Replication of parainfluenza type-3 virus and bovine respiratory syncytial virus in isolated bovine type-II alveolar epithelial cells.

The objectives of our research were to determine whether bovine pulmonary type-II alveolar epithelial cells could be isolated from bovine lung and maintained in tissue culture and to determine whether isolated bovine type-II alveolar epithelial cells would support productive viral replication of bovine parainfluenza type-3 virus and bovine respiratory syncytial virus. Type-II alveolar epithelial cells were isolated from lungs of 4- to 7-day-old male Holstein calves by enzymatic dissociation of pulmonary tissue with trypsin and by separation of cells with the use of filtration and centrifugation on continuous Percoll gradients. Cells were further separated by panning on IgG-coated plastic plates and by lectin binding. Isolated type-II alveolar cells were maintained on basement membrane-coated tissue cultured plates. In culture, type-II cells formed alveolar structures and maintained other cytologic features of type-II cells, including osmiophilic lamellar inclusions. Cell cultures were inoculated with and supported productive replication of bovine parainfluenza type-3 virus and bovine respiratory syncytial virus. This was determined by recovery of infectious viruses from inoculated cell cultures and by identification of viral structures in type-II alveolar epithelial cells by transmission electron microscopy.     

THE DURATION OF THE RESPONSE OF CATTLE TO INOCULATION WITH ATYPICAL MYCOBACTERIA

SUMMARY Each of 4 strains of atypical mycobacteria was inoculated into 2 cattle and the responses of the cattle were studied over the following 52 weeks. Each strain was injected subcutaneously into one animal and into a mesenteric lymph node of another. Within 7 days palpable lesions were produced at the sites of subcutaneous inoculation in response to all the strains. After intervals varying from 3 to 26 weeks, lesions due to 3 of the strains were no longer palpable. The lesion produced in response to the fourth strain, a non-agglutinable serotype of Mycobacterium intracellulare, was still palpable at necropsy, 52 weeks post-inoculation (PI). Of the 8 cattle inoculated with mycobacteria, the latter was the only animal that had a lesion with features consistent with a mycobacterial infection and from which mycobacteria were isolated. The inoculated cattle and 4 uninoculated control cattle were turberculin tested on 8 occasions during the post-inoculation period. Bovine purified protein derivative (PPD), avian PPD and PPD tuberculins prepared from each of the atypical mycobacteria were used. In inoculated cattle, sensitivity to both avian and bovine PPD was short lived, significant levels not persisting in any animal beyond 16 weeks PI. From the results of intradermal tests on the control cattle, a 95% confidence interval for their response to any of the 6 tuberculins used, was found to be ±1.36mm. On this basis all inoculated cattle developed sensitivity to the homologous tuberculin. The animal with mycobacterial granuloma at the subcutaneous inoculation site at necropsy had never developed significant levels of sensitivity to bovine PPD, had not shown significant levels of avian sensitivity after week 16 PI nor had it shown homologous sensitivity after week 22 PI. In all animals the level of sensitivity to bovine PPD decreased between successive tests. This fact could be used to clarify the status of a reactor if non-specific bovine sensitivity was suspected. Alternatively, the comparative intradermal tuberculin test using both bovine and avian PPD may be employed.     

THE EFFECT OF BOVINE EPHEMERAL FEVER VIRUS ON THE BOVINE FOETUS

Six pregnant heifers that were experimentally infected with bovine ephemeral fever virus produced normal calves. Foetuses of 8 heifers that were immune to bovine ephemeral fever were inoculated with bovine ephemeral fever virus. One was aborted after 36 days, but the cause of abortion could not be determined. One was sacrificed after 28 days. The foetus and its membranes were normal and neither virus nor antibody was demonstrated. The other 6 heifers produced normal calves. One calf, 501, inoculated after 160 days gestation, contained high levels of neutralising antibody in serum before ingestion of colostrum. The others, inoculated at gestational ages of 52 days to 157 days showed no evidence of neutralising antibody in blood samples collected at birth.     

Development of interspecies cloned monkey embryos reconstructed with bovine enucleated oocytes

This study was carried out to determine whether culture media reconstructed with bovine enucleated oocytes and the expression pattern of Oct-4 could support dedifferentiaton of monkey fibroblasts in interspecies cloned monkey embryos. In this study, monkey and bovine skin fibroblasts were used as donor cells for reconstruction with bovine enucleated oocytes. The reconstructed monkey interspecies somatic cell nuclear transfer (iSCNT) embryos were then cultured under six different culture conditions with modifications of the embryo culture media and normal bovine and monkey specifications. The Oct-4 expression patterns of the embryos were examined at the two-cell to blastocyst stages using immunocytochemistry. The monkey iSCNT embryos showed similar cleavage rates to those of bovine SCNT and bovine parthenogenetic activation (PA). However, the monkey iSCNT embryos were not able to develop beyond the 16-cell stage under any of the culture conditions. In monkey and bovine SCNT embryos, Oct-4 could be detected from the two-cell to blastocyst stage, and in bovine PA embryos, Oct-4 was detectable from the morula to blastocyst stage. These results suggested that bovine ooplasm could support dedifferentiation of monkey somatic cell nuclei but could not support embryo development to either the compact morula or blastocyst stage. In conclusion, we found that the culture conditions that tend to enhance monkey iSCNT embryo development and the expression pattern of Oct-4 in cloned embryos (monkey iSCNT and bovine SCNT) are different than in bovine PA embryos.     

HOW THE MACH PHENOMENON AND SHAPE AFFECT THE RADIOGRAPHIC APPEARANCE OF SKELETAL STRUCTURES

The shape of skeletal structures and their position relative to the x-ray beam have a considerable effect on their radiographic appearance. Depending on the thickness of the cortical or subchondral bone, skeletal structures display the characteristics of either homogeneous or compound lamellar structures. Convex homogeneous structures are associated with a negative Mach line, and concave homogeneous structures are associated with a positive Mach line. Convex compound lamellar structures are associated with a negative Mach band and visualization of the lamina (subchondral or cortical bone) is reduced. Concave compound lamellar structures are associated with a positive Mach band and visualization of the lamina is enhanced. The combined effect of Mach phenomenon, shape, and thickness enhances visualization of some skeletal surfaces and make others imperceptible. These principles are very useful to correctly identify complex skeletal structures and avoid misinterpretations.     

The timeline of lamellar basement membrane changes during equine laminitis development

Reasons for performing study: The timing of lamellar basement membrane (BM) changes occurring during laminitis development is incompletely understood. Objectives: To determine the temporal progression of lamellar BM changes and whether laminin‐332 (Ln‐332) γ2 cleavage products are generated during laminitis development. Methods: Eight clinically normal Standardbred horses were allocated into treatment (n = 5) or sham (n = 3) groups. The treatment group received, via nasogastric intubation, an oligofructose (OF) bolus (10 g/kg bwt) while the sham group was given water. Laminitis induction proceeded for 48 h followed by euthanasia. Lamellar biopsies were obtained prior to dosing and at intervals during the treatment period for analysis (at 12, 18, 24, 30 and 36 h and at 48 h following euthanasia). Results: Changes in lamellar collagen type IV and Ln‐332 were first observed at 12 h post dosing. A unique pattern of reactivity for the Ln‐332 γ2 antibody D4B5 occurred, in which reactivity was observed only in lamellar tissue affected by laminitis. No bioactive Ln‐332 γ2 proteolytic fragments were detected in lamellar samples. Conclusions: Basement membrane changes occurred early during the laminitis process. Direct Ln‐332 γ2 cleavage to release biologically active products did not appear to occur. Thus loss of stability or protein interaction of the BM is probably responsible for the γ2 specific reactivity observed. Potential relevance: Basement membrane changes may a first step in lamellar failure occurring prior to detection with conventional methods. Thus, more sensitive detection methods of BM changes are required to study laminitis development.     

Epidemiology of brucellosis among cattle in Korea from 2001 to 2011

In the present study, the outbreak patterns of bovine brucellosis in Korea from 2000 to 2011 were analyzed to understand the epidemiological evolution of this disease in the country. A total of 85,521 brucella reactor animals were identified during 14,215 outbreaks over the 12-year study period. The number of bovine brucellosis cases increased after 2003 and peaked in 2006 before decreasing thereafter. The majority of the bovine brucellosis cases were Korean native cattle, Han Woo. The numbers of human brucellosis cases and cattle outbreaks increased and decreased in the same pattern. The correlation coefficient for human and bovine cases per year was 0.96 (95% confidence interval = 0.86~0.99; p < 10^-3). The epidemiological characteristics of bovine brucellosis appeared to be affected by the intensity of eradication programs that mainly involved a test-and-slaughter policy. Findings from the present study were based on freely available statistics from web pages maintained by government agencies. This unlimited access to information demonstrates the usefulness of government statistics for continually monitoring the health of animal populations.     

Harlequin ichthyosis in two greater kudu (Tragelaphus strepsiceros)

Two greater kudu calves (Tragelaphus strepsiceros) born 7 years apart were found with fissures and thickened, scaly, cutaneous plates covering over 80% of their bodies. One was dead at presentation, and the other was euthanized shortly after birth. Both animals shared a common sire. On necropsy, chemosis, ectropion, eclabium, and bilateral valgus deformities of the tarsal joints were observed in one calf, presumed to be secondary to the plates restricting normal fetal development. The principal microscopic lesion was severe lamellar orthokeratosis, with focal mild parakeratosis. Ultrastructural epidermal lesions included the absence of normal lamellar granules, large dilated endoplasmic reticulum, and abnormal retention of organelles and vesicles. Gross, histopathologic, and electron microscopic findings in both kudu calves were consistent with those of harlequin ichthyosis, a rare dermatosis of humans believed to have an autosomal recessive inheritance pattern. The underlying genetic and molecular abnormality and heritability of this condition in this greater kudu herd were not determined.     

Differences in fluorescent antibody staining of bovine respiratory syncytial virus-infected cells by ovine and bovine sera

In indirect fluorescent antibody tests in which sera from cattle and sheep with respiratory disease problems were used to stain foetal bovine lung cells infected with a bovine respiratory syncytial virus strain, differences were noted in the pattern of fluorescence produced by some sheep sera and that produced by positive bovine sera. In serum neutralisation tests, also using a bovine respiratory syncytial virus strain, 4 of 7 sera giving this atypical pattern of fluorescence had very low neutralising antibody titres (highest 1/4), and 3 were negative. It is suggested that two related but antigenically distinguishable respiratory syncytial virus types are present in sheep, one of which is similar to bovine strains.     

Equine laminitis model: Lamellar histopathology seven days after induction with oligofructose

Reasons for performing study: The histopathology of laminitis during its transition from the acute to the chronic phase has not been previously documented. Studying hoof lamellar tissues 7 days after induction of laminitis may provide insight into the intractable nature of the chronic phase of the disease. Objectives: To induce laminitis and investigate hoof wall lamellar tissues 7 days after dosing. Methods: Laminitis was induced using oligofructose in 6 normal Standardbred horses. The dorsal hoof lamellar tissues of these and 12 normal horses were processed and examined by light microscopy. Serial sections of a lamellar tip affected by laminitis were used to create a 3 dimensional reconstruction. Results: Transverse sections of dorsal hoof wall lamellae were significantly longer than normal. Many secondary epidermal lamellae were not connected to primary lamellae and existed as spherical or ovoid, discrete islands isolated in the lamellar dermis. The lamellar basement membrane was intact. Conclusions: Lamellar tissue has the ability to reorganise rapidly following an episode of acute laminitis. Although histopathological evidence of ongoing acute laminitis was absent by 7 days, there was marked disruption of lamellar architecture. Potential relevance: The architecture and subsequent strength of the resultant lamellar interface could be greatly influenced for the better by strategies that minimise mechanical displacement during the acute phase of laminitis.     

Effect of bovine adenovirus 3 on mitochondria

Viruses alter the structure and the function of mitochondria for survival. Electron microscopy analysis of the cells infected with bovine adenovirus 3 revealed extensive damage to the inner mitochondrial membrane characterized by dissolution of the cristae and amorphous appearance of mitochondrial matrix with little or no damage to the outer mitochondrial membrane. There were fewer cristae with altered morphology. Potential patches of protein synthesis machinary around mitochondria could be observed at 12 hours post infection (hpi). At 24 hpi, the multi vascular bodies were evident throughout the infected cell. ATP production, mitochondrial Ca²⁺ and mitochondrial membrane potential (MMP) peaked at 18 hpi but decreased significantly at 24 hpi. This decrease coincided with the increased production of superoxide (SO) and reactive oxygen species (ROS), at 24 hpi indicating acute oxidative stress in the cells and suggesting a complete failure of the cellular homeostatic machinary. The results reveal an intericate relationship between Ca²⁺ homeostasis, the ATP generation ability of cells, SO and ROS production, and regulation of MMP following infection by bovine adenovirus 3.     

交替浸渍矿化丝胶膜表面沉积的矿化物及对丝胶膜结构的改变

丝胶蛋白中含有的大量羧基、羟基等活性基团与生物矿化有着密切的联系。以家蚕丝胶膜为模板,采用氯化钙和磷酸氢二钠溶液交替浸渍的方法,研究经不同矿化周期处理丝胶膜上的矿化物沉积及对丝胶膜结构的改变情况。通过红外吸收光谱(FTIR)仪、X射线衍射(XRD)仪、扫描电子显微镜(SEM)和X射线能谱(EDX)仪对表面沉积矿化物的丝胶膜进行微观形貌与结构表征,可见矿化物能快速有效地在丝胶膜表面沉积和生长,随着矿化周期的增加,矿化物的沉积量不断增加,丝胶膜表面的结晶度略有提高,沉积的矿化物呈龟裂、薄板状,主要成分为无定形磷酸钙盐。采用氯化钙和磷酸氢二钠溶液交替矿化的方法并增加矿化周期可促进无定形磷酸钙盐在丝胶膜表面的沉积,有望进一步开发出丝胶蛋白/磷酸钙盐复合支架材料应用于骨组织工程。     

Equine laminitis: Ultrastructural lesions detected in ponies following hyperinsulinaemia

Reasons for performing study: Anatomical changes in the hoof lamellar tissue induced by prolonged hyperinsulinaemia have not been described previously. Analysis of the induced lesions may promote understanding of hyperinsulinaemic laminitis pathogenesis and produce clinical benefit. Objectives: To use light and transmission electron microscopy (TEM) to document hoof lamellar lesions in ponies clinically lame after prolonged hyperinsulinaemia. Methods: Nine clinically normal, mature ponies were allocated randomly to either a treatment group (n = 5) or control group (n = 4). The treatment group received insulin via a modified, prolonged euglycaemic hyperinsulinaemic clamp technique (EHCT) and were subjected to euthanasia when clinical signs of Obel grade II laminitis occurred. The control group was sham treated with an equivalent volume of 0.9% saline and killed at 72 h. Lamellar tissues of the right front feet were harvested and processed for TEM. Results: Lamellae from insulin treated ponies were attenuated and elongated with many epidermal basal cells (EBC) in mitosis. Unlike carbohydrate induced laminitis in horses there was no global separation at the lamellar dermal/epidermal interface among ponies. Sporadic EBC basement membrane (BM) separation was associated with the proximity of infiltrating leucocytes. In 2 ponies, the lamellar BM was thickened. The number of hemidesmosomes/μm of BM was decreased in all insulin treated ponies. Conclusions: Prolonged hyperinsulinaemia causes unique lamellar lesions normally characteristic of acute and chronic laminitis. Lamellar proliferation may be an insulin effect through its mitogenic pathway. Aberrant lamellar mitosis may lengthen and weaken the lamellar, distal phalanx attachment apparatus and contribute to the clinical signs that developed. Potential relevance: The study shows that insulin alone, in higher than normal circulating concentrations, induces profound, changes in lamellar anatomy. Medical control of insulin resistance and hyperinsulinaemia may ameliorate lesions and produce clinical benefit.     

牛体内囊胚全转录组模式解析

旨在解析牛体内囊胚的全转录组模式。本研究选择3头15月龄以上、健康状况良好的泌乳奶牛，采用超排获得体内囊胚，采用单细胞全转录组测序技术检测囊胚mRNA、lncRNA、circRNA转录模式，并采用生物信息学工具对其进行分析。试验获得牛体内囊胚约19 975个mRNAs、12 715个novel lncRNAs及887个circRNAs。mRNA有12种可变剪切方法，以转录起始区域可变剪切（TSS）为主。采用基因本体（GO）和相关信号通路（KEGG）对其分析发现，生物功能主要富集在新陈代谢、细胞膜和蛋白结合、细胞通讯、细胞组织成分、生长发育等功能，Pathway主要富集在细胞周期、膜运输、染色体组织调控等通路；circRNA有6种类型，其中CLASSIC类型circRNA数量最多，这种剪切方式会形成成环的外显子circRNA （EcircRNA）。本研究阐明了牛体内囊胚的全转录组模式，为全面了解牛体内囊胚提供了新的思路。     

RADIOGRAPHY OF THE BOVINE CRANIOVENTRAL ABDOMEN

This study consists of a review of 115 consecutively accrued bovine cranial abdomial radiographs. The sensitivity and specificity of radiography in detecting traumatic reticuloperitonitis or pericarditis was 83% and 90%, respectively. Increased reticular size was associated with vagal indigestion. Increased reticulo-diaphragmatic separation did not correlate with a specific disease process, 25/35 (71%) cattle presented for radiographic examination with a 1 cm or longer metallic reticular foreign body unattached to a magnet had traumatic reticuloperitonitis. Standing lateral abdominal radiographs were determined to be a valuable tool in the diagnosis of cranial abdominal disorders in the bovine.     

成年型牛淋巴肉瘤淋巴样细胞的细胞化学研究

应用过氧化物酶-抗过氧化物酶(PAP)免疫酶标技术及酸性α-萘酯乙酸酯酶(ANAE)、Mg~( )-ATP酶技术等细胞化学方法,研究了10例乳牛成年型淋巴肉瘤淋巴样细胞的细胞化学特性。结果显示,绝大多数淋巴样细胞具有表面膜免疫球蛋白(Smlg)及膜Mg~( )-ATP酶活性,证明南京及皖南地区乳牛地方流行性白血病系B淋巴细胞增生。肿瘤组织中淋巴样细胞以弥散性增生为特证,在形态上存在大小两种细胞类型,虽均带有Smlg,但小型细胞又多呈ANAE阳性,提示后者是起源于B淋巴细胞的ANAE活性异常升高的类型,ANAE技术不适用于牛白血病T、B淋巴细胞的鉴别。     

Development,validation, and pilot application of a semiquantitative Western blot analysis and an ELISA for bovine adiponectin

Adiponectin is an adipose tissue-derived glycoprotein circulating as highly abundant multimers. It regulates glucose metabolism and insulin sensitivity. In ruminants, valid data about serum concentrations and tissue-specific protein expression are lacking, and we, therefore, aimed to generate a polyclonal antibody against bovine adiponectin to apply it in immunodetection. The specificity of the purified anti-adiponectin antibody was established by Western blot analysis with the use of reducing and denaturing conditions applied to both the purified protein and the bovine serum samples. Besides bovine serum, the applicability of the antibody for immunodetection of adiponectin was confirmed for the supernatant fluid of in vitro–differentiated bovine adipocytes, for protein extracts from bovine adipose tissue, and also in a multispecies comparison: bands comparable in size with monomeric bovine adiponectin were obtained under denaturing conditions in serum of camel, horse, human, mouse, pig, roe deer, and sheep. In addition, when used in immunohistochemistry on bovine adipose tissue sections, a characteristic adipocyte-specific staining pattern was obtained with this antibody. The antibody was used for establishing a semiquantitative Western blot procedure and the development of an ELISA. Both methods were extensively validated and were first applied to characterize the serum adiponectin concentrations in multiparous dairy cows during the transition from pregnancy to lactation, that is, 3 wk before until 5 wk after calving. With both assays a time effect (P = 0.017, P = 0.026, respectively) with lowest values at the day of parturition was observed. We thus established 2 useful tools to validly assess bovine adiponectin at the protein level.     

Hepatocellular carcinoma with metastasis to the spleen in a Holstein cow

Hepatocellular carcinoma (HCC) with metastasis to the spleen in a Holstein cow was studied by histopathologic and immunohistochemical methods. The tumor was characterized by a pseudoglandular (acinar) pattern with an associated fibrous stroma. Individual cells often had a "hepatoid" appearance but were interspersed with scattered cells exhibiting a clear, periodic acid-Schiff (PAS)-positive cytoplasm and small eccentric nuclei. This pattern was present in nodules found in both liver and spleen. Moreover, hepatoid tumor cells were positive for alpha-fetoprotein. Immunohistochemical studies suggest that myofibroblasts were responsible for the production of fibrous septa surrounding the pseudoglandular structures of bovine HCC. In summary, our histologic and immunohistochemical findings support a diagnosis of primary HCC with splenic metastasis. Furthermore, the associated stromal response appears to be of a myofibroblast origin. The primary etiology of bovine HCC and the significance of the intralesional, PAS-positive clear cells remain undetermined.     

Isolation and comparative restriction endonuclease DNA fingerprinting of equine herpesvirus-1 from cattle

Deoxyribonucleic acid fingerprinting analyses with 4 restriction endonucleases (EcoRI, BamHI, BglII, and HindIII) and serotest results have definitively indicated that 5 herpesviruses isolated from 1974 to 1986 from aborted bovine fetuses and from bovine tissues and nasal secretions were abortigenic subtypes of equine herpesvirus type 1 (EHV-1). The herpesviruses, designated BH1247, 3M20-3, G118, H1753, and 9BSV4, were neutralized by EHV-1-specific antiserum and could be propagated in cultures of either bovine or equine cells. Only minor differences in restriction endonuclease patterns were detected from the pattern of an Army 183 isolate of EHV-1 subtype 1 that had been passaged only in equine cells and from that of an attenuated EHV-1 subtype 1 (RQ) strain that had been passaged several hundred times in non-equine cells. The individual differences in the restriction endonuclease fragments of the 5 bovine isolates and the Army 183 and RQ strains mainly were attributable to alterations in the terminally repeated and the unique short nucleotide sequences of the EHV-1 genomes, which are known to be hot spots for deletions and tandem repeats. The BamHI restriction endonuclease pattern of the 1977 bovine isolate H1753 was identical to that of EHV-1 subtype-1 strains responsible for most of the virus abortions in vaccinated horses since 1981. Abortigenic EHV-1 strains have the ability to infect cattle and cause disease under natural conditions.