Bovine herpesvirus-5 infection in a rabbit experimental model: Immunohistochemical study of the cellular response in the CNS

Since little information is available regarding cellular antigen mapping and the involvement of non-neuronal cells in the pathogenesis of bovine herpesvirus type 5 (BHV-5) infection, it were determined the BHV-5 distribution, the astrocytic reactivity, the involvement of lymphocytes and the presence of matrix metalloproteinase (MMP)-9 in the brain of rabbits experimentally infected with BHV-5. Twelve New Zealand rabbits that were seronegative for BHV-5 were used for virus inoculation, and five rabbits were used as mock-infected controls. The rabbits were kept in separate areas and were inoculated intranasally with 500 μl of virus suspension (EVI 88 Brazilian isolate) into each nostril (virus titer, 107.5 TCID50). Control rabbits were inoculated with the same volume of minimum essential medium. Five days before virus inoculation, the rabbits were submitted to daily administration of dexamethasone. After virus inoculation, the rabbits were monitored clinically on a daily basis. Seven rabbits showed respiratory symptoms and four animals exhibited neurological symptoms. Tissue sections were collected for histological examination and immunohistochemistry to examine BHV-5 antigens, astrocytes, T and B lymphocytes and MMP-9. By means of immunohistochemical and PCR methods, BHV-5 was detected in the entire brain of the animals which presented with neurological symptoms, especially in the trigeminal ganglion and cerebral cortices. Furthermore, BHV-5 antigens were detected in neurons and/or other non-neural cells. In addition to the neurons, most infiltrating CD3 T lymphocytes observed in these areas were positive for MMP-9 and also for BHV-5 antigen. These infected cells might contribute to the spread of the virus to the rabbit brain along the trigeminal ganglia and olfactory nerve pathways. © 2013 Elsevier Ltd.

Novel microbicidal agents derived from Naja atra cardiotoxin 1 (CTX-1)

During my PhD course, I focused my research on antimicrobial peptides (AMPs), in particular on the aspects of their computational design and development. This work led to the development of a new family of AMPs that I designed, starting from the amino acid sequence of a snake venom toxin, the cardiotoxin 1 (CTX-1) of Naja atra. Naja atra atra cardiotoxin 1, produced by Chinese cobra snakes belonging to Elapidae family, is included in the three-finger toxin family and exerts high cytotoxicity and antimicrobial activity too. This toxin family is characterized by specific folding of three beta-sheet loops (“fingers”) extending from the central core and by four conserved disulfide bridges. Using as template the first loop of this toxin, different sequences of 20 amino acids linear cationic peptides have been designed in order to avoid toxic effects but to maintain and strengthen the antimicrobial activity. As a result, the sequence NCP-0 (Naja Cardiotoxin Peptide-0) was designed as ancestor and subsequently other 4 variant sequences of NCP0 were developed. These variant sequences have shown microbicidal activity towards a panel of reference strains of Gram-positive and Gram-negative bacteria, fungi and an enveloped virus. In particular, the sequence designed as NCP-3 (Naja Cardiotoxin Peptide-3) and its variants NCP-3a and NCP-3b have shown the best antimicrobial activity together with low cytotoxicity against eukaryotic cells and low hemolytic activity. Bactericidal activity has been demonstrated by minimum bactericidal concentration (MBC) assay at values below 10 μg/ml for Pseudomonas aeruginosa ATCC 27853, Acinetobacter baumannii ( clinical isolates), Moraxella catharralis ATCC 25238, MRSA ATCC 43400, while towards Staphylococcus aureus ATCC 25923, Enterococcus hirae ATCC 10541 and Streptococcus agalactiae ATCC 13813 the bactericidal activity was demonstrated even below 1.6 μg/ml concentration. This potent antimicrobial activity was confirmed even for unicellular fungi Candida albicans, Candida glabrata and Malassezia pachydermatis (MBC 32.26-6.4 μg/ml), and also against the fast-growing mycobacteria Mycobacterium smegmatis DSMZ 43756 and Mycobacterium fortuitum DSMZ 46621 (MBC 100 μg/ml). Moreover, NCP-3 has shown a virucidal activity on the enveloped virus Bovine Herpesvirus 1 (BoHV1) belonging to herpesviridae family. The bactericidal activity is maintained in a high salt concentration (125 and 250 mM NaCl) medium and PB +20% Mueller Hinton Medium for E. coli, MRSA and Pseudomonas aeruginosa reference strains. Considering these in vitro obtained data, we propose NCP-3 and its variants NCP-3a and NCP-3b as promising antimicrobial candidates. For this reason, the whole novel AMPs family has been protected by a national patent (n°102015000015951).

Associations between exposure to viruses and bovine respiratory disease in Australian feedlot cattle

Bovine respiratory disease (BRD) is the most important cause of clinical disease and death in feedlot cattle. Respiratory viral infections are key components in predisposing cattle to the development of this disease. To quantify the contribution of four viruses commonly associated with BRD, a case-control study was conducted nested within the National Bovine Respiratory Disease Initiative project population in Australian feedlot cattle. Effects of exposure to Bovine viral diarrhoea virus 1 (BVDV-1), Bovine herpesvirus 1 (BoHV-1), Bovine respiratory syncytial virus (BRSV) and Bovine parainfluenza virus 3 (BPIV-3), and to combinations of these viruses, were investigated. Based on weighted seroprevalences at induction (when animals were enrolled and initial samples collected), the percentages of the project population estimated to be seropositive were 24% for BoHV-1, 69% for BVDV-1, 89% for BRSV and 91% for BPIV-3. For each of the four viruses, seropositivity at induction was associated with reduced risk of BRD (OR: 0.6–0.9), and seroincrease from induction to second blood sampling (35–60 days after induction) was associated with increased risk of BRD (OR: 1.3–1.5). Compared to animals that were seropositive for all four viruses at induction, animals were at progressively increased risk with increasing number of viruses for which they were seronegative; those seronegative for all four viruses were at greatest risk (OR: 2.4). Animals that seroincreased for one or more viruses from induction to second blood sampling were at increased risk (OR: 1.4–2.1) of BRD compared to animals that did not seroincrease for any viruses. Collectively these results confirm that prior exposure to these viruses is protective while exposure at or after feedlot entry increases the risk of development of BRD in feedlots. However, the modest increases in risk associated with seroincrease for each virus separately, and the progressive increases in risk with multiple viral exposures highlights the importance of concurrent infections in the aetiology of the BRD complex. These findings indicate that, while efficacious vaccines could aid in the control of BRD, vaccination against one of these viruses would not have large effects on population BRD incidence but vaccination against multiple viruses would be expected to result in greater reductions in incidence. The findings also confirm the multifactorial nature of BRD development, and indicate that multifaceted approaches in addition to efficacious vaccines against viruses will be required for substantial reductions in BRD incidence.

Equine herpesvirus infections in yearlings in South-East Queensland.

Twelve nasal swabs were collected from yearling horses with respiratory distress and tested for equid herpesvirus 1 (EHV-1) and equid herpesvirus 4 (EHV-4) by real-time PCR targeting the glycoprotein B gene. All samples were negative for EHV-1; however, 3 were positive for EHV-4. When these samples were tested for EHV-2 and EHV-5 by PCR, all samples were negative for EHV-2 and 11 were positive for EHV-5. All three samples that were positive for EHV-4 were also positive for EHV-5. These three samples gave a limited CPE in ED cells reminiscent of EHV-4 CPE. EHV-4 CPE was obvious after 3 days and was characterised by syncytia. None of the samples produced cytopathic effect (CPE) on African green monkey kidney (Vero) cells or hamster kidney (BSR) cells. Four of the samples, which were positive in the EHV-5 PCR, produced CPE on rabbit kidney (RK13) cells and equine dermis (ED) cells. EHV-5 CPE on both cell lines was slow and was apparent after four 7-day passages. On RK13 cells, the CPE was characteristic of equid herpesvirus, with the formation of syncytia. However, in ED cells, the CPE was characterised by ring-shaped syncytia. For the first time, a case of equine respiratory disease involving dual infection with EHV-4 and EHV-5 has been reported in Queensland (Australia). This was shown by simultaneously isolating EHV-4 and EHV-5 from clinical samples. EHV5 was recovered from all samples except one, suggesting that EHV5 was more prevalent in young horses than EHV2.

Studies on interaction of colloidal Ag nanoparticles with Bovine Serum Albumin (BSA)

Biofunctionalization of noble metal nanoparticles like Ag, Au is essential to obtain biocompatibility for specific biomedical applications. Silver nanciparticles are being increasingly used in bio-sensing applications owing to excellent optoelectronic properties. Among the serum albumins, the most abundant proteins in plasma, a wide range of physiological functions of Bovine Serum Albumin (BSA) has made it a model system for biofunctionalization. In absence of adequate prior reports, this study aims to investigate the interaction between silver nanoparticles and BSA. The interaction of BSA [0.05-0.85% concentrations] with Ag nanoparticles [50 ppm concentration] in aqueous dispersion was Studied through UV-vis spectral changes, morphological and surface structural changes. At pH 7, which is More than the isoelectric point of BSA, a decrease in absorbance at plasmon peak of uninteracted nanciparticles (425 mn) was noted till 0.45% BSA, beyond that a blue shift towards 410 urn was observed. The blue shift may be attributed to enhanced electron density on the particle surfaces. Increasing pH to 12 enhanced the blue shift further to 400 rim. The conformational changes in BSA at alkaline pH ranges and consequent hydrophobic interactions also played an important role. The equilibrium adsorption data fitted better to Freundlich isotherm compared to Langmuir Curve. The X-ray diffraction study revealed complete coverage of Ag nanoparticles by BSA. The scanning electron microscopic study of the interacted nanoparticles was also carried Out to decipher morphological changes. This study established that tailoring the concentration of BSA and pH of the interaction it was possible to reduce aggregation of nanoparticles. Biofunctionalized Ag nanoparticles with reduced aggregation will be more amenable towards bio-sensing applications. (C) 2009 Elsevier B.V. All rights reserved.

AgriSense: Multichannel disposable sensors for animal health disease diagnostics

Rapid and sensitive detection of viral infections associated with Bovine Respiratory Disease (BRD) in live animals is recognized as key to minimizing the impact of this disease. ELISA-based testing is limited as it typically relies on the detection of a single viral antibody subtype within an individual test sample and testing is relatively slow and expensive. We have recently initiated a new project entitled AgriSense to develop a novel low-cost and label-free, integrated bimodal electronic biosensor system for BRD. The biosensor system will consist of an integrated multichannel thin-film-transistor biosensor and an electrochemical impedance spectroscopy biosensor, interfaced with PDMS-based microfluidic sample delivery channels. By using both sensors in tandem, nonspecific binding biomolecules must have the same mass to charge ratio as the target analyte to elicit equivalent responses from both sensors. The system will target simultaneous multiplexed sensing of the four primary viral agents involved in the development of BRD: bovine herpesvirus-1 (BHV-1), bovine parainfluenza virus-3 (BPIV-3), bovine respiratory syncytial virus (BRSV), and bovine viral diarrhea (BVD). Optimized experimental conditions derived through model antigen-antibody studies will be applied to the detection of serological markers of BRD-related infections based on IgG interaction with a panel of sensor-immobilized viral proteins. This rapid, “cowside” multiplex sensor capability presents a major step forward in disease diagnosis, helping to ensure the integrity of the agri-food supply chain by reducing the risk of disease spread during animal movement and transport.

Identification of a novel herpesvirus associated with cutaneous ulcers in a fisher (Martes pennanti).

On December 8, 2008, a male fisher (Martes pennanti) housed in a quarantine enclosure at the St-Félicien Zoo was found dead with multiple skin ulcers on the muzzle and plantar pads. At necropsy, no major findings were found, and a specific cause of death was not determined microscopically. However, at the borders of ulcerated sites, there were increased numbers of koilocytes, with perinuclear vacuolation and nuclear enlargement. A pan-herpesvirus nested polymerase chain reaction (PCR) assay was conducted, and an expected PCR product of 230 nucleotides was obtained within tissues collected from around the skin ulcers. Other tissues, including intestines and pool of lung, liver, and kidney, tested negative. The obtained PCR amplicon was sequenced and was highly related to the partial viral DNA polymerase (DPOL) gene of Mustelid herpesvirus 1. Virus isolation was negative, and no virion was detected by electron microscopy. The pathogenic potential of this novel herpesvirus and its role in the death of the fisher are unknown.

Effects of bovine somatotropin injection on serum concentrations of progesterone in non-lactating dairy cows

The objective of this experiment was to evaluate the effects of bovine somatotropin administration on serum concentrations of glucose, insulin, NEFA, IGF-I, and progesterone (P4) in ovariectomized non-lactating dairy cows receiving exogenous P4, as a model to estimate treatment effects on hepatic P4 degradation. Ten non-lactating, non-pregnant, and ovariectomized Gir×Holstein cows were assigned to the experiment (d -14 to 27). On d 0, cows were ranked by BW and BCS, and randomly assigned to one of two treatments: (1) bovine somatotropin (BST; n=5) or (2) saline control (control; n=5). Cows assigned to the BST treatment were administered s.c. injections containing 500. mg of sometribove zinc on d 0, 9, and 18 of the experiment, whereas control cows concurrently received a 10-mL s.c. injection of 0.9% saline. On d -2, cows were inserted with an intravaginal releasing device containing 1.9. g of P4, which remained in the cows until the end the experiment (d 27). Cow BW and BCS were assessed on d -14, 0, and 27. Blood samples were collected daily from d 0 to d 27, at 0 (immediately before), 1, and 2. h relative to concentrate feeding for determination of serum glucose, insulin, NEFA, P4, and IGF-I concentrations. Concentrations of glucose, NEFA, and insulin obtained prior to feeding (0. h) were used to determine pre-prandial revised quantitative insulin sensitivity check index (RQUICKI). No treatment effects were detected for BW (P=0.72) and BCS change (P=0.79) during the experiment. Beginning on d 2 of the experiment, BST cows had greater (P≤0.01) serum IGF-I concentrations compared with control cohorts (treatment×day interaction; P<0.01). Cows receiving BST had greater (P≤0.05) insulin concentrations compared with control cohorts from d 8 to d 11, d 16 and 17, as well as from d 19 to d 21 of the experiment (treatment×day interaction; P<0.01). Cows receiving BST had greater (P≤0.01) mean glucose and NEFA concentrations, as well as reduced (P<0.01) mean RQUICKI during the experiment compared with control cohorts. No treatment effects, however, were detected (P=0.73) for serum P4 concentrations. In conclusion, results from this experiment indicate that hepatic P4 catabolism is not directly regulated by circulating IGF-I, whereas BST administration decreases insulin sensitivity in non-lactating dairy cows in adequate nutritional status. © 2013 Elsevier B.V.

Detecção herpesvírus bovino tipo 5 em cortes histológicos e fragmentos de encéfalo congelado pela reação em cadeia de polimerase

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Parâmetros clínicos, alterações sorológicas e qualidade do sêmen de touros jovens infectados experimentalmente com herpesvírus bovino tipo 1

Pós-graduação em Medicina Veterinária - FCAV

Prevalência dos principais vírus respiratórios em bovinos da raça holandesa, no estado do Paraná

Pós-graduação em Medicina Veterinária - FMVZ

Epizootiologic Investigations of Selected Abortive Agents in Free-Ranging Alpine Ibex (Capra Ibex Ibex) in Switzerland

In the early 2000s, several colonies of Alpine ibex (Capra ibex ibex) in Switzerland ceased growing or began to decrease. Reproductive problems clue to infections with abortive agents might have negatively affected recruitment. We assessed the presence of selected agents of abortion in Alpine ibex by serologic, molecular, and culture techniques and evaluated whether infection with these agents might have affected population densities. Blood and fecal samples were collected from 651 ibex in 14 colonies throughout the Swiss Alps between 2006 and 2008. All samples were negative for Salmonella. spp., Neospora caninum, and Bovine Herpesvirus-1. Antibodies to Coxiella burnetii, Leptospira spp., Chlamydophila abortus, Toxoplasma gondii, and Bovine Viral Diarrhea virus were detected in at least one ibex. Positive serologic results for Brucella spp. likely were false. Overall, 73 samples (11.2%) were antibody-positive for at least one abortive agent. Prevalence was highest for Leptospira spp. (7.9%, 95% CI=5.0-11.7). The low prevalences and the absence of significant differences between colonies with opposite population trends suggest these pathogens do not play a significant role in the population dynamics of Swiss ibex. Alpine ibex do not seem to be a reservoir for these abortive agents or an important source of infection for domestic livestock in Switzerland. Finally, although interactions on summer pastures occur frequently, spillover from infected livestock to free-ranging ibex apparently is uncommon.

Tissue neogenesis and STRO-1 expression in immature and mature articular cartilage

This study determined the potential for neotissue formation and the role of STRO-1+ cells in immature versus mature articular cartilage. Cartilage explants from immature and mature bovine knee joints were cultured for up to 12 weeks and stained with safranin-O, for type II collagen and STRO-1. Bovine chondrocyte pellet cultures and murine knee joints at the age of 2 weeks and 3 months, and surgically injured cartilage, were analyzed for changes in STRO-1 expression patterns. Results show that immature explants contained more STRO-1+ cells than mature explants. After 8 weeks in culture, immature explants showed STRO-1+ cell proliferation and newly formed tissue, which contained glycosaminoglycan and type II collagen. Mature cartilage explants showed only minimal cell expansion and neotissue formation. Pellet cultures with chondrocytes from immature cartilage showed increased glycosaminoglycan synthesis and STRO-1+ staining, as compared to pellets with mature chondrocytes. The frequency of STRO-1+ cells in murine knee joints significantly declined with joint maturation. Following surgical injury, immature explants had higher potential for tissue repair than mature explants. In conclusion, these findings suggest that the high percentage of STRO-1+ cells in immature cartilage changes with joint maturation. STRO-1+ cells have the potential to form new cartilage spontaneously and after tissue injury. (c) 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

Crystallization and preliminary X-ray analysis of a Kunitz-type inhibitor, textilinin-1 from Pseudonaja textilis textilis

Textilinin-1 (Txln-1), a Kunitz-type serine protease inhibitor, is a 59-amino-acid polypeptide isolated from the venom of the Australian Common Brown snake Pseudonaja textilis textilis. This molecule has been suggested as an alternative to aprotinin, also a Kunitz-type serine protease inhibitor, for use as an anti-bleeding agent in surgical procedures. Txln-1 shares only 47% amino-acid identity to aprotinin; however, six cysteine residues in the two peptides are in conserved locations. It is therefore expected that the overall fold of these molecules is similar but that they have contrasting surface features. Here, the crystallization of recombinant textilinin-1 (rTxln-1) as the free molecule and in complex with bovine trypsin (229 amino acids) is reported. Two organic solvents, phenol and 1,4-butanediol, were used as additives to facilitate the crystallization of free rTxln-1. Crystals of the rTxln-1-bovine trypsin complex diffracted to 2.0 angstrom resolution, while crystals of free rTxln-1 diffracted to 1.63 angstrom resolution.

Discovery and characterization of IGFBP-mediated endocytosis in the human retinal pigment epithelial cell line ARPE-19

Insulin-like growth factor binding proteins (IGFBPs) are prime regulators of IGF-action in numerous cell types including the retinal pigment epithelium (RPE). The RPE performs several functions essential for vision, including growth factor secretion and waste removal via a phagocytic process mediated in part by vitronectin (Vn). In the course of studying the effects of IGFBPs on IGF-mediated VEGF secretion and Vn-mediated phagocytosis in the RPE cell line ARPE-19, we have discovered that these cells avidly ingest synthetic microspheres (2.0 μm diameter) coated with IGFBPs. Given the novelty of this finding and the established role for endocytosis in mediating IGFBP actions in other cell types, we have explored the potential role of candidate cell surface receptors. Moreover, we have examined the role of key IGFBP structural motifs, by comparing responses to three members of the IGFBP family (IGFBP-3, IGFBP-4 and IGFBP-5) which display overlapping variations in primary structure and glycosylation status. Coating of microspheres (FluoSpheres®, sulfate modified polystyrene filled with a fluorophore) was conducted at 37 °C for 1 h using 20 μg/mL of test protein, followed by extensive washing. Binding of proteins was confirmed using a microBCA assay. The negative control consisted of microspheres treated with 0.1% bovine serum albumin (BSA), and all test samples were post-treated with BSA in an effort to coat any remaining free protein binding sites, which might otherwise encourage non-specific interactions with the cell surface. Serum-starved cultures of ARPE-19 cells were incubated with microspheres for 24 h, using a ratio of approximately 100 microspheres per cell. Uptake of microspheres was quantified using a fluorometer and was confirmed visually by confocal fluorescence microscopy. The ARPE-19 cells displayed little affinity for BSA-treated microspheres, but avidly ingested large quantities of those pre-treated with Vn (ANOVA; p < 0.001). Strong responses were also observed towards recombinant formulations of non-glycosylated IGFBP-3, glycosylated IGFBP-3 and glycosylated IGFBP-5 (all p < 0.001), while glycosylated IGFBP-4 induced a relatively minor response (p < 0.05). The response to IGFBP-3 was unaffected in the presence of excess soluble IGFBP-3, IGF-I or Vn. Likewise, soluble IGFBP-3 did not induce uptake of BSA-treated microspheres. Antibodies to either the transferrin receptor or type 1 IGF-receptor displayed slight inhibitory effects on responses to IGFBPs and Vn. Heparin abolished responses to Vn, IGFBP-5 and non-glycosylated IGFBP-3, but only partially inhibited the response to glycosylated IGFBP-3. Our results demonstrate for the first time IGFBP-mediated endocytosis in ARPE-19 cells and suggest roles for the IGFBP-heparin-binding domain and glycosylation status. These findings have important implications for understanding the mechanisms of IGFBP actions on the RPE, and in particular suggest a role for IGFBP-endocytosis.