First characterization of major histocompatibility complex class II DRB3 diversity in cattle breeds raised in Egypt.

Genes encoding bovine leukocyte antigen (BoLA) enable the immune system to identify pathogens. Therefore, these genes have been used as genetic markers for infectious and autoimmune diseases as well as for immunological traits in cattle. Although BoLA polymorphisms have been reported in various cattle breeds worldwide, they have not been studied in cattle populations in Egypt. In this study, we characterized BoLA-DRB3 in two local Egyptian populations and one foreign population using polymerase chain reaction-sequence-based typing (PCR-SBT) method. Fifty-four previously reported BoLA-DRB3 alleles and eight new alleles (BoLA-DRB3*005:08, *015:07, *016:03, *017:04, *020:02:02, *021:03, *164:01, and *165:01) were identified. Alignment analysis of the eight new alleles revealed 90.7-98.9 %, and 83.1-97.8 % nucleotide and amino acid identities, respectively, with the BoLA-DRB3 cDNA clone NR-1. Interestingly, BoLA-DRB3 in Egyptian cattle showed a high degree of allelic diversity in native (na = 28, hE > 0.95), mixed (na = 61, hE > 0.96), and Holstein (na = 18, hE > 0.88) populations. BoLA-DRB3*002:01 (14.3 %), BoLA-DRB3*001:01 (8.5 %), and BoLA-DRB3*015:01 (20.2 %) were the most frequent alleles in native, mixed, and Holstein populations, respectively, indicating that the genetic profiles differed in each population. Based on the allele frequencies of BoLA-DRB3, genetic variation among Egyptian, Asian, African, and American breeds was examined using Nei's distances and principal component analysis. The results suggested that native and mixed cattle populations were most closely associated with African breeds in terms of their gene pool, whereas Holstein cattle were more distinct from the other breeds and were closely related to Holstein cattle populations from other countries.

Single-cell transcriptomic and chromatin accessibility analyses of dairy cattle peripheral blood mononuclear cells and their responses to lipopolysaccharide.

BACKGROUND: Gram-negative bacteria are important pathogens in cattle, causing severe infectious diseases, including mastitis. Lipopolysaccharides (LPS) are components of the outer membrane of Gram-negative bacteria and crucial mediators of chronic inflammation in cattle. LPS modulations of bovine immune responses have been studied before. However, the single-cell transcriptomic and chromatin accessibility analyses of bovine peripheral blood mononuclear cells (PBMCs) and their responses to LPS stimulation were never reported. RESULTS: We performed single-cell RNA sequencing (scRNA-seq) and single-cell sequencing assay for transposase-accessible chromatin (scATAC-seq) in bovine PBMCs before and after LPS treatment and demonstrated that seven major cell types, which included CD4 T cells, CD8 T cells, and B cells, monocytes, natural killer cells, innate lymphoid cells, and dendritic cells. Bioinformatic analyses indicated that LPS could increase PBMC cell cycle progression, cellular differentiation, and chromatin accessibility. Gene analyses further showed significant changes in differential expression, transcription factor binding site, gene ontology, and regulatory interactions during the PBMC responses to LPS. Consistent with the findings of previous studies, LPS induced activation of monocytes and dendritic cells, likely through their upregulated TLR4 receptor. NF-κB was observed to be activated by LPS and an increased transcription of an array of pro-inflammatory cytokines, in agreement that NF-κB is an LPS-responsive regulator of innate immune responses. In addition, by integrating LPS-induced differentially expressed genes (DEGs) with large-scale GWAS of 45 complex traits in Holstein, we detected trait-relevant cell types. We found that selected DEGs were significantly associated with immune-relevant health, milk production, and body conformation traits. CONCLUSION: This study provided the first scRNAseq and scATAC-seq data for cattle PBMCs and their responses to the LPS stimulation to the best of our knowledge. These results should also serve as valuable resources for the future study of the bovine immune system and open the door for discoveries about immune cell roles in complex traits like mastitis at single-cell resolution.

Development and Characterization of New Monoclonal Antibodies Against Porcine Interleukin-17A and Interferon-Gamma.

Current research efforts require a broad range of immune reagents, but those available for pigs are limited. The goal of this study was to generate priority immune reagents for pigs and pipeline them for marketing. Our efforts were aimed at the expression of soluble swine cytokines and the production of panels of monoclonal antibodies (mAbs) to these proteins. Swine interleukin-17A (IL-17A) and Interferon-gamma (IFNγ) recombinant proteins were produced using yeast expression and used for monoclonal antibody (mAb) production resulting in panels of mAbs. We screened each mAb for cross-species reactivity with orthologs of IL-17A or IFNγ and checked each mAb for inhibition by other related mAbs, to assign mAb antigenic determinants. For porcine IL-17A, the characterization of a panel of 10 mAbs identified eight different antigenic determinants; interestingly, most of the mAbs cross-reacted with the dolphin recombinant ortholog. Likewise, the characterization of a panel of nine anti-PoIFNγ mAbs identified four different determinants; most of the mAbs cross-reacted with dolphin, bovine, and caprine recombinant orthologs. There was a unique reaction of one anti-PoIFNγ mAb that cross-reacted with the zebrafish recombinant ortholog. The αIL-17A mAbs were used to develop a quantitative sandwich ELISA detecting the yeast expressed protein as well as native IL-17A in stimulated peripheral blood mononuclear cell (PBMC) supernatants. Our analyses showed that phorbol myristate acetate/ionomycin stimulation of PBMC induced significant expression of IL-17A by CD3+ T cells as detected by several of our mAbs. These new mAbs expand opportunities for immunology research in swine.

Effects of intra-articular inoculation with Mycoplasma bovis on immunological responses in calf joints.

Mycoplasma arthritis that caused by Mycoplasma bovis exhibit severe lameness. This disease is difficult to cure with antibiotics, but the detailed pathological mechanisms have not been fully clarified. In this study, we examined the effects of intra-articular inoculation with M. bovis on immunological responses in calf joints. We inoculated three calves each with M. bovis or phosphate buffer saline (control) into the right stifle joint and dissected them at 15 days postinoculation. Mycoplasma bovis-inoculated calves exhibited swelling of the stifle joint, increases in synovial fluid, fibrin deposition, and cartilage thinning. Intracellular M. bovis was detected in synovial tissues analyzed by immunohistochemistry and transmission electron microscopy. Messenger RNA expressions of interleukin (IL)-1ß, IL-6, IL-8, IL-12p40, and IL-17A in synovial fluid cells and synovial tissues from M. bovis-inoculated calves were significantly higher than those from control calves. Protein levels of these cytokines in synovial fluid from M. bovis-inoculated calves were markedly higher than those from control calves. Our study clarified that inoculation with M. bovis into the stifle joint induced the production of inflammatory cytokines by synovial fluid cells and synovial tissues, causing a severe inflammatory response in joints. Additionally, M. bovis could invade cells in synovial tissues, which may have aided it in evading antibiotics and host immune surveillance.

Preliminary Evidence of Endotoxin Tolerance in Dairy Cows during the Transition Period.

The blastogenic response of bovine peripheral blood mononuclear cells (PBMCs) to lipopolysaccharides (LPS) has been investigated for a long time in our laboratories. In particular, a possible correlation between the blastogenic response to LPS and the disease resistance of dairy cows has been suggested in previous studies. Isolated PBMCs from eight cows at three different time points during the transition period (T0 = 15 days before calving; T1 = 7 days post-calving; T2 = 21 days post-calving) were cultured in the presence or absence of LPS, and the blastogenic response was assayed 72 h after in vitro stimulation. Moreover, the gene expression of proinflammatory cytokines and kynurenine pathway molecules was investigated by real-time RT-PCR on both unstimulated and stimulated PBMCs. The cows were retrospectively divided into healthy and diseased, based on the development of peripartum diseases (subclinical ketosis and placenta retention). The comparison between healthy and diseased cows suggested that healthy animals seemed to better control the response to LPS. On the contrary, diseased animals showed a much higher inflammatory response to LPS. Moreover, cows were retrospectively classified as high and low responders based on the in vitro proliferative response of PBMCs to LPS, using the median value as a threshold. Unstimulated PBMCs of low responders showed higher expression of the proinflammatory cytokines Interleukin 1-ß (IL-1ß), Interleukin 6 (IL-6) and Tumor Necrosis Factor-α (TNF-α), compared to high responders. Our preliminary data suggest that, during the peripartum period, high responders seem to be more tolerant to endotoxins and develop a lower inflammatory response to different stressors. Instead, low responders could be more prone to the development of unwanted inflammatory conditions in response to mild/moderate stressors.

Effects of Deoxynivalenol and Fumonisins Fed in Combination to Beef Cattle: Immunotoxicity and Gene Expression.

We evaluated the effects of a treatment diet contaminated with 1.7 mg deoxynivalenol and 3.5 mg fumonisins (B1, B2 and B3) per kg ration on immune status and peripheral blood gene expression profiles in finishing-stage Angus steers. The mycotoxin treatment diet was fed for a period of 21 days followed by a two-week washout period during which time all animals consumed the control diet. Whole-blood leukocyte differentials were performed weekly throughout the experimental and washout period. Comparative profiles of CD4+ and CD8+ T cells, along with bactericidal capacity of circulating neutrophils and monocytes were evaluated at 0, 7, 14, 21 and 35 days. Peripheral blood gene expression was measured at 0, 7, 21 and 35 days via RNA sequencing. Significant increases in the percentage of CD4-CD8+ T cells were observed in treatment-fed steers after two weeks of treatment and were associated with decreased CD4:CD8 T-cell ratios at this same timepoint (p ≤ 0.10). No significant differences were observed as an effect of treatment in terms of bactericidal capacity at any timepoint. Dietary treatments induced major changes in transcripts associated with endocrine, metabolic and infectious diseases; protein digestion and absorption; and environmental information processing (inhibition of signaling and processing), as evaluated by dynamic impact analysis. DAVID analysis also suggested treatment effects on oxygen transport, extra-cellular signaling, cell membrane structure and immune system function. These results indicate that finishing-stage beef cattle are susceptible to the immunotoxic and transcript-inhibitory effects of deoxynivalenol and fumonisins at levels which may be realistically encountered in feedlot situations.

Low serum vitamin D concentrations in Spring-born dairy calves are associated with elevated peripheral leukocytes.

A role for vitamin D in the immune system is emerging from human research but data in the bovine is limited. In the current study, 48 Holstein-Friesian calves were randomly assigned to one of 4 groups designed to expose calves to divergent vitamin D levels for a 7 month period and to determine its effects on circulating immunity in young calves. Concentrations of circulating 25-hydroxyvitamin D (25OHD) was measured in serum using a commercial ELISA with validated bovine standards. Results showed that mean circulating concentrations of 25OHD at birth was 7.64 ± 3.21 ng/ml indicating vitamin D deficiency. Neither the injection of Vit D3 at birth nor the elevated levels in milk replacer yield discernible changes to pre-weaning circulating concentration of 25OHD. No calf reached the recommended level of vitamin D immune sufficiencyof 30 ng/ml of 25OHD until at least 3 months of age (T4). Increasing dietary Vit D3 via ration in the post-weaning period significantly elevated 25OHD concentrations in serum in VitD-In calves. Maximal levels of circulating 25OHD were achieved in VitD-Out calves, reaching 60.86 ± 7.32 ng/ml at 5 months of age (T7). Greatest divergence in haematology profile was observed between Ctl-In vs VitD-In groups with Ctl-In calves showing an elevated count of neutrophils, eosinophils, and basophils associated with reduced 25OHD concentrations. Neither IL-8 expression nor ROS production in serum were significantly different between calves with high and low 25OHD, indicating that other vitamin D-dependent mechanisms may contribute to the divergent circulating cellular profiles observed. This novel data on the vitamin D status of neonatal calves identifies a significant window of vitamin D insufficiency which is associated with significant differences in circulating immune cell profiles. Vitamin D insufficiency may therefore exacerbate pre-weaning disease susceptibility, and further work in now warranted.

Bovine innate immune phenotyping via a standardized whole blood stimulation assay.

Cattle vary in their susceptibility to infection and immunopathology, but our ability to measure and longitudinally profile immune response variation is limited by the lack of standardized immune phenotyping assays for high-throughput analysis. Here we report longitudinal innate immune response profiles in cattle using a low-blood volume, whole blood stimulation system-the ImmunoChek (IChek) assay. By minimizing cell manipulation, our standardized system minimizes the potential for artefactual results and enables repeatable temporal comparative analysis in cattle. IChek successfully captured biological variation in innate cytokine (IL-1ß and IL-6) and chemokine (IL-8) responses to 24-hr stimulation with either Gram-negative (LPS), Gram-positive (PamCSK4) bacterial or viral (R848) pathogen-associated molecular patterns (PAMPs) across a 4-month time window. Significant and repeatable patterns of inter-individual variation in cytokine and chemokine responses, as well as consistent high innate immune responder individuals were identified at both baseline and induced levels. Correlation coefficients between immune response read-outs (IL-1ß, IL-6 and IL-8) varied according to PAMP. Strong significant positive correlations were observed between circulating monocytes and IL-6 levels for null and induced responses (0.49-0.61) and between neutrophils and cytokine responses to R848 (0.38-0.47). The standardized assay facilitates high-throughput bovine innate immune response profiling to identify phenotypes associated with disease susceptibility and responses to vaccination.

Effect of in-vitro heat stress challenge on the function of blood mononuclear cells from dairy cattle ranked as high, average and low immune responders.

BACKGROUND: The warming climate is causing livestock to experience heat stress at an increasing frequency. Holstein cows are particularly susceptible to heat stress because of their high metabolic rate. Heat stress negatively affects immune function, particularly with respect to the cell-mediated immune response, which leads to increased susceptibility to disease. Cattle identified as having enhanced immune response have lower incidence of disease. Therefore, the objective of this study was to evaluate the impact of in vitro heat challenge on blood mononuclear cells from dairy cattle, that had previously been ranked for immune response, in terms of heat shock protein 70 concentration, nitric oxide production, and cell proliferation. RESULTS: Blood mononuclear cells from dairy cattle classified as high immune responders, based on their estimated breeding values for antibody and cell-mediated responses, produced a significantly greater concentration of heat shock protein 70 under most heat stress treatments compared to average and low responders, and greater cell-proliferation across all treatments. Similarly, a trend was observed where high responders displayed greater nitric oxide production compared to average and low responders across heat treatments. CONCLUSION: Overall, these results suggest that blood mononuclear cells from high immune responder dairy cows are more thermotolerant compared to average and low immune responders.

Bovine Immunology: Implications for Dairy Cattle.

The growing world population (7.8 billion) exerts an increased pressure on the cattle industry amongst others. Intensification and expansion of milk and beef production inevitably leads to increased risk of infectious disease spread and exacerbation. This indicates that improved understanding of cattle immune function is needed to provide optimal tools to combat the existing and future pathogens and improve food security. While dairy and beef cattle production is easily the world's most important agricultural industry, there are few current comprehensive reviews of bovine immunobiology. High-yielding dairy cattle and their calves are more vulnerable to various diseases leading to shorter life expectancy and reduced environmental fitness. In this manuscript, we seek to fill this paucity of knowledge and provide an up-to-date overview of immune function in cattle emphasizing the unresolved challenges and most urgent needs in rearing dairy calves. We will also discuss how the combination of available preventative and treatment strategies and herd management practices can maintain optimal health in dairy cows during the transition (periparturient) period and in neonatal calves.

Expression characteristics of immune factors in the yak (Bos grunniens) testis.

The goal of this study was to characterize and evaluate the main markers of macrophages, T lymphocytes, B lymphocytes and plasmocytes in the testis of juvenile and adult yaks by quantitative real-time polymerase chain reaction and immunohistochemistry. Within the same age group, the mRNA expression of CD68 was always highest, followed by that of CD3ε, CD79α, IgG and IgA. Moreover, CD68, CD3, CD79α, IgA and IgG positive cells were all located in the testicular interstitial tissues of juvenile and adult yaks. In the same age group, the frequency of CD68 positive macrophages was higher than that of CD3 positive T lymphocytes, which was followed by that of CD79α positive B lymphocytes and IgA and IgG positive plasmocytes. No significant difference was observed between the B lymphocyte and plasmocyte frequencies in yak testes. Furthermore, CD68, CD3ε, CD79α, IgA and IgG mRNA expression levels and the frequencies of CD68, CD3, CD79α, IgA and IgG positive cells increased from juveniles to adults. Similarly, the frequencies of CD68, CD3, CD79α, IgA and IgG positive cells also increased with age. These results suggest that in the yak testis, the immune defence system against pathogens might primarily comprise macrophages and T lymphocytes in the testicular interstitial tissue. Moreover, the testicular immune environment may mature and expand to a fully functional state in adult yaks. The low frequencies of B lymphocyte and plasmocyte in yaks, differing from those in rodents and humans, might be related to the fact that yaks live in low-oxygen plateaus.

Development of a Potential Yeast-Based Vaccine Platform for Theileria parva Infection in Cattle.

East Coast Fever (ECF), caused by the tick-borne apicomplexan parasite Theileria parva, remains one of the most important livestock diseases in sub-Saharan Africa with more than 1 million cattle dying from infection every year. Disease prevention relies on the so-called "Infection and Treatment Method" (ITM), which is costly, complex, laborious, difficult to standardise on a commercial scale and results in a parasite strain-specific, MHC class I-restricted cytotoxic T cell response. We therefore attempted to develop a safe, affordable, stable, orally applicable and potent subunit vaccine for ECF using five different T. parva schizont antigens (Tp1, Tp2, Tp9, Tp10 and N36) and Saccharomyces cerevisiae as an expression platform. Full-length Tp2 and Tp9 as well as fragments of Tp1 were successfully expressed on the surface of S. cerevisiae. In vitro analyses highlighted that recombinant yeast expressing Tp2 can elicit IFNγ responses using PBMCs from ITM-immunized calves, while Tp2 and Tp9 induced IFNγ responses from enriched bovine CD8+ T cells. A subsequent in vivo study showed that oral administration of heat-inactivated, freeze-dried yeast stably expressing Tp2 increased total murine serum IgG over time, but more importantly, induced Tp2-specific serum IgG antibodies in individual mice compared to the control group. While these results will require subsequent experiments to verify induction of protection in neonatal calves, our data indicates that oral application of yeast expressing Theileria antigens could provide an affordable and easy vaccination platform for sub-Saharan Africa. Evaluation of antigen-specific cellular immune responses, especially cytotoxic CD8+ T cell immunity in cattle will further contribute to the development of a yeast-based vaccine for ECF.

Subpopulations of swine γδ T cells defined by TCRγ and WC1 gene expression.

γδ T cells constitute a major portion of lymphocytes in the blood of both ruminants and swine. Subpopulations of swine γδ T cells have been distinguished by CD2 and CD8α expression. However, it was not clear if they have distinct expression profiles of their T-cell receptor (TCR) or WC1 genes. Identifying receptor expression will contribute to understanding the functional differences between these subpopulations and their contributions to immune protection. Here, we annotated three genomic assemblies of the swine TCRγ gene locus finding four gene cassettes containing C, J and V genes, although some haplotypes carried a null TRGC gene (TRGC4). Genes in the TRGC1 cassette were homologs of bovine TRGC5 cassette while the others were not homologous to bovine genes. Here we evaluated three principal populations of γδ T cells (CD2+/SWC5-, CD2-/SWC5+, and CD2-/SWC5-). Both CD2- subpopulations transcribed WC1 co-receptor genes, albeit with different patterns of gene expression but CD2+ cells did not. All subpopulations transcribed TCR genes from all four cassettes, although there were differences in expression levels. Finally, the CD2+ and CD2- γδ T-cell populations differed in their representation in various organs and tissues, presumably at least partially reflective of different ligand specificities for their receptors.

Prevalence of failure of passive transfer of immunity in dairy calves in a Mediterranean pasture-based production system of the south-west region of Western Australia.

The objective of this study was to estimate the prevalence of failure of passive transfer of immunity (FPTI) in dairy calves in the south-west region of Western Australia herds. A cross-sectional study was conducted in 26/140 dairy farms and serum samples were collected from 495 healthy 2-7 day-old calves. A radial immunodiffusion (RID) test was used to determine the concentration of serum IgG and calves were classified as having FPTI if the IgG concentration was less than 10 mg/mL. Estimation of FPTI was also assessed using two indirect methods using serum total protein (STP) and a brix refractometer. The estimated prevalence of FPTI was found to be 8.7% (43 calves out of 495) by RID with the concentration of IgG ranging between 0 and 6.2 mg/ml. The STP was found to vary from 46 to 96 mg/mL and using a cut-off point of 55 mg/mL the calf level prevalence was estimated as 7.1% (33 calves). Using the brix refractometer, the prevalence was found to be 13.1% (65 calves) with the refractometer reading ranging 6-14% of IgG. In the present study there was no association between calf-level factors (age, sex and breed) and FPTI. There was a higher correlation of the RID test results and the STP results compared to the RID and brix refractometer results. It is concluded that the prevalence of FPTI in dairy calves in the south-west region of Western Australia is low (8.7%) and the brix refractometer is not a reliable indirect method for determining passive transfer of immunity to calves.

Weaning at 30, 75 and 180 days: Comparison between immune responses of beef calves.

The interruption of the maternal-filial bond in young calves can alter the physiological responses due to increased stress at this moment. Therefore, we aimed to analyze the association between age at weaning and the immune responses of 64 beef calves weaned abruptly. The animals were distributed in three weaning (W) treatments: hyper-early (W30), early (W75) and conventional (W180). The treatments agree with the weaning ages used in cow-calf systems in South America. Blood samples were collected of all experimental animals on the day of weaning and days 1, 2 and 7 post-weaning day of the three treatments. Blood samples were used to analyze hematological variables, total plasma proteins (TPP), fibrinogen, serum amyloid A protein (SAA) and cortisol. The variables analyzed were influenced by the sampling day (P < 0.001) and the calf gender (P < 0.05), and an interaction between treatment x sampling day was observed. Over the study period, high serum cortisol levels were observed for W30 calves. The serum amyloid A protein was significantly influenced by sampling day. When compared to W180 calves, W30 and W75 animals showed prolonged concentrations in plasma fibrinogen. In addition, they presented neutrophilia and lymphopenia during the 180-187 days period, which resulted in a greater neutrophil:lymphocyte ratio (N:L ratio). Calf gender resulted in changes in the number of blood leukocytes (P < 0.05). This result provides an alert for weaning at an early age, indicating that different management strategies must be tested to minimize physiological instabilities in critical periods of the life of animals.

Peripheral leucocyte molecular indicators of inflammation and oxidative stress are altered in dairy cows with embryonic loss.

Objective of experiment was to determine whether oxidative stress (OS) and inflammation altered embryonic loss in dairy cows. Blood samples were collected at days 0, 16, 32 and 60 after timed (AI) from 200 Holstein cows to determine embryonic loss based on interferon-stimulated gene-15 (ISG15) mRNA expression (day 16) and ultrasound at day 32 and day 60. Leucocyte expressions of mRNA TLR2, TLR4, TNF-α, IL1B, IL10, STAT3 (inflammation), PTGS2, PTGES (prostaglandin synthesis), and PLA2G4A and ALOX5AP (eicosanoid metabolism) at days 0 and 16 were determined. Plasma redox status for antioxidant enzymatic activities of glutathione peroxidase (GPX), superoxide dismutase (SOD), total antioxidant capacity (TAC), and concentrations of malondialdehyde (MDA) were determined at days 0, 16, 32 and 60. All antioxidant-redox responses were beneficially significant in pregnant cows diagnosed pregnant at day16 and sustained pregnancy to day 60 compared to non-pregnant cows at day16 or pregnant at day16 and lost embryos by days 32 or 60. The leucocyte mRNA expressions of TLR2, TLR4, STAT 3, IL1B, PTGS2, PLA2G4A and ALOX5AP were greater and PTGES was lower at day16 in pregnant cows that lost embryos early (P < 0.05). In conclusion peripheral leucocyte molecular indicators of inflammation and plasma indicators of OS were altered in pregnant cows undergoing embryonic losses compared to cows with a sustained pregnancy.

Effects of an oral hydro-ethanolic purple coneflower extract on performance, clinical health and immune parameters in calves.

The objective of this randomized, placebo-controlled, double-blinded field trial was to investigate the effects of oral administration of purple coneflower (Echinacea purpurea L. (EP)) on performance, health and immune parameters in calves. Calves (n = 27) were enrolled to three groups (9 calves per group): 0.5 g EP/calf per day (ECL), 5 g EP/calf per day (ECH) or placebo. Calves were vaccinated with Bluetongue-Virus (BTV) serotype 4 vaccine to investigate EPs effects on seroconversion. Clinical and performance parameters, inter alia body weight, health and milk intake were recorded for 57 days. Blood samples were analyzed for BTV antibodies and IgG by ELISA, white and red blood cell counts by flow cytometry and mRNA abundance of various inflammatory markers in leukocytes (IL-1ß, IL-8, tumor necrosis factor α (TNFα), cyclooxygenase 2 (Cox-2) and prostaglandin E synthase) was studied. The findings demonstrated no differences between groups regarding performance parameters. In all groups, calves suffered from diarrhea for a minimum of 2 days, but EP reduced the number of diarrhea days by 44% in ECL and increased the body temperature. Interestingly, ECL resulted in an increased number of respiratory disease days during the follow-up period. EP did not change blood cell and IgG counts, whereas eosinophil granulocytes were reduced in ECL. Decreased levels of hemoglobin and hematocrit were found in ECH. Prostaglandin E synthase levels in leukocytes were higher in ECL and ECH, whereas no differences were obtained for IL-1ß, IL-8, TNFα and Cox-2. Due to the unexpected occurrence of BTV seropositive calves before the first vaccination, 13 calves were excluded from the evaluation on seroconversion and no statistical analyses could be performed regarding antibody production. BTV-4 antibodies were not produced in 4 placebo-calves, whereas 4 of 5 and 1 of 6 ECL- and ECH-calves produced antibodies. Further investigations are needed to draw final conclusions on mode of action and efficacy of EP in calves.

Effect of IL8 haplotype on immunological traits in periparturient dairy cows.

Interleukin 8 (IL8) is a major mediator of the innate immune response. Polymorphisms in this gene are associated with susceptibility to inflammatory disease in humans. Two major promoter polymorphic haplotypes (IL8-h1 and IL8-h2) segregating in cattle populations have shown a significant effect on the immune response profile in calves but their implications for transition cow immunity have not been established. The aims of this study were to assess functional relevance of the IL8 haplotypes on the immunological traits of periparturient cows (n = 32) belonging to three genetic groups: Holstein (HO), Simmental (SI) and their crosses (CR) and to evaluate the frequency of IL8 haplotypes in the HO (dairy) and SI (dual purpose) pure breeds. IL8 haplotypes showed a significant effect on circulating number of both T helper lymphocytes (P = 0.0133) and T cytotoxic lymphocytes (P = 0.0024). Differences in percentage of CD14+ monocytes and T lymphocyte subsets were found between haplotype groups at different time points. Plasma concentrations of Serum Amyloid A (SAA) and Haptoglobin (Hp) were enhanced at calving in IL8-h2 (P = 0.0019, P = 0.0029) and IL8-het (P = 0.050 and P = 0.052) respectively, compared with IL8-h1 cows. In contrast, significantly lower levels of reactive oxygen metabolites (d-ROMs) activation were identified in IL8-h2 and IL8-het cows after calving compared with IL8-h1 cows. Furthermore, genotyping results showed that SI cows have a high frequency of the homozygous IL8-h2 haplotype compared to the HO cows (87.5 % vs 40 %) which reflects the different selective pressure between the two pure breeds. In conclusion, our preliminary data suggests that IL8 promoter haplotype is associated with significant and dynamic changes in immunological traits during peripartum and early lactation period. Future work will focus on a more comprehensive assessment of immune changes in additional cows.

Evaluation of Brix refractometer as an on-farm tool for colostrum IgG evaluation in Italian beef and dairy cattle.

In this study it is hypothesized that there are differences between immunoglobulin G (IgG) content in colostrum from beef (Chianina, Podolica) and dairy (Holstein Friesian) cows and that variables such as breed, and parity can influence IgG content. The further objective was to determine if these factors may vary in terms of sensitivity, specificity and the cut point when data obtained with the digital Brix refractometer is compared with the gold standard radial immunodiffusion assay (RID). A total of 90 samples of first-milking colostrum were collected within 2 h after parturition. IgG concentration was determined indirectly by digital Brix refractometer and directly by RID. Results obtained by RID were compared among breed and parity. For the digital Brix refractometer, sensitivity and specificity to detect colostrum with an IgG concentration lower than 50 g/l were calculated and the optimal cut-point was selected for each breed. Samples containing less than <50 g/l IgG accounted for 15.9% of the total. Parity influenced colostral IgG concentration and beef cows had a higher mean concentration of IgG (101.1 g/l in Chianina and 90.6 g/l in Podolica) than dairy cows (71.1 g/l in Holstein Friesian) First parity Chianina cows had the highest IgG mean content (116.1 g/l). At the optimal cut-point for Brix refractometer (20%) sensitivity and specificity were 0.93 (0.84-0.97) and 0.81 (0.70-0.88), however, a breed-related cut-point could be used to reduce evaluation error. Linear regression modeling showed that refractometer data were related to RID (r = 0.78). Results obtained suggest that breed and parity can influence IgG content of colostrum and, despite the Brix refractometer being an excellent on-farm tool, a breed-based definition of optimal cut point is needed.

Predicting colostrum and calf blood components based on refractometry.

Provision of good quality colostrum is essential for the passive immunity and nutrition of newborn calves. In order to better predict the quality of colostrum and the transfer of passive immunity, the relationships between colostrum components and between calf serum components were examined in this study. Samples of bulk tank milk, colostrum pooled from several cows 0-4 d postpartum, and colostrum collected from individual cows twice daily for 3 d post-partum were compared. With the exception of fat percentage, there were strong correlations between the levels of the components in the pooled colostrum and in the individual cow colostrum collected 0-1 d postpartum. The correlations between total solids as measured by Brix refractometry and total protein, immunoglobulin G (IgG), lactose % and protein % in colostrum within 1 d postpartum and pooled colostrum were 0.92, 0.90, -0.88 and 0.98, respectively. These high correlations enabled these colostrum components to be accurately predicted from Brix % and therefore, the volume of colostrum required to feed neonate calves can be optimised based on Brix refractometry to avoid failure of passive immunity transfer. To assess whether the components obtained from colostrum were correlated in calf blood, newborn calves were separated from their dams before suckling and blood sampled before feeding (day 0), and on days 1 and 7, after receiving colostrum or milk twice a day. The correlations between glucose, total protein, IgG, and gamma-glutamyl transferase (GGT) levels in the calf blood were lower than the correlations observed between the colostrum components. The highest correlation was between serum protein measured by refractometer and serum IgG within one week postpartum. GGT activity was not a good indicator of serum IgG levels. However, serum protein refractometer measurements predicted serum IgG level with high accuracy, providing an on-farm test to determine that calves have received sufficient passive immunity and colostrum components.