Toxocara tanuki larval distribution in mice and the infectivity of tissue larvae.

Toxocara tanuki is a common large roundworm in raccoon dogs. Experimental infection studies of T. tanuki in mice were conducted to clarify the distribution and infectivity of larvae in tissue. Groups of BALB/c and C57BL/6 mice (n = 5 mice/group) were each inoculated with 1000 embryonated T. tanuki eggs and necropsied at 7, 31, 91, and 182 days post inoculation (dpi). The number of larvae in the central nervous system, heart, lungs, kidneys, spleen, gastrointestinal tract, liver, and carcass was examined. Larvae obtained from the aforementioned mice on different days of the necropsy were orally inoculated into four groups of ICR mice (n = 6 mice/group) that were then necropsied at 21 dpi. Larvae were recovered from all mice. In the BALB/c and C57BL/6 mice, most of the larvae (> 88.7%) were recovered from the liver and the remainder from other tissues. The total number of larvae recovered from C57BL/6 mice was significantly higher than that from BALB/c mice, but no difference in the relative larval distribution within the viscera between the two mouse strains was observed. The mean recovery percentage of larvae from ICR mice infected with 182-day-old tissue larvae was 3.3%. Our findings showed that T. tanuki larvae migrated predominantly to the liver of mice and that the larvae maintained their infectivity for at least half a year.

Trichinella pseudospiralis infection in red-eared slider, Trachemys scripta, influenced by environmental temperature.

Experimental infection with Trichinella pseudospiralis larvae in red-eared slider Trachemys scripta was carried out. Ten turtles were divided into 3 groups and kept at different temperature (38, 32 and 28 °C). The turtles were fed mice muscles which were infected with T. pseudospiralis larvae (444 - 23,013 larvae/mouse), kept in the aforementioned temperature and necropsied at day 30 after infection. At necropsy, the tongue, forelimbs, hindlimbs and spinal column muscles of turtles were removed separately for pepsin digestion, and the larvae counted. Larvae were found from all 3 turtles kept at 38 °C. Larvae per gram of muscle were highest in the tongue. No larvae were recovered from turtles kept at 32 and 28 °C. The result suggested that T. pseudospiralis is able to infect the red-eared slider in a high temperature environment. We speculated that environmental temperature play an important role in altering the physiological condition of the turtle to facilitate the infection of T. pseudospiralis.

Detection of larvae of Toxocara cati and T. tanuki from the muscles of free-ranging layer farm chickens.

Although raw or undercooked livestock meat or viscera has been suggested to be a source of human toxocariasis, there have been few reports on the prevalence of Toxocara larvae in the tissue of livestock animals. To investigate the presence of Toxocara larvae in chickens, we examined 50 culled chickens from a commercial layer farm. The liver, breast meat, and thigh meat were separated individually and artificially digested to examine for the presence of larvae. Nematode larvae were detected in 2 out of 50 chickens. One larva was detected from the breast meat, and it was molecularly identified as Toxocara tanuki. The other from the thigh meat of another chicken was molecularly identified as Toxocara cati. The present study demonstrated for the first time that T. tanuki larvae do infect chickens in the natural environment. The fact that Toxocara spp. larvae were found in muscles of farm chickens suggests that consumption of raw or undercooked chicken meat may present a risk for human toxocariasis.

Toxocara cati larval migration to mouse fetuses through transplacental infection.

To examine the Toxocara cati larval migration in mouse fetuses through vertical transmission, 7 pregnant mice were orally inoculated with 5000 embryonated eggs at day 4 and again with the same dose at day 9 of gestation (total 10,000 eggs / mouse). Seven non-pregnant mice of the same age were also inoculated likewise. All mice were necropsied 12 days after the first inoculation with eggs. The carcass, viscera, uterus, placenta and fetuses were individually removed and digested with pepsin for larval counts. Larvae were recovered from all pregnant and non-pregnant mice. The majority of the larvae were recovered from the carcass, and some from the viscera, uterus, placenta and fetuses. There were no differences between the pregnant and non-pregnant mice for the number of larvae in the carcass, viscera and uterus. Of a total of 103 fetuses from the 7 pregnant mice, larvae were recovered from 74 fetuses (71.8 %). The mean number of larvae recovered per infected fetus was 2.6 (n = 103), and the maximum number of larvae recovered in a fetus was 9. This study showed that most mouse fetuses were infected with T. cati larvae through intrauterine transmission, although the number of larvae was low. Since no significant difference in the number of larvae recovered from the pregnant and non-pregnant mice was observed, it is suggested that pregnancy does not directly affect the vertical transmission of T. cati larvae.

Prevalence of Blatticola blattae (Thelastomatidae) in German cockroaches Blattella germanica in Japan.

A survey for nematode infection in German cockroaches captured in restaurants in various areas of Japan as well as the laboratory-bred colony was carried out. The nematodes were then identified morphologically and molecularly. Of the 320 German cockroaches collected at 79 restaurants in 26 prefectures in Japan, 66.6% (213/320) were found to be parasitized by a single species of pinworm in the hindgut. The mean number of pinworms per cockroach was less than 1.6. Of the three laboratory-bred lines of cockroaches examined, 2 lines (NIID and NK) were found to be infected with a single species of nematode. The prevalence was 93.0% (40/43) and 84.8% (39/46), respectively. The other laboratory line (WAT) was found to be free of the nematode infection. The nematode detected in this study was identified as Blatticola blattae. This is the first report of B. blattae infection in German cockroaches in Japan. Our study showed that B. blattae is distributed all over Japan together with its host Blattella germanica. An experimental infection with B. blattae in nematode-free cockroach by contaminating the rearing environment with infected-cockroach feces showed that once the environment of the cockroach is contaminated with B. blattae eggs, the pinworm infection could spread easily.

Possible mechanisms of action of clarithromycin and its clinical application as a repurposing drug for treating multiple myeloma.

Clarithromycin (CAM), a semisynthetic macrolide antibiotic, is a widely used antibacterial drug. Recently, the efficacy of CAM as an add-on drug for treating multiple myeloma (MM) has been noted. Its effect on treating MM has been confirmed in combination chemotherapies that include CAM. However, a single treatment of CAM has no efficacy for treating MM. Many myeloma growth factors (MGFs) including interleukin (IL)-6 are known to be closely involved in the development of MM. CAM has been shown to suppress many MGFs, particularly IL-6. The possible mechanisms of action of CAM in treating MM have been suggested to include its immunomodulatory effect, autophagy inhibition, reversibility of drug resistance, steroid-sparing/enhancing effect and suppression of MGFs. In addition, MM is characterised by uncontrolled cell growth of monoclonal immunoglobulin (Ig)-producing neoplastic plasma cells. Large quantities of unfolded or misfolded Ig production may trigger considerable endoplasmic reticulum stress. Thus, MM is originally a fragile neoplasm particularly susceptible to autophagy-, proteasome- and histone deacetylase 6-inhibitors. Taken together, CAM plays an important role in MM treatments through its synergistic mechanisms. In addition, CAM with its pleiotropic effects on cytokines including IL-6 and indirect antiviral effects might be worth a try for treating COVID-19.

Effects of anthelmintics on the pinworm Blatticola blattae in laboratory-reared German cockroaches Blattella germanica.

The efficacy of pyrantel pamoate, pyrvinium pamoate, ivermectin, and piperazine citrate against pinworm in cockroach was evaluated. Laboratory-reared German cockroaches naturally infected with Blatticola blattae were treated with the anthelmintics and necropsied at 3 to 35 days after treatment. Ivermectin at over 5 ppm and piperazine citrate at over 2000 ppm killed all the treated cockroaches. Pinworms were still detected in cockroaches given lower concentration of the aforementioned drugs. Administration of pyrantel pamoate (100-1000 ppm) and pyrvinium pamoate (2000 ppm) did not kill the cockroaches, and no pinworms were detected at 3 and 17 days after treatment. Thus, pyrantel pamoate and pyrvinium pamoate were found to be effective for deworming B. blattae in the German cockroaches, without causing mortality for the host. Our results showed that anthelmintics selection is essential for eradication of pinworms in cockroaches because of the toxicity for the host such as ivermectin or piperazine citrate. This is the first report of piperazine citrate toxicity in cockroaches.

The role of the American cockroach (Periplaneta americana) as transport host of Eimeria tenella to chickens.

The role of the American cockroach, Periplaneta americana as transport host for Eimeria tenella was evaluated. Twenty-four cockroaches were orally fed with sporulated oocysts of E. tenella. Their feces and digestive tract were examined for oocysts by sugar centrifugal flotation technique and PCR. Infectivity of the oocysts recovered from the digestive tract of infected cockroaches as well as from their feces was evaluated by orally inoculating them into Boris Brown chickens. E. tenella oocysts were found in the digestive tract and feces of infected cockroaches up to day 4 after ingestion of oocysts. Furthermore, oocysts that were recovered from the digestive tract and feces of cockroaches remained infective for 4 and 3 days after ingestion of oocysts, respectively. Presence of oocysts in the feces of chicken that had been inoculated with either digestive tract or feces of P. americana demonstrated the infectivity of E. tenella oocysts from digestive tract or feces, suggesting that P. americana may play a role in the transmission of E. tenella among chicken and between chicken flocks.

Eimeria tenella oocyst excretion and riboflavin supplement in infected chicken.

The effect of riboflavin supplement in Eimeria tenella-infected chickens was evaluated. Mortality, fecal consistency, and oocysts per gram of feces were monitored for groups of E. tenella-infected chickens administered a basal diet supplemented with either riboflavin, the anti-coccidial drug amprolium, or with both compounds. The number of oocysts shed per chicken in the riboflavin-treated group was significantly higher than the positive non-treated control group. No significant difference in oocyst number between the amprolium-treated group and riboflavin plus amprolium-treated group was observed. Thus, the addition of 0.8 g/kg of riboflavin to basal diet can increase oocyst number in E. tenella-infected chicken, but has no effect on the efficacy of amprolium.

Dynamics of liver enzymes in rabbits experimentally infected with Fasciola sp. (Intermediate form from Japan).

Dynamics of serum liver enzymes in rabbits experimentally infected with metacercariae of Fasciola sp. (intermediate form between Fasciola hepatica and F. gigantica) were monitored. Gradual increase of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were observed from 3 weeks post-inoculation (WPI) and peaked at 6 WPI, which corresponded well to the period of migration and development of juvenile fluke in the liver parenchyma and the time when the young adult flukes migrated to the bile duct. However, no significant increase in serum gamma-glutamyl transferase (GGT) and alkaline phosphatase (ALP) were observed. This could reflect reduced or minimal injury of bile ducts and biliary epithelia as the flukes had reached the adult stage. Alpha- fetoprotein (AFP) and carcinoembryonic antigen (CEA) were not detected in the infected rabbit during the course of the experiment. Serum liver enzymes monitoring might be useful for understanding the host-parasite relationship in fascioliasis.

Invalidation of Diphyllobothrium hottai (Cestoda: Diphyllobothriidae) based on morphological and molecular phylogenetic analyses.

Diphyllobothrium hottai Yazaki, Fukumoto & Abe, 1988 was described based on the morphology of adult worms recovered from golden hamsters that had been experimentally infected with plerocercoids obtained from Japanese surf smelts (Hypomesus pretiosus japonicus) and olive rainbow smelts (Osmerus eperlanus mordax). Although D. hottai was considered to be distinct from Diphyllobothrium ditremum (Creplin, 1825), their taxonomic relationship requires further clarification. In our study, D. hottai and D. ditremum obtained from hamsters experimentally infected with plerocercoids isolated from Japanese surf smelts were compared using morphological and molecular methods. The criterion usually used to differentiate between D. hottai and D. ditremum is the difference in the angle between the long axis of the cirrus sac and that of the seminal vesicle. However, we found variation of the angle within the same individual and, one specimen showed both of the different angles that were supposedly unique to each of the species. Furthermore, phylogenetic analysis of the complete sequences of the mitochondrial cytochrome c oxidase subunit 1 and cytochrome b genes revealed that both species were genetically indistinguishable. Therefore, D. hottai is considered to be a junior synonym of D. ditremum.

Molecular identification of Heterakis spumosa obtained from brown rats (Rattus norvegicus) in Japan and its infectivity in experimental mice.

Heterakis spumosa is a nematode of invasive rodents, mainly affiliated with Rattus spp. of Asian origin. Despite the ecological importance and cosmopolitan distribution, little information is available on the genetic characteristics and infectivity to experimental animals of this roundworm. Heterakis isolates obtained from naturally infected brown rats caught in 2007 in the city of Sagamihara, east central Honshu, Japan, and maintained by laboratory passages were subjected to mitochondrial sequence analysis and experimental infection in mice. Sequencing of the cox1 gene revealed that nucleotides of H. spumosa and previously examined Heterakis isolonche isolates from gallinaceous birds in Japan differed by 11.2-12.2% that conforms to the range expected for interspecific differences. The two H. spumosa isolates differed by a single 138T/C non-synonymous substitution in the 393-bp mt sequence. In a dendrogram, the H. spumosa samples formed a subcluster with members of the nematode superfamily Heterakoidea, H. isolonche and Ascaridia galli. In an experimental infection study, ICR, AKR, B10.BR and C57BL/6 mice strains were inoculated with 200 H. spumosa eggs/head and necropsied at 14 and 90 days post-inoculation (DPI) when the number of worms was recorded. Eggs were initially detected in faeces from 32-35 DPI in ICR, AKR and B10.BR mice and the highest mean number of eggs per gram of faeces (EPG) was 4,800 at 38 DPI, 2,200 at 58 DPI and 800 at 44 and 72 DPI in ICR, AKR and B10.BR mice, respectively. No eggs were observed in faeces of the C57BL/6 mouse strain during the experiment. A similar number of juvenile worms were isolated from all mouse strains at 14 DPI, whereas no adult worms were detected in C57BL/6 mice at 90 DPI.

Morphogenetic study on the maturation of osteoblastic cell as induced by inorganic polyphosphate.

Since inorganic polyphosphates [poly(P)] have an activity to induce bone differenciation in vitro and in vivo, we examined an effect of poly(P) on organelle by light microscopy and electron microscopy in Murine MC3T3-E1 osteoblastic cells. The MC3T3-E1 cells were ultrastructurally observed to possess morphological characteristics of osteoblasts. Cells cultured with poly(P) were strongly stained with an anti-collagen type I antibody but not in those cultured without poly(P). Ultrastructural analysis of cells cultured with poly(P) revealed a well-developed Golgi apparatus, swollen and elongated rough endoplasmic reticulum, large mitochondria and many coated pits. Since MC3T3-E1 cells can be transformed from a resting phase to an active blastic cell phase after supplementation with poly(P), it implies that poly(P) can be an effective material for bone regeneration.

Sexual differentiation and developmental stage identification of the Indian cockroach, Pycnoscelus indicus (Blattodea: Blaberidae).

We found that sexual differentiation of all the nymphal stages of Pycnoscelus indicus (Fabricius) was possible by observing the developmental features of their posterior abdominal segments. Using this observation, the sex of even the 1(st) stage instar nymph could be determined. The female of the 1(st) to 6(th) stage instar nymph possess a V-shaped notch at the middle of the posterior edge of the 9(th) sternite. This notch is not seen in the male nymph. In the female 7(th) stage (final stage) instar nymph, the styli were not apparent and, the 8(th) and 9(th) sternites became degenerated and were covered over by the profoundly developed 7(th) sternite. In contrast, all stages of the male nymph showed the presence of styli. Thus, it is possible to differentiate the sex of all the stages, from 1(st) to 7(th), of the nymph of P. indicus taxonomically. Moreover, it is also possible to identify the various specimens as to which stage the nymphal instar belong to, by counting the number of cercal segments from the ventral view.

Cockroach fauna in the Ogasawara Chain Islands of Japan and analysis of their habitats.

A survey of cockroach fauna was carried out on the 3 inhabited islands of the Ogasawara chain island of Japan, namely, Chichijima island, Hahajima island and Iwo island. Seven species, namely, Periplaneta americana (Linnaeus, 1758), Periplaneta australasiae (Fabricius, 1775), Blattella lituricollis (Walker, 1868), Onychostylus vilis (Brunner von Wattenwyl, 1865), Supella longipalpa (Fabricius, 1798), Pycnoscelus surinamensis (Linnaeus, 1758) and Opisthoplatia orientalis (Burmeister, 1838), were collected on Chichijima island. Four species, namely, P. americana, P. australasiae, O. vilis and P. surinamensis were collected on Hahajima island and 6 species, namely, P. americana, P. australasiae, B. lituricollis, O. vilis, P. surinamensis and Neostylopyga rhombifolia were collected on Iwo island. This is the first record of N. rhombifolia and Onychostylus orientalis on the Ogasawara chain islands. Our study increases the recorded taxon of cockroaches on the Ogasawara from 3 families, 5 genera 10 species to 4 families, 7 genera, 12 species. A list of the cockroach species on Ogasawara islands reported to date as well as a key for their identification is also presented. Periplaneta americana and P. australasiae, being the dominant species, together with S. longipalpa, were collected mostly in the indoor environment, indicating their preference for this habitat. Pycnoscelus surinamensis, which is considered as an outdoor insect has been found in semi-household environments such as greenhouse and shed, indicating their new adaptation to the changing environment.

Historical overview of Taenia asiatica in Taiwan.

An overview of the epidemiological, biological, and clinical studies of Taenia and taeniasis in Taiwan for the past century is presented. The phenomenal observations that led to the discovery of Taenia asiatica as a new species, which differ from Taenia solium and Taenia saginata, are described. Parasitological surveys of the aborigines in Taiwan revealed a high prevalence of taeniasis, which might be due to the culture of eating raw liver of hunted wild boars. Chemotherapeutic deworming trials involving many patients with taeniasis were discussed. Praziquantel was found to be very effective, but sometimes complete worms could not be recovered from the feces after treatment, probably due to the dissolution of the proglottids. Atabrine, despite some side effects, can still be used, in properly controlled dosages, as the drug of choice for human T. asiatica infection if we need to recover the expelled worms for morphological examinations. Research results on the infection of T. asiatica eggs from Taiwan aborigines in experimental animals were also noted. Since the pig serve as the natural intermediate host of T. asiatica and the predilection site is the liver, a differential comparison of other parasitic pathogens that might cause apparently similar lesions is also presented.

Metagonimus yokogawai: metacercariae survey in fishes and its development to adult worms in various rodents.

A parasitological survey for Metagonimus yokogawai metacercariae was carried out by examining a total of 321 freshwater fish comprising of 7 species. Of the 321 fish samples examined, 182 (56.7%) were found to be infected with M. yokogawai metacercariae. The prevalence of M. yokogawai metacercariae in Opsariichthys pachycephalus was 93.4% (86/92), Zacco platypus 75.0% (30/40), Distoechodon turmirostris 61.3% (38/62), Varicorhinus barbatulus 56.5% (13/23), Hemibarbus labeo 33.3% (1/3), Acrossocheilus formosanus 15.9% (14/88), and 0% in Sinibrama macrops (0/13), respectively. This is the first record of M. yokogawai infection in Z. platypus, D. turmirostris, V. barbatulus, and H. labeo in Taiwan. The major site of predilection of the metacercariae in the fishes was in the scale, but some metacercariae were also observed in the flesh and fins. The M. yokogawai metacercariae were orally inoculated into mice, rat, gerbil, and golden hamster to study their infectivity and also to obtain the adult worms for taxonomic study. Worm recovery in hamsters was 75.3%, in mice was 70.0%, in rats was 23.3%, and in gerbils was 6.0%, respectively. Moreover, larger worms were recovered from the golden hamster. Golden hamster was thus found to be the most susceptible experimental rodent host for the infectivity study of Metagonimus. Besides M. yokogawai, metacercariae of Centrocestus formosanus was also observed in the fishes examined.

Molecular identification of Anisakis type I larvae isolated from hairtail fish off the coasts of Taiwan and Japan.

Anisakid nematodes are known to cause the zoonotic disease, anisakiasis, through the consumption of raw or undercooked fish. The parasites most frequently associated with the disease in humans are categorized as Anisakis type I, which comprise several species of the genus Anisakis. The larvae show primitive forms and lack the detailed morphological characteristics required for precise species identification. Thus, molecular characterization is necessary for determining the species of Anisakis type I larvae and acquiring important clinical and epidemiological information. In this study, we isolated Anisakis type I larvae from hairtail fish caught off the coasts of Taiwan and Japan. The ribosomal DNA (rDNA) internal transcribed spacer (ITS) region was sequenced, and restriction fragment length polymorphism (RFLP) analyses using HinfI and HhaI was carried out for species identification. Most larvae isolated from hairtail caught in Taiwan were Anisakis typica (84%), while those isolated from hairtail caught in Japan were almost exclusively identified either as Anisakis simplex sensu stricto (65%) or Anisakis pegreffii (33%). This is the first report of A. typica in fish obtained from Taiwan. Our results shed the light on the epidemiology of Anisakis type I larvae, which is a potential cause of human anisakiasis in Taiwan and Japan.

Infectivity and development of Metagonimus yokogawai in experimentally infected domestic ducks (Cairina moschata).

Information concerning whether fowl such as duck is a suitable reservoir host of Metagonimus yokogawai is largely unclear to date. In the present study, the growth and development of M. yokogawai metacercaria (Mc) in domestic duck (Cairina moschata) was determined by worm recovery rate (WRR) and morphological changes e.g., the size of fluke's body as well as their internal organs was assessed by using Semichon's acetocarmine staining. Each duck was orally inoculated with 50 Mcs of M. yokogawai and infected ducks were deeply anesthetized with ether and killed at 1, 2, 3, 4, 5, 6, 7, and 14 days post-infection (DPI). On each date, two infected ducks were killed and the small intestines of each duck were separated into four parts then they were opened longitudinally to harvest the flukes. Results revealed that WRR of M. yokogawai from inoculated ducks increased during early infection with a peak as seen at 4 DPI (28.5+/-6.9%); thereafter it gradually decreased and a drastic decline was observed in 14 DPI (2.0+/-1.1%) in the trial. The preference sites for M. yokogawai were low portions of the small intestine; nevertheless the size of fluke's body and organs developed increasingly with time and they maturated to produce ova from 4 DPI onward in the trial. However, present results indicated that ducks, based on the findings of this study, are not suitable hosts for establishment of M. yokogawai infection because most flukes were expelled from duck's intestine within 14 days. Nevertheless, it was proposed that ducks might play a certain role in transmitting M. yokogawai when they deposited the ova via feces into marsh where snails and fish were abundant since they could presumably establish transient and possibly patent infections with this parasite.

Fecal occult blood manifestation of intestinal Eimeria spp. infection in rabbit.

The relationship between oocysts production and fecal occult blood manifestation was studied in rabbits experimentally infected with a single species of Eimeria. Pure single Eimeria species infection in non-infected rabbits for 6 species of Eimeria, namely: Eimeria media, Eimeria magna, Eimeria perforans, Eimeria coecicola, Eimeria piriformis, and Eimeria exigua were produced using a single oocyst. Fecal samples from these single Eimeria species infected rabbits were collected daily from day 1 through day 23 post-infection (DPI) to observe for oocysts shedding and also to test for presence of fecal occult blood (FOB). Rabbits infected with E. perforans were positive for FOB test (FOBT) reaction when their OPG exceeded 2.2x105 at 11 and 12 DPI. FOBT positive reactions were also seen in naturally infected rabbits with concurrent infections, of E. perforans and E. media as well as of E. perforans and E. magna. Thus, E. perforans was observed to cause intestinal hemorrhage during the period of massive oocyst shedding. This phenomenon was not seen in the other Eimeria species that we had examined. This is also the first report on the association of E. perforans infection with intestinal hemorrhage in rabbit.