Ultrastructure of the hypodermis during cuticle formation in the third molt of the nematode Nippostrongylus brasiliensis

Summary The fine structure of the hypodermis of Nippostrongylus brasiliensis, a parasitic nematode of the rat, was studied in free-living third-stage filariform (infective) larvae, and in parasitic forms undergoing the third molt. In filariform larvae the hypodermis displayed a comparatively poorly developed RER, and few Golgi complexes, mitochondria, and vesicles. Nuclei contained large amounts of heterochromatin and no nucleoli. After worms reached the lungs and the third molt began, the RER, Golgi complexes, mitochondria, multivesicular bodies, and coated vesicles greatly increased in amounts. Nuclei displayed less heterochromatin and contained prominent nucleoli. These morphological changes were associated with transition from the free-living to the parasitic mode in life. The results were correlated with the fact that the hypodermis in N. brasiliensis is actively synthesizing culticular collagen during the third molt.This investigation was supported, in part, by awards 1-F02-AI3750-02 and AI-09625 from the National Institutes of Health, United States Public Health Service.We are grateful to Lida Petruniak and Dora Lou for technical assistance.     

The reversibility of intestinal immune expulsion effects on adult Nippostrongylus brasiliensis

An attempt has been made to study the extent and nature of the damage occurring in adult Nippostrongylus brasiliensis undergoing immune expulsion from the rat. It was found that worms are not killed nor irreparably damaged when being rejected. On transfer into naive second recipient rats the rate of re-establishment of worms previously incubated in immune rat recipients for 4-17 hr was high (68-69%) and comparable to that shown by worms from normal recipient rats (48-56%). Similarly, worms taken on days 10, 11, and 12 of a primary infection, already passed to the distal half of the small intestine due to immune expulsion effects, on transfer into naive recipient rats re-established themselves well (rates varying from 62 to 80%) compared to those harvested from their normal habitat in the proximal half of the small intestine (rates varying from 44 to 87%). Worm damage is associated with decreased motility and impaired locomotion capacity. The phenomenon of mucosal trapping occurs during expulsion, but merely to the extent of some 30% of the worm population. It is suggested that in principle, worms subjected to immune expulsion are in a state of acute, transient metabolic crisis. The present results support the enteroallergic indirect mechanism for worm rejection.     

Nippostrongylus brasiliensis: feeding activity in the mouse

Nippostrongylus brasiliensis incorporated the fluorescent dye, Rhodamine B, while feeding in vivo. Uptake of dye in both sexes of helminth increased linearly from 30 to 120 min after the hosts were given dye per os. Feeding by female N. brasiliensis significantly exceeded that of the male at 4 and 5 days postinfection. Feeding declined in older helminths of both sexes. The density of helminths had no effect on their incorporation of dye in vivo. Feeding in male- and female-only groups of worms was similar to that seen in populations of mixed sexes. Feeding by helminths decreased during the first 36 h of food deprivation in the host, but increased during subsequent fasting of the host. Both sexes of N. brasiliensis resumed feeding within 15 min after the fasted hosts were fed. Growth of N. brasiliensis increased linearly from 4 to 7 days postinfection, based on dry weight. Seven-day-old and older females were significantly heavier than males.     

Nippostrongylus brasiliensis: in vitro feeding activity

Nippostrongylus brasiliensis incorporated the fluorescent dye, Rhodamine B, while feeding in vitro. Uptake of dye in both sexes of helminth was time-dependent with maximal incorporation occurring after 30 min. Feeding activity was elevated as the time of removal from the host increased from 1 to 8 h. Feeding was reduced in 4- or 5-day-old helminths when compared to 7-day-old or older worms. Feeding activity increased also after temperature increases, pH decreases and solute decreases. Feeding was similar under conditions of both light and dark. Feeding by males and females of N. brasiliensis decreased as the density of worms in single- or mixed-sex groups of worms was increased. Females fed significantly more than males under crowded conditions.     

Carboxypeptidase-like activity during development and aging of Nippostrongylus brasiliensis

An acid carboxypeptidase whose activity, based on several biochemical parameters, was determined to be similar to mammalian lysosomal carboxypeptidase was maximal at pH 5.0 during the early parasitic stages of development of the nematode Nippostrongylus brasiliensis. No such enzyme activity could be detected in the early free-living larval stages of this nematode. Carboxypeptidase activity declined as the age of the parasitic adult increased. Timing of maximum activity corresponds to the shift from free-living to parasitic stages of the life cycle.     

Changes in the adenylate energy charge of Nippostrongylus brasiliensis and Nematodirus battus during the development of immunity to these nematodes in their host

Infection of rats with 2000 infective juveniles of Nippostrongylus brasiliensis and of lambs with 60 000 infective juveniles of Nematodirus battus results in a well-marked immunity to these nematodes in their respective host. There is a fall in the adenylate energy charge value of these nematodes during the course of these infections, reaching values of 0.37 in males and 0.27 in females of N. brasiliensis, and 0.31 in males and 0.23 in females of N. battus towards the end of the infections. In hosts given relatively small numbers of infective juveniles, the values for the nematodes removed from the hosts late in the infection remain at a relatively high level. These results indicate that the immune response of the host may affect the energy status of these nematodes, and this could help to explain their subsequent expulsion from the immune host.     

Nippostrongylus brasiliensis: malabsorption in experimentally infected rats

Glucose absorption and net small intestinal water movement were examined in rats infected with Nippostrongylus brasiliensis at Days 4, 6, 9, 13, and 19 after inoculation. Rats were infected with 4 X 10(3) N. brasiliensis third stage larvae. The entire small intestine was divided into three segments and each segment perfused simultaneously in vivo with Krebs-Ringer phosphate buffer containing 80 mM glucose, 6 X 10(5) dpm/ml [3H]glucose, and 6.2 X 10(3) dpm/ml [14C]polyethylene glycol. Rats perfused on Days 6, 9, 13, and 19 after inoculation showed a significant (P less than 0.05) decrease in glucose absorption rates from all three segments of the small intestine when compared to uninfected controls. In the three segments of uninfected rat small intestine and those perfused on Days 4, 13, and 19 after inoculation, net absorption of water occurred. However, in the proximal and distal segments perfused on Day 6 and the proximal segment perfused on Day 9, net water movement into the lumen occurred. This is the first report of depressed glucose absorption along the entire length of the small intestine during nippostrongylosis and contradicts previous reports of unaltered net glucose absorption in response to this parasite.     

Biogenic amines and gaba in the larval and adult forms of the nematode Nippostrongylus brasiliensis

1. Simultaneous detection (HPLC and electrochemical detection) of biological extracts of larval and adult stages of Nippostrongylus brasiliensis was performed in order to assay biogenic amines.2. Gamma-amino-butyric acid was assayed in the same samples.3. Tryptophan, 5-hydroxyindoleacetic acid were at the same level in adults and larvae.4. 5-Hydroxytryptophan, serotonin, dihydroxyphenylalanine and dopamine were significantly higher in larvae in which gamma-amino-butyric acid was not detected.     

Intestinally implanted Nippostrongylus brasiliensis adult worms decrease serum paraoxonase-1 activity in rats

We have previously reported a significant decrease in serum PON1 activity after Nippostrongylus brasiliensis infection in Wistar rats in association with the inflammatory response mounted against the parasite in the migratory phase of infection. However, the roles of intestinal phase and the associated oxidative stress during N. brasiliensis infection on PON1 activity have not yet been elucidated. In the present study, we observed a significant reduction in serum paraoxonase and arylesterase activity on days 6 and 9 post-implantation with N. brasiliensis adult worms in the absence of a significant increase in various serum pro-inflammatory cytokines. In addition, provision of the antioxidant butylated hydroxyanisole (BHA) to adult worm-implanted rats did not ameliorate the reduction in PON1 activity. Due to the prolonged intestinal phase of gastrointestinal nematode infections, alterations in PON1 activity during this phase need to be further examined to elucidate the mechanism of alteration in PON1 activity.     

Nippostrongylus brasiliensis: occurrence of multiple protein kinases

The presence of cyclic AMP-dependent protein kinase and phosvitin kinases, with activity independent of cyclic nucleotides, was shown in the intestinal nematode Nippostrongylus brasiliensis. The activity of the cyclic AMP-dependent protein kinase was found to be enhanced about 8-fold in the presence of 10(-7) M cyclic AMP; the apparent Km values were determined to be 20 microM and 80 microM for ATP and kemptide, respectively. The molecular weight of the holoenzyme was about 170 000. Two phosvitin kinases could be isolated and distinguished by their molecular weights of 600 000 and 40 000. The activity of the high-molecular-weight phosvitin kinase was effectively inhibited by suramin and heparin. The apparent Km values were found to be 30 microM and 0.1 mg/ml for ATP and phosvitin, respectively. In the case of the low-molecular-weight phosvitin kinase the apparent Km values for ATP and phosvitin were found to be 30 microM and 0.6 mg/ml, respectively. The investigation of different developmental stages of N. brasiliensis revealed a marked higher level of protein kinase activity in the L4 larvae compared to L3 larvae and adults.