Salceda Fernández-Barredo

Practical Approach for Typing Strains of Leishmania infantum by Microsatellite Analysis

ABSTRACT Currently the universally accepted standard procedure for characterizing and identifying strains of Leishmania is isoenzyme analysis. However, in the Mediterranean area, despite their very wide geographical distribution, most Leishmania infantum strains belong to zymodeme MON-1. In order to increase our understanding of polymorphism in strains of L. infantum, we developed PCR assays amplifying 10 microsatellites and sequenced PCR products. The discriminative power of microsatellite analysis was tested by using a panel of 50 L. infantum strains collected from patients and dogs from Spain, France, and Israel, including 32 strains belonging to zymodeme MON-1, 8 strains belonging to zymodemes MON-24, MON-29, MON-33, MON-34, or MON-80, and 10 untyped strains. Five of the microsatellites were polymorphic, revealing 22 genotypes, whereas the five remaining microsatellites were not variable. In particular, MON-1 strains could be separated into 13 different closely related genotypes. MON-33 and MON-34 strains also gave two additional genotypes closely related to MON-1, while MON-29, MON-24, and MON 80 strains exhibited more divergent genotypes. Among the foci examined, the Catalonian focus displayed a high polymorphism, probably reflecting isoenzyme polymorphism, while the Israeli focus exhibited a low polymorphism that could be consistent with the recent reemergence and rapid spread of canine leishmaniasis in northern and central Israel. The strains originating from the south of France and the Madrid, Spain, area displayed significant microsatellite polymorphism even though they were monomorphic by isoenzyme analysis. In conclusion, microsatellite polymorphism exhibits a high discriminative power and appears to be suitable for characterization of closely related strains of L. infantum in epidemiological studies.

Detection of hepatitis E virus shedding in feces of pigs at different stages of production using reverse transcription-polymerase chain reaction.

The aim of this study was to determine at which production stages hepatitis E virus (HEV) is shed by the highest number of pigs and to estimate the relative risk associated with each stage. For this purpose, 146 fecal samples of pigs from 21 farms were studied. In addition, 1 sample from the manure ditch and another sample of drinking water, collected directly from the trough located in the pen, were taken from 16 farms. HEV RNA was detected in fecal samples from 34 pigs (23.29%). The production stages in which most pigs excreted HEV were weaners (41.7%) and pigs in the first month of feeding (60%). The results of the statistical analysis showed that the principal significant risk stage in HEV shedding was the first month of feeding (odds ratio [OR] 19.5, 95% CI 3.59–106.07, P = 0.001) followed by the weaners stage (OR 9.3, 95% CI.78–48.42, P = 0.008). In 8 out of 16 farms tested (50%) HEV RNA was detected in raw manure and in the water trough of only 1. Detection of HEV in manure ditches raises the concern of how to deal with manure of swine origin, because it is used as soil fertilizer.

Diagnostic Techniques To Detect Cryptic Leishmaniasis in Dogs

ABSTRACT This study of several techniques for detecting cryptic leishmaniasis in dogs from areas in Spain where Leishmania infantum is highly endemic concludes that immunological techniques (enzyme-linked immunosorbent assay, immunofluorescence antibody test, Western blotting, delayed-type hypersensitivity reaction, and in vitro lymphocyte proliferation assay) do not clearly differentiate between noninfected and infected asymptomatic dogs and that culture and PCR are more reliable diagnostic tools.

Fulminant Hepatitis E in a Woman Taking Oral Contraceptive Medication

We describe a fulminant autochthonous hepatic failure caused by hepatitis E (HEV) in a patient admitted in our hospital for liver-transplant evaluation. The only risk factor recorded for this severe course was the use of oral contraceptives that are known to mimic a hormonal status similar to pregnancy. The diagnosis was based on the presence of IgG and IgM anti-HEV in the serum of the patient and confirmed by the isolation of a strain of HEV genotype 3f from a blood sample obtained the fourth day after hospital admission. HEV genotype 3 is present in human and swine populations in Spain. The patient began to recover while waiting for a liver transplant. To our knowledge, this is the first report of fulminant hepatitis E in a non-pregnant European patient on oral contraceptives.

Multilocus genotyping of Giardia duodenalis in lambs from Spain reveals a high heterogeneity

Fecal specimens from 120 lambs in Valencia (Spain) were analyzed for Giardia duodenalis by IFA and nested-PCR using the beta giardin (bg), glutamate dehydrogenase (gdh), triose phosphate isomerase (tpi) and small subunit ribosomal RNA (ssurRNA) genes. The highest prevalence was obtained using the ssurRNA gene (89.2%), whereas values from other techniques ranged from 64.1% to 69.2%. Sequences of the ssurRNA showed a high proportion of assemblage A or mixed assemblage A/E samples (55.1% and 25.2%, respectively). When the other 3 loci were analyzed, between 6.5% and 15.4% were found to be assemblage A or A/E, respectively. Nested PCR for the tpi gene was the most variable of the targets employed. Twelve new sequences of gdh and tpi for G. duodenalis from sheep were found. Multilocus genotyping resulted in 63 patterns from the 71 samples sequenced at the four loci. This high variability among isolates possibly reflects the high frequency of mixed infections.

Occurrence and genotypes of Giardia isolated from lambs in Spain

Three hundred and eighty six faecal specimens were randomly collected from 1- to 3-month-old lambs from 16 farms in Spain to investigate the presence of different genotypes of Giardia duodenalis. Individual specimens were examined by IFA (Immunofluorescence assay) and beta-giardin PCR polymerase chain reaction. Cysts of G. duodenalis were shed by lambs in every flock analyzed, showing a prevalence by farms of 100%. The average prevalence of G. duodenalis for the 386 specimens was 42%, ranging from 8.3 to 80% depending on the farm. beta-giardin PCR positive samples were sequenced to determine the genotypes present at each farm and seven new subtypes of beta-giardin Assemblage E are reported in this study. In each farm, one to six different beta-giardin subtypes were found, showing the high variability of the target. Also, one flock had the zoonotic Assemblage A. This is the first report of Giardia subgenotype A-1 in sheep in Spain.

Detection of hepatitis E virus (HEV) through the different stages of pig manure composting plants

Hepatitis E virus (HEV) is an increasing cause of acute hepatitis in industrialized countries. The aim of this study was to evaluate the presence of HEV in pig manure composting plants located in Spain. For this purpose, a total of 594 samples were taken in 54 sampling sessions from the different stages of composting treatment in these plants as follows: slurry reception ponds, anaerobic ponds, aerobic ponds, fermentation zone and composting final products. HEV was detected by reverse transcription polymerase chain reaction (RT‐nested PCR) in four (80%) of five plants studied, mainly in the first stages of the process. HEV was not detected in any final product (compost) sample, destined to be commercialized as a soil fertilizer, suggesting that composting is a suitable method to eliminate HEV and thus, to reduce the transmission of HEV from pigs to humans.

Development of a specific polymerase chain reaction assay for the detection of Basidiobolus

The etiology of chronic diarrhea is complex in humans and animals. It is always necessary to evaluate a list of differential diagnosis, including bacteria, protozoa and fungi. Basidiobolomycosis is a fungal disease reported sporadically worldwide, mainly caused by B. ranarum, a frequent organism found in soil or in the intestine and skin of lizards and frogs. It is an opportunistic pathogen that causes infections characterized by granulomatous lesions in the subcutaneous tissues as well as in the intestinal wall in humans and animals. In this work we have developed a PCR technique to differentiate Basidiobolus from other causes of intestinal disease in dogs and humans. To test the specificity of the PCR assay we included closely related organisms, common intestinal microbiota and pathogenic organisms, such as Aspergillus, Candida, Cryptosporidium, Escherichia, Giardia, Mucor, Proteus, Rhizopus and Salmonella. Pythium insidiosum, which cause clinically similar disease in dogs but require a different treatment. Only Basidiobolus was positive to the PCR assay.