Chronic Toxoplasma gondii infection enhances susceptibility to colitis.

Oral infection with Toxoplasma gondii results in dysbiosis and enteritis, both of which revert to normal during chronic infection. However, whether infection leaves a lasting impact on mucosal responses remains uncertain. Here we examined the effect of the chemical irritant dextran sodium sulfate (DSS) on intestinal damage and wound healing in chronically infected mice. Our findings indicate that prior infection with T. gondii exacerbates damage to the colon caused by DSS and impairs wound healing by suppressing stem cell regeneration of the epithelium. Enhanced tissue damage was attributable to inflammatory monocytes that emerge preactivated from bone marrow, migrate to the intestine, and release inflammatory mediators, including nitric oxide. Tissue damage was reversed by neutralization of inflammatory monocytes or nitric oxide, revealing a causal mechanism for tissue damage. Our findings suggest that chronic infection with T. gondii enhances monocyte activation to increase inflammation associated with a secondary environmental insult.

TIDE Analysis of Cryptosporidium Infections by gp60 Typing Reveals Obscured Mixed Infections.

BACKGROUND: Cryptosporidiosis is a parasitic disease associated with potentially fatal diarrhea. The most used method in Cryptosporidium subtyping is based on the glycoprotein gene gp60. Each infection can represent a parasite population, and it is important to investigate the influence on transmission and virulence, as well as any impact on public health investigations. However, an easy-to-use method for detection is lacking. METHODS: Here we report on the use of the bioinformatic program TIDE for deconvolution of gp60 chromatograms. A combination of single oocyst analysis and cloning successfully confirmed the within-sample parasite population diversity. Retrospective sample analysis was conducted on archived chromatograms. RESULTS: For Cryptosporidium parvum, 8.6% multistrain infections (13 of 152) obscured by currently used consensus base calling were detected. Importantly, we show that single oocysts can harbor a mixed population of sporozoites. We also identified a striking dominance of unappreciated polymerase stutter artefacts in all 218 chromatograms analyzed, challenging the uncritical use of gp60 typing. CONCLUSIONS: We demonstrate the value of a new, easy-to-use analytical procedure for critical characterization of C. parvum and Cryptosporidium hominis in epidemiological investigations, also applicable retrospectively. Our findings illuminate the hidden parasite diversity with important implications for tracing zoonotic and person-to-person transmissions.

Fatal Diplostomum phoxini infection in captive Atlantic puffin Fratercula arctica chicks following ingestion of infected European minnows Phoxinus phoxinus.

Conservation of endangered animal species is a major task of zoos. Husbandry and breeding of Atlantic puffins Fratercula arctica in captivity is challenging. In 2019, the entire chick population (n = 4 chicks) in Berne Animal Park's Atlantic puffin colony (Bern, Switzerland) died within 7 d. Due to supply constraints, the chicks had been fed with wild-caught European minnows Phoxinus phoxinus. At necropsy, the main pathological finding in all deceased puffin chicks was a multifocal, moderate to severe subacute heterophilic and granulomatous enteritis with intralesional adult trematodes and eggs. Metacercariae surrounded by few necrotic cells and scattered macrophages were found in the brain and spinal cord of the food fish. Additional microbiological analyses of both the puffin chicks and fish were unremarkable. Diplostomum phoxini DNA could be identified in formalin-fixed paraffin-embedded tissue from the small intestine of all puffin chicks and European minnows following PCR and sequencing of the 18S ribosomal RNA gene and the internal transcribed spacer (ITS1) region. This report illustrates the importance of intensive health checks of food fish for animal species kept in captivity.

Toxoplasma gondii effector TgIST blocks type I interferon signaling to promote infection.

In contrast to the importance of type II interferon-γ (IFN-γ) in control of toxoplasmosis, the role of type I IFN is less clear. We demonstrate here that TgIST, a secreted effector previously implicated in blocking type II IFN-γ signaling, also blocked IFN-ß responses by inhibiting STAT1/STAT2-mediated transcription in infected cells. Consistent with a role for type I IFN in cell intrinsic control, ∆Tgist mutants were more susceptible to growth inhibition by murine and human macrophages activated with IFN-ß. Additionally, type I IFN was important for production of IFN-γ by natural killer (NK) cells and recruitment of inflammatory monocytes at the site of infection. Mice lacking type I IFN receptors (Ifnar1-/-) showed increased mortality following infection with wild-type parasites and decreased virulence of ∆Tgist parasites was restored in Ifnar1-/- mice. The findings highlight the importance of type I IFN in control of toxoplasmosis and illuminate a parasite mechanism to counteract the effects of both type I and II IFN-mediated host defenses.

Vertebral fracture due to Actinobacillus pleuropneumoniae osteomyelitis in a weaner.

BACKGROUND: Osteomyelitis is relatively frequent in young pigs and a few bacterial species have been postulated to be potential causative agents. Although Actinobacillus (A.) pleuropneumoniae has been sporadically described to cause osteomyelitis, typically, actinobacillosis is characterized by respiratory symptoms. Nevertheless, subclinical infections are a challenging problem in pig herds. To the authors' knowledge, this is the first case description that reports clinical, diagnostic imaging, pathological and histopathological findings of vertebral osteomyelitis in a pig and first describes A. pleuropneumoniae as the causative agent identified by advanced molecular methods. CASE PRESENTATION: An eight-week-old female weaner was presented with a non-ambulatory tetraparesis. The neurological signs were consistent with a lesion in the C6-T2 spinal cord segments. Imaging studies revealed a collapse of the seventh cervical vertebral body (C7) with a well demarcated extradural space-occupying mass ventrally within the vertebral canal severely compressing the spinal cord. Post-mortem examination identified an abscess and osteomyelitis of C7 and associated meningitis and neuritis with subsequent pathological fracture of C7 and compression of the spinal cord. In the microbiological analysis, A. pleuropneumoniae was identified using PCR and DNA sequence analysis. CONCLUSIONS: A. pleuropneumoniae can be responsible for chronic vertebral abscess formation with subsequent pathological fracture and spinal cord compression in pigs.

Toxoplasma Effectors Targeting Host Signaling and Transcription.

Early electron microscopy studies revealed the elaborate cellular features that define the unique adaptations of apicomplexan parasites. Among these were bulbous rhoptry (ROP) organelles and small, dense granules (GRAs), both of which are secreted during invasion of host cells. These early morphological studies were followed by the exploration of the cellular contents of these secretory organelles, revealing them to be comprised of highly divergent protein families with few conserved domains or predicted functions. In parallel, studies on host-pathogen interactions identified many host signaling pathways that were mysteriously altered by infection. It was only with the advent of forward and reverse genetic strategies that the connections between individual parasite effectors and the specific host pathways that they targeted finally became clear. The current repertoire of parasite effectors includes ROP kinases and pseudokinases that are secreted during invasion and that block host immune pathways. Similarly, many secretory GRA proteins alter host gene expression by activating host transcription factors, through modification of chromatin, or by inducing small noncoding RNAs. These effectors highlight novel mechanisms by which T. gondii has learned to harness host signaling to favor intracellular survival and will guide future studies designed to uncover the additional complexity of this intricate host-pathogen interaction.

Functional Analysis of the Role of Toxoplasma gondii Nucleoside Triphosphate Hydrolases I and II in Acute Mouse Virulence and Immune Suppression.

Bioluminescent reporter assays have been widely used to study the effect of Toxoplasma gondii on host gene expression. In the present study, we extend these studies by engineering novel reporter cell lines containing a gamma-activated sequence (GAS) element driving firefly luciferase (FLUC). In RAW264.7 macrophages, T. gondii type I strain (GT1) infection blocked interferon gamma (IFN-γ)-induced FLUC activity to a significantly greater extent than infection by type II (ME49) and type III (CTG) strains. Quantitative trait locus (QTL) analysis of progeny from a prior genetic cross identified a genomic region on chromosome XII that correlated with the observed strain-dependent phenotype. This QTL region contains two isoforms of the T. gondii enzyme nucleoside triphosphate hydrolase (NTPase) that were the prime candidates for mediating the observed strain-specific effect. Using reverse genetic analysis we show that deletion of NTPase I from a type I strain (RH) background restored the higher luciferase levels seen in the type II (ME49) strain. Rather than an effect on IFN-γ-dependent transcription, our data suggest that NTPase I was responsible for the strain-dependent difference in FLUC activity due to hydrolysis of ATP. We further show that NTPases I and II were not essential for tachyzoite growth in vitro or virulence in mice. Our study reveals that although T. gondii NTPases are not essential for immune evasion, they can affect ATP-dependent reporters. Importantly, this limitation was overcome using an ATP-independent Gaussia luciferase, which provides a more appropriate reporter for use with T. gondii infection studies.

Parasite distribution and early-stage encephalitis in Sarcocystis calchasi infections in domestic pigeons (Columba livia f. domestica).

Pigeon protozoal encephalitis is a biphasic, neurologic disease of domestic pigeons (Columba livia f. domestica) caused by the apicomplexan parasite Sarcocystis calchasi. Despite severe inflammatory lesions of the brain, associated parasitic stages have only rarely been identified and the cause of the lesions is still unclear. The aim of this study was therefore to characterize the tissue distribution of S. calchasi within pigeons between the two clinical phases and during the occurrence of neurological signs. For this purpose, a semi-quantitative real-time polymerase chain reaction (PCR) was developed. Forty-five domestic pigeons were infected orally (via a cannula into the crop) with 200 S. calchasi sporocysts and euthanized in groups of three pigeons at intervals of 2 to 10 days over a period of 61 days. Tissue samples including brain and skeletal muscle were examined by histology, immunohistochemistry, and PCR. Schizonts were detected in the liver of one pigeon at day 10 post infection. A mild encephalitis was detected at day 20 post infection, around 4 weeks before the onset of neurological signs. At the same time, immature sarcocysts were present in the skeletal muscle. In seven pigeons a few sarcocysts were identified in the brain, but not associated with any lesion. These results suggest that the encephalitis is induced at a very early stage of the S. calchasi lifecycle rather than in the chronic phase of pigeon protozoal encephalitis. Despite the increasing severity of lesions in the central nervous system, the amount of sarcocysts did not increase. This supports the hypothesis of a delayed-type hypersensitivity response as the cause of the encephalitis. The study also demonstrated that S. calchasi DNA is detectable in tissues negative by histological methods, indicating a higher sensitivity of the real-time PCR.

Pathogenesis of histomonosis in experimentally infected specific-pathogen-free (SPF) layer-type chickens and SPF meat-type chickens.

Several studies have shown differences in the course of histomonosis, the infection with the trichomonad parasite Histomonas meleagridis, in different chicken breeds. In the present study, 10 specific-pathogen-free (SPF) layer-type (LT) chickens and twelve SPF meat-type (MT) chickens were infected intracloacally with 200,000 H. meleagridis trophozoites. One and two weeks postinfection (p.i.), three birds of each group were euthanatized. The remaining birds were euthanatized 3 wk p.i. Infected birds showed severe gross lesions typical for histomonosis in ceca at the first and second week p.i., while livers showed necrotic foci at 2 and 3 wk p.i., but only very rarely at 1 wk p.i. Differences between groups in the severity of lesions were statistically insignificant. In histopathology, LT chickens showed a significantly more-severe necrosis and ablation of the cecal epithelium 1 wk p.i. Parasites without inflammation were also found in most investigated spleens and lungs but only in a few kidneys. Investigation of these organs for histomonal DNA by real-time PCR confirmed these results. In addition, the humoral immune response against histomonal actinin 1 and 3 was measured by an ELISA. The humoral immune response against actinin 1 started sooner and was significantly higher in LT chickens than in MT chickens. In conclusion, the results of the present study suggest that the genetic background of the birds influences the reaction to infection with H. meleagridis.

TurboID mapping reveals the exportome of secreted intrinsically disordered proteins in the transforming parasite Theileria annulata.

Theileria annulata is a tick-transmitted apicomplexan parasite that gained the unique ability among parasitic eukaryotes to transform its host cell, inducing a fatal cancer-like disease in cattle. Understanding the mechanistic interplay between the host cell and malignant Theileria species that drives this transformation requires the identification of responsible parasite effector proteins. In this study, we used TurboID-based proximity labeling, which unbiasedly identified secreted parasite proteins within host cell compartments. By fusing TurboID to nuclear export or localization signals, we biotinylated proteins in the vicinity of the ligase enzyme in the nucleus or cytoplasm of infected macrophages, followed by mass spectrometry analysis. Our approach revealed with high confidence nine nuclear and four cytosolic candidate parasite proteins within the host cell compartments, eight of which had no orthologs in non-transforming T. orientalis. Strikingly, all eight of these proteins are predicted to be highly intrinsically disordered proteins. We discovered a novel tandem arrayed protein family, nuclear intrinsically disordered proteins (NIDP) 1-4, featuring diverse functions predicted by conserved protein domains. Particularly, NIDP2 exhibited a biphasic host cell-cycle-dependent localization, interacting with the EB1/CD2AP/CLASP1 parasite membrane complex at the schizont surface and the tumor suppressor stromal antigen 2 (STAG2), a cohesion complex subunit, in the host nucleus. In addition to STAG2, numerous NIDP2-associated host nuclear proteins implicated in various cancers were identified, shedding light on the potential role of the T. annulata exported protein family NIDP in host cell transformation and cancer-related pathways.IMPORTANCETurboID proximity labeling was used to identify secreted proteins of Theileria annulata, an apicomplexan parasite responsible for a fatal, proliferative disorder in cattle that represents a significant socio-economic burden in North Africa, central Asia, and India. Our investigation has provided important insights into the unique host-parasite interaction, revealing secreted parasite proteins characterized by intrinsically disordered protein structures. Remarkably, these proteins are conspicuously absent in non-transforming Theileria species, strongly suggesting their central role in the transformative processes within host cells. Our study identified a novel tandem arrayed protein family, with nuclear intrinsically disordered protein 2 emerging as a central player interacting with established tumor genes. Significantly, this work represents the first unbiased screening for exported proteins in Theileria and contributes essential insights into the molecular intricacies behind the malignant transformation of immune cells.

A novel high-resolution melt PCR assay discriminates Anaplasma phagocytophilum and "Candidatus Neoehrlichia mikurensis".

"Candidatus Neoehrlichia mikurensis" (Anaplasmataceae) is an emerging pathogen transmitted by Ixodes ticks. Conventional PCR and the newly developed high-resolution melt PCR were used to detect and discriminate "Candidatus Neoehrlichia mikurensis" and Anaplasma phagocytophilum. Both bacterial species were frequently found in Ixodes ricinus and Ixodes hexagonus but virtually absent from Dermacentor reticulatus. In rodents, "Candidatus N. mikurensis" was significantly more prevalent than A. phagocytophilum, whereas in cats, only A. phagocytophilum was found.

Modulation of the host Th1 immune response in pigeon protozoal encephalitis caused by Sarcocystis calchasi.

Pigeon protozoal encephalitis (PPE) is an emerging central-nervous disease of domestic pigeons (Columba livia f. domestica) reported in Germany and the United States. It is caused by the apicomplexan parasite Sarcocystis calchasi which is transmitted by Accipter hawks. In contrast to other members of the Apicomplexa such as Toxoplasma and Plasmodium, the knowledge about the pathophysiology and host manipulation of Sarcocystis is scarce and almost nothing is known about PPE. Here we show by mRNA expression profiling a significant down-modulation of the interleukin (IL)-12/IL-18/interferon (IFN)-γ axis in the brains of experimentally infected pigeons during the schizogonic phase of disease. Concomitantly, no cellular immune response was observed in histopathology while immunohistochemistry and nested PCR detected S. calchasi. In contrast, in the late central-nervous phase, IFN-γ and tumor necrosis factor (TNF) α-related cytokines were significantly up-modulated, which correlated with a prominent MHC-II protein expression in areas of mononuclear cell infiltration and necrosis. The mononuclear cell fraction was mainly composed of T-lymphocytes, fewer macrophages and B-lymphocytes. Surprisingly, the severity and composition of the immune cell response appears unrelated to the infectious dose, although the severity and onset of the central nervous signs clearly was dose-dependent. We identified no or only very few tissue cysts by immunohistochemistry in pigeons with severe encephalitis of which one pigeon repeatedly remained negative by PCR despite severe lesions. Taken together, these observations may suggest an immune evasion strategy of S. calchasi during the early phase and a delayed-type hypersensitivity reaction as cause of the extensive cerebral lesions during the late neurological phase of disease.

Adenoviral gizzard erosion in broiler chickens in Germany.

Avian adenovirus infections cause important disease complexes in chickens, but many of the viruses also infect chickens without resulting in overt disease. Previously several outbreaks of gizzard erosions caused by a fowl adenovirus A serotype-1 (FAdV-1) were reported from Japan. Here we report an outbreak of gizzard erosions in 12 broiler flocks in Germany in 2011. Chickens had a reduced daily weight gain and a higher total mortality rate of up to 8%. The birds showed a severe detachment of the koilin layer and ulcerative to necrotizing lesions of the underlying mucosa. Histopathologically, necrotizing ventriculitis with basophilic, intranuclear inclusion bodies in epithelial cells was diagnosed. Immunohistochemistry, egg culture, and electron microscopic examination revealed adenovirus-like particles in the samples. No concurrent infectious agent could be identified. The virus was genotyped as FAdV-1 by PCR and subsequent sequencing. Phylogenetic analysis of the hexon loop L1 gene yielded 100% sequence identity to the chicken embryo lethal orphan strain. These findings suggest that outbreaks of adenoviral gizzard erosion can lead to significant economic losses in Germany and may be caused by an unusual virulent FAdV-1 strain.

Murine infection models for Aspergillus terreus pulmonary aspergillosis reveal long-term persistence of conidia and liver degeneration.

Aspergillus terreus is emerging as a causative agent of life-threatening invasive aspergillosis. Prognosis for affected patients is often worse than for A. fumigatus infections. To study A. terreus-mediated disease, we developed 3 infection models. In embryonated hen's eggs and leucopenic mice, the outcome of invasive aspergillosis was similar to that described for A. fumigatus. However, 10(2)- and 10(3)-fold higher conidia concentrations were required for 100% lethality. In corticosteroid-treated mice, only 50% mortality was observed, although bioluminescence imaging revealed transient disease in all infected animals. In surviving animals, we observed persistence of ungerminated but viable conidia. Cytokine levels in these mice were comparable to uninfected controls. In contrast to A. fumigatus infections, all mice infected with A. terreus developed fatty liver degeneration, suggesting the production of toxic secondary metabolites. Thus, at least in mice, persistence and subclinical liver damage are unique features of A. terreus infections.

A parasite DNA binding protein with potential to influence disease susceptibility acts as an analogue of mammalian HMGA transcription factors.

Intracellular pathogens construct their environmental niche, and influence disease susceptibility, by deploying factors that manipulate infected host cell gene expression. Theileria annulata is an important tick-borne parasite of cattle that causes tropical theileriosis. Excellent candidates for modulating host cell gene expression are DNA binding proteins bearing AT-hook motifs encoded within the TashAT gene cluster of the parasite genome. In this study, TashAT2 was transfected into bovine BoMac cells to generate three expressing and three non-expressing (opposite orientation) cell lines. RNA-Seq was conducted and differentially expressed (DE) genes identified. The resulting dataset was compared with genes differentially expressed between infected cells and non-infected cells, and DE genes between infected cell lines from susceptible Holstein vs tolerant Sahiwal cattle. Over 800 bovine genes displayed differential expression associated with TashAT2, 209 of which were also modulated by parasite infection. Network analysis showed enrichment of DE genes in pathways associated with cellular adhesion, oncogenesis and developmental regulation by mammalian AT-hook bearing high mobility group A (HMGA) proteins. Overlap of TashAT2 DE genes with Sahiwal vs Holstein DE genes revealed that a significant number of shared genes were associated with disease susceptibility. Altered protein levels encoded by one of these genes (GULP1) was strongly linked to expression of TashAT2 in BoMac cells and was demonstrated to be higher in infected Holstein leucocytes compared to Sahiwal. We conclude that TashAT2 operates as an HMGA analogue to differentially mould the epigenome of the infected cell and influence disease susceptibility.

Direct analysis and identification of pathogenic Lichtheimia species by matrix-assisted laser desorption ionization-time of flight analyzer-mediated mass spectrometry.

Zygomycetes of the order Mucorales can cause life-threatening infections in humans. These mucormycoses are emerging and associated with a rapid tissue destruction and high mortality. The resistance of Mucorales to antimycotic substances varies between and within clinically important genera such as Mucor, Rhizopus, and Lichtheimia. Thus, an accurate diagnosis before onset of antimycotic therapy is recommended. Matrix-assisted laser desorption ionization (MALDI)-time of flight (TOF) mass spectrometry (MS) is a potentially powerful tool to rapidly identify infectious agents on the species level. We investigated the potential of MALDI-TOF MS to differentiate Lichtheimia species, one of the most important agents of mucormycoses. Using the Bruker Daltonics FlexAnalysis (version 3.0) software package, a spectral database library with m/z ratios of 2,000 to 20,000 Da was created for 19 type and reference strains of clinically relevant Zygomycetes of the order Mucorales (12 species in 7 genera). The database was tested for accuracy by use of 34 clinical and environmental isolates of Lichtheimia comprising a total of five species. Our data demonstrate that MALDI-TOF MS can be used to clearly discriminate Lichtheimia species from other pathogenic species of the Mucorales. Furthermore, the method is suitable to discriminate species within the genus. The reliability and robustness of the MALDI-TOF-based identification are evidenced by high score values (above 2.3) for the designation to a certain species and by moderate score values (below 2.0) for the discrimination between clinically relevant (Lichtheimia corymbifera, L. ramosa, and L. ornata) and irrelevant (L. hyalospora and L. sphaerocystis) species. In total, all 34 strains were unequivocally identified by MALDI-TOF MS with score values of >1.8 down to the generic level, 32 out of 34 of the Lichtheimia isolates (except CNM-CM 5399 and FSU 10566) were identified accurately with score values of >2 (probable species identification), and 25 of 34 isolates were identified to the species level with score values of >2.3 (highly probable species identification). The MALDI-TOF MS-based method reported here was found to be reproducible and accurate, with low consumable costs and minimal preparation time.

Infections with Sarcocystis wenzeli are prevalent in the chickens of Yunnan Province, China, but not in the flocks of domesticated pigeons or ducks.

The distribution and prevalence of infections with species of Sarcocystis in domestic fowl in Asia are poorly known. Here, ducks, pigeons, and chickens from Yunnan Province, China were examined for evidence of parasitic infection with Sarcocystis spp. One hundred and ninety one chickens, 514 ducks, and nine pigeons were investigated. Whereas the ducks and pigeons lacked tissue cysts in their muscle, brain or peripheral nervous system, cysts of Sarcocystis wenzeli were identified in 17 of 191 chickens (8.9%). Morphologically, the cysts were thread-like, ranging in size from 334-3169 × 41-117 µm (mean 1093 × 65 µm). Cysts were septate with dense, short finger-like protrusions which appeared radially striated. The cyst wall was 1.4-3.5 µm (mean 2.4 µm) thick. The bradyzoites were lancet shaped and measured 12.2-17.7 × 1.8-2.9 µm (mean 14.6 × 2.5 µm). Ultrastucturally, the primary sarcocyst wall had stubby villar protrusions, corresponding to the 'type 9' class previously designated. The protrusions measured 0.87-1.89 × 0.47-0.91 µm (mean 1.27 × 0.59 µm; n = 57). These findings confirm previous work from the vicinity of Kunming concerning the occurrence of S. wenzeli in chickens, and its use of both cats and dogs as definitive hosts, but indicate that corresponding infections may not occur in the regional domestic flocks of other types of fowl.

Single- and duplex TaqMan-quantitative PCR for determining the copy numbers of integrated selection markers during site-specific mutagenesis in Toxoplasma gondii by CRISPR-Cas9.

Herein, we developed a single and a duplex TaqMan quantitative PCR (qPCR) for absolute quantification of copy numbers of integrated dihydrofolate reductase-thymidylate synthase (mdhfr-ts) drug selectable marker for pyrimethamine resistance in Toxoplasma gondii knockouts (KOs). The single TaqMan qPCR amplifies a 174 bp DNA fragment of the inserted mdhfr-ts and of the wild-type (WT) dhfr-ts (wtdhfr-ts) which is present as single copy gene in Toxoplasma and encodes a sensitive enzyme to pyrimethamine. Thus, the copy number of the dhfr-ts fragment in a given DNA quantity from KO parasites with a single site-specific integration should be twice the number of dhfr-ts copies recorded in the same DNA quantity from WT parasites. The duplex TaqMan qPCR allows simultaneous amplification of the 174 bp dhfr-ts fragment and the T. gondii 529-bp repeat element. Accordingly, for a WT DNA sample, the determined number of tachyzoites given by dhfr-ts amplification is equal to the number of tachyzoites determined by amplification of the Toxoplasma 529-bp, resulting thus in a ratio of 1. However, for a KO clone having a single site-specific integration of mdhfr-ts, the calculated ratio is 2. We then applied both approaches to test T. gondii RH mutants in which the major surface antigen (SAG1) was disrupted through insertion of mdhfr-ts using CRISPR-Cas9. Results from both assays were in correlation showing a high accuracy in detecting KOs with multiple integrated mdhfr-ts. Southern blot analyses using BsaBI and DraIII confirmed qPCRs results. Both TaqMan qPCRs are needed for reliable diagnostic of T. gondii KOs following CRISPR-Cas9-mediated mutagenesis, particularly with respect to off-target effects resulting from multiple insertions of mdhfr-ts. The principle of the duplex TaqMan qPCR is applicable for other selectable markers in Toxoplasma. TaqMan qPCR tools may contribute to more frequent use of WT Toxoplasma strains during functional genomics.

Fatal spirorchiidosis in European pond turtles (Emys orbicularis) in Switzerland.

Infections with intravascular digenean trematodes of the Spirorchiidae family (spirorchiidoses) are of great conservation concern both in marine and freshwater turtles due to their pathogenic potential. Between 2014 and 2021, Spirorchis sp. infections associated with granulomatous inflammation and sudden death were detected in European pond turtles (Emys orbicularis) from three conservation breeding facilities in Switzerland. Blood fluke eggs associated with lesions were found in the intestine, spleen, testis, skeletal musculature, heart, kidneys, stomach, pancreas, liver, lung, and meninges from nine pond turtles submitted for necropsy and in the intestinal content from five of these animals. Two novel polymerase chain reactions (PCRs) targeting the 28S ribosomal RNA gene and the ITS2 region and subsequent sequencing revealed 100% nucleotide identity with a Spirorchis sp. previously isolated from an Escambia map turtle (Graptemys ernsti) in the USA. Our findings suggest a spill-over event secondary to direct or indirect contact with invasive North American turtle species in Switzerland. We describe the clinical, haematological, ultrasonographical, endoscopical, parasitological, pathological, and molecular findings associated with spirorchiid blood fluke infections of the Spirorchis genus in E. orbicularis, as well as the biosecurity measures that were developed to prevent the spread of this parasite among breeding and highly endangered free-ranging E. orbicularis populations in Switzerland.

Fungal species identification from avian lung specimens by single hypha laser microdissection and PCR product sequencing.

Accurate species diagnosis in cases of fungal pneumonia may be hampered by environmental contamination and colonization resulting in false-positive results. Our novel approach for fungal species diagnostics combines fluorescent staining of mounted cryosections with the optical brightener Blankophor, laser capture microdissection and PCR amplification with subsequent sequencing of the first internal transcribed spacer region (ITS-1). Using clinical specimens from infected birds, we show that the procedure is suitable for species identification from single hyphae of intralesional filamentous fungi. Our data also suggest that multiple Aspergillus fumigatus strain infections may occur frequently in pulmonary aspergillosis of birds.