed for CD11b (P < 0.10). However, no difference in CD62 downregulation was observed with respect to genotype. These results provide initial evidence for a phenotypic association between a single nucleotide polymorphism and neutrophil function in dairy cows, as well as potential insight into specific mechanisms affected in cows more susceptible to mastitis.

Association of CXCR2 polymorphisms with subclinical and clinical mastitis in dairy cattle.

The ability to identify objectively cows that are more or less susceptible to mastitis has been a long-standing goal. Genetic markers associated with inflammatory responses during mastitis could aid in selection of these cattle. One potential marker is CXCR2, a chemokine receptor required for neutrophil migration to infection sites, which contains single nucleotide polymorphisms (SNP) within the gene. The objective of this experiment was to evaluate the association of CXCR2 SNP genotypes with subclinical and clinical mastitis. Thirty-seven Holstein and 42 Jersey cows that completed at least 2 full lactations were used. Quarter foremilk samples were collected for bacteriological examination quarterly and when cows exhibited clinical mastitis. Subclinical mastitis was defined as the presence of the same pathogen in the same quarter in at least 2 of 3 consecutive samples. A significant association was detected between CXCR2 SNP +777 genotype and percentages of subclinical mastitis cases in Holsteins. Holsteins expressing genotype GG had decreased percentages of subclinical mastitis, but genotype CC cows had increased percentages of subclinical mastitis. Significant differences in clinical mastitis incidence were not detected between genotypes for either breed. This approach of genetically identifying mastitis resistant cows may represent an effective means of marker-assisted selection for mastitis and other inflammatory diseases involving neutrophils.

Novel single nucleotide polymorphisms and haplotypes within the bovine CXCR2 gene.

The objectives of this study were to identify single nucleotide polymorphisms (SNPs) and resulting haplotypes in the bovine CXCR2 gene. A 311-bp segment of the bovine CXCR2 gene was amplified and sequenced. Five SNPs at positions 612, 684, 777, 858, and 861 were expressed in both Holstein and Jersey dairy cattle. Four SNPs resulted in synonymous substitutions, while a non-synonymous switch at position 777 (G-->C) resulted in a glutamine to histidine substitution at amino acid residue 245. Strong linkage disequilibrium was exhibited for both breeds among all five loci (P<0.001). Both allele and genotype frequencies differed significantly with respect to breed at four of the five loci (P<0.001). The five polymorphisms generated ten distinct haplotypes. Six haplotypes were common between the two breeds, while Holsteins and Jerseys each uniquely expressed two haplotypes. Of the six common haplotypes, two represented 83% of the Jersey population; whereas four of these haplotypes represented 95% of the Holstein population.

Dynamics of leukocytes and cytokines during experimentally induced Streptococcus uberis mastitis.

Streptococcus uberis causes a significant proportion of clinical and subclinical intramammary infections (IMI) in lactating and non-lactating dairy cows. In spite of this, its pathogenesis is incompletely understood. A study was conducted to determine leukocyte and cytokine dynamics during experimentally induced S. uberis mastitis. Five Jersey and five Holstein cows were challenged via intramammary inoculation of S. uberis into two uninfected mammary glands. Sixteen of 20 challenged mammary glands developed clinical mastitis with peak clinical signs observed at 144 h. The number of S. uberis in milk increased (P<0.05) 48 h after challenge, in spite of an increase in milk somatic cells that began at 18 h (P<0.001) and remained elevated throughout the study. Increased tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and interleukin-8 (IL-8) in milk were detected 66 h after challenge (P<0.05). Peak TNF-alpha and IL-8 concentrations occurred 120 h after challenge and preceded peak clinical signs. Experimental S. uberis IMI induced local production of TNF-alpha, IL-1beta and IL-8, which may play a role in the pathogenesis of S. uberis mastitis. Other mediators may be involved in initial leukocyte recruitment to the mammary gland, since increases in milk somatic cells occurred earlier than cytokine production.

Therapeutic treatment of DMBA-induced mammary tumors with PPAR ligands.

The objective of this study was to evaluate the ability of troglitazone (a thiazolidinedione) and Wy-14,643 (a clofibrate) to inhibit progression of non-detectable and detectable mammary tumors in rats induced by 7,12 dimethylbenz(a)anthracene (DMBA) when compared to those receiving no treatment or tamoxifen. Although not as effective as tamoxifen in decreasing overall tumor incidence, Wy-14,643 reduced the percentage and number of malignant tumors that developed when compared to both troglitazone and control. Treatment of detectable tumors with either Wy-14,643 or troglitazone induced regression or stasis of total tumor volume in 40-50% of the animals, compared to only 10% in control and 65% in tamoxifen treated animals. Moreover, each PPAR ligand was as effective as tamoxifen in preventing additional tumor development. In summary, both PPAR ligands were more effective than no treatment in preventing tumor progression once detected. However, only the PPAR-alpha activator, Wy-14,643 was able to reduce the development of malignant tumors when administered prior to detection.

Selenium and vitamin E deficiency impair transferrin receptor internalization but not IL-2, IL-2 receptor, or transferrin receptor expression.

Vitamin E and Se deficiency increase the risk of disease by impairing the immune response. To aid in the understanding of how vitamin E and Se deficiency reduce immune competence, this study examined several mechanisms necessary for lymphocyte proliferation. Weanling rats were fed a vitamin E-deficient, selenium-deficient, or control diet for 8 weeks. At this time splenic mononuclear cells were isolated and stimulated with concanavalin A for 48 h. Although the percentage of lymphocytes and monocytes capable of proliferating were consistent among the dietary groups, lymphocyte proliferation was decreased significantly in vitamin E- and selenium-deficient rats. This decrease in proliferation was not associated with alterations in interleukin-2, interleukin-2 receptor, or transferrin receptor expression. However, stimulated cells from vitamin E- and Se-deficient rats internalized few if any transferrin receptors. Reduced transferrin receptor internalization may limit lymphocyte expansion by depleting the intracellular iron stores needed for cellular function and proliferation.

Expression of peroxisome proliferator activated receptor mRNA in normal and tumorigenic rodent mammary glands.

The peroxisome proliferator activated receptors (PPARs) alpha, beta/delta, and gamma are novel nuclear hormone receptors activated by long chain fatty acids and synthetic ligands and which regulate lipid metabolism. Recent studies have detected PPARgamma mRNA in human mammary tumor cell lines. The current study examined the expression profile of PPAR mRNAs in normal and malignant rodent mammary tissues. Virgin murine mammary glands contained PPAR alpha, beta/delta, and gamma mRNAs based on northern blot analysis. The PPARgamma isoform was predominantly gamma2 based on quantitative PCR analysis. During pregnancy and lactation, the PPARalpha and gamma mRNAs decreased while the PPAR beta/delta mRNA remained relatively unchanged. NMuMG cells, an epithelial line derived from normal murine mammary gland, expressed PPAR alpha, beta/delta, and gamma mRNAs, independent of the presence or absence of compounds modifying PPAR activity. In rats, the physiologic expression pattern of PPARgamma mRNA paralleled the murine model; levels were detected in virgin but not lactating mammary glands. In addition, the PPARgamma mRNA was not detected in several histologically distinct 7,12-dimethylbenz(a)anthracene induced mammary tumors. These findings suggest that PPARs may regulate mammary epithelial and stromal cell function in response to physiologic or pathologic stimuli that profoundly alter lipid metabolism.

Diminished mammary gland lymphocyte functions parallel shifts in trafficking patterns during the postpartum period.

Once activated, lymphocytes can regulate both specific and nonspecific immune responses. Alterations in lymphocyte function may increase the host's vulnerability to bacterial infections such as mastitis. Susceptibility to mastitis as well as diminished leukocyte functional capabilities have been shown to be influenced by lactational stage. Therefore, the present study characterized the phenotypes and functions of several bovine lymphoid populations at two points in the lactational cycle. Mononuclear cells were isolated from peripheral blood, supramammary lymph nodes, and mammary parenchyma of mid-lactating and postpartum dairy cows. The phenotypic composition, proliferative ability, cytokine secretion, and cytotoxic activity of isolated leukocytes were assessed with respect to lactational stage and tissue source. Lower percentages of T lymphocytes were consistent with diminished mitogen-stimulated proliferation and spontaneous cytotoxic activity by lymphocytes isolated from postpartum compared with mid-lactating animals. Stimulation with interleukin-2 did not enhance the cytotoxic activity or proliferative ability of lymphocytes isolated postpartum to similar levels observed for those isolated from mid-lactating animals. These data indicate that certain diminished lymphocyte functions observed during the postpartum period may result from shifts in leukocyte trafficking patterns.

Specific immune responses of dairy cattle after primary inoculation with recombinant bovine interferon-gamma as an adjuvant when vaccinating against mastitis.

OBJECTIVE: To examine whether specific immunity in the mammary gland and blood of dairy cattle was enhanced after primary inoculation with a test antigen keyhole limpet hemocyanin (KLH) and recombinant bovine interferon-gamma (rBoIFN-gamma) as an adjuvant. DESIGN: Holstein dairy cows received IM injections of KLH in conjunction with saline solution (n = 4), Freund's incomplete adjuvant (FIA; n = 4), or rBoIFN-gamma (n = 3). Milk and blood samples were collected during a 1-month period and analyzed for KLH antibody content. Isolated blood mononuclear cells were examined for their ability to proliferate and produce interleukin 2 (IL-2) after mitogen and/or KLH stimulation in vitro. The phenotype of isolated blood mononuclear cells also was characterized through flow cytometric analysis. RESULTS: The adjuvant rBoIFN-gamma induced serum KLH antibody titers similar to those induced by administration of FIA. However, FIA induced significantly more KLH antibodies in milk. Administration of rBoIFN-gamma enhanced T-lymphocyte activity, as indicated by the greater expression of high-affinity IL-2 receptors and the increased response to the mitogens concanavalin A, phytohemagglutinin, and IL-2, compared with FIA or saline treatment. Lymphocyte and monocyte movement from the blood also was altered after rBoIFN-gamma treatment, which can have a profound influence on secondary immune responses, such as antibody production. CONCLUSIONS: rBoIFN-gamma may safely enhance specific immunity in the bovine mammary gland and may be an effective adjuvant in mastitis immunization protocols.

Enhanced production of bovine tumor necrosis factor-alpha during the periparturient period.

This study examined the production of tumor necrosis factor (TNF-alpha) by mononuclear cells isolated from peripheral blood and supramammary lymph nodes of periparturient and mid to late lactating dairy cows. Monocyte-enriched cell populations were stimulated with lipopolysaccharide (LPS) and analyzed for TNF-alpha concentrations. Flow cytometric analysis was performed to determine the frequencies of relevant cell populations. Isolated mononuclear cells from periparturient dairy cows produced significantly higher levels of TNF-alpha than mid to late lactating dairy cows regardless of tissue location. A corresponding increase in the frequency of monocytes also was observed in tissue samples obtained from periparturient animals. The higher proportion of monocytes capable of producing TNF-alpha in the periparturient dairy cow may account for the increased levels of this potent mediator. Within the periparturient period, peripheral blood mononuclear cells were found to produce significantly less TNF-alpha than cells isolated from mammary lymph nodes. However, flow cytometric analysis revealed similar monocyte concentrations in both the peripheral blood and mammary lymph node. This indicates that the differences in cytokine production may be due to variations in monocyte activation state with respect to tissue location. It is possible that greater potential to produce TNF-alpha during the periparturient period may contribute to the severe acute phase response of the mammary gland to coliform infections during this time. Limiting TNF-alpha production by monocytes, particularly within the mammary gland, may reduce the severity of clinical coliform mastitis in periparturient dairy cattle.

Enhanced antigen-specific responses in bovine mammary glands following administration of interleukin-2.

This study examined the feasibility of using interleukin-2 as a vaccine adjuvant to enhance specific immunity in the bovine mammary gland. In trial 1, cows were immunized by intramammary infusion of keyhole limpet hemocyanin with either saline (n = 3) or interleukin-2 (n = 3) as adjuvants. In trial 2, cows were immunized by intramuscular injection near the supramammary lymph node with keyhole limpet hemocyanin in conjunction with saline (n = 4). Freund's incomplete adjuvant (n = 4), or interleukin-2 (n = 4). Local immunization with interleukin-2 as an adjuvant significantly increased antibody titers in milk over preimmunization levels and levels in saline-treated cows. The use of Freund's incomplete adjuvant and interleukin-2 as adjuvants in cows that were immunized systemically enhanced both sera and lacteal antibodies to keyhole limpet hemocyanin. However, cows that were administered interleukin-2 responded more quickly than those given Freund's incomplete adjuvant. Only those cows that received interleukin-2 as an adjuvant demonstrated significant increases in blood lymphocyte proliferation to keyhole limpet hemocyanin, pokeweed mitogen, and interleukin-2. The results of these two trials suggest that immunization with interleukin-2 as an adjuvant may be more effective than Freund's incomplete adjuvant in enhancing specific immunity in the bovine mammary gland and may possibly be an effective adjuvant in mastitis immunization protocols.

Altered interleukin-2 production by lymphocyte populations from bovine leukemia virus-infected cattle.

The effects of bovine leukosis virus (BLV) on the phenotypic and functional characteristics of peripheral blood mononuclear cells were investigated. Whole blood differentials showed that persistently lymphocytotic (BLV+PL) dairy cattle had more lymphocytes and fewer neutrophils than the aleukemic seropositive (BLV+AL) or seronegative (BLV-) animals. Flow cytometric analyses of peripheral blood mononuclear cells indicated that the BLV+PL animals had more B lymphocytes, with a concomitant decrease in CD2 positive cells when compared with the BLV- group. Mononuclear cells from the BLV+AL animals also had fewer CD2 positive cells, but no differences in B lymphocytes were observed when compared with BLV- cattle. Peripheral blood mononuclear cells were used in blastogenesis assays to assess the functional ability of lymphocytes. Lymphocytes from BLV+PL animals had lower proliferative responses to concanavalin A and pokeweed mitogen when compared with cells from the BLV- or BLV+AL groups. The level of spontaneous blastogenesis in the absence of mitogenic stimulation was high for lymphocytes obtained from BLV+AL cattle. Cultures of lymphocytes obtained from BLV+PL animals produced greater amounts of interleukin-2 (IL-2) than BLV+AL and BLV- groups, although no differences were observed in the expression of IL-2 receptors. The development of uncontrolled lymphocytosis in BLV-infected cattle may result from an altered responsiveness to IL-2-regulated B-lymphocyte proliferation.