Toxoplasma gondii in animal and human hosts

The protozoan parasite Toxoplasma gondii (T. gondii) is an important zoonotic pathogen, which has the ability to infect all warm blooded mammals including humans, with approximately one third of the human population predicted to be infected. Transmission of the parasite to the foetus during pregnancy can result in miscarriage, however, a child infected during pregnancy may go on to develop clinical symptoms such as retinochoroiditis (ocular toxoplasmosis), hydrocephalus or learning difficulties in later life. Post-natally acquired infection in humans is generally asymptomatic, however, individuals who are immunocompromised may develop ocular toxoplasmosis or toxoplasmic encephalitis. T. gondii type II is reported to be the predominant genotype in Europe and the United States, but currently very little information exists about the prevalence and genotypes present within Great Britain. Consumption of T. gondii tissue cysts from raw or undercooked meat is a main source of infection for humans, with infected pork being considered a high risk. Currently the “gold standard” for assessing the viability of infective T. gondii tissue cysts is by an in vivo mouse bioassay. However, more recent ethical requirements to reduce, refine or replace experimental animals raises the question as to whether molecular technologies could be incorporated into these studies to reduce mouse numbers. The main aims of this PhD were to: (i) determine the prevalence and genotypes of T. gondii within different wildlife populations and humans in Great Britain; (ii) determine whether vaccination of pigs with a live attenuated strain of T. gondii would reduce the load of viable T. gondii tissue cysts within this species; (iii) study the viability and dissemination of tissue cysts from oocyst and bradyzoite infected pigs and (iv) to compare mouse bioassay with molecular detection of T. gondii DNA from experimentally infected pigs. The main findings of this work show that the prevalence of T. gondii within carnivorous wildlife varied from 6.0% to 44.4% depending on the host species with type II being the predominant lineage identified, however, type III and two alleles for type I were also present. In humans, serological detection of the parasite from a group of Scottish blood donors from Glasgow and Dundee (n=1403) was determined at 13.0%, molecular detection of T. gondii in human brains (n=151) from the Sudden Death Brain Bank show a prevalence of 17.9%. A correlation between increasing age and an increase in the detection of parasite was identified from both study groups. T. gondii strain genotyping using DNA extracted from human brains identified alleles for type I and III, however, no direct link between cause of death and detection of parasite DNA could be made. Live vaccination and subsequent oocyst challenge of pigs showed a significant reduction in the establishment of viable T. gondii tissue cysts. Mouse bioassay clearly demonstrates this result, where 100% of mice that were inoculated with homogenised tissues from vaccinated/challenged pigs survived, compared to the survival of only 51% of mice, which received homogenised tissues from non-vaccinated/oocyst challenged animals. In addition, porcine tissues from pigs challenged with either oocysts or bradyzoites did not show a significant difference in mouse survival following bioassay of these tissues. Challenge with either stage of the parasite (oocysts or bradyzoites) showed a preference to form tissue cysts in brains and highly vascular muscles (tongue, diaphragm, heart or masseter) of pigs. The findings, comparing mouse bioassay with molecular detection of parasite DNA from homogenised porcine tissue (prior to inoculation into mice), showed similar levels of detection. However, mouse bioassay was more sensitive and also provides evidence of parasite viability. In conclusion, this research not only provides current figures for prevalence and genotypes of T. gondii in both wildlife and humans in Great Britain, it also successfully answers the question as to whether live vaccination of pigs with the S48 strain can reduce the tissue cyst burden. These promising results show the potential of a vaccine against T. gondii in producing safer pork for human consumption. Although the mouse bioassay still remains the most sensitive method for the detection and viability assessment of tissue cysts, further research should be carried out in this area, perhaps incorporating a technique such as magnetic capture qPCR, to enable an effective in vitro technique to be developed

Increased Toxoplasma gondii positivity relative to age in 125 Scottish sheep flocks; evidence of frequent acquired infection

Toxoplasma gondii seroprevalence was determined in 3333 sheep sera from 125 distinct sheep flocks in Scotland, with the majority of flocks being represented by 27 samples, which were collected between July 2006 and August 2008. The selected farms give a representative sample of 14 400 sheep holdings identified in the Scottish Government census data from 2004. Overall T. gondii seroprevalence, at individual sheep level, was determined to be 56.6%; each flock tested, had at least a single positive animal and in four flocks all ewes tested positive. The seroprevalence of sheep increased from 37.7% in one year old stock to 73.8% in ewes that were older than six years, showing that acquired infections during the life of the animals is frequent and that environmental contamination by T. gondii oocysts must be significant. The median within-flock seroprevalence varied significantly across Scotland, with the lowest seroprevalence of 42.3% in the South and the highest seroprevalence of 69.2% in the far North of Scotland and the Scottish Islands, while the central part of Scotland had a seroprevalence of 57.7%. This distribution disequilibrium may be due to the spread and survival of oocysts on pasture and lambing areas. A questionnaire accompanying sampling of flocks identified farms that used Toxovax®, a commercial vaccine that protects sheep from abortion due to T. gondii infection. Only 24.7% of farmers used the vaccine and the vaccine did not significantly affect the within flock seroprevalence for T. gondii. The implications for food safety and human infection are discussed

Analysis of Jmjd6 Cellular Localization and Testing for Its Involvement in Histone Demethylation

BACKGROUND: Methylation of residues in histone tails is part of a network that regulates gene expression. JmjC domain containing proteins catalyze the oxidative removal of methyl groups on histone lysine residues. Here, we report studies to test the involvement of Jumonji domain-containing protein 6 (Jmjd6) in histone lysine demethylation. Jmjd6 has recently been shown to hydroxylate RNA splicing factors and is known to be essential for the differentiation of multiple tissues and cells during embryogenesis. However, there have been conflicting reports as to whether Jmjd6 is a histone-modifying enzyme. METHODOLOGY/PRINCIPAL FINDINGS: Immunolocalization studies reveal that Jmjd6 is distributed throughout the nucleoplasm outside of regions containing heterochromatic DNA, with occasional localization in nucleoli. During mitosis, Jmjd6 is excluded from the nucleus and reappears in the telophase of the cell cycle. Western blot analyses confirmed that Jmjd6 forms homo-multimers of different molecular weights in the nucleus and cytoplasm. A comparison of mono-, di-, and tri-methylation states of H3K4, H3K9, H3K27, H3K36, and H4K20 histone residues in wildtype and Jmjd6-knockout cells indicate that Jmjd6 is not involved in the demethylation of these histone lysine residues. This is further supported by overexpression of enzymatically active and inactive forms of Jmjd6 and subsequent analysis of histone methylation patterns by immunocytochemistry and western blot analysis. Finally, treatment of cells with RNase A and DNase I indicate that Jmjd6 may preferentially associate with RNA/RNA complexes and less likely with chromatin. CONCLUSIONS/SIGNIFICANCE: Taken together, our results provide further evidence that Jmjd6 is unlikely to be involved in histone lysine demethylation. We confirmed that Jmjd6 forms multimers and showed that nuclear localization of the protein involves association with a nucleic acid matrix

Early immune responses and parasite tissue distribution in mice experimentally infected with oocysts of either archetypal or non-archetypal genotypes of Toxoplasma gondii

In most of the world Toxoplasma gondii is comprised of archetypal types (types I, II and III); however, South America displays several non-archetypal strains. This study used an experimental mouse model to characterize the immune response and parasite kinetics following infection with different parasite genotypes. An oral inoculation of 50 oocysts per mouse from T. gondii M4 type II (archetypal, avirulent), BrI or BrIII (non-archetypal, virulent and intermediate virulent, respectively) for groups (G)2, G3 and G4, respectively was used. The levels of mRNA expression of cytokines, immune compounds, cell surface markers and receptor adapters [interferon gamma (IFNγ), interleukin (IL)-12, CD8, CD4, CD25, CXCR3 and MyD88] were quantified by SYBR green reverse transcription-quantitative polymerase chain reaction. Lesions were characterized by histology and detection by immunohistochemistry established distribution of parasites. Infection in G2 mice was mild and characterized by an early MyD88-dependent pathway. In G3, there were high levels of expression of pro-inflammatory cytokines IFNγ and IL-12 in the mice showing severe clinical symptoms at 8–11 days post infection (dpi), combined with the upregulation of CD25, abundant tachyzoites and tissue lesions in livers, lungs and intestines. Significant longer expression of IFNγ and IL-12 genes, with other Th1-balanced immune responses, such as increased levels of CXCR3 and MyD88 in G4, resulted in survival of mice and chronic toxoplasmosis, with the occurrence of tissue cysts in brain and lungs, at 14 and 21 dpi. Different immune responses and kinetics of gene expression appear to be elicited by the different strains and non-archetypal parasites demonstrated higher virulence

The Development of Ovine Gastric and Intestinal Organoids for Studying Ruminant Host-Pathogen Interactions

Gastrointestinal (GI) infections in sheep have significant implications for animal health, welfare and productivity, as well as being a source of zoonotic pathogens. Interactions between pathogens and epithelial cells at the mucosal surface play a key role in determining the outcome of GI infections; however, the inaccessibility of the GI tract in vivo significantly limits the ability to study such interactions in detail. We therefore developed ovine epithelial organoids representing physiologically important gastric and intestinal sites of infection, specifically the abomasum (analogous to the stomach in monogastrics) and ileum. We show that both abomasal and ileal organoids form self-organising three-dimensional structures with a single epithelial layer and a central lumen that are stable in culture over serial passage. We performed RNA-seq analysis on abomasal and ileal tissue from multiple animals and on organoids across multiple passages and show the transcript profile of both abomasal and ileal organoids cultured under identical conditions are reflective of the tissue from which they were derived and that the transcript profile in organoids is stable over at least five serial passages. In addition, we demonstrate that the organoids can be successfully cryopreserved and resuscitated, allowing long-term storage of organoid lines, thereby reducing the number of animals required as a source of tissue. We also report the first published observations of a helminth infecting gastric and intestinal organoids by challenge with the sheep parasitic nematode Teladorsagia circumcincta, demonstrating the utility of these organoids for pathogen co-culture experiments. Finally, the polarity in the abomasal and ileal organoids can be inverted to make the apical surface directly accessible to pathogens or their products, here shown by infection of apical-out organoids with the zoonotic enteric bacterial pathogen Salmonella enterica serovar Typhimurium. In summary, we report a simple and reliable in vitro culture system for generation and maintenance of small ruminant intestinal and gastric organoids. In line with 3Rs principals, use of such organoids will reduce and replace animals in host-pathogen research

NKp46⁺CD3⁺ Cells: A Novel Nonconventional T Cell Subsetin Cattle Exhibiting Both NK Cell and T Cell Features

The NKp46 receptor demonstrates a high degree of lineage-specificity, being expressed almost exclusively in natural killer cells. Previous studies have demonstrated NKp46 expression by T-cells, but NKp46(+)CD3(+) cells are rare and almost universally associated with NKp46 acquisition by T-cells following stimulation. In this study we demonstrate the existence of a population of NKp46(+)CD3(+) cells resident in normal bovine PBMC which include cells of both the αβ TCR(+) and γδ TCR(+) lineages and is present at a frequency of 0.1-1.7%. NKp46(+)CD3(+) cells express transcripts for a broad repertoire of both natural killer (NKR) and T-cell receptors (TCR) and also the CD3ζ, DAP10 and FcεR1γ but not DAP12 adaptor proteins. In vitro functional analysis of NKp46(+)CD3(+) cells confirm that NKp46, CD16 and CD3 signalling pathways are all functionally competent and capable of mediating-re-direct cytolysis. However, only CD3 cross-ligation elicits IFN-γ release. NKp46(+)CD3(+) cells exhibit cytotoxic activity against autologous Theileria parva infected cells in vitro and during in vivo challenge with this parasite an expansion of NKp46(+)CD3(+) cells was observed in some animals, indicating the cells have the potential to act as an anti-pathogen effector population. The results presented herein identifies and describes a novel non-conventional NKp46(+)CD3(+) T-cell subset that is phenotypically and functionally distinct from conventional NK and T-cells. The ability to exploit both NKR and TCR suggests these cells may fill a functional niche at the interface of innate and adaptive immune responses

Vaccination of pigs with the S48 strain of Toxoplasma gondii

Acknowledgements This project was partially funded by a transnational access project funded through the European Union Seventh Framework Network of Animal Disease Infectiology Research Facilities (NADIR; reference number FP7-228394). AB was funded by the Moredun Foundation. FK, PMB, MN, JT, FC, EAI were supported by the Scottish Government, Rural and Environmental Sciences and Analytical Services (RESAS). JB was supported by CSIC and financed in part by European Social Fund (ESF). JLG was financed by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, BEX 10259-/12-0), Brazil and GC by the Instituto Nacional de Tecnologia Agropecuaria (INATA), Argentina.Peer reviewedPublisher PD

Developing vaccines to control protozoan parasites in ruminants: dead or alive?

Protozoan parasites are among some of the most successful organisms worldwide, being able to live and multiply within a very wide range of hosts. The diseases caused by these parasites cause significant production losses in the livestock sector involving reproductive failure, impaired weight gain, contaminated meat, reduced milk yields and in severe cases, loss of the animal. In addition, some protozoan parasites affecting livestock such as Toxoplasma gondii and Cryptosporidium parvum may also be transmitted to humans where they can cause serious disease. Data derived from experimental models of infection in ruminant species enables the study of the interactions between parasite and host. How the parasite initiates infection, becomes established and multiplies within the host and the critical pathways that may lead to a disease outcome are all important to enable the rational design of appropriate intervention strategies. Once the parasites invade the hosts they induce both innate and adaptive immune responses and the induction and function of these immune responses are critical in determining the outcome of the infection. Vaccines offer green solutions to control disease as they are sustainable, reducing reliance on pharmacological drugs and pesticides. The use of vaccines has multiple benefits such as improving animal health and welfare by controlling animal infections and infestations; improving public health by controlling zoonoses and food borne pathogens in animals; solving problems associated with resistance to acaricides, antibiotics and anthelmintics; keeping animals and the environment free of chemical residues and maintaining biodiversity. All of these attributes should lead to improved sustainability of animal production and economic benefit. Using different protozoan parasitic diseases as examples this paper will discuss various approaches used to develop vaccines to protect against disease in livestock and discuss the relative merits of using live versus killed vaccine preparations. A range of different vaccination targets and strategies will be discussed to help protect against: acute disease, congenital infection and abortion, persistence of zoonotic pathogens in tissues of food animals and passive transfer of immunity to neonates