Increased susceptibility to infection with Brugia pahangi in aged female jirds (Meriones unguiculatus)

Female Mongolian jirds, Meriones unguiculatus, from 5 age groups of 2, 12, 16, 21, and 28 months, were infected with Brugia pahangi. Infections were followed for 125 days by weekly bleedings beginning 55 days postinoculation. Jirds were then killed and adult parasites recovered. Results showed a significant shortening of the prepatent period in the 12-, 21-, and 28-month-old groups. The proportion of gravid female worms did not vary significantly among the 5 groups. Similarly, the ratio of females to male worms showed little variation from group to group. Microfilaremia data for the 5 infection groups show an age-associated increase in numbers of circulating microfilariae. Some individuals in the 28-month group demonstrated 1,500 to 3,000 microfilariae per 0.25 ml peripheral blood, a level that was not approached by young females.     

Attempted vaccination of jirds (Meriones unguiculatus) against Brugia pahangi with radiation attenuated infective larvae

Jirds were vaccinated by three to five subcutaneous (SC) injections of infective larvae of Brugia pahangi which had been irradiated at 25, 45 or 90 krads from a 60Co source. They were challenged either SC or intraperitoneally. Vaccination with four doses of 50 larvae irradiated with 25 krads produced 49.3% resistance to IP challenge worms and 39.8% against SC challenge worms. Five doses of larvae irradiated with 45 krads produced 62% resistance to SC challenge. Three doses of larvae irradiated with 90 krads produced 74.9% resistance to SC challenge and five doses produced 76.2% resistance. The reasons why irradiated larvae produce resistance whereas normal larvae do not are discussed.     

Brugia pahangi: infections and their effect on the lymphatic system of Mongolian jirds (Meriones unguiculatus)

Brugia pahangi has been found to be primarily a lymphatic-dwelling parasite in jirds when infections are induced by the subcutaneous injection of infective larvae or by allowing infected Aedes aegypti to feed.Migration to the regional lymphatics occurred as early as 1–4 days. Although some injected larvae remained in the skin for as long as 30 days and some became localized in the heart, lungs, pleural cavity, or peritoneal cavity, about three-fourths of the recovered filariae were found in the regional lymphatics. In contrast, when larvae were injected peritoneally they remained largely in the peritoneal cavity for at least 30 days.The relevant lymphatics and their drainage patterns in jirds have been described.The major pathological changes noted in jirds involved the regional lymphatic vessels and nodes, which were severely affected when they contained dead worms. Pulmonary granulomas due to dead microfilariae and occasionally to dead larvae or adult worms were noted.Observations are included on the susceptibility and course of B. pahangi infections in jirds.     

Induction of protective immunity to Brugia pahangi in jirds by drug-abbreviated infection.

Protective immunity of homologous challenge infection was examined in jirds after drug-abbreviated infection with Brugia pahangi. Mebendazole (MBZ) treatment at the early prepatent (5-7 weeks of post infection) or the late prepatent (7-9 weeks of post infection) period was highly effective in causing almost complete eradication of the primary infection. After challenge infection, the worm burden was significantly reduced 19% (31.1 in average) and 77% (9.5) to that of the controls (38.8 and 41.7), respectively. The magnitude of eosinophil response paralleled the degree of protection. No or only a few microfilariae were seen after challenge infection in jirds treated during the prepatent periods. They were also resistant to intravenous challenge with the microfilariae of B. pahangi. MBZ treatment at the patent period was, on the contrary, incomplete against primarily infected adult worms, and was not able to induce either significant protection (30.1 vs 33.1 in control) or eosinophil response to the challenge infection.     

Effect of immunostimulatory oligodeoxynucleotides on host responses and the establishment of Brugia pahangi in Mongolian gerbils (Meriones unguiculatus)

Infection of humans with filarial parasites has long been associated with the maintenance of a dominant Th2-type host immune response. This is reflected by increases in interleukin (IL)-4- and IL-5-producing T cells, elevated immunoglobulin (Ig)E and IgG4 levels, and a pronounced eosinophilia. The Mongolian gerbil (Meriones unguiculatus) is permissive for the filarial nematodes Brugia malayi and B. pahangi. As in humans, persistent microfilaremic infections of gerbils with Brugia spp. results in increases in Th2 cytokines such as IL-4 and IL-5. The association of dominant Th2 cytokine profiles with the maintenance of infection suggests that the introduction of Brugia spp. into a strongly Th1-biased environment may adversely affect parasite establishment. Indeed, studies conducted in mice with B. malayi suggest that depleting Th1 effectors such as interferon (IFN)-gamma and nitric oxide results in increased worm recoveries. In the present studies, the Mongolian gerbil was used as a model to investigate the effect of a dominant Th1 cytokine environment on the establishment of B. pahangi. Intraperitoneal (i.p.) administration of immunostimulatory oligodeoxynucleotide (IS ODN) induced the production of IFN-gamma in the peritoneal exudate cells and spleen of gerbils. The presence of IFN-gamma at the time of B. pahangi infection did result in an altered host immune response to B. pahangi. Gerbils that received IS ODN before i.p. B. pahangi infections showed lower levels of the Th2-type cytokines IL-4 and IL-5, compared with animals that received B. pahangi alone (0 + Bp). This alteration in cytokine profile, however, did not alter the establishment or development of B. pahangi in the peritoneal cavity. Furthermore, there was no difference in the granulomatous response of gerbils to soluble adult B. pahangi antigen bound to beads embolized in their lungs, regardless of treatment group, suggesting that IL-4 and IL-5 are not essential contributors to the systemic host inflammatory response to B. pahangi in this model.     

Differential susceptibility to Brugia pahangi infection in Mongolian gerbils (Meriones unguiculatus) of different coat colour

The influence of intraspecific host variables on the response to parasitic infections is an important aspect of host-parasite relationships, yet little is known about this aspect of filariasis for lack of a model. This study presents coat colour mutants of the Mongolian gerbil (Meriones unguiculatus) as potential new models for research into the effects of host genetic variation on response to filarial infection. Peak level of microfilaraemia, eosinophil response, body weight and degree of splenomegaly in gerbils infected with Brugia pahangi varied with agouti, albino, and black coat colour. These results suggested that coat colour-related genes might influence host immune response to developmental stages of the parasite and eosinophil-mediated reaction might cause host damage.     

Temporary shift of microfilariae of Brugia pahangi from the lungs to muscles in Mongolian jirds, Meriones unguiculatus, after a single injection of diethylcarbamazine

A single-dose treatment with diethylcarbamazine (DEC) reduced microfilaria (mf) counts of Brugia pahangi by >90% at 30 min post-treatment in Mongolian jirds (Meriones unguiculatus). The reduction was followed by a rapid increase in microfilaremia, with the count reaching pretreatment level in 3 hr. The mechanisms behind this temporary reduction of mf were investigated. Without treatment, mf accumulated in the lungs. At 30 min post-treatment, they had moved from the lungs and accumulated in the muscle. At the same time, electron microscopy revealed many mf in the muscle interstitium. DEC concentrations at 30 min were much lower in the muscle (12.2 microg/g of tissue) than in the lungs, liver, and kidneys (19.8-40.7 microg/g), all of which declined to < 0.6 microg/g by 3 hr. The presence of mf in the muscle would be advantageous for avoiding high DEC concentrations, and their extravascular location could prevent attack by host effector cells.     

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.     

Duration of infection of jirds (Meriones unguiculatus) with Trichostrongylus colubriformis

Jirds (Meriones unguiculatus) were infected with 700 infective larvae (L3) of Trichostrongylus colubriformis. The fecal egg counts (epg) were followed regularly until 340 days postinoculation, and worm numbers were determined on necropsy at 315 and 350 days postinoculation. The epg increased to a peak of 2,600 on day 24 postinoculation. Thereafter, the epg showed continuous decrease from day 31 until day 318 postinoculation. The rates of inoculum recovery were 0.29-1.43% on day 315 and 0.142-0.428% on day 350 postinoculation. On day 315 postinoculation, 12 male worms were recovered from 2 of 3 animals necropsied. On day 350 postinoculation, all 6 animals were infected, and 6 female and 4 male worms were recovered. The systemic IgM, IgG, and intestinal mucous IgA responses were significantly higher in infected groups compared with negative control groups necropsied on 315 and 350 days postinoculation (P < 0.001). These results confirm that the T. colubriformis-jird system is a useful model for studies on the various host-parasite relationships of chronic trichostrongylosis.     

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 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.     

Induction of lymphatic lesions by Brugia pahangi in jirds with large and small preexisting homologous intraperitoneal infections

The effect of intraperitoneal (i.p.) infections induced by inoculations of 30 or 150 Brugia pahangi third-stage larvae (L3) on the development of infections and lymphatic lesions induced by subsequent homologous subcutaneous (s.c.) inoculations were compared in the present study. Lymphatic lesion severity, as judged by the numbers of lymph thrombi present, and lymphatic lesion scores were significantly reduced in both groups of jirds with existing i.p. infections. The numbers of adult worms that developed, locations of these worms, and the subsequent microfilaremias did not differ significantly between groups. All jirds with i.p. infections developed similar antibody titers to crude somatic adult antigen as measured by ELISA. These levels did not change following s.c. infections. Immediate and delayed footpad swelling responses were also similar in all groups. Results of these experiments support and extend previous studies indicating that i.p. infections of B. pahangi induce a hyporesponsive state in jirds to subsequent s.c. infections without significantly affecting the subsequent parasite burden. This effect appears to be independent of the numbers of L3 inoculated i.p. prior to lymphatic-induced infection. Circulating antibody titers and footpad swelling responses to B. pahangi antigen were not reduced in jirds with the hyporesponsive lymphatic inflammatory response and do not correlate with this condition.     

Degenerative changes in lymphatic endothelium of jirds infected with Brugia pahangi

The quantitative changes of cytoplasmic vesicles and vacuoles in lymphatic endothelial cells of the mongolian jirds associated with Brugia pahangi infections were observed by transmission electron microscopy. The present study revealed a decrease in the proportion of cytoplasm occupied by vesicles and in the number of cytoplasmic vesicles in endothelial cells from lymphatic vessels harboring B. pahangi at 3, 4, and 10 mo after infection (3.55, 3.36, and 2.55 vesicles/micron 2, respectively) when compared with cells from uninfected control vessels (7.03 vesicles/micron 2). On the contrary, there was an increase in the area of vacuoles in endothelial cells of jirds at 3, 4, and 10 mo postinfection. The mean +/- SD diameter of vesicles in cells from lymphatic vessels at 10 mo after infection was significantly smaller (78.6 +/- 5.6 nm) compared to vesicles in uninfected vessels (87.5 +/- 9.7 nm).     

Assessment of Blood Collection from the Lateral Saphenous Vein for Microfilaria Counts in Mongolian Gerbils (Meriones unguiculatus) Infected with Brugia pahangi

The NIH guidelines for survival bleeding of mice and rats note that using the retroorbital plexus has a greater potential for complications than do other methods of blood collection and that this procedure should be performed on anesthetized animals. Lateral saphenous vein puncture has a low potential for complications and can be performed without anesthesia. Mongolian gerbils (Meriones unguiculatus) are the preferred rodent model for filarial parasite research. To monitor microfilaria counts in the blood, blood sampling from the orbital plexus has been the standard. Our goal was to refine the blood collection technique. To determine whether blood collection from the lateral saphenous vein was a feasible alternative to retroorbital sampling, we compared microfilaria counts in blood samples collected by both methods from 21 gerbils infected with the filarial parasitic worm Brugia pahangi. Lateral saphenous vein counts were equivalent to retroorbital counts at relatively high counts (greater than 50 microfilariae per 20 µL) but were significantly lower than retroorbital counts when microfilarial concentrations were lower. Our results indicate that although retroorbital collection may be preferable when low concentrations of microfilariae need to be enumerated, the lateral saphenous vein is a suitable alternative site for blood sampling to determine microfilaremia and is a feasible refinement that can benefit the wellbeing of gerbils.Abbreviations: FR3, Filariasis Research Reagent Resource CenterLymphatic filariasis a major threat to human health worldwide. More than one billion people in more than 90 countries around the globe are at risk from lymphatic filariasis, and between 120 and 150 million people are infected.^9,11,25 Infection with the filarioid parasitic worms Brugia malayi and Wuchereria bancrofti can result in severe sequelae, including elephantiasis and hydrocoele formation.^3,11,15,25 In addition to the clinical manifestations of filariasis are the potential associated psychologic, social, and cultural effects in persons exhibiting visible signs of infection.^9,23,34The life cycle of filarioid nematodes requires an arthropod intermediate host and a definitive vertebrate host. Within the definitive host, dioecious adult filarial nematodes reproduce sexually. Inseminated adult female worms then release live, sheathed microfilariae into the lymph that circulate in the peripheral blood.²¹ In the case of B. malayi and W. bancrofti, the intermediate host is the mosquito.²¹ When an uninfected mosquito ingests a blood meal from an infected human, ingested microfilariae unsheathe to penetrate the midgut of the mosquito to reach the thoracic muscles and molt twice, to become the infectious third-stage larvae. The third-stage larvae then migrate to the mosquito''s proboscis and can infect another human when the mosquito takes a blood meal.^10,11 The third-stage larvae enter the new host''s lymphatic system which is their final location, where they undergo 2 molts into adults.Because of the complexity of filarioid life cycles, research involving these parasites can be logistically challenging. Although mice can be infected with W. bancrofti, they do not maintain the infection.³⁵ Furthermore, there is no suitable nonhuman host that can maintain a patent infection, with the exception of the silvered leaf monkey (Trachypithecus cristatus).⁹ Because the closely related parasites B. malayi and B. pahangi have more extensive host ranges than does W. bancrofti, they are easier to maintain in a research setting. Domestic cats (Felis catus) can be experimentally infected with B. malayi and develop a patent infection, and both domestic cats and dogs (Canis familiaris) can be experimentally infected with B. pahangi^13,29,37 and are suitable for obtaining microfilaremic blood for experimental feeding of mosquitoes. The Mongolian gerbil can be infected with B. pahangi. Because replacing a phylogenetically higher species with a lower species is preferable³⁶ and because performing experiments involving dogs and cats can be logistically difficult and cost-prohibitive, many researchers prefer a rodent model, specifically gerbils.The Filariasis Research Reagent Resource Center (FR3) is an NIH center whose mandate is to support filariasis research worldwide. The FR3 provides parasitic and molecular resources, as well as training in animal procedures, to researchers from many nations. The FR3 maintains both B. malayi and B. pahangi, and researchers occasionally require gerbils with patent infections. Because the required level of microfilaria counts varies among investigators, an accurate microfilaria count must be obtained prior to the shipment of gerbils. For example, some experiments require that live mosquitoes feed directly on infected gerbils, and when the microfilaria level is too low, the mosquitoes do not become infected. Conversely when the level is too high, the migration of microfilariae and the later larval stages can kill the mosquitoes. Historically, the FR3 has used retroorbital sampling under general anesthesia to obtain the blood for microfilaria counts.²⁸ Although this method has been fairly successful, the FR3 has encountered occasional complications secondary to the procedure, including exophthalmia and, rarely, death under anesthesia. The NIH guidelines for survival bleeding of mice and rats notes that compared with other blood collection methods, retroorbital sampling has a greater potential for complications. The guidelines recommend a 10- to 14-d period between retroorbital blood collections and state that the procedure is “…best conducted under general anesthesia.”³¹ By comparison, collecting blood from the lateral saphenous vein is considered to have a low potential for complications or tissue damage, can be performed without general anesthesia,^12,18,31 and can be performed repeatedly, even daily.³¹In the current study, we proposed to refine the blood collection method being used by FR3 by developing sampling from the lateral saphenous vein as the new standard blood-collection method for monitoring microfilaremia. Our goal was to assess blood collection from the lateral saphenous vein as a feasible refinement technique to potentially replace retroorbital sampling by determining whether the microfilaria counts in blood collected from the lateral saphenous vein without anesthesia were sufficiently similar to those from retroorbital blood sampling with anesthesia to provide adequate information about the microfilaremia level.     

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.     

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.     

Qualitative characterization of antibody responses to single and multiple Brugia pahangi infections in jirds

Antibody responses of jirds, singly and multiply inoculated with Brugia pahangi infective larvae (L3), to soluble somatic extracts of adult parasites were characterized by western blot analysis. Forty-two protein bands ranging in molecular weight from 12 to 160 kDa were recognized by sera from infected jirds. Antibody recognition of individual B. pahangi antigen bands in this assay appears to be independent of antibody enzyme-linked immunosorbent assay (ELISA) titers to crude parasite extract, severity of lymphatic lesions, levels of microfilaremia, numbers of L3 inoculated, or numbers of adult parasites in individual jirds. Antibody recognition of protein bands with molecular weights of 37 kDa, 21 kDa, and 17 kDa, however, did temporally correspond with certain parasitological and pathologic events. Antibody against the 37-kDa protein band first was identified at the onset of patency, reaching a 90% prevalence rate by 90 days postinfection (DPI). The prevalence of this antibody remained high. Antibody recognition of the 21-kDa protein band first occurred at 90 DPI and gradually increased in prevalence during the course of infection temporally similar to the increase in microfilaremia. Recognition of the 17-kDa protein band first occurred at 48 DPI, reached a maximum prevalence of 80% at 90 DPI, and decreased to a minimal prevalence by 160 DPI. Prevalence of antibody responses to the 17-kDa protein band corresponded temporally with the kinetics of the rise and fall of numbers of intralymphatic thrombi. The patterns of antibody response to these 3 bands were similar in both singly and multiply inoculated animals.(ABSTRACT TRUNCATED AT 250 WORDS)