RESUMO
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.
Assuntos
Mel , Animais , Antibacterianos/farmacologia , Bactérias Gram-Negativas , Bactérias Gram-Positivas , Testes de Sensibilidade Microbiana/veterinária , Animais de EstimaçãoRESUMO
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.
RESUMO
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.
RESUMO
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.
RESUMO
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.
RESUMO
The bacterium Pseudomonas aeruginosa (PA) and the yeast Candida albicans (CA) are pathogens that cohabit the mucosa of the respiratory tracts of animals and humans. Their virulence is largely determined by chemical communication driven by quorum sensing systems (QS), and the cross perception of their quorum sensing molecules (QSM) can modulate the prevalence of one microorganism over the other. Aiming to investigate whether some of the protein components dissolved in the mucus layering the respiratory mucosa might interfere with virulence and cross-communication of these, and eventually other microorganisms, ligand binding assays were carried out to test the scavenging potential of the bovine and porcine forms of the Lipocalin odorant binding protein (OBP) for several QSMs (farnesol, and acylhomoserine lactones), and for pyocyanin, a toxin produced by PA. In addition, the direct antimicrobial activity of the OBPs was tested by time kill assay (TKA) against CA, PA and other bacteria and yeasts. The positivity of all the ligand binding assays and the antimicrobial activity determined for CA, and for some of the other microorganisms tested, let hypothesize that vertebrate OBPs might behave as humoral components of innate immunity, active against pathogenic bacteria and fungi. In addition, TKAs with mutants of bovine OBP with structural properties different from those of the native form, and with OBP forms tagged with histidines at the amino terminal, provided information about the mechanisms responsible of their antimicrobial activity and suggested possible applications of the OBPs as alternative or co-adjuvants to antibiotic therapeutic treatments.
Assuntos
Anti-Infecciosos/imunologia , Candida albicans , Imunidade Inata , Pseudomonas aeruginosa , Receptores Odorantes/imunologia , Animais , Anti-Infecciosos/metabolismo , Candida albicans/crescimento & desenvolvimento , Candida albicans/imunologia , Bovinos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/imunologia , Receptores Odorantes/metabolismo , SuínosRESUMO
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.
Assuntos
Anti-Infecciosos/farmacologia , Proteínas Cardiotóxicas de Elapídeos/química , Peptídeos/farmacologia , Sequência de Aminoácidos , Animais , Anti-Infecciosos/química , Candida/efeitos dos fármacos , Bovinos , Dicroísmo Circular , Hemólise/efeitos dos fármacos , Herpesvirus Bovino 1/efeitos dos fármacos , Malassezia/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Mycobacterium/efeitos dos fármacos , Naja naja , Peptídeos/química , Conformação Proteica , Ovinos , Staphylococcus aureus/efeitos dos fármacosRESUMO
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.
Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla , Peptídeos/farmacologia , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/veterinária , Pseudomonas aeruginosa/efeitos dos fármacos , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/ultraestrutura , Parede Celular/efeitos dos fármacos , Parede Celular/ultraestrutura , Cães , Humanos , Viabilidade Microbiana/efeitos dos fármacos , Microscopia Eletrônica de Varredura , Pseudomonas aeruginosa/isolamento & purificaçãoRESUMO
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.
Assuntos
Antibacterianos/uso terapêutico , Peptídeos Catiônicos Antimicrobianos/uso terapêutico , Clorexidina/análogos & derivados , Doenças do Cão/microbiologia , Ácido Edético/análogos & derivados , Ácido Edético/farmacologia , Otite Externa/veterinária , Pseudomonas aeruginosa/efeitos dos fármacos , Trometamina/análogos & derivados , Trometamina/farmacologia , Animais , Clorexidina/administração & dosagem , Clorexidina/uso terapêutico , Cães , Géis , Otite Externa/microbiologiaRESUMO
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.
RESUMO
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.
Assuntos
Bactérias/isolamento & purificação , Infecções Bacterianas/veterinária , Conjuntivite/veterinária , Tartarugas/microbiologia , Animais , Bactérias/classificação , Infecções Bacterianas/microbiologia , Estudos de Casos e Controles , Conjuntivite/microbiologiaRESUMO
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.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Doenças dos Bovinos/tratamento farmacológico , Doenças do Cão/tratamento farmacológico , Farmacorresistência Bacteriana Múltipla , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Doenças dos Suínos/tratamento farmacológico , Animais , Bovinos , Doenças dos Bovinos/microbiologia , Doenças do Cão/microbiologia , Cães , Suínos , Doenças dos Suínos/microbiologiaRESUMO
Among 97 bacterial isolates, 74 strains of Staphylococcus spp developed from 95 swabs taken from skin lesions in dogs. Twenty-eight staphylococcal strains resistant to methicillin and/or oxacillin were identified and mecA expression was confirmed for 14 of these strains. S. aureus and S. intermedius group (SIG) strains were particularly relevant in our cases due to their antibiotic resistance leading to an increased veterinary and public health risk. We suggest a diagnostic protocol based on cytological examination, bacterial identification to species level, and antibiotic sensitivity testing prior to prescribing antibiotic treatment for canine skin diseases.
Assuntos
Antibacterianos/farmacologia , Dermatite , Doenças do Cão/tratamento farmacológico , Doenças do Cão/microbiologia , Infecções Cutâneas Estafilocócicas , Staphylococcus , Animais , Proteínas de Bactérias/genética , Dermatite/tratamento farmacológico , Dermatite/microbiologia , Dermatite/veterinária , Cães , Farmacorresistência Bacteriana/genética , Proteínas de Ligação às Penicilinas , Infecções Cutâneas Estafilocócicas/tratamento farmacológico , Infecções Cutâneas Estafilocócicas/microbiologia , Infecções Cutâneas Estafilocócicas/veterinária , Staphylococcus/efeitos dos fármacos , Staphylococcus/genética , Staphylococcus/isolamento & purificação , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Staphylococcus aureus/isolamento & purificação , Staphylococcus epidermidis/efeitos dos fármacos , Staphylococcus epidermidis/genética , Staphylococcus epidermidis/isolamento & purificação , Staphylococcus hominis/efeitos dos fármacos , Staphylococcus hominis/genética , Staphylococcus hominis/isolamento & purificação , Staphylococcus intermedius/efeitos dos fármacos , Staphylococcus intermedius/genética , Staphylococcus intermedius/isolamento & purificaçãoRESUMO
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.
Assuntos
Anticorpos Antivirais/sangue , Vírus da Diarreia Viral Bovina/imunologia , Vetores Genéticos , Herpesvirus Bovino 4/genética , Células Estromais/imunologia , Vacinação/métodos , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/sangue , Antígenos Virais/genética , Antígenos Virais/imunologia , Vírus da Diarreia Viral Bovina/genética , Herpesvirus Bovino 4/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Células Estromais/metabolismo , Suínos , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/genéticaRESUMO
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.
Assuntos
Aborto Espontâneo/etiologia , Aborto Animal/etiologia , Absidia/isolamento & purificação , Mucormicose/complicações , Mucormicose/microbiologia , Aborto Espontâneo/microbiologia , Aborto Animal/microbiologia , Animais , Bovinos , Doenças dos Bovinos/etiologia , Doenças dos Bovinos/microbiologia , Doenças dos Bovinos/patologia , Feminino , Mucormicose/patologia , Placenta/microbiologia , Placenta/patologia , GravidezRESUMO
In a previous study, an apathogenic strain of bovine herpesvirus 4 (BoHV-4) cloned as a bacterial artificial chromosome and expressing a chimeric peptide (gE2/gD) as a secreted form was described. Recombinant virus-inoculated animals produced antibodies against bovine viral diarrhea virus (BVDV) gE2 and BoHV-1 gD. However, neutralizing antibodies were produced only against BVDV, not against BoHV-1. In the present work a recombinant BoHV-4 expressing a membrane-linked form of gE2/gD chimeric peptide was constructed, and inoculated rabbits produced serum-neutralizing antibodies against both BVDV and BoHV-1. Protein cell sorting and targeting are a very important issue when immunodominant antigens are engineered for recombinant virus vaccine development.
Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Antígenos Heterófilos/metabolismo , Herpesvirus Bovino 4/imunologia , Proteínas Virais/metabolismo , Vacinas Virais/imunologia , Animais , Antígenos Heterófilos/imunologia , Vírus da Diarreia Viral Bovina/genética , Vírus da Diarreia Viral Bovina/imunologia , Herpesvirus Bovino 4/genética , Transporte Proteico , Coelhos , Proteínas Virais/imunologiaRESUMO
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-IgKE2-14 Delta TK) 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-IgKE2-14 Delta TK 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-IgKE2-14 Delta TK, 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.
Assuntos
Vírus da Diarreia Viral Bovina/fisiologia , Vetores Genéticos/genética , Herpesvirus Bovino 4/genética , Imunização/métodos , Transfecção/métodos , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Animais , Coelhos , OvinosRESUMO
Two farms in the Belgrade area have experienced serious problems with postpartal metritis. Serological examination of BoHV-4 infection was done using ELISA test and vaginal swabs were used for virus isolation. Average seroprevalence of BoHV-4 in these farms was 84.37%. BoHV-4 isolation was successful from three vaginal swabs on the MDBK cell line. Rising values of BoHV-4 antibodies were recorded in nine of ten cows with clinical signs of postpartal metritis. PCR and restriction analysis were used for better characterisation and isolate classification. Two isolates showed similarity with MOVAR 33/63 virus type, but one differed in polyrepetitive and other parts of DNA. This was the first isolation and characterisation of BoHV4 from Serbian herds.
Assuntos
Doenças dos Bovinos/epidemiologia , Surtos de Doenças , Endometrite/veterinária , Infecções por Herpesviridae/veterinária , Herpesvirus Bovino 4/isolamento & purificação , Período Pós-Parto , Animais , Anticorpos Antivirais/sangue , Bovinos , Doenças dos Bovinos/virologia , Endometrite/virologia , Feminino , Infecções por Herpesviridae/virologia , Herpesvirus Bovino 4/genética , Herpesvirus Bovino 4/imunologia , Reação em Cadeia da Polimerase , Gravidez , Mapeamento por Restrição , Iugoslávia/epidemiologiaRESUMO
Coxiella burnetii, the agent of Q fever in humans, has been associated with abortion in cattle. In this study 650 sera from cattle with abortion and 600 randomly-selected control sera were examined for antibodies to C. burnetii by ELISA. Two hundred and ninety-two (44.9%) out of 650 animals which experienced abortion were seropositive versus 132 (22%) out of 600 of the control group. A statistically significant difference resulted from the comparison of the seroprevalence of aborted cattle with that of controls (p < 0.001). Moreover, a significant higher prevalence was disclosed in cattle which aborted during late gestation (p < 0.002) and in the autumn (p < 0.001).
Assuntos
Aborto Animal/microbiologia , Doenças dos Bovinos/microbiologia , Coxiella burnetii/isolamento & purificação , Aborto Animal/epidemiologia , Animais , Anticorpos Antibacterianos/sangue , Bovinos , Doenças dos Bovinos/epidemiologia , Coxiella burnetii/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Itália/epidemiologia , Gravidez , Estudos SoroepidemiológicosRESUMO
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.