Antibacterial activities of Manuka and Honeydew honey-based membranes against bacteria that cause wound infections in animals.

INTRODUCTION: In this study, membranes composed of honey (Manuka or Honeydew) and pectin were developed, and the ISO 22196 method was used to evaluate their antibacterial activities against multidrug-resistant bacteria (i.e., Staphylococcus pseudointermedius, Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa) that cause wound infection in animals. The results demonstrated that both Manuka and Honeydew honey-based membranes had strong antibacterial activities against the strain of methicillin-resistant S. pseudointermedius tested. Specifically, membranes composed of Manuka honey were effective in inhibiting the growth of Gram-negative bacteria within 3 h, whereas those composed of Honeydew honey needed 24 h to neutralise bacterial growth. The antimicrobial activities of both membranes developed in this study suggest that they can be effectively used as wound dressing in veterinary clinical medicine.

Dans le cadre de cette étude, on a fabriqué des membranes à base de miel (miel de Manuka et miel de miellat) et de pectine et on a testé, selon le processus ISO 22196, leur activité antibactérienne sur des germes multirésistants provenant de blessures d'animaux (Staphylococcus pseudointermedius, E. coli, Proteus mirabilis und Pseudomonas aeruginosa). Les résultats montrent que les deux types de membranes ont une forte activité bactéricide sur les souches de Staphylococcus pseudointermedius résistantes à la méthicilline. Les membranes à base de miel de Manuka étaient également actives contre tous les germes gram négatifs ét réduisaient leur nombre en 3 heures, alors qu'un contact de 24 heures était nécessaire pour que les membranes à base de miel de miellat réduisent la croissance bactérienne. L'activité antibactérienne des membranes utilisées dans la présente étude justifie leur emploi dans la médecine vétérinaire clinique.

Evaluation of antimicrobial activity of Italian honey for wound healing application in veterinary medicine.

INTRODUCTION: Honey as a topical treatment for infected wounds dates back to ancient times. However, few studies have been reported concerning the medical properties of Italian honey. In this study, the microbial contamination, the antimicrobial activity and the antibiotic residues of 6 different varieties of Piedmont honeys were evaluated. The antimicrobial activity of honeys was tested by agar well diffusion method and 1 honey for each variety has been selected and tested by broth micro-dilution test to determine Minimum Inhibitory Concentrations (MICs) and evaluated by Minimum Bactericidal Concentrations (MBCs). The honeys with a high level of antibacterial activity were analyzed for the presence of tetracyclines, sulfonamides and macrolide residues. The agar well diffusion method showed the greatest antimicrobial activity for honeydew, chestnut and lime tree honeys. The MICs and MBCs identified the close similarity to the medical manuka honey of honeydew, polyfloral and chestnut honey. The levels of antibiotic residues on these honeys were below the limit of quantification. Based on our results the Italian variety of honeydew showed the best antimicrobial activity and can be considered for the treatment of infected wounds in animals.

INTRODUCTION: L'utilisation du miel pour le traitement des plaies infectées remonte à loin dans l'antiquité. Dans le présent travail, on étudie les contaminations microbiennes, l'activité antimicrobienne et les résidus d'antibiotiques dans 6 sortes de miels différentes provenant du Piémont. L'activité antimicrobienne a été mesurée au moyen d'une méthode de diffusion sur gel d'agar et un échantillon de chaque sorte de miel a été examiné quant à sa concentration minimale inhibitrice (CMI) et sa concentration minimale bactéricide (CMB) au moyen d'un test de micro-dilution. Les échantillons présentant une haute activité antibactérienne ont été analysés quant à la présence de tétracycline, de sulfamidés et de macrolides. Au test de diffusion sur agar, le miel de miellat ainsi que ceux de châtaignier et de tilleul ont démontré la plus grande activité antimicrobienne. Les CIM et CBM permettent de reconnaitre une grande similitude entre les miels de miellat, de nectar et de tilleul avec le miel de Manuka utilisé à des fins thérapeutiques. Les résidus d'antibiotiques de ces échantillons se situaient en dessous des limites de détection. Sur la base de ces constatations, les divers miels de miellat italiens présentent la plus grande activité antimicrobienne et peuvent être utilisés pour le traitement de plaies infectées chez les animaux.

Cloning, expression, and antigenic characterization of recombinant protein of Mycoplasma gallisepticum expressed in Escherichia coli.

Mycoplasma gallisepticum (MG) is a member of the most important avian mycoplasmas, causing chronic respiratory disease in chickens and leading to important economic losses in the poultry industry. Recombinant technology represents a strategic approach used to achieve highly reliable and specific diagnostic tests in veterinary diseases control: in particular this aspect is crucial for confirming mycoplasma infection and for maintaining mycoplasma-free breeder flocks. In this study, we identified a component of the pyruvate dehydrogenase dihydrolipoamide acetyltransferase (i.e., E2) protein by 2-dimensional electrophoresis (2-DE), characterized it in immunoblotting assays, and analyzed its recombinant (r-E2) in a rec-ELISA test. For full-length protein expression in Escherichia coli (EC) a point mutation was introduced. A rabbit antiserum produced against r-E2 was tested in a Western Blot using different samples of Mycoplasma species. The results showed the applicability of site-directed mutagenesis, with a good yield of the r-E2 after purification. Also, anti-E2 serum reacted with all the tested MG strains showing no cross reaction with other mycoplasmas. The developed E2 ELISA test was capable of detecting MG antibodies in the sera examined. Those results demonstrate the antigenic stability of the E2 protein which could represent a recombinant antigen with potential diagnostic applications.

Occurrence and functionality of cycle inhibiting factor, cytotoxic necrotising factors and cytolethal distending toxins in Escherichia coli isolated from calves and dogs in Italy.

Escherichia coli isolated from animals up to three months of age, with diarrhea (255 calves and 29 dogs (pups)), without diarrhea (21 calves and 11 pups, used as controls), and 58 adult dogs with cystitis were tested to investigate the occurrence and functional expression of cyclomodulins cycle inhibiting factor (CIF), cytotoxic necrotizing factors (CNFs) and cytolethal distending toxins (CDTs). In cyclomodulin-positive isolates the association was assessed with other virulence genotypes and phylogenetic groups. Of 374 E. coli isolates, 80 (21.4%) were positive for at least one cyclomodulin and 14 of the latter (3.7%) showed different combinations of more than one. cif-positive isolates showed a low number of additional virulence factors, and were commonly associated with phylogroup B1, while cnf- and cdt-positive isolates, harboring many extraintestinal virulence factors, belonged to phylogroups B2 and D. Almost all isolates showed an irreversible cytopathic effect (CPE), displaying functionality of cyclomodulins. Five isolates that presented a mutation of cif were CPE-negative.

Prevalence of Campylobacter jejuni, Campylobacter coli and enteric Helicobacter in domestic and free living birds in North-Western Italy.

In order to investigate the prevalence of some thermophilic Campylobacter (C. jejuni and C. coli) and enteric Helicobacter (H. pullorum and H. canadensis) in domestic and wild birds, a total of 278 bird caecal samples were analyzed over a 2 year period in North-Western Italy. Samples were collected from poultry raised in intensive farming at the slaughterhouse (n=102, group A) and in small scale rural farms (n=60, group B) as well as from wild birds (n=116, group C). PCR amplifications were carried out on DNA extracted from caecal samples. Molecular assays targeted the hipO gene for C. jejuni, the asp gene for C. coli and the 16S rRNA gene of H. pullorum/H. canadensis. To differentiate H. pullorum from H. canadensis, PCR products were subjected to an ApaLI digestion assay. Prevalence of thermophilic Campylobacter and enteric Helicobacter was significantly different among groups (p<0.0001). Campylobacter infections were detected in all three bird groups (78.4% group A, 18.3% group B and 38.8% group C, respectively), Helicobacter infections were only detected in poultry, with H. pullorum infecting 68.6% of group A and 21.7% of group B birds. H. canadensis was detected in Guinea fowls (group A) and for the first time in pheasants (group B). Mixed infections by enteric Campylobacter and Helicobacter were shown in 53.9% of group A and in 5.0 % of group B. Our results show that both microorganisms commonly infect poultry, especially intensive farming animals. Only hooded crows among the wild bird group (group C), proved to be highly sensitive to Campylobacter infection.

Observations on Antricola ticks: small nymphs feed on mammalian hosts and have a salivary gland structure similar to ixodid ticks.

Ticks use bloodmeals as a source of nutrients and energy to molt and survive until the next meal and to oviposit, in the case of females. However, only the larvae of some tick species are known to feed upon bats; females are obligatorily autogenous, and nymphal stages are believed to not feed. We investigated the presence of blood in a natural population of nymphal Antricola delacruzi ticks collected from bat guano; their ability to feed upon laboratory hosts; and the microscopic structure of both salivary glands and gut. DNA amplification of gut contents of freshly collected material was positive for a mammal in 4 of 11 first instar nymphs, but we were unsuccessful in the amplification of host bloodmeal DNA from late instar nymphs. All early nymphal stages (n = 10) fed on rabbits, and host DNA was detected and sequenced from gut contents. However, all the large nymphs (n = 10) rejected feeding, and host DNA remained undetected in these ticks. All stages of A. delacruzi have salivary glands similar in morphology to the ixodid agranular Type I salivary gland acini and to granular Type II or Type B acini. All stages of A. delacruzi had a similar gut structure, consisting of digestive cells in the basal portion that contained hematin granules. Neither regenerative nor secretory cell traces were observed in the sections of gut.

Phylogenetic background of attaching and effacing Escherichia coli isolates from animals.

Detection and distribution of eae gene in forty-four attaching and effacing Escherichia coli (AEEC) strains of animal origin were investigated. Association of distinct intimin alleles with phylogenetic background were assessed among strains in comparison with different serogroups. Phylogenetic analysis showed that 31 EHEC/eae+ STEC strains belong to groups A, B1 and E, 13 EPEC strains segregated in B1 and B2. Moreover, group A possessed the eae gamma2/theta type, group B1 the eae beta1, eae kappa, eae zeta, and eae epsilon types, group B2 the eae alpha1, eae alpha2 and eae iota types, while the group E possessed the eae gamma1 type. The presence of numerous eae-types show that EPEC and EHEC/eae+ STEC tested have a high genetic homology within each phylogenetic group.

Identification of intimin alleles in pathogenic Escherichia coli by PCR-restriction fragment length polymorphism analysis.

A rapid two-step identification method based on PCR-RFLP analysis of the intimin gene was developed to differentiate specific alleles in pathogenic Escherichia coli. This technique, tested on isolates eae-positive, accurately detects eae and resolves alleles encoding the alpha1, alpha2, beta, gamma1, gamma2/theta, kappa, epsilon, zeta, and iota intimin variants.

Identification of enteric Helicobacter in avian species.

The presence of enteric Helicobacter species was investigated in poultry (n=130) and in pet and ornamental birds (n=50) using a PCR sequencing method which permits the differentiation of many Helicobhacter species derived from animal tissues. All samples were of Italian origin, except for 21 Guinea fowl from a French flock. About 80% of poultry (chickens, laying hens, Guinea fowl) were positive to Helicobacter DNA. H. pullorum was most frequently (62.1%) identified whereas H. pylori and 3 H. sp. hamster B strains were seen in only 3 cases each. Pet and ornamental birds were all negative. H. canadensis was found in all Guinea fowl from a French farm. This is the first report on the occurrence of this bacterium in poultry.

Isolation of Acinetobacter lwoffii from a lovebird (Agapornis roseicollis) with severe respiratory symptoms.

Although Acinetobacter lwoffii is generally considered an ubiquitous and opportunistic bacterium, this germ has been isolated from the pulmonary and abdominal air sac swabs obtained from a Lovebird (Agapornis roseicollis), which died of a severe respiratory disease. Bacteriological tests (phenotypic and genotypic) led to the identification of A. lwoffii in pure culture. All the other parrots in the breeding centre were treated orally with oxytetracycline for 14 days and 3 months later no bird showed any signs of respiratory symptoms.