Quantifying plasmid dynamics using single-cell microfluidics and image bioinformatics.

Multicopy plasmids play an important role in bacterial ecology and evolution by accelerating the rate of adaptation and providing a platform for rapid gene amplification and evolutionary rescue. Despite the relevance of plasmids in bacterial evolutionary dynamics, evaluating the population-level consequences of randomly segregating and replicating plasmids in individual cells remains a challenging problem, both in theory and experimentally. In recent years, technological advances in fluorescence microscopy and microfluidics have allowed studying temporal changes in gene expression by quantifying the fluorescent intensity of individual cells under controlled environmental conditions. In this paper, we will describe the manufacture, experimental setup, and data analysis pipeline of different microfluidic systems that can be used to study plasmid dynamics, both in single-cells and in populations. To illustrate the benefits and limitations of microfluidics to study multicopy plasmid dynamics, we will use an experimental model system consisting on Escherichia coli K12 carrying non-conjugative, multicopy plasmids (19 copies per cell, in average) encoding different fluorescent markers and ß-lactam resistance genes. First, we will use an image-based flow cytometer to estimate changes in the allele distribution of a heterogeneous population under different selection regimes. Then we will use a mothermachine microfluidic device to obtain time-series of fluorescent intensity of individual cells to argue that plasmid segregation and replication dynamics are inherently stochastic processes. Finally, using a microchemostat, we track thousands of cells in time to reconstruct bacterial lineages and evaluate the allele frequency distributions that emerge in response to a range of selective pressures.

Older age of childhood type 1 diabetes onset is associated with islet autoantibody positivity >30 years later: the Pittsburgh Epidemiology of Diabetes Complications Study.

AIMS: To examine the association between islet autoantibody positivity and clinical characteristics, residual ß-cell function (C-peptide) and prevalence of complications in a childhood-onset (age <17 years), long-duration (≥32 years) type 1 diabetes cohort. METHODS: Islet autoantibodies (glutamic acid decarboxylase, insulinoma-associated protein 2 and zinc transporter-8 antibodies) were measured in the serum of participants who attended the 2011-2013 Pittsburgh Epidemiology of Diabetes Complications study follow-up examination (n=177, mean age 51 years, diabetes duration 43 years). RESULTS: Prevalences of islet autoantibodies were: glutamic acid decarboxylase, 32%; insulinoma-associated protein 2, 22%; and zinc transporter-8, 4%. Positivity for each islet autoantibody was associated with older age at diabetes onset (glutamic acid decarboxylase antibodies, P=0.03; insulinoma-associated protein 2 antibodies, P=0.001; zinc transporter-8 antibodies, P<0.0001). Older age at onset was also associated with an increasing number of autoantibodies (P = 0.001). Glutamic acid decarboxylase antibody positivity was also associated with lower HbA1c (P = 0.02), insulinoma-associated protein 2 antibody positivity was associated with lower prevalence of severe hypoglycaemic episodes (P=0.02) and both distal and autonomic neuropathy (P=0.04 for both), and zinc transporter-8 antibody positivity was associated with higher total and LDL cholesterol (P=0.01). No association between autoantibody positivity and C-peptide was observed. CONCLUSIONS: The strong association between islet autoantibody positivity and older age at type 1 diabetes onset supports the hypothesis of a less aggressive, and thus more persistent, immune process in those with older age at onset. This observation suggests that there may be long-term persistence of heterogeneity in the underlying autoimmune process.

Resistance profile and molecular characterization of pyrethroid resistance in a Rhipicephalus microplus strain from Colombia.

Intensive use of chemical acaricides for the control of cattle ticks (Rhipicephalus microplus) has led to the development of multiple acaricide resistance in Colombia. The present study aimed to characterize, using toxicological bioassays and molecular biology techniques, the resistance profile of a tick strain isolated from the Arauca state, Northeast Colombia. Commercial acaricides were used in adult immersion tests to determine its in vitro efficacies. Deltamethrin showed very low activity (4-7.3%), a mixture of cypermethrin and chlorpyrifos had intermediate efficacy (64-75.2%), and ethion presented the highest activity (88.5-100%). A colony (Arauquita strain) was established and larval immersion tests confirmed high resistance level to deltamethrin (241-fold) and susceptibility to ivermectin. A quantitative polymerase chain reaction-high resolution melt technique was used to identify single nucleotide polymorphisms (SNPs) in the para-sodium channel gene. All of the genotyped individuals were mutant, presenting one (n = 7), two (n = 7) or three (n = 9) SNPs previously associated with pyrethroid resistance. Sequencing revealed a novel mutation (F712L), that was found for the first time in R. microplus ticks from South America. This is the first description of mutations associated with pyrethroid resistance in R. microplus from Colombia. The acaricide resistance pattern found in the Arauquita strain is similar to other parts of Colombia.

Importance of tissue sampling, laboratory methods, and patient characteristics for detection of Pneumocystis in autopsied lungs of non-immunosuppressed individuals.

To understand the epidemiological significance of Pneumocystis detection in a lung tissue sample of non-immunosuppressed individuals, we examined sampling procedures, laboratory methodology, and patient characteristics of autopsy series reported in the literature. Number of tissue specimens, DNA-extraction procedures, age and underlying diagnosis highly influence yield and are critical to understand yield differences of Pneumocystis among reports of pulmonary colonization in immunocompetent individuals.

Expansion of the Mexican Rice Borer (Lepidoptera: Crambidae) into Rice and Sugarcane in Louisiana.

The Mexican rice borer, Eoreuma loftini (Dyar) (Lepidoptera: Crambidae), is an invasive pest of sugarcane, Saccharum spp., rice, Oryza sativa L., and other graminaceous crops in the Gulf Coast region of the United States. Traps baited with E. loftini female sex pheromones were used to document establishment and distribution of E. loftini near sugarcane, rice, and noncrop hosts in seven southwest Louisiana parishes from 2009 to 2013. Additional field surveys documented larval infestations in commercial sugarcane and rice. After its initial detection in 2008, no E. loftini were detected in Louisiana in 2009 and only two adults were captured in 2010. Trapping documented range expansion into Cameron, Beauregard, and Jefferson Davis parishes in 2011 and Allen, Acadia, and Vermilion parishes in 2013. During the course of this study, E. loftini expanded its range eastward into Louisiana 120 km from the Texas border (≈22 km/yr). Surveys of larval infestations provided the first record of E. loftini attacking rice and sugarcane in Louisiana. Infestations of E. loftini in rice planted without insecticidal seed treatments in Calcasieu Parish reached damaging levels.

Estimation of genetic structure of a Mycosphaerella musicola population using inter-simple sequence repeat markers.

Among the diseases affecting banana (Musa sp), yellow Sigatoka, caused by the fungal pathogen Mycosphaerella musicola Leach, is considered one of the most important in Brazil, causing losses throughout the year. Understanding the genetic structure of pathogen populations will provide insight into the life history of pathogens, including the evolutionary processes occurring in agrosystems. Tools for estimating the possible emergence of pathogen variants with altered pathogenicity, virulence, or aggressiveness, as well as resistance to systemic fungicides, can also be developed from such data. The objective of this study was to analyze the genetic diversity and population genetics of M. musicola in the main banana-producing regions in Brazil. A total of 83 isolates collected from different banana cultivars in the Brazilian states of Bahia, Rio Grande do Norte, and Minas Gerais were evaluated using inter-simple sequence repeat markers. High variability was detected between the isolates, and 85.5% of the haplotypes were singletons in the populations. The highest source of genetic diversity (97.22%) was attributed to variations within populations. Bayesian cluster analysis revealed the presence of 2 probable ancestral groups, however, showed no relationship to population structure in terms of collection site, state of origin, or cultivar. Similarly, we detected noevidence of genetic recombination between individuals within different states, indicating that asexual cycles play a major role in M. musicola reproduction and that long-distance dispersal of the pathogen is the main factor contributing to the lack of population structure in the fungus.

Analysis of FokI polymorphism of vitamin D receptor gene in intervertebral disc degeneration.

AIM: We have hypothesized a possible relationship between disc degeneration (DD) and VDR FokI/T2C polymorphism. METHODS: A case-control study was performed comprising 121 Brazilian patients with confirmed DD by nuclear magnetic resonance and a control group consisting of 131 healthy patients without a history of disc cysts of the lumbar spine. Detection of VDR FokI/T2C polymorphism was performed using restriction fragment length polymorphism-polymerase chain reaction. The chi-square test was used to compare allele and genotype frequencies between groups, and a p-value of <0.05 was considered statistically significant. RESULTS: The results disclosed statistical difference between allele distribution among cases and controls (p=0.025, odds ratio=1.58, confidence interval=1.07-2.32) considering VDR FokI/T2C polymorphism. CONCLUSION: The results showed a positive association between VDR FokI/T2C polymorphism and DD in Brazilian patients tested.

Acaricide and ivermectin resistance in a field population of Rhipicephalus microplus (Acari: Ixodidae) collected from red deer (Cervus elaphus) in the Mexican tropics.

In the Neotropics the control of tick infestations in red deer (Cervus elaphus) is achieved primarily through the use of acaricides and macrocyclic lactones. In Mexico, resistance to one or multiple classes of acaricides has been reported in Rhipicephalus microplus infesting cattle, but information on acaricide susceptibility in R. microplus infesting red deer is lacking. In this study we report the level of resistance to different classes of acaricides and ivermectin in R. microplus collected from red deer in the Mexican tropics. Engorged R. microplus females were collected from a red deer farm in Yucatan, Mexico. The larval packet test was used to detect resistance to the organophosphates (OPs) chlorpyrifos and coumaphos, synthetic pyrethroids (SPs) cypermethrin and permethrin, and the phenylpyrazol, fipronil. Resistance to the formamidine amitraz (Am), and ivermectin was ascertained using the larval immersion test. Data were subjected to probit analysis to determine lethal concentrations and resistance ratios to kill 50% (RR50) and 99% (RR99) of the tick population under evaluation in relation to susceptible reference strains. Additionally, allele specific polymerase chain reaction was used to detect the sodium channel F1550I mutation associated with SP resistance in R. microplus. The R. microplus population from red deer in Yucatan showed very high resistance to the two SPs evaluated (RRs>72.2 for cypermethrin; RR for permethrin resistance was so high a dose-response curve was not possible). All individual larvae tested to detect the sodium channel F1550I mutation associated with SP resistance in R. microplus were homozygous. The same tick population showed different levels of resistance to OPs (chlorpyrifos: RR50=1.55, RR99=0.63; coumaphos: RR50=6.8, RR99=5.9), fipronil (RR50=1.8, RR99=0.9), and amitraz (RR50=2.3, RR99=4.4). Resistance to ivermectin was regarded as moderate (RR50=7.1, RR99=5.0). This is the first report of R. microplus ticks collected from red deer in Mexico with different levels of resistance to four acaricide groups and ivermectin.

In vitro and in vivo evaluation of cypermethrin, amitraz, and piperonyl butoxide mixtures for the control of resistant Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) in the Mexican tropics.

A study was conducted to evaluate the efficacy of cypermethrin, amitraz, and piperonyl butoxide (PBO) mixtures, through in vitro laboratory bioassays and in vivo on-animal efficacy trials, for the control of resistant Rhipicephalus (Boophilus) microplus on cattle in the Mexican tropics. Also, to examine mechanisms of resistance to cypermethrin in this tick population, the frequency of a mutated sodium channel gene (F1550I) was determined using a PCR assay. Results of laboratory bioassays using modified larval packet tests revealed that cypermethrin toxicity was synergized by PBO (from 46.6-57.0% to 83.7-85.0% larval mortality; P<0.05). The cypermethrin and amitraz mixture showed an additive effect (from 46.6-57.0% to 56.0-74.3% larval mortality). Strong synergism was observed with the mixture of cypermethrin+amitraz+PBO and this mixture was the most effective killing resistant tick larvae in vitro (96.7-100% of larval mortality). Tick larvae surviving exposure to cypermethrin or mixtures either with amitraz and PBO in vitro showed 2.9-49.6 higher probability to present the mutated allele than those killed by acaricide treatment (P<0.05). In the in vivo trial, the mixtures containing cypermethrin+PBO (80.6-97.3%), and cypermethrin+amitraz (87.0-89.7%) were more efficacious than cypermethrin alone (76.3-80.5%). The highest level of efficacy was obtained with the mixture of cypermethrin+amitraz+PBO, which yielded >95% control that persisted for 28 days post-treatment against R. microplus infesting cattle when tested under field conditions in the Mexican tropics. Although this mixture is a potentially useful tool to combat pyrethroid resistance, a product based on an acaricide mixture like the one tested in this study has to be used rationally.

Establishing the Jamaica lupus registry: report of patients with systemic lupus erythematosus attending a major referral hospital in Jamaica.

BACKGROUND: Systemic lupus erythematosus (SLE) is an autoimmune disorder characterized by multisystem microvascular inflammation with the generation of autoantibodies. There are reports on demographic data and clinical manifestation of lupus in the United States of America and some other developed countries. There is a single study that has reported on the clinical and immunological features of SLE patients in Jamaica and another that reported that the prevalence of SLE in Jamaica was 5-17/100,000 in 1979. METHOD: A Jamaican lupus registry was established in 2008 at the Department of Medicine, The University of the West Indies. Data were collected using patient records and interview of patients fulfilling the American College of Rheumatology revised diagnostic criteria for SLE. Information on demographics, presence of diagnostic criteria for SLE, presence of complications and other clinical parameters were collected. RESULTS: There were a total of 107 patients that met the criteria for diagnosis of SLE at the referral centre, 96.3% of them female. Positive ANA (90.7%), arthritis (70.0%), malar rash (53.5%) and a positive dsDNA (40.1%) were the more frequent manifestations and diagnostic indices of the disease. Up to 41.7% of the SLE population suffered some form of complication. CONCLUSIONS: The initiation of a lupus registry has allowed for reporting ofpreliminary demographic, clinical and serological data and identifying of disease burden.

New microsatellite markers for bananas (Musa spp).

Thirty-four microsatellite markers (SSRs) were identified in EST and BAC clones from Musa acuminata burmannicoides var. Calcutta 4 and validated in 22 Musa genotypes from the Banana Germplasm Bank of Embrapa-CNPMF, which includes wild and improved diploids. The number of alleles per locus ranged from 2 to 14. The markers were considered highly informative based on their polymorphism information content values; more than 50% were above 0.5. These SSRs will be useful for banana breeding programs, for studies of genetic diversity, germplasm characterization and selection, development of saturated genetic linkage maps, and marker assisted selection.

Non-FDG PET in oncology.

Although FDG PET and PET/CT have a well established role in the management of most cancer patients, they also have some limitations. For the last 15-20 years a growing number of non-FDG PET tracers have been used in research. Many of these new PET tracers are being investigated for the non-invasive assessment of different biologic functions in cancer cells. This unique information should contribute to making personalized cancer therapy a reality. This paper reviews the non-FDG PET tracers that are most likely to find clinical application, some of them in the near future.

Radical prostatectomy outcomes at the University Hospital of the West Indies: 2000-2007.

OBJECTIVES: Prostate cancer is the commonest cancer in Jamaican men with an age-specific incidence of 65.5 per 100 000 and also the commonest cause of male cancer death. This study reports on the oncological outcome and morbidity after radical retropubic prostatectomy. SUBJECTS AND METHODS: The records of 116 patients with clinically localized prostate cancer (cT1c- T2) who underwent radical retropubic prostatectomy at the University Hospital of the West Indies from January 2000 to December 2007 were examined. Preoperative Prostate specific antigen (PSA), clinical stage and Gleason score were recorded. Operative time, blood loss, hospital stay and complications were assessed. Oncological outcome was assessed using biochemical progression. Disease progression was defined by PSA value of 0.4 ng/ml or greater. RESULTS: Mean patient age was 61 (43-75) years. The mean presenting PSA was 10.1 (2-25.1) ng/ml. Mean Gleason score on preoperative biopsy was 6. The commonest clinical stage was T1c (68%). Nodal involvement was seen in only one patient. The positive surgical margin rate was 15.5%. Mean operating time was 246 minutes and mean estimated blood loss was 1.44 L. The mean hospital stay was 6.9 days and 17% of patients developed minor complications, with no treatment or disease related deaths. Five-year biochemical-free survival was 78.4%. CONCLUSIONS: Oncological outcomes after radical retropubic prostatectomy in Jamaica appear to meet global standards with acceptable morbidity.

Lipid oxidation stability of omega-3- and conjugated linoleic acid-enriched sous vide chicken meat.

Lipid oxidation is known to occur rather rapidly in cooked chicken meat containing relatively high amounts of polyunsaturated fatty acids. To assess the lipid oxidation stability of sous vide chicken meat enriched with n-3 and conjugated linoleic acid (CLA) fatty acids, 624 Cobb × Ross broilers were raised during a 6-wk feeding period. The birds were fed diets containing CLA (50% cis-9, trans-11 and 50% trans-10, cis-12 isomers), flaxseed oil (FSO), or menhaden fish oil (MFO), each supplemented with 42 or 200 mg/kg of vitamin E (dl-α-tocopheryl acetate). Breast or thigh meat was vacuum-packed, cooked (74°C), cooled in ice water, and stored at 4.4°C for 0, 5, 10, 15, and 30 d. The lipid oxidation development of the meat was estimated by quantification of malonaldehyde (MDA) values, using the 2-thiobarbituric acid reactive substances analysis. Fatty acid, nonheme iron, moisture, and fat analyses were performed as well. Results showed that dietary CLA induced deposition of cis-9, trans-11 and trans-10, cis-12 CLA isomers, increased the proportion of saturated fatty acids, and decreased the proportions of monounsaturated and polyunsaturated fatty acids. Flaxseed oil induced higher deposition of C18:1, C18:2, C18:3, and C20:4 fatty acids, whereas MFO induced higher deposition of n-3 fatty acids, eicosapentaenoic acid (C20:5), and docosahexaenoic acid (C22:6; P < 0.05). Meat lipid oxidation stability was affected by the interaction of either dietary oil or vitamin E with storage day. Lower (P < 0.05) MDA values were found in the CLA treatment than in the MFO and FSO treatments. Lower (P < 0.05) MDA values were detected in meat samples from the 200 mg/kg of vitamin E than in meat samples from the 42 mg/kg of vitamin E. Nonheme iron values did not affect (P > 0.05) lipid oxidation development. In conclusion, dietary CLA, FSO, and MFO influenced the fatty acid composition of chicken muscle and the lipid oxidation stability of meat over the storage time. Supranutritional supplementation of vitamin E enhanced the lipid oxidation stability of sous vide chicken meat.

Radical prostatectomy outcomes at the university hospital of the West Indies: 2000-2007 / Evolución clínica de la prostatectomía radical en el hospital universitario de West Indies: 2000-2007

OBJECTIVES: Prostate cancer is the commonest cancer in Jamaican men with an age-specific incidence of 65.5 per 100 000 and also the commonest cause of male cancer death. This study reports on the oncological outcome and morbidity after radical retropubic prostatectomy. SUBJECTS AND METHODS: The records of 116 patients with clinically localized prostate cancer (cT1c-T2) who underwent radical retropubic prostatectomy at the University Hospital of the West Indies from January 2000 to December 2007 were examined. Preoperative Prostate specific antigen (PSA), clinical stage and Gleason score were recorded. Operative time, blood loss, hospital stay and complications were assessed. Oncological outcome was assessed using biochemical progression. Disease progression was defined by PSA value of 0.4 ng/ml or greater. RESULTS: Mean patient age was 61 (43-75) years. The mean presenting PSA was 10.1 (2-25.1) ng/ml. Mean Gleason score on preoperative biopsy was 6. The commonest clinical stage was T1c (68%). Nodal involvement was seen in only one patient. The positive surgical margin rate was 15.5%. Mean operating time was 246 minutes and mean estimated blood loss was 1.44 L. The mean hospital stay was 6.9 days and 17% of patients developed minor complications, with no treatment or disease related deaths. Five-year biochemical-free survival was 78.4%. CONCLUSIONS: Oncological outcomes after radical retropubic prostatectomy in Jamaica appear to meet global standards with acceptable morbidity.

OBJETIVOS: El cáncer de la próstata es el cáncer más común entre los hombres jamaicanos con una incidencia específica por edad de 65.5 por 100 000, y es también la causa más común de la muerte por cáncer entre los hombres. Este estudio reporta la evolución clínica oncológica y la morbilidad tras la prostatectomía radical retropúbica. MÉTODOS: Se examinaron las historias clínicas de 116 pacientes con cáncer de próstata clínicamente localizado (cT1c - T2), sometidos a prostatectomía radical retropúbica en el Hospital Universitario de West Indies de enero de 2000 a diciembre 2007. Se registraron el antígeno específico de próstata (AEP) preoperativo, la etapa clínica y la puntuación de Gleason. Se evaluaron el tiempo operativo, la pérdida de sangre, la estadía hospitalaria, y las complicaciones. Se evaluó la evolución clínica oncológica usando la progresión bioquímica. La progresión de la enfermedad se definió por el valor del AEP de 0.4 ng/ml o mayor. RESULTADOS: La edad promedio de los pacientes fue 61 (43-75) años. El AEP promedio fue PSA 10.1 (2-25.1) ng/ml. La puntuación promedio Gleason en la biopsia preoperativa fue 6. La etapa clínica más común fue T1c (68%). Se observó compromiso de nódulos en sólo un paciente. La tasa de márgenes quirúrgicos positivos fue 15.5%. El tiempo promedio de operación fue 246 minutos y la pérdida de sangre promedio estimada fue 1.44 L. La estadía hospitalaria promedio fue de 6.9 días y 17% de los pacientes desarrollaron complicaciones menores, sin tratamiento o enfermedad relacionados con muertes. La supervivencia quinquenal libre del uso de productos bioquímicos fue 78.4%. CONCLUSIONES: Los resultados oncológicos después de la prostatectomía retropúbica radical en Jamaica muestran estar en correspondencia con los estándares globales con una morbilidad aceptable.

Dietary lipid source and vitamin E effect on lipid oxidation stability of refrigerated fresh and cooked chicken meat.

The fatty acid composition of chicken muscle may affect the lipid oxidation stability of the meat, particularly when subjecting the meat to thermal processing and storage. The objective of this study was to evaluate the diet effect on lipid oxidation stability of fresh and cooked chicken meat. Six hundred broilers were raised for a 6-wk feeding period and were assigned to 8 treatments with 3 repetitions. Broilers were fed a basal corn-soybean meal diet, including 5% of either animal-vegetable, lard, palm kernel, or soybean (SB) oil, each supplemented with a low (33 mg/kg) or high (200 to 400 mg/kg) level of vitamin E. Fresh breast and thigh meat and skin were packaged and refrigerated (4°C) for 15 d. Breast and thigh meat were frozen (-20°C) and stored for ~6 mo and then thawed, deboned, ground, and formed into patties of 150 g each. Patties were cooked (74°C), cooled, packaged, and stored in refrigeration for 6 d. The lipid oxidation development of the products was determined using the TBA reactive substances analysis. The results showed that the lipid oxidation development, in both fresh chicken parts and cooked meat patties, was influenced by the interaction of either dietary lipid source or vitamin E level with storage time. Fresh breast meat showed no susceptibility to lipid oxidation, but thigh meat and skin presented higher (P < 0.05) malonaldehyde values in the SB oil treatment, starting at d 10 of storage. In cooked patties, during the entire storage time, the SB oil showed the highest (P < 0.05) lipid oxidation development compared with the other treatments. Regarding vitamin E, in both fresh parts and cooked meat patties, in most sampling days the high supplemented level showed lower (P < 0.05) malonaldehyde values than the control treatment. In conclusion, the lipid oxidation stability of chicken meat is influenced by the lipid source and vitamin E level included in the diet upon storage time and processing of the meat.

Soybean, palm kernel, and animal-vegetable oils and vitamin E supplementation effect on lipid oxidation stability of sous vide chicken meat.

There is an increasing demand in precooked chicken meat products for restaurants and catering services. Because cooked chicken meat develops lipid oxidation relatively fast, sous vide chicken meat was studied to assess its shelf-life. Six hundred Cobb x Ross broilers were fed for 6 wk with a basal corn-soybean meal diet including soybean, palm kernel, or animal-vegetable oil, each supplemented with 33 or 200 mg/kg of dl-alpha-tocopheryl acetate. Broilers were randomly assigned into 6 treatments and 4 repetitions with 25 birds each. Boneless breast or thigh muscle pieces were dissected into 5 x 5 x 5 cm cubes, vacuum-packed, cooked in water bath (until 74 degrees C internal temperature), chilled, and stored at 4 degrees C for 1, 5, 10, 25, and 40 d. For each storage day, each pouch contained 3 pieces of meat, either breast or thigh. Thiobarbituric acid reactive substances analysis, to quantify malonaldehyde (MDA) values, was conducted to estimate the lipid oxidation development. Nonheme iron values of cooked meat were analyzed. Fatty acid methyl esters analysis was performed in chicken muscle to determine its fatty acid composition. There was no interaction between dietary fat and vitamin E level in all of the variables studied except in nonheme iron. Dietary fat significantly influenced the fatty acid composition of the muscle (P < 0.01), but it did not affect the MDA values, regardless of differences in the muscle fatty acid composition between treatments. Supplementation of the high level of vitamin E significantly reduced the MDA values in both breast and thigh meat (P < 0.01). The maximum MDA values were observed at d 40 of storage in thigh and breast meat in animal-vegetable and soybean oil treatments with the low levels of vitamin E, 0.91 and 0.70 mg/kg, respectively. Nonheme iron values in thigh meat differed between treatments at 1 or 25 d of storage but not in breast meat. In conclusion, refrigerated sous vide chicken meat has a prolonged shelf-life, which is enhanced by dietary supranutritional supplementation of vitamin E.

Purification and characterization of a liver-derived beta-N-Acetylhexosaminidase from marine mammal Sotalia fluviatilis.

A beta-N-Acetylhexosaminidase (EC 3.2.1.52) was purified from hepatic extracts of Sotalia fluviatilis, order Cetacea. The protein was purified by using ammonium sulfate fractionation and four subsequent chromatographies (Biogel A 1.5 m, Chitin, Deae-Biogel and hydroxyapatite resins). After these purification steps, the enzyme was purified 380.5-fold with an 8.4% yield. The molecular mass (10 kDa) was estimated by SDS-PAGE and MALDI-TOF analysis. A Km of 2.72 mM and Vmax 9.5 x 10(-6) micromol/(min x mg) were found for this enzyme, determined by p-nitrophenyl-beta-D: -hexosaminide substrate digestion. Optimal pH and temperature for beta-N-Acetylhexosaminidase activity were 5.0 and 60 degrees C, respectively. Enzyme activity was inhibited by sodium selenate (Na(2)SeO(4)), mercuric chloride (HgCl(2)) and sodium dodecyl sulfate (C(12)H(25)SO(4)Na), and activated by zinc, calcium, barium and lithium ions. Characterization of the beta-N-Acetylhexosaminidase in Sotalia fluviatilis can be a basis for physiological studies in this species.

First report of the cattle tick Rhipicephalus microplus resistant to ivermectin in Mexico.

Three cattle farms with ticks, Rhipicephalus microplus, thought to be resistant to ivermectin in Yucatan, Mexico were studied (SFDO, SPN, LUADY). Each field-population was collected and tested twice several months apart. The larval immersion test was used on the progeny of collected adult females to test the susceptibility to ivermectin. Dose-mortality regressions, lethal concentrations (LC), their confidence intervals and slope were estimated by probit analysis. Resistance ratios (RR) were determined in the three investigated populations at the LC(50) and LC(99) estimates. The LUADY (RR(50): 2.04 and 2.29, RR(99): 2.67 and 3.55), SPN (RR(50): 3.55 and 3.68, RR(99): 8.19-11.06) and SFDO (RR(50): 6.84 and 8.59, RR(99): 54.17 and 87.86) ticks had significantly higher LC(50)/LC(99) than the reference susceptible Deutch strain, demonstrating resistance in the field-collected populations. Furthermore, there was significant difference between LC(50)/LC(99) of the SFDO, SPN and LUADY tick populations, which indicates not only the presence of resistant populations, but also different levels of resistance to ivermectin in the field populations studied. There was no difference observed at the LC(50) nor LC(99) estimates at two different times of collection from any of the three populations studied. In conclusion, we report for the first time field populations of R. microplus resistant to ivermectin in Mexico.

DNA identification and characterization of Campylobacter jejuni and Campylobacter coli isolated from caecal samples of chickens in Grenada.

AIMS: To speciate Campylobacter strains from the caeca of chickens in Grenada using PCR and to evaluate DNA-based typing methods for the characterization of these isolates. METHODS AND RESULTS: Isolates were speciated with two multiplex PCR assays and were typed with flaA-RFLP, pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Results confirmed that Campylobacter coli strains were more predominant than Campylobacter jejuni strains. From 56 isolates, 18 were misidentified using biochemical tests. PFGE typing gave the highest discriminatory power among the methods used (Simpson's index of diversity, D=0.9061). However, the combination of flaA-RFLP, PFGE and MLST results gave the highest discrimination for subtyping of these isolates (D=0.9857). A band position tolerance of 4% in BioNumerics was the most appropriate for the analysis of this database. MLST profiles were generally concordant with PFGE and/or flaA-RFLP types. Several isolates exhibited new MLST sequence types (STs), and 43 of the 49 Camp. coli strains belonged to the ST-828 clonal complex. CONCLUSIONS: Campylobacter coli was the most prevalent species isolated from broilers and layers in Grenada, and a combination of restriction and sequence methods was most appropriate for the typing of Camp. coli isolates. Campylobacter coli STs clustered with described poultry-associated Camp. coli STs by phylogenetic analysis. SIGNIFICANCE AND IMPACT OF THE STUDY: Further studies to understand the predominance of Camp. coli within Campylobacter spp. from chickens in Grenada may help elucidate the epidemiology of these pathogens in chickens.