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Objective In evaluation of systemic lupus erythematosus (SLE), anti–double-stranded DNA antibodies (anti-dsDNA) play a significant role in diagnosis, monitoring SLE activity, and assessing prognosis. However, evaluations of the performance and limitations for recently developed methods for anti-dsDNA assessment are sparse. Methods Specimens used for antinuclear antibody testing (n = 129) were evaluated for anti-dsDNA assay comparability across 4 medical centers in the United States. The methods compared were Werfen Quanta Lite dsDNA, Zeus Scientific dsDNA Enzyme Immunoassay, Bio-Rad multiplex immunoassay (MIA) dsDNA, ImmunoConcepts Crithidia, and Bio-Rad Laboratories Crithidia. Results For quantitative anti-dsDNA measurements, Spearman's correlation coefficient was highest between Zeus and Werfen (ρ = 0.86; CI, 0.81-0.90; P <.0001). Comparison of MIA to Werfen or Zeus yielded similar results to each other (ρ = 0.58; CI, 0.44-0.68; P <.0001; and ρ = 0.59; CI, 0.46-0.69; P <.0001, respectively), but lower than the correlation between Zeus and Werfen. Positive concordance between assays ranged from 31.4% to 97.1%, and negative concordance between assays ranged from 58.5% to 100%. The detection of anti-dsDNA in those with SLE diagnosis ranged from 50.9% to 77.4% for quantitative assays and 15.1% to 24.5% for Crithidia assays. Conclusion Current quantitative anti-dsDNA assays are not interchangeable for patient follow-up. Crithidia -based assays demonstrate high negative concordance and lack positive concordance among the methods. [ABSTRACT FROM AUTHOR]

This article explores the field of immunology and its connection to inborn errors of immunity (IEI) and protozoa. IEI refers to a range of diseases caused by genetic defects that affect the immune system, making individuals more susceptible to infections. The article emphasizes the need to understand the characteristics of the disease and the infective agent to identify compromised immune mechanisms. It also discusses the global health challenges posed by neglected tropical diseases (NTDs) and their impact on marginalized populations. The article presents a case study of a child with an inborn error of immunity and unusual infections, highlighting the need for innovative diagnostic assays and treatment protocols. The case underscores the importance of collaboration among immunologists in Brazil to enhance diagnostic capabilities and improve patient care for uncommon infections. [Extracted from the article]

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.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The prevalence of autochthonous leishmaniasis in Thailand is increasing but the natural vectors that are responsible for transmission remain unknown. Experimental in vivo infections in Culicoides spp. with Leishmania (Mundinia) martiniquensis and Leishmania (Mundinia) orientalis, the major causative pathogens in Thailand, have demonstrated that biting midges can act as competent vectors. Therefore, the isolation and detection of Leishmania and other trypanosomatids were performed in biting midges collected at a field site in an endemic area of leishmaniasis in Tha Ruea and a mixed farm of chickens, goats, and cattle in Khuan Phang, Nakhon Si Thammarat province, southern Thailand. Results showed that Culicoides peregrinus was the abundant species (>84%) found in both locations and only cow blood DNA was detected in engorged females. Microscopic examination revealed various forms of Leishmania promastigotes in the foregut of several C. peregrinus in the absence of bloodmeal remnants, indicating established infections. Molecular identification using ITS1 and 3'UTR HSP70 type I markers showed that the Leishmania parasites found in the midges were L. martiniquensis. The infection rate of L. martiniquensis in the collected flies was 2% in Tha Ruea and 6% in Khuan Phang, but no L. orientalis DNA or parasites were found. Additionally, organisms from two different clades of Crithidia, both possibly new species, were identified using SSU rRNA and gGAPDH genes. Choanomastigotes and promastigotes of both Crithidia spp. were observed in the hindgut of the dissected C. peregrinus. Interestingly, midges infected with both L. martiniquensis and Crithidia were found. Moreover, four strains of Crithidia from one of the clades were successfully isolated into culture. These parasites could grow at 37°C in the culture and infect BALB/c mice macrophages but no multiplication was observed, suggesting they are thermotolerant monoxenous trypanosomatids similar to Cr. thermophila. These findings provide the first evidence of natural infection of L. martiniquensis in C. peregrinus supporting it as a potential vector of L. martiniquensis. [ABSTRACT FROM AUTHOR]

Recent declines of wild pollinators and infections in honey, bumble and other bee species have raised concerns about pathogen spillover from managed honey and bumble bees to other pollinators. Parasites of honey and bumble bees include trypanosomatids and microsporidia that often exhibit low host specificity, suggesting potential for spillover to co-occurring bees via shared floral resources. However, experimental tests of trypanosomatid and microsporidial cross-infectivity outside of managed honey and bumble bees are scarce. To characterize potential cross-infectivity of honey and bumble bee-associated parasites, we inoculated three trypanosomatids and one microsporidian into five potential hosts - including four managed species - from the apid, halictid and megachilid bee families. We found evidence of cross-infection by the trypanosomatids Crithidia bombi and C. mellificae, with evidence for replication in 3/5 and 3/4 host species, respectively. These include the first reports of experimental C. bombi infection in Megachile rotundata and Osmia lignaria, and C. mellificae infection in O. lignaria and Halictus ligatus. Although inability to control amounts inoculated in O. lignaria and H. ligatus hindered estimates of parasite replication, our findings suggest a broad host range in these trypanosomatids, and underscore the need to quantify disease-mediated threats of managed social bees to sympatric pollinators.

Abstract Background Trypanosomatid parasites are widely distributed in nature and can have a monoxenous or dixenous life-cycle. These parasites thrive in a wide number of insect orders, some of which have an important economic and environmental value, such as bees. The objective of this study was to develop a robust and sensitive real-time quantitative PCR (qPCR) assay for detecting trypanosomatid parasites in any type of parasitized insect sample. Methods A TaqMan qPCR assay based on a trypanosomatid-conserved region of the α-tubulin gene was standardized and evaluated. The limits of detection, sensitivity and versatility of the α-tubulin TaqMan assay were tested and validated using field samples of honeybee workers, wild bees, bumblebees and grasshoppers, as well as in the human infective trypanosomatid Leishmania major. Results The assay showed a detection limit of 1 parasite equivalent/µl and successfully detected trypanosomatids in 10 different hosts belonging to the insect orders Hymenoptera and Orthoptera. The methodology was also tested using honeybee samples from four apiaries (n = 224 worker honeybees) located in the Alpujarra region (Granada, Spain). Trypanosomatids were detected in 2.7% of the honeybees, with an intra-colony prevalence of 0% to 13%. Parasite loads in the four different classes of insects ranged from 40.6 up to 1.1 × 108 cell equivalents per host. Conclusions These results show that the α-tubulin TaqMan qPCR assay described here is a versatile diagnostic tool for the accurate detection and quantification of trypanosomatids in a wide range of environmental settings. Graphical Abstract

Background: Trypanosomatid parasites are widely distributed in nature and can have a monoxenous or dixenous life-cycle. These parasites thrive in a wide number of insect orders, some of which have an important economic and environmental value, such as bees. The objective of this study was to develop a robust and sensitive real-time quantitative PCR (qPCR) assay for detecting trypanosomatid parasites in any type of parasitized insect sample. Methods: A TaqMan qPCR assay based on a trypanosomatid-conserved region of the α-tubulin gene was standardized and evaluated. The limits of detection, sensitivity and versatility of the α-tubulin TaqMan assay were tested and validated using field samples of honeybee workers, wild bees, bumblebees and grasshoppers, as well as in the human infective trypanosomatid Leishmania major. Results: The assay showed a detection limit of 1 parasite equivalent/µl and successfully detected trypanosomatids in 10 different hosts belonging to the insect orders Hymenoptera and Orthoptera. The methodology was also tested using honeybee samples from four apiaries (n = 224 worker honeybees) located in the Alpujarra region (Granada, Spain). Trypanosomatids were detected in 2.7% of the honeybees, with an intra-colony prevalence of 0% to 13%. Parasite loads in the four different classes of insects ranged from 40.6 up to 1.1 × 108 cell equivalents per host. Conclusions: These results show that the α-tubulin TaqMan qPCR assay described here is a versatile diagnostic tool for the accurate detection and quantification of trypanosomatids in a wide range of environmental settings. [ABSTRACT FROM AUTHOR]

The prevalence of autochthonous leishmaniasis in Thailand is increasing but the natural vectors that are responsible for transmission remain unknown. Experimental in vivo infections in Culicoides spp. with Leishmania (Mundinia) martiniquensis and Leishmania (Mundinia) orientalis, the major causative pathogens in Thailand, have demonstrated that biting midges can act as competent vectors. Therefore, the isolation and detection of Leishmania and other trypanosomatids were performed in biting midges collected at a field site in an endemic area of leishmaniasis in Tha Ruea and a mixed farm of chickens, goats, and cattle in Khuan Phang, Nakhon Si Thammarat province, southern Thailand. Results showed that Culicoides peregrinus was the abundant species (>84%) found in both locations and only cow blood DNA was detected in engorged females. Microscopic examination revealed various forms of Leishmania promastigotes in the foregut of several C. peregrinus in the absence of bloodmeal remnants, indicating established infections. Molecular identification using ITS1 and 3’UTR HSP70 type I markers showed that the Leishmania parasites found in the midges were L. martiniquensis. The infection rate of L. martiniquensis in the collected flies was 2% in Tha Ruea and 6% in Khuan Phang, but no L. orientalis DNA or parasites were found. Additionally, organisms from two different clades of Crithidia, both possibly new species, were identified using SSU rRNA and gGAPDH genes. Choanomastigotes and promastigotes of both Crithidia spp. were observed in the hindgut of the dissected C. peregrinus. Interestingly, midges infected with both L. martiniquensis and Crithidia were found. Moreover, four strains of Crithidia from one of the clades were successfully isolated into culture. These parasites could grow at 37°C in the culture and infect BALB/c mice macrophages but no multiplication was observed, suggesting they are thermotolerant monoxenous trypanosomatids similar to Cr. thermophila. These findings provide the first evidence of natural infection of L. martiniquensis in C. peregrinus supporting it as a potential vector of L. martiniquensis.

Gut symbionts can augment resistance to pathogens by stimulating host-immune responses, competing for space and nutrients, or producing antimicrobial metabolites. Gut microbiota of social bees, which pollinate many crops and wildflowers, protect hosts against diverse infections and might counteract pathogen-related bee declines. Bumble bee gut microbiota, and specifically abundance of Lactobacillus Firm-5 bacteria, can enhance resistance to the trypanosomatid parasite Crithidia bombi. However, the mechanism underlying this effect remains unknown. We hypothesized that the Firm-5 bacterium Lactobacillus bombicola, which produces lactic acid, inhibits C. bombi via pH-mediated effects. Consistent with our hypothesis, L. bombicola spent medium inhibited C. bombi growth via reduction in pH that was both necessary and sufficient for inhibition. Inhibition of all parasite strains occurred within the pH range documented in honey bees, though sensitivity to acidity varied among strains. Spent medium was slightly more potent than HCl, d- and l-lactic acids for a given pH, suggesting that other metabolites also contribute to inhibition. Results implicate symbiont-mediated reduction in gut pH as a key determinant of trypanosomatid infection in bees. Future investigation into in vivo effects of gut microbiota on pH and infection intensity would test the relevance of these findings for bees threatened by trypanosomatids.

Background and Aim: Cutaneous leishmaniasis (CL) is an important health problem in Iran and one of the world ̛s neglected diseases which is caused by Leishmania spp. The aim of this study was to investigate the epidemiology of cutaneous leishmaniasis and its associated risk factors in an endemic center in Golestan Province. Materials and Methods: This descriptive cross-sectional study included patients with suspected CL who had been referring to the health centers of Gonbad–e Qabus town, between 2018 and 2020. Demographic and epidemiological data of CL patients were recorded and diagnosis of Leishmania spp. was made by Giemsa staining of direct smear. PCR was also used to determine and confirm the parasite species. Results: From 1979 suspected CL lesions, 1704(87.4%) had positive smears. 898 (52.7%) patients were male and 806 (47.3%) were female. The highest incidence rate was observed in the patients of less than 5 (25.93%) and those between 25 and 45 years of age (25.41%). In all samples in this study, Leishmania major was the only Leishmania spp detected by ITS-PCR. Also, Critidia fasciulata was identified in three patients. Conclusion: The study provided valuable data concerning the epidemiologic aspects of the CL in an old endemic focus in north of Iran, which can be used as the basis for planning future control strategies.

Competition between organisms is often mediated by environmental factors, including temperature. In animal intestines, nonpathogenic symbionts compete physically and chemically against pathogens, with consequences for host infection. We used metabolic theory-based models to characterize differential responses to temperature of a bacterial symbiont and a co-occurring trypanosomatid parasite of bumblebees, which regulate body temperature during flight and incubation. We hypothesized that inhibition of parasites by bacterial symbionts would increase with temperature, due to symbionts having higher optimal growth temperatures than parasites. We found that a temperature increase over the range measured in bumblebee colonies would favour symbionts over parasites. As predicted by our hypothesis, symbionts reduced the optimal growth temperature for parasites, both in direct competition and when parasites were exposed to symbiont spent medium. Inhibitory effects of the symbiont increased with temperature, reflecting accelerated growth and acid production by symbionts. Our results indicate that high temperatures, whether due to host endothermy or environmental factors, can enhance the inhibitory effects of symbionts on parasites. Temperature-modulated manipulation of microbiota could be one explanation for fever- and heat-induced reductions of infection in animals, with consequences for diseases of medical and conservation concern.

Bats are infected with several trypanosomatid species; however, assessing the diversity of this interaction remains challenging since there are species apparently unable to grow in conventional culture media. Accordingly, the ecology and biology of the Molecular Operational Taxonomic Units (MOTUs) Trypanosoma spp. Neobats are unknown. Therefore, we performed the molecular characterization targeting the 18S small subunit rDNA from the blood clot of 280 bats of three Brazilian regions (Paraíba, Rio de Janeiro and Acre states), bypassing the selective pressure of hemoculture. From 68 (24%) positive blood clot samples, we obtained 49 satisfactory sequences. Of these successfully sequenced results, T. spp. Neobats (1, 3 and 4) represented 67%, with the most abundant T. sp. Neobat 4 (53%). Our results show: (1) high abundance and wide geographic range of T. sp. Neobat 4, restricted to Carollia bats; (2) high infection rate of T. sp. Neobat 4 in Carollia perspicillata populations (mean 26%); (3) infection with the monoxenous Crithidia mellificae; and (4) a new MOTU (T. sp. Neobat 5) in Artibeus cinereus, positioning in the Trypanosoma wauwau clade. These data corroborate the importance of bats as hosts of many Trypanosoma species and C. mellificae. They also show that the diversity of the T. wauwau clade is underestimated and warn about the high magnitude of trypanosomes we overpass with the hemoculture. Our findings combined with previous data show that T. spp. Neobats include host-specific and host-generalist species, probably playing different ecological roles: T. sp. Neobat 1 shows broad host range; T. spp. Neobat 3 and 4 are restricted to Artibeus and Carollia, respectively. Finally, T. Neobat 4 seems to be a well-succeeded parasite, especially within C. perspicillata metapopulations across a wide geographical distribution. This work is a step forward to understand the biology and life history of T. spp. Neobats.

Monoxenous (insect-restricted) trypanosomatids are highly diverse and abundant in nature. While many papers focus on the taxonomy and distribution of these parasites, studies on their biology are still scarce. In particular, this concerns trypanosomatids inhabiting the ubiquitous mosquitoes. To shed light on the circulation of monoxenous trypanosomatids with the participation of mosquitoes, we performed a multifaceted study combining the examination of naturally- and experimentally-infected insects using light and electron microscopy and molecular identification of parasites. Our examination of overwintering mosquitoes (genera Culex and Culiseta) revealed that their guts contained living trypanosomatids, which can be spread during the next season. Experimental infections with Crithidia spp. demonstrated that imagines represent permissive hosts, while larvae are resistant to these parasites. We argue that for the parasites with wide specificity, mosquitoes act as facultative hosts. Other trypanosomatids may have specific adaptations for vertical transmission in these insects at the expense of their potential to infect a wider range of hosts and, consequently, abundance in nature. [ABSTRACT FROM AUTHOR]

Crithidia fasiculata belongs to the trypanosomatidae order of protozoan parasites, bearing close relation to other kinetoplastid parasites such as Trypanosoma brucei and Leishmania spp. As an early diverging lineage of eukaryotes, the study of kinetoplastid parasites has provided unique insights into alternative mechanisms to traditional eukaryotic metabolic pathways. Crithidia are a monogenetic parasite for mosquito species and have two distinct lifecycle stages both taking place in the mosquito gut. These consist of a motile choanomastigote form and an immotile amastigote form morphologically similar to amastigotes in Leishmania. Owing to their close relation to Leishmania, Crithidia are a growing research tool, with continuing interest in its use as a model organism for kinetoplastid research with the added benefit that they are non-pathogenic to humans and can be grown with no special equipment or requirements for biological containment. Although comparatively little research has taken place on Crithidia, similarities to other kinetoplast species has been shown in terms of energy metabolism and genetics. Crithidia also show similarities to kinetoplastids in their production of the monosaccharide D-arabinopyranose similar to Leishmania, which is incorporated into a lipoarabinogalactan a major cell surface GPI-anchored molecule. Additionally, Crithidia have been used as a eukaryotic expression system to express proteins from other kinetoplastids and potentially other eukaryotes including human proteins allowing various co- and post-translational protein modifications to the recombinant proteins. Despite the obvious usefulness and potential of this organism very little is known about its lipid metabolism. Here we describe a detailed lipidomic analyses and demonstrate the possible placidity of Crithidia’s lipid metabolis. This could have important implications for biotechnology approaches and how other kinetoplastids interact with, and scavenge nutrients from their hosts.

Objective:The relationship between drug resistance and the expression of hexokinase (HK) has been indicated in leishmaniasis. According to the prolonged treatment period in cutaneous leishmaniasis (CL) patients co-infected with Crithidia in Iran, this study aims to investigate the expression of HK in the proteome of Leishmania major and Crithidia using a proteomic approach.Methods:A total of 205 samples were removed from the lesions of patients in Fars province, Iran, for the characterization of L. major and Crithidia using polymerase chain reaction (PCR). After protein extraction, two-dimensional gel electrophoresis was employed for protein separation. Several spots were isolated for HK determination in the proteomes of L. major and Crithidia using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF/TOF MS).Results:The PCR results showed 5 positive cases for Crithidia and 96 positive cases for L. major. MALDI TOF/TOF MS indicated HK as a common protein in the proteome of L. major and Crithidia. HK was up-regulated in the Crithidia proteome in comparison with the L. major proteome.Conclusion:Since a relationship between HK expression and drug resistance has been indicated in leishmaniasis, the overexpression of HK in Crithidia might be related to the increased duration of the treatment period in CL patients co-infected with Crithidia.

Commercial bumblebees have become popular models to understand stressors and solutions for pollinator health, but few studies test whether results translate to other pollinators. Consuming sunflower pollen dramatically reduces infection by the gut parasite Crithidia bombi in commercially reared Bombus impatiens. We assessed the effect of sunflower pollen on infection in wild B. impatiens, Bombus griseocollis, Bombus bimaculatus and Bombus vagans. We also asked how pollen diet (50% sunflower pollen versus wildflower pollen) and infection (yes/no) affected performance in wild B. impatiens microcolonies. Compared to controls, sunflower pollen dramatically reduced Crithidia infection in commercial and wild B. impatiens, had similar but less dramatic effects in B. bimaculatus and B. vagans, and no effect in B. griseocollis. Bombus impatiens, B. bimaculatus and B. vagans are in the same subgenus, suggesting that responses to sunflower pollen may be phylogenetically conserved. In microcolonies, 50% sunflower pollen reduced infection compared to wildflower pollen, but also reduced reproduction. Sunflower pollen could control Crithidia infections in B. impatiens and potentially close relatives, but may hinder reproduction if other resources are scarce. We caution that research using managed bee species, such as B. impatiens, be interpreted carefully as findingsmay not relate to all bee species. [ABSTRACT FROM AUTHOR]

Bats are infected with several trypanosomatid species; however, assessing the diversity of this interaction remains challenging since there are species apparently unable to grow in conventional culture media. Accordingly, the ecology and biology of the Molecular Operational Taxonomic Units (MOTUs) Trypanosoma spp. Neobats are unknown. Therefore, we performed the molecular characterization targeting the 18S small subunit rDNA from the blood clot of 280 bats of three Brazilian regions (Paraíba, Rio de Janeiro and Acre states), bypassing the selective pressure of hemoculture. From 68 (24%) positive blood clot samples, we obtained 49 satisfactory sequences. Of these successfully sequenced results, T. spp. Neobats (1, 3 and 4) represented 67%, with the most abundant T. sp. Neobat 4 (53%). Our results show: (1) high abundance and wide geographic range of T. sp. Neobat 4, restricted to Carollia bats; (2) high infection rate of T. sp. Neobat 4 in Carollia perspicillata populations (mean 26%); (3) infection with the monoxenous Crithidia mellificae ; and (4) a new MOTU (T. sp. Neobat 5) in Artibeus cinereus , positioning in the Trypanosoma wauwau clade. These data corroborate the importance of bats as hosts of many Trypanosoma species and C. mellificae. They also show that the diversity of the T. wauwau clade is underestimated and warn about the high magnitude of trypanosomes we overpass with the hemoculture. Our findings combined with previous data show that T. spp. Neobats include host-specific and host-generalist species, probably playing different ecological roles: T. sp. Neobat 1 shows broad host range; T. spp. Neobat 3 and 4 are restricted to Artibeus and Carollia , respectively. Finally, T. Neobat 4 seems to be a well-succeeded parasite, especially within C. perspicillata metapopulations across a wide geographical distribution. This work is a step forward to understand the biology and life history of T. spp. Neobats. [Display omitted] • Trypanossoma spp. Neobats 1–4 show distinct host ranges. • High abundance and wide geographic range of T. sp. Neobat 4, restricted to Carollia bats. • Discovery of a new MOTU within T. wauwau clade, detected in Artibeus cinereus. • Infection with Crithidia mellificae in bats from different localities. • Identification of mixed infections. [ABSTRACT FROM AUTHOR]

Background. Glyphosate is the world's most used pesticide and it is used without the mitigation measures that could reduce the exposure of pollinators to it. However, studies are starting to suggest negative impacts of this pesticide on bees, an essential group of pollinators. Accordingly, whether glyphosate, alone or alongside other stressors, is detrimental to bee health is a vital question. Bees are suffering declines across the globe, and pesticides, including glyphosate, have been suggested as being factors in these declines. Methods. Here we test, across a range of experimental paradigms, whether glyphosate impacts a wild bumble bee species, Bombus terrestris. In addition, we build upon existing work with honey bees testing glyphosate-parasite interactions by conducting fully crossed experiments with glyphosate and a common bumble bee trypanosome gut parasite, Crithidia bombi. We utilised regulatory acute toxicity testing protocols, modified to allow for exposure to multiple stressors. These protocols are expanded upon to test for effects on long term survival (20 days). Microcolony testing, using unmated workers, was employed to measure the impacts of either stressor on a proxy of reproductive success. This microcolony testing was conducted with both acute and chronic exposure to cover a range of exposure scenarios. Results. We found no effects of acute or chronic exposure to glyphosate, over a range of timespans post-exposure, on mortality or a range of sublethal metrics. We also found no interaction between glyphosate and Crithidia bombi in any metric, although there was conflicting evidence of increased parasite intensity after an acute exposure to glyphosate. In contrast to published literature, we found no direct impacts of this parasite on bee health. Our testing focussed on mortality and worker reproduction, so impacts of either or both of these stressors on other sublethal metrics could still exist. Conclusions. Our results expand the current knowledge on glyphosate by testing a previously untested species, Bombus terrestris, using acute exposure, and by incorporating a parasite never before tested alongside glyphosate. In conclusion our results find that glyphosate, as an active ingredient, is unlikely to be harmful to bumble bees either alone, or alongside Crithidia bombi. [ABSTRACT FROM AUTHOR]

Background Glyphosate is the world’s most used pesticide and it is used without the mitigation measures that could reduce the exposure of pollinators to it. However, studies are starting to suggest negative impacts of this pesticide on bees, an essential group of pollinators. Accordingly, whether glyphosate, alone or alongside other stressors, is detrimental to bee health is a vital question. Bees are suffering declines across the globe, and pesticides, including glyphosate, have been suggested as being factors in these declines. Methods Here we test, across a range of experimental paradigms, whether glyphosate impacts a wild bumble bee species, Bombus terrestris. In addition, we build upon existing work with honey bees testing glyphosate-parasite interactions by conducting fully crossed experiments with glyphosate and a common bumble bee trypanosome gut parasite, Crithidia bombi. We utilised regulatory acute toxicity testing protocols, modified to allow for exposure to multiple stressors. These protocols are expanded upon to test for effects on long term survival (20 days). Microcolony testing, using unmated workers, was employed to measure the impacts of either stressor on a proxy of reproductive success. This microcolony testing was conducted with both acute and chronic exposure to cover a range of exposure scenarios. Results We found no effects of acute or chronic exposure to glyphosate, over a range of timespans post-exposure, on mortality or a range of sublethal metrics. We also found no interaction between glyphosate and Crithidia bombi in any metric, although there was conflicting evidence of increased parasite intensity after an acute exposure to glyphosate. In contrast to published literature, we found no direct impacts of this parasite on bee health. Our testing focussed on mortality and worker reproduction, so impacts of either or both of these stressors on other sublethal metrics could still exist. Conclusions Our results expand the current knowledge on glyphosate by testing a previously untested species, Bombus terrestris, using acute exposure, and by incorporating a parasite never before tested alongside glyphosate. In conclusion our results find that glyphosate, as an active ingredient, is unlikely to be harmful to bumble bees either alone, or alongside Crithidia bombi.

Monoxenous (insect-restricted) trypanosomatids are highly diverse and abundant in nature. While many papers focus on the taxonomy and distribution of these parasites, studies on their biology are still scarce. In particular, this concerns trypanosomatids inhabiting the ubiquitous mosquitoes. To shed light on the circulation of monoxenous trypanosomatids with the participation of mosquitoes, we performed a multifaceted study combining the examination of naturally- and experimentally-infected insects using light and electron microscopy and molecular identification of parasites. Our examination of overwintering mosquitoes (genera Culex and Culiseta) revealed that their guts contained living trypanosomatids, which can be spread during the next season. Experimental infections with Crithidia spp. demonstrated that imagines represent permissive hosts, while larvae are resistant to these parasites. We argue that for the parasites with wide specificity, mosquitoes act as facultative hosts. Other trypanosomatids may have specific adaptations for vertical transmission in these insects at the expense of their potential to infect a wider range of hosts and, consequently, abundance in nature.

Flowers can be transmission platforms for parasites that impact bee health, yet bees share floral resources with other pollinator taxa, such as flies, that may be hosts or non-host vectors (i.e., mechanical vectors) of parasites. Here, we assessed whether the fecal-orally transmitted gut parasite of bees, Crithidia bombi, can infect Eristalis tenax flower flies. We also investigated the potential for two confirmed solitary bee hosts of C. bombi, Osmia lignaria and Megachile rotundata, as well as two flower fly species, Eristalis arbustorum and E. tenax, to transmit the parasite at flowers. We found that C. bombi did not replicate (i.e., cause an active infection) in E. tenax flies. However, 93% of inoculated flies defecated live C. bombi in their first fecal event, and all contaminated fecal events contained C. bombi at concentrations sufficient to infect bumble bees. Flies and bees defecated inside the corolla (flower) more frequently than other plant locations, and flies defecated at volumes comparable to or greater than bees. Our results demonstrate that Eristalis flower flies are not hosts of C. bombi, but they may be mechanical vectors of this parasite at flowers. Thus, flower flies may amplify or dilute C. bombi in bee communities, though current theoretical work suggests that unless present in large populations, the effects of mechanical vectors will be smaller than hosts. [ABSTRACT FROM AUTHOR]

Background: Leishmaniasis is caused by the protozoan Leishmania spp. In some loci from Iran, some isolates have ITS1 like the one in Crithidia spp. Therefore, in this study, we analyzed molecular characters of the mentioned isolates. Methods: DNA was extracted from all clinical isolates harboring ITS1 like the one in Crithidia spp. Amplification was performed using the specific primer pair of 13A/13B and followed by sequencing. The sequences were assessed using BLASTn, multiple alignment, T-coffee, and Vector NTI. Phylogenic tree was used for grouping and analyzing the isolates. Findings: Sequencing analyzing showed low heterogeneity among the mentioned isolates, but high heterogeneity between the mentioned isolates and the ones in genbank, therefore the mentioned isolates were grouped separately. Moreover, although ITS1 from the mentioned isolates were homolog to the ones in Crithodia spp., but 13A/13B loci on mitochondria showed homology with Leishmania. Conclusion: As Crithidia is a monoxenous, and its natural host is flies, finding its genes in the agents of cutaneous leishmaniasis may show these parasites may meet each other in sandfly. Till now, no sexual reproduction has been reported in Leishmania spp., therefore it is likely recombinant.

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., (© 2024. The Author(s).)

1. Social insect castes and sexes differ in many ways, including morphology, behavior, and sometimes ploidy level. Recent studies have found that consuming sunflower pollen reduces the gut pathogen Crithidia bombi in workers of the common eastern bumble bee (Bombus impatiens). Here, this work is extended to the reproductive individuals that represent colony fitness – males and queens – to assess if the medicinal effects of sunflower pollen vary with bee caste and sex. 2. This study examined the effect of sunflower pollen compared to a diverse wildflower pollen mix on infection in worker, male, and daughter queen commercial B. impatiens. Bees were infected, fed either sunflower pollen or wildflower pollen for 7 days, and then infection levels were assessed. 3. Compared to wildflower pollen, sunflower pollen dramatically reduced Crithidia infection in workers and daughter queens, but not males. Infection levels were very low for both diets in males; this could be due to low pollen consumption or other mechanisms. 4. Reducing Crithidia infection in young queens before they undergo hibernation is important for population dynamics since infected queens are less likely to survive hibernation, and those that do are less likely to successfully establish a nest the following spring. Because sunflowers bloom in late summer when new queens are emerging, sunflowers could provide an important dietary component for queens during this critical life stage. Deepening our understanding of how diet impacts pathogens in reproductive bees, as well as workers, is crucial to maintain healthy pollinator populations. [ABSTRACT FROM AUTHOR]

Abstract The trypanosomatid parasites continue their killing spree resulting in significant annual mortality due to the lack of effective treatments and the prominence of these diseases in poorer countries. These dimorphic parasites thrive unchecked in the host system, outsmarting the immune mechanisms. An understanding of biology of these parasitic forms will help in the management and elimination of these fatal diseases. Investigation of various metabolic pathways in these parasites has shed light in the understanding of the unique biology of the trypansomatids. An understanding of these pathways have helped in tracing the soft targets in the metabolic pathways, which could be used as effective drug targets which would further impact the therupeutic implications. Pyrimidine pathway is a vital metabolic pathway which yields in the formation of pyrimidines, which are then integrated in nucleic acids (DNA and RNA) in sugars (UDP sugars) and lipids (CDP lipids). A wealth of data and information has been generated in the past decades by in-depth analyses of pyrimidine pathway in the trypanosomatid parasites, which can aid in the identification of anomalies between the parasitic and host counterpart which could be further harnessed to develop therapeutic interventions for the treatment of parasitic diseases. This review presents an updated and comprehensive detailing of the pyrimidine metabolism in the trypansomatids, their uniqueness and their distinctions, and its possible outcomes that would aid in the eradication of these parasitic diseases.

Objective: To explore the co-detection of natural infection of Trypanosomatidae parasites such as Leishmania and Crithidia in reservoir hosts of leishmaniasis. Methods: Rodent populations were monitored in two endemic foci of cutaneous leishmaniasis of Fars province, southern Iran from March to October 2016. Rodents were trapped alive in several parts of Shiraz and Kharameh cities. Afterwards, their organs were prepared for detection of Leishmania and Crithidia species by molecular, microscopic, and culture methods. Results: Totally, 115 rodents of five species; Tatera indica (T. indica) (85), Rattus rattus (12), Meriones libycus (9), Mus musculus (7), and Rattus norvegicus (2), were trapped alive and their tissue samples were examined using microscopic, cultivation, and molecular assays. Overall, 59 (51.3%) rodents were positive for Leishmania or Crithidia parasites. The highest rate (61.2%; 52/85) of Leishmania infection was related to the T. indica population. The cultivation, and molecular observations showed that two (2.4%; 2/85) of T. indica (foot-pad, and spleen samples) were positive to Crithidia. Conclusions: This is the first report of Crithidia infection in T. indica in Iran. Consequently, more epidemiological and ecological studies are needed to understand the role of Crithidia and Leishmania in T. indica.

Bumble bees (Bombus Latrielle: Apidae) are important pollinators; however, declines of several species have been documented worldwide. Although pathogens have been linked to some declines, the biology, distribution, and impacts of most pathogens are poorly understood. Here, we report the first record of a recently characterized protozoan pathogen, Crithidia expoeki Schmid-Hempel & Tognazzo (Trypanosomatida: Trypanosomatidae), from bumble bees in Canada. This provides further insight on its global distribution and importance as a threat to bumble bees in Canada. [ABSTRACT FROM AUTHOR]

Numerous studies have reported a diversity of stressors that may explain continental-scale declines in populations of native pollinators, particularly those in the genus Bombus. However, there has been little focus on the identification of the local-scale dynamics that may structure currently impoverished Bombus communities. For example, the historically diverse coastal-zone communities of New England (USA) now comprise only a few species and are primarily dominated by a single species, B. impatiens. To better understand the local-scale factors that might be influencing this change in community structure, we examined differences in the presence of parasites in different species of Bombus collected in coastal-zone communities. Our results indicate that Bombus species that are in decline in this region were more likely to harbor parasites than are B. impatiens populations, which were more likely to be parasite-free and to harbor fewer intense infections or co-infections. The contrasting parasite burden between co-occurring winners and losers in this community may impact the endgame of asymmetric contests among species competing for dwindling resources. We suggest that under changing climate and landscape conditions, increasing domination of communities by healthy, synanthropic Bombus species (such as B. impatiens) may be another factor hastening the further erosion of bumble bee diversity.

ABSTRACT In this study, we sequenced and analyzed the genomes of 40 strains, in addition to the already-reported two type strains, of two Crithidia species infecting bumblebees in Alaska and Central Europe and demonstrated that different strains of Crithidia bombi and C. expoeki vary considerably in terms of single nucleotide polymorphisms and gene copy number. Based on the genomic structure, phylogenetic analyses, and the pattern of copy number variation, we confirmed the status of C. expoeki as a separate species. The Alaskan populations appear to be clearly separated from those of Central Europe. This pattern fits a scenario of rapid host-parasite coevolution, where the selective advantage of a given parasite strain is only temporary. This study provides helpful insights into possible scenarios of selection and diversification of trypanosomatid parasites. IMPORTANCE A group of trypanosomatid flagellates includes several well-studied medically and economically important parasites of vertebrates and plants. Nevertheless, the vast majority of trypanosomatids infect only insects (mostly flies and true bugs) and, because of that, has attracted little research attention in the past. Of several hundred trypanosomatid species, only four can infect bees (honeybees and bumblebees). Because of such scarcity, these parasites are severely understudied. We analyzed whole-genome information for a total of 42 representatives of bee-infecting trypanosomatids collected in Central Europe and Alaska from a population genetics point of view. Our data shed light on the evolution, selection, and diversification in this important group of trypanosomatid parasites.

Background: Leishmania major and Leishmania tropica are two main species causing cutaneous leishmaniasis (CL) in Iran. Recently, Crithidia spp. has also been reported in the wound of patients with CL. In this study, we determined the species causing CL in the southern of Iran and the role of Crithidia spp. in creating skin ulcers. Methods: In this cross-sectional study from Apr to Sep 2016, 66 patients with CL referred to Diagnostic Lab of Leishmaniasis, Valfajr Health Center, Shiraz, Iran, were selected. After DNA extraction from the Giemsa stained smears, all samples were amplified in two separate steps using specific primers, firstly, to differentiate Leishmania species and then to identify Crithidia spp. Results: Two species L. major and L. tropica were responsible for 60 and 6 cases, respectively. Moreover, in two patients, mixed infection with Crithidia was confirmed. In mix infection cases, the morphology of the cutaneous ulcers was not different from the wounds of other patients. Conclusion: Leishmania major is responsible for the most common CL in southern Iran. In addition, in two patients with L. major and L. tropica, mix infection with Crithidia was confirmed. The potential role of Crithidia as the main factor for CL and the probability of this parasite to have synergistic effects on Leishmania, as a hypothesis, requires more comprehensive researches on the ambiguity of this protozoon. [ABSTRACT FROM AUTHOR]

Avian malaria (Plasmodium spp.) and kinetoplastid (Trypanosoma spp.) parasites are common vector-borne pathogens in birds worldwide; however, knowledge about vector competence of different mosquito species is currently lacking. For a pilot project examining vector competence of mosquitoes of the Culex pipiens complex and Culex torrentium for protozoan parasites in the city of Vienna, 316 individual mosquitoes were sampled in the months June-August 2017 around the campus of the Veterinary University of Vienna. Since vector competence for avian Plasmodium can only be ascertained by finding infectious sporozoites in mosquito salivary glands, special emphasis was on examining these, or at least insect thoraxes, which contain the salivary glands. After species identification, the mosquitoes were processed in three different ways to determine the best method of visually detecting protozoan parasites in salivary glands: (1) microscopic examination of individual, fixed and Giemsa-stained salivary glands, (2) microscopic examination of stained sections of individually fixed and embedded mosquito thoraxes and (3) stained sections of individual whole insects. Material from all three groups was also subjected to PCR to detect avian haemosporidian and trypanosomatid parasite DNA. PCR was performed on all 316 collected mosquitoes, with 37 pools (n = 2-10) of 263 individuals and 53 single individuals in all together 90 PCR reactions. Avian Plasmodium was found in 18 (20%) and trypanosomatid parasites were found in 10 (11.1%) of the examined samples and pools yielded a higher proportion of positives than did individual samples. Six different species of protozoan parasites were identified, namely Plasmodium vaughani SYAT05 which was the most common, P. elongatum GRW6, P. relictum SGS1, Trypanosoma avium, T. culicavium and Crithidia dedva. Seventy-seven mosquito salivary glands were dissected and stained with Giemsa solution. Of these, one (1.3%) featured sporozoites and one (1.3%) trypanosomatid parasites. While the trypanosomes were identified as T. avium, the avian Plasmodium species were present in a mixed infection with P. vaughani SYAT05 as the dominant species. In conclusion, mosquitoes of the Culex pipiens complex are very likely vectors of different avian Plasmodium and Trypanosoma species and PCR was the most successful and reliable method for parasite detection in mosquito samples, delivering higher rates and more accurate results. The visual detection of parasite stages in the salivary glands was more difficult and only a few specimens were detected using Giemsa stain and chromogenic in situ hybridization. For further studies on vector competence of different protozoan parasites in mosquitoes, the use of PCR-based methods would be preferable. [ABSTRACT FROM AUTHOR]

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The role of population and demographic factors in influencing the transmission and establishment parasites infecting multiple hosts is poorly understood. We assessed the effects of these factors on parasite prevalence in a model system – the intestinal protozoan Crithidia bombi (Trypanosomatidae) infecting a range of bumblebee species (Bombus spp.). We used microsatellite markers and sibship reconstruction to infer genetic diversity and the density of host populations to infer their relative contributions to parasite prevalence. We established the prevalence, type (single- vs. multiple-strain infection) and intensity of C. bombi infections in workers and males of three common bumblebee species (B. terrestris, B. lapidarius and B. pascuorum) at 30 locations across Germany. We found evidence that colony density promoted prevalence, while increased genetic diversity lowered prevalence in B. terrestris. The effect size for genetic diversity was much larger than for colony density. Thus, genetic factors affected the prevalence, while demographic and life-history traits (e.g. population density and seasonal cycle of development) were additional factors shaping the spread and establishment of a multi-host parasite. Bombus lapidarius possessed characteristics that indicate it might act as a reservoir species spreading disease to other species. [ABSTRACT FROM AUTHOR]

Phlebotominae sand flies are involved in human diseases, such as leishmaniasis, and cause a considerable number of deaths every year. Besides, some of them have been identified as allergen sources or the potential mechanical vectors related to nosocomial infections. The present study aimed to assess the monthly activity, fauna, and detection of protozoan agents in phlebotomine sand flies using polymerase chain reaction (PCR) in re-emerging zoonotic cutaneous leishmaniasis foci of Shiraz and Kharameh in Fars province, southern Iran during 2016-2017. To determine the monthly activity, sand flies were caught from indoors and outdoors of both studied areas. Afterward, all female phlebotomine sand flies were processed for DNA extraction and PCR assays for Leishmania and Crithidia detections. During the study, 6975 sand flies of 16 species (eight Phlebotomus and eight Sergentomyia species) were caught in both foci. Sand flies' monthly activities started in early April and terminated in late November and October. Additionally, two active peaks of sand flies were observed in both foci; first in June and second in August to September. Phlebotomus papatasi (47.1%) was the most dominant species in out/indoors of both Shiraz (31.1%) and Kharameh (16.0%). It was also the only species which was found infected with Leishmania major, indeed, 2.68% and 2.53% of P. papatasi were infected to L. major in Kharameh and Shiraz, respectively. However, none of the female sand flies was positive for Crithidia spp. Despite various control strategies, especially against Leishmania, considerable cases of leishmaniasis are recorded from Iran every year. Phlebotomine plays the main role in transmission of Leishmania in these foci. Therefore, further studies are needed to determine the role of different phlebotomine species in epidemiological aspects of leishmaniasis. [ABSTRACT FROM AUTHOR]

The kinetoplastids are protozoa characterized by the presence of a distinctive organelle, called the kinetoplast, which contains a large amount of DNA (kinetoplast DNA (kDNA)) inside their single mitochondrion. Kinetoplastids of medical and veterinary importance include Trypanosoma spp. (the causative agents of human and animal African Trypanosomiasis and of Chagas disease) and Leishmania spp. (the causative agents of the various forms of leishmaniasis). These neglected diseases affect millions of people across the globe, but drug treatment is hampered by the challenges of toxicity and drug resistance, among others. Propolis (a natural product made by bees) and compounds isolated from it are now being investigated as novel treatments of kinetoplastid infections. The anti-kinetoplastid efficacy of propolis is probably a consequence of its reported activity against kinetoplastid parasites of bees. This article presents a review of the reported anti-kinetoplastid potential of propolis, highlighting its anti-kinetoplastid activity in vitro and in vivo regardless of geographical origin. The mode of action of propolis depends on the organism it is acting on and includes growth inhibition, immunomodulation, macrophage activation, perturbation of the cell membrane architecture, phospholipid disturbances, and mitochondrial targets. This gives ample scope for further investigations toward the rational development of sustainable anti-kinetoplastid drugs.

Alien insect species may present a multifaceted threat to ecosystems into which they are introduced. In addition to the direct damage they may cause, they may also bring novel diseases and parasites and/or have the capacity to vector microorganisms that are already established in the ecosystem and are causing harm. Damage caused by ectoparasitic larvae of the invasive fly, Philornis downsi (Dodge and Aitken) to nestlings of endemic birds in the Galapagos Islands is well documented, but nothing is known about whether this fly is itself associated with parasites or pathogens. In this study, diagnostic molecular methods indicated the presence of insect trypanosomatids in P. downsi; to our knowledge, this is the first record of insect trypanosomatids associated with Philornis species. Phylogenetic estimates and evolutionary distances indicate these species are most closely related to the Crithidia and Blastocrithidia genera, which are not currently reported in the Galapagos Islands. The prevalence of trypanosomatids indicates either P. downsi arrived with its own parasites or that it is a highly suitable host for trypanosomatids already found in the Galapagos Islands, or both. We recommend further studies to determine the origin of the trypanosomatid infections to better evaluate threats to endemic fauna of the Galapagos Islands.

Glycoalkaloids are important secondary metabolites accumulated by plants as protection against pathogens. One of them, α-tomatine, is found in high concentrations in green tomato fruits, while in the ripe fruits, its aglycone form, tomatidine, does not present a protective effect, and it is usual to find parasites of tomatoes like Phytomonas serpens in these ripe fruits. To investigate the sensitivity of trypanosomatids to the action of α-tomatine, we used logarithmic growth phase culture of 20 trypanosomatids from insects and plants and Trypanosoma cruzi. The lethal dose 50% (LD50) was determined by mixing 107 cells of the different isolates with α-tomatine at concentrations ranging from 10-3 to 10-8 M for 30 min at room temperature. The same tests performed with the tomatidine as a control showed no detectable toxicity against the same trypanosomatid cultures. The tests involved determination of the percentage (%) survival of the protozoan cultures in a Neubauer chamber using optical microscopy. The LD50 values varied from 10-4 to 10-6 M α-tomatine. Slight differences were detected among the LD50 values of the analyzed samples, and none of them showed evidence of resistance to the action of tomatinase, as shown by some pathogenic fungi.

Background: Leishmania major and Leishmania tropica are two main species causing cutaneous leishmaniasis (CL) in Iran. Recently, Crithidia spp. has also been reported in the wound of patients with CL. In this study, we determined the species causing CL in the southern of Iran and the role of Crithidia spp. in creating skin ulcers. Methods: In this cross-sectional study from Apr to Sep 2016, 66 patients with CL referred to Diagnostic Lab of Leishmaniasis, Valfajr Health Center, Shiraz, Iran, were selected. After DNA extraction from the Giemsa stained smears, all samples were amplified in two separate steps using specific primers, firstly, to differentiate Leishmania species and then to identify Crithidia spp. Results: Two species L. major and L. tropica were responsible for 60 and 6 cases, respectively. Moreover, in two patients, mixed infection with Crithidia was confirmed. In mix infection cases, the morphology of the cutaneous ul­cers was not different from the wounds of other patients. Conclusion: Leishmania major is responsible for the most common CL in southern Iran. In addition, in two patients with L. major and L. tropica, mix infection with Crithidia was confirmed. The potential role of Crithidia as the main factor for CL and the probability of this parasite to have synergistic effects on Leishmania, as a hypothesis, requires more comprehensive researches on the ambiguity of this protozoon.

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., Competing Interests: Competing interests: JPB is an Editorial Board Member at Lupus Science & Medicine. DZ, HMB, AS and PI have no competing interests., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

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.

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., Competing Interests: Competing interests: The last author (AJR) has participated as a speaker in three scientific conferences sponsored by Thermo Fisher Scientific., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)