Detection of Hemopathogens in Chelonoidis carbonaria: Microscopic, Molecular, Hematological, and Clinical Biochemistry Aspects.

Background: The growing contact between men and wild animals, caused by the increase in the population in urban centers and the destruction of the habitat of these animals, has been leading to a greater circulation of pathogens between humans and wildlife. Chelonoidis carbonaria, a tortoise found throughout South America, is one of the animals most rescued from animal trafficking and illegal breeding. Considering this situation, this study aimed to verify the occurrence of hemoparasites in C. carbonaria. Materials and Methods: Blood samples from 73 C. carbonaria were collected from animals located in (1) a rural commercial breeding unit, (2) an urban zoo, and (3) a center of rescued animal screening. Genomic DNA was extracted from these animals and used in PCRs to detect specific genomic fragments of haemogregarines (i.e., Hepatozoon and Hemolivia), and members of the Anaplasmataceae Family (i.e., Ehrlichia sp. and Anaplasma sp.). Blood samples were screened for hemopathogens by direct microscopy and were used for hematological assays, and serum samples were analyzed to determine the concentration of serum components. Results: It was found that 34.2% of the tortoises presented Sauroplasma sp. in their blood samples; these animals showed clinical biochemistry changes that indicate altered liver function. Two zoo animals were positive for Ehrlichia sp. in PCR, and also presented clinical biochemistry and hematological changes. Conclusion: The present project is pioneer in the detection of Ehrlichia sp. in C. carbonaria, and was able to identify changes in clinical biochemistry that can be a result of the infection by hemopathogens in this species.

Comparative antimicrobial activity of four different endodontic sealers.

Dental cements are widely used in the clinical routine, specifically for root canal sealing. Within this context, it is expected that these materials present antimicrobial activity, since it would help in the prevention of apical and periapical infections. The present study aimed to comparatively verify the antimicrobial activity of four dental cements against microorganisms that are routinely isolated from endodontic infections. Reference strains of Enterococcus faecalis, Candida albicans and Escherichia coli were submitted to the agar diffusion test and to modified direct contact test using four different sealers: an eugenol zinc oxide compound, an epoxy resin associated to calcium hydroxide and bismuth, a mineral trioxide aggregate (MTA) and a bioceramics. Different E. coli, C. albicans and E. faecalis growth inhibition profiles were observed in the agar diffusion assay. In the direct contact test, the bioceramics presented a higher microbicide activity on all microorganisms tested herein. Dental cements have different antimicrobial activities, being that the bioceramics present the most consistent antimicrobial activity, and that the direct contact test presented more uniform results than the agar diffusion test. This study reveals the antimicrobial activities of different cements and allow dentists to decide which material to employ in their daily practice.

Pharmacokinetics of Carboplatin in Combination with Low-Dose Cyclophosphamide in Female Dogs with Mammary Carcinoma.

This prospective study aimed to evaluate the effect of metronomic cyclophosphamide on carboplatin's tolerability, efficacy, and pharmacokinetics in dogs with mammary carcinoma. Sixteen female dogs with mammary carcinoma were divided into groups: 300 mg/m2 intravenous (i.v.) carboplatin therapy (G1 = 8) or 300 mg/m2 i.v. carboplatin which was associated with 12.5 mg/m2 oral cyclophosphamide in a metronomic regimen (G2 = 8). The investigated animals underwent a clinical evaluation, a mastectomy, a carboplatin chemotherapy, and serial blood sampling for the pharmacokinetic analysis. The adverse events and survival rates were monitored. A non-compartmental analysis was applied to calculate the pharmacokinetic parameters of carboplatin in the 2nd and 4th chemotherapy cycles. Carboplatin PK showed high interindividual variability with a 10-fold variation in the area under the plasma concentration−time curve (AUC) in G1. The systemic plasma exposure to carboplatin was equivalent in both of the treatments considering the AUC and maximum plasma concentration (Cmax) values. Although the red blood cells (p < 0.0001), platelets (p = 0.0005), total leukocytes (p = 0.0002), and segmented neutrophils (p = 0.0007) were reduced in G2, the survival rate increased (p = 0.0044) when it was compared to G1. In conclusion, adding low daily doses of cyclophosphamide to a carboplatin therapy showed promising outcomes in female dogs with mammary tumors.

Exploring the MALDI Biotyper for the Identification of Corynebacterium pseudotuberculosis biovar Ovis and Equi.

Biochemical, serological, and molecular methods have been developed for the laboratory diagnosis of diseases caused by C. pseudotuberculosis (CP), but the identification of the pathogen and biovars differentiation may be time-consuming, expensive, and confusing compared with other bacteria. This study aimed to evaluate MALDI Biotyper and Overall Genome Relatedness Index (OGRI) analysis to optimize the identification and differentiation of biovars of C. pseudotuberculosis. Out of 230 strains isolated from several hosts and countries, 202 (87.8%) were precisely classified using MALDI Biotyper and the BioNumerics platform. The classification accuracies for the Ovis and Equi biovars were 80 (88.75%) and 82 (92.68%), respectively. When analyzing a sampling of these strains by Average Nucleotide Identity based on BLAST and TETRA analyses using genomic sequence data, it was possible to differentiate 100% of the strains in Equi and Ovis. Our data show that MALDI Biotyper and OGRI analysis help identify C. pseudotuberculosis at the species and biovar levels.

Proteomics and immunoblotting analyses reveal antigens that optimize the immunodiagnosis of the infection by Toxocara spp.

Toxocariasis is an infection caused by the round worms Toxocara canis and Toxocara cati. It occurs worldwide though it is more prevalent in developing countries. For the diagnosis of toxocariasis, the most used method is the indirect enzyme-linked immunosorbent assay (indirect ELISA), based on the detection of specific antibodies using the excreted/secreted products from T. canis larvae (TES) as antigens, but it cross-reacts with several helminth infections. For this reason, there is a need to investigate species-specific immunoreactive proteins, which can be used for the development of a more sensitive and specific diagnosis. This study aims to investigate immunoreactive protein candidates to be used for the development of a more sensitive and specific diagnosis of Toxocara spp. infection in humans. We have used immunoblotting and mass spectrometry to select four Toxocara canis immunoreactive proteins that were recombinantly expressed in bacteria and evaluated as potential new diagnostic antigens (rMUC3, rTES 26, rTES32 and rCTL4). The recognition of these recombinant proteins by total serum IgG and IgG4 was assayed using the purified proteins in an isolated manner or in combination. The IgG ELISAs performed with individual recombinant antigens reached values of sensitivity and specificity that ranged from 91.7% to 97.3% and 94.0% to 97.9%, respectively. Among the analyses, the IgG4 immunoassay was proven to be more effective, revealing a sensitivity that ranged from 88.8% to 98.3% and a specificity of 97.8%-97.9%. The IgG4 ELISA was shown to be more effective and presented no cross-reactivity when using combinations of the rTES 26 and rCTL4 recombinant proteins. The combination of these two molecules achieved 100% sensitivity and specificity. The use of only two recombinant proteins can contribute to improve the current panorama of toxocariasis immunodiagnosis for, with a better optimization and reduced cost.

Activity of Fusarium oxysporum-Based Silver Nanoparticles on Candida spp. Oral Isolates.

Candida spp. resistant to commercially available antifungals are often isolated from patients with oral candidiasis, a situation that points to the need for the development of new therapies. Thus, we evaluated the activity of Fusarium oxysporum-based silver nanoparticles (AgNPs) on Candida spp. isolated from denture stomatitis lesions. Candida isolates were molecularly identified and submitted to susceptibility assays using AgNPs and commercial fungicides. The interference on biofilm formation and the mechanisms of action of AgNPs on Candida spp. were also investigated. Scanning electron microscopy was used to evaluate the morphology of AgNP-treated Candida. Candida albicans was the most frequent species isolated from denture stomatitis cases. All Candida spp. were susceptible to AgNPs at low concentrations, except Candida parapsilosis. AgNPs caused surface damage, cell disruption, and biofilm formation inhibition. The ergosterol supplementation protected C. albicans against the AgNP action. AgNPs are effective against Candida spp. and can be faced as a promising new therapeutic agent against oral candidiasis.

Correlation between the natural infection by Corynebacterium pseudotuberculosis in goats and serum progesterone levels and corpus luteum development.

Caseous Lymphadenitis (CLA) is an infectious disease that affects small ruminants, and the bacterium Corynebacterium pseudotuberculosis is its etiologic agent. This disease presents a high morbidity and a great economic impact on goat farming, leading to reduced milk and meat production and reproductive losses. The available data about the influence of C. pseudotuberculosis on the reproductive system were obtained after experimental inoculations. In this way, this study aimed to evaluate the influence of the natural infection by C. pseudotuberculosis on the luteal function and serum progesterone (P4) levels in goats. Sixteen female goats were diagnosed for the presence of C. pseudotuberculosis specific antibodies and divided into 2 groups: CLA positive and negative animals. They were submitted to estrous synchronization, followed by controlled mating. Corpus luteum and serum levels of P4 were evaluated on the 7th and 20th days after mating (D7 and D20). The ultrasonographic results were not statistically different between the 2 groups, as well as serum P4 levels on D7. However, a significant increase in serum P4 levels on the CLA positive group was identified on D20, along with a significant correlation between C. pseudotuberculosis specific antibody production, and P4 serum levels. The result of this study indicates that the infection by C. pseudotuberculosis may influence the reproductive status of female goats through an enhanced production of progesterone.

Applications of Silver Nanoparticles in Dentistry: Advances and Technological Innovation.

Silver nanoparticles (AgNPs) have been successfully applied in several areas due to their significant antimicrobial activity against several microorganisms. In dentistry, AgNP can be applied in disinfection, prophylaxis, and prevention of infections in the oral cavity. In this work, the use of silver nanoparticles in dentistry and associated technological innovations was analyzed. The scientific literature was searched using PubMed and Scopus databases with descriptors related to the use of silver nanoparticles in dentistry, resulting in 90 open-access articles. The search for patents was restricted to the A61K code (International Patent Classification), using the same descriptors, resulting in 206 patents. The results found were ordered by dental specialties and demonstrated the incorporation of AgNPs in different areas of dentistry. In this context, the search for patents reaffirmed the growth of this technology and the dominance of the USA pharmaceutical industry over AgNPs product development. It could be concluded that nanotechnology is a promising area in dentistry with several applications.

Comparative Proteomic Analyses Between Biofilm-Forming and Non-biofilm-Forming Strains of Corynebacterium pseudotuberculosis Isolated From Goats.

Caseous lymphadenitis (CLA) is a chronic disease that affects small ruminants and causes economic losses in the associated breeding system. The causative agent of CLA is Corynebacterium pseudotuberculosis, a Gram-positive bacterium that exhibits tropism for external and internal lymph nodes and induces abscess formation in the host. Bacterial communities often produce a biofilm matrix that serves various functions, including protection against hostile environmental conditions, antibiotics, and the host immune response. Although biofilm formation has been reported for C. pseudotuberculosis, not all strains demonstrate this property in culture. In this work, we report the first comparative proteomic analysis of one biofilm-forming (CAPJ4) and one biofilm-non-forming strain (CAP3W) of C. pseudotuberculosis isolated from goats. Bacterial whole cell protein extracts were obtained for mass spectrometry analyses. Using LC-MS/MS, our studies reveal three and four proteins exclusively found in the CAPJ4 and CAP3W proteome, respectively. In addition, label-free quantitative analysis identified 40 proteins showing at-least 2-fold higher values in CAPJ4 compared CAP3W proteome Notably, CAPJ4 differentially synthesized the penicillin-binding protein, which participates in the formation of peptidoglycans. CAPJ4 also exhibited upregulation of N-acetylmuramoyl-L-alanine amidase and galactose-1-phosphate uridylyltransferase, which are involved in biofilm formation and exopolysaccharide biosynthesis. Here, we demonstrate that biofilm formation in C. pseudotuberculosis is likely associated with specific proteins, some of which were previously shown to be associated with virulence and biofilm formation in other organisms. Our findings may drive studies related to the bacterial mechanisms involved in the biofilm formation, in addition to providing targets for the treatment of CLA.

Rabbit as an Animal Model for Ocular Surface Disease, Tear Osmolarity, Electrolyte, and Tear Ferning Profiles.

SIGNIFICANCE: The tear film promotes ocular surface health and protection through its various constituents' functions. The application of methods for ocular surface examination is essential in the research of diseases that affect the tear film. Rabbits have been used as a model to study some human ocular diseases and to test ophthalmic products. PURPOSE: The aim of the present study was to determine the biochemical profile, osmolarity, and tear ferning patterns of rabbit tears. METHODS: Ten rabbits (Oryctolagus cuniculus) were evaluated for tear osmolarity, tear ferning types and grades (using Rolando and Masmali scales), and biochemical analysis of total protein, urea, and electrolytes (chloride, phosphorus, iron, sodium, potassium, and calcium). RESULTS: Median ± semi-interquartile range for tear osmolarity was 283.5 ± 7.5 mOsm/L. Tear ferning test grades were type 1.0 ± 0.5 on the Rolando scale and 1.0 ± 0.5 on the Masmali scale. Type I and grade 1 were the most commonly observed ferning classifications (60 and 50%, respectively) for the rabbits' tears. Results for electrolytes and other biochemical compounds were as follows: total protein 4.40 g/dL, urea 130.60 mg/dL, chloride 196.51 mEq/L, phosphate 7.35 mg/dL, iron 95.76 µg/dL, sodium 202.04 mmol/L, potassium 12.74 mmol/L, and calcium 11.53 mg/dL. CONCLUSIONS: The results of the various tests described herein may serve as a basis for research using rabbits as an ophthalmic disease model and in the development of diagnostic and therapeutic agents used for ocular health.

Comparative Analysis of Tear Composition in Humans, Domestic Mammals, Reptiles, and Birds.

Tears are an important component of the ocular surface protection mechanism and are in close contact with the corneal epithelium and the environment. Their composition is well-known in humans; however, there are few investigations on the composition and function of tears in reptiles, birds and others mammals, which would elucidate the mechanisms governing the maintenance of ocular homeostasis. In this work, electrophoretic profiles and an evaluation of total protein, albumin, urea, glucose, and cholesterol concentrations in tears of semi-aquatic, terrestrial, and marine reptiles (Caiman latirostris, Chelonia mydas, Caretta caretta, Eretmochelys imbricata, Lepidochelys olivacea, and Chelonoidis carbonaria), birds (Tyto furcata, Rupornis magnirostris and Ara ararauna), and mammals (Equus caballus and Canis lupus familiaris) were apresented. Human tear components and respective blood serum samples were used as references. The electrophoretic analysis revealed similarities whithin same Classes. The results of the tear-blood serum relationship and the comparison to human tear components showed particularities that are potentially derived from a homeostatic response to the environment. When the tear compositions of animals belonging to different ecological clusters were compared, marked differences were observed in total protein and urea concentrations. Thus, reptile, bird, and mammalian tears are complex fluids with differing concentrations of biochemical components that are potentially a result of the animals' adaptation to different environments.

Lipid and metabolic profiles in female dogs with mammary carcinoma receiving dietary fish oil supplementation.

BACKGROUND: Dyslipidemias induce angiogenesis and accelerate the development and in vitro growth of breast tumors. The aim of this study was to assess the lipid and metabolic profile of female dogs with mammary carcinomas and their correlations with body condition score and degree of tumor malignancy, as well as to study the effect of dietary fish oil supplementation on these animals. RESULTS: Overweight or obese dogs had more aggressive carcinomas and higher triglyceride (p = 0.0363), VLDL (p = 0.0181), albumin (p = 0.0188), globulin (p = 0.0145) and lactate (p = 0.0255) concentrations. There was no change in the lipid profile after supplementation with fish oil at any concentration. However, in relation to the metabolic profile, glucose (p = 0.0067), total protein (p = 0.0002) and globulin (p = 0.0002) concentrations were increased when 90% omega-3 fish oil was used as a dietary supplement. CONCLUSION: Obese dogs showed altered lipid and metabolic profiles and more aggressive tumors, suggesting an important relationship between dyslipidemia and tumor aggressiveness. Supplementation with fish oil, rich in omega-3 fatty acids, may alter metabolic parameters in cancer patients.

Leishmania infantum-derived lipophosphoglycan as an antigen in the accurate serodiagnosis of canine leishmaniasis.

Lipophosphoglycan (LPG) is the major surface glycoconjugate of Leishmania protozoan and has an important biological role in host-parasite interactions both in the midgut epithelium of the sand fly vector and in the vertebrate macrophages. Canine leishmaniasis (CanL) is a chronic infectious disease predominantly caused by Leishmania infantum. An early and accurate immunodiagnosis of the disease is crucial for veterinary clinical practice and for disease control. In this work, we evaluated L. infantum LPG as an antigen in an indirect enzyme-linked immunosorbent assay (ELISA) for CanL immunodiagnosis (LPG-ELISA) by testing serum samples from 97 naturally infected dogs with diverse clinical presentations ranging from subclinical infection to severe disease, as evaluated by veterinarian infectologists. Serum samples from healthy dogs from non-endemic areas (n = 68) and from dogs with other infectious diseases (n = 64) were used as controls for assay validation. The performance of the LPG-ELISA was compared with that of an ELISA using the soluble fraction of L. infantum total lysate antigen (TLA). LPG-ELISA presented a superior performance in comparison to TLA-ELISA, with 91.5% sensitivity, 98.5% specificity and 99.7% accuracy. A distinguishing feature of the LPG-ELISA compared to the TLA-ELISA was its higher ability to identify subclinical infection in clinically healthy dogs, in addition to the absence of cross-reactivity with other canine infectious diseases. Finally, LPG-ELISA was compared to TR DPP visceral canine leishmaniasis test, the immunochromatographic test recommended by the Brazilian Ministry of Agriculture. LPG-ELISA exhibited higher values of specificity (98.5% versus 93.1%) and sensitivity (91.5% versus 90.6%) compared to TR DPP. In conclusion, L. infantum-derived LPG was recognized by antibodies elicited during CanL in different infection stages and was shown to be a suitable antigen for specific clinical settings of veterinary diagnosis and for public health usage.

A panel of recombinant proteins for the serodiagnosis of caseous lymphadenitis in goats and sheep.

Caseous lymphadenitis (CLA) is a small ruminant disease characterized by the development of granulomatous lesions in superficial and internal lymph nodes, as well as in some organs, and causes significant economic losses worldwide. The aetiological agent of CLA is the bacterium Corynebacterium pseudotuberculosis; however, the commercially available diagnostic tools present problems with regard to specificity, which can lead to false-negative results. This study aimed to develop an indirect enzyme-linked immunosorbent assay (ELISA) for the detection of specific immunoglobulins in goats and sheep using recombinant C. pseudotuberculosis PLD, CP40, PknG, DtxR and Grx proteins. For validation of the ELISAs, 130 goat serum samples and 160 sheep serum samples were used. The best ELISA for goats was developed using a combination of PLD and CP40 as antigens at a 1:1 ratio, which presented 96.9% sensitivity and 98.4% specificity. The most effective ELISA for sheep presented 91% sensitivity and 98.7% specificity when recombinant PLD alone was used as the antigen. These ELISAs can be used as highly accurate tools in epidemiological surveys and for the serodiagnosis of C. pseudotuberculosis infection in goats and sheep.

The effect of naltrexone as a carboplatin chemotherapy-associated drug on the immune response, quality of life and survival of dogs with mammary carcinoma.

The objective of this study was to evaluate the effect of low-dose naltrexone (LDN) as a carboplatin chemotherapy-associated drug in female dogs with mammary carcinoma in benign mixed tumors (MC-BMT) after mastectomy and to assess its association with quality of life and survival rates. Sixty female dogs were included in this study, all of which had histopathological diagnosis of MC-BMT and were divided into three groups: G1 (control), consisting of animals submitted only to mastectomy with or without regional metastasis; G2, composed of treated animals that did not present with metastasis; and G3, treated dogs that presented with metastasis. G2 and G3 were also subdivided according to the treatment administered: chemotherapy alone (MC-BMT(-) C/MC-BMT(+) C) or LDN and chemotherapy (MC-BMT(-) C+LDN/MC-BMT(+) C+LDN). All animals were subjected to clinical evaluation, mastectomy, peripheral blood lymphocyte immunophenotyping, beta-endorphin and met-enkephalin quantification, and evaluation of survival rates and quality of life scores. The results showed higher serum concentrations of beta-endorphin and met-enkephalin, fewer chemotherapy-related side effects, and better quality of life and survival rates in the LDN-treated groups than in LDN-untreated groups (P < 0.05). Evaluation of clinical and pathological parameters indicated a significant association between the use of LDN and both prolonged survival and enhanced quality of life. These results indicate that LDN is a viable chemotherapy-associated treatment in female dogs with MC-BMT, maintaining their quality of life and prolonging survival rates.

Evaluation of lacrimal production, osmolarity, crystallization, proteomic profile, and biochemistry of capuchin monkeys' tear film.

BACKGROUND: To evaluate the composition and characteristics of capuchin monkey (CM) tear film. METHODS: Eleven CM (Sapajus sp.) was evaluated. Strip meniscometry test (SMT), osmolarity, and tear ferning test (TFT) (by Rolando and Masmali scales) were assessed. Tear protein profile (SDS-PAGE), and total protein, albumin, urea, glucose, and cholesterol levels in tear film and blood serum were screened. RESULTS: Median ± semi-interquatil range for SMT and osmolarity values were 8.0 ± 1.625 and 303.0 ± 9.875, respectively. TFT for Rolando was 2.0 ± 0.5, and Masmali was 2.0 ± 0.0. Monkeys's tear obtained type II and III for Rolando, and 1 and 2 for Masmali. Tear components showed protein bands among 23-217 kDa, and presence of albumin, urea, glucose, and cholesterol. CONCLUSIONS: The results of SMT, osmolarity, TFT, SDS-PAGE, and tear biochemistry may serve as a reference baseline for CM, and the data may serve as a basis for future experimental model evaluations.

Association of Corynebacterium pseudotuberculosis recombinant proteins rCP09720 or rCP01850 with rPLD as immunogens in caseous lymphadenitis immunoprophylaxis.

Caseous lymphadenitis (CLA) is a chronic disease responsible for significant economic losses in sheep and goat breeding worldwide. The treatment for this disease is not effective, and an intense vaccination schedule would be the best control strategy. In this study, we evaluated the associations of rCP09720 or rCP01850 proteins from Corynebacterium pseudotuberculosis with recombinant exotoxin phospholipase D (rPLD) as subunit vaccines in mice. Four experimental groups (10 animals each) were immunized with a sterile 0.9% saline solution (G1), rPLD (G2), rPLD + rCP09720 (G3), and rPLD + rCP01850 (G4). The mice received two doses of each vaccine at a 21-day interval and were challenged 21 days after the last immunization. The animals were evaluated daily for 40 days after the challenge, and mortality rate was recorded. The total IgG production level increased significantly in the experimental groups on day 42 after the first vaccination. Similarly, higher levels of specific IgG2a were observed in experimental groups G2, G3, and G4 compared to the IgG1 levels on day 42. G4 showed a significant (p < .05) humoral response against both antigens of the antigenic formulations. The cellular immune response induced by immunization was characterized by a significant (p < .05) production of interferon-γ compared to that in the control, while the concentrations of interleukin (IL)-4 and IL-12 were not significant in any group. A significant increase of tumor necrosis factor was observed only in G4. The survival rates after the challenge were 30% (rPLD), 40% (rPLD + rCP09720), and 50% (rPLD + rCP01850). Thus, the association of rCP01850 with rPLD resulted in the best protection against the challenge with C. pseudotuberculosis and induced a more intense type 1 T-helper cell immune response.