Antibody frequency for Toxoplasma gondii and Neospora spp. in domiciliated and stray cats from Araguaína, Tocantins, Eastern Amazonia

Toxoplasma gondii and Neospora spp. are protozoa that have a significant impact on animal health due to the diseases they cause in domestic and wild animals. The aim of the present study was to investigate the presence of antibodies against T. gondii and Neospora spp. in cats from northern Brazil. Serum samples were collected from 180 cats in the municipality of Araguaína, Tocantins and used to evaluate the presence of anti-T. gondii and anti-Neospora spp. antibodies using the indirect fluorescent antibody test, with a cutoff of 1:64 and 1:25, respectively. The association between infection and individual animal characteristics (age, sex, origin, breed, and clinical signs) was tested using univariate analysis, followed by multivariate logistic regression. We found that 48.3% (87/180) of the animals had anti-T. gondii (95% CI: 40.8%–55.90%) and 3.9% (7/180) had anti-Neospora spp. (95% CI: 1.6%–7.8%) antibodies. There was no association between age, sex, breed origin, clinical signs, and seropositivity for T. gondii. Cats of defined breeds were more likely to be infected by Neospora spp. (OR = 10.7). Therefore, we found a high rate of seropositivity for T. gondii and a high rate of occurrence of Neospora infections in cats from the Araguaína region. The exposure of the feline population to the studied coccidia indicates the need to monitor the feline population for these infections and underscores the importance of effective sanitary measures against such pathogens.

A comparison of assays for the detection of Cryptosporidium parvum in the feces of scouring calves

Cryptosporidium parvum is a zoonotic, protozoan parasite that causes potentially life-threatening diarrhea in the host and can be transmitted via the fecal-oral route. C. parvum can infect cattle and may be detected in their feces using a variety of tests. We compared the level of agreement, ease of procedure, and cost among PCR, lateral flow immunoassay, fluorescent antibody, and Kinyoun acid-fast stain direct smear tests. Over the course of 9 mo, 74 calf fecal samples were submitted and tested for C. parvum using all 4 tests. A Fleiss kappa value of 0.813 was obtained, indicating an excellent level of agreement among tests. Overall, the best test based on cost and ease of procedure was the Kinyoun acid-fast stain direct smear.

Identification and Characterization of P0 Protein as a Vaccine Candidate Against Babesia divergens, Blood Parasite of Veterinary and Zoonotic Importance

The molecular identification and antigenic characterization of P0 protein in Babesia divergens, a blood parasite of veterinary and zoonotic importance, were carried out in this study for use in developing subunit vaccines against B. divergens infection. Recombinant protein encoding P0 (BdP0) was developed in Escherichia coli, and its antiserum was generated in mice for further molecular characterization. Anti-rBdP0 serum had a specific interaction with the corresponding legitimate B. divergens protein, as confirmed by Western blotting and indirect fluorescent antibody tests. ELISA was used to assess the immunogenicity of BdP0 in a group of 68 bovine field samples, and significant immunological reactivity was found in 19 and 20 positive samples of rBdp0 and B. divergens lysate, respectively. The in vitro growth of B. divergens cultures treated with anti-rBdP0 serum was significantly inhibited (p < 0.05). Furthermore, after 6 h of incubation with 2 mg/ml anti-rBdP0 serum, the ability of pre-incubated free merozoites to invade bovine erythrocytes was reduced by 59.88%. The obtained data suggest the possible use of rBdP0 as diagnostic antigen and may serve as a vaccine candidate against babesiosis caused by B. divergens either in animal or human.

Use of a Direct, Rapid Immunohistochemical Test for Diagnosis of Rabies Virus in Bats

Rabies, a zoonotic encephalitis due to transmission of a lyssavirus, such as rabies virus (RABV), has the highest case fatality of any infectious disease. A global program for the elimination of human rabies caused by dogs is proposed for realization by 2030. Sensitive, specific, and inexpensive diagnostic tests are necessary for enhanced surveillance to detect infection, inform public health and veterinary professionals during risk assessments of exposure, and support overall programmatic goals. Multiple laboratory techniques are used to confirm a suspect case of rabies. One method for the detection of lyssavirus antigens within the brain is the direct rapid immunohistochemical test (dRIT), using light microscopy, and suitable for use under field conditions. Besides dogs, other major RABV reservoirs reside among mammalian mesocarnivores and bats. To date, use of the dRIT has been applied primarily for the diagnosis of RABV in suspect mesocarnivores. The purpose of this study was to assess the usefulness of the dRIT to the diagnosis of rabies in bats, compared to the gold-standard, the direct fluorescent antibody test (DFAT). Brains of 264 suspect bats, consisting of 21 species from Arizona and Texas, were used in the evaluation of the dRIT. The overall sensitivity of the dRIT was 100% (0.969–1.0, 95% CI) and the specificity was 94.6% (0.896–0.976, 95% CI), comparable to the DFAT. This preliminary study demonstrated the utility of the dRIT in the confirmation of RABV infection in bats. Future studies should include additional geographic, lyssavirus, and mammalian species representations for broader application during enhanced rabies surveillance, with incorporation of any potential adjustments to standard protocols, as needed.

Post-Translational Tubulin Modifications in Differentiated Human Neural Stem Cells

The tubulin protein fulfills a variety of cellular functions that range from chromosomal separation to locomotion. The functional diversity of tubulin is achieved through the expression of specific tubulin isotypes in different cell types or developmental time periods. Post-translational modifications (PTMs) of tubulin also are vital for specific intracellular tasks, such as binding and recruiting motor proteins. In neurons, the isotypic expression profile for tubulin is well characterized, and the importance of PTMs for proper neuronal function has gained recent attention due to their implication in neurodegenerative disorders. In contrast, the role of tubulin specializations in the specification of neural cell fate has received minimal attention and studies of tubulin PTMs and isotypes in neuroglia such as astrocytes are relatively few. To bridge this knowledge gap, we undertook an analysis of PTMs in neurons and astrocytes derived from the federally approved H9 hESC-derived human neural stem cell (hNSC) line. In hNSCs, basal cells can be directed to assume neural fate as neurons or astrocytes by specifying different media growth conditions. Immunocytochemical methods, fluorescent antibody probes, and confocal microscopy facilitated image acquisition of fluorescent signals from class III β- tubulin (βIII-tubulin), acetylated tubulin, and polyglutamylated tubulin. Fluorescent probe intensities were assessed with the EBImage package for the statistical programming language R and compared using Student's t-tests. Qualitative analysis indicated that βIII-tubulin, acetylated tubulin, and polyglutamylated tubulin were expressed to some degree in basal hNSCs and their media-differentiated hNSC neuronal and astroglial progeny. In media-differentiated hNSC astrocyte progeny, quantification and statistical analysis of fluorescence probe intensity showed that acetylated tubulin/ βIII-tubulin ratios were greater than the ratio for polyglutamylated tubulin/ βIII-tubulin. These findings represent a snapshot of the dynamic and varied changes tubulin expression profile during the specification of neural cell fate. Results imply that investigations of tubulin PTMs have the potential to advance our understanding of the generation and regeneration of nervous tissue.

Viremia in Equine Herpes Virus-1 infection and a possible link to Transient Protective Immunity

Equine herpes virus (EHV-1) causes respiratory infections in equine, and results in abortion, paresis, neonatal death, and retinopathy and the virus may become latent following initial infection. Virus entry is via the respiratory route, and the virus replicates in the host in ciliated and non-ciliated epithelial cells of the respiratory tract and in Type 1 and Type 2 pneumocytes in the lung parenchyma. After viral replication in the respiratory system, the virus can become disseminated to other parts of body via viraemic cells. The virus also can cross the placenta which leads to abortion of live or dead fetuses without premonitory signs. Infected horses show transient immunity after natural or experimental infection and immune responses to EHV-1, but the immunoprotective status begins to decline after a few months of active infection. Due to the transient immune response, recovered horses are not immunoprotected and thus are prone to subsequent re-infection. Immunity is not long lived after experimental or natural infection, and as a result the development of an effective vaccine has remained a challenge. In this study viraemic cells were studied in a murine EHV-1 infection model. Mice were infected intranasally and viraemic cells were studied on days three and five which occurs during the peak of the infection. The results of this study may help to identify the nature of viraemic cells and their role in the transient immune response to infection. Buffy coat cells and lungs were removed and stained with a fluorescent antibody test for EHV-1 antigen, and lung specimens were subjected to transmission electron microscopy. Both techniques confirmed the presence of viraemic cells in lung tissues. These viraemic cells were further stained for EHV-1 antigen, and for CD4 or CD8 biomarkers and results are discussed re: pathogenesis of EHV-1 infection, identification of viraemic cells in a murine model and possible link of viraemia to transient immune responses in EHV-1 infection, which demonstrate the validity of this murine model for the investigation of the cytopathologic mechanism and sequelae of EHV manifestation in this model.

PREVALENCE OF TOXOPLASMOSIS AMONG SHEEP AND GOATS IN BAGHDAD AREA

The complement fixation test (CFT) and the direct fluorescent antibody test were used for detecting anti Toxoplasma gondii antibodies in sera obtained from 143 sheep and 44 goats. Complement fixing antibodies were detected in 38 (26.2%) for sheep sera and 24 (54.5%) of goat sera tested by CFT. On the other hand, 26(18.2%) of sheep sera were positive by the FAT. The combined use of CFT and FAT allows the differentiation between an acute or latent T.gondii infection.

New Functions of Low-Molecular-Weight Hyaluronic Acid on Epidermis Filaggrin Production and Degradation

Hyaluronic acid (HA) is a high-molecular-weight polysaccharide with high moisturizing power. It is composed of repeating disaccharides of N-acetyl-D-glucosamine and D-glucuronic acid. Low-molecular-weight hyaluronan (LMHA) is obtained by changing the molecular weight or modifying the functional groups of HA and is commonly used together with HA in cosmetics. The objective of this study was to determine whether LMHA promotes the synthesis of filaggrin (FLG). We also investigated whether LMHA activates FLG-degrading enzymes. Three-dimensional (3D) models of the human epidermis were cultured with LMHA. Real-time PCR was used to quantify the mRNA levels of profilaggrin (proFLG), involucrin (IVL), and FLG-degrading enzymes. FLG protein levels were measured by fluorescent antibody staining and Western blotting. The mRNA was quantified using a 3D epidermis model, and it was observed that the mRNA levels of proFLG, IVL, caspase-14 (CASP14), and bleomycin hydrolase were increased by the application of LMHA. Immunofluorescence results showed an increase in FLG proteins, and results from experiments using 3D epidermis models showed that LMHA increased the activity of CASP14. This suggests that the topical application of LMHA would result in an increase in natural moisturizing factor and promote moisturization of the stratum corneum.

Whole Genome Sequencing and Phylogenetic Analysis of Rabies Viruses from Bats in Connecticut, USA, 2018–2019

We performed whole genome sequencing and genetic characterization of rabies viruses (RABV) detected in bats submitted to the Connecticut Veterinary Medical Diagnostic Laboratory (CVMDL) during 2018-2019. Among 88 bats submitted to CVMDL, six brain samples (6.8%, 95% confidence interval: 1.6% to 12.1%) tested positive by direct fluorescent antibody test. RABVs were detected in big brown bats (Eptesicus fuscus, n = 4), a hoary bat (Lasiurus cinereus, n = 1), and an unidentified bat species (n = 1). Complete coding sequences of four out of six detected RABVs were obtained. In phylogenetic analysis, the RABVs (18-62, 18-4347, and 19-2274) from big brown bats belong to the bats EF-E1 clade, clustering with RABVs detected from the same bat species in Pennsylvania and New Jersey. The bat RABV (19-2898) detected from the migratory hoary bat belongs to the bats LC clade, clustering with the eleven viruses detected from the same species in Arizona, Washington, Idaho, and Tennessee. The approach used in this study generated novel data regarding genetic relationships of RABV variants, including their reservoirs, and their spatial origin and it would be useful as reference data for future investigations on RABV in North America. Continued surveillance and genome sequencing of bat RABV would be needed to monitor virus evolution and transmission, and to assess the emergence of genetic mutations that may be relevant for public health.

Lateral flow devices for samples collected by straw sampling method for postmortem canine rabies diagnosis

The direct fluorescent antibody test (dFAT) using brain sample after opening the skull is the standard rabies diagnostic test in animal rabies. However, it is not feasible in many resource-limited settings. Lateral flow devices (LFD) combined with a simple sampling methodology is quicker, simpler, and less hazardous than the standard test and can be a useful tool. We conducted a prospective on-site study to evaluate the diagnostic accuracy of the LFD with the straw sampling method compared with that of the dFAT with the skull opening procedure for post-mortem canine rabies diagnosis. We collected 97 rabies-suspected animals between December 1, 2020 and March 31, 2021. Among the 97 samples, 53 and 50 cases were positive tests for dFAT and LFD, respectively. The sensitivity and specificity of LFD with straw sampling method were 94.3% (95% confidence interval [CI], 84.3–98.8%) and 100% (95% CI, 92.0–100%), respectively. The performance of LFD by the straw sampling method showed relatively high sensitivity and 100% specificity compared with that of dFAT performed on samples collected after opening the skull. This methodology can be beneficial and is a strong tool to overcome limited animal surveillance in remote areas. However, because of our limited sample size, more data using fresh samples on-site and the optimizations are urgently needed for the further implementation in endemic areas.