Bivalent Vaccine against Streptococcus agalactiae and Aeromonas hydrophila in Nile Tilapia (Oreochromis niloticus): A Laboratory-Phase and Large-Scale Study.

One of the main factors limiting tilapia's production is the occurrence of infections caused by Aeromonas and Streptococcus species. This work intended to evaluate a bivalent vaccine against A. hydrophila and S. agalactiae by intraperitoneal (i.p) administration in Nile tilapia (Oreochromis niloticus) in Brazil. The study was carried out in two phases: one in the laboratory, on a small scale, and from the results obtained, the study was expanded to a large scale in a production system in cages. The vaccine proved to be safe and effective in laboratory tests, with a vaccine efficacy (VE) of 93.66%. However, in large-scale tests with 12,000 tilapias, the VE was 59.14%, with a better food conversion ratio (1.54 kg) in the vaccinated group compared to the control group (1.27 kg). These results corroborate the efficiency of this tested vaccine; however, they indicate the need for field tests to attest to real protection.

Polymeric Delivery Systems as a Potential Vaccine against Visceral Leishmaniasis: Formulation Development and Immunogenicity.

Recent studies suggest that the association of antigens in microparticles increases the anti-Leishmania vaccine immunogenicity. This study aims to investigate the in situ effect of the adjuvant performance consisting of chitosan-coated poly(D,L-lactic) acid submicrometric particles (SMP) and analyze the inflammatory profile and toxicity. Two formulations were selected, SMP1, containing poly(D,L-lactide) (PLA) 1% wt/v and chitosan 1% wt/v; and SMP2, containing PLA 5% wt/v and chitosan 5% wt/v. After a single dose of the unloaded SMP1 or SMP2 in mice, the SMPs promoted cell recruitment without tissue damage. In addition, besides the myeloperoxidase (MPO) activity having demonstrated similar results among the analyzed groups, a progressive reduction in the levels of N-acetyl-ß-D-glucosaminidase (NAG) until 72 h was observed for SMPs. While IL-6 levels were similar among all the analyzed groups along the kinetics, only the SMPs groups had detectable levels of TNF-α. Additionally, the Leishmania braziliensis antigen was encapsulated in SMPs (SMP1Ag and SMP2Ag), and mice were vaccinated with three doses. The immunogenicity analysis by flow cytometry demonstrated a reduction in NK (CD3-CD49+) cells in all the SMPs groups, in addition to impairment in the T cells subsets (CD3+CD4+) and CD3+CD8+) and B cells (CD19+) of the SMP2 group. The resulting data demonstrate that the chitosan-coated SMP formulations stimulate the early events of an innate immune response, suggesting their ability to increase the immunogenicity of co-administered Leishmania antigens.

Cross-sectional spatial and epidemiological analysis of canine visceral leishmaniasis cases in the triple border region, Brazil, Argentina and Paraguay, between 2015 and 2020.

Visceral Leishmaniasis is one of the most important vector-borne zoonoses in the world. In 2013, the first autochthonous canine case of the disease in the state of Paraná, southern region of Brazil, was reported in Foz do Iguaçu, on the triple border between Brazil, Argentina and Paraguay. In 2015, the first human case was related. Once the endemic was confirmed, the Zoonoses Control Center (ZCC), an agency of the Municipal Health Department, started actions to implement the Human Visceral Leishmaniasis (HVL) Surveillance and Control Program (VLSCP), of the Ministry of Health. Between 2015 and 2020, 12,205 dog samples were analyzed for the diagnosis of the disease. A prevalence of 37.94% (4,630 samples) was found: 2016 had the highest prevalence, with 46.25%, and the year with the lowest prevalence was 2020, with 25.98%. Possible risk factors for dogs were analyzed, and the results obtained were: whether the request for the exam was performed by the ZCC was a significant protective factor, with a lower prevalence (37.5%) than dogs coming from private clinics (OR = 0.89, p-value = 0.016). Males were significantly more infected than females, with 41.1% and 35.7% positivity, respectively (OR = 1.24, p < 0.0001). Companion dogs and mixed breed dogs were significantly less affected than the other groups tested (OR = 0.44, p < 0.001; OR = 0.79, p = 0.012, respectively). The dogs' dark coat color was a significant risk factor with respect to the other color categories. Short and medium coat sizes were significantly considered risk factors, with 41.3% and 31.3% positivity. Long-haired dogs had only 22.7% positivity. In univariate analyses, giant, large and medium dogs were significantly more affected than small dogs. Dogs up to four years of age were significantly less affected than those in other age groups. There was a coincidence of human and canine cases in the spatial distribution. However, according to the literature, a higher incidence would be expected in humans, due to the high prevalence found in dogs. Therefore, further studies should be carried out to understand the dynamics of the disease in this region.

Immunogenicity of HLA-DR1 and HLA-A2 peptides derived from Leishmania major Gp63 in golden hamsters.

AIMS: This study aimed to evaluate the toxicity and humoral and cellular immune response of three heterologous vaccines against Leishmania infantum, yet containing synthetic peptides from Leishmania major in the experimental model in hamsters. METHODS AND RESULTS: Through bioinformatics analyses, two Leishmania major Gp63 peptides were predicted and selected for vaccine formulations. Hamsters were divided into four groups, with each group receiving doses of three vaccine formulations containing HLA-DR1 or HLA-A2 peptides plus MontanideTM or both associated with the adjuvant. The animals received three vaccine doses and were evaluated for toxicity after each dose, in addition to being analysed for the production of antibodies and lymphoproliferation on day 211 after the last vaccine dose. Peptides predicted in association with oily adjuvant induced a humoral response and strong lymphoproliferation to Leishmania infantum antigen-specific stimulation.

Neutrophil properties in healthy and Leishmania infantum-naturally infected dogs.

Visceral leishmaniasis is a chronic disease that affects humans and dogs as well. Dogs, the domestic reservoir of Leishmania, play a central role in the transmission of visceral leishmaniasis, the most severe form of this disease. Neutrophils are the most abundant leukocytes in blood and interact with the parasite after infection. Here, we evaluate the effector properties of neutrophils from healthy and naturally Leishmania infantum-infected dogs. Our results showed that the parasite induced neutrophil extracellular trap (NET) release from neutrophils in both groups. Additionally, phagocytosis and NETs contributed differently to parasite killing by neutrophils from healthy and infected animals, and IFN-γ, IL-8, IL-4 and TNF-α production by neutrophils from both groups were differentially modulated by the parasite. Our results contribute to a better understanding of the complex role played by neutrophils in canine visceral leishmaniasis, which may favor the development of more effective therapies.

Characterization of the Catalytic Structure of Plant Phytase, Protein Tyrosine Phosphatase-Like Phytase, and Histidine Acid Phytases and Their Biotechnological Applications.

Phytase plays a prominent role in monogastric animal nutrition due to its ability to improve phytic acid digestion in the gastrointestinal tract, releasing phosphorus and other micronutrients that are important for animal development. Moreover, phytase decreases the amounts of phytic acid and phosphate excreted in feces. Bioinformatics approaches can contribute to the understanding of the catalytic structure of phytase. Analysis of the catalytic structure can reveal enzymatic stability and the polarization and hydrophobicity of amino acids. One important aspect of this type of analysis is the estimation of the number of ß-sheets and α-helices in the enzymatic structure. Fermentative processes or genetic engineering methods are employed for phytase production in transgenic plants or microorganisms. To this end, phytase genes are inserted in transgenic crops to improve the bioavailability of phosphorus. This promising technology aims to improve agricultural efficiency and productivity. Thus, the aim of this review is to present the characterization of the catalytic structure of plant and microbial phytases, phytase genes used in transgenic plants and microorganisms, and their biotechnological applications in animal nutrition, which do not impact negatively on environmental degradation.

Application of rapid in vitro co-culture system of macrophages and T-cell subsets to assess the immunogenicity of dogs vaccinated with live attenuated Leishmania donovani centrin deleted parasites (LdCen-/-).

BACKGROUND: Live attenuated Leishmania donovani parasites as LdCen(-/-) were shown to confer protective immunity against Leishmania infection in mice, hamsters, and dogs. Strong immunogenicity in dogs vaccinated with LdCen(-/-) has been previously reported, including increased antibody response favoring Th1 response lymphoproliferative responses, CD4(+) and CD8(+) T-cells activation, increased levels of Th1 and reduction of Th2 cytokines, in addition to a significant reduction in parasite burden after 18 and 24 months post virulent parasite challenge. METHODS: Aimed at validating a new method using in vitro co-culture systems with macrophages and purified CD4(+) or CD8(+) or CD4(+):CD8(+) T-cells of immunized dogs with both LdCen(-/-) and Leishmune® to assess microbicide capacity of macrophages and the immune response profile as the production of IFN-γ, TNF-α, IL-12, IL-4 and IL-10 cytokines. RESULTS AND DISCUSSION: Our data showed co-cultures of macrophages and purified T-cells from dogs immunized with LdCen(-/-) and challenged with L. infantum were able to identify high microbicidal activity, especially in the co-culture using CD4(+) T-cells, as compared to the Leishmune® group. Similarly, co-cultures with CD8(+) T-cells or CD4(+):CD8(+) T-cells in both experimental groups were able to detect a reduction in the parasite burden in L. infantum infected macrophages. Moreover, co-cultures using CD4(+) or CD8(+) or CD4(+):CD8(+) T-cells from immunized dogs with both LdCen(-/-) and Leishmune® were able to identify higher levels of IFN-γ and IL-12 cytokines, reduced levels of IL-4 and IL-10, and a higher IFN-γ/IL-10 ratio. While the highest IFN-γ levels and IFN-γ/IL-10 ratio were the hallmarks of LdCen(-/-) group in the co-culture using CD4(+) T-cells, resulting in strong reduction of parasitism, the Leishmune® immunization presented a differential production of TNF-α in the co-culture using CD4(+):CD8(+) T-cells. CONCLUSION: The distinct conditions of co-culture systems were validated and able to detect the induction of immune protection. The method described in this study applied a new, more accurate approach and was able to yield laboratory parameters useful to test and monitor the immunogenicity and efficacy of Leishmania vaccines in dogs.

Insecticidal and repellent activity of Siparuna guianensis Aubl. (Negramina) against Aedes aegypti and Culex quinquefasciatus.

This study investigated the toxic effects of essential oils isolated from Siparuna guianensis against Aedes aegypti, Culex quinquefasciatus (eggs, larvae, pupae, and adult) and Aedes albopictus (C6/36) cells. The oviposition-deterring activity, egg viability, and repellence activity in the presence of different essential oils concentrations were determined. The essential oils showed high toxicity to all developmental stages of A. aegypti and C. quinquefasciatus. Furthermore, the oils also showed high repellent activity towards the adult stage of mosquitoes (0.025 to 0.550 µg/cm2 skin conferred 100% repellence up to 120 min) and in contact with cultured insect cells (C6/36) induced death possibly by necrosis. The results presented in this work show the potential of S. guianensis essential oils for the development of an alternative and effective method for the natural control of mosquitoes in homes and urban areas.

Cytokine and nitric oxide patterns in dogs immunized with LBSap vaccine, before and after experimental challenge with Leishmania chagasi plus saliva of Lutzomyia longipalpis.

In the studies presented here, dogs were vaccinated against Leishmania (Leishmania) chagasi challenge infection using a preparation of Leishmania braziliensis promastigote proteins and saponin as adjuvant (LBSap). Vaccination with LBSap induced a prominent type 1 immune response that was characterized by increased levels of interleukin (IL-) 12 and interferon gamma (IFN-γ) production by peripheral blood mononuclear cells (PBMC) upon stimulation with soluble vaccine antigen. Importantly, results showed that this type of responsiveness was sustained after challenge infection; at day 90 and 885 after L. chagasi challenge infection, PBMCs from LBSap vaccinated dogs produced more IL-12, IFN-γ and concomitant nitric oxide (NO) when stimulated with Leishmania antigens as compared to PBMCs from respective control groups (saponin, LB- treated, or non-treated control dogs). Moreover, transforming growth factor (TGF)-ß decreased in the supernatant of SLcA-stimulated PBMCs in the LBSap group at 90 days. Bone marrow parasitological analysis revealed decreased frequency of parasitism in the presence of vaccine antigen. It is concluded that vaccination of dogs with LBSap vaccine induced a long-lasting type 1 immune response against L. chagasi challenge infection.

Analysis using canine peripheral blood for establishing in vitro conditions for monocyte differentiation into macrophages for Leishmania chagasi infection and T-cell subset purification.

Canine visceral leishmaniasis (CVL) is a parasitic disease endemic in many countries, and dogs present as the major natural reservoir of the parasite, Leishmania chagasi (syn. L. infantum). Biomarkers in the canine immune system is an important technique in the course of developing vaccines and treatment strategies against CVL. New methodologies for studying the immune response of dogs during Leishmania infection and after receiving vaccines and treatments against CVL would be useful. In this context, we used peripheral blood mononuclear cells (PBMCs) from healthy dogs to evaluate procedures related to (i) establishment of in vitro conditions of monocytes differentiated into macrophages infected with L. chagasi and (ii) purification procedures of T-cell subsets (CD4(+) and CD8(+)) using microbeads. Our data demonstrated that after 5 days of differentiation, macrophages were able to induce significant phagocytic and microbicidal activity after L. chagasi infection and also showed increased frequency of parasitism and a higher parasite load. Although N-acetyl-ß-d-glucosaminidase (NAG) levels presented similar levels of macrophage culture and L. chagasi infection, a progressive decrease in myeloperoxidase (MPO) levels was a hallmark over 5 days of culture. High purity levels (>90%) of CD4 and CD8 T cells were obtained on a magnetic separation column. We concluded that monocytes differentiated into macrophages at 5 days and displayed an intermediate frequency of parasitism and parasite load 72 h after L. chagasi infection. Furthermore, the purification system using canine T-lymphocyte subsets obtained after 5 days of monocyte differentiation proved efficient for CD4 or CD8 T-cell purification (≥90%). The in vitro analysis using L. chagasi-infected macrophages and purified T cells presented a prospective methodology that could be incorporated in CVL vaccine and treatment studies that aim to analyze the microbicidal potential induced by specific CD4(+) and/or CD8(+) T cells.

Leishmania (Viannia) braziliensis: immunoblotting analysis for the detection of IgG subclasses in the diagnosis of symptomatic and asymptomatic dogs.

During a seroepidemiological survey 2004-2006 from areas in Brazil endemic for American cutaneous leishmaniasis (ACL), serum samples from 10 dogs with ulcerated cutaneous lesions (S-ACL) and 52 asymptomatic dogs (AS-ACL) of unknown age and breed living in areas endemic for ACL were monitored for 1 year for ulcerated cutaneous lesions and immunoblotting using peroxidase-conjugated secondary anti-IgG, anti-IgG1 and anti-IgG2 dog antibodies. We reported that antibodies against Leishmania (Viannia) braziliensis in the sera of 22/52 dogs with asymptomatic disease showed intense reactivity to peptides larger than 66 kDa. We believe that dogs harboring subclinical amastigotes show an immunoblotting profile similar to that of symptomatic animals because a dog with self-healing presented antigens greater than 66 kDa. Such patterns can be exploited for diagnostic and epidemiological research for leishmaniasis.

Comparação da contagem de células somáticas em leite cru por quatro métodos de coloração / Comparison of somatic cell count in raw milk by four staining methods

A contagem de células somáticas (CCS) determina a quantidade de leucócitos e células epiteliais presentes no leite. Em caso de inflamação (mastite subclínica ou clínica), há um aumento considerável na CCS, principalmente de neutrófilos. É importante quantificar e qualificar os tipos celulares presentes para conhecer o grau de inflamação e caracterizar como aguda ou crônica. O objetivo deste trabalho foi testar quatro métodos de coloração de células somáticas; broadhurst-Paley, hematoxilina-eosina, corante Panótico® e May Grunwald-Giemsa por meio da microscopia óptica e determinar a mais adequada para nossas condições de laboratório. Diante dos dados obtidos, elegeu-se a coloração de associação de eosina e azul de metileno (May Grunwald-Giemsa) com a qual se obteve resultados variando entre 60.000 e 58.690.000 células/mL em diferentes amostras de leite bovino. Não foi possível quantificar as células pelos outros três métodos de coloração devido às alterações morfológicas exacerbadas das mesmas.

The somatic cell count (SCC) determines the amount of leukocytes and epithelial cells present in milk. In case of inflammation (subclinical or clinical), there is a considerable increase in SCC, especially neutrophils. It is important to quantify and qualify the cell types present to know the degree of inflammation and characterized as acute or chronic. The objective was to test four staining methods of somatic cells; Broadhurst-Paley, hematoxylin-eosin stain Panoptic and May Grunwald-Giemsa by light microscopy and determine the most suitable for our laboratory conditions. Considering the available data, was elected the association of eosin staining and methylene blue (May-Grunwald Giemsa) with which they obtained results ranging from 60,000 to 58,690,000 cells / ml in different samples of bovine milk. It was not possible to quantify the cells by the other three staining methods due to morphological changes exacerbated them.

Frequência de anticorpos anti-Brucella abortus em rebanhos bovinos de aptidão leiteira no município de Alegre, Estado do Espírito Santo / Frequency of anti-Brucella abortus antibodies in cattle from Alegre municipality, Espírito Santo state, Brazil

A brucelose é uma doença infecto-contagiosa causada por bactérias do gênero Brucella spp acometendo várias espécies de animais, assim como o homem. Nos bovinos, a principal manifestação é o aborto que ocorre em torno do sétimo mês de gestação. Após a infecção, o aborto ocorre quase sempre na primeira gestação, mas em decorrência do desenvolvimento da imunidade celular, é pouco frequente na segunda gestação após a infecção, e muito raro nas subsequentes. É estimado que a brucelose cause perdas de 20 a 25% na produção leiteira, devido aos abortos e aos problemas de fertilidade. Foram coletadas e avaliadas pelo teste do antígeno acidificado tamponado (TAAT) 114 amostras de soro de fêmeas bovinas em lactação, distribuídas em 19 propriedades do município de Alegre no sul do Estado do Espírito Santo durante o ano de 2007. Das 114 amostras testadas, foram observadas quatro sendo reagentes positivas oriundas de três propriedades rurais.

Brucellosis is an infectious and contagious disease caused by bacteria from the genus Brucella spp, that affects various species of animals and the humans. In cattle the main manifestation is the abortion that occurs around the seventh month of pregnancy. After infection, abortion often is in the first pregnancy, but due to the development of cellular immunity, it is rare in the second pregnancy after infection, and in the subsequent pregnancies are very rare. It is estimated that brucellosis causes losses of 20 to 25% in milk production due to miscarriages and fertility problems. Were collected and evaluated by the antigen test acidified buffered (TAAT) 114 serum samples from female cattle in milk, distributed in 19 properties in the municipality of Alegre in southern state of Espírito Santo during the year of 2007. On the 114 samples tested were found four positive reagents from three different farms.