A transgenic male-only strain of the New World screwworm for an improved control program using the sterile insect technique.

BACKGROUND: The New World screwworm, Cochliomyia hominivorax, is a devastating pest of livestock endemic to subtropical and tropical regions of the Western hemisphere. The larvae of this species feed on the tissue of living animals, including man, and can cause death if untreated. Over 60 years ago, the sterile insect technique (SIT) was developed with the aim of eradicating this pest, initially from Florida but subsequently from all of North and Central America. From the outset it was appreciated that SIT would be more efficient if only sterile males were released in the field, but this was not possible until now. RESULTS: Here, we report on the development and evaluation of the first sexing strains of C. hominivorax that produce only males when raised on diet without tetracycline. Transgenic lines have been developed that possess a tetracycline repressible female-lethal genetic system. Ten of these lines show high female lethality at the late larval/pupal stages and three of them present dominant female lethality. Most of the lines were comparable to the wild type parental strain in several fitness parameters that are relevant to mass rearing in a production facility. Further, three lines performed well in male mating success and male competition assays, suggesting they would be sexually competitive in the field. Consequently, one transgenic line has been selected by the New World Screwworm Program for evaluation under mass rearing conditions. CONCLUSIONS: We conclude that the promising characteristics of the selected sexing strains may contribute to reduce production costs for the existing eradication program and provide more efficient population suppression, which should make a genetic control program more economical in regions were C. hominivorax remains endemic.

An alternative chicken-based diet for mass-rearing screwworm flies.

Screwworm flies are mass-reared and released along the Panama-Colombia border to prevent reinfestation of Central and North America. The cost of the production facility, labor, and diet materials makes mass-rearing the most expensive component of the program. The mass-rearing diet has a large impact on the quality and quantity of insects produced, both of which are necessary for the successful implementation of the sterile insect technique. The diet currently used to rear screwworm flies in Panama contains dried bovine red blood cells, dried bovine plasma, egg powder, milk replacement powder, cellulose (thickening agent), formaldehyde (antimicrobial), and water. Here, we tested an alternative diet containing 2 chicken by-products, which cost less and are locally available, to replace the egg powder and milk replacement powder currently used in the diet. We used 2 screwworm colony strains in our test, the current production strain (Jamaica) and an early female-lethal strain. The chicken diet performed similarly to the production diet with the Jamaica strain, while further optimization will likely be needed for transgenic strain. Finally, nutritional analysis conducted on 7 diet ingredients will assist with diet optimization and the identification of alternative diet ingredients.

Doxycycline is a viable alternative to tetracycline for use in insect Tet-Off transgenic sexing systems, as assessed in the blowflies Cochliomyia hominivorax and Lucilia cuprina (Diptera: Calliphoridae).

Transgenic insect strains with tetracycline repressible (Tet-Off) female-lethal genes provide significant advantages over traditional sterile insect techniques for insect population control, such as reduced diet and labor costs and more efficient population suppression. Tet-Off systems are suppressed by tetracycline-class antibiotics, most commonly tetracycline (Tc) or doxycycline (Dox), allowing for equal sex ratio colonies of transgenic insects when reared with Tc or Dox and male-only generations in their absence. Dox is a more stable molecule and has increased uptake than Tc, which could be advantageous in some insect mass-rearing systems. Here, we evaluated the suitability of Dox for rearing Tet-Off female-lethal strains of Australian sheep blowfly, Lucilia cuprina (Wiedemann, 1830) (Diptera: Calliphoridae), and New World screwworm, Cochliomyia hominivorax (Coquerel, 1858) (Diptera: Calliphoridae), and the effects of dosage on strain performance. For both species, colonies were able to be maintained with mixed-sex ratios at much lower dosages of Dox than Tc. Biological yields of C. hominivorax on either antibiotic were not significantly different. Reduction of Dox dosages in C. hominivorax diet did not affect biological performance, though rearing with 10 or 25 µg/mL was more productive than 50 µg/mL. Additionally, C. hominivorax mating performance and longevity were equal on all Dox dosages. Overall, Dox was a suitable antibiotic for mass-rearing Tet-Off female-lethal L. cuprina and C. hominivorax and was functional at much lower dosages than Tc.

The microbiome of wild and mass-reared new world screwworm, Cochliomyia hominivorax.

Insect population control through continual releases of large numbers of sterile insects, called sterile insect technique (SIT), is only possible if one can mass-rear large quantities of healthy insects. Adaptation of insect stocks to rearing conditions and artificial feeding systems can have a multitude of negative effects such as inbreeding depression, reduced compatibility with wild strains, unintentional selection for traits that lower fitness after release, and an altered microbiome. Changes to insect microbiomes can have many effects on insects ranging from a reduction in sex pheromones or reduced fitness. Thus understanding these systems is important for mass rearing and the performance of the sterile insect control programs. In this study we explored the microbiome of the New World screwworm, Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae) an economically important parasite of warm-blooded animals. Samples from myiases in cows and wild adults were compared to and mass-reared flies used by the SIT program. Significant differences were observed between these treatments, with wild captured flies having a significantly more diverse microbial composition. Bacteria known to stimulate oviposition were found in both wild and mass-reared flies. Two bacteria of veterinary importance were abundant in wild flies, suggesting screwworm is a potential vector of these diseases. Overall, this study provides the screwworm eradication program a platform to continue exploring the effects associated bacteria have on screwworm fitness.

Genomic analyses of a livestock pest, the New World screwworm, find potential targets for genetic control programs.

The New World Screwworm fly, Cochliomyia hominivorax, is a major pest of livestock in South America and Caribbean. However, few genomic resources have been available for this species. A genome of 534 Mb was assembled from long read PacBio DNA sequencing of DNA from a highly inbred strain. Analysis of molecular evolution identified 40 genes that are likely under positive selection. Developmental RNA-seq analysis identified specific genes associated with each stage. We identify and analyze the expression of genes that are likely important for host-seeking behavior (chemosensory), development of larvae in open wounds in warm-blooded animals (heat shock protein, immune response) and for building transgenic strains for genetic control programs including gene drive (sex determination, germline). This study will underpin future experiments aimed at understanding the parasitic lifestyle of the screwworm fly and greatly facilitate future development of strains for efficient systems for genetic control of screwworm.

Specific Gene Disruption in the Major Livestock Pests Cochliomyia hominivorax and Lucilia cuprina Using CRISPR/Cas9.

Cochliomyia hominivorax and Lucilia cuprina are major pests of livestock. Their larvae infest warm-blooded vertebrates and feed on host's tissues, resulting in severe industry losses. As they are serious pests, considerable effort has been made to develop genomic resources and functional tools aiming to improve their management and control. Here, we report a significant addition to the pool of genome manipulation tools through the establishment of efficient CRISPR/Cas9 protocols for the generation of directed and inheritable modifications in the genome of these flies. Site-directed mutations were introduced in the Chominivorax and Lcuprina yellow genes (ChY and LcY) producing lightly pigmented adults. High rates of somatic mosaicism were induced when embryos were injected with Cas9 ribonucleoprotein complexes (RNPs) pre-assembled with guide RNAs (sgRNAs) at high concentrations. Adult flies carrying disrupted yellow alleles lacked normal pigmentation (brown body phenotype) and efficiently transmitted the mutated alleles to the subsequent generation, allowing the rapid creation of homozygous strains for reverse genetics of candidate loci. We next used our established CRISPR protocol to disrupt the Chominivorax transformer gene (Chtra). Surviving females carrying mutations in the Chtra locus developed mosaic phenotypes of transformed ovipositors with characteristics of male genitalia while exhibiting abnormal reproductive tissues. The CRISPR protocol described here is a significant improvement on the existing toolkit of molecular methods in calliphorids. Our results also suggest that Cas9-based systems targeting Chtra and Lctra could be an effective means for controlling natural populations of these important pests.

A comparison between polymerase chain reaction (PCR) and traditional techniques for the diagnosis of leptospirosis in bovines.

Leptospirosis is caused by Leptospira, gram negative spirochaetes whose microbiologic identification is difficult due to their low rate of growth and metabolic activity. In Colombia leptospirosis diagnosis is achieved by serological techniques without unified criteria for what positive titers are. In this study we compared polymerase chain reaction (PCR) with microbiological culture and dark field microscopy for the diagnosis of leptospirosis. Microbiological and molecular techniques were performed on 83 samples of urine taken from bovines in the savannahs surrounding Bogotá in Colombia, with presumptive diagnosis of leptospirosis. 117 samples of urine taken from healthy bovines were used as negative controls. 83 samples were MAT positive with titers ≥ 1:50; 81 with titers ≥ 1:100; and 66 with titers ≥ 1:200. 36% of the total samples (73/200) were Leptospira positives by microbiological culture, 32% (63/200) by dark field microscopy and 37% (74/200) by PCR. Amplicons obtained by PCR were 482 base pair long which are Leptospira specific. An amplicon of 262 base pairs typical of pathogenic Leptospira was observed in 71 out of the 74 PCR positive samples. The remaining 3 samples showed a 240 base pair amplicon which is typical of saprophytic Leptospira. PCR as a Leptospira diagnosis technique was 100% sensitive and 99% specific in comparison to microbiological culture. Kappa value of 0.99 indicated an excellent concordance between these techniques. Sensitivity and specificity reported for MAT when compared to microbiological culture was 0.95 and 0.89 with a ≥ 1:50 cut off. PCR was a reliable method for the rapid and precise diagnosis of leptospirosis when compared to traditional techniques in our study. The research presented here will be helpful to improve diagnosis and control of leptospirosis in Colombia and other endemic countries.

Measurement of depletion region width in poled silica.

The width of the depletion region in fused-silica samples thermally poled during various periods of time is investigated experimentally with four previously reported characterization techniques in an attempt to unify their findings. Although all measurements give a similar width of the depletion region, it is shown that the determination of the profile of chi(2) is also required for a good estimate of the nonlinearity induced by poling.

Aislamiento y susceptibilidad de especies bacterianas sin reporte previo como causantes de infección ocular en segmento anterior / Isolation and susceptibility in bacterial species without a Previous report as the cause of eye infection in the anterior Segment

A nivel ocular, gran cantidad de microorganismo generan infección; la falta de cultivos bacteriológicos en la práctica oftalmológica, puede conllevar a un subregistro de los agentes etiológicos bacterianos causantes de las infecciones extra oculares, debido a que estos no se identifican. En este artículo se reportan seis especies bacterianas aisladas de pacientes con infección extra ocular, algunas sin reporte previo; se relaciona el diagnostico clínico presuntivo y la susceptibilidad encontrada frente a los antibióticos comúnmente utilizados en el campo oftalmológico. Para esto se evaluaron bacteriológicamente 286 muestras oculares, de pacientes con diagnostico clínico de conjuntivitis, se realizó coloración de Gram, cultivos bacterianos y pruebas de susceptibilidad. El análisis estadístico se realizó en los programas EPI-INFO y ESTATA 6,0. De los 286 cultivos realizados se aislaron 177 microorganismos, de los cuales se identificaron 6 géneros microbianos, poco asociados a infecciones del segmento anterior algunos sin reporte previo en la literatura, Enterococcus (n=6) y S. Grupo D no enterococo (n=3), Alcaligenes feacalis (n=3), Citrobacter sp (n=2), Kluyvcra ascorbata (n=2) y Chryseobacterium meningosepticum (n=1). Con respecto a la resistencia, Trimethoprim Sulfamethoxazole (SXT), Cephalothin (CF) y Tobramicina (NN) fueron los antibióticos frente a los cuales se observó una mayor resistencia. A partir de los resultados obtenidos se hace necesario implementar cultivos y antibiogramas en la práctica oftálmica con el fin de identificar y documentar estos microorganismos, y poder relacionarlos, según su frecuencia, con diversas patologías oculares, que además de determinar la implicación definitiva como patógenos oculares también contribuya al estudio de los cambios en la epidemiologia de las infecciones, y al monitoreo de las variaciones en la sensibilidad y resistencia bacteriana.

A great number of microorganisms produce infections in the eyes. The lack of bacteriologiccultures in the ophthalmic practice may lead to anunder registration of bacterial etiological agentswhich cause infections outside the eyes, due to theyare not identified. This article reports six isolatedbacterial species in patients with infections outsidethe eyes, some without previous report; and it alsomentions the presumptive clinic diagnosis and thesusceptibility found in front of the commonly usedantibiotics in the ophthalmic field. 286 eye sampleswere bacteriologically evaluated from patients witha clinic diagnosis of conjunctivitis; Gram coloration,bacterial cultures and susceptibility tests were made.The statistical analysis was made in EPI-INFO andESTATA 6.0 programs. Out of the 286 cultures, 177microorganisms were isolated, from which 6microbial genres were identified, not very associatedto infections in the anterior segment, some withoutany pervious report in literature, Enterococcus(n=6), S.Group D no enterococcus (n=3), Alcaligenesfeacalis (n=3), Citrobacter sp (n=2), Kluyveraascorbata (n=2) and Chryseobacterium meningosepticum(n=1). Regarding resistance, Trimethroprimsulfamethoxazole (SXT), Cephalotin (CF), and Tobramicina(NN) were the antibiotics which organismspresented a higher resistance. Based on the results,it is necessary to implement cultures andantibiograms in the ophthalmic practice in order toidentify and document these microorganisms; to beable to relate them with different eye pathologies byits frequency; to determine the definite implicationas eye pathogens; and to contribute also to the studyof the changes in the epidemiology of infections andto the monitoring of sensibility variations andbacterial resistance.

Aislamiento e identificacion de 10 cepas bacterianas desnitrificantes a partir de un suelo agricola contaminado con abonos nitrogenados proveniente de una finca productora de cebolla en la laguna de Tota, Boyaca Colombia / Isolation and identification of ten bacterial denitrifying stocks from a planting soil contaminated with nitrogen rich fertilizer from an onion farm in Laguna de Tota Boyaca Colombia

Este articulo presenta los resultados de la versatilidad adaptativa y tolerancia de una consorcio bacterianoconstituido por 10 cepas bacterianas endémicas de un suelo agrícola contaminado, proveniente de una fincaproductora de cebolla ubicada en la laguna de Tota en Aquitania, Boyacá, Colombia, expuesto al uso incontroladode fertilizantes nitrogenados. La consorcio bacteriano aislado del suelo está conformado por: Bacillusmegaterium, Bacillus licheniformis, Pseudomonas stutzeri, Acinetobacter sp, Propionibacterium sp, Peptoestreptococcus sp, Sthaphylococcus coagulasa negativa, Corynebacterium sp, Clostridium sp y Actinomyces sp. Se demostró que los microorganismos aislados tienen capacidad desnitrificante in vitro,transformando el nitrato a nitrógeno molecular. La consorcio bacteriano aislado se constituye en una alternativa de biorremediación para recuperar suelo agrícola contaminado con exceso de fertilizantes nitrogenados.

Estudio serológico de infección ocular por toxoplasma gondii en pacientes que conviven o no con animales / Serological study of ocular infection by gondii toxoplasm in patients who live with animals

El Toxoplasma gondii, se encuentra como parásito del gato y algunos felinos. En las materias fecales de los gatos salen los ooquistes los cuales son infectantes. Los parásitos posteriormente desarrollan quistes que contienen en su interior numerosos bradizoitos. La formación de quistes hace que la infección se vuelva crónica y permanezca en forma latente por muchos años. En la fase de reproducción por taquizoitos hay daño celular y reacción inflamatoria. Por diseminación hemática o linfática hay invasión hacia todo el organismo. Las lesiones oculares por Toxoplasma gondii tanto en la forma congénita como en la adquirida constituyen una uveítis granulomatosa.Se estudiaron 25 pacientes del Instituto de Investigaciones Optométricas de la Universidad de la Salle, y pacientes captados en brigadas de salud realizadas en las veredas a las afueras de la ciudad de Bogotá.Se tomaron muestras serológicas para determinar los títulos de anticuerpos isotipo IgG e IgM contra T. gondii mediante la técnica de MICROELISA. (Diagnostics systems).Con relación a las pruebas serológicas para infección intraocular por Toxoplasma gondii se detectaron títulos de anticuerpos anti-Toxoplasma en el 92 por ciento (23/25) de los casos. Se descarto infección reciente en el 86 por ciento (20/23) de las pruebas positivas ya que los valores de IgM fueron negativos y solo en el 13 por ciento (3/23) se detectó una infección reciente. De los casos positivos el 62 por ciento presentó uveítis y el 34 por ciento retinocoroiditis. En este estudio al comparar la presencia de anticuerpos contra T. gondii con la convivencia con animales y la zona de procedencia se observó que 11 pacientes (91 por ciento) con títulos positivos conviven con animales y su procedencia es urbana, 10 pacientes (83 por ciento) que habitan en zona urbana no conviven con animales y 2 conviven con animales y habitan en zona rural (100 por ciento), por lo tanto no existe asociación estadísticamente significativa entre la convivencia con animales y la zona de procedencia.

Alteraciones oculares en diabetes / Ocular alterations in diabetes

Muchas alteraciones oculares son ocasionadas por los cambios bioquímicos generados en la Diabetes. La diabetes es una entidad patológica con múltiples complicaciones, siendo el daño ocular una de las más importantes, ya que se presentan problemas visuales y retinopatía. Aproximadamente el 25 por ciento de los diabéticos generen ceguera por alteraciones en retina lo que ocasiona discapacidad en los pacientes y disminuye su calidad de vida. La retinopatía es característica de personas con diabetes tipo 1 y 2, según estudios realizados por Dinneen S et al, 1998 en Estados Unidos es la causa principal de ceguera en adultos. En Colombia no se han realizado investigaciones en estricto sentido donde se busque la asociación entre parámetros bioquímicos, genéticos, oftalmológicos y optométricos. Las investigaciones realizadas se enfocan en un seguimiento refractivo a ciertos pacientes que presentan diabetes, pero no se han asociado estas alteraciones y otras con marcadores biológicos, por consiguiente, es importante realizar estudios donde se correlacionen aspectos bioquímicos, genéticos, oftalmológicos y optométricos para generar medidas que permitan mejorar las condiciones de las personas que tienen esta patología. Esta revisión pretende presentar algunos aspectos bioquímicos relacionados con alteraciones oculares.

Many ocular alterations are you caused by the biochemistry changes generated in the Diabetes. The diabetes is a pathological company with multiple complications, being the ocular damage one of the most important, since visual problems are presented and retinopathy. Approximately the 25 percent of the diabetic they generate blindness by alterations in retaining what causes disability in the patients and diminishes its quality of life. The retinopathy is characteristic of persons with diabetes type 1 and 2, according to studies carried out by Dinne S et al, 1998 in the United States is the main cause of blindness in adults. In Colombia itself not investigations in strict sense have been carried out where the association among parameters be sought biochemistry, genetic, ophthalmologic and optometric. The investigations carried out are focused in a monitoring refractive to certain patients that present diabetes, but themselves not these alterations they have associated and other with biological scoreboards, consequently, is important to carry out studies where be correlated aspects biochemistry, genetic, ophthalmologic and optometric to generate measures that permit to improve the conditions of the persons that have this pathology. This revision intends to present some aspects biochemistry related to ocular alterations.