Ecological and social factors influence interspecific pathogens occurrence among bees.

The interspecific transmission of pathogens can occur frequently in the environment. Among wild bees, the main spillover cases are caused by pathogens associated with Apis mellifera, whose colonies can act as reservoirs. Due to the limited availability of data in Italy, it is challenging to accurately assess the impact and implications of this phenomenon on the wild bee populations. In this study, a total of 3372 bees were sampled from 11 Italian regions within the BeeNet project, evaluating the prevalence and the abundance of the major honey bee pathogens (DWV, BQCV, ABPV, CBPV, KBV, Nosema ceranae, Ascosphaera apis, Crithidia mellificae, Lotmaria passim, Crithidia bombi). The 68.4% of samples were positive for at least one pathogen. DWV, BQCV, N. ceranae and CBPV showed the highest prevalence and abundance values, confirming them as the most prevalent pathogens spread in the environment. For these pathogens, Andrena, Bombus, Eucera and Seladonia showed the highest mean prevalence and abundance values. Generally, time trends showed a prevalence and abundance decrease from April to July. In order to predict the risk of infection among wild bees, statistical models were developed. A low influence of apiary density on pathogen occurrence was observed, while meteorological conditions and agricultural management showed a greater impact on pathogen persistence in the environment. Social and biological traits of wild bees also contributed to defining a higher risk of infection for bivoltine, communal, mining and oligolectic bees. Out of all the samples tested, 40.5% were co-infected with two or more pathogens. In some cases, individuals were simultaneously infected with up to five different pathogens. It is essential to increase knowledge about the transmission of pathogens among wild bees to understand dynamics, impact and effects on pollinator populations. Implementing concrete plans for the conservation of wild bee species is important to ensure the health of wild and human-managed bees within a One-Health perspective.

A case study of the diet-microbiota-parasite interplay in bumble bees.

AIMS: Diets and parasites influence the gut bacterial symbionts of bumble bees, but potential interactive effects remain overlooked. The main objective of this study was to assess the isolated and interactive effects of sunflower pollen, its phenolamides, and the widespread trypanosomatid Crithidia sp. on the gut bacterial symbionts of Bombus terrestris males. METHODS AND RESULTS: Bumble bee males emerged in microcolonies fed on either (i) willow pollen (control), (ii) sunflower pollen, or (iii) willow pollen spiked with phenolamide extracts from sunflower pollen. These microcolonies were infected by Crithidia sp. or were pathogen-free. Using 16S rRNA amplicon sequencing (V3-V4 region), we observed a significant alteration of the beta diversity but not of the alpha diversity in the gut microbial communities of males fed on sunflower pollen compared to males fed on control pollen. Similarly, infection by the gut parasite Crithidia sp. altered the beta diversity but not the alpha diversity in the gut microbial communities of males, irrespective of the diet. By contrast, we did not observe any significant alteration of the beta or alpha diversity in the gut microbial communities of males fed on phenolamide-enriched pollen compared to males fed on control pollen. Changes in the beta diversity indicate significant dissimilarities of the bacterial taxa between the treatment groups, while the lack of difference in alpha diversity demonstrates no significant changes within each treatment group. CONCLUSIONS: Bumble bees harbour consistent gut microbiota worldwide, but our results suggest that the gut bacterial communities of bumble bees are somewhat shaped by their diets and gut parasites as well as by the interaction of these two factors. This study confirms that bumble bees are suitable biological surrogates to assess the effect of diet and parasite infections on gut microbial communities.

Clinical implications of discordance between anti-dsDNA antibodies by multiplex flow immunoassay and Crithidia luciliae assay in a multiethnic racial cohort of patients with SLE.

OBJECTIVE: Anti-dsDNA antibodies (anti-dsDNA) are a component of all classification schemes in SLE and comprise one of the domains in validated activity indices. Anti-dsDNA is frequently measured commercially by an enzyme immunoassay (EIA) or Crithidia luciliae immunofluorescence test (CLIFT). To address the clinical impact of measuring these antibodies by two different assays, this study leveraged a well-phenotyped multiethnic/racial cohort. METHODS: All patients fulfilled the classification criteria for SLE by at least one of the validated schemes: American College of Rheumatology, Systemic Lupus Erythematosus International Collaborating Clinics and/or American College of Rheumatology/European League Against Rheumatism classification criteria. Patients with one or more simultaneously paired anti-dsDNA by multiplex EIA and CLIFT were identified. Analysis of concordance or discordance, titre comparability of assays and association with hybrid SLE Disease Activity Index score, prevalence of lupus nephritis (LN), ability to predict a flare and classification criteria was performed. RESULTS: 207 patients were simultaneously tested by EIA and CLIFT at least once for anti-dsDNA, generating 586 paired results. 377 pairs were concordant and 209 were discordant. 41 of 207 patients always had discordant paired results and 39 patients always had results with titre discordance. In 100 patients with LN, 60 were positive by EIA and 72 by CLIFT. Sensitivities and specificities for patients with LN versus patients without LN were EIA 60% and 47%, and CLIFT 72% and 37%, respectively. 42 patients had flare assessment within 90 days of their paired result. Six of seven patients with mild flares and all four patients with severe flares had concordant positive results. CONCLUSION: Our data demonstrate that discordance of positivity between both assays for anti-dsDNA is relatively common, occurring in a fifth of patients overall and a third of visits. EIA positivity is associated with LN less often than CLIFT positivity. With the significant discordance of results between anti-dsDNA assays, obtaining both CLIFT and EIA assays may be beneficial for classification and routine monitoring of SLE.

Methods matter: the influence of method on infection estimates of the bumblebee parasite Crithidia bombi.

The bumblebee gut parasite, Crithidia bombi, is widespread and prevalent in the field. Its interaction with Bombus spp. is a well-established epidemiological model. It is spread faecal-orally between colonies via the shared use of flowers when foraging. Accurately measuring the level of infection in bumblebees is important for assessing its distribution in the field, and also when conducting epidemiological experiments. Studies generally use 1 of 2 methods for measuring infection. One approach measures infection in faeces whereas the other method measures infection in guts. We tested whether the method of measuring infection affected the estimation of infection. Bumblebees were inoculated with a standardized inoculum and infection was measured 1 week later using either the faecal or gut method. We found that when the gut method was used to measure infection intensity estimates were significantly different to and approximately double those from the faecal method. These results have implications for the interpretation of previous study results and for the planning of future studies. Given the importance of bumblebees as pollinators, the impact of C. bombi on bumblebee health, and its use as an epidemiological model, we call on researchers to move towards consistent quantification of infections to enable future comparisons and meta-analyses of studies.

Longitudinal study of patients with discrepant results in CLIFT and a solid-phase dsDNA antibody assay: does a gold standard dsDNA assay exist?

OBJECTIVE: Antidouble-stranded DNA (dsDNA) antibodies are essential for diagnosis and follow-up of systemic lupus erythematous (SLE). To ensure the best diagnostic approach, most healthcare laboratories opt for a combination of highly sensitive methods, such as solid-phase immunoassays, and highly specific methods, such as the Crithidia luciliae indirect immunofluorescence test (CLIFT). Even so, discordant results are common, thus hindering the diagnostic process. Therefore, this study aimed to characterise a cohort of patients with discrepant results for a dsDNA fluorescence enzyme immunoassay (FEIA) and CLIFT during 2016-2018 and to follow patients up until December 2021. METHODS: We performed an observational, longitudinal and retrospective study on 417 samples from 257 patients who had been referred for suspected connective tissue diseases or followed up after diagnosis. All of them were positive for antinuclear antibodies (ANAs) using an indirect immunofluorescence assay (IFA) on Hep-2 cells, the entry criterion in our laboratory, and positive for FEIA dsDNA. Samples were then tested with CLIFT according to our routine protocol, which includes CLIFT testing after FEIA dsDNA results ≥10 UI/ml. After the assessment of data quality, the final analysis was based on 222 patients. RESULTS: Eighty-three patients (37.4%) had positive results in both tests and met the diagnostic criteria for SLE. However, 139 patients (62.6%) had discrepant results (FEIA+, CLIFT-). Of these, 58 patients (41.7%) had a diagnosis of SLE, with 47 (33.8%) having been previously diagnosed and under treatment. The remaining 11 patients (7.9%) had a new diagnosis of SLE, which was made up within 4 years of the initial screening. A total of 81 of the 139 patients (57.5%) with discrepant results did not meet lupus criteria during the follow-up period. CONCLUSIONS: The study showed that CLIFT could be negative in both treated and newly diagnosed SLE, thus underlining the importance of follow-up of dsDNA-positive results using solid-phase tests. Therefore, quantitative tests such as FEIA could add value to the diagnosis and management of patients with suspected SLE.

Gut Transplants from Bees Fed an Antipathogenic Pollen Diet Do Not Confer Pathogen Resistance to Recipients.

Pollinators are threatened by diverse stressors, including microbial pathogens such as Crithidia bombi. Consuming sunflower pollen dramatically reduces C. bombi infection in the bumble bee Bombus impatiens, but the mechanism behind this medicinal effect is unclear. We asked whether diet mediates resistance to C. bombi through changes in the gut microbiome. We hypothesized that sunflower pollen changes the gut microbiome, which in turn reduces Crithidia infection. To test this, we performed a gut transplant experiment. We fed donor bees either a sunflower pollen treatment or buckwheat pollen as a control treatment and then inoculated recipient bees with homogenized guts from either sunflower-fed or buckwheat-fed donor bees. All recipient bees were then fed a wildflower pollen diet. Two days after the transplant, we infected recipients with C. bombi, and 2 days later, we provided another donor gut transplant. To quantify infection, we performed both fecal screens and dissections of the recipient bees. We found no significant differences in C. bombi infection intensity or presence between bees that received sunflower-fed microbiomes versus buckwheat-fed microbiomes. This suggests that sunflower pollen's effects on pathogen resistance are not mediated by gut microbiota.

Trypanosomatids in honey bee colonies in Spain: A new specific qPCR method for specific quantification of Lotmaria passim, Crithidia mellificae and Crithidia bombi.

Bee trypanosomatids have not been widely studied due to the original belief that these organisms were not pathogenic to honey bees. However, trypanosomatids have been linked to increased winter mortality in honey bee colonies in recent years and it has been shown that these pathogens can shorten a honey bee worker's lifespan in laboratory conditions. These studies found that this mortality corresponded to dose-dependent infection. Although Lotmaria passim is the most prevalent species worldwide, the natural load in colonies remains poorly investigated. Here we describe a new highly specific and sensitive qPCR method that allows the differentiation and quantification of the parasitic load of each of the three most common trypanosomatid species described to date in honey bee colonies: L. passim, Crithidia mellificae, and Crithidia bombi. We have used this new method to analyze honey bee colonies in central Spain and confirm that L. passim is the most common species and the one with higher parasitic loads in the colonies, which increased over the years, being higher in spring than in autumn. Crithidia mellificae was present along the study, with the highest prevalence in autumn 2019 and lately it was only found in non-quantifiable loads. Crithidia bombi was not detected in any of the colonies analyzed.

RNA viruses of Crithidia bombi, a parasite of bumblebees.

Leishbuviridae (Bunyavirales) are a diverse monophyletic group of negative-sense single-stranded RNA virus infecting parasitic flagellates of the family Trypanosomatidae. The presence of RNA viruses in trypanosomatids can influence the virulence of the latter. Here, we performed a screening for viruses in Crithidia bombi - a common parasite of important pollinators Bombus spp. (bumblebees) that negatively affects its host in stressful conditions. The majority (8/10) of C. bombi isolates collected in Europe and North America were positive for a virus that we named Crithidia bombi leishbuvirus 1 with high conservation of amino acid sequences between isolates. The results of our comparative phylogenetic analyses of the trypanosomatids and their viruses suggest that the high mobility of bumblebees and frequent coinfections by different strains of C. bombi determine an extensive viral exchange between the latter.

Neither sulfoxaflor, Crithidia bombi, nor their combination impact bumble bee colony development or field bean pollination.

Many pollinators, including bumble bees, are in decline. Such declines are known to be driven by a number of interacting factors. Decreases in bee populations may also negatively impact the key ecosystem service, pollination, that they provide. Pesticides and parasites are often cited as two of the drivers of bee declines, particularly as they have previously been found to interact with one another to the detriment of bee health. Here we test the effects of an insecticide, sulfoxaflor, and a highly prevalent bumble bee parasite, Crithidia bombi, on the bumble bee Bombus terrestris. After exposing colonies to realistic doses of either sulfoxaflor and/or Crithidia bombi in a fully crossed experiment, colonies were allowed to forage on field beans in outdoor exclusion cages. Foraging performance was monitored, and the impacts on fruit set were recorded. We found no effect of either stressor, or their interaction, on the pollination services they provide to field beans, either at an individual level or a whole colony level. Further, there was no impact of any treatment, in any metric, on colony development. Our results contrast with prior findings that similar insecticides (neonicotinoids) impact pollination services, and that sulfoxaflor impacts colony development, potentially suggesting that sulfoxaflor is a less harmful compound to bee health than neonicotinoids insecticides.

The effect of sunflower pollen age and origin on pathogen infection in the common eastern bumble bee (Apidae: Hymenoptera).

Bumble bees are globally important pollinators, contributing hundreds of millions of dollars annually in crop pollination services. Several species are in decline, making it paramount to understand how pathogens and nutrition shape bee health. Previous work has shown that consuming sunflower pollen (Helianthus annuus) dramatically reduces infection by the trypanosomatid gut pathogen, Crithidia bombi, in the common eastern bumble bee (Bombus impatiens). Sunflower pollen may therefore be useful as a dietary supplement for reducing this pathogen in managed bumble bee colonies. Here, we assessed the efficacy of freezer-stored sunflower pollen that was collected in different years and locations for reducing pathogen infection. We tested sunflower pollen that was 1, 3, 4, or 5 yr old and from sunflowers grown in the United States or China against a control of 1-yr-old buckwheat pollen from China, since buckwheat pollen results in high infection. We hypothesized that older pollen would have weaker medicinal effects due to degradation of pollen quality. We found that all sunflower pollen treatments significantly decreased Crithidia infection compared to controls. These results suggest that sunflower pollen can be freezer-stored for up to 5 yr and sourced from a wide range of geographic areas and still maintain its medicinal effects against Crithidia in the common eastern bumble bee. This is helpful information for stakeholders who might administer sunflower pollen as a dietary supplement to manage Crithidia in commercial bumble bee colonies.

Frequent Parasitism of Apis mellifera by Trypanosomatids in Geographically Isolated Areas with Restricted Beekeeping Movements.

Trypanosomatids form a group of high prevalence protozoa that parasitise honey bees, with Lotmaria passim as the predominant species worldwide. However, the knowledge about the ecology of trypanosomatids in isolated areas is limited. The Portuguese archipelagos of Madeira and Azores provide an interesting setting to investigate these parasites because of their geographic isolation, and because they harbour honey bee populations devoid of two major enemies: Varroa destructor and Nosema ceranae. Hence, a total of 661 honey bee colonies from Madeira and the Azores were analysed using different molecular techniques, through which we found a high prevalence of trypanosomatids despite the isolation of these islands. L. passim was the predominant species and, in most colonies, was the only one found, even on islands free of V. destructor and/or N. ceranae with severe restrictions on colony movements to prevent the spread of them. However, islands with V. destructor had a significantly higher prevalence of L. passim and, conversely, islands with N. ceranae did not shown any significant correlation with the trypanosomatid. Crithidia bombi was detected in Madeira and on three islands of the Azores, almost always coincident with L. passim. By contrast, Crithidia mellificae was not detected in any sample. A high-throughput sequencing analysis distinguished two main haplotypes of L. passim, which accounted for 98% of the total sequence reads. This work suggests that L. passim and C. bombi are parasites that have been associated with honey bees predating the spread of V. destructor and N. ceranae.

No impacts of glyphosate or Crithidia bombi, or their combination, on the bumblebee microbiome.

Pesticides are recognised as a key threat to pollinators, impacting their health in many ways. One route through which pesticides can affect pollinators like bumblebees is through the gut microbiome, with knock-on effects on their immune system and parasite resistance. We tested the impacts of a high acute oral dose of glyphosate on the gut microbiome of the buff tailed bumblebee (Bombus terrestris), and glyphosate's interaction with the gut parasite (Crithidia bombi). We used a fully crossed design measuring bee mortality, parasite intensity and the bacterial composition in the gut microbiome estimated from the relative abundance of 16S rRNA amplicons. We found no impact of either glyphosate, C. bombi, or their combination on any metric, including bacterial composition. This result differs from studies on honeybees, which have consistently found an impact of glyphosate on gut bacterial composition. This is potentially explained by the use of an acute exposure, rather than a chronic exposure, and the difference in test species. Since A. mellifera is used as a model species to represent pollinators more broadly in risk assessment, our results highlight that caution is needed in extrapolating gut microbiome results from A. mellifera to other bee species.

Minimal sharing of nosematid and trypanosomatid parasites between honey bees and other bees, but extensive sharing of Crithidia between bumble and mason bees.

We document gut parasites in co-occurring Apis, Bombus, and Osmia spp. in the Northern Virginia region, USA. Trypanosomatidea occurred in sixty percent of specimens and 13% carried Nosematidae. We found strong host partitioning: Lotmaria passim and Vairimorpha (Nosema) ceranae predominated in Apis, and Crithidia bombi and V. bombi in Bombus. We did not detect pathogen spread from Apis to Bombus but did detect sharing of C. bombi between Bombus and Osmia, higher parasite levels in Apis at sites with apiaries, and clustering of Vairimopha infection. Given the presence of C. bombi in Osmia, we suggest disease sharing across taxa be monitored.

A comparison of the chemiluminescence immunoassay and Crithidia luciliae immunofluorescence test in detecting anti-dsDNA antibodies and assessing the activity of systemic lupus erythematosus.

OBJECTIVES: This study aimed to compare the YHLO chemiluminescence immunoassay (CLIA) with the Crithidia luciliae immunofluorescence test (CLIFT) to detect anti-dsDNA antibodies and its correlation with disease activity in systemic lupus erythematosus (SLE). METHOD: In total, 208 patients diagnosed with SLE, 110 other autoimmune patients, 70 infectious disorders patients, and 105 healthy people were enrolled in this study. Serum samples were tested using CLIA in a YHLO chemiluminescence system and CLIFT. RESULTS: The overall agreement between YHLO CLIA and CLIFT was 76.9% (160/208), with a moderate correlation (kappa = 0.530, p < 0.001). The sensitivity of YHLO CLIA and CLIFT were 58.2% and 55.3%, respectively. The specificity of YHLO CLIA and CLIFT were 95.1% and 99.3%, respectively. The sensitivity of YHLO CLIA was increased to 66.8% with a specificity of 93.6% when the cut-off value was set at 24 IU/mL. Spearman's correlation coefficient between the quantitative results of YHLO CLIA and the titers of CLIFT was 0.59 (p < .01). A significant correlation was found between the anti-dsDNA results detected by YHLO CLIA and the SLE Disease Activity Index 2000 (SLEDAI-2K). Spearman's correlation coefficient between YHLO CLIA and SLEDAI-2K (r = 0.66, p < .01) was higher than that of CLIFT (r = 0.60, p < .01). CONCLUSIONS: Good correlation and agreement were found between YHLO CLIA and CLIFT. In addition, there was a significant correlation between YHLO CLIA and the SLE Disease Activity Index, which was superior to that of CLIFT. The YHLO chemiluminescence system is recommended for the assessment of disease activity.

Co-infection of Leishmania infantum and a Crithidia-related species in a case of refractory relapsed visceral leishmaniasis with non-ulcerated cutaneous manifestation in Brazil.

We report a refractory and relapsed visceral leishmaniasis case in a male child patient followed from 2016 to 2020, whose clinical isolates from multiple relapses were analyzed at the genome level. To the best of our knowledge, it is the first report that both visceral leishmaniasis and non-ulcerated cutaneous leishmaniasis have concomitantly manifested in the same patient. Importantly, sequence analysis revealed that the patient was co-infected with Leishmania infantum and a Crithidia-related parasite, which was previously found in a fatal case of visceral leishmaniasis from the same endemic region.

Sunflower plantings reduce a common gut pathogen and increase queen production in common eastern bumblebee colonies.

Community diversity can reduce the prevalence and spread of disease, but certain species may play a disproportionate role in diluting or amplifying pathogens. Flowers act as both sources of nutrition and sites of pathogen transmission, but the effects of specific plant species in shaping bee disease dynamics are not well understood. We evaluated whether plantings of sunflower (Helianthus annuus), whose pollen reduces infection by some pathogens when fed to bees in captivity, lowered pathogen levels and increased reproduction in free-foraging bumblebee colonies (Bombus impatiens). Sunflower abundance reduced the prevalence of a common gut pathogen, Crithidia bombi, and reduced infection intensity, with an order of magnitude lower infection intensity at high sunflower sites compared with sites with little to no sunflower. Sunflower abundance was also positively associated with greater queen production in colonies. Sunflower did not affect prevalence of other detected pathogens. This work demonstrates that a single plant species can drive disease dynamics in foraging B. impatiens, and that sunflower plantings can be used as a tool for mitigating a prevalent pathogen while also increasing reproduction of an agriculturally important bee species.

Differential bumble bee gene expression associated with pathogen infection and pollen diet.

BACKGROUND: Diet and parasitism can have powerful effects on host gene expression. However, how specific dietary components affect host gene expression that could feed back to affect parasitism is relatively unexplored in many wild species. Recently, it was discovered that consumption of sunflower (Helianthus annuus) pollen reduced severity of gut protozoan pathogen Crithidia bombi infection in Bombus impatiens bumble bees. Despite the dramatic and consistent medicinal effect of sunflower pollen, very little is known about the mechanism(s) underlying this effect. However, sunflower pollen extract increases rather than suppresses C. bombi growth in vitro, suggesting that sunflower pollen reduces C. bombi infection indirectly via changes in the host. Here, we analyzed whole transcriptomes of B. impatiens workers to characterize the physiological response to sunflower pollen consumption and C. bombi infection to isolate the mechanisms underlying the medicinal effect. B. impatiens workers were inoculated with either C. bombi cells (infected) or a sham control (un-infected) and fed either sunflower or wildflower pollen ad libitum. Whole abdominal gene expression profiles were then sequenced with Illumina NextSeq 500 technology. RESULTS: Among infected bees, sunflower pollen upregulated immune transcripts, including the anti-microbial peptide hymenoptaecin, Toll receptors and serine proteases. In both infected and un-infected bees, sunflower pollen upregulated putative detoxification transcripts and transcripts associated with the repair and maintenance of gut epithelial cells. Among wildflower-fed bees, infected bees downregulated immune transcripts associated with phagocytosis and the phenoloxidase cascade. CONCLUSIONS: Taken together, these results indicate dissimilar immune responses between sunflower- and wildflower-fed bumble bees infected with C. bombi, a response to physical damage to gut epithelial cells caused by sunflower pollen, and a strong detoxification response to sunflower pollen consumption. Identifying host responses that drive the medicinal effect of sunflower pollen in infected bumble bees may broaden our understanding of plant-pollinator interactions and provide opportunities for effective management of bee pathogens.

Biogeography, climate, and land use create a mosaic of parasite risk in native bumble bees.

Host-parasite interactions are crucial to the regulation of host population growth, as they often impact both long-term population stability and ecological functioning. Animal hosts navigate a number of environmental conditions, including local climate, anthropogenic land use, and varying degrees of spatial isolation, all of which can mediate parasitism exposure. Despite this, we know little about the potential for these environmental conditions to impact pathogen prevalence at biogeographic scales, especially for key ecosystem service-providing animals. Bees are essential pollination providers that may be particularly sensitive to biogeography, climate, and land-use as these factors are known to limit bee dispersal and contribute to underlying population genetic variation, which may also impact host-parasite interactions. Importantly, many native bumble bee species have recently shown geographic range contractions, reduced genetic diversity, and increased parasitism rates, highlighting the potential importance of interacting and synergistic stressors. In this study, we incorporate spatially explicit environmental, biogeographic, and land-use data in combination with genetically derived host population data to conduct a large-scale epidemiological assessment of the drivers of pathogen prevalence across >1000 km for a keystone western US pollinator, the bumble bee Bombus vosnesenskii. We found high rates of infection from Crithidia bombi and C. expoekii, which show strong spatial autocorrelation and which were more prevalent in northern latitudes. We also show that land use barriers best explained differences in parasite prevalence and parasite community composition, while precipitation, elevation, and B. vosnesenskii nesting density were important drivers of parasite prevalence. Overall, our results demonstrate that human land use can impact critical host-parasite interactions for native bees at massive spatial scales. Further, our work indicates that disease-related survey and conservation measures should take into account the independent and interacting influences of climate, biogeography, land use, and local population dynamics.

Determination of specific autoantibodies in patients with systemic lupus erythematosus by Line immunoassay, ELISA and CLIF assay.

BACKGROUND: Detection of specific antinuclear-antibodies is very importance in term of diagnosis, prognosis and management of patients with systemic lupus erythematosus (SLE). To date, Line immunoassay (LIA), enzyme-linked immunosorbent assay (ELISA) and Crithidia luciliae indirect immunofluorescence (CLIF) assay are commonly used for detection of specific antinuclear-antibodies. OBJECTIVE: To determine the performance of LIA, ELISA and CLIF for the detection of anti-double-stranded DNA (dsDNA), anti-nucleosome, and anti-extractable nuclear antigens (ENA) antibodies in patients with SLE. METHODS: A total 100 sera from 50 patients with SLE, 25 patients with disease control and 25 healthy control subjects were tested for anti-dsDNA, anti-nucleosome, and anti-ENA antibodies by LIA, ELISA, and CLIF assay. Agreement and diagnostic performance of each assay were analyzed using Cohen's kappa coefficient and receiver operating characteristic curve analysis. RESULTS: For the detection of anti-dsDNA antibody, ELISA had a substantial agreement with CLIF assay (? = 0.74) but LIA had a fair agreement with ELISA and CLIF assay (? = 0.37, and 0.35 respectively). For the detection of anti-nucleosome, anti-nRNP/Sm, anti-Sm, anti-SSA, and anti-SSB antibodies, LIA had a substantial to perfect agreement with ELISA (? = 0.64, 0.78, 0.68, 0.91, and 0.74, respectively). Anti-dsDNA-NcX ELISA and anti-dsDNA CLIF assay had equally diagnostic performance (sensitivity, 66% vs. 68%, and specificity, 96% vs. 94%, respectively) whereas, anti-dsDNA LIA has low sensitivity (22%) but high specificity (100%). CONCLUSIONS: LIA, ELISA, and CLIF demonstrated comparable performance for the detection of specific antinuclear-antibodies. However, there were some discrepancy between assays particularly in the detection of anti-dsDNA antibody.

Molecular detection of Lotmaria passim in honeybees in Japan.

Crithidia mellificae (C. mellificae) and Lotmaria passim (L. passim) are trypanosomatids that infect Apis mellifera. We analyzed the prevalence of C. mellificae and L. passim in six regions of Japan from 2018 to 2019. The detection rate of C. mellificae was 0.0% in all regions, whereas L. passim was detected in 16.7%-66.7% of the honeybees. L. passim was detected at a significantly lower rate in the Cyugoku-Shikoku region than in other regions. Furthermore, phylogenetic analysis of the internal transcribed spacer 1 (ITS1) locus of related species was performed. All the samples in this study could be assigned to the L. passim clade. This study reveals that L. passim infection is predominantly prevalent in Japan. Further epidemiological surveys are needed to clarify the prevalence of C. mellificae infection in honeybees in Japan.