The cloning of the virus envelope glycoprotein F of canine distemper virus expressed in Pichia pastoris.

Canine distemper virus (CDV) is a pathogen which affects members of the Canidae family, causing an acute, often fatal, systemic disease. CDV is an RNA virus of the family Paramyxoviridae that contains two envelope glycoproteins: F and HA. In this study, we focused on the envelope glycoprotein F as the main target for neutralizing antibodies produced after infection or vaccination. The complete coding region of the protein (60 kDa) was expressed in the methylotrophic yeast Pichia pastoris, obtained in a recombinant form and secreted to the culture medium. Later, to analyze its immunogenicity, the protein was combined with an oily adjuvant and used to inoculate mice. The results provide evidence supporting a potential application of this recombinant protein as a subunit vaccine.

Detection and molecular characterization of porcine parvovirus in fetal tissues from sows without reproductive failure in Argentina.

Porcine parvovirus (PPV) is one of many pathogens responsible for reproductive failure in pregnant sows. Several studies have reported the appearance of new PPV strains that differ from previous isolates both genetically and antigenically. Thus, the protective effects of commercially inactivated vaccines could not be complete. In South America, the information about PPV is limited. Thus, the aim of the present study was to detect and characterize the PPV strains present in 131 mummies or stillbirths from normal deliveries in sows from a commercial swine farm of Argentina that uses the commercial vaccine. PCR results showed that 17/131 were positive to PPV. Ten of these viruses were isolated and sequenced. All viruses were related to the PPV1 sequence (NADL-2), maintaining the amino acid differences in positions 436 (S-P) and 565 (R-K). This study is the first to report the isolation of PPV in Argentina and the results suggest that PPV can cross the placenta even in vaccinated sows, thus affecting some of the fetuses and being able to cause fetal death in sows without reproductive failure. The results also suggest that vaccination only reduces clinical signs and reproductive disorders and may thus not be a perfect tool to manage PPV infection. This study provides information that needs to be studied in depth to improve strategies to prevent and control PPV infection in swine farms.

Evaluation of apoptosis markers in different cell lines infected with equine arteritis virus.

Equine arteritis virus (EAV) induces apoptosis in infected cells. Cell death caused by EAV has been studied mainly using three cell lines, BHK-21, RK-13 and Vero cells. The mechanism of apoptosis varies among cell lines and results cannot be correlated owing to differences in EAV strains used. We evaluated different markers for apoptosis in BHK-21, RK-13 and Vero cell lines using the Bucyrus EAV reference strain. Acridine orange/ethidium bromide staining revealed morphological changes in infected cells, while flow cytometry indicated the extent of apoptosis. We also observed DNA fragmentation, but the DNA ladder was detected at different times post-infection depending on the cell line, i.e., 48, 72 and 96 h post-infection in RK-13, Vero and BHK-21 cells, respectively. Measurement of viral titers obtained with each cell line indicated that apoptosis causes interference with viral replication and therefore decreased viral titers. As an unequivocal marker of apoptosis, we measured the expression of caspase-3 and caspases-8 and -9 as extrinsic and intrinsic markers of apoptosis pathways, respectively. Caspase-8 in BHK-21 cells was the only protease that was not detected at any of the times assayed. We found that Bucyrus EAV strain exhibited a distinctive apoptosis pathway depending on the cell line.

Triatominae in furnariid nests of the Argentine Gran Chaco.

Triatomines (Hemiptera, Reduviidae) are bloodsucking insects involved in the transmission of Trypanosoma cruzi, the causative agent of Chagas disease, an important public health problem in Latin America. The triatomine species found in sylvatic habitats generally play a limited epidemiological role compared to domestic species, but they may act as a reinfestation source of dwellings after insecticide spraying and have to be carefully considered in control strategies of Chagas disease transmission. The objectives of this work were to carry out a survey of the sylvatic triatomine species colonizing Furnariidae nests in a typical area of the Chaco region of Argentina during the winter and to study the parasites and natural enemies associated with the collected triatomines. Sixty-three triatomine specimens were collected from Furnariidae nests (Coryphistera alaudina and Phacellodomus sibilatrix) randomly selected within the study area. Fifty-four were identified as Psammolestes coreodes, seven as Triatoma platensis, and two as Triatoma infestans. Specimens of T. infestans and T. platensis were found in one nest. The first finding of instar nymphs of T. infestans x T. platensis in a sylvatic habitat is reported. For the first time, sylvatic collected specimens of T. platensis were found infected by T. cruzi. Triatoma virus was found in one Ps. coreodes specimen.

The equine arteritis virus isolate from the 2010 Argentinian outbreak.

A semen sample from a stallion infected during the 2010 equine arteritis virus (EAV) outbreak was received for viral isolation prior to castration of the animal. The virus was identified using a polyclonal antibody immunofluorescence test. Reverse-transcription polymerase chain reaction (RT-PCR) was used to amplify a region of the GP5 gene with primers GL105F and GL673R. The PCR products were purified and sequences of both strands were determined in a MegaBACE™1000 with inner primers CR2 and EAV32. A phylogenetic dataset was built with the previously reported sequences of five strains isolated in Argentina, together with a group of selected sequences obtained from GenBank. The unrooted neighbour-joining tree was constructed using molecular evolutionary genetic analysis (MEGA) and bootstrap analyses were conducted using 1,000 replicate datasets. Evolutionary distances were computed using the maximum composite likelihood method. A NetNGlyc server analysis at the Technical University of Denmark (www.cbs.dtu.dk/services/NetNGlyc/) was used to predict N-glycosylation in GP5 sequences. The phylogenetic analysis revealed that the new strain GLD-LP-ARG), together with other strains previously isolated, belongs to the European group EU-1 but in a different branch. The new strain shows 99% nucleotide identity with strain Al1and 98.1% with the Belgian strain 08P178. Persistently infected stallions and their cryopreserved semen constitute a reservoir of EAV, which ensures its persistence in the horse population around the world. These findings reinforce the importance of careful monitoring of persistently infected stallions, as well as semen straws, by RT-PCR or test mating, in accordance with national regulations.

ELA-DRA polymorphisms are not associated with Equine Arteritis Virus infection in horses from Argentina.

Polymorphisms at Major Histocompatibility Complex (MHC) genes have been associated with resistance/susceptibility to infectious diseases in domestic animals. The aim of this investigation was to evaluate whether polymorphisms of the DRA gene the Equine Lymphocyte Antigen is associated with susceptibility to Equine Arteritis Virus (EAV) infection in horses in Argentina. The equine DRA gene was screened for polymorphisms using Pyrosequencing® Technology which allowed the detection of three ELA-DRA exon 2 alleles. Neither allele frequencies nor genotypic differentiation exhibited any statistically significant (P-values=0.788 and 0.745) differences between the EAV-infected and no-infected horses. Fisher's exact test and OR calculations did not show any significant association. As a consequence, no association could be established between the serological condition and ELA-DRA.

Eca20 microsatellite polymorphisms in equine viral arteritis-infected horses from Argentina.

We investigated the association of equine arteritis virus (EAV) infection and three short tandem repeat (STR) polymorphisms located within or in close proximity to equine lymphocyte antigen (ELA) region. We used a case-control design as a first approach before proceeding to select candidate genes. One hundred and sixty-five Silla Argentino horses were taken in 2002 from positive serological detections of EAV in Argentina, to determine whether STR genotypes were correlated to genetic susceptibility to EVA. Allele frequency distribution did not show significant differences between both groups (P = 0.0781). However, in particular alleles, Fisher exact test and odds ratio calculations showed significant values >1 for TKY08 and LEX52, and <1 for UM011, TKY08, LEX52 and VHL20. Interestingly, TKY08 STR is located in ELA class I region.

Phylogenetic analysis of Suid Herpesvirus 1 isolates from Argentina.

Argentinean Suid Herpesvirus 1 isolates were compared with reference strains and sequences available at GenBank and phylogenetically analyzed. A short fragment of the gE gene of the immunodominant epitopes was used for preliminary grouping of isolates by phylogenetic analysis. The analysis of the partial gC gene provided more precise genetic typing and segregation into the main genotypes I and II. Results confirmed that the Argentinean genotype I isolates predominate in our country. The topology of the partial gC gene was similar to that previously reported. The Argentinean type I isolates belonged to one cluster and grouped together with NIA-3 and American and Brazilian genotype I strains. However, the results obtained by the algorithms allow inferring that the Yamagata S-81 and Mer (genotype II) strains are grouped together.

Characterization of Suid herpesvirus 1 field isolates from Argentina.

The genomic characterization of Suid herpesvirus 1 (SHV-1) isolates from Argentina was accomplished by restriction pattern analysis using the BamHI, BstEII and XhoI enzymes. Type II genome has been described only once in Argentina. This study revealed considerable homogeneity of BamHI endonuclease sites in all the strains analyzed, according to the number and size of the fragments. No deletion of BamHI fragment #7 among the Argentinean isolates suggests that these strains are wild-type. In addition, the main antigenic domain of glycoprotein E of all the Argentinean strains, as well as the reference strains and sequences available in the GenBank, were characterized. The similarity percent oscillated between 99 and 100%.

An atypical clinical presentation for the first isolation of canid herpesvirus 1 in Argentina / Uma apresentação clínica atípica para o primeiro isolamento do herpesvirus canino 1 na Argentina

Relatam-se o primeiro isolamento de herpesvirus canino 1 (CaHV-1) e a localização atípica das lesões vesiculares associadas a este vírus na Argentina. A amostra foi recuperada de lesões vesiculares, localizadas na parte interna da coxa direita, em uma fêmea de raça Labrador. A cadela tinha quatro anos de idade e era de propriedade privada. O primeiro diagnóstico foi realizado pela reação em cadeia da polimerase e, posteriormente, o vírus foi isolado e sua identificação confirmada por imunofluorescência indireta e pelo teste de neutralização viral.

An atypical clinical presentation for the first isolation of Canid herpesvirus 1 in Argentina / Uma apresentação clínica atípica para o primeiro isolamento do herpesvirus canino 1 na Argentina

Relatam-se o primeiro isolamento de herpesvirus canino 1 (CaHV-1) e a localização atípica das lesões vesiculares associadas a este vírus na Argentina. A amostra foi recuperada de lesões vesiculares, localizadas na parte interna da coxa direita, em uma fêmea de raça Labrador. A cadela tinha quatro anos de idade e era de propriedade privada. O primeiro diagnóstico foi realizado pela reação em cadeia da polimerase e, posteriormente, o vírus foi isolado e sua identificação confirmada por imunofluorescência indireta e pelo teste de neutralização viral.(AU)

Evaluation of neutralization patterns of the five unique Argentine equine arteritis virus field strains reported.

Equine viral arteritis (EVA) is a contagious viral disease that frequently causes mild or subclinical infections in adult horses. Only one EAV serotype has been described. However, there are differences in antigenicity, pathogenicity and neutralization characteristics of virus field strains. The interaction of two viral proteins, GP5 and M, is critical for infectivity and amino acid changes in the GP5 sequences have an effect on the neutralizing phenotype, regardless the effects of other viral proteins. The objective of the present study was to evaluate the neutralization phenotypes of the 5 unique Argentine EAV strains reported and to compare them with the neutralization phenotypes of the EAV-UCD reference strain, with special emphasis on the analysis of M and GP5 proteins. The strains had a similar neutralization phenotype pattern when anti-EAV serum, derived from EAV seropositive horses, was used in the analysis. Meanwhile, low titers were observed when equine polyclonal anti-EAV reference sera were used in the assay. Argentine strains have almost the same amino acid substitutions, with the exception of LP01 strain, that mainly involves the first variable region V1, especially in neutralization sites B and C. However, they are fairly different from the EAV-UCD strain. Nevertheless, the nucleotide and amino acid differences observed among the Argentine strains LP02/R, LP02/C, LP02/P and LP-LT-ARG did not show any variations in the neutralization phenotype.

Evaluation of neutralization patterns of the five unique Argentine equine arteritis virus field strains reported / Evaluación de los patrones de neutralización de las únicas cinco cepas argentinas descritas de arteritis viral equina

Equine viral arteritis (EVA) is a contagious viral disease that frequently causes mild or subclinical infections in adult horses. Only one EAV serotype has been described. However, there are differences in antigenicity, pathogenicity and neutralization characteristics of virus field strains. The interaction of two viral proteins, GP5 and M, is critical for infectivity and amino acid changes in the GP5 sequences have an effect on the neutralizing phenotype, regardless the effects of other viral proteins. The objective of the present study was to evaluate the neutralization phenotypes of the 5 unique Argentine EAV strains reported and to compare them with the neutralization phenotypes of the EAV-UCD reference strain, with special emphasis on the analysis of M and GP5 proteins. The strains had a similar neutralization phenotype pattern when anti-EAV serum, derived from EAV seropositive horses, was used in the analysis. Meanwhile, low titers were observed when equine polyclonal anti-EAV reference sera were used in the assay. Argentine strains have almost the same amino acid substitutions, with the exception of LP01 strain, that mainly involves the first variable region V1, especially in neutralization sites B and C. However, they are fairly different from the EAV-UCD strain. Nevertheless, the nucleotide and amino acid differences observed among the Argentine strains LP02/R, LP02/C, LP02/P and LP-LT-ARG did not show any variations in the neutralization phenotype.

La arteritis viral equina (AVE) ocasiona infecciones, en su mayoría subclínicas, pero puede causar abortos y enfermedad respiratoria. Si bien se ha descrito un solo serotipo de AVE, existen diferencias en cuanto a la antigenicidad, patogenicidad y patrones de neutralización en las cepas de campo. Los ORF5 y ORF6 del virus codifican las proteínas de envoltura GP5 y M; la interacción entre estas proteínas es crítica para la infectividad. Los cambios en las secuencias de aminoácidos en la proteína GP5, especialmente en la región V1, afectan el fenotipo neutralizante, sin tener en cuenta variaciones aminoacídicas de otras proteínas virales. En este estudio evaluamos los fenotipos neutralizantes de las 5 únicas cepas de arteritis viral equina aisladas en Argentina y los comparamos con los de la cepa de referencia EAV-UCD por virus neutralización cruzada y análisis de secuencias aminoacídicas de las proteínas M y GP5. Las cepas argentinas presentaron un patrón de neutralización similar cuando se utilizaron sueros positivos del banco de sueros, mientras que fueron neutralizadas en menor medida por los sueros policlonales de referencia anti-AVE. A excepción de la cepa LP01, las cepas argentinas tienen casi las mismas sustituciones aminoacídicas en la primera región variable V1 de la proteína GP5, específicamente en los sitios neutralizantes B y C, pero difieren en gran medida respecto de la cepa de referencia EAV-UCD. Las diferencias encontradas en los aislamientos LP02/R, LP02/C, LP02/P y LT-LP-ARG no se reflejaron en variaciones en el fenotipo neutralizante.

Development of an ELA-DRA gene typing method based on pyrosequencing technology.

The polymorphism of equine lymphocyte antigen (ELA) class II DRA gene had been detected by polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP) and reference strand-mediated conformation analysis. These methodologies allowed to identify 11 ELA-DRA exon 2 sequences, three of which are widely distributed among domestic horse breeds. Herein, we describe the development of a pyrosequencing-based method applicable to ELA-DRA typing, by screening samples from eight different horse breeds previously typed by PCR-SSCP. This sequence-based method would be useful in high-throughput genotyping of major histocompatibility complex genes in horses and other animal species, making this system interesting as a rapid screening method for animal genotyping of immune-related genes.

[PCR-RFLP for Campylobacter jejuni subtyping]. / Aplicación de PCR-RFLP para subtipificar Campylobacter jejuni.

Ten Campylobacter jejuni isolates, 8 identified as C. jejuni biotype II of Lior and 2 as C. jejuni biotipe I, were recovered from aborted pig fetuses. In order to discriminate among strains, restriction fragment length polymorphism (RFLP) using Ddel of polymerase chain reaction (PCR) products of flaA gen was used. C. jejuni biotype II strains could be diferenciated in 6 by PCR-RFLP, and one subtype was obtained from C. jejunibiotype I. Although there is great variability of molecular techniques applied to the Campylobacter epidemiological studies, PCR-RFLP demonstrated to be a simple and accessible technique to discriminate Campylobacter jejuni isolates.

Aplicación de PCR-RFLP para subtipificar Campylobacter jejuni / [PCR-RFLP for Campylobacter jejuni subtyping]

Ten Campylobacter jejuni isolates, 8 identified as C. jejuni biotype II of Lior and 2 as C. jejuni biotipe I, were recovered from aborted pig fetuses. In order to discriminate among strains, restriction fragment length polymorphism (RFLP) using Ddel of polymerase chain reaction (PCR) products of flaA gen was used. C. jejuni biotype II strains could be diferenciated in 6 by PCR-RFLP, and one subtype was obtained from C. jejunibiotype I. Although there is great variability of molecular techniques applied to the Campylobacter epidemiological studies, PCR-RFLP demonstrated to be a simple and accessible technique to discriminate Campylobacter jejuni isolates.

Aplicación de PCR-RFLP para subtipificar Campylobacter jejuni. / [PCR-RFLP for Campylobacter jejuni subtyping]

Ten Campylobacter jejuni isolates, 8 identified as C. jejuni biotype II of Lior and 2 as C. jejuni biotipe I, were recovered from aborted pig fetuses. In order to discriminate among strains, restriction fragment length polymorphism (RFLP) using Ddel of polymerase chain reaction (PCR) products of flaA gen was used. C. jejuni biotype II strains could be diferenciated in 6 by PCR-RFLP, and one subtype was obtained from C. jejunibiotype I. Although there is great variability of molecular techniques applied to the Campylobacter epidemiological studies, PCR-RFLP demonstrated to be a simple and accessible technique to discriminate Campylobacter jejuni isolates.

The first isolation of equine arteritis virus in Argentina.

This paper describes the first isolation of equine arteritis virus (EAV) in Argentina. The virus was isolated from the semen of an imported seropositive stallion held in isolation at a breeding farm in Tandil in the Buenos Aires Province. In addition, viral nucleic acid was detected in seminal plasma using the reverse-transcription polymerase chain reaction. The isolated virus was propagated in cell cultures and confirmed as EAV by indirect immunofluorescence and virus neutralisation, using a serum specific for the reference Bucyrus strain of EAV. As far as the authors are aware, this is the first time that EAV has been isolated in South America. The equine industry is very important for Argentina and international movement of horses is very intensive. This finding may have effects on the international trade of horses and semen from Argentina.

[Campylobacter jejuni and C. coli in aborted swine: comparison between phenotypic identification and polyacrylamide gel protein profiles]. / Campylobacter jejuni y C. coli en suinos abortados: comparación entre la identificación fenotípica y los perfiles proteicos en geles de poliacrilamida.

Campylobacter jejuni and Campylobacter coli were isolated from aborted pig fetuses which proceeded from different animals and farms between February 2000 and March 2001. Seven Campylobacter jejuni biotype II, three biotype I and one Campylobacter coli biotype I were identified by phenotypic tests and Lior's scheme. To corroborate and compare the phenotypic results, 7.5, 10 and 12.5% polyacrilamide gel electrophoresis (SDS-PAGE) were used under reducing conditions. Characteristic bands of hypervariable dense zone within C. jejuni and C. coli species were observed in all the whole cell protein extracts with differences in mobility. It was possible to establish differences between identical phenotypic Campylobacter isolates and different protein profile from fetuses of the same litter. SDS-PAGE is a stable and reproducible method to establish differences between Campylobacter strains and is considered applicable for the differentiation of the wide variability of Campylobacter species for epidemiologic purposes.

Campylobacter jejuni y C. coli en suinos abortados: comparación entre la identificación fenotípica y los perfiles proteicos en geles de poliacrilamida. / [Campylobacter jejuni and C. coli in aborted swine: comparison between phenotypic identification and polyacrylamide gel protein profiles]

Campylobacter jejuni and Campylobacter coli were isolated from aborted pig fetuses which proceeded from different animals and farms between February 2000 and March 2001. Seven Campylobacter jejuni biotype II, three biotype I and one Campylobacter coli biotype I were identified by phenotypic tests and Liors scheme. To corroborate and compare the phenotypic results, 7.5, 10 and 12.5

polyacrilamide gel electrophoresis (SDS-PAGE) were used under reducing conditions. Characteristic bands of hypervariable dense zone within C. jejuni and C. coli species were observed in all the whole cell protein extracts with differences in mobility. It was possible to establish differences between identical phenotypic Campylobacter isolates and different protein profile from fetuses of the same litter. SDS-PAGE is a stable and reproducible method to establish differences between Campylobacter strains and is considered applicable for the differentiation of the wide variability of Campylobacter species for epidemiologic purposes.