A comparative infection study was performed to investigate the clinical outcome and pathogen shedding patterns in calves experimentally infected with Cryptosporidium parvum and/or bovine coronavirus (BCoV). A total of 15 calves were enrolled and assigned into three infection groups (each n = 5) within 12 h of birth. Group 1 (G1) was experimentally infected with C. parvum, group 2 (G2) was experimentally infected with C. parvum and BCoV, and group 3 (G3) was experimentally infected with BCoV. Clinical outcomes were registered, and feces and nasal swabs were collected daily for 28 days. Prior to experimental infection, all calves were found negative for BCoV RNA in fecal and upper respiratory samples, but 12/15 calves had detectable C. parvum DNA in feces, meaning all groups were already naturally infected with C. parvum. Calves double infected with C. parvum (G1 and G2; experimentally and naturally infected) showed more clinical signs over a longer period of time, compared with calves experimentally infected with BCoV and also naturally infected with C. parvum (G3). The shedding pattern of Cryptosporidium DNA in feces was similar in all groups. The peak shedding period of C. parvum correlated with the days when calves displayed the most clinical signs. However, a significant increase in fecal BCoV RNA occurred after the period where calves displayed the most clinical signs. In this study, a double infection of neonatal calves with C. parvum (G1 and G2) resulted in a more severe clinical outcome than a mixed infection with C. parvum and BCoV.

Toxoplasma gondii (T. gondii), a zoonotic parasite, can cause severe disease in warm-blooded animals. Pathological changes in murine tissues infected with different T. gondii isolates were studied to establish factors influencing lesion severity and parasite burden. In Study A, mice were orally inoculated with genotype #3, #6 or #8 oocysts. In Study B, mice were inoculated intraperitoneally with genotype #1, #3, #6, #13, #141 or #265 tachyzoites. Mice were euthanised serially and tissues processed for histopathology. In Study A, genotype #6 caused pathology in the liver, brain, lung, intestine and kidney, predominantly associated with tachyzoites, while #8 caused mainly moderate pathology in the brain, lung and liver, usually associated with tissue pseudocysts/cysts. Genotype #3 occasionally caused mild pathology, but the parasite was not visible in examined tissues. In Study B, genotypes #13 and #6 caused systemic infections associated with tachyzoites. Genotypes #3, #141 and #265 caused moderate pathology associated with pseudocysts/cysts in the brain and tachyzoites in peripheral organs. Genotype #1 caused mild pathology associated with pseudocysts/cysts in organs assessed. Comparison of genotype #6 between studies showed parasite stage and inoculation method did not affect the severity of pathology, but for #3, pathology was more severe when mice were inoculated intraperitoneally with tachyzoites compared to those inoculated orally with oocysts. This study confirmed route of infection, T. gondii strain, life stage and dose influence infection outcome and ultimately contributes to the refinement of T. gondii pathogenesis knowledge, which is fundamental for toxoplasmosis management and treatment.

Cryptosporidium is an apicomplexan parasite that causes diarrhoeal disease. The species C. parvum is zoonotic and causes significant morbidity and mortality for both humans and farm animals; most commonly, calves and lambs. A One Health approach that integrates human, animal and environmental health perspectives is required to tackle this disease. Current treatments are limited and ineffective, meaning there is an urgent need to develop new anti-cryptosporidials both for human and animal health. The neonatal calf model is a natural model of infection employed as a tool for drug discovery or generating parasite material. However, the model is seldom utilised to investigate host-parasite interaction. Fundamental information about this model, including the location of the parasite in the gut, is lacking. It is also unclear how the more commonly utilised immunocompromised mouse models of cryptosporidiosis compare to the neonatal calf model. To address this, we established an acute, moderate experimental C. parvum infection in neonatal calves. Using transgenic parasites, we created a tissue atlas of infection for neonatal calf gut and immunocompromised mouse models and mapped and quantified infection to draw robust comparisons between models. Cryptosporidium infection was observed at high levels throughout the neonatal calf gastrointestinal tract and was not limited to the ileal-cecal junction, as previously suggested. This infection pattern is most similar to the acute cryptosporidiosis mouse model, interferon-gamma knockout mice (IFNγKO). Infection with transgenic parasites allowed us to perform in vivo and ex vivo tissue imaging of the chronic cryptosporidiosis mouse model, NOD SCID Gamma KO (NSG) mice. In contrast, in NSG mice infection is low in the small intestines and highest in the caecum and colon. Understanding the true distribution of infection in the gastrointestinal tract of these three key animal models provides new perspectives on how to interpret and design drug efficacy studies and provides new insight into host-pathogen interaction. ### Competing Interest Statement Huvepharma donated Parofor Crypto for propagation studies, namely passage 1 and 2 in neonatal calves. They sponsored FK to speak about Cryptosporidium at 2 events and a webinar organised by Huvepharma but neither the company nor their employees had any input in the study design, interpretation of the results for any studies, and were not involved in the preparation of this manuscript. Royal Society, https://ror.org/03wnrjx87, 213469/Z/18/Z Wellcome Trust, https://ror.org/029chgv08, 213469/Z/18/Z, 203134/Z/16/Z, 223608/Z/21/Z, 223952/Z/21/Z, 204816/Z/16/Z UKRI Biotechnology and Biological Sciences Research Council (BBSRC), BB/T00875X/1 UKRI Medical Research Council (MRC), MR/R015791/1 Scottish Government, Rural and Environment Science and Analytical Services Division (RESAS), Underpinning National Capacity and the Moredun Foundation

Cryptosporidium spp. and Giardia duodenalis are zoonotic pathogens that cause enteric disease in humans and animals. This study aimed to identify the species/assemblages of Cryptosporidium and Giardia duodenalis in Scottish wild deer populations. Faecal samples (n = 990) were collected from across mainland Scotland from red deer, roe deer, sika deer and fallow deer. The oocysts/cysts were concentrated, DNA was extracted and then analysed by PCR (Cryptosporidium SSU-rRNA and Giardia duodenalis β-giardin, glutamate dehydrogenase (gdh), triosephosphate isomerase (tpi) DNA). All positive PCR amplicons were sequenced to determine Cryptosporidium species and Giardia assemblage. Overall, 12.3 % (122/990) of samples were positive for Cryptosporidium and identified C. ryanae (77/122), C. parvum (22/122) and C. ubiquitum (13/122). Rates of Cryptosporidium infection varied significantly (P = 0.009) between the deer species. Cryptosporidium was also significantly (P = 0.001) more likely to be detected in male deer compared to females and in seasons with high rainfall (P = 0.002). Giardia was detected in 7.9 % (76/962) samples, and sequencing identified assemblage A (62/76), assemblage B (4/76), assemblage D (2/76) and assemblage E (5/76). The assemblages of 3/76 positive samples were unidentifiable. Detection of Giardia was significantly higher (P < 0.001) in roe deer than the other deer species. During this study we also demonstrate that deer calves had significantly higher rates of infection for both Cryptosporidium (P = 0.001) and Giardia (P < 0.001), than other age groups. Co-infection with both pathogens was rare (14/962 samples). These findings confirm that wild Scottish deer harbour both Cryptosporidium and Giardia; however, their role in transmission to humans or livestock remains unclear.

Cryptosporidium spp., particularly Cryptosporidium parvum, pose a significant threat to raw water quality and public health. Cryptosporidiosis, a gastrointestinal zoonotic disease, causes diarrhoea in dairy and beef production systems worldwide. Infected calves shed Cryptosporidium spp. oocysts in faeces, posing risks of contaminating surface water sources. Understanding the dynamics of Cryptosporidium contamination is crucial for effective water quality management. This study investigated Cryptosporidium spp. prevalence in neonatal calves and its potential impact on water quality before abstraction and treatment. The study analysed faecal samples from 1-3-week-old calves on two English dairy farms upstream of a water abstraction point. Initial screening used C. parvum immune chromatographic assays (ICT) on 47 faecal samples. This was followed by DNA extraction and species identification, with gp60 subtyping. Raw and treated water quality data were analysed to determine Cryptosporidium oocyst counts. Cryptosporidium parvum was the predominant species in calves at both farms, with gp60 subtype IIaA17G2R1 being the only subtype detected. Some calf samples revealed mixed infections with C. parvum and C. ryanae. Raw-water samples (n = 214) revealed a 50.00% positivity rate for Cryptosporidium oocysts, with 22.43% (24/107) containing C. parvum/C. hominis and the remainder 77.57% (83/107) C. andersoni. Water treatment significantly reduced oocyst counts (P < 0.001); however, due to the increase in popularity of bathing in rivers, oocysts present in raw river water may still prove a public health risk.

The objective of this study was to characterise a Toxoplasma gondii-induced abortion outbreak on a goat farm in the State of Paraíba, Northeast Region of Brazil. From a herd of 10 does, seven experienced abortions and one gave birth to twins (one stillborn and the other weak and underdeveloped). Serum samples from all of the does were analysed by indirect fluorescent antibody test (IFAT). Samples of colostrum and placenta from two does, along with lung, heart, brain and umbilical cord samples from four of the foetuses, were screened by nested ITS1 PCR specific for T. gondii. The positive samples were then analysed by multiplex nested PCR-RFLP. All ten does tested positive by IFAT for anti-T. gondii IgG (titrations ranging from 1:4096 to 1:65,536). The ITS1 PCR screening revealed T. gondii DNA in the placenta (2/2), colostrum (2/2), umbilical cord (2/4), lung (1/4), heart (1/4), and brain (1/4). Four samples produced complete RFLP genotyping results, identifying a single genotype, ToxoDB #13. In conclusion, we demonstrated a high rate of abortion caused by T. gondii in a goat herd, highlighting the pathogenicity of genotype #13, one of the most prevalent genotypes of T. gondii in Brazil.

This study aimed to genotype isolates of Toxoplasma gondii obtained from samples of brain, diaphragm and heart of goats and sheep intended for human consumption in the State of Paraíba, Brazil. Tissue samples from 14 animals, goats (n = 5) and lambs (n = 9), were sourced from public slaughterhouses in seven cities and bio-assayed in mice. The brains of the mice were utilized for DNA extraction. Genotyping was carried out by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) using 10 markers (SAG1, SAG2, SAG3, BTUB, c22-8, PK1, GRA6, L358, c-29-2 and Apico). A total of 10 isolates were fully genotyped (i.e. at all loci), three from goats and seven from sheep, revealing five distinct genotypes: #13 (n = 4); #48 (n = 3); #57 (n = 1); #273 (n = 1); and one new genotype that had not been previously described. Genotype #13 is frequently found in the Northeast of Brazil and represents a clonal lineage circulating in this region and was the most prevalent genotype identified (n = 4). Moreover, in the present study genotypes #13, #48, #57, and #273 were documented for the first time in sheep from Brazil, and the novel genotype was isolated from a goat. Our findings align with previous studies on T. gondii from Brazil, where new genotypes are continuously being identified, highlighting a high level of genetic diversity of T. gondii isolates in the country.

Cryptosporidium parvum is a causative agent of cryptosporidiosis, an infectious gastroenteritis in neonatal ruminants, which can be fatal in severe cases. The aim of this study was to determine the prevalence of infections in dairy cattle/calves during the early stages of a calving season and the species/genotypes of the Cryptosporidium present. Faecal samples collected from pre- and post-partum dams (n = 224) as well as calves from age ∼1 day onwards (n = 312) were examined. Oocysts were concentrated, DNA extracted and tested by Cryptosporidium 18S rRNA gene PCR and sequencing, while genotypes of C. parvum were determined by gp60 and VNTR analysis. Results showed that 31.3% and 30.4% of pre- and post-partum dams tested positive for Cryptosporidium, respectively. In the adults, C. parvum (n = 52), C. bovis (n = 4) and C. andersoni (n = 19) were identified, while in the calves 248 out of 312 (79.5%) were PCR-positive for C. parvum. The proportion of positive calf samples was significantly higher (P < 0.0001) than the proportion of positive adult cattle during the first seven weeks of the calving season. In adult cattle, three distinct gp60 genotypes were identified, a predominant genotype IIaA15G2R1 (n = 36) and genotypes IIaA15R1 (n = 2) and IIaA14G2R1 (n = 1). In the calves, only genotype IIaA15G2R1 was detected (n = 125). Although C. parvum was observed in adult cattle two weeks after the start of the calving season, the predominant genotypes were not detected until Week 4 in both adults and calves, meaning it is still unclear whether adult cattle are the initial source of C. parvum infections on the farm. Historically calves on this dairy farm demonstrated the IIaA19G2R1 genotype, which, has now clearly been replaced with the IIaA15G2R1 genotype that is now found in both adults and calves. During the study season, significantly higher levels of neonatal calf mortality were observed compared to the seasons before (P = 0.046) and after (P = 0.0002). This study has shown comparable levels of C. parvum infection in both pre- and post-partum dams but higher levels of infection in neonatal calves.

Ovine toxoplasmosis caused by Toxoplasma gondii is a zoonotic disease responsible for triggering reproductive problems in sheep with great public health impact. The zoonotic risk for humans occurs through the ingestion of meat contaminated with bradyzoites, water and vegetables contaminated with oocysts, or by ingesting unprocessed sheep milk contaminated with tachyzoites. The study aimed to determine the seroprevalence, isolate, and genetically characterize T. gondii from sheep in northeastern Brazil. Blood and tissue samples, including the liver, heart, brain, lung, and diaphragm, were collected from 256 sheep in slaughterhouses. The presence of anti-T. gondii IgG antibodies was assessed by the indirect fluorescent antibody test (IFAT), and sera with titers ≥ 1: 64 were considered positive. Tissue samples from seropositive sheep were digested in acidic pepsin solution and inoculated into two Swiss mice for T. gondii isolation and further characterized by molecular methods. The prevalence of anti-T. gondii antibodies in sheep was 31.64

This study aimed to evaluate the presence and viability of Toxoplasma gondii in chickens intended for human consumption in the Pernambuco State, Brazil. Blood and tissue samples were collected from 25 chickens sold in markets in Recife, Pernambuco. Samples were evaluated by indirect immunofluorescence assay (IFA) to detect antibodies to T. gondii. Pools of brain and heart of seropositive chickens were subjected to bioassay in two Swiss Webster mice, which were evaluated for 45 days then tested by IFA to detect seroconversion. The mice were euthanized, and their brains were evaluated for cysts. Peritoneal lavage was also conducted in mice that exhibited clinical signs. Brains containing cysts or peritoneal lavage with tachyzoites were inoculated into MA-104 cells. Brains of mice inoculated with the same tissue were pooled and analysed by ITS1-PCR. We obtained a frequency of antibodies to T. gondii of 68.00% (17/25) in chickens, and a seroconversion rate of 70.58% (24/34) in mice. Detection of Toxoplasma ITS1 DNA confirmed an isolation rate of 41.1% (7/17). Three isolates were characterized by mnPCR-RFLP as genotypes ToxoDB#36 and ToxoDB#114. We highlight the occurrence of ToxoDB#36 in chickens in Pernambuco State and the parasitesʼ viability in chickens intended for human consumption.

A wide spectrum of disease severity associated with cryptosporidiosis has been described, ranging from asymptomatic to fatal in both human and animal hosts. The reasons for the variations in severity are likely to be multifactorial, involving environmental, host and parasite factors. This paper describes two experimental infection trials in lambs, a symptomatic host for the parasite, to investigate variation in the clinical manifestations following infection with two distinct isolates of Cryptosporidium parvum. In the first experiment, groups of naïve lambs were challenged with one of two isolates (CP1 or CP2) at ​< ​1 week of age, to test the effect of the isolates on disease outcome. In a second experiment one group of lambs challenged at < 1 week of age (CP1) was then re-challenged with the same isolate at 6 weeks of age (CP1), while a second group was challenged for the first time at 6 weeks of age (CP1). This experiment examined age-related disease symptoms, oocyst shedding and the effect of prior exposure to the parasite on a subsequent homologous challenge. The two isolates were associated with significant differences in the demeanour of the animals and in the numbers of oocysts shed in the faeces. There were also differences in the duration and severity of diarrhoea, though these were not significant. The age of the lamb, at the time of a primary challenge (<1 week or 6 weeks), also resulted in differences in clinical outcomes, with younger lambs showing more severe clinical disease than the older lambs (feeding profiles and presentation of diarrhoea), while older lambs showed virtually no signs of infection but still produced large numbers of oocysts.

Eosinophilic myositis in bovine striated muscle thought to be caused by a hypersensitivity reaction to the degradation of Sarcocystis tissue cysts, is a rare reason for carcase condemnation in the United Kingdom. This paper describes the identification of Sarcocystis cruzi associated with lesions of generalised eosinophilic myositis in three English beef carcases, by gross and histopathological examination followed by PCR with subsequent sequencing. Samples from two unaffected animals were also examined. Although sarcocystosis caused by S.cruzi is not considered a public health risk, the clinically affected carcases were deemed unfit for human consumption due to the extensive lesions affecting meat quality. We believe this to be the first report from the UK describing the molecular-based identification of Sarcocystis cruzi in meat affected and unaffected with eosinophilic myositis.

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.

Background: A rescued, captive, adult, male, inland bearded dragon (Pogona vitticeps) was presented with chronic swelling of the right upper and lower eyelids, with intermittent blepharospasm. Case description: Histopathology revealed a proliferative conjunctivitis, including inflammation of the nictitating membrane, along with a mixed, chronic, inflammatory response. Located on the epithelial surface, were numerous, surface oriented apicomplexan organisms, histologically compatible with Cryptosporidium sp. Molecular characterisation revealed these organisms to be most closely related to C. avium based on amplification and sequencing of the 18S rRNA gene. Conclusions and case relevance: This report describes the diagnosis of spontaneous conjunctivitis involving Cryptosporidium sp. in a reptile; C. avium has not been implicated previously in extra-enteric disease in a non-avian species. Infection by C. avium has only once been reported in lizards and this study provides further evidence that its host range may be broader than anticipated. Cryptosporidiosis should be considered as a differential diagnosis for refractory conjunctivitis in lizards.

Neospora caninum is a commonly diagnosed cause of reproductive losses in farmed ruminants worldwide. This study examined 495 and 308 samples (brain, heart and placenta) which were collected from 455 and 119 aborted cattle and sheep fetuses, respectively. DNA was extracted and a nested Neospora ITS1 PCR was performed on all samples. The results showed that for bovine fetuses 79/449 brain [17.6% (14.2-21.4)], 7/25 heart [28.0% (12.1-49.4)] and 5/21 placenta [23.8% (8.2-47.2)] were PCR positive for the presence of Neospora DNA. Overall 82/455 [18.0% (14.6-21.7)] of the bovine fetuses tested positive for the presence of N. caninum DNA in at least one sample. None (0/308) of the ovine fetal samples tested positive for the presence of Neospora DNA in any of the tissues tested. The results show that N. caninum was associated with fetal losses in cattle (distributed across South-West Scotland), compared to sheep in the same geographical areas where no parasite DNA was found. Neospora is well distributed amongst cattle in South-West Scotland and is the potential cause of serious economic losses to the Scottish cattle farming community; however, it does not appear to be a problem amongst the Scottish sheep flocks.

This study aimed to determine the prevalence and assemblages of Giardia duodenalis present in Scottish beef and dairy cattle at different ages, to try to ascertain if cattle could play a role in the spread of zoonotic assemblages of Giardia. A total of 388 fecal samples (128 beef and 253 dairy, seven of unknown breed) were collected from 19 farms in Scotland. Samples were sub-divided by host age, 1, 2, 3, 4, 5 and 6, 7-24 and > 25 weeks. DNA was extracted and tested by PCR to detect G. duodenalis DNA. Of the 388 samples, 126 tested positive, giving an overall prevalence of 32.5%, with positive samples being observed in all age groups tested. The prevalence in dairy cattle was 44.7% (113/235), which was significantly higher (P < 0.001) than the prevalence in beef cattle 10.1% (13/128). Sequence analysis demonstrated the presence of assemblage E (77.2%, sequence types E-S1-E-S5), assemblage B (18.2%) and assemblage A (sub-assemblages AI-AII) (4.6%). These data demonstrate that G. duodenalis is found routinely in both dairy and beef cattle throughout Scotland; the presence of assemblages A and B also indicates that cattle may play a role in the spread of potentially zoonotic assemblages of Giardia.

BACKGROUND:Toxoplasma gondii is a zoonotic parasite of global importance. The outcome of infection in humans can depend on a number of factors including the infecting stage of the parasite, inoculating dose and virulence of the infecting strain. Molecular epidemiological studies have demonstrated an abundance of atypical strains of T. gondii in South America, many of which have been associated with more severe sequelae of infection. The aim of this study was to compare the virulence of T. gondii strains isolated in the Caribbean to a virulent Brazilian strain and an avirulent European strain.METHODS:One hundred and twenty Swiss CD-1 mice were split into 8 groups of 15 mice and each group was inoculated with 200 tachyzoites of one of 8 isolates, comprising ToxoDB genotypes #1, #141, #265, #13, #3 and #6. Five mice per group were euthanized at day 8 post-inoculation (p.i.) and parasite burden was determined in heart, lungs and eyes using quantitative PCR. Lungs and brain were also examined by histopathology and immunohistochemistry. The remaining 10 mice per group were part of a survival experiment to assess virulence. DNA was extracted from tachyzoites of each of the 8 T. gondii isolates and genotyped at four ROP gene loci, including ROP5, ROP16, ROP17 and ROP18 to look for association with markers of virulence.RESULTS:Infection with ToxoDB genotype #13 from the Caribbean resulted in 100% of mice being euthanized which was comparative to infection with the virulent Brazilian strain (ToxoDB genotype #6). Significantly higher parasite burdens were recorded in the lungs and eyes of mice infected with ToxoDB genotypes #13 and #6. Genotyping of ROP loci revealed that the virulent Caribbean isolates had a different ROP18/ROP5 allelic profile (3/1) to the virulent Brazilian isolate (1/3); however, the avirulent Caribbean isolate (ToxoDB genotype #1) had the same ROP18/ROP5 profile as the avirulent European isolate (ToxoDB #3) (both 2/2). Caribbean isolates of intermediate virulence (ToxoDB #141 and #265) all had the same ROP18/ROP5 allelic profile (2/2).CONCLUSIONS:Isolates from the Caribbean with ToxoDB genotype #13 were acutely virulent for mice and comparable to a known virulent Brazilian isolate. The ROP protein allelic profile of the virulent Caribbean and Brazilian isolates differed indicating that perhaps other factors are involved in predicting virulence. Understanding virulence is important for predicting disease outcome in humans and may also aid vaccine design as well as drug discovery.

Toxoplasma gondii has a worldwide distribution and can infect almost all warm blooded animals including pigs and humans. This study aims to examine the immune responses induced in pigs following vaccination (live S48 tachyzoites) and/or challenge with T. gondii oocysts, through the examination of changes in levels of transcription in CD4, CD8 alpha, IFN-gamma, IL-12p35, CXCR3, MyD88. The experiment involved four groups of animals; pigs in group 1 (Challenged) (Chal) were challenged orally with (1 x 10(3) oocysts) on day 28 of the experiment. Pigs in group 2 (Vaccinated /Challenged) (Vac/Chal) were vaccinated (S48 isolate tachyzoites) on day 0, then challenged on day 28. The group 3 (Vaccinated) (Vac) animals were vaccinated (S48 isolate tachyzoites) on day 0 of the experiment. Finally the group 4 (control) pigs remained non-vaccinated and non-challenged. All animals were culled 6 weeks post challenge. At post mortem samples of retropharyngeal lymph node (RLN), mesenteric LN (MLN) and spleen were collected, RNA was extracted and cDNA synthesised. The results showed significant increases in IFN-gamma expression in samples from groups 1 (Chal) and 2 (Vac/Chal) (RLN) and groups 1, 2 and 3 (Vac) (spleen) and in MyD88 expression (RLN) in samples from groups 1, 2 and 3 compared to the group 4 (control) animals. Significant increases were also observed in CD8 alpha expression in group 1 (Chal) (RLN) and groups 1 and 2 (Vac/Chal) (RLN and MLN) compared against group 4 (control) and group 3 (Vac) respectively. Conversely, significant down regulation of CD4 and/or IL-12p35 transcription was found in at least one sample from groups 1 (Chal), 2 (Vac/Chal) and 3 (Vac) compared to group 4 (control) pigs. This study demonstrates that cell mediated and innate immune responses are generated in pigs following exposure to T. gondii parasites (oocysts or tachyzoites), key amongst them appear to be IFN-gamma, MyD88 and CD8 alpha.

Background: Although the detection of Toxoplasma gondii in bovine tissues is rare, beef might be an important source of human infection. The use of molecular techniques, such as magnetic capture qPCR (MC-qPCR), in combination with the gold standard method for isolating the parasite (mouse bioassay), may increase the sensitivity of T. gondii detection in infected cattle. The risk of transmission of the parasite to humans from undercooked/raw beef is not fully known and further knowledge about the predilection sites of T. gondii within cattle is needed. In the current study, six Holstein Friesian calves (Bos taurus) were experimentally infected with 106 T. gondii oocysts of the M4 strain and, following euthanasia (42 dpi), pooled tissues were tested for presence of the parasite by mouse bioassay and MC-qPCR. Results: Toxoplasma gondii was detected by both MC-qPCR and mouse bioassay from distinct pools (100 g) of tissues comprising: liver, tongue, heart, diaphragm, semitendinosus (hindlimb), longissimus dorsi muscle (sirloin) and psoas major muscle (fillet). When a selection of individual tissues which had been used for mouse bioassay were examined by MC-qPCR, parasite DNA could only be detected from two animals, despite all calves showing seroconversion after infection. Conclusions: It is apparent that one individual test will not provide an answer as to whether a calf harbours T. gondii tissue cysts. Although the calves received a known number of infectious oocysts and highly sensitive methods for the detection of the parasite within bovine tissues were applied (mouse bioassay and MC-qPCR), the results confirm previous studies which report low presence of viable T. gondii in cattle and no clear predilection site within bovine tissues.

The work describes a case of Sarcocystis gigantea infection in a 3-years-old Corriedale ewe from Buenos Aires Province, Argentina. The ewe was found dead with a poor body condition. Pathological and molecular studies were carried out in order to try and confirm the causative agent of the infection. At necropsy, approx. 100 whitish elliptic (3-5 mm to 5-8 mm) macrocysts with a hard consistency were observed along the esophageal and pharyngeal muscular layers. Microscopically, the macrocysts consisted of an eosinophilic wall, internal septa originated from the eosinophilic wall and basophilic parasitic cells were located among the septa. The sarcocysts were identified molecularly through PCR amplification and sequencing of a short segment of the 18S rRNA gene. Sequence analysis of the amplified DNA demonstrated 100% identity to S. gigantea sequences previously published. To our knowledge this is the first molecular confirmation of S. gigantea infection in sheep in the Americas.