Testing the impact of a single nucleotide polymorphism in a Plasmodium berghei ApiAP2 transcription factor on experimental cerebral malaria in mice.

Cerebral malaria (CM) is the deadliest form of severe Plasmodium infections. Currently, we have limited understanding of the mechanisms by which Plasmodium parasites induce CM. The mouse model of CM, experimental CM (ECM), induced by infection with the rodent parasite, Plasmodium berghei ANKA (PbANKA) has been extensively used to study the pathophysiology of CM. Recent genomic analyses revealed that the coding regions of PbANKA and the closely related Plasmodium berghei NK65 (PbNK65), that does not cause ECM, differ in only 21 single nucleotide polymorphysims (SNPs). Thus, the SNP-containing genes might contribute to the pathogenesis of ECM. Although the majority of these SNPs are located in genes of unknown function, one SNP is located in the DNA binding site of a member of the Plasmodium ApiAP2 transcription factor family, that we recently showed functions as a virulence factor alternating the host's immune response to the parasite. Here, we investigated the impact of this SNP on the development of ECM. Our results using CRISPR-Cas9 engineered parasites indicate that despite its immune modulatory function, the SNP is neither necessary nor sufficient to induce ECM and thus cannot account for parasite strain-specific differences in ECM phenotypes.

Global changes in nitration levels and DNA binding profile of Trypanosoma cruzi histones induced by incubation with host extracellular matrix.

Adhesion of T. cruzi trypomastigotes to components of the extracellular matrix (ECM) is an important step in mammalian host cell invasion. We have recently described a significant increase in the tyrosine nitration levels of histones H2A and H4 when trypomastigotes are incubated with components of the ECM. In this work, we used chromatin immunoprecipitation (ChIP) with an anti-nitrotyrosine antibody followed by mass spectrometry to identify nitrated DNA binding proteins in T. cruzi and to detect alterations in nitration levels induced upon parasite incubation with the ECM. Histone H1, H2B, H2A and H3 were detected among the 9 most abundant nitrated DNA binding proteins using this proteomic approach. One nitrated tyrosine residue (Y29) was identified in Histone H2B in the MS/MS spectrum. In addition, we observed a significant increase in the nitration levels of histones H1, H2B, H2A and H4 upon parasite incubation with ECM. Finally, we used ChIP-Seq to map global changes in the DNA binding profile of nitrated proteins. We observed a significant change in the binding pattern of nitrated proteins to DNA after parasite incubation with ECM. This work provides the first global profile of nitrated DNA binding proteins in T. cruzi and additional evidence for modification in the nitration profile of histones upon parasite incubation with ECM. Our data also indicate that the parasite interaction with the ECM induces alterations in chromatin structure, possibly affecting nuclear functions.

Toxoplasma gondii causes increased ICAM-1 and serotonin expression in the jejunum of rats 12 h after infection.

OBJECTIVE: To analyze the remodeling dynamics of total collagen, type I and III, the expression of ICAM-1 and 5-HT in the jejunum of rats. METHODS: Twenty-eight Wistar rats were randomly assigned to two experimental groups: the control group (CG, n = 7) and the infected group (receiving 5,000 sporulated T. gondii oocysts - ME49 strain, genotype II, n = 21). Seven infected rats each at 6 (G6), 12 (G12), and 24 (G24) hours post infection were sacrificed and segments of jejunum were collected for standard histological, histochemical, and immunohistochemistry processing techniques. RESULTS: The infection promoted ICAM-1 and 5-HT expression, type III collagen, and total mast cell increases. However, it also caused a reduction in the area occupied by type I collagen fibers, and in submucosa thickness, and caused ganglion and peri-ganglion alterations. CONCLUSION: The structural damage caused by toxoplasmic infection is intense during the first 24 h post inoculation. At peak dissemination, from 12 to 24 h, there is an increase in ICAM-1 and 5-HT expression, with intense migration of mast cells to the site of infection. There was also a reduction in submucosa thickness, and an effective loss of extracellular matrix (ECM) organization, which included changes in the dynamics of type I and III total collagen deposition.

Reprogramming of Trypanosoma cruzi metabolism triggered by parasite interaction with the host cell extracellular matrix.

Trypanosoma cruzi, the etiological agent of Chagas' disease, affects 8 million people predominantly living in socioeconomic underdeveloped areas. T. cruzi trypomastigotes (Ty), the classical infective stage, interact with the extracellular matrix (ECM), an obligatory step before invasion of almost all mammalian cells in different tissues. Here we have characterized the proteome and phosphoproteome of T. cruzi trypomastigotes upon interaction with ECM (MTy) and the data are available via ProteomeXchange with identifier PXD010970. Proteins involved with metabolic processes (such as the glycolytic pathway), kinases, flagellum and microtubule related proteins, transport-associated proteins and RNA/DNA binding elements are highly represented in the pool of proteins modified by phosphorylation. Further, important metabolic switches triggered by this interaction with ECM were indicated by decreases in the phosphorylation of hexokinase, phosphofructokinase, fructose-2,6-bisphosphatase, phosphoglucomutase, phosphoglycerate kinase in MTy. Concomitantly, a decrease in the pyruvate and lactate and an increase of glucose and succinate contents were detected by GC-MS. These observations led us to focus on the changes in the glycolytic pathway upon binding of the parasite to the ECM. Inhibition of hexokinase, pyruvate kinase and lactate dehydrogenase activities in MTy were observed and this correlated with the phosphorylation levels of the respective enzymes. Putative kinases involved in protein phosphorylation altered upon parasite incubation with ECM were suggested by in silico analysis. Taken together, our results show that in addition to cytoskeletal changes and protease activation, a reprogramming of the trypomastigote metabolism is triggered by the interaction of the parasite with the ECM prior to cell invasion and differentiation into amastigotes, the multiplicative intracellular stage of T. cruzi in the vertebrate host.

Invadosome-Mediated Human Extracellular Matrix Degradation by Entamoeba histolytica.

Entamoeba histolytica is a protozoan parasite that causes invasive amoebiasis when it invades the human colon. Tissue invasion requires a shift from an adhesive lifestyle in the colonic lumen to a motile and extracellular matrix (ECM) degradative lifestyle in the colonic tissue layers. How the parasite regulates these two lifestyles is largely unknown. Previously, we showed that silencing the E. histolytica surface metalloprotease EhMSP-1 results in parasites that are hyperadherent and less motile. To better understand the molecular mechanism of this phenotype, we now show that the parasites with EhMSP-1 silenced cannot efficiently form specialized dot-like polymerized actin (F actin) structures upon interaction with the human ECM component fibronectin. We characterized these F actin structures and found that they are very short-lived structures that are the sites of fibronectin degradation. Motile mammalian cells form F actin structures called invadosomes that are similar in stability and function to these amoebic actin dots. Therefore, we propose here that E. histolytica forms amoebic invadosomes to facilitate colonic tissue invasion.

Morphophysiological changes in the splenic extracellular matrix of Leishmania infantum-naturally infected dogs is associated with alterations in lymphoid niches and the CD4+ T cell frequency in spleens.

The spleen is one of the main affected organs in canine visceral leishmaniasis (CVL). Disorganization of the splenic white pulp (SWP) has been associated with immunosuppression and disease progression. This study aims to assess structural and cellular changes in the splenic extracellular matrix of dogs with CVL, correlating these changes with the parasite load and clinical signs. Splenic fragments were collected from 41 naturally infected animals for parasite load quantification by quantitative PCR, histopathological analysis and immunohistochemistry for CD3+, CD4+, and CD8+ T cells; CD21+ B cells; Ki-67+, IFN-γ+, and IL-10+ cells; and the MMP-9 and ADAM-10 enzymes. Laminin, collagen and fibronectin deposition were also evaluated. The animals were grouped according to the level of SWP organization. SWP disorganization was accompanied by a reduction in the quantity of lymphoid follicles/mm2 (p > 0.0001). Animals with moderate to intense SWP disorganization showed more clinical signs (p = 0.021), higher laminin (p = 0.045) and collagen deposition (p = 0.036), higher MMP-9 expression (p = 0.035) and lower numbers of CD4+ T cells (p = 0.027) in the spleen than the animals with organized SWP. These data suggest that splenic structure and function are drastically altered and compromised during CVL.

Transcriptome modulation of bovine trophoblast cells in vitro by Neospora caninum.

Neospora caninum is one of the most efficient transplacentally transmitted pathogens in cattle and is a cause of abortion in this domestic species. The invasion and proliferation of Neospora caninum in the placenta and its dissemination to the foetus are crucial events in the outcome of an infection. In the bovine placenta, the placentomes are formed by maternal caruncles, which are delimited by a maternal epithelium and foetal cotyledons, which are delimited by an epithelial layer named the trophoblast. These epithelia form a physical barrier against foetal infection. Furthermore, trophoblast cells act as an innate immune defence at the foetal-maternal interface. Neospora caninum invades and proliferates in trophoblast cells in vitro, but it is unknown whether host cell modulation events, which affect the immune response and other processes in the trophoblast, occur. In this work, we investigated the transcriptomic modulation by Neospora caninum infection in the bovine trophoblast cell line F3. In addition, two Neospora caninum isolates with marked differences in virulence, Nc-Spain1H and the Nc-Spain7, were used in this study to investigate the influence of these isolates in F3 modulation. The results showed a clear influence on extracellular matrix reorganisation, cholesterol biosynthesis and the transcription factor AP-1 network. Interestingly, although differences in the transcriptome profiles induced by each isolate were observed, specific isolate-modulated processes were not identified, suggesting very similar regulation in both isolates. Differential expression of the N. caninum genes between both isolates was also investigated. Genes involved in host cell attachment and invasion (SAG-related and microneme proteins), glideosome, rhoptries, metabolic processes, cell cycle and stress response were differentially expressed between the isolates, which could explain their variability. This study provides a global view of Neospora caninum interactions with bovine trophoblast cells and of the intra-specific differences between two Neospora caninum isolates with biological differences.

RNA-seq analysis of early enteromyxosis in turbot (Scophthalmus maximus): new insights into parasite invasion and immune evasion strategies.

Enteromyxum scophthalmi, an intestinal myxozoan parasite, is the causative agent of a threatening disease for turbot (Scophthalmus maximus, L.) aquaculture. The colonisation of the digestive tract by this parasite leads to a cachectic syndrome associated with high morbidity and mortality rates. This myxosporidiosis has a long pre-patent period and the first detectable clinical and histopathological changes are subtle. The pathogenic mechanisms acting in the early stages of infection are still far from being fully understood. Further information on the host-parasite interaction is needed to assist in finding efficient preventive and therapeutic measures. Here, a RNA-seq-based transcriptome analysis of head kidney, spleen and pyloric caeca from experimentally-infected and control turbot was performed. Only infected fish with early signs of infection, determined by histopathology and immunohistochemical detection of E. scophthalmi, were selected. The RNA-seq analysis revealed, as expected, less intense transcriptomic changes than those previously found during later stages of the disease. Several genes involved in IFN-related pathways were up-regulated in the three organs, suggesting that the IFN-mediated immune response plays a main role in this phase of the disease. Interestingly, an opposite expression pattern had been found in a previous study on severely infected turbot. In addition, possible strategies for immune system evasion were suggested by the down-regulation of different genes encoding complement components and acute phase proteins. At the site of infection (pyloric caeca), modulation of genes related to different structural proteins was detected and the expression profile indicated the inhibition of cell proliferation and differentiation. These transcriptomic changes provide indications regarding the mechanisms of parasite attachment to and invasion of the host. The current results contribute to a better knowledge of the events that characterise the early stages of turbot enteromyxosis and provide valuable information to identify molecular markers for early detection and control of this important parasitosis.

Phosphatidylinositol 3-Kinase γ is required for the development of experimental cerebral malaria.

Experimental cerebral malaria (ECM) is characterized by a strong immune response, with leukocyte recruitment, blood-brain barrier breakdown and hemorrhage in the central nervous system. Phosphatidylinositol 3-kinase γ (PI3Kγ) is central in signaling diverse cellular functions. Using PI3Kγ-deficient mice (PI3Kγ-/-) and a specific PI3Kγ inhibitor, we investigated the relevance of PI3Kγ for the outcome and the neuroinflammatory process triggered by Plasmodium berghei ANKA (PbA) infection. Infected PI3Kγ-/- mice had greater survival despite similar parasitemia levels in comparison with infected wild type mice. Histopathological analysis demonstrated reduced hemorrhage, leukocyte accumulation and vascular obstruction in the brain of infected PI3Kγ-/- mice. PI3Kγ deficiency also presented lower microglial activation (Iba-1+ reactive microglia) and T cell cytotoxicity (Granzyme B expression) in the brain. Additionally, on day 6 post-infection, CD3+CD8+ T cells were significantly reduced in the brain of infected PI3Kγ-/- mice when compared to infected wild type mice. Furthermore, expression of CD44 in CD8+ T cell population in the brain tissue and levels of phospho-IkB-α in the whole brain were also markedly lower in infected PI3Kγ-/- mice when compared with infected wild type mice. Finally, AS605240, a specific PI3Kγ inhibitor, significantly delayed lethality in infected wild type mice. In brief, our results indicate a pivotal role for PI3Kγ in the pathogenesis of ECM.

Contribution of myeloid cell subsets to liver fibrosis in parasite infection.

Accumulation of extracellular matrix components secreted by fibroblasts is a normal feature of wound healing during acute inflammation. However, during most chronic/persistent inflammatory diseases, this tissue repair mechanism is incorrectly regulated and results in irreversible fibrosis in various organs. Fibrosis that severely affects tissue architecture and can cause organ failure is a major cause of death in developed countries. Organ-recruited lymphoid (mainly T cells) and myeloid cells (eosinophils, basophils, macrophages and DCs) have long been recognized in their participation to the development of fibrosis. In particular, a central role for recruited monocyte-derived macrophages in this excessive connective tissue deposit is more and more appreciated. Moreover, the polarization of monocyte-derived macrophages in classically activated (IFNγ-dependent) M1 cells or alternatively activated (IL-4/IL-13) M2 cells, that mirrors the Th1/Th2 polarization of T cells, is also documented to contribute differentially to the fibrotic process. Here, we review the current understanding of how myeloid cell subpopulations affect the development of fibrosis in parasite infections.

Role of matrix metalloproteinases 2 and 9 in ex vivo Trypanosoma cruzi infection of human placental chorionic villi.

BACKGROUND: Chagas' disease is caused by the haemophlagelated protozoan Trypanosoma cruzi (T. cruzi). During congenital transmission the parasite breaks down the placental barrier. In the present study we analyzed the participation of matrix metalloproteases (MMPs) in the extracellular matrix (ECM) remodeling during T. cruzi ex vivo infection of human placental chorionic villi explants. METHODS: Chorionic villi from healthy woman placentas were incubated in the presence or absence of 105 or 106 T. cruzi trypomastigotes (Y strain) with or without the MMPs inhibitor doxycycline. Effective infection was tested measuring parasite DNA by real time PCR (qPCR). MMP-2 and MMP-9 expression were determined by western blotting and immunohistochemistry and their activities were measured by zymography. The effect of MMPs on ECM structure was analyzed histochemically. RESULTS: T. cruzi induces the expression and activity of MMP-2 and MMP-9 in chorionic villi. Inhibition of the MMPs prevents the tissue damage induced by T. cruzi and partially decreases the ex vivo infection of the chorionic villi. CONCLUSION: MMPs are partially responsible for the ECM changes observed in human chorionic villi during T. cruzi infection and participate in tissue invasion. On the other hand, MMPs may be part of a local placental antiparasitic mechanism.

Shedding of TRAP by a rhomboid protease from the malaria sporozoite surface is essential for gliding motility and sporozoite infectivity.

Plasmodium sporozoites, the infective stage of the malaria parasite, move by gliding motility, a unique form of locomotion required for tissue migration and host cell invasion. TRAP, a transmembrane protein with extracellular adhesive domains and a cytoplasmic tail linked to the actomyosin motor, is central to this process. Forward movement is achieved when TRAP, bound to matrix or host cell receptors, is translocated posteriorly. It has been hypothesized that these adhesive interactions must ultimately be disengaged for continuous forward movement to occur. TRAP has a canonical rhomboid-cleavage site within its transmembrane domain and mutations were introduced into this sequence to elucidate the function of TRAP cleavage and determine the nature of the responsible protease. Rhomboid cleavage site mutants were defective in TRAP shedding and displayed slow, staccato motility and reduced infectivity. Moreover, they had a more dramatic reduction in infectivity after intradermal inoculation compared to intravenous inoculation, suggesting that robust gliding is critical for dermal exit. The intermediate phenotype of the rhomboid cleavage site mutants suggested residual, albeit inefficient cleavage by another protease. We therefore generated a mutant in which both the rhomboid-cleavage site and the alternate cleavage site were altered. This mutant was non-motile and non-infectious, demonstrating that TRAP removal from the sporozoite surface functions to break adhesive connections between the parasite and extracellular matrix or host cell receptors, which in turn is essential for motility and invasion.

Extracellular matrix alterations in experimental Leishmania amazonensis infection in susceptible and resistant mice.

Leishmania is inoculated, by the bite of an infected sandfly, into the skin of the host, where the promastigotes are phagocyted by dermal macrophages. The dermal region comprises cells and abundant extracellular matrix. Studies show that matrix metalloproteinases play an important role in host defense responses against pathogens in mammals and that their activities lead to the production of antimicrobial peptides. The aim of this study is to evaluate the changes in the distribution of fibronectin and laminin as well as in the elastic system fibres during the course of infection caused by Leishmania amazonensis in mice with distinct genetic backgrounds of susceptibility to this parasite. The results showed that BALB/c presented an enhancement of fibronectin during the course of infection when compared to their control group while the infected or non-infected C3H.He showed a decrease of this protein at end of the experiment. Laminin, on the other hand, remained unaltered in both strains. Also in both BALB/c and C3H.He mice the elastic and elaunin fibres remained unchanged while the oxytalan fibres decreased along the experiment. Ninety days after the infection C3H.He mice had recovered their capacity to produce oxytalan fibres.

Molecular mechanisms of host cell invasion by Trypanosoma cruzi.

The protozoan parasite Trypanosoma cruzi, the etiologic agent of Chagas disease, is an obligate intracellular protozoan pathogen. Overlapping mechanisms ensure successful infection, yet the relationship between these cellular events and clinical disease remains obscure. This review explores the process of cell invasion from the perspective of cell surface interactions, intracellular signaling, modulation of the host cytoskeleton and endosomal compartment, and the intracellular innate immune response to infection.

Acanthamoeba culbertsoni: analysis of amoebic adhesion and invasion on extracellular matrix components collagen I and laminin-1.

Acanthamoeba are free-living amoebae found in most environments that can cause brain and corneal infections. To infect humans, these pathogens must interact with host cells and the extracellular matrix (ECM). In order to define the mode by which amoebae recognize ECM components and process this recognition, we analyzed Acanthamoeba culbertsoni attachment and invasion, respectively, on collagen I and laminin-1 and on tridimensional collagen I and matrigel matrices. We determined that amoebae surface proteins are involved in adhesion, that exogenous sugars can decrease adhesion and invasion, and that adhesion and invasion are dependent on microfilament reorganization. In addition, we determined the role of serine- and metallo-proteases on invasion and found that adhesion was blocked when amoebae were treated with a metallo-protease inhibitor. Collectively, these results suggest that adhesion and invasion are protease- and microfilament-dependent events in which amoebic surface proteins play a pivotal role.

Entamoeba histolytica: lipid rafts are involved in adhesion of trophozoites to host extracellular matrix components.

Adhesion is an important virulence function for Entamoeba histolytica, the causative agent of amoebic dysentery. Lipid rafts, cholesterol-rich domains, function in compartmentalization of cellular processes. In E. histolytica, rafts participate in parasite-host cell interactions; however, their role in parasite-host extracellular matrix (ECM) interactions has not been explored. Disruption of rafts with a cholesterol extracting agent, methyl-beta-cyclodextrin (MbetaCD), resulted in inhibition of adhesion to collagen, and to a lesser extent, to fibronectin. Replenishment of cholesterol in MbetaCD-treated cells, using a lipoprotein-cholesterol concentrate, restored adhesion to collagen. Confocal microscopy revealed enrichment of rafts at parasite-ECM interfaces. A raft-resident adhesin, the galactose/N-acetylgalactosamine-inhibitable lectin, mediates interaction to host cells by binding to galactose or N-acetylgalactosamine moieties on host glycoproteins. In this study, galactose inhibited adhesion to collagen, but not to fibronectin. Together these data suggest that rafts participate in E. histolytica-ECM interactions and that raft-associated Gal/GalNAc lectin may serve as a collagen receptor.

Fibrosis and hypertrophy induced by Trypanosoma cruzi in a three-dimensional cardiomyocyte-culture system.

Cardiac damages caused by in vivo infection with Trypanosoma cruzi are still not fully clarified. Here we describe for the first time an in vitro model of fibrosis, hypertrophy, and remodeling induced by T. cruzi in cardiomyocyte spheroids (cardiac microtissues). In this new 3-dimensional system, cardiac spheroids showed spontaneous contractility, with typical cardiac morphology and production of extracellular matrix components. There were 4- and 6-fold increases, respectively, in the area and the volume of T. cruzi-infected cardiomyocytes and whole microtissues, together with a 50% reduction of the cell population. Immunofluorescence showed increased expression of fibronectin, collagen IV, and laminin in the microtissues 144 h after infection. T. cruzi infection induced an increase in both the cellular area and the extracellular matrix components in cardiac spheroids, which contributed to an increase in total microtissue volume, making this a powerful 3-dimensional in vitro model for the study of cardiac-tissue hypertrophy, fibrosis, and remodeling.

Altered thymocyte migration during experimental acute Trypanosoma cruzi infection: combined role of fibronectin and the chemokines CXCL12 and CCL4.

We previously showed migration disturbances in the thymus during experimental infection with Trypanosoma cruzi, the causative agent of Chagas disease. These changes were related to the enhanced expression of extracellular matrix ligands and receptors, leading to the escape of immature cells to the periphery. Here, we analyzed the expression and role of selected chemokines (CXCL12 and CCL4) and their receptors (CXCR4 and CCR5) in regulating thymocyte migration in conjunction with extracellular matrix during acute T. cruzi infection. We found increased chemokine deposition in the thymus of infected mice when compared to controls, accompanied by enhanced co-localization with fibronectin as well as up-regulated surface expression of CXCR4 and CCR5 in thymocytes. We also noticed altered thymocyte migration towards the chemokines analyzed. Such an enhancement was even more prominent when fibronectin was added as a haptotatic stimulus in combination with a given chemokine. Our findings suggest that thymocyte migration results from a combined action of chemokines and extracellular matrix (ECM), which can be altered during pathological conditions such as T. cruzi infection, and may be at the origin of the changes in the T cell repertoire seen in this pathological process.

Alteration of extracellular collagen matrix in the myocardium of canines infected with Dirofilaria immitis.

The heart consists of cardiocytes and the interstitial extracellular matrix (ECM), which is made up mainly of collagens. The ECM has been suggested to be important in maintaining the structure and function of the heart. This investigation attempted to elucidate the changes in the ECM collagens in the hearts of canines with dirofilariasis. The ECM collagen fibrils of the heart are grouped into endomysial struts, epimysial weaves, and perimysial coils. In the present study, we used the modified silver impregnation technique to stain paraffin-embedded sections to demonstrate three types of ECM. The results revealed that the ECM content of the heart was significantly reduced in heartworm-infected dogs, and became fragmented and dissociated. In addition, the amounts of collagen in the septum (Sep), RVs and LVs in canines with dirofilariasis (Sep=11.55+/-0.65, RV=12.07+/-0.59, LV=11.72+/-0.62 microg/mg, n=24) were significantly lower (p<0.01) than that in the normal canines (Sep=15.09+/-0.72, RV=15.16+/-0.83, LV=14.91+/-0.89 microg/mg, n=8). These results indicated that heartworm infection induced the remodeling of the extracellular matrix, thus markedly altering the architecture and function of the heart.

Extracellular matrix alterations in experimental murine Leishmania (L.) amazonensis infection.

Here we describe extracellular matrix alterations in footpad lesions and draining lymph nodes caused by Leishmania (L.) amazonensis in mouse strains with distinct susceptibilities to this parasite: BALB/c (susceptible), C57BL/6 (intermediate), and DBA/2 (resistant). Changes in ECM were observed mainly in BALB/c mice that, in general, presented tissue damage associated with high parasite burden. Under polarized light, Sirius Red revealed type I collagen that was predominant in the primary lesion in all strains studied at the early phase of infection, but gradually decreased and was replaced by abundant type III collagen fibres in chronic phase lesions. The presence of type III collagen seemed to provide support to inflammatory cells, mainly vacuolated and parasitized macrophages. Laminin expression was not altered during infection by L. (L.) amazonensis in any of the mouse strains studied. Furthermore, the decreased fibronectin expression, in all strains, in areas where amastigotes have been found, indicated that this decline was also not related to the genetic background.