Oral transmission of Brugia pahangi and Dipetalonema viteae to adult and neonatal jirds

Sullivan J. J. and Chernin E. 1976. Oral transmission of Brugia pahangi and Dipetalonema viteae to adult and neonatal jirds. International Journal for Parasitology6: 75–78. Confirming previous studies, anaesthetized adult jirds became infected after oral doses of 100 infective larvae of B. pahangi; 4 of 5 jirds became microfilaria-positive and all 5 harbored adult worms. Among 7 unanaesthetized adult jirds similarly exposed, none developed microfilaraemia although 5 each harbored a few adult worms. In these unanaesthetized jirds, presumably, rapid passage of the inoculum through the mouth permitted fewer larvae to penetrate the mucosa, and the rest were probably killed in the stomach. Unanaesthetized 4-day old jirds proved highly and equally susceptible to oral or subcutaneous infection with B. pahangi as indicated by microfilaraemias and large worm-burdens. Direct and indirect evidence suggest that the baby's stomach and small intestine are not inimical to swallowed larvae, thus accounting for the relatively numerous mature worms in the peritoneal cavity. Third-stage larvae of D. viteae, readily infective subcutaneously, succeeded relatively infrequently in maturing when given orally to anaesthetized adult or to unanaesthetized baby jirds. Consistent oral infectivity may thus be a feature of filariae more closely related to B. pahangi.     

Comparative susceptibility to anthelmintics of Brugia pahangi in jirds infected by different methods

It is possible to infect jirds with Brugia pahangi by three methods. Infective larvae (L3) can be injected either intraperitoneally (ip), when adults develop in the peritoneal cavity, or sub-cutaneously (sc), when they develop in the lymphatics or the heart and blood vessels associated with the lungs. Alternatively adult worms which have been grown in the peritoneal cavities of jirds can be implanted into the peritoneal cavities of other jirds. This latter system has been widely used for screening for new filaricides. We have compared the activity of 9 macrofilaricidal compounds against these 3 types of infection. Mebendazole and albendazole were more active against implanted adults than against L3 induced adults in the peritoneal cavity. Oxibendazole, flubendazole, CGP24588A and oxfendazole were equally active against both types of worm. CGP20376, Mel Ga and Mel Ni were more active against adult worms derived from inoculated L3 than implanted worms. When comparing intra-lymphatic and ip adults (both derived from L3 infections and in the same jirds) albendazole and CGP20376 were active at the same levels against both types of infection. Mebendazole, flubendazole, oxfendazole, CGP24588A, Mel Ga and Mel Ni were more active against ip adults than intra-lymphatic adults. No drug was more active against intra-lymphatic adults than against adults.     

The anthelmintic activity of a novel organic arsenical, R7/45, upon Brugia pahangi in Meriones unguiculatus

The new organic arsenical R7/45 is a rapidly acting and very potent anthelmintic against adult Brugia pahangi in jirds. Against adult worms implanted into the peritoneal cavity 5 subcutaneous (SC) injections at 2.5 mg/kg of R7/45 killed 100% of adult worms. A single dose SC of 20 mg/kg was 100% effective and 10 mg/kg 76% effective against adult worms. When jirds were autopsied at different times after treatment at 20 mg/kg SC 89% of worms were dead within three days. R7/45 was not active when given by stomach intubation. Pretreatment of jirds with R7/45 had no effect on adult worms subsequently implanted into jirds. R7/45 was highly active against third and fourth stage larvae of B. pahangi in jirds.     

Studies with Brugia pahangi. I. Parasitological observations on primary infections of cats (Felis catus)

The large majority of cats given a single inoculation of third stage larvae of Brugia pahangi became microfilaraemic. Some cats had microfilariae in their blood 53 or 54 days after infection and most had become positive before 72 days after infection. In the majority of cats microfilarial counts remained very steady between 2 and 10 microfilariae per mm³ for long periods. At autopsy 10·7% of the infective larvae injected were recovered as adult worms. The recovery of adult worms was directly related to the number of larvae injected. The microfilarial level did not increase significantly with an increase in the number of adult worms.     

Brugia pahangi: comparative susceptibility of the Mongolian jird, Meriones unguiculatus, and the PD4 inbred hamster, Mesocricetus auratus

The susceptibility of Mongolian jirds, Meriones unguiculatus, and PD4 hamsters, Mesocricetus auratus, to Brugia pahangi was compared based on the percentage adult worm recoveries, mean microfilaremia levels, and adult worm lengths. Fourteen male jirds and seventeen male PD4 hamsters were each inoculated subcutaneously in the left inguinal region with 90-100 L3 of B. pahangi and necropsied 130-150 days after inoculation. There were no significant differences between jirds and hamsters in mean adult worm recoveries (24.7 vs 25.4%) and prepatent periods (69.9 vs 77 days after inoculation). In hamsters, 85% of recovered worms were found in the heart and lungs and 15% were found in genital lymphatic vessels. In jirds, distribution of recovered worms was 66% in genital lymphatics, 23% in the heart and lungs, 8% in the peritoneal cavity, and 3% in lymphatic vessels in other sites. The mean microfilaremia level in jirds (16.5/20 microliter) was significantly higher than in hamsters (8.7/20 microliter. Female worms in the genital lymphatics of jirds were significantly longer than female worms in the genital lymphatics of PD4 hamsters (33.5 vs 27.3 mm). Lengths of worms in other locations were similar between the two species.     

Brugia pahangi: susceptibility and macroscopic pathology of golden hamster.

Twenty male hamsters were inoculated with 95 to 150 infective larvae of B. pahangi via the subcutaneous route. Worms recovered from 19 hamsters averaged 14% (0–32) from 11 hamsters killed at 105–195 days after infection and 16% (5–19) from 8 hamsters examined at 23–45 days after infection. Approximately one-half of the worms recovered were from the lymphatic vessels of the testes, epididymis, and spermatic cord. A few were found in afferent or efferent vessels of regional lymph nodes. The remaining worms were from the heart and lungs. Low-level microfilaremias were observed in 10 of 12 hamsters held for over 100 days. The average prepatent period was 89 days (65–128). Worms were recovered for up to 3 weeks following inoculation of nine hamsters via the intraperitoneal route with 100–400 infective larvae of B. pahangi.Gross lymphatic pathologic lesions consisted of moderate to marked dilation of lymphatic vessels, enlargement of regional lymph nodes, and numerous lymphthrombi and emboli. Macroscopic changes were most consistent and severe in the lymphatic vessels of the testes, epididymis, and spermatic cord and were noted less frequently in the afferent or efferent vessels of various regional lymph nodes. Areas of reddish discoloration were observed frequently on the serosal surface of the lung in infected hamsters.     

Brugia pahangi: Immunization with early L3 ES alters parasite migration,and reduces microfilaremia and lymphatic lesion formation in gerbils (Meriones unguiculatus)

Previous studies have shown that intradermally (ID) injected Brugia pahangi L3s migrate through various tissues and into the lymphatics of gerbils in a distinct pattern. Excretory/secretory products (ES) produced at the time of invasion of B. pahangi are likely to be important in this early migration phase of the parasite life cycle in their rodent host. Hence, early L3 ES was collected from 24 h in vitro cultures of B. pahangi L3 larvae and used in immunization experiments to investigate the effect of immunity to early L3 ES on worm migration, survival and development of B. pahangi. Immunization of gerbils with ES in RIBI adjuvant produced antibodies to numerous ES proteins eliciting a strong humoral response to ES and indirect fluorescent antibody (IFA) assay using anti-ES serum recognized the ES proteins on the surface of B. pahangi L3 larvae. Following ES immunization, gerbils were challenged either ID or intraperitoneally (IP) with 100 L3s of B. pahangi and euthanized at 3 or 106 days post inoculation (DPI). Immunization with early ES slowed the migration of ID inoculated L3 at 3 DPI and significantly altered the locations of adult worms at 106 DPI. Immunization did not induce protection in any treatment group. However, immunized animals had significantly fewer microfilariae per female worm suggesting the antigens in ES are important in microfilariae development or survival in the host. The number of lymphatic granulomas was also significantly reduced in ES immunized animals. It is important to note that microfilariae serve as a nidus in these granulomas. Our results shows immunization with early Brugia malayi L3 ES alters the worm migration, affects circulating microfilarial numbers and reduces lymphatic granulomas associated with B. pahangi infection in gerbils.     

Litomosoides carinii: retardation of worm growth and of migration of challenge infections in jirds (Meriones unguiculatus)

Inbred jirds (Meriones unguiculatus) were divided into three groups; each animal in two of the groups was infected with 30 infective larvae (L3) of Litomosoides carinii. When these infections were patent, the jirds of one of the two infected groups plus those of the third group were injected with 30 L3 L. carinii each. All animals were killed either on day 14 or 24 after the second infection for the recovery, enumeration and measurement of all worms and developing larvae. Challenge larvae were stunted (smaller) and fewer than control larvae. Additionally, fewer challenge larva were recovered on day 14 than on day 24, indicating that migration to the pleural cavity was retarded.     

Brugia pahangi: effects of duration of infection and parasite burden on lymphatic lesion severity, granulomatous hypersensitivity, and immune responses in jirds (Meriones unguiculatus)

The effects of Brugia pahangi infection duration and parasite burden on parasite-associated inflammatory and immune responses were determined over a 181-day period in jirds receiving from one to eight inoculations of infective larvae. Multiple infections did not produce a protective resistance to reinfection as determined by adult worm recovery at necropsy. Intralymphatic granulomatous lesions, lymph thrombi, were first seen at 48 days post initial inoculation (DPI). The numbers of lymph thrombi reached peak levels in singly inoculated jirds at 90 DPI and significantly decreased to low levels by 160 DPI. The ratio of lymph thrombi to adult worms recovered from the spermatic cord lymphatics followed a similar pattern. Sizes of renal lymph nodes, which drain lymphatics containing parasites, followed a temporal pattern of increase and decrease similar to that of lymph thrombi numbers. Peak granuloma areas around antigen-coated beads embolized in lungs were seen at 27 DPI. Granuloma areas around antigen-coated beads began to decrease after 69 DPI and reached sizes not significantly different from uninfected controls by 118 DPI. Multiple inoculations of infective larvae and increasing worm burdens did not affect the pattern of granulomatous response to antigen-coated beads. Eosinophilia of singly and multiply infected jirds peaked at 26 DPI. Eosinophilia of singly infected jirds returned to normal levels by 103 DPI but those of multiply infected jirds remained elevated until 160 DPI. Lymph node cell blastogenic responses to antigen were greater than those of splenocytes at all time intervals measured. However, significant differences in stimulation indexes between groups with different infection durations were not seen with either cell type. Antibody responses to somatic adult worm antigen as measured by ELISA reached near peak levels by 48 DPI and remained elevated for the course of the study in all infected jirds. The decrease in lymphatic lesion severity seen in chronically infected jirds temporally corresponds to the decrease in granulomatous reactivity measured around antigen-coated beads embolized in the lungs. This observation suggests that host and/or parasite factors associated with these two phenomena may be similar. Although these decreases may be the result of down-regulated immune responses, corresponding decreases in antibody levels and blastogenesis of lymphocytes stimulated by crude worm extracts were not observed in chronic infections.     

Studies with Brugia pahangi. II. The effect of repeated infection on parasite levels in cats

Denham D. A., Ponnudurai T., Nelson G. S., Rogers Rosemary and Guy Frances 1972. Studies with Brugia pahangi—II. The effect of repeated infection on parasite levels in cats. International Journal for Parasitology2: 401–407. 21 cats were given a primary infection of 100–200 infective larvae of Brugia pahangi followed, some time later, by repeated challenge with 50 larvae per time at 10-day intervals. In most cats the microfilarial levels increased considerably but in a minority the levels remained the same as those seen in cats given only one infection. Adult worm recoveries were very much higher than after a single infection but after about 20 challenges there was no further increase in the number of worms establishing an infection. After a long series of challenge infections, the microfilarial counts of some cats suddenly fell and the blood became free of microfilariae.     

Brugia pahangi: Histopathological study of golden hamsters

Sixteen male hamsters were inoculated subcutaneously with 95 to 150 infective larvae of B. pahangi and were examined for histopathologic lesions at 39–109 days postinfection. The basic microscopic lesion observed was obstructive granulomatous lymphangitis. Analogous lymph node changes sometimes occurred along with lymphoreticular hyperplasia and increased numbers of eosinophils. Cellular infiltration of perivascular and perinodal tissues was common, with plasma cells and eosinophils predominating. Genital lesions included funiculitis, epididymitis, and mild orchitis. Live and dead worms were found in the testicular parenchyma. Pulmonary changes in hamsters infected more than 105 days included multiple small, granulomatous foci and periarteriolar, peribronchiolar, and subpleural cellular accumulations of plasma cells and eosinophils. Granulomatous obstruction of pulmonary arteries associated with dead worms was observed in two hamsters infected for 39–45 days and in one hamster infected for 109 days. Small liver granulomas were common. Disintegrating microfilariae occurred within giant cells in the lymph nodes, spleen, lungs, and testes.     

The granulomatous inflammatory response in jirds, Meriones unguiculatus, to Brugia pahangi: an ultrastructural and histochemical comparison of the reaction in the lymphatics and peritoneal cavity

A granulomatous inflammatory response develops in jirds with lymphatic or intraperitoneal infections of Brugia pahangi. Light, histochemical, and ultrastructural microscopy were used for comparative studies of the reactions in these 2 locations. The reactions observed were categorized into 3 types: (1) an initial response in which lymphocytes, monocytes, macrophages, and eosinophils were present; (2) an intermediate one which consisted of macrophages, epithelioid cells, lymphocytes, eosinophils, collagen, and mesothelial/endothelial cells with central areas of necrosis; and (3) a terminal reaction consisting of degenerating, necrotic cells. Microfilariae and adult worms were associated with these reactions. Macrophages were the predominant cell type in the lesion and were often found attached to the surface of the parasite. The inflammatory responses to B. pahangi in the lymphatics and in the peritoneal cavity appear to be similar, and thus, the peritoneal cavity may be useful in studying specific cell-parasite interactions to further define the pathogenesis of filarial disease.     

Dipetalonema viteae: primary, secondary and tertiary infections in hamsters.

Hamsters were given primary infections of 100, 200, and 300 D. viteae larvae and groups killed at various intervals after infection. In addition, hamsters were sequentially infected with 100, 200, and 300 larvae and groups killed at 100 or 75 days after the secondary and tertiary infection, respectively. Blood microfilariae were detected on Day 60 following a primary infection, reached a maximum on Day 75, declined to low levels by Day 105, and were negative on Day 120. No microfilariae reappeared in the blood of hamsters given secondary or tertiary infections.Between 20–30% of the infecting larval dose had reached the adult stage by Days 75 or 100 postinfection in hamsters given primary, secondary, or tertiary infections. There was no evidence of arrested larval development in hamsters receiving a second or third challenge infection. Almost half of the tertiary infection hamsters developed subcutaneous nodules and their numbers varied greatly among individual animals. The nodules variously contained living worms, pus, and fragmented worms, or pus only. Hamsters given primary infections of 100, 200, or 300 larvae and killed 375 days after infection had no subcutaneous nodules; however, hamsters given the 200 and 300 larval infections were seen to have dead worms in the subcutaneous tissues. No stunting of adult worms was noted and all female worms had uteri packed with microfilariae.     

A Repurposing Strategy for Hsp90 Inhibitors Demonstrates Their Potency against Filarial Nematodes

Novel drugs are required for the elimination of infections caused by filarial worms, as most commonly used drugs largely target the microfilariae or first stage larvae of these infections. Previous studies, conducted in vitro, have shown that inhibition of Hsp90 kills adult Brugia pahangi. As numerous small molecule inhibitors of Hsp90 have been developed for use in cancer chemotherapy, we tested the activity of several novel Hsp90 inhibitors in a fluorescence polarization assay and against microfilariae and adult worms of Brugia in vitro. The results from all three assays correlated reasonably well and one particular compound, NVP-AUY922, was shown to be particularly active, inhibiting Mf output from female worms at concentrations as low as 5.0 nanomolar after 6 days exposure to drug. NVP-AUY922 was also active on adult worms after a short 24 h exposure to drug. Based on these in vitro data, NVP-AUY922 was tested in vivo in a mouse model and was shown to significantly reduce the recovery of both adult worms and microfilariae. These studies provide proof of principle that the repurposing of currently available Hsp90 inhibitors may have potential for the development of novel agents with macrofilaricidal properties.     

Taenia crassiceps: Fate of cysticerci following ingestion by the mouse

Cysticerci of Taenia crassiceps were administered to mice by gavage to determine whether enteral or parenteral infections would establish consistently. Some worms survived in the small intestine up to 16 days, whereas others penetrated through the gut wall into the peritoneal cavity within 24 hr. Similar proportions of different doses of worms reached the peritoneal cavity regardless of the size of the inoculum and sex or strain of mice used. In addiiton, it was shown that mice may acquire an intraperitoneal infection with T. crassiceps by eating the carcass of an infected mouse.     

Brugia pahangi and Dirofilaria immitis: experimental infections in the ferret, Mustela putorius furo.

Ferrets were inoculated with 160 third-stage larvae of the filarial nematode Brugia pahangi, followed 23 days later by 15 larvae of another filarial nematode, Dirofilaria immitis. Other ferrets received only one of these species. Microfilaremia developed in some ferrets with single infections of each species and in some ferrets with dual infections. The nature of the experiment did not permit a thorough study of microfilaremia, but B. pahangi microfilariae were found in numbers as high as 15,650/ml. At necropsy, approximately 8 months after inoculation, adult B. pahangi were recovered from the lymphatic vessels of all 8 ferrets inoculated only with that species, the recovery rate (based on 6 animals only) varying from 2 to 50% of the inoculum (mean 25%). Adult D. immitis were recovered from the heart of all three ferrets inoculated only with that species, the recovery rate being 7, 47, and 60% (mean 38%) of the inoculum. All 5 ferrets inoculated with both species yielded both adult B. pahangi (6 to 23%, mean 16% of inoculum) and adult D. immitis (13 to 67%, mean 37% of inoculum). It is concluded that the ferret is highly susceptible to both species and that concurrent infections with both species may readily be established.     

Delayed macrofilaricidal activity of diethylcarbamazine against Brugia pahangi in Mongolian jirds

The macrofilaricidal activity of diethylcarbamazine (DEC) was confirmed in jirds infected with Brugia pahangi. Seventy jirds were inoculated subcutaneously with 100 infective larvae. At 20 weeks post-infection, the microfilaraemic jirds were divided into two groups, untreated and treated. For the treated group, 200 mg kg(-1) of DEC was injected intraperitoneally for 5 consecutive days. One, 4, 8, 12, 16 and 27 weeks after the final treatment, 4-7 jirds in each group were sacrificed to measure adult worm burdens. The number of adult worms recovered from treated jirds was comparable to controls at earlier necropsy (1 and 4 weeks post-treatment). However, at late necropsy (8 weeks and later) the recovery rate of adult worms in treated jirds was significantly lower than that in untreated controls, indicating an adultcidal effect of DEC. The present study demonstrates that DEC requires 8 weeks to kill B. pahangi adult worms in jirds and that the Mongolian jird is a useful model for screening antifilarial activity.     

Possible direct effect of diethylcarbamazine on the infective larvae of Brugia pahangi

The direct action of diethylcarbamazine (DEC) on the infective larvae of Brugia pahangi was studied. The larvae were cultured in RPMI 1640 supplemented with foetal bovine serum and antibiotics for 22 days. Most of the larvae remained alive for 8 days, but survival rate of larvae decreased rapidly from day 10 onwards. The larvae did not grow in the culture system. The addition of DEC did not affect the morbidity of the larvae and no difference was observed in the morphological characteristics between the larvae cultured in the presence or absence of DEC. The infective larvae were cultured in vitro for 5 days in the presence or absence of DEC, and inoculated into jirds. The animals were necropsied at intervals, and developing larvae and adult worms were recovered. When the larvae were cultured without DEC and then inoculated subcutaneously into jirds, 29.8% of the inoculum was recovered 3-15 days, and 25% 19-22 weeks, post-inoculation. However, when the larvae were exposed to DEC in vitro and inoculated into jirds, the rate of recovery was reduced to 25% 3-15 days post-inoculation and 2% after 19-22 weeks. When the control larvae cultured in vitro were inoculated intraperitoneally into jirds, 41.3% of inoculum was recovered 3-15 days, and 42.8% 19-22 weeks, post-inoculation. Again the corresponding value for larvae exposed to DEC in vitro was reduced to 19.8% 3-15 days, and 8% 19-22 weeks, post-inoculation. It was observed that the larvae exposed to DEC in vitro were retarded in their development in jirds.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of primary and secondary infections with Strongyloides ratti in mice

Dawkins H. J. S. and Grove D. I. 1981 Kinetics of primary and secondary infections with Strongyloides ratti in mice. International journal for Parasitology11: 89–96. The kinetics of infection with S. ratti were quantitated in normal and previously exposed C57B1 /6 mice. In primary infections, larvae penetrated the skin rapidly and were seen in peak numbers 12 h after infection. By 24 h after infection, larval numbers had declined appreciably and there was a slow decrease in numbers thereafter. Larvae were first observed in the lungs at 24 h and maximal recovery occurred at 48 h. It is thought that larval migration through the lungs is rapid. Worms were first seen in the intestines two days after infection. Maximum numbers were seen on the fifth day and worm expulsion was complete by day 10. Two moults took place in the small intestine during days 3 and 4 after infection. Rhabditiform larvae were first noted on the fourth day after infection. Mice exposed to S. ratti four weeks previously had significantly less larvae in the skin 4 and 12 h after infection but by 24 h there was no difference when compared with mice with primary infections. Peak recovery of larvae from the lungs occurred 24 h after infection; significantly less larvae were recovered on days 2 and 3 when compared with normal mice. There was a marked reduction in the adult worm burden in the gut; the number of worms recovered was less than one fifth of that seen in primary infections. Those worms which did mature were less fecund and were expelled from the intestines within 7 days of infection. It is suggested that in previously exposed animals, the migration of larvae from the skin is hastened, many of these larvae are destroyed in the lungs and that expulsion of worms which do mature in the intestines is accelerated.     

Brugia pahangi: effects upon the flight capability of Aedes aegypti.

Aedes aegypti mosquitoes were adversely affected by infections of the filarial worm Brugia pahangi. Infected mosquitoes flew significantly shorter distances and showed marked reductions in total flight time during 24-hr flight mill tests compared to uninfected controls. Total flight range and duration flown by infected mosquitoes remained relatively constant throughout the infection process, while control mosquitoes flew further and longer with increasing time after their blood meal. Furthermore, a significantly greater number of infected mosquitoes either died or were rendered incapable of flight. Of flying and nonflying mosquitoes with 6-day-old or older infections dissected for parasite burdens, the nonflying group contained significantly more worms. Results of this study indicate that developing filarial larvae within this mosquito vector reduce its ability to survive and to transmit its infection by reducing its flight capabilities. Conclusions from this study relate only to A. aegypti homozygous for the gene fm which is fully susceptible to this filarial parasite.