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Mycoplasma arthritis in calves caused by M. bovis exhibits joint swelling, lameness, and immobility. In contrast to M. bovis, M. arginini, and M. californicum which were similarly isolated from the affected joints, only induced mild inflammation. The changes in pathogenesis that depended on species, however, remained unknown. This investigation aims to examine the characteristics of immune responses to M. bovis, M. arginini, and M. californicum in synovial cells. Intracellular M. bovis was detected by gentamicin assay, but M. arginini and M. californicum were not detected. M. bovis-infected synovial cells were encouraged to proliferate and had their apoptosis suppressed. We suggest that M. bovis invaded and inhibited apoptosis of synovial to evade host immunity, which led to long term survival in joints. M. bovis infection significantly increased IL-6 mRNA expression compared to control, although M. arginini and M. californicum infection were comparable to control. We suggest that M. arginini and M. californicum have low abilities to induce inflammation in joints and therefore do not cause severe pathology. Our findings are the first to show the variations in synovial cell immune responses to M. bovis, M. arginini, and M. californicum, which are thought to be related to the pathogenicity of arthritis., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflicts of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

This study aimed to examine the antimicrobial susceptibility of bovine mastitis pathogens in Japan and develop criteria for testing antimicrobial susceptibility using the simplified agar disk diffusion (ADD) method that is currently being used in clinical practice. Milk samples from 1,349 dairy cows with clinical mastitis were collected and cultured. The minimum inhibitory concentrations (MICs) of the antimicrobials were determined for 504 strains of 28 bacteria. Of the gram-positive bacteria, most Staphylococcus spp. were susceptible to penicillin G (PCG), kanamycin (KM), oxytetracycline (OTC), cefazolin (CEZ), pirlimycin, enrofloxacin, and marbofloxacin. Streptococcus spp. and Trueperella pyogenes showed resistance to OTC and KM. Most gram-negative bacteria were resistant to OTC and CEZ and particularly susceptible to fluoroquinolones. To develop the criteria for a disk diffusion test of the simplified ADD method, the relationships between MICs and diameters of inhibition zones (DIZs) were analyzed and compared with the conventional method. The susceptibility breakpoints of several antimicrobials were lower for both gram-positive and gram-negative bacteria. Particularly for gram-positive bacteria, the application of the new criteria lowers the breakpoint for PCG, suggesting that the use of PCG instead of CEZ may increase. The results suggest that use of these criteria for the simplified ADD method may lead to appropriate antimicrobial choice and consequently the appropriate use of antimicrobials in clinical practice.

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)-1β, 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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

A bacterial culture of milk is the most common test to determine the presence of mastitis-causing pathogens, which informs appropriate treatment. However, a certain proportion of clinical mastitis milk shows no growth of any mastitis-causing pathogens. We hypothesized that bacterial culture-negative clinical mastitis milk is associated with the activity of antimicrobial components contained in the milk. In this study, the differences in antimicrobial components (lactoferrin, transferrin, lysozyme, lactoperoxidase, and lingual antimicrobial peptide [LAP]) between bacterial culture-positive and culture-negative bovine clinical mastitis milk were investigated using Holstein cows. Our results showed that 37 out of 71 samples of clinical mastitis milk had negative bacterial cultures. The LAP concentration in bacterial culture-negative milk was lower than that in positive milk (31.95 ± 1.64 nM vs. 42.85 ± 4.01 nM). In contrast, the lysozyme concentration in bacterial culture-negative milk was higher than that in positive milk (0.76 ± 0.15 μg/ml vs. 0.42 ± 0.06 μg/ml). In conclusion, the concentration of antimicrobial components was different between bacterial culture-positive and culture-negative bovine clinical mastitis milk, which suggests that antimicrobial components are related to bacterial culture results., (© 2022 Japanese Society of Animal Science.)

Intramammary infusion of Bifidobacterium breve (B. breve)-induced somatic cell (SC) counts, chemiluminescent response (CL), lactoferrin (LF) concentrations and mastitis-causing pathogens from quarters with subclinical mastitis were measured to evaluate innate immune response of mammary glands in dairy cows at 3 to 4 weeks before drying off. SC counts in 7 quarters of 7 control cows and 5 quarters of 6 cows with mastitis increased markedly on day 1 and SC values in control cows were significantly (P<0.05) increased and returned to pre-infusion levels on day 5 after B. breve-infusion. CL values in both groups increased markedly on day 1 and then decreased after B. breve-infusion; however, CL values in cows with mastitis did not return to normal levels on day 5 and at postpartum. The CL values were highly correlated with their SC counts in milk from both groups. LF concentrations increased toward day 3 after B. breve-infusion and were higher in cows with mastitis. B. breve-infusion eliminated 16.6% (1/6) of pathogens from 6 quarters with chronic subclinical mastitis. B. breve-induced SC responses in quarters from 3 cows with mastitis showed characteristic patterns of recovery, persistent and new infections. B. breve-induced SC counts in quarters from the cows in the pre-drying off were lower (25.7-70.6%) than those of the cows in mid-lactation. The intrinsic innate immune response in cows on pre-drying off may be decreased and appears to be insufficient to eliminate pathogens from mammary gland in the pre-drying off.

Mycoplasma bovis (M. bovis) is a significant worldwide pathogen of cattle. Neutrophils have an important role in the innate immune response during infection with M. bovis. However, even though neutrophils accumulate in M. bovis infection, the interaction of M. bovis and neutrophils has not been fully elucidated. We attempted to elucidate the innate immune response of neutrophils stimulated with M. bovis and evaluate the transcriptome and functional analysis of bovine neutrophils stimulated with M. bovis. Proinflammatory cytokines, such as inducible nitric oxide (iNOS), which was the most increased gene in transcriptome analysis, were increased in quantitative polymerase chain reaction analysis of bovine neutrophils stimulated with live or heat-killed M. bovis. Nitric oxide and intracellular reactive oxygen species production of neutrophils stimulated with M. bovis was significantly increased. Neutrophils stimulated with M. bovis showed an increased ratio of nonapoptotic cell death compared to unstimulated controls. We demonstrated that neutrophil extracellular traps (NETs) formation was not recognized in neutrophils stimulated with live M. bovis. However, heat-killed M. bovis induced NETs formation. We also showed the interaction with M. bovis and bovine neutrophils regarding proinflammatory cytokine gene expression and functional expression related to NETs formation. Live and killed M. bovis induced innate immune responses in neutrophils and had the potential to induce NETs formation, but live M. bovis escaped NETs.

Mycoplasma bovis is a pathogenic bacterium in bovines that causes huge global economic losses. Numerous factors play important roles in M. bovis pathogenesis; however, the host immune response involved in M. bovis infection has not been fully elucidated. We aimed to determine the characteristics of the host immune response to Mycoplasma infection. We evaluated the responsiveness of bovine peripheral blood mononuclear cells (PBMCs) stimulated with M. bovis via microarray analysis. The transcriptional abundance of innate immune-related genes IL-36A, IL-27, IFN-γ, and IL-17 in PBMCs increased after M. bovis exposure. Upon M. bovis infection, there was increased expression of the lymphocyte activated genes basic leucine zipper transcription factor (BATF) and signaling lymphocytic activation molecule family members 1 and 7 (SLAMF 1 and SLAMF 7) in PBMCs compared with that in unstimulated cells. The study revealed that the transcriptional abundance of innate immunity genes in PBMCs increased during M. bovis infection. This induced the activation of PBMCs, giving rise to an immune response, which is followed by the development of the inflammatory response. The results from this study could be used as the basis for the development of novel vaccine candidates against M. bovis., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Bovine Mycoplasma arthritis (MA) is caused by Mycoplasma bovis and exhibits severe clinical symptoms. However, the pathophysiology of bovine MA is incompletely understood. In this study, we examined the cytokine mRNA expression of synovial fluid (SF) cells and cytokine concentrations in the SF of MA calves. The SF was isolated from five clinically healthy (control) and seven MA calves. mRNA and protein levels of interleukin (IL)-1β, IL-6, IL-8, IL-12, and IL-17 in the SF from MA calves were significantly higher than those from control calves. Our results indicate that SF cells produce inflammatory cytokines, which mainly contribute to the severe inflammatory response in the joints of the MA calves.

Mycoplasma bovis causes chronic arthritis in calves, presenting as osteolysis in affected joints. Matrix metalloproteinase-3 (MMP-3), an enzyme involved in cartilage degradation, is produced by synovial cells. Production of this proteinase is regulated by interleukin (IL)-1β, which is produced by mononuclear cells. Both factors are known to play important roles in osteolysis in human autoimmune and bacterial arthritis. However, the pathophysiology of Mycoplasma arthritis (MA) has not been elucidated. In this study, we evaluated the levels of MMP-3 and IL-1β in synovial fluid (SF) from MA calves and examined the effect of IL-1β on MMP-3 expression in bovine synovial cells in vitro. Levels of MMP-3 and IL-1β in SF from MA calves were significantly higher than those of clinically healthy calves. Mycoplasma bovis induced significant increases in the expression of IL-1β mRNA and protein in mononuclear cells, compared with cells not exposed to M. bovis. Interestingly, the supernatant of mononuclear cells stimulated with M. bovis contained high levels of IL-1β, which induced higher expression of MMP-3 mRNA and protein in synovial cells than direct stimulation by M. bovis. Recombinant bovine IL-1β also induced increased MMP-3 mRNA and protein expression in synovial cells. Our results indicate that M. bovis induces IL-1β expression by bovine mononuclear cells, and this cytokine then promotes MMP-3 production by synovial cells. These findings suggest that MMP-3 and IL-1β are key factors in the development of osteolysis in MA calves., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The immune related factors of peripheral blood mononuclear cells (PBMC) were analyzed in the clinical cases with Mycoplasma (M.) bovis infection. Seventy-eight Holstein calves in one farm were used. These calves were divided into three groups; the calves with M. bovis infection of poor outcome after treatment (Non-Recovery Group), the calves with M. bovis infection recovered (Recovery Group) and clinically healthy calves (Control Group). Blood samples were collected at days of the first medical treatment and the final treatment or euthanasia. IL-17A levels in the Non-Recovery Group were higher than those in the Recovery Group on both days. Our result suggested that the IL-17A of PBMC is an important factor to affect outcome of the calves with M. bovis infection.

Mycoplasma mastitis is a dairy herd health problem with growing concern in Japan. To complement the lack of epidemiological knowledge of the disease, we conducted estimation of herd-level prevalence and risk factor analysis for Mycoplasma bovis (M. bovis) mastitis using data collected from Tokachi region, one of the nation's largest milk producing area, in Hokkaido Prefecture in 2015. The herd-level prevalence was estimated at 3.8% (95% confidence interval (CI): 2.6%, 5.4%) across the region with municipality-specific prevalence ranging from 0% to 14.3%. In this study, identified risk factors for the presence of with M. bovis infection on farms were corporation-type farms and purchased cattle, after controlling for the herd size. Corporation-type farms may reflect higher frequencies of moving cows and fomites to and from other farms, which increases the risk of pathogen introduction. Purchased cattle was considered as one of the major pathways of the disease incursion, and this finding highlighted the importance of more stringent separation or quarantine protocols when introducing cattle from outside in Tokachi region. Due to the limited information available in this study and inherent nature of the study designs, these results should be interpreted with caution and further research is needed., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Staphylococcus aureus is a major pathogen that causes subclinical mastitis associated with huge economic losses to the dairy industry. A few vaccines for bovine mastitis are available, and they are expected to induce the production of S. aureus-specific antibodies that prevent bacterial adherence to host cells or promote opsonization by phagocytes. However, the efficacy of such vaccines are still under debate; therefore, further research focusing on improving the current vaccines by seeking additional mechanisms of action is required to reduce economic losses due to mastitis in the dairy industry. Here, we generated S. aureus-specific bovine IgG antibodies (anti-S. aureus) that directly inhibited bacterial growth in vitro. Inhibition depended on specificity for anti-S. aureus, not the interaction between Protein A and the fragment crystallizable region of the IgG antibodies or bacterial agglutination. An in vitro culture study using S. aureus strain JE2 and its deletion mutant JE2ΔSrtA, which lacks the gene encoding sortase A, revealed that the effect of anti-S. aureus was sortase-A-independent. Sortase A is involved in the synthesis of cell-wall-associated proteins. Thus, other surface molecules, such as membrane proteins, cell surface polysaccharides, or both, may trigger the inhibition of bacterial growth by anti-S. aureus. Together, our findings contribute insights into developing new strategies to further improve the available mastitis vaccine by designing a novel antigen on the surface of S. aureus to induce inhibitory signals that prevent bacterial growth.

Introduction: Bovine mycoplasma, chiefly Mycoplasma bovis, is a pathogen that causes pneumonia, mastitis, arthritis, and otitis media in cattle. This pathogen exerts immunosuppressive effects, such as the inhibition of interferon production. However, the mechanisms involved in bovine mycoplasmosis have not been fully elucidated. In this study, we investigated the role of the programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1) pathway in immunosuppression in bovine mycoplasmosis., Methods: In the initial experiments, we used enzyme-linked immunosorbent assay to measure interferon-γ (IFN-γ) from peripheral blood mononuclear cells (PBMCs) isolated from cattle with mycoplasmosis., Results: Expectedly, IFN-γ production significantly decreased in cattle with mycoplasmosis compared with that in clinically healthy cattle. Concomitantly, flow cytometric analysis revealed that the proportions of PD-1 + CD4 + and PD-L1 + CD14 + cells significantly increased in peripheral blood of the infected cattle. Interestingly, the number of PD-1 + CD4 + and PD-1 + CD8 + T cells were negatively correlated with IFN-γ production from PBMCs in bovine mycoplasmosis. Additionally, blockade of the PD-1/PD-L1 pathway in vitro by anti-bovine PD-1- and anti-bovine PD-L1 antibodies significantly upregulated the production of IFN-γ from anti-mycoplasma-specific cells., Conclusions: These results suggest that the PD-1/PD-L1 pathway could be involved in immune exhaustion of bovine mycoplasma-specific T cells. In conclusion, our study opens up a new perspective in the therapeutic strategy for bovine mycoplasmosis by targeting the immunoinhibitory receptor pathways., (© 2017 The Authors. Immunity, Inflammation and Disease Published by John Wiley & Sons Ltd.)

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., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Bovine mastitis due to Mycoplasma californicum is often accompanied by huge economic losses, and the disease spreads very quickly. An appropriate molecular epidemiological analysis is needed to prevent and control infectious disease, but molecular epidemiological analysis methods for M. californicum have not yet been reported. Here we developed a combination of multiple-locus variable-number tandem repeat analysis (MLVA) and pulsed-field gel electrophoresis (PFGE) methods, which are common genotyping methods for various bacteria, for M. californicum. The MLVA is based on four interspersed repeat units that were found in the M. californicum genome data. The MLVA using these repeat units showed sufficient discriminatory power for a molecular epidemiological analysis; i.e., a Hunter-Gaston diversity index (HGDI) of 0.949, against M. californicum strains in Japan and M. californicum strain ATCC 33461. The PFGE for M. californicum also showed sufficient discriminatory power, with an HGDI of 0.985. Strain ATCC 33461 showed MLVA profiles and pulsotypes that differed greatly from those of strains from Japan. These results indicate that MLVA and PFGE are good tools for identifying M. californicum transmission events more accurately. Our combined MLVA and PFGE analysis suggests the persistence of M. californicum infection among herds in a specific area for a long period of time, as well as the movement of cows and heifers accompanying the expansion of M. californicum infection. Failure to identify asymptomatic infected cows is suspected as one of the central causes of the present M. californicum infection scenario in Japan., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

This study investigated the incidence of mycoplasma infection in the uterus of postpartum Holstein dairy cows and its relationship to the occurrence of endometritis. The genital tracts of 209 cows from three dairy farms in the Iwate Prefecture, Japan, were examined at Weeks 5 and 7 postpartum. The condition of the cervicovaginal mucus was assessed using a Metricheck device and assigned a score from 0 (clear mucus) to 4 (purulent material with fetid odor). Intrauterine samples (N = 418) were collected at Weeks 5 and 7 postpartum using a cytobrush. After its withdrawal, swab samples were placed in mycoplasma culture broth at 37 °C for 72 hours. A novel and rapid polymerase chain reaction was used to detect seven mycoplasma species (Mycoplasma bovis, M. arginini, M. bovigenitalium, M. californicum, M. bovirhinis, M. alkalescens, and M. canadense). The cytobrush was also rolled gently along the length of a glass slide for subsequent polymorphonuclear neutrophil count. The diagnostic criteria for cytological endometritis were 6% or more and 4% or more polymorphonuclear neutrophils at Weeks 5 and 7, respectively. From a subset of cows, additional swabs were rolled against the cytobrush and then placed in transport medium. These samples were then plated on specific agar plates and cultured under aerobic and anaerobic conditions to identify other bacteria present. The incidence of dystocia at the last calving was compared in mycoplasma positive and negative cows. Of the seven mycoplasma species, only M. bovigenitalium was detected; it was detected in 31 of the 418 uterine swabs (7.4%). Twenty-four cows were positive for M. bovigenitalium (eight cows at Week 5, nine cows at Week 7, and seven cows at both Weeks 5 and 7). The incidence of dystocia was higher (P < 0.0001) in mycoplasma positive (7/24; 29.2%) compared with mycoplasma negative (4/185; 2.2%) cows. However, there was no significant association between dystocia at last calving and subsequent uterine infection with other bacteria. In addition, the incidence of cytologic endometritis was higher (P < 0.05) in mycoplasma positive (8/16; 50%) than in mycoplasma negative (47/193; 24.4%) cows at Week 7. Therefore, we concluded that M. bovigenitalium infection in the uterus might be associated with recent dystocia and with cytologic endometritis in postpartum dairy cows., (Copyright © 2013 Elsevier Inc. All rights reserved.)

We investigated the contribution of quinolone resistance-determining region (QRDR) mutations to fluoroquinolone (enrofloxacin, orbifloxacin and danofloxacin) susceptibility in 58 Mycoplasma bovis isolates from dairy cattle in Japan. Fluoroquinolone non-resistant isolates (fluoroquinolone-MICs≤2 μg/ml) possessed no QRDR mutations (19 isolates) or Ser83Leu in GyrA (32 isolates). Fluoroquinolone-resistant isolates (fluoroquinolone-MICs≥4 μg/ml) possessed Ser83Leu in GyrA and Ser81Pro in ParC (3 isolates) or Ser83Phe in GyrA and Ser80Ile in ParC (4 isolates). Laboratory-derived fluoroquinolone-resistant mutants selected from 2 isolates (possessing Ser83Leu in GyrA) had an amino acid substitution in ParC at the same position (Ser80Ile or Ser81Tyr) as fluoroquinolone-resistant isolates, suggesting a concurrent amino acid substitution in ParC (Ser80 or Ser81) is important in fluoroquinolone resistance in M. bovis isolates.

The aim of this study was to evaluate the reliability and effectiveness of direct determination of trace and major element concentrations in bronchoalveolar lavage fluid samples from Holstein calves with Mycoplasma bronchopneumonia (n = 21) and healthy controls (n = 20). The samples were obtained during bronchoscopy using a standard examination method. A total of 18 elements (aluminum, bromine, calcium, chlorine, chromium, copper, iron, potassium, magnesium, manganese, molybdenum, nickel, phosphorous, sulfur, silicon, strontium, titanium, and zinc) were detected by particle-induced X-ray emission. The average bromine, iron, potassium, magnesium, and phosphorous concentrations were higher in calves with bronchopneumonia than in controls (p < 0.05). They were found to have higher amounts of calcium and zinc, and a higher zinc-copper ratio than that in healthy calves (p < 0.001). Based on the receiver operating characteristics curves, we propose a diagnostic cutoff point for zinc-copper ratio for identification of Mycoplasma pneumonia of 8.676. Our results indicate that assessment of the elemental composition of broncholaveolar lavage fluid is a promising diagnostic tool for Mycoplasma bronchopneumonia.

The carrier rates of Complex Vertebral Malformation (CVM) in 9 Holstein dairy herds in Hokkaido, number of usages of CVM carrier semen for breeding and gene frequencies of CVM carriers were measured. The mean CVM carrier rates of 140 cows from 4 herds in 1994 and 315 cows from 5 herds in 2003 were 10.8%(range 4.7-30.0%) and 5.1%(range 0.0-6.1%), respectively. The rate of use of CVM carrier semen in the Hokkaido district was 5.6% in 2002. The gene frequencies calculated from CVM carriers among the 315 cows and number of CVM carrier semen samples used were 0.032 and 0.028, and the occurrence of homozygous CVM in 2003 was estimated to be 0.1% in the local districts of Hokkaido, Japan.

The effects of ammonia and hydrogen sulfide on the physical and biochemical properties of the claw horn of Holstein cows were evaluated. Significant (P < 0.05, 0.01) decreases in hardness and elasticity were found in claw horns soaked in ammonia (NH(3)) and hydrogen sulfide (H(2)S) solutions compared with those that were soaked in water for 12, 24, and 48 h. Water absorption rate, as a indicator of permeability barrier function, increased significantly (P < 0.05) over time during the soaking period and was found to be dependent on the concentrations of NH(3) and H(2)S in the solutions. The contents of ceramide, the main lipid component for the permeability barrier system of the stratum corneum, were significantly decreased in claw horns soaked in NH(3) and H(2)S solutions compared with the values before soaking. Quantities of eluted protein released from claw horns treated with NH(3) and H(2)S solutions were approximately 20 times and 30 to 40 times greater than those released from claw horns treated with water alone. Interestingly, the quantities of cytokeratin 10, the main cytoskeletal protein of the stratum corneum, eluted from claw horns treated with NH(3) and H(2)S solutions were markedly greater than the quantity released from horns soaked in water. Our results suggest that abnormal changes in the physical property of claw horn caused by NH(3) and H(2)S treatment are due to disruption of the biochemical property of the claw horn induced by these chemical agents derived from slurry.

Quantities and types of ceramides and their relationships to physical properties of the horn covering the claws of clinically normal cows and cows with subclinical laminitis were investigated. Total ceramide content of the horn covering the sole and wall from cows with subclinical laminitis was 872.2 +/- 146.6 microg/g and 528.6 +/- 61.3 microg/g, respectively, and was significantly (P < 0.01, 0.05) lower than that from clinically normal cows. The mean moisture content in the claws from cows with subclinical laminitis (43.5% +/- 4.3%) was significantly (P < 0.05) higher than that in the claws from clinically normal cows. The hardness of claws from cows with subclinical laminitis (35.2 +/- 3.5) was significantly (P < 0.05) less than that of claws from clinically normal cows. Significant correlations between ceramides and moisture content (P < 0.001) and between ceramide and hardness (P < 0.001) were found in clinically normal cows and cows with subclinical laminitis. Our results indicate that decreases in ceramide contents may be related to changes in physical properties of the horn covering the claw in cows with subclinical laminitis.

Effects of dietary biotin supplementation on serum biotin levels and physical properties of sole horn of 40 Holstein cows were evaluated. The mean serum biotin level in biotin-supplemented cows after 10 mo of biotin supplementation (1163.2 +/- 76.2 pg/mL) was significantly higher (P = 0.007) than that in control cows (382.0 +/- 76.2 pg/mL). The sole horn of biotin-supplemented cows was significantly harder (P = 0.026) and had a significantly lower moisture content (P = 0.021) than that of control cows. No morphologic differences in horn tubules or intertubular horn were found between the biotin-supplemented and control cows. The total lipid content of sole horn was significantly higher (P = 0.030) in the biotin-supplemented cows than in the control cows. These results suggest that dietary biotin supplementation causes increases in serum biotin levels and changes in physical properties and fat content of sole horn.

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.

A female stillborn Holstein calf with shortened cervical and thoracic regions, protrusion of the tongue, and bilateral symmetric flexural contraction of the anterior limbs was delivered on gestation day 281. Multiple hemivertebrae, fused and misshaped vertebrae, synostosis and scoliosis of cervical, thoracic and lumbar vertebral column were found in the affected calf by radiographic and computed tomographic (CT) analysis. Ten pairs of ribs were present and the sternum consisted of 9 sternebrae. Multiple morphologic abnormalities including fusion, malformation, and displacement, were found in the ribs and sternum. Cardiac anomalies, including atrial septal defect and hypertrophy of right ventricle, were observed. DNA-polymerase chain reaction (PCR) analysis demonstrated that amplified product from the liver DNA of the affected calf had identical pattern to that associated with complex vertebral malformation (CVM) of Holstein calves and that her dam was a heterozygous carrier of CVM. The affected calf was diagnosed as having CVM based on the DNA-PCR results and the characteristic findings, and was recorded as a first documentation of CVM confirmed in a Holstein calf in Japan.

Serum cytokine levels and their expression of mRNA on neutrophils from a bone marrow (BM) transplanted heifer with leukocyte adhesion deficiency were evaluated. The clinical condition of the affected heifer was relatively stable after BM-transplantation. Persistent hyper gamma-globulinemia and increased serum immunoglobulin G (IgG) concentrations were monitored longitudinally. The concentration of interleukin (IL)-1beta in serum from the affected heifer ranged from 15.8 to 321.7 ng/ml, and maximum concentration occurred at the time which coincided with peak IL-6. Serum levels of IL-6 ranged from 0.32 to 27.9 ng/m l, and they appeared to be associated with the increment of serum IgG in the affected heifer. mRNAs for IL-1beta, IL-6, IL-8 and granulocyte and macrophage colony stimulating factor (GM-CSF) were increased in neutrophils from the affected heifer compared to controls. Persistent hyper gamma-globulinemia of the affected heifer appeared to be associated with enhanced mRNA expression for IL-6 and its serum levels. These findings suggest that humoral immunity of the affected heifer is activated and the production of neutrophils appears to be enhanced under the incapability of beta(2) integrin-mediated functions of phagocytic cells.

Histopathologic and immunohistochemical examinations were conducted on a 5-year-old Holstein-Friesian cow with systemic kappaAL amyloidosis associated with bovine leukocyte adhesion deficiency. Amyloid deposits were present in the perivascular and intercellular spaces of the visceral organs, such as the liver, kidneys, pancreas, adrenal glands, and upper alimentary tract. Amyloid was stained positively with Congo red with or without 5% potassium permanganate pretreatment and had green birefringence observed under polarized light. Immunohistochemically, amyloid reacted strongly against anti-bovine IgG (H+L) and anti-bovine kappa-light chain and reacted weakly against bovine X-light chain antibodies but was negative for anti-human amyloid AA antibody. This is the first description of AL amyloidosis immunohistochemically related to immunoglobulin kappa-light chains of precursor protein in cattle.

Bone marrow transplantation (BMT) was performed in a 9-month-old heifer with bovine leucocyte adhesion deficiency (BLAD). Clinical and haematological findings, selected neutrophil function and CD18 expression of neutrophils in a B2 integrin-deficient heifer were examined. Twelve months after BMT, a small fluorescent region in the CD18-positive area of a flow cytometric profile was demonstrated and estimated to represent 0.3-0.5% of the CD18-positive neutrophils as measured by flow cytometric analysis following immunomagnetic separation. The animal's clinical condition appeared to improve and stabilize over our observation period of 28 months following BMT. Newly expressed CD18 seemed to play an important role in ameliorating the clinical signs of BLAD in this heifer.

The emigration of leukocytes from calves with beta 2 integrin deficiency (BLAD) into bronchoalveolar spaces and scraped tissues was compared to that of normal calves. Polymorphonuclear neutrophils were found in bronchoalveolar lavage fluid from BLAD-affected calves showing chronic pneumonia. The neutrophils were complement receptor type 3 (CR3)-negative when characterized by flow cytometric analysis using anti-CD18 monoclonal antibody. Chemiluminescent response mediated by CR3 in neutrophils isolated from bronchoalveolar lavage fluid from BLAD-calves showed similar findings obtained from CR3-deficient neutrophils. Neutrophils from normal calves migrated into scraped tissue which was prepared in an upper gluteal surface area, whereas few leukocytes from calves with BLAD migrated to the scraped tissue, evaluated by skin window (Rebuck) method. These findings confirmed the extravasation of CR3-deficient leukocytes into bronchoalveolar lumen in BLAD calves, and demonstrated in vivo characteristics of extravasating property of normal and CR3-deficient neutrophils into scraped tissues.

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.

1Biosynthesis of CD11/CD18 in bovine leucocytes, intracellular Ca2+ ([Ca2+]i) signalling, chemiluminescent responses and membrane fluidity of neutrophils and the effects of D-mannose on neutrophils from control heifers and a heifer with bovine leucocyte adhesion deficiency (BLAD) were measured. The synthesis of CD11/CD18 complex was clearly detected in leucocytes from a normal heifer, but not in a BLAD-affected heifer. The transient phase of increased [Ca2+]i was clearly detected in neutrophils from a heifer with BLAD stimulated with opsonised zymosan, aggregated bovine immunoglobulin G or concanavalin A, whereas the sustained phase was deficient or significantly decreased compared with control heifers. [Ca2+]i signalling of neutrophils from control heifers and a heifer with BLAD stimulated with phorbol myristate acetate via an 11b/CD18-independent pathway showed no transient phase, and the subsequent increase in [Ca2+]i was almost identical in neutrophils from affected and control heifers. [Ca2+]i concentration and chemiluminescent responses of neutrophils from a control heifer were clearly decreased by treatment with anti-CD18 and anti-IgG antibodies. No differences in membrane fluidity were detected between neutrophils derived from control and CD18-deficient cattle. D-mannose binds mainly to Fc rather than CD18 receptors, and decreased Agg-IgG induced [Ca2+]i and the chemiluminescent response of neutrophils. The [Ca2+]i responses and Agg-IgG induced chemiluminescent responses of neutrophils from control heifers and a BLAD-affected heifer were inhibited by D-mannose. The characteristic changes of [Ca2+]i signalling and functional responses of B2-integrin-deficient neutrophils were demonstrated.

The expression of Fc receptors for immunoglobulin G(IgG) and concanavalin A (con A)-binding receptors, luminol-dependent chemiluminescent (LDCL) responses, and the effect of anti-bovine IgG on LDCL responses were evaluated in neutrophils from Holstein calves with leukocyte adhesion deficiency (BLAD). Neutrophils from affected calves showed a 2.1- to 2.5-fold increase in Fc receptor expression compared with those of control calves by flow cytometric analysis. Con A-binding activities of neutrophils from affected calves were similar to those of control calves. Neutrophils from a calf with BLAD, when stimulated with zymosan opsonized with bovine serum (OPZ), heat-aggregated bovine IgG (Agg-bovine IgG), sheep red blood cells (SRBC) sensitized with anti-SRBC antibody (SRBC-anti-SRBC Ab), or con A had LDCL responses of 36 (P < 0.05), 77, 126 and 119% of peak LDCL values of controls, respectively. The NBT-reducing value of neutrophils from a calf with BLAD when stimulated with Agg-bovine IgG after pretreatment with anti-bovine IgG was 116.5% of the values of neutrophils from control calves, but the difference was not significant. The LDCL responses of neutrophils from a control calf and a calf with BLAD stimulated with OPZ were inhibited markedly by pre-incubation with anti-bovine IgG antiserum at concentrations ranging from 1.25 to 20 or 40 micrograms/ml. Although an increase in Fc receptor expression on neutrophils from calves with BLAD was observed, the LDCL responses stimulated with SRBC-anti-SRBC Ab and NBT-reducing activity stimulated with Agg-bovine IgG after pretreatment with anti-bovine IgG did not correlate significantly with the increased Fc receptor expression. These results support that neutrophil functions mediated by the Fc receptors are associated synergistically with the presence of the complement receptor type 3 (CR3)(CD11b/CD18).