Antibiotic resistance of Aeromonas ssp. strains isolated from Sparus aurata reared in Italian mariculture farms.

Selective pressure in the aquatic environment of intensive fish farms leads to acquired antibiotic resistance. This study used the broth microdilution method to measure minimum inhibitory concentrations (MICs) of 15 antibiotics against 104 Aeromonas spp. strains randomly selected among bacteria isolated from Sparus aurata reared in six Italian mariculture farms. The antimicrobial agents chosen were representative of those primarily used in aquaculture and human therapy and included oxolinic acid (OXA), ampicillin (AM), amoxicillin (AMX), cephalothin (CF), cloramphenicol (CL), erythromycin (E), florfenicol (FF), flumequine (FM), gentamicin (GM), kanamycin (K), oxytetracycline (OT), streptomycin (S), sulfadiazine (SZ), tetracycline (TE) and trimethoprim (TMP). The most prevalent species selected from positive samples was Aeromonas media (15 strains). The bacterial strains showed high resistance to SZ, AMX, AM, E, CF, S and TMP antibiotics. Conversely, TE and CL showed MIC90 values lower than breakpoints for susceptibility and many isolates were susceptible to OXA, GM, FF, FM, K and OT antibiotics. Almost all Aeromonas spp. strains showed multiple antibiotic resistance. Epidemiological cut-off values (ECVs) for Aeromonas spp. were based on the MIC distributions obtained. The results showed a high frequency of Aeromonas spp. contamination in Sparus aurata reared on the Italian coast and an elevated biodiversity in isolated bacterial strains. Aeromonas isolates comprise potentially pathogenic species for humans, often resistant to several antibiotics and able to transfer the genes responsible for antibiotic resistance to microorganisms pathogenic for humans throughout the food chain. The few ECV studies available on many antibiotics against Aeromonas spp. strains isolated from the aquaculture environment highlight the need for further research in this area, while regular monitoring programmes should be stepped up to check for antibiotic resistance.

Development and validation of a novel SNP panel for the genetic characterization of Italian chicken breeds by next-generation sequencing discovery and array genotyping.

The aim of this study was to compare the intra and inter genetic variability and population structure of 7 indigenous chicken breeds of the Veneto region, through a novel panel of 64 SNP, each located in an exonic region and mostly on different chromosomes. A total of 753 blood samples from 7 local chicken breeds (Ermellinata di Rovigo, Millefiori di Lonigo, Polverara, Pepòi, Robusta Lionata, Robusta Maculata, and Padovana) was collected and analyzed. Two strains of Polverara (Nera and Bianca) and Padovana (Dorata and Camosciata) were included in the study. The observed heterozygosity ranged from 0.124 (Pèpoi) to 0.244 (Ermellinata di Rovigo), and the expected heterozygosity varied from 0.132 (Millefiori di Lonigo) to 0.300 (Ermellinata di Rovigo). Global FIS results (0.114) indicated a low-medium inbreeding effect, with values ranging from 0.008 (Millefiori di Lonigo) to 0.223 (Ermellinata di Rovigo). Pairwise FST values (0.167) for all populations ranged from 0.020 (Polverara Nera and Polverara Bianca) to 0.193 (Robusta Lionata and Polverara Nera), indicating that the studied breeds were genetically highly differentiated. The software STRUCTURE was used to detect the presence of population substructures, and the most probable number of clusters (K) of the 10 chicken populations was at K = 8. The affiliation was successful in all Veneto chicken breeds. The present SNP marker results, compared with previous data obtained using microsatellites, provided a reliable estimate of genetic diversity within and between the studied breeds, and demonstrated the utility of the proposed panel as a rapid, efficient, and cost-effective tool for periodical monitoring of the genetic variability among poultry populations. In addition, the present SNP panel could represent a resource for a systematic approach with relevant impact on breeding program decisions and could turn out to be a reliable tool for genetic traceability of indigenous chicken meat. Adoption of a periodical monitoring system of genetic diversity is a fundamental tool in conservation actions and should increase the value of typical and niche products.

The microbiological conditions of carcasses from large game animals in Italy.

This study investigates the microbiological conditions of large game animal carcasses following evisceration. Carcasses of animals (N=291) hunted in the Upper Susa Valley (Italian Alps) were analysed for pH, Aerobic Viable Count (AVC), Enterobacteriaceae, Yersinia spp., Listeria monocytogenes and Salmonella spp. After shooting, evisceration occurred within 60 min in 90.7% of animals and sampling within 90 min in 88.3% of animals. Mean pH values (5.97: ruminants; 5.77: wild boar) were similar to those of regularly slaughtered domestic species. AVC values were highest in animals shot in the abdomen. Within species, AVC and Enterobacteriaceae values did not differ across different shooting-evisceration/sampling times. However, these counts exceeded 5 and 2.5 log, respectively, in 18% of wild boar and 39% of ruminants; the highest values were detected in wild boar. No pathogens were detected in any species. These results reveal inadequate hygiene in game meat handling/harvesting, implicating the need for improved practices.

Interleukin-4 induces lipogenesis in porcine endothelial cells, which in turn is critical for induction of protection against complement-mediated injury.

Interleukin (IL)-4 has been shown to induce protection in porcine vascular endothelial cells (ECs) from killing by human complement. This protection is dependent on the PI3K/Akt signaling pathway. In this study, we investigated mechanisms downstream of Akt and found that activation of the lipid biosynthesis pathway is required for protection from complement in ECs treated with IL-4. Cells incubated with IL-4 for 48 hours contained increased fatty acids and phospholipids but cholesterol was not increased when compared with medium-treated controls. The transcription factor SREBP-1, which regulates fatty acid synthesis, was found to be activated in extracts of ECs incubated with IL-4 for 6 hours. Finally, induction of protection from complement killing with IL-4 was fully prevented by the presence of the SREBP inhibitor 25-OH cholesterol. This study showed that IL-4 induces lipid biosynthesis in porcine ECs through activation of SREBP-1 and that the activation of this pathway is critical for IL-4 to induce protection of porcine ECs from killing by human complement. Further study of these mechanisms may provide new strategies for the prevention of complement-mediated vascular injury as it occurs in xenograft rejection.

Development of a multiplex PCR assay for the identification of pathogenic genes of Escherichia coli in milk and milk products.

A multiplex PCR for the simultaneous detection of some pathogenic genes of enteropathogenic, enterotoxigenic and verocytotoxin-producing Escherichia coli was developed. In this study primers found in literature as well as primers to the purpose designed were used. In this way, it was possible to generate specific fragments of 96, 170, 229, 285, 348, 414 and 510 bp for Hlya, St, EaeA, Lt, Vt1, UidA and Vt2 genes, respectively. When applied to bacterial strains experimentally inoculated in milk and milk products, the proposed PCR showed a detection limit of 5 x 10(4)CFU/ml for Hyla, St, Eaea, Vt1 primers, while for Lt and Vt2 primers the limit resulted of 10(6)CFU/ml.

A multiplex PCR assay for the identification of animal species in feedstuffs.

A multiplex Polymerase Chain Reaction (PCR) assay was applied to feedstuff analysis for the identification of the most used species in rendering plants (ruminant, poultry, fish and pork materials). Primers were designed in different regions of mitochondrial DNA (12S rRNA, tRNA Val and 16S rRNA) after alignment of the available sequences in the GenBank database. The primers generated specific fragments of 104-106, 183, 220-230 and 290 bp length for ruminants, poultry, fish and pork, respectively. The detection limit was 0.004% for fish primers and 0.002% for ruminants, poultry and pork primers. The multiplex PCR proposed in this study can be considered a valid alternative to the microscopic method for the detection of animal derived materials banned by a European Union Regulation as a preventive measure against the spread of Bovine Spongiform Encephalopathy.

A recombinant soluble chimeric complement inhibitor composed of human CD46 and CD55 reduces acute cardiac tissue injury in models of pig-to-human heart transplantation.

BACKGROUND: Inasmuch as complement plays a critical role in many pathological processes and in xenograft rejection, efficient complement inhibitors are of great interest. Because the membrane-associated complement inhibitors are very effective, recombinant soluble molecules have been generated. METHODS: We tested the efficacy of complement activation blocker-2 (CAB-2), a recombinant soluble chimeric protein derived from human decay accelerating factor (DAF, CD55) and membrane cofactor protein (MCP, CD46), in two models of pig-to-human xenotransplantation in which tissue injury is complement mediated. The in vitro model consisted of porcine aortic endothelial cells and human serum, and the ex vivo model consisted of a porcine heart perfused with human blood. RESULTS: In vitro, addition of CAB-2 to serum inhibited cytotoxicity and the deposition of C4b and iC3b on the endothelial cells. Ex vivo, addition of CAB-2 to human blood prolonged organ survival from 17.3 +/- 6.4 min in controls to 108 +/- 55.6 min with 910 nM (100 microg/ml) CAB-2 and 219.8 +/- 62.7 min with 1820 nM (200 microg/ml) CAB-2. CAB-2 also retarded the onset of increased coronary vascular resistance. The complement activity of the perfusate was reduced by CAB-2, as was the generation of C3a and SC5b-9. The myocardial tissues had similar deposition of IgG, IgM, and Clq; however, CAB-2 reduced the deposition of C3, C4, and C9. Hearts surviving >240 min demonstrated trace to no deposition of C9 and normal histologic architecture. CONCLUSION: These results indicate that CAB-2 can function as an inhibitor of complement activation and markedly reduce tissue injury in models of pig-to-human xenotransplantation and thus may represent a useful therapeutic agent for xenotransplantation and other complement-mediated conditions.

Transgenic swine lungs expressing human CD59 are protected from injury in a pig-to-human model of xenotransplantation.

BACKGROUND: Pulmonary xenotransplantation is currently limited by hyperacute rejection mediated in part by xenoreactive natural antibody and complement. Transgenic swine organs that express the human complement regulatory protein CD59 have demonstrated improved survival in models of pig-to-primate xenotransplantation. OBJECTIVE: The purpose of this study was to evaluate transgenic swine lungs that express the human complement regulatory protein CD59 in a model of pig-to-human xenotransplantation. METHODS: Transgenic swine lungs (n = 5, experimental group) and outbred swine lungs (n = 6, control group) were perfused with fresh, whole human blood through a centrifugal pump on an ex vivo circuit. Functional data were collected throughout perfusion. Immunoglobulin and complement studies were performed on perfusate samples, and both histologic and immunofluorescent analyses were performed on tissue sections. RESULTS: Mean lung survival for the experimental group was increased when compared with controls, 240 +/- 0 minutes versus 35.3 +/- 14.5 minutes, respectively, with a P value of less than.01. A decreased rise in pulmonary vascular resistance at 15 minutes was observed in the experimental group (343 +/- 87 mm Hg. L(-1). min(-1), in contrast to the control group (1579 +/- 722 mm Hg. L(-1). min(-1); P <.01). Pulmonary compliance at 15 minutes was improved for the experimental group versus control group (9.31 +/- 1.41 mL. cm(-2) H(2)O and 4.11 +/- 2.84 mL. cm(-2) H(2)O, respectively; P <.01). SC5b-9 generation in the plasma perfusate was delayed for the experimental group versus the control group. Immunofluorescent examination of tissue sections demonstrated equivalent deposition of immunoglobulin G, immunoglobulin M, C1q, and C3 in both groups, with reduced deposition of C9 in the experimental group. CONCLUSIONS: Transgenic swine pulmonary xenografts that express the human complement regulatory protein CD59 demonstrated improved function and survival in an ex vivo model of pig-to-human xenotransplantation.

Resistance against the membrane attack complex of complement induced in porcine endothelial cells with a Gal alpha(1-3)Gal binding lectin: up-regulation of CD59 expression.

Endothelial cells (EC) play central roles in vascular physiology and pathophysiology. EC activation results in proinflammatory activities with production of cytokines and expression of adhesion molecules. However, we have shown before in a model of xenotransplantation that prolonged stimulation of porcine EC with human anti-porcine IgM natural Abs can activate the cells to become resistant against cytotoxicity by the membrane attack complex of complement (MAC). Now we report the major characteristics of induction and maintenance of resistance elicited in porcine EC with Bandeiraea simplicifolia lectin that binds terminal gal alpha(1-3)gal. Lectin-treated cells underwent little or no cytotoxicity and PGI2 release when exposed to MAC. Induction of resistance required incubation of the EC with lectin for 4 h but was not fully manifested until 16 h later. Most of the initially bound lectin remained on the cell surface for >60 h. EC-bound lectin did not inhibit binding of IgM natural Abs or activation and binding of C components, including C9, but a C-induced permeability channel of reduced size was present. Induction of resistance required protein synthesis, developed slowly, and was associated with up-regulation of expression of mRNA for the MAC inhibitor CD59 and membrane-associated CD59 protein. Resistance lasted at least 3 days, and the cells regained normal morphology and were metabolically active. This induced resistance may have a physiologic counterpart that might be amenable to pharmacologic manipulation in vascular endothelium pathophysiology.

Virulence and molecular diversity of parthenogenetic root-knot nematodes, Meloidogyne spp.

Root-knot nematodes (RKN) are sedentary endoparasites causing severe damage to a wide variety of crops, including tomato. Among them, the parthenogenetic species Meloidogyne arenaria, M. incognita and M. javanica are of particular economic importance. The genetic diversity and relationships of 17 populations belonging to these three major species, either avirulent or virulent against the tomato Mi resistance gene, were investigated in order to determine whether (a)virulence of the nematodes could be related to their molecular fingerprints. Genomic polymorphisms between populations were assessed by using amplified fragment length polymorphism (AFLP) markers, and data were treated by means of a multiple correspondence analysis. A total of 1550 polymorphic amplified DNA fragments were identified and used to compute the relationships between the populations. As expected, the three RKN species were clearly distributed into distinct groups, but combination of data for virulence phenotypes and DNA markers showed that clustering of populations was not associated with their (a)virulence against the tomato Mi resistance gene. Such a lack of correlation indicates that most of the observed DNA polymorphism is independent of virulence, which is presumably under host selection. This result demonstrates that virulent populations do not share a common origin, and strongly suggests that they might have appeared late after the establishment of these clonal lineages, as the result of independent mutational events.

Dyspepsia is associated with CagA-positive Helicobacter pylori.

OBJECTIVE: The role of Helicobacter pylori in nonulcer dyspepsia is controversial. Speculation has arisen that only strains of H. pylori carrying the CagA virulence factor are important in the development of dyspepsia. The objective of this study was to determine whether nonulcer dyspepsia correlated with CagA-positive H. pylori infection. METHODS: A total of 435 healthy blood donors and 102 general medicine clinic respondents completed the Bowel Disease Questionnaire and the PRIME-MD survey, a validated screen for common psychiatric disorders. Subjects were classified as cases of nonulcer dyspepsia if they reported pain in the upper abdomen more than six times in the previous year and denied a past or current history of peptic ulcer disease. Study participants were tested for IgG antibodies to H. pylori and the CagA protein. RESULTS: Clinic respondents were more likely than healthy blood donors to meet the case definition for nonulcer dyspepsia (34% vs 13%, p < 0.001), to be seropositive for H. pylori (54% vs 18%, p < 0.001), and to be CagA seropositive (41% vs 10%, p = 0.01). Logistic regression identified CagA seropositivity (p = 0.03), race (p = 0.001), and positive screens for depression (p = 0.007) or somatization (p < 0.001) as variables independently associated with nonulcer dyspepsia. CONCLUSION: Infection with a CagA-positive strain of H. pylori is associated with a clinical diagnosis of nonulcer dyspepsia. However, nonulcer dyspepsia was also strongly and independently associated with positive screens for depression or somatization disorder as well as with ethnicity. These potential sources of variance should be considered in the design of future studies evaluating nonulcer dyspepsia.

Killing of Streptococcus pneumoniae by capsular polysaccharide-specific polymeric IgA, complement, and phagocytes.

The role of IgA in the control of invasive mucosal pathogens such as Streptococcus pneumoniae is poorly understood. We demonstrate that human pneumococcal capsular polysaccharide-specific IgA initiated dose-dependent killing of S. pneumoniae with complement and phagocytes. The majority of specific IgA in serum was of the polymeric form (pIgA), and the efficiency of pIgA-initiated killing exceeded that of monomeric IgA-initiated killing. In the absence of complement, specific IgA induced minimal bacterial adherence, uptake, and killing. Killing of S. pneumoniae by resting phagocytes with immune IgA required complement, predominantly via the C2-independent alternative pathway, which requires factor B, but not calcium. Both S. pneumoniae-bound IgA and complement were involved, as demonstrated by a 50% decrease in killing with blocking of Fcalpha receptor (CD89) and CR1/CR3 (CD35/CD11b). However, IgA-mediated killing by phagocytes could be reproduced in the absence of opsonic complement by pre-activating phagocytes with the inflammatory products C5a and TNF-alpha. Thus, S. pneumoniae capsule-specific IgA may show distinct roles in effecting clearance of S. pneumoniae in the presence or absence of inflammation. These data suggest mechanisms whereby pIgA may serve to control pneumococcal infections locally and upon the pathogen's entry into the bloodstream.

Mouse-to-rabbit xenotransplantation: a new small animal model of hyperacute rejection mediated by the classical complement pathway.

BACKGROUND: Hyperacute rejection of porcine organs transplanted into primate recipients is initiated by the binding of preformed xenoreactive natural antibodies to the vascular endothelium of the graft and activation of the classical complement pathway. Several small animal models are currently employed to study various aspects of xenograft rejection; however, none has been shown to manifest hyperacute rejection mediated by the classical pathway of complement activation. METHODS: We performed heterotopic mouse heart transplants into weanling rabbits, adult rabbits, and C6-deficient rabbits. The recipients received no immunosuppression. Rejected grafts were subjected to histologic analysis and immunofluorescence staining for rabbit IgG, IgM, and C3. Levels of preexisting cytotoxic antibodies as well as classical and alternative complement pathway activities were determined in rabbit serum using mouse red cells as targets. RESULTS: Mean graft survival was 37+/-9.6 min for mouse-to-weanling rabbit transplants (n=10), and 40+/-11.1 min for mouse-to-adult rabbit transplants (n=5). Rejected grafts showed diffuse interstitial hemorrhage, endothelial cell damage, myocyte necrosis, moderate diffuse deposition of rabbit IgG, and dense deposition of rabbit IgM and C3 on the vascular endothelium of the graft, consistent with hyperacute rejection. One mouse-to-C6-deficient rabbit transplant was rejected at 21 hr with severe interstitial hemorrhage, cellular necrosis and a moderate cellular infiltrate consisting primarily of neutrophils and some mononuclear cells. A second transplant in a C6-deficient rabbit was functioning when the recipient died at 6.5 hr as a result of complications of surgery; the graft had normal myocytes and vasculature with minimal spotty interstitial hemorrhage. Both weanling and adult rabbit serum were found to have high titers of cytotoxic IgM anti-mouse antibodies and strong classical complement pathway activity with minimal alternative pathway activity towards mouse red cells. CONCLUSIONS: The mouse-to-rabbit species combination manifests hyperacute xenograft rejection. In vitro studies suggest that this process is mediated by IgM anti-mouse natural antibodies and activation of the classical pathway of complement.

Spectrum of resistance to root-knot nematodes and inheritance of heat-stable resistance in in pepper (Capsicum annuum L.).

Capsicum annuum L. has resistance to root-knot nematodes (RKN) (Meloidogyne spp.), severe polyphagous pests that occur world-wide. Several single dominant genes confer this resistance. Some are highly specific, whereas others are effective against a wide range of species. The spectrum of resistance to eight clonal RKN populations of the major Meloidogyne species, M. arenaria (2 populations), M. incognita (2 populations), M. javanica (1 population), and M. hapla (3 populations) was studied using eight lines of Capsicum annuum. Host susceptibility was determined by counting the egg masses (EM) on the roots. Plants were classified into resistant (R; EM ≤ 5) or susceptible (H; EM >5) classes. The french cultivar Doux Long des Landes was susceptible to all nematodes tested. The other seven pepper lines were highly resistant to M. arenaria, M. javanica and one population of M. hapla. Variability in resistance was observed for the other two populations of M. hapla. Only lines PM687, PM217, Criollo de Morelos 334 and Yolo NR were resistant to M. incognita. To investigate the genetic basis of resistance in the highly resistant line PM687, the resistance of two progenies was tested with the two populations of M. incognita: 118 doubled-haploid (DH) lines obtained by androgenesis from F(1) hybrids of the cross between PM687 and the susceptible cultivar Yolo Wonder, and 163 F(2) progenies. For both nematodes populations, the segregation patterns 69 R / 49 S for DH lines and 163 R / 45 S for F(2) progenies were obtained at 22°C and at high temperatures (32°C and 42°C). The presence of a single dominant gene that totally prevented multiplication of M. incognita was thus confirmed and its stability at high temperature was demonstrated. This study confirmed the value of C. annuum as a source of complete spectrum resistance to the major RKN.

RPE, a plant gene involved in early developmental steps of nematode feeding cells.

Sedentary plant-parasitic nematodes are able to induce the redifferentiation of root cells into multinucleate nematode feeding sites (NFSs). We have isolated by promoter trapping an Arabidopsis thaliana gene that is essential for the early steps of NFS formation induced by the root-knot nematode Meloidogyne incognita. Its pattern of expression is similar to that of key regulators of the cell cycle, but it is not observed with the cyst nematode. Later in NFS development, this gene is induced by both root-knot and cyst nematodes. It encodes a protein similar to the D-ribulose-5-phosphate 3-epimerase (RPE) (EC 5.1.3.1), a key enzyme in the reductive Calvin cycle and the oxidative pentose phosphate pathway (OPPP). Quantitative RT-PCR showed the accumulation of RPE transcripts in potato, as in Arabidopsis NFS. Homozygous rpe plants have a germination mutant phenotype that can be rescued in dwarf plants on sucrose-supplemented medium. During root development, this gene is expressed in the meristems and initiation sites of lateral roots. These results suggest that the genetic control of NFSs and the first stages of meristem formation share common steps and confirms the previous cytological observations which indicate that root cells undergo metabolic reprogramming when they turn into NFSs.