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Background: Detection of Leishmania RNA virus (LRV) in Old World Leishmania species and their possible role in the disease prognosis requires sensitive and specific methods, preferably independent of the viral genome. We aimed to develop an indirect immunofluorescence antibody (IFA) assay to detect LRV in the Old World Leishmania parasites. Methods: Clinical samples were collected from 86 cutaneous leishmaniasis (CL) patients in different endemic areas of CL in Iran, during 2017-2019. For antibody preparation, the viruses were obtained from sediment of an LRV-infected L. major culture-using freeze and thaw cycles followed by gradient cesium chloride centrifugation. The purified viruses were used to immunize a male 3-4 months rabbit. Various dilutions of the LRV-immunized rabbit's serum and a conjugated antibody were deployed to detect LRV in 48 isolates by IFA assay. Results: LRV virus was detected in four of the 48 CL cases using IFA method. Amplification of a partial fragment of RNA-dependent RNA polymerase (RdRp) gene from the isolates confirmed the IFA results. In phylogeny, the generated RdRp sequences from four isolates were grouped with the other Old World LRVs, but separate from L. aethiopica LRVs, which appeared as a highly supported distinct clade. Conclusion: Further optimization of this approach to detect the LRV directly in lesion scrapings can make it a more reliable tool for field studies and disclosing the virus's possible role in disseminating and unusual clinical features.

Background Porcine parainfluenza virus 1 (PPIV-1) is a respiratory virus in the family Paramyxoviridae and genus Respirovirus . It is closely related to bovine parainfluenza virus 3, human parainfluenza virus 1, and Sendai virus. Recent reports suggest PPIV-1 is widespread in swine herds in the United States and abroad. However, seroprevalence studies and the ability to evaluate cross neutralization between heterologous strains is not possible without validated antibody assays. This study describes the development of an indirect fluorescence antibody (IFA) assay, a whole virus enzyme-linked immunosorbent assay (wv-ELISA) and a serum virus neutralization (SVN) assay for the detection of PPIV-1 antibodies using 521 serum samples collected from three longitudinal studies and two different challenge strains in swine. Results The area under the curve (AUC) of the wv-ELISA (95% CI, 0.93–0.98) was significantly higher ( p  = 0.03) compared to the IFA (95% CI, 0.90–0.96). However, no significant difference was observed between the IFA and wv-ELISA when compared to the SVN (95% CI, 0.92–0.97). All three assays demonstrated relatively uniform results at a 99% true negative rate, with only 11 disagreements observed between the IFA, wv-ELISA and SVN. Conclusions All three serology assays detected PPIV-1 antibody in swine serum of known status that was collected from experimental studies. The SVN detected seroconversion earlier compared to the IFA and the wv-ELISA. Both the wv-ELISA and the SVN had similar diagnostic performance, while the IFA was not as sensitive as the wv-ELISA. All three assays are considered valid for routine diagnostic use. These assays will be important for future studies to screen seronegative swine for research, determine PPIV-1 seroprevalence, and to evaluate vaccine efficacy against PPIV-1 under experimental and field conditions.

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.

Background: Our knowledge of the epidemiology of rodents’ parasitic agents in Iran is scarce, although some of these pathogens play an important role in human and veterinary medicine, such as Toxoplasma gondii and Neospora caninum. The purpose of this study was to determine the seroprevalence of Toxoplasma gondii and Neospora caninum in rodents of northwestern Iran between Mar and Dec 2015. Methods: Overall, 157 serum samples from rodents (101 Meriones persicus, 41 Mus musculus, and 15 Cricetulus migratorius) were assayed by the indirect fluorescence antibody test (IFAT) for antibodies to T. gondii and N. caninum Results: We found a prevalence of 20.38% (32/157) for N. caninum, 35% (55/157) for T. gondii. Co-presence of antibodies to N. caninum and T. gondii was found in 10 (6.36%) rodents. A significant association was found between the rodents species and seropositivity to N. caninum (P<0.05) but there was no association with rodents species for T. gondii. The overall prevalence of the aforementioned parasites was higher in male versus female rodents. Conclusion: The high seroprevalence of toxoplasmosis and neosporosis in rodents in the study area has implications for translocation of these infections across wider geographical regions since these rodents are mostly preyed on by cats or dogs; hence, which can transfer the parasite to other hosts.

The prevalence of equine piroplasmosis caused by Theileria equi and Babesia caballi in Nigde, in central Anatolia, Turkey has remained unknown. Serum samples were obtained from a total of 125 horses and were tested for antibodies to T. equi and B. caballi using the Indirect Fluorescence Antibody Test (IFAT). Twenty-three (18.4%) horses were seropositive for equine piroplasmosis. Anti-T. equi was observed in 16 horses (12.8%) while anti-B. caballi was detected in 12 horses (9.6%). In addition, 5 serum samples were positive for both parasites. The prevalence rates of antibodies to T. equi and B. caballi for female and male horses were statistically indifferent (p = 0.19 and 0.90). The difference between the seropositivity rates to T. equi among age groups was statistically insignificant (p = 0.44) while the difference to B. caballi among age groups is statistically significant (p = 0.01). Seropositivity rates ranged from 2.9% to 25.7% for T. equi and 2.9% to 14.3% for B. caballi from the selected districts in Nigde. A statistically significant difference on seropositivity rates for the study sites was observed for only T.equi (p = 0.03). This study indicates that T. equi is higher than B. caballi in Nigde.

The objectives of this study were to determine the individual and herd-level prevalence and genotype of Cryptosporidium and to identify putative risk factors associated with Cryptosporidium spp. infections in water buffaloes in northeast Thailand. Fecal samples from 600 water buffaloes of 287 farms in six provinces were collected and tested using DMSO-modified acid-fast staining and polymerase chain reaction. The overall prevalence of Cryptosporidium infections in buffaloes was 5.7 and 8.7 % among individual animals and herds, respectively. The provinces with highest infected Cryptosporidium were located in the Sakon Nakhon Basin in the northern part of the region. In addition, higher herd prevalence was observed among farms with more than five buffaloes (30 %) than those with five or less animals (16.2 %). Thirty (88.2 %) of the 34 Cryptosporidium-positive samples were Cryptosporidium parvum and four (11.8 %) were Cryptosporidium ryanae.

Being able to visualize protein localizations within cells and tissues by means of immuno-fluorescence microscopy has been key to developments in cell biology and beyond. Gut et al. present a high-throughput method that achieves the detection of more than 40 different proteins in biological samples across multiple spatial scales. This allows the simultaneous quantification of their expression levels in thousands of single cells; captures their detailed subcellular distribution to various compartments, organelles, and cellular structures within each of these single cells; and places all this information within a multicellular context. Such a scale-crossing dataset empowers artificial intelligence–based computer vision algorithms to achieve a comprehensive profiling of intracellular protein maps to measure their responses to different multicellular, cellular, and pharmacological contexts, and to reveal new cellular states. Science , this issue p. eaar7042 Multiplexed (40-plex) indirect immunofluorescence quantifies subcellular protein organization on cellular and multicellular scales. Obtaining highly multiplexed protein measurements across multiple length scales has enormous potential for biomedicine. Here, we measured, by iterative indirect immunofluorescence imaging (4i), 40-plex protein readouts from biological samples at high-throughput from the millimeter to the nanometer scale. This approach simultaneously captures properties apparent at the population, cellular, and subcellular levels, including microenvironment, cell shape, and cell cycle state. It also captures the detailed morphology of organelles, cytoskeletal structures, nuclear subcompartments, and the fate of signaling receptors in thousands of single cells in situ. We used computer vision and systems biology approaches to achieve unsupervised comprehensive quantification of protein subcompartmentalization within various multicellular, cellular, and pharmacological contexts. Thus, highly multiplexed subcellular protein maps can be used to identify functionally relevant single-cell states.

Despite the advantages of automated systems for antinuclear antibody (ANA) analysis, the prediction of end-point titers avoiding serial dilutions is still in progress. The aims of this study were to set a conversion table providing discriminant ranges of fluorescence signal intensity values (FI) corresponding to the end-point titers and validate this tool in a real-life laboratory setting. Eight hundred ninety-four serum samples were analyzed for ANA using Image Navigator System. In order to classify FI into non-overlapping groups corresponding to conventional end-point titers, statistical discriminant analysis was used. Validation study was performed calculating agreement and error rates between visual readings and conversion table of 1119 routine ANA positive samples. Setting of FI ranges corresponding to the end-point titers for different staining patterns was computed. For samples showing single pattern, the overall agreement between visual readings and conversion table was 98.4% for all titers ranging from 1:160 to 1:2560, of which 68.0% had the same titer and 30.4% were within ± one titer difference. Concordance rates according to ANA patterns were as follows: (1) nuclear 98.4%, of which 67.0% had the same titer and 31.4% ± one titer; (2) cytoplasmic 100%, of which 72.7% had the same titer and 27.3% than ± one titer; (3) mitotic 66.6%, of which 33.3% had more ± one titer. Our study developed a quantification method for autoantibodies titers assessment based on just one single sample dilution instead of traditional serial dilution approach, providing significant advantages in routine laboratory in terms of reduction in hand-on time and harmonization of results.

The immunofluorescence assay is widely used for cellular biology and diagnosis applications. Such an antigen–antibody detection system enables the assessment and visualization of the expression and localization of target proteins. In the classical indirect immunofluorescence assay, secondary antibodies are conjugated to fluorophores. However, conventional secondary antibodies have limited applications due to their large size (150 kDa). Moreover, as animal-derived products, secondary antibodies are associated with ethical concerns and batch-to-batch variability. In this study, we developed fluorescence-labeled recombinant nanobodies as secondary antibodies by utilizing previously established anti–mouse and anti–rabbit IgG secondary nanobodies in combination with the self-labeling SNAP-tag. Nanobodies, which are significantly smaller (15 kDa), are capable to detect primary antibodies produced in mice and rabbits. The SNAP-tag (20 kDa) enables site-specific binding of various O6-benzylguanine (BG)-modified fluorophores to the recombinant nanobodies. These recombinant nanobodies were produced using mammalian cell expression system, and their specific binding to mouse or rabbit antibodies was validated using flow cytometry and multi-color fluorescence microscopy. The low cost, easy of expression, purification and site-specific conjugation procedures for these anti–mouse and anti–rabbit IgG secondary nanobodies make them an attractive alternative to traditional secondary antibodies for indirect immunofluorescence assays.