Development of a novel rodent model for dog heartworm microfilaremia using the severe-combined immunodeficiency mouse.

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.

Effect of E-64 Supplementation during In Vitro Maturation on the Developmental Competence of Bovine OPU-Derived Oocytes.

Recovery of bovine oocytes using the ovum pick-up (OPU) technique offers the advantage of rapid genetic improvement through propagation of desired genes from animals with high genetic qualities. However, the developmental competence of OPU-derived immature oocytes remains relatively poor. We previously found that cathepsin B gene expression and activity are increased in poor quality oocytes and embryos compared to good quality ones. In this study, we investigated the effect of E-64 (cathepsin B inhibitor) supplementation during in vitro maturation (IVM) on the developmental competence of OPU-derived immature oocytes and the quality of the produced blastocysts. Our results showed that supplementation of IVM medium with E-64 significantly improved the developmental competence of OPU-derived immature oocytes as evidenced by the significant increase of the blastocyst rate. Importantly, the presence of E-64 during IVM also significantly improved blastocyst quality by increasing the total cell number and decreasing the percentage of TUNEL positive cells. These results indicate that E-64 supplementation during IVM is a promising tool to improve the efficiency of OPU-IVF program by improving the developmental competence of OPU-derived immature oocytes.

Molecular detection of Blastocystis sp. subtype 14 in the Yezo sika deer (Cervus nippon yesoensis) in Hokkaido, Japan.

This study describes the first report of Blastocystis sp. colonization in the sika deer (Cervus nippon) in Japan and in other animals in Hokkaido, Japan. Blastocystis sp. is one of the most widespread intestinal protist in a wide range of animals. Blastocystis sp. isolated from mammalian and avian species have been classified into 17 subtypes (STs). Some of the STs are zoonotic. The aim of this study was to evaluate Blastocystis sp. colonization in the Yezo sika deer (Cervus nippon yesoensis) in Hokkaido, Japan. The Yezo sika deer are currently overabundant and they are expanding their habitat to humans and livestock. A total of 132 deer fecal samples were subjected for molecular detection of Blastocystis sp. Of these, 60 (45.5%) samples were positive using PCR, which targets the small subunit ribosomal RNA gene sequence. All Blastocystis sp. DNA sequences from the Yezo sika deer were genotyped into ST14, which were originally reported in cattle. These findings indicate that the current public health risks of Blastocystis sp. from the Yezo sika deer is low, although more detailed future analysis is required.

Glyoxalase pathway is required for normal liver-stage proliferation of Plasmodium berghei.

The glyoxalase system is a ubiquitous detoxification pathway of methylglyoxal, a cytotoxic byproduct of glycolysis. Actively proliferating cells, such as cancer cells, depend on their energy metabolism for glycolysis. Therefore, the glyoxalase system has been evaluated as a target of anticancer drugs. The malaria sporozoite, which is the infective stage of the malaria parasite, actively proliferates and produces thousands of merozoites within 2-3 days in hepatocytes. This is the first step of infection in mammalian hosts. The glyoxalase system appears to play an important role in this active proliferation stage of the malaria parasite in hepatocytes. In this study, we aimed to dissect the role of the glyoxalase system in malaria parasite proliferation in hepatocytes to examine its potential as a target of malaria prevention using a reverse genetics approach. The malaria parasite possesses a glyoxalase system, comprised of glyoxalases and GloI-like protein, in the cytosol and apicoplast. We generated cytosolic glyoxalase II (cgloII) knockout, apicoplast targeted glyoxalase gloII (tgloII) knockout, and cgloII and tgloII double-knockout parasites and performed their phenotypic analysis. We did not observe any defects in the cgloII or tgloII knockout parasites. In contrast, we observed approximately 90% inhibition of the liver-stage proliferation of cgloII and tgloII double-knockout parasites in vivo. These findings suggest that although the glyoxalase system is dispensable, it plays an important role in parasite proliferation in hepatocytes. Additionally, the results indicate a complementary relationship between the cytosolic and apicoplast glyoxalase pathways. We expect that the parasite utilizes a system similar to that observed in cancer cells to enable its rapid proliferation in hepatocytes; this process could be targeted in the development of novel strategies to prevent malaria.

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

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 third-stage larvae (L3) after 13 days 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 A. 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 study of macrocyclic lactone resistance in heartworms and the education of veterinarians.

Development and evaluation of a novel loop-mediated isothermal amplification (LAMP) method targeting Theileria parasites infecting Yezo sika deer.

The increasing Yezo sika deer (Cervus nippon yesoensis) population is creating a large problem. Yezo sika deer are an important blood meal source, and these deer contribute to the maintenance of tick populations. Theileria spp. infections in Yezo sika deer and T. orientalis infections in cows occur at high frequencies, and the same tick species infests both deer and cows. Therefore, a specific detection method to identify deer Theileria spp. is important. In this study, we establish a novel molecular detection method for identifying Theileria spp. from deer and tick samples using loop-mediated isothermal amplification (LAMP). This method targets a metalloprotease/cell division cycle protein gene homologue. Our LAMP protocol was able to detect deer Theileria and did not show cross reactivity with other closely related protozoan parasites, including T. orientalis. The LAMP method showed sensitivity and specificity equivalent to those of nested PCR performed on the same field samples from deer and ticks. These results demonstrate the applicability of LAMP to field surveys in which the detection of deer Theileria spp. is required. In conclusion, due to its simplicity, specificity, and reliability, we suggest our LAMP protocol as an appropriate method for routine surveys to detect Yezo sika deer and ticks infected with deer Theileria spp. parasites. Additionally, this LAMP method offers great promise as a useful tool to distinguish Yezo sika deer Theileria from related Theileria parasites present in livestock.

Prevalence and phylogenetic analysis of Cryptosporidium infections in Yezo sika deer (Cervus nippon yesoensis) in the Tokachi sub-prefecture of Hokkaido, Japan.

The Yezo sika deer (Cervus nippon yesoensis) on the island of Hokkaido, Japan are currently recognized as overabundant. Hunting is used to control the deer population, and this has increased the supply of game meat, which is associated with a high risk of various food-borne infections. Additionally, the sub-prefecture Tokachi has a dense population of livestock, which are potentially at risk of cross-species infections from the deer. In this study, we undertook the first analysis of the incidence of Cryptosporidium infection in the Yezo sika deer in the Tokachi area using polymerase chain reaction testing and phylogenetic analysis. Polymerase chain reaction analysis showed Cryptosporidium species present in 7.5% of fecal samples (13/173) collected from deer hunted between 2016 and 2017. However, the zoonotic Cryptosporidium paruvm parasite was not detected in the phylogenetic analysis; when sequenced, all species in the positive samples matched the Cryptosporidium deer genotype. However, deer may act as a reservoir of the zoonotic Cryptosporidium parvum parasite, which affects both humans and livestock. Therefore, we recommend the continuation of surveys of the incidence of Cryptosporidium infections in Yezo sika deer.

Improvement of an in vitro drug selection method for generating transgenic Plasmodium berghei parasites.

BACKGROUND: Reverse genetics approaches have become powerful tools to dissect the biology of malaria parasites. In a previous study, development of an in vitro drug selection method for generating transgenic parasite of Plasmodium berghei was reported. Using this method, two novel and independent selection markers using the P. berghei heat shock protein 70 promoter was previously established. While the approach permits the easy and flexible genetic manipulation of P. berghei, shortcomings include a low variety in promoter options to drive marker gene expression and increased complexity of the selection procedure. In this study, addressing these issues was attempted. METHODS: To secure a variety of promoters, the use of a P. berghei elongation factor-1α promoter for marker gene expression was attempted. To simplify the procedure of in vitro selection, the establishment of a two cell-cycle culture method and its application for drug selection were attempted. RESULTS: The P. berghei elongation factor-1α (pbef-1α) promoter, which is commonly used to drive marker gene expression, was successfully applied as an alternative promoter model for marker gene expression, using the parasite's codon-optimized marker sequence. To simplify the in vitro selection method, a two cell-cycle culture method in which the merozoite was released by filtration of the culture containing matured schizont-infected erythrocytes was also developed and successfully applied for drug selection. CONCLUSION: The pbef-1α promoter was successfully applied in an in vitro selection system. The in vitro selection procedure also could be simplified for practical use using a two cell-cycle culture method. These improvements provide a more versatile platform for the genetic manipulation of P. berghei.

High frequencies of F1534C and V1016I kdr mutations and association with pyrethroid resistance in Aedes aegypti from Somgandé (Ouagadougou), Burkina Faso.

BACKGROUND: Resistance to pyrethroid insecticides involving kdr mutations is widespread in Aedes aegypti (L.) (Diptera: Culicidae) and potentially could impact control efforts in endemic countries. Dengue cases had been sporadic in Burkina Faso for over a decade prior to the 2016-2017 outbreak that resulted in 15,074 suspected cases and 36 deaths, mainly in Ouagadougou. These outbreaks highlighted the lack of information on numerous aspects of the biology, behaviour and insecticide status of local dengue vector populations that are fundamental to vector control. RESULTS: We investigated the insecticide resistance profiles and the kdr mutations involved in pyrethroid resistance of Ae. aegypti from Somgandé, a district of Ouagadougou. WHO bioassays revealed that the local Ae. aegypti populations were highly resistant to pyrethroids with mortalities of 15% for permethrin and 37% for deltamethrin. Resistance to carbamates was also detected with mortalities of 55% for propoxur and 90% for bendiocarb, but high mortalities (> 97%) to organophosphates (malathion and fenitrothion) indicated susceptibility. Allele-specific PCR and voltage-gated sodium channel gene sequencing showed a very high frequency (97%) of the F1534C kdr allele whilst the V1016I kdr mutation frequency was 46%. Association of dual-locus kdr mutations was detected for permethrin resistance. CONCLUSION: We conclude that in this locality of Burkina Faso, Ae. aegypti is resistant to pyrethroid and carbamate insecticides but remains susceptible to organophosphates, providing useful information for possible future control.

Involvement of interferon-tau in the induction of apoptotic, pyroptotic, and autophagic cell death-related signaling pathways in the bovine uterine endometrium during early pregnancy.

Interferon-tau (IFNT), a type I interferon (IFN), is known as pregnancy recognition signaling molecule secreted from the ruminant conceptus during the preimplantation period. Type I IFNs, such as IFN-alpha and IFN-beta, are known to activate cell-death pathways as well as induce apoptosis. In cows, induction of apoptosis with DNA fragmentation is induced by IFNT in cultured bovine endometrial epithelial cells. However, the status of cell-death pathways in the bovine endometrium during the preimplantation period still remains unclear. In the present study, we investigated the different cell-death pathways, including apoptosis, pyroptosis, and autophagy, in uterine tissue obtained from pregnant cows and in vitro cultured endometrial epithelial cells with IFNT stimulation. The expression of CASP7, 8, and FADD (apoptosis-related genes) was significantly higher in pregnant day 18 uterine tissue in comparison to non-pregnant day 18 tissue. The expression of CASP4, 11, and NLRP3 (pyroptosis-related genes) was significantly higher in the pregnant uterus in comparison to non-pregnant uterus. In contrast, autophagy-related genes were not affected by pregnancy. We also investigated the effect of IFNT on the expression of cell-death pathway-related genes, as well as DNA fragmentation in cultured endometrial epithelial cells. Similar to its effects in pregnant uterine tissue, IFNT affected the increase of apoptosis-related (CASP8) and pyroptosis-related genes (CASP11), but did not affect autophagy-related gene expression. IFNT also increased γH2AX-positive cells, which is a marker of DNA fragmentation. These results suggest that apoptosis- and pyroptosis-related genes are induced by IFNT in the pregnant bovine endometrial epithelial cells.

Hot topic: Pregnancy-induced expression of interferon-stimulated genes in the cervical and vaginal mucosal membranes.

In ruminants, IFN-tau (IFNT) is a pregnancy recognition signal secreted by the embryonic trophectoderm before implantation, and it induces the expression of IFN-stimulated genes (ISG) in the uterine endometrium and blood leukocytes. The expression of ISG in blood leukocytes could indicate the presence of a viable conceptus before return of the next estrus; however, expression levels have high variation for confirming pregnancy. We hypothesized that the secreted IFNT in the uterus would affect ISG expression in cervical and vaginal tissues because they are directly adjacent to the uterus. To prove the hypothesis, we investigated the expression of 3 ISG (ISG15, MX1, and MX2) in cervical and vaginal mucosal membranes collected from pregnant (n = 12) and nonpregnant (n = 11) lactating Holstein cows at 17 to 18 d after artificial insemination. Mucosal membrane samples of the cervical canal near the external os (cervix) and deep vaginal wall surrounding the external os (vagina) were collected separately by simply scraping with a curette on d 17 or 18 of pregnancy (d 1 = ovulation), at which time IFNT secretion into the maternal uterus is maximal. After pregnancy diagnosis on d 30 and 60, separately collected samples confirmed as pregnant and nonpregnant were used for evaluation of the expression of IFN-stimulated protein 15 kDa (ISG15) and myxovirus-resistance protein 1 and 2 (MX1, MX2) with quantitative real-time PCR. The collected mucosal membrane samples from cervix contained mostly cell clots showing membrane structure and a low content of blood cells. The expression levels of all 3 genes were significantly increased in pregnant cows compared with nonpregnant cows in both cervical and vaginal samples. These results suggest that increased expression of ISG in the cervix and vagina is a pregnancy-associated phenomenon and is highly affected by IFNT secreted from the conceptus through the uterus.

Activation of lysosomal cathepsins in pregnant bovine leukocytes.

In ruminants, interferon-tau (IFNT)-mediated expression of interferon-stimulated genes in peripheral blood leukocytes (PBLs) can indicate pregnancy. Recently, type 1 IFN-mediated activation of lysosomes and lysosomal cathepsins (CTSs) was observed in immune cells. This study investigated the status of lysosomal CTSs and lysosomes in PBLs collected from pregnant (P) and non-pregnant (NP) dairy cows, and conducted in vitro IFNT stimulation of NP blood leukocytes. Blood samples were collected 0, 7, 14 and 18 days post-artificial insemination, and the peripheral blood mononuclear cells (PBMCs) and polymorphonuclear granulocytes (PMNs) separated. The fluorescent activity of CTSB and CTSK in PMNs significantly increased with the progress of pregnancy, especially on day 18. In vitro supplementation of IFNT significantly increased the activities of CTSB and CTSK in NP PBMCs and PMNs. CTSB expression was significantly higher in PBMCs and PMNs collected from P day-18 cows than from NP cows, whereas there was no difference in CTSK expression. IFNT increased CTSB expression but did not affect CTSK expression. Immunodetection showed an increase of CTSB in P day-18 PBMCs and PMNs. In vitro stimulation of IFNT increased CTSB in NP PBMCs and PMNs. Lysosomal acidification showed a significant increase in P day-18 PBMCs and PMNs. IFNT also stimulated lysosomal acidification. Expressions of lysosome-associated membrane protein (LAMP) 1 and LAMP2 were significantly higher in P day-18 PBMCs and PMNs. The results suggest that pregnancy-specific activation of lysosomal functions by CTS activation in blood leukocytes is highly associated with IFNT during maternal and fetal recognition of pregnancy.

Estrous cycle stage-dependent manner of type I interferon-stimulated genes induction in the bovine endometrium.

Interferon tau (IFN-τ) is a ruminant-specific type I IFN secreted by a conceptus before its attachment to the uterus. IFN-τ induces the expression of IFN-stimulated genes (ISGs) via the type I IFN receptor (IFNAR), which is composed of IFNAR1 and IFNAR2 subunits in the endometrium. However, expression patterns of IFNARs during the estrous cycle have not been reported. We hypothesized that the response to a type I IFN changes along with IFNARs and the IFN-regulatory factors (IRFs) driving transcription of IFN signal-related genes and modulating a type I IFN signal during the estrous cycle. We investigated the estrous cycle stage-dependent type I IFN induction of ISGs and expression patterns of IFN signal-related genes in bovine endometrial tissues. Endometrial tissue pieces collected from bovine uteri at each estrous stage (early, mid, and late) were cultured with or without recombinant bovine IFN-α or concentrated pregnant uterine flushing (PUF) on day 18 after confirming the presence of a conceptus. IFN-α and PUF each significantly increased the expression of ISGs in endometrial tissues. The induction levels of the typical ISGs (MX1-a and ISG15) were significantly higher at the mid stage and correlated with high expression of IRFs at the mid stage. The immunostaining of IFNARs showed strong fluorescence intensities in luminal and glandular epithelia at the early and mid stages. Collectively, these results suggest that the endometrium exhibits estrous cycle stage-dependent responsiveness to type I IFN that may be associated with the expression of IFNARs and IRFs for pregnancy recognition.

Expression dynamics of bovine MX genes in the endometrium and placenta during early to mid pregnancy.

MX belongs to a family of type I interferon (IFN)-stimulated genes, and the MX protein has antiviral activity. MX has at least two isoforms, known as MX1 and MX2, in mammals. Moreover, bovine MX1 has been found to have alternative splice variants-namely, MX1-a and MX1B. In ruminants, IFN-τ-a type I IFN-is temporarily produced from the conceptus before implantation and induces MX expression in the endometrium. However, the expression dynamics of MX after implantation are not clear. In the present study, we investigated the expression of MX1-a, MX1B and MX2 in the endometrium and placenta before and after implantation along with the expression of IFN-α, type I receptors (IFNAR1 and IFNAR2) and interferon regulatory factors (IRF3 and IRF9). Pregnant uterine samples were divided into five groups according to pregnancy days 14-18, 25-40, 50-70, 80-100, and 130-150. Tissue samples were collected from the intercaruncular endometrium (IC), caruncular endometrium (C) and fetal placenta (P). Although all the MX expressions were significantly higher in the IC and C at days 14-18, presumably caused by embryo-secreted IFN-τ stimulation, their expressions were also detectable in the IC, C and P after implantation. Furthermore, IFN-α expression was significantly higher in the IC. RT-PCR indicated IFNAR1, IFNAR2, IRF3 and IRF9 mRNA in all the tissues during pregnancy. These results suggest that all the MX genes are affected by the type I IFN pathway during pregnancy and are involved in an immune response to protect the mother and fetus.