Inhibitory effects of epigallocatechin gallate on the propagation of bovine coronavirus in Madin-Darby bovine kidney cells

Epigallocatechin gallate (EGCg) is the main active component of tea polyphenol and shows several biological activities, such as antimicrobial, antitumor‐promoting, anti‐inflammatory and anti‐oxidative activities. In the present study, the inhibitory effect of EGCg on bovine coronavirus (BCV) propagation in Madin‐Darby bovine kidney (MDBK) cells was investigated. EGCg at concentrations of less than 10 µg/mL did not show any cytotoxicity to MDBK cells. BCV propagation was significantly inhibited by pretreatment of the virus with EGCg (0.5–10 µg/mL) before virus inoculation in dose‐dependent, incubation time‐dependent and temperature‐dependent manners. The antiviral effect of pretreating MDBK cells with EGCg on BCV propagation was much weaker than that of pretreating BCV with EGCg. The hemagglutination activity of BCV was also reduced by EGCg in a dose‐dependent manner. These results demonstrate that EGCg possesses a distinct anti‐BCV activity and strongly suggest that EGCg interferes with the adsorption of BCV to MDBK cells by the interaction of EGCg with BCV particles. EGCg may therefore be a useful candidate for controlling BCV infection more effectively.     

A continuous bovine kidney cell line for routine assays of foot-and-mouth disease virus

A continuous bovine kidney cell line, LF-BK, arose from primary bovine calf kidney cells that survived infection with a temperature-sensitive mutant of foot-and-mouth disease virus. No virus was recovered after the first passage. Cells of Passage 48 were inoculated into two steers which remained healthy and did not develop neutralizing antibodies to the virus. The karyotype of cells of the 53rd and 87th passages was similar and revealed that the cells were markedly transformed. The modal number of diploid chromosomes was 52 at both passage levels. LF-BK cells and primary bovine kidney cells were equally susceptible in plaque assays to each of the 7 types of foot-and-mouth disease virus. The cell line and primary bovine kidney cells were less susceptible than primary bovine thyroid cells to several subtypes of the virus in suspensions of tongue epithelium. The LF-BK continuous cell line is recommended for routine plaque assays or plaque neutralization tests as a substitute for primary bovine kidney cells.     

Susceptibility of cell line cultures of bovine kidney origin to infectious bovine rhinotracheitis and parainfluenza-3 viruses

A comparative study was carried out on the susceptibility of primary bovine embryo kidney (PBEK) cell cultures, and that of AUBEK and MDBK cell lines to infectious bovine rhinotracheitis (IBR) and Parainfluenza-3 (PI-3) viruses.

The cytopathic effects induced by the two viruses were rather inconsistent, based on observations of unstained preparations. On the other hand, there was no significant difference between the susceptibility of the PBEK cultures and the cell line cultures to infection with either virus on the basis of the lesions detected in stained preparations, and of the growth curve patterns.

It is concluded that PBEK cell cultures are more sensitive for isolating IBR or PI-3 viruses than are the AUBEK and MDBK cell lines. However, the latter appear to be satisfactory for studies of these two viruses.     

Generation of recombinant bovine interferon tau in the human embryonic kidney cell line and its biological activity

The objective of this study was to generate recombinant bovine interferon tau (rbIFNT) in mammalian hosts. The complementary DNA encoding bovine IFNT2 was cloned for the construction of pRcRSV‐bIFNT2 expression vector. The expression vector was transfected to 293 cells. Transfected cells harboring expression vector were selected with G418. Highly expressing clonal line was adapted to serum‐free suspension culture in a spinner flask. The recombinant protein had 24 kDa apparent molecular mass, suggesting being expressed as a glycoprotein, and was purified from serum‐free conditioned medium by the combination of Diethylaminoethanol Sepharose ion exchange and Sephacryl S‐200 HR gel filtration. A total of 7.3 mg rbIFNT was obtained from 13.5 L conditioned medium. Generated rbIFNT was biologically active in terms of antiviral activity measured by the plaque inhibition assay with Madin‐Darby bovine kidney cells and the vesicular stomatitis virus. The recombinant protein was also utilized for immunization to raise antibodies in the rabbit. The generated antibody was capable of use in both Western blotting and the binding assay. The results in the present study suggest that a certain amount of rbIFNT is raised in mammalian hosts by using conventional plasmid vector and its antibody provides useful tools for studies in the biology of bovine IFNT.     

Bovine costimulator. II. Generation and maintenance of a bovine costimulator-dependent bovine lymphoblastoid cell line

Bovine peripheral blood leukocytes, activated with conconavalin A, were cultured in bovine costimulator-containing conditioned medium prepared in a totally defined, serum-free medium. A population of leukocytes subsequently grew exponentially. These bovine cells had the morphology of lymphoblasts, were negative for chloroacetate esterase, slightly positive for nonspecific esterases, and highly peanut agglutinin-positve. These data suggested that the bovine leukocytes were of the T-cell lineage and that the active factor in the costimulator-containing conditioned medium might be the bovine equivalent of Interleukin 2. A quantitative microassay, subsequently developed, revealed that the lymphoblastoid cell line was costimulator-dependent and lectin-independent. Further utilization of the microassay supported this contention and strengthened the concept of a bovine Interleukin 2-dependent bovine T-cell line: Phytohemagglutin-M, phytohemagglutinin-P, and concanovalin A induced active factor from peripheral blood leukocytes, while lipopolysaccharide, a potent inducer of Interleukin 1 in other systems, failed to induce activity; and both T-cells and macrophages were required for optimal factor activity. Finally, a means by which to optimize production of the active moiety, utilizing lymph node cells, as opposed to peripheral blood leukocytes, was examined.     

Cytogenetic study of bovine leukosis

Nine dairy cows aged four to eleven years were subjected to examination by chromosomal analysis. These cows had been found, by haematological examination, to suffer from leucosis. The study also covered one ten-day-old calf - heifer. The test group included two dam-daughter pairs. The animals belonged to the Black-Pied Lowland breed. The blood was sampled from vena jugularis and the karyotypes were processed and evaluated by the method after Moorhead et al. (1960), modified by Lojda et al. (1974). A list was kept for each animal. The tested animals were included in classes by the percentages of the chromosome aberrations: class I - two animals (up to 10% of aberrations), class II - seven animals (from 10% to 20% of aberrations), class III - one animal (above 20% of aberrations). Hyposomy was found in all cases, polysomy and hyperploidy in four cases. Structural aberrations were observed in nine cases, breaks being the most frequent anomalies (7 cases). Breaks on sexual chromosomes were observed in five cases, including the dam-daughter pairs; centric fusion occurred in one case and mixed aberrations in two cases.     

Development of a cell culture line of bovine fetal endometrial cells.

Monolayers of bovine fetal endometrial cells were established as primary culture cells within 1 to 2 weeks. After the 2nd passage, these cells were inoculated with bovine viral diarrhea virus. Effects of the virus were observed each day with a light microscope. Specific cytopathic effects consisting of degeneration and sloughing of the cells and a well-defined pattern of cytoplasmic vacuolation were observed at 5 days after inoculation.     

Partial characterization of bovine leukemic cell populations

Peripheral blood lymphocytes (LL) from cows with chronic lymphocytic leukemia (CLL) have been studied using a number of surface markers. Cell populations were obtained by partial enrichment and depletion using Sephadex G-200-anti-F(ab')2 and Sephadex G-200-anti-LL-Ig immunoadsorbent columns. It was shown that cell populations having different markers, i.e., surface immunoglobulins (sIg) and leukemia-associated antigens (LAA), are characterized by similar parameters. Thus the adherent cells obtained by both fractionation methods formed 1.1-4.0% of rosettes with sheep red blood cells, while 90.6-94.3% were sIg positive cells. The cytotoxicity test (CTT) performed with anti-B and anti-LL sera indicated that a vast majority of adherent cells reacted to the sera. Thus the adherent cells represent a population with a high percentage of B-cells.     

A sensitive plaque assay for bovine rotavirus in cultures of the bovine cell line GBK

The Lincoln strain of bovine rotavirus was found to replicate with cytopathic effects in cultures of GBK cells, a stable cell line derived from bovine kidney, when the cultures were maintained in the presence of trypsin. The virus was readily passaged and the infected cells were shown to contain specific viral antigen by indirect immunofluorescent staining. The virus formed plaques in GBK cell monolayers, when trypsin was incorporated in the agar overlay medium. The plaque count increased about twofold when diethylaminoethyl dextran was further included in the overlay medium. Plaque assay in GBK cells was more sensitive than that in MA-104 cells previously reported by Matsuno et al. The specificity of plaques was confirmed by specific inhibition with antiserum against the Lincoln strain.     

Cytogenetic study of in vitro-derived bovine embryos

A cytogenetic study of early bovine embryos (2-16 blastomeres) produced in vitro was conducted to determine the incidence of embryos carrying chromosome anomalies. The embryos were produced from immature oocytes matured in vitro and fertilized by sperm prepared using the Percoll density gradient method. Slides were prepared according to an 'air drying' technique and the chromosomal complement of embryos was studied by Giemsa-staining. Approximately 57% of prepared embryos were suitable for analysis. The results revealed that 18% of cytogenetically analysed embryos presented chromosomal anomalies, including haploidy (8%), aneuploidy (2%) and polyploidy (8%). Our results were compared to the results of other studies in cattle and other domestic animals.     

Protection against bovine leukosis virus infection in sheep with the BL 20 bovine lymphoblastoid cell line

The bovine lymphoblastoid BL 20 cell line derived from a case of sporadic bovine leukosis when inoculated into sheep did not induce an antibody response directed against bovine leukosis virus (BLV) structural proteins. Sheep were inoculated twice with the BL 20 cell line and then challenged with BLV infected lymphocytes. Three out of four sheep challenged four weeks after BL 20 inoculation did not develop BLV antibodies. Of the 12 sheep challenged later, three sheep did not develop BLV antibodies. BLV was isolated from all the seropositive animals and from none of the seronegative animals.     

Evaluation of the protective effect of bovine lactoferrin against lipopolysaccharides in a bovine mammary epithelial cell line

Lactoferrin (Lf) is a non-haem iron-binding glycoprotein with a molecular weight of about 80 kDa, synthesized by glandular epithelial cells and stored in the secondary granules of neutrophils. The physiological significance of Lf is related to non-specific immune defence against pathogens, immunomodulatory activity, iron homeostasis, antioxidant properties and regulation of cell growth. Lf is a bioactive component of the mammary secretions and its modulatory and defensive functions do affect the newborn and the mammary gland as well. In this work a bovine mammary epithelial cell line (BME-UV1) was used as an in vitro model of the bovine mammary epithelium to examine the protective role of exogenous bovine Lf (bLf) against the cytotoxic damage induced by bacterial lipopolysaccharides (LPS) and the endogenous bLf mRNA expression after LPS exposure. In the in vitro model used, exogenous bLf exerts a protective effect against endotoxin cytotoxicity, which could be mediated by the LPS-neutralizing capability of bLf. In addition, in BME-UV1 cells the response to LPS exposure does not involve bLf mRNA expression, suggesting that this cell line lack of functional LPS-responsive elements.     

Cytogenetic characterization of a canine haemangiopericytoma

A 15-year-old dachshund bitch developed a haemangiopericytoma in the perineal region. The cytogenetic evaluation of the tumour cells showed a chromosome number of 74. The following abnormalities were found: an intersitially deleted chromosome no. 1 and centric fusions 5/6, 5/14, 7/15 and 9/17.     

Isolation and propagation of infectious bursal disease virus using the ovine kidney continuous cell line.

Twenty-six samples known to contain infectious bursal disease virus (IBDV) were examined by virus-isolation attempts on ovine kidney (OK) cell line, Vero cell line, and chicken embryo fibroblast (CEF) cultures. Virus was isolated from two of 26 samples, three of 26 samples, and three of 25 samples on OK, Vero, and CEF cultures, respectively. However, in contrast to IBDV replication in Vero and CEF cultures, isolated virus was unable to induce serially sustained cytopathic effects (CPE) during successive passages in the OK cell line, unless cell lysates were treated with chloroform between every other passage. The cytopathogenicity of the untreated virus passaged in OK cells was revived and maintained upon passage in Vero cells. An initial single passage of laboratory or field material in OK cells followed by further passages in Vero cells resulted in virus isolation from six of 26 samples, which was better virus recovery than when either cell line was used alone or when CEF cultures were used. Twenty of the 26 test samples were originally positive when examined by nucleic acid hybridization with radiolabeled IBDV cDNA, indicating that some of the samples that were negative upon virus isolation using OK and Vero cells may have contained inactivated virus.     

Amyloidosis in the bovine kidney

After a survey of the most important literature on amyloidosis the results of the authors own studies on amyloidosis in the bovine kidney are reported. The macroscopical and microscopical features of the various types of bovine renal amyloidosis are described. In most cases predominantly medullary depositions occurred (types A, B, C and D). In a series of over 1000 cases only six kidneys with glomerular amyloid were found (type E). All these, however, contained some medullary amyloid as well. It is questionable whether the different types of kidney lesions contain different forms of amyloid or represent variants of the same disorder. Clinico-pathological studies may solve this problem.Medullary amyloid may be a subclinical disease. In clinically normal slaughter cattle it occurred in 2.7% of 1326 animals. In clinically normal dairy cattle (including young animals) it occurred in 2.6% of 378 animals and the incidence appeared to increase with age. In the medullary amyloid deposits different amyloid-containing cell types were found. The intracellular amyloid was arranged in parallel fibrils and was localized in lysosomes. Induced phagocytosis of bovine amyloid by rat macrophages appeared to result in similar vacuoles, indicating that phagocytosis may occur in the bovine cells. In the literature parallel intralysosomal amyloid has been interpreted as formation of amyloid in these vacuoles.An attempt is made to elucidate the pathogenesis of amyloidosis.

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Kurzfassung Nach der Beschreibung der wichtigsten Literaturangaben von Amyloidose werden die eigene Befunde von Amyloidose der Niere des Rindes besprochen. Die makroskopischen und mikroskopischen Eigenschaften der Nierenamyloidose des Rindes werden beschrieben. In den meisten Fällen gab es vor allem medulläre Ablagerungen (Typ A, B, C, und D). Bei über 1000 untersuchten Rindernieren wurden nur 6 Nieren mit hauptsächlich glomerulärem Amyloid gefunden (Typ E). Dieser Typ enthielt ausserdem ein wenig medulläres Amyloid in allen Fällen. Es ist die Frage, ob die verschiedenen Typen von Nierenlaesionen verschiedene Amyloidformen enthalten oder Varianten der selben Krankheit sind. Klinisch-pathologische Untersuchungen müssen diese Frage zu lösen versuchen.Medulläres Amyloid könnte eine subklinische Krankheit darstellen: Bei klinisch gesunden Schlachtrindern kam es in 2.7% von 1326 Tieren vor. Bei klinisch gesundem Milchvieh (einschliesslich Jungvieh) kam es in 2.6% von 378 Tieren vor, wobei mit steigendem Alter eine Zunahme gefunden wurde.Bei den medullären Amyloidablagerungen wurden mehrere amyloid-enthaltende Zelltypen gefunden. Das intrazelluläre Amyloid zeigte eine paralle Anordnung der Fibrillen und war lokalisiert in Lysosomen. Bei durch Injektion von Rinderamyloid bei Ratten induzierte Phagocytose zeigten sich die gleichen Vakuolen. Dieser Befund weist darauf hin dass Phagozytose auch bei Rindern eine Möglichkeit sein kann. In der Literatur wird paralleles intralysosomales Amyloid als örtlich entstanden erklärt. Schliesslich werden einige Untersuchungsrichtungen gezeigt, die möglicherweise zur pathogenese der Amyloidose führen könnten.

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Resume Après avoir résumé les points les plus importants de l'amyloidose relevés dans la littérature, l'auteur présente le résultat de ses observations. Il décrit l'aspect macroscopique et microscopique de l'amyloidose rénale des bovins. Dans la plupart des cas prédominent les dépôts médullaires (types A, B, C, D). Sur 1000 cas, il a trouvé 6 reins avec dépôts glomérulaires (type E), et partiellement médullaires.Une question se pose: les différents types de lésions rénales contiennent-ils diverses substances amyloides ou bien répresententils des variants d'un même affection. Des recherches clinico-histopathologiques devraient pouvoir apporter une réponse.L'amyloidose medullaire peut être une maladie sub-clinique. 2,7% de cas ont été décelés sur 1326 bovins de boucherie cliniquement sains et 2,6% sur 378 vaches laitières (y compris les jeunes) cliniquement saines.Les différents types de cellules contenant la substance amyloide existent dans les dépôts médullaires. La substance amyloide intracellulaire se présente en arrangements parallèles et est localisée dans les lysosomes. La phagocytose induits de la substance amyloide par les macrophages de Rat apparait sous la forme de vacuoles semblables, indiquant que la phagocytose peut se manifester dans les cellules bovines. Dans la littérature, la substance amyloid intra-lysosomale parailèle a été interpretée comme étant une formation de la substance amyloide de ces vacuoles.L'auteur suggere finalement quelques recherches pour élucider la pathogénie de l'amyloidose.

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Riassunto L'autore presenta il risultato delle sue osservazioni dopo aver riassunto i punti piu importanti dell'amiloidosi riportati in litteratura. Descrive sia l'aspetto macroscopico che microscopico dell'amiloidosi. Nella maggior parte dei casi precominano i depositi midollari (tipi A, B, C, D). Su 1.000 casi, ha trovato sei reni con depositi glomerulari (tipo E) e parzialmente midollari. Si pone la domanda se i diversi tipi di lesioni renali contangano sostanze amiloidi diverse, oppure se rappresentano dell varianti di una stessa affezione. Ricerche clinico-isopatologiche dovrebbero poter dare una risposta.L'amiloidosi midollare puo essere una malattia sub-clinica: il 2,7% dei casi e stato rilevato su 1.326 bovini da macello clinicamente sani, e su 378 vacche da latte clinicamente sane (ivi comprese le giovani), il tasso rilevato e del 2,6%. Nei depositi amilodali midollari si sono trovato vari tipi cellulari contenenti l'amiloide.L'amiloide intracellulare, che si localizza nei lisosomi, si presenta in strutture parallele. La fagocitosi della sostanza amiloide indotta dae microfaghi di ratto compare sotto forma di vacuoli simili, indicando che la fagocitosi puo manifestarsi nelle cellule bovine. In litterature, la sostanza amiloidale intralysomale parallela e stata interpretata come una formazione di sostanza amiloidale in questi vacuoli. Infine, l'autore suggerisce di fare alcune ricerche per chiarire la patogenesi dell'amiloidosi.

     

Establishment and characterization of a chicken mononuclear cell line

A new chicken mononuclear cell line (MQ-NCSU) has been established. The starting material used to initiate this cell line was a transformed spleen from a female Dekalb XL chicken which had been experimentally challenged with the JM/102W strain of the Marek's disease virus. After homogenization, a single cell suspension of splenic cells was cultured using L.M. Hahn medium supplemented with 10 microM 2-mercaptoethanol. Under these culture conditions, a rapidly proliferating cell was observed and then expanded after performing limiting dilution cultures. These cells were moderately adherent and phagocytic for sheep red blood cells and Salmonella typhimurium. When tested against a panel of monoclonal antibodies (mAb) using the flow cytometry, MQ-NCSU cells stained readily with anti-chicken monocyte specific (K-1) mAb but did not stain with mAb detecting T-helper, T-cytotoxic/suppressor, and NK cells. MQ-NCSU cells expressed very high levels of Ia antigens and transferrin receptors. In addition, cell-free supernatant obtained from MQ-NCSU culture contained a factor which exhibited cytolytic activity against tumor cell targets. Based on their cultural, morphological, and functional characteristics and mAb reactivity profile, we conclude that MQ-NCSU cell line represents a malignantly-transformed cell which shares features characteristic of cells of the mononuclear phagocyte lineage.     

Establishment and characterization of canine rhabdomyosarcoma cell line CMS-C

A novel canine tumor cell line designated as the CMS-C cell line was established from pleomorphic rhabdomyosarcoma (RMS) raised in the prostate gland of a 14-year-old intact male mixed-breed dog. CMS-C cells displayed the same immunohistochemical characteristics (positive for vimentin and desmin and negative for cytokeratin and smooth muscle actin) as the original tumor cells and express myoD1 and UCP3, known as striated muscle-specific molecules, as shown by RT-PCR assay. Therefore, the established CMS-C cell line appears to be of rhabdomyoblast cell origin. The CMS-C cell line established from pleomorphic RMS will be a useful tool for further studies about canine RMS.