In defense of children's brain: reshuffling the laboratory toolbox for the diagnosis of congenital toxoplasmosis.

For decades, an immunosorbent agglutination assay (ISAGA) has been considered the gold standard method for the detection of Toxoplasma gondii-specific IgM in infants for the diagnosis of congenital toxoplasmosis (CT). The Toxoplasma IgM ISAGA was consistently reported as having superior sensitivity. Unfortunately, the commercial kit for the detection of Toxoplasma IgM ISAGA will no longer be available in 2024 and alternatives will only be available at a handful of reference laboratories as in-house or laboratory-developed tests. In a recent study, S. Arkhis, C. Rouges, N. Dahane, H. Guegan, et al. (J Clin Microbiol 62:e01222-23, 2024, https://doi.org/10.1128/jcm.01222-23), reported that the performance of the PLATELIA Toxo IgM was comparable to that of the ISAGA method for the diagnosis of CT. A second study revealing similar results supports the PLATELIA Toxo IgM as the new gold standard for the detection of T. gondii-specific IgM in infants. Although the laboratory toolbox for CT diagnosis has been reshuffled successfully, it is by universally implementing all available serological and molecular tools at the earliest possible time during gestation that we can best defend children's brain from the potential harm caused by trans-placentally transmitted T. gondii.

A novel syphilis Treponema pallidum lipoprotein peptide antigen diagnostic assay using red cell kodecytes in routine blood centre column agglutination testing platforms.

BACKGROUND AND OBJECTIVES: The detection of treponemal antibodies, which are used to make a diagnosis of syphilis, is important both for diagnostic purposes and as a mandatory blood donor test in most countries. We evaluated the feasibility of using Kode Technology to make syphilis peptide red cell kodecytes for use in column agglutination serologic platforms. MATERIALS AND METHODS: Candidate Kode Technology function-spacer-lipid (FSL) constructs were made for the Treponema pallidum lipoprotein (TmpA) of T. pallidum, using the peptide and FSL selection algorithms, and then used to make kodecytes. Developmental kodecytes were evaluated against a large range of syphilis antibody reactive and non-reactive samples in column agglutination platforms and compared against established methodologies. Overall, 150 reactive and 2072 non-reactive Syphicheck assay (a modified T. pallidum particle agglutination) blood donor samples were used to evaluate the agreement rate of the developed kodecyte assay. RESULTS: From three FSL-peptide candidate constructs, one was found to be the most suitable for diagnostics. Of 150 Syphicheck assay reactive samples, 146 were TmpA-kodecyte reactive (97.3% agreement), compared with 58.0% with the rapid plasmin reagin (RPR) assay for the same samples. Against the 2072 expected syphilis non-reactive samples the agreement rate for TmpA-kodecytes was 98.8%. CONCLUSION: TmpA-kodecytes are viable for use as cost-effective serologic reagent red cells for the detection of treponemal antibodies to diagnose syphilis with a high level of specificity in blood centres. This kodecyte methodology also potentially allows for introduction of the reverse-algorithm testing into low-volume laboratories, by utilizing existing transfusion laboratory infrastructure.

Diagnosis of human leptospirosis: systematic review and meta-analysis of the diagnostic accuracy of the Leptospira microscopic agglutination test, PCR targeting Lfb1, and IgM ELISA to Leptospira fainei serovar Hurstbridge.

BACKGROUND: Leptospirosis is an underdiagnosed infectious disease with non-specific clinical presentation that requires laboratory confirmation for diagnosis. The serologic reference standard remains the microscopic agglutination test (MAT) on paired serum samples. However, reported estimates of MAT's sensitivity vary. We evaluated the accuracy of four index tests, MAT on paired samples as well as alternative standards for leptospirosis diagnosis: MAT on single acute-phase samples, polymerase chain reaction (PCR) with the target gene Lfb1, and ELISA IgM with Leptospira fainei serovar Hurstbridge as an antigen. METHODS: We performed a systematic review of studies reporting results of leptospirosis diagnostic tests. We searched eight electronic databases and selected studies that tested human blood samples and compared index tests with blood culture and/or PCR and/or MAT (comparator tests). For MAT selection criteria we defined a threshold for single acute-phase samples according to a national classification of leptospirosis endemicity. We used a Bayesian random-effect meta-analysis to estimate the sensitivity and specificity of MAT in single acute-phase and paired samples separately, and assessed risk of bias using the Quality Assessment of Studies of Diagnostic Accuracy Approach- 2 (QUADAS-2) tool. RESULTS: For the MAT accuracy evaluation, 15 studies were included, 11 with single acute-phase serum, and 12 with paired sera. Two included studies used PCR targeting the Lfb1 gene, and one included study used IgM ELISA with Leptospira fainei serovar Hurstbridge as antigen. For MAT in single acute-phase samples, the pooled sensitivity and specificity were 14% (95% credible interval [CrI] 3-38%) and 86% (95% CrI 59-96%), respectively, and the predicted sensitivity and specificity were 14% (95% CrI 0-90%) and 86% (95% CrI 9-100%). Among paired MAT samples, the pooled sensitivity and specificity were 68% (95% CrI 32-92%) and 75% (95% CrI 45-93%) respectively, and the predicted sensitivity and specificity were 69% (95% CrI 2-100%) and 75% (2-100%). CONCLUSIONS: Based on our analysis, the accuracy of MAT in paired samples was not high, but it remains the reference standard until a more accurate diagnostic test is developed. Future studies that include larger numbers of participants with paired samples will improve the certainty of accuracy estimates.

Development of toluidine red particle agglutination-based turbidimetric immunoassay for anticardiolipin antibody detection in syphilis.

INTRODUCTION: Serological tests of non-treponemal and treponemal types are the most frequently used for syphilis diagnosis. Nontreponemal tests are used to monitor disease activity. Toluidine red unheated serum test (TRUST), as one of nontreponemal tests, is generally applicable to hospitals at different levels. However, accurate judgment of TRUST results is inseparable from an experienced and accurate operator. To reduce current shortcomings of manual TRUST method, we attempted to convert the manual TRUST test into automatic TRUST test, that is, to determine the degree of aggregation of toluidine red particles by detecting the absorbance value of serum after reaction with toluidine red particles. METHODS: 50 µL of serum sample and 80 µL toluidine red particles were added to 96-well plate. Then, the 96-well plate was placed on a microplate reader at medium grade for 8 min to mix. Then, plasma reagin reacted with toluidine red particles and promoted the aggregation of toluidine red particles to form a large clot, which eventually caused a decrease in the absorbance at 540 nm. RESULTS: The results showed that the specificity of the automatic TRUST test was 100%, the sensitivity was 87%. And this method showed 93.5% correlation with manual TRUST test. The developed method is simple and involves less subjectivity in reading results, opening new avenues for syphilis diagnostic testing. CONCLUSION: Turbidimetric immunoassay can avoid the shortcomings of subjective interpretation, time-consuming and manual operation of manual TRUST method, and is more suitable for large-scale screening in health examination.

A Quantitative Human Red Blood Cell Agglutination Assay for Characterisation of Galectin Inhibitors.

Galectins are a family of beta-galactoside-binding proteins that are characterised by their carbohydrate recognition domain (CRD) and include galectin-1 and galectin-3. These galectins have been implicated in numerous diseases due to their pleiotropic nature, including cancer and fibrosis, with therapeutic inhibitors being clinically developed to block the CRD. One of the early methods developed to characterise these galectins was the hemagglutination of red blood cells. Although it is insightful, this approach has been hampered by a lack of sensitivity and accurate quantification of the agglutination observed. In this study, we aimed to validate a more precise and quantitative method to enable the further investigation of differences between galectins in respect to agglutination induction in different blood groups, as well as the characterisation of small molecule inhibitors. Quantification of hemagglutination was shown to be optimal using U-bottom plates imaged and analysed with FIJI ImageJ rather than flat-bottom plates read for absorbance on an optical density plate reader. Galectin-3-induced red blood cell agglutination efficacy increased significantly from blood group O to A to B. However, for both the galectin-1 monomer and concatemer, a more comparable effect was observed between blood group B and O, but with more potent effects than in blood group A. Inhibition assays for both galectin-3 and galectin-1 induced-hemagglutination were able to demonstrate clear concentration responses and expected selectivity profiles for a set of small-molecule glycomimetics, confirming the historical profiles obtained in biochemical binding and functional cellular assays.

Leptospira infection and carrier survey on rats from wet market areas in Kuala Lumpur, Malaysia.

BACKGROUND OBJECTIVES: Leptospirosis is an important zoonotic infection that has caused significant mortality and morbidity worldwide. This disease is endemic in Malaysia and as a developing tropical country, leptospirosis is concerning as it threatens Malaysian public health and the country's economic sectors. However, there is limited information on leptospirosis in Malaysia, especially regarding leptospiral seroepidemiology among carriers in Malaysia. Therefore, more epidemiological information on the source of the disease and reservoir are needed for better disease control and source intervention. The objectives of this study are to gather information on Leptospira infection and the carrier status of rats captured from selected wet markets of Kuala Lumpur metropolitan city in Malaysia. METHODS: Live rat trappings were performed in four major wet markets in Kuala Lumpur, namely, Pudu, Chow Kit, Datuk Keramat, and Petaling Street. Animal samplings were performed for 12 months in 2017, where blood and kidney samples were collected and tested for anti-leptospiral antibodies via Microscopic Agglutination Test (MAT) and pathogenic Leptospira screening via Polymerase Chain Reaction (PCR) amplification offlaB gene. RESULTS: MAT showed that 34.7% (n = 50/144) of the captured rats were positive for anti-leptospiral antibody of which the most prominent serovar was Malaya followed by a local strain, IMR LEP 175. In parallel, 50 rats were also positive for pathogenic Leptospira DNA. INTERPRETATION CONCLUSION: This study showed that there are persistent Leptospira infections among rats in Kuala Lumpur wet markets and these rats are important reservoir hosts for the bacteria.

Objectification of evaluation criteria in microscopic agglutination test using deep learning.

We aim to objectify the evaluation criteria of agglutination rate estimation in the Microscopic Agglutination Test (MAT). This study proposes a deep learning method that extracts free leptospires from dark-field microscopic images and calculates the agglutination rate. The experiments show the effect of objectification with real pictures.

Toxoplasma gondii Survey in Waterfowl and Gulls from Eight USA States.

Sera from 391 waterbirds from eight USA states were tested for Toxoplasma gondii antibodies using the modified agglutination test. Fifteen different waterbird species (26.6%; n=104) were seropositive. Of the adults, 25.4% (n=52) showed a significantly higher T. gondii seroprevalence compared with juveniles (13.4%; n=17); however, sex was not a significant factor.

Levels of Cytokines in Leptospirosis Patients with Different Serovars and rfb Locus.

Leptospirosis has a wide spectrum of clinical manifestations ranging from mild to severe disease. The cytokine response is considered one of the key drivers for this varying manifestation. The different cytokine response observed in patients with leptospirosis could be due to the variation of infecting serovars. Since the rfb locus codes for the lipopolysaccharide synthesis of the bacterial cell wall, which also determines the serovar, this locus may play a role in driving a specific cytokine response in the host. We investigated 12 commonly used cytokine profiles in serum samples of culture, microscopic agglutination test (MAT), or polymerase chain reaction (PCR)-positive patients with leptospirosis. The sequences of the rfb locus in culture-positive samples were generated from whole genome sequencing and serovar status was drawn from original data published. Isolated cultures were subjected to whole genome sequencing using the PacBio RS II system, and the resulting data were used to determine the species. The recovered genomic data were annotated with the Rapid Annotation using Subsystem Technology (RAST) subsystem, and the rfb locus was extracted. The cytokine analysis was carried out using the Qiagen human ELISA kit. Eighteen samples were found to be positive by culture, while the other 7 samples were positive by PCR or MAT. Infections from Leptospira interrogans serovar Autumnalis (5), Pyrogens (3), Icterohaemorrhagiae (1) Leptospira borgpetersenii (all 7 samples clustered in same clonal group with serovar status not determined), Leptospira weilii (1 with serovar status not determined), and Leptospira kirschneri serovar Grippotyphosa (1) were included in the analysis. Three patients [infected with Leptospira interrogansserovar Autumnalis (2) and Pyrogens (1)] and 2 MAT-positive patients (highest titer against serovar Bratislava of L.interrognas) were reported to have severe clinical manifestations, while the rest had mild to moderate symptoms. Although the serum cytokine concentration of patients with severe clinical manifestation was comparatively higher, a statistically significant difference was observed only for interleukin (IL)-1ß (P < 0.05). IL-10/tumor necrosis factor-alpha (TNF-α) ratio was high in patients with severe complications. In general, patients infected with L. interrogans showed higher concentration of cytokines compared to L. borgpetersenii.

Diagnosis of human brucellosis: Systematic review and meta-analysis.

BACKGROUND: Brucellosis, a widely spread zoonotic disease, poses significant diagnostic challenges due to its non-specific symptoms and underreporting. Timely and accurate diagnosis is crucial for effective patient management and public health control. However, a comprehensive comparative review of available diagnostic tests is lacking. METHODOLOGY/PRINCIPAL FINDINGS: This systematic review addressed the following question: 'What is the accuracy of the available tests to confirm human brucellosis?' Two independent reviewers examined articles published up to January 2023. The review included original studies reporting symptomatic patients with brucellosis suspicion, through any index test, with sensitivity and/or specificity as outcomes. As exclusion criteria were considered: sample size smaller than 10 patients, studies focusing on complicated brucellosis, and those lacking essential information about index or comparator tests. Sensitivity and specificity were assessed, with consideration for the index test, and 'culture' and 'culture and standard tube agglutination test (SAT)' were used as reference standards. Bias assessment and certainty of evidence were carried out using the QUADAS-2 and GRADE tools, respectively. A total of 38 studies reporting diagnostic test performance for human brucellosis were included. However, the evidence available is limited, and significant variability was observed among studies. Regarding the reference test, culture and/or SAT are deemed more appropriate than culture alone. Rose Bengal, IgG/IgM ELISA, and PCR exhibited equally high performances, indicating superior overall diagnostic accuracy, with very low certainty of the evidence. CONCLUSIONS/SIGNIFICANCE: This systematic review underscores the potential of the Rose Bengal test, IgG/IgM ELISA, and PCR as promising diagnostic tools for brucellosis. However, the successful implementation and recommendations for their use should consider the local context and available resources. The findings highlight the pressing need for standardization, improved reporting, and ongoing advancements in test development to enhance the accuracy and accessibility of brucellosis diagnosis.

Investigation of anti-Leptospira spp. antibodies and leptospiruria in cats attended to a veterinary teaching hospital in southern Brazil.

Leptospirosis is a bacterial zoonosis that affects both humans and animals worldwide. Currently, it is known that cats may be susceptible to infection. This study aims to investigate the presence of anti-Leptospira spp. antibodies and leptospiruria in cats, using Microscopic Agglutination Test (MAT) and Real-time Polymerase Chain Reaction (PCR) techniques, respectively. A total of 76 cats, undergoing comprehensive anamnesis, general physical examination, and complementary exams were included in the investigation. Among the 76 cats tested, 9.2% (7/76) exhibited the presence of anti-Leptospira spp. antibodies, while Leptospira spp. DNA was detected in at 1.3% (1/76) of the evaluated urine samples. No significant associations were observed between the serological and molecular diagnostic results and the assessed variables, including clinical data and laboratory results of cats testing positive. This study provides insight into the occurrence of Leptospira spp. infection and leptospiruria in cats treated at a veterinary teaching hospital in southern Brazil.

Study on the interference of vaccine antibodies with the serological diagnosis of leptospirosis in cattle.

A simple IgG-specific ELISA for Leptospira spp. was compared with the microscopic agglutination test (MAT) to detect IgG antibody responses to a commercial vaccine in cattle. We used an enzyme-linked immunosorbent assay (ELISA) with sonicated Leptospira interrogans serovar copenhageni M 20. After initial vaccination, specific antibodies against Leptospira spp. were detected in 90 % of the animals by IgG-ELISA and 60 % by MAT, while after booster, antibodies were detected in 100 % and 80 % of the animals by IgG-ELISA and MAT, respectively. Both serological MAT and ELISA tests revealed interferences of vaccine antibodies. Disease diagnosis with ELISA and MAT methods should be made two and a half months and four months, respectively, after vaccination to avoid interference of vaccine antibodies. On the other hand, our results suggest that IgG-ELISA may be a useful method to assess the development of IgG antibodies induced by Leptospira vaccine.

RAPID POINT-OF-CARE TESTING FOR DETECTION OF ANTIBODIES TO TOXOPLASMA GONDII IN BLACK VULTURES AND RING-BILLED GULLS FROM PENNSYLVANIA.

Toxoplasma gondii is a zoonotic protozoan parasite that infects most warm-blooded animals, including birds. Scavenging birds are epidemiologically important hosts because they can serve as indicators of environmental T. gondii levels. A rapid point-of-care (POC) test that detects antibodies to T. gondii in humans is commercially available. In this research, we assessed the ability of the human POC test to detect anti-T. gondii antibodies in 106 black vultures (Coragyps atratus) and 23 ring-billed gulls (Larus delawarensis) from Pennsylvania, USA. Serum samples were tested with the POC test and compared to the modified agglutination test (MAT) in a blinded study. Overall, anti-T. gondii antibodies were detected in 2.8% (3/106) of black vultures and 60.9% (14/23) of ring-billed gulls by the POC test. One false-positive POC test occurred in a black vulture that was negative by MAT. False-negative results were obtained in 2 black vultures and 4 ring-billed gulls that had MAT titers of 1:25 or 1:50. The sensitivity and specificity of the POC for both black vultures and ring-billed gulls combined were 95.7% and 95.5%, respectively. This is the first study using human POC tests to detect antibodies to T. gondii in birds. Further study of the rapid test as a screening tool for serological surveillance of T. gondii in birds is warranted.

Identification of Dominant Leptospira Serogroups among Leptospirosis Cases and Their Clinical Outcomes: A Prospective Hospital-Based Study in Mangaluru, India.

Leptospirosis is a reemerging zoonotic disease of worldwide significance, endemic to the southern region of India, with clinical manifestations similar to other febrile illnesses; hence, it is often misdiagnosed and underreported. Inadequate information about the disease burden and the regional circulating serogroups contributes to its neglected disease status. This study aimed to identify the infecting Leptospira serogroup in the coastal region of Mangaluru and study the clinical symptoms and outcome among leptospirosis patients. Serum samples were collected from 30 patients with confirmed leptospirosis admitted to a tertiary care center in Mangaluru and screened by microscopic agglutination test (MAT) for the infecting serogroup. The clinical profile of these cases was reviewed, and data regarding epidemiological factors such as age, sex, complications, and mortality were recorded. The MAT identified a higher occurrence of serogroup Bataviae (n = 7, 43.75%) and serogroup Australis (n = 5, 31.25%) compared with other serogroups screened in this study population. Patients were aged 16 to 65 years, with a predominance of males. The clinical presentation of leptospirosis ranged from a mild febrile illness to multiorgan failure. Fever (n = 29, 96%) was the common clinical presentation, followed by myalgia, nausea, and abdominal pain. Acute kidney injury, acute respiratory distress syndrome, and multiple organ dysfunction syndrome were the common complications observed. Determining the circulating serogroup is necessary to understand the epidemiology and diversity of Leptospira serogroups among animals and humans to strategize appropriate preventive measures.

Detection of tetanus toxoid with iron magnetic nanobioprobe.

Diagnosis of diseases with low facilities, speed, accuracy and sensitivity is an important matter in treatment. Bioprobes based on iron oxide nanoparticles are a good candidate for early detection of deadly and infectious diseases such as tetanus due to their high reactivity, biocompatibility, low production cost and sample separation under a magnetic field. In this study, silane groups were coated on surface of iron oxide nanoparticles using tetraethoxysilane (TEOS) hydrolysis. Also, NH2groups were generated on the surface of silanized nanoparticles using 3-aminopropyl triethoxy silane (APTES). Antibody was immobilized on the surface of silanized nanoparticles using TCT trichlorothriazine as activator. Silanization and stabilized antibody were investigated by using of FT-IR, EDX, VSM, SRB technique. UV/vis spectroscopy, fluorescence, agglutination test and ELISA were used for biosensor performance and specificity. The results of FT-IR spectroscopy showed that Si-O-Si and Si-O-Fe bonds and TCT chlorine and amine groups of tetanus anti-toxoid antibodies were formed on the surface of iron oxide nanoparticles. The presence of Si, N and C elements in EDX analysis confirms the silanization of iron oxide nanoparticles. VSM results showed that the amount of magnetic nanoparticles after conjugation is sufficient for biological applications. Antibody stabilization on nanoparticles increased the adsorption intensity in the uv/vis spectrometer. The fluorescence intensity of nano bioprobe increased in the presence of 10 ng ml-1. Nanobio probes were observed as agglomerates in the presence of tetanus toxoid antigen. The presence of tetanus antigen caused the formation of antigen-nanobioprobe antigen complex. Identification of this complex by HRP-bound antibody confirmed the specificity of nanobioprobe. Tetanus magnetic nanobioprobe with a diagnostic limit of 10 ng ml-1of tetanus antigen in a short time can be a good tool in LOC devices and microfluidic chips.

Seroprevalence of leptospirosis among stray dogs in Bosnia and Herzegovina.

Leptospirosis is a (re) emerging zoonosis that occurs worldwide. This study aimed to assess seroprevalence of leptospirosis and to identify the most common reactive serovars and risk factors for seropositivity in apparently healthy stray dogs of unknown vaccination status in the Sarajevo region of Bosnia and Herzegovina. Positive microscopic agglutination test titres (≥ 1:25) were detected in 3.87% (156/4028) of samples and most of the sera reacted against one serovar (85.9%). Dogs were most commonly reactive to Canicola (40.4%) and Hardjo (33.3%), followed by Pomona (15.4%) Tarassovi (14.7%), Icterohaemorrhagiae (8.3%), Grippotyphosa (5.8%), Bratislava (1.3%) and Saxkoebing (0.6%). Dogs older than one year had higher odds of seropositivity compared to younger dogs. The seropositivity was higher in spring and autumn than in summer. These results advocate for the need of a control strategy for this zoonosis in the country, which should include sero-surveillance, monitoring, and the inclusion of additional serovars in the testing.

Leptospira infection in bats in Vietnam.

Bats from three provinces in Vietnam (Lai Chau, Son La, and Dong Thap) were examined for the presence of pathogenic Leptospira or specific antibodies using polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), and microscopic agglutination test (MAT). Tissue specimens from 298 bats belonging to 11 species were analyzed using a real-time PCR assay specific for leptospires of pathogenic species. Leptospiral DNA was identified in 40 bats from following species: Rousettus amplexicaudatus (5/9; 55.5 %), Rousettus leschenaultii (17/42; 40.4 %), Myotis hasseltii (8/25; 32 %), Taphozous longimanus (3/12; 25 %), and Eonycteris spelaea (7/32; 21.9 %). Based on secY phylogeny, sequences from M. hasseltii bore a strong resemblance to L. borgpetersenii. Sequences from other species revealed unique lineages: one of them resembled Leptospira sp., previously identified in Rousettus madagascariensis (Madagascar) and Rousettus aegyptiacus (South Africa); the second lineage showed close relation to L. kirshneri; and the third held an intermediary position between L. noguchii and L. interrogans. Through ELISA, anti-Leptospira antibodies were found in 83 of 306 bats, with the highest seroprevalence observed in R. leschenaultii (44/48; 91.6 %), R. amplexicaudatus (6/8; 75 %), and E. spelaea (19/25; 76 %). 66 of these ELISA-positive samples were tested using MAT; 41 of them were confirmed in MAT as positive. The predominant serogroups in our study were Tarassovi and Mini.

A novel Bayesian Latent Class Model (BLCM) evaluates multiple continuous and binary tests: A case study for Brucella abortus in dairy cattle.

Bovine brucellosis, primarily caused by Brucella abortus, severely affects both animal health and human well-being. Accurate diagnosis is crucial for designing informed control and prevention measures. Lacking a gold standard test makes it challenging to determine optimal cut-off values and evaluate the diagnostic performance of tests. In this study, we developed a novel Bayesian Latent Class Model that integrates both binary and continuous testing outcomes, incorporating additional fixed (parity) and random (farm) effects, to calibrate optimal cut-off values by maximizing Youden Index. We tested 651 serum samples collected from six dairy farms in two regions of Henan Province, China with four serological tests: Rose Bengal Test, Serum Agglutination Test, Fluorescence Polarization Assay, and Competitive Enzyme-Linked Immunosorbent Assay. Our analysis revealed that the optimal cut-off values for FPA and C-ELISA were 94.2 mP and 0.403 PI, respectively. Sensitivity estimates for the four tests ranged from 69.7% to 89.9%, while specificity estimates varied between 97.1% and 99.6%. The true prevalences in the two study regions in Henan province were 4.7% and 30.3%. Parity-specific odds ratios for positive serological status ranged from 1.2 to 2.2 for different parity groups compared to primiparous cows. This approach provides a robust framework for validating diagnostic tests for both continuous and discrete tests in the absence of a gold standard test. Our findings can enhance our ability to design targeted disease detection strategies and implement effective control measures for brucellosis in Chinese dairy farms.

Equine brucellosis in Iran: serological, bacteriological and molecular analysis.

Equine brucellosis significantly impacts the health and functionality of horses, leading to complications such as bursitis infection, septic tenosynovitis, septic arthritis, and non-specific lameness resulting from joint infections. In the present study, we used the Rose Bengal plate agglutination test (RBPT), serum agglutination test (SAT), and the 2-mercaptoethanol (2-ME) assays to find equine brucellosis. From June 2018 to September 2022, 876 blood samples were randomly taken from apparently healthy racing horses in certain parts of Iran, such as Kerman, Isfahan, Tehran, Qom, and Kurdistan. DNA extraction was carried out directly on all 63 serum samples identified as seropositive through RBPT. An additional 30 seronegative serum samples were also randomly chosen for study. Bacterial culture was also done on milk, blood, and vaginal swabs taken from seropositive horses.The bacteria that were found in the samples were then put through Bruce-ladder PCR. Our results indicated that 63 (7.1%), 21 (2.3%), and 2 (0.2%) of horses were seropositive using RBPT, SAT, and 2-ME, respectively. Also, none of the 30 DNA-extracted serum samples from seronegative horses tested positive for Brucella DNA, while 44.5% (28/63) of the DNA samples from seropositive horses yielded positive results for Brucella DNA. Out of the seropositive samples, 26 had DNA from Brucella abortus and 2 had DNA from Brucella melitensis. Also, B. melitensis biovar 1 was found in two milk samples from mares in the provinces of Kerman and Isfahan. It was identified using classical biotyping, and molecular assays. It was seen that some of healthy racing horses in some parts of Iran had antibodies against Brucella. The bacteriology and PCR methodologies provide a more comprehensive and reliable means of identifying Brucella spp. infections in horse, especially when the RBPT test came back positive. This underscores the imperative for employing molecular, bacterial, and serological methods in the diagnosis and monitoring of this zoonotic infection. Additionally, this finding suggests that Brucella is being transmitted to equine hosts as a result of its presence in ruminants. The mechanism of transmission may involve interactions between infected ruminants and susceptible equines. This discovery is significant as it underscores the potential cross-species transmission of Brucella and highlights the importance of understanding and managing the spread of the pathogen in both ruminant and equine populations.

Detection of anti-Toxoplasma gondiiantibodies in wild free-living birds and mammals from the northwest region of São Paulo state, Brazil / Detecção de anticorpos anti-Toxoplasma gondiiem aves e mamíferos silvestres de vida livre da região noroeste do estado de São Paulo, Brasil

Toxoplasmosis is a protozoonosis caused by an obligate intracellular parasite named Toxoplasma gondii, which can infect humans and a large number of homeothermic animal species with worldwide distribution. The present study aimed to detect anti-T. gondii antibodies from serological samples of free-living wild animals from the northwest region of São Paulo state, Brazil. Thirty-two samples (eight from birds and 24 from mammals) were analyzed by the modified agglutination test (MAT) using 5 cut-off points for birds and 25 for mammals. Seropositivity was observed in 25% (2/8) of birds, including the species Rupornis magnirostris (roadside hawk) and Caracara plancus (southern caracara), and 29.2% (7/24) animals were seropositive among mammals, including one hoary fox (Lycalopex vetulus), two maned wolves (Chrysocyon brachyurus), one black howler monkey (Alouatta caraya), two crab-eating foxes (Cerdocyon thous) and one gray brocket deer (Mazama gouazoubira). The results obtained with the present study indicate the exposure to T. gondiiof free-living wild animals from the northwest region of São Paulo state and, therefore, that they probably play a role in the transmission and maintenance of T. gondii in the environment they inhabit. Thus, identification of the infection in several animal species in the region indicates the environmental contamination of the area. Studies of this nature may help to understand the importance of the prevention and control of this disease in Brazil.(AU)

A toxoplasmose é uma protozoonose causada por um parasita intracelular obrigatório denominado Toxoplasma gondii, que pode infectar os humanos e um vasto número de espécies animais homeotérmicas, apresentando distribuição mundial. O presente estudo objetivou a detecção de anticorpos anti-T. gondii a partir de amostras sorológicas de animais silvestres de vida livre da região noroeste do estado de São Paulo. Foram analisadas 32 amostras (oito de aves e 24 de mamíferos) por meio do teste de aglutinação modificado (MAT), utilizando ponto de corte 5 para as aves e 25 para os mamíferos. Soropositividade foi observada em 25% (2/8) das aves, incluindo as espécies Rupornis magnirostris (gavião-carijó) e Caracara plancus (carcará); entre os mamíferos, 29,2% (7/24) foram soropositivos incluindo uma raposa-do-campo (Lycalopex vetulus), dois lobos-guará (Chrysocyon brachyurus), um bugio-preto (Alouatta caraya), dois cachorros-do-mato (Cerdocyon thous) e um veado-catingueiro (Mazama gouazoubira). Os resultados obtidos com o presente estudo indicam a exposição dos animais selvagens de vida livre a T. gondii na região noroeste do estado de São Paulo e, portanto, que provavelmente apresentam papel na transmissão e manutenção de T. gondii no meio ambiente em que vivem. Assim, a identificação da infecção em várias espécies de animais na região indica a contaminação ambiental da área. Estudos dessa natureza podem ajudar no entendimento sobre a prevenção e o controle dessa importante doença no Brasil.(AU)