Enhancing chlamydospore production in Duddingtonia flagrans on solid substrate: The impact of mannitol and varied cultivation conditions.

Duddingtonia flagrans is a nematophagous fungus which has shown promising results as a non-chemical parasitic control tool. The fungus disrupts the parasite's life cycle by trapping larvae in the environment through the networks generated from chlamydospores, thus preventing the reinfection of animals. One barrier to the development of a commercial product using this tool is the need to increase chlamydospore production in the laboratory for its administration to livestock. The purpose of this study was to evaluate the addition of mannitol to an enriched culture medium and the effect of adverse cultivation conditions on chlamydospore production. D. flagrans was cultivated on Petri dishes with corn agar for 4 weeks at 27 °C and 70% relative humidity (RH). Four groups were then formed, all with Sabouraud agar as a base, to which different growth inducers were added: GSA (glucose Sabouraud agar), GSA-MI (glucose Sabouraud agar + meso inositol), GSA-E (enriched glucose Sabouraud agar), and AE-M (enriched agar + mannitol). After 4 weeks, chlamydospores were recovered by washing the surface of each plate with distilled water and then quantified. The medium that yielded the highest amount of chlamydospores was subjected to different cultivation conditions: NC (normal conditions): 70% RH and 27 °C, AC (adverse conditions) 1: 20% RH and 40 °C, CA2: 60% RH and 27 °C, and CA3: 55% RH and 24 °C. It was determined that mannitol increases chlamydospore production (65x106 chlamydospores/plate), and when reducing humidity by 10% under cultivation conditions it resulted in an approximately 10% increase in chlamydospore production compared to the control group. These results suggest that the addition of polyols, as well as its cultivation under certain environmental conditions, can improve chlamydospore production on a laboratory scale.

Synergism of small molecules targeting VDAC with sorafenib, regorafenib or lenvatinib on hepatocarcinoma cell proliferation and survival.

Voltage dependent anion channels (VDAC) in the outer mitochondrial membrane regulate the influx of metabolites that sustain mitochondrial metabolism and the efflux of ATP to the cytosol. Free tubulin and NADH close VDAC. The VDAC-binding small molecules X1 and SC18 modulate mitochondrial metabolism. X1 antagonizes the inhibitory effect of tubulin on VDAC. SC18 occupies an NADH-binding pocket in the inner wall of all VDAC isoforms. Here, we hypothesized that X1 and SC18 have a synergistic effect with sorafenib, regorafenib or lenvatinib to arrest proliferation and induce death in hepatocarcinoma cells. We used colony formation assays to determine cell proliferation, and a combination of calcein/propidium iodide, and trypan blue exclusion to assess cell death in the well differentiated Huh7 and the poorly differentiated SNU-449 cells. Synergism was assessed using the Chou-Talalay method. The inhibitory effect of X1, SC18, sorafenib, regorafenib and lenvatinib was concentration and time dependent. IC50s calculated from the inhibition of clonogenic capacity were lower than those determined from cell survival. At IC50s that inhibited cell proliferation, SC18 arrested cells in G0/G1. SC18 at 0.25-2 IC50s had a synergistic effect with sorafenib on clonogenic inhibition in Huh7 and SNU-449 cells, and with regorafenib or lenvatinib in SNU-449 cells. X1 or SC18 also had synergistic effects with sorafenib on promoting cell death at 0.5-2 IC50s for SC18 in Huh7 and SNU-449 cells. These results suggest that small molecules targeting VDAC represent a potential new class of drugs to treat liver cancer.

Effect of macrocyclic lactones on nontarget coprophilic organisms: a review.

Macrocyclic lactones are frequently used dewormers in livestock farms around the world. Due to their wide spectrum of action against nematodes and arthropods and their practicality of application at very low doses, their use has become massive since their discovery. These compounds are eliminated in a large percentage in the feces of animals, causing adverse effects on coprophilic fauna. Several research groups around the world have been devoted to evaluating these effects on this fauna. The aim of this review is to register the adverse effects of the concentrations in which macrocyclic lactones are eliminated in the feces of domestic animals and the importance of the coprophilic and edaphilous fauna on the degradation of the feces of the animals. The documented data shows that the use of macrocyclic lactones has a high toxicological risk for the different species that colonize the dung, thus causing an adverse effect on its disintegration and its subsequent incorporation into the soil. Even so, more studies at the regional level and their standardization are necessary to make the comparison between different areas possible.

Development of a construct-based risk assessment framework for genetic engineered crops.

Experience gained in the risk assessment (RA) of genetically engineered (GE) crops since their first experimental introductions in the early nineties, has increased the level of familiarity with these breeding methodologies and has motivated several agencies and expert groups worldwide to revisit the scientific criteria underlying the RA process. Along these lines, the need to engage in a scientific discussion for the case of GE crops transformed with similar constructs was recently identified in Argentina. In response to this need, the Argentine branch of the International Life Sciences Institute (ILSI Argentina) convened a tripartite working group to discuss a science-based evaluation approach for transformation events developed with genetic constructs which are identical or similar to those used in previously evaluated or approved GE crops. This discussion considered new transformation events within the same or different species and covered both environmental and food safety aspects. A construct similarity concept was defined, considering the biological function of the introduced genes. Factors like environmental and dietary exposure, familiarity with both the crop and the trait as well as the crop biology, were identified as key to inform a construct-based RA process.

Rectal pre-treatment with ozonized oxygen (O3) aggravates clinic status in septic rats treated with amoxicillin/clavulanate.

INTRODUCTION: Despite the advanced antibiotic therapies, sepsis continues being a clinical entity with high morbidity and mortality. The ozone/oxygen mixture (O3/O2) has been reported to exhibit positive effects on immunity. The aim of our study was to analyze whether (O3/O2) combined with amoxicillin/clavulanate has any influence on the morbidity and mortality of septic rats. METHODS: We used 48 Sprague-Dawley rats randomly allocated to 6 groups (n=8): healthy (C), septic (I), healthy+ozone therapy (O3), septic+amoxicillin/clavulanate (AMC), septic+amoxicillin/clavulanate+ozone therapy (AMC/O3) and septic+ozone therapy (I/O3). O3/O2 was administered rectally at increasing O3 concentrations during 10 days prior to the onset of sepsis model (intraperitoneally injection of fecal material) or saline administration in healthy control rats. Later (post-inoculation), 3 days per week, O3 was also administered. Vital signs were recorded, and microbiological, hematological and histopathological studies were performed. RESULTS: The number of surviving animal/total was higher in AMC (8/8) than in AMC/O3 (4/8) p=0.077. The percentage of surviving animals with pneumonia was higher in AMC/O3 than in AMC (100% vs 37.5%). In dead animals, AMC/O3 rats had a significantly higher percentage of lesions: Cardiac lesions, pulmonary hemorrhages and pleuritis (100%) and serositis/peritonitis (75%). Only Escherichia coli (2 different biotypes) was isolated from blood and/or peritoneal fluid from all infected groups. A significant decrease in the percentage of band neutrophils from the surviviors belonging to AMC/O3vs AMC was observed (p<0.05). CONCLUSION: Rectal pre-treatment with O3/O2 aggravates clinic status in septic rats treated with amoxicillin/clavulanate.

Control and eventual eradication of Trypanosoma evansi infection in dromedary camels after an episodic outbreak in mainland Spain: an example in a non-endemic area.

In 2008, Trypanosoma evansi was detected on a camel farm in mainland Spain. The animals were isolated, confined in a closed stable, and treated twice with melarsamine (Cymelarsan(®), Merial, Lyon, France) with an interval of 1 month. Clinical and laboratory examinations by means of parasitological, serological, and molecular procedures (polymerase chain reaction (PCR)) were carried out regularly for 6 years. After the treatment, all parasitemic camels were cleared of parasites, and in the seropositive camels a progressive decrease in antibody levels was observed, with complete disappearance of antibodies between 15 and 21 months, except in one animal which showed doubtful Ag-Ab reaction at 21 months post treatment. In the next assessment, 6 months later, the diagnostic tests conducted on all animals had a negative result. The diagnostic and therapeutic tools recently developed against T. evansi will evidence new and alternative approaches after the parasite is detected, particularly if outbreak occurs in geographically localized areas in territories free of the disease.

Adenomatosis erosiva de pezón: reporte de un caso / Erosive adenomatosis of the nipple: a case report

La adenomatosis erosiva del pezón es un tumor raro y benigno que se origina a partir de los conductores lactíferos del pezón y afecta principalmente a mujeres de mediana edad. Clínicamente se presenta como eritema, costras, erosiones, ulceraciones o nódulos, con o sin exudación serosa o sanguinolenta, pudiendo ser asintomática o presentar prurito, sensibilidad o dolor. La biopsia de piel es necesaria para llegar al diagnóstico, pues clínicamente puede ser indistinguible de la enfermedad de Paget. Histológicamente, su principal diagnóstico diferencial es el de adenocarcinoma de mama. El tratamiento es usualmente quirúrgico. Presentamos el caso de un paciente de 32 años de edad con una lesión inicialmente eritematosa y luego costrosa en el pezón derecho de 8 años de evolución.

Erosive adenomatosis of the nipple is a rare and benign tumor of the lactiferous ducts of the breast that affects primarily middle-aged women. Erythema, crusts, erosions, ulcerations or nodules, with or without serous or bloody discharge, are common clinical findings; pruritus, tenderness or pain may occur or it can be asymptomatic. Since the lesions are clinically indistinguishable from PagetÆs disease of the breast, a skin biopsy is mandatory for diagnosis. Adenocarcinoma of the breast is the main histologically differential diagnosis. Surgical resection is the treatment of choice. We present such a case in a 32-year-old woman with an 8-year history that begun as an erythematous, crusty lesion on her right nipple.

Cross-sectional study on prevalence of Trypanosoma evansi infection in domestic ruminants in an endemic area of the Canary Islands (Spain).

Trypanosoma evansi is the most widely spread of the pathogenic African trypanosomes of animals. The disease (surra) was first diagnosed in the Canary Islands in a dromedary camel in 1997; thus, a control plan was implemented achieving the eventual eradication of T. evansi from most of the infected areas in the Archipelago. However, a little area remains still infected despite the use of the same control measures. To evaluate possible reservoirs in the area a representative sample of domestic ruminants was examined by serological, parasitological and molecular tests. Of a total of 1228 ruminants assessed, 61 (5%) were serologically positive (7 cattle, 21 goats, 33 sheep), but T. evansi could be demonstrated in none of them. According to FreeCalc assessment, cattle and goat populations would be free from disease; however, the results from sheep are not adequate to conclude that the population would be free from disease. As a conclusion, surveillance must be exercised on ruminant farms in the surroundings of the infected area in order to evaluate the possible extension of the disease and their potential role as reservoirs of T. evansi.

Morphological and biometrical features of Trypanosoma evansi isolates from an outbreak in mainland Spain.

According to several authors, Trypanosoma evansi is a monomorphic trypanosome found exclusively in slender intermediate forms, although additional studies have revealed that many strains present stumpy forms on rare occasions. In a recent T. evansi outbreak in mainland Spain, several atypical forms were observed in blood smear examinations. Molecular procedures were then necessary to confirm the causal agent. Morphological and biometric measures were taken to characterize the different forms of T. evansi. In contrast to published information, the results of this study would indicate that biometrically distinct T. evansi could also be found in the same farm and even in the same animal species. These data could be useful for many trypanosomes endemic areas of the world where molecular methods are not commonly available.

The role of wild rodents in the transmission of Trypanosoma evansi infection in an endemic area of the Canary Islands (Spain).

Trypanosoma evansi was diagnosed for the first time in camels in the Canary Islands in 1997. Several sanitary measures including treatment of infected animals were taken; however, nowadays a little area is still infected. In order to determine possible reservoirs 138 wild rodents were trapped, 64 of them in the infected farms and the remaining 74 in other areas. The captured species were Rattus rattus (24), Rattus norvegicus (69) and Mus musculus domesticus (45). Serological (CATT/T. evansi), parasitological (micro-Hematocrit Centrifugation technique and stained smears) and molecular (PCR) methods for T. evansi and T. lewisi were used as diagnostic methods. None of the examined rodents was positive for T. evansi; 18, however, showed motile trypanosomes at micro-Hematocrit Centrifugation technique and resulted positive for T. lewisi by PCR. The results would suggest that the studied rodent species would not play a relevant role in the epidemiology of T. evansi infection in Canaries.

Antimicrobial susceptibility of Enterococcus strains isolated from poultry faeces.

We have investigated the resistance of Enterococcus isolated from poultry faeces to antibiotics commonly used as therapy of enterococcal infections. Identification was made by the method of Facklam and Collins. Minimal inhibitory concentrations of penicillin, ampicillin, vancomycin and teicoplanin were determined and high level aminoglycoside resistance was investigated. Genes codifying high level aminoglycoside resistance (HLAR) were determined by PCR. Fifty five Enterococcus strains were isolated (63.6% E. faecalis, 12.7% E. mundtii, 9.1% E. faecium, 7.3% E. casseliflavus, 3.7% E. durans and 3.6% E. hirae). None of the strains were resistant to VAN, TEC, P or AM. HLAR was found in 34.5% of strains for SM, 27.3% for KM and 7.3% for GM. The gene for the bifunctional enzyme was found only in one strain, that showed HLAR to GM and KM. Fourteen strains harboured the gene aph(3')-III, being 11 resistant to KM and STR, and three resistant to GM, KM and STR. The remaining six strains showed HLAR to STR, but were negative for the three genes tested by PCR. The gene ant(4'4") was not detected in any of the strains. No unexpected vancomycin resistance was detected. The resistance rates among poultry strains were lower than those found among human strains isolated from hospital patients in recent Canary studies.

Group 1 beta-lactamases of Aeromonas caviae and their resistance to beta-lactam antibiotics.

The contribution of beta-lactamase production to beta-lactam antibiotic resistance was examined in an Aeromonas caviae mutant strain, selected in vitro by cefotaxime and derived from a wild-type strain isolated in our laboratory from crude sewage. Both strains produced beta-lactamase. The mutant strain (AC7m) produced beta-lactamase constitutively, in contrast to the parental strain (AC7), which was inducible by cefoxitin. AC7m was regarded as a mutant from AC7, which over-expressed beta-lactamase. The mutant strain showed a remarkable reduction in sensitivity to most of the beta-lactam antibiotics tested, such as (i) aminopenicillins and their combinations with clavulanic acid and sulbactam, (ii) carboxypenicillins, (iii) ureidopenicillins, and (iv) cephalosporins. This strain remained susceptible to ceftazidime, imipenem, and aztreonam. Isoelectric focusing of sonic extracts revealed that both strains AC7 and AC7m shared a common major beta-lactamase band at pI 6.5. The plasmid DNA assays showed that the beta-lactamases expressed by each A. caviae strain were chromosomally encoded. Based on substrate and inhibitor profiles determined in sonic extracts for AC7 and AC7m, the enzymes displayed on isoelectric focusing at pI 6.5 were assigned to chromosomal Group 1 beta-lactamases. Imipenem would therefore be the appropriate choice for therapy of infections caused by A. caviae beta-lactamase over-expressing mutants.

Identification and antimicrobial susceptibility of coagulase positive staphylococci isolated from healthy dogs and dogs suffering from otitis externa.

A total of 67 strains of coagulase positive staphylococci isolated from healthy dogs and dogs suffering from otitis externa were studied. Twenty-two isolates were from healthy dogs (five from hound dogs and 17 from companion dogs) and 45 from dogs suffering otitis externa (14 from hound dogs and 31 from companion dogs). Presumptive identification was attempted using the following tests: production of acetoin, anaerobic utilization of mannitol, acid production from mannitol, presence of beta-galactosidase, and growth on P agar supplemented with different concentrations of acriflavine. Susceptibility of staphylococci to 16 antibiotics was determined. Most effective antibiotics were imipenem, amoxycillin/clavulanic acid, ciprofloxacin, tobramycin, gentamicin and marbofloxacin. Penicillin, ampicillin and polymyxin B showed the lowest activity. There were no significant differences in antimicrobial susceptibility among isolates from healthy dogs and dogs suffering from otitis externa.

Bioavailability of solid and non-aqueous phase liquid (NAPL)-dissolved phenanthrene to the biosurfactant-producing bacterium Pseudomonas aeruginosa 19SJ.

The biodegradation of phenanthrene by the biosurfactant-producing strain Pseudomonas aeruginosa 19SJ was investigated in experiments with the compound present either as crystals or dissolved in non-aqueous phase liquids (NAPLs). Growth on solid phenanthrene exhibited an initial phase not limited by dissolution rate and a subsequent, carbon-limited phase caused by exhaustion of the carbon source. Rhamnolipid biosurfactants were produced from solid phenanthrene and appeared in solution and particulate material (cells and phenanthrene crystals). During the carbon-limited phase, the concentration of rhamnolipids detected in culture exceeded the critical micelle concentration (CMC) determined with purified rhamnolipids. The biosurfactants caused a significant increase in dissolution rate and pseudosolubility of phenanthrene, but only at concentrations above the CMC. Externally added rhamnolipids at a concentration higher than the CMC increased the biodegradation rate of solid phenanthrene. Mineralization curves of low concentrations of phenanthrene initially dissolved in two NAPLs [2,2,4,4,6,8,8-heptamethylnonane and di(2-ethylhexyl)phthalate] were S-shaped, although no growth was observed in the population of suspended bacteria. Biosurfactants were not detected in solution under these conditions. The observed mineralization was attributed not only to suspended bacteria, but also to bacterial populations growing at the NAPL-water interface, mineralizing the compound at higher rates than predicted by abiotic partitioning. We suggest that rhamnolipid production and attachment increased the bioavailability of phenanthrene, so promoting biodegradation activity.

Identification and antibiotic resistance of faecal enterococci isolated from water samples.

We have isolated 47 strains of presumptive faecal streptococci from different water samples. Identification was made by the method of Facklam et al. (1989). Antibiotic resistance was studied on Mueller-Hinton Agar. Twelve antibiotics were tested. High-level aminoglycoside resistance (HLAR) and resistance to glycopeptides were studied. Biochemical identification of presumptive faecal streptococci isolates gave the following results: 19 Enterococcus faecalis, 12 E. faecium, 8 E. hirae, 4 E. durans and 4 E. mundtii. E. mundtii is not included among faecal enterococci. None of the strains were resistant to glycopeptides (vancomycin and teicoplanin). Three strains of Enterococci showed HLAR. Two of them were isolated from coastal bathing waters and the other from wastewater. This suggests that water could contribute to spread of HLAR enterococci and it should be a matter of concern for public health authorities.

Changes in thyroid function in puppies fed a high iodine commercial diet.

Abnormally low(131)I uptakes were noticed in dogs fed with commercial diets at the University Animal Clinic in Buenos Aires, but the total iodine content of eight different commercial diets examined was found to provide an iodine intake above daily requirements. To investigate this anomaly, 18 dogs were distributed into three groups, fed either: (1) a home-prepared diet; (2) a commercial diet; (3) a home-prepared diet supplemented with potassium iodide equivalent to that found in the commercial diet. The(131)I uptake in dogs of groups B and C was significantly decreased, as was basal serum thyroxine (T(4)) and free thyroxine (FT(4)), whereas urinary iodide excretion and serum thyroid stimulating hormone (TSH), were increased. The thyroid releasing hormone (TRH)-TSH test showed an increased response in dogs from group B, while the TRH-T(4)test was inhibited in both groups B and C. The results demonstrate that the excessive amount of iodine present in some commercial diets in Argentina causes a significant impairment of thyroid function and hypothyroidism.

Antibacterial susceptibility patterns of Pseudomonas strains isolated from chronic canine otitis externa.

Development of antibiotic resistance in bacteria is a problem of great concern. It is important to establish the convenience of antimicrobial susceptibility tests in animal infections. The aim of this study was to test the susceptibility to antibiotics of Pseudomonas strains isolated from chronic canine otitis externa. We tested 23 strains of Pseudomonas: 19 Ps. aeruginosa, three Ps. fluorescens and one Pseudomonas spp. The most effective antibiotics were tobramycin (100% susceptible), marbofloxacin (91.3%) and ceftazidime (91.3%). Ticarcillin and gentamicin, commonly used for the treatment of otitis externa also showed good results (susceptibility of strains was 86 and 65.2% respectively). Lower susceptibility was found using enrofloxacin (52.1%) probably due to its indiscriminate use. We emphasize the need for a rational policy of antibiotic prescribing in order to prevent the selection of resistant strains.

Aminoglycoside resistance mechanisms in clinical isolates of Pseudomonas aeruginosa from the Canary Islands.

Strains of Pseudomonas aeruginosa resistant to aminoglycoside antibiotics were selected from 152 clinical isolates. We identified two patterns of resistance correlating with the resistance mechanism characterized by changes in permeability, enzymatic modification due to the acetylating enzyme, AAC(6')-II, or a combination of both. We detected enzymatic activity of the phosphorylase enzyme, APH(3'), in all the isolates. We compared the mechanisms of resistance detected by three methods i.e., radioenzymatic assay, phenotype of resistance and DNA probes. The phenotype of resistance was tested using a kit developed by Schering-Plough Corp. Hybridization was made with 18 DNA probes for the most frequent genes encoding for aminoglycoside-modifying enzymes. All isolates with AAC(6') activity hybridized with the aac(6')-Ib probes and to a minor degree, with the aac(6')-IIb probe. None of the isolates showed hybridization with aph(3')-I, aph(3')-II, or aph(3')-III DNA probes. Serotyping of the strains showed that the O:11 serotype was the most frequent one in strains whose resistance was due to the AAC(6') enzyme. The O:6 serotype was associated with changes in permeability. Encoding of the resistance mechanism seemed to be chromosomal in all the strains.

[Treatment with low-protein diet and caloric supplements in patients with chronic kidney failure in predialysis. Comparative study]. / Tratamiento con dieta hipoproteica y suplementos calóricos en pacientes con insuficiencia renal crónica en prediálisis. Estudio comparativo.

The low-protein diet (LPD) is used in patients with advanced chronic renal failure (CRF) to improve their symptoms and decrease the progression of CRF. LPD entails the risk for caloric malnutrition, which increases protein catabolism. Two groups were obtained from a total of 33 patients with CRF with LPD (0.6 g protein/kg/day): control group (group C), which went on with the same diet, and a group S, in which a portion of proteins and calories were provided through a low-protein and hypercaloric supplement (Suplena). During 6 months the protein intake and the evolution of the nutritional status and renal function were studied and compared between both groups. Additionally, tolerance and secondary effects of the supplement were studied in group S. Twenty-two patients (eleven in each group) completed the six month follow-up. At the end of the study, group S had the nutritional parameters better preserved, came closer to the low-protein diet objective, had a better compliance with therapy and had a less marked decrease in renal function--as measured by creatinine clearance--than group C. Tolerance to supplement was good in more than 70% of patients and secondary effects--nausea, vomiting and loss of appetite--occurred in 18% of patients at the end of the 6 months. We conclude that the use of this supplement in an LPD is usually well tolerated, enhances the compliance with the diet and can be of benefit for the mebacolic-nutritional status.