Marcelo Ricardo Pecoraro

First report of Israeli acute paralysis virus in asymptomatic hives of Argentina

Honey bee mortality has recently been associated with Israeli acute paralysis virus (IAPV), a proposed etiological agent for a new syndrome known as Colony Collapse Disorder. Bees infected with this virus show shivering wings, progress into paralysis, and finally die outside the hive. During the last years, honey bee mortality became a serious problem for Argentinean beekeepers. We herein report the preliminary results of a survey carried out to detect IAPV in samples taken from several Argentine provinces, by using a reverse transcription Polymerase Chain Reaction assay. Our data indicate the existence of high frequency of IAPV in asymptomatic hives of Argentina.

First report of viral infections that affect argentine honeybees

Honey is one of the most important agricultural products for export in Argentina. In fact, more than 3.5 million beehives and 50 000 beekeepers are related with this production, mainly located in Buenos Aires province. Honeybee mortality is a serious problem that beekeepers in Argentina have had to face during the last 3 years. It is known that the consequence of the complex interactions between environmental and beekeeping parameters added to the effect of different disease agents such as viruses, bacteria, fungi and parasitic mites may result in a sudden collapse of the colony. In addition, multiple viral infections are frequently detected concomitantly in bee colonies. We describe here the preliminary results of a survey of three honeybee-pathogenic viruses, acute bee paralysis viruses (ABPV), chronic bee paralysis viruses (CBPV) and Sacbrood viruses (SBV) detected during a screening of 61 apiaries located in the main honey producer province using a RT-PCR assay. This is the first molecular report of the presence of these viruses in Argentine apiaries.

Simultaneous detection of bee viruses by multiplex PCR.

Honey bee mortality is a serious problem that beekeepers in Argentina have had to face during the last 3 years. It is known that the consequence of the complex interactions between environmental and beekeeping parameters added to the effect of different disease agents such as viruses, bacteria, fungi and parasitic mites may result in a sudden collapse of the colony. In addition, multiple viral infections are detected frequently concomitantly in bee colonies. The aim of this study was to establish a multiplex polymerase chain reaction method for rapid and simultaneous detection of the most prevalent bee viruses. This multiplex PCR assay will provide specific, rapid and reliable results and allow for the cost effective detection of a particular virus as well as multiple virus infections in a single reaction tube. This method could be a helpful tool in the surveillance of the most frequently found bee viruses and to study the dynamics and the interactions of the virus populations within colonies.

First molecular detection of co-infection of honey bee viruses in asymptomatic Bombus atratus in South America

Pollination is critical for food production and has the particularity of linking natural ecosystems with agricultural production systems. Recently, losses of bumblebee species have been reported worldwide. In this study, samples from a commercial exploitation of bumblebees of Argentina with a recent history of deaths were studied using a multiplex PCR for the detection of the honey bee viruses most frequently detected in South America. All samples analysed were positive for co-infections with Deformed wing virus, Black queen cell virus and Sacbrood virus. This is the first report of infection of Bombus atratus with honey bee viruses. A better understanding of viral infections in bumblebees and of the epidemiology of viruses could be of great importance as bumblebees can serve as possible viral reservoirs, resulting in pathogen spillover towards honey bees and native bumblebees.

Production of equine herpesvirus 1 recombinant glycoprotein D and development of an agar gel immunodiffusion test for serological diagnosis.

Equine herpesvirus 1 and 4 (EHV-1 and 4) infect most of the worlds horses, causing serious clinical illness. Viral glycoproteins have been identified as the immunodominant antigens that generate the antiviral serological responses to EHV-1 and EHV-4 in infected horses. Here, glycoprotein D of EHV-1 was expressed by a recombinant baculovirus, purified and evaluated by a simple agar gel immunodiffusion test (AGID). Compared with virus neutralization, serological analysis by AGID showed good specificity (100%) and sensitivity (99.5%). The estimated Kappa values for repeatability and reproducibility were satisfactory. Thus, this rapid, inexpensive, simple and highly specific AGID test seems to be a valuable alternative tool for serological detection of antibodies against both EHV-1 and EHV-4.

Feline immunodeficiency virus infection: a comparative study of different diagnostic techniques

This study evaluated an indirect immunofluorescence assay (IFA) to detect feline immunodeficiency virus infection (FIV) antibody in a comprehensive epidemiological survey of FIV in Argentina. IFA modified in our laboratory, was compared with two other immunoassays, western blot (WB) and a sandwich immunochromatographic commercial kit (SI), and also with a direct polymerase chain reaction (PCR) method that detects proviral DNA. IFA showed to be a test with high sensitivity and specificity, and could be useful as a diagnostic tool in epidemiological studies. The presence of a low percentage of results with non-specific reactivity in the IFA could be resolved with further testing or use of an alternative method.

An effective and simplified DO-stat control strategy for production of rabies glycoprotein in Pichia pastoris

The glycoprotein (G-protein) of rabies virus is responsible for viral attachment to the host cell surface and induces virus neutralization antibodies. In the present study, the G-protein gene of rabies virus CVS strain was cloned, sequenced and expressed in the yeast, Pichia pastoris, as a secreted protein, using a simplified DO-stat control feeding strategy. This strategy involves the addition of methanol when the dissolved oxygen (DO) level rises above the setpoint avoiding methanol accumulation and oxygen limitation. The G-protein expression was evaluated by SDS-PAGE, ELISA, and western blot assays. Like native G-protein, the recombinant G-protein was found reactive when it was challenged against specific antibodies. The data indicate that the recombinant G-protein can be easily expressed and isolated, and may be useful as a safe source in the production of diagnostic kits and subunit vaccines to prevent rabies.

Genomic study of Argentinean Equid herpesvirus 1 strains

Equid herpesvirus 1 (EHV-1) infection has a significant economic impact on equine production, causing abortion, respiratory disease, neonatal death and neurological disorders. The identification of specific EHV-1 genes related to virulence and pathogenicity has been the aim of several research groups. The purpose of the present study was to analyze different genomic regions of Argentinean EHV-1 strains and to determine their possible relationship with virulence or clinical signs. Twenty-five EHV-1 Argentinean isolates recovered from different clinical cases between 1979 and 2007 and two reference strains were amplified and sequenced. The sequence alignments were carried out using Clustal X version 1.92 and the putative amino acid sequences were deduced using Bio-Edit version 7.05. Minor changes were observed. No changes that could be involved in the different virulence in the mouse model of three EHV-1 Argentinean strains were found. No genetic variants were observed. The genomic regions analyzed are unsuitable for differentiation between abortigenic strains and those isolated from neonatal deaths.

Aggregation of mummified adult crabeater seals (Pinnipedia: Phocidae) in the eastern Antarctic Peninsula: age and sex structure, taphonomy and cause of death

Abstract In Antarctica, crabeater seals tend to strand as immature animals with disorientation, due to their inexperience, given as the probable cause. In 2012 and 2013, we examined a group of 80 mummified crabeater seals on Seymour Island (Marambio). The age and gender of 28 seals was determined, and virology and stomach content analyses were performed in order to determine the cause of stranding. Around 82% of the seals examined were adults and 79% were females, some of which were pregnant. All of the seals sampled tested negative for Morbillivirus, suggesting that the stranding was not related to the mass mortality event reported in the 1950s in the region. Most seals had empty stomachs and thin blubber suggesting that they died from starvation. The state of the carcasses suggests multiple stranding events. Most of the seals were located along an ice-covered stream, suggesting that this may act as a ‘natural trap’, isolating the seals from the open ocean. This is exceptional as it is the first report of mostly adult female seals to strand in Antarctica and refutes the theory that only young animals are prone to stranding.

Systemic infection induced by intranasal inoculation of Bovine herpesvirus 1.1 in pregnant and non-pregnant rabbits

Bovine herpesvirus (BoHV) type 1.1 (BoHV-1.1) causes repeated outbreaks of upper respiratory disease and abortion in cattle. The systemic effects of BoHV-1.1 in rabbits, using intranasal inoculation are reported. Female rabbits were divided into four groups and inoculated with the virus 10 days before mating, and at 15 or 22 days of pregnancy. Studies of the clinical signs, antibody production, virus isolation, and DNA detection as well as histological and immunohistochemical studies were carried out on lungs, kidneys, spleen, placentas, uteri and foetal tissues. All virus-inoculated animals developed respiratory clinical signs and a humoral response. BoHV-1.1 was isolated from nasal swabs and plasma rich in leukocytes, and viral DNA was detected in blood, dead foetuses and placentas. Histopathological lesions were found in the respiratory tract and some placentas and foetuses were immunohistochemically positive. Intranasal inoculation might be useful to study the systemic effects of BoHV-1.1 infection in the rabbit model.