RESUMEN
Insertional mutagenesis is an important tool for functional genomics in Drosophila melanogaster. The insertion site in the KG00562 mutant fly line has been mapped to the CG8709 (herein named DmLpin) locus and to the 3’ of kermit (also called dGIPC). This mutant line presents a high lethality rate resulting from a gain of function. To obtain some insight into the biological role of the mutated locus, we have characterized the mutation and its relation to the high mortality of the KG00562 fly line. In this mutant, we did not detect one of the DmLpin transcripts, namely DmLpinK, but we did detect an unusual 2.3-kb mRNA (LpinK-w). Further investigation revealed that the LpinK-w transcript results from an aberrant splicing between the untranslated first exon of DmLpinK and the mini-white marker gene. Lack of DmLpinK or LpinK-w expression does not contribute to lethality, since heterozygous KG00562/Def7860 animals presented lethality rates comparable to those of the wild type. In contrast, the overexpression of kermit was associated with lethality of the KG00562 fly line. Significantly higher levels of kermit were detected in the Malpighian tubules of KG00562/+ flies that presented higher lethality rates than wild-type or KG00562/Def7860 animals, in which the lethality was rescued. In agreement with a recently reported study, our data support the hypothesis that misexpression of kermit/dGIPC could interfere with Drosophila development, with further investigations being needed in this direction.
Asunto(s)
Animales , Proteínas Portadoras/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Expresión Génica/genética , Mutación/genética , Transcripción Genética/genética , Proteínas Portadoras/metabolismo , Proteínas de Drosophila/metabolismo , Túbulos de Malpighi/química , Fenotipo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transformación GenéticaRESUMEN
Insertional mutagenesis is an important tool for functional genomics in Drosophila melanogaster. The insertion site in the KG00562 mutant fly line has been mapped to the CG8709 (herein named DmLpin) locus and to the 3' of kermit (also called dGIPC). This mutant line presents a high lethality rate resulting from a gain of function. To obtain some insight into the biological role of the mutated locus, we have characterized the mutation and its relation to the high mortality of the KG00562 fly line. In this mutant, we did not detect one of the DmLpin transcripts, namely DmLpinK, but we did detect an unusual 2.3-kb mRNA (LpinK-w). Further investigation revealed that the LpinK-w transcript results from an aberrant splicing between the untranslated first exon of DmLpinK and the mini-white marker gene. Lack of DmLpinK or LpinK-w expression does not contribute to lethality, since heterozygous KG00562/Def7860 animals presented lethality rates comparable to those of the wild type. In contrast, the overexpression of kermit was associated with lethality of the KG00562 fly line. Significantly higher levels of kermit were detected in the Malpighian tubules of KG00562/+ flies that presented higher lethality rates than wild-type or KG00562/Def7860 animals, in which the lethality was rescued. In agreement with a recently reported study, our data support the hypothesis that misexpression of kermit/dGIPC could interfere with Drosophila development, with further investigations being needed in this direction.
Asunto(s)
Proteínas Portadoras/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Expresión Génica/genética , Mutación/genética , Transcripción Genética/genética , Animales , Proteínas Portadoras/metabolismo , Proteínas de Drosophila/metabolismo , Túbulos de Malpighi/química , Fenotipo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transformación GenéticaRESUMEN
The prevalence of Toxoplasma gondii in 118 unwanted dogs from São Paulo City, São Paulo State, Brazil, was determined. Antibodies to T. gondii were assayed by the modified agglutination test and found in 42 (35.8%) dogs, with titers of 1:20 in 10, 1:40 in 6, 1:80 in 5, 1:160 in 5, 1:320 in 6, 1:640 in 7, and 1:1,280 or higher in 3. Hearts and brains of 36 seropositive dogs were bioassayed in mice, or cats, or both. Tissues from 20 seropositive dogs were fed to 20 T. gondii-free cats. Feces of cats were examined for oocysts. Toxoplasma gondii was isolated from 15 dogs by a bioassay in mice, from the brain alone of 1, from the heart alone of 4, and from both brains and hearts of 10. All infected mice from 5 of 15 isolates died of toxoplasmosis during primary infection. Four additional isolates were obtained by bioassay in cats. Genotyping of these 19 T. gondii isolates using polymorphisms at 10 nuclear markers including SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and a new SAG2 (an apicoplast marker Apico) revealed 12 genotypes. One isolate had Type III alleles at all 11 loci, and the remaining 18 isolates contained a combination of different alleles and were divided into 11 genotypes. The absence of Type II in Brazil was confirmed. The result supports previous findings that T. gondii population genetics is highly diverse in Brazil.
Asunto(s)
Enfermedades de los Perros/parasitología , Variación Genética , Toxoplasma/clasificación , Toxoplasmosis Animal/parasitología , Alelos , Animales , Anticuerpos Antiprotozoarios/sangre , Bioensayo/métodos , Bioensayo/veterinaria , Encéfalo/parasitología , Brasil/epidemiología , Gatos , ADN Protozoario/análisis , Enfermedades de los Perros/epidemiología , Perros , Heces/parasitología , Femenino , Marcadores Genéticos/genética , Genotipo , Corazón/parasitología , Ratones , Prevalencia , Toxoplasma/genética , Toxoplasma/inmunología , Toxoplasmosis Animal/epidemiologíaRESUMEN
The prevalence of Toxoplasma gondii in free-ranging chickens is a good indicator of the prevalence of T. gondii oocysts in the soil because chickens feed from the ground. The prevalence of T. gondii in 50 free-range chickens (Gallus domesticus) from Amazon, Brazil, was determined. Antibodies to T. gondii were assayed by the modified agglutination test (MAT) and found in 33 (66%) chickens with titers of 1:5 in 3, 1:10 in 2, 1:20 in 1, 1:40 in 1, 1:80 in 2, 1:160 in 5, 1:200 in 9, 1:400 in 5, 1:800 in 2, 1:1,600 in 2, and 1:3,200 or higher in 1. Hearts and brains of 33 seropositive chickens were bioassayed individually in mice. Tissues from 17 seronegative chickens were pooled and fed to 2 T. gondii-free cats. Feces of cats were examined for oocysts, but none was found. Toxoplasma gondii was isolated from 24 chickens with MAT titers of 1:5 or higher. Genotyping of these 24 T. gondii isolates by polymorphisms at the SAG2 locus indicated that 14 were type I, and 10 were type III; the absence of type II strains from Brazil was confirmed. Fifty percent of the infected mice died of toxoplasmosis, irrespective of the genotype.
Asunto(s)
Pollos/parasitología , Enfermedades de las Aves de Corral/parasitología , Toxoplasma/aislamiento & purificación , Toxoplasmosis Animal/parasitología , Pruebas de Aglutinación/veterinaria , Animales , Anticuerpos Antiprotozoarios/sangre , Bioensayo/métodos , Bioensayo/veterinaria , Encéfalo/parasitología , Brasil , Gatos , Heces/parasitología , Genotipo , Corazón/parasitología , Ratones , Oocistos/aislamiento & purificación , Toxoplasma/inmunología , Toxoplasma/patogenicidadRESUMEN
The prevalence of Toxoplasma gondii in free-ranging chickens is a good indicator of the prevalence of T. gondii oocysts in the soil because chickens feed from the ground. The prevalence of T. gondii in 77 free-range chickens (Gallus domesticus) from Colombia, South America was determined. Antibodies to T. gondii were assayed by the modified agglutination test (MAT), and found in 32 (44.4%) of 72 chickens with titers of 1:5 in 4, 1:10 in 3, 1:20 in 1, 1:40 in 1, 1:80 in 8, 1:160 in 8, 1:320 in 3, and 1:640 or higher in 4. Hearts and brains of 31 seropositive chickens were pooled and bioassayed in mice. Tissues from 32 (16+16) seronegative chickens were pooled and fed to two, T. gondii-free cats, and tissues from nine chickens without matching sera were fed to one T. gondii-free cat. Feces of cats were examined for oocysts. T. gondii oocysts were excreted by a cat that was fed tissues of 16 seronegative chickens. T. gondii was isolated by bioassay in mice from 23 chickens with MAT titers of 1:20 or higher. All infected mice from 16 of the 23 isolates died of toxoplasmosis. Overall, 82 (81.1%) of 101 mice that became infected after inoculation with chicken tissues died of toxoplasmosis. Genotyping of these 24 isolates using polymorphisms at the SAG2 locus indicated that seven T. gondii isolates were Type I, 17 were Type III, and none was Type II. Phenotypically, T. gondii isolates from chickens from Colombia were similar to isolates from Brazil but different from the isolates from North America; most isolates from chickens from Brazil and Colombia were lethal for mice whereas isolates from North America did not kill inoculated mice. Genetically, none of the T. gondii isolates from Colombia and Brazil was SAG2 Type II, whereas most isolates from chickens from North America were Type II. This is the first report of genetic characterization of T. gondii isolates from Colombia, South America.
Asunto(s)
Pollos , Enfermedades de las Aves de Corral/parasitología , Toxoplasma/genética , Toxoplasmosis Animal/parasitología , Pruebas de Aglutinación/veterinaria , Animales , Anticuerpos Antiprotozoarios/sangre , Antígenos de Protozoos/química , Antígenos de Protozoos/genética , Bioensayo/veterinaria , Encéfalo/parasitología , Gatos , Colombia , ADN Protozoario/química , ADN Protozoario/genética , Heces/parasitología , Femenino , Corazón/parasitología , Ratones , Reacción en Cadena de la Polimerasa/veterinaria , Polimorfismo de Longitud del Fragmento de Restricción , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Población Rural , Toxoplasma/clasificación , Toxoplasma/aislamiento & purificación , Toxoplasmosis Animal/sangreRESUMEN
The prevalence of Toxoplasma gondii in free-range chickens is a good indicator of the prevalence of T. gondii oocysts in the soil because chickens feed from the ground. The prevalence of T. gondii in 102 free-range chickens (Gallus domesticus) from Grenada was determined. Antibodies to T. gondii were assayed by the modified agglutination test (MAT). Antibodies were found in 53 (52%) chickens with titers of 1:5 in 6, 1:10 in 4, 1:20 in 4, 1:40 in 4, 1:80 in 15, 1:160 in 9, 1: 320 in 5, 1:640 in 4, and 1:1,280 or greater in 2. Hearts, pectoral muscles, and brains of 43 seropositive chickens with MAT titers of 1:20 or greater were bioassayed individually in mice. Tissues of each of 10 chickens with titers of 1:5 and 1:10 were pooled and bioassayed in mice. Tissues from the remaining 49 seronegative chickens were pooled and fed to 4 T. gondii-free cats. Feces of cats were examined for oocysts; they did not shed oocysts. T. gondii was isolated from 35 of 43 chickens with MAT titers of 1:20 or greater; from the hearts, brains, and pectoral muscles of 2, hearts and brains of 20, from the hearts alone of 11, and brains alone of 2. T. gondii was isolated from 1 of 10 chickens with titers of 1:5 or 1:10. All 36 T. gondii isolates were avirulent for mice. Genotyping of these 36 isolates using polymorphisms at the SAG2 locus indicated that 29 were Type III, 5 were Type I, 1 was Type II, and 1 had both Type I and Type III. Genetically, the isolates from Grenada were different from those from the United States; Type II was the predominant type from the United States. Phenotypically, all isolates from Grenada were avirulent for mice, whereas those from Brazil were mouse-virulent. This is the first report of isolation of T. gondii from chickens from Grenada, West Indies.
Asunto(s)
Pollos/parasitología , Enfermedades de las Aves de Corral/epidemiología , Toxoplasma/aislamiento & purificación , Toxoplasmosis Animal/epidemiología , Pruebas de Aglutinación/veterinaria , Animales , Anticuerpos Antiprotozoarios/sangre , Bioensayo/veterinaria , Encéfalo/parasitología , Gatos , ADN Protozoario/química , Femenino , Genotipo , Grenada/epidemiología , Corazón/parasitología , Ratones , Músculos Pectorales/parasitología , Reacción en Cadena de la Polimerasa/veterinaria , Polimorfismo de Longitud del Fragmento de Restricción , Enfermedades de las Aves de Corral/parasitología , Prevalencia , Toxoplasma/genética , Toxoplasma/inmunología , Toxoplasmosis Animal/parasitologíaRESUMEN
Attempts were made to isolate Neospora caninum from naturally infected water buffaloes (Bubalus bubalis) from Brazil. Brains from six buffaloes with indirect fluorescent antibodies (>1:100) to N. caninum were used to isolate the parasite by bioassay in dogs and gerbils followed by in vitro culture. Shedding of Neospora-like oocysts was noticed in dogs fed brains from three buffaloes (isolate designation NcBrBuf-1, 2 and 4). Two more isolates (NcBrBuf-3 and 5) were obtained by in vitro culture of the brains of gerbils previously infected with brains of two other buffaloes. The identity of the isolates was confirmed by biological and molecular methods. The isolates were found to be non-pathogenic to gerbils. All five isolates amplified the gene 5 amplicons using Neospora-specific PCR assay. The sequences of gene 5 fragments and the common toxoplasmatiid ITS-1 fragments were analyzed. The dynamics of oocyst production in the dogs indicate that water buffaloes are natural intermediate hosts for N. caninum. This is the first report of isolation of N. caninum from water buffaloes.
Asunto(s)
Búfalos/parasitología , Coccidiosis/veterinaria , Neospora/aislamiento & purificación , Animales , Anticuerpos Antiprotozoarios , Bioensayo/veterinaria , Encéfalo/parasitología , Brasil , Coccidiosis/parasitología , Perros , Heces/parasitología , Técnica del Anticuerpo Fluorescente Indirecta/veterinaria , Gerbillinae , Masculino , Ratones , Neospora/patogenicidad , Oocistos/aislamiento & purificaciónRESUMEN
Cats are important in the epidemiology of Toxoplasma gondii because they are the only hosts that can excrete environmentally resistant oocysts. The prevalence of T. gondii was determined in 58 domestic cats from 51 homes from Santa Isabel do Ivai, Parana State, Brazil where a water-associated outbreak of acute toxoplasmosis had occurred in humans. Antibodies to T. gondii were found with the modified agglutination test in 49 of 58 (84.4%) cats at a serum dilution of 1:20. Tissues (brain, heart, and skeletal muscle) of 54 of these cats were bioassayed in T. gondii-free, laboratory-reared cats; T. gondii oocysts were excreted by 33 cats that were fed feline tissues. Brains from these 54 cats were bioassayed in mice; T. gondii was isolated from 7. Skeletal muscles and hearts of 15 cats were also bioassayed in mice; T. gondii was isolated from skeletal muscles of 9 and hearts of 13. The results indicate that T. gondii localizes in muscle tissue more than the brains of cats. In total there were 37 T. gondii isolates from 54 cats. Most isolates of T. gondii were virulent for mice. Genotyping of the 37 isolates of T. gondii, using the SAG2 locus, revealed that 15 isolates were type I and 22 were type III. The absence of type II genotype in cats in this study is consistent with the previous studies on T. gondii isolates from Brazil and is noteworthy because most T. gondii isolates from the United States are type II. These findings support the view that Brazilian and North American T. gondii isolates are genetically distinct. This is the first report of genotyping of T. gondii isolates from the domestic cat.
Asunto(s)
Enfermedades de los Gatos/parasitología , Toxoplasma/aislamiento & purificación , Toxoplasmosis Animal/parasitología , Pruebas de Aglutinación/veterinaria , Animales , Anticuerpos Antiprotozoarios/sangre , Bioensayo/veterinaria , Encéfalo/parasitología , Brasil/epidemiología , Enfermedades de los Gatos/epidemiología , Gatos , ADN Protozoario/análisis , Heces/parasitología , Femenino , Genotipo , Corazón/parasitología , Masculino , Ratones , Músculo Esquelético/parasitología , Estudios Seroepidemiológicos , Toxoplasma/genética , Toxoplasma/inmunología , Toxoplasma/patogenicidad , Toxoplasmosis Animal/epidemiología , Virulencia , Abastecimiento de AguaRESUMEN
Canine isolates of Hammondia heydorni from Argentina, Brazil, and the United States were analysed for genetic diversity. A total of 14 isolates were tested for their ability to produce amplification using three PCR assays, one targeting the common toxoplasmatiid ITS-1 region and 2 amplifying novel, H. heydorni-specific loci, HhAP7 and HhAP10. While the ITS-1 fragments could be amplified from all isolates, only six isolates were capable of amplifying the fragments from the novel loci. The PCR products were further investigated for genetic diversity using restriction fragment length polymorphism (RFLP) and single strand conformation polymorphism (SSCP) techniques. Polymorphism in the digestion pattern was evident only at the HhAP10 locus, differentiating two of the Argentinean isolates from the remainder. Mobility shifts on SSCP gels revealed that the two Argentinean isolates were not only different from the other four isolates, but also differed from each other, both at the HhAP7 and HhAP10 loci. The ITS-1 fragments of all isolates were identical by RFLP. However, two distinct mobility patterns resulted when the products were electrophoresed on SSCP gels. Based on the sequence data from the ITS-1 and the two random loci, the isolates could be broadly classified into two distinct groups, within which minor polymorphisms were evident. In contrast, very little heterogeneity occurred in the sequences of corresponding ITS-1 regions of Neospora caninum and Toxoplasma gondii isolates. Thus, it is concluded that there is a considerable degree of microheterogeneity among isolates of H. heydorni. This diversity should be taken into consideration while attempting to elucidate the systematics, diagnostics, and biology of H. heydorni in relation to N. caninum.