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Experimental Infection of Sheep with Bovine Leukemia Virus (BLV)

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+Model RAM-326; No. of Pages 8 ARTICLE IN PRESS Rev Argent Microbiol. 2019;xxx(xx):xxx---xxx R E V I S T A A R G E N T I N A D E MICROBIOLOGÍA www.elsevier.es/ram ORIGINAL ARTICLE Experimental infection of sheep with Bovine leukemia virus (BLV): Minimum dose of BLV-FLK cells and cell-free BLV and neutralization activity of natural antibodies a,b a,b,∗ c Natalia Gabriela Porta , Irene Alvarez , Guillermo Suarez Archilla , Vanesa a,b c a,b Ruiz , Alejandro Abdala , Karina Trono a Instituto Nacional de Tecnología Agropecuaria (INTA) --- Instituto de Virología, Centro de Investigaciones en Ciencias Veterinarias y Agronómicas, Nicolas Repetto y de los Reseros s/n (1686), Hurlingham, Buenos Aires, Argentina b Consejo Nacional de Investigaciones Científicas y Técnicas, CONICET, Godoy Cruz 2290 (C1425FQB), CABA, Argentina c Instituto Nacional de Tecnología Agropecuaria (INTA) --- Laboratorio de Sanidad Animal, Estación Experimental Agropecuaria Rafaela, Ruta 34 Km 227 (2300) Rafaela, Santa Fe, Argentina Received 23 July 2018; accepted 8 January 2019 KEYWORDS Abstract Bovine leukemia virus (BLV) is an important cattle pathogen that causes major eco- nomic losses worldwide, especially in dairy farms. The use of animal models provides valuable Bovine leukemia virus; insight into the pathogenesis of viral infections. Experimental infections of sheep have been conducted using blood from BLV-infected cattle, infectious BLV molecular clones or tumor- Animal model; Experimental derived cells. The Fetal Lamb Kidney cell line, persistently infected with BLV (FLK-BLV), is one infection; of the most commonly used long-term culture available for the permanent production of virus. Sheep; FLK-BLV cells or the viral particles obtained from the cell-free culture supernatant could be used as a source of provirus or virus to experimentally infect sheep. In this report, we aimed FLK-BLV cells to determine the minimum amount of FLK-BLV cells or cell-free supernatant containing BLV needed to produce infection in sheep. We also evaluated the amount of antibodies obtained from a naturally-infected cow required to neutralize this infection. We observed that both sheep experimentally inoculated with 5000 FLK-BLV cells became infected, as well as one of the sheep receiving 500 FLK-BLV cells. None of the animals inoculated with 50 FLK-BLV cells showed evi- dence of infection. The cell-free FLK-BLV supernatant proved to be infective in sheep up to a 1:1000 dilution. Specific BLV antibodies showed neutralizing activity as none of the sheep became infected. Conversely, the animals receiving a BLV-negative serum showed signs of BLV infection. These results contribute to the optimization of a sheep bioassay which could be useful to further characterize BLV infection. © 2019 Asociacion´ Argentina de Microbiolog´ıa. Published by Elsevier Espana,˜ S.L.U. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by- nc-nd/4.0/). ∗ Corresponding author. E-mail address: [email protected] (I. Alvarez). https://doi.org/10.1016/j.ram.2019.01.004 0325-7541/© 2019 Asociacion´ Argentina de Microbiolog´ıa. Published by Elsevier Espana,˜ S.L.U. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Please cite this article in press as: Porta NG, et al. Experimental infection of sheep with Bovine leukemia virus (BLV): Minimum dose of BLV-FLK cells and cell-free BLV and neutralization activity of natural antibodies. Rev Argent Microbiol. 2019. https://doi.org/10.1016/j.ram.2019.01.004 +Model RAM-326; No. of Pages 8 ARTICLE IN PRESS 2 N.G. Porta et al. Infección experimental en ovinos con el virus de leucosis bovina: dosis mínima de PALABRAS CLAVE células BLV-FLK y virus libre de células, y actividad neutralizante de anticuerpos Virus de la leucemia bovina; naturales Modelo animal; Resumen El virus de la leucosis bovina (bovine leukemia virus [BLV]) es un importante agente Infección patógeno del ganado que causa importantes pérdidas económicas en todo el mundo, especial- experimental; mente en los rodeos lecheros. El uso de modelos animales proporciona información valiosa sobre Ovino; la patogénesis de las infecciones virales. Se realizaron infecciones experimentales en ovejas Células FLK-BLV usando sangre de bovinos infectados con BLV, clones moleculares de BLV infecciosos o células derivadas de tumores. La línea celular Fetal Lamb Kidney, persistentemente infectada con el BLV (FLK-BLV), es uno de los cultivos a largo plazo más utilizados para la producción permanente de virus. Las células FLK-BLV o las partículas virales obtenidas del sobrenadante del cultivo libre de células podrían usarse como fuente de provirus o de virus para infectar experimentalmente ovejas. En este trabajo, nuestro objetivo fue determinar la cantidad mínima de células FLK-BLV o de sobrenadante libre de células que contiene BLV necesaria para producir infección en ove- jas. También evaluamos la cantidad de anticuerpos bovinos anti-BLV necesaria para neutralizar la infección. Observamos que las dos ovejas inoculadas experimentalmente con 5000 células FLK-BLV se infectaron, y que una de las dos ovejas que recibieron 500 células FLK-BLV se infectó. Ninguno de los animales inoculados con 50 células FLK-BLV mostró evidencia de infección. El sobrenadante FLK-BLV libre de células demostró ser infectivo en ovejas hasta la dilución 1:1000. Los anticuerpos BLV específicos mostraron actividad neutralizante, ya que ninguna de las ovejas se infectó. Por el contrario, los animales que recibieron un suero BLV negativo mostraron signos de infección por BLV. Estos resultados contribuyen a la optimización de un bioensayo en ovejas útil para caracterizar la infección por BLV. © 2019 Asociacion´ Argentina de Microbiolog´ıa. Publicado por Elsevier Espana,˜ S.L.U. Este es un art´ıculo Open Access bajo la licencia CC BY-NC-ND (http://creativecommons.org/licenses/by- nc-nd/4.0/). Introduction BLV has an intense transmission dynamics. All practices carried out without blood transmission control, such as blood extraction, vaccination, castration, dehorning, injection of Bovine leukemia virus (BLV) is a retrovirus that belongs medications, rectal palpation, tattooing, etc., are routes to the Deltaretrovirus genus and the causative agent of of potential infection, and animals with higher levels of enzootic bovine leukosis (EBL) in cattle and water buffaloes, 2 provirus in blood are those that represent a higher risk for their natural hosts . The infection is distributed worldwide transmission8,21. and the individual prevalence levels of infection vary among Most of the infected animals are asymptomatic carriers and within countries. In the European Union, measures of since they do not exhibit clinical changes and their leuko- control and eradication were successful in most Western 1,22,25 cyte count remains normal. Between 20 and 30% of infected European countries . Something similar occurred in New 5,6 animals develop persistent lymphocytosis (PL) , which is Zealand and Australia, which are BLV-free since 2008 and 12 characterized by a permanent and stable increase in the 2013 respectively . In the Americas, there are different lev- number of circulating peripheral CD4 + IgM + B cells. These els of BLV prevalence; in the case of Argentina, more than 32 animals do not show clinical symptoms, but an elevated 30% of cattle and over 80% of dairy farms are infected white blood cell count can be observed at the hematologi- with an individual endemicity higher than 80% in areas of 37 19 cal level . Of all the infected animals, only 1---5% of them intense production . BLV infection is a serious concern since develop fatal lymphoma. The animals develop tumors of the it causes major economic losses in the dairy productive sys- 9,30,32 lymphatic system by proliferation of B-cells, mostly affect- tem and in international trade . 7 ing adult cattle ( 1 --- 8 years old) . The virus can infect different cells of the immune system Cattle and water buffaloes are the natural hosts of BLV, but has a preferential tropism for B lymphocytes. There- but there are some species that can be experimentally fore, it is a blood borne pathogen as Human T-lymphotropic 2,31 infected with different clinical, hematological and immuno- virus 1 and 2 (HTLV-I and HTLV-II) , pathogens from the logical manifestations. The use of animal models is very same viral family with genetic and antigenic similarities. important as they allow to study the different stages of Active viral expression occurs during the first stages of viral infection along with the progression of the disease, the infection, after which the virus remains in a silent as well as the characterization of samples for their infec- state in the infected host, permanently integrated into the 2,13 tivity, protective potential, among others. In rabbits, the cell genome as provirus DNA . This produces a persistent infection is in most cases evidenced by the manifestation immune response in the host without evidence of active viral circulation. of circulating specific antibodies and the presence of the Please cite this article in press as: Porta NG, et al. Experimental infection of sheep with Bovine leukemia virus (BLV): Minimum dose of BLV-FLK cells and cell-free BLV and neutralization activity of natural antibodies. Rev Argent Microbiol. 2019. https://doi.org/10.1016/j.ram.2019.01.004 +Model RAM-326; No. of Pages 8 ARTICLE IN PRESS Experimental infection of sheep with Bovine leukemia virus (BLV) 3 BLV genome. In some rabbits, a decreased T-cell responsive- in RPMI 1640 medium (Corning, NY, USA) supplemented with ness to phytolectin stimulation and signs of clinical disease, 5% of inactivated fetal bovine serum (FBS, Internegocios, such as conjunctivitis, rhinitis, severe weight loss and unex- Buenos Aires, Argentina) until the formation of a conflu- 28,39 pected death, can be also observed .
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    Sánchez García et al. 23 ○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○ Morena Gallega cattle breeds with limited numbers: origin, productive characteristics and conservation programmes L. Sánchez García1, A. Iglesias1, A. Fernández1, J.L. Viana1 & M. Vallejo2 1Departamento de Anatomía y Producción Animal, Facultad de Veterinaria, Universidad de Santiago de Compostela, 27002 Lugo, Spain 2Departamento de Producción Animal, Facultad de Veterinaria, Universidad Complutense, 28040 Madrid, Spain Resumen not sufficient to survive). In order to prevent eventual extinction, an Operative Programme to maintain a germplasm bank (semen and En los últimos años las poblaciones embryos) and animals in situ (cattle farms and autóctonas de bovinos gallegos han natural reserves) has been established to disminuido hasta alcanzar una situacion de ensure preservation and facilitate study from vulnerabilidad, que va de un estado crítico de the ethnological, productive and reproductive menos de 100 hembras de raza con una point of view. Five herds of Cachena, variabilidad genética muy reducida, y un Caldelana, Frieiresa, Limiana and Vianesa estado de peligro con 100 a 1 000 hembras de breeds, have been studied for census, habitat, raza que no bastan para la supervivencia. Al morphological characters, animal breeding, objeto de prevenir una eventual extinción, se productive character, seminal quality of bulls estableció un Programa Operativo para el and reproductive performances. mantenimiento de un banco de germoplasma (semen y embriones) y de animales
  • A List, Bibliography, and Index of the Fossil Vertebrates of Louisiana and Mississippi Daryl P

    A List, Bibliography, and Index of the Fossil Vertebrates of Louisiana and Mississippi Daryl P

    A LIST, BIBLIOGRAPHY, AND INDEX OF THE FOSSIL VERTEBRATES OF LOUISIANA AND MISSISSIPPI DARYL P. DOMNING1 Tulane University New Orleans, Louisiana ABSTRACT Species of fossil vertebrates reported from Louisiana and Mississippi are listed. The bibliography consists of 167 titles and contains detailed annotations on vertebrates from those states. Both systematic and chronologic-geographic indexes are provided. A BRIEF HISTORY OF these deposits had been generally recognized. In 1828 VERTEBRATE PALEONTOLOGY Richard Harlan, a Philadelphia physician and anatomist IN LOUISIANA AND MISSISSIPPI and the American Philosophical Society's expert on fossil bones, described the skull of a sperm whale which had Louisiana and Mississippi are not usually thought of as been disinterred near the mouth of the river some time areas possessing significant numbers of fossil vertebrates. before. (According to Harlan it had previously been Compared with many other parts of the United States, described as a giant reptile, "Megistosaurus", by John they seem to be among the least productive in this regard; Davidson Godman, in a paper which apparently was never but it would be more accurate to say they are among the published.) A very similar find was that of a large baleen most neglected. In fact, this region contains quite respect­ whale skull unearthed a few miles above New Orleans and able fossil faunas — principally Cretaceous, Eocene, and described in 1837 in two articles by A. E. A. Riviere. J. Pleistocene in age — and includes several classic localities E. DeKay, in 1842, illustrated a rorqual skull found about of considerable interest in the history of the science as a 1837, said to be from the mouth of the Mississippi, but whole.

  • Combining Multiple Autosomal Introns for Studying Shallow Phylogeny And

    Molecular Phylogenetics and Evolution 66 (2013) 766–775 Contents lists available at SciVerse ScienceDirect Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev Combining multiple autosomal introns for studying shallow phylogeny and taxonomy of Laurasiatherian mammals: Application to the tribe Bovini (Cetartiodactyla, Bovidae) ⇑ Alexandre Hassanin a,b, , Junghwa An a,b, Anne Ropiquet c, Trung Thanh Nguyen a, Arnaud Couloux d a Muséum national d’Histoire naturelle (MNHN), Département Systématique et Evolution, UMR 7205 – Origine, Structure et Evolution de la Biodiversité, 75005 Paris, France b MNHN, UMS 2700, Service de Systématique Moléculaire, 75005 Paris, France c Department of Conservation Ecology and Entomology, Stellenbosch University, Private Bag X1, Matieland 7602, Western Cape, South Africa d Genoscope, Centre National de Séquençage, 91057 Evry, France article info abstract Article history: Mitochondrial sequences are widely used for species identification and for studying phylogenetic rela- Received 15 May 2012 tionships among closely related species or populations of the same species. However, many studies of Revised 27 September 2012 mammals have shown that the maternal history of the mitochondrial genome can be discordant with Accepted 1 November 2012 the true evolutionary history of the taxa. In such cases, the analyses of multiple nuclear genes can be Available online 15 November 2012 more powerful for deciphering interspecific relationships. Here, we designed primers for amplifying 13 new exon-primed intron-crossing (EPIC) autosomal loci Keywords: for studying shallow phylogeny and taxonomy of Laurasiatherian mammals. Three criteria were used Laurasiatheria for the selection of the markers: gene orthology, a PCR product length between 600 and 1200 nucleotides, Bovinae Nuclear introns and different chromosomal locations in the bovine genome.