Runting and stunting syndrome (RSS) is an enteric viral disease in commercial poultry that directly affects gut health; however, its influence on gut microbiota remains unknown. This study aimed to investigate the compositional changes in the bacterial community of the ileum of 7-day-old broiler chicks naturally affected and not affected by RSS, using next-generation sequencing (NGS) technology. Twenty-one samples were obtained from the ileal contents and mucosa of 11 chicks with RSS and 10 healthy chicks, raised in a dark house system located on a farm in the state of Minas Gerais, Brazil. The results revealed overall changes in the gut microbiota of the chicks with RSS, including a decrease in microbial richness and diversity. In particular, there was a decrease in Lactobacillus and an increase in Candidatus Arthromitus and Clostridium sensu stricto 1. These results indicate a relationship between viral infection and the gut microbial composition, which can cause gut dysbiosis and may influence inflammation in this organ.RESEARCH HIGHLIGHTSRSS causes dysbiosis of the gut microbiota of the ilea of chicks.A difference was found in gut microbiota between chicks with and without RSS.Candidatus Arthromitus was predominant in chicks with RSS.Clostridium sensu stricto 1 was strictly associated with chicks with RSS.

Proliferative enteropathy is an enteric disease caused by the bacterium Lawsonia intracellularis, which affects several species of domestic and wild animals. The mechanisms underlying the mechanisms employed by L. intracellularis to cause host cell proliferation are poorly understood, mostly because this bacterium is extremely difficult to isolate and propagate in vitro. Comparative genomics methods for searching for genes orthologous to genes known to be associated with pathogenesis allow identification of genes potentially involved in pathogenesis by the pathogen of interest. The goal of this study was to carry out in silico research on L. intracellularis genes orthologous to genes required for intracellular invasion and survival present in other pathogenic bacteria, particularly Brucella abortus, B. melitensis, B. suis, Listeria monocytogenes, Mycobacterium tuberculosis, Mycobacterium avium subspecies paratuberculosis, Salmonella enterica, Yersinia pestis, Y. enterocolitica, and Y. pseudotuberculosis. A total of 127 genes associated with invasion and intracellular survival from five known intracellular bacteria were mapped against the predicted proteomes of all L. intracellularis strains publicly available on GenBank, using the OrthoFinder program. A total of 45 L. intracellularis genes were orthologous to genes associated with pathogenesis of other intracellular bacteria. Genes putatively associated with signal the transduction of chemotaxis and cell motility were identified. Genes related to DNA binding and repair were also identified, with some of them supporting a possible association of bacteria with macrophages or inducing pro-inflammatory responses. The homology-based identification of these genes suggests their potential involvement in the virulence and pathogenicity of L. intracellularis, opening avenues for future research and insights into the molecular mechanisms of Lawsonia-elicited proliferative enteropathy.

Studies investigating the pathogenesis of Lawsonia intracellularis often require bacterial quantification in suspension. However, due to the organism’s fastidious growth requirements—being both intracellular and microaerophilic—traditional quantification methods, such as colony-forming unit counting, are not feasible. Currently, the only widely available method for quantifying L. intracellularis is real-time quantitative PCR (RT-qPCR). Unfortunately, the time required to perform RT-qPCR is incompatible with the bacterium’s limited survival outside its intracellular and microaerophilic environment. As a result, bacterial suspensions are typically quantified subjectively, based on the researcher’s experience for immediate use, with RT-qPCR conducted afterward. Optical density (OD) spectrophotometry is a rapid, although indirect, method of estimating bacterial concentrations in suspension, and it has been applied successfully to fast-growing prokaryotic species. Therefore, the objective of this study was to determine the correlation between RT-qPCR results and the optical density of L. intracellularis suspensions, with the goal of enabling the use of spectrophotometry for immediate bacterial quantification in experimental settings. Optical densities (ODs) were measured at 405 nm and 450 nm, using either a cuvette or microplate, while RT-qPCR was employed to establish a standard curve from samples of known concentration and to quantify the concentration of L. intracellularis in the test suspensions. Four comparison variations between OD and RT-qPCR were evaluated: (1) spectrophotometry at 405 nm using a cuvette vs. RT-qPCR; (2) spectrophotometry at 405 nm using a microplate vs. RT-qPCR; (3) spectrophotometry at 450 nm using a cuvette vs. RT-qPCR; and (4) spectrophotometry at 450 nm using a microplate vs. RT-qPCR. The tests were conducted in two independent replications, with each sample analyzed in duplicate. In all variations, the correlation between the bacterial concentrations determined by RT-qPCR and those estimated by OD was greater than 80%, with a statistical significance of p < 0.05. The following OD conversion equations for determining the number of microorganisms/mL were obtained: (1) f(x) = −7.438 × 10⁸ + 1.797 × 10¹⁰. x; (2) f(x) = 3.255 × 10⁸ + 3.003 × 10⁹. x; (3) f(x) = −8.006 × 10⁸ + 2.169 × 10¹⁰. x; (4) f(x) = 3.107 × 10⁸ + 3.758 × 10⁹. x. Here, “X” is the Ct value obtained by RT-qPCR. These findings enable researchers to improve the accuracy of their L. intracellularis experiments by utilizing optical spectrometry—a straightforward method that provides immediate results for determining bacterial concentration in suspensions.

The venom of Loxosceles spiders is characterized by a complex protein composition, which underlies the clinical condition known as loxoscelism. This syndrome is characterized mainly by gravitational spreading dermonecrotic lesions, but also, in rare cases, it can involve severe systemic complications, such as renal failure and hematological disorders. Although the pathophysiological mechanism of cutaneous loxoscelism is widely investigated, the neurological manifestations associated with the venom are rare and poorly researched. The present study aimed to investigate the histopathological alterations in guinea pigs' (Cavia porcellus) liver, kidney, and central nervous system (CNS) inoculated with Loxosceles intermedia venom. Sixteen guinea pigs were challenged with venom doses ranging from 11.627 to 350 μg/animal intradermally in the intrascapular region, while two additional animals were used as controls, receiving only 0.9 % saline solution in the same application region. Microscopic evaluation of hepatic and renal tissues demonstrated hepatic necrosis and acute renal tubular necrosis, respectively. In the CNS, lesions compatible with lymphoplasmacytic and histiocytic encephalitis, moderate focal gliosis, and neutrophilic margination in certain regions of the brain were identified. These neurological findings in guinea pigs exposed to L. intermedia venom are unprecedented, providing new evidence of CNS susceptibility to Loxosceles venom.

Background: Bites from spiders of the genus Loxosceles, popularly known as the brown recluse spider, can cause a syndrome called loxoscelism, which is classified into cutaneous and viscerocutaneous forms. The cutaneous form, which is more common, causes the development of a necrotizing lesion with gravitational spread at the bite site. The viscerocutaneous form, which is rarer, includes systemic symptoms such as intravascular hemolysis, thrombocytopenia, and acute kidney injury, which can lead to death. However, clinical reports suggest a potential cardiotoxic role of Loxosceles venom, although this has been little studied. It should be noted that phospholipase-D is the main toxin present in the venom and has been implicated as the factor responsible for the development of cardiac dysfunction. Given this context, the present study sought to deepen the understanding of the effects of L. intermedia spider venom on the hearts of guinea pigs, using a detailed approach involving cardiac biomarkers, electrocardiograms, and histopathological analysis through optical and electron microscopy. Materials, Methods & Results: Eighteen male guinea pigs (Cavia porcellus) weighing 600 g were used. Of these, 16 animals received intradermal injections of Loxosceles intermedia venom at doses ranging from 11.6 to 350 μg diluted in 0.5 mL of 0.9% saline, and 2 animals received only 0.5 mL of 0.9% saline, serving as controls. Cardiac function was assessed by serial electrocardiograms and by measuring serum cardiac biomarkers, such as creatine kinase (CK) and its MB fraction (CK-MB), lactate dehydrogenase (LDH), troponin I (TnI), N-terminal fragment of B-type natriuretic peptide (NT-ProBNP) and D-dimer at 24 and 72 h post-injection. After 72 h, the animals were euthanized for histopathological and ultrastructural analysis of cardiac tissue using optical and transmission electron microscopy. Survival time varied inversely with the venom dose. Electrocardiograms recorded before envenomation showed no abnormalities. However, after Loxosceles envenomation, ECG changes were observed, including cardiac arrhythmias such as junctional beats and supraventricular extrasystoles, paired ventricular extrasystoles associated with isolated junctional beats, isolated junctional beats and atrioventricular block, junctional arrhythmia, and isolated atrial extrasystole. Elevated CK, CK-MB, LDH, and TnI levels were observed. Histopathological changes included diffuse cardiac congestion and focal hemorrhage, while ultrastructural analysis revealed autophagic vacuoles and mitochondrial damage in cardiac fibers. Discussion: This research was the 1st to conduct a clinical study on systemic loxoscelism and its cardiovascular impacts. The data obtained demonstrate that loxoscelism envenomation can cause significant cardiac and biochemical alterations, with rapidly manifesting effects, such as atrial and ventricular premature beats. Atrial premature beats result from early atrial ectopic beats, and ventricular premature beats present as beats originating early in the ventricle, with a post-extrasystolic pause. In envenomations, ventricular premature beats are associated with damage to cardiac muscle fibers due to myotoxic activity and impaired perfusion of cardiac tissue. Junctional beats, which are early ectopic beats originating in the atrioventricular junction, were also observed. Therefore, these results indicate that loxoscelism venom can directly interfere with cardiac electrophysiology, causing conduction disturbances and arrhythmias. Furthermore, the venom was capable of generating histological lesions consistent with myocardial damage that may also indirectly contribute to impaired cardiac function through mechanisms such as hypoxia and hemorrhages. Keywords: Loxosceles intermedia, loxoscelism, cardiotoxicity, cardiac biomarkers, electrocardiogram.