Effects of fixation and demineralization on bone collagen D-spacing as analyzed by atomic force microscopy

Abstract

Purpose/Aim: Collagen’s role in bone is often considered secondary. As increased attention is paid to collagen, understanding the impact of tissue preservation is important in interpreting experimental results. The goal of this study was to test the hypothesis that bone fixation prior to demineralization would maintain its collagen ultrastructure in an undisturbed state when analyzed using Atomic Force Microscopy (AFM).

Materials/Methods: The anterior diaphysis of a pig femur was cut into 6?mm pieces along its length. Samples were mounted, polished and randomly assigned to control or fixation groups (n?=?5/group). Fixation samples were fixed for 24?h prior to demineralization. All samples were briefly demineralized to expose collagen, and imaged using AFM. Mouse tail tendons were also analyzed to explore effects of dehydration and fixation. Measurements from each bone sample were averaged and compared using a Mann–Whitney U-test. Tendon sample means were compared using RMANOVA. To investigate differences in D-spacing distributions, Kolmogorov–Smirnov tests were used.

Results: Fixation decreased D-spacing variability within and between bone samples and induced or maintained a higher average D-spacing versus control by shifting the D-spacing population upward. Tendon data indicate that fixing and drying samples leaves collagen near its undisturbed and hydrated native state.

Discussion: Fixation in bone prior to demineralization decreased D-spacing variability. D-spacing was shifted upward in fixed samples, indicating that collagen is stretched with mineral present and relaxes upon its removal. The ability to decrease variability in bone suggests that fixation might increase the power to detect changes in collagen due to disease or other pressures.     

晚期糖基化终末产物对骨组织细胞代谢的影响

文题释义： 晚期糖基化终末产物：是还原糖(如葡萄糖)和某些代谢产物(如甲基已二醛)与蛋白质氨基经过非酶促反应生成的多种化合物,可以积聚在骨组织中,影响骨组织的结构和力学性能,导致骨强度显著下降。 骨胶原交联：骨胶原分子交联包括有利的酶催化交联(即未成熟的二价交联、成熟的三价交联)和不利的非酶催化交联。在骨组织发育过程中,胶原分子在酶催化的情况下,形成不成熟的二价交联,其中一部分二价交联进一步成熟形成三价交联;而在无酶催化情况下交联反应可形成晚期糖基化终末产物。 背景：随着骨组织工程学的研究和发展,发现晚期糖基化终末产物可以在骨组织中积累,影响骨骼的结构及生物力学性能。目前许多研究发现晚期糖基化终末产物/晚期糖基化终末产物受体通过特殊的作用机制后能够引起以成骨细胞、破骨细胞及骨细胞为主的骨组织细胞发生病理改变,导致骨重建失衡,骨骼强度下降,骨折发生率增加。 目的：综述晚期糖基化终末产物对骨骼生物力学的影响以及晚期糖基化终末产物/晚期糖基化终末产物受体对骨组织细胞的作用机制。 方法：由第一作者检索2005年1月至 2019年 7 月在PubMed、Web of Science 和 Medline数据库发表的有关晚期糖基化终末产物/晚期糖基化终末产物受体对骨组织细胞代谢的影响的文章,检索结果限于英文文献。 结果与结论：最终选取具有代表性的54篇文献进行归纳总结。晚期糖基化终末产物对骨胶原交联的影响,使得骨强度显著下降;晚期糖基化终末产物/晚期糖基化终末产物受体通过使骨组织细胞发生病理机制改变影响骨代谢,使得骨组织细胞发生本质改变。最终导致骨代谢平衡紊乱,骨骼脆性增加。骨质疏松症的发生与骨代谢相关的细胞活力改变有着直接关系,但具体相关作用机制需进一步研究,而这种特殊机制的改变在今后有可能为骨质疏松症提供独特的病理机制、诊断思维和相关治疗及预防策略。 中国组织工程研究杂志出版内容重点：人工关节;骨植入物;脊柱;骨折;内固定;数字化骨科;组织工程     

Improved methods for immunohistochemical detection of BrdU in hard tissue

Bromodeoxyuridine (BrdU) is used to label synthesizing DNA and to chase label-retaining cell (LRC). As stem cells divide slowly in adult tissues, they can be visualized as LRCs. In order to identify LRCs in hard tissue, we examined optimal conditions of fixation, demineralization, and DNA denaturation/antigen retrieval for immunohistochemistry of BrdU in hard tissues including bone, tooth, and periodontal ligament. Mice were subcutaneously injected with BrdU (50 µg/g body weight) twice a day from the postnatal day 11 to day 15 and sacrificed at 2 h after the last injection. Dissected maxillae were fixed (Bouin's solution or 4% paraformaldehyde), demineralized (Morse's solution or EDTA), and embedded in paraffin. Antigen retrieval procedures were performed before incubation with primary antibody. When sections were treated with HCl for DNA denaturation, the staining intensity of BrdU positive cells was not affected by difference of fixatives. Higher sensitivity was obtained by demineralization with Morse than with EDTA. Although heat-induced antigen retrieval techniques in citrate buffer (pH 6.0) showed as well or better sensitivity than acid pretreatment, heating caused tissue damage specifically to tooth dentine and the surrounding tissue. When the LRCs at four weeks after the last injection of BrdU were compared, much more LRCs were observed in specimen demineralized with Morse than with 10% EDTA. Our data suggest that demineralization with Morse with Bouin fixative plus HCl pretreatment gives rise to the optimal results for BrdU immunodetection in hard tissue.     

Trabecular Shear Stresses Predict <Emphasis Type="Italic">In Vivo</Emphasis> Linear Microcrack Density but not Diffuse Damage in Human Vertebral Cancellous Bone

Linear microcracks and diffuse damage (staining over a broad region) are two types of microscopic damage known to occur in vivo in human vertebral trabecular bone. These damage types might be associated with vertebral failure. Using microcomputed tomography and finite element analysis for specimens of cancellous bone, we estimated the stresses in the trabeculae of human vertebral tissue for inferosuperior loading. Microdamage was quantified histologically. The density of in vivo linear microcracks was, but the diffuse damage area was not, related to the estimates of von Mises stress distribution in the tissue. In vivo linear microcrack density increased with increasing coefficient of variation of the trabecular von Mises stress and with increasing average trabecular von Mises stress generated per superoinferior apparent axial stress. Nonlinear increase in linear crack density, similar to the increase of the coefficient of variation of trabecular shear stresses, with decreasing bone stiffness and bone volume fraction suggests that damage may accumulate rather rapidly in diseases associated with low bone density due to the dramatic increase of shear stresses in the tissue. © 2003 Biomedical Engineering Society. PAC2003: 8719Rr, 8719Xx, 8759Ls, 8759Fm, 8710+e     

利用光学相干断层图像平均灰度值定量研究牛恒牙早期人工脱矿

目的 研究经酸蚀脱矿后牛恒牙光学相干断层图像平均灰度值的变化,探讨平均灰度值作为一个参数指标定量检测牛恒牙早期脱矿的可行性.方法 以正常离体牛恒牙为样本,采用凝胶酸蚀法制备人工脱矿模型;用光学相干断层摄影术对人工脱矿模型扫描成像,利用Matlab软件计算不同酸蚀时间脱矿区牙体层110 μm范围内的平均灰度值.将测得图像灰度和归一化后图像灰度,分别进行单因素方差分析.结果 牙体表层110 μm范围内不同时间酸蚀脱矿区的平均灰度值从大到小依次为30 min酸蚀脱矿区、1O min酸蚀脱矿区、0 min酸蚀脱矿区,图像灰度归一化后做单因素方差分析,P＜0.05.结论 牙体表层110 μm范围内不同酸蚀时间脱矿区平均灰度值差异有统计学意义,并随脱矿时间增加而增大,即平均灰度值可以用于光学相干断层摄影术定量检测牙齿的早期脱矿.光学相干断层图像灰度归一化使得不同酸蚀脱矿区的平均灰度值更有可比性,减小了由于样本之间差异引起的误差,提高了平均灰度值作为参数指标定量检测牙齿早期脱矿的可靠性.     

HCIF/胶原复合物组织结构的显微观察

目的采用组织学方法,观察HCIF/胶原蛋白复合物的生物学特性。方法取HCIF、HCIF/胶原蛋白复合物及未经任何处理的牛骨（作对照）三个样品,行组织学切片,对其进行组织学观察和研究。结果HCIF试样与对照试样相比,镜下骨板、骨单位组织结构无明显改变,而骨基质伊红染色明显变浅;而HCIF/胶原蛋白复合物的骨基质伊红染色与对照试样相近。结论理化方法能破坏牛骨组织中的胶原基质,复合物能恢复牛骨组织中的胶原基质。     

Effects of dehydration-induced structural and material changes on the apparent modulus of cancellous bone

Dehydration is known to cause an increase in the elastic modulus of bone tissue. However, it also causes structural changes (i.e. shrinkage) which can themselves significantly alter the mechanical properties, particularly in cancellous bone. The current study attempts to estimate the contribution of these two competing factors to the net change of dehydration on the apparent modulus of bovine cancellous bone. Cylindrical cores from the lumbar vertebrae were tested in tension, while hydrated and again after dehydration. The bone volume fractions (BV/TV) were measured in both conditions. The results indicate that the average overall increase in the apparent modulus after dehydration is 14 ± 14% (mean ± SD), which represents the net effect of a 27% increase in modulus due to increased tissue modulus offset by a modulus decrease of 13% due to reductions in bone volume fraction. These observations underscore the need to consider both structural and material changes when comparing hydrated and dehydrated mechanical behaviour.     

Comparison of in vitro techniques to controllably decrease bone mineral density of cancellous bone for biomechanical compressive testing

It is not surprising that an orthopedic device used with poorly mineralized bone can have lower mechanical fixation strength than the same device with well-mineralized bone. As new devices are being designed and tested, it is important to develop a controllable technique to decrease the bone mineral density of bone in vitro, so the fixation strength of the devices can be better modeled. Several different bone demineralization techniques have been established, but some use caustic chemicals and comparisons of their rates of demineralization have not been performed. In this study, a total of 120 cancellous bone cores were excised from ovine vertebra, scanned using a pico dual energy X-ray absorptiometry system to determine bone mineral density, then placed into one of five solutions (0.9% saline, 0.5 M hydrochloric acid, 0.5 M ethylenediaminetetraacetic acid, 0.5 M formic acid, and 5% acetic acid). For each solution, 12 time periods ranging from 0 to 144 h were investigated. After demineralization, all cores were rescanned and biomechanically loaded in compression to failure. Based on the rate of demineralization, the ease of use, the availability, and the correlation with the compressive bone strength, it was determined that the 5% acetic acid was the optimal demineralization solution to controllably decrease the bone mineral density of cancellous bone.     

Correlation between ultrasound velocity and densitometry in fresh and demineralized cortical bone

OBJECTIVE:To compare ultrasound propagation velocity with densitometry in the diaphyseal compact cortical bone of whole sheep metatarsals.METHODS:The transverse ultrasound velocity and bone mineral density of 5-cm-long diaphyseal bone segments were first measured. The bone segments were then divided into four groups of 15 segments each and demineralized in an aqueous 0.5 N hydrochloric acid solution for 6, 12, 24 or 36 hours. All measurements were repeated after demineralization for each time duration and the values measured before and after demineralization were compared.RESULTS:Ultrasound velocity and bone mineral density decreased with demineralization time, and most differences in the pre- and post-demineralization values within each group and between groups were significant: A moderate correlation coefficient (r=0.75956) together with a moderate agreement was determined between both post-demineralization parameters, detected by the Bland-Altman method.CONCLUSION:We conclude that both ultrasound velocity and bone mineral density decrease as a result of demineralization, thus indicating that bone mineral content is of great importance for maintaining the acoustic parameters of cortical bone, as observed for cancellous bone. Ultrasound velocity can be used to evaluate both compact cortical bone quality and bone mineral density.     

Evaluation of the mechanical properties of rat bone under simulated microgravity using nanoindentation

Exposure to microgravity causes a decrease in bone mass and altered bone geometry due to the lack of weight-bearing forces on the skeleton. The mechanical properties of bone are due not only to the structure and geometry, but also to the tissue properties of the bone material itself. To study the effects of microgravity on bone tissue, the mechanical properties of tail suspension rat femurs were investigated. Twelve Sprague–Dawley rats were randomly divided into two groups, tail suspension (TS) and control (CON). On days 0 and 14, the bone mineral density (BMD) of the femurs was determined by Dual Energy X-ray Absorptiometry. After 14 days, three-point bending was used to test the mechanical properties of the whole femur and nanoindentation was used to measure the mechanical properties of the bone materials. The BMD of femurs in TS was significantly lower than that in CON. In the three-point bending testing, the breaking load, stiffness and energy absorption all decreased significantly in the TS group. In the nanoindentation tests, there was no significant difference between TS and CON in elastic modulus (E), while hardness (H) was significantly decreased and E/H significantly increased in TS. Weightlessness affects the intrinsic mechanical properties of bone at the bone material level. It is necessary to investigate further the effect of microgravity on the collagen bone matrix. Nanoindentation is a relatively new technique that is useful for investigating the above changes induced by microgravity and for assessing the efficacy of intervention.     

Changes of zero-bending-moment states and structures of rat arteries in response to a step lowering of the blood pressure

There are many papers on tissue remodeling of blood vessels in hypertension, but there are few documents describing the tissue remodeling of the blood vessels following a step lowering of the blood pressure. The present article presents data on the opening angle, the vessel wall thickness, and the thicknesses of the intima-media and adventitia layers of the blood vessels of the lower body (the abdominal aorta, and the common iliac, femoral, saphenous branch, medial plantar, and plantar metatarsal arteries) of the rat after a step lowering of the blood pressure and flow by a controlled constriction of the aorta below both renal arteries. We found a pattern of changes that depend on space (location on the vascular tree), time (after the blood pressure change), and the intensity of disturbance. We model mathematically the dynamics shown by the experimental results by means of the indicial response functions, which are defined as the morphometric changes in response to a step decrease of blood pressure or blood flow. Under the hypothesis that there is a range of linearity between the degree of tissue remodeling and the amplitude of the pressure change, we can use the indicial functions to predict the remodeling of the vessel under an arbitrary history of decreasing blood pressure; and conversely, we can compute the indicial response functions from pertinent results of a single experiment. The totality of all our experiments is consistent with the linearity hypothesis within the range of the experiment. The mathematical analysis and the formulas are presented in the Appendix. © 2002 Biomedical Engineering Society. PAC2002: 8719Uv, 8719Rr     

Nitrite-Induced Cross-Linking Alters Remodeling and Mechanical Properties of Collagenous Engineered Tissues

Cumulative damage to long-lived connective tissue proteins play a key role in the development of age-related human diseases such as cardiovascular stiffening and age-related macular degeneration. The processes that result in the accumulation of increasingly insoluble, undigestible damaged collagen are only partially known. Nonenzymatic glycation (NEG) is one such process and has been linked to the development of diabetic-related complications and aging. An additional novel mechanism particularly relevant to smoking- and inflammation-related diseases involves the nonenzymatic nitrite (NEN) modification of connective tissue proteins. The present study was undertaken to examine the effects of NEN of fibrillar type I collagen on cell-mediated remodeling and mechanical properties of collagenous tissues. Using a modification of an in vitro fibroblast-populated collagen gel model system developed in our laboratory, we tested two hypotheses: NEN reduces the ability of primary adult cardiac fibroblasts to remodel type I collagen gels; NEN reduces the deformability of type I collagen gels subjected to mechanical testing. The results show that NEN impairs both cell-mediated remodeling and mechanical deformability in collagenous engineered tissues. Furthermore, these mechanical changes correlate with the degree of cross-linking as determined by SDS-PAGE. Thus, we concluded that NEN reactions may contribute to alterations in the biomechanical properties of collagen-containing tissues consistent with the age-related functional decline observed in human disease.     

Comparison of novel xenograft (bovine fetal growth plate) and allograft effects on experimental bone defect healing in rabbit

Large bone defects resulting from trauma, tumors, osteitis, implant loosening, or corrective osteotomies require surgical therapy because spontaneous regeneration is limited to relatively small defects. Currently, transplantation of autografts or allografts, mineral bone substitutes, and callus distraction are the most commonly used techniques for skeletal reconstruction. Each method has significant limitations, e.g., availability and biological or biomechanical reasons. This study was designed to evaluate allograft and new xenograft (bovine fetal growth plate) effects on the bone healing process. Twenty male New Zealand White rabbits were used in this study. In the allograft group, the defect was filled by fresh allogeneic cortical graft; in the xenograft group, the defect was filled by a segment of bovine fetal growth plate and was fixed by cerclage wire. Radiological, histopathological, and biomechanical evaluations were performed and results were scored and analyzed statistically. Statistical tests did not support significant differences between the two groups radiographically at the 14th postoperative day (P > 0.05). There was a significant difference in bone formation at the 28th, 42nd, and 56th postoperative days. There were significant radiological differences for bone union and remodeling by the 42nd day postoperatively (P < 0.05). The xenograft was superior to the allograft by the 56th postoperative day for radiological bone formation (P < 0.03); histopathological and biomechanical evaluation revealed no significant differences between the two groups. It can be concluded that the superior bone healing process in the xenograft group was due to the presence of some osteoinduction proteins in bovine fetal growth plate.     

An in vitro Model System to Study the Damaging Effects of Prolonged Mechanical Loading of the Epidermis

Pressure ulcers are areas of soft tissue breakdown that result from sustained mechanical loading of the skin and underlying tissues. Today, little is known with respect to the aetiology of these ulcers. This study introduces an in vitro model system to study the effects of clinically relevant loading regimes on damage progression in the epidermis, the uppermost skin layer. Engineered epidermal equivalents (EpiDerm) were subjected to 6.7 and 13.3 kPa for either 2 or 20 h using a custom-built loading device. Tissue damage was assessed by (1) histological examination, (2) tissue viability evaluation, and (3) by the release of a pro-inflammatory mediator, interleukin-1α (IL-1α). Loading the EpiDerm samples for 2 h increased the IL-1α release, although no visible tissue damage was observed. However, in the 20 h loading experiments visible tissue damage and a small decrease in tissue viability were observed. Furthermore, in these experiments the IL-1α release increased with magnitude of loading. It is concluded that this in vitro model system can be applied to improve insight in the epidermal damage process due to prolonged mechanical loading and can serve as a sound basis for effective clinical identification and prevention of pressure ulcers.     

Mathematical Model of Diffusion-Limited Evolution of Multiple Gas Bubbles in Tissue

Models of gas bubble dynamics employed in probabilistic analyses of decompression sickness incidence in man must be theoretically consistent and simple, if they are to yield useful results without requiring excessive computations. They are generally formulated in terms of ordinary differential equations that describe diffusion-limited gas exchange between a gas bubble and the extravascular tissue surrounding it. In our previous model (Ann. Biomed. Eng. 30: 232–246, 2002), we showed that with appropriate representation of sink pressures to account for gas loss or gain due to heterogeneous blood perfusion in the unstirred diffusion region around the bubble, diffusion-limited bubble growth in a tissue of finite volume can be simulated without postulating a boundary layer across which gas flux is discontinuous. However, interactions between two or more bubbles caused by competition for available gas cannot be considered in this model, because the diffusion region has a fixed volume with zero gas flux at its outer boundary. The present work extends the previous model to accommodate interactions among multiple bubbles by allowing the diffusion region volume of each bubble to vary during bubble evolution. For given decompression and tissue volume, bubble growth is sustained only if the bubble number density is below a certain maximum. © 2003 Biomedical Engineering Society. PAC2003: 8719Uv, 8710+e, 8715Vv, 8719Xx     

Similar damage initiation but different failure behavior in trabecular and cortical bone tissue

The mechanical properties of bone tissue are reflected in its micro- and nanostructure as well as in its composition. Numerous studies have compared the elastic mechanical properties of cortical and trabecular bone tissue and concluded that cortical bone tissue is stiffer than trabecular bone tissue. This study compared the progression of microdamage leading to fracture and the related local strains during this process in trabecular and cortical bone tissue. Unmachined single bovine trabeculae and similarly-sized cortical bovine bone samples were mechanically tested in three-point bending and concomitantly imaged to assess local strains using a digital image correlation technique. The bone whitening effect was used to detect microdamage formation and propagation. This study found that cortical bone tissue exhibits significantly lower maximum strains (trabecular 36.6%±14% vs. cortical 22.9%±7.4%) and less accumulated damage (trabecular 16100±8800 pix/mm2 vs. cortical 8000±3400 pix/mm2) at failure. However, no difference was detected for the maximum local strain at whitening onset (trabecular 5.8%±2.6% vs. cortical 7.2%±3.1%). The differences in elastic modulus and mineral distribution in the two tissues were investigated, using nanoindentation and micro-Raman imaging, to explain the different mechanical properties found. While cortical bone was found to be overall stiffer and more highly mineralized, no apparent differences were noted in the distribution of modulus values or mineral density along the specimen diameter. Therefore, differences in the mechanical behavior of trabecular and cortical bone tissue are likely to be in large part due to microstructural (i.e. orientation and distribution of cement lines) and collagen related compositional differences.     

基于损伤修复的骨重建数值模拟

目的 研究不同损伤情况下的骨重建行为。方法 提出一种疲劳机制作用下的骨重建模型,通过建立股骨近端三维有限元模型,并结合有限元法,分别模拟3种载荷工况下的骨重建情况,分析股骨近端的力学性能及密度变化。结果 通过增加载荷循环次数,使损伤不断增加。在不同损伤情况下,骨展现出不同的骨重建行为。骨重建作为一种修复机制,在一定范围内可以弥补由于疲劳损伤造成的骨量丢失。结论 提出的损伤自适应重建模型可以模拟不同损伤情况下的骨重建行为,以及由于载荷循环次数过大引起的过载吸收。研究疲劳损伤作用下的骨重建行为,可以为骨折预防以及术后康复治疗提供参考。     

Dependence of the velocity and attenuation of ultrasound in bone on the mineral content.

Measurements of the attenuation and velocity of ultrasound from 0.3 to 0.8 MHz have been performed on a number of bovine cancellous bone samples. The influence of bone mineral content has been isolated by measuring the acoustic properties of the samples at various stages of demineralization resulting from controlled nitric acid attack. The correlation coefficient r, between the attenuation at different frequencies and bone density was found to be in the range 0.68-0.97. Broadband ultrasonic attenuation (BUA) was also calculated and produced r values between 0.84 and 0.99. The velocity measurements indicated a correlation greater than 0.97 in all cases. Thus velocity is the parameter most sensitive to changes in bone mineral density alone. Attenuation and BUA are less well correlated presumably because of a sensitivity to minor structural change.     

Stimulation of Reparative Osteogenesis after Xenotransplantation of Human Prenatal Mesenchymal Stem Cells and Chondroblasts

Regeneration of the bone tissue after unilateral xenogeneic transplantation of a suspension of human mesenchymal cells and chondroblasts was studied in rats with experimental damage to both femurs. The state of animals was satisfactory and non-depressed in the early and late postoperation period. No local pathological reactions and complications were seen. Administration of mesenchymal stem cells and chondroblasts into the area of bone defect accelerated regeneration on days 10 and 30 (compared to the control group). Implantation of mesenchymal stem cells more significantly stimulated reparative osteogenesis compared to treatment with chondroblasts. This was mainly associated with increased ratio of mature lamellar bone tissue. Unilateral transplantation of the cell suspension stimulated regeneration in the contralateral limb due to accelerated maturation of the bone tissue. The bone tissue formed after transplantation of mesenchymal stem cells and chondroblasts was integrated into bone organ and underwent complete remodeling. Xenotransplantation of prenatal mesenchymal stem cells and chondroblasts without immunosuppression was not followed by the development of early and delayed complications or local reactions of graft rejection.__________Translated from Kletochnye Tekhnologii v Biologii i Meditsine, No. 2, pp. 75–83, 2005     

Apoptosis: a possible tactic ofHaemophilus somnusfor evasion of killing by bovine neutrophils?

Haemophilus somnusis an important veterinary pathogen that causes respiratory disease, arthritis, septicaemia and abortion in cattle and sheep. In the present study we investigated the possibility thatH. somnusresists killing by bovine neutrophils, by causing the latter to undergo morphological changes consistent with apoptosis. Both serum-sensitive and serum-resistant strains ofH. somnusenhanced bovine neutrophil chromatin condensation and shape change (i.e. zeiosis)in vitro, suggesting that the cells were undergoing apoptosis. Heat-killed or formalin-killedH. somnushad less effect than viableH. somnus.Chromatin margination of neutrophils was greater whenH. somnuswas opsonized with adult bovine serum, which facilitates phagocytosis of the bacteria.H. somnusculture filtrates did not cause bovine neutrophil chromatin condensation. These findings suggest that direct contact withH. somnusis required for the maximal effect on bovine neutrophils. Apoptosis was confirmed by flow cytometry, using propidium iodide staining to detect DNA fragmentation. These findings suggest thatH. somnuscan evade killing by bovine neutrophils, in part, by inducing these cells to undergo apoptosis.