The In Vitro Antibacterial Activity of Phytogenic and Acid-Based Eubiotics against Major Foodborne Zoonotic Poultry Pathogens.

The aim of the study was to investigate in vitro the antibacterial activity of 8 commercial drinking water additives against major zoonotic poultry pathogens (Campylobacter spp., Escherichia coli, Salmonella Typhimurium, Staphylococcus aureus and Listeria spp.). We tested two essential oil-based phytogenics (Phyto CSC Liquide B, AEN 350 B Liquid), two acid-based eubiotics (Salgard® liquid, Intesti-Flora), and four blends of essential oils and organic acids (ProPhorceTM SA Exclusive, Herbal acid, Rigosol-N and Eubisan 3000). The antibacterial activity was determined by estimating the minimum inhibitory concentration (MIC) using a microdilution method. The MICs of the products against Campylobacter spp. ranged from 0.071% to 0.568% v/v, in which Herbal acid, a blend rich in lactic and phosphoric acids, also containing thyme and oregano oils, exhibited the highest efficacy (MIC: 0.071% v/v) against all the tested strains. The MICs of the tested products against Escherichia coli ranged between 0.071% and 1.894% v/v. Specifically, the MIC of Rigosol-N, a blend of high concentrations of lactic and acetic acid, was 0.142% v/v for both tested strains, whereas the MICs of Intesti-Flora, a mixture rich in lactic and propionic acid, ranged from 0.284% to 0.568% v/v. The MICs of the products against Salmonella Typhimurium were between 0.095% and 1.894% v/v. Specifically, the MIC of Eubisan 3000, a blend rich in oregano oil, was 0.284% v/v. The MICs against Staphylococcus aureus were between 0.142% and 9.090% v/v. The MICs of Phyto CSC Liquide B, which is rich in trans-cinnamaldehyde, were between 3.030% and 9.090% v/v, showing the highest MIC values of all tested products. Finally, the MIC values of the tested commercial products against Listeria spp. were 0.095% to 3.030% v/v. The MICs of ProPhorceTM SA Exclusive, a highly concentrated blend of formic acid and its salts, were 0.095-0.142% v/v against Listeria spp., while the MICs of AEN 350 B Liquid were between 0.284% and 1.894% exhibiting high Listeria spp. strain variability. In conclusion, all the selected commercial products exhibited more or less antibacterial activity against pathogenic bacteria and, thus, can be promising alternatives to antibiotics for the control of zoonotic poultry pathogens and the restriction of antimicrobial-resistant bacteria.

Effects of Dietary Supplementation of Essential Oils, Lysozyme, and Vitamins' Blend on Layer Hen Performance, Viral Vaccinal Response, and Egg Quality Characteristics.

Maintaining respiratory tract health is crucial for layers, impacting gut health, laying performance, and egg quality. Viral diseases and standard vaccinations can compromise tracheal epithelium function, leading to oxidative stress. This study assessed the impact of a blend of feed additives, predominantly lysozyme (L), essential oils (EO), and vitamins (VIT) (referred to as L + EO + VIT), on young layers during an oral vaccination schedule. The supplementation significantly enhanced antibody titers for Newcastle Disease Virus (NDV) and Infectious Bronchitis Virus (IBV) after vaccination, trachea functionality and intestinal health in the jejunum, increased egg production, and exhibited a trend toward higher egg weight. Although feed intake showed no significant difference, egg quality remained consistent across experimental groups. Moreover, L + EO + VIT supplementation elevated total phenolic content in eggs, improving oxidative stability in both fresh and stored eggs, particularly under iron-induced oxidation. Notably, it substantially reduced yolk lipid peroxidation and albumen protein carbonyls. In conclusion, water supplementation with L + EO + VIT may enhance humoral immune response to IBV and NDV, positively impacting hen productivity. These findings indicate improved tracheal function and enhanced oxidative stability, emphasizing the potential of this blend in promoting overall health and performance in layers.

Investigation of Potential Gut Health Biomarkers in Broiler Chicks Challenged by Campylobacter jejuni and Submitted to a Continuous Water Disinfection Program.

The exploration of novel biomarkers to assess poultry health is of paramount importance, not only to enhance our understanding of the pathogenicity of zoonotic agents but also to evaluate the efficacy of novel treatments as alternatives to antibiotics. The present study aimed to investigate potential gut health biomarkers in broiler chicks challenged by Campylobacter jejuni and subjected to a continuous water disinfection program. A total of 144 one-day-old hatched broiler chicks were randomly allocated to four treatment groups with four replicates each, according to the following experimental design: Group A received untreated drinking water; Group B received drinking water treated with 0.01-0.05% v/v Cid 2000™ (hydrogen peroxide, acetic acid and paracetic acid); Group C was challenged by C. jejuni and received untreated drinking water; and Group D was challenged by C. jejuni and received drinking water treated with 0.01-0.05% v/v Cid 2000™. The use of Cid 2000™ started on day 1 and was applied in intervals until the end of the experiment at 36 days, while the C. jejuni challenge was applied on day 18. Potential biomarkers were investigated in serum, feces, intestinal tissue, intestinal content, and liver samples of broilers. Statistical analysis revealed significant increases (p < 0.001) in serum cortisol levels in C. jejuni-challenged broilers. Serum fluorescein isothiocyanate dextran (FITC-d) increased significantly (p = 0.004) in broilers challenged by C. jejuni and treated with drinking water disinfectant, while fecal ovotransferrin concentration also increased significantly (p < 0.001) in broilers that received the drinking water disinfectant alone. The gene expression levels of occludin (p = 0.003) and mucin-2 (p < 0.001) were significantly upregulated in broilers challenged by C. jejuni, while mucin-2 significantly increased in birds that were challenged and received the drinking water disinfectant (p < 0.001). TLR-4 expression levels were significantly (p = 0.013) decreased in both groups that received the drinking water disinfectant, compared to the negative control group. Finally, the C. jejuni challenge significantly increased (p = 0.032) the crypt depth and decreased (p = 0.021) the villus height-to-crypt-depth ratio in the ileum of birds, while the tested disinfectant product increased (p = 0.033) the villus height in the jejunum of birds. Furthermore, the counts of C. jejuni in the ceca of birds (p = 0.01), as well as its translocation rate to the liver of broilers (p = 0.001), were significantly reduced by the addition of the water disinfectant. This research contributes to novel insights into the intricate interplay of water disinfection and/or C. jejuni challenge with potential intestinal biomarkers. In addition, it emphasizes the need for continued research to unveil the underlying mechanisms, expands our understanding of broiler responses to these challenges and identifies breakpoints for further investigations.

In Vitro Investigation of the Antibacterial Activity of Nine Commercial Water Disinfectants, Acidifiers, and Glyceride Blends against the Most Important Poultry Zoonotic Bacteria.

Identifying and monitoring the efficiency of alternative biocides that are presently used in livestock is gaining vast attention. The objective of this study was to determine, in vitro, the antibacterial activity of nine commercial water disinfectants, acidifiers, and glyceride blends against clinical isolates or reference strains of zoonotic pathogens belonging to the genera Escherichia spp., Salmonella spp., Campylobacter spp., Listeria spp., and Staphylococcus spp. For each product, the antibacterial activity was tested in concentrations ranging from 0.002 to 1.136% v/v and expressed as the minimum concentration of the product that inhibits bacterial growth (MIC). Water disinfectants Cid 2000™ and Aqua-clean® recorded MICs ranging from 0.002 to 0.142% v/v, while the lowest MICs were recorded at two strains of Campylobacter (0.002-0.004% v/v). Virkon® S displayed various MICs (0.013-0.409% w/v) and was highly effective at suppressing the growth of Gram-positive bacteria such as S. aureus (0.013-0.026% w/v). The MICs of water acidifiers (Agrocid Super™Oligo, Premium acid, and Ultimate acid) and glyceride blends (CFC Floramix, FRA®LAC34, and FRA®Gut Balance) ranged from 0.036 to 1.136% v/v, and for most of these products, MICs were closely correlated by their ability to modify the pH of the culture medium close to 5. In conclusion, most of the tested products showed promising antibacterial activity; as a result, they would be good candidates for pathogen control in poultry farms and for reducing the emergence of antimicrobial resistance. However, further in vivo studies are recommended to provide relevant information for the underlying mechanisms, as well as for the establishment of the optimal dosage scheme for each product and their possible synergies.

Characterization of Extended Spectrum Cephalosporin-Resistant Escherichia coli Strains Isolated from Raw Poultry Carcasses in Catering Services in Northern Greece.

Antimicrobial resistance is considered a topic of utmost interest under the concept of "One Health", having severe implications in both human and veterinary medicine. Among the antibiotic-resistant bacteria, gram-negative bacteria, especially those belonging to the order of Enterobacterales (such as Escherichia coli), hold a prominent position in terms of both virulence and possessing/disseminating antimicrobial resistance (AMR) traits. The aim of this study was to examine the presence of extended-spectrum ß-lactamase producing E. coli isolates in raw poultry carcasses collected from a university club. Five hundred raw poultry skin samples were collected from the Aristotle University of Thessaloniki (AUTh) club in Thessaloniki, Greece. A total of 64% of the samples were positive for the presence of extended-spectrum ß-lactamase (ESBL)-producing E. coli. The isolates were further examined for their susceptibility to selected antibiotics by the disc diffusion method and were characterized as true ESBL, as producing class C cephalosporinases (AmpC) or "of unknown etiology" by the combination disc test. The 86 of the 120 isolates (71.67%) were classified as true ESBL, 24 (20.00%) as AmpC, and 10 (8.33%) as "of unknown etiology". The isolates were screened for the occurrence of ß-lactamase genes (blaTEM, blaCTX-M, blaSHV, and blaOXA). Thirty-six isolates (32 ESBL- and 4 AmpC-phenotype) harbored both blaTEM and blaCTX-M genes, twenty-two isolates (among which 19 ESBL-phenotype and 2 AmpC-phenotype) harbored blaCTX-M only, whereas twenty-six (14 ESBL- and 12 AmpC-phenotype) isolates harbored blaTEM alone. No isolate harboring blaSHV or blaOXA was detected. The results demonstrate the existence of E. coli isolates producing extended-spectrum ß-lactamases in poultry carcasses from Greece, pausing a risk for antibiotic resistance transfer to humans.

Investigation of the Effect of Three Commercial Water Acidifiers on the Performance, Gut Health, and Campylobacter jejuni Colonization in Experimentally Challenged Broiler Chicks.

This study investigated the effect of three commercial water acidifiers on the performance, gut health, and C. jejuni colonization in experimentally challenged broiler chicks. A total of 192 one-day-old broiler chicks (Ross 308®) were randomly allocated into 6 treatment groups with 4 replicates according to the following experimental design: group A, birds were not challenged and received tap water; group B, birds were challenged and received tap water; groups C, D, E, and F, birds were challenged and received tap water treated with 0.1% v/v SPECTRON®, with 0.1-0.2% v/v ProPhorce™ SA Exclusive, with 0.1-0.2% v/v Premium acid, and with 0.1-0.2% v/v Salgard® Liquid, respectively. The continuous water acidification evoked undesirable effects on broilers' performance and to an increased number of birds with ulcers and erosions in the oral cavity and the upper esophageal area. ProPhorce™ SA Exclusive and Premium acid significantly reduced the C. jejuni counts in the crop, whereas Salgard® Liquid significantly reduced the C. jejuni counts in the ceca of birds. At slaughter age, only Premium acid significantly reduced C. jejuni counts in the ceca of birds. All the tested products ameliorated the changes induced by C. jejuni infection in the pH in the ceca of birds. It can be concluded that besides the effectiveness of the tested products in controlling C. jejuni in broilers, their continuous application evoked undesirable effects on broilers' performance, leading to the need to modify the dosage scheme in future investigations.

First Detection and Identification of FAdV-8b as the Causative Agent of an Outbreak of Inclusion Body Hepatitis in a Commercial Broiler Farm in Greece.

Inclusion body hepatitis (IBH) is an economically important disease of chickens, with a worldwide distribution, caused by Fowl Aviadenoviruses (FAdVs). Currently, the increased number of cases, the virulence of the isolate strains, as well as the lack of cross-species protection highlight that detailed in-field data are fundamental for the development of successful control strategies. This case report provides a detailed clinicopathological investigation of an unusual IBH outbreak in a commercial broiler farm in the region of Macedonia, Greece. The farm consisted of 64,000 birds, originated from the same breeder stock and placed in three different houses (Flock A-C). At 20 days of age, a sudden increase in daily mortality was recorded in Flock A. It is worth mentioning that, although all flocks were serologically (indirect ELISA) and molecularly (RT-PCR) positive for FAdV, the mortality rate, attributed to IBH, was much higher in Flock A compared to others. The clinical manifestation included non-specific symptoms such as depression, inappetence, yellowish mucoid diarrhea, and lack of uniformity. At necropsy, typically, enlarged, pale, and friable livers were dominant, while sporadically lesions were recorded in the pancreas, kidneys, skeletal muscles, and lymphoid organs. The histopathological examination of liver samples showed multifocal inflammation, necrosis, and the presence of basophilic/ eosinophilic inclusion bodies in hepatocytes. In addition, the loss of the architecture of pancreatic lobules and the presence of fibrosis and foci of mononuclear cell aggregates were suggestive of chronic pancreatic inflammation. PCR analysis confirmed the presence of FAdV, belonging to species E, serotype FAdV-8b. Performance and financial calculations revealed that IBH increased Feed Conversion Ratio (FCR), feed cost/chick as well as feed cost/kg live weight, whereas the Livability (%) and the European Production Efficiency Factor (EPEF) were decreased in the most severely affected flocks (Flock A). This study is the first report of the detection and identification of FAdV serotypes associated with IBH in commercial broiler flocks in Greece. However, there is still a lack of information about the circulating FAdV serotypes in the country, and therefore epidemiological studies are needed to establish control strategies for IBH.

A Case of Infectious Laryngotracheitis in an Organic Broiler Chicken Farm in Greece.

Infectious laryngotracheitis is an economically significant viral disease of chickens, that mainly affects the upper respiratory tract, and is present worldwide. This case reports the first outbreak of infectious laryngotracheitis in a four-week-old organic broiler farm and surrounding flocks in Greece, with typical clinical symptoms and lesions, allegedly provoked by a wild strain of infectious laryngotracheitis virus. Our findings contradict the general perception indicating that the disease appears mainly in older birds and that vaccine strains are the primary cause of infectious laryngotracheitis outbreaks in most continents. A recombinant vectored vaccine was administered, supplementary to biosecurity measures, containing the viral spread. The responsible strain was potentially circulating in the area; therefore, an industry-wide holistic approach was applied, including the vaccination of neighboring broilers and breeders with the same vaccine, the rapid molecular diagnosis of the disease, and strict biosecurity protocols. The results of this holistic effort were effective because, following the application of vaccine and management protocols, manifestations of the disease in regional flocks dropped significantly, and there was no recurrence to date. These findings suggest that vaccination protocols should be modified, especially for organic broilers, to include vaccination against infectious laryngotracheitis.

Avian Mycobacteriosis and Molecular Identification of Mycobacterium avium Subsp. avium in Racing Pigeons (Columba livia domestica) in Greece.

In this report, cases of avian mycobacteriosis in two lofts of racing pigeons are described. Three racing pigeons of 2-year old from the first loft (A) and four racing pigeons of 4-5 years old from the second loft (B) were submitted to the Unit of Avian Medicine for clinical examination and necropsy. In the case history chronic and debilitating disease was reported. The clinical signs included emaciation, depression, lameness, periorbital swelling and diarrhea, although the appetite was normal. Post mortem lesions involved an enlarged spleen with multiple different sized yellow nodules. Similar lesions were also observed in the liver, conjunctiva of the inferior eyelids and in the femoral bone marrow. The suspicion of avian mycobacteriosis was based on history, clinical signs and typical lesions. In order to confirm the diagnosis, histopathology was performed on tissue sections and revealed the presence of multiple granulomas with central necrosis. In addition, Ziehl-Neelsen positive bacilli were observed in histological sections and smears from the granulomas of the affected tissues. Molecular analysis identified the causative agent as Mycobacterium avium subsp. avium. This is the first case report of avian mycobacteriosis in Greece, which describes the presence of granulomatous conjunctivitis and the molecular identification of M. avium subsp. avium as the causative agent in racing pigeons.

Investigation of a Novel Multicomponent Mycotoxin Detoxifying Agent in Amelioration of Mycotoxicosis Induced by Aflatoxin-B1 and Ochratoxin A in Broiler Chicks.

The present study was designed to determine the efficacy of a novel multicomponent mycotoxin detoxifying agent (MMDA) containing modified zeolite (Clinoptilolite), Bacillus subtilis, B. licheniformis, Saccharomyces cerevisiae cell walls and silymarin against the deleterious effects of Aflatoxin B1 (AFB1) and Ochratoxin A (OTA) in broiler chicks. A total of 160 one-day-old Ross 308® broiler chicks were randomly allocated in four treatment groups, with four replicates, according to the following experimental design for 42 days. Group A received a basal diet; Group B received a basal diet contaminated with AFB1 and OTA at 0.1 mg/kg and 1 mg/kg, respectively; Group C received a basal diet contaminated with AFB1 and OTA and MMDA at 1 g/kg feed, and Group D received a basal diet contaminated with AFB1 and OTA and MMDA at 3 g/kg feed. Results showed that ingested mycotoxins led to significant (p ≤ 0.05) reduction in body weight and feed conversion from 25 days of age, induced histopathological changes, increased the pH of the intestinal content, and altered the biochemical profile of birds with significantly (p ≤ 0.05) increased aspartate aminotransferase (AST) values (p ≤ 0.05). On the other hand, the supplementation of MMDA significantly (p ≤ 0.05) improved the feed conversion ratio (FCR) during the second part of the study, diminished biochemical alterations, reduced pH in jejunal and ileal content, and E. coli counts in the caeca of birds (p ≤ 0.05). It may be concluded that the dietary supplementation of the MMDA partially ameliorated the adverse effects of AFB1 and OTA in broilers and could be an efficient tool in a mycotoxin control program.