Characterization of BoHV-4 ORF45.

Bovine herpesvirus 4 (BoHV-4) is a Gammaherpesvirus belonging to the Rhadinovirus genus. The bovine is BoHV-4's natural host, and the African buffalo is BoHV-4's natural reservoir. In any case, BoHV-4 infection is not associated with a specific disease. Genome structure and genes are well-conserved in Gammaherpesvirus, and the orf 45 gene and its product, ORF45, are one of those. BoHV-4 ORF45 has been suggested to be a tegument protein; however, its structure and function have not yet been experimentally characterized. The present study shows that BoHV-4 ORF45, despite its poor homology with other characterized Rhadinovirus ORF45s, is structurally related to Kaposi's sarcoma-associated herpesvirus (KSHV), is a phosphoprotein, and localizes in the host cell nuclei. Through the generation of an ORF45-null mutant BoHV-4 and its pararevertant, it was possible to demonstrate that ORF45 is essential for BoHV-4 lytic replication and is associated with the viral particles, as for the other characterized Rhadinovirus ORF45s. Finally, the impact of BoHV-4 ORF45 on cellular transcriptome was investigated, an aspect poorly explored or not at all for other Gammaherpesvirus. Many cellular transcriptional pathways were found to be altered, mainly those involving p90 ribosomal S6 kinase (RSK) and signal-regulated kinase (ERK) complex (RSK/ERK). It was concluded that BoHV-4 ORF45 has similar characteristics to those of KSHV ORF45, and its unique and incisive impact on the cell transcriptome paves the way for further investigations.

Survey on Carbapenem-Resistant Bacteria in Pigs at Slaughter and Comparison with Human Clinical Isolates in Italy.

This study is focused on resistance to carbapenems and third-generation cephalosporins in Gram-negative microorganisms isolated from swine, whose transmission to humans via pork consumption cannot be excluded. In addition, the common carriage of carbapenem-resistant (CR) bacteria between humans and pigs was evaluated. Sampling involved 300 faecal samples collected from slaughtered pigs and 300 urine samples collected from 187 hospitalised patients in Parma Province (Italy). In swine, MIC testing confirmed resistance to meropenem for isolates of Pseudomonas aeruginosa and Pseudomonas oryzihabitans and resistance to cefotaxime and ceftazidime for Escherichia coli, Ewingella americana, Enterobacter agglomerans, and Citrobacter freundii. For Acinetobacter lwoffii, Aeromonas hydrofila, Burkolderia cepacia, Corynebacterium indologenes, Flavobacterium odoratum, and Stenotrophomonas maltophilia, no EUCAST MIC breakpoints were available. However, ESBL genes (blaCTXM-1, blaCTX-M-2, blaTEM-1, and blaSHV) and AmpC genes (blaCIT, blaACC, and blaEBC) were found in 38 and 16 isolates, respectively. P. aeruginosa was the only CR species shared by pigs (4/300 pigs; 1.3%) and patients (2/187; 1.1%). P. aeruginosa ST938 carrying blaPAO and blaOXA396 was detected in one pig as well as an 83-year-old patient. Although no direct epidemiological link was demonstrable, SNP calling and cgMLST showed a genetic relationship of the isolates (86 SNPs and 661 allele difference), thus suggesting possible circulation of CR bacteria between swine and humans.

Antimicrobial activity of a standardized medical honey on bacterial isolates from infected skin lesions of non-traditional companion animals.

In recent years, due to the growing phenomenon of antimicrobial resistance, the search for alternative strategies to antibiotic treatments is increasing and a considerable interest for the use of medical honey in clinical practice has emerged. Honey has been used for the treatment of skin lesions, in both humans and animals. However, knowledge concerning the use of medical honey in non­traditional companion animals is scarce. The aim of this study was to assess the antibacterial activity of a standardized medical honey (Revamil, BFactory) against bacterial strains isolated from skin lesions of non­traditional companion animals. The minimum bactericidal concentration (MBC) of Revamil honey against seventeen clinical isolates and three reference strains was established.The medical honey showed antimicrobial activity against both Gram­positive and Gram­negative bacteria. Growth was inhibited for all the strains at concentrations of medical honey ranging from 10 to 40%. Pseudomonas oryzihabitans and Alcaligenes faecalis showed the lowest MBC (10%). The reference strain Staphylococcus aureus ATCC25923 showed a higher sensitivity to 20% honey compare to the corresponding clinical isolate (P = 0.001). The observed results suggest that Revamil could represent an effective therapeutic aid, useful for the reduction of antibiotic use, in case of pathological skin infections in non­traditional companion animals.

Leptospira Seroprevalence in Colombian Dairy Herds.

Leptospirosis in cattle has important economic effects on the infected farms. Moreover, livestock farming is considered a major occupational risk factor for the transmission of Leptospira infection to humans. A survey was performed to determine the overall and within-herd seroprevalence and mapping of different Leptospira serovars in dairy cattle from farms located in some municipalities of the Colombian department of Boyacá. Nine hundred and fifty-nine animals, from 20 unvaccinated and one vaccinated herd, were included in the study. Anti-Leptospira serum antibodies were detected by the microscopic agglutination test (MAT). Only one herd was seronegative. Overall seroprevalence to at least one serovar of Leptospira was 24.1% for unvaccinated animals and 62.3% for animals from the vaccinated herd. A very high within-herd seroprevalence (>60%) was present in 20% of the unvaccinated herds. The presence in the vaccinated herd of 20/398 animals showing high titers, between 1000 and 4000, to at least one serovar of Leptospira suggest that some animals could have been infected. Moreover, due to the presence of seronegative animals, a failure of vaccination immunity or the presence of unvaccinated animals in the vaccinated herd cannot be excluded. In all farms, domestic animals other than cattle were present. Considering the farming practices occurring on dairy farms in the study area, higher hygienic standards and stricter biosecurity measures are suggested.

Wild Micromammals as Bioindicators of Antibiotic Resistance in Ecopathology in Northern Italy.

Antimicrobial resistance (AMR) is an increasing threat to human health and an important issue also in the natural environment. For this study, an ecopathological approach was applied to the monitoring of the antimicrobial resistance in the province of Parma, Northern Italy. Fourteen monitoring sites and seventy-four faecal samples from four species of wild micromammals (Apodemus sylvaticus, Microtus savii, Mus domesticus and Suncus etruscus) were collected. Samples were subjected to bacteriological examination and antimicrobial susceptibility testing. Antibiotics belonging to 13 different antibiotic classes were tested. Collected data showed a prevalence of multi-drug resistant (MDR) strains of 55.13% and significant differences in the prevalence of MDR strains among the different micromammal species, while sex, age and anthropization level did not significantly affected MDR strains prevalence. Moreover, a high prevalence of bacterial strains resistant to colistin (95%), gentamicin (87%) and amikacin (83%) was observed. To our knowledge, this is the first report on antibiotic resistance in wild micromammals in the province of Parma.

Leptospira Seroprevalence in Bardigiano Horses in Northern Italy.

A cross-sectional study was carried out in Bardigiano horses in the Province of Parma, Northern Italy, to assess the seroprevalence of Leptospira spp. and to investigate risk factors associated with the infection. A representative sample of 134 horses from 43 farms was selected by stratified systematic randomization. Blood sera were examined by MAT for the presence of antibodies against seven Leptospira serovars. Ninety animals (67.2%; 95% Confidence Interval 63.2-71.1) and 41 farms (95.3%; 95% CI 92.2-98.5%) were found positive to at least one of the serovars. The most frequently detected reactions were against serovar Bratislava (41.8%), followed by Canicola (36.6%), Tarassovi (28.4%), Copenhageni (17.9%), Pomona (10.4%) and Hardjo (2.2%). None of the sera reacted against serovar Grippothyphosa. Forty-eight horses (53.3% of the seropositives) were positive for more than one serovar and 21 (15.7% of the seropositives) had serum titres ≥ 1000. Bratislava was the serovar providing the highest antibody titres. Prevalence was significantly higher between adult horses and in farms lacking rodent control (p = 0.006 and p = 0.025, respectively). No significant gender or housing-related difference in seroprevalence was found. The anamnestic data suggest that the infection in Bardigiano horses is subclinical in most of the cases. The high seroprevalence indicates that Bardigiano horses living in the investigated area are at high risk of exposure and infection by Leptospira spp.

The prevalence of Pseudomonas aeruginosa and multidrug resistant Pseudomonas aeruginosa in healthy captive ophidian.

BACKGROUND: Snakes are globally considered as pet animals, and millions of ophidians are bred in captivity. Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterium that can act as an opportunistic pathogen of man and animals and is frequently present in the oral and cloacal microbiota of healthy ophidians. It can cause severe clinical diseases and often shows antibiotic resistance. The aim of this study was to evaluate the prevalence and antibiotic resistance profiles of P. aeruginosa isolated from the cloacal microbiota of a large population sample of healthy captive ophidians and to evaluate the statistical associations with farming conditions. METHODS: A total of 419 cloacal swabs were collected from snakes belonging to the Boidae (n = 45), Colubridae (n = 48) and Pythonidae (n = 326) families and inoculated onto complete culture media. Food, water and bedding samples were also analyzed. The antimicrobial susceptibility of P. aeruginosa isolates was evaluated through the Kirby-Bauer agar diffusion test. Statistical analyses were performed with the chi-square test. RESULTS: The prevalence of P. aeruginosa was 59.9%, and 35.5% of these strains were multidrug resistant (MDR). The prevalence of MDR P. aeruginosa was significantly higher in adult samples than in young samples, and widespread resistance to Cephalosporins, Polymyxins and Sulfonamides was observed. Statistically significant differences in the prevalence of P. aeruginosa were observed depending on the farm size and snake family. Feeding thawed prey was associated with a higher P. aeruginosa and MDR P. aeruginosa prevalence. Moreover, snakes fed home-raised prey had a significantly higher MDR P. aeruginosa prevalence than snakes fed commercially available feed. Less frequent terrarium cleaning was associated with a higher MDR P. aeruginosa prevalence. On the other hand, snake reproductive status was not significantly associated with P. aeruginosa or MDR P. aeruginosa prevalence. All food, water and bedding samples were negative for P. aeruginosa presence. DISCUSSION: The overall P. aeruginosa prevalence found in this study was lower than that found by other authors, but a high proportion of the isolates were MDR. This study highlighted the presence of constitutive (such as age and taxonomic family) and managerial (farm size, cleaning cycle frequency and food type) factors associated with P. aeruginosa and/or MDR P. aeruginosa prevalence. Good breeding management and proper antibiotic treatment of P. aeruginosa infections could help reduce the presence of P. aeruginosa and MDR P. aeruginosa in the gut microbiota of snakes and consequently reduce the risk to public health.

Capacity to Elicit Cytotoxic CD8 T Cell Activity Against Mycobacterium avium subsp. paratuberculosis Is Retained in a Vaccine Candidate 35 kDa Peptide Modified for Expression in Mammalian Cells.

Studies focused on development of an attenuated vaccine against Mycobacterium avium subsp. paratuberculosis (Map), the causative agent of paratuberculosis (Ptb) in cattle and other species, revealed that deletion of relA, a global gene regulator, abrogates the ability of Map to establish a persistent infection. In the absence of relA, cattle develop CD8 cytotoxic T cells (CTL) with the ability to kill intracellular bacteria. Analysis of the recall response to a relA mutant, Map/ΔrelA, with cells from a vaccinated steer demonstrated that a 35-kDa membrane peptide (MMP) is one of the targets of the response. This observation suggested that it might be possible to develop a peptide-based vaccine. As reported here, the gene encoding the hypothetical MMP ORF, MAP2121c, was modified for expression in mammalian cells as a first step in developing an expression cassette for incorporation into a mammalian expression vector. The modified sequence of MMP, tPA-MMP, was mutated to generate two additional sequences for the study, one with substitutions to replace five potential residues that could be glycosylated, tPA-MMP-5mut, and one with substitutions to replace the first two potential residues that could be glycosylated, tPA-MMP-2mut. The sequences were placed in an expression cassette to produce peptides for analysis. An ex vivo platform was used with flow cytometry and a bacterium viability assay to determine if modifications in the gene encoding MMP for expression in mammalian cells altered its capacity to elicit development of CD8 CTL, essential for its use in a peptide-based vaccine. Monocyte-depleted PBMC (mdPBMC) were stimulated with antigen-presenting cells (APC) pulsed with different MMP constructs. CD4 and CD8 T cells proliferated in response to stimulation with MMP (control) expressed in Escherichia coli (eMMP), tPA-MMP, and tPA-MMP-2mut. CD8 T cells retained the capacity to kill intracellular bacteria. The tPA-MMP-5mut failed to elicit a proliferative response and was not included in further studies. The data show that the expression cassettes containing MMP and MMP-2mut can be used to screen and select a mammalian expression vector for the development of an efficacious peptide-based vaccine against Ptb.

Bovine Herpesvirus-4-Based Vector Delivering Peste des Petits Ruminants Virus Hemagglutinin ORF Induces both Neutralizing Antibodies and Cytotoxic T Cell Responses.

Peste des Petits Ruminants Virus (PPRV) is an extremely infective morbillivirus that primarily affects goats and sheep. In underdeveloped countries where livestock are the main economical resource, PPRV causes considerable economic losses. Protective live attenuated vaccines are currently available but they induce antibody responses similar to those produced in PPRV naturally infected animals. Effective vaccines able to distinguish between vaccinated and naturally infected animals are required to PPRV control and eradication programs. Hemagglutinin (H) is a highly immunogenic PPRV envelope glycoprotein displaying both hemagglutinin and neuraminidase activities, playing a crucial role in virus attachment and penetration. In this study, a recombinant Bovine Herpesvirus-4 (BoHV-4)-based vector delivering an optimized PPRV-Hemagglutinin expression cassette, BoHV-4-A-PPRV-H-ΔTK, was assessed in immunocompetent C57BL/6 mice. BoHV-4-A-PPRV-H-ΔTK-immunization elicited both cellular and humoral immune responses with specific T cell, cytotoxic T lymphocyte, and sero-neutralizing antibody against PPRV. These data suggest recombinant BoHV-4-A-PPRV-H-ΔTK as an effective vaccine candidate to protect against PPRV herd infection and potentially applicable for eradication programs.

Novel Naja atra cardiotoxin 1 (CTX-1) derived antimicrobial peptides with broad spectrum activity.

Naja atra subsp. atra cardiotoxin 1 (CTX-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. Using as template mainly the tip and the subsequent ß-strand of the first "finger" of this toxin, different sequences of 20 amino acids linear peptides have been designed in order to avoid toxic effects but to maintain or even strengthen the partial antimicrobial activity already seen for the complete toxin. As a result, the sequence NCP-0 (Naja Cardiotoxin Peptide-0) was designed as ancestor and subsequently 4 other variant sequences of NCP-0 were developed. These synthesized variant sequences have shown microbicidal activity towards a panel of reference and field strains of Gram-positive and Gram-negative bacteria. The sequence named NCP-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 most of the tested bacterial strains. This potent antimicrobial activity was confirmed even for unicellular fungi Candida albicans, Candida glabrata and Malassezia pachydermatis (MBC 50-6.3 µg/ml), and against the fast-growing mycobacteria Mycobacterium smegmatis and Mycobacterium fortuitum. Moreover, NCP-3 has shown virucidal activity on Bovine Herpesvirus 1 (BoHV1) belonging to Herpesviridae family. The bactericidal activity is maintained even in a high salt concentration medium (125 and 250 mM NaCl) and phosphate buffer with 20% Mueller Hinton (MH) medium against E. coli, methicillin resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa reference strains. Considering these in vitro obtained data, the search for active sequences within proteins presenting an intrinsic microbicidal activity could provide a new way for discovering a large number of novel and promising antimicrobial peptides families.

Induction of Antihuman C-C Chemokine Receptor Type 5 Antibodies by a Bovine Herpesvirus Type-4 Based Vector.

Bovine herpesvirus 4 (BoHV-4) is a promising vector for the delivery and intracellular expression of recombinant antigens and can thus be considered as a new prototype vaccine formulation system. An interesting, and actively pursued, antigen in the context of human immunodeficiency virus (HIV) infection prophylaxis (and therapy) is the C-C chemokine receptor type 5 (CCR5) co-receptor, whose blockage by specific antibodies has been shown to inhibit both viral entry and cell-to-cell transmission of the virus. Building on our previous work on the BoHV-4 vector system, we have engineered and tested a replication-competent derivative of BoHV-4 (BoHV-4-CMV-hCCR5ΔTK) bearing a human CCR5 (hCCR5) expression cassette. We show here that CCR5 is indeed expressed at high levels in multiple types of BoHV-4-CMV-hCCR5ΔTK-infected cells. More importantly, two intravenous inoculations of CCR5-expressing BoHV-4 virions into rabbits led to the production of anti-CCR5 antibodies capable of reacting with the CCR5 receptor exposed on the surface of HEK293T cells through specific recognition of the amino-terminal region (aa 14-34) of the protein. Given the growing interest for anti-CCR5 immunization as an HIV control strategy and the many advantages of virus-based immunogen formulations (especially for poorly immunogenic or self-antigens), the results reported in this study provide preliminary validation of BoHV-4 as a safe viral vector suitable for CCR5 vaccination.

BoHV-4-based vector delivering Ebola virus surface glycoprotein.

BACKGROUND: Ebola virus (EBOV) is a Category A pathogen that is a member of Filoviridae family that causes hemorrhagic fever in humans and non-human primates. Unpredictable and devastating outbreaks of disease have recently occurred in Africa and current immunoprophylaxis and therapies are limited. The main limitation of working with pathogens like EBOV is the need for costly containment. To potentiate further and wider opportunity for EBOV prophylactics and therapies development, innovative approaches are necessary. METHODS: In the present study, an antigen delivery platform based on a recombinant bovine herpesvirus 4 (BoHV-4), delivering a synthetic EBOV glycoprotein (GP) gene sequence, BoHV-4-syEBOVgD106ΔTK, was generated. RESULTS: EBOV GP was abundantly expressed by BoHV-4-syEBOVgD106ΔTK transduced cells without decreasing viral replication. BoHV-4-syEBOVgD106ΔTK immunized goats produced high titers of anti-EBOV GP antibodies and conferred a long lasting (up to 6 months), detectable antibody response. Furthermore, no evidence of BoHV-4-syEBOVgD106ΔTK viremia and secondary localization was detected in any of the immunized animals. CONCLUSIONS: The BoHV-4-based vector approach described here, represents: an alternative antigen delivery system for vaccination and a proof of principle study for anti-EBOV antibodies generation in goats for potential immunotherapy applications.

In vitro antimicrobial activity of a gel containing antimicrobial peptide AMP2041, chlorhexidine digluconate and Tris-EDTA on clinical isolates of Pseudomonas aeruginosa from canine otitis.

BACKGROUND: Pseudomonas aeruginosa (PA) may cause suppurative otitis externa with severe inflammation and ulceration in dogs. Multidrug resistance is commonly reported for this organism, creating a difficult therapeutic challenge. OBJECTIVE: The aim of this study was to evaluate the in vitro antimicrobial activity of a gel containing 0.5 µg/mL of antimicrobial peptide AMP2041, 0.07% chlorhexidine digluconate (CLX), 0.4% Tris and 0.1% EDTA on 30 clinical isolates of PA from canine otitis externa. MATERIALS AND METHODS: Antimicrobial activity was evaluated through minimal bactericidal concentration (MBC). Standardized bacterial suspensions were incubated with different concentrations of the gel at 37°C for 30 min and plated for colony forming unit (CFU) counts. Time-to-kill kinetics were evaluated with the undiluted product and at MBC for each PA strain at 30 s, 1, 5, 10, 15, 30 min, 24 and 48 h. RESULTS: The MBC was 1:64 for two of 30 strains, 1:128 for 15 of 30 strains and 1:256 for 13 of 30 strains. The geometric mean was 1:165, equivalent to a concentration of 0.003 µg/mL AMP2041 + 0.0004% CLX + 0.0024%Tris + 0.0006% EDTA. Time-to-kill assays with the undiluted product showed complete bactericidal effect within 30 s for all isolates, whereas at the MBC this effect was reached within 5 min for 20 of 30 isolates and within 30 min for all isolates. Bactericidal activity was maintained after 48 h for all isolates. CONCLUSION: This gel has shown rapid, complete and long-lasting activity against a panel of 30 PA isolates from cases of canine otitis externa.

BoHV-4 immediate early 1 gene is a dispensable gene and its product is not a bone marrow stromal cell antigen 2 counteracting factor.

BACKGROUND: Bovine herpesvirus 4 (BoHV-4) is a gammaherpesvirus whose genome was cloned as Bacterial Artificial Chromosome (BAC) and exploited as a gene delivery vector for vaccine purposes. Although BoHV-4 genome has been completely sequenced and its open reading frames (ORFs) structurally defined in silico, most of them are not functionally characterized. In BoHV-4 genome two major immediate early genes (IE) are present, IE1 and IE2. IE2 is an essential gene because its removal from the viral genome renders the virus unable to replicate, whereas for IE1 no many functional information are available. RESULTS: In this work, IE1 contribution in initiating and maintaining BoHV-4 lytic replication was assessed generating a recombinant BoHV-4 genome lacking of IE1 gene, BoHV-4ΔIE1. In contrast to BoHV-4IE2 deleted mutant, BoHV-4ΔIE1 infectious replicating viral particles (IRVPs) could be reconstituted following viral DNA electroporation in permissive cells. However the titer of BoHV-4ΔIE1 IRVPs produced into the cell supernatant and BoHV-4ΔIE1 plaques size were reduced respect to BoHV-4 undeleted control. Further the impaired BoHV-4ΔIE1 IRVPs produced into the cell supernatant could be rescued by expressing IE1 gene product in trans, confirming the implication of IE1 in BoHV-4 lytic replication. Next, the possible role of BoHV-4IE1 as bone marrow stromal cell antigen 2 (BST-2) counteracting factor, as hypothesized by IE1 amino-terminal gene product homology with Kaposi Sarcoma Associated Herpesvirus (KSHV) K5, was excluded too. CONCLUSIONS: Although the real function of BoHV-4IE1 is still elusive, a new BoHV-4 genome gene locus as a target site for the insertion of foreign DNA and resulting in the attenuation of the virus has been revealed. These data can be considered of relevance to improve BoHV-4 gene delivery properties.

Conjunctival flora of clinically normal and diseased turtles and tortoises.

BACKGROUND: In captive breed turtles and tortoises conjunctival disease is common. Our aim was to investigate the bacterial and fungal flora present in the eyes of healthy and pathological chelonians and to compare findings in turtles with those in tortoises. RESULTS: Samples were taken from the conjunctival sacs of 34, diseased and healthy, chelonians (18 tortoises and 16 turtles) and submitted to bacterial and fungal investigation. All samples showed bacterial growth. Thirteen animals (38%), harboured a single bacterial species as sole isolate and twenty-one animals (62%) harboured more than one species. Detection of multiple bacterial infection was clearly greater in tortoises compared to turtles. Most frequently isolated bacterial species were Bacillus spp. (13 isolates), Staphylococcus xylosus (10 isolates), Sphingomonas paucimobilis (6 isolates), Staphylococcus sciuri and Aeromonas hydrophila/caviae (each 5 isolates), Ochrobactrum anthropi (3 isolates), Citrobacter freundii, Enterobacter cloacae and Pseudomonas luteola (each 2 isolates). Only one isolate of Kocuria varians/rosea, Staphylococcus aureus, Staphylococcus auricularis, Staphylococcus haemolyticus, Staphylococcus lentus, Morganella morganii, Pasteurella multocida, Pasteurella pneumotropica/haemolytica, Proteus spp., Pseudomonas putida, Salmonella enterica ssp. arizonae, Stenotrophomonas maltophilia and Vibrio parahaemolyticus was evidenced. The presence in 8 animals of Mycoplasma spp. and in 1 animal with severe conjunctivitis of Chlamydia spp. was detected by PCR. Candida spp. was also isolated from two healthy animals. CONCLUSIONS: A clear predominance of Gram positive isolates in tortoises and Gram negative isolates in turtles was found. However, we cannot ascribe the observed difference to the diversity of animal species, as other factors, including especially different characteristics of the living environments, may play a role. Almost all bacterial species isolated may have clinical significance, mostly as opportunistic pathogens, both for humans and animals. That chelonians are often carrier of bacteria with zoonotic potential is a well-known fact, in particular with regard to Salmonella spp. Therefore, it is not surprising the detection of a strain of Salmonella enterica ssp. arizonae in the eye of one of the animals tested. Worthy of note is the finding of Chlamydia spp. in a severe case of conjunctivitis, though we cannot epidemiologically assess a cause-effect relationship between presence of chlamydia and disease.

Interferon gamma-mediated BoHV-4 replication restriction in bovine endometrial stromal cells is host IDO1 gene expression independent and BoHV-4 IE2 gene expression dependent.

In the present work the interaction between bovine herpesvirus 4 (BoHV-4)-infected bovine endometrial stromal cells (BESCs) and interferon gamma (IFNG) was investigated. Starting from the particular tropism of BoHV-4 toward BESCs, a pure population of these cells, free of CD45-positive cells, was prepared and proven to have a bona fide mesenchymal derivation as shown by vimentin-positive and cytokeratin-negative staining. BESCs expressed functional IFNG receptors (IFNGR) 1 and 2 but not IFNG ligand. BESCs transfected with a new reporter construct made by cloning the bovine indoleamine 2, 3-dioxygenase 1 (IDO1) promoter in front of the luciferase reporter gene responded to exogenous IFNG treatment. Further, IFNG-treated or constitutively secreting IFNG BESCs strongly restricted BoHV-4 replication and consequent cytopathic effect. IDO1 expression in BESCs was tightly induced by IFNG and IDO1 was previously shown to be the mediator for some of the IFNG pathogenostatic effects. However, IDO1 inhibitors and IDO1 constitutive expression could not respectively abrogate or recapitulate IFNG effect on BoHV-4-infected BESCs, whereas BoHV-4 immediate early (IE2) gene expression was transcriptionally depressed by IFNG axis activation independently from IDO1 expression; this was further confirmed by revealing a BoHV-4 IE2 gene promoter area containing potential responsive elements interacting with inhibitory transcription factors induced by IFNG in BESCs. The data achieved in this work highlight at least two issues: first, the role of BESCs as target/effector cells for the IFNG; second, the importance of uterine IFNG integrity to control BoHV-4 infection recrudescence from a persistent/latent state to a chronic disease, endometritis.

In vivo image analysis of BoHV-4-based vector in mice.

Due to its biological characteristics bovine herpesvirus 4 (BoHV-4) has been considered as an appropriate gene delivery vector. Its genomic clone, modified as a bacterial artificial chromosome (BAC), is better genetically manipulable and can be used as an efficient gene delivery and vaccine vector. Although a large amount of data have been accumulated in vitro on this specific aspect, the same cannot be asserted for the in vivo condition. Therefore, here we investigated the fate of a recombinant BoHV-4 strain expressing luciferase (BoHV-4-A-CMVlucΔTK) after intraperitoneal or intravenous inoculation in mice, by generating a novel recombinant BoHV-4 expressing luciferase (BoHV-4-A-CMVlucΔTK) and by following the virus replication through in vivo imaging analysis. BoHV-4-A-CMVlucΔTK was first characterized in vitro where it was shown, on one hand that its replication properties are identical to those of the parental virus, and on the other that the transduced/infected cells strongly express luciferase. When BoHV-4-A-CMVlucΔTK was inoculated in mice, either intraperitoneally or intravenously, BoHV-4-A-CMVlucΔTK infection/transduction was exclusively localized to the liver, as detected by in vivo image analysis, and in particular almost exclusively in the hepatocytes, as determined by immuno-histochemistry. These data, that add a new insight on the biology of BoHV-4 in vivo, provide the first indication for the potential use of a BoHV-4-based vector in gene-transfer in the liver.

Broad-spectrum activity of a novel antibiotic peptide against multidrug-resistant veterinary isolates.

The emergence of multidrug resistant (MDR) and extensively drug resistant (XDR) bacteria has become a medical and veterinary problem. Antimicrobial peptides (AMPs) show potential to overcome antibiotic resistance and could be used therapeutically. A novel AMP (AMP2041) was developed in silico and its microbiocidal activity against MDR clinical strains isolated from cattle (n=6), dogs (n=8), and pigs (n=20) was evaluated. AMP2041 showed strong antimicrobial activity against all Gram-positive and Gram-negative MDR clinical strains tested. Within 20 min of incubation, there was complete killing of Pseudomonas aeruginosa ATCC 27953 and a 90% reduction of colony count for Escherichia coli ATCC 25922. For Staphylococcus aureus ATCC 25923, a 90% reduction of colony count was observed within 120 min of incubation.

Bovine herpesvirus 4 glycoprotein B is indispensable for lytic replication and irreplaceable by VSVg.

BACKGROUND: Bovine herpesvirus 4 (BoHV-4) is a gammaherpesvirus, belonging to Rhadinovirus genus, with no clear association with disease. However, there is increasing evidence of its secondary pathogenic role in cases of post-partum metritis in cattle. BoHV-4 Open Reading Frame 8 (ORF8) codifies for glycoprotein B (gB) that shows a heterodimeric structure, composed of two subunits and covalently linked by disulfide bonds and responsible for host cell adhesion through binding to heparan sulfates associated with cellular proteoglycans. Here we describe the generation of several tagged soluble forms of gB ectodomain, in order to test their ability to neutralize BoHV-4 infection. RESULTS: The results show, however, that none of these soluble forms are able to block viral infectivity. To better understand the role of gB during BoHV-4 lytic replication, a recombinant BoHV-4 was generated by homologous recombination from a BoHV-4 cloned as a Bacterial artificial chromosome (BAC) (pBAC-BoHV-4-A), in which most of the BoHV-4 gB ORF was substituted by the insertion of a DNA stuffer selectable cassette. The resulting recombinant BoHV-4 genome (pBAC-BoHV-4-AΔgB-KanaGalK) was completely unable to reconstitute infectious replicating viral particles (Infectious Replicating Viral Particles, IRVPs) and to replicate when transfected in permissive cell lines in comparison to its revertant clone (pBAC-BoHV-4-ΔgB-Rev) or pBAC-BoHV-4-A parental clone. CONCLUSION: This demonstrates that the BoHV-4 replicating cycle is dependent on gB. Moreover, when gB was deleted from a recombinant BoHV-4 genome delivering an heterologous glycoprotein, Vesicular Stomatitis Virus Glycoprotein (VSVg), VSVg was unable to complement gB. This study provides direct evidence that gB is necessary for BoHV-4 lytic replication.

Clinical protection of goats against CpHV-1 induced genital disease with a BoHV-4-based vector expressing CpHV-1 gD.

Caprine herpesvirus type 1 (CpHV-1) is an alphaherpesvirus causing genital disease leading to abortion in adult pregnant goats and a systemic disease with high morbility and mortality in kids. Further, Caprine herpesvirus 1 infection represents a valuable large animal model for human herpesvirus induced genital disease, exploitable for pathogenic studies, new vaccines and antiviral molecules testing. Here, the bovine herpesvirus 4 (BoHV-4) based vector derived from an apathogenic isolate of BoHV-4 and expressing the immunodominant CpHV-1 glycoprotein D (BoHV-4-A-gD(cp)gD(106)ΔTK) was constructed and its ability to protect goats against CpHV-1 induced genital disease evaluated. The subcutaneous route of recombinant BoHV-4 administration was first tested in vivo/ex vivo by in vivo image analysis and in vitro by goat skin primary cultures preparation and transduction. Next, an exploratory immunization and safety study in goats was performed with two recombinant BoHV4, BoHV-4-A-gD(cp)gD(106)ΔTK or BoHV-4-CMV-IgK-gE2gD-TM. In both cases no clinical signs were evident but a good titer of serum neutralizing antibodies was produced in all inoculated animals. When a challenge experiment was performed in a new group of animals using a highly pathogenic dose of CpHV-1, all the vaccinated goats with BoHV-4-A-gD(cp)gD(106)ΔTK were protected toward CpHV-1 induced genital disease respect to the unvaccinated control which showed typical vaginal lesions with a high grade of clinical score as well as a long lasting viral shedding. In summary, the data acquired in the present study validate BoHV-4-based vector as a safe and effective viral vector for goat vaccination against CpHV-1 induced genital disease and pave the way for further applications.