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

Targeting histone deacetylase 3 (HDAC3) in the bone marrow microenvironment inhibits multiple myeloma proliferation by modulating exosomes and IL-6 trans-signaling

Multiple myeloma (MM) is an incurable cancer that derives pro-survival/proliferative signals from the bone marrow (BM) niche. Novel agents targeting not only cancer cells, but also the BM-niche have shown the greatest activity in MM. Histone deacetylases (HDACs) are therapeutic targets in MM and we previously showed that HDAC3 inhibition decreases MM proliferation both alone and in co-culture with bone marrow stromal cells (BMSC). In this study, we investigate the effects of HDAC3 targeting in BMSCs. Using both BMSC lines as well as patient-derived BMSCs, we show that HDAC3 expression in BMSCs can be induced by co-culture with MM cells. Knock-out (KO), knock-down (KD), and pharmacologic inhibition of HDAC3 in BMSCs results in decreased MM cell proliferation; including in autologous cultures of patient MM cells with BMSCs. We identified both quantitative and qualitative changes in exosomes and exosomal miRNA, as well as inhibition of IL-6 trans-signaling, as molecular mechanisms mediating anti-MM activity. Furthermore, we show that HDAC3-KD in BM endothelial cells decreases neoangiogenesis, consistent with a broad effect of HDAC3 targeting in the BM-niche. Our results therefore support the clinical development of HDAC3 inhibitors based not only on their direct anti-MM effects, but also their modulation of the BM microenvironment

A Relay Pathway between Arginine and Tryptophan Metabolism Confers Immunosuppressive Properties on Dendritic Cells

Arginase 1 (Arg1) and indoleamine 2,3-dioxygenase 1\ua0(IDO1) are immunoregulatory enzymes catalyzing the degradation of L-arginine and L-tryptophan, respectively, resulting in local amino acid deprivation. In addition, unlike Arg1, IDO1 is also endowed with non-enzymatic signaling activity in dendritic cells (DCs). Despite considerable knowledge of their individual biology, no integrated functions of Arg1 and IDO1 have been reported yet. We found that IDO1 phosphorylation and consequent activation of IDO1 signaling in DCs was strictly dependent on prior expression of Arg1 and Arg1-dependent production of polyamines. Polyamines, either produced by DCs or released by bystander Arg1+ myeloid-derived suppressor cells, conditioned DCs toward an IDO1-dependent, immunosuppressive phenotype via activation of the Src kinase, which has IDO1-phosphorylating activity. Thus our data indicate that Arg1 and IDO1 are linked by an entwined pathway in immunometabolism and that their joint modulation could represent an important target for effective immunotherapy in several disease settings

An explainable model of host genetic interactions linked to COVID-19 severity

We employed a multifaceted computational strategy to identify the genetic factors contributing to increased risk of severe COVID-19 infection from a Whole Exome Sequencing (WES) dataset of a cohort of 2000 Italian patients. We coupled a stratified k-fold screening, to rank variants more associated with severity, with the training of multiple supervised classifiers, to predict severity based on screened features. Feature importance analysis from tree-based models allowed us to identify 16 variants with the highest support which, together with age and gender covariates, were found to be most predictive of COVID-19 severity. When tested on a follow-up cohort, our ensemble of models predicted severity with high accuracy (ACC = 81.88%; AUCROC = 96%; MCC = 61.55%). Our model recapitulated a vast literature of emerging molecular mechanisms and genetic factors linked to COVID-19 response and extends previous landmark Genome-Wide Association Studies (GWAS). It revealed a network of interplaying genetic signatures converging on established immune system and inflammatory processes linked to viral infection response. It also identified additional processes cross-talking with immune pathways, such as GPCR signaling, which might offer additional opportunities for therapeutic intervention and patient stratification. Publicly available PheWAS datasets revealed that several variants were significantly associated with phenotypic traits such as "Respiratory or thoracic disease", supporting their link with COVID-19 severity outcome.A multifaceted computational strategy identifies 16 genetic variants contributing to increased risk of severe COVID-19 infection from a Whole Exome Sequencing dataset of a cohort of Italian patients

Carriers of ADAMTS13 Rare Variants Are at High Risk of Life-Threatening COVID-19

Thrombosis of small and large vessels is reported as a key player in COVID-19 severity. However, host genetic determinants of this susceptibility are still unclear. Congenital Thrombotic Thrombocytopenic Purpura is a severe autosomal recessive disorder characterized by uncleaved ultra-large vWF and thrombotic microangiopathy, frequently triggered by infections. Carriers are reported to be asymptomatic. Exome analysis of about 3000 SARS-CoV-2 infected subjects of different severities, belonging to the GEN-COVID cohort, revealed the specific role of vWF cleaving enzyme ADAMTS13 (A disintegrin-like and metalloprotease with thrombospondin type 1 motif, 13). We report here that ultra-rare variants in a heterozygous state lead to a rare form of COVID-19 characterized by hyper-inflammation signs, which segregates in families as an autosomal dominant disorder conditioned by SARS-CoV-2 infection, sex, and age. This has clinical relevance due to the availability of drugs such as Caplacizumab, which inhibits vWF-platelet interaction, and Crizanlizumab, which, by inhibiting P-selectin binding to its ligands, prevents leukocyte recruitment and platelet aggregation at the site of vascular damage

Gain- and Loss-of-Function CFTR Alleles Are Associated with COVID-19 Clinical Outcomes

Carriers of single pathogenic variants of the CFTR (cystic fibrosis transmembrane conductance regulator) gene have a higher risk of severe COVID-19 and 14-day death. The machine learning post-Mendelian model pinpointed CFTR as a bidirectional modulator of COVID-19 outcomes. Here, we demonstrate that the rare complex allele [G576V;R668C] is associated with a milder disease via a gain-of-function mechanism. Conversely, CFTR ultra-rare alleles with reduced function are associated with disease severity either alone (dominant disorder) or with another hypomorphic allele in the second chromosome (recessive disorder) with a global residual CFTR activity between 50 to 91%. Furthermore, we characterized novel CFTR complex alleles, including [A238V;F508del], [R74W;D1270N;V201M], [I1027T;F508del], [I506V;D1168G], and simple alleles, including R347C, F1052V, Y625N, I328V, K68E, A309D, A252T, G542*, V562I, R1066H, I506V, I807M, which lead to a reduced CFTR function and thus, to more severe COVID-19. In conclusion, CFTR genetic analysis is an important tool in identifying patients at risk of severe COVID-19

Valutazione della qualità del pet food e analisi del microbiota intestinale in cani con differente condizione corporea

A complete and balanced diet is essential to maintain animals in good health. Most dogs and cats in Europe, the United States and other developed countries are fed commercially manufactured pet foods, whose market is still growing every year. Commercial pet diets should provide safe and nutritionally balanced foods to meet the animal’s nutritional needs; therefore, pet food manufacturers are required to follow the recommended allowances presented by the European Pet Food Industry Federation. European feed law also establishes rules and requirements for labelling to provide adequate information for consumers. At the same time, we are seeing an increasing popularity of commercial raw pet food diets, as many owners choose to feed a more natural or ancestral diet to their pets due to anecdotal health benefits. However, since pet food labels lack precise information and there is a scarcity of regular controls, concerns may arise about the quality of commercially available pet diets, both extruded and raw. The overall aim of this project was therefore to provide greater insight into the nutrient composition of commercial pet food and the microbiological quality of raw meat‐based products. First, a general introduction about pet foods’ features and sales is provided in Chapter 1. Chapter 2 explores the amino acid composition of 80 complete pet foods. Dietary proteins should provide all the essential amino acids that animals cannot synthesize, and whether the intake is sufficient to meet their requirement, depends on the protein quality included. The amino acid composition of the selected pet food showed limited variability among different categories, and some products displayed certain essential amino acids deficiency. Chapter 3 is centered on the fatty acid profile of 22 complete and 21 complementary pet foods. Fat is an important component of pet food which provides essential fatty acids and improves the palatability. Moreover, specific omega‐3&6 supplements are formulated to improve the pet’s health in certain disease conditions. The lipid composition of the analyzed products showed great variability, mainly depending on the ingredients included, and unacceptable discordances were found among labeled and analyzed values. 5 The aim of the study reported in Chapter 4 was to shed light upon the problem of contamination in novel protein diets and hydrolyzed protein diets testing for the presence of 19 animal species. Elimination diets are common tools for the diagnosis of adverse food reaction in dogs and cats, but the presence of undeclared ingredients may negatively interfere with the trial and prevent the veterinarian from making an appropriate diagnosis. Of the 40 analyzed products, only ten presented a content that correctly matched the label. In Chapter 5 the evaluation of the microbiological quality of raw‐meat pet foods purchased online is reported. It has been well documented that feeding raw diets increases exposure to dangerous bacteria for both pets and humans, raising questions about the quality of raw meat‐based products intended for dogs and cats. The collected products were largely contaminated by microbes and more than half of the samples resulted positive for potentially zoonotic bacteria. Finally, the last chapter of this PhD thesis deals with the gut microbiota instead. Previous work has shown obesity to be associated with changes in intestinal microbiota; however, while obesity has become a health epidemic in pets, limited information is available about the role of the canine gut microbiota. The aim of the study described in Chapter 6 was to investigate whether alterations in the intestinal microbiota may be associated with canine obesity. Using 16S rRNA gene sequencing, the composition of the fecal microbiota in 15 lean and 14 overweight pet dogs was evaluated. The gut microbiota of the recruited dogs showed considerable interindividual variation, but no significant differences based on body condition.Una dieta completa e bilanciata è essenziale per mantenere gli animali in buona salute. La maggior parte di cani e gatti in Europa, Stati Uniti e altri paesi sviluppati consuma pet food industriali il cui mercato è in costante crescita ogni anno. Gli alimenti commerciali per pets devono essere sicuri e nutrizionalmente bilanciati per soddisfare i loro fabbisogni; pertanto, ai produttori è richiesto di seguire le raccomandazioni fornite dalla Federazione europea dell'industria alimentare per animali domestici. La legge europea stabilisce inoltre regole per l’etichettatura allo scopo di fornire informazioni adeguate ai consumatori. Allo stesso tempo si sta assistendo ad una aumentata popolarità delle diete crude commerciali per pet, poiché molti proprietari decidono di alimentare i propri animali con una dieta più naturale o ancestrale. Poiché nelle etichette mancano informazioni precise e i controlli regolari scarseggiano, possono insorgere dei dubbi circa la qualità delle diete commerciali per pet, sia estruse che crude. L’obiettivo generale di questo progetto è stato quello di fornire maggiori dettagli circa il valore nutritivo del pet food e circa la qualità microbiologica dei prodotti a base di carne cruda. Una introduzione generale riguardo alle caratteristiche e al mercato del pet food è fornita nel Capitolo 1. Il Capitolo 2 illustra la composizione amminoacidica di 80 pet food completi. La proteina dietetica dovrebbe fornire tutti gli aminoacidi essenziali che gli animali non possono sintetizzare, e un l’apporto sufficiente a garantire i loro fabbisogni dipende dalla sua qualità. Il pet food selezionato ha dimostrato una variabilità limitata fra categorie differenti, e alcuni prodotti hanno dimostrato alcune carenze di determinati amminoacidi essenziali. Il Capitolo 3 è incentrato sul profilo lipidico di 22 mangimi completi e 21 complementari. I grassi sono una componente importante del pet food in quanto fornisce acidi grassi essenziali e ne aumenta l’appetibilità. Inoltre, specifici integratori a base di omega‐3&6 vengono formulati per migliorare la condizione di salute in animali affetti da certe patologie. I prodotti analizzati hanno mostrato un’ampia variabilità, dipesa soprattutto dagli ingredienti inclusi, e discordanze inaccettabili sono state trovate fra valori i riportati in etichetta e quelli analizzati. L’obiettivo dello studio riportato nel Capitolo 4 è stato quello di far luce sul problema della contaminazione in diete novel protein e idrolizzate testando la presenza di 19 specie animali. Le diete di eliminazione sono uno strumento valido per la diagnosi di reazioni avverse al cibo in cani e gatti, ma la presenza di ingredienti non dichiarati può impedire al veterinario di formulare una diagnosi appropriata. Dei 40 prodotti analizzati, solo dieci presentavano un’etichettatura coerente con il contenuto. Nel Capitolo 5 è riportata la valutazione della qualità microbiologica di alimenti a base di carne cruda per pet acquistati online. È ampiamente documentato il fatto che le diete crude aumentino l’esposizione sia di animali che di umani a batteri pericolosi, e i prodotti analizzati erano abbondantemente contaminati da microrganismi, più della metà risultatando positiva per batteri potenzialmente zoonotici. Infine, l’ultimo capitolo di questa tesi di Dottorato riguarda il microbiota intestinale. L’obiettivo dello studio descritto nel Capitolo 6 è stato quello di indagare se delle alterazioni nel microbiota intestinale potessero essere associate all’obesità nel cane. Mediante il sequenziamento del gene 16S rRNA, è stata valutata la composizione del microbiota fecale in 15 cani normopeso e 14 sovrappeso. Il microbiota dei cani esaminati ha mostrato una considerevole variazione interindividuale, ma non delle differenze significative per quanto concerne la condizione corporea

Valutazione della qualità del pet food e analisi del microbiota intestinale in cani con differente condizione corporea

A complete and balanced diet is essential to maintain animals in good health. Most dogs and cats in Europe, the United States and other developed countries are fed commercially manufactured pet foods, whose market is still growing every year. Commercial pet diets should provide safe and nutritionally balanced foods to meet the animal’s nutritional needs; therefore, pet food manufacturers are required to follow the recommended allowances presented by the European Pet Food Industry Federation. European feed law also establishes rules and requirements for labelling to provide adequate information for consumers. At the same time, we are seeing an increasing popularity of commercial raw pet food diets, as many owners choose to feed a more natural or ancestral diet to their pets due to anecdotal health benefits. However, since pet food labels lack precise information and there is a scarcity of regular controls, concerns may arise about the quality of commercially available pet diets, both extruded and raw. The overall aim of this project was therefore to provide greater insight into the nutrient composition of commercial pet food and the microbiological quality of raw meat‐based products. First, a general introduction about pet foods’ features and sales is provided in Chapter 1. Chapter 2 explores the amino acid composition of 80 complete pet foods. Dietary proteins should provide all the essential amino acids that animals cannot synthesize, and whether the intake is sufficient to meet their requirement, depends on the protein quality included. The amino acid composition of the selected pet food showed limited variability among different categories, and some products displayed certain essential amino acids deficiency. Chapter 3 is centered on the fatty acid profile of 22 complete and 21 complementary pet foods. Fat is an important component of pet food which provides essential fatty acids and improves the palatability. Moreover, specific omega‐3&6 supplements are formulated to improve the pet’s health in certain disease conditions. The lipid composition of the analyzed products showed great variability, mainly depending on the ingredients included, and unacceptable discordances were found among labeled and analyzed values. 5 The aim of the study reported in Chapter 4 was to shed light upon the problem of contamination in novel protein diets and hydrolyzed protein diets testing for the presence of 19 animal species. Elimination diets are common tools for the diagnosis of adverse food reaction in dogs and cats, but the presence of undeclared ingredients may negatively interfere with the trial and prevent the veterinarian from making an appropriate diagnosis. Of the 40 analyzed products, only ten presented a content that correctly matched the label. In Chapter 5 the evaluation of the microbiological quality of raw‐meat pet foods purchased online is reported. It has been well documented that feeding raw diets increases exposure to dangerous bacteria for both pets and humans, raising questions about the quality of raw meat‐based products intended for dogs and cats. The collected products were largely contaminated by microbes and more than half of the samples resulted positive for potentially zoonotic bacteria. Finally, the last chapter of this PhD thesis deals with the gut microbiota instead. Previous work has shown obesity to be associated with changes in intestinal microbiota; however, while obesity has become a health epidemic in pets, limited information is available about the role of the canine gut microbiota. The aim of the study described in Chapter 6 was to investigate whether alterations in the intestinal microbiota may be associated with canine obesity. Using 16S rRNA gene sequencing, the composition of the fecal microbiota in 15 lean and 14 overweight pet dogs was evaluated. The gut microbiota of the recruited dogs showed considerable interindividual variation, but no significant differences based on body condition

Valutazione della qualit\ue0 del pet food e analisi del microbiota intestinale in cani con differente condizione corporea

A complete and balanced diet is essential to maintain animals in good health. Most dogs and cats in Europe, the United States and other developed countries are fed commercially manufactured pet foods, whose market is still growing every year. Commercial pet diets should provide safe and nutritionally balanced foods to meet the animal’s nutritional needs; therefore, pet food manufacturers are required to follow the recommended allowances presented by the European Pet Food Industry Federation. European feed law also establishes rules and requirements for labelling to provide adequate information for consumers. At the same time, we are seeing an increasing popularity of commercial raw pet food diets, as many owners choose to feed a more natural or ancestral diet to their pets due to anecdotal health benefits. However, since pet food labels lack precise information and there is a scarcity of regular controls, concerns may arise about the quality of commercially available pet diets, both extruded and raw. The overall aim of this project was therefore to provide greater insight into the nutrient composition of commercial pet food and the microbiological quality of raw meat‐based products. First, a general introduction about pet foods’ features and sales is provided in Chapter 1. Chapter 2 explores the amino acid composition of 80 complete pet foods. Dietary proteins should provide all the essential amino acids that animals cannot synthesize, and whether the intake is sufficient to meet their requirement, depends on the protein quality included. The amino acid composition of the selected pet food showed limited variability among different categories, and some products displayed certain essential amino acids deficiency. Chapter 3 is centered on the fatty acid profile of 22 complete and 21 complementary pet foods. Fat is an important component of pet food which provides essential fatty acids and improves the palatability. Moreover, specific omega‐3&6 supplements are formulated to improve the pet’s health in certain disease conditions. The lipid composition of the analyzed products showed great variability, mainly depending on the ingredients included, and unacceptable discordances were found among labeled and analyzed values. 5 The aim of the study reported in Chapter 4 was to shed light upon the problem of contamination in novel protein diets and hydrolyzed protein diets testing for the presence of 19 animal species. Elimination diets are common tools for the diagnosis of adverse food reaction in dogs and cats, but the presence of undeclared ingredients may negatively interfere with the trial and prevent the veterinarian from making an appropriate diagnosis. Of the 40 analyzed products, only ten presented a content that correctly matched the label. In Chapter 5 the evaluation of the microbiological quality of raw‐meat pet foods purchased online is reported. It has been well documented that feeding raw diets increases exposure to dangerous bacteria for both pets and humans, raising questions about the quality of raw meat‐based products intended for dogs and cats. The collected products were largely contaminated by microbes and more than half of the samples resulted positive for potentially zoonotic bacteria. Finally, the last chapter of this PhD thesis deals with the gut microbiota instead. Previous work has shown obesity to be associated with changes in intestinal microbiota; however, while obesity has become a health epidemic in pets, limited information is available about the role of the canine gut microbiota. The aim of the study described in Chapter 6 was to investigate whether alterations in the intestinal microbiota may be associated with canine obesity. Using 16S rRNA gene sequencing, the composition of the fecal microbiota in 15 lean and 14 overweight pet dogs was evaluated. The gut microbiota of the recruited dogs showed considerable interindividual variation, but no significant differences based on body condition