Prevalence of trichomonads in the cloaca of wild wetland birds in the Netherlands.

Severe granulomatosis in productive layer chickens due to Tetratrichomonas gallinarum strain 13/16632 infection occurred in 2013 and 2017 on farms situated in a wetland area in the Netherlands. We hypothesized that wetland birds could be the source of the infection. Therefore, a prevalence study on trichomonads was performed by analysing cloaca swabs of 526 birds belonging to 13 species of wetland birds. The number of birds sampled ranged from 1 to 275 per species. Birds were sampled at 15 locations in the Netherlands. DNA extracted from the cloaca swabs was subjected to nested PCR using trichomonad-specific primers targeting the internal transcribed spacer 1 (ITS1)-5.8S rRNA-ITS2 region followed by cloning and sequencing. In nine bird species, trichomonads were detected; the overall prevalence was 9% (47/526), while the prevalence in the five species for which a substantial number of birds were examined (at least 39 per species) ranged from 4% to 24%. Three trichomonad species were found: T. gallinarum, Trichomonas tenax and Simplicimonas sp. of which T. gallinarum dominated. The virulent T. gallinarum strain 13/16632 was not detected, but closely related strains were. Phylogenetic analysis revealed that all T. gallinarum isolates belonged to two clusters within lineage 15 of Tetratrichomonas lineages. All T. tenax isolates were identical and clustered with reference strain H95, while Simplicimonas sp. isolates showed large genetic diversity. Some isolates may represent a new species of the genus Simplicimonas. We conclude that trichomonads are widespread amongst wetland birds, raising the question, amongst others, of their relevance for commercial poultry.RESEARCH HIGHLIGHTSTrichomonads occur among wild wetland birds in the Netherlands.T. gallinarum is the dominant trichomonad species in the cloaca of wetland birds.Some T. gallinarum isolates are closely related to a strain causing granulomas in layer chickens.Some isolates may represent a new species of the genus Simplicimonas.

Emergence of Aspergillus fumigatus azole resistance in azole-naïve patients with chronic obstructive pulmonary disease and their homes.

Azole-resistant Aspergillus fumigatus (ARAF) has been reported in patients with chronic obstructive pulmonary disease (COPD) but has not been specifically assessed so far. Here, we evaluated ARAF prevalence in azole-naïve COPD patients and their homes, and assessed whether CYP51A mutations were similar in clinical and environmental reservoirs. Sixty respiratory samples from 41 COPD patients with acute exacerbation and environmental samples from 36 of these patient's homes were prospectively collected. A. fumigatus was detected in respiratory samples from 11 of 41 patients (27%) and in 15 of 36 domiciles (42%). Cyp51A sequencing and selection on itraconazole medium of clinical (n = 68) and environmental (n = 48) isolates yielded ARAF detection in 1 of 11 A. fumigatus colonized patients with COPD (9%) and 2 of 15 A. fumigatus-positive patient's homes (13%). The clinical isolate had no CYP51A mutation. Two environmental isolates from two patients harbored TR34 /L98H mutation, and one had an H285Y mutation. Coexistence of different cyp51A genotypes and/or azole resistance profiles was detected in 3 of 8 respiratory and 2 of 10 environmental samples with more than one isolate, confirming the need for a systematic screening of all clinically relevant isolates. The high prevalence of ARAF in patients with COPD and their homes supports the need for further studies to assess the prevalence of azole resistance in patients with Aspergillus diseases in Northern France.

Assessment of microscopic and molecular tools for the diagnosis and follow-up of cryptosporidiosis in patients at risk.

Cryptosporidiosis is an important though underreported public health concern. Molecular tools might be helpful in improving its diagnosis. In this study, ZR Fecal DNA MiniPrep™ Kit (ZR) and NucliSens® easyMAG® (EM) were compared using four Cryptosporidium-seeded feces and 29 Cryptosporidium-positive stools. Thereafter, ZR was selected for prospective evaluation of Cryptosporidium detection by 18S rDNA and LAXER quantitative PCR (qPCR) in 69 stools from 56 patients after Cryptosporidium detection by glycerin, modified Ziehl-Neelsen (ZN) and auramine-phenol (AP) stainings. The combination of any of the two extraction methods with 18S qPCR yielded adequate detection of Cryptosporidium in seeded stools, but the ZR kit showed the best performance. All 29 Cryptosporidium-positive samples were positive with 18S qPCR, after both ZR and EM extraction. However, false-negative results were found with LAXER qPCR or nested PCR. Cryptosporidiosis was diagnosed in 7/56 patients. All the microscopic methods enabled the initial diagnosis, but Cryptosporidium was detected in 12, 13, and 14 samples from these seven patients after glycerin, ZN, and AP staining respectively. Among these samples, 14 and 12 were positive with 18S and LAXER qPCR respectively. In two patients, Cryptosporidium DNA loads were found to be correlated with clinical evolution. Although little known, glycerin is a sensitive method for the initial detection of Cryptosporidium. When combined with 18S qPCR, ZR extraction, which had not been evaluated so far for Cryptosporidium, was an accurate tool for detecting Cryptosporidium and estimating the oocyst shedding in the course of infection.

Granuloma disease in flocks of productive layers caused by Tetratrichomonas gallinarum.

In 2013, seven outbreaks of granuloma disease occurred in Dutch flocks of productive layers housed on different farms. These outbreaks were characterized by increased mortality and high incidence of granulomas, mainly in caeca (340/408 hens = 83%) and livers (69/408 hens = 17%). Mortality started to increase between 21 and 35 weeks of age and reached 3.7% to 11.0% exceeding the breeder's norm in periods ranging from 9 to 48 weeks. Some flocks also showed decreased egg production and/or loss of mean egg weight. All affected flocks were linked to one rearing farm, which therefore seemed to be the source of the disease. However, no signs of disease had been observed at this rearing farm. Sentinel hens placed in one of the affected flocks to determine whether the disease had an infectious nature developed granulomas identical to those seen in the outbreaks. Next, by fulfilling Koch's postulates it was shown that Tetratrichomonas gallinarum was the aetiological agent of the granuloma disease. The condition was reproduced in mature specified pathogen free White Leghorn hens (GD - Animal Health, Deventer, the Netherlands) by inoculation via both an artificial and a natural route with a well-defined axenic T. gallinarum isolate obtained from one of the affected flocks. Other causes of granuloma disease were excluded.

On Blastocystis secreted cysteine proteases: a legumain-activated cathepsin B increases paracellular permeability of intestinal Caco-2 cell monolayers.

Blastocystis spp. pathogenic potential remains unclear as these anaerobic parasitic protozoa are frequently isolated from stools of both symptomatic and asymptomatic subjects. In silico analysis of the whole genome sequence of Blastocystis subtype 7 revealed the presence of numerous proteolytic enzymes including cysteine proteases predicted to be secreted. To assess the potential impact of proteases on intestinal cells and gut function, we focused our study on two cysteine proteases, a legumain and a cathepsin B, which were previously identified in Blastocystis subtype 7 culture supernatants. Both cysteine proteases were produced as active recombinant proteins. Activation of the recombinant legumain was shown to be autocatalytic and triggered by acidic pH, whereas proteolytic activity of the recombinant cathepsin B was only recorded after co-incubation with the legumain. We then measured the diffusion of 4-kDa FITC-labelled dextran across Caco-2 cell monolayers following exposition to either Blastocystis culture supernatants or each recombinant protease. Both Blastocystis culture supernatants and recombinant activated cathepsin B induced an increase of Caco-2 cell monolayer permeability, and this effect was significantly inhibited by E-64, a specific cysteine protease inhibitor. Our results suggest that cathepsin B might play a role in pathogenesis of Blastocystis by increasing intestinal cell permeability.

Molecular subtyping of Blastocystis spp. using a new rDNA marker from the mitochondria-like organelle genome.

Blastocystis spp. are common anaerobic intestinal protozoa found in both human and animals. They are characterized by a high genetic diversity with at least 17 subtypes (STs) that have been described on the basis of a 600 bp 'barcoding region' from the 18S rDNA gene. However, analysis of the recently sequenced genome of a Blastocystis ST7 isolate (strain B) revealed the presence of multiple variable copies of the 18S rDNA gene, with 17 completely assembled copies. Comparison of the barcoding region from these 17 copies allowed us to classify the 18S rDNA sequences into 6 clusters, each cluster containing identical sequences. Surprisingly, 4 of these clusters had the highest homology with 18S rDNA sequences from 2 other Blastocystis ST7 isolates referred as QQ98-4 and H. These results suggest that the 18S rDNA gene is not the marker of choice to discriminate between strains within STs. In the present study, we identified a single-copy subtyping rDNA marker in the genome of the mitochondria-like organelles (MLOs). Using a partial sequence of the MLO rDNA, we successfully subtyped 66 isolates from both human and animals belonging to Blastocystis ST1 to ST10. Our results also indicate that this mitochondrial marker could be useful to detect co-infections by different isolates of a same ST.

Tetratrichomonas gallinarum granuloma disease in a flock of free range layers.

Granuloma disease in a flock of free range productive layers in the Netherlands in 2017 is described. The disease resembled granuloma outbreaks in layers caused by Tetratrichomonas gallinarum in 2013 and occurred in the same area in which the rearing farm considered as the source of the 2013 outbreaks was located. Between 55 and 84 weeks of age mortality was 20.3% (breeder's norm 3.9%). All dead hens examined (n = 20) showed granulomas especially in liver and ceca. Nine hens with or without liver and/or ceca granulomas were examined for trichomonads in mentioned organs by in situ hybridization (ISH), nested PCR, and cloning and sequencing. Ceca were also examined by culture. T. gallinarum ISH was positive in all livers and ceca with granulomas and negative in case granulomas were absent. T. gallinarum strain 13/16632, which caused the 2013 outbreaks was found in 4/8 hens with granulomas. Moreover, other trichomonads were detected: a T. gallinarum strain GPO-like and a Simplicimonas sp. strain GABC1-like. Mixed infections also occurred. Infectious causes of granuloma disease other than the afore-mentioned trichomonads could be excluded. Trichomonad DNA was not detected in environmental samples and wild ducks originating from the farm of concern, except for one duck in which the same Simplicimonas sp. as in hens was detected, leaving the source of the T. gallinarum infection in hens unknown. It is concluded that the herein described granuloma disease likely was caused by T. gallinarum strain 13/16632. However, the pathogenicity of the other trichomonads found remains to be clarified.

Prevalence and genetic diversity of Campylobacter spp. in the production chain of broiler chickens in Lebanon and its association with the intestinal protozoan Blastocystis sp.

Campylobacter jejuni is recognized as the most common foodborne pathogen associated with human gastroenteritis worldwide. Broilers are frequently infected by the bacteria and are considered the main source of exposure to humans. However, despite its public health impact, no recent data are currently available in Lebanon about Campylobacter spp. in poultry and human population. Therefore, this study aimed to determine the prevalence and genetic diversity of Campylobacter spp. in 227 ceca and on 227 carcasses of broiler chickens collected in Lebanese slaughterhouses. Overall, the prevalence of Campylobacter was shown to reach 67.0% in ceca and 17.2% on carcasses of Lebanese poultry. The only 2 Campylobacter species identified were C. jejuni and C. coli, with a slightly higher prevalence of C. coli in ceca and of C. jejuni on carcasses. A high level of genetic diversity was reported among the 51 C. jejuni isolates selected, since 25 distinct profiles were identified according to the comparative genomic fingerprinting typing method based on a subset of 40 genes using the 90% similarity threshold. Predominant clusters observed in Lebanese poultry isolates were also frequently found among French human clinical cases, highlighting that broiler chickens represent a potential reservoir for human campylobacteriosis. In addition, a significantly higher prevalence of Campylobacter spp. was found in slaughterhouse workers than in a cohort of hospitalized patients with no contact with poultry, confirming that contaminated broiler chickens in slaughterhouse appeared to be a non-negligible source of Campylobacter spp. transmission. Interestingly, a significant association between Campylobacter spp. and Blastocystis sp. has been observed. This correlation suggested that the presence of Campylobacter spp. would be favored when Blastocystis sp. is present and, similarly, the absence of one would favor the absence of the other. This is the first large-scale investigation focusing on the impact of Campylobacter spp. in broiler chickens in Lebanon and confirmed the need to implement prevention and control measures in the poultry production to reduce the burden of campylobacteriosis in the human population.

A review of methods for nematode identification.

Nematodes are non-segmented roundworms found in soil, aquatic environment, plants, or animals. Either useful or pathogenic, they greatly influence environmental equilibrium, human and animal health, as well as plant production. Knowledge on their taxonomy and biology are key issues to answer the different challenges associated to these organisms. Nowadays, most of the nematode taxonomy remains unknown or unclear. Several approaches are available for parasite identification, from the traditional morphology-based techniques to the sophisticated high-throughput sequencing technologies. All these techniques have advantages or drawbacks depending on the sample origin and the number of nematodes to be processed. This review proposes an overview of all newly available methods available to identify known and/or unknown nematodes with a specific focus on emerging high-throughput molecular techniques.

Molecular cloning, expression analysis and iron metal cofactor characterisation of a superoxide dismutase from Toxoplasma gondii.

A genomic region of 12 kb encompassing the gene encoding the superoxide dismutase (SOD) of Toxoplasma gondii has been cloned. The gene contains four exons of 121, 42, 381 and 59 bp which are separated by three introns of 321, 202, and 577 bp, respectively. The open reading frame can be translated into a protein of 201 amino acids with a molecular mass of 22.6 kDa. Alignment indicated that it is a FeSOD, a type only found in bacteria, protozoa and chloroplast of higher plants. Recombinant SOD was expressed in a Escherichia coli double mutant lacking both MnFeSOD and FeSODs. The presence of iron as metal cofactor was confirmed by measurements of iron by absorption mass spectrometry and electron paramagnetic resonance studies. Semi-quantitative reverse transcribed polymerase chain reaction experiments showed a similar amount of SOD transcripts in two developmental stages of T. gondii. Antibodies raised against the purified recombinant protein detected SOD protein in both bradyzoite and tachyzoite forms suggesting this SOD might be essential for the intracellular growth of both developmental stages. Southern blot analysis indicated that SOD occured as a single copy gene in T. gondii genome.

New Insights into the Phylogeny of Trichomonads Inferred from Small Subunit rRNA Sequences.

Small subunit ribosomal DNA sequences were obtained by polymerase chain reaction from four trichomonad species: a frog endosymbiont Trichomitus batrachorum, an intestinal endosymbiont of a squamate reptile, Hypotrichomonas acosta and two free-living isolates, Monotrichomonas carabina and Monotrichomonas sp. Molecular trees inferred by distance, parsimony and likelihood techniques identify three well-resolved clusters within the trichomonads, however bootstrap values do not strongly support a particular branching order for these lineages. The first cluster includes the Devescovinidae and the Calonymphidae. The second clade unites Trichomitus batrachorum and Hypotrichomonas acosta. The third cluster embraces all known free-living genera, including Monotrichomonas, and various members of the Trichomonadinae subfamily such as Trichomonas vaginalis, and Pentatrichomonoides scroa. Neither Monocercomonadidae nor the Trichomonadidae as envisaged are monophyletic. Most of the monocercomonads, which possess a rudimentary cytoskeleton, were likely descendants of more complex forms. The study also suggests that the genus Trichomitus is currently polyphyletic, partly explaining the discordant positions of this genus in previous molecular analyses.

Analysis of genetic diversity at the iron-containing superoxide dismutase locus in Plasmodium falciparum wild isolates.

In order to investigate the genetic diversity of iron-containing superoxide dismutase (FeSOD) from Plasmodium falciparum, a potential anti-malarial therapeutic target, we cloned and sequenced Plasmodium FeSOD from 26 blood samples from non-infected patients. Fifteen clones had the same nucleotide sequence as that of the FeSOD gene of the P. falciparum strain HB3 cultivated in vitro. The other 11 clones presented mutations responsible for punctual amino acid changes which did not modify key residues for the function or the structure of the enzyme. The high sequence conservation between FeSOD from the isolates confirms that this enzyme could represent a therapeutic target.

Cloning and expression of an iron-containing superoxide dismutase in the parasitic protist, Trichomonas vaginalis.

A superoxide dismutase (SOD) gene of the parasitic protist Trichomonas vaginalis was cloned, sequenced, expressed in Escherichia coli, and its gene product characterized. It is an iron-containing dimeric protein with a monomeric mass of 22,067 Da. Southern blots analyses suggested the presence of seven iron-containing (FeSOD) gene copies. Hydrophobic cluster analysis revealed some peculiarities in the 2D structure of the FeSOD from T. vaginalis and a strong structural conservation between prokaryotic and eukaryotic FeSODs. Phylogenetic reconstruction of the SOD sequences confirmed the dichotomy between FeSODs and manganese-containing SODs. FeSODs of protists appeared to group together with homologous proteobacterial enzymes suggesting a possible origin of eukaryotic FeSODs through an endosymbiotic event.

Cytoskeleton in trichomonads: I. Immunological and biochemical comparative study of costal proteins in the genus Tritrichomonas.

Proteins of the whole cytoskeleton fraction obtained by Triton X-100 action on several Tritrichomonas species have been analyzed by gel electrophoresis. In addition to tubulins, several major protein components with molecular weights between 100 and 150 kDa were separated and presumably represent costal proteins. The partial purification of the costae from the whole cytoskeleton fraction of Tritrichomonas foetus treated with 0.3 M KI confirmed the presence of costal proteins in the 100-150 kDa zone. Costa fibres could be solubilized in 8 M urea. These characteristics indicate that costal proteins may represent a novel class of striated root proteins. A library of 7 monoclonal antibodies (MAbs) raised in mice immunized with the whole cytoskeleton fraction of Tritrichomonas foetus labelled the costa by immunofluorescence and recognize five polypeptides at 135,127,114, 88 and 47 kDa by immunoblotting. Two of these MAbs cross-react by immunofluorescence and immunoblotting with the three other Tritrichomonas species tested, i.e. T. mobilensis, T. augusta, T. muris. However, these 7 MAbs do not show immunological cross-reactivity with other trichomonad genera indicating that the costae are not identical in their biochemical composition; this corresponds to the differences observed in their respective fine structure. Nonetheless, a polyclonal antibody produced against the 118 kDa protein of the costa of Trichomonas vaginalis also labels a 118 kDa protein and the costa by IF in Tritrichomonas species indicating common epitopes.

Cytoskeleton in trichomonads: II. Immunological and biochemical characterization of the preaxostylar fibres and undulating membrane in the genus Tritrichomonas.

The production of 3 monoclonal antibodies (MAbs) and the use of immunocytochemical techniques such as immunofluorescence (IF), immunoblotting (IB) and immunogold staining (IGS) reveal that the preaxostylar fibres of Tritrichomonas foetus are composed of at least 3 polypeptides of 55, 53 and 46 kDa. Two of these MAbs cross-react with Tritrichomonas mobilensis and one with Tritrichomonas augusta and Tritrichomonas muris on polypeptides with very similar molecular weights (M.W.) However, no cross-reactivity is seen with the preaxostylar fibres of other trichomonad species tested. These cross-reactivities restricted to the Tritrichomonas genus are similar to those observed with the costa and several explanations are proposed. Similarly, 5 MAbs obtained against Tritrichomonas foetus cytoskeleton label the undulating membrane (UM) by IF and IGS. IB identifies 5 polypeptides of very different M.W. (148, 72, 39, 33 and 23 kDa) in Tritrichomonas foetus. Among them, 2 cross-react by IF and IB in Tritrichomonas mobilensis, one in Tritrichomonas augusta and none in Tritrichomonas muris. These results are in agreement with the electron microscopy studies which have shown that the UM ultrastructure of Tritrichomonas foetus, Tritrichomonas mobilensis and Tritrichomonas augusta are very similar and different from that of Tritrichomonas muris. The lack of cross-reactivity with the lamellar type UM of the Trichomonadinae genera which is very different from the Tritrichomonadinae UM is also demonstrated.

Molecular evolution inferred from small subunit rRNA sequences: what does it tell us about phylogenetic relationships and taxonomy of the parabasalids?

The Parabasala are a primitive group of protists divided into two classes: the trichomonads and the hypermastigids. Until recently, phylogeny and taxonomy of parabasalids were mainly based on the comparative analysis of morphological characters primarily linked to the development of their cytoskeleton. Recent use of molecular markers, such as small subunit (SSU) rRNA has led to now insights into the systematics of the Parabasala and other groups of prolists. An updated phylogeny based on SSU rRNA is provided and compared to that inferred from ultrastructural data. The SSU rRNA phylogeny contradicts the dogma equating simple characters with pumitive characters. Hypermastigids, possessing a hyperdeveloped cytoskeleton, exhibit the most basal emergence in the parabasalid lineage. Other observations emerge from the SSU rRNA analysis, such as the secondary loss of some cytoskeleton structures in all representatives of the Monocercomonadidae, the existence of secondarily free living taxa (reversibility of parasitism) and the evidence against the co-evolution of the endobiotic parabasalids and their animal hosts. According to phylogenies based on SSU rRNA, all the trichomonad families are not monophyletic groups, putting into question the validity of current taxonomic assignments. The precise branching order of some taxa remains unclear, but this issue can possibly be addressed by the molecular analysis of additional parabasalids. The goal of such additional analyses would be to propose, in a near future, a revision of the taxonomy of this group of protists that takes into account both molecular and morphological data.

Monitoring of four DNA extraction methods upstream of high-throughput sequencing of Anisakidae nematodes.

Different methods were evaluated to extract DNA from pooled nematodes belonging to Anisakis, Contracaecum, Pseudoterranova and Hysterothylacium genera isolated from edible fish. Pooled DNA extraction is the first and compulsory step to allow the identification of a large number of samples through high-throughput DNA sequencing with drastic time and cost reductions.

The genotypic characterisation of Acanthamoeba isolates from human ocular samples.

BACKGROUND: We characterised 37 amoebae cultured from corneal scrapings, contact lenses or lens case solutions of patients with suspected Acanthamoeba keratitis. METHODS: The isolates were identified by their morphology and by PCR targeting the Acanthamoeba nuclear small-subunit rRNA gene. Acanthamoeba isolates were genotyped by DNA sequence analysis. RESULTS: The 37 isolates comprised 35 Acanthamoeba, one Hartmannella and one Vahlkampfia. Ten Acanthamoeba isolates were shown to be responsible for keratitis. CONCLUSION: Genotype T4 was the only Acanthamoeba genotype responsible for keratitis in this study, and represented 79% of non-pathogenic isolates.

Striated fibers in trichomonads: costa proteins represent a new class of proteins forming striated roots.

The production of monoclonal antibodies and the use of biochemical techniques revealed that B-type costa proteins in trichomonads are composed of several major polypeptides with molecular weight detected between 100 and 135 kDa similar to those found in the A-type costae. Although differences were observed between the two types in their fine structure, we tested whether proteins composing the two costa types belong to the same protein family. A polyclonal antibody produced against the 118 kDa costa protein of Trichomonas vaginalis also recognized a 118 kDa costa protein in all other trichomonad genera studied so far whether they have A- or B-type costae. Moreover biochemical characteristics of costa proteins indicated that these proteins might represent a novel class of striated root-forming proteins in addition to centrin, giardin, and assemblin.