Disposable electrochemical platform based on solid-binding peptides and carbon nanomaterials: an alternative device for leishmaniasis detection.

Neglected tropical diseases are those caused by infectious agents or parasites and are considered endemic in low-income populations. These diseases also have unacceptable indicators and low investment in research, drug production, and control. Tropical diseases such as leishmaniasis are some of the main causes of morbidity and mortality around the globe. Electrochemical immunosensors are promising tools for diagnostics against these diseases. One such benefit is the possibility of assisting diagnosis in isolated regions, where laboratory infrastructure is lacking. In this work, different peptides were investigated to detect antibodies against Leishmania in human and canine serum samples. The peptides evaluated (395-KKG and 395-G) have the same recognition site but differ on their solid-binding domains, which ensure affinity to spontaneously bind to either graphene oxide (GO) or graphene quantum dots (GQD). Cyclic voltammetry and differential pulse voltammetry were employed to investigate the electrochemical behavior of each assembly step and the role of each solid-binding domain coupled to its anchoring material. The graphene affinity peptide (395-G) showed better reproducibility and selectivity when coupled to GQD. Under the optimized set of experimental conditions, negative and positive human serum samples responses were distinguished based on a cut-off value of 82.5% at a 95% confidence level. The immunosensor showed selective behavior to antibodies against Mycobacterium leprae and Mycobacterium tuberculosis, which are similar antibodies and potentially sources of false positive tests. Therefore, the use of the graphene affinity peptide as a recognition site achieved outstanding performance for the detection of Leishmania antibodies.

Gold-binding peptide as a selective layer for electrochemical detection of SARS-CoV-2 antibodies.

Electrochemical immunosensors are excellent alternatives to prepare portable platforms used for rapid and inexpensive diagnostic of infectious diseases such as the recently emerged COVID-19. Incorporating synthetic peptides as selective recognition layers combined with nanomaterials such as gold nanoparticles (AuNPs) can significantly enhance the analytical performance of immunosensors. In the present study, an electrochemical immunosensor based on solid-binding peptide was built and evaluated towards SARS-CoV-2 Anti-S antibodies detection. The peptide used as recognition site has two important portions: one based on the viral receptor binding domain (RBD), capable of recognizing antibodies of the spike protein (Anti-S), and another suitable for interacting with gold nanoparticles. Gold-binding peptide (Pept/AuNP) dispersion was used directly to modify a screen-printed carbon electrode (SPE). The voltammetric behavior of the [Fe(CN)6]3-/4- probe after every construction and detection step was recorded using cyclic voltammetry by assessing the stability of the Pept/AuNP as a recognition layer onto the electrode surface. Differential pulse voltammetry was used as a detection technique, and a linear working range from 75 ng mL-1 to 15 µg mL-1 was established, with 1.059 µA dec-1 of sensitivity and R2 = 0.984. The response selectivity against SARS-CoV-2 Anti-S antibodies was investigated in presence of concomitant species. The immunosensor was used to detect SARS-CoV-2 Anti-spike protein (Anti-S) antibodies in human serum samples, successfully differentiating between negative and positive responses of samples at a 95% confidence level. Therefore, the gold-binding peptide is a promising tool to be applied as a selective layer for antibody detection.

Effects of a microbial muramidase on the growth performance, intestinal permeability, nutrient digestibility, and welfare of broiler chickens.

The objective of these studies was to evaluate the inclusion of a microbial muramidase (MUR) in the diets of broiler chickens on the growth performance, intestinal permeability (IP), total blood carotenoid content, apparent ileal digestibility (AID), and foot pad dermatitis (FPD). In Experiment 1, a total of 1,000 one-day-old chicks were placed in floor-pens with reused litter, and randomly distributed into 4 treatments with 10 replicates each. Treatments were a basal diet (control), or basal diet supplemented with 15,000; 25,000 or 35,000 LSU (F)/kg of MUR. Feed intake (FI), body weight gain (BWG), and feed conversion ratio (FCR) were evaluated at d 21 and 43. Intestinal permeability was evaluated on d 35 by FITC-d, and FPD and AID on d 43. In Experiment 2, a total of 800 one-day-old chicks were placed in floor-pens with fresh litter, and randomly distributed into 4 treatments with 8 replicates each. Treatments were a basal diet (control), or basal diet supplemented with 25,000 or 35,000 LSU (F)/kg of MUR, and a fourth group where the basal diet was supplemented with enramycin. The birds were induced to a mild intestinal challenge. Feed intake, BWG, and FCR were evaluated on d 21 and d 42, and total blood concentration of carotenoids was evaluated on d 28. In experiment 1, 35,000 LSU (F)/kg of MUR promoted the best FCR (P < 0.05). Muramidase supplementation linearly increased the AID of dry matter, ash, and fat (P < 0.01), and regardless of the dose, MUR decreased the IP (P < 0.05). In Experiment 2, the supplementation of 35,000 LSU (F)/kg of MUR improved BWG and FCR in the entire cycle (1-42 d) and increased the concentration of carotenoids in the blood on d 28 compared to the control group (P < 0.05). These studies show that MUR improves growth performance of broilers by improving intestinal permeability, digestibility of dry matter, ash and fat, absorption of carotenoids, and reducing FPD.

Graphene-Binding Peptide in Fusion with SARS-CoV-2 Antigen for Electrochemical Immunosensor Construction.

The development of immunosensors to detect antibodies or antigens has stood out in the face of traditional methods for diagnosing emerging diseases such as the one caused by the SARS-CoV-2 virus. The present study reports the construction of a simplified electrochemical immunosensor using a graphene-binding peptide applied as a recognition site to detect SARS-CoV-2 antibodies. A screen-printed electrode was used for sensor preparation by adding a solution of peptide and reduced graphene oxide (rGO). The peptide-rGO suspension was characterized by scanning electron microscopy (SEM), Raman spectroscopy, and Fourier transform infrared spectroscopy (FT-IR). The electrochemical characterization (electrochemical impedance spectroscopy-EIS, cyclic voltammetry-CV and differential pulse voltammetry-DPV) was performed on the modified electrode. The immunosensor response is based on the decrease in the faradaic signal of an electrochemical probe resulting from immunocomplex formation. Using the best set of experimental conditions, the analytic curve obtained showed a good linear regression (r2 = 0.913) and a limit of detection (LOD) of 0.77 µg mL-1 for antibody detection. The CV and EIS results proved the efficiency of device assembly. The high selectivity of the platform, which can be attributed to the peptide, was demonstrated by the decrease in the current percentage for samples with antibody against the SARS-CoV-2 S protein and the increase in the other antibodies tested. Additionally, the DPV measurements showed a clearly distinguishable response in assays against human serum samples, with sera with a response above 95% being considered negative, whereas responses below this value were considered positive. The diagnostic platform developed with specific peptides is promising and has the potential for application in the diagnosis of other infections that lead to high antibody titers.

Novel approach based on GQD-PHB as anchoring platform for the development of SARS-CoV-2 electrochemical immunosensor.

In the present work, we report an innovative approach for immunosensors construction. The experimental strategy is based on the anchoring of biological material at screen-printed carbon electrode (SPE) modified with electrodeposited Graphene Quantum Dots (GQD) and polyhydroxybutyric acid (PHB). It was used as functional substract basis for the recognition site receptor-binding domain (RBD) from coronavirus spike protein (SARS-CoV-2), for the detection of Anti-S antibodies (AbS). SEM images and EDS spectra suggest an interaction of the protein with GQD-PHB sites at the electrode surface. Differential pulse voltametric (DPV) measurements were performed before and after incubation, in presence of the target, shown a decrease in voltametric signal of an electrochemical probe ([Fe(CN)6]3/4-). Using the optimal experimental conditions, analytical curves were performed in PBS and human serum spiked with AbS showing a slight matrix effect and a relationship between voltametric signal and AbS concentration in the range of 100 ng mL-1 and 10 µg mL-1. The selectivity of the proposed sensor was tested against yellow fever antibodies (YF) and the selective layer on the electrode surface did not interact with these unspecific antibodies. Eight samples of blood serum were analyzed and 87.5% of these total investigated provided adequate results. In addition, the present approach showed better results against traditional EDC/NHS reaction with enhancements in time and the possibility to develop an immunosensor in a single drop, since the proteins can be anchored prior to the electrode modification step.

Recombinant human interferon-α14 for the treatment of canine allergic pruritic disease in eight dogs.

BACKGROUND: Allergic pruritic diseases are increasingly common in dogs. This group of conditions hampers life quality as pruritus progressively interferes with normal behaviours. Therefore, new treatment modalities for canine allergic pruritic diseases are necessary. While novel drugs have recently reached the market, there is still the need for other therapeutic approaches. Some dogs are refractory even to the newer compounds, and cost is also an important issue for these. Older therapeutic modalities are only moderately successful or have considerable secondary effects, as is the case with glucocorticoids. OBJECTIVES: Report on the use of recombinant human interferon-α14 (rhIFN-α14) for the treatment of canine allergic pruritus. Following the experience with a similar compound in the Japanese market, it was expected that rhIFN-α14 could alter the Th1/Th2 disbalance that drives these diseases. METHODS: Here, we present an uncontrolled trial in which eight dogs with clinical diagnosis of allergic pruritus were treated with rhIFN-α14, either orally or via subcutaneous injections. Skin condition, microbiota and anti-interferon antibody levels were assessed. RESULTS: The parenteral use of interferon induced hypersensitivity in two of the three dogs in which it was used. The oral administration was consistently safe and could reduce signs of the allergic condition in three of the five treated animals. Treatment also altered the skin microbiota, as verified by next-generation sequencing. CONCLUSION: The present results indicate that rhIFN-α14 is a viable candidate for the treatment of canine allergic pruritus. Future controlled studies are needed, and the oral route is indicated for further trials.

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.

Effects of aroA deleted E. coli vaccine on intestinal microbiota and mucosal immunity.

E. coli infection of broilers can result in systemic diseases and productivity losses. Use of antimicrobials against this condition is common but other approaches, such as vaccination, are gaining ground. Anecdotal field reports indicate that intestinal health is improved unspecifically following E. coli live vaccination. We hypothesized that the intestine may be an important site for the functionality of the vaccine. Vaccine effects on the intestine were assessed. Spray vaccination induced marked alterations of the caecum microbiota of broilers within 3 days, and this effect gradually waned. However, T cell activation occurred in the spleen, but not in caecal tonsils, and anti-E. coli IgA was concentrated in the respiratory mucosae. Accordingly, IL-6 mRNA was produced in the lungs following immunization. Overall, these data are an initial indication that any vaccine-induced effects on the intestine are greatly associated with the microbiota. However, immunity conferred by vaccination is not primarily induced in gut-associated lymphoid tissues.

Fish oil supplementation enhances colon recovery after experimental colitis.

INTRODUCTION: Fish oil (FO) has an anti-inflammatory and pro-resolution activity and it has been used to restore physiological disturbances on inflammatory conditions. Here, we investigate whether FO supplementation could, acutely, prevent or restore inflammatory damages on experimental colitis. METHODS: Wistar rats orally received 2 g.kg-1.day-1 of FO for 30 days before induction of experimental colitis. Specimens were collected on the 2nd and 7th days after colitis-induction and intestinal mucus, inflammatory activity and colon integrity were determined. RESULTS: Experimental colitis did cause colon disruption and FO, acutely, did not prevent the loss of intestinal and fecal mucus, neither the increase of inflammatory activity and intestinal permeability. On the 7th day of colitis, FO soften the perturbations of experimental colitis, increasing histological and fecal mucus and, also decreased inflammatory activity, but this was not accompanied by intestinal permeability. CONCLUSION: FO did not protect, acutely, intestinal damages from experimental colitis, but at long run promotes higher intestinal recovery.

Cyclophosphamide Increases Lactobacillus in the Intestinal Microbiota in Chickens.

Recent data in humans indicate that immunosuppression is correlated with shifts in the intestinal microbiota. However, the relationship between immunosuppression and intestinal microbiota has not been studied in chickens. Thus, we investigated the correlations between immune cells and intestinal microbiota by massive parallel sequencing of the 16S rRNA bacterial gene in chickens immunosuppressed with cyclophosphamide. The results showed correlations between peripheral immune cells and intestinal microbiota. Surprisingly, an increase in the abundance of intestinal Lactobacillus in the immunosuppressed chickens was observed. These birds also had low intestinal IgA antibody levels among other alterations in the microbiota. These shifts indicate a role of the immunity system in controlling the microbiota of birds.IMPORTANCE Poultry production is a very intensive industry. Due to the substantial number of animals being raised by any one producer, even small variations in productivity lead to important economical outcomes. The intestinal microbiota of birds is under intense scrutiny by the immune system. Therefore, it is a factor that can influence the states of health and disease of the host. The body of knowledge on the interactions between these systems is gradually bringing practical guidance for poultry production.

A blocking antibody against canine CSF-1R maturated by limited CDR mutagenesis.

CSF-1R is a receptor mostly associated with the mononuclear phagocytic system. However, its expression within tumors has been linked with poor prognosis in both humans and dogs. Accordingly, several reports have demonstrated the beneficial effects of blocking CSF-1R in model systems of cancer. In this study, we generated a monoclonal antibody that could block CSF-1R in dogs as the first step to develop an anticancer drug for this species. Initially, an antibody was raised by the hybridoma methodology against the fragment responsible for receptor dimerization. mAb3.1, one of the resulting hybridoma clones, was able to bind macrophages in fixed tissues and was shown to inhibit cells of the mononuclear phagocytic line. Nevertheless, mAb 3.1 could not bind to some glycoforms of the receptor in its native form, while also demonstrating cross-reactivity with other proteins. To enhance binding properties of the mAb, five amino acids of the complementarity-determining region 2 of the variable heavy chain of mAb3.1 were mutated by PCR, and the variant scFv clones were screened by phage display. The selected scFv clones demonstrated improved binding to the native receptor as well as increased anti-macrophage activity. The resulting scFv antibody fragment presented here has the potential for use in cancer patients and in inflammatory diseases. Furthermore, this work provides insights into the use of such restricted mutations in antibody engineering.

Effect of an Enterococcus faecium probiotic on specific IgA following live Salmonella Enteritidis vaccination of layer chickens.

Probiotics and immunization are being widely adopted by the poultry industry with the goal of controlling Salmonella enterica. However, the interaction between these two management protocols has been sparsely studied. The present study aimed to understand the role of an Enterococcus faecium probiotic in the production of salmonella-specific IgA in layers immunized with a live vaccine. Four groups were used: "Control" (no vaccine or probiotic); "Probiotic" (which received an E. faecium product); "Vaccine" (immunized with two doses of a live attenuated S. Enteritidis vaccine); and "Vaccine + probiotic". Faecal salmonella-specific IgA was analysed 7 and 20 days post-vaccination (dpv) boost. At 7 dpv, the "Vaccine" and "Vaccine + probiotic" groups had similar IgA levels. However, at 20 dpv, IgA levels were two times higher in the "Vaccine + probiotic" group compared to the "Vaccine" group. To understand the role of the intestinal microbiota in this finding, bacterial diversity in faeces was analysed by 16S rRNA gene sequencing. The improvement in IgA production in probiotic-treated birds was accompanied by marked changes in the faecal microbiome. Some of the main differences between the "Vaccine" and "Vaccine + probiotic" groups included reduction of Escherichia-Shigella and increases in Blautia, Anaerotruncus and Lactobacillus in the latter group. Although no direct causal link can be established from this study design, it is possible that the E. faecium probiotic induces improved antibody production following vaccination via modulation of the intestinal microbiota.

Challenges and opportunities for monoclonal antibody therapy in veterinary oncology.

Monoclonal antibodies (mAbs) have come to dominate the biologics market in human cancer therapy. Nevertheless, in veterinary medicine, very few clinical trials have been initiated using this form of therapy. Some of the advantages of mAb therapeutics over conventional drugs are high specificity, precise mode of action and long half-life, which favour infrequent dosing of the antibody. Further advancement in the field of biomedical sciences has led to the production of different forms of antibodies, such as single chain antibody fragment, Fab, bi-specific antibodies and drug conjugates for use in diagnostic and therapeutic purposes. This review describes the potential for mAbs in veterinary oncology in supporting both diagnosis and therapy of cancer. The technical and financial hurdles to facilitate clinical acceptance of mAbs are explored and insights into novel technologies and targets that could support more rapid clinical development are offered.

Immunohistochemical Expression of CCR2, CSF1R and MMP9 in Canine Inflammatory Mammary Carcinomas.

BACKGROUND: Canine inflammatory mammary cancer (IMC) and its human counterpart, inflammatory breast cancer, are extremely aggressive types of cancer. Our aim was to characterize immunohistochemical expression of C-C chemokine receptor 2, colony stimulating factor 1 receptor and metalloproteinase-9 in canine IMC versus non-IMC and to analyze associations with clinicopathological variables. MATERIALS AND METHODS: Immunohistochemical staining of CCR2, CSF1R and MMP9 was performed in a series of 25 IMC and 15 non-IMC tumors. RESULTS: No differences in the expression of these biomarkers between IMC and non-IMC were observed. Distinct nuclear subcellular expression of CCR2 was observed in IMC (p<0.001). For IMC, higher CCR2 expression was associated with increased nuclear grade (p=0.037), and higher neoplastic MMP9 expression was associated with fewer mitoses (p=0.022), higher nuclear grade (p=0.047) and increased CSF1R expression (p=0.025). CONCLUSION: Expression of CCR2, CSF1R and MMP9 in canine IMC could contribute to increased nuclear pleomorphism, but the biological mechanisms involved warrant further investigation.

Canine mammary cancer cells direct macrophages toward an intermediate activation state between M1/M2.

BACKGROUND: Canine mammary carcinoma is the most common cancer in female dogs and is often fatal due to the development of distance metastasis. The microenvironment of a tumour often contains abundant infiltrates of macrophages called tumour-associated macrophages (TAMs). TAMs express an activated phenotype, termed M2, which sustains proliferation of cancer cells, and has been correlated with poor clinical outcomes in human cancer patients. Cancer cells themselves have been implicated in stimulating the conversion of macrophages to a TAM with an M2 phenotype. This process has yet to be fully elucidated. Here we investigate the interplay between cancer cells and macrophages in the context of canine mammary carcinoma. RESULTS: We show that cancer cells inhibit lipopolysaccharide (LPS)-induced macrophage activation. Further, we show that macrophage associated proteins, colony-stimulating factor (CSF)-1 and C-C motif ligand (CCL)-2, stimulate macrophages and are responsible for the effects of cancer cells on macrophages. We suggest the existence of a feedback loop between macrophages and cancer cells; while cancer cells influence the phenotype of the TAMs through CSF-1 and CCL2, the macrophages induce canine mammary cancer cells to upregulate their own expression of the receptors for CSF-1 and CCL2 and increase the cancer cellular metabolic activity. However, these cytokines in isolation induce a phenotypic state in macrophages that is between M1 and M2 phenotypes. CONCLUSIONS: Overall, our results demonstrate the extent to which canine mammary carcinoma cells influence the macrophage phenotype and the relevance of a feedback loop between these cells, involving CSF-1 and CCL2 as important mediators.

Effect of spray Escherichia coli vaccine on the immunity of poultry.

Avian pathogenic Escherichia coli is a current problem in the poultry industry, causing mortality and economic losses. This paper evaluates the dynamics in immune response after the use of spray vaccination against E. coli and, thereby, seeks to understand how the vaccine can provide protection. During the early stages of response to vaccination the presence of antigen-presenting cells is predominant, but these diminish within the first 7 days after vaccination. The immune correlate of protection of vaccination using the E. coli vaccine Poulvac E. coli (aroA-deficient mutant strain) probably does not depend on the production of circulating antibodies (as assessed through the presence of B lymphocytes) and is linked to the presence of CD4+TCRVbeta1+. These cells act on mucosa tissue stimulating the production of immunoglobulin A. Vaccination stimulated a high state of immunocompetence, as assessed by measurement of several cellular subsets. This state of "immune alertness," however, may be associated with reduced weight gain. The high presence of naive and memory CD8 cells in the vaccinated group at 14 and 21 days postvaccination may indicate greater ability in the future to prevent tissue invasion by E. coli, based on the possibility that these cells will proliferate rapidly to a new stimulus. The simultaneous use of vaccine with the antibiotic ceftiofur sodium interferes with the immune response obtained through vaccination. In combination, the data obtained in this study indicate that the immune response produced by a spray vaccine against E. coli is mainly a cellular response, especially relevant to the sites in contact with the pathogen. It is suggested that there is a strong cell migration to the mucous membranes, where macrophages act first and then lymphocytes take part to protect the host. It is believed that recruited lymphocytes will act in the production of secreted IgA, which probably plays a greater role in the defense when compared with circulating immunoglobulins. The assessment of cellular dynamics by flow cytometry made it possible to elucidate the operation mechanism of the live E. coli vaccine.

Comportamento de células do sistema imune frente ao desafio com Salmonella Enteritidis em aves tratadas e não tratadas com ácidos orgânicos / Behavior of cells of immune system to the challenge with Salmonella Enteritidis in birds treated and untreated with organic acids

A Salmonelose é uma importante zoonose, considerada a principal causa de infecções bacterianas, sendo associada ao consumo de produtos avícolas. Como alternativa de controle, ácidos orgânicos têm sido amplamente usados. No entanto, pouco se conhece sobre o estado imunológico de aves de produção, e uma avaliação deste status é necessária para proteger frente a enfermidades e para garantir à aplicação segura de agentes terapêuticos ou imunização profilática. Este trabalho teve como objetivo verificar o comportamento do sistema imunológico das aves previamente infectadas com Salmonella Enteritidis (SE) tratadas com um composto de ácidos orgânicos em diferentes concentrações administrado via água e ração comparando com as aves infectadas e não tratadas. Foram inoculados 120 frangos de corte com 1mL de SE, via oral, na concentração de 1,0 x 108 UFC/mL, no 1º e 2º dia de idade, divididos em seis tratamentos com duas repetições, utilizando 200, 400, 500 e 1000ppm do ácido orgânico. Aos 35 dias de vida das aves, foram coletados, de todos os grupos, alíquotas de sangue de 3mL em tubo contendo EDTA para a avaliação das células imunes através de citometria de fluxo. Foram analisadas as porcentagens circulantes de células CD4+, CD8β+, MHC I+, MHC II+, TCRVβ1+, TCRVβ2+ e CD28+. Para análise microbiológica foram coletadas tonsilas cecais destas aves. Observou-se com esse estudo que os ácidos orgânicos nas dosagens 1000ppm na água e 500ppm na ração durante, dois e sete dias respectivamente antes do abate, foram eficazes na redução da infecção por SE em frangos de corte, comprovadas pelo método microbiológico e demonstradas através do comportamento das células do sistema imune. No presente estudo as aves infectadas apresentaram uma proporção menor de células T auxiliares circulantes quando comparadas às aves infectadas, mas tratadas com o AO ou com o grupo não infectado. A mesma tendência pode ser observada para as células CD28+, TCRVβ1+ e MHC IIbright+, e, com menor resolução, para CD8β+.

Salmonellosis is an important zoonosis, considered the leading cause of bacterial infections, and is associated with the consumption of poultry products. As alternative control, organic acids have been widely used. However, little is known about the immune status of poultry production, and an evaluation of this status is necessary to protect against disease and to ensure the safe application of therapeutic agents or prophylactic vaccination. This study aimed to verify the behavior of the immune system of birds previously infected with Salmonella Enteritidis (SE) treated with a compound of organic acids in different concentrations administered via water and food, compared with the infected birds and untreated. One hundred and twenty broilers were orally inoculated with 1ml of SE at a concentration of 1.0x108 CFU/mL, at 1 and 2-days-old and divided into six treatments with two repetitions of 200, 400, 500 and 1000ppm organic acid. From 35-days-old birds of all groups were collected aliquots of 3mL of blood into a tube containing EDTA for the evaluation of immune cells by flow cytometry. We then analyzed the percentages of circulating CD4+, CD8β+, MHC I+ MHC II+, TCRVβ1+, CD28+ + and TCRVβ2. For microbiological analysis were collected caecal tonsils of these birds. We found that organic acids in dosages 1000ppm 500ppm in water and in feed for 2 to 7 days before slaughter, respectively, were effective in reducing SE infection in broilers, proven by microbiological method and demonstrated through the behavior of immune cells. The infected birds showed a lower proportion of circulating T helper cells compared with infected poultry, but treated with AO or with the uninfected group. The same trend can be observed for CD28+ cells, and MHC IIbright+ TCRVβ 1+, and with lower resolution, for CD8β+.

Avaliação microbiológica, histológica e imunológica de frangos de corte desafiados com Salmonella Enteritidis e Minnesota e tratados com ácidos orgânicos / Microbiology, histology and immunology evaluation of broiler chickens challenged against Salmonella Enteritidis and Minnesota and treated with organic acids

Dois experimentos foram desenvolvidos para avaliar a eficiência de ácidos orgânicos frente a Salmonella enterica enterica sorovar Enteritidis (SE) e Minnesota (SM) em frangos. No primeiro experimento foram avaliados 3 tratamentos: T1 - ração adicionada de ácido orgânico, T2 - ração adicionada de ácido orgânico e ácido orgânico na água de bebida, T3 - grupo controle. Todos os animais foram inoculados com SE, via oral. A utilização de ácidos orgânicos na ração (T1) e na ração e na água (T2) diminuíram a excreção de Salmonella no papo e no ceco 7 dias pós inoculação com SE e houve redução de células CD3+ no jejuno dos frangos. No segundo experimento foram avaliados 4 tratamentos sendo T1 - controle, T2 - controle inoculado via oral com Salmonella Minnesota (SM), T3 - animais inoculados via oral com SM e ácidos orgânicos na ração e T4 - animais inoculados via oral com SM e ácidos orgânicos na ração e na água de bebida. Ácidos orgânicos a ração (T3) e na ração e na água (T4) reduziram a excreção de SM em papo de frangos de corte desafiados, 7 dias após inoculação. O uso de ácidos orgânicos na ração e na ração e na água foram mais eficientes em reduzir SE do que SM.

Two experiments were carried out to evaluate effectiveness of organic acids against Salmonella enterica enterica serovars Enteritidis (SE) and Minnesota (SM) in broilers. In the first experiment three treatments were evaluated: T1 - feeding with organic acids, T2 - feeding with organic acids and organic acids in drink water, and T3 - control group. All animals were oral challenged with SE. Organic acids in diet (T1) and organic acids in diet and drink water (T2) reduced the shadding of Salmonella in crop and cecum 7 days post challenged with SE and reduced the CD3+ cells in jejunal mucosa of broilers. In the second experiment four treatments were evaluated, T1 - control group, T2 - control group oral challenged with Salmonella Minnesota (SM); T3 - oral challenged animals with SM and treated with organic acids in diet; T4 - oral challenged animals with SM and treated with organic acids in diet and in drink water. Organic acids in diet (T3) and organic acids in diet and in drink water (T4) reduced the shadding of SM in crop of challenged broilers, 7 days post inoculation The use of organic acids in diet and in water was more effective to control SE than SM.