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

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.

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

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.

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

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.

The effect of catecholamines and catecholamine antagonists on the third larval moult of Dirofilaria immitis in vitro

1992 ◽  
Vol 66 (4) ◽  
pp. 273-278 ◽  
Author(s):  
E. V. Warbrick ◽  
S. A. Ward
Keyword(s):  
Dirofilaria Immitis ◽  
Parasitic Nematode ◽  
The Third ◽  
Adrenergic Antagonist ◽  
Third Stage ◽  
Larval Moult

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.

scholarly journals Oxygen and Carbon Dioxide Transport Characteristics of the Blood of the Nile Monitor Lizard (Varanus Niloticus)

1987 ◽  
Vol 130 (1) ◽  
pp. 27-38
Author(s):  
JAMES W. HICKS ◽  
ATSUSHI ISHIMATSU ◽  
NORBERT HEISLER
Keyword(s):  
Carbon Dioxide ◽  
Haemoglobin Concentration ◽  
Oxygen Affinity ◽  
Total Blood ◽  
Carbon Dioxide Transport ◽  
Monitor Lizard ◽  
The Right ◽  
Dissociation Curves

Oxygen and carbon dioxide dissociation curves were constructed for the blood of the Nile monitor lizard, Varanus niloticus, acclimated for 12h at 25 and 35°C. The oxygen affinity of Varanus blood was low when Pco2 w a s in the range of in vivo values (25°C: P50 = 34.3 at PCOCO2 = 21 mmHg; 35°C: P50 = 46.2 mmHg at PCOCO2 = 35 mmHg; 1 mmHg = 133.3 Pa), and the oxygen dissociation curves were highly sigmoidal (Hill's n = 2.97 at 25°C and 3.40 at 35°C). The position of the O2 curves was relatively insensitive to temperature change with an apparent enthalpy of oxygenation (ΔH) of −9.2kJ mol−1. The carbon dioxide dissociation curves were shifted to the right with increasing temperature by decreasing total CCOCO2 at fixed PCOCO2, whereas the state of oxygenation had little effect on total blood CO2 content. The in vitro buffer value of true plasma (Δ[HCO3−]pl/-ΔpHpl) rose from 12.0 mequiv pH−1−1 at 25°C to 17.5 mequiv pH−11−1 at 35°C, reflecting a reversible increase of about 30% in haemoglobin concentration and haematocrit levels during resting conditions in vivo.

Ivermectin-Induced Hypertrophic Changes in Adult Canine Heartworm (Dirofilaria Immitis) Gut Epithelium

1998 ◽  
Vol 4 (S2) ◽  
pp. 1144-1145
Author(s):  
W. L. Steffens ◽  
J. W. McCall
Keyword(s):  
Dirofilaria Immitis ◽  
Muscle Paralysis ◽  
Somatic Muscle ◽  
Diverse Range ◽  
Gut Epithelium ◽  
Transmission Electron ◽  
And Control ◽  
Hypertrophic Changes

Ivermectin is a drug widely utilized for its anthelminthic activity over a diverse range of animal parasites. It has proved to be particularly useful in the prophylaxis of infection by the heartworm (Dirofilaria immitis) in dogs and cats. Although its application in this respect has been as a filaricide in preventing early growth and maturation of naturally acquired larvae, it is known to have activity against young adults as well. Previous studies have shown that in vitro exposure to ivermectin induces somatic muscle paralysis in the nematode Haemonchus contortus, resulting in pharyngeal dysfunction and disruption of normal ingestion. Experiments were performed to determine the effect of in vivo exposure of adult canine heartworms to this drug.Adult heartworms were harvested from groups of dogs treated monthly with ivermectin beginning four to five months after inoculation of infective larvae and from untreated control dogs. Live worms from both experimental and control dogs were fixed, embedded, and sectioned for conventional transmission electron microscopy.

Effects of four tanniferous plant extracts on thein vitroexsheathment of third-stage larvae of parasitic nematodes

Parasitology ◽  
2006 ◽  
Vol 132 (4) ◽  
pp. 545-554 ◽  
Author(s):  
D. BAHUAUD ◽  
C. MARTINEZ-ORTIZ DE MONTELLANO ◽  
S. CHAUVEAU ◽  
F. PREVOT ◽  
F. TORRES-ACOSTA ◽  
...  
Keyword(s):  
Secondary Metabolites ◽  
Plant Extracts ◽  
Castanea Sativa ◽  
Nematode Species ◽  
Parasitic Nematodes ◽  
Rye Grass ◽  
Pine Tree ◽  
Third Stage

The anthelmintic properties of tanniferous plants and of their secondary metabolites represent one possible alternative to chemotherapy that is currently being explored as a means of achieving sustainable control of gastrointestinal nematodes in ruminants. Previousin vivoandin vitroresults suggest that tanniferous plants can have direct anti-parasitic effect against different stages of nematodes. However, the mode of action of the bioactive plant compounds remains obscure. The objectives of the current study were (1) to examine the hypothesis that extracts of tanniferous plants might interfere with the exsheathment of third-stage infective larvae (L3); (2) to assess the role of tannins in the process by examining the consequence of adding an inhibitor of tannins (polyethylene glycol: PEG) to extracts. The effects of 4 tanniferous plant extracts on exsheathment have been examined on L3 ofHaemonchus contortusandTrichostrongylus colubriformis. Artificial exsheathment was inducedin vitroby adding hypochloride solution to larval suspension. The evolution of exsheathment with time was measured by repeated observations at 10-min interval for 60 min. The selected plants were: genista (Sarothamnus scoparius), heather (Erica erigena), pine tree (Pinus sylvestris), and chestnut tree (Castanea sativa), with tannin contents ranging from 1·5 to 24·7% of DM. Extracts of a non-tanniferous plant (rye grass, tannin content: 0·3% of DM) were included in the assay as negative controls. The extracts were tested at the concentration of 600 μg/ml and the effects were compared to the rate of exsheathment of control larvae in PBS. No statistical differences in the pattern of exsheathment was observed after addition of rye grass or genista extracts for both nematode species and with heather extracts forT. colubriformis. In contrast, pine tree extracts on larvae of both species and heather extracts withH. contortusinduced a significant delay in exsheathment. Last, contact with chest nut extracts led to a total inhibition of the process for both nematodes. These results suggest that extracts of tanniferous plants might affect a key process in the very early stages of larval invasion of the host. In most cases, the addition of PEG led to a total or partial restoration towards control values. This suggests that tannins are largely involved in the inhibitory process. However, other secondary metabolites may also interfere with the process that would help to explain some of the differences in response observed between the two nematode species.

External monitoring of 203Hg-labelled Dirofilaria immitis microfilariae in mice and dogs

Parasitology ◽  
1986 ◽  
Vol 92 (2) ◽  
pp. 463-469 ◽  
Author(s):  
F. W. Lengemann ◽  
R. B. Grieve ◽  
M. Chmielewicz ◽  
F. A. Kallfelz ◽  
J. R. Georgi
Keyword(s):  
Dirofilaria Immitis ◽  
Half Time ◽  
Beagle Dogs ◽  
External Monitoring

SUMMARYMicrofilariae of Dirofliaria immitis were labelled with 203Hg 2+in vitro and injected into irradiated mice and Beagle dogs. With irradiated mice it was possible to demonstrate microfilariae present in the blood and to detect 203Hg by external counting as long as 28 days after dosing. The 203Hg 2+ label had a half-time of 4–5 days; the amount of stable mercury in the labelling medium strongly influenced the survival of microfilariae in vivo. In dogs, external counting showed the lungs to be a major location of the microfilariae soon after reinjection into the host. Evidence was obtained that labelled microfilariae can circulate; however, the detection of dispersed microfilariae is difficult because of the relative insensitivity of the detecting system. For radiomercury the accumulation of the inorganic form in the liver and kidneys limits the long-term usefulness of 203Hg 2+ as a label if the organism being studied also accumulates in these organs.

scholarly journals Interleukin-10 and Antigen-Presenting Cells Actively Suppress Th1 Cells in BALB/c Mice Infected with the Filarial Parasite Brugia pahangi

1999 ◽  
Vol 67 (4) ◽  
pp. 1599-1605 ◽  
Author(s):  
Julie Osborne ◽  
Eileen Devaney
Keyword(s):  
Interleukin 10 ◽  
Interleukin 4 ◽  
Th1 Cells ◽  
Spleen Cells ◽  
Third Stage ◽  
Antigen Presenting ◽  
Th1 Cytokines

ABSTRACT Infection with the third-stage larvae (L3) of the filarial nematodeBrugia results in a Th2-biased immune response in mice and humans. Previously we have shown that the production of interleukin 4 (IL-4) is critical for down-regulating polyclonal Th1 responses in L3-infected mice. However, the in vitro neutralization of IL-4 did not fully recover the defective polyclonal Th1 responses, nor did it result in the production of any antigen (Ag)-specific Th1 cytokines, suggesting that perhaps infection with L3 does not result in priming of Th1 cells in vivo. In this study, we analyzed the role of IL-10 and Ag-presenting cells (APCs) in the spleen as additional factors controlling the Th2 bias in infected mice. Our data show that IL-10 and APCs also contribute to the suppression of mitogen-driven Th1 responses of spleen cells from infected mice. In addition, the neutralization of IL-10 or the replacement of the resident APC population from spleen cell cultures resulted in the production of Ag-specific Th1 cytokines. Irradiated spleen cells from either L3-infected or uninfected mice were able to restore Ag-specific Th1 responses in vitro. Therefore, it appears that Brugia-reactive Th1 cells are primed following infection with L3, but are actively suppressed in vivo by a mechanism that involves IL-10 and the resident APC population, but not IL-4. These results indicate that a complex interplay of cytokines and cell populations underscores the Th2-polarized response in L3-infected mice.

Chemical composition and antioxidant activity of Carob pulp (Ceratonia Siliqua L.) extracts

2015 ◽  
Vol 5 (2) ◽  
pp. 76-83
Author(s):  
Yasmina Mokhtaria Boufadia ◽  
Manel Elaoufi ◽  
Fatiha Tabet ◽  
Mohamed Benali ◽  
Ali Riazi
Keyword(s):  
Antioxidant Activity ◽  
Food Additives ◽  
Ethanolic Extract ◽  
Natural Food ◽  
Ceratonia Siliqua ◽  
Total Blood ◽  
Pre Treatment ◽  
Ceratonia Siliqua L

  The role of polyphenols and flavonoids in prevention of cellular damages carried out with the oxidative stress is well documented. In the present experiment, we determined the polyphenols and flavonoids concentrations of three extracts (ethanol 70%: V/V, methanol 80%: V/V and distilled water extracts) of carob pulp mature (Ceratonia siliqua L.) and their in vitro and in vivo antioxidant activity. A significant dose-dependent anti-free radical activ-ity of ethanolic extract of carob (EEC) was related to the highest polyphenol content (44.74 mg QE/mL) and to the in vitro antioxidant activity (EC50 = 0.28 mg/mL) of this extract. LC-MS /MS analysis of the EEC have shown the presence of several phenolic compounds as well as many types of flavonoids. The in vivo experiment brought out the significant effect of the daily EEC oral pre-treatment of rats, 30 min before AlCl3 administration for 28 d on the improvement of the total blood antioxidant capacity. These results suggest that the carob pulp contain many substances having in vitro and in vivo antioxidant effects, and which could be used as natural food additives in order to preserve food quality.

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