New records of Trichinella patagoniensis from Argentina.

Trichinella spiralis was long considered the sole Trichinella species in Argentina. However, since 2004, various Trichinella species, including the encapsulated Trichinella patagoniensis and Trichinella britovi, as well as the unencapsulated Trichinella pseudospiralis, have been detected in the country. The present study aimed to identify Trichinella ML at the species level from cougars naturally infected from Argentina. To this end, muscle tissue samples from one cougar each from Córdoba, Neuquén, and Santa Cruz Provinces were individually analysed using the artificial digestion technique. DNA extraction and molecular identification of Trichinella species were conducted on individual muscle larvae by PCR amplification of the ESV region and subsequent PCR amplification and sequencing of the COI gene. Morphological analysis revealed muscle larvae with characteristics consistent with Trichinella genus. PCR revealed a single band of approximately 127 bp for each individual muscle larva. PCR amplification of the COI gene from each isolate generated a 309 bp band. Sequencing of the mitochondrial COI gene confirmed the identity of the parasite as T. patagoniensis. The present study documents new occurrences of T. patagoniensis in Puma concolor from Argentina, including the first detection of T. patagoniensis in Puma concolor from Córdoba and Neuquén Province. These findings expand the limited knowledge of T. patagoniensis distribution in Argentina.

Study of Trichinella patagoniensis in wild boars.

Trichinellosis is a zoonotic disease, which represents a significant public health concern in some South American countries, such as Argentina and Chile. Its impact is essentially due to absence of adequate control measures on meat from game animals, as well as the presence of illegal slaughterhouses and the trade of meat products without being tested for this parasite. In Argentina, trichinellosis is an endemic disease. At present, Trichinella spiralis, Trichinella patagoniensis, Trichinella pseudospiralis, and Trichinella britovi have been detected in animals from Argentina. Until now, T. patagoniensis had only been found in mountain cougars (Puma concolor) in Argentina but there is limited information available. The present study intends to determine susceptibility, serological response and distribution of muscle larvae in wild boars infected with T. patagoniensis, T. spiralis and T. pseudospiralis. For each of the Trichinella species five wild boars were inoculated with 20,000 muscle larvae. Except for two specimens which died during the experiment, the animals were euthanized 19 weeks post infection (pi). Blood samples were collected throughout the study in order to determine the antibody kinetics. Also, nine muscle samples from each specimen were taken and analysed for determination of larval distribution. Additionally, four muscle samples were used to obtain muscle juices. Wild boars infected with T. patagoniensis showed little to no larvae in the muscle samples analysed while animals infected with T. spiralis and T. pseudospiralis had a significantly high larval load in all the samples analysed. Optical density (OD) values remained above the cut-off value throughout the experiment. This is the first study to characterize the biological aspects of T. patagoniensis in wild boars.

Trichinella spiralis in a South American sea lion (Otaria flavescens) from Patagonia, Argentina.

Trichinella spp. from a sylvatic cycle has been found in several animal species such as pumas (Puma concolor), armadillos (Chaetophractus villosus), rats (Rattus norvegicus), and wild boars (Sus scrofa) in Argentina. Moreover, Trichinella infection has been detected in a wide range of marine mammals around the world, including polar bears (Ursus maritimus) and walruses (Odobenus rosmarus). Until the present time, Trichinella spp. infection has not been detected in marine mammals of South America. Samples from four South American sea lions (Otaria flavescens) found dead in Rio Negro, Argentina, were analyzed by artificial digestion, and in the case of one animal, Trichinella larvae were identified at the species level by nested multiplex PCR as Trichinella spiralis. This is the first report of a Trichinella species infecting marine mammals from South America.

Measurement of serum bilirubin and its mono- and diconjugates: application to patients with hepatobiliary disease.

A technique has recently been described by Blanckaert and his colleagues that specifically and accurately quantifies unconjugated bilirubin, diconjugated bilirubin, and the C-8 and C-12 isomers of monoconjugated bilirubin. This technique has now been used to determine the distribution pattern of bilirubin and its ester conjugates in 91 sera from 65 patients with hepatobiliary disease, and the results were compared with two conventional diazo assays. Both diazo assays yielded values for total bilirubin concentration that were markedly and unpredictably higher than those obtained by the new technique, and the direct-reacting fraction by diazo assay showed little or no agreement with the fraction of total ester conjugates determined by the new method. Previous studies using the new method had shown that bilirubin conjugates are undetectable in sera from healthy adults or individuals with Gilbert's syndrome, but they were found in 89 of the 91 present patient sera. The fraction of total serum bilirubin represented by C-8 monoconjugates, C-12 monoconjugates, diconjugates, and total ester conjugates was higher in patients with biliary obstruction than in those with parenchymal liver disease, but extensive overlap between groups prevented determination of these conjugated species from being diagnostically useful. Overall, bilirubin ester conjugates in serum consisted of 30% C-8 monoconjugates, 37% C-12 monoconjugates, and 33% diconjugates, while urine contained predominantly diconjugates.

Measurement of bilirubin and its monoconjugates and diconjugates in human serum by alkaline methanolysis and high-performance liquid chromatography.

No accurate methods are available for specific determination of unconjugated and conjugated bilirubin. Using a novel approach, we now have developed such an assay which permits direct individual measurement of bilirubin and its isomeric monoconjugates and diconjugates in serum. The monosugar and disugar conjugates are quantitatively converted to the corresponding methyl esters, which are readily extractable into chloroform. These monomethyl and dimethyl esters and unconjugated bilirubin are then separated by HPLC and detected spectrophotometrically in the effluent. Use of an internal standard and calibration of the method with crystalline reference bilirubin and bilirubin methyl esters permit direct measurement of the individual pigment fractions in the sample. The acuracy of this procedure was verified by a radioisotope dilution method. In sera of 22 healthy adults and six patients with Gilbert syndrome, only unconjugated bilirubin was detected. In 42 serum samples of jaundiced patients with hepatobiliary disease, unconjugated and all conjugated bilirubin fractions were increased, with the monoconjugates generally predominating. The total concentration of bilirubin and its carbohydrate conjugates, as determined by the new method, was considerably lower than the TB obtained with conventional diazo procedures. Contrariwise, both the new method and the diazo procedures gave comparable resuts when normal serum enriched with purified bilirubin glucuronides was assayed. Our findings thus indicate that unidentified, diazo-positive compounds distinct from bilirubin and its ester conjugates are present in pathlogical serum samples. The reported asssay is expectd to serve as a reference method for measurement of bilirubin and its carbohydrate conjugates in serum and to find general application in the study of bilirubin metabolism.

Simultaneous determination of phosphatidylglycerol and the lecithin/sphingomyelin ratio in amniotic fluid.

We describe a single-dimension thin-layer chromatographic method by which one can simultaneously determine the lecithin/sphingomyelin ratio and the proportion of phosphatidylglycerol in amniotic fluid. The phospholipids are conveniently detected by an improved molybdenum-blue spray reagent, which immediately produces blue spots on a white background. The stained phospholipids are stable for at least 90 min after spraying, and densitometry results in symmetrical peaks that are easily quantitated.