Mariano Carossino

Development and evaluation of a reverse transcription-insulated isothermal polymerase chain reaction (RT-iiPCR) assay for detection of equine arteritis virus in equine semen and tissue samples using the POCKIT™ system.

Equine arteritis virus (EAV) is the causative agent of equine viral arteritis (EVA), a respiratory and reproductive disease of horses. Most importantly, EAV induces abortion in pregnant mares and can establish persistent infection in up to 10-70% of the infected stallions, which will continue to shed the virus in their semen. The objective of this study was to develop and evaluate a reverse transcription insulated isothermal polymerase chain reaction (RT-iiPCR) for the detection of EAV in semen and tissue samples. The newly developed assay had a limit of detection of 10 RNA copies and a 10-fold higher sensitivity than a previously described real-time RT-PCR (RT-qPCR). Evaluation of 125 semen samples revealed a sensitivity and specificity of 98.46% and 100.00%, respectively for the RT-qPCR assay, and 100.00% and 98.33%, respectively for the RT-iiPCR assay. Both assays had the same accuracy (99.2%, k=0.98) compared to virus isolation. Corresponding values derived from testing various tissue samples (n=122) collected from aborted fetuses, foals, and EAV carrier stallions are as follows: relative sensitivity, specificity, and accuracy of 88.14%, 96.83%, and 92.62% (k=0.85), respectively for the RT-qPCR assay, and 98.31%, 92.06%, and 95.08% (k=0.90), respectively for the RT-iiPCR assay. These results indicate that RT-iiPCR is a sensitive, specific, and a robust test enabling detection of EAV in semen and tissue samples with very considerable accuracy. Even though the RT-qPCR assay showed a sensitivity and specificity equal to virus isolation for semen samples, its diagnostic performance was somewhat limited for tissue samples. Thus, this new RT-iiPCR could be considered as an alternative tool in the implementation of EAV control and prevention strategies.

Epidemiological and virological findings during multiple outbreaks of equine influenza in South America in 2012.

In 2012, equine influenza (EI) virus was confirmed as the cause of outbreaks of respiratory disease in horses throughout South America. In Uruguay and Argentina, hundreds of vaccinated thoroughbred horses in training and racing facilities were clinically affected.

Equine Arteritis Virus Has Specific Tropism for Stromal Cells and CD8+ T and CD21+ B Lymphocytes but Not for Glandular Epithelium at the Primary Site of Persistent Infection in the Stallion Reproductive Tract

ABSTRACT Equine arteritis virus (EAV) has a global impact on the equine industry as the causative agent of equine viral arteritis (EVA), a respiratory, systemic, and reproductive disease of equids. A distinctive feature of EAV infection is that it establishes long-term persistent infection in 10 to 70% of infected stallions (carriers). In these stallions, EAV is detectable only in the reproductive tract, and viral persistence occurs despite the presence of high serum neutralizing antibody titers. Carrier stallions constitute the natural reservoir of the virus as they continuously shed EAV in their semen. Although the accessory sex glands have been implicated as the primary sites of EAV persistence, the viral host cell tropism and whether viral replication in carrier stallions occurs in the presence or absence of host inflammatory responses remain unknown. In this study, dual immunohistochemical and immunofluorescence techniques were employed to unequivocally demonstrate that the ampulla is the main EAV tissue reservoir rather than immunologically privileged tissues (i.e., testes). Furthermore, we demonstrate that EAV has specific tropism for stromal cells (fibrocytes and possibly tissue macrophages) and CD8+ T and CD21+ B lymphocytes but not glandular epithelium. Persistent EAV infection is associated with moderate, multifocal lymphoplasmacytic ampullitis comprising clusters of B (CD21+) lymphocytes and significant infiltration of T (CD3+, CD4+, CD8+, and CD25+) lymphocytes, tissue macrophages, and dendritic cells (Iba-1+ and CD83+), with a small number of tissue macrophages expressing CD163 and CD204 scavenger receptors. This study suggests that EAV employs complex immune evasion mechanisms that warrant further investigation. IMPORTANCE The major challenge for the worldwide control of EAV is that this virus has the distinctive ability to establish persistent infection in the stallions reproductive tract as a mechanism to ensure its maintenance in equid populations. Therefore, the precise identification of tissue and cellular tropism of EAV is critical for understanding the molecular basis of viral persistence and for development of improved prophylactic or treatment strategies. This study significantly enhances our understanding of the EAV carrier state in stallions by unequivocally identifying the ampullae as the primary sites of viral persistence, combined with the fact that persistence involves continuous viral replication in fibrocytes (possibly including tissue macrophages) and T and B lymphocytes in the presence of detectable inflammatory responses, suggesting the involvement of complex viral mechanisms of immune evasion. Therefore, EAV persistence provides a powerful new natural animal model to study RNA virus persistence in the male reproductive tract.

Detection of equine arteritis virus by two chromogenic RNA in situ hybridization assays (conventional and RNAscope®) and assessment of their performance in tissues from aborted equine fetuses

Equine arteritis virus (EAV) is the causative agent of equine viral arteritis, a respiratory and reproductive disease of equids. EAV infection can induce abortion in pregnant mares, fulminant bronchointerstitial pneumonia in foals, and persistent infection in stallions. Here, we developed two RNA in situ hybridization (ISH) assays (conventional and RNAscope® ISH) for the detection of viral RNA in formalin-fixed paraffin-embedded (FFPE) tissues and evaluated and compared their performance with nucleocapsid-specific immunohistochemistry (IHC) and virus isolation (VI; gold standard) techniques. The distribution and cellular localization of EAV RNA and antigen were similar in tissues from aborted equine fetuses. Evaluation of 80 FFPE tissues collected from 16 aborted fetuses showed that the conventional RNA ISH assay had a significantly lower sensitivity than the RNAscope® and IHC assays, whereas there was no difference between the latter two assays. The use of oligonucleotide probes along with a signal amplification system (RNAscope®) can enhance detection of EAV RNA in FFPE tissues, with sensitivity comparable to that of IHC. Most importantly, these assays provide important tools with which to investigate the mechanisms of EAV pathogenesis.

Reproductive effects of arteriviruses: equine arteritis virus and porcine reproductive and respiratory syndrome virus infections

Equine arteritis virus (EAV) and porcine reproductive and respiratory syndrome virus (PRRSV) are the most economically important members of the family Arteriviridae. EAV and PRRSV cause reproductive and respiratory disease in equids and swine, respectively and constitute a significant economic burden to equine and swine industries around the world. Furthermore, they both cause abortion in pregnant animals and establish persistent infection in their natural hosts, which fosters viral shedding in semen leading to sexual transmission. The primary focus of this article is to provide an update on the effects of these two viruses on the reproductive tract of their natural hosts and provide a comparative analysis of clinical signs, virus-host interactions, mechanisms of viral pathogenesis and viral persistence.

Equine arteritis virus elicits a mucosal antibody response in the reproductive tract of persistently infected stallions

ABSTRACT Equine arteritis virus (EAV) has the ability to establish persistent infection in the reproductive tract of the stallion (carrier) and is continuously shed in its semen. We have recently demonstrated that EAV persists within stromal cells and a subset of lymphocytes in the stallion accessory sex glands in the presence of a significant local inflammatory response. In the present study, we demonstrated that EAV elicits a mucosal antibody response in the reproductive tract during persistent infection with homing of plasma cells into accessory sex glands. The EAV-specific immunoglobulin isotypes in seminal plasma included IgA, IgG1, IgG3/5, and IgG4/7. Interestingly, seminal plasma IgG1 and IgG4/7 possessed virus-neutralizing activity, while seminal plasma IgA and IgG3/5 did not. However, virus-neutralizing IgG1 and IgG4/7 in seminal plasma were not effective in preventing viral infectivity. In addition, the serological response was primarily mediated by virus-specific IgM and IgG1, while virus-specific serum IgA, IgG3/5, IgG4/7, and IgG6 isotype responses were not detected. This is the first report characterizing the immunoglobulin isotypes in equine serum and seminal plasma in response to EAV infection. The findings presented herein suggest that while a broader immunoglobulin isotype diversity is elicited in seminal plasma, EAV has the ability to persist in the reproductive tract, in spite of local mucosal antibody and inflammatory responses. This study provides further evidence that EAV employs complex immune evasion mechanisms during persistence in the reproductive tract that warrant further investigation.

Detection, molecular characterization and phylogenetic analysis of G3P[12] and G14P[12] equine rotavirus strains co-circulating in central Kentucky

Equine rotavirus A (ERVA) is the leading cause of diarrhea in neonatal foals and a major health problem to the equine breeding industry worldwide. The G3P[12] and G14P[12] ERVA genotypes are the most prevalent in foals with diarrhea. Control and prevention strategies include vaccination of pregnant mares with an inactivated vaccine containing a prototype ERVA G3P[12] strain with limited and controversial field efficacy. Here, we performed the molecular characterization of ERVA strains circulating in central Kentucky using fecal samples collected during the 2017 foaling season. The data indicated for the first time that the G14P[12] genotype is predominant in this region in contrast to a previous serotyping study where only G3 genotype strains were reported. Overall, analysis of antigenic sites in the VP7 protein demonstrated the presence of several amino acid substitutions in the epitopes exposed on the surface including a non-conserved N-linked glycosylation site (D123N) in G14P[12] strains, while changes in antigenic sites of VP8* were minor. Also, we report the successful isolation of three ERVA G14P[12] strains which presented a high identity with other G14 strains from around the world. These may constitute ideal reference strains to comparatively study the molecular biology of G3 and G14 strains and perform vaccine efficacy studies following heterologous challenge in the future.

Infectious Diseases in Donkeys and Mules: An Overview and Update

&NA; The global equine population comprises a total of 114 million equids, which includes an estimate of 44 million donkeys (Equus asinus) and 11 million mules. Donkey populations have gradually expanded throughout the globe, particularly in developing countries. In spite of their relevance in several countries around the world, the infectious diseases affecting this group of equids are largely unknown; consequently, the available information regarding infectious diseases affecting working equids is limited. The scope of this article is to provide a brief update on the most relevant infectious diseases affecting donkeys and mules worldwide. HighlightsThe impact of different infectious diseases on working equids is largely unknown.Differences in disease outcome are observed between working equids and horses.Working equids play a critical epidemiological role for several infectious diseases.