Detection of early pregnancy-specific proteins in Holstein milk

Bovine pregnancy is commonly diagnosed by rectal palpation or ultrasonography and changes in progesterone concentration. To determine a simpler and less expensive diagnostic method, we sought to identify early pregnancy-specific proteins in bovine milk by comparing samples collected from pregnant and non-pregnant Holstein cattle. Of the 600-700 protein spots visible on 2-DE gel images, 39 were differentially expressed in milk from pregnant and non-pregnant cattle. Antibodies generated against synthetic peptides of milk whey proteins expressed specifically during pregnancy were used to confirm protein expression patterns. Western blot analysis showed that the levels of expression of lactoferrin (lactotransferrin) and alpha1G T-type calcium channel subunit (alpha-1G) were higher in samples from pregnant than non-pregnant cattle. These findings suggest that assays for pregnancy-specific milk proteins may be used to diagnose pregnancy in cattle.     

In-depth analysis of low abundant proteins in bovine colostrum using different fractionation techniques

Bovine colostrum is well known for its large content of bioactive components and its importance for neonatal survival. Unfortunately, the colostrum proteome is complicated by a wide dynamic range, because of a few dominating proteins that hamper sensitivity and proteome coverage achieved on low abundant proteins. Moreover, the composition of colostrum is complex and the proteins are located within different physical fractions that make up the colostrum. To gain a more exhaustive picture of the bovine colostrum proteome and gather information on protein location, we performed an extensive pre-analysis fractionation of colostrum prior to 2D-LC-MS/MS analysis. Physical and chemical properties of the proteins and colostrum were used alone or in combination for the separation of proteins. ELISA was used to quantify and verify the presence of proteins in colostrum. In total, 403 proteins were identified in the nonfractionated colostrum (NF) and seven fractions (F1-F7) using six different fractionation techniques. Fractionation contributed with 69 additional proteins in the fluid phase compared with NF. Different fractionation techniques each resulted in detection of unique subsets of proteins. Whey production by high-speed centrifugation contributed most to detection of low abundant proteins. Hence, prefractionation of colostrum prior to 2D-LC-MS/MS analysis expanded our knowledge on the presence and location of low abundant proteins in bovine colostrum.     

Immunodepletion of albumin and immunoglobulin G from bovine plasma

Current MS-based proteomics has facilitated the identification of large numbers of proteins from complex mixtures. The bovine plasma proteome has the potential to provide a wealth of information concerning the biological state of an animal. However, during MS-based experiments, higher abundance proteins such as albumin and immunoglobulin G (IgG) can hinder the identification of potentially important proteins that are present in much lower abundance. While a variety of readily available technologies exist for the depletion of multiple high-abundance proteins from human, mouse and rat samples, there are few available for bovine. In this study, we report the depletion of >97% of albumin and >92% of IgG from bovine plasma.     

A genome-wide association study of immune response traits in Canadian Holstein cattle

Background

Breeding for enhanced immune response (IR) has been suggested as a tool to improve inherent animal health. Dairy cows with superior antibody-mediated (AMIR) and cell-mediated immune responses (CMIR) have been demonstrated to have a lower occurrence of many diseases including mastitis. Adaptive immune response traits are heritable, and it is, therefore, possible to breed for improved IR, decreasing the occurrence of disease. The objective of this study was to perform genome-wide association studies to determine differences in genetic profiles among Holstein cows classified as High or Low for AMIR and CMIR. From a total of 680 cows with immune response phenotypes, 163 cows for AMIR (81 High and 82 Low) and 140 for CMIR (75 High and 65 Low) were selectively genotyped using the Illumina Bovine SNP50 BeadChip. Results were validated using an unrelated population of 164 Holstein bulls IR phenotyped for AMIR and 146 for CMIR.

Results

A generalized quasi likelihood score method was used to determine single nucleotide polymorphisms (SNP) and chromosomal regions associated with immune response. After applying a 5% chromosomal false discovery rate, 186 SNPs were significantly associated with AMIR. The majority (93%) of significant markers were on chromosome 23, with a similar peak found in the bull population. For CMIR, 21 SNP markers remained significant. Candidate genes within 250,000 base pairs of significant SNPs were identified to determine biological pathways associated with AMIR and CMIR. Various pathways were identified, including the antigen processing and presentation pathway, important in host defense. Candidate genes included those within the bovine Major Histocompatability Complex such as BoLA-DQ, BoLA-DR and the non-classical BoLA-NC1 for AMIR and BoLA-DQ for CMIR, the complement system including C2 and C4 for AMIR and C1q for CMIR, and cytokines including IL-17A, IL17F for AMIR and IL-17RA for CMIR and tumor necrosis factor for both AMIR and CMIR. Additional genes associated with CMIR included galectins 1, 2 and 3, BCL2 and β-defensin.

Conclusions

The significant genetic variation associated with AMIR and CMIR in this study may imply feasibility to include immune response in genomic breeding indices as an approach to improve inherent animal health.     

Impact of volume,immunoglobulin G concentration,and feeding method of colostrum product on neonatal nursing behavior and transfer of passive immunity in beef calves

One-third of beef calves fail to achieve adequate transfer of passive immunity (TPI) through timely ingestion of colostrum, which substantially increases their risk of preweaning morbidity and mortality. Two randomized clinical trials were designed to assess the impact of volume, immunoglobulin G (IgG) concentration, and feeding method of colostrum product on neonatal nursing behavior and TPI. In Trial 1, 47 calves were randomly assigned to receive one of three colostrum interventions by oro-esophageal tube feeder (OET): 1 L with 100 g/L IgG, 1.4 L with 70 g/L IgG, or 2 L with 100 g/L IgG. In Trial 2, 29 calves were randomly assigned to be fed 1 L of colostrum product with 100 g/L IgG by either nipple bottle (NB) or OET. Colostrum intervention (i.e. feeding of colostrum product) occurred within 60 minutes of birth. Cow-calf pairs were monitored by video surveillance in individual stalls for 24 h. Dam colostrum was collected at 10 minutes and calf serum was collected at 24–36 h after birth to assess IgG concentration. Differences among colostrum intervention groups on latency to stand and nurse were analyzed using Kaplan-Meier survival curves and Cox proportional hazard models. The impact of colostrum intervention group on TPI was assessed using multivariable linear regression modeling. In Trial 1, calves fed 1.4 L with 70 g/L IgG by OET nursed from their dams statistically significantly earlier compared to calves fed 1 L with 100 g/L IgG (P = 0.003) and calves fed 2 L with 100 g/L IgG (P = 0.008). Six of the 15 calves in the NB group in Trial 2 refused to consume part of the colostrum feeding offered by bottle and required follow-up tube feeding of the remaining volume. These calves were analyzed as a separate group (NB + OET). Calves fed 1 L by NB stood and nursed statistically significantly earlier than calves fed by OET (P = 0.005) or a combination of NB + OET (P = 0.003). Calf serum IgG concentrations were not statistically significantly different among colostrum intervention groups (P > 0.1). Overall, the colostrum interventions assessed in this study led to only one calf with failed TPI. While statistically significant differences in serum IgG concentrations were not detected in this study, subsequent nursing behavior did vary and was improved by feeding a moderate volume (1.4 L with 70 g/L IgG) of colostrum when using an OET, and by using the NB when feeding a smaller volume (1 L with 100 g/L IgG).     

Relationship between serum nonesterified fatty acids at calving and the incidence of periparturient diseases in Holstein dairy cows

The objective was to describe the relationship between concentration of serum nonesterified fatty acids (NEFAs) at calving and the incidence of periparturient disorders in Chilean Holstein dairy cows (Bos taurus). The study was conducted at two dairies (central Chile) with 700 milking cows each and similar management. Between July 2006 and March 2007, 350 cows were selected, and concentrations of serum NEFAs were determined at calving. The incidence of milk fever (MF), retained fetal membranes (RFMs), metritis, and clinical mastitis from calving to 100 d in lactation were consistently recorded. The relationship between concentration of serum NEFAs at calving and the incidence of periparturient diseases was determined using logistic regression. The main explanatory variable was concentration of serum NEFAs at calving. The incidence of MF, RFM, metritis, and mastitis was 5.4%, 15.6%, 10.8%, and 14.4%, respectively. There was no association between concentration of NEFAs at calving and the incidence of these conditions when the median value of NEFAs (0.9 mEq/L) was used as a cutoff. However, when the 75th percentile (1.2 mEq/L) was used as the cutoff, cows with values <1.2 mEq/L were 0.45 and 0.32 times as likely to develop clinical mastitis and MF, respectively, compared with cows with values ≥1.2 mEq/L. When the 90th percentile (1.6 mEq/L) was used as a cutoff, cows with values <1.6 mEq/L were 0.25 times as likely to develop clinical mastitis compared with cows with values ≥1.6 mEq/L. As a continuous variable, for every 0.1 mEq/L increment in NEFAs at calving, cows were 1.11 times more likely to experience clinical mastitis. In conclusion, cows with NEFA concentrations ≥1.2 mEq/L had a higher incidence of clinical mastitis and MF than that of cows with values <1.2 mEq/L.     

Identification of antigenic proteins from Neospora caninum recognized by bovine immunoglobulins M, E, A and G using immunoproteomics

Antigenic proteins of Neospora caninum (N. caninum) against bovine immunoglobulins M, E, A, and G were investigated by using immunoproteomics. Proteins of N. caninum (KBA-2) tachyzoite lysates separated by two-dimensional gel electrophoresis were transferred to polyvinylidene difluoride (PVDF) membranes, probed with different bovine immunoglobulin class and classified. Antigenic spots recognized were also identified by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) analysis. 132, 84, 4, and 40 antigenic protein spots were recognized on N. caninum immunoblot profiles against bovine IgM, IgE, IgA, and IgG, respectively. Of these protein spots, the antigenic proteins recognized by either IgM, IgE, and IgG, or IgM and IgG were HSP70, pyruvate kinase, actin, NCDG-1, tubulin alpha-chain, and putative ribosomal protein S2. On the other hand, IgM, IgE, and IgA reacted with NTPase, HSP60, tubulin beta-chain, putative protein disulfide isomerase, enolase, lactate dehydrogenase, serine-threonine phosphatase, 14-3-3 protein homologue, and GRA2 protein. Most of the antigenic proteins identified were associated with the process of invasion, proliferation, and egression of apicomplexans. In our study, HSP70, actin, NTPase, HSP60, pyruvate kinase, enolase, putative ribosomal protein S2, NCDG-1, and GRA2 proteins were found to be immunodominant proteins, which may contribute to the development of diagnostic markers and vaccine.     

Prevalence and molecular subtyping of Blastocystis from dairy cattle in Kanagawa,Japan

Blastocystis is an intestinal protist, commonly found in the human population and in a wide range of animals globally. Currently, isolates from mammalian and avian hosts are classified into 17 subtypes (STs) based on phylogeny of the small subunit rRNA gene (SSU rDNA), of which ten (ST1-9, 12) are reported in humans. ST10 is a major ST reported from livestock cattle. However, other STs including ST1, 3, 4, 5, and 6, which have the potential to be transmitted to humans, are also reported from cattle in several countries. Although a survey has been conducted previously in western Japan for livestock cattle, there is no information available regarding other parts of Japan. Therefore, this study surveyed the prevalence of Blastocystis and its STs in cattle from Kanagawa prefecture, eastern Japan. Fecal specimens, collected from 133 dairy cattle on four different farms, were subjected to a short-term xenic in vitro culture and Blastocystis were identified by microscopic examination. Seventy-two cattle were positive for Blastocystis (54.1%). Direct sequences for the partial SSU rDNA were obtained for 45 samples. Based on nucleotide sequence homology search and phylogenetic analysis, 44 isolates were identified as ST14 and one as ST10. Our study confirms the presence of these STs in dairy cattle in Japan for the first time. The STs identified here, ST10 and ST14, support previous findings that Bovidae may be the natural host for both STs.     

Relationship between fructosamine with serum protein, albumin and glucose concentrations in dairy ewes

The aim of the study was to investigate the changes in blood fructosamine concentrations of ewes during late pregnancy and lactation. The relationship of serum fructosamine to changes in the serum glucose and blood protein and albumin concentrations were measured in 10 crossbred dairy ewes. Blood was sampled 10 days prior to lambing (D − 10), on day 10 (D + 10), day 20 (D + 20), day 90 (D + 90) and on day 130 post partum (D + 130). The concentration of serum fructosamine significantly decreased on D + 10, then significantly increased on D + 20 (p < 0.05), remaining on the same level until D + 130. The concentration of serum glucose was highest on D − 10, and gradually decreased, being significantly lower on D + 130 post partum, compared to D − 10. Similarly the serum albumin and total protein concentrations were highest on D − 10, and significantly decreased on D + 10. The concentration of both parameters increased by D + 20, but then decreased on D + 90 and D + 130. The significant decrease in fructosamine concentration observed on D + 10 probably resulted from the substantial and rapid decrease in total serum protein and albumin concentrations, also established during the same period. The results confirm that in ewes, similar to other species, the shorter serum protein life span during the transition period has an influence on the decrease in blood fructosamine concentrations. These should be considered during the interpretation of serum fructosamine concentration as part of blood biochemical findings during the pre and post partum period.     

Effects of bovine colostrum, foal serum immunoglobulin concentration and intravenous plasma transfusion on chemiluminescence response of foal neutrophils

Summary. The effects of bovine colostrum, absorption of equine colostral immunoglobulins and age on phagocytic and serum opsonizing activity of nine clinically healthy foals were examined. Cells and serum were collected prior to suckling and at 7, 14 and 28 days of age. Seven foals had serum IgG concentrations >600mg/dl whereas two foals had <350mg of IgG/dl. Phagocytic and serum opsonic activity of eight clinically ill foals with <400mg of IgG/dl of serum were also examined before and after plasma transfusion. Phagocytic and serum opsonizing activities were evaluated by an assay for chemiluminescence (CL) after addition of opsonized streptococci. Results showed that bovine colostrum stimulated CL of foal neutrophils. Preliminary characterization of opsonins in bovine colostrum by ammonium sulphate fractionating and heat inactivation indicated that opsonins generating CL were mainly associated with immunoglobulin G. Chemiluminescence generated by foal neutrophils varied with age with foal neutrophils collected at day 14 producing more CL than adult neutrophils ( P <0.05). Foal serum opsonizing activity was similar to adult opsonizing activity if serum IgG concentrations were >600mg/dl but it was less if IgG concentration was <350mg/dl ( P <0.05). Chemiluminescence generated by foal and adult neutrophils was higher when post-transfusion foal serum was used as the source of opsonin than when pre-transfusion foal serum was used ( P <0.05). When adult serum was the opsonin, chemiluminescence of foal neutrophils collected before and after plasma transfusion did not differ. The increase in CL following plasma transfusion was probably due to an increase in serum opsonizing activity.     

Immunohistochemical localization of immunoglobulins A, G and M in the mouse female genital tract

The localization of immunoglobulins A, G and M (IgA, IgG, IgM) in the mouse genital tract was studied by immunoperoxidase techniques at oestrus, on the day of mating and at the time of implantation. In the horns and body of the uterus, IgA and IgG were located in plasma cells in the endometrium surrounding uterine glands and in the gland lumina. The numbers of these plasma cells increased markedly between Day 1 and Days 4 and 5 of pregnancy and the ratio of plasma cells containing IgA and IgG was about 3 or 4 to 1 at all stages. Area measurements indicated that the increased number of plasmacytes was not due to an increase in the amount of endometrial, myometrial or glandular tissues. Plasma cells were not detected in the cervix and vaginal fornix at oestrus and Day 1, but a few were present on Day 5. In the oviduct, plasma cells containing IgA and IgG were present only in the preampulla and both immunoglobulins were present in the extracellular space of the lamina propria only in this region. No IgM was detected in any part of the reproductive tract at any of the times studied. Uteri on Day 1 of pregnancy contained bacteria of several kinds, some of which were aggregated and coated with IgA. This suggests that the uterine lumen at this time may contain specific anti-bacterial IgA antibodies. Our observations indicate that the horns and body of the uterus and the preampulla of the oviduct are major sites of a local immune system in the female mouse genital tract.     

The effects of bovine serum albumin, bovine uterine fluid, and heat-treated bovine serum on in vitro mouse embryo development

Two-cell mouse embryos were cultured in Whitten's medium with one of three supplements: bovine serum albumin (WM + BSA), heat-treated bovine serum (WM + HTBS) or bovine uterine fluid (WM + BUF). Protein concentrations for cultures of WM + BSA were 50.2, 100.5, 251.2, 502.5, and 1005.0 mug/ml and for WM + HTBS were 70.4, 105.1, 269.0, 524.5 and 1193.9 mug/ml. Protein concentrations ranged from 56.9 to 739.1 mug/ml for 22 WM + BUF samples. Embryo development in all media was significantly correlated with the log total protein concentration. When compared to WM + BSA, development was not significantly inhibited or stimulated in any WM + BUF cultures or in WM with 70.4, 524.5 and 1193.9 mug/ml HTBS. Development was enhanced in WM with 105.1 and 269.0 mug/ml HTBS (P<0.05). The results suggest that at the protein concentrations used, culture media supplemented with BUF and BSA support similar mouse embryo development. Culture medium supplemented with HTBS supported embryo development more than medium with BSA. Uterine factors in the bovine capable of enhancing or inhibiting early embryo development were not detected.     

In vitro development of bovine morulae in bovine serum albumin, normal steer serum and uterine flushings

One hundred fifty-three excellent and good bovine morulae were cultured in Ham's F-10, supplemented with 10 % steer serum, bovine serum albumin, or uterine flushings (64 mg protein/ml) to compare embryo development. Embryos were observed every 12 h in culture. Treatment differences were evaluated by assigning a numerical value of 0 to 4 to each embryo representing the stage of development reached in vitro. The final morphological developmental score of the embryos was comparable for steer serum (2.66) and bovine serum albumin (2.50), but it differed significantly for uterine flushings obtained from ovariectomized, steroid-supplemented cows (< 0.1) or heat-treated uterine flushings (0.07). Since albumin alone was able to support development, it suggests that the albumin component of steer serum may be responsible for the observed development. Uterine fluids were unable to support growth of embryos, suggesting that incompatibility may be due to asynchrony between the early bovine embryo and uterine constituents, or a concentration of uterine components may exacerbate actions of inhibitory substances.     

Localization of immunoglobulins G, A and M in glial cells of reactive and neoplastic origin

The localization of immunoglobulins G, A and M in glial cells of neoplastic and reactive origin have been investigated by the use of the PAP (peroxidase-antiperoxidase) method on paraffin embedded tissue previously fixed in calcium formol. It has been found, that some glial cells of astrocyte type showed a very intense staining when oligoclonal antibodies to human immunoglobulins G, A and M specific for gamma, alpha, and mu chains were used. The localization of immunoglobulins was disclosed in astrocytes of various morphology; astrocytes with well developed processes, gemistocyte type cells without or only with short and thick cell processes and in small cells with scanty cytoplasm. The number of cells with immunoglobulins localized is very small. No positive results have been noted if the normal brain tissue is concerned. The specificity of the method is discussed.     

Bound class M, A and G immunoglobulins in the thymus of myasthenia gravis patients]

Deposits of granular material containing IgM, IgA, and IgG were revealed in the thymus of patients with myasthenia by direct immunofluorescence. Treatment of the thymus sections by unlabeled preparations against individual classes of human immunoglobulins inhibited the reaction of the granular material with the homologous labeled preparations. Disappearance of fluorescence of these deposits was also seen in treatment of the sections with glycine-HCl-buffer, pH 2.8. These data permitted a suggestion that granular material represented immune complexes where IgM, IgA, and IgG served as antibody, and thymus tissue components--as an antigen. The presence of bound immunoglobulin in the thymus indicated that an autoimmune process directed against the tissues of this organ occurred in myasthenia.     

Performance and financial consequences of stillbirth in Holstein dairy cattle

Stillbirth is an economically important trait on dairy farms. Knowledge of the consequences of, and the economic losses associated with stillbirth can help the producer when making management decisions. The objectives of this study were to determine the effects of stillbirth on productive and reproductive performance as well as financial losses due to stillbirth incidence in Iranian Holstein dairy farms. Economic and performance data were collected from nine Holstein dairy farms in Isfahan and Khorasan provinces of Iran from March 2008 to December 2013. The final data set included 160 410 calving records from 53 265 cows. A linear mixed model was developed to evaluate the effects of stillbirth on performance of primiparous and multiparous cows separately and overall. An economic model was used to estimate the economic losses due to stillbirth. The incidence of stillbirth cases per cow per year was 4.2% on average (3.4% to 6.8% at herd level). The least square means results showed that a case of stillbirth significantly (P<0.05) reduced 305-day milk production in multiparous cows and overall, but had no significant effects on primiparous cows production performance (P>0.05). Overall, a case of stillbirth reduced 305-day milk yield by 544.0±76.5 kg/cow per lactation. Stillbirth had no significant effects on 305-day fat and protein percentages in either primiparous or multiparous cows. Overall, cows that gave birth to stillborn calves had significantly increased days open by 14.6±2.6 days and the number of inseminations per conception by 0.2 compared with cows that gave birth to live calves (P<0.01). In general, the negative productive and reproductive effects associated with stillbirth were smaller and non-significant for primiparous cows compared with multiparous cows. The financial losses associated with stillbirth incidence averaged US$ 938 per case (range from $US 767 to $US 1189 in the nine investigated farms). The loss of a calf was not the only cost associated with stillbirth, as it accounted for 71.0% of the total cost. The costs of dystocia (7.6%) and culling and replacement expenses (6.3%) were the next most important costs associated with stillbirth. These results can be used to assess the potential return from management strategies to reduce the occurrence of stillbirths.