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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 (&gt, 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.

Simple Summary In recent years, animal and human health have been linked in a “One Health” approach. Wildlife could act as a reservoir for different antibiotic-resistant pathogens, representing an issue for human and domestic animal health. The aim of this study was to evaluate the presence and circulation of antimicrobial-resistant bacterial species in wild micromammals in the province of Parma, Northern Italy. Multi-drug-resistant strains and a high prevalence of resistance to critically important antibiotics were detected. Furthermore, resistance to commonly used antibiotics was detected in a large percentage of isolates. Considering that micromammals are good bioindicators, obtained results highlighted a high prevalence of strains resistant to antimicrobials of critical importance for human and animals in the investigated areas, thus representing a public health hazard. Abstract 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.

BackgroundSnakes are globally considered as pet animals, and millions of ophidians are bred in captivity.Pseudomonas aeruginosais 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 ofP. aeruginosaisolated from the cloacal microbiota of a large population sample of healthy captive ophidians and to evaluate the statistical associations with farming conditions.MethodsA 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 ofP. aeruginosaisolates was evaluated through the Kirby-Bauer agar diffusion test. Statistical analyses were performed with the chi-square test.ResultsThe prevalence ofP. aeruginosawas 59.9%, and 35.5% of these strains were multidrug resistant (MDR). The prevalence of MDRP. aeruginosawas 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 ofP. aeruginosawere observed depending on the farm size and snake family.Feeding thawed prey was associated with a higherP. aeruginosaand MDRP. aeruginosaprevalence. Moreover, snakes fed home-raised prey had a significantly higher MDRP. aeruginosaprevalence than snakes fed commercially available feed. Less frequent terrarium cleaning was associated with a higher MDRP. aeruginosaprevalence. On the other hand, snake reproductive status was not significantly associated withP. aeruginosaor MDRP. aeruginosaprevalence. All food, water and bedding samples were negative forP. aeruginosapresence.DiscussionThe overallP. aeruginosaprevalence 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 withP. aeruginosaand/or MDRP. aeruginosaprevalence. Good breeding management and proper antibiotic treatment ofP. aeruginosainfections could help reduce the presence ofP. aeruginosaand MDRP. aeruginosain the gut microbiota of snakes and consequently reduce the risk to public health.

Antimicrobial resistance (AMR) in a wide range of infectious agents is a growing public health threat. Birds of prey are considered indicators of the presence of AMR bacteria in their ecosystem because of their predatory behaviour. Only few data are reported in the literature on AMR strains isolated from animals housed in zoos and none about AMR in raptors housed in zoological gardens. This study investigated the antibiotic sensitivity profile of the isolates obtained from the conjunctival and cloacal bacterial flora of 14 healthy birds of prey, 6 Accipitriformes, 3 Falconiformes and 5 Strigiformes, housed in an Italian zoological garden. Staphylococcus spp. was isolated from 50% of the conjunctival swabs, with S. xylosus as the most common species. From cloacal swabs, Escherichia coli was cultured from all animals, while Klebsiella spp. and Proteus spp. were isolated from a smaller number of birds. Worthy of note is the isolation of Escherichia fergusonii and Serratia odorifera, rarely isolated from raptors. Staphylococci were also isolated. All the isolates were multidrug resistant (MDR). To the author's knowledge, this is the first report regarding the presence of MDR strains within raptors housed in a zoological garden. Since resistance genes can be transferred to other pathogenic bacteria, this represents a potential hazard for the emergence of new MDR pathogens. In conclusion, the obtained data could be useful for ex‐situ conservation programmes aimed to preserve the health of the endangered species housed in a zoo.

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 β-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.

Background Antimicrobial resistance is a growing threat to public health. Pseudomonas aeruginosa is a relevant pathogen causing human and animal infections, frequently displaying high levels of resistance to commonly used antimicrobials. The increasing difficulty to develop new effective antibiotics have discouraged investment in this area and only a few new antibiotics are currently under development. An approach to overcome antibiotic resistance could be based on antimicrobial peptides since they offer advantages over currently used microbicides. Methods The antimicrobial activity of the synthetic peptide AMP2041 was evaluated against 49 P. aeruginosa clinical strains with high levels of antimicrobial resistance, isolated from humans (n = 19) and animals (n = 30). In vitro activity was evaluated by a microdilution assay for lethal dose 90% (LD90), while the activity over time was performed by time-kill assay with 12.5 µg/ml of AMP2014. Evidences for a direct membrane damage were investigated on P. aeruginosa ATCC 27853 reference strain, on animal isolate PA-VET 38 and on human isolate PA-H 24 by propidium iodide and on P. aeruginosa ATCC 27853 by scanning electron microscopy. Results AMP2041 showed a dose-dependent activity, with a mean (SEM) LD90 of 1.69 and 3.3 µg/ml for animal and human strains, respectively. AMP2041 showed microbicidal activity on P. aeruginosa isolates from a patient with cystic fibrosis (CF) and resistance increased from first infection isolate (LD90 = 0.3 μg/ml) to the mucoid phenotype (LD90 = 10.4 μg/ml). The time-kill assay showed a time-dependent bactericidal effect of AMP2041 and LD90 was reached within 20 min for all the strains. The stain-dead assay showed an increasing of membrane permeabilization and SEM analysis revealed holes, dents and bursts throughout bacterial cell wall after 30 min of incubation with AMP2041. Conclusions The obtained results assessed for the first time the good antimicrobial activity of AMP2041 on P. aeruginosa strains of human origin, including those deriving from a CF patient. We confirmed the excellent antimicrobial activity of AMP2041 on P. aeruginosa strains derived from dog otitis. We also assessed that AMP2041 antimicrobial activity is linked to changes of the P. aeruginosa cell wall morphology and to the increasing of membrane permeability.

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&ndash, 71.1) and 41 farms (95.3%, 95% CI 92.2&ndash, 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 &ge, 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 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.

Antimicrobial-peptide-based therapies could represent a reliable alternative to overcome antibiotic resistance, as they offer potential advantages such as rapid microbicidal activity and multiple activities against a broad spectrum of bacterial pathogens. Three synthetic antimicrobial peptides (AMPs), AMP72, AMP126, and also AMP2041, designed by using ad hoc screening software developed in house, were synthesized and tested against nine reference strains. The peptides showed a partial β-sheet structure in 10-mM phosphate buffer. Low cytolytic activity towards both human cell lines (epithelial, endothelial, and fibroblast) and sheep erythrocytes was observed for all peptides. The antimicrobial activity was dose dependent with a minimum bactericidal concentration (MBC) ranging from 0.17 to 10.12 μM (0.4–18.5 µg/ml) for Gram-negative and 0.94 to 20.65 μM (1.72-46.5 µg/ml) for Gram-positive bacteria. Interestingly, in high-salt environment, the antibacterial activity was generally maintained for Gram-negative bacteria. All peptides achieved complete bacterial killing in 20 min or less against Gram-negative bacteria. A linear time-dependent membrane permeabilization was observed for the tested peptides at 12.5 µg/ml. In a medium containing Mg2+ and Ca2+, the peptide combination with EDTA restores the antimicrobial activity particularly for AMP2041. Moreover, in combination with anti-infective agents (quinolones or aminoglycosides) known to bind divalent cation, AMP126 and AMP2041 showed additive activity in comparison with colistin. Our results suggest the following: (i) there is excellent activity against Gram-negative bacteria, (ii) there is low cytolytic activity, (iii) the presence of a chelating agent restores the antimicrobial activity in a medium containing Mg2+ and Ca2+, and (iv) the MBC value of the combination AMPs–conventional antibiotics was lower than the MBC of single agents alone. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.

Increasingly effective vaccination strategies are needed to counteract the high incidence of contagious diseases associated with intensive swine breeding. Recombinant viral vaccines are a promising new avenue in this direction. Key features of viral vectors suitable for immunoprophylaxis are safety, ease of manipulation and the ability to replicate in a variety of hosts. Most of the above requirements are met by bovine herpesvirus 4 (BoHV-4), a non-pathogenic dsDNA virus capable of infecting a broad range of cell types in vitro. Here we report the results of an exploratory study using an engineered BoHV-4 virus (eBoHV-4) expressing two unrelated glycoprotein antigens from bovine viral diarrhea virus (BVDV) and bovine herpesvirus 1 (BoHV-1), to assess the potential of recombinant BoHV-4 as a self-adjuvanted immunogen in pigs. Free eBoHV-4 virions and virions preloaded into homologous swine adipose-derived stromal cells (SADSC) were tested. Neither virus formulation elicited neutralizing anti-BoHV-4 antibodies, nor any disease symptom, yet both induced specific immune responses against the heterologous antigens. However, a much earlier (18 vs 28 days post-infection) and more robust neutralizing response against BVDV and BoHV-1 viruses was elicited by eBoHV-4-preinfected SADSCs compared to free virions. The data validate BoHV-4 as a safe and effective heterologous antigen carrier/producer and identify SADSCs as helpful tools for the formulation of increasingly efficacious recombinant immunogens for pig vaccination.

Bovine herpesvirus 4 (BoHV-4) is a gammaherpesvirus with no clear disease association, it establishes persistent infections in its natural host, the bovine, and in an experimental host, the rabbit. BoHV-4 immediate early 2 (IE2) RNA is the less abundant, spliced, 1.8 kb RNA. The predicted amino acid sequence, of the IE2 protein, reveals that it could encode a 61 kDa protein with amino acid sequence homology to the Epstein-Barr virus (EBV) transactivator R protein and its homologues including, herpesvirus saimiri (HVS), equine herpesvirus 2 (EHV-2), murine herpesvirus 68 and Kaposi's sarcoma-associated herpesvirus (KSHV). We examined recently the interaction of BoHV-4 with a human rhabdomyosarcoma cell line, RD-4, and found that although some infectious viruses can be produced, no cytopathic effect (CPE) was observed [J. Gen. Virol. 81 (2000) 1807]. Because IE2 could play a critical role in BoHV-4 productive infection and its overexpression in RD-4 cells could switch the non-permissive RD-4 status to a permissive one. RD-4 cells expressing stably BoHV-4 IE2 gene were generated. BoHV-4 IE2 induced an increased production of infectious viral particles sufficient to obtain an apparent cytopathic effect. It is concluded that BoHV-4 IE2 is a key factor in determining the outcome of BoHV-4 infection.

Bovine herpesvirus 4 (BoHV-4) is a gammaherpesvirus with no clear disease association. Although BoHV-4 is not considered a neurotropic virus, it has been detected in peripheral and/or central nervous system tissues during persistent infection (Lopez et al, 1996, Microb Pathogen 21: 47-58; Yamamoto et al, 2000, Arch Virol 145: 2363-2370; Asano et al, 2003, J Vet Med Sci 65: 87-93). However, the direct interaction between BoHV-4 and neurons has not been studied so far. The authors investigated the interaction of BoHV-4 with N2a (neuroblastoma cell line) cells through the use of two recombinant viruses (BoHV-4/26A3 neo and BoHV-4EGFP?TK). Because of the unique biological characteristics of N2a cells, which differentiate in neuron-like cells producing dendrites, axon, and specific neuronal markers, the authors found that BoHV-4 infects differentiated N2a cells and a persistent infection can be established. BoHV-4 persistently infected N2a cells produce infectious viral particles, which do not interfere with cellular differentiation.

The aims of the study were to identify microbial flora in boar semen under field conditions in northern Italy, to investigate antibiotic resistance and sensitivity of isolated bacteria, and to evaluate elimination of bacteria after storage in two types of extenders added with different antibiotics (amikacin vs gentamicin). A total of 60 boars were collected in 13 pig farms. Bacteriological and mycological investigations were performed immediately on raw semen samples, then at 48 and 120 h of storage on semen diluted randomly in a new short-term modified extender (ME-S) or in a commercial one (CRONOSTM). Bacterial contamination was found in 63% of raw semen samples and different bacterial species were isolated: E.coli, Serratia marcescens, Staphylococcus epidermidis and aureus, Proteus spp., Streptococcus spp. and Pseudomonas aeruginosa. E. coli was the most isolated contaminant (53%); Pseudomonas aeruginosa was found only in one semen sample. The analysis of variance of factors affecting contamination levels was significant for the farm of origin (P

Although the seroprevalence of Chlamydia psittaci is widespread in Italian dairy herds, its role in inducing genital disorders has not been elucidated. We therefore set up a case-control study to compare seroprevalence to C. psittaci in an aborted-cow population and in a randomly selected control group in the province of Parma (the Po Valley of northern Italy). The true seroprevalence (45%) in aborted cows was significantly higher than that in the control group (24%) (adjusted odds ratio=2.53).

The study is aimed at assessing and comparing the immune response to BoHV-1 elicited by an inactivated marker vaccine in buffaloes and cattle. Vaccination did not produced any local or general reactions in buffaloes. Seroneutralizing antibodies and cellular response by IFN-γ- test have been detected in buffaloes and cattle after a prime/ booster vaccination strategy. Humoral and cellular responses were significantly higher in cattle than in buffaloes. Data pointed out the possibility to use the marker vaccine in buffaloes. However, further studies must be planned to assess the immune pressure of marker vaccines in terms of IBR eradicative attitude in infected buffalo herds.

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.

THE number of pet reptiles is steadily increasing in some European countries, as well as in the USA, ([Mermin and others 1997][1], [Editorial Team and others 2008][2]) and green iguanas ( Iguana iguana ) are frequently being kept as pets. Green iguanas are native to regions extending from southern

Background Lichtheimia corymbifera (previously Absidia corymbifera) is a filamentous zygomycetes belonging to the order Mucorales and to the family Lichtheimiaceae. Members of genus Lichtheimia spp. are cosmopolitan and ubiquitous in nature. Lichtheimia corymbifera is a recognized agent of diseases in man and animals. In cattle it causes abortion and mastitis. Three cases of bovine abortion occurred in a herd located in the Po Valley. Serological examinations were performed on fetal and mother's blood. One of the aborted fetus was referred to our laboratory. The paper describes the isolation and characterization of Lichtheimia corymbifera from a bovine aborted fetus. Methods Serological examinations were performed on fetal and mother's blood. Lesions on fetal tissues and placenta leaded the diagnostic suspect towards a mycotic aetiology. Tissues were then put in culture, and at the same time an histological examination was performed, together with bacteriological and virological tests. The isolate from placenta and fetal tissues was identified and characterized by PCR and RFLP, using the ITS region as a target sequence and AclI restriction site within the amplicon to distinguish Lichtheimia corymbifera among the other fungi. Results Serological, bacteriological and virological tests gave aspecific results. Histological examination evidenced numerous PAS positive hyphae within the necrotic cotiledons and numerous fungal nonseptate hyphae to the GMS stain. Colonies with typical morphological features of fungi grew up on Sabouraud agar from fetal skin and placenta. On the developed colonies the microscopic examination has shown a large number of nonseptate hyphae and sporangia consistent with Mucorales. PCR and RFLP allowed the identification of the isolate as Lichtheimia corymbifera. Conclusion The present report describes the isolation and the molecular characterisation of a fungal isolate from bovine aborted fetus and placenta. The diagnostic protocol allowed to identify and characterise the strain. This is the first isolation in Italy of Lichtheimia corymbifera in a bovine aborted fetus.

A bovine herpesvirus 4 was isolated from the milk cell fraction of a healthy cow and his full genome cloned as a bacterial artificial chromosome. So cloned viral genome was used as a vector platform to deliver in vitro and in vivo an optimized secreted chimeric peptide obtained by the fusion of the bovine viral diarrhoea virus glycoprotein E2 ectodomain with the bovine herpesvirus 1 glycoprotein D ectodomain. Recombinant virus infected cells robustly expressed and secreted the chimeric peptide into the culture medium and inoculated animals with the recombinant virus successfully responded toward antigens, gE2 and gD. Thus, this work has implications for the development of safe and effective polyvalent vaccines.

Background The biological characteristics of BoHV-4 make it a good candidate as a gene delivery vector for vaccination purposes. These characteristics include little or no pathogenicity, unlikely oncogenicity, the capability to accommodate large amounts of foreign genetic material, the ability to infect several cell types from different animal species, and the ability to maintain transgene expression in both undifferentiated and differentiated cells. Results A recombinant bovine herpesvirus 4 (BoHV-4CMV-IgKE2-14ΔTK) expressing an enhanced secreted form of the bovine viral diarrhea virus (BVDV) structural glycoprotein E2 (gE2-14), obtained by the removal of the putative transmembrane domain and addition of a 14 amino acids peptide at its carboxyl terminal and an immunoglobulin K signal peptide to the amino terminal, was successfully constructed using a Recombineering (recombination -mediated genetic engineering) approach on BoHV-4 cloned as bacterial artificial chromosome. The galactokinase – based recombineering system was modified by the introduction of a kanamycin expression cassette and a kanamycin selection step that allowed a significant reduction of the untargeted background clones. BoHV-4CMV-IgKE2-14ΔTK infected cell lines highly expressed gE2-14, which maintained native antigenic properties in a serum neutralization inhibition test. When rabbits and sheep were immunized with BoHV-4CMV-IgKE2-14ΔTK, high levels of serum neutralized antibodies against BVDV were generated. Conclusion This work highlights the engineerization of BoHV-4 genome as a vector for vaccine purposes and may provide the basis for BVDV vaccination exploiting the BoHV-4- based vector that delivers an improved secreted version of the BVDV structural glycoprotein E2.

Gene transfer into hepatocytes is highly desirable for the long-term goal of replacing deficient proteins and correcting metabolic disorders. Bovine herpesvirus 4 (BoHV-4) based vector capability to transduce rat liver cells in vitro and in vivo was assessed. For the in vitro study, a buffalo rat liver cell line was successfully transduced by BoHV-4 and although did not show toxicity, the immediate early two viral gene was transcribed and cells harboring the intact viral genome could be pharmacologically selected, but no viral replication took place. For the in vivo study, adult male rats were inoculated intraportally and intraparenchimally with a BoHV-4 expressing enhanced green fluorescent protein and liver sections were analyzed through fluorescent microscopy. Although the liver parenchyma could not be transduced, the endothelial layer of the liver vasculature showed a robust transgene expression without toxicity. Successful BoHV-4 based vector transduction of primary cultures of rat hepatocytes suggests that extrinsic factors, and not hepatocytes per se, are the cause of such lack of transducibility. The present study serves as a starting point for study of the use of BoHV-4 based vectors to target gene delivery to vascular endothelial cells.

In cattle with endemic N. caninum infection, reactivation of latent infection frequently occurs during pregnancy and is followed by vertical transmission of the pathogen to the foetus, which can cause abortion or birth of congenitally infected calves (Dubey and Lindsay, 1996). The N. caninum infection elicits an antibody response (Bjorkman and Uggla, 1999). Serological investigations have shown that N. caninum is widespread among Italian cattle, even in the absence of abortion (Ferrari et al., 1997; Magnino et al., 2000) or contact with dogs (Cabassi et al., 2001) which are the definitive host of the parasite. Immunodepressive events occurring in cattle with congenital infection can be considered as risk factors for the reactivation of the N. caninum infection, which is followed by seroconversion. To support such a hypothesis, a correlation between N. caninum active infection and Bovine Viral Diarrhea Virus, an immunosuppressive agent, has been shown (Bjorkman et al., 2000). Therefore, seroconversion to N. caninum can be considered an indicator of immune failure in cattle (Hemphill and Gottstein, 2000). Due to the degree of attenuation of Blue Tongue (BT) vaccines, the duration of postvaccinal viremia (Monaco et al., 2003) and the virus tropism for lymphocytes suggest that vaccination can alter the immune omeostasis, eliciting the reactivation of latent infections, such as neosporosis. On this basis, a study aimed at demonstrating an association between N. caninum antibody response and BT vaccination has been performed. Starting from 2002, BT vaccination was performed in Italian epidemic areas using monovalent (serotype 2) or bivalent (serotypes 2–9) vaccines, depending on epidemiological status.

Three commercially available assays, designed to specifically detect the presence of Mycobacterium avium subsp. paratuberculosis (MAP) in fecal samples by IS900-PCR, were compared with a conventional culture method. Fecal samples from 100 dairy cows were tested. Fifty-four (67.5%) of 80 culture-positive samples were positive for an assay that detects MAP DNA by dot spot hybridization of polymerase chain reaction products (kit A), 48 (60%) were positive by an assay using ethidium bromide staining for agar gel visualization of amplification products (kit B), and 49 (61.3%) were positive by an assay in which amplified products are detected by a colorimetric detection system (kit C). Relative sensitivity of all tests increased in proportion to the presence of MAP in fecal samples. Specificity was 100% based on results from 20 culture-negative samples from an MAP-free herd.

From January 2001 to December 2002, 543 ostrich eggs were submitted for bacteriologic investigation. The eggs were laid by 387 domesticated ostriches that suffered fertility disorders and that came from 44 farms located in different areas of Northern and Central Italy. Microbiologic investigations showed bacterial isolation in 105 (19.3%) of 543 eggs examined, with a high prevalence of enterobacteria from albumen and yolk. In only a few cases did bacterial isolation result from yolk or albumen alone. An antibiotic sensitivity test was conducted on isolates by the Kirby-Bauer disc diffusion method. This is the first report regarding the microbiologic status of eggs from ostrich farms located in different Italian regions.