Deformed wing virus type a and b in managed honeybee colonies of Argentina.

Apis mellifera is infected by more than 24 virus species worldwide, mainly positive-sense, single-stranded RNA viruses of the Dicistroviridae and Iflaviridae families. Among the viruses that infect honey bees, Deformed wing virus is the most prevalent and is present as three master variants DWV-A, B, and C. Given that the ectoparasitic mite Varroa destructor vectors these virus variants, recombination events between them are expected, and variants and their recombinants can co-exist in mites and honeybees at the same time. In this study, we detect, through RT-qPCR, the presence of DWV-A and B in the same samples of adult bees from colonies of Argentina. Total RNA was extracted from pools of ten adult bees from 45 apiaries distributed across the main beekeeping Provinces of Argentina (Buenos Aires, Santa Fe, Córdoba, Santiago del Estero, Río Negro, and Mendoza); then RT-qPCR reactions were performed to detect DWV-A and B, with specific primer pairs. After the amplifications, PCR products (204 and 660 bp amplicons for DWV-B, and ~250 bp for DWV-A) were purified and sequenced to verify that they corresponded to reported sequences, analyzing them using the Blast software. Of the 45 samples analyzed by RT-qPCR, over 90% were infected with DWV-A and 47% were also positive for DWV-B, where it was found in high prevalence specifically in colonies of A. mellifera of the Buenos Aires Province. Future studies will determine the impact of this type of the virus and its ability to recombine with the other DWV types in the apiaries of our country.

Heat shock proteins in Varroa destructor exposed to heat stress and in-hive acaricides.

Varroa destructor is one of the major pests that affect honeybees around the world. Chemical treatments are common to control varroosis, but mites possess biochemical adaptive mechanisms to resist these treatments, enabling them to survive. So far, no information is available regarding whether these pesticides can induce the expression of heat shock protein (Hsp) as a common protective mechanism against tissue damage. The aims of this study were to determine differences in heat shock tolerance between mites collected from brood combs and phoretic ones, and to examine patterns of protein expression of Hsp70 that occur in various populations of V. destructor after exposure to acaricides commonly employed in beekeeping, such as flumethrin, tau-fluvalinate and coumaphos. Curiously, mites obtained from brood cells were alive at 40 °C, unlike phoretic mites that reached 100% mortality, demonstrating differential thermo-tolerance. Heat treatment induced Hsp70 in mites 4 × more than in control mites and no differences in response were observed in phoretic versus cell-brood-obtained mites. Dose-response assays were carried out at increasing acaricide concentrations. Each population showed a different stress response to acaricides despite belonging to the same geographic region. In one of them, coumaphos acted as a hormetic stressor. Pyrethroids also induced Hsp70, but mite population seemed sensitive to this treatment. We concluded that Hsp70 could represent a robust biomarker for measuring exposure of V. destructor to thermal and chemical stress, depending on the acaricide class and interpopulation variability. This is relevant because it is the first time that stress response is analyzed in this biological model, providing new insight in host-parasite-xenobiotic interaction.

Genetic structure of Varroa destructor populations infesting Apis mellifera colonies in Argentina.

Although mitochondrial DNA mapping of Varroa destructor revealed the presence of several haplotypes, only two of them (Korean and Japanese haplotypes) were capable to infest Apis mellifera populations. Even though the Korean haplotype is the only one that has been reported in Argentina, these conclusions were based on mites sampled in apiaries from a specific geographical place (Buenos Aires province). To study mites from several sites of Argentina could reveal the presence of the Japanese genotype, especially considering sites near to Brazil, where Japanese haplotype was already detected. The aim of this work was to study the genetic structure of V. destructor populations from apiaries located in various provinces of Argentina, in order to determine the presence of different haplotypes. The study was carried out between January 2006 and December 2009. Phoretic adult Varroa mites were collected from honey bee workers sampled from colonies of A. mellifera located in Entre Ríos, Buenos Aires, Corrientes, Río Negro, Santa Cruz and Neuquén provinces. Twenty female mites from each sampling site were used to carry out the genetic analysis. For DNA extraction a nondestructive method was used. DNA sequences were compared to Korean haplotype (AF106899) and Japanese haplotype (AF106897). All DNA sequences obtained from mite populations sampled in Argentina, share 98% of similitude with Korean Haplotype (AF106899). Taking into account these results, we are able to conclude that Korean haplotype is cosmopolite in Argentina.

Synergistic effect of surfactin from Bacillus subtilis C4 and Achyrocline satureioides extracts on the viability of Paenibacillus larvae.

The aim of this work was to determine the in vitro effect of the mixture between the lipopeptide surfactin, synthesized by Bacillus subtilis C4 (strain isolated from honey) and the most active vegetal extract from Achyrocline satureioides, a traditional medicinal plant, on local strains of Paenibacillus larvae, the agent of American Foulbrood in honeybees. Five P. larvae strains isolated in Córdoba, Argentina, were phenotypically characterized. These and 12 other P. larvae strains from different regions of Argentina were analysed. The antimicrobial activities of the essential oil, hexane (HE) and benzene extracts from A. satureioides were assessed against P. larvae and the HE showed the highest anti-P. larvae activity. A combination of the biosurfactant surfactin, produced by B. subtilis C4, and the HE of A. satureioides revealed a synergistic action on P. larvae. The effective surfactin concentration in the mixture decreased from 32 to 1 µg ml(-1) and the HE concentration from 32 to 4 µg ml(-1), values similar or equal to minimal inhibitory concentrations observed for oxytetracycline. The fractional inhibitory concentration index confirmed synergism in 4 strains and partial synergism in one strain. The combination of surfactin synthesized by B. subtilis C4 and the HE from A. satureioides could be a natural alternative to help beekeepers to combat the American foulbrood agent P. larvae.

Pathogens Detection in the Small Hive Beetle (Aethina tumida (Coleoptera: Nitidulidae)).

Aethina tumida Murray is currently a worldwide emergent pest of Apis mellifera L. hives. Although the damaging effect on the colony stores and brood is well known, the possible role of these beetles as a disease carrier is not clear. This is the first report of DNA presence of the trypanosome honeybee parasite Lotmaria passim and Crithidia bombi, and the Apis mellifera filamentous virus (AmFV) in A. tumida. Further studies will be needed to determine if A. tumida is indeed a mechanical or biological vector of these pathogens.

Evaluation of culture media for Paenibacillus larvae applied to studies of antimicrobial activity.

This study was conducted to compare different liquid culture media for Paenibacillus larvae growth in order to find the best one to be used in studies on activity of antimicrobial substances, such as essential oils. P. larvae presented poor growth in usual broths such as Mueller-Hinton, commonly employed in antimicrobial activity assays. Growth in liquid media was evaluated using Paenibacillus larvae strains isolated from hives located in different geographical zones. The MYT medium (Mueller-Hinton broth, yeast extract and thiamine) was selected out of the eight liquid media analyzed, as it proved to be the most adequate due to its higher absorbance at 620 nm. The following mean values were obtained from the four P. larvae strains: 0.227 +/- 0.016 for the Cobo strain, 0.279 +/- 0.015 for La Plata strain, 0.758 +/- 0.020 for Mechongué strain and 0.244 +/- 0.0079 for Sierra de los Padres strain, respectively.

Lactobacillus kunkeei strains decreased the infection by honey bee pathogens Paenibacillus larvae and Nosema ceranae.

Due to their social behaviour, honey bees can be infected by a wide range of pathogens including the microsporidia Nosema ceranae and the bacteria Paenibacillus larvae. The use of probiotics as food additives for the control or prevention of infectious diseases is a widely used approach to improve human and animal health. In this work, we generated a mixture of four Lactobacillus kunkeei strains isolated from the gut microbial community of bees, and evaluated its potential beneficial effect on larvae and adult bees. Its administration in controlled laboratory models was safe for larvae and bees; it did not affect the expression of immune-related genes and it was able to decrease the mortality associated to P. larvae infection in larvae and the counts of N. ceranae spores from adult honey bees. These promising results suggest that this beneficial microorganism's mixture may be an attractive strategy to improve bee health. Field studies are being carried out to evaluate its effect in naturally infected colonies.

[Inhibition of Paenibacillus larvae employing a mixture of essential oils and thymol]. / Inhibición de Paenibacillus larvae empleando una mezcla de aceites esenciales y timol.

In vitro antimicrobial activity of a mixture of two essential oils and thymol against Paenibacillus larvae, causal agent of American Foulbrood (AFB), was evaluated. The essential oils were extracted from cinnamon (Cinnamomum zeylanicum) and thyme (Thymus vulgaris). The third component used, thymol, is the major component of the essential oil of thyme which contains 39.9% of thymol. Minimal inhibitory concentration (MIC) in Mueller-Hinton broth by the tube dilution method and minimal bactericide concentration (MBC) on MYPGP agar were evaluated. Thyme registered MIC values of 150-250 microg/ml and MBC values of 200-300 microg/ml, while the MIC and MBC values obtained for cinnamon were of 50-100 microg/ml and 100-125 microg/ml. Thymol showed similar MIC and MBC values of 100-150 microg/ml. No significant differences between the bacterial strains were detected, but significant differences between essential oils and thymol activity were registered (P<0,01). An inhibitory synergetic effect on AFB was observed reducing MIC and MBC values due to the use of a mixture of 62.5% of thyme, 12.5% of cinnamon and 25% of thymol.

Mitral and aortic valve decalcification by ultrasonic energy. Experimental report.

In this investigation we used ultrasonic energy to decalcify 10 mitral and six aortic valves after they had been surgically removed. The calcium was disintegrated in all cases and normal valvular tissue was preserved. The fragments of disintegrated calcium were collected in a suction bottle, which prevented peripheral embolism. The ultrasonic urologic probe is an unwieldy instrument for cardiac surgery, and we suggest the manufacture of a new probe for clinical use in our specialty.

Conservative operation for mitral stenosis with densely fibrosed or partially calcified valves. An eight-year evaluation.

Of 284 patients undergoing open mitral commissurotomy for mitral stenosis from January 1978 to December 1985, 81 patients had a densely scarred or partly calcified valve. In this study, we evaluated the postoperative results in these 81 patients. There were no operative or late deaths. Seventy-seven patients (95%) are in Functional Class I or II. Three patients required reoperation 3, 30, and 50 months after the initial procedure because of moderate or severe residual mitral regurgitation. After valvotomy there were four episodes of embolism. The actuarial rate of freedom from any complication (mortality, reoperation, congestive heart failure, and thromboembolic events) was 89.3% +/- 3.9% (mean +/- standard error of the mean) 8 years after operation. We conclude that for a follow-up period of 8 years, the stenotic mitral valve with anatomical deformities can be salvaged with satisfactory results. Therefore, we believe that this approach, at present, is a better alternative than prosthetic replacement with any type of valve presently available.

Early open mitral commissurotomy: long-term results.

It has frequently been suggested that early mitral commissurotomy could improve long-term results in patients with severe mitral stenosis. However, the real advantages of this procedure have yet to be demonstrated. To evaluate this hypothesis, we retrospectively studied 397 patients who underwent operation for mitral stenosis in our unit between 1978 and 1988. Forty of these patients (group I) fulfilled the criteria for early mitral commissurotomy: being young (average age 33 years), being asymptomatic or showing few symptoms, and being in sinus rhythm. The remaining 357 patients who underwent operation during the same period of time served as control (group II). Mitral valve replacement (p < 0.05) and associated tricuspid annuloplasty (p < 0.05) occurred less frequently in patients from group I than it did in patients from group II. Survival after 11 years for patients with early mitral commissurotomy was 100%, 90% of whom were in functional class I, were in sinus rhythm, and were receiving no medication whatsoever. Ninety-six percent of these patients were free of complications after 11 years, compared with 73% of patients in group II (p < 0.05). In our opinion, these results support the use of early mitral commissurotomy for young patients in sinus rhythm, who are symptom free, who are and who have a mitral valve area is 1.3 cm2 or less.

A comparison of repair and replacement for mitral stenosis with partially calcified valve.

From January 1978 to December 1987 we operated on 135 patients with calcified mitral stenosis. In 60 patients a conservative operation was performed (group I). Nine patients required mitral annuloplasty associated with the commissurotomy. The other 75 patients underwent mitral valve replacement (group II). In 37 patients a mechanical prosthesis was used and in 38 a biologic one. The patients given mitral valve replacement had a more heavily calcified valve than those undergoing a conservative procedure. Twenty-one patients (12 from group I and 13 from group II) required associated tricuspid annuloplasty. The mean follow-up time was 69.1 months (1 months to 10 years). There were no significant differences between the two groups in terms of operative death (0% and 4%, respectively), postoperative functional class, actuarial survival rate at 10 years (84% and 96%, respectively), and probability of freedom from thromboemboli at 10 years (98% and 96%, respectively). However, the probability of freedom from reoperation at 10 years significantly favored the conservative surgery group (84% and 69%, respectively, p less than 0.01). Finally, the probability of freedom from complications at 10 years was also significantly higher in the conservative surgery group (82% and 64%, respectively, p less than 0.005). Because of these results we believe that conservative surgery is, at present, a better alternative than mitral valve replacement for patients with partially calcified mitral stenosis.

Intraoperative contrast two-dimensional echocardiography. Evaluation of the presence and severity of aortic and mitral regurgitation during cardiac operations.

We have used contrast two-dimensional echocardiography in the intraoperative evaluation of aortic and mitral regurgitation in 35 patients undergoing cardiac operations. All of them underwent previous cardiac catheterization in order to document the presence and severity of regurgitation. With the pericardium open, a catheter was introduced into the left ventricle (to document mitral regurgitation) or into the ascending aorta (to document aortic regurgitation). The two-dimensional echocardiographic probe was placed on the anterior surface of the right ventricle to obtain a basal image, equivalent to a conventional parasternal longitudinal view. Dextrose in water (5 ml) was rapidly hand-injected through the catheter, while echocardiograms were recorded on videotape. The observation of contrast medium (microbubbles) flowing in the retrograde direction through the incompetent valve was carefully evaluated with the same scoring system used in the hemodynamic laboratory. In 34 cases there was agreement between angiographic and echocardiographic evaluation of the presence and severity of mitral and aortic regurgitation. Only one case was evaluated as mild aortic regurgitation by angiography and moderate aortic regurgitation by echocardiography. There were no false positives or false negatives in the study. In view of the high degree of correlation between contrast two-dimensional echocardiography and hemodynamic data, we suggest that our method is an important tool for the cardiac surgeon. In addition, the present approach overcame the disadvantages of the conventional intraoperative methods, most of which are performed in a nonbeating or fibrillating heart.

Conservative operation for mitral stenosis. Independent determinants of late results.

A retrospective study was designed to define the independent determinants of late results in 282 consecutive patients operated on for mitral stenosis between 1978 and 1985. A total of 25 variables were investigated by multivariate discriminant analysis for their possible influence on symptomatic outcome, subsequent reoperation, postoperative thromboembolism, and death. All the patients underwent a conservative mitral valve operation. In addition, 25 patients required concomitant tricuspid annuloplasty. The surgical mortality rate was 1.4% and the late mortality, reoperation, and thromboembolism rates were, respectively, 0.14%, 0.80%, and 0.95%/pt-yr. Actuarial probability of complication-free survival at 8 years was 83% +/- 3.7%. Left atrial size on the M-mode echocardiograms, tricuspid annuloplasty, mitral annuloplasty, presence of left atrial thrombus, male gender, and separation of subvalvular apparatus portended a significantly higher likelihood of poor postoperative symptomatic status (p less than 0.0005). Preoperative mild mitral regurgitation, E to F slope on the M-mode echocardiogram, and tricuspid annuloplasty had significant influence on the need for late reoperation (p less than 0.05). Postoperative atrial fibrillation, mitral valve amplitude on the M-mode echocardiogram, preoperative embolism, and residual mitral incompetence had an independent predictive power for postoperative thromboembolism (p less than 0.05). Finally, preoperative atrial fibrillation, preoperative cardiothoracic ratio, postoperative left atrial size, and postoperative atrial fibrillation all influenced the probability of long-term complications. These results suggest that earlier operation should be considered in patients with mitral stenosis, to increase the complication-free survival rate.

Aberrant right subclavian artery associated with common carotid trunk. A rare cause of vascular ring.

We have reviewed four cases of incomplete vascular rings caused by the presence of a common carotid trunk from which arose both carotid arteries associated with an aberrant right subclavian artery. The patients were aged between 3 and 9 months. All patients presented with recurrent respiratory tract infections. Three patients showed signs of malnutrition and failure to thrive caused by episodes of bronchial aspiration from extrinsic compression of the oesophagus. One patient presented with dyspnoea induced by feeding and another had stridor. A common carotid trunk associated with an aberrant subclavian artery was confirmed in all cases. No other associated anomaly was observed in any patient. Ligature and section of the anomalous right subclavian artery was performed in all patients. The low incidence of this type of vascular ring, its physiopathological mechanism and surgical management are discussed.

Accurate length adjustment of right or circumflex coronary artery bypass grafts.

A simple method is described for the accurate adjustment of the length of right and circumflex coronary grafts. The essential feature of the technique is the use of a marking suture initially positioned in the pericardium for future reference. The technique has been adopted and successfully used by us over the past year.

Plasma levels of glucose and insulin during deep hypothermia with varying degrees of hemodilution in dogs.

The variations of plasma glucose and insulin levels were studied during the course of deep hypothermia with cardiocirculatory arrest of 60 minutes in 3 experimental groups of dogs using a pump mixture of homologous blood and Ringer's lactate solution at 33%, 50%, and 100% hemodilution. Insulin levels decreased in all groups during the cooling period and remained stable throughout the rest of the experiments, showing a slight significant increase only at the end of rewarming after a temperature of 30 degrees C was reached. Glucose levels reacted similarly except during rewarming, where an important increase in glucose concentration greatly preceded the rise in insulin. We stress the importance of this dissociation in view of the possible clinical implications that may exist.

Pulmonary valve endocarditis during puerperal sepsis.

A case of pulmonary valve endocarditis caused by Staphylococcus aureus during puerperal sepsis in a female patient is reported. The M-mode and two dimensional echocardiographic finding are described. A review of the literature shows that this entity is rare. A large vegetation in the leaflet of the pulmonary valve was excised and the patient recovered after a full course of antibiotics.

Anoxic cardiac arrest: Its effect on myocardial mitochondrial metabolism.

Forty eight healthy dogs are subjects to cardiopulmonary bypass and divide into two groups according to whether the perfusion is performed in normothermia or moderate hypothermia (30 degrees C). The effects on oxidative phosphorylation and mitochondrial respiration, produced by increasing periods of myocardial anoxia from aortic cross clamping, are studied. A clear correlation is found between the disturbances of mitochondrial metabolism and the future cardiac recovery. Hypothermia shows a protective effect on the anoxic myocardium. It is concluded that the period of absolute safety in cardiac anoxia, as far as mitochondrial function is concerned, is 15 minutes in normal thermic perfusion and 90 minutes in moderately hypothermic ones.

Budd-Chiari syndrome due to obstruction of the inferior vena cava. A report of two cases.

Two cases of Budd-Chiari syndrome caused by membranous occlusion of the proximal portion of the inferior vena cava are presented. Both were treated surgically, using the modified technique of Kimura in one case, and a resection with direct vision and extracorporeal circulation in the other. Both patients progressed well. We discuss the etiology and pathogenesis of the syndrome, the diagnostic means that can be used, and the importance of early treatment.