AAV Vectors Pseudotyped with Capsids from Porcine and Bovine Species Mediate In Vitro and In Vivo Gene Delivery.

Adeno-associated virus (AAV) vectors are among the most widely used delivery vehicles for in vivo gene therapy as they mediate robust and sustained transgene expression with limited toxicity. However, a significant impediment to the broad clinical success of AAV-based therapies is the widespread presence of pre-existing humoral immunity to AAVs in the human population. This immunity arises from the circulation of non-pathogenic endemic human AAV serotypes. One possible solution is to use non-human AAV capsids to pseudotype transgene-containing AAV vector genomes of interest. Due to the low probability of human exposure to animal AAVs, pre-existing immunity to animal-derived AAV capsids should be low. Here, we characterize two novel AAV capsid sequences: one derived from porcine colon tissue and the other from a caprine adenovirus stock. Both AAV capsids proved to be effective transducers of HeLa and HEK293T cells in vitro. In vivo, both capsids were able to transduce the murine nose, lung, and liver after either intranasal or intraperitoneal administration. In addition, we demonstrate that the porcine AAV capsid likely arose from multiple recombination events involving human- and animal-derived AAV sequences. We hypothesize that recurrent recombination events with similar and distantly related AAV sequences represent an effective mechanism for enhancing the fitness of wildtype AAV populations.

The effect of aerosolized bacterial lysate on experimentally induced Mannheimia haemolytica pneumonia in calves.

Bovine respiratory disease (BRD) often occurs during specific periods of increased susceptibility when stress, viral infection, or reduced air quality are thought to suppress respiratory defences. The innate immune system is rapidly responsive and broadly protective and could be a target for preventing BRD during these periods of increased susceptibility. This study tested the hypothesis that stimulation of pulmonary innate immune responses by aerosol delivery of a lysate of killed Escherichia coli and Staphylococcus aureus bacteria would protect calves against Mannheimia haemolytica pneumonia. Ten clean-catch colostrum-deprived Holstein calves were randomly assigned to receive either aerosolized bacterial lysate or saline 24 hours before M. haemolytica challenge. Effects of this treatment on clinical, hematologic, microbiologic, and pathologic outcomes were assessed. Compared to controls, lysate-treated calves had lower serum haptoglobin and blood leukocyte and neutrophil concentrations following M. haemolytica challenge. There were no differences in temperature, heart and respiratory rates, clinical scores, ultrasound lesions, or number of M. haemolytica in the nasal cavity or lung. Thus, treatment with bacterial lysate prior to M. haemolytica challenge appeared to ameliorate early measures of inflammation but did not provide sufficient protection to substantially alter the course of disease.

La maladie respiratoire bovine (BRD) survient souvent pendant des périodes spécifiques de sensibilité accrue lorsque le stress, une infection virale ou une qualité de l'air réduite sont censés supprimer les défenses respiratoires. Le système immunitaire inné est rapidement réactif et largement protecteur et pourrait être une cible pour prévenir la BRD pendant ces périodes de sensibilité accrue. Cette étude a testé l'hypothèse selon laquelle la stimulation des réponses immunitaires innées pulmonaires par la délivrance d'aérosols d'un lysat de bactéries Escherichia coli et Staphylococcus aureus tuées protégerait les veaux contre la pneumonie à Mannheimia haemolytica. Dix veaux Holstein dont on a limité la contamination bactérienne et privés de colostrum ont été répartis au hasard pour recevoir soit un lysat bactérien en aérosol, soit une solution saline 24 heures avant une infection défi par M. haemolytica. Les effets de ce traitement sur les résultats cliniques, hématologiques, microbiologiques et pathologiques ont été évalués. Comparativement aux témoins, les veaux traités au lysat présentaient des concentrations sériques d'haptoglobine et de leucocytes et de neutrophiles sanguins plus faibles après la provocation par M. haemolytica. Il n'y avait aucune différence dans la température, les fréquences cardiaques et respiratoires, les scores cliniques, les lésions échographiques ou le nombre de M. haemolytica dans la cavité nasale ou les poumons. Ainsi, le traitement avec un lysat bactérien avant la provocation par M. haemolytica a semblé améliorer les réactions précoces de l'inflammation mais n'a pas fourni une protection suffisante pour modifier substantiellement l'évolution de la maladie.(Traduit par Docteur Serge Messier).

Development of an aerosolized Mannheimia haemolytica experimental pneumonia model in clean-catch colostrum-deprived calves.

Mannheimia haemolytica is an important cause of bovine respiratory disease (BRD). BRD is usually a multifactorial disease with host factors and viral infections influencing pathogenesis. Previous studies that have attempted to experimentally induce pneumonia using aerosolized M. haemolytica alone have produced inconsistent results, yet an aerosol model would be useful to study the details of early infection and to investigate the role of innate defences in pathogenesis. The objective of these studies was to develop and characterize an aerosolized M. haemolytica disease model. In an initial study, conventionally raised calves with higher levels of antibody against M. haemolytica leukotoxin developed acute respiratory distress and diffuse alveolar damage, but did not develop bronchopneumonia, following challenge with M. haemolytica serotype 1. Clean-catch colostrum-deprived calves challenged with 1 × 1010 colony forming units of M. haemolytica serotype 1 consistently developed bronchopneumonia, with elevations in rectal temperature, serum haptoglobin, plasma fibrinogen, and blood neutrophils. Mannheimia haemolytica serotype 1 was consistently isolated from the nasal cavities and lungs of challenged calves. Despite distribution of aerosol and isolation of M. haemolytica in all lung lobes, gross lesions were mainly observed in the cranioventral area of lung. Gross and histologic lesions included neutrophilic bronchopneumonia and fibrinous pleuritis, with oat cells (necrotic neutrophils with streaming nuclei), and areas of coagulative necrosis, which are similar to lesions in naturally occurring BRD. Thus, challenge with M. haemolytica serotype 1 and use of clean-catch colostrum-deprived calves with low or absent antibody titres allowed development of an effective aerosol challenge model that induced typical clinical disease and lesions.

Resilience to infection by Mycobacterium avium subspecies paratuberculosis following direct intestinal inoculation in calves.

Mycobacterium avium subspecies paratuberculosis (Map) is the cause of Johne's disease, a chronic enteritis of cattle. A significant knowledge gap is how persistence of Map within the intestinal tract after infection contributes to progression of disease. To address this, we exposed calves to Map by direct ileocecal Peyer's patch injection. Our objective was to characterize the persistence of Map in tissues, associated intestinal lesions, fecal Map shedding, and serum antibody responses, through the first 28-weeks post-inoculation (wpi). Previous work using this model showed 100% rate of Map infection in intestine and lymph node by 12 wpi. We hypothesized that direct inoculation of Map into the distal small intestine would induce intestinal Map infection with local persistence and progression towards clinical disease. However, our data show decreased persistence of Map in the distal small intestine and draining lymph nodes. We identified Map in multiple sections of distal ileum and draining lymph node of all calves at 4 and 12 wpi, but then we observed reduced Map in distal ileum at 20 wpi, and by 28 wpi we found that 50% of animals had no detectable Map in intestine or the lymph node. This provides evidence of resilience to Map infection following direct intestinal Map inoculation. Further work examining the immune responses and host-pathogen interactions associated with this infection model are needed to help elicit the mechanisms underlying resilience to Map infection.

Seroconversion of sheep experimentally infected with enzootic nasal tumor virus.

BACKGROUND: Enzootic nasal tumor virus (ENTV-1) is an exogenous betaretrovirus of sheep that transforms epithelial cells lining the ethmoid turbinates leading to a disease called enzootic nasal adenocarcinoma (ENA). A unique feature of ENA is the apparent absence of a specific humoral immune response to the virus, despite the highly productive infection in nasal tumors. The sheep genome contains approximately 27 copies of endogenous ovine betaretroviral sequences (enJSRVs) and expression of enJSRVs in the ovine placenta and uterine endometrium throughout gestation is thought to induce immunological tolerance to exogenous ovine betaretroviruses, a factor that may influence the likelihood of exogenous ENTV infection and disease outcome. Nevertheless, we recently demonstrated the presence of neutralizing antibodies directed against the ENTV-1 envelope glycoprotein in sheep naturally exposed to ENTV-1. FINDINGS: Here, we employed an ENTV-1 envelope glycoprotein surface subunit specific ELISA and a virus neutralization assay to monitor serum antibody responses to ENTV-1 in a group of lambs experimentally infected with ENTV-1 virus containing filtered ENA tumor homogenate. Seroconversion and development of neutralizing antibodies was detected in one of six experimentally infected lambs. CONCLUSIONS: Our results demonstrate that sheep can respond immunologically and seroconvert following ENTV-1 infection suggesting that anti-viral immune responses may play a role in the development of ENA.

Development of an ante-mortem diagnostic test for enzootic nasal tumor virus and detection of neutralizing antibodies in host serum.

Enzootic nasal adenocarcinoma (ENA) is a contagious neoplasm of the nasal mucosa of sheep and goats and is associated with enzootic nasal tumour virus (ENTV). As ENA is a common disease in North America and there are no vaccines against ENTV-1, diagnostic tests that can identify infected animals and assist with eradication and disease surveillance efforts are greatly needed. In this study, we endeavoured to develop a novel, non-invasive diagnostic tool that could be used not only to validate clinical signs of ENA but also to detect ENTV-1 infection prior to the onset of disease signs (i.e. pre-clinical diagnosis). Cytology, serology and reverse transcription (RT)-PCR-based diagnostic methods were investigated. Although the cytology-based assay was able to detect ENTV-1 infection in some animals, it had poor sensitivity and specificity and thus was not developed further as an ante-mortem diagnostic method. Three different assays, including ELISA, Western blotting and virus neutralization, were developed to detect the presence of ENTV-1-specific antibodies in sheep serum. Whilst a surprisingly large number of sheep mounted an antibody-mediated immune response against ENTV-1, and in some cases neutralizing, correlation with disease status was poor. In contrast, RT-PCR on RNA extracted from nasal swabs reliably detected exogenous ENTV-1 sequences, did not amplify endogenous ovine betaretroviral sequences, demonstrated high concordance with immunohistochemical staining for ENTV-1 envelope protein, and had perfect sensitivity and specificity. This report describes a practical and highly specific RT-PCR technique for the detection of clinical and pre-clinical ENA that may prove beneficial in future control or eradication programmes.