Harnessing probiotics capability to combat Salmonella Heidelberg and improve intestinal health in broilers.

The poultry industry faces significant challenges in controlling Salmonella contamination while reducing antibiotic use, particularly with the emergence of Salmonella Heidelberg (SH) strains posing risks to food safety and public health. Probiotics, notably lactic acid bacteria (LAB) and Saccharomyces boulardii (SB) offer promising alternatives for mitigating Salmonella colonization in broilers. Understanding the efficacy of probiotics in combating SH and their impact on gut health and metabolism is crucial for improving poultry production practices and ensuring food safety standards. This study aimed to assess the inhibitory effects of LAB and SB against SH both in vitro and in vivo broilers, while also investigating their impact on fecal metabolites and caecal microbiome composition. In vitro analysis demonstrated strong inhibition of SH by certain probiotic strains, such as Lactiplantibacillus plantarum (LP) and Lacticaseibacillus acidophilus (LA), while others like SB and Lactobacillus delbrueckii (LD) did not exhibit significant inhibition. In vivo testing revealed that broilers receiving probiotics had significantly lower SH concentrations in cecal content compared to the positive control (PC) at all ages, indicating a protective effect of probiotics against SH colonization. Metagenomic analysis of cecal-content microbiota identified predominant bacterial families and genera, highlighting changes in microbiota composition with age and probiotic supplementation. Additionally, fecal metabolomics profiling showed alterations in metabolite concentrations, suggesting reduced oxidative stress, intestinal inflammation, and improved gut health in probiotic-supplemented birds. These findings underscore the potential of probiotics to mitigate SH colonization and improve broiler health while reducing reliance on antibiotics.

Chicken embryos are a valuable model for the selection of Bacillus subtilis for probiotic purposes.

Bacillus subtilis (BS) has been used as an excellent probiotic; however, some BS strains seem to be opportunist pathogens or do not present inhibitory effects in the pathogenic bacteria, so the characterization of BS strains for use in animals is mandatory. This study aimed to select nonpathogenic strains of BS, which can inhibit Salmonella spp., avian pathogenic Escherichia coli (APEC), and Campylobacter jejuni (CJ) using a chicken embryo as a model. We tested nine (9) strains of BS isolated from several sources (named A to I) in in vitro by tests of mucin degradation activity, haemolytic activity, apoptosis, and necrosis in fibroblasts from chickens. After the in vitro test, we tested the remaining seven (7) strains (strains A to G) in a chicken embryo (CE) as an in vivo model and target animal. We inoculated 3 log CFU/CE of each strain via allantoic fluid at the 10th day postincubation (DPI). Each treatment group consisted of eight CEs. At the 17th DPI we checked CE mortality, gross lesions, CE weight, and whether BS strains were still viable. To perform the cytokine, total protein, albumin, and reactive C protein analysis, we collected the CE blood from the allantoic vessel and intestine fragments in the duodenum portion for histomorphometric analysis. After the results in CEs, we tested the inhibition capacity of the selected BS strains for diverse strains of Salmonella  Heidelberg (SH), S. Typhimurium (ST), S. Enteritidis (SE), S. Minnesota (SM), S. Infantis (SI), Salmonella var. monophasic (SVM), APEC and C. jejuni. After the in vitro trial (mucin degradation activity, haemolytic activity, apoptosis, and necrosis), we removed two (2) strains (H and I) that showed ß-haemolysis, mucin degradation, and/or high apoptosis and necrosis effects. Although all strains of BS were viable in CEs at the 17th DPI, we removed four (4) strains (A, B, D, F) once they led to the highest mortality in CEs or a high albumin/protein ratio. C. jejuni inoculated with strain G had greater weight than the commercial strain, which could be further used for egg inoculation with benefits to the CE. From the tests in CEs, we selected the strains C, E, and G for their ability to inhibit pathogenic strains of relevant foodborne pathogens. We found that the inhibition effect was strain dependent. In general, strains E and/or G presented better or similar results than commercial control strains in the inhibition of SH, ST, SI, APEC, and two (2) strains of CJ. In this study, we selected BS strains C, E and G due to their in vitro and in vivo safety and beneficial effects. In addition, we emphasize the value of CE as an in vivo experimental model for assessing BS's safety and possible benefits for poultry and other animals.

A Ternary Copper (II) Complex with 4-Fluorophenoxyacetic Acid Hydrazide in Combination with Antibiotics Exhibits Positive Synergistic Effect against Salmonella Typhimurium.

Salmonella spp. continues to figure prominently in world epidemiological registries as one of the leading causes of bacterial foodborne disease. We characterised 43 Brazilian lineages of Salmonella Typhimurium (ST) strains, characterized drug resistance patterns, tested copper (II) complex as control options, and proposed effective antimicrobial measures. The minimum inhibitory concentration was evaluated for seven antimicrobials, isolated and combined with the copper (II) complex [Cu(4-FH)(phen)(ClO4)2] (4-FH = 4-fluorophenoxyacetic acid hydrazide and phen = 1,10-phenanthroline), known as DRI-12, in planktonic and sessile ST. In parallel, 42 resistance genes were screened (PCR/microarray). All strains were multidrug resistant (MDR). Resistance to carbapenems and polymyxins (86 and 88%, respectively) have drawn attention to the emergence of the problem in Brazil, and resistance is observed also to CIP and CFT (42 and 67%, respectively), the drugs of choice in treatment. Resistance to beta-lactams was associated with the genes blaTEM/blaCTX-M in 39% of the strains. Lower concentrations of DRI-12 (62.7 mg/L, or 100 µM) controlled planktonic and sessile ST in relation to AMP/SUL/TET and AMP/SUL/TET/COL, respectively. The synergistic effect provided by DRI-12 was significant for COL/CFT and COL/AMP in planktonic and sessile ST, respectively, and represents promising alternatives for the control of MDR ST.

First Report of Genetic Variability of Erysipelothrix sp. Strain 2 in Turkeys Associated to Vero Cells Morphometric Alteration.

Erysipelas is a disease caused by the Erysipelothrix genus, whose main species is the E. rhusiopathiae, the causative agent of animal erysipelas and human erysipeloid. We isolated Erysipelothrix sp. strain 2 (ES2) from turkey's organs during an outbreak in Brazilian commercial and breeder flocks with sepsis and high mortality levels. We studied 18 flocks, accounting for 182 samples, being eight flocks (84 samples) as ES2 positive with individuals demonstrating clinical symptoms and high mortality. We obtained the genetic variability of 19 samples with PFGE and found two clones, both from the same flock but different samples, and two clusters. Interestingly, we found 15 strains with high genetic variability among and within flocks. We have found a positive association between the proximity of ES2 positive turkey flocks and commercial swine sites through epidemiological analysis. We infected Vero cells with two different isolates and three distinct concentrations of ES2. After performing the morphometry, we recorded enlargement of the nucleus and nucleolus. Moreover, we performed fluorescence assays that resulted in apoptotic and necrotic cells. We demonstrated that ES2 could multiply in the extracellular medium and invade and survive inside Vero cells. For the first time, our finds show that ES2 may have similar behavior as E. rhusiopathiae as a facultative intracellular microorganism, which may represent a hazard for humans.

Comparative genomics of a novel clade shed light on the evolution of the genus Erysipelothrix and characterise an emerging species.

Erysipelothrix sp. isolates obtained from a deadly outbreak in farmed turkeys were sequenced and compared to representatives of the genus. Phylogenetic trees-supported by digital DNA:DNA hybridization and Average Nucleotide Identity-revealed a novel monophyletic clade comprising isolates from pigs, turkeys, and fish, including isolates previously described as E. sp. Strain 2. Genes coding for the SpaC protein, typically found in E. sp. Strain 2, were detected in all isolates of the clade. Therefore, we confirm E. sp. Strain 2 represents a unique species that may be isolated from a broad host range, and the name "Erysipelothrix takahashiae" is suggested. Core genome analysis showed that the pathogenic species of this genus, E. rhusiopathiae and the clade E. sp. Strain 2, are enriched in core functionalities related to nutrient uptake and transport, but not necessarily homologous pathways. For instance, whereas the aerobic DctA transporter may uptake C4-dicarboxylates in both species, the anaerobic DcuC transporter is exclusive of the E. sp. Strain 2. Remarkably, the pan-genome analysis uncovered that genes related to transport and metabolism, recombination and repair, translation and transcription in the fish isolate, within the novel clade, have undergone a genomic reduction through pseudogenization. This reflects distinct selective pressures shaping the genome of species and strains within the genus Erysipelothrix while adapting to their respective niches.

Fatal onion (Allium cepa) toxicosis in water buffalo (Bubalus bubalis).

Toxicosis caused by the ingestion of onion (Allium cepa) by 5 water buffalo (Bubalus bubalis) occurred in the district of Caçador, Santa Catarina, Brazil. The water buffalo died after ingestion of a large quantity of onion that had been left in the pasture. Clinical signs started 8 days postingestion and were characterized by pale mucous membranes, lethargy, and dark urine. At necropsy, pieces of onions were found in the rumen of 1 animal. The carcass smelled strongly of onion, and the kidneys and urine were dark brown. Microscopic renal lesions included tubular degeneration and necrosis with deposits of eosinophilic material in the cytoplasm of renal tubular epithelial cells and tubular lumina. These changes were consistent with hemoglobinuric nephrosis. Centrilobular coagulation necrosis was observed in the liver accompanied by hemorrhage and macrophages containing brown cytoplasmic pigment. A diagnosis of hemolytic anemia caused by onion toxicosis was based on the epidemiological data, clinical signs, macroscopic changes, and histological lesions.