Genotyping of European Toxoplasma gondii strains by a new high-resolution next-generation sequencing-based method.

PURPOSE: A new high-resolution next-generation sequencing (NGS)-based method was established to type closely related European type II Toxoplasma gondii strains. METHODS: T. gondii field isolates were collected from different parts of Europe and assessed by whole genome sequencing (WGS). In comparison to ME49 (a type II reference strain), highly polymorphic regions (HPRs) were identified, showing a considerable number of single nucleotide polymorphisms (SNPs). After confirmation by Sanger sequencing, 18 HPRs were used to design a primer panel for multiplex PCR to establish a multilocus Ion AmpliSeq typing method. Toxoplasma gondii isolates and T. gondii present in clinical samples were typed with the new method. The sensitivity of the method was tested with serially diluted reference DNA samples. RESULTS: Among type II specimens, the method could differentiate the same number of haplotypes as the reference standard, microsatellite (MS) typing. Passages of the same isolates and specimens originating from abortion outbreaks were identified as identical. In addition, seven different genotypes, two atypical and two recombinant specimens were clearly distinguished from each other by the method. Furthermore, almost all SNPs detected by the Ion AmpliSeq method corresponded to those expected based on WGS. By testing serially diluted DNA samples, the method exhibited a similar analytical sensitivity as MS typing. CONCLUSION: The new method can distinguish different T. gondii genotypes and detect intra-genotype variability among European type II T. gondii strains. Furthermore, with WGS data additional target regions can be added to the method to potentially increase typing resolution.

Monitoring fibrillation in the mechanical production of lignocellulosic micro/nanofibers from bleached spruce thermomechanical pulp.

The present work aims at assessing the main characteristics of lignocellulosic micro/nanofibers (LCMNF) from bleached thermomechanical pulp (BTMP) from spruce while glimpsing the suitability of cationic demand (CD) as effective monitoring parameter of the fibrillation process. For this, BTMP was mechanically refined at different times in a Valley beater, aiming at determining the required refining time and fiber length to be later fibrillated in a high-pressure homogenizer. It was found that 150 min treatment is required to avoid clogging in the pressure chambers of the homogenizer. The mechanically treated BTMP was gradually passed through a high-pressure homogenizer, leading to four LCMNF with different fibrillation degree. The main characteristics of the LCMNF were determined, as well as the effect that high-pressure homogenization may generate onto the LCMNF structure. It was observed that CD is a robust parameter to monitor the fibrillation process, as it is a good indicator of the LCMNF characteristics. In addition, it was found that WRV may not be a good indicator of the extent of fibrillation for LCMNF, as the lignin content varies with the homogenization intensity. Finally, the limitations of CD as monitoring parameter and perspectives on this regard are provided to the reader.

Correlations Between Clinicopathological Features and the Vegetative Nervous System in Gastric Cancer.

Gastric cancer currently represents one of the most important public health problems. Recent studies have demonstrated the existence of strong correlations between the vegetative nervous system and the role it plays in the initiation of the oncogenetic process and the progression of cancer. The purpose of this paper is to demonstrate the involvement of the sympathetic and parasympathetic vegetative nervous system in the evolution of gastric cancer, according to the stage of tumor differentiation. In this current paper we have included a number of four patients diagnosed with gastric cancer post UGI (Upper Gastrointestinal Endoscopy) and have analyzed relations that exist between the tumor differentiation degree and the metanephrine and normetanephrine serum level in the blood of the patients. Following the research, we have observed an increased value of the metanephrine and normetanephrine serum level in the patient which displayed the lowest degree of differentiation.

Metabolic and tissular effects of artemisinin supplemented diets in broiler chicken.

Artemisinin is a powerful antimalarial drug, useful in the treatment of many diseases, including chickens coccidiosis. Its toxic effects have been well studied in humans and experimental animals, but not sufficiently in broiler chickens. Therefore, in the present study, we aimed to assess the side effects of artemisinin in chickens, by measuring the serum level of proteins and enzymes (ALT, AST, GGT, ALP, CK), by histopathological examination and by the evaluation of relative weight of organs (liver, kidney, heart). Artemisinin was administered in the standard feed for chickens in three different concentrations: 5, 50 and 500 ppm. Each concentration of artemisinin increased the total serum proteins, gamma-globulins and the serum activity of CK and decreased the serum ALP level. The values of ALT and GGT were higher in the chickens treated with 50 and 500 ppm of artemisinin. Multifocal liver necrosis and inflammatory infiltrate were detected in the chickens that received the 50 and 500 ppm dosage of artemisinin. Minimal tubular necrosis, renal tubular epithelium vacuolation, multifocal interstitial nephritis and mild uric nephrosis were detected in chickens treated with the drug. Artemisinin administration produced no significant changes in the organs relative weight. Artemisinin, at a concentration of 5 mg/kg of feed is well tolerated by broiler chickens, but the concentrations of 50 and 500 mg/ kg feed can produce toxic effects.

The relationship between the presence of antibodies and direct detection of Toxoplasma gondii in slaughtered calves and cattle in four European countries.

In cattle, antibodies to Toxoplasma gondii infection are frequently detected, but evidence for the presence of T. gondii tissue cysts in cattle is limited. To study the concordance between the presence of anti-T. gondii IgG and viable tissue cysts of T. gondii in cattle, serum, liver and diaphragm samples of 167 veal calves and 235 adult cattle were collected in Italy, the Netherlands, Romania and the United Kingdom. Serum samples were tested for anti-T. gondii IgG by the modified agglutination test and p30 immunoblot. Samples from liver were analyzed by mouse bioassay and PCR after trypsin digestion. In addition, all diaphragms of cattle that had tested T. gondii-positive (either in bioassay, by PCR on trypsin-digested liver or serologically by MAT) and a selection of diaphragms from cattle that had tested negative were analyzed by magnetic capture quantitative PCR (MC-PCR). Overall, 13 animals were considered positive by a direct detection method: seven out of 151 (4.6%) by MC-PCR and six out of 385 (1.6%) by bioassay, indicating the presence of viable parasites. As cattle that tested positive in the bioassay tested negative by MC-PCR and vice-versa, these results demonstrate a lack of concordance between the presence of viable parasites in liver and the detection of T. gondii DNA in diaphragm. In addition, the probability to detect T. gondii parasites or DNA in seropositive and seronegative cattle was comparable, demonstrating that serological testing by MAT or p30 immunoblot does not provide information about the presence of T. gondii parasites or DNA in cattle and therefore is not a reliable indicator of the risk for consumers.

Reproductive behaviour of couples at risk for sickle cell disease in Cuba: a follow-up study.

Three hundred and forty-three Cuban women at risk of having children with sickle cell anaemia or sickle cell-haemoglobin C disease were interviewed 2-8 years after the index pregnancy (that in which their risk was detected). The aim was to collect information on their attitude towards prenatal diagnosis in subsequent pregnancies. Twenty-two per cent (75/343) had dissolved their marriage and 9 per cent of these (7/75) considered that their at-risk status had influenced the separation. Sixty-three per cent of stable couples at risk (168/268) had decided to have no further children; 27 per cent of these (45/168) were afraid of having an affected child and 4 per cent (7/168) were afraid of the obstetric procedure. Nineteen per cent of the stable couples at risk (52/268) had had at least one further child or pregnancy. Of these, 44 per cent (23/52) requested prenatal diagnosis early and spontaneously, and a further 44 per cent (23/52) requested prenatal diagnosis but after re-identification by screening and recounselling. Only 12 per cent (6/52) did not request prenatal diagnosis. Attitude towards prenatal diagnosis was most positive among more educated women. The general perception of the prevention programme was good.