Indirect Immunofluorescent Antibody Test Research Articles

Detection of Anaplasma spp. and Ehrlichia spp. in dogs from a veterinary teaching hospital in Italy: a retrospective study 2012-2020.

Anaplasma phagocytophilum, Anaplasma platys and Ehrlichia canis, responsible of diseases in dogs, are tick-borne pathogens with a proven or potential zoonotic role that have shown increasing prevalence worldwide. The aims of this retrospective study were to assess the frequency of Anaplasma spp. and Ehrlichia spp. exposure in dogs tested in a veterinary teaching hospital in Italy over a 9-year period, to compare the performance of the diagnostic tests used, to evaluate correlations with clinical data, and to genetically analyse the identified bacteria. During the study period, 1322 dogs tested by at least one of the rapid immunoenzymatic test, indirect immunofluorescent antibody test or end-point PCR assay for Anaplasmataceae detection were included. Dogs were tested if they had clinical signs or clinicopathological alteration or risk factors related to infection, and if they were potential blood-donor animals. Ninety-four of 1322 (7.1%) dogs tested positive for at least one pathogen: 53 (4.3%) for A. phagocytophilum, one (0.1%) for A. platys and 63 (4.6%) for E. canis. The number of dogs tested increased and the positivity rate progressively declined over the years. Comparison of tests showed a near-perfect agreement between serological tests and a poor agreement between PCR and indirect assays. A breed predisposition has been highlighted for A. phagocytophilum infection in hunting breed dogs and for E. canis infection in mixed breed dogs. Phylogeny confirmed potential zoonotic implications for A. phagocytophilum and showed no correlation of the identified bacteria with the geographical origin. Our study provides new insights into possible risk factors in dogs and evidenced discordant results between different tests, suggesting that a combination of serological and molecular assays is preferable for a correct diagnosis.

Toxoplasmosis Behind Bars: One Health Approach on Serosurvey Dynamics and Associated Risk Factors for Women Inmates, Correctional Officers, and In-Prison Feral Cats

Brazil holds the third highest general and fifth female incarcerated population worldwide. Despite the incarceration ecosystem that may favor the spreading of zoonotic diseases, particularly when unattended animals are present, no comprehensive study has focused on toxoplasmosis dynamics in such environment. Accordingly, the present study has aimed to serologically assess anti-Toxoplasma gondii (IgG) antibodies by indirect immunofluorescent antibody test in inmates, correctional officers, and feral cats at the Women’s State Penitentiary of Parana, southern Brazil. In overall, 230/506 (45.5%; CI 95%: 41.2–49.8) incarcerated women, 31/91 (34.1%; 95% CI: 25.2–44.3) correctional officers, and 23/39 (59.0%; CI 95%: 43.2–72.9) cats were seropositive to anti-T. gondii antibodies. Logistic regression revealed that seropositivity likelihood increased with consumption of raw meat (p=0.040) and decreased with elementary educational level (p=0.001). No statistical difference was found comparing seropositivity between inmates and correctional officers (p=0.057). As women inmates have been considered among the most vulnerable groups in disease morbidity and mortality, seropositivity observed herein may be directly related to vulnerability and high T. gondii oocyst exposure dispersed in cat feces during incarceration.

Spontaneous splenic rupture associated with scrub typhus: a case report

BackgroundScrub typhus, an acute febrile disease with mild to severe, life-threatening manifestations, potentially presents with a variety of complications, including pneumonia, acute respiratory distress syndrome, cardiac arrhythmias (such as atrial fibrillation), myocarditis, shock, peptic ulcer, gastrointestinal bleeding, meningitis, encephalitis, and renal failure. Of the various complications associated with scrub typhus, splenic rupture has rarely been reported, and its mechanisms are unknown. This study reports a case of scrub typhus-related spontaneous splenic rupture and identifies possible mechanisms through the gross and histopathologic findings.Case presentationA 78-year-old man presented to our emergency room with a 5-day history of fever and skin rash. On physical examination, eschar was observed on the left upper abdominal quadrant. The abdomen was not tender, and there was no history of trauma. The Orientia tsutsugamushi antibody titer using the indirect immunofluorescent antibody test was 1:640. On Day 6 of hospitalization, he complained of sudden-onset left upper abdominal quadrant pain and showed mental changes. His vital signs were a blood pressure of 70/40 mmHg, a heart rate pf 140 beats per min, and a respiratory rate of 20 breaths per min, with a temperature of 36.8 °C. There were no signs of gastrointestinal bleeding, such as hematemesis, melena, or hematochezia. Grey Turner's sign was suspected during an abdominal examination. Portable ultrasonography showed retroperitoneal bleeding, so an emergency exploratory laparotomy was performed, leading to a diagnosis of hemoperitoneum due to splenic rupture and a splenectomy. The patient had been taking oral doxycycline (100 mg twice daily) for 6 days; after surgery, this was discontinued, and intravenous azithromycin (500 mg daily) was administered. No arrhythmia associated with azithromycin was observed. However, renal failure with hemodialysis, persistent hyperbilirubinemia, and multiorgan failure occurred. The patient did not recover and died on the fifty-sixth day of hospitalization.ConclusionsClinicians should consider the possibility of splenic rupture in patients with scrub typhus who display sudden-onset abdominal pain and unstable vital signs. In addition, splenic capsular rupture and extra-capsular hemorrhage are thought to be caused by splenomegaly and capsular distention resulting from red blood cell congestion in the red pulp destroying the splenic sinus.Graphical abstract

Seroprevalence of Toxoplasma gondii in wild boars (Sus scrofa) hunted in Ukraine

Toxoplasma gondii is an important zoonotic parasite worldwide, but it has received limited attention in Ukraine. A seroepidemiological study was conducted and samples from 452 wild boars that had been hunted in 2006–2011 in 23 of the 25 regions of Ukraine were tested to estimate T. gondii seroprevalence. A locally available commercial enzyme-linked immunosorbent assay (ELISA) was used for the investigation. Additionally, we tested 92 of the sera using a widely used commercial multi-species ELISA and an indirect immunofluorescence antibody test (IFAT). With the locally available ELISA, 35 of the 452 wild boars tested positive, yielding a seroprevalence estimate of 7.7% (95% confidence interval 5.5–10.5). The seropositive wild boars originated from eight of the regions. Using the majority criteria, 10/92 samples tested using both ELISAs and the IFAT were considered positive, yielding an estimated seroprevalence of 10.9% within the subset of samples. The highest seroprevalence was observed in wild boars hunted in Luhans'k (30.0%), Odesa (17.7%) and Kharkiv (12.7%). Seroprevalence was higher in older animals (13.3% for age group >12 months and 7.7% for age group ≤12 months). This is the first seroepidemiological study of T. gondii in wild boars in Ukraine. Assuming that seropositivity indicates presence of infectious parasites in the tissues, eating undercooked meat of wild boars hunted in Ukraine could be a potential source of infection to other hosts, including humans.

Comparison of laboratory methods for diagnosis of human visceral leishmaniasis in the Center-West Region of São Paulo State, Brazil

Visceral leishmaniasis (VL) is a neglected disease and represents an important public health problem. The present study aimed to compare the conventional laboratory methods with the molecular method for VL diagnosis in human blood samples. Direct parasitological test of bone marrow aspirate (direct parasitological test), rapid immunochromatographic test (RIT), indirect immunofluorescent antibody test (IFAT) and polymerase chain reaction (PCR) were used to evaluate 26 human samples with clinical suspicion of VL. The positivity rates obtained for direct parasitological test, RIT, IFAT and PCR were 80.8%, 80.8%, 73.1% and 84.6%, respectively. PCR had greater positivity and agreement with the other methodologies. Therefore, the use of PCR in the diagnostic routine should be encouraged to complement the serological results.

Identification of specific antigens between Toxoplasma gondii and Neospora caninum and application of potential diagnostic antigen TgGRA54.

Toxoplasma gondii is a zoonotic parasite that is very common in livestock. Meat products from livestock infected with T. gondii are one of the important transmission routes of toxoplasmosis. Rapid and reliable diagnosis is a prerequisite for the prevention and control of toxoplasmosis. Neospora caninum and T. gondii are similar in morphology and life history, and there are a large number of cross antigens between them, making clinical diagnosis of toxoplasmosis more difficult. In this study, immunoprecipitation-mass spectrometry (IP-MS) was used to screen for T. gondii-specific antigens, and the specific antigen was cloned and expressed in Escherichia coli. The specific antigen was then used to establish an indirect ELISA diagnostic method. A total of 241 specific antigens of T. gondii and 662 cross antigens between T. gondii and N. caninum were screened by IP-MS. Through bioinformatics analysis and homology comparison, seven proteins were selected for gene cloning and prokaryotic expression, and the most suitable antigen, TgGRA54, was selected to establish an indirect ELISA for T. gondii. Compared with the indirect immunofluorescent antibody test (IFAT), the positive coincidence rate of the ELISA based on rTgGRA54 was 100% (72/72) and the negative coincidence rate was 80.95% (17/21). The indirect ELISA method based on TgGRA54 recombinant protein was established to detect T. gondii antibodies in bovine sera, and the recombinant protein reacted well with T. gondii positive sera from sheep, mouse, and swine, indicating that the recombinant protein is a good diagnostic antigen for T. gondii.

Anti-Leishmania spp. antibody detection in domestic cats from a visceral leishmaniasis transmission area.

Feline leishmanial infection is reported worldwide, but the epidemiological role of domestic cats in the leishmaniasis cycle remains unclear, and cats might act as cryptic reservoir hosts in endemic areas with no feline leishmaniosis cases. Considering that, a serological screening for anti-Leishmania spp. antibodies was performed by indirect immunofluorescence antibody test (IFAT) in 389 necropsied cats' serum samples from a new visceral leishmaniasis transmission area with no feline leishmanial infection reported to unveil if the cats are being exposed to the parasite. The overall seroprevalence for Leishmania spp. was 11.05% (43/389). No association was found between sex, neutering status, age group, breed, coat length, feline immunodeficiency virus (FIV) infection, and Leishmania spp. antibody detection. A positive association was found with coat color (cats within the orange spectrum with white [particolor]) (OR = 2.47, CI 95% 1 - 6.13, P = 0.044) and a negative association (OR = 0.38, CI 95% 0.18 - 0.79, P = 0.01) between feline leukemia virus (FeLV) infection and IFAT positivity for Leishmania spp. Therefore, it is concluded that the seroprevalence found was greater than 10%, indicating contact of the protozoan with cats in the region served.

One Health Approach in Serosurvey of Toxoplasma gondii in Former Black Slave (Quilombola) Communities in Southern Brazil and Among Their Dogs.

Brazilian quilombos are rural semi-isolated remnant communities of former black slaves and their descendants who traditionally maintained themselves through archaic subsistence livestock and agriculture practices and historically lacked specific public health policies. Although such individuals and their dogs may be exposed to zoonotic pathogens such as Toxoplasma gondii, no study to date has assessed these human-animal populations together. Populations in four different Brazilian quilombos in southern Brazil were evaluated. Overall, 93/208 people (44.7%) and 63/100 dogs (63.0%) were seropositive for IgG anti-T. gondii antibodies by indirect immunofluorescent antibody test (IFAT), 4/208 (1.9%) human samples seropositive for IgM anti-T. gondii antibodies, with a human-dog seropositivity ratio for IgG of 0.71. Quilombola individuals ingesting game meat were 2.43-fold more likely (95% CI: 1.05-5.9) to be seropositive. No risk factors were associated with seropositivity among dogs, thus suggesting that their exposure to T. gondii was random. Surprisingly, our research group had previously found an inverted human-dog ratio for T. gondii seropositivity of 2.54 in the urban area of a nearby major city. Because consumption of raw/undercooked game meat by quilombola individuals may have contributed to higher exposure, higher overall seroprevalence among dogs may have also indicated interaction with wildlife. Although these dogs may hunt wildlife without their owners' awareness, the higher dog seropositivity may also be related to feeding from discarded food in the community or backyard livestock animals and drinking surface water contaminated with oocysts. Thus, wildlife cannot be singled out as the reason, and future studies should consider sampling water, soil, wildlife, and livestock tissues, to fully establish the source of infection in dogs herein.

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.

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.

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.

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.

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.

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.

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.

Evaluation of monoclonal antibodies in immunofluorescence assay for rapid quality control of Peste des petits ruminants (PPR) vaccine

Peste des petits ruminants (PPR) is an acute viral disease of small ruminants with morbidity and mortality rates as high as 90 % and 100 % respectively. Vaccination using a homologous live-attenuated vaccine is the main control strategy available in endemic countries, and ensuring the quality of these vaccines is of utmost importance for PPR control. Currently, the quality control test to determine vaccine potency is the time-consuming and subjective tissue culture infectious dose (TCID50) assay involving titration on Vero cell line and reading the cytopathic effect (CPE) using light microscopy. In this study, an indirect immunofluorescent antibody test (IFAT) was evaluated for PPR vaccines potency testing. Accuracy and repeatability of the IFAT were also determined. Six PPR vaccine batches were produced and tested simultaneously by CPE reading and the IFAT. Vero cells were seeded in 96-well plates, and infected with 10-fold serial dilutions of the vaccines. CPEs were read continuously from day 3–14 after infection, and titers expressed in TCID50/ml. From 3–7 days post-infection, cells were fixed, incubated with anti-N and anti-H monoclonal antibodies (mAbs), and stained with a secondary antibody labeled with a fluorescent tag. Fluorescent foci of infection were detected using a fluorescence microscope, and titers were calculated as TCID50/ml. The results indicated that from day 4, the IFAT gave similar results to CPE readings of day 14 (intraclass correlation coefficient, ICC = 0.957, P < 0.0001). This is a three-fold reduction in time and can be used for rapid PPR vaccine potency evaluation. However, there was no difference observed between the results of the two mAbs (ICC = 0.910, P < 0.0001) indicating that both are equally sensitive and rapid. This result indicates that the IFAT may be more suited for potency testing of PPR vaccines as it saves time and minimizes analyst-related variability associated with CPE reading.