Undeclared animal species in dry and wet novel and hydrolyzed protein diets for dogs and cats detected by microarray analysis

Abstract Background Although the European Pet Food Industry Federation (FEDIAF) stated that labels must be accurate and provide detailed information on the ingredients, mislabeling of pet food has been documented by several authors. This phenomenon is of particular concern when related to products used as elimination diets for the diagnosis of adverse food reaction (AFR) in dogs and cats because the presence of undeclared ingredients may negatively interfere with the trial and prevent the veterinarian from making an appropriate diagnosis. The aim of this study was to shed light upon the problem of contamination and mislabeling in both dry and wet novel protein diets (NPDs) and hydrolyzed protein diets (HPDs) using a microarray-based commercial kit which tests for the presence of 19 animal species. Results Of the 40 analyzed products (9 dry NPDs, 22 wet NPDs, 6 dry HPDs and 3 wet HPDs), ten presented a content that correctly matched the label, while five did not contain the declared animal species, twenty-three revealed the presence of undeclared animal species, and two had a vague label that did not allow the evaluation of its accuracy. The most frequently contaminants identified in both dry and wet pet foods were pork, chicken and turkey. The presence of undeclared animal species was higher in dry than wet pet foods; furthermore, a lower number of contaminating animal species was identified in HPDs than NPDs (4 vs 10), and a lower number of contaminated HPDs (6 out of 9, 67%) than contaminated NPDs was detected (24 out of 31, 77%). Thirteen out of 14 brands tested presented at least one mislabeled product. Conclusions Mislabeling seems to be a widespread issue in pet foods used as elimination diets. Contamination can occur in all types of products used for the purpose, although dry NPDs are the main issue. Due to the high risk of contamination, particular attention should be given to both the selection of raw material suppliers and the production process

Occurrence, Diversity of Listeria spp. Isolates from Food and Food-Contact Surfaces and the Presence of Virulence Genes

This study evaluates the hazards posed by foodborne bacteria of the Listeria genus by analyzing the occurrence, diversity and virulence of Listeria spp.in food and food-manufacturing plants. Seventy-five isolates obtained from the routine analysis of 653 samples taken by three diagnostic laboratories in Northern Italy were genotypically differentiated by Repetitive Extragenic Palindrome (rep) PCR, with the GTG5 primer identified by sequencing the 16S rRNA gene and examined by specific PCR tests for the presence of L. monocytogenes virulence determinants occasionally found to occur in other species of the genus. Within this sample, 76% (n = 57) isolates were identified as L. innocua, 16% (n = 12) as L. monocytogenes, 6.6% (n = 5) as L. welshimeri and 1.3% (n = 1) as L. seeligeri. All L. monocytogenes isolates belonged to the serotype 1/2a and were predicted to be virulent for the presence of the inlJ internalin gene. Potentially virulent strains of L. innocua, L. seeligeri and L. welshimeri, carrying the L. monocytogenesinlA gene and/or hly gene, were identified, and most isolates were found to possess the toxin–antitoxin system mazEF for efficient adaptation to heat shock. Results indicated the need to reinforce food-contamination-prevention measures against all Listeria species by defining efficiently their environmental distribution

Assessment of chicken breast shelf life based on bench-top and portable near-infrared spectroscopy tools coupled with chemometrics

Abstract Objectives Near-infrared (NIR) spectroscopy is a rapid technique able to assess meat quality even if its capability to determine the shelf life of chicken fresh cuts is still debated, especially for portable devices. The aim of the study was to compare bench-top and portable NIR instruments in discriminating between four chicken breast refrigeration times (RT), coupled with multivariate classifier models. Materials and Methods Ninety-six samples were analysed by both NIR tools at 2, 6, 10 and 14 days post mortem. NIR data were subsequently submitted to partial least squares discriminant analysis (PLS-DA) and canonical discriminant analysis (CDA). The latter was preceded by double feature selection based on Boruta and Stepwise procedures. Results PLS-DA sorted moderate separation of RT theses, while shelf life assessment was more accurate on application of Stepwise-CDA. Bench-top tool had better performance than portable one, probably because it captured more informative spectral data as shown by the variable importance in projection (VIP) and restricted pool of Stepwise-CDA predictive scores (SPS). Conclusions NIR tools coupled with a multivariate model provide deep insight into the physicochemical processes occurring during storage. Spectroscopy showed reliable effectiveness to recognise a 7-day shelf life threshold of breasts, suitable for routine at-line application for screening of meat quality

Tolerance to biocides, persistence and nanoparticles: a molecular view in Listeria monocytogenes and Salmonella enterica.

The widespread distribution of resistant and multidrug resistant bacteria is one of the main issues of the current scientific world. The distribution of bacterial strain resistant to the compounds used in the clinical practice and for environmental decontamination is a threat for the public health. Infections caused by multidrug resistant bacteria have a serious impact on productivity and cost of medical care. The aim of the present project was to investigate this issue exploring the selective pressure of use and misuse of biocides in meat processing plants on Listeria monocytogenes tolerance development; investigating the distribution of genetic determinants of resistance and persistence in the population of Salmonella, and to investigate the possible future application of innovative compounds, such as nanoparticles, for their biocidal effect on foodborne pathogens. Paper I investigated the real-life scenario of pork meat industries in the light of biocides use for environmental decontamination. Five meat processing plants were evaluated according to their application of Cleaning and Disinfection (C&D) procedures. Several environmental samples were performed on different surface both before and after the C&D procedures. The samples were investigated for the presence of Listeria monocytogenes. A collection of the strains was typed according to the Multi Locus Sequence Type (MLST) protocol, their antibiotic resistance profile was phenotypically evaluated with the Minimum Inhibitory Concentration (MIC) method, and the presence of genetic resistance determinants was assessed. The results presented a high similarity between the collected strains. In order to evaluate their behavior under stress, the gene expression profile was performed on two strains that presented the also the same Enterobacterial Repetitive Intergenic Consensus (ERIC) profile, retrieved on the same surface before and after the C&D procedures. The strains were incubated with different concentrations of a quaternary ammonium compound (QAC) for 24 hours. At different time point the RNA was extracted from the strains and retrotranscribed in order to evaluate the expression of two genes coding for two aspecific efflux pumps (mdrL and lde). Lde was expressed with a dose-dependent trend mainly by the strain isolated before the C&D procedures in the presence of the biocide. The strain isolated after the C&D procedures displayed a low and constant trend of lde expression with a slight increase in the presence of the maximum biocide dose. The mdrL gene expression was different from the other gene. For mdrL, the two strains presented a similar expression profile with the maximum expression during the absence of the biocide and a strong reduction under subinhibitory stress. The gene showed a slight increase of expression during the exposure under the maximum dose of the biocide. So, it was concluded that lde exerted a protective role for the strain isolated before the C&D procedures, while the other strain seemed to be more resistant without the activation of the gene. The strain collected after the C&D procedures expressed the lde gene only under a bigger stress, highlighting the role of the gene and the different intrinsic ability to resist of the strain. On the other hand, the other gene (mdrL) showed not significant influence in the resistance of the strains under this type of stress. Finally, this paper highlighted the importance of an efficient application of the C&D procedures in order to avoid the selection for more resistant strains in the environment. In Paper II the distribution of Toxin-Antitoxin (TA) modules and antibiotic resistance profile were investigated in Salmonella spp. A collection of the most prevalent serovars in Europe (S. Enteritidis, S. Typhimurium, and S. 4,[5],12:i:-, S. Gallinarum) and a collection of rare strains (S. Tennessee, S. Lawndale and S. Alachua) were tested. TA modules support bacterial adaptability to unfavorable environmental conditions, contributing to the generation of non-growing cells as stress response, leading to the protection of the bacteria by making them less sensitive to harmful environments. The TA modules investigated were vapBC and ccdAB, belonging to the type II TA modules (both toxin and antitoxin are proteins). In this study the constitutive occurrence of vapBC was observed in all isolates. CcdAB was present in the most prevalent serovars but it was absent in the rare strains. The common strains presented a multidrug resistance profile against antibiotics, while the rare strains were not resistant to the antibiotics. The presence of both the TA modules in the common strains may represent a selective advantage for their successful diffusion. Furthermore, a point mutation in the ccdAB gene was found after the sequencing of the gene. This point mutation inactivates the Toxin CcdB, the TA module become ineffective for segregational killing. For this reason, the presence in the genome, despite the absence of mechanisms of selective selection, may arise the possibility that the gene is important for the persisters formation in the bacteria population. In Paper III the effect of new antimicrobial agents against Listeria monocytogenes was assessed in vitro. Silver nanoparticles (AgNPs) were tested at different concentration against Listeria monocytogenes at different time points. In the light of the discovery of new active agents against pathogenic bacteria, nanomaterials are interesting compounds. The nanoparticles small dimension make them suitable for a lot of different use. In the study, the effect of AgNPs against Gram positive bacteria was assessed in parallel to the effect of silver nitrate. Silver nitrate exerted an early inhibitor effect after 4 hours of incubation. On the contrary, the AgNPs exerted their full inhibitor effect after 24 hours of incubation. The ionic release of both the compounds was assessed. The ions released were comparable to the effect of inhibition of both the compounds. For this reason, it was supposed a dependence of efficacy based on the time of releasing of silver ions from the compounds. The possible application of the delayed effect was speculated to be useful in the food industry for long term storage and to keep low the bacteria population. To conclude, during the PhD project different aspects of foodborne pathogens adaptation under selective pressure were addressed, leading to the production of three scientific articles that provide deep insights into the issue, pointing out possible mitigation strategies.L’elevata distribuzione a livello mondiale di batteri resistenti e multi resistenti agli antibiotici è uno dei più importanti problemi che il mondo scientifico stia affrontando. La diffusione di ceppi batterici resistenti ai composti usati nella pratica clinica e nella decontaminazione ambientale è un pericolo per la salute pubblica. Le infezioni causate da batteri multi resistenti hanno un importante impatto sulla produttività e sui costi del servizio sanitario. Lo scopo di questo progetto è stato quello di approfondire il problema, valutando la pressione selettiva per lo sviluppo di tolleranze in Listeria monocytogenes data dall’uso e dall’uso improprio dei biocidi usati negli impianti di trasformazione della carne; studiando la distribuzione dei determinanti genetici di resistenza e persistenza nella popolazione di Salmonella; e valutando le possibili applicazioni future di composti innovativi, come le nano particelle, per il loro effetto antimicrobico sui patogeni di origine alimentare. Nell’Articolo I lo scenario reale delle industrie di trasformazione della carne di suino è stato studiato valutando l’uso dei biocidi per la decontaminazione ambientale. Sono state valutate cinque industrie di lavorazione della carne secondo la loro applicazione delle procedure di pulizia e disinfezione. È stata effettuata una serie di campioni su diverse superfici, sia prima che dopo le procedure di pulizia e disinfezione, che sono stati testati per la presenza di Listeria monocytogenes. Usa serie di isolati è stata selezionata per essere tipizzata secondo un protocollo di Multi Locus Sequence Type (MLST). La resistenza fenotipica agli antibiotici è stata valutata usando il metodo di Minimum Inhibitory Concentration (MIC) e sono stati ricercati con PCR i determinanti genetici di resistenza. I risultati hanno presentato un alto grado di similarità tra i ceppi raccolti. Per valutare la risposta batterica allo stress, è stata effettuata l’analisi dell’espressione genetica di due ceppi identici da un punto di vista genetico (secondo il profilo di Enterobacterial Repetitive Intergenic Consensus, ERIC), che sono stati raccolti sulla stessa superficie prima e dopo le procedure di pulizia e disinfezione. I ceppi sono stati incubati con diverse concentrazioni di un sale d’ammonio quaternario (QAC) per 24 ore. L’RNA è stato estratto a differenti time point, per essere poi retro trascritto per la valutazione dell’espressione di due geni codificanti per due pompe aspecifiche di efflusso (mdrL e lde). Il gene lde è stato espresso, durante lo stress antimicrobico, con una tendenza dose-dipendente, dal ceppo isolato prima delle procedure di pulizia e disinfezione; invece il ceppo isolato dopo le procedure di pulizia e disinfezione ha presentato un’espressione di lde bassa e costante, con un leggero incremento in presenza della massima concentrazione di disinfettante. Per quanto riguarda mdrL, la sua espressione è stata diversa da quella di lde. L’espressione di mdrL è stata simile tra i due ceppi: una massima espressione senza lo stress del biocida, una forte riduzione con lo stress sub inibitorio e un leggero aumento durante l’esposizione alla massima concentrazione. Quindi si è potuto notare come lde sembri svolgere una funzione protettiva per il ruolo che ha esercitato nel ceppo isolato prima delle operazioni di pulizia e disinfezione. Il ceppo isolato dopo le procedure ha espresso lde solo sotto forte stress antimicrobico, mettendo in evidenza il ruolo del gene sotto stress ambientale e le diverse caratteristiche di tolleranza tra i due ceppi. Invece il gene mdrL è sembrato non presentare una particolare induzione all’espressione sotto lo stress antimicrobico testato. Infine, questo articolo evidenzia l’importanza dell’applicazione efficiente delle procedure di pulizia e disinfezione per evitare la selezione nell’ambiente di ceppi batterici resistenti. Nell’Articolo II è stata studiata la distribuzione di moduli Tossina- Antitossina (TA) e i profili di antibiotico resistenza in diverse specie di Salmonella. È stata testata una selezione dei serovar più prevalenti (S. Enteritidis, S. Typhimurium, and S. 4,[5],12:i:-, S. Gallinarum) e più rari (S. Tennessee, S. Lawndale and S. Alachua) che circolano a livello europeo. I moduli TA predispongono l’adattabilità batterica per la sopravvivenza in condizioni ambientali non favorevoli, contribuendo all’induzione di quiescenza cellulare come risposta allo stress, dando così una protezione ai batteri che diventano meno sensibili ad ambienti sfavorevoli. I moduli TA investigati sono stati vapBC e ccdAB, rappresentanti dei moduli TA di tipo II (in cui sia tossina che antitossina sono proteine). In questo studio, è stata osservata la prevalenza costitutiva del gene vapBC in tutti i ceppi testati, mentre il gene ccdAB è stato ritrovato solo nei serovar più prevalenti e non in quelli rari. I ceppi più comuni hanno presentato un profilo di multi resistenza agli antibiotici, mentre i ceppi rari non hanno mostrato particolari resistenze agli antibiotici. La presenza di entrambi i moduli TA nei ceppi più prevalenti rappresenta un vantaggio importante che potrebbe contribuire al successo della loro distribuzione. Inoltre è stata osservata, in seguito al sequenziamento dell’amplicone, una mutazione puntiforme nel gene ccdAB. Tale mutazione inattiva la tossina CcdB, bloccando l’effetto di segregationa killing del modulo. Quindi, mancando tale meccanismo di selezione del gene, si rende evidente la possibilità che il modulo TA sia importante per la probabile insorgenza di ceppi persistenti nella popolazione batterica. Nell’Articolo III è stato studiato in vitro l’effetto antimicrobico di un nuovo composto su Listeria monocytogenes. Le nano particelle di argento (AgNPs) sono state usate a diverse concentrazioni contro una collezione di ceppi di Listeria monocytogenes valutandone l’efficacia a diversi tempi di incubazione. I nano materiali sono interessanti composti per quanto riguarda la scoperta di nuovi agenti attivi contro i batteri patogeni. Le piccole dimensioni delle nano particelle le rendono utilizzabili per molti scopi, in quanto presentano diverse caratteristiche chimico-fisiche rispetto al materiale nella forma normale. In questo studio, l’effetto delle nano particelle di argento contro un batterio Gram positivo è stato valutato in parallelo all’effetto esercitato dal nitrato d’argento. L’argento nitrato ha esercitato un suo effetto inibitore precoce, presentando inibizione batterica dopo 4 ore di incubazione. Invece le nano particelle di argento hanno esercitato il loro effetto inibitore dopo 24 ore di incubazione. Per comprendere meglio questa tendenza è stato effettuato un test di rilascio ionico su entrambi i composti. Il rilascio degli ioni d’argento è risultato essere sovrapponibile all’effetto inibitorio dei due composti, in quanto ad alto rilascio ionico è corrisposta un’alta inibizione batterica. Per questo motivo è stata supposta una dipendenza di efficacia tra tempo di risposta e tempo di rilascio ionico. Le possibili applicazioni delle nano particelle potrebbero consistere nello sfruttare l’effetto antimicrobico ritardato per mantenere bassa la popolazione batterica durante i periodi di lunga conservazione. In conclusione, durante il progetto di dottorato sono stati studiati diversi aspetti dell’adattabilità dei patogeni di origine alimentare durante una pressione selettiva ambientale; questo ha portato alla stesura di tre articoli scientifici che approfondiscono l’argomento, mettendo in luce anche possibili strategie di azione verso tale sfida

Resistance to biocides in Listeria monocytogenes collected in meat-processing environments

The emergence of microorganisms exerting resistance to biocides is a challenge to meat-processing environments. Bacteria can be intrinsically resistant to biocides but resistance can also be acquired by adaptation to their sub-lethal concentrations. Moreover, the presence of biocide resistance determinants, which is closely linked to antibiotic resistance determinants, could lead to co-selection during disinfection practices along the food chain, and select cross-resistant foodborne pathogens. The purpose of this work was to test the resistance of wild strains of Listeria monocytogenes, isolated from pork meat processing plants, toward benzalkonium chloride (BC), used as proxy of quaternary ammonium compounds. Furthermore, the expression of two non-specific efflux pumps genes (lde and mdrL) under biocide exposure was evaluated. L. monocytogenes were isolated from five processing plants located in the Veneto region (northeast of Italy) before and after cleaning and disinfection (C&D) procedures. A total of 45 strains were collected: 36 strains before and nine after the C&D procedures. Collected strains were typed according to MLST and ERIC profiles. Strains sampled in the same site, isolated before, and after the C&D procedures and displaying the same MLST and ERIC profiles were tested for their sensitivity to different concentrations of BC, in a time course assay. The expression of non-specific efflux pumps was evaluated at each time point by qPCR using tufA gene as housekeeping. A differential expression of the two investigated genes was observed: lde was found to be more expressed by the strains isolated before C&D procedures while its expression was dose-dependent in the case of the post C&D procedures strain. On the contrary, the expression of mdrL was inhibited under low biocidal stress (10 ppm BC) and enhanced in the presence of high stress (100 ppm BC). These findings suggests a possible role for C&D procedures to select L. monocytogenes persisters, pointing out the importance of dealing with the identification of risk factors in food plants sanification procedures that might select more tolerant strains

Heavy Metal Presence in Two Different Types of Ice Cream: Artisanal Ice Cream (Italian Gelato) and Industrial Ice Cream

none5noIce cream, a popular product worldwide, is usually a milk-based product with other types of ingredients (fruit, eggs, cocoa, dried fruit, additives, and others). Different materials are used to obtain the desired taste, texture, consistency, and appearance of the final product. This study surveyed ice cream products available in Italy for heavy metals (lead, cadmium, chromium, tin, and arsenic). The differences between artisanal and industrial ice cream were also investigated because of the importance in the Italian diet and the diffusion of this ready-to-eat food. Ice cream sampling was performed between October 2010 and February 2011 in the northeast of Italy. A total of 100 samples were randomly collected from different sources: 50 industrial samples produced by 19 different brands were collected in coffee bars and supermarkets; 50 artisanal ice cream samples were gathered at nine different artisanal ice cream shops. Ten wooden sticks of industrial ice cream were analyzed in parallel to the ice cream. All samples were negative for arsenic and mercury. None of the artisanal ice cream samples were positive for lead and tin; 18% of the industrial ice cream samples were positive. All positive lead samples were higher than the legal limit stated for milk (0.02 mg/kg). All industrial ice cream samples were negative for cadmium, but cadmium was present in 10% of the artisanal ice cream samples. Chromium was found in 26% of the artisanal and in 58% of the industrial ice cream samples. The heavy metals found in the wooden sticks were different from the corresponding ice cream, pointing out the lack of cross-contamination between the products. Considering the results and the amount of ice cream consumed during the year, contamination through ice cream is a low risk for the Italian population, even though there is need for further analysis.noneConficoni, D; Alberghini, L; Bissacco, E; Ferioli, M; Giaccone, VConficoni, Daniele; Alberghini, Leonardo; Bissacco, E; Ferioli, M; Giaccone, Valeri

A limited survey of heavy metal concentrations in fresh and frozen cuttlefish ink and mantle used as food

none5nonedaniele conficoni, leonardo alberghini, elisa bissacco, barbara contiero, valerio giacconeConficoni, Daniele; Alberghini, Leonardo; Bissacco, Elisa; Contiero, Barbara; Giaccone, Valeri

Investigating the Determinants of Toxoplasma gondii Prevalence in Meat: A Systematic Review and Meta-Regression.

BACKGROUND:Toxoplasma gondii is one of the most widespread parasites in humans and can cause severe illness in immunocompromised individuals. However, its role in healthy people is probably under-appreciated. The complex epidemiology of this protozoan recognizes several infection routes but consumption of contaminated food is likely to be the predominant one. Among food, consumption of raw and undercooked meat is a relevant route of transmission, but the role of different meat producing animal species and meats thereof is controversial. OBJECTIVES:The aim of the present work is to summarize and analyse literature data reporting prevalence estimates of T. gondii in meat animals/meats. DATA SOURCES:We searched Medline, Web of Science, Science Direct (last update 31/03/2015). ELIGIBILITY CRITERIA:Relevant papers should report data from primary studies dealing with the prevalence of T. gondii in meat from livestock species as obtained through direct detection methods. Meta-analysis and meta-regression were performed. RESULTS:Of 1915 papers screened, 69 papers were included, dealing mainly with cattle, pigs and sheep. Pooled prevalences, based on random-effect models, were 2.6% (CI95 [0.5-5.8]) for cattle, 12.3% (CI95 [7.6-17.8]) for pigs and 14.7% (CI95 [8.9-21.5]) for sheep. Due to the high heterogeneity observed, univariable and multivariable meta-regression models were fitted showing that the geographic area for cattle (p = 0.032), the farming type for pigs (p = 0.0004) and the sample composition for sheep (p = 0.03) had significant effects on the prevalences of Toxoplasma detected/estimated. Moreover, the role of different animal species was dependent on the geographic location of animals' origin. LIMITATIONS:Limitations were due mainly to a possible publication bias. CONCLUSIONS AND IMPLICATIONS:The present work confirms the role of meat, including beef, as T. gondii sources, and highlights the need for a control system for this parasite to be implemented along the meat production chain. Moreover, consumer knowledge should be strengthened in order to reduce the impact of disease