BackgroundEimeria species are parasitic protozoa that cause coccidiosis, an intestinal disease commonly characterised by malabsorption, diarrhoea and haemorrhage that is particularly important in chickens. Vaccination against chicken coccidiosis is effective using wild-type or attenuated live parasite lines. The development of protocols to express foreign proteins in Eimeria species has opened up the possibility of using Eimeria live vaccines to deliver heterologous antigens and function as multivalent vaccine vectors that could protect chickens against a range of pathogens.ResultsIn this study, genetic complementation was used to express immunoprotective virus antigens in Eimeria tenella. Infectious bursal disease virus (IBDV) causes Gumboro, an immunosuppressive disease that affects productivity and can interfere with the efficacy of poultry vaccination programmes. Infectious laryngotracheitis virus (ILTV) causes a highly transmissible respiratory disease for which strong cellular immunity and antibody responses are required for effective vaccination. Genes encoding the VP2 protein from a very virulent strain of IBDV (vvVP2) and glycoprotein I from ILTV (gI) were cloned downstream of 5’Et-Actin or 5’Et-TIF promoter regions in plasmids that also contained a mCitrine fluorescent reporter cassette under control of the 5’Et-MIC1 promoter. The plasmids were introduced by nucleofection into E. tenella sporozoites, which were then used to infect chickens. Progeny oocysts were sorted by FACS and passaged several times in vivo until the proportion of fluorescent parasites in each transgenic population reached ~20 % and the number of transgene copies per parasite genome decreased to < 10. All populations were found to transcribe and express the transgene and induced the generation of low titre, transgene-specific antibodies when used to immunise chickens.ConclusionsE. tenella can express antigens of other poultry pathogens that are successfully recognised by the chicken immune system. Nonetheless, further work has to be done in order to improve the levels of expression for its future use as a multivalent vaccine vector.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1756-2) contains supplementary material, which is available to authorized users.

Trypanosoma brucei relies on an essential Variant Surface Glycoprotein (VSG) coat for survival in the mammalian bloodstream. High VSG expression within an expression site body (ESB) is mediated by RNA polymerase I (Pol I), which in other eukaryotes exclusively transcribes ribosomal RNA genes (rDNA). As T. brucei is reliant on Pol I for VSG transcription, we investigated Pol I transcription inhibitors for selective anti-trypanosomal activity. The Pol I inhibitors quarfloxin (CX-3543), CX-5461, and BMH-21 are currently under investigation for treating cancer, as rapidly dividing cancer cells are particularly dependent on high levels of Pol I transcription compared with nontransformed cells. In T. brucei all three Pol I inhibitors have IC50 concentrations for cell proliferation in the nanomolar range: quarfloxin (155 nM), CX-5461 (279 nM) or BMH-21 (134 nM) compared with IC50 concentrations in the MCF10A human breast epithelial cell line (4.44 μM, 6.89 μM or 460 nM, respectively). T. brucei was therefore 29-fold more sensitive to quarfloxin, 25-fold more sensitive to CX-5461 and 3.4-fold more sensitive to BMH-21. Cell death in T. brucei was due to rapid inhibition of Pol I transcription, as within 15 minutes treatment with the inhibitors rRNA precursor transcript was reduced 97-98% and VSG precursor transcript 91-94%. Incubation with Pol I transcription inhibitors also resulted in disintegration of the ESB as well as the nucleolus subnuclear structures, within one hour. Rapid ESB loss following the block in Pol I transcription argues that the ESB is a Pol I transcription nucleated structure, similar to the nucleolus. In addition to providing insight into Pol I transcription and ES control, Pol I transcription inhibitors potentially also provide new approaches to treat trypanosomiasis.

Eimeria is a genus of apicomplexan parasites that contains a large number of species, most of which are absolutely host‐specific. Seven species have been recognized to infect chickens. Infection of susceptible chickens results in an intestinal disease called coccidiosis, characterized by mucoid or hemorrhagic enteritis, which is associated with impaired feed conversion or mortality in severe cases. Intensive farming practices have increased the significance of coccidiosis since parasite transmission is favored by high‐density housing of large numbers of susceptible chickens. Routine chemoprophylaxis and/or vaccination with live parasite vaccines provides effective control of Eimeria, although the emergence of drug resistance and the relative cost and production capacity of current vaccine lines can prove limiting. As pressure to reduce drug use in livestock production intensifies, novel vaccination strategies are needed. Development of effective protocols supporting genetic complementation of Eimeria species has until recently been hampered by their inability to replicate efficiently in vitro. Now, the availability of such protocols has raised the prospect of generating transgenic parasite lines that function as vaccine vectors to express and deliver heterologous antigens. For example, this technology has the potential to streamline the production of live anticoccidial vaccines through the generation of parasite lines that co‐express immunoprotective antigens derived from multiple Eimeria species. In this paper we describe detailed protocols for genetic manipulation, laboratory growth, and in vivo propagation of Eimeria tenella parasites, which will encourage future work from other researchers to expand biological understanding of Eimeria through reverse genetics. © 2019 by John Wiley & Sons, Inc.

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There are increasing concerns of infections by enteroviruses (EVs) causing severe disease in humans. EV diagnostic laboratory methods show differences in sensitivity and specificity as well as the level of genetic information provided. We examined a detection method for EVs based on next generation sequencing (NGS) analysis of amplicons covering the entire capsid coding region directly synthesized from clinical samples. One hundred and twelve clinical samples from England; previously shown to be positive for EVs, were analyzed. There was high concordance between the results obtained by the new NGS approach and those from the conventional Sanger method used originally with agreement in the serotypes identified in the 83 samples that were typed by both methods. The sensitivity and specificity of the NGS method compared to those of the conventional Sanger sequencing typing assay were 94.74% (95% confidence interval, 73.97% to 99.87%) and 97.85% (92.45% to 99.74%) for Enterovirus A, 93.75% (82.80% to 98.69%) and 89.06% (78.75% to 95.49%) for Enterovirus B, 100% (59.04% to 100%) and 98.10% (93.29% to 99.77%) for Enterovirus C, and 100% (75.29% to 100%) and 100% (96.34% to 100%) for Enterovirus D. The NGS method identified five EVs in previously untyped samples as well as additional viruses in some samples, indicating co-infection. This method can be easily expanded to generate whole-genome EV sequences as we show here for EV-D68. Information from capsid and whole-genome sequences is critical to help identifying the genetic basis for changes in viral properties and establishing accurate spatial-temporal associations between EV strains of public health relevance.

Infection with enterovirus D68 (EV-D68) has been linked with severe neurological disease such as acute flaccid myelitis (AFM) in recent years. However, active surveillance for EV-D68 is lacking, which makes full assessment of this association difficult. Although a high number of EV-D68 infections were expected in 2020 based on the EV-D68′s known biannual circulation patterns, no apparent increase in EV-D68 detections or AFM cases was observed during 2020. We describe an upsurge of EV-D68 detections in wastewater samples from the United Kingdom between July and November 2021 mirroring the recently reported rise in EV-D68 detections in clinical samples from various European countries. We provide the first publicly available 2021 EV-D68 sequences showing co-circulation of EV-D68 strains from genetic clade D and sub-clade B3 as in previous years. Our results show the value of environmental surveillance (ES) for the early detection of circulating and clinically relevant human viruses. The use of a next-generation sequencing (NGS) approach helped us to estimate the prevalence of EV-D68 viruses among EV strains from other EV serotypes and to detect EV-D68 minor variants. The utility of ES at reducing gaps in virus surveillance for EV-D68 and the possible impact of nonpharmaceutical interventions introduced to control the COVID-19 pandemic on EV-D68 transmission dynamics are discussed.

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