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The prompt and accurate identification of the etiological agents of viral respiratory infections is a critical measure in mitigating outbreaks. In this study, we developed and clinically evaluated a novel melting-curve-based multiplex real-time PCR (M-m-qPCR) assay targeting the RNA-dependent RNA polymerase (RdRp) and nucleocapsid phosphoprotein N of SARS-CoV-2, the Matrix protein 2 of the Influenza A virus, the RdRp domain of the L protein from the Human Respiratory Syncytial Virus, and the polyprotein from Rhinovirus B genes. The analytical performance of the M-m-qPCR underwent assessment using in silico analysis and a panel of reference and clinical strains, encompassing viral, bacterial, and fungal pathogens, exhibiting 100% specificity. Moreover, the assay showed a detection limit of 10 copies per reaction for all targeted pathogens using the positive controls. To validate its applicability, the assay was further tested in simulated nasal fluid spiked with the viruses mentioned above, followed by validation on nasopharyngeal swabs collected from 811 individuals. Among them, 13.4% (109/811) tested positive for SARS-CoV-2, and 1.1% (9/811) tested positive for Influenza A. Notably, these results showed 100% concordance with those obtained using a commercial kit. Therefore, the M-m-qPCR exhibits great potential for the routine screening of these respiratory viral pathogens.

IgY antibodies are found in the blood and yolk of eggs. Several studies show the feasibility of utilising IgY for immunotherapy and immunodiagnosis. These antibodies have been studied because they fulfil the current needs for reducing, replacing, and improving the use of animals. Affinity and avidity represent the strength of the antigen-antibody interaction and directly influence antibody action. The aim of this review was to examine the factors that influence the affinity and avidity of IgY antibodies and the methodologies used to determine these variables. In birds, there are few studies on the maturation of antibody affinity and avidity, and these studies suggest that the use of an adjuvant-type of antigen, the animal lineage, the number of immunisations, and the time interfered with the affinity and avidity of IgY antibodies. Regarding the methodologies, most studies use chaotropic agents to determine the avidity index. Studies involving the solution phase and equilibrium titration reactions are also described. These results demonstrate the need for the standardisation of methodologies for the determination of affinity and avidity so that further studies can be performed to optimise the production of high avidity IgY antibodies.

Ochratoxin A (OTA) is a mycotoxin produced by species of Penicillium and Aspergillus that can contaminate products of plant origin that are used as animal feed. Through oral exposure, this mycotoxin primarily affects the chicken gastrointestinal system. The present study evaluated the intestinal toxic effects of OTA and the introduction of L-tryptophan to alleviate these effects in chickens. One-day-old chicks were exposed to a single OTA dose (1.4 mg/kg body weight-b.w.) and treated with or without four daily doses of L-tryptophan (100 mg/kg b.w.). Duodenal villus height/crypt depth, fecal immunoglobulin A/immunoglobulin Y (IgA/IgY) levels, and duodenal positive immunoglobulin A cells (IgA + ) were evaluated by histology, ELISA, and immunohistochemistry, respectively, on the 14th day. There were significant changes in the duodenal villus height, crypt depth, and levels of fecal IgA/IgY and duodenal IgA + cells ( p < 0.05) in groups exposed to OTA. On the other hand, groups exposed to OTA and treated with L-tryptophan showed similar levels of villus height, IgA/IgY levels, and duodenal IgA + cells to those of the control group ( p > 0.05). In conclusion, exposure to a single dose of OTA orally induces changes in intestinal morphology, levels of IgA/IgY antibodies, and IgA + cells. Thus, treatment with L-tryptophan may be a valid alternative means to reduce the harmful effects of OTA on the intestinal mucosa, which requires further study.

Ochratoxin A (OTA), an immunosuppressive mycotoxin, can increase the risk of many infectious diseases and contribute to economic losses to the poultry industry. The immunosuppressive effect has mainly been investigated through oral exposure; however, birds may also be contaminated through skin absorption. The present study investigated the influence of OTA exposure on the defense system of broiler chicks through the subcutaneous route and including low doses. Groups of broiler chicks (Cobb), 05 days old, were exposed to subcutaneous inoculation of OTA at concentrations of 0.1; 0.5; 0.9; 1.3; and 1.7 mg OTA/kg body weight. The size of the lymphoid organs, circulating immune cells, and total IgY and IgA levels were evaluated 21 days post inoculation. Subcutaneous OTA exposure decreased the weight of the thymus, spleen, and bursa of Fabricius, and leukocytopenia ( p < 0.05) was detected in chicks of the OTA treated groups. In a dose-dependent way, decreased levels of circulating lymphocytes and heterophils ( p < 0.05), and increased levels of monocytes ( p < 0.05) were detected. Decreased IgY and IgA serum concentrations were noted in the OTA treated groups ( p < 0.05). In conclusion, subcutaneous OTA exposure induces immunosuppression even at low levels.

Fermented products and components of Saccharomyces cerevisiae have been widely used in animal nutrition to promote the development and quality of broilers. This study aims to evaluate different levels of inclusion (0, 250, 750, 1,500 g/t) of S. cerevisiae fermentation product (SCFP) in broiler feed to gauge its effect on carcass characteristics and cuts beyond the quality of breast meat. For analyses of carcass yield, cuts, and meat quality, 16 broilers per treatment were slaughtered. The meat quality analyses were performed 24 h after slaughter and evaluated color, pH, water holding capacity, cooking loss, and shear force. Lipid oxidation was determined in frozen breast samples stored at -20°C for 45 d. The results indicate that different levels of inclusion of SCFP provided no changes in carcass yield, color, water holding capacity, cooking loss, and shear force; however, inclusion of 1,500 g/t of SCFP increased leg yield and reduced pH. The inclusion of 750 g/t of SCFP decreased the lipid oxidation of breast meat (P < 0.05). This study concluded that inclusion of SCFP may improve leg yield and the lipid oxidation of breast meat.

The mycotoxin, ochratoxin-A (OTA), produced by some fungi, and is a natural contaminant of many foods and animal feeds worldwide. Due to its toxic effects, the recommended maximum daily intake of OTA for poultry feeds is 0.1 mg OTA/kg (ECR2006/575/EC); this dose does not induce changes in hepatic/renal parameters, but decreases thymus size and serum globulin concentrations. Accordingly, in this study, we assessed quantitatively the total circulating IgY and IgA serum levels, in chicks consuming a 0.1 mg OTA/kg diet (limit) and higher doses (0.3⁻1.1 mg OTA/kg diet) for 14 or 21 days. We also evaluated other immunological parameters (thymus, bursa of Fabricius, and spleen weights and leukocyte profiles) at day 21. Decreased IgY serum levels were observed in all OTA-treated groups ( p < 0.05). In the low-dose group, IgA levels were decreased on day 21, but not on day 14. The size of the thymus and the bursa of Fabricius was decreased in all OTA-treated groups ( p < 0.05), whereas reduced spleen size and altered leukocyte profiles were detected only in the high-dose group ( p < 0.05). We concluded that chronic exposure to OTA, even at the recommended highest dose, affected IgY and IgA production in chicks.

Introduction: Herein, we report a one-tube, semi-nested-polymerase chain reaction (OTsn-PCR) assay for the detection of Paracoccidioides brasiliensis., Methods: We developed the OTsn-PCR assay for the detection of P. brasiliensis in clinical specimens and compared it with other PCR methods., Results: The OTsn-PCR assay was positive for all clinical samples, and the detection limit was better or equivalent to the other nested or semi-nested PCR methods for P. brasiliensis detection., Conclusions: The OTsn-PCR assay described in this paper has a detection limit similar to other reactions for the molecular detection of P. brasiliensis, but this approach is faster and less prone to contamination than other conventional nested or semi-nested PCR assays.

Phospholipase is an important virulence factor for pathogenic fungi. In this study, we demonstrate the following: (i) the Paracoccidioides brasiliensis pld gene is preferentially expressed in mycelium cells, (ii) the plb1 gene is mostly up-regulated by infection after 6 h of co-infection of MH-S cells or during BALB/c mice lung infection, (iii) during lung infection, plb1, plc and pld gene expression are significantly increased 6-48 h post-infection compared to 56 days after infection, strongly suggesting that phospholipases play a role in the early events of infection, but not during the chronic stages of pulmonary infection by P. brasiliensis.

Propolis is a honeybee product showing several biological properties that enhance the immune response, depending on the concentration and intake period. Because propolis possesses an immunomodulatory action on mammals, the objective of our study was to investigate the effects of propolis on the humoral immune response of laying hens by evaluating antibody production. Laying hens (ISA Brown) were divided into 5 groups with 7 birds each. Group 1 was a nonimmunized control, whereas birds in group 2 were immunized intravenously with SRBC, and those in groups 3, 4, and 5 were treated intraperitoneally with propolis (2, 10, and 50 mg/kg, respectively) on 3 consecutive days and then inoculated intravenously with SRBC. Hematological and serological analyses were carried out on d 0, 3, and 38. Natural and specific antibody levels were determined by hemagglutination with rabbit red blood cells and SRBC, respectively. Propolis-treated birds (50 mg/kg) showed a significant decline in heterophils and in the heterophil:lymphocyte ratio. After SRBC immunization, significant increases in levels of IgG were observed in groups 4 and 5. Furthermore, higher levels of natural antibodies were observed in propolis-treated laying hens. The administration of propolis to laying hens increased the production of IgG specific to SRBC and natural antibodies, and could be used to increase antigen-specific antibody responses to vaccines.

Introduction: Paracoccidioidomycosis is a systemic infection caused by Paracoccidioides brasiliensis., Methods: In this study, a semi-nested PCR for paracoccidioidomycosis diagnosis was developed. The primers ITS1 and ITS4 were used in the first reaction, while the primers MJ03 and ITS1 primer were used in the second reaction. The semi-nested PCR was used to investigate biopsies of five patients with oral lesions that resembled paracoccidioidomycosis., Results: The semi-nested PCR was positive for four samples and negative for a sample from a patient later diagnosed with leishmaniasis., Conclusions: The new semi-nested PCR describe is useful for paracoccidioidomycosis diagnosis.

The dimorphic fungus Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis, a human granulomatous disease. Recently the first case of natural disease in dogs was reported. The complement system is an important effector component of humoral immunity against infectious agents. Therefore, the aim of this study was to evaluate the activation of the dog alternative complement pathway by P. brasiliensis. Initially, the ability of erythrocytes of guinea pig, rabbit, sheep, chicken and swine to activate the dog alternative pathway was evaluated. The guinea pig erythrocytes showed the greatest capacity to activate dog alternative pathway. The alternative (AH50) hemolytic activity was evaluated in 27 serum samples from healthy dogs and the mean values were 87.2 AH50/ml. No significant differences were observed in relation to sex and age. The alternative pathway activation by P. brasiliensis was higher in serum samples from adult dogs when compared to puppies and aged dogs (p ≤ 0.05). This is the first report of dog alternative complement pathway activation by P. brasiliensis and suggests that it may play a protective role in canine paracoccidioidomycosis.

We have amplified and sequenced the 5.8S and 28S ribosomal DNA genes and intergenic regions of Paracoccidioides brasiliensis, strain Pb01. Using primers specifically designed for both ribosomal DNA regions, we were able to discriminate between P. brasiliensis and other human pathogenic fungi by PCR. The use of this molecular marker could be important for paracoccidiodomycosis diagnosis and ecological and molecular epidemiological studies of P. brasiliensis in Latin America.