Indirect Fluorescent Antibody Test Research Articles

Risk factors for Toxoplasma gondii infection in dogs: a serological survey

BackgroundDogs, as well as a wide variety of other warm-blooded animals, act as intermediate host of Toxoplasma gondii. In dogs, most cases of toxoplasmosis are subclinical, although clinical disease has been sporadically reported. Beyond its role in diagnostic pathways, seropositivity also functions as a reflection of the parasite’s spread within the dog’s living environment. The aim of the present study was to evaluate the possible risk factor associated with seropositivity to T. gondii in dogs in Central-Northern Italy, analysing 120 dogs sera for the presence of IgG antibodies by indirect fluorescence antibody test (IFAT).ResultsThe population examined was composed of 54.2% hunting dogs, 24.2% companion dogs, 14.2% truffle dogs and 7.5% watchdogs. Thirty-four (29.2%) dogs tested positive for T. gondii IgG, with titres ranging from 1:40 to 1:1280. Seroprevalence and antibodies titres were not related to dog gender, age or function. The logistic regression and ordered logistic regression results indicated that seroprevalence, and antibody titres were significantly higher in dogs cohabiting with cats, exhibiting coprophagy habits, and living constantly outdoors. Notably, the lifestyle factor showed the highest odds-ratios in the study: dogs living constantly outdoors were found to be at approximately 5 times greater risk of testing positive and having higher antibody titres compared to dogs living both indoors and outdoors.ConclusionBoth logistic and ordered logistic regression results support the key role of living with cats, engaging in coprophagy behaviours, and maintaining an outdoor lifestyle in increasing the risk of T. gondii infection in dogs. These identified risk factors collectively suggest that both ingesting oocysts, as observed through cat cohabitation and coprophagy, and engaging in predatory behaviours, as possible for outdoor living dogs, are indicating likely sources of T. gondii infection in this host species.

Specificity and sensitivity of an indirect fluorescent antibody test to detect antibodies against bovine viral diarrhea virus.

Since being reported in 1979 and 2006, indirect fluorescent antibody (IFA) tests have not been reported to detect bovine viral diarrhea virus (BVDV) antibodies to our knowledge. Thus, we re-evaluated the efficacy and usefulness of IFA tests for BVDV serology. We tested 4 combinations of 2 antibody conjugates (fluorescein isothiocyanate [FITC]-conjugated rabbit IgG anti-bovine IgG; rabbit IgG F(ab')2 fragment anti-bovine IgG [F(ab')2 FITC-IgG]) and 2 washing solutions (PBS; carbonate-bicarbonate-buffered saline [CBBS]) to evaluate the specificity of an IFA test for BVDV. We compared the sensitivity of the optimal combination with virus neutralization (VN) tests and an ELISA, and compared IFA with VN titers against different genotype (subgenotype) strains. For the F(ab')2 FITC-IgG/CBBS combination, only 1 of the 156 (0.6%) 4-fold diluted cattle sera resulted in a nonspecific reaction; other combinations led to a much higher incidence (22.9-37.2%). For the F(ab')2 FITC-IgG/CBBS combination, IFA detection rates were identical (36 of 59) for BVDV1 and BVDV2 genotypes, and IFA titers against them were strongly correlated (r = 0.99). The antibody-detection rates of the IFA tests were almost identical to those of VN tests and the ELISA (κ: 0.96 and 0.89, respectively). The IFA titers against 4 strains (BVDV1a, BVDV1j, BVDV2a, and an unidentified strain) were similar, 1,024 to ≥4,096, although the VN titers were different. Thus, our IFA tests were specific and sensitive, and more useful than VN tests given that the IFA tests could evaluate the immune status of cattle using a representative strain, regardless of genotype (subgenotype).

Observational longitudinal study on Toxoplasma gondii infection in fattening beef cattle: serology and associated haematological findings

Toxoplasmosis, caused by the protozoan parasite Toxoplasma gondii, is a globally distributed zoonotic infection with significant implications for human and animal health. This study investigated the prevalence of T. gondii infection in a population of beef cattle at three different stages of their productive lifespan and examined the impact of T. gondii serological status on blood parameters. A commercial beef fattening unit in Italy was the setting for this research, which involved a biosecurity assessment upon cattle arrival, blood sampling at three time points and Toxoplasma-specific serological testing using indirect fluorescent antibody tests (IFAT). Results revealed a dynamic pattern of T. gondii seropositivity in cattle, with an initial prevalence of 30.6% at arrival (T0) that increased to 44.6% at 14 days (T1) and then decreased slightly to 39.3% at slaughter after 5 months (T2). Interestingly, seroconversion was observed during the study, indicating ongoing infections, and antibody waning occurred in some animals. In terms of blood parameters, seropositive cattle exhibited significantly lower mean corpuscular volume (MCV) and a higher neutrophil–lymphocyte (N/L) ratio, suggesting an activation of the innate immune response. Furthermore, cattle with higher antibody titres displayed higher neutrophil counts. However, all blood parameters with a statistical significance were within the reference range. This study provides for the first time a longitudinal investigation on the serological status for T. gondii in naturally exposed beef cattle. These findings provide valuable insights into the clinico-pathological aspects of natural T. gondii exposure in cattle and underscore the importance of monitoring and managing T. gondii infection in livestock production systems.

Sero-epidemiological investigation on Toxoplasma gondii infection in Apennine wolf (Canis lupus italicus) and wild boar (Sus scrofa) in Italy

BackgroundThe wild boar (Sus scrofa) and the Apennine wolf (Canis lupus italicus) are two wild species that have both increased their presence in the Italian territory, albeit in varying numbers. They can be occasionally found in peri-urban areas as well. Both of these species can serve as intermediate hosts for Toxoplasma gondii, as they can become infected either through the consumption of oocysts found in water, soil, or on vegetables, or through the ingestion of meat containing bradyzoites. Consequently, these animals can be regarded as key indicators of Toxoplasma presence in the wild or peri-urban environment. In our study, we examined a total of 174 wild boar meat juice and 128 wolf sera from Italy for the detection of T. gondii IgG using the indirect fluorescent antibody test (IFAT).ResultsThe results showed that 40 (22.6%) of the wild boar meat juice and 34 (26.6%) of the wolf serum samples tested positive. Interestingly, there were no significant differences in seropositivity with respect to gender, age group, or the region of origin in both species.ConclusionsOverall the results indicate a moderate exposure in both the species under investigation, highlighting the spread of T. gondii in sylvatic and periurban environments. The prevalence of T. gondii in wild boar is consistent with findings from other studies conducted in Europe. Our study, with a considerably larger sample size compared to the available research in European context, provides valuable data on the seroprevalence of T. gondii in wolves.

Evaluation of Immunodiagnostic Performances of Neospora caninum Peroxiredoxin 2 (NcPrx2), Microneme 4 (NcMIC4), and Surface Antigen 1 (NcSAG1) Recombinant Proteins for Bovine Neosporosis.

Bovine neosporosis is among the main causes of abortion in cattle worldwide, causing serious economic losses in the beef and dairy industries. A highly sensitive and specific diagnostic method for the assessment of the epidemiology of the disease, as well as it surveillance and management, is imperative, due to the absence of an effective treatment or vaccine against neosporosis. In the present study, the immunodiagnostic performance of Neospora caninum peroxiredoxin 2 (NcPrx2), microneme 4 (NcMIC4), and surface antigen 1 (NcSAG1) to detect IgG antibodies against N. caninum in cattle were evaluated and compared with that of the indirect fluorescent antibody test (IFAT). The results revealed that NcSAG1 had the highest sensitivity and specificity, with values of 88.4% and 80.7%, respectively, followed by NcPrx2, with a high sensitivity of 87.0% but a low specificity of 67.0%, whereas NcMIC4 showed sensitivity and specificity of 84.1% and 78.9%, respectively, when compared with IFAT. A high degree of agreement was observed for NcSAG1 (k = 0.713) recombinant protein, showing the highest diagnostic capability, followed by NcMIC4 (k = 0.64) and NcPrx2 (k = 0.558). The present study demonstrates that NcSAG1 is helpful as an antigen marker and also demonstrates the potential immunodiagnostic capabilities of NcPrx2 and NcMIC4, which could serve as alternative diagnostic markers for detecting N. caninum infection in cattle. These markers may find utility in future treatment management, surveillance, and risk assessment of neosporosis in livestock or other animal host species. Further research should be directed toward understanding the in vivo immune response differences resulting from immunization with both recombinant proteins.

Comparative Study of a Novel Lateral Flow Rapid Test with Conventional Serological Test Systems for the Diagnosis of Canine Leishmaniosis in Croatia and Brazil.

Control of canine infections with Leishmania infantum (L. infantum), a major zoonotic disease in Brazil and southern Europe, is becoming increasingly important due to its close proximity to humans, the increasing import of dogs from endemic regions and the impact of climate change on vector spreading. Simple, rapid and reliable diagnostic tests are therefore needed to detect infected dogs. Here, we re-evaluated different serological methods for the diagnosis of canine leishmaniosis (CanL) in Croatia and Brazil. The diagnostic performance of the indirect fluorescent antibody test (IFAT) and the VetLine® Leishmania ELISA (GSD Frankfurt, Germany) was compared with three rKLi8.3-based diagnostic test systems, the rKLi8.3 ELISA (GSD Frankfurt, Germany), the INgezim® Leishma CROM (GSD Madrid, Spain) lateral flow test (LFT) and the VetBlot®Leishmania LineBlot (GSD Frankfurt, Germany). CanL symptomatic dogs were efficiently diagnosed by all tests, except the VetLine® Leishmania ELISA, which is based on whole Leishmania antigens. The advantage of rKLi8.3 was also observed in oligo- and asymptomatic dogs from Brazil and Croatia, although with reduced diagnostic efficiency compared to symptomatic dogs. Similar to IFAT and rKLi8.3 ELISA, the LFT did not cross-react with other common canine pathogens; it showed very high specificity for healthy dogs from endemic regions in both countries and did not react with healthy, vaccinated dogs in Brazil. In conclusion, serodiagnostic tests based on the rKLi8.3 antigens are superior to whole parasite antigens, and the LFT has the advantage of providing a laboratory-independent, rapid and specific diagnosis of CanL.

Detection of Rabies IgG and IgM Antibodies Using the Rabies Indirect Fluorescent Antibody Test.

The rabies indirect fluorescent antibody (IFA) test was developed to detect various rabies-specific antibody isotypes in sera or cerebral spinal fluid. This test provides rapid results and can be used to detect rabies antibodies in several different scenarios. The rabies IFA test is especially useful for the quick and early detection of antibodies to evaluate the immune response in a patient who has developed rabies. Although other methods for antemortem rabies diagnosis take precedence, this test may be utilized to demonstrate recent rabies virus exposure through antibody detection. The IFA test does not provide a virus-neutralizing antibody (VNA) titer, but the pre-exposure prophylaxis (PrEP) response can be evaluated through positive or negative antibody presence. This test can be utilized in various situations and can provide results for a number of different targets. In this study, we used several paired serum samples from individuals who received PrEP and demonstrated their rabies antibody presence over time using the IFA test.

Neospora caninum surface antigen 1 is a major determinant of the pathogenesis of neosporosis in nonpregnant and pregnant mice.

NcSAG1 is one of most widely investigated antigens of Neospora caninum in various research fields. Such studies demonstrated the proficiency of NcSAG1 in the regulatory process of parasite adhesion and invasion of host cells. Accordingly, the contribution of NcSAG1 to the pathogenesis of neosporosis can undoubtedly be extrapolated, but direct evidence is lacking. Herein, we provide the first successful attempt at the gene disruption of NcSAG1 and novel data on the invasion and virulence potentials of N. caninum in vitro and in vivo. The disruption of the NcSAG1 gene was applied using the CRISPR/Cas9 system and confirmed by PCR, western blot and indirect fluorescent antibody tests as NcSAG1 knockout parasites (NcSAG1KO). Then, we investigated the role of NcSAG1 in the growth kinetics of the parasite in vitro. The deletion of the NcSAG1 gene significantly decreased the infection rate and reduced the egress rate of the parasite. An in vivo study using nonpregnant female and male BALB/c mice revealed a significantly higher survival rate and lower body weight change in the group infected with the NcSAG1KO parasite than in the parental strain (Nc-1)-infected group. Regarding the vertical transmission model of BALB/c mice, the absence of the NcSAG1 gene significantly enhanced the survival of pups and greatly lowered the parasite burden in the brains of pups. In conclusion, our study suggested NcSAG1 as a key molecule in the pathogenesis of N. caninum.

Coinfection with Leishmania infantum and Toxoplasma gondii in Domestic Cats from a Region with a High Prevalence of Feline Immunodeficiency Virus.

The aim of this study was to investigate the coinfection of feline retroviruses (feline immunodeficiency virus-FIV, and the feline leukemia virus-FeLV) with Leishmania infantum and Toxoplasma gondii and the factors associated with these pathogens in domestic cats from Mossoró, a city endemic for canine and human leishmaniasis situated in the semiarid region of Northeast Brazil. Blood samples from 120 cats were collected, and an epidemiological questionnaire was applied to investigate the risk factors associated with the infections. Retroviruses, L. infantum, and T. gondii infections were assessed using a point-of-care ELISA and quantitative PCR (qPCR), indirect fluorescent antibody test (IFAT) and qPCR, and IFAT, respectively. The overall seroprevalences observed were 35% (95% CI = 27.0-43.8%) for FIV, 0.8% (95% CI = 0.1-4.5%) for FeLV, 25.8% (95% CI = 18.8-34.3%) for T. gondii, and 4.2% (95% CI = 1.7-9.3%) for L. infantum. Coinfection with FIV and L. infantum was observed in 2.5% (3/120) of the assessed cats, while 12.5% (15/120) were coinfected with FIV and T. gondii. No significant association was found among the investigated agents (p > 0.05). The factors associated with FIV infection in the multivariable analysis were male sex and age above 78 months. The findings of this study demonstrated a high rate of FIV infection in cats from the Brazilian semiarid region and the exposure of these animals to zoonotic and opportunistic agents. Due to the immunosuppressive potential of FIV, cats infected with this retrovirus should be screened for coinfections with L. infantum and T. gondii, and preventative measures should be adopted.

Evaluation of Four Adult Visceral Leishmaniasis Cases.

Leishmania infantum is the species responsible for visceral leishmaniasis [(VL), kala-azar], which is observed sporadically mainly in pediatric age groups in the Aegean, Mediterranean and Central Anatolian regions of Türkiye. The aim of this study is to evaluate the diagnosis, clinic, laboratory results and treatments of four adult patients with VL who applied to our hospital. The patients were referred to our hospital to investigate hematological malignancy. In the study, the data of four patients (three men, one woman; age range: 30-40 years) who were diagnosed with VL and treated in the infectious diseases clinic of our hospital between January 2022 and April 2022 were evaluated retrospectively. The diagnosis of VL was made according to appropriate clinical and physical examination findings, biochemical and serological tests (indirect fluorescent antibody test and rK39 rapid antigen test) and polymerase chain reaction (PCR) results, as well as the presence of amastigote forms of the parasite in bone marrow samples. Serology positivity was found in all patients, and bone marrow positivity was found in two patients. According to the results of RT-PCR in all patients, it was determined that the species causing the disease was L. infantum/L. donovani. Initially, the most common symptoms were fever, fatigue, and abdominal distension. None of the patients had an immunosuppressive condition. It was understood that all the patients lived in the rural area of Syria's Idlib province. Hepatosplenomegaly, increased erythrocyte sedimentation rate, anemia, leukopenia and thrombocytopenia were found in all patients. The patients were treated with liposomal amphotericin-B (L-AMB). One patient did not come for follow-ups, the other three patients were found to have completely recovered in their follow-up. No recurrence was observed in any of the patients. In conclusion, VL should be considered in patients who apply to health institutions with complaints of fever, hepatosplenomegaly, increased erythrocyte sedimentation rate, anemia, leukopenia and thrombocytopenia.

An abortion storm in a goat farm in the Northeast Region of Brazil was caused by the atypical Toxoplasma gondii genotype #13

The objective of this study was to characterise a Toxoplasma gondii-induced abortion outbreak on a goat farm in the State of Paraíba, Northeast Region of Brazil. From a herd of 10 does, seven experienced abortions and one gave birth to twins (one stillborn and the other weak and underdeveloped). Serum samples from all of the does were analysed by indirect fluorescent antibody test (IFAT). Samples of colostrum and placenta from two does, along with lung, heart, brain and umbilical cord samples from four of the foetuses, were screened by nested ITS1 PCR specific for T. gondii. The positive samples were then analysed by multiplex nested PCR-RFLP. All ten does tested positive by IFAT for anti-T. gondii IgG (titrations ranging from 1:4096 to 1:65,536). The ITS1 PCR screening revealed T. gondii DNA in the placenta (2/2), colostrum (2/2), umbilical cord (2/4), lung (1/4), heart (1/4), and brain (1/4). Four samples produced complete RFLP genotyping results, identifying a single genotype, ToxoDB #13. In conclusion, we demonstrated a high rate of abortion caused by T. gondii in a goat herd, highlighting the pathogenicity of genotype #13, one of the most prevalent genotypes of T. gondii in Brazil.

Detection of IgG Anti-Giardia duodenalis Antibodies in Sera by Indirect Immunofluorescence and Western Blotting Assays.

Serological assays are alternative laboratory tools for the diagnosis of parasitic infections. The aim of this work was to evaluate the performance of the indirect fluorescent antibody test (IFAT) and Western blotting (WB) for the detection of IgG anti-Giardia antibodies in human sera. Sera from individuals infected with Giardia duodenalis, other parasites or non-parasitized were selected for serological assays. Ninety-seven sera were tested by IFAT at 1:20 and 1:40 dilutions and of these, 40 samples were also analyzed by WB. The sensitivity and specificity of the IFAT was 97% and 46.9% at 1:20 sera dilution, and 39.4% and 59.4% at 1:40 sera dilution. The low molecular weight polypeptides fractions of 25kDa, 27-31kDa and 45-55kDa were the most frequently identified by the sera of individuals infected with G. duodenalis, along with low cross-reactivity, presenting an individual sensitivity of 42.8%, 50.0% and 57.1%, and specificity of 83.3%, 83.3% and 91.7%, respectively. The highest overall sensitivity of WB (85.7%) was based on the immunoreactivity of sera with at least one of those proteins. The concordance between the detection of G. duodenalis in feces by microscopy and the WB results was considered substantial (Kappa = 0.61). Constant exposure to Giardia infection throughout a lifetime can maintain high levels of specific antibodies in serum, even without active infection. Moreover, proteins found in intestinal amoebas may hinder the serological diagnosis of giardiasis in endemic areas due to cross-reactivity, which can be partially solved using Giardia low molecular weight proteins.

EVALUATION OF CANINE TOXOPLASMOSIS SEROPREVALENCE IN ANIMALS FROM GREATER VITÓRIA-ES

Toxoplasmosis is a zoonosis of worldwide distribution, and dogs, despite intermediate hosts, participate in the epidemiological chain of transmission, both to other animals and humans. As an epidemiological surveillance activity, a serological research was carried out for antibodies of the IgG class anti-Toxoplasma gondii in serum samples obtained from 150 dogs, from January to March 2022, belonging to the serum bank of the Veterinary Clinical Laboratory, of the Faculty of Veterinary Medicine of UVV, Vila Velha-ES. The indirect fluorescent antibody test (IFAT-IgG) was used, evaluating the results in relation to the titers obtained, age, and gender. Sixteen (10.66%) of the samples were positive, with variable titers between 16 and 256, 15 (93.75%) with title 16, 11 (68.75%) 64, and three (18.75%) 256. Regarding gender, of the 16 positive animals, nine (56.25%) were females, and seven (43.75%) were males. Related to age, positive animals ranged from three to 15 years, and animals with higher titles were between 10 and 12 years old. It is concluded that despite the low seroprevalence, T. gondii dispersion occurs in the studied region and that surveillance measures should be established to control infection and disease in the canine and human populations.

Molecular and serological prevalence of corridor disease (buffalo associated Theileria parva infection) in cattle populations at the livestock/game interface of KwaZulu-Natal province, South Africa

Theileria parva are intracellular protozoal parasites responsible for three disease syndromes in cattle, namely East Coast fever (ECF), Corridor disease (CD) and Zimbabwean theileriosis. The increase in reports of CD outbreaks in recent years has raised questions about the probability of adaptation of buffalo-derived T. parva strains in cattle herds adjacent to game reserves. A cross-sectional study was conducted from March 2016 to December 2018 to investigate the extent of occurrence of T. parva infections in cattle in the CD-controlled area of KwaZulu-Natal Province. Blood samples were collected from 1137 cattle from 14 herds and analysed by quantitative real-time PCR (qPCR) and indirect fluorescent antibody test (IFAT) to determine the prevalence of T. parva. A total of 484 samples from 4 of the 14 herds were further tested on qPCR for the presence of T. taurotragi infections. The data were analysed using descriptive statistics and a chi-square test was used to assess association between variables. The overall prevalence of T. parva was 1.3% (95%CI:1–2%) and 19.9% (95%CI:17–22%) on qPCR and IFAT, respectively. The qPCR positive samples were detected in March and May while IFAT positive samples were detected in all seasons sampled, with higher numbers during summer months. The Pearson Chi-squared test showed that T. parva prevalence rates based on both qPCR and IFAT were positively associated with herds with previous history of CD outbreaks (χ2 = 8.594, p = 0.003; χ2 = 69.513, p < 0.001, respectively). The overall prevalence of T. taurotragi was 39.4% (95% CI: 35–44%) with the herd-level prevalence ranging between 35.0% and 43.4%. Possible cross-reaction of T. parva IFAT to T. taurotragi was detected on few samples, however, there was no significant association between T. taurotragi infections and IFAT positivity (χ2 = 0.829, p = 0.363). Results from this study demonstrated the extent of occurrence of subclinical carriers and the level of exposure to T. parva infections in cattle populations at a livestock/game interface area of KwaZulu-Natal Province. The molecular and seroprevalence rates were low when compared with other areas where cattle-adapted T. parva infections are endemic. The adaptation of buffalo-derived T. parva in cattle population resulting in cattle-cattle transmissions seem to be unlikely under the current epidemiological state.

Dynamics of anti-Toxoplasma gondii antibodies and seroconversion during pregnancy and lactation in naturally infected goats

The objective of this work was to evaluate the dynamics of anti-T. gondii antibodies and seroconversion in naturally infected goats from the last third of pregnancy to 100 days of lactation and relate it to hematological and dehydration parameters. Blood samples were obtained from 56 goats in the different physiological states (pregnancy, kidding and lactation) as in different years (2019, 2021 and 2022). A total of 266 serum samples were obtained and evaluated by indirect fluorescent antibody test (IFAT) to end titer. The overall T. gondii seropositivity was 80.4% (45/56), with titers ranging from 100 to 25.600. The goats older than 3 years (4967 ± 1329) had significantly higher IFAT titers than the younger goats (2705 ± 681). The highest rate of positive seroconversion 31.1% (14/45) was found between kidding and 70 days of lactation; and of negative seroconversion 28.9% (13/45) between late pregnancy and kidding. The highest proportion of slightly dehydrated animals was found in the last third of pregnancy (14/25) and kidding (9/28). The correlation between seroconversion and T. gondii antibody titers was negative to the established dehydration index. These data suggest that in all physiological states and at different ages of goats, there is seroconversion which is not related to hydration status. Pregnancy, kidding and peak of lactation are stressful physiological periods, facilitating the reactivation of chronic T. gondii infections which are expressed by higher antibodies titers.

B-110 Validation of Digital and Automated anti-dsDNA IFA Interpretations Using the dIFine Automated Fluorescent Microscope System

Abstract Background Detection of anti-double stranded DNA antibodies (anti-dsDNA) by the indirect fluorescent antibody (IFA) assay is a well-established tool for aiding in the diagnosis of systemic lupus erythematosus (SLE). ZEUS Scientific has manufactured an FDA-cleared anti-dsDNA IFA test system for decades, which relies upon manual interpretation of qualitative results using a standard fluorescent microscope. This same anti-dsDNA IFA test system was recently utilized to train software, in conjunction with a specialized fluorescent microscope (collectively termed dIFine), towards automating qualitative result interpretation. The objective of this study was to validate the reproducibility, sensitivity, and specificity of the ZEUS anti-dsDNA IFA test system, in conjunction with the dIFine automated microscope. Methods Multi-site reproducibility study–2 low-positive, 2 mid-positive, 2 high-positive, and 2 negative serum samples were assembled to create a reproducibility panel. The sample identities of the panel members were scrambled prior to testing. The panel was assayed via the ZEUS anti-dsDNA-IFA test system at a 1:10 screening dilution, in triplicate, twice per day, on five days, at three different laboratories (including ZEUS Scientific). At each laboratory, the processed slides were interpreted manually by two operators, then scanned by dIFine to generate digital images of the wells, as well as an automated qualitative result interpretation. The digital images of each slide well were also interpreted by two operators, resulting in 3 independent interpretations per sample, for each operator and instrument (Manual, Digital, Automated). The number of slide wells processed for interpretation at each laboratory was: 8 samples × 30 replicates each = 240 total. Clinical study–300 serum samples derived from donors previously diagnosed with SLE were assembled. A control cohort consisting of 360 samples from donors previously diagnosed with other rheumatic and non-rheumatic diseases was also assembled. The sample identities were scrambled for both cohorts prior to testing. The 660 samples were assayed at a 1:10 screening dilution using the ZEUS anti-dsDNA IFA test system at ZEUS Scientific. Manual, digital, and automated interpretations were carried out at ZEUS Scientific as described above. Results Reproducibility (Actual/Expected): [Site 1 - Operator A - Manual (240/240), Digital (240/240); Site 1 - Operator B - Manual (240/240), Digital (240/240); Site 1 - Automated - (231/240)]. [Site 2 - Operator A - Manual (240/240), Digital (240/240); [Site 2 - Operator B - Manual (240/240), Digital (240/240); Site 2 - Automated - (236/240)]. [Site 3 - Operator A - Manual (240/240), Digital (238/240); Site 3 - Operator B - Manual (240/240), Digital (238/240); Site 3 - Automated - (231/240)]. Sensitivity for SLE cohort (Positive/Total, %): [Operator A - Manual, (79/300, 26.3%), Digital (79/300, 26.3%)]; [Operator B - Manual, (79/300, 26.3%), Digital (79/300, 26.3%), Automated (81/300, 27.0%)]. Specificity for control cohort (Negative/Total): [Operator A - Manual, (358/360, 99.4%), Digital (358/360, 99.4%)]; [Operator B - Manual, (358/360, 99.4%), Digital (358/360, 99.4%), Automated (358/360, 99.4%)]. Conclusions These validation studies demonstrate that the ZEUS anti-dsDNA-IFA test system, in conjunction with the automated dIFine microscope, yields highly reproducible and accurate results derived from digital and automated interpretations.

Evidence of Horse Exposure to Anaplasma phagocytophilum, Borrelia burgdorferi, and Leishmania infantum in Greece through the Detection of IgG Antibodies in Serum and in an Alternative Diagnostic Sample-The Saliva.

Among the various zoonotic pathogens that infect horses, Anaplasma phagocytophilum, Borrelia spp. and Leishmania spp. have gained scientific interest, and relevant molecular and serological studies in horses have been conducted worldwide. Moreover, human and veterinary medicine have extensively applied alternatives to serum diagnostic samples-such as saliva-for detecting pathogens or antibodies. In this study, we investigated the exposure of horses in Greece to A. phagocytophilum, B. burgdorferi, and L. infantum, and we assessed the diagnostic accuracy of saliva compared to serum in detecting IgG antibodies against the abovementioned pathogens. Paired saliva and serum samples were collected from 317 horses from different regions in Greece. The paired samples were examined using the indirect fluorescent antibody test (IFAT) for detecting IgG antibodies against A. phagocytophilum, B. burgdorferi, and L. infantum. Sensitivity, specificity, positive likelihood ratio (PLR), and negative likelihood ratio (NLR) were determined to assess the validity of saliva as an alternative to serum. The receiver operating characteristic (ROC) curve revealed that the optimal cut-off value for detecting antibodies against all the examined pathogens in saliva was 1/10. Higher seropositivity rates were found for B. burgdorferi (15.14%) and A. phagocytophilum (14.19%) compared to L. infantum (1.26%). The detection of IgG antibodies using IFAT in saliva samples had a good test performance compared to serum. The two sample types had a substantial to almost perfect agreement. Although the sensitivity was moderate (70.83-75.56%) in all cases, the specificity was almost perfect to perfect (99.63-100%). This study provides the first evidence that horses in Greece are exposed to A. phagocytophilum and B. burgdorferi and confirms that the seroprevalence of L. infantum in horses in Greece remains low. Our findings suggest that saliva sampling coupled with IFAT could be successfully applied for detecting IgG antibodies against these important zoonotic pathogens in large-scale epidemiological studies in horses, at the population level, as an alternative to serum.

Validation of a multiplex microsphere immunoassay for detection of antibodies to Trypanosoma cruzi in dogs.

The vector-borne protozoan parasite Trypanosoma cruzi causes Chagas disease in humans, dogs, and many other mammalian hosts. Canine Chagas disease is increasingly diagnosed in dogs of the southern United States where triatomine insect vectors occur, and there are limited veterinary testing options; only the indirect fluorescent antibody (IFA) test is offered at a single accredited diagnostic laboratory. We evaluated a multiplex microsphere immunoassay (MIA) for the detection of antibodies against T. cruzi in dogs and compared it with existing serologic methods to establish cutoff values and relative sensitivity and specificity. We tested 135 canine sera that had been characterized using the IFA and off-label use of 2 commercial rapid assays with our multiplex MIA against 12 antigens: 9 T. cruzi antigens, a negative control recombinant protein (green fluorescent protein, GFP), a Leishmania antigen, and a canine parvovirus antigen (used as an antibody control given near-ubiquitous parvoviral vaccination). The median fluorescence intensity (MFI) ratio between each T. cruzi antigen and GFP was calculated for every sample. Samples with an antigen:GFP MFI ratio > 4 SDs above the mean of 25 known-negative sera were considered positive to that antigen. Samples testing positive to ≥ 2 antigens were considered positive for T. cruzi antibodies. Compared to the IFA, our multiplex MIA had a relative sensitivity of 100% and specificity of 97.0%. Given its precision, high-throughput format, potential for automation, and lack of subjective interpretation, our multiplex MIA should be considered a valid and improved assay for T. cruzi antibodies in dogs.

Worldwide Overview of Neospora spp. Infection in Equids Diagnosed by Serological Tests: Systematic Review and Meta-Analysis

Equids can be infected by Neospora spp., and its infection is associated with neurological and reproductive disorders. This systematic review and meta-analysis aimed to assess the prevalence of anti-Neospora caninum antibodies in horses, donkeys and mules worldwide between 2012 and 2022. Five scientific databases were consulted: MEDLINE, ScienceDirect, PubMed, LILACS and SciELO, using the descriptors “protozoan” or “neospora” AND “horses” or “donkeys” or “mules”. A total of 1909 studies were screened, and 25 studies met the inclusion criteria. The pooled prevalence of Neospora spp. infection in equids, based on a total of 11,076, was 8.34% (95% CI: 4.78–14.17%). Further analysis by species revealed no statistically significant difference in the prevalence of Neospora spp. infection across equids: 8.5% (95% CI 5.03–13.99%) in horses, 7.36% (CI 2.08–22.94%) in donkeys and 6.07% (95% CI 0.71–36.83%) in mules. Regarding diagnostic tests, the prevalence of Neospora spp. infection determined by the indirect fluorescent antibody test (IFAT) was 8.08% (95% CI: 3.74–16.61%), by the enzyme-linked immunosorbent assay (ELISA) was 7.91% (95% CI: 3.71–16.08%), and by the modified agglutination test (MAT) was 20% (95% CI: 15.37–25.60%). This meta-analysis provided valuable and comprehensive insights into the prevalence of Neospora spp. infection in horses, donkeys, and mules using serological detection methods. The results highlight the wide distribution of Neospora spp. infection in equids across all continents, indicating the importance of this infection. Transmission of the parasite can occur both horizontally (directly between animals) and vertically (from mother to offspring), further emphasizing the significance of understanding and managing this infection in equid populations.

Prevalence and incidence of canine visceral leishmaniasis and its clinical-immunological features in an endemic area of the Brazilian Amazon.

A cohort study for 2 years period analysed the prevalence, incidence and clinical-immunological features of canine Leishmania (L.) chagasi-infection in 316 mongrel dogs in a visceral leishmaniasis-endemic area in Pará State, Brazil. Diagnosis of infection was performed by the indirect fluorescent antibody test (IFAT-IgG), the leishmanin skin test (LST) and a parasite search (from the popliteal lymph node aspiration) at the beginning of the study and at 6, 12 and 24 months intervals. IFAT/LST revealed three immune profiles of infection: (I) IFAT(+) /LST(-) (81), (II) IFAT(-) /LST(+) (17) and (III) IFAT(+) /LST(+) (13). Prevalence of profiles I, II and III were 25.6, 5.4 and 4.1%, and an overall prevalence 35.1%. Incidence of profiles I, II and III were 5.4, 0.3 and 0.0%, and an overall incidence 5.7% dogs per month. Incidence at the age ranges <1 year, ≥1 year, <7 years and ≥7 years evidenced a highest rate in the age range <1 year (6.6% dogs per month). Parasitological diagnosis was positive in 19% dogs at the prevalence (85.7% profile I), and in 11% at the incidence (100% profile I). The clinical picture of 179 infected dogs showed 145 (81%) of profile I (82% subclinical); 21 (11.7%) of profile II (100% subclinical); and 13 (7.3%) of profile III (84.6% subclinical). Conversion from subclinical to sick dogs was higher (p<0.05) in profile I (40.2%) than in profiles II (5.8%) and III (9%). Immunological conversion showed that only 3.2% of profile I dogs (prevalence) converted to LST(+) (two at the end of the first 6 months and 1 after 24 months), while 82.3% of profile II dogs converted to IFAT(+) (11 in the first 6 months, whereas three after 12 months). A 100% death rate was observed in dogs from profile I alone. These results reinforce the need of adopting preventive strategies against CVL as early as in the first semester of the dog's life.