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Visceral leishmaniasis (VL) is a neglected disease considered a serious public health problem, especially in endemic countries. Several studies have discovered monoxenous trypanosomatids ( Leptomonas and Crithidia ) in patients with VL. In different situations of leishmaniasis, investigations have examined cases of co-infection between Leishmania spp. and Crithidia spp. These coinfections have been observed in a wide range of vertebrate hosts, indicating that they are not rare. Diagnostic techniques require improvements and more robust tools to accurately detect the causative agent of VL. This study aimed to develop a real-time quantitative dye-based PCR (qPCR) assay capable of distinguishing Leishmania infantum from Crithidia -related species and to estimate the parasite load in samples of VL from humans and animals. The primer LinJ31_2420 targets an exclusive phosphatase of L. infantum ; the primer Catalase_LVH60-12060_1F targets the catalase gene of Crithidia . Therefore, primers were designed to detect L. infantum and Crithidia sp. LVH60A (a novel trypanosomatid isolated from VL patients in Brazil), in samples related to VL. These primers were considered species-specific, based on sequence analysis using genome data retrieved from the TriTryp database and the genome assembling of Crithidia sp. LVH60A strain, in addition to experimental and clinical data presented herein. This novel qPCR assay was highly accurate in identifying and quantifying L. infantum and Crithidia sp. LVH60A in samples obtained experimentally (in vitro and in vivo) or collected from hosts (humans, dogs, cats, and vectors). Importantly, the screening of 62 cultured isolates from VL patients using these primers surprisingly revealed that 51 parasite cultures were PCR+ for Crithidia sp. In addition, qPCR assays identified the co-infection of L. infantum with Crithidia sp. LVH60A in two new VL cases in Brazil, confirming the suspicion of co-infection in a previously reported case of fatal VL. We believe that the species-specific genes targeted in this study can be helpful for the molecular diagnosis of VL, as well as for elucidating suspected co-infections with monoxenous-like trypanosomatids, which is a neglected fact of a neglected disease.

Immunoglobulin G (IgG) antibodies play an important role in the immune response against viruses such as SARS-CoV-2. As the effector functions of IgG are modulated by N -glycosylation of the Fc region, the structure and possible function of the IgG N -glycome has been under investigation in relation to divergent COVID-19 disease courses. Through LC-MS analysis we studied both total IgG1 and spike protein-specific IgG1 Fc glycosylation of 129 German and 163 Brazilian COVID-19 patients representing diverse patient populations. We found that hospitalized COVID-19 patients displayed decreased levels of total IgG1 bisection and galactosylation and lowered anti-S IgG1 fucosylation and bisection as compared to mild outpatients. Anti-S IgG1 glycosylation was dynamic over the disease course and both anti-S and total IgG1 glycosylation were correlated to inflammatory markers. Further research is needed to dissect the possible role of altered IgG glycosylation profiles in (dys)regulating the immune response in COVID-19., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Siekman, Pongracz, Wang, Nouta, Kremsner, da Silva-Neto, Esen, Kreidenweiss, Held, Trapé, Fendel, de Miranda Santos, Wuhrer and ImmunoCovid Consortium.)

Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The major histocompatibility complex (MHC) contains many genes that play key roles in initiating and regulating immune responses. This includes the polymorphic MHCI and MHCII genes that present epitopes to CD8+ and CD4+ T-cells, respectively. Consequently, the characterisation of the repertoire of MHC genes is an important component of improving our understanding of the genetic variation that determines the outcomes of immune responses. In cattle, MHC (BoLA) research has predominantly focused on Holstein-Friesian animals (as the most economically important breed globally), although the development of high-throughput approaches has allowed the BoLA-DRB3 repertoire to be studied in a greater variety of breeds. In a previous study we reported on the development of a MiSeq-based method to enable high-throughput and high-resolution analysis of bovine MHCI repertoires. Herein, we report on the expansion of this methodology to incorporate analysis of the BoLA-DRB3 and its application to analyse MHC diversity in a large cohort of cattle from Brazil (>500 animals), including representatives from the three major Bos indicus breeds present in Brazil - Guzerat, Gir and Nelore. This large-scale description of paired MHCI-DRB3 repertoires in Bos indicus cattle has identified a small number of novel DRB3 alleles, a large number of novel MHCI alleles and haplotypes, and provided novel insights into MHCI-MHCII association - further expanding our knowledge of bovine MHC diversity., (© 2021 The Authors. HLA: Immune Response Genetics published by John Wiley & Sons Ltd.)

The cattle tick, Rhipicephalus microplus, is a monoxenous tick that co-evolved with indicine cattle on the Indian subcontinent. It causes massive damage to livestock worldwide. Cattle breeds present heritable, contrasting phenotypes of tick loads, taurine breeds carrying higher loads of the parasite than indicine breeds. Thus, a useful model is available to analyze mechanisms that determine outcomes of parasitism. We sought to gain insights on these mechanisms and used RNA sequencing and Multidimensional Protein Identification Technology (MudPIT) to generate a transcriptome from whole larvae and salivary glands from nymphs, males and females feeding on genetically susceptible and resistant bovine hosts and their corresponding proteomes. 931,698 reads were annotated into 11,676 coding sequences (CDS), which were manually curated into 116 different protein families. Male ticks presented the most diverse armamentarium of mediators of parasitism. In addition, levels of expression of many genes encoding mediators of parasitism were significantly associated with the level and stage of host immunity and/or were temporally restricted to developmental stages of the tick. These insights should assist in developing novel, sustainable technologies for tick control.

Ticks cause massive damage to livestock and vaccines are one sustainable alternative for the acaricide poisons currently heavily used to control infestations. An experimental vaccine adjuvanted with alum and composed by four recombinant salivary antigens mined with reverse vaccinology from a transcriptome of salivary glands from Rhipicephalus microplus ticks was previously shown to present an overall efficacy of 73.2% and cause a significant decrease of tick loads in artificially tick-infested, immunized heifers; this decrease was accompanied by increased levels of antigen-specific IgG1 and IgG2 antibodies, which were boosted during a challenge infestation. In order to gain insights into the systemic effects induced by the vaccine and by the tick challenge we now report the gene expression profile of these hosts' whole-blood leukocytes with RNA-seq followed by functional analyses. These analyses show that vaccination induced unique responses to infestations; genes upregulated in the comparisons were enriched for processes associated with chemotaxis, cell adhesion, T-cell responses and wound repair. Blood transcriptional modules were enriched for activation of dendritic cells, cell cycle, phosphatidylinositol signaling, and platelets. Together, the results indicate that by neutralizing the tick's salivary mediators of parasitism with vaccine-induced antibodies, the bovine host is able to mount normal homeostatic responses that hinder tick attachment and haematophagy and that the tick otherwise suppresses with its saliva., Competing Interests: Competing interestsThe authors declare no competing interests., (© The Author(s) 2019.)

Given the difficulties of controlling the tick Rhipicephalus microplus due to acaricide resistance, this study aimed to ascertain whether shearing could reduce infestation in cattle. 17 taurine cattle were sheared on the anterior third of one randomly selected side. Shearing was undertaken using a machine with a blade, leaving coats with a thickness of 1 mm. Subsequently, eight evaluations were performed once a week, counting adult females of R. microplus with a diameter > 4.5 mm on the anterior third of both sides (shorn and unshorn). The coat length was also monitored by taking five hair samples from each animal fortnightly (1, 15, 29, 43 and 57 days post shorn) from a central area of both shoulders (shorn and unshorn). The tick counts and hair length data were transformed for normalisation and were analysed using mixed models. The tick and hair length means were significantly higher for the unshorn side. Tick counts were significantly lower on the sheared side until the fifth evaluation, with the final three presenting no differences between the sides. The hair length was significantly lower for the sheared side during the five evaluations. We conclude that as the hair length increased, there was also an increase in the number of ticks on the sheared side. Although this method is not practical for large herds, it can be deemed an option in extreme conditions of tick infestation. In addition, the study reinforces the suggestion that the selection and/or use of cattle with shorter hairs may contribute to reduced tick infestation.

Background: Visceral Leishmaniasis in humans presents with fever, anemia, and splenomegaly and can be lethal if not treated. Nevertheless, the majority of Leishmania infantum-infected individuals does not manifest symptoms and remain so provided they are not immunosuppressed. In this work, the performance of different tests was evaluated to detect asymptomatic individuals who were living in Teresina, Piauí state, Brazil, an endemic area for VL., Methodology: L. infantum-specific antibodies were detected by ELISA and two different rapid immunochromatographic (IC) diagnostic tests, Kalazar Detect and OnSite, and parasitic loads were detected by real time PCR [qPCR]. Additionally, we measured levels of the biomarkers monokine induced by IFN-γ (MIG) and IFN-γ-induced protein 10 (IP-10) before and after stimulation of whole blood with soluble Leishmania antigen [SLA]., Principal Findings: Kalazar Detect and OnSite detected, respectively, 76% and 64% of patients presenting with active Visceral Leishmaniasis; 50% and 57% of patients remained positive in these tests, respectively, after treatment. Of the healthy participants in the study who were living in the endemic area, only 1.7% were positive with both of the IC tests. On the other hand, reactivity in ELISA tests revealed that 13% of these individuals presented asymptomatic infections; among VL patients, 84% presenting with active disease were reactive in ELISA, and after treatment, 55.5% were seropositive. L. infantum DNA was present in the blood of 37.9% of infected individuals living in the endemic area, while IP-10 and MIG biomarkers were detected in 26.7% of them. The greatest concordance of positivity occurred between ELISA and qPCR., Conclusion: The association of different techniques can detect asymptomatic infections, however, more research is necessary to develop ideal biomarkers that are simple to use in the clinic and in field studies in areas endemic for Visceral Leishmaniasis., Competing Interests: The authors have declared that no competing interests exist.

Background: It has recently been demonstrated that saliva from Rhipicephalus sanguineus ticks contains adenosine (ADO) and prostaglandin E2 (PGE2), two non-protein molecules that have significant immunomodulatory properties. These molecules can inhibit cytokine production by dendritic cells (DCs), while also reducing the expression of CD40 in these cells. However, more studies are needed for a better understanding of their participation in the feeding of ticks in vivo. This work, therefore, evaluated the importance of ADO during tick infestations. Mice were infested with adult ticks (3 couples/mouse), and their skin was collected at the tick-infested site (3rd and 7th day), and mRNA for receptors of ADO was quantified by real-time PCR., Results: Tick infestation increased by four and two times the expression of the A2b and A3v1 receptors on day 3, respectively, while expression of other ADO receptors was unaltered. In addition, we treated mice (n = 10/group) daily with 8-(p-Sulfophenyl)theophylline, 8-pSPT, 20 mg/kg, i.p.), a non-selective antagonist of ADO receptors, and evaluated the performance of ticks during infestations. Female ticks fed on 8-pSPT-treated mice presented a reduction in their engorgement, weight and hatching rates of egg masses, and survival times of larvae compared to the same parameters presented by ticks in the control group. To investigate if these 8-pSPT-treated mice presented altered immune responses, we performed three tick infestations and collected their lymph node cells to determine the percentages and activation state of DCs and cytokine production by lymphocytes by flow cytometry (Cytometric Bead Array technique, CBA). Our data showed that 8-pSPT-treated mice presented an increase in the percentage of DCs as well as of their stimulatory and co-stimulatory molecules (CD40, CD80 and MHCII). Regarding production of T cell cytokines, we observed a significant increase in the levels of IL-2 and a significant decrease in IL-10, IL-17, TNF-α and IFN-γ cytokines., Conclusions: These results suggest that ADO produced by ticks helps them feed and reproduce and that this effect may be due to modulation of host DCs and T cells.

Background: Infections with parasites of the Leishmania donovani complex result in clinical outcomes that range from asymptomatic infection to severe and fatal visceral leishmaniasis (VL). Neutrophils are major players of the immune response against Leishmania, but their contribution to distinct states of infection is unknown. Gene expression data suggest the activation of the NETosis pathway during human visceral leishmaniasis. Thus, we conducted an exploratory study to evaluate NET-related molecules in retrospective sera from VL patients, asymptomatic individuals and uninfected endemic controls., Results: We demonstrate that VL patients and asymptomatic individuals exhibit differential regulation of molecules associated with neutrophil extracellular traps (NET). These differences were observed at the transcriptional level of genes encoding NET-associated proteins; in quantifications of cell free DNA and metalloproteinase 9; and in enzymatic activity of DNAse and elastase. Moreover, multivariate analysis resulted in class-specific signatures, and ROC curves demonstrate the ability of these molecules in discriminating asymptomatic infection from uninfected controls., Conclusion: Molecules that are associated with NETs are differentially regulated between distinct states of infection with L. infantum, suggesting that NETs might have distinct roles depending on the clinical status of infection. Although unlikely to be exclusive for VL, these signatures can be useful to better characterize asymptomatic infections in endemic regions of this disease.

Mogiana tick virus (MGTV) is a segmented jingmenvirus isolated in 2011 from cattle ticks in Brazil. Here, we present a complete coding genome sequence for MGTV isolate MGTV/V4/11, including all four segments. MGTV is evolutionarily related to the Jingmen tick virus isolates SY84 and RC27., (Copyright © 2017 Villa et al.)

Background: Ticks cause massive damage to livestock and vaccines are one sustainable substitute for the acaricides currently heavily used to control infestations. To guide antigen discovery for a vaccine that targets the gamut of parasitic strategies mediated by tick saliva and enables immunological memory, we exploited a transcriptome constructed from salivary glands from all stages of Rhipicephalus microplus ticks feeding on genetically tick-resistant and susceptible bovines., Results: Different levels of host anti-tick immunity affected gene expression in tick salivary glands; we thus selected four proteins encoded by genes weakly expressed in ticks attempting to feed on resistant hosts or otherwise abundantly expressed in ticks fed on susceptible hosts; these sialoproteins mediate four functions of parasitism deployed by male ticks and that do not induce antibodies in naturally infected, susceptible bovines. We then evaluated in tick-susceptible heifers an alum-adjuvanted vaccine formulated with recombinant proteins. Parasite performance (i.e. weight and numbers of females finishing their parasitic cycle) and titres of antigen-specific antibodies were significantly reduced or increased, respectively, in vaccinated versus control heifers, conferring an efficacy of 73.2%; two of the antigens were strong immunogens, rich in predicted T-cell epitopes and challenge infestations boosted antibody responses against them., Conclusion: Mining sialotranscriptomes guided by the immunity of tick-resistant hosts selected important targets and infestations boosted immune memory against salivary antigens.