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Background In pigs, modified live virus (MLV) vaccines against porcine reproductive and respiratory syndrome virus (PRRSV) are commonly used and administered by intramuscular (IM) injection. In contrast, PRRSV, as a primary respiratory pathogen, is mainly transmitted via the intranasal (IN) route. The objective of this study was to evaluate the efficacy of a commonly used commercial PRRSV MLV delivered IN compared to the IM route. Methods Fifty-four pigs were divided into five treatment groups. All vaccinated groups received the same MLV vaccine but administered via different routes. Group IN-JET-VAC was vaccinated with an automated high pressure prototype nasal jet device (IN-JET-VAC, n = 12), group IN-MAD-VAC was vaccinated with a mucosal atomization device (IN-MAD-VAC, n = 12), group IM-VAC was vaccinated intramuscularly (IM-VAC; n = 12) according to label instructions, while the NEG-CONTROL (n = 6) and the POS-CONTROL (n = 12) groups were both unvaccinated. At 28 days post vaccination all vaccinated groups and the POS-CONTROL pigs were challenged with a pathogenic US PRRSV isolate. Blood and nasal swabs were collected at regular intervals, and all pigs were necropsied at day 10 post challenge (dpc) when gross and microscopic lung lesions were assessed. Results Prior to challenge most vaccinated pigs had seroconverted to PRRSV. Clinical signs (fever, inappetence) were most obvious in the POS-CONTROL group from dpc 7 onwards. The vaccinated groups were not different for PRRSV viremia, seroconversion, or average daily weight gain. However, IN-JET-VAC and IN-MAD-VAC had significantly higher neutralizing antibody levels against the vaccine virus at challenge. Conclusions Comparable vaccine responses were obtained in IN and IM vaccinated pigs, suggesting the intranasal administration route as an alternative option for PRRSV vaccination.

Comparative studies of naturally occurring canine cancers have provided new insight into many areas of cancer research. Development and validation of circulating tumor DNA (ctDNA) analysis in pet dogs can help address diagnostic needs in veterinary as well as human oncology. Dogs have high incidence of naturally occurring spontaneous cancers, demonstrate molecular heterogeneity and clonal evolution during therapy, allow serial sampling of blood from the same individuals during the course of disease progression, and have relatively compressed intervals for disease progression amenable to longitudinal studies. Here, we present a feasibility study of ctDNA analysis performed in 48 dogs including healthy dogs and dogs with either benign splenic lesions or malignant splenic tumors (hemangiosarcoma) using shallow whole genome sequencing (sWGS) of cell-free DNA. To enable detection and quantification of ctDNA using sWGS, we adapted two informatic approaches and compared their performance for the canine genome. At the time of initial clinical presentation, mean ctDNA fraction in dogs with malignant splenic tumors was 11.2%, significantly higher than dogs with benign lesions (3.2%; p = 0.001). ctDNA fraction was 14.3% and 9.0% in dogs with metastatic and localized disease, respectively (p = 0.227). In dogs treated with surgical resection of malignant tumors, mean ctDNA fraction decreased from 11.0% prior to resection to 7.9% post-resection (p = 0.047 for comparison of paired samples). Our results demonstrate that ctDNA analysis is feasible in dogs with hemangiosarcoma using a cost-effective approach such as sWGS. Additional studies are needed to validate these findings, and determine the role of ctDNA to assess burden of disease and treatment response in dogs with cancer.

Fragmentation patterns observed in plasma DNA reflect chromatin accessibility in contributing cells. Since DNA shed from cancer cells and blood cells may differ in fragmentation patterns, we investigated whether analysis of genomic positioning and nucleotide sequence at fragment ends can reveal the presence of tumor DNA in blood and aid cancer diagnostics. We analyzed whole genome sequencing data from >2700 plasma DNA samples including healthy individuals and patients with 11 different cancer types. We observed higher fractions of fragments with aberrantly positioned ends in patients with cancer, driven by contribution of tumor DNA into plasma. Genomewide analysis of fragment ends using machine learning showed overall area under the receiver operative characteristic curve of 0.96 for detection of cancer. Our findings remained robust with as few as 1 million fragments analyzed per sample, suggesting that analysis of fragment ends can become a cost-effective and accessible approach for cancer detection and monitoring.One-sentence summaryAnalyzing the positioning and nucleotide sequence at fragment ends in plasma DNA may enable cancer diagnostics.

Comparative studies of naturally-occurring canine cancers have provided new insight into many areas of cancer research. The inclusion of pet dogs in the development and validation of circulating tumor DNA (ctDNA) diagnostics may be uniquely informative for human translation for many reasons, including: high incidence of certain spontaneous cancers, repeated access to blood and tumor from the same individuals during the course of disease progression, and molecular heterogeneity of naturally-occurring cancers in dogs. Here, we present a feasibility study of ctDNA analysis performed in 9 healthy dogs and 39 dogs with either benign or malignant splenic tumors (hemangiosarcoma) using shallow whole genome sequencing (sWGS) of cell-free DNA. To enable detection and quantification of ctDNA using sWGS, we adapted two informatic approaches and compared their performance for the canine genome. At presentation, mean ctDNA tumor fraction in dogs with malignant splenic tumors was 11.2%, significantly higher than dogs with benign lesions (3.2%; p 0.001), achieving an AUC of 0.84. ctDNA tumor fraction was 14.3% and 9.0% in dogs with metastatic and localized disease, respectively although this difference was not statistically significant (p 0.227). In paired analysis, ctDNA fraction decreased from 11.0% to 7.9% after resection of malignant tumors (p 0.047). Our results demonstrate that ctDNA analysis is feasible in dogs with hemangiosarcoma using a cost-effective approach such as sWGS. Future studies are underway to validate these findings, and further evaluate the role of ctDNA to assess burden of disease and treatment response during drug development.

A influenza equina (EI) é uma enfermidade respiratória viral aguda que acomete equídeos de todas as idades. A doença é causada pelo vírus da influenza equina (EIV do inglês Equine Influenza Virus), que podem ser do subtipo viral H3N8 (antigo Equi-2), mundialmente distribuído, e o H7N7 (antigo Equi-1), considerado extinto desde 1980. Os EIVs pertencem à família Orthomyxoviridae, gênero Influenza A, possuem genoma RNA fita simples segmentado de senso negativo e são envelopados. A EI tem alta morbidade e baixa mortalidade leva a grandes perdas econômicas no ramo equestre. Os animais acometidos ficam impedidos de serem transportados ou de participarem de eventos equestres no Brasil. A rápida e fácil disseminação do EIV é bastante característico em surtos de EI. Surtos de EI foram descritos em 2012 em diversos países, incluindo o Brasil. Em 2015, um surto de EIV ocorreu em um Hospital Veterinário na cidade de São Paulo-SP. Os objetivos do presente trabalho foram: a) obter isolados do vírus da influenza equina de surtos recentes do estado de São Paulo, Brasil; b) realizar o sequenciamento dos EIVs isolados; c) promover a análise filogenética e evolutiva desses EIVs. Foram isolados 12 EIVs de um surto de EI em um hospital veterinário na cidade de São Paulo em 2015. Promoveu-se o sequenciamento dos oito genes virais dos isolados de 2015 e de uma estirpe de EIV de um surto brasileiro de 2012, e da hemaglutinina de outras duas estirpes do mesmo surto de 2012. Os vírus do surto de 2012 foram cedidos pelo Laboratório de Raiva e Encefalites Virais do Instituto Biológico de São Paulo para as análises. O sequenciamento dos genes HA e NA indicaram que os EIVs de 2012 e 2015 brasileiros são do subtipo H3N8 e pertencem à sublinhagem Flórida 1 e tiveram maiores identidades com os vírus de 2012 da América do Sul, Estados Unidos e Dubai de 2012. Os vírus de 2015 formaram um clado separado dos demais EIV Flórida 1, tanto na análise filogenética quanto na análise de relógio molecular. As análises de mutações de nucleotídeos e aminoácidos, associados à análise de perfil de hidrofobicidade sugerem mudanças em estruturas antigênicas que poderiam acarretar no maior distanciamento em relação à estirpe vacinal A/equine/South Africa/4/03. The equine influenza (EI) is a viral acute respiratory disease that can affect equines of any age. The disease is caused by the equine influenza virus (EIV) and has two different subtypes H3N8 (formerly Equi-2) that occurs worldwide and H7N7 (formerly Equi-1), considered extinct since 1980. EIVs belong to the Orthomyxoviridae family, Influenza A genus, are enveloped and has negative segmented single strand RNA genome. EI causes high morbidity and low mortality and drives to high economic losses in equestrian industry. In Brazil, the transport and participation in equestrian events are not allowed when horses have EIV. The quickly spread pattern is easily seen in EIV outbreaks. EI outbreaks occurred in 2012 in several countries, including Brazil. In 2015, an outbreak occurred in a Veterinarian Hospital in São Paulo-SP. The objectives of the present study were: a) to obtain isolates of equine influenza virus from recent outbreaks in the State of São Paulo, Brazil; b) to promote the sequencing of these EIVs; c) to perform the phylogenetic and evolutive analysis of these EIVs. Twelve EIVs were isolated from an outbreak in a veterinarian hospital in the State of São Paulo in 2015. The eight viral genes were sequenced from EIVs of 2015 and from one EIV of a Brazilian outbreak, 2012. Also, the HA gene from two other EIVs from the same 2012-outbreak were sequenced. The viruses from the 2012-outbreak were from the Laboratory of Rabies and Viral Encephalitis, Instituto Biológico de São Paulo. Sequencing on HA and NA genes indicates that the Brazilian 2012 and 2015 EIVs are H3N8 subtype, belong to the Florida 1 sublineage and were more identical to the 2012 EIVs from South America, United States and Dubai. In the phylogenetic tree and the molecular clock the EIVs from 2015 grouped in a cluster that was separeted from other Florida 1 EIVs. The nucleotide, amino acid and hydrophobicity analysis suggest changes in antigenic structures that could lead to differences from the vaccine strain A/equine/South Africa/4/03.

Equine influenza virus (EIV) (H3N8 and H7N7) is the causative agent of equine influenza, or equine flu. The H7N7 subtype has been considered to be extinct worldwide since 1980. Affected animals have respiratory symptoms that can be worsened by secondary bacterial respiratory infection, thereby leading to great economic losses in the horse-breeding industry. In Brazil, equine influenza outbreaks were first reported in 1963 and studies on hemagglutination antibodies against viral subtypes in Brazilian horses have been conducted since then. The objective of the present review was to present the history of the emergence of EIV around the world and in Brazil and the studies that have thus far been developed on EIV in Brazilian equines. O vírus da influenza equina (EIV) (H3N8 e H7N7) é o agente causador da influenza equina, ou gripe equina. O subtipo viral H7N7 é considerado como mundialmente extinto desde 1980. Os animais afetados têm sintomas respiratórios característicos que podem ser agravados por uma infecção respiratória bacteriana secundária causando grandes prejuízos no ramo equestre. No Brasil, os surtos da EI têm sido relatados desde 1963 e desde então vem sendo efetuados estudos sobre a presença de anticorpos hemaglutinantes contra os subtipos virais nos equídeos brasileiros. O presente artigo tem o objetivo de apresentar um histórico sobre o surgimento do EIV no mundo e no Brasil destacando os estudos conduzidos no Brasil até o momento acerca da infecção pelo EIV nos equídeos brasileiros.

Oral tolerance can be defined as the inability of an adult animal to produce specific antibodies or cellular immune responses upon conventional immunization, after oral antigenic administration. Recently, the oral administration of antigens has gained renewed interest because of the possibility of inducing tolerance in nonimmunized adult animals and, consequently, opening up the theoretical possibility of preventing or treating diseases caused by malfunction of the immune system. This strategy has been proven to be useful in the prevention of allergic and autoimmune diseases in rodents, as well as in the amelioration of certain autoimmune diseases in humans. Although there is experimental and clinical evidence for the usefulness of oral tolerance in medical practice, the mechanisms responsible for this phenomenon are still poorly understood, and the results obtained are not always satisfactory. Herein, we show that the thymus is required for the induction and maintenance of oral tolerance, providing evidence that it is not a pure form of clonal deletion-based peripheral tolerance. Oral tolerance could therefore depend on the formation and release to the periphery of regulatory T cells, such as cd or ab T cells, by the thymus. This finding may have profound implications for the treatment of autoimmune diseases, since most of them are associated with thymic hypofunction. On the other hand, due to so far unknown mechanisms, the intraperitoneal co-administration of normal IgG to mice orally treated with tolerogen leads to a sustained and intense immunological tolerance, both in euthymic and thymectomized mice, including those of the lupus erythematosus-prone NZB · NZW lineage. This approach for inducing and maintaining tolerance in thymus-deficient conditions is discussed and put forth herein as a new evidence-based proposition for the therapy of autoimmune diseases.

Equine influenza is one of the major respiratory infectious diseases in horses. An equine influenza virus outbreak was identified in vaccinated and unvaccinated horses in a veterinary school hospital in São Paulo, SP, Brazil, in September 2015. The twelve equine influenza viruses isolated belonged to Florida Clade 1. The hemagglutinin and neuraminidase amino acid sequences were compared with the recent isolates from North and South America and the World Organisation for Animal Health recommended Florida Clade 1 vaccine strain. The hemagglutinin amino acid sequences had nine substitutions, compared with the vaccine strain. Two of them were in antigenic site A (A138S and G142R), one in antigenic site E (R62K) and another not in antigenic site (K304E). The four substitutions changed the hydrophobicity of hemagglutinin. Three distinct genetic variants were identified during the outbreak. Eleven variants were found in four quasispecies, which suggests the equine influenza virus evolved during the outbreak. The use of an out of date vaccine strain or updated vaccines without the production of protective antibody titers might be the major contributing factors on virus dissemination during this outbreak.