Hidetoshi Higuchi

Detection of serum haptoglobin by enzyme-linked immunosorbent assay in cows with fatty liver.

Haptoglobin cannot be detected in the serum of healthy cattle by the haemoglobin-binding assay or single radial immunodiffusion. The present study was designed to examine whether an enzyme-linked immunosorbent assay could be applied to measure serum haptoglobin concentrations in healthy cows and in cows with fatty liver. When either purified cow haptoglobin or haptoglobin-positive serum were used as the antigen in the assay, the standard curves obtained were distinctly different, and haptoglobin in serum was detected more sensitively than the purified protein. The addition of bovine serum albumin to purified haptoglobin shifted the curve towards that obtained with haptoglobin-positive serum, suggesting that an interaction with serum albumin was partly responsible for the different standard curves. By use of the standard curve for haptoglobin in serum, the mean (SEM) concentration of haptoglobin in samples from four cows with fatty liver was 466 (147) micrograms ml-1, but the sera from four apparently healthy cows contained less than 0.01 microgram ml-1 haptoglobin.

Antibacterial activity of bovine lactoferrin hydrolysate against mastitis pathogens and its effect on superoxide production of bovine neutrophils.

Antibacterial activity of bovine lactoferrin hydrolysates (LFH) on microorganisms isolated from bovine mastitis, and superoxide () production of bovine neutrophils were evaluated. Antibacterial effects of LFH were measured in vitro against Staphylococcus aureus, coagulase‐negative staphylococci, Streptococci, Enterococci, Escherichia coli, Klebsiella pneumoniae, yeast‐like fungi and Prototheca zopfii isolated from clinical cases of bovine mastitis. To compare susceptibilities against LFH, minimal inhibitory concentration (MIC) values were determined by a micro‐plate assay method. Most organisms were sensitive to LFH. Prototheca zopfii was highly sensitive to LFH; the growth of the microorganism was inhibited completely even at 1 μg/ml. Staphylococcus aureus and Escherichia coli were resistant to LFH. The production of by bovine neutrophils was used to evaluate the effect of LFH administration on functional activity. Increase in production by bovine neutrophils occurred upon addition of LFH to neutrophils. These results demonstrate that LFH possesses antibacterial activity against pathogens that cause mastitis and activates neutrophil superoxide production.

Physiological changes in the concentrations of biotin in the serum and milk and in the physical properties of the claw horn in Holstein cows.

Physiological changes in the concentrations of biotin in the serum and milk and in the physical properties of the claw horn were examined in Holstein cows. A lower concentration of biotin in the serum and a higher concentration of biotin in milk were found during early and late lactation and during the dry period, and a significant (p<0.05) inverse correlation was found between serum and milk biotin concentrations. A high moisture content and a low level of hardness of the claw horn were found during mid-lactation. Our results indicate that change in the serum biotin concentration probably results from the loss of biotin in the milk of cows during each stage of lactation and also confirm that the moisture content and hardness of the claw horn undergo physiological changes.

Oral administration of IL-1β enhanced the proliferation of lymphocytes and the O2− production of neutrophil in newborn calf

Recently, we demonstrated the presence of IL-1 beta in the colostral whey from dairy cows. Here, authors examined oral transmission of colostral IL-1 beta and its immunological effects on the neonatal calves. Biotin-labeled recombinant bovine (rb) IL-1 beta was administered orally to newborn calves and monitored in the serum. The results disclosed the passive transfer of colostral cytokines via the oral route, and a potent increase in white blood cell (WBC) count was observed in all calves administered with rbIL-1 beta. Oral administration of IL-1 beta significantly increased the proliferation of peripheral blood mononuclear cells (PBMCs) stimulated with concanavalin A, and the O(2)(-) production of stimulates neutrophils in newborn calves. These results suggest that the oral administration of IL-1 beta has an immunostimulatory activity in the newborn calf.

Suppressive effects of neutrophil by Salp16-like salivary gland proteins from Ixodes persulcatus Schulze tick

Salp16, a 16‐kDa tick salivary gland protein, is known to be the molecule involved in the transmission of Anaplasma phagocytophilum, an obligate intracellular pathogen causing zoonotic anaplasmosis, from its mammalian hosts to Ixodes scapularis. Recently, the presence of A. phagocytophilum was documented in Japan and Ixodes persulcatus was identified as one of its vectors. The purpose of this study was to identify Salp16 genes in I. persulcatus and characterize their function. Two cDNA clones encoding the Salp16‐like sequences were obtained from the salivary glands of fed female I. persulcatus ticks and designated Salp16 Iper1 and Iper2. Gene expression analyses showed that the Salp16 Iper genes were expressed specifically in the salivary glands and were up‐regulated by blood feeding. These proteins attenuated the oxidative burst of activated bovine neutrophils and inhibited their migration induced by the chemoattractant interleukin‐8 (IL‐8). These results demonstrate that Salp16 Iper proteins contribute to the establishment of blood feeding as an immunosuppressant of neutrophil, an essential factor in innate host immunity. Further examination of the role of Salp16 Iper in the transmission of pathogens, including A. phagocytophilum, will increase our understanding of the tick–host–pathogen interface.

A simplified PCR assay for fast and easy mycoplasma mastitis screening in dairy cattle

A simplified polymerase chain reaction (PCR) assay was developed for fast and easy screening of mycoplasma mastitis in dairy cattle. Species of major mycoplasma strains [Mycoplasma (M.) bovis, M. arginini, M. bovigenitalium, M. californicum, M. bovirhinis, M. alkalescens and M. canadense] in cultured milk samples were detected by this simplified PCR-based method as well as a standard PCR technique. The minimum concentration limit for detecting mycoplasma by the simplified PCR was estimated to be about 2.5 × 103 cfu/mL and was similar to that of the standard PCR. We compared the specificity and sensitivity of the simplified PCR to those of a culture method. Out of 1,685 milk samples cultured in mycoplasma broth, the simplified PCR detected Mycoplasma DNA in 152 that were also positive according to the culture assay. The sensitivity and specificity of the simplified PCR were 98.7% and 99.7%, respectively, for detecting mycoplasma in those cultures. The results obtained by the simplified PCR were consistent with ones from standard PCR. This newly developed simplified PCR, which does not require DNA purification, can analyze about 300 cultured samples within 3 h. The results from our study suggest that the simplified PCR can be used for mycoplasma mastitis screening in large-scale dairy farms.

Complement Receptor Type 3 (CR3)- and Fc Receptor (FcR)-Mediated Matrix Metalloproteinase 9 (MMP-9) Secretion and Their Intracellular Signalling of Bovine Neutrophils

Complement receptor type 3 (CR3)- and Fc receptor (FcR)-mediated metalloproteinase-9 (MMP-9) secretion and their intracellular signalling of bovine neutrophils were evaluated. Relative density of MMP-9 secreted by neutrophils stimulated with opsonized zymosan (OPZ, stimulant for CR3) was significantly (p < 0.05) increased when the OPZ concentration was increased from 0 to 0.4 mg/ml. Similar results were obtained for neutrophils stimulated with heat-aggregated IgG (Agg-IgG, stimulant for Fc receptor) at concentrations from 0 to 0.40 mg/ml. Preincubation of neutrophils with 1–30 nmol/L wortmannin (phosphoinositide 3-kinase inhibitor) resulted in inhibition of MMP-9 secretion induced by stimulation with OPZ and Agg-IgG in a concentration-dependent manner, 30 nmol/L wortmannin causing complete inhibition. Similarly, preincubation of neutrophils with 0–100 μmol/L genistein (tyrosine kinase inhibitor) also resulted in inhibition of OPZ- and Agg-IgG-induced MMP-9 secretion in a concentration-dependent manner, with 100 μmol/L genistein causing complete inhibition. Significant (p < 0.05) positive correlations were found between MMP-9 and luminal-dependent chemiluminescent response (LDCL) in the case of stimulation with OPZ (r = 0.754) and in the case of stimulation with Agg-IgG (r = 0.728). Our findings suggested that CR3 and FcR play a critical role in production of MMP-9 and may be regulated by intracellular signal transduction, including that by phosphoinositide 3-kinase (PI3K) and tyrosine kinase (TK).

Relationship between concentration of lingual antimicrobial peptide and somatic cell count in milk of dairy cows

Lingual antimicrobial peptide (LAP) belongs to the β-defensin family in cattle and is found in milk. LAP concentrations increase in milk from mastitic udders; however, the relationship between LAP concentrations and the somatic cell count (SCC) in milk remains to be elucidated in detail. Therefore, the present study was undertaken to investigate the relationship between LAP concentrations and the SCC in bovine milk to assess whether LAP may be used as an indicator of SCC. Milk was collected from 66 udders showing various SCCs. The SCC and LAP concentrations were measured in the milk. A significantly higher LAP concentration was observed in milk having 500-5000 × 10(3)cells/ml and >5000 × 10(3)cells/ml SCC groups than in lower SCC groups (<50 × 10(3)cells/ml and 50-500 × 10(3)cells/ml). A significantly positive correlation between LAP concentrations and SCCs in milk was observed (r=0.68). In milk samples with >26 nM of LAP, 92.0% of milk samples had high SCCs (>200 × 10(3)cells/ml). The concentration of LAP in milk infected with Staphylococcus aureus, Streptococcus bovis, Streptococcus dysgalactiae, and Escherichia coli was significantly higher than that in uninfected milk. These results suggest that the concentration of LAP can be a useful indicator of the SCC in dairy cows.

Prevalence of Mycoplasma species in bulk tank milk in Japan

Mycoplasma species are highly contagious pathogens and their ability to cause intramammary infection is a serious problem on dairy farms (Nicholas and Ayling 2003). Since the cure rate of clinical mastitis caused by Mycoplasma species is very low due to difficulties in antibiotic therapy, Mycoplasma -infected cows on farms must be culled in an emergency to prevent outbreaks of Mycoplasma mastitis (Nicholas and Ayling 2003). Bovine Mycoplasma mastitis was first reported in 1962 (Hale and others 1962). However, little is known about the prevalence of Mycoplasma mastitis on dairy farms in Japan. In this study, the prevalence of Mycoplasma species in bulk tank milk from dairy farms in Japan was investigated. A total of 1241 commercial dairy farms (n=45 to 1125 cows/farm) were randomly selected for bulk tank milk screening. The samples were collected from April to September 2010. Each bulk tank contained milk from two days of production. Milk samples were aseptically collected into 50 ml tubes. One hundred microlitres …

Cytokine mRNA profiling and the proliferative response of bovine peripheral blood mononuclear cells to Mycoplasma bovis.

Mycoplasma bovis is known as a significant pathogen and cause of large economic losses in beef and dairy calves worldwide. Numerous factors appear to play an important role in the development of disease during infection with M. bovis, e.g., inhibition of immune cell proliferation and induction of lymphocyte apoptosis. However, the mechanisms involved in M. bovis infections have not been explored and remain incompletely understood. We investigated the major cytokine mRNA expression in bovine PBMC stimulated with M. bovis, for comparison, Staphylococcus aureus and Escherichia coli, which are the representative mastitis-causing pathogens. Here we demonstrated that live M. bovis significantly induced tumor necrosis factor alpha (TNF-α), interleukin 12p40 (IL-12), and interferon gamma (IFN-γ) mRNA expression in bovine peripheral blood mononuclear cells (PBMC) at a multiplicity of infection (MOI) of 1000 but not at an MOI of 10 and 100. Live M. bovis at MOIs of 1, 10, and 100 induced significant bovine PBMC proliferative responses compared with unstimulated bovine PBMC. Furthermore, we showed that the cultural supernatant of M. bovis induced a significant increase in TNF-α, IL-6, and IL-10 mRNA expression in bovine PBMC. Our results suggest that M. bovis weakly affects the cellular integrity of bovine PBMC and induces clear proliferative responses and associated cytokine production in them. However, large numbers of live M. bovis are required to induce an immune response in bovine PBMC.