In pigs, three different trichomonad species (Tritrichomonas foetus, Tetratrichomonas buttreyi and Tritrichomonas rotunda) have been described as commensals in the large intestine. The aim of this study was to gain further knowledge on the prevalence and pathogenicity of trichomonads in pigs by using a morphology-based approach. Chromogenic in situ hybridization (ISH) is a technique which allows direct localization of the protozoa in the intestinal tissue and correlation of the infection with pathologic changes. In the present study paraffin-wax embedded colon and ileum samples of 192 pigs were analyzed with this method. Using a probe specific for all known members of the order Trichomonadida (OT) 100 of the 192 pigs were tested positive. Thereof, about 10% showed moderate to high-grade parasitic load with trichomonads invading the lamina propria. Partial 18S ribosomal RNA gene sequencing of six of those animals showed a 100% sequence identity with T. foetus sequences. The majority of these animals were also tested positive for other enteropathogenic agents, such as Brachyspira sp., Lawsonia intracellularis, Escherichia coli, and porcine circovirus type 2. All OT-positive samples were further examined with another probe complementary to all known Tritrichomonas species sequences including T. foetus, T. augusta, T. mobilensis and T. nonconforma resulting in only 48 positives. These results suggest that T. foetus may not only be considered as an intestinal commensal but rather a facultative pathogen of pigs with a tendency for tissue invasion in the presence of other agents. Furthermore, the existence of other - yet to be identified - trichomonad species in the colon of pigs was shown.

Haplotypes with reduced or missing homozygosity may harbor deleterious alleles that compromise juvenile survival. A scan for homozygous haplotype deficiency revealed a short segment on bovine chromosome 19 (Braunvieh haplotype 2, BH2) that was associated with high juvenile mortality in Braunvieh cattle. However, the molecular genetic underpinnings and the pathophysiology of BH2 remain to be elucidated.

Three different parasites of the phylum Parabasala (Tritrichomonas foetus, Trichomitus rotunda and Tetratrichomonas buttreyi) have been described in pigs. In a previous study (Mostegl et al., 2011) approximately 47% of 91 paraffin wax-embedded intestinal samples of pigs which were Trichomonas-positive by in situ hybridization using a probe with a broad reactivity spectrum contained other species than T. foetus. Out of these, intestinal trichomonads from three pigs (pigs 1-3) were further analyzed by gene sequencing of a part of the 18S ribosomal RNA (rRNA) gene using primer walking. Subsequently, the partial sequences achieved by the different primer pairs were combined to a sequence of about 1000 bp for each trichomonad. In all three pigs unique sequences were acquired which showed only moderate similarities to sequences available in the GenBank. Alignments and the BLAST analysis showed a high degree of homology between sequences of trichomonads from pig 1 and pig 3 with only 1% difference. These sequences were found to be 92% similar to Hypotrichomonas acosta, a trichomonad isolated from squamate reptiles. The trichomonad sequence detected in the intestine of pig 2 showed about 10% nucleotide differences compared to pigs 1 and 3. This sequence was 97% similar to two Trichomitus batrachorum (a frog symbiont) sequences. A phylogenetic analysis using the neighbor-joining and maximum likelihood methods supported the data of the BLAST analysis. These results suggest the presence of at least two as yet undescribed trichomonad species in the intestinal contents of pigs.

In this retrospective study 102 cats were analyzed for the presence of trichomonads in intestinal tissue sections using chromogenic in situ hybridization (CISH). Two intestinal trichomonad species are described in cats: Pentatrichomonas hominis and Tritrichomonas foetus. While P. hominis is considered a mere commensal, T. foetus has been found to be the causative agent of feline large-bowel diarrhea. For the detection of both agents within intestinal tissue CISH assays using three different probes were performed. In the first CISH run a probe specific for all relevant members of the order Trichomonadida (OT probe) was used. In a second CISH run all positive samples were further examined on three consecutive tissue sections using the OT probe, a probe specific for the family of Tritrichomonadidae (Tritri probe) and a newly designed probe specifically detecting P. hominis (Penta hom probe). In total, four of the 102 cats were found to be positive with the OT probe. Thereof, one cat gave a positive reaction with the P. hominis probe and three cats were positive with the T. foetus probe. All Trichomonas-positive cats were pure-bred and between 8 and 32 weeks of age. In one cat positive for T. foetus large amounts of parasites were found in the gut lumen and invading the intestinal mucosa. The species of the detected trichomonads were confirmed by polymerase chain reaction and nucleotide sequencing of a part of the 18S ribosomal RNA gene. In this study, the usefulness of CISH to detect intestinal trichomonads within feline tissue samples was shown. Additionally, the specific detection of P. hominis using CISH was established. Generally, it was shown that CISH is well suited for detection and differentiation of trichomonosis in retrospective studies using tissue samples.

In the present study, we report the occurrence of several outbreaks of hepatitis in flocks of young pheasants in France, between 2017 and 2021. The disease was characterized by prostration, apathy and a median cumulative mortality of 12%, with the birds presenting multifocal to coalescing necrotizing hepatitis on necropsy. Severe extensive areas of degeneration and necrosis were observed in the liver, with degenerative hepatocytes presenting large amphophilic to acidophilic intranuclear inclusion bodies. Transmission electron microscopy examination of liver samples showed the presence of parvovirus-like virions of 21-24 nm, a finding already reported decades ago. Further investigations by Next Generation Sequencing and PCR revealed the complete genome of a novel species of parvovirus, here designated Phasianus chaphamaparvovirus 1 (PhChPV-1), that belongs to the new genus Chaphamaparvovirus in the Hamaparvovirinae subfamily. In situ hybridization and real-time PCR confirmed the etiology of the outbreaks, demonstrating the viral genome in the lesions. The findings establish the etiology of a pathology first described in pheasants 50 years ago and pave the way for a targeted protection strategy.

Mitochondria are fundamental for life and require balanced ion exchange to maintain proper functioning. The mitochondrial cation exchanger LETM1 sparks interest because of its pathophysiological role in seizures in the Wolf Hirschhorn Syndrome (WHS). Despite observation of sleep disorganization in epileptic WHS patients, and growing studies linking mitochondria and epilepsy to circadian rhythms, LETM1 has not been studied from the chronobiological perspective. Here we established a viable letm1 knock-out, using the diurnal vertebrate Danio rerio to study the metabolic and chronobiological consequences of letm1 deficiency. We report diurnal rhythms of Letm1 protein levels in wild-type fish. We show that mitochondrial nucleotide metabolism is deregulated in letm1-/- mutant fish, the rate-limiting enzyme of NAD+ production is up-regulated, while NAD+ and NADH pools are reduced. These changes were associated with increased expression amplitude of circadian core clock genes in letm1-/- compared with wild-type under light/dark conditions, suggesting decreased NAD(H) levels as a possible mechanism for circadian system perturbation in Letm1 deficiency. Replenishing NAD pool may ameliorate WHS-associated sleep and neurological disorders.

Shiga toxin (Stx) producing Escherichia coli (E. coli) (STEC) is the most frequent cause of diarrhoea-positive haemolytic uraemic syndrome (D + HUS) in humans. In 2011, a huge outbreak with an STEC O104:H4 strain in Germany highlighted the limited possibilities for causative treatment of this syndrome. The responsible STEC strain was found to combine Stx production with adherence mechanisms normally found in enteroaggregative E. coli (EAEC). Pathotypes of E. coli evolve and can exhibit different adhesion mechanisms. It has been shown previously that neonatal gnotobiotic piglets are susceptible for infection with STEC, such as STEC O157:H7 as well as for EAEC, which are considered to be the phylogenetic origin of E. coli O104:H4. This study was designed to characterise the host response to infection with the STEC O104:H4 outbreak strain in comparison to an STEC O157:H7 isolate by evaluating clinical parameters (scoring) and markers of organ dysfunction (biochemistry), as well as immunological (flow cytometry, assessment of cytokines/chemokines and acute phase proteins) and histological alterations (light- and electron microscopy) in a gnotobiotic piglet model of haemolytic uraemic syndrome.

In 2013, several Austrian piglet-producing farms recorded outbreaks of action-related repetitive myoclonia in newborn piglets ("shaking piglets"). Malnutrition was seen in numerous piglets as a complication of this tremor syndrome. Overall piglet mortality was increased and the number of weaned piglets per sow decreased by more than 10% due to this outbreak. Histological examination of the CNS of affected piglets revealed moderate hypomyelination of the white substance in cerebellum and spinal cord. We detected a recently discovered pestivirus, termed atypical porcine pestivirus (APPV) in all these cases by RT-PCR. A genomic sequence and seven partial sequences were determined and revealed a 90% identity to the US APPV sequences and 92% identity to German sequences. In confirmation with previous reports, APPV genomes were identified in different body fluids and tissues including the CNS of diseased piglets. APPV could be isolated from a "shaking piglet", which was incapable of consuming colostrum, and passaged on different porcine cells at very low titers. To assess the antibody response a blocking ELISA was developed targeting NS3. APPV specific antibodies were identified in sows and in PCR positive piglets affected by congenital tremor (CT). APPV genomes were detected continuously in piglets that gradually recovered from CT, while the antibody titers decreased over a 12-week interval, pointing towards maternally transmitted antibodies. High viral loads were detectable by qRT-PCR in saliva and semen of infected young adults indicating a persistent infection.

The interaction between bacteriophages, bacteria and the human host as a tripartite system has recently captured attention. The taxonomic diversity of bacteriophages, as a natural parasite of bacteria, still remains obscure in human body biomes, representing a so-called "viral dark matter." Here, we isolated and characterized coliphages from blood, urine and tracheal aspirates samples collected at a tertiary care hospital in Austria. Phages were more often isolated from blood, followed by urine and tracheal aspirates. Phylogenetic analysis and genome comparisons allowed the identification of phages belonging to the Tunavirinae subfamily, and to the Peduovirus and Tequintavirus genera. Tunavirinae phages cluster together and are found in samples from 14 patients, suggesting their prevalence across a variety of human samples. When compared with other phage genomes, the highest similarity level was at 87.69% average nucleotide identity (ANI), which suggests that these are in fact a newly isolated phage species. Tequintavirus phages share a 95.90% with phage 3_29, challenging the ANI threshold currently accepted to differentiate phage species. The isolated phages appear to be virulent, with the exception of the Peduovirus members, which are integrative and seem to reside as prophages in bacterial genomes.

In captive penguins, avian malaria due to Plasmodium parasites is a well-recognized disease problem as these protozoa may cause severe losses among valuable collections of zoo birds. In blood films from naturally infected birds, identification and differentiation of malaria parasites based on morphological criteria are difficult because parasitaemia is frequently light and blood stages, which are necessary for identification of parasites, are often absent. Post-mortem diagnosis by histological examination of tissue samples is sometimes inconclusive due to the difficulties in differentiating protozoal tissue stages from fragmented nuclei in necrotic tissue. The diagnosis of avian malaria would be facilitated by a technique with the ability to specifically identify developmental stages of Plasmodium in tissue samples. Thus, a chromogenic in-situ hybridization (ISH) procedure with a digoxigenin-labelled probe, targeting a fragment of the 18S rRNA, was developed for the detection of Plasmodium parasites in paraffin wax-embedded tissues. This method was validated in comparison with traditional techniques (histology, polymerase chain reaction), on various tissues from 48 captive penguins that died at the zoological garden Schönbrunn, Vienna, Austria. Meronts of Plasmodium gave clear signals and were easily identified using ISH. Potential cross-reactivity of the probe was ruled out by the negative outcome of the ISH against a number of protozoa and fungi. Thus, ISH proved to be a powerful, specific and sensitive tool for unambiguous detection of Plasmodium parasites in paraffin wax-embedded tissue samples.

Intensive care units (ICUs) are critical locations for the transmission of pathogenic and opportunistic microorganisms. Bacteria may develop a synergistic relationship with bacteriophages and more effectively resist various stresses, enabling them to persist despite disinfection and antimicrobial treatment. We collected 77 environmental samples from the surroundings of 12 patients with infection/colonizations by Escherichia coli, Staphylococcus aureus or Klebsiella spp in an ICU in Austria. Surface swabs were tested for lytic phages and bacterial isolates for mitomycin C-inducible prophages. No lytic bacteriophages were detected, but S. aureus was isolated from the surroundings of all patients. About 85% of the colonies isolated from surface samples were resistant to antimicrobials, with 94% of them multidrug resistant. Two inducible temperate bacteriophages-myovirus vB_EcoM_P5 and siphovirus vB_SauS_P9-were recovered from two clinical isolates. Staphylococci phage vB_SauS_P9 lysed S. aureus isolates from the surface swabs collected from the surroundings of three patients. No transductants were obtained on propagation in phage-sensitive antimicrobial-resistant isolates. The two phages were sensitive to 0.25% (v/v) of the disinfectant TPH Protect, which eliminated viable phages after 15 min. Coliphage vB_EcoM_P5 was inactivated at 70 °C and staphylococci phage vB_SauS_P9 at 60 °C after 60 min.

Species of avian malaria parasites (Plasmodium) are widespread, but their virulence has been insufficiently investigated, particularly in wild birds. During avian malaria, several cycles of tissue merogony occur, and many Plasmodium spp. produce secondary exoerythrocytic meronts (phanerozoites), which are induced by merozoites developing in erythrocytic meronts. Phanerozoites markedly damage organs, but remain insufficiently investigated in the majority of described Plasmodium spp. Avian malaria parasite Plasmodium (Giovannolaia) homocircumflexum (lineage pCOLL4) is virulent and produces phanerozoites in domestic canaries Serinus canaria, but its pathogenicity in wild birds remains unknown. The aim of this study was to investigate the pathology caused by this infection in species of common European birds.

Mitochondrial Ca2+ ions are crucial regulators of bioenergetics and cell death pathways. Mitochondrial Ca2+ content and cytosolic Ca2+ homeostasis strictly depend on Ca2+ transporters. In recent decades, the major players responsible for mitochondrial Ca2+ uptake and release have been identified, except the mitochondrial Ca2+ /H+ exchanger (CHE). Originally identified as the mitochondrial K+ /H+ exchanger, LETM1 was also considered as a candidate for the mitochondrial CHE. Defining the mitochondrial interactome of LETM1, we identify TMBIM5/MICS1, the only mitochondrial member of the TMBIM family, and validate the physical interaction of TMBIM5 and LETM1. Cell-based and cell-free biochemical assays demonstrate the absence or greatly reduced Na+ -independent mitochondrial Ca2+ release in TMBIM5 knockout or pH-sensing site mutants, respectively, and pH-dependent Ca2+ transport by recombinant TMBIM5. Taken together, we demonstrate that TMBIM5, but not LETM1, is the long-sought mitochondrial CHE, involved in setting and regulating the mitochondrial proton gradient. This finding provides the final piece of the puzzle of mitochondrial Ca2+ transporters and opens the door to exploring its importance in health and disease, and to developing drugs modulating Ca2+ exchange.