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Experimental Infection of Sheep with Bovine Leukemia Virus (BLV)

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Experimental Infection of Sheep with Bovine Leukemia Virus (BLV) +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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