Indirect Immunofluorescent Antibody Research Articles

Antinuclear antibody staining patterns by indirect immunofluorescence assay observed in patients from a tertiary health center in Latin America

Introduction/ObjectiveThis study aimed to describe the frequency of antinuclear antibody (ANA) staining patterns by indirect immunofluorescence assay observed in patients from a tertiary health center in Latin America. Materials and methodsThis retrospective, descriptive, and observational study evaluated data from all patients undergoing antinuclear antibody indirect immunofluorescence assay from a single-tertiary center (University Hospital Fundación Valle del Lili, Cali-Colombia) in 2020. ResultsOne thousand and eight patients met the inclusion criteria. The median patient age was 47 (34–59.2) years, and most were female (769, 75.3%). A positive ANA immunofluorescence assay was observed in approximately two-thirds of patients (664, 65.8%). ANA test results were primarily used to exclude a suspected diagnosis in approximately half of the patients (466, 46.2%). Thirty-seven percent (250/664) of the cohort with ANA-positive titers had a systemic autoimmune rheumatic disease (SARD). The most prevalent SARDs included rheumatoid arthritis (RA) (55, 8.2%) followed by systemic lupus erythematosus (SLE) (37, 5.5%). The vast majority of ANA-positive patients had a reported speckled pattern (anti-cell [AC]-2,4,5; 269; 40.5%) followed by homogenous (AC-1; 266; 40%), nucleolar (AC-8,9,10; 46; 6.9%), and centromere (AC-3; 16; 2.4%). The most frequent patterns observed among SLE patients included homogenous (AC-1) patterns in 17 (45.9%) patients, speckled (AC-2,4,5) nuclear patterns in 11 (29.7%) patients, mixed patterns in 7 (18.9%) patients, and reticular/anti-mitochondrial antibody (AMA, AC-21) cytoplasmic patterns in 2 (5.4%) patients. ConclusionThis study is the first to describe ANA patterns in a Colombian population. Speckled and homogenous patterns were predominant in patients with SARDs.

Detection of bornavirus-reactive antibodies and BoDV-1 RNA only in encephalitis patients from virus endemic areas: a comparative serological and molecular sensitivity, specificity, predictive value, and disease duration correlation study

PurposeHuman Borna disease virus (BoDV-1) encephalitis is an emerging disease in Germany. This study investigates the spectrum of human BoDV-1 infection, characterizes anti-BoDV-1-antibodies and kinetics, and compares laboratory test performances.MethodsThree hundred four encephalitis cases, 308 nation-wide neuropsychiatric conditions, 127 well-defined psychiatric cases from Borna disease-endemic areas, and 20 persons with contact to BoDV-1 encephalitis patients or animals were tested for BoDV-1 infections by serology and PCR.ResultsBoDV-1 infections were only found in encephalitis patients with residence in, or recent travel to, virus-endemic areas. Antibodies were detected as early as 12 days after symptom onset. Serum antibody levels correlated with disease duration. Serology was ordered after 50% of the disease duration had elapsed, reflecting low awareness. BoDV-1-antibodies were of IgG1 subclass, and the epitope on BoDV-1 antigens was determined. Specificity of the indirect immunofluorescence antibody test (IFAT) and lineblot (LB) from serum and cerebrospinal fluid (CSF), as well as PCR testing from CSF, was 100%. Sensitivity, depending on first or all samples, reached 75–86% in serum and 92–94% in CSF for the IFAT, and 33–57% in serum and 18–24% in CSF for the LB. Sensitivity for PCR in CSF was 25–67%. Positive predictive values were 100% each, while negative predictive values were 99% (IFAT), 91–97% (LB), and 90% (PCR). ConclusionsThere is no hint that BoDV-1 causes other diseases than encephalitis in humans. Awareness has to be increased in virus-endemic areas. Tests are robust but lack sensitivity. Detection of IgG1 against specific peptides may facilitate diagnosis. Screening of healthy individuals is likely not beneficial.

Toxoplasma gondii exposure in Brazilian indigenous populations, their dogs, environment, and healthcare professionals

Although Toxoplasma gondii exposure has been reported in indigenous populations worldwide, a One Health approach has not been applied to date. This study concurrently assessed T. gondii exposure in indigenous populations, and their dogs, environment, and indigenous or non-indigenous healthcare professionals (HPs). Human and dog serum samples from 9 indigenous communities in Brazil were assessed by indirect immunofluorescence antibody test for anti-T. gondii antibodies. Soil samples (30 per community) were processed with PCR to amplify T. gondii DNA. Associated risk factors and seroprevalence were analyzed using logistic regression models. Human seropositivity and type of water source were assessed by generalized linear mixed model (GLMM) with binomial error distribution, and game meat consumption with chi-squared test. Overall, 225/463 (49%) indigenous persons were seropositive for anti-T. gondii antibodies. Of all the HPs, 67/168 (40%) were positive, and included 54/147 (37%) positive non-indigenous HPs. Indigenous persons more likely to be seropositive compared with non-indigenous HPs (OR: 1.63; 95% CI: 1.11–2.39). A total of 97/253 (38%) dogs were seropositive and highly associated with seropositive owners (p < 0.001). Based on univariate analysis for indigenous individuals, state location of community (p < 0.001), ethnicity (p < 0.001), consumption of game meat (p < 0.001), type of water source (p < 0.001), and educational level (p = 0.026) were associated with seropositivity. Logistic regression showed that indigenous seropositivity was associated with eating game meat (p = 0.002), drinking water from rivers (p < 0.001), and inversely proportional to the educational level. According to univariate analysis for non-indigenous HP, age (p = 0.005), frequency of visits to the indigenous populations (p < 0.001), consumption of water at the indigenous communities (p < 0.001), and ingestion of raw meat (p = 0.023) were associated with T. gondii seropositivity. Logistic regression revealed living outdoors (p = 0.042), habit of hunting (p = 0.008), and drinking river water (p = 0.007) as risk factors associated to seropositivity in dogs. In addition, indigenous communities lacking water treatment had higher seroprevalence for all groups including indigenous persons (GLMM; z = −7.153; p < 0.001), their dogs (GLMM; z = −2.405; p = 0.0162), and all HPs (GLMM; z = −2.420; p = 0.0155). Human seropositivity was associated with that of their dogs (p < 0.001). A single soil sample, out of 270 (0.37%), was positive for T. gondii by PCR. Our results indicate water source is a risk for human and dog toxoplasmosis in indigenous communities; both share similar exposure. Moreover, quality water access was shown to be crucial to prevent toxoplasmosis in both total and non-indigenous HPs who work in these indigenous communities.

An Investigation of the Relationship Between Isolated Anti-dense Fine-Speckled 70 Autoantibodies and Allergen-Specific Immunoglobulin E.

Background Although the mechanisms of the formation of anti-dense fine-speckled 70 (anti-DFS70) antibodies are not fully known, there is evidence in the literature that allergic reactions may play a role in their formation. Immunoglobulin E (IgE)-mediated immunopathological mechanisms are increasingly being elucidated in diseases such as atopic dermatitis and urticaria-related diseases. We aimed to reveal its relationship with anti-DFS70 in allergen-sensitive patients with positive specific IgE (sIgE) levels. Methodology The study included samples of 758 patients who underwent antinuclear antibody (ANA) screening and allergen-sIgE testing between January 2019 and January 2022. Patients' clinical diagnoses were retrospectively obtained from the hospital information management system. ANA was tested according to the instructions of the manufacturer by the indirect immunofluorescent antibody method using HEp-2 cell substrates (Euroimmun Luebeck, Germany). Allergen-sIgE was determined by chemiluminescence on the Immulite 2000 XPI system (Siemens Healthcare Diagnostics Products GmbH, Marburg, Germany) according to the instructions of the manufacturer. Results ANA pattern was detected in 74 samples included in the study. ANA-positive patients were divided into DFS70 (+) and DFS70 (-) groups. A statistically significant increase in the DFS70 pattern was observed in patients with a positive allergen-sIgE test (p < 0.0001). Both allergen-sIgE and DFS70 positivity were statistically significant in younger age groups (p < 0.05). The most common diagnosis was urticaria-related conditions in 23 (31%) patients with a positive allergy test. Conclusions Our study shows that the positivity of the DFS70 pattern is increased in allergen-sensitive patients. Therefore, the allergen-sIgE-mediated allergic disease should be considered in patients with isolated anti-DSF70. Studies with related disease groups are needed to determine whether there is a relationship between anti-DFS70 and allergy-related disease in these patients. If an immunopathological mechanism is not found, these false-positive results can be considered clinically insignificant, and unnecessary consultations can be avoided.

Epidemiological characteristics of bovine besnoitiosis (Besnoitia besnoiti) in a beef cattle farm: a cross-sectional serological assessment.

Bovine besnoitiosis is an emerging disease caused by the protozoa Besnoitia besnoiti that can have a serious economic impact on affected farms. The fact that there is no effective vaccine nor treatment, along with the lack of consistent epidemiologic data, renders the implementation of preventive medicine and control strategies much harder. A cross-sectional serological assessment was performed to better understand the distribution and prevalence of this parasite in a large beef cattle farm in Portugal and to establish some epidemiological characteristics of besnoitiosis. A random blood sampling of 450 animals from a farm that keeps around 2,000 cattle head was performed and sera were submitted to an indirect immunofluorescent antibody test (IFAT). Data on breed, age, sex, and birthplace of the tested animals and their mothers were recorded. The overall prevalence of positive animals was 16.89%, with significant differences between under 1-year-old calves (4.8%) and adults (19.67%). A higher antibody prevalence was shown in animals 1-2 years and >7 years old, in Salers breed and in cows imported from France or whose mothers had come from this country. Calves under 1 year old and crossbreed animals with ancestry born in the current farm presented the lowest antibody prevalence. The most significant risk factors revealed were age (>7 years old) and breed (Salers). Genetic studies should be carried out in order to confirm whether indeed there is a breed susceptibility to bovine besnoitiosis. We suggest that similar studies should be performed across southern Europe to establish strong epidemiologic data that would allow a rigorous transnational control program to be launched.

Evidence of intrathecally-derived antibodies against Toxoplasma gondii in horses suspected of neurological disease consistent with equine protozoal myeloencephalitis

Among the recognized neurologic diseases in horses, equine protozoal myeloencephalitis (EPM) has been reported around the world and still presents challenges in diagnosis and treatment. Horses can present with clinical neurologic signs consistent with EPM while testing negative for the two main causative agents, Sarcocystis neurona or Neospora hughesi, and may still be clinically responsive to anti-parasitic drug therapy. This context led to our hypothesis that another protozoal parasite, Toxoplasma gondii, which is known to cause toxoplasmosis in other mammalian species, is a potential pathogen to cause neurologic disease in horses. To evaluate this hypothesis, serum and cerebrospinal fluid (CSF) were collected from 210 horses presenting with clinical signs compatible with EPM, and the indirect immunofluorescent antibody test (IFAT) was used to detect antibody titers for T. gondii, S. neurona, and N. hughesi. Additionally, the serum to CSF titer ratio was calculated for T. gondii, S. neurona, and N. hughesi infections, suggesting intrathecally-derived antibodies for each of the three agents if the serum:CSF ratio was ≤ 64. There were 133 (63.3%) horses positive for serum T. gondii antibodies using a cutoff titer of 160, and 31 (14.8%) positive for CSF T. gondii antibodies using a cutoff titer of 5. Overall, 21 (10.0%) of EPM-suspect horses had a serum:CSF ratio ≤ 64 for antibodies for T. gondii, while 43 (20.5%) and 8 (3.8%) horses had a serum to CSF ratio ≤ 64 for antibodies for S. neurona and N. hughesi, respectively. A total of 6 (2.9%) animals presented evidence of concurrent intrathecally-derived antibodies for T. gondii and at least one other apicomplexan parasite in this study. Signalment and clinical signs were not different across the groups aforementioned. These data provide evidence of intrathecal production of anti-T. gondii antibodies, indicative of T. gondii infection in the brain and/or spinal cord of horses with EPM-like disease.

Seroprevalence of and Risk Factors for Toxoplasma gondii Infection in Cats from Greece

Toxoplasmosis is one of the most important protozoan diseases with a global impact on the health of domestic cats and with zoonotic significance. The aims of this study were to determine the prevalence of seropositivity for Toxoplasma gondii in different populations of cats in Greece and to assess risk factors for seropositivity. A total of 457 cats were prospectively enrolled, and a commercially available indirect immunofluorescence antibody testing (IFAT) kit was used for the detection of anti-T. gondii immunoglobulin G (IgG) in serum. Overall, 95 (20.8%) of the 457 cats were seropositive for T. gondii. Based on multivariate analysis, factors associated with seropositivity included older age [Odds ratio (OR), 1.33; p < 0.001]; a history of cat-fight trauma (OR, 3.88; p = 0.004); and lack of vaccination against calicivirus, herpesvirus-1, panleukopenia, and rabies (OR, 10; p = 0.002). This study shows a high prevalence of seropositivity for T. gondii in cats in Greece. This implies that toxoplasmosis is still a major public health concern and that optimal strategies for the prevention of infection with T. gondii in cats should be established.

Seroprevalence of severe fever with thrombocytopenia syndrome using specimens from the Korea National Health & Nutrition Examination Survey.

Severe fever with thrombocytopenia syndrome (SFTS) is an acute febrile disease caused by bites from ticks infected with the SFTS virus. In Korea, SFTS patients are observed nationwide, including Jeju Island, but there are currently no data regarding the national prevalence of SFTS, including that of residents of 16 cities and provinces. This study aimed to investigate the seroprevalence of SFTS in Korea. A total of 1500 participants were selected through random sampling from the 2014-2015 Korea National Health and Nutrition Examination Survey (KNHANES). An indirect immunofluorescence antibody assay (IFA) was performed to assess immunoglobulin (Ig) G and IgM antibody titers against SFTS virus. Of the 1500 participants, 55 (3.7%) tested positive for IgG and 1 (0.1%) tested positive for IgM, with antibody titer of ≥ 1:32. Approximately 3.9% and 2.5% of participants in urban and rural areas, respectively, had a positive titer of ≥ 1:32. There was a significant correlation between SFTS incidence per 100,000 population and seroprevalence using an IgG titer ≥ 1:64 as the cut-off value. This is the first study to investigate national SFTS seroprevalence in all 16 cities and provinces representing Korea. Our study will also provide useful guidelines for the development of preventive measures against SFTS.

Increased seroprevalence of anti-Toxoplasma gondii antibodies in dogs in southern Brazil after an outbreak of human toxoplasmosis

Toxoplasmosis is a worldwide disease caused by Toxoplasma gondii, which can infect diverse hosts, including dogs. Although T. gondii infection in dogs is usually subclinical, they are susceptible to infection and develop a specific immune response to the parasite. In 2018, the largest outbreak of human toxoplasmosis in the world occurred in Santa Maria, in southern Brazil; however, the impact of this outbreak on other hosts was not investigated at the time. Considering that dogs often share the same environmental sources of infection as humans, mainly water sources, and that in Brazil, the detection rates of anti-T. gondii immunoglobulin G (IgG) in dogs is very high, this study investigated the frequency of anti-T. gondii IgG in dogs in Santa Maria before and after the outbreak. A total of 2.245 serum samples were analyzed, 1159 collected before the outbreak and 1086 collected after the outbreak. Serum samples were tested for anti-T. gondii antibodies using an indirect immunofluorescence antibody test (IFAT). The infection detection of T. gondii was 16% (185/1159) before the outbreak and 43% (466/1086) after the outbreak. These results showed the infection of dogs with T. gondii and the high frequency of anti-T. gondii antibodies in dogs after the outbreak in humans in 2018, reinforcing water as a possible source of infection and the importance of including toxoplasmosis in the differential diagnosis of dogs.

Evaluation of an immunochromatographic serologic test to detect the presence of anti-Toxoplasma gondii antibodies in cats.

Toxoplasmosis is a protozoan disease caused by Toxoplasma gondii. Different T. gondii confirmatory techniques, including serologic methods, are available to detect the presence of the parasite. Among serology techniques, immunochromatographic rapid testing could be a reliable alternative to serologic laboratory techniques. This study evaluated a commercial immunochromatographic test (FASTest TOXOPLASMA g) in seronegative and seropositive cats. Two indirect immunofluorescence antibody reference tests, an in-house technique, and a commercial test were used to classify 292 feline serum samples. The rapid test was evaluated in different groups of cats, including healthy seronegative cats (n=121), seropositive cats with variable anti-Toxoplasma antibodies (n=146), and cats with positive serologic results for other pathogens (n=25). The sensitivity, specificity, accuracy, receiver operating characteristic curves, and kappa statistics were analyzed as performance measures. Of the 292 samples, 146 were classified as T. gondii seropositive and 146 as T. gondii seronegative. Concordant results were obtained for all samples using immunofluorescence antibody tests. The diagnostic measures of this rapid test showed 98.63% sensitivity and 100% specificity, and 99.32% accuracy. The kappa statistics value was 0.986, and the area under the receiver operating characteristic curve was 0.993. This rapid test showed diagnostic measurements similar to those of traditional quantitative serologic methods. In situations where laboratory techniques are not available, this test, under clinical conditions, could be a useful alternative to obtain accurate results rapidly.

SARS-CoV-2 infection of kidney tissues from severe COVID-19 patients.

Coronavirus disease 2019 (COVID-19) caused by infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) manifests diverse clinical pathologies involving multiple organs. While the respiratory tract is the primary SARS-CoV-2 target, acute kidney injury is common in COVID-19 patients, displaying as acute tubular necrosis (ATN) resulting from focal epithelial necrosis and eosinophilia, glomerulosclerosis, and autolysis of renal tubular cells. However, whether any renal cells are infected by SARS-CoV-2 and the mechanism involved in the COVID-19 kidney pathology remain unclear. Kidney tissues obtained at autopsy from four severe COVID-19 patients and one healthy subject were examined by hematoxylin and eosin staining. Indirect immunofluorescent antibody assay was performed to detect SARS-CoV-2 spike protein S1 and nonstructural protein 8 (NSP8) together with markers of different kidney cell types and immune cells to identify the infected cells. Renal parenchyma showed tissue injury comprised of ATN and glomerulosclerosis. Positive staining of S1 protein was observed in renal parenchymal and tubular epithelial cells. Evidence of viral infection was also observed in innate monocytes/macrophages and NK cells. Positive staining of NSP8, which is essential for viral RNA synthesis and replication, was confirmed in renal parenchymal cells, indicating the presence of active viral replication in the kidney. In fatal COVID-19 kidneys, there are SARS-CoV-2 infection, minimally infiltrated innate immune cells, and evidence of viral replication, which could contribute to tissue damage in the form of ATN and glomerulosclerosis.

Detection of anti-Sarcocystis spp. antibodies in domestic cats, in southern Brazil.

Parasites of the genus Sarcocystis can infect several species of animals and cause multiple diseases such as equine protozoal myeloencephalitis. Felines are considered hosts of this protozoa; therefore, the present study aimed to detect anti-Sarcocystis spp.-specific antibodies in domestic cats that were under clinical evaluation, using the indirect immunofluorescence antibody test. Anti-Sarcocystis-specific immunoglobulin Gs were detected in 24 out of 497 (4.82%) cat serum samples. These findings support the fact that natural Sarcocystis infections do occur in cats. Furthermore, it highlights the importance of domestic cats as both intermediate and definitive hosts in the Sarcocystis life cycle, maintaining the parasite and serving as a source of infection for various other animals. To the best of our knowledge, this is the first study to identify antibodies against the genus Sarcocystis in cats from a region in southern Brazil.

Уровни регуляторных Т-лимфоцитов и B-клеток у пациенток с болезнью Грейвса после тиреоидэктомии

Aim: To study the levels of regulatory T lymphocytes (Treg) and B-cells in peripheral blood in patients with Graves' disease in dynamics after thyroidectomy. Design: Single center, observational, prospective, cohort, open, controlled study. Materials and methods. The study included 96 patients with Graves' disease, mean age 42.86 ± 10.81 years. Clinical, hormonal and immunological examinations were performed against the background of persistent drug-induced euthyroidism, before surgery, and also 1, 3, and 6 months after thyroidectomy. The levels of Treg and B-cells in the blood was examined by flow cytometry using direct immunofluorescence and monoclonal antibodies. The level of antibodies to thyroid-stimulating hormone receptors (rTSH) was assessed by enzyme immunoassay. The control group consisted of 85 healthy women of the same age. Results. In patients with Graves' disease in the dynamics of the postoperative period, there was a consistent statistically significant decrease in the titer of antibodies to rTSH: from 14.69 (8.67–19.81) to 0.81 (0.59–0.93) IU/l. The absolute amount of Treg in their blood was reduced relative to the control values already in the preoperative period and decreased even more at 6 months after thyroidectomy. The proportion of CD19+CD5+- and CD19+CD5–-cells in the examined patients before surgery was significantly higher than in the control group. The content of CD19+CD5+ cells decreased as early as 1 month after thyroidectomy and remained at the level of control values. The сontent of CD19+CD27–-cells in patients with Graves' disease in the preoperative period was higher than in the control group, decreased to the control values 1 month after thyroidectomy and remained in this range until the end of the follow-up. The content of CD19+CD27+ cells in patients before thyroidectomy was lower compare to control, in the period of 1–3 months it corresponded to the control values, but decreased again on the 6th month after surgery. Conclusion. The reduction of Treg and B-memory cells in the peripheral blood in patients with Graves' disease six months after thyroidectomy remains of immunosuppressive regulatory mechanisms with an increase in the migration activity of immune cells for a long time. Keywords: Graves' disease, thyroidectomy, regulatory T lymphocytes, memory B cells, target therapy.

Treatment-naïve pemphigus patients showing IgG tissue deposits have serum IgG antibodies against desmoglein 1 detected more often than those against desmoglein 3: a monocentre laboratory experience.

Pemphigus is a heterogeneous group of autoimmune acantholytic diseases. To check whether there is a relationship between detecting IgG deposits in the direct immunofluorescence (DIF) and finding IgG antibodies against particular desmoglein (DSG) isoforms in ELISA techniques in patients with pemphigus. Single-step DIF for revealing the deposits of IgA, IgM, IgG, IgG1, IgG4 and C3, and monoanalyte ELISAs or the multiplex ELISA were used for diagnosis. The Z test for two independent proportions was used for the statistical analysis. We evaluated 19 consecutive treatment-naïve pemphigus patients, who exhibited IgG deposits, accompanied by other types of immunoreactants in various combinations, in DIF. Serum IgG antibodies against DSG1 were detected in 18 patients, whereas serum IgG antibodies against DSG3 were found in 10 patients. The statistical analysis showed that the proportion of anti-DSG1 antibody-positive individuals (18 of 19, 94.74%) was statistically significantly higher than the proportion of anti-DSG3 antibody-positive ones (10 of 19, 52.63%) (p = 0.0099). IgG deposition in the pemphigus pattern seems to be related to the presence of serum IgG antibodies against DSG1 rather than against DSG3. DSG1 may bind IgG more efficiently than DSG3 since DSG1 has a longer cytoplasmic region compared to that of DSG3.

Occurrence of IgG antibodies against Toxoplasma gondii, Neospora caninum, and Leptospira spp. in goats and sheep from an indigenous village in Pernambuco, Brazil.

This study aimed to determine the occurrence of anti-Toxoplasma gondii, Neospora caninum, and Leptospira spp. antibodies in sheep and goats raised in villages of the Xukuru do Ororubá indigenous community, Pernambuco, Brazil. A total of 180 serum samples from sheep and 108 serum samples from goats of both sexes and different ages were analyzed. For antibody research, indirect immunofluorescence antibody test (IFAT) were used for the protozoa T. gondii and N. caninum, and microscopic agglutination test (MAT) for Leptospira spp., with a cutoff titer of 1:64, 1:50 and 1:100, respectively. The frequency of anti-T. gondii antibodies was 16.6% (30/180) for sheep and 11.1% (12/108) for goats. The frequency of anti-N. caninum antibodies was 10.55% (19/180) for sheep, and 20.37% (22/108) for goats, while for Leptospira spp., 2.2% (4/180) of sheep and 1.85% (2/108) of goats reacted positively. The results obtained in this study are unprecedented in indigenous communities in the country and serve as an alert for monitoring goats and sheep from the Xukuru do Ororubá indigenous village regarding the occurrence and productive impact of infections by T. gondii, N. caninum, and Leptospira spp., in addition to the occurrence of the zoonosis toxoplasmosis and leptospirosis in the indigenous community.

Feline leishmaniosis: hematological and biochemical analysis.

One hundred and sixty-six cats from two animal shelters were subjected to enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence antibody test (IFAT), conventional polymerase chain reaction (cPCR), quantitative PCR (qPCR) and parasitological tests (PA) for the diagnosis of Leishmania spp. Among them, 15% (25/166), 53.6% (89/166), 3.6% (06/166) and 1.8% (03/166) were positive by ELISA, IFAT, both PCRs and PA, respectively. The sequencing of ITS-1 PCR amplicons revealed a 100% match with Leishmania infantum. After the Leishmania spp. survey, 12 cats were selected and divided into two groups for clinical, hematological, and biochemical analysis: six L. infantum positive cats (G1) and six Leishmania spp. negative cats (G2). All the cats were negative for feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV). A statistical analysis indicated significantly low platelet counts and significant hyperproteinemia associated with hypoalbuminemia in positive cats (p<0.05). Our results suggest that in endemic areas, cats with clinical signs of feline leishmaniosis (such as skin lesions, weight loss and/or enlarged lymph nodes) and that exhibit hematological and biochemical changes, such as low platelet counts and hyperproteinemia with hypoalbuminemia, should be tested for Leishmania spp. infection.

Designing a Recombinant Multi-epitope DNA Vaccine as Candidate for Protection against Pathogenic Leptospira Infection in Animals

Leptospirosis can cause severe disease and probable death in humans. Antigenic epitopes from pathogenic strains of the bacteria have shown potential for serving as vaccine candidates and play a key role in the sensitivity and specificity of immunodiagnostic tests. This in-vitro analysis was undertaken to develop a prototype recombinant DNA vaccine using in-silico epitope prediction method. Epitope prediction software programs predicted the most antigenic linear B-cell epitopes of OmpL1, LipL32, LipL41, Loa22, and LigA. Thirteen epitopes were predicted, connected by the Gly-Ser linker, and synthesized. The purity of the concentrated recombinant multi-epitope protein was assessed by restriction enzyme digestion and gel electrophoresis. In-vitro expression on mammalian Chinese Hamster Ovary cell line indicated strong cytoplasmic fluorescence produced based on an indirect immunofluorescence antibody test. The green color of the cytoplasm indicates successful transcribed and translated DNA as against the blue-stained nucleus observed in the un-transfected control group based on the indirect immunofluorescence antibody test. The findings of the current study showed high antibody binding potentials of the vaccine constructs, which could be used for diagnostic applications or as polyvalent vaccine candidates.

Seroepidemiology of Toxoplasma gondii infection and parasite DNA in free-range chickens in Aguascalientes, Mexico

The objectives of the study were to estimate the prevalence of anti-Toxoplasma gondii antibodies in free-range chickens in Aguascalientes, Mexico, its association with certain risk factors, and the frequency ofparasite DNA in the heart. Eighty-one small rural family farms were included, and blood and heartsamples were taken from 150 clinically healthy, adult, free-range chickens. Serum samples were processed by indirect immunofluorescence antibody test considering a dilution of 1:16 as a positive reaction, while the DNA detection was done by PCR. The correlation between the presence of antibodies and the potential risk factors was estimated with logistic regression. The overall seroprevalence in the examined populations was 67%, with a range from 33% to 100% among municipalities, and 78% of the farms having at least one seropositive bird. The PCR test identified the presence of parasite DNA for one case only. Among the variables, the presence of cats and the presence of other animal species on the farm were the ones that had the highest values, while the presence of feral cats and the presence of noxious fauna were also identified as potential risk factors. The results indicate high soil contamination by T. gondii oocysts, which implies the active role of cats living on the farms.

Detecção sorológica de Rickettsiae do grupo da Febre Maculosa Brasileira em cavalos de Brasília, Distrito Federal, Brasil

Brazilian spotted fever (BSF) is a zoonosis caused by spotted-fever group Rickettsiae that is transmitted to humans through tick bites. Dogs and horses are considered to be sentinel animals for this disease. In this study, a serological survey was carried out among horses apprehended by the governmental animal control service of the Federal District, Brazil. Serum samples were obtained from 122 horses over a one-year period. Through the indirect immunofluorescence antibody assay (IFA) for Rickettsia rickettsii anti-IgG antibodies, 21 samples were found to be positive, with titers ranging from 1:64 to 1:512. This demonstrates that the agent presents silent circulation in the Federal District.

Influence of the polymorphisms of the melatonin receptor 1A (MTNR1A) gene on the distribution of MT1 and MT2 melatonin receptors in ram sperm

Melatonin can exert some of its functions by binding to specific membrane receptors, mainly MT1 and MT2 in mammals. The melatonin receptor 1A (MTNR1A) gene exhibits several polymorphic sites. Certain RsaI genotypes have been related to an advance in the mating activity of ram-lambs or to a more intense reproductive behavior in adult rams. However, the influence of those genotypes in the location of melatonin receptors in ram spermatozoa remains unknown. The aim of this work was to evaluate whether RsaI polymorphisms of the MTNR1A gene influence the cellular distribution of MT1 and MT2 receptors in ram sperm. For this purpose, ejaculates from 18 previously genotyped Rasa Aragonesa rams were collected, and the location of melatonin receptors was assessed by indirect immunofluorescence, using anti-MT1 (Abnova Corporation, Cat# H00004543- A01, RRID: AB_462681) and anti-MT2 (OriGene, Cat#AP01322PU-N, RRID: AB_1619198) antibodies. Statistical analysis (χ²) was performed using SPSS Statistics v.26. For the MT1 receptor, G/A rams had a higher percentage of type I spermatozoa (labelling all over the head and tail) but lower rates of immunotypes II (reactivity at the equatorial and post-acrosomal regions, neck and tail) and III (only the equatorial region and tail labelled) when compared to G/G and A/A rams (P<0.01). Immunotype IV (only the flagelum stained) was also lower in G/A rams than in G/G (P<0.05). For the MT2 receptor, G/A rams had higher percentages of type AP spermatozoa (acrosome and post- acrosome equally stained), and lower percentages of type A (more intensity in acrosome than post-acrosome) when compared to G/G and A/A rams (P<0.001). Furthermore, G/G rams had a higher percentage of type P spermatozoa (more intense staining on post- acrosome than acrosome) than G/A and A/A rams (P<0.01). In conclusion, these results suggested that polymorphisms of the MTNR1A gene could influence the distribution of MT1 and MT2 receptors in ram sperm, which could have consequences on the direct effects of melatonin on spermatozoa. [Research supported by PID2020-113239RB-I00 and A07_20R. V.P-D. has a predoctoral contract from the Diputación General de Aragón]