RESUMEN
The recent identification of isolates of D. immitis with confirmed resistance to the macrocyclic lactone preventatives presents an opportunity for comparative genomic studies using these isolates, and examining the genetic diversity within and between them. We studied the genomes of Wolbachia endosymbionts of five isolates of D. immitis maintained at the University of Georgia. Missouri and Georgia-2 are maintained as drug susceptible isolates, and JYD-27, Yazoo-2013 and Metairie-2014 are resistant to the macrocyclic lactone preventatives. We used whole genome amplification followed by Illumina-based sequencing from 8 to 12 individual microfilariae from each of the five isolates, obtaining a depth of coverage of approximately 40-75 fold for each. The Illumina sequences were used to create new genome assemblies for all the Wolbachia isolates studied. Comparisons of the Wolbachia sequences revealed more than 3000 sequence variations in each isolate. We identified 67 loci specific in resistant isolates but not in susceptible isolates, including 18 genes affected.Phylogenetic analysis suggested that the endosymbionts of the drug-susceptible isolates are more closely related to each other than to those from any of the resistant parasites. This level of variation in the Wolbachia endosymbionts of D. immitis isolates suggests a potential for selection for resistance against drugs targeting them.
Asunto(s)
Dirofilaria immitis/efectos de los fármacos , Resistencia a Medicamentos , Variación Genética , Genoma Bacteriano , Lactonas/farmacología , Wolbachia/genética , Animales , Dirofilaria immitis/microbiología , Compuestos Macrocíclicos/farmacologíaRESUMEN
Cardiopulmonary dirofilariosis caused by Dirofilaria immitis produces inflammation, blood vessel obstruction and hypoxia, which are required conditions for the beginning of the process of neovascularization. Since D. immitis harbours intracellular symbiotic Wolbachia bacterium, the global understanding of the angiogenic process requires the analysis of the effect of the parasite molecules, but also that of Wolbachia. Canine primary lung microvascular endothelial cells were treated with the recombinant Wolbachia surface protein (rWSP) and the expression of angiogenic factors like Vascular Endothelial Growth Factor-A (VEGF-A), sFlt, membrane Endoglin (mEndoglin) and soluble Endoglin (sEndoglin), as well as the in vitro formation of pseudocapillaries, were measured. The analyses showed a significant increase in the expression of pro-angiogenic VEGF-A and anti-angiogenic sEndoglin, together with a significant decrease in both pro-angiogenic mEndoglin and pseudocapillary formation, compared to untreated controls. Due to the complexity of the angiogenic process and its relationship with other physiological processes like inflammation and fibrinolysis, these results might suggest that rWSP participate in various mechanisms related to each other and its effects might depend either on the balance between them or on the moment of their occurrence.
Asunto(s)
Inductores de la Angiogénesis/química , Proteínas Bacterianas/química , Dirofilariasis/complicaciones , Proteínas de la Membrana/química , Wolbachia/química , Animales , Células Cultivadas , Dirofilaria immitis/microbiología , Dirofilariasis/microbiología , Perros , Células Endoteliales/microbiología , Corazón/parasitología , Humanos , Inflamación , Pulmón/citología , Pulmón/parasitología , SimbiosisRESUMEN
Wolbachia can reduce the capability of mosquitoes to transmit infectious diseases to humans and is currently exploited in campaigns for the control of arboviruses, like dengue and Zika. Under the assumption that Wolbachia-mediated activation of insect immunity plays a role in the reduction of mosquito vectorial capacity, we focused our attention on the Wolbachia surface protein (WSP), a potential inductor of innate immunity. We hypothesized that the heterologous expression of this protein in gut- and tissue-associated symbionts may reduce parasite transmission. We thus engineered the mosquito bacterial symbiont Asaia to express WSP (AsaiaWSP). AsaiaWSP induced activation of the host immune response in Aedes aegypti and Anopheles stephensi mosquitoes, and inhibited the development of the heartworm parasite Dirofilaria immitis in Ae. aegypti. These results consolidate previous evidence on the immune-stimulating property of WSP and make AsaiaWSP worth of further investigations as a potential tool for the control of mosquito-borne diseases.
Asunto(s)
Acetobacteraceae/metabolismo , Aedes/microbiología , Anopheles/microbiología , Proteínas de la Membrana Bacteriana Externa/metabolismo , Dirofilaria immitis/microbiología , Proteínas de la Membrana/metabolismo , Wolbachia/metabolismo , Acetobacteraceae/genética , Aedes/inmunología , Animales , Anopheles/parasitología , Proteínas de la Membrana Bacteriana Externa/genética , Dirofilaria immitis/crecimiento & desarrollo , Interacciones Huésped-Parásitos , Proteínas de la Membrana/genética , Fagocitosis , Simbiosis , Wolbachia/genéticaRESUMEN
BACKGROUND: Angiogenesis can occur under pathological conditions when stimuli such as inflammation, vascular obstruction or hypoxia exist. These stimuli are present in cardiopulmonary dirofilariosis (Dirofilaria immitis). The aim of this study was to analyze the capacity of D. immitis antigens to modify the expression of angiogenic factors and trigger the formation of pseudocapillaries (tube-like structures) in an in vitro model of endothelial cells. METHODS: The expression of VEGF-A, sFlt, mEndoglin and sEndoglin in cultures of canine microvascular endothelial cells stimulated with extract of adult worms of D. immitis obtained from an untreated dog (DiSA) and from a dog treated for 15 days with doxycycline (tDiSA), was determined by using commercial kits. The capacity of pseudocapillary formation was evaluated analyzing cell connections and cell groups in Matrigel cell cultures stimulated with DiSA and tDiSA. In both cases non-stimulated cultures were used as controls. RESULTS: First, we demonstrated that worms obtained from the dog treated with doxycycline showed a significantly lower amount of Wolbachia (less than 60%) than worms removed from the untreated dog. Only DiSA was able to significantly increase the expression of the proangiogenic factor VEGF-A in the endotelial cells cultures. None of the D. immitis extracts modified the expression of sFlt. tDiSA extract was able to modify the expression of the endoglins, significantly decreasing the expression of the pro-angiogenic mEndoglin and increasing the anti-angiogenic sEndoglin. The formation of pseudocapillaries was negatively influenced by tDiSA, which reduced the organization and number of cellular connections. CONCLUSIONS: The ability of antigens from adult D. immitis worms to modify the expression of pro and anti-angiogenic factors in endotelial cell cultures was demonstrated, as well as the trend to form pseudocapillaries in vitro. The capacity of stimulation may be linked to the amount of Wolbachia present in the antigenic extracts.
Asunto(s)
Antígenos Helmínticos/farmacología , Dirofilaria immitis/química , Células Endoteliales/efectos de los fármacos , Neovascularización Fisiológica/efectos de los fármacos , Animales , Antígenos Bacterianos/farmacología , Capilares/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Dirofilaria immitis/microbiología , Perros , Inflamación , Wolbachia/química , Wolbachia/genéticaRESUMEN
BACKGROUND: The American Heartworm Society currently recommends the use of a monthly macrocyclic lactone, a 28-day course of 10 mg/kg doxycycline BID, and the 3-dose protocol of melarsomine dihydrochloride for the treatment of canine heartworm disease. Doxycycline is necessary for the reduction of the bacterium Wolbachia, found in all heartworm life-stages. Previous price increases and decreasing availability prompted us to evaluate alternative tetracycline antibiotics, i.e. minocycline, for the reduction of Wolbachia during canine heartworm treatment. METHODS: Thirty-two heartworm-positive dogs were randomized to receive 10 mg/kg or 5 mg/kg of either doxycycline or minocycline for 28 days BID, for a total of 8 dogs per experimental group. All dogs received 6 months of Heartgard Plus® (ivermectin/pyrantel) and the 3-dose protocol of 2.5 mg/kg melarsomine dihydrochloride. Blood samples were collected prior to the initiation of treatment, every 7 days throughout tetracycline treatment, and then monthly thereafter until the dog tested negative for the presence of heartworm antigen. DNA was isolated from circulating microfilarial samples and qPCR was performed on each sample. RESULTS: A greater number of dogs in the 10 mg/kg doxycycline and minocycline treated groups experienced gastrointestinal side effects as compared to the 5 mg/kg doxycycline and minocycline treated groups. All eight dogs in the 10 mg/kg doxycycline-treated group tested negative for the presence of Wolbachia DNA by 28 days post-tetracycline treatment. A total of two dogs in both the 5 mg/kg doxycycline- and 10 mg/kg minocycline-treated groups and three dogs in the 5 mg/kg minocycline-treated group remained positive for the presence of Wolbachia DNA by the end of tetracycline treatment. CONCLUSIONS: No lung pathology was assessed in this clinical trial, therefore the clinical effect of the remaining Wolbachia DNA in the 10 mg/kg minocycline-, 5 mg/kg doxycycline- and 5 mg/kg minocycline-treated groups cannot be determined. Owner compliance in the proper administration of these tetracyclines may be impacted by the increased severe gastrointestinal side effects reported for the 10 mg/kg doxycycline- and minocycline-treated groups. We recommend that veterinarians prescribe the recommended 10 mg/kg doxycycline for canine heartworm treatment and reduce the dosage to 5 mg/kg in cases of severe gastrointestinal side effects in order to improve owner compliance in administration of medications.
Asunto(s)
Antibacterianos/administración & dosificación , Arsenicales/administración & dosificación , Dirofilaria immitis/efectos de los fármacos , Dirofilariasis/tratamiento farmacológico , Enfermedades de los Perros/tratamiento farmacológico , Doxiciclina/administración & dosificación , Filaricidas/administración & dosificación , Minociclina/administración & dosificación , Triazinas/administración & dosificación , Wolbachia/efectos de los fármacos , Animales , Dirofilaria immitis/microbiología , Dirofilaria immitis/fisiología , Dirofilariasis/parasitología , Enfermedades de los Perros/parasitología , Perros , Doxiciclina/efectos adversos , Quimioterapia Combinada , Femenino , Masculino , Minociclina/efectos adversos , Wolbachia/genética , Wolbachia/fisiologíaRESUMEN
BACKGROUND: Doxycycline has been considered the first drug of choice for treating Wolbachia, a member of the Rickettsiaceae, which has a symbiotic relationship with filarial worms, including heartworms. Wolbachia, is susceptible to tetracyclines, which have been used as adjunctive treatments for heartworm disease. Treatment with doxycycline reduces Wolbachia numbers in all stages of heartworms and improves outcomes and decreased microfilaremia in dogs treated for heartworm disease. The American Heartworm Society recommends treatment with doxycycline in dogs diagnosed with heartworm disease at a dose of 10 mg/kg twice daily for 28 days. If doxycycline is not available, minocycline can be considered as a substitute. However, minocycline has not undergone an evaluation in dogs with heartworm disease, nor has an effective dose been established. Minocycline is an attractive option because of the higher cost of doxycycline and new pharmacokinetic information for dogs that provides guidance for appropriate dosage regimens to achieve pharmacokinetic-pharmacodynamic (PK-PD) targets. RESULTS: Published reports from the Anti-Wolbachia Consortium (A-WOL) indicate superior in vitro activity of minocycline over doxycycline. Studies performed in mouse models to measure anti-Wolbachia activity showed that minocycline was 1.7 times more effective than doxycycline, despite a 3-fold lower pharmacokinetic exposure. To achieve the same exposure as achieved in the mouse infection model, a pharmacokinetic-pharmacodynamic (PK-PD) analysis was conducted to determine optimal dosages for dogs. The analysis showed that an oral minocycline dose of 3.75 to 5 mg/kg administered twice daily would attain similar targets as observed in mice and predicted for human infections. CONCLUSIONS: There are potentially several advantages for use of minocycline in animals. It is well absorbed from oral administration, it has less protein binding than doxycycline (65% vs 92%) allowing for better distribution into tissue, and it is approximately two times more lipophilic than doxycycline, which may result in better intracellular penetration. More work is needed to document efficacy of minocycline for treating canine heartworm disease.
Asunto(s)
Antibacterianos/administración & dosificación , Dirofilaria immitis/microbiología , Dirofilariasis/tratamiento farmacológico , Enfermedades de los Perros/tratamiento farmacológico , Doxiciclina/administración & dosificación , Minociclina/administración & dosificación , Wolbachia/efectos de los fármacos , Animales , Antibacterianos/farmacocinética , Dirofilaria immitis/fisiología , Dirofilariasis/parasitología , Enfermedades de los Perros/parasitología , Perros , Doxiciclina/farmacocinética , Ratones , Minociclina/farmacocinética , Wolbachia/fisiologíaRESUMEN
Dirofilaria immitis is a mosquito-borne parasite that produces an inflammatory process in the wall of the blood vessels of its definitive host during cardiopulmonary dirofilariosis, known as proliferative endarteritis. Parasite antigens participate in the appearance of this inflammatory event, among other mechanisms through the over-activation of the host fibrinolytic system. Since Wolbachia, endosymbiont bacteria of filarial nematodes, is released into the vertebrate host when worms die, the aim of this work was to analyse the interaction between this bacteria and the host fibrinolytic system to complete the study of this part of the host-parasite relationships. For that purpose, the recombinant form of the major Wolbachia surface protein (rWSP) was cloned, sequenced and expressed and then, its ability to bind plasminogen and enhance the generation of plasmin was assessed. We demonstrated that rWSP is a conserved antigen within the family Onchocercidae with ability to bind plasminogen and stimulate plasmin generation in a tissue-plasminogen activator (t-PA) and lysine residues of the rWSP-dependent manner. These results indicate that the recruitment of plasminogen by Wolbachia and the possible excess of plasmin generated could contribute to exacerbate the pathological events occurred at the vascular level during cardiopulmonary dirofilariosis, as well as in other diseases caused by filarial nematodes that harbour Wolbachia, when the bacteria is released after the death of the worms.
Asunto(s)
Dirofilaria immitis/microbiología , Dirofilariasis/parasitología , Enfermedades de los Perros/parasitología , Proteínas de la Membrana/metabolismo , Activador de Tejido Plasminógeno/metabolismo , Wolbachia/fisiología , Secuencia de Aminoácidos , Animales , Perros , Fibrinolisina/metabolismo , Fibrinólisis , Interacciones Huésped-Parásitos , Humanos , Proteínas de la Membrana/genética , Modelos Moleculares , Proteínas Recombinantes , Alineación de Secuencia/veterinaria , SimbiosisRESUMEN
The fauna of bloodsucking mosquitoes in the Nizhny Novgorod Region is represented by 11 species from 5 genera of the family Culicidae. During 2014-2015, the predominant species were Ochlerotatus cantans and Aedes cinereus mosqui- toes in both a population aggregate and woodland. The infected mosquitoes accounted for 1.3% of their total number and were registered only in the village of Fokino. The investigators identified two human pathogenic nematode species: Diro- filaria immits and Dirofilaria repens (0.9% and 0.4% respectively). The effective carriers of Dirofilaria in the examined area can be Ae.cinereus and Och.cantans as only these species were found to have an invasive stage of the parasite. The symbiotic bacterium Wolbachia was detected in the mosquitoes that were not infected with dirofilariasis. This is the first study in Russia to investigate the effects of Wolbachia on the susceptibility of dirofilariasis vectors to infection.
Asunto(s)
Dirofilariasis/microbiología , Dirofilariasis/parasitología , Mosquitos Vectores/microbiología , Mosquitos Vectores/parasitología , Simbiosis , Aedes/microbiología , Aedes/parasitología , Animales , ADN de Helmintos/genética , Dirofilaria immitis/microbiología , Dirofilaria immitis/patogenicidad , Dirofilaria repens/microbiología , Dirofilaria repens/patogenicidad , Perros , Humanos , Mosquitos Vectores/genética , Wolbachia/aislamiento & purificación , Wolbachia/patogenicidadRESUMEN
Wolbachia is an obligatory intracellular endosymbiotic bacterium, present in over 20% of all insects altering insect reproductive capabilities and in a wide range of filarial worms which is essential for worm survival and reproduction. In Egypt, no available data were found about Wolbachia searching for it in either mosquitoes or filarial worms. Thus, we aimed to identify the possible concurrent presence of Wolbachia within different mosquitoes and filarial parasites, in Assiut Governorate, Egypt using multiplex PCR. Initially, 6 pools were detected positive for Wolbachia by single PCR. The simultaneous detection of Wolbachia and filarial parasites (Wuchereria bancrofti, Dirofilaria immitis, and Dirofilaria repens) by multiplex PCR was spotted in 5 out of 6 pools, with an overall estimated rate of infection (ERI) of 0.24%. Unexpectedly, the highest ERI (0.53%) was for Anopheles pharoensis with related Wolbachia and W. bancrofti, followed by Aedes (0.42%) and Culex (0.26%). We also observed that Wolbachia altered Culex spp. as a primary vector for W. bancrofti to be replaced by Anopheles sp. Wolbachia within filaria-infected mosquitoes in our locality gives a hope to use bacteria as a new control trend simultaneously targeting the vector and filarial parasites.
Asunto(s)
Culicidae/microbiología , ADN Bacteriano/análisis , Dirofilaria immitis/microbiología , Dirofilaria repens/microbiología , Wolbachia/aislamiento & purificación , Wuchereria bancrofti/microbiología , Animales , Culicidae/parasitología , ADN Bacteriano/genética , Egipto , Femenino , Reacción en Cadena de la Polimerasa Multiplex , Wolbachia/genéticaRESUMEN
Dirofilaria immitis, the cause of canine and feline heartworm disease, was the first filarial nematode described to harbour the bacterial endosymbiont Wolbachia. This ground-breaking discovery has led to intense research aimed at unravelling the nature of the endosymbiotic relationship; genomic studies have revealed how the bacteria may interact with the parasite and help explain why each is so dependent on the other. Analysis of the immune response to these bacteria may elucidate the mechanisms through which filarial parasites are able to survive for long periods of time in otherwise immune-competent hosts. Finally, studies aimed at the removal of the bacteria using specific antibiotic treatment in infected hosts is leading to the development of novel approaches for interrupting the transmission cycle and for the treatment and control of heartworm disease.
Asunto(s)
Dirofilaria immitis/microbiología , Dirofilariasis/tratamiento farmacológico , Dirofilariasis/microbiología , Doxiciclina/uso terapéutico , Wolbachia/fisiología , Animales , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Antinematodos/farmacología , Antinematodos/uso terapéutico , Dirofilaria immitis/efectos de los fármacos , Dirofilariasis/transmisión , Doxiciclina/farmacología , Sinergismo Farmacológico , Humanos , Ivermectina/uso terapéutico , Wolbachia/inmunologíaRESUMEN
The heartworm Dirofilaria immitis is an important parasite of dogs. Transmitted by mosquitoes in warmer climatic zones, it is spreading across southern Europe and the Americas at an alarming pace. There is no vaccine, and chemotherapy is prone to complications. To learn more about this parasite, we have sequenced the genomes of D. immitis and its endosymbiont Wolbachia. We predict 10,179 protein coding genes in the 84.2 Mb of the nuclear genome, and 823 genes in the 0.9-Mb Wolbachia genome. The D. immitis genome harbors neither DNA transposons nor active retrotransposons, and there is very little genetic variation between two sequenced isolates from Europe and the United States. The differential presence of anabolic pathways such as heme and nucleotide biosynthesis hints at the intricate metabolic interrelationship between the heartworm and Wolbachia. Comparing the proteome of D. immitis with other nematodes and with mammalian hosts, we identify families of potential drug targets, immune modulators, and vaccine candidates. This genome sequence will support the development of new tools against dirofilariasis and aid efforts to combat related human pathogens, the causative agents of lymphatic filariasis and river blindness.
Asunto(s)
Antihelmínticos/farmacología , Dirofilaria immitis/genética , Dirofilariasis/parasitología , Enfermedades de los Perros/parasitología , Genoma de los Helmintos , Vacunas/inmunología , Animales , Antihelmínticos/uso terapéutico , Dirofilaria immitis/efectos de los fármacos , Dirofilaria immitis/inmunología , Dirofilaria immitis/microbiología , Dirofilariasis/tratamiento farmacológico , Dirofilariasis/prevención & control , Enfermedades de los Perros/tratamiento farmacológico , Enfermedades de los Perros/prevención & control , Perros , Femenino , Variación Genética , Genoma Bacteriano , Masculino , Filogenia , Proteoma , ARN de Helminto/química , Simbiosis , Transcriptoma/genética , Wolbachia/genética , Wolbachia/fisiologíaRESUMEN
A fundamental role for the endosymbiotic bacteria Wolbachia pipientis in the pathogenesis of Dirofilaria immitis infections has emerged in recent years. Diagnostic opportunities arising from this breakthrough have not yet been fully exploited. This study was aimed at developing conventional and real-time PCR assays to carry out a molecular survey in a convenience sample of cats living in an area where D. immitis is endemic and to evaluate the detection of bacterial DNA in blood as a surrogate assay for diagnosing filaria-associated syndromes in cats. COI and FtsZ loci were used as targets for D. immitis and Wolbachia PCR assays, respectively, and real-time TaqMan PCR assays were used only for Wolbachia. A convenience sample of 307 disease-affected or healthy cats examined at a University facility were PCR tested, and their medical records were investigated. Conventional nested PCR for Wolbachia amplified the endosymbionts of both D. immitis and D. repens, while real-time PCR was highly specific only for the former. Observed prevalences of 0.3 and 10.4% were found using conventional nested PCR assays for D. immitis and real-time PCR for Wolbachia, respectively. Similar prevalences were established using the Wolbachia nested PCR (98% concordance with real-time PCR). The group of Wolbachia-positive samples had a significantly higher proportion of subjects with respiratory signs (29.0% versus 9.7%; P = 0.002). The findings of this study indicate that a highly sensitive PCR assay can be used to detect the Wolbachia organism in the peripheral blood of cats with respiratory signs.
Asunto(s)
Sangre/microbiología , Enfermedades de los Gatos/diagnóstico , ADN Bacteriano/aislamiento & purificación , Dirofilaria immitis/microbiología , Filariasis/veterinaria , Reacción en Cadena de la Polimerasa/métodos , Wolbachia/aislamiento & purificación , Animales , Proteínas Bacterianas/genética , Gatos , ADN Bacteriano/genética , Filariasis/diagnóstico , Sensibilidad y Especificidad , Medicina Veterinaria/métodos , Wolbachia/genéticaRESUMEN
Canine heartworm disease wreaks havoc inside canines all throughout the modern world, including the USA. Any region where mosquitoes thrive will provide efficient dog-to-dog transportation for the microfilaria of the infectious nematode Dirofilaria immitis. Veterinary scientists have recently discovered both phylogenetic and biochemical evidence for the obligate symbiosis of D. immitis and the bacteria Wolbachia pipientis. As a result, veterinarians have initiated testing of antibiotic therapies either instead of, or together with, the currently utilized antiparasitic treatments for canine heartworm. The toxicity of melarsomine adulticidal therapies has prompted an abundance of new research involving doxycycline and other antibiotics, which will be addressed in this review. As our knowledge of the Wolbachia endosymbiont expands, so will our abilities to minimize toxicity and maximize efficiency of heartworm treatments.
Asunto(s)
Dirofilaria immitis/microbiología , Dirofilaria immitis/patogenicidad , Dirofilariasis/parasitología , Enfermedades de los Perros/parasitología , Simbiosis , Wolbachia/fisiología , Animales , Antibacterianos/uso terapéutico , Dirofilaria immitis/fisiología , Dirofilariasis/tratamiento farmacológico , Enfermedades de los Perros/tratamiento farmacológico , Perros , Doxiciclina/uso terapéutico , Resultado del Tratamiento , Estados UnidosRESUMEN
Heartworm infection (Dirofilaria immitis) can cause kidney damage due to the presence of circulating microfilariae (mf) that contribute to the production and deposit of immune complexes. It has been shown that mf are a major source of Wolbachia antigen during active infection. Here the authors compared urine samples from 19 naturally infected dogs with (mf+) and 12 without (mf-) microfilariae for the presence of proteinuria and anti-Wolbachia Surface Protein (-WSP) IgG in ELISA. Kidneys from 6 mf+ and 3 mf- dogs were also examined by anti-WSP immuno-histochemistry. All infected dogs showed proteinuria, but mf+ dogs had significantly higher values compared to mf-dogs. Mf+ dogs had optical density values for anti-WSP IgG consistently higher than established cut-off values and were significantly higher than values for mf- dogs. Kidneys from mf+ dogs showed Wolbachia+ mf in glomerular capillaries. Results strongly suggest that Wolbachia associated with circulating mf may contribute to immune-mediated kidney disease in dogs with heartworm infection.
Asunto(s)
Anticuerpos Antibacterianos/orina , Proteínas de la Membrana Bacteriana Externa/inmunología , Dirofilaria immitis , Dirofilariasis/inmunología , Enfermedades de los Perros/inmunología , Wolbachia/inmunología , Animales , Dirofilaria immitis/inmunología , Dirofilaria immitis/microbiología , Dirofilariasis/sangre , Dirofilariasis/orina , Enfermedades de los Perros/sangre , Enfermedades de los Perros/orina , Perros , Microfilarias/inmunología , Microfilarias/microbiologíaRESUMEN
Wolbachia is an obligate intracellular endosymbiont and likely mutualist living within the heartworm Dirofilaria immitis and a number of other filarial nematodes in the family Onchocercidae. The bacterial infection is passed from worm to worm transovarially; the organisms are in ovarian cells, the developing microfilariae, and multiply and persist in all later developmental stages through the mosquito and into the next host. Besides being present in the ovaries of the adult worms, they also are present in large numbers within the hypodermal tissues of the nematode. It is now know that these bacteria that were first observed in heartworms more than 30 years ago are actually related to similar Wolbachia bacteria that are found in arthropods. Wolbachia is an alpha-proteobacteria, and this group includes a number of important arthropod-transmitted bacterial agents of dogs and cats: Rickettsia rickettsii, R. felis, Anaplasma platys, Ehrlichia canis, E. chaffeensis, and E. ewingii. Alpha-proteobacteria are also important as obligate intracellular mutualists in plants in which they are responsible for nitrogen fixation. Recent work on the treatment of heartworms in dogs with doxycycline stems from related work with the human filarial nematode Onchocerca volvulus that causes river blindness in people.
Asunto(s)
Dirofilaria immitis/microbiología , Dirofilariasis/tratamiento farmacológico , Doxiciclina/uso terapéutico , Filaricidas/uso terapéutico , Anaplasma/crecimiento & desarrollo , Animales , Bartonella/crecimiento & desarrollo , Brucella/crecimiento & desarrollo , Enfermedades de los Gatos/tratamiento farmacológico , Enfermedades de los Gatos/parasitología , Gatos , Dirofilaria immitis/efectos de los fármacos , Dirofilariasis/parasitología , Enfermedades de los Perros/tratamiento farmacológico , Enfermedades de los Perros/parasitología , Perros , Ehrlichia/crecimiento & desarrollo , Rickettsia/crecimiento & desarrollo , Simbiosis , Wolbachia/crecimiento & desarrolloRESUMEN
Drug treatments for heartworm disease have not changed significantly in the last decade. Due to concerns about possible drug resistance and their lower efficacy against adult worms, there is a need for the development of new antifilarial drug therapies. The recent availability of genomic sequences for the related filarial parasite Brugia malayi and its Wolbachia endosymbiont enables genome-wide searching for new drug targets. Phosphoglycerate mutase (PGM) enzymes catalyze the critical isomerization of 3-phosphoglycerate (3-PG) and 2-phosphoglycerate (2-PG) in glycolytic and gluconeogenic metabolic pathways. There are two unrelated PGM enzymes, which are structurally distinct and possess different mechanisms of action. The mammalian enzyme requires 2,3-bisphosphoglycerate as a cofactor (dependent PGM or dPGM), while the other type of PGM does not (independent PGM or iPGM). In the present study, we have determined that Dirofilaria immitis and its Wolbachia endosymbiont both possess active iPGM. We describe the molecular characterization and catalytic properties of each enzyme. Our results will facilitate the discovery of selective inhibitors of these iPGMs as potentially novel drug treatments for heartworm disease.
Asunto(s)
Dirofilaria immitis/enzimología , Fosfoglicerato Mutasa/metabolismo , Wolbachia/enzimología , 2,3-Difosfoglicerato/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Clonación Molecular , ADN Complementario/química , ADN Complementario/aislamiento & purificación , Dirofilaria immitis/genética , Dirofilaria immitis/microbiología , Femenino , Expresión Génica , Ácidos Glicéricos/metabolismo , Proteínas del Helminto/genética , Proteínas del Helminto/aislamiento & purificación , Proteínas del Helminto/metabolismo , Datos de Secuencia Molecular , Fosfoglicerato Mutasa/química , Fosfoglicerato Mutasa/genética , Fosfoglicerato Mutasa/aislamiento & purificación , Filogenia , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Simbiosis , Wolbachia/genética , Wolbachia/fisiologíaRESUMEN
BACKGROUND AND AIMS: Many species of filarial nematodes are infected with Wolbachia pipientis, a maternally inherited endosymbiont. In addition to manipulating host reproduction, these bacteria also affect the evolution of the mitochondrial DNA with which they are transmitted. Selective sweeps can establish a single mitochondrial lineage within a Wolbachia-infected population and purge genetic diversity. While this phenomenon has been studied in insect model systems, it has not been thoroughly examined in a filarial nematode. MATERIALS AND METHODS: Patterns of mitochondrial diversity were examined in Dirofilaria immitis, a Wolbachia-infected species. RESULTS: The levels of genetic diversity observed in canine heartworm were much lower than those in related species not known to be hosts of Wolbachia. CONCLUSION: RESULTS suggest that a maternally inherited endosymbiont can depress mitochondrial diversity in a filarial host.
Asunto(s)
ADN Mitocondrial/genética , Dirofilaria immitis/genética , Dirofilaria immitis/microbiología , Variación Genética , Wolbachia/fisiología , Animales , Carnívoros/parasitología , ADN de Helmintos/genética , Dirofilaria immitis/citología , América del NorteRESUMEN
Heartworm disease was first recognized in dogs more than 100 years ago and is still prevalent among dogs and found in cats worldwide. The complications of heartworm disease can be devastating, and treatment carries risks. Wolbachia spp are gram-negative bacteria that infect filarial nematodes, including Dirofilaria immitis, and elicit an inflammatory response in cats and dogs. Antimicrobial therapy directed against these bacteria has resulted in decreased microfilarial loads, inhibition of the development of larval worms, female worm infertility, and reduced numbers of Wolbachia organisms. Antimicrobial therapy against Wolbachia spp may be useful in treating heartworm disease in cats and dogs, but further research is needed.
Asunto(s)
Enfermedades de los Gatos/microbiología , Dirofilaria immitis/microbiología , Dirofilariasis/microbiología , Enfermedades de los Perros/microbiología , Wolbachia/patogenicidad , Animales , Antibacterianos/efectos adversos , Antibacterianos/uso terapéutico , Enfermedades de los Gatos/tratamiento farmacológico , Gatos , Enfermedades de los Perros/tratamiento farmacológico , Perros , Wolbachia/efectos de los fármacosRESUMEN
Dirofilaria immitis carries intracellular endosymbiotic bacteria of the genus Wolbachia, known to be vital for the worms and sensitive to tetracycline antibiotics. With the purpose of studying the interaction between D. immitis and the endosymbiont Wolbachia sp., heartworm naturally infected microfilaremic or antigenemic dogs were treated with doxycycline (10mg/kg/day of the drug in three cycles of 21 days each, with 6-month intervals). Blood samples were collected on days 0, 7 and 21 of each treatment as well as on day 111 after the beginning of each cycle. A final sample was collected on day 723 from the beginning of the first treatment. The samples were examined for the presence and number of microfilariae and the presence of antigen as well as the presences of D. immitis and Wolbachia sp. DNA using PCR (polymerase chain reaction). With this approach, an evaluation of the effect of doxycycline on antigenemia and on the presence of Wolbachia sp. DNA in dogs with heartworm infection was possible. Doxycycline treatment did not alter the detection of adult parasite antigens with the exception of two animals, though the number of animals carrying Wolbachia sp. DNA decreased, despite the presence of the microfilariae. The effect of the antibiotic therapy on the worms may have interfered with the transmission of heartworm disease because the population of microfilariae and the number of microfilaremic dogs were reduced and the microfilariae positive samples that were found did not test positive for Wolbachia sp. in many cases. These findings suggest that in areas were doxycycline is extensively used D. immitis transmission may be impaired by the reduction on the number of microfilariae and on the endosymbiotic bacteria in the larvae turning them incapable of completing development once they infected a new host.
Asunto(s)
Antibacterianos/farmacología , Dirofilaria immitis/microbiología , Enfermedades de los Perros/microbiología , Enfermedades de los Perros/parasitología , Doxiciclina/farmacología , Wolbachia/efectos de los fármacos , Animales , Antígenos Bacterianos/sangre , ADN Bacteriano/sangre , Dirofilaria immitis/efectos de los fármacos , Perros , Factores de TiempoRESUMEN
Dirofilaria immitis is the causative agent of heartworm disease in canines and felines, and pulmonary dirofilariasis in man. It harbors a symbiotic intracellular bacterium from the genus Wolbachia that plays an important role in its biology and contributes to the inflammatory pathology of the heartworm. This endosymbiont is sensitive to the tetracycline family of antibiotics prompting its use in the treatment of filariasis. To track Wolbachia during treatment, primers were designed based on the FtsZ gene from Wolbachia. These primers amplify a single PCR product with the expected size from DNA samples derived from various species of worms that harbor Wolbachia (D. immitis, Brugia malayi and Brugia pahangy). The detection limit of Wolbachia DNA in the assay was 80 pg of D. immitis DNA. Furthermore, the primer set successfully amplified the expected PCR product using blood samples from dogs harboring the heartworm and circulating microfilariae.