The primers traditionally used to detect Plasmodium ovale infections are known for not binding all P. ovale parasites within the small-subunit rRNA gene when used alone. We describe a simple, cost-and time-efficient multiplex nested PCR and a nested PCR using a novel set of primers for the simultaneous detection of P. ovale curtisi and P. ovale wallikeri. Genes of the small-subunit (SSU) rRNA are highly conserved regions that not only allow the discrimination of different Plasmodium species but can also be used for the phylogenetic characterization of a wide range of different malaria parasites (11). For the diagnosis of human malaria parasites, a nested PCR technique (NP-1993 protocol) that binds the SSU rRNA gene was originally developed in the early 1990s (12). This technique soon became one of the most widely used and standardized PCR techniques for the detection and differentiation of human malaria parasites. The lower limits of detection were reported to be between a single parasite in 10 l blood (0.000002% parasitemia) and six parasites in 1 l blood (9, 11). Several modifications of the NP-1993 protocol followed (2,8,9). It soon became obvious that the NP-1993 protocol had some limitations for the diagnosis of Plasmodium ovale (primers rOVA1/rOVA2). Some patient samples that were positive for P. ovale by microscopy gave negative results with the nested PCR, and finally an updated protocol was described in 2002 (11). The primers for the Nest2 analysis of P. ovale were changed to genus-specific primer rPLU2 combined with rOVA1.Until 2005, more than 14 different protocols had been published specifically for the diagnosis of P. ovale. On the basis of the availability of more specific diagnostic tools, P. ovale was divided into the classic and variant types. In 2005, the NP-2005 protocol using primers rOVA1v and rOVA2v to detect variant P. ovale parasites was presented (1).Subsequent studies showed that the differences between the classic and variant types of P. ovale are not limited to the SSU rRNA gene. The P. ovale reticulocyte binding protein 2 gene (porbp2) and tryptophan-rich antigen gene (potra) are other examples of genetic differences. The perfect linkage between the two dimorphic forms of P. ovale finally led to the introduction of P. ovale curtisi (the former classic type) and P. ovale wallikeri (the former variant type) (13). Recent studies documented the sympatric distribution of P. ovale curtisi and P. ovale wallikeri in Africa and Asia and that they are morphologically indistinguishable (5, 7, 13).The combination of NP-1993 primers rOVA1 and rOVA2 for the diagnosis of P. ovale curtisi and the NP-2005 primer rOVA1v and rOVA2v has previously been published but still requires two separate PCRs (5).The principal aim of this study was to simplify the methodology for differentiating P. ovale curtisi and wallikeri (and thereby the methodology of adequate P. ovale molecular diagnosis) by developing a single PCR. We designed (i) new primers binding within the SSU rRNA gene that are highly specific for b...

The novel pestivirus species known as lateral-shaking inducing neuro-degenerative agent (LINDA) virus emerged in 2015 in a piglet-producing farm in Austria. Affected piglets showed strong congenital tremor as a result of severe lesions in the central nervous system. Here, we report the results of a controlled animal infection experiment. Post-weaning piglets were infected with LINDA to determine the susceptibility of pigs, the clinical consequences of infection and the humoral immune response against LINDA. No clinically overt disease signs were observed in the piglets. Viremia was hardly detectable, but LINDA was present in the spleen and several lymphatic organs until the end of the experiment on day 28 post-infection. Oronasal virus shedding together with the infection of one sentinel animal provided additional evidence for the successful replication and spread of LINDA in the piglets. Starting on day 14 post-infection, all infected animals showed a strong humoral immune response with high titers of neutralizing antibodies against LINDA. No cross-neutralizing activity of these sera with other pestiviral species was observed. According to these data, following postnatal infection, LINDA is a rather benign virus that can be controlled by the pig’s immune system. However, further studies are needed to investigate the effects of LINDA on the fetus after intrauterine infection.

Among the many diseases compromising the well-being of the honey bee (Apis mellifera) the chronic paralysis syndrome of adult honey bees is one of the best described. The causative agent, chronic bee paralysis virus (CBPV), is a positive sense, single-stranded RNA virus with a segmented genome. Segment 1 encodes three putative open reading frames (ORFs), including the RNA-dependent RNA polymerase and other non-structural protein coding regions. Segment 2 encodes four putative ORFs, which contain the genes of supposed structural proteins. In this study, we established a reverse genetic system for CBPV by molecular cloning of DNA copies of both genome segments. CBPV rescue was studied in imago and honey bee pupae infection models. Virus replication and progeny virus production was only initiated when capped RNAs of both genome segments were injected in honey bees. As injection of these clonal RNAs caused clinical symptoms similar to wild-type CBPV infection, we conclude that the novel molecular clone fulfilled Koch’s postulates. Our virus clone will enable in-depth analysis of CBPV pathogenesis and help to increase knowledge about this important honey bee disease.