Real-Time PCR for Molecular Detection of Zoonotic and Non-Zoonotic Giardia spp. in Wild Rodents.

Giardiasis in humans is a gastrointestinal disease transmitted by the potentially zoonotic Giardia duodenalis genotypes (assemblages) A and B. Small wild rodents such as mice and voles are discussed as potential reservoirs for G. duodenalis but are predominantly populated by the two rodent species Giardia microti and Giardia muris. Currently, the detection of zoonotic and non-zoonotic Giardia species and genotypes in these animals relies on cumbersome PCR and sequencing approaches of genetic marker genes. This hampers the risk assessment of potential zoonotic Giardia transmissions by these animals. Here, we provide a workflow based on newly developed real-time PCR schemes targeting the small ribosomal RNA multi-copy gene locus to distinguish G. muris, G. microti and G. duodenalis infections. For the identification of potentially zoonotic G. duodenalis assemblage types A and B, an established protocol targeting the single-copy gene 4E1-HP was used. The assays were specific for the distinct Giardia species or genotypes and revealed an analytical sensitivity of approximately one or below genome equivalent for the multi-copy gene and of about 10 genome equivalents for the single-copy gene. Retesting a biobank of small rodent samples confirmed the specificity. It further identified the underlying Giardia species in four out of 11 samples that could not be typed before by PCR and sequencing. The newly developed workflow has the potential to facilitate the detection of potentially zoonotic and non-zoonotic Giardia species in wild rodents.

Resistance to intranasal infection with Balamuthia mandrillaris amebae is T-cell dependent.

T and B cell-deficient BALB/c SCID mice become severely ill and die of amebic encephalitis after intranasal infection with Balamuthia mandrillaris, while adult immunocompetent BALB/c wild-type (WT) mice are resistant. To further investigate the role of lymphocytes in protection from Balamuthia amebic encephalitis (BAE), SCID mice were reconstituted with and WT mice selectively depleted of lymphocytes before infection. Reconstitution of SCID mice with whole spleen cells from WT mice rendered the recipients as resistant to BAE as WT mice. SCID mice that had received spleen cells depleted of CD4(+) T cells remained susceptible. When adult WT mice were depleted of both CD4(+) and CD8(+) T cells or of CD4(+) T cells alone, these mice also became susceptible to BAE. Depletion of CD8(+) T cells alone increased susceptibility only marginally. All morbidity and mortality data were underpinned by histological analysis of the brain.

Balamuthia and Acanthamoeba-binding antibodies in West African human sera.

Little is known about the prevalence of Balamuthia mandrillaris amoebae and Balamuthia amoebic encephalitis in Africa. As an approach, relative concentrations of amoebae-binding serum antibodies (Ab) were assessed by flow cytometry using formaldehyde-fixed B. mandrillaris, Acanthamoeba lenticulata 72-2 and Acanthamoeba castellanii 1BU amoebae for specific Ab capture (B.m.-Ab, A.l.-Ab, A.c.-Ab). One hundred and ninety-two sera from West African (Côte d'Ivoire) donors aged 11-95years (mean 38 a; 51% males), and living in villages surrounded by rainforest near the Liberian border, were tested and related to reference sera from Berlin. While B.m.-Ab tended to increase with donor age, A.l.-Ab and A.c.-Ab did not. Accordingly, B.m.-Ab correlated only weakly with A.l.-Ab or A.c.-Ab. Of the nine individuals with the highest B.m.-Ab concentrations, most were elderly (mean 58 a), male (78%), and professed intensive outdoor activity (hunting, farming). Only three of these sera also showed elevated A.l.-Ab, and none elevated A.c.-Ab.

Assessment of Balamuthia mandrillaris-specific serum antibody concentrations by flow cytometry.

A flow cytometry (fluorescence-activated cell sorter)-based assay was adapted to detect and quantify antibodies to Balamuthia mandrillaris, a causative agent of fatal amoebic encephalitis (BAE), and to Acanthamoeba species. With sera from BAE patients for positive and a group of inconspicuous volunteers for negative reference, most of the 237 sera from random blood donors, patients with atypical encephalitis, atypical pneumonitis, visceral amoebiasis and toxoplasmosis and from subjects working with primates and other mammals were rated negative, 19% elevated and of these 2% high. In comparison, 23 of 25 West Africans living in rural areas revealed elevated, of these 15 high, and one very high B. mandrillaris-binding antibody titers, the latter well in the range of BAE patients. To date, none of the tested individuals have developed symptoms indicative of BAE. Criss-cross analysis with rabbit hyper immune sera against B. mandrillaris, Acanthamoeba comandoni (group 1), Acanthamoeba castellanii (group 2) and Acanthamoeba lenticulata (group 3) confirmed that cross-reactivity between B. mandrillaris and Acanthamoeba sp. is negligible while accentuating antigenic differences between the three morphological groups of Acanthamoeba.

Detection of Balamuthia mandrillaris DNA by real-time PCR targeting the RNase P gene.

BACKGROUND: The free-living amoeba Balamuthia mandrillaris may cause fatal encephalitis both in immunocompromised and in - apparently - immunocompetent humans and other mammalian species. Rapid, specific, sensitive, and reliable detection requiring little pathogen-specific expertise is an absolute prerequisite for a successful therapy and a welcome tool for both experimental and epidemiological research. RESULTS: A real-time polymerase chain reaction assay using TaqMan probes (real-time PCR) was established specifically targeting the RNase P gene of B. mandrillaris amoebae. The assay detected at least 2 (down to 0.5) genomes of B. mandrillaris grown in axenic culture. It did not react with DNA from closely related Acanthamoeba (3 species), nor with DNA from Toxoplasma gondii, Leishmania major, Pneumocystis murina, Mycobacterium bovis (BCG), human brain, various mouse organs, or from human and murine cell lines. The assay efficiently detected B. mandrillaris DNA in spiked cell cultures, spiked murine organ homogenates, B. mandrillaris-infected mice, and CNS tissue-DNA preparations from 2 patients with proven cerebral balamuthiasis. This novel primer set was successfully combined with a published set that targets the B. mandrillaris 18S rRNA gene in a duplex real-time PCR assay to ensure maximum specificity and as a precaution against false negative results. CONCLUSION: A real-time PCR assay for B. mandrillaris amoebae is presented, that is highly specific, sensitive, and reliable and thus suited both for diagnosis and for research.

Oral infection of immunocompetent and immunodeficient mice with Balamuthia mandrillaris amebae.

Balamuthia mandrillaris is an opportunistic agent of lethal granulomatous amebic encephalitis (GAE). In mice, we have shown that intranasally instilled B. mandrillaris amebae infect the brain via the olfactory nerve pathway. In this study, we raised the question whether this ameba might also reach the brain after an oral/gastrointestinal infection. Immunocompetent (WT) and immunodeficient (RAG) mice received B. mandrillaris amebae by gavage into the esophagus. Mice of both groups became ill and some died (WT 20%, RAG 40%) within 42 days. All orally infected mice revealed B. mandrillaris amebae in the central nervous system. Outwardly intact amebae and/or specific antigen were found widely distributed in various organs and the stool. The data indicate that oral infection with B. mandrillaris leading to GAE is possible. Exit from the gastrointestinal tract and dissemination remains unresolved. Though stool cultures were negative, transmission of this highly pathogenic ameba via stool cannot be ruled out.

Cytopathogenicity of Balamuthia mandrillaris, an opportunistic causative agent of granulomatous amebic encephalitis.

Balamuthia mandrillaris is a free-living ameba and an opportunistic agent of lethal granulomatous amebic encephalitis in humans and other mammals. Balamuthia mandrillaris is highly cytopathic but, in contrast to the related Acanthamoeba, does not feed on bacteria and seems to feed only on eukaryotic cells instead. Most likely, the cytopathogenicity of B. mandrillaris is inseparable from its infectivity and pathogenicity. To better understand the mechanisms of B. mandrillaris cytopathogenicity, an assay for measuring amebic cytolytic activity was adapted that is based on the release of a reporter enzyme by damaged target cells. The ameba is shown to lyse murine mastocytoma cells very efficiently in a time- and dose-related manner. Furthermore, experiments involving semipermeable membranes and phagocytosis inhibitors indicate that the cytolytic activity of B. mandrillaris is essentially cell contact-dependent. Standard and fluorescence light microscopy, as well as scanning and transmission electron microscopy support and extend these findings at the ultrastructural level.