Molting and Exsheathment In vitro of Third-Stage Dirofilaria immitis

ABSTRACTVarious catecholamines and catecholamine antagonists have been examined for their effects on the third larval moult of the parasitic nematode, Dirofilaria immitis, cultured in vitro. The non-selective α and β agonist, noradrenaline, and the β agonist, isoprenaline, had no effect on the timing of the third stage moult when used at a concentration of 10−5M. The α-adrenergic antagonist. phentolamine, resulted in worm mortality at 10−5M. At 10−7M, both phentolamine and the β-antagonist, propranolol caused a significant reduction in the numbers of larvae capable of completing the third stage moult. Idazoxan, an a2-antagonist, at 10−5M did not affect worm mortality but did completely prevent ecdysis. The potential of these compounds as possible filaricides is discussed.

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Secretory microRNA Profiles of Third- and Fourth-Stage Dirofilaria immitis Larvae with Different Macrocyclic Lactone Susceptibility: In Search of Biomarkers for Early Detection of Infection

Pathogens ◽

10.3390/pathogens10070786 ◽

2021 ◽

Vol 10 (7) ◽

pp. 786

Author(s):

Lucienne Tritten ◽

Erica J. Burkman ◽

Tobias Clark ◽

Guilherme G. Verocai

Keyword(s):

Early Detection ◽

Dirofilaria Immitis ◽

Macrocyclic Lactone ◽

The United States ◽

Fourth Stage ◽

Diagnostic Biomarkers ◽

Medical Interventions ◽

Third Stage ◽

Heartworm Infection

The canine heartworm, Dirofilaria immitis, is among the most important parasites of dogs in the United States and worldwide, and may cause severe and potentially fatal disease. Current diagnostic recommendations rely on serological detection of an adult female antigen, and visualization of microfilariae in the blood. Therefore, a reliable diagnosis can be only performed approximately six months post-infection. There is a growing need to characterize novel diagnostic markers that are capable of detecting the early stages of heartworm infection, in special markers associated with third-stage larvae (L3) and fourth-stage larvae (L4). The early detection of infection would guide medical interventions that could impede the development of patent infections and further parasite transmission. We cultured D. immitis L3 and L4 of two laboratorial strains with different susceptibility statuses to macrocyclic lactone drugs in vitro. Excretory/secretory microRNAs were sequenced and analyzed. We identified two miRNA novel candidates secreted abundantly by both L3 and L4 of both strains. These candidates were previously detected in the secretions of other D. immitis stages and one of them was found in the blood of D. immitis-infected dogs. These miRNAs have not been found in the secretions of other nematodes and could be D. immitis-specific diagnostic biomarkers, which could allow for the early detection of infection.

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Cryopreservation of Dirofilaria immitis microfilariae and third-stage larvae

Journal of Helminthology ◽

10.1017/s0022149x00011019 ◽

1983 ◽

Vol 57 (4) ◽

pp. 319-324 ◽

Cited By ~ 11

Author(s):

J. B. Lok ◽

M. Mika-Grieve ◽

R. B. Grieve

Keyword(s):

Hydroxyethyl Starch ◽

Dirofilaria Immitis ◽

Fourth Stage ◽

The Third ◽

Third Stage

AbstractMicrofilariae of Dirofilaria immitis retained their infectivity for susceptible mosquitoes after cooling to −196°C in the presence of 5% dimethylsulphoxide (Me2SO) using a two-step cooling sequence. Motility and in vitro development of cryopreserved microfilariae also compared favourably with unfrozen controls. Third-stage larvae frozen by the same cooling sequence in the presence of either 5% Me2SO or 16% hydroxyethyl starch were motile upon thawing. Thawed larvae completed the third- to fourth-stage moult in vitro at a frequency approximately 5 to 10% of that seen in unfrozen controls.

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Identification and validation of a commercial cryopreservation medium for the practical preservation of Dirofilaria immitis microfilaria

10.21203/rs.3.rs-17649/v3 ◽

2020 ◽

Author(s):

Takahiro Shirozu ◽

Akira Soga ◽

Shinya Fukumoto

Keyword(s):

Dirofilaria Immitis ◽

Preservation Solution ◽

Total Blood ◽

Infected Blood ◽

Preservation Method ◽

Heartworm Disease ◽

Third Stage ◽

Concentrated Solution

Abstract Background Dirofilaria immitis is a parasitic nematode transmitted by mosquitoes and the cause of heartworm disease in dogs and dirofilariasis in humans and other mammals. The parasite is endemic worldwide. Vector stage research requires a reliable supply of D. immitis microfilariae (mf). It is believed that cryopreserved mf would retain viability and provide a powerful tool for vector stage research. However, reports on cryopreservation of D. immitis mf are limited. Therefore, this study aimed to validate commercial cryopreservation media to establish a practical, convenient, and reproducible storage procedure for D. immitis mf. Methods Six different commercially available cryopreservation media were compared with the traditional polyvinylpyrrolidone-dimethyl sulfoxide (PVP-DMSO) preservation solution. In vitro viability of purified D. immitis mf and mf-infected total blood was analyzed using a motility assay and propidium iodide staining. In vivo infectivity of Aedes aegypti mosquitoes with cryopreserved mf was assessed using a mosquito survival test and quantifying the number of infective third stage larvae (L3) after 13 d post-infection. Results Purified mf cryopreserved in CultureSure showed the best viability when compared to mf cryopreserved in the remaining five commercially available media and PVP-DMSO. Viability of mf in mf-infected total blood cryopreserved in CultureSure varied with the ratio of infected blood to CultureSure. Optimum results were obtained with 200 µL mf-infected blood:800 µl CultureSure. CultureSure was also the optimum medium for cryopreserving mf prior to infectivity of Ae. aegypti. The number of L3 was approximately the same for CultureSure cryopreserved mf (3×-concentrated solution) and non-cryopreserved fresh mf. Conclusions CultureSure is an optimal commercial cryopreservation solution for the storage of D. immitis purified mf, mf-infected total blood, and mf used for in vivo mosquito experiments. Furthermore, this study describes an easy preservation method for clinical D. immitis -infected blood samples facilitating vector stage studies, as well as the education of veterinarians.

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Identification and validation of a commercial cryopreservation medium for the practical preservation of Dirofilaria immitis microfilaria

10.21203/rs.3.rs-17649/v2 ◽

2020 ◽

Author(s):

Takahiro Shirozu ◽

Akira Soga ◽

Shinya Fukumoto

Keyword(s):

Dirofilaria Immitis ◽

Preservation Solution ◽

Total Blood ◽

Infected Blood ◽

Preservation Method ◽

Heartworm Disease ◽

Third Stage ◽

Concentrated Solution

Abstract Background Dirofilaria immitis is a parasitic nematode transmitted by mosquitoes and the cause of heartworm disease in dogs and dirofilariasis in humans and other mammals. The parasite is endemic worldwide. Vector stage research requires a reliable supply of D. immitis microfilariae (mf). It is believed that cryopreserved mf would retain viability and provide a powerful tool for vector stage research. However, reports on cryopreservation of D. immitis mf are limited. Therefore, this study aimed to validate commercial cryopreservation media to establish a practical, convenient, and reproducible storage procedure for D. immitis mf. Methods Six different commercially available cryopreservation media were compared with the traditional polyvinylpyrrolidone-dimethyl sulfoxide (PVP-DMSO) preservation solution. In vitro viability of purified D. immitis mf and mf-infected total blood was analyzed using a motility assay and propidium iodide staining. In vivo infectivity of Aedes aegypti mosquitoes with cryopreserved mf was assessed using a mosquito survival test and quantifying the number of infective third stage larvae (L3) after 13 d post-infection. Results Purified mf cryopreserved in CultureSure showed the best viability when compared to mf cryopreserved in the remaining five commercially available media and PVP-DMSO. Viability of mf in mf-infected total blood cryopreserved in CultureSure varied with the ratio of infected blood to CultureSure. Optimum results were obtained with 200 µL mf-infected blood:800 µl CultureSure. CultureSure was also the optimum medium for cryopreserving mf prior to infectivity of Ae. aegypti. The number of L3 was approximately the same for CultureSure cryopreserved mf (3×-concentrated solution) and non-cryopreserved fresh mf. Conclusions CultureSure is an optimal commercial cryopreservation solution for the storage of D. immitis purified mf, mf-infected total blood, and mf used for in vivo mosquito experiments. Furthermore, this study describes an easy preservation method for clinical D. immitis -infected blood samples facilitating vector stage studies, as well as the education of veterinarians.

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Identification and validation of a commercial cryopreservation medium for the practical preservation of Dirofilaria immitis microfilaria

10.21203/rs.3.rs-17649/v1 ◽

2020 ◽

Author(s):

Takahiro Shirozu ◽

Akira Soga ◽

Shinya Fukumto

Keyword(s):

Dirofilaria Immitis ◽

Preservation Solution ◽

Total Blood ◽

Infected Blood ◽

Preservation Method ◽

Heartworm Disease ◽

Third Stage ◽

Concentrated Solution

Abstract Background Dirofilaria immitis is a parasitic nematode transmitted by mosquitoes and the cause of heartworm disease in dogs and dirofilariasis in humans and other mammals. The parasite is endemic worldwide. Vector stage research requires a reliable supply of D. immitis microfilariae (mf). It is believed that cryopreserved mf would retain viability and provide a powerful tool for vector stage research. However, reports on cryopreservation of D. immitis mf are limited. Therefore, this study aimed to validate commercial cryopreservation media to establish a practical, convenient, and reproducible storage procedure for D. immitis mf.Methods Six different commercially available cryopreservation media were compared with the traditional polyvinylpyrrolidone-dimethyl sulfoxide (PVP-DMSO) preservation solution. In vitro viability of purified D. immitis mf and mf-infected total blood was analyzed using a motility assay and propidium iodide staining. In vivo infectivity of Aedes aegypti mosquitoes with cryopreserved mf was assessed using a mosquito survival test and quantifying the number of infective third stage larvae (L3) after 13 d post-infection.Results Purified mf cryopreserved in CultureSure showed the best viability when compared to mf cryopreserved in the remaining five commercially available media and PVP-DMSO. Viability of mf in mf-infected total blood cryopreserved in CultureSure varied with the ratio of infected blood to CultureSure. Optimum results were obtained with 200 µL mf-infected blood:800 µl CultureSure. CultureSure was also the optimum medium for cryopreserving mf prior to infectivity of Ae. aegypti. The number of L3 was approximately the same for CultureSure cryopreserved mf (3×-concentrated solution) and non-cryopreserved fresh mf.Conclusions CultureSure is an optimal commercial cryopreservation solution for the storage of D. immitis purified mf, mf-infected total blood, and mf used for in vivo mosquito experiments. Furthermore, this study describes an easy preservation method for clinical D. immitis -infected blood samples facilitating vector stage studies, as well as the education of veterinarians.

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Dirofilaria immitis: Heartworm Products Contract Rat Trachea in Vitro

Experimental Parasitology ◽

10.1006/expr.1994.1007 ◽

1994 ◽

Vol 78 (1) ◽

pp. 76-84 ◽

Cited By ~ 5

Author(s):

J.M. Collins ◽

J.F. Williams ◽

L. Kaiser

Keyword(s):

Dirofilaria Immitis ◽

Rat Trachea

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A rapid, parasite-dependent cellular response to Dirofilaria immitis in the Mongolian jird (Meriones unguiculatus)

Parasites & Vectors ◽

10.1186/s13071-020-04455-x ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Christopher C. Evans ◽

Katherine M. Day ◽

Yi Chu ◽

Bridget Garner ◽

Kaori Sakamoto ◽

...

Keyword(s):

Cellular Response ◽

Dirofilaria Immitis ◽

Cell Attachment ◽

Early Response ◽

Meriones Unguiculatus ◽

Filarial Parasite ◽

Immune Mediated ◽

Secretory Products ◽

Species Specific

Abstract Background The Mongolian jird (Meriones unguiculatus) has long been recognized as a permissive host for the filarial parasite Brugia malayi; however, it is nonpermissive to another filarial parasite, canine heartworm (Dirofilaria immitis). By elucidating differences in the early response to infection, we sought to identify mechanisms involved in the species-specific clearance of these parasites. We hypothesized that the early clearance of D. immitis in intraperitoneal infection of the jird is immune mediated and parasite species dependent. Methods Jird peritoneal exudate cells (PECs) were isolated and their attachment to parasite larvae assessed in vitro under various conditions: D. immitis and B. malayi cultured separately, co-culture of both parasites, incubation before addition of cells, culture of heat-killed parasites, and culture with PECs isolated from jirds with mature B. malayi infection. The cells attaching to larvae were identified by immunohistochemistry. Results In vitro cell attachment to live D. immitis was high (mean = 99.6%) while much lower for B. malayi (mean = 5.56%). This species-specific attachment was also observed when both filarial species were co-cultured, with no significant change from controls (U(9, 14) = 58.5, p = 0.999). When we replicated these experiments with PECs derived from jirds subcutaneously infected with B. malayi, the results were similar (99.4% and 4.72% of D. immitis and B. malayi, respectively, exhibited cell attachment). Heat-killing the parasites significantly reduced cell attachment to D. immitis (mean = 71.9%; U(11, 14) = 7.5, p < 0.001) while increasing attachment to B. malayi (mean = 16.7%; U(9, 15) = 20, p = 0.002). Cell attachment to both species was reduced when larvae were allowed a 24-h pre-incubation period prior to the addition of cells. The attaching cells were identified as macrophages by immunohistochemistry. Conclusions These results suggest a strongly species-dependent response from which B. malayi could not confer protection by proxy in co-culture. The changes in cell attachment following heat-killing and pre-incubation suggest a role for excretory/secretory products in host immune evasion and/or antigenicity. The nature of this attachment is the subject of ongoing study and may provide insight into filarial host specificity.

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Angiostrongylus cantonensis (Nematode: Metastrongiloidea): In Vitro Cultivation of Infective Third-Stage Larvae to Fourth-Stage Larvae

PLoS ONE ◽

10.1371/journal.pone.0072084 ◽

2013 ◽

Vol 8 (8) ◽

pp. e72084 ◽

Cited By ~ 3

Author(s):

Rong-Jyh Lin ◽

Jie-Wen He ◽

Li-Yu Chung ◽

June-Der Lee ◽

Jiun-Jye Wang ◽

...

Keyword(s):

Angiostrongylus Cantonensis ◽

In Vitro Cultivation ◽

Fourth Stage ◽

Third Stage

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Leishmania infantum and Dirofilaria immitis coinfection in dogs in Greece

Parasitology Open ◽

10.1017/pao.2016.15 ◽

2016 ◽

Vol 2 ◽

Cited By ~ 1

Author(s):

PANTELIS NTAIS ◽

VASILIKI CHRISTODOULOU ◽

EMMANOUIL DOKIANAKIS ◽

MARIA ANTONIOU

Keyword(s):

Leishmania Infantum ◽

Dirofilaria Immitis ◽

Low Cost ◽

Blood Cultures ◽

Molecular Techniques ◽

Immunological Methods ◽

Parasitic Diseases ◽

Survival Potential ◽

Knott’S Test

SUMMARYLeishmaniasis and dirofilariasis are parasitic diseases of humans and dogs, worldwide, and they are often found as coinfections in endemic areas. Cases of human and canine dirofilariasis have being reported in Greece and leishmaniasis is endemic in most prefectures in humans and dogs. In most cases, dirofilariasis is established by parasitological (the modified Knott's test) and/or immunological methods, whilst for leishmaniasis molecular techniques and culture are also used. During an epidemiological study in Greece, 22·1% of the 5772 dogs studied were found positive by serology forLeishmania.Blood cultures of 165 (12·94%) of these animals producedLeishmaniapromastigotes and 26 (2·03%)Dirofilariamicrofilariae (L1), whilst only in two (0·16%) bothLeishmaniaandDirofilariaL1 appeared. The aim was to assess coinfections by the two parasites in dogs in Greece, the isolation and survival ofDirofilariamicrofilariae andLeishmaniapromastigotes using clotted blood (a fast, simple and low-cost method) and the survival potential of the two parasites in coexistence,in vitro.

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