Development and diagnostic validation of an ELISA based on an antigenic mixture for the detection of bovine tuberculosis.

Programs for the eradication of bovine tuberculosis (bTB) focus on the tuberculin skin test (TST) and slaughter of reactor cattle. However, the disease remains an animal health concern in several countries and improving the efficiency of the TST has become a critical issue. The detection of Mycobacterium bovis antibodies in serum, within weeks after the TST, may be a rapid and inexpensive way to improve bTB control. This study reports the validation of an enzyme-linked immunosorbent assay (ELISA) to detect bovine tuberculosis as an ancillary test to TST in dairy farms in Argentina. The estimated validation parameters were within the established requirements of the World Organization for Animal Health (OIE). The test demonstrated high repeatability, with coefficients of variation <25%. High test reproducibility through interlaboratory testing was also found, with an estimated Pearson coefficient of 0.9648 (95% confidence intervals 0.9315-0.9820). The ELISA detected tuberculous cattle unidentified by the TST. Of 43 animals sent to slaughterhouses that were ELISA positive 15-17 days after a negative TST, 36 were confirmed as infected with M. bovis by histopathology and IS6110 PCR. According to ROC curve analysis of results of 145 cattle from M. bovis-free herds and the 36 M. bovis-infected cattle, at a corrected optical density cut-off point of 0.3853, specificity was 95.95% and the positive predictive value at this cut-off was 83.72%. The ELISA detection test validated in this study could be readily applied in dairy farms, to complement a prior TST and improve livestock health.

Calodium hepaticum (Trichuroidea: Capillariidae) Infection In Akodon azarae: First Description of Histopathological Lesions In Sigmodontinae.

Calodium hepaticum (Trichinellida: Capillaridae) is a parasitic nematode of mammals distributed worldwide. Although this parasite can infect the liver of a wide diversity of mammals (including humans), it is mostly associated with Muroidea hosts. Sigmodontinae rodents were recently recognized as important hosts of this parasite in Argentina, but the impact of this parasitism on these hosts has not been established. Here we report results of histopathological analyses of 40 livers of Akodon azarae infected with C. hepaticum. Lesions were classified into 4 categories: level 0, absence of lesions; level 1, with focal granulomatous hepatitis; level 2, presence of multifocal granulomatous hepatitis, fibrosis and focal necrosis with neutrophils, and level 3, absence of intact adult parasites, diffuse distribution, necrosis, and fibrosis. Most samples presented lesions of level 2 (55%), but all categories of lesions were found. This is the first study to describe the lesions caused by C. hepaticum in the liver of Sigmodontinae rodents, and the results suggest that infection by this parasite is costly to A. azarae populations.

Molecular markers located on the DGAT1, CAST, and LEPR genes and their associations with milk production and fertility traits in Holstein cattle.

The objective of the present study was to investigate single nucleotide polymorphisms (SNPs) located in three candidate genes previously reported to have effects on fertility and milk production traits in a population of 123 Holstein cows. The milk production traits evaluated included lifetime averages of milk yield, protein concentration, and fat concentration. Fertility traits evaluated included lifetime averages of services per conception and days-open. Candidate genes included those encoding diacylglycerol acyltransferase (DGAT1), leptin receptor (LEPR), and calpastatin (CAST). A total of 60 SNPs were selected (20 per gene) at equidistant locations on each candidate gene to identify potential linkage with causative mutations. Four SNPs were identified as being significantly associated with the evaluated fertility traits. Specifically, SNPs rs109663724 and rs137673193 were significantly associated with lifetime average days-open, while rs109663724 and rs135560721 were significantly associated with lifetime average number of services per conception. Five SNP (rs109663724, rs132699547, rs135423283, rs135576599, and rs13675432) were significantly associated with lifetime averages of milk protein concentration and milk fat concentration, with only one SNP (rs109663724) being significantly associated with the average lifetime milk yield. Although multiple SNPs were identified in the current study as being significantly associated with milk production and fertility traits, it is essential that these SNPs are validated in larger populations, under more diverse environments, and that additional SNPs and candidate genes are evaluated prior to their implementation into selection strategies.

Genome-wide linkage analysis and tumoral characterization reveal heterogeneity in familial colorectal cancer type X.

BACKGROUND: Familial colorectal cancer type X (FCCTX) fulfils clinical criteria defining Lynch syndrome (LS), but is not related to germline mutations in DNA mismatch-repair genes. Its aetiology remains unexplained and there is little evidence of involvement of the common colorectal carcinogenetic pathways. We aimed to identify susceptibility loci and gain insights into carcinogenic pathways involved FCCTX tumour development. METHODS: We performed a linkage analysis in 22 FCCTX families. We also constructed a tissue microarray in order to define an immunohistochemical (IHC) profile for FCCTX tumours (N = 27) by comparing them to three other types of colorectal tumors: LS (N = 18), stable early-onset (N = 31) and other sporadic disease (N = 80). Additionally, we screened for BRAF/KRAS mutations and determined CpG island methylator phenotype (CIMP) status for all FCCTX tumours. RESULTS: We found suggestive evidence of linkage at four chromosomal regions; 2p24.3, 4q13.1, 4q31.21 and 12q21.2-q21.31. We screened genes in 12q21 and ruled out the implication of RASSF9 and NTS, good candidates due to their potential involvement in carcinogenesis and colorectal epithelium development. Based on IHC profiles FCCTX tumours did not form a single, exclusive cluster. They were clearly different from LS, but very similar to stable early onset tumours. The CIMP and chromosomal instability pathways were implicated in one-third and one-quarter of FCCTX cases, respectively. The remaining cases did not have alterations in any known carcinogenic pathways. CONCLUSIONS: Our results highlight the heterogeneity of FCCTX tumours and call into question the utility of using only clinical criteria to identify FCCTX cases.

Identification of a founder EPCAM deletion in Spanish Lynch syndrome families.

Germline deletions at the 3'-end of EPCAM have been involved in the etiology of Lynch syndrome (LS). The aim of this study was to characterize at the molecular level Spanish families harboring EPCAM deletions. Non-commercial multiplex ligation-dependent probe amplification (MLPA) probes and long-range polymerase chain reaction (PCR) amplification were used to characterize each deletion. Haplotyping was performed by analyzing eight microsatellite markers and five MSH2single nucleotide polymorphisms (SNPs). Methylation of MSH2 was analyzed by methylation specific-MLPA. Tumors diagnosed in seven Spanish families harboring EPCAM deletions were almost exclusively colorectal. Mosaicism in MSH2 methylation was observed in EPCAM deletion carrier samples, being average methylation levels higher in normal colon and colorectal tumors (27.6% and 31.1%), than in lymphocytes and oral mucosa (1.1% and 0.7%). Three families shared the deletion c.858 + 2568_*4596del, with a common haplotype comprising 9.9 Mb. In two families the novel EPCAM deletion c.858 + 2488_*7469del was identified. This study provides knowledge on the clinical and molecular characteristics of mosaic MSH2 epimutations. The identification of an EPCAM founder mutation has useful implications for the molecular diagnosis of LS in Spain.

Innovative methods to assess upper limb strength and function in non-ambulant Duchenne patients.

Upper limb assessment in non-ambulant patients remains a challenge. We have designed new tools to precisely assess pinch (MyoPinch), grip (MyoGrip), wrist flexion and extension (MyoWrist) strength. We have also designed a new tool to assess the ability of patients to produce repetitive flexion/extension movements of wrist and fingers (MoviPlate). We have assessed the feasibility and reliability of these new tools in 30 non-ambulant patients with Duchenne muscular dystrophy and in 30 age-matched male controls. Existing measures, such as Motor Function Measure, Tapping, and the Brooke Upper Extremity Functional Rating Scale were also performed. Results demonstrated that assessments were feasible in nearly all upper limbs tested for MyoGrip, MyoPinch and MoviPlate. The reliability of all tests, including MyoWrist which was not feasible in the patients presenting with contractures, was excellent in patients as in controls. Motor capacities decrease with the number of months spent in the wheelchair. The scores in the tests were partially correlated with each other, and with clinical measures such as vital capacity, Motor Function Measure, functional hand scale and Brooke score. This study validates a panel of upper limb muscle strength and function measures for Duchenne Muscular Dystrophy which can be applied from controls to extremely weak patients.

Embolización bronquial en bronquiectasias / Bronchial artery embolization for bronchiectasias

Se presenta nuestra experiencia en el tratamiento de la hemoptisis con embolización bronquial en pacientes con bronquiectasias, desde el 2001 al 2008, en el Hospital Italiano de Buenos Aires. Veinte pacientes fueron tratados con este método: 12 mujeres y 8 hombres con una edad media de 51 años (15-83 años). Las causas de bronquiectasias fueron secundarias a infecciones inespecíficas en el 40%; otro 40% secuelas de tuberculosis y 20% por enfermedad fibroquística. La indicación de embolización fue hemoptisis mayor a 300 ml/24 hs o hemoptisis persistentes durante más de 3 días consecutivos con requerimiento de hospitalización. En el 85% de los casos se efectuó embolización bronquial y en el resto (15%) bronquial y mamaria. Este procedimiento fue bilateral en el 60% de los pacientes, 20% solo en el lado derecho y 20% del lado izquierdo. En 18 pacientes se logró oclusión vascular completa de todos los territorios pulmonares patológicos. Control agudo del sangrado fue obtenido en todos los pacientes (100%) y sólo dos presentaron nuevo sangrado a los 8 y 12 meses respectivamente. Como efecto colateral al tratamiento se observó dolor torácico leve y transitorio en dos pacientes. Concluimos que la embolización de las arterias bronquiales constituye un tratamiento adecuado y seguro para el control agudo de la hemoptisis en pacientes con bronquiectasias.

We present our experience on arterial embolization for the treatment of hemoptysis in patients with bronchiectasis between 2001 and 2008 at the Hospital Italiano in Buenos Aires. Twenty patients were treated with this method: 12 women and 8 men with a median age of 51 years (15-83 years). Bronchiectasis was secondary to bacterial infections in 40% and secondary to tuberculosis infection in 40% of the patients; 20% of the patients had cystic fibrosis. Embolization was prescribed when the hemoptysis was above 300 ml/24 hours or the hemoptysis was persistent for more than 3 consecutive days and the patient’s hospitalization was required. In 85% of the cases embolization was done only in bronchial arteries and in the remaining 15% in bronchial and mammary arteries. The procedure was bilateral in 60% of the patients, only on the right side in 20% and only on the left side in 20%. In 18 patients complete occlusion was achieved. Complete control of the bleeding was obtained in 100% of patients. Only two patients had a new bleeding, 8 and 12 months later respectively. The treatment side effects were slight and transitory thoracic pain in two patients. We concluded that the embolization of the bronchial arteries is a suitable and safe treatment to control hemoptysis in patients with bronchiectasis.

Extramammary Paget's disease--occupational exposure to used engine oil and a new skin grafting technique.

SUMMARY: Extramammary Paget's disease (EMPD) is a rare intraepithelial adenocarcinoma, affecting mainly 50-70-year-olds with a female preponderance of 3:1. Exposure to polyaromatic hydrocarbons (PAHs) is associated with a substantial risk of skin cancer. We report a case of 64-year-old male with longstanding occupational exposure to used engine oil presenting with EMPD of the left scrotum and groin.

Malaria deaths as sentinel events to monitor healthcare delivery and antimalarial drug safety.

OBJECTIVES: To identify case management, health system and antimalarial drug factors contributing to malaria deaths. METHOD: We investigated malaria-related deaths in South Africa's three malaria endemic provinces from January 2002 to July 2004. Data from healthcare facility records and a semi-structured interview with patients' contacts were reviewed by an expert panel, which sought to reach consensus on factors contributing to the death. This included possible health system failures, adverse reactions to antimalarials, inappropriate medicine use and failing to respond to treatment. RESULTS: Approximately 177 of 197 cases met inclusion criteria for the study. Delay in seeking formal health care was significantly longer for patients who sought traditional health care [median 4; inter-quartile range (IQR) 3-7 days] than for patients who did not (median 3; IQR 1-5 days; P = 0.033). Patients with confirmed or suspected HIV/AIDS were significantly more likely to use traditional approaches (25%) than those with other comorbidities (0%; P = 0.002). Malaria was neither suspected nor tested for at a primary care facility in 23% of cases with adequate records. Initial hospital assessment was considered inadequate in 74% of cases admitted to hospital and in-patient monitoring and management was adequate in only 27%. There were 32 suspected adverse reactions to antimalarial therapy. CONCLUSION: A confidential enquiry into malaria-related deaths is a useful tool for identifying preventable factors, health system failures and adverse events affecting malaria case management.

Results and complications in dorsal and volar wrist Ganglia arthroscopic resection.

The authors present the procedure and results of five years of arthroscopic treatment of wrist radiocarpal and midcarpal ganglia. Thirty cases of dorsal ganglia and seventeen cases of volar ganglia were operated on arthroscopically. The technique was easy to perform in all the radiocarpal ganglia, not easy in midcarpal dorsal ganglia and very difficult in midcarpal volar ganglia. The results were recorded with a mean follow-up of 15 months. Twenty-seven cases of dorsal ganglia and twelve cases of volar ganglia had excellent results with active motion recovery, no complications, absence of scars and no recurrence. Two cases had a recurrence. There were four complications: a case of injury of a radial artery branch, a case of extensive haematoma, and two cases of neuropraxia. In three cases the procedure was converted into open surgery: they had a longer time of healing and a residual scar. The arthroscopic resection has been in our experience effective and safe for the treatment of all radiocarpal ganglia. Good results have been obtained also in the treatment of dorsal midcarpal ganglia. Concerning the uncommon cases of volar midcarpal (STT) ganglia, an open approach seems still indicated.

Antibody dynamics in Holstein Friesian heifers vaccinated with Brucella abortus strain 19, using seven serological tests.

The serological response induced by Brucella abortus strain 19 was evaluated in 52 Holstein females from a brucellosis-free herd using seven serological tests. Each calf was vaccinated at an age of 4 and 8 months old with 3 x 10(10) CFU B. abortus S19 and the antibody response was determined as the proportion of positive results to each test. The antibody dynamics, measured with the buffered plate antigen (BPA) test and the rapid automated presumptive (RAP) test, were similar. The proportion of positive reactions in these tests reached 100% one week after vaccination and remained at this level for seven weeks, after which the proportion of positive samples slowly declined to 8% (BPA) and 2% (RAP) at week 50. The response in the indirect enzyme immunoassay (i-ELISA) was similar, but shorter than that observed with the BPA/RAP. The antibody dynamic, measured using the seroagglutination test (SAT) in parallel with the 2-mercaptoethanol (2-Me) test and the complement fixation test (CFT) were similar to the RAP/BPA, but of slightly shorter duration. The competitive ELISA (c-ELISA) was positive in all animals for 3 weeks, followed by a rapid decline. The fluorescence polarization assay (FPA) reached a maximum of 68.5% positive animals at week 4 and then declined. Based on these data, the c-ELISA and FPA discriminated residual antibody activity due to vaccination more efficiently than the other tests.

Transcoronary sinus delivery of autologous bone marrow and angiogenesis in pig models with myocardial injury.

PURPOSE: The aim of the investigation is to study myocardial injury on pig model with two objectives: (1) feasibility of stimulating angiogenesis with fresh autologous bone marrow; (2) administration of the same fresh autologous bone marrow via coronary sinus with transitory occlusion. METHODS: A controlled study was done in animal model with three phases, in a study group of 12 pigs (bone marrow administration) as well as in control group of 4 pigs (saline administration). Phase 1-production of coronary stenosis and myocardial injury; Phase 2-two weeks later, administration of bone marrow through coronary sinus with 10 min occlusion in the study group and saline solution in the control group. Phase 3-two weeks later, histological staining with hematoxylin-eosin and inmunohistochemical staining with monoclonal antibody for smooth muscle alpha-actin were conducted on both study and control groups. RESULTS: The percentage of angionenesis observed in the study group was 91% and 0% in control group. Counting of positive actin in affected and control areas showed statistically significant differences in relation to both groups: study group (1.37 vs. 0.79) and control group (0.47 vs. 0.51). The percentage of mononuclear immature cells observed in the myocardium in the study group was 25% and in the control group was 0%. There was no increment in the coronary collateral circulation when comparing coronary angiography. CONCLUSIONS: Autologous bone marrow in animal model with experimental myocardial injury enhances angiogenesis, as well as vessels with smooth muscles. The transitory occlusion of the coronary sinus might be an effective way to administer cells as those from the bone marrow.

[Identification of fungi in tissues and cultures: the importance of argirophilic substances on its cellular walls]. / Identificación de hongos en tejidos y cultivos: importancia de sustancias argirófilas en su estructura.

An experimental development based on the combination of microwaves action with one of the methods of silver staining by Del Río Hortega is presented. Material from pathological tissues and culture of fungi were studied. Besides morphological studies, were considered the causes of reduction from ionic to metalic silver, some characteristics of the silver reagent and its relationship with histochemical constitution of cellular walls. It is pointed the rapidity in fungi demonstration, the satisfactory definition of affected tissues, the advantages of working with a stable reagent, the omission of carcinogenetic substances, the possibility of stain fungical structures in previously stained materials with anilinic techniques, and the extent of the method to cultured materials without necessity of previous formaldehidic fixation.

Identificación de hongos en tejidos y cultivos: importancia de sustancias argirófilas en su estructura. / [Identification of fungi in tissues and cultures: the importance of argirophilic substances on its cellular walls]

An experimental development based on the combination of microwaves action with one of the methods of silver staining by Del Río Hortega is presented. Material from pathological tissues and culture of fungi were studied. Besides morphological studies, were considered the causes of reduction from ionic to metalic silver, some characteristics of the silver reagent and its relationship with histochemical constitution of cellular walls. It is pointed the rapidity in fungi demonstration, the satisfactory definition of affected tissues, the advantages of working with a stable reagent, the omission of carcinogenetic substances, the possibility of stain fungical structures in previously stained materials with anilinic techniques, and the extent of the method to cultured materials without necessity of previous formaldehidic fixation.

Spermatozoal methylene blue reduction: an indicator of mitochondrial function and its correlation with motility.

Methylene blue reduction rates (MeBRR) were evaluated spectrophotometrically to study bovine spermatozoal mitochondrial function and its relation to motility. A chemical reaction (H2SO4, methylene blue solution, and zinc powder) was used to quantify methylene blue reduction. Absorbance measurements were made for 10 min at 609 nm in a narrow band spectrophotometer. In a second experiment, fresh ejaculates were assessed for concentration and motility evaluated with phase microscopy (37 degrees C). Semen was diluted to 100 million cells/mL in a sodium citrate-glucose buffer and methylene blue. Absorbance and motility were evaluated every 30 min for 2.5 h in a water-jacketed cuvette (41 degrees C). Methylene blue reduction rates and motility decreased at each subsequent period. Methylene blue reduction rates were correlated to sperm motility. Lastly, the methylene blue reduction rate was measured with a broad band spectrophotometer and compared with motility using similar conditions. Motility estimates were made on sperm from the cuvettes. Sperm motility was correlated to methylene blue reduction rates measured spectrophotometrically.

Bovine spermatozoal head size variation and evaluation of a separation technique based on this size.

The objectives of this study were to determine the variation of head areas of normal spermatozoa attributable to breed, individual bull and ejaculate and to verify separation of X and Y chromosome-bearing spermatozoa and separation effectiveness. Spermatozoa were evaluated using video enhanced contrast microscopy combined with video intensified fluorescent microscopy and the polymerase chain reaction (PCR). In Experiment 1, spermatozoal head areas were measured from 2 ejaculates collected from bulls of 3 beef and 2 dairy breeds. No differences in head areas were found between breeds or between bulls within breeds; variation was observed among ejaculates from individual bulls across breeds. In Experiment 2, spermatozoa from 5 ejaculates were separated on individual SEPDEVICEs (Patented). Head area, fluorescent intensity and PCR of spermatozoa retained in the SEPDEVICEs suggested a separation based on size in 1 of 5 samples. Ejaculate variation in head areas affected separation efficiency.

A technique for the evaluation of sperm penetrating ability and quality of bovine semen processed in an extender made with Brackett-Oliphant medium and egg yolk.

Egg yolk-sodium citrate (EYC) semen extender was compared with an extender made of Brackett-Oliphant medium and egg yolk (BOEY). Ejaculates were divided into equal portions, processed and frozen. Semen was thawed and evaluated for quality. Additional semen was thawed, stained with Hoechst 33342 and the spermatozoa capacitated, after which they were co-incubated with zona-free hamster oocytes to determine their penetrating ability. Sperm penetration of non-compressed, unfixed oocytes was evaluated using an optical sectioning technique on a standard research microscope. Sperm penetration was considered successful if a fluorescing sperm head was observed within the living oocyte in a hanging drop of fertilization medium. There were small differences in percentage of secondary abnormalities and percentage of progressive motility immediately after thawing between spermatozoa extended in EYC or BOEY diluent. There were no differences due to by extender composition in percentage of spermatozoa with intact acrosomes or percent of progressively motile after a 3 h incubation at 37 degrees C, nor the percentage of spermatozoa with head abnormalities. While there were significant correlations between all seminal quality characteristics, no quality measurements were correlated to percentage of oocyte penetration. The new penetration evaluation method allowed for examination of the fertilized oocytes using fluorescent microscopy initially and again after re-incubation for further development.

[Consensus guidelines for respiratory endoscopy issued by the Argentine Society of Bronchoesophagology]. / Normas de consenso para la realización de la endoscopia respiratoria de la Sociedad Argentina de Broncoesofagología.

The international consensus is that guidelines for respiratory endoscopy are inadequate, regarding such issues as institutional requirements, benefits, risks and limitations of the procedure, training programs and accreditation, with the result that the way or performing endoscopy varies according to how an operator was trained. The absence of precise recommendations means that practice is highly diverse and inappropriate use of the procedure has increased. The Argentine Consensus Group for Normalization of Respiratory Endoscopy was created in 1995 to unify criteria for several aspects of endoscopic practice. The official recommendations of the Group and of the Argentine Society of Bronchoesophagology define the indications (diagnostic, therapeutic and investigative) and contraindications (absolute, relative and high risk) for bronchoscopy. Required pre-bronchoscopic studies for routine and special cases are defined, as are indications for premedication, intubation and general anesthesia. Requirements for the setting, support personal and instruments are specified. Guidelines for topical anesthesia and techniques for insertion of the endoscope are suggested. The technique, indications and limitations of bronchoalveolar lavage, bronchial brushing and biopsy and transbronchial needle biopsy are defined. The utility and limitations of the various therapeutic techniques of bronchoscopy (laser, radiotherapy and stents) are defined. Norms to safeguard the patient, instruments and operator are emphasized.

Leukocyte infiltration and collagenolysis in cervical tissue from intrapartum sheep.

Sheep uterine cervices and cervical mucus were heavily infiltrated by neutrophils during labour, whereas samples of cervices obtained from non-pregnant controls had no infiltrate. The neutrophilic infiltrate of the sheep uterine cervix at term was not homogeneously distributed throughout the organ: luminal mucus contained more neutrophils than tissues which, in turn, displayed a differential distribution, the superficial subepithelial layer being more heavily infiltrated than the deeper submucous layers. A widespread collagenolysis was observed in the sheep uterine cervix at term. The homogeneous morphological aspect of degradation of collagen fibres throughout the whole cervical stroma contrasted with the above-mentioned differential distribution of neutrophils. On the basis of previous reports showing that collagenolysis follows the leukocytic invasion of human and rat cervices at term, a possible role for the neutrophilic infiltrate of the sheep uterine cervix is discussed.