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 8­11 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.

Detection and dissemination of Toxoplasma gondii in experimentally infected calves, a single test does not tell the whole story.

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.

Occurrence of abortions induced by Neospora caninum in dairy cattle from Santa Catarina, southern Brazil / Ocorrência de abortamentos induzidos por Neospora caninum em vacas de leite de Santa Catarina, sul do Brasil

Abstract The aim of the present study was to investigate the occurrence of N. caninum associated with abortions of dairy cattle from Santa Catarina state, southern Brazil by using enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (IHC), and polymerase chain reaction (PCR). Blood from dairy cows that aborted along with intrathoracic fluid and tissue samples (brain, heart, liver, and lung) from their fetuses were collected and used for serology; PCR, histopathological, and immunohistochemistry (IHC) evaluations were also conducted. Twenty-one cows (51.2%) out of 41, and eight fetuses (26.7%) out of 30 were ELISA (HerdCheck, IDEXX) positive for N. caninum. Dams > 36 months of age had a higher risk of being serum positive than younger animals. PCR and IHC revealed that 38.8% (14/36) and 25.0% (9/36) of the fetuses were positive for N. caninum, respectively for each of the tests. Seropositive cows had a higher frequency of fetuses that were also positive by either intrathoracic fluid, PCR, or IHC. In summary, the present study observed a high frequency of N. caninum in abortions from dairy cows from southern Brazil, with a higher N. caninum prevalence found in cows that were older than 36 months. In addition, serology, PCR, and IHC should be used all together for better diagnosis of neosporosis in cattle.

Resumo O objetivo deste estudo foi avaliar a ocorrência de N. caninum associado a abortamentos em vacas de leite do estado de Santa Catarina, sul do Brasil pelo uso das técnicas de ELISA (HerdCheck, IDEXX), reação em cadeia pela polimerase (PCR) e imunohistoquímica (IHC). O sangue das vacas leiteiras que abortaram, bem como, o líquido intratorácico e amostras de tecidos (cérebro, coração, fígado e pulmão) de seus fetos foram coletados e usados para sorologia, PCR (Np21+ e Np6+), e IHC. Vinte e uma vacas (51,2%) de um total de 41, bem como, oito fetos (26,7%) de um total de 30 foram positivos no ELISA (IDEXX) para N. caninum. As vacas > 36 meses de idade tiveram um maior risco de serem soropositivas do que os animais mais jovens. PCR e IHC revelaram que 38,8% e 25,0% dos fetos foram positivos para N. caninum, respectivamente para cada um dos testes. As vacas soropositivas tiveram uma maior frequência de fetos que também foram positivos no fluído intratorácico, na PCR ou na IHC. Em resumo, o presente estudo observou uma alta frequência de N. caninum em abortos de vacas leiteiras na região estudada, com maior prevalência de N. caninum em vacas com mais de 36 meses de idade. Além disso, sorologia, PCR e IHC deveriam ser utilizadas ​​conjuntamente para melhor diagnóstico de neosporose em bovinos.

Vaccination of pigs with the S48 strain of Toxoplasma gondii--safer meat for human consumption.

As clinical toxoplasmosis is not considered a problem in pigs, the main reason to implement a control strategy against Toxoplasma gondii (T. gondii) in this species is to reduce the establishment of T. gondii tissue cysts in pork, consequently reducing the risk of the parasite entering the human food chain. Consumption of T. gondii tissue cysts from raw or undercooked meat is one of the main sources of human infection, with infected pork being considered a high risk. This study incorporates a mouse bioassay with molecular detection of T. gondii DNA to study the effectiveness of vaccination (incomplete S48 strain) in its ability to reduce tissue cyst burden in pigs, following oocyst (M4 strain) challenge. Results from the mouse bioassay show that 100% of mice which had received porcine tissues from vaccinated and challenged pigs survived compared with 51.1% of mice which received tissues from non-vaccinated and challenged pigs. The presence (or absence) of T. gondii DNA from individual mouse brains also confirmed these results. This indicates a reduction in viable T. gondii tissue cysts within tissues from pigs which have been previously vaccinated with the S48 strain. In addition, the study demonstrated that the main predilection sites for the parasite were found to be brain and highly vascular muscles (such as tongue, diaphragm, heart and masseter) of pigs, while meat cuts used as human food such as chop, loin, left tricep and left semitendinosus, had a lower burden of T. gondii tissue cysts. These promising results highlight the potential of S48 strain tachyzoites for reducing the number of T. gondii tissues cysts in pork and thus improving food safety.

Immunization of lambs with the S48 strain of Toxoplasma gondii reduces tissue cyst burden following oral challenge with a complete strain of the parasite.

This study evaluates the influence of immunizing lambs with the incomplete S48 strain of Toxoplasma gondii, on parasite dissemination following a live oral challenge with a complete strain of T. gondii (M4). Lambs were culled at 14, 28 and 42 days post challenge. Parasite DNA was detected at significantly (p<0.0001) lower levels in samples from the vaccinated/challenged group (0% in heart and 5.9% in skeletal muscles), when compared to the non-vaccinated/challenged animals (75% heart, 87.9% skeletal muscle). S48 T. gondii DNA was found in muscle or lymph nodes until 42 days post infection, suggesting that parasite DNA or tachyzoites could persist longer after immunization than previously thought. Non-vaccinated/challenged animals showed more frequent lesions in muscles and central nervous system than the vaccinated animals. These results demonstrate that vaccination of lambs with the incomplete S48 T. gondii strain, can protect against establishment of tissue cysts following challenge with a complete strain of T. gondii. Consumption of undercooked meat containing T. gondii cysts is a major route of transmission to people, therefore vaccination of food animals may improve the safety of meat for human consumption.

Production of a bovine IL-12p40 probe and application using in situ hybridization on ruminant fixed tissues.

Pro-inflammatory cytokines (particularly IL-12) are important for initiating protective T helper 1 (Th1)-type immune responses and hence vital for combating intracellular infections and tumours. In situ hybridization (ISH) provides a powerful diagnostic tool allowing the identification and localization of cells producing these mediators in fixed tissues. The objective of this work was to produce a bovine IL-12p40 probe that allows detection of IL-12p40 mRNA in fixed tissues from different ruminant species. The RNA probe sequence is 447bp in length and from a region with high cross-species-sequence homology (>97.3% homology) to the ovine, cervine, caprine and bubaline IL-12p40 genes. ISH was carried out on paraformaldehyde fixed tissues collected from cattle, sheep and goats. The probe was efficient in identifying IL-12p40 expressing cells in fixed tissues from all these species. In conclusion, the IL-12p40 probe was efficient in identifying and localizing cells that express IL-12p40, and provides a good immuno-diagnostic technique to characterize immune responses in fixed tissues.

Factors influencing temporal variation of scrapie incidence within a closed Suffolk sheep flock.

Several studies have shown that transmission of natural scrapie can occur vertically and horizontally, and that variations in scrapie incidence between and within infected flocks are mostly due to differences in the proportion of sheep with susceptible and resistant PRNP genotypes. This report presents the results of a 12-year period of scrapie monitoring in a closed flock of Suffolk sheep, in which only animals of the ARQ/ARQ genotype developed disease. Among a total of 120 of these, scrapie attack rates varied between birth cohorts from 62.5 % (5/8) to 100 % (9/9), and the incidence of clinical disease among infected sheep from 88.9 % (8/9) to 100 % (in five birth cohorts). Susceptible sheep born to scrapie-infected ewes showed a slightly higher risk of becoming infected (97.2 %), produced earlier biopsy-positive results (mean 354 days) and developed disease at a younger age (median 736 days) than those born to non-infected dams (80.3 %, 451 and 782 days, respectively). Taken together, this was interpreted as evidence of maternal transmission. However, it was also observed that, for the birth cohorts with the highest incidence of scrapie (90-100 %), sheep born to infected and non-infected dams had a similar risk of developing scrapie (97.1 and 95.3 %, respectively). Compared with moderate-attack-rate cohorts (62.5-66.7 %), high-incidence cohorts had greater numbers of susceptible lambs born to infected ewes, suggesting that increased rates of horizontal transmission in these cohorts could have been due to high levels of environmental contamination caused by infected placentas.

Urinary alpha1-antichymotrypsin: a biomarker of prion infection.

The occurrence of blood-borne prion transmission incidents calls for identification of potential prion carriers. However, current methods for intravital diagnosis of prion disease rely on invasive tissue biopsies and are unsuitable for large-scale screening. Sensitive biomarkers may help meeting this need. Here we scanned the genome for transcripts elevated upon prion infection and encoding secreted proteins. We found that alpha(1)-antichymotrypsin (alpha(1)-ACT) was highly upregulated in brains of scrapie-infected mice. Furthermore, alpha(1)-ACT levels were dramatically increased in urine of patients suffering from sporadic Creutzfeldt-Jakob disease, and increased progressively throughout the disease. Increased alpha(1)-ACT excretion was also found in cases of natural prion disease of animals. Therefore measurement of urinary alpha(1)-ACT levels may be useful for monitoring the efficacy of therapeutic regimens for prion disease, and possibly also for deferring blood and organ donors that may be at risk of transmitting prion infections.