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.

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.

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 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.

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.

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.

Efficacy of a Dietary Polyherbal Formula on the Performance and Gut Health in Broiler Chicks after Experimental Infection with Eimeria spp.

One-hundred and fifty, one-day-old Ross-308 female chicks were randomly allocated to five equal treatments: NCONTR negative control-not challenged; PCONTR positive control-challenged; PHERB1 and PHERB2 diets were supplemented with phytogenic formula (1 and 2 g/kg feed, respectively)-challenged; PSALIN diet was supplemented with salinomycin (60 mg/kg feed)-challenged. Challenge was made by oral inoculation with 3.5 × 104 E. acervulina, 7.0 × 103 E. maxima and 5.0 × 103 E. tenella oocysts, at 14 days of age. One week post inoculation, bloody diarrhea, oocysts numbers, and intestinal lesions were evaluated, along with intestinal microbiota, viscosity, and pH of digesta, and histopathology. PHERB2 had a comparable (p ≤ 0.001) growth performance and feed conversion ratio to PSALIN. PHERB1 and PHERB2 had similar (p ≤ 0.001) oocyst counts to PSALIN and lower than PCONTROL. PHERB2 and PSALIN had lower (p ≤ 0.001) jejunal, ileal, and cecal lesion scores compared to PCONTR. PHERB1 and PHERB2 had higher (p ≤ 0.001) jejunal and cecal lactobacilli and lower (p ≤ 0.001) coliform counts compared to other treatments. PCONTR had lower (p ≤ 0.001) jejunum villus height, height to crypt ratio, and villus goblet cells. Breast and thigh meat resistance to oxidation was improved (p ≤ 0.001) in PHERB1 and PHERB2 compared to the PCONTR. The polyherbal formula exerted a substantial improvement on growth performance and intestinal health of the Eimeria-challenged birds.

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.

Molecular epidemiology of fowl adenoviruses in Greece.

Outbreaks of inclusion body hepatitis (IBH) and adenoviral gizzard erosion have been anecdotally reported in Greece since approximately 2011. However, a relevant increase in clinical outbreaks compatible with IBH has been described since 2014. Unfortunately, with limited exceptions, only serological assays were performed, and involved strains were not properly characterized. In the present study, 35 outbreaks were investigated in the period between July 2017 and February 2018 in Greece. In addition to clinical and histopathological diagnosis, fowl adenovirus (FAdV) presence was investigated by PCR and sequencing. Thirty-four out of 35 samples tested FAdV positive. Twenty-nine (85.29%) and 5 (14.71%) strains were classified as FAdV-E and FAdV-D, respectively. Fowl adenovirus-E strains were genetically homogeneous and formed an independent cluster of Greek-only sequences, including the sole previously available sequence, suggesting the prolonged circulation of this species in Greece. On the contrary, FAdV-D strains were more heterogeneous and closely related to strains sampled in other European countries, testifying the occurrence of multiple introduction events. The evaluation of phylogenetic relationships, geographic clustering, age of infection, and origin of the broiler breeder flocks suggests that both vertical and horizontal transmission are important in FAdV epidemiology in Greece and highlights the limited efficacy of currently implemented control measures. Of note, a significantly higher mortality was observed in precociously infected flocks, likely because of the higher susceptibility of younger animals. This evidence stresses the need of preventing vertical and/or early infection to limit the economic impact of adenovirus-induced diseases.

Dimethylglycine Supplementation in Reduced Energy Broilers' Diets Restores Performance by Improving Nutrient Digestibility.

Reducing the energy content of broiler diets could lead to the formulation of diets with reduced production cost. Dimethylgycine (DMG) has been used as a dietary supplement to enhance dietary fat utilization in poultry. The objective of the present study was to investigate the effects of DMG supplementation in reduced energy diets on performance and nutrient digestibility in broiler chickens. Four hundred and eighty day-old broilers were randomly allocated to three dietary treatments: a standard energy diet (PC treatment), a reduced energy diet by 66 kcal/kg (NC treatment) and the reduced energy diet supplemented with 500 mg/kg of DMG (DMG treatment). Fat digestibility was significantly higher in DMG group, compared to PC and NC groups. Intestines and gizzard lesion scores were found to be lower in the DMG group compared to PC. DMG supplementation resulted in lower jejunum pH and ileum viscosity in broilers. Overall, the present study showed that DMG supplementation in reduced energy broiler diets restored growth performance to the levels obtained with a standard diet. This result was probably mediated by the positive effects on the gastrointestinal function of the broilers after DMG supplementation, as evidenced by the improved nutrient digestibility, the reduced gross lesion scores and the lower values in intestinal pH and viscosity.

Poultry management: a useful tool for the control of necrotic enteritis in poultry.

The intestinal ecosystem of poultry has been inevitably changed as a result of the ban of antimicrobial growth promoters. The re-emergence of necrotic enteritis has been the most significant threat for the poultry industry, which, in clinical form, causes high mortality and in subclinical forms, affects growth and feed conversion. It is one of the most common and economically devastating bacterial diseases in modern broiler flocks in terms of performance, welfare and mortality. Necrotic enteritis is a multi-factorial disease process, in which a number of co-factors are usually required to precipitate an outbreak of the disease. Although, Clostridium perfringens has been identified as the aetiological agent of the disease, the predisposing factors that lead to over-proliferation of C. perfringens and the subsequent progression to disease are poorly understood. Any factor that causes stress in broiler chicks could suppress the immune system and disturb the balance of the intestinal ecosystem, in such a way that the risk of a necrotic enteritis (NE) outbreak increases. Poultry management could significantly affect the pathogenesis of NE. In particular, feed restriction and coccidiosis vaccination can protect against NE, while extreme house temperature, feed mycotoxins and high stocking density predispose to NE. It becomes really important to understand the pathogenesis of the disease, as well as to clarify the interactions between husbandry, nutritional and infectious factors and the outbreak of necrotic enteritis. This is necessary and extremely important in order to develop managerial strategies at the farm level to control the incidence and severity of the disease in the post-antibiotic era.

The potential role of migratory birds in the transmission of zoonoses.

The instinct for survival leads migratory birds to exploit seasonal opportunities for breeding habitats and food supplies. Consequently, they travel across national and international borders. These birds are distinguished in local migrants, short-distance, long-distance and vagrant and nomadic migrants. They can transfer micro-organisms across the globe and play a significant role in the ecology and circulation of pathogenic organisms. They are implicated in the transmission of zoonoses as biological and mechanical carriers and as hosts and carriers of infected ectoparasites. They can cause water-borne, tick-borne and insect-borne diseases. Favourable agents, such as seasonality and stress due to migration, influence the transmission of pathogens. The migration of birds is a natural phenomenon that is followed by the unavoidable repercussions of the participation of these birds as carriers or hosts in the transmission of pathogens. It is not possible to interrupt this sequence but risks can be minimised by controlling and preventing perilous situations. Surveillance of wetlands, 'stopovers', places of destination and wintering regions can be done. Furthermore, the implementation strict biosecurity measures that reduce contact with migratory birds will limit the transmission of pathogens.