First autochthonous Dirofilaria immitis (Leidy, 1856) infection in a dog in Hungary

AbstractA 4 year-old, male Hungarian Vizsla dog which had never been abroad was referred with poor general condition, decrease in body weight, haematemesis and jaundice to the Central Clinic of Veterinary Science University, Budapest. After symptomatic treatment abdominal ultrasonography and diagnostic laparatomy were carried out. The dog was humanely euthanized two days later following owner's consent because of sudden worsening of clinical conditions. Two adult heartworms (Dirofilaria immitis) were found in the right ventricle partially coiling around the tricuspid valve. PCR on blood was positive for both D. immitis and Dirofilaria repens while only D. repens microfilariae were found by modified Knott's test and the serological test was negative for D. immitis antigens. This is the first, confirmed report of autochthonous canine heartworm infection in Hungary

Enhanced diagnostic protocol to identify E.coli VTEC from milk filters

The EHEC (enterohemorrhagic E. coli) are a subgroup of VTEC with strong pathogenicity. The most well-known EHEC serotype is E. coli O157:H7, which has been implicated in many large outbreaks of deadly human diseases. However, EHEC strains of other serotypes have increasingly been implicated in sporadic cases and outbreaks of serious illness in humans, e.g., serotypes O26, O45, O103, O111, O121 and O145. Epidemics studied from 1982 to date have shown that ruminants, and in particular bovine, appear almost always involved in the transmission of these bacteria to humans through direct or indirect fecal contamination of foods. Unpasteurized milk and milk products are considered minor, but important sources of infection. The possible ways to the entrance of VTEC in milk are fecal contamination and mammary excretion during E.coli mastitis. Between the two ways, the first is considered much more frequent in practice, but it cannot be excluded that a small part of EHEC found in milk resulting from mammary gland, as reported. Previous investigation suggested that milk filters used in milking machines could be a useful control point to identify the presence of EHEC in dairy herds. However, conventional methods to identify the presence of EHEC have a poor sensitivity due the high content of fecal bacteria of these filters. In order to set up a monitoring scheme to identify herds at risk, we developed and tested a diagnostic protocol involving VIDAS\uae UP E.coli serogroups (ESPT) which is a method using phage recombinant proteins for the immuno-concentration (IC) of E.coli serogroups O157, O26, O103, O111, O145, O45 and O121 from food, multiplex PCR and high resolution melting analysis (HRMA). Practically, bulk tank milk or washing solution obtained from milk filters after stomacher mixing were analyzed by Vidas ESPT. After incubation, the solution obtained was analyzed by multiplex PCR based on serotype-specific primers coding for O-antigen regions of the seven major VTEC serogroups available in literature. If PCR was positive for any of the seven serogroups, a HRMA-based protocol to detect virulence-predictive SNPs, as discovered by Norman et al., 2012, was applied to confirm the presence of a EHEC strain. The protocol was preliminary validated by inoculation of milk and milk filters with a known concentration of the seven EHEC serotypes (O26, O45, O103, O111, O121, O145, O157). The results confirmed that this protocol was able to identify as low as 101 UFC in both milk and milk filters. The protocol applied to milk and milk filters obtained from 70 dairy herds allow to identify 2 EHEC from milk and 17 from milk filters for one or more of the EHEC serogroups considered. The proposed protocol confirmed to be useful in detecting the presence of EHEC and that milk filters are an important critical control point to identify herd at risk

A new integrated approach to analyze bulk tank milk and raw milk filters for the presence of the E. coli serogroups frequently associated with VTEC status

We optimized a combination of microbiological and molecular methods to quickly identify the presence of the O157 and the six non-O157 serogroups (O26, O45, O103, O111, O121 and O145) most frequently associated with VTEC status, at herd level. The lower detection limit of this methodology is 101 CFU/ml for each of the serogroups tested. We tested 67 bulk tank milk (BTM) and raw milk filters (RMF) derived from dairy herds located in Lombardy and Trentino Alto Adige. We identified 3 positive samples and 20 positive samples out of 67 respectively in the BTM and RMF. Interestingly, several samples showed positivity for more than one serogroups at the same time. We also identified the presence of E. coli O45 and O121 for the first time in raw milk and raw milk filters. Once screened the seven serogroups of interest in our samples, we evaluated the real pathogenicity of our positive, non-O157 samples through two parallel molecular biology methods: virulence gene research by PCR, and HRMA and sequencing. The most frequently isolated serogroups in milk were O157 (2.64%), O103 (2.11%), and O145 (1.06%), while in RMF the frequencies were, respectively 14.92%, 4.48%, and 2.98%. Moreover, this is the first published report in Italy of positive recovery of O45 and O121 serogroups in milk and milk filters. The new diagnostic approach proposed investigate the presence of the O157 and big six non-O157 serogroups at farm level and not to identify VTEC hazard only once the product is processed and/or is ready to be consumed

Evaluation of virulence factors profiles and antimicrobials resistance of Escherichia coli isolated from bulk tank milk and raw milk filters

Data on the presence of pathogenic Escherichia coli in bulk tank milk (BTM) and raw milk filters (RMF) are not available in Italy and there are few studies worldwide. Therefore, a study under field condition was conducted to assess the presence of E.coli pathogenic and commensal (CoEC) strains in BTM and RMF samples and their associated AMR pattern. One hundred forty-nine E.coli isolates were characterized. Among all the isolates, 53 (35.6%) were classified as pathogenic while the other ones were classified as CoEC. Among the pathogenic ones, 23 (54.7%) were classified as enterotoxigenic E.coli (ETEC), 6 (11.3%) as enteroinvasive E.coli (EIEC), 2 (3.8%) as enteroaggregative E.coli (EAEC), 12 (22.6%) harboured virulence factors (VF) common to ETEC+EIEC, and 2 (3.8%) common to ETEC+EAEC. To our knowledge, it is the first time that ETEC isolates harboring VF associated with EAEC or EIEC are observed in raw milk. These data support the presence of transmission of VFs genes among isolates. None of the isolates showed resistance to three or more antimicrobials. The CoEC role as a vector of AMR was confirmed by the presence of 18% ampicillin- and cephalexin-resistant isolates. The presence of AMR in CoEC supports the role of these bacteria as source of resistance genes. Monitoring raw milk by either BTM or RMF analysis, and the relatively cheap procedure applied to identify E.coli pathotypes can be useful to identify hazards related to the spread of enteric diseases and antimicrobial resistance

Quantitative miRNA Expression Analysis Using Fluidigm Microfluidics Dynamic Arrays

MicroRNA (miRNA) is a small non-coding RNA that can regulate gene expression in both plants and animals. Studies showed that miRNAs play a critical role in human cancer by targeting messenger RNAs that are positive or negative regulators of cell proliferation and apoptosis. Here, we evaluated miRNA expression in formalin fixed, paraffin embedded (FFPE) samples and fresh frozen (FF) samples using a high throughput qPCR-based microfluidic dynamic array technology (Fluidigm). We compared the results to hybridization-based microarray platforms using the same samples. We obtained a highly correlated Ct values between multiplex and single-plex RT reactions using standard qPCR assays for miRNA expression. For the same samples, the microfluidic technology (Fluidigm 48.48 dynamic array systems) resulted in a left shift towards lower Ct values compared to those observed by standard TaqMan (ABI 7900HT, mean difference, 3.79). In addition, as little as 10ng total RNA was sufficient to reproducibly detect up to 96 miRNAs at a wide range of expression values using a single 96-multiplexing RT reaction in either FFPE or FF samples. Comparison of miRNAs expression values measured by microfluidic technology with those obtained by other array and Next Generation sequencing platforms showed positive concordance using the same samples but revealed significant differences for a large fraction of miRNA targets. The qPCRarray based microfluidic technology can be used in conjunction with multiplexed RT reactions for miRNA gene expression profiling. This approach is highly reproducible and the results correlate closely with the existing singleplex qPCR platform while achieving much higher throughput at lower sample input and reagent usage. It is a rapid, cost effective, customizable array platform for miRNA expression profiling and validation. However, comparison of miRNA expression using different platforms requires caution and the use of multiple platforms

Comparison among simultaneous confidence regions for nonlinear diffusion models

Accuracy measures for parameter estimates represent a tricky issue in nonlinear models. Practitioners often use separate marginal confidence intervals for each parameter in place of a simultaneous confidence region (sCR). However, this can be extremely misleading due to the curvature of the parameter space of the nonlinear model. For low parameter dimensions, routines for evaluating approximate sCRs are available in the most common software programs; however, the degree of accuracy depends on the curvature of the parameter space and the sample size. Exact sCRs are computationally intensive, and for this reason, in the past, they did not receive much attention. In this paper, we perform a comparison among exact, asymptotic exact, approximate sCRs, and marginal confidence intervals. More modern regions based on bootstrap are also examined as an alternative approach (both parametric and nonparametric). Their degree of accuracy is compared with both real data and simulation results. Among the nonlinear models, in this paper, the focus is on two of the most widespread diffusion models of products and technologies, that is, the Bass and Generalized Bass models. Three different empirical studies are analyzed here. Simulation studies are also performed for lifecycles with the same diffusion characteristics as those of the empirical studies. Our results show that, as the parameter dimension increases, overlapping among the alternative sCRs reduces. The approximate sCR shows inadequate values of overlapping with the exact sCR, even for moderate parameter dimension. Bootstrap regions also exhibit good performance in describing the shape of the exact region when curvature is present, but they fail to spread up to its boundary. The coverage probability of each region is assessed with simulations. We observe that the coverage probability of the approximate sCR decreases rapidly, even for moderate parameter dimension, and it is smaller than the nominal level for bootstrap regions