Identification of Oocyst-Driven Toxoplasma gondii Infections in Humans and Animals through Stage-Specific Serology—Current Status and Future Perspectives

The apicomplexan zoonotic parasite Toxoplasma gondii has three infective stages: sporozoites in sporulated oocysts, which are shed in unsporulated form into the environment by infected felids; tissue cysts containing bradyzoites, and fast replicating tachyzoites that are responsible for acute toxoplasmosis. The contribution of oocysts to infections in both humans and animals is understudied despite being highly relevant. Only a few diagnostic antigens have been described to be capable of discriminating which parasite stage has caused an infection. Here we provide an extensive overview of the antigens and serological assays used to detect oocyst-driven infections in humans and animals according to the literature. In addition, we critically discuss the possibility to exploit the increasing knowledge of the T. gondii genome and the various ‘omics datasets available, by applying predictive algorithms, for the identification of new oocyst-specific proteins for diagnostic purposes. Finally, we propose a workflow for how such antigens and assays based on them should be evaluated to ensure reproducible and robust results.Peer Reviewe

The Protozoan Neospora caninum Directly Triggers Bovine NK Cells To Produce Gamma Interferon and To Kill Infected Fibroblasts

Natural killer (NK) cells are considered to be key players in the early innate responses to protozoan infections, primarily indirectly by producing gamma interferon (IFN-γ) in response to cytokines, like interleukin 12 (IL-12). We demonstrate that live, as well as heat-inactivated, tachyzoites of Neospora caninum, a Toxoplasma-like protozoan, directly trigger production of IFN-γ from purified, IL-2-activated bovine NK cells. This response occurred independently of IL-12 but was increased by the addition of the cytokine. A similar IFN-γ response was measured in cocultures of NK cells and N. caninum-infected autologous fibroblasts. However, no NK cell-derived IFN-γ response was detected when cells were cultured with soluble antigens from the organism, indicating that intact tachyzoites or nonsoluble components are necessary for NK cell triggering. Furthermore, N. caninum-infected autologous fibroblasts had increased susceptibility to NK cell cytotoxicity compared to uninfected fibroblasts. This cytotoxicity was largely mediated by a perforin-mediated mechanism. The activating receptor NKp46 was involved in cytotoxicity against fibroblasts but could not explain the increased cytotoxicity against infected targets. Interestingly, N. caninum tachyzoites were able to infect cultured NK cells, in which tachyzoites proliferated inside parasitophorous vacuoles. Together, these findings underscore the role of NK cells as primary responders during a protozoan infection, describe intracellular protozoan infection of NK cells in vitro for the first time, and represent the first functional study of purified bovine NK cells in response to infection

Seroprevalence of Hepatitis E Virus in Moose (<i>Alces alces</i>), Reindeer (<i>Rangifer tarandus</i>), Red Deer (<i>Cervus elaphus</i>), Roe Deer (<i>Capreolus capreolus</i>), and Muskoxen (<i>Ovibos moschatus</i>) from Norway

Hepatitis E virus (HEV), a major cause of viral hepatitis worldwide, is considered an emerging foodborne zoonosis in Europe. Pigs (Sus scrofa domestica) and wild boars (S. scrofa) are recognized as important HEV reservoirs. Additionally, HEV infection and exposure have been described in cervids. In Norway, HEV has been identified in pigs and humans; however, little is known regarding its presence in wild ungulates in the country. We used a species-independent double-antigen sandwich ELISA to detect antibodies against HEV in the sera of 715 wild ungulates from Norway, including 164 moose (Alces alces), 186 wild Eurasian tundra reindeer (Rangifer tarandus tarandus), 177 red deer (Cervus elaphus), 86 European roe deer (Capreolus capreolus), and 102 muskoxen (Ovibos moschatus). The overall seroprevalence was 12.3% (88/715). Wild reindeer had the highest seropositivity (23.1%, 43/186), followed by moose (19.5%, 32/164), muskoxen (5.9%, 6/102), and red deer (4%, 7/177). All roe deer were negative. According to our results, HEV is circulating in wild ungulates in Norway. The high seroprevalence observed in wild reindeer and moose indicates that these species may be potential reservoirs of HEV. To the authors’ knowledge, this is the first report of HEV exposure in reindeer from Europe and in muskoxen worldwide

Detection of serum neutralizing antibodies to Simbu sero-group viruses in cattle in Tanzania

Background: Orthobunyaviruses belonging to the Simbu sero-group occur worldwide, including the newly recognized Schmallenberg virus (SBV) in Europe. These viruses cause congenital malformations and reproductive losses in ruminants. Information on the presence of these viruses in Africa is scarce and the origin of SBV is unknown. The aim of this study was to investigate the presence of antibodies against SBV and closely related viruses in cattle in Tanzania, and their possible association with reproductive disorders. Results: In a cross-sectional study, serum from 659 cattle from 202 herds collected in 2012/2013 were analyzed using a commercial kit for SBV ELISA, and 61 % were positive. Univariable logistic regression revealed significant association between ELISA seropositivity and reproductive disorders (OR = 1.9). Sera from the same area collected in 2008/2009, before the SBV epidemic in Europe, were also tested and 71 (54.6 %) of 130 were positive. To interpret the ELISA results, SBV virus neutralization test (VNT) was performed on 110 sera collected in 2012/2013, of which 51 % were positive. Of 71 sera from 2008/2009, 21 % were positive. To investigate potential cross reactivity with related viruses, 45 sera from 2012/2013 that were positive in SBV ELISA were analyzed in VNTs for Aino, Akabane, Douglas, Peaton, Sabo, SBV, Sathuperi, Shamonda, Simbu and Tinaroo viruses. All 45 sera were positive for one or more of these viruses. Twenty-nine sera (64.4 %) were positive for SBV, and one had the highest titer for this virus. Conclusions: This is the first indication that Aino, Akabane, Douglas, Peaton, Sabo, SBV, Sathuperi, Shamonda and Tinaroo viruses circulate and cause negative effect on reproductive performance in cattle in Tanzania. SBV or a closely related virus was present before the European epidemic. However, potential cross reactivity complicates the interpretation of serological studies in areas where several related viruses may circulate. Virus isolation and molecular characterization in cattle and/or vectors is recommended to further identify the viruses circulating in this region. However, isolation in cattle is difficult due to short viremic period of 2 to 6 days, and isolation in vectors does not necessarily reflect the situation in cattle

First isolation, identification, phenotypic and genotypic characterization of Brucella abortus biovar 3 from dairy cattle in Tanzania

Background: Brucellosis is a disease of worldwide public health and economic importance. Successful control is based on knowledge of epidemiology and strains present in an area. In developing countries, most investigations are based on serological assays. This study aimed at investigating a dairy herd experiencing abortions in order to establish within-herd seroprevalence to Brucella spp., identify, characterize Brucella strains by Multiple Loci Variable Number of Tandem Repeats Analysis (MLVA-VNTR) and investigate possible spillover to other species. Results: The within-herd seroprevalence in cattle (n = 200) was 48 % (95 % CI 41–55), using an indirect ELISA, while the Rose Bengal Test (RBT) yielded lower prevalence (21.5 %; 95 % CI 16–27). Two sheep (n = 35) and one goat (n = 50) were seropositive using ELISA while none of the dogs (n = 6) was positive with the RBT. Three Brucella were isolated from an aborted fetus and associated membranes. Real time PCR (IS711), Bruce-ladder and classical biotyping classified the isolates as B. abortus biovar 3. MLVA-VNTR revealed two different but closely related genotypes. The isolates showed unique profiles, providing the first genotypic data from Tanzania. These genotypes were not related to B. abortus biovar 3 reference strain Tulya originally isolated from a human patient in Uganda in 1958, unlike the genotypes isolated and characterized recently in Kenya. High within-herd prevalence, isolation of the pathogen and abortion confirm that B. abortus is circulating in this herd with cattle as reservoir hosts. A low seroprevalence in sheep and goats suggests a spillover of B. abortus from cattle to small ruminants in the herd. Conclusions: This is the first isolation and characterization of B. abortus biovar 3 from a dairy cow with abortion in Tanzania. The origin of the Tanzanian genotypes remain elusive, although they seem to be related to genotypes found in Europe, Turkey and China but not related to B. abortus biovar 3 reference strain or genotypes from Kenya. Importantly, replacement heifers are commonly sourced from large farms like this to smallholder farmers, which poses risk of spread of bacteria to other herds. B. abortus is a significant zoonotic risk and animal health problem in this production system, therefore further studies on humans is recommended