Epidemic of cutaneous fowlpox in a naïve population of chickens and turkeys in Austria: Detailed phylogenetic analysis indicates co-evolution of fowlpox virus with reticuloendotheliosis virus.

Cutaneous fowlpox is a disease of chickens and turkeys caused by the fowlpox virus (FWPV), characterized by the development of proliferative lesions and scabs on unfeathered areas. FWPVs regularly carry an integrated, active copy of the reticuloendotheliosis virus (REV), and it has been hypothesized that such FWPVs are more problematic in the field. Extensive outbreaks are usually observed in tropical and sub-tropical climates, where biting insects are more difficult to control. Here, we report an epidemic of 65 cutaneous fowlpox cases in Austria in layer chickens (91% of the cases) and broiler breeders and turkeys, all of them unvaccinated against the disease, from October 2018 to February 2020. The field data revealed appearance in flocks of different sizes ranging from less than 5000 birds up to more than 20,000 animals, with the majority raised indoors in a barn system. The clinical presentation was characterized by typical epithelial lesions on the head of the affected birds, with an average decrease of 6% in egg production and an average weekly mortality of 1.2% being observed in the flocks. A real-time multiplex polymerase chain reaction (PCR) confirmed the presence of FWPV-REV DNA, not only in the lesions but also in the environmental dust from the poultry houses. The integration of the REV provirus into the FWPV genome was confirmed by PCR, and revealed different FWPV genome populations carrying either the REV long terminal repeats (LTRs) or the full-length REV genome, reiterating the instability of the inserted REV. Two selected samples were fully sequenced by next generation sequencing (NGS), and the whole genome phylogenetic analysis revealed a regional clustering of the FWPV genomes. The extensive nature of these outbreaks in host populations naïve for the virus is a remarkable feature of the present report, highlighting new challenges associated with FWPV infections that need to be considered.

Microsatellite Analysis of Geographically Close Isolates of Cystoisospora suis.

Microsatellites are short repetitive DNA sequences of 2-6 repeats interspersed in the genome that display a rapid mutation rate and consequently show high variation between individuals or populations. They have been used to characterize population diversity and structure and the level of variation between different isolates of a number of different organisms, including apicomplexan protozoa. Currently nothing is known about the genetic variability and population structure of Cystoisospora suis (Apicomplexa: Coccidia), the causative agent of piglet coccidiosis, and we made use of the recently available genome of C. suis (strain Wien-I) to amplify microsatellite regions (ca. 300-550 bp) and evaluate the applicability of fluorescence-labeled primers to investigate amplicon length variation at high resolution using capillary electrophoresis (CE). Two phenotypically characterized isolates (Wien-I, toltrazuril susceptible; Holl 1 toltrazuril resistant) and six field isolates from Europe were compared by conventional PCR followed by agar-gel electrophoresis, Sanger sequencing, and CE (fluorescence labeling and fragment length analysis) to evaluate the applicability of the method. Four primer pairs could be identified that amplified bands of the expected size and were labeled for CE analysis. High resolution CE for size determination of PCR amplicons proved to be a reliable and simple method. It revealed high diversity of the analyzed strains, with marked differences even between two strains from neighboring swine farms. In follow-up studies, adaptation of the PCR assay to multiplexing and amplification of small DNA quantities will provide a cost-effective tool to analyse field strains to reveal geographic diversity that could be mapped to phenotypic traits.

Comparison of an injectable toltrazuril-gleptoferron (Forceris®) and an oral toltrazuril (Baycox®) + injectable iron dextran for the control of experimentally induced piglet cystoisosporosis.

BACKGROUND: Cystoisospora suis causes diarrhoeal disease and reduced weight gain in suckling piglets, and a toltrazuril-based oral suspension is available for treatment. Recently a combinatorial product with toltrazuril plus iron has been developed for parenteral application. In this study we compared the efficacy of the injectable product with the oral suspension against experimentally induced piglet cystoisosporosis. METHODS: In a randomised controlled study, three groups of piglets (n = 10-13) were treated either with a fixed dose of 45 mg toltrazuril + 200 mg gleptoferron i.m. per piglet (Forceris®) on the second day of life (study day 2; SD 2) or with 20 mg toltrazuril/kg body weight as an oral suspension (Baycox® 5%) on SD 4 or left untreated (Control group). The Baycox® and the Control group received 200 mg of iron dextran/piglet on SD 2. All piglets were infected with 1000 sporulated C. suis oocysts on SD 3. Faecal samples were taken daily from SD 7 to SD 20 to determine faecal consistency, oocyst shedding and other diarrhoeal pathogens. Body weight was recorded on SD 1 and then weekly until SD 29. Animals were observed daily for general health and after treatment for possible adverse events. RESULTS: In the Control group all animals shed oocysts for 3.1 days on average and all animals showed diarrhoea for an average of five days. Excretion peaked on SD 9 (max. 48,618 oocysts per gram of faeces). Treatment with Forceris® completely suppressed oocyst excretion. In the Baycox® group, low levels of excretion could be detected. Diarrhoea was reduced to single piglets in the treated groups. Body weight development was reduced in the Control group compared to the treated groups. Enteropathogenic bacteria (Escherichia coli, Clostridium perfringens) could be detected. All parameters related to oocyst excretion, faecal consistency and weight gain were significantly improved in the treated groups compared to the Control group without significant differences between the treated groups. Both products were safe to use. CONCLUSIONS: Treatment with both the injectable (Forceris®) and the oral (Baycox®) formulation of toltrazuril in the prepatent period were safe and highly effective against experimental infection with C. suis in newborn piglets.

Cloning, expression and molecular characterization of a Cystoisospora suis specific uncharacterized merozoite protein.

BACKGROUND: The genome of the apicomplexan parasite Cystoisospora suis (syn. Isospora suis) has recently been sequenced and annotated, opening the possibility for the identification of novel therapeutic targets against cystoisosporosis. It was previously proposed that a 42 kDa uncharacterized merozoite protein, encoded by gene CSUI_005805, might be a relevant vaccine candidate due to its high immunogenic score, high expression level and species-specificity as determined in silico. METHODS: The 1170 bp coding sequence of the CSUI_005805 gene was PCR amplified and cloned into the bacterial expression vector pQE-31. The specificity of the expressed recombinant protein was evaluated in an immunoblot, and relative levels of expression in different developmental stages and subcellular localization were determined by quantitative real-time PCR and indirect immunofluorescence assay, respectively. RESULTS: The CSUI_005805 gene encoded for a 389 amino acid protein containing a histidine-rich region. Quantitative RT-PCR showed that CSUI_005805 was differentially expressed during the early development of C. suis in vitro, with higher transcript levels in merozoites compared to sporozoites. The recombinant protein was specifically recognized by sera from chicken immunized with recombinant CSUI_005805 protein and sera from piglets experimentally infected with C. suis, all of which suggested that despite prokaryotic expression, the recombinant CSUI_005805 protein maintained antigenic determinants and could elicit an immune response in the host. Immunofluorescence labelling and confocal microscopy revealed localization primarily at the surface of the parasite. CONCLUSIONS: The results suggest that CSUI_005805 is highly expressed in merozoites and might thus be critical for their survival and establishment inside host cells. Owing to its specificity, localization and expression pattern, CSUI_005805 could be exploited as an attractive candidate for alternative control strategies against C. suis such as vaccines.

The genome of the protozoan parasite Cystoisospora suis and a reverse vaccinology approach to identify vaccine candidates.

Vaccine development targeting protozoan parasites remains challenging, partly due to the complex interactions between these eukaryotes and the host immune system. Reverse vaccinology is a promising approach for direct screening of genome sequence assemblies for new vaccine candidate proteins. Here, we applied this paradigm to Cystoisospora suis, an apicomplexan parasite that causes enteritis and diarrhea in suckling piglets and economic losses in pig production worldwide. Using Next Generation Sequencing we produced an ∼84Mb sequence assembly for the C. suis genome, making it the first available reference for the genus Cystoisospora. Then, we derived a manually curated annotation of more than 11,000 protein-coding genes and applied the tool Vacceed to identify 1,168 vaccine candidates by screening the predicted C. suis proteome. To refine the set of candidates, we looked at proteins that are highly expressed in merozoites and specific to apicomplexans. The stringent set of candidates included 220 proteins, among which were 152 proteins with unknown function, 17 surface antigens of the SAG and SRS gene families, 12 proteins of the apicomplexan-specific secretory organelles including AMA1, MIC6, MIC13, ROP6, ROP12, ROP27, ROP32 and three proteins related to cell adhesion. Finally, we demonstrated in vitro the immunogenic potential of a C. suis-specific 42kDa transmembrane protein, which might constitute an attractive candidate for further testing.

Cystoisospora suis - A Model of Mammalian Cystoisosporosis.

Cystoisospora suis is a coccidian species that typically affects suckling piglets. Infections occur by oral uptake of oocysts and are characterized by non-hemorrhagic transient diarrhea, resulting in poor weight gain. Apparently, primary immune responses to C. suis cannot readily be mounted by neonates, which contributes to the establishment and rapid development of the parasite, while in older pigs age-resistance prevents disease development. However, the presence of extraintestinal stages, although not unequivocally demonstrated, is suspected to enable parasite persistence together with the induction and maintenance of immune response in older pigs, which in turn may facilitate the transfer of C. suis-specific factors from sow to offspring. It is assumed that neonates are particularly prone to clinical disease because infections with C. suis interfere with the establishment of the gut microbiome. Clostridia have been especially inferred to profit from the altered intestinal environment during parasite infection. New tools, particularly in the area of genomics, might illustrate the interactions between C. suis and its host and pave the way for the development of new control methods not only for porcine cystoisosporosis but also for other mammalian Cystoisospora infections. The first reference genome for C. suis is under way and will be a fertile ground to discover new drugs and vaccines. At the same time, the establishment and refinement of an in vivo model and an in vitro culture system, supporting the complete life cycle of C. suis, will underpin the functional characterization of the parasite and shed light on its biology and control.

Recombinant activated factor VII for hemostatic cover of orthopedic interventions in a girl with thrombocytopenia with absent radii syndrome.

Over the past 10 years recombinant activated factor VIIa (rFVIIa) has been successfully used for treatment and prophylaxis of bleeding in patients with platelet defects, including thrombocytopenia and congenital and acquired platelet function abnormalities. Most reported data concern patients with Glanzmann's thrombasthenia and the information available is still limited, especially for surgery. We report on a 15-year-old girl with thrombocytopenia ( approximately 60,000/microl) and platelet dysfunction (bleeding time 30 min, absent platelet aggregation and ATP secretion in response to collagen), related to thrombocytopenia with absent radii syndrome, undergoing two surgical interventions on the upper limbs due to forearm deformities, with prolonged postoperative revisions. In both surgeries rFVIIa was successfully employed as a bolus administration (80 microg/kg every 4 h during the first day, then every 6 h over the following 5 and 3 days, respectively; tranexamic acid was associated from the second day, administered for 2 weeks), avoiding the need for blood products. This report highlights rFVIIa as an attractive, alternative approach to secure hemostasis in patients with platelet defects; on the other hand, the heterogeneity of reported rFVIIa treatment regimens and, in particular, the lack of definite and easily available parameters (or assays) for monitoring rFVIIa efficacy and safety are the main open issues in this setting.