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Viability of cryopreserved D. immitis microfilariae (mf )-infected total blood. a In vitro motility was scored over a range of blood volumes (X-axis) in CultureSure (1 ml total volume) and compared to purified mf at 0, 1 and 3 days post-thawing (dpt). Scoring was at four levels as follows: inactive or dead (-), less active (+), moderately active (++) and highly active (+++). An asterisk indicates a significant difference (P < 0.05) by one-way ANOVA, followed by Tukey's multiple comparisons test. Error bars indicate ± SD. b Percent positive PI staining of mf-infected total blood on 3 dpt. An asterisk indicates a significant difference by Chi-square test and adjusted residual (AR). AR ≥ 1.96 was considered significant (P < 0.05). c Fluorescent images of propidium iodide (PI) staining of mf-infected total blood on 3 dpt (exposure, 100 ms; gain, 0.5). PI image (left) and bright field (BF) image (right). Abbreviation: PC, positive control. Scale-bars: 100 µm
Source publication
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 viabilit...
Contexts in source publication
Context 1
... compared with traditional PVP-DMSO cryopreservation. Non-cryopreserved, fresh mf were used as a positive control. The survival rate of mosquitoes fed LaboBanker cryopreserved mf was significantly higher compared to mosquitoes fed CultureSure (χ 2 = 4.761, df = 1, P = 0.029) or PVP-DMSO (χ 2 = 4.086, df = 1, P = 0.043) cryopreserved mf on 13 dpi (Fig. 3a). The number of L3 in mosquitoes fed CultureSure cryopreserved mf tended to be higher than that of the L3 for mf cryopreserved in PVP-DMSO (t = 1.728, df = 53.64, P = 0.090) and fresh (t = 1.897, df = 56.14, P = 0.063) mf (Fig. 3b). Although different mosquito colonies and mf infectious inocula were used, the mf infectivity reported ...
Context 2
... CultureSure (χ 2 = 4.761, df = 1, P = 0.029) or PVP-DMSO (χ 2 = 4.086, df = 1, P = 0.043) cryopreserved mf on 13 dpi (Fig. 3a). The number of L3 in mosquitoes fed CultureSure cryopreserved mf tended to be higher than that of the L3 for mf cryopreserved in PVP-DMSO (t = 1.728, df = 53.64, P = 0.090) and fresh (t = 1.897, df = 56.14, P = 0.063) mf (Fig. 3b). Although different mosquito colonies and mf infectious inocula were used, the mf infectivity reported here was similar to the one previously reported for PVP-DMSO cryopreserved mf [6]. The ratios of mean L3 numbers in individual mosquitoes/feeding inoculum concentration were 0.85 and 0.77 for PVP-DMSO cryopreserved mf in our study and ...
Citations
... The dog was housed in an individual cage under standard conditions at 23 °C with a light cycle of 06:00 to 18:00 h. The Aedes aegypti Liverpool-OB strain, susceptible to D. immitis infection 9 , was kept at 27 °C and > 80% humidity on a diet of 5% fructose solution supplemented with 0.05% para-aminobenzoic acid (PABA) under a 12-h light and 12-h dark photoperiod. All animal experiments complied with the Guide for Laboratory Animals of the Obihiro University of Agriculture and Veterinary Medicine and in accordance with ARRIVE guidelines. ...
... However, the viability of cryopreserved microfilariae after thawing remains a substantial challenge 11 . In our earlier investigation, we found that thawed microfilariae had inferior fitness, despite approximately 80% of the microfilariae surviving 3 days after thawing in vitro 9 . Furthermore, the number of L3 larvae recovered from mosquitoes infected with cryopreserved microfilariae was approximately one-third of that recovered from mosquitoes infected with an equal number of non-cryopreserved fresh microfilariae 9 . ...
... In our earlier investigation, we found that thawed microfilariae had inferior fitness, despite approximately 80% of the microfilariae surviving 3 days after thawing in vitro 9 . Furthermore, the number of L3 larvae recovered from mosquitoes infected with cryopreserved microfilariae was approximately one-third of that recovered from mosquitoes infected with an equal number of non-cryopreserved fresh microfilariae 9 . In this study, we used SCID mice to recover the cryopreserved microfilariae. ...
Dirofilaria immitis is a mosquito-borne parasitic nematode that causes fatal heartworm disease in canids. The microfilariae are essential for research, including drug screening and mosquito-parasite interactions. However, no reliable methods for maintaining microfilaria long-term are currently available. Therefore, we used severe combined immunodeficiency (SCID) mice to develop a reliable method for maintaining D. immitis microfilaria. SCID mice were injected intravenously with microfilariae isolated from a D. immitis-infected dog. Microfilariae were detected in blood collected from the tail vein 218 days post-inoculation (dpi) and via cardiac puncture 296 dpi. Microfilariae maintained in and extracted from SCID mice showed infectivity and matured into third-stage larvae (L3s) in the vector mosquito Aedes aegypti. L3s can develop into the fourth stage larvae in vitro. Microfilariae from SCID mice respond normally to ivermectin in vitro. The microfilariae in SCID mice displayed periodicity in the peripheral circulation. The SCID mouse model aided in the separation of microfilariae from cryopreserved specimens. The use of SCID mice enabled the isolation and sustained cultivation of microfilariae from clinical samples. These findings highlight the usefulness of the SCID mouse model for studying D. immitis microfilaremia in canine heartworm research.
... Consequently, a detailed understanding of the D. immitis transmission mechanisms in mosquito vectors is essential for such research projects. In this context, a steady supply of ne micro lariae is critical because damaged micro lariae, such as cryopreserved micro lariae, cannot infect mosquitoes normally [9]. ...
... However, in vitro culture can only sustain D. immitis micro lariae for less than two months [10], and cryopreservation substantially reduces the viability of recovered micro lariae compared to fresh micro laria [9,11]. The rodent micro laria model is regarded as a viable alternative to D. immitis-infected dogs. ...
... Mice were housed under standard conditions at 22 ºC with a light cycle of 08:00 to 20:00 h. A six-monthold female beagle dog (KITAYAMA LABES Co., Nagano, Japan) was subcutaneously infected with 40 L3s of the D. immitis SF1 strain, initially isolated in Japan [9,13] from the dorsal neck region. The dog was housed in an individual cage under standard conditions at 23 ºC with a light cycle of 06:00 to 18:00 h. ...
Dirofilaria immitis is a mosquito-borne parasitic nematode that causes fatal heartworm disease in canids. The fine microfilariae are essential for research, including drug screening and mosquito-parasite interactions. However, no reliable methods for maintaining fine microfilaria long-term are currently available. Therefore, we used severe combined immunodeficiency (SCID) mice to develop a reliable method for maintaining D. immitis microfilaria. SCID mice were injected intravenously with microfilariae isolated from a D. immitis -infected dog. Microfilariae were detected in blood collected from the tail vein 218 days post-inoculation (dpi) and via cardiac puncture 296 dpi. Microfilariae maintained in and extracted from SCID mice showed infectivity and matured into third-stage larvae (L3s) in the vector mosquito Aedes aegypti. L3s can develop into the fourth stage larvae in vitro. Microfilariae from SCID mice respond normally to ivermectin in vitro. The microfilariae in SCID mice displayed periodicity in the peripheral circulation. The SCID mouse model aided in the separation of superior microfilariae from cryopreserved specimens. The use of SCID mice enabled the isolation and sustained cultivation of microfilariae from clinical samples. These findings highlight the usefulness of the SCID mouse model for studying D. immitis microfilaremia in canine heartworm research.
... Cryopreservation protocols for parasitic worms can be found in James (1985James ( , 1990James ( , 2004 and Eckert (1988). Other protocols have been developed for specific genera such as Heterorhabditis (Nugent et al. 1996), Schistosomula (James 1982), Dirofilaria (Shirozu et al. 2020;Zinser et al. 2021), and Pristionchus plus other free-living nematodes (Matthias Herrmann, pers. comm.). ...
... Cryopreservation protocols for parasitic worms can be found in James (1985James ( , 1990James ( , 2004 and Eckert (1988). Other protocols have been developed for specific genera such as Heterorhabditis (Nugent et al. 1996), Schistosomula (James 1982), Dirofilaria (Shirozu et al. 2020;Zinser et al. 2021), and Pristionchus plus other free-living nematodes (Matthias Herrmann, pers. comm.). ...
Mosquito-borne diseases such as dengue and filariasis are a growing public health concern in endemic countries. Biological approaches, such as the trans-infection of Wolbachia pipientis in mosquitoes, are an alternative vector control strategy, especially for arthropod-borne viruses such as dengue. In the present study, the effect of Wolbachia (wMel strain) on the vectorial capacity of Aedes aegypti for Dirofilaria immitis was studied. Our results showed that Wolbachia does not affect the phenotype of mosquito survival or the prevalence, number, and molting rate of third-stage larvae in both susceptible and resistant strains of Ae. aegypti. RNA-seq analysis of Malpighian tubules at 2 days post-infection with D. immitis showed the differentially expressed genes (DEGs) with and without wMel infection. No characteristic immune-related gene expression patterns were observed among the DEGs. No significant change in the amount of Wolbachia was observed in the Ae. aegypti after D. immitis infection. Our results suggest that infection of D. immitis in Ae. aegypti populations will not interfere with Wolbachia-based vector control strategies in dengue-endemic areas where cases of D. immitis are present. This study demonstrated the veterinary medical validity of a dengue control program using Wolbachia.
In recent years, the interest in media representations of migrants and the media as a space for participation has increased within the
field of migration studies. Yet, most scholars’ attention is focused on immigrants and the media in destination countries, while less attention
is paid to origin countries and emigrants’ representation. Taking advantage of the increased attention paid to migrants and migration during
the first phase of the COVID-19 pandemic, we investigated the media representations of Romanian migrants in agriculture who work in other
European countries and interpreted how their voices could be heard through media accounts. Through content analysis, we investigated a
sample of 297 articles published between 1st April and 31st May 2020 on the websites of the six most visible Romanian media outlets.
This study contributes to the existing knowledge on media representations of Romanian migrants by documenting a series of tendencies,
including an event-oriented approach, oversimplified representations of migration, massification and schematisation of migrant representations, and the high sensitivity to reports from destination countries’ media on Romanian migrants.
Our analysis reveals that the approach taken to reporting on migration during the COVID-19 pandemic, at least during its first phase, highly
depended on the existing, institutionalised modes of media reporting on migration.
Background aims
The use of effective methods for the cryopreservation of hematopoietic stem cells (HSCs) is vital to retain the maximum engraftment activity of cord blood units (CBUs). Current protocols entail the use of dimethyl sulfoxide (DMSO) as intracellular cryoprotective agent (CPA) and dextran and plasma proteins as extracellular CPAs, but DMSO is known to be cytotoxic, and its infusion in patients is associated with mild to moderate side effects. However, new, commercially available, DMSO-free cryopreservation solutions have been developed, but their capacity to protect HSCs remains poorly investigated.
Methods
Herein the authors compared the capacity of four DMSO-free freezing media to cryopreserve cord blood (CB) HSCs: CryoProtectPureSTEM (CPP-STEM), CryoScarless (CSL), CryoNovo P24 (CN) and Pentaisomaltose (PIM). Clinical-grade DMSO/dextran solution was used as control.
Results
Of the four cryopreservation solutions tested, the best post-thaw cell viability, recovery of viable CD45+ and CD34+ cells and potency were achieved with CPP-STEM, which was equal or superior to that seen with the control DMSO. CSL provided the second best post-thaw results followed by PIM, whereas CN was associated with modest viability and potency. Further work with CPP-STEM revealed that CB CD34-enriched HSCs and progenitors cryopreserved with CPP-STEM maintained high viability and growth expansion activity. In line with this, a pilot transplantation assay confirmed that CPP-STEM-protected CB grafts supported normal short- and long-term engraftment kinetics.
Conclusions
The authors’ results suggest that new, valuable alternatives to DMSO are now available for the cryopreservation of HSCs and grafts, including CBUs.
