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.     

Nippostrongylus brasiliensis: effects of testosterone on reproduction and establishment

Gonadectomy of the host reduced the establishment of Nippostrongylus brasiliensis in male mice, but elevated the recovery of worms from female hosts. Similarly, production of eggs by individual females of N. brasiliensis decreased and increased in gonadectomized male and female animals, respectively. Implantation of testosterone-filled capsules caused a dosage-dependent release of eggs by helminths in gonadectomized hosts of both sexes. Maximal production of eggs by nematodes was found when plasma testosterone in the host exceeded 20 ng/ml. Treatment of gonadectomized mice of both sexes with testosterone implants gave recoveries of worms that were similar to the number of helminths in intact male mice. Ligation of the bile duct of male mice that were implanted with testosterone reduced both establishment and reproduction of N. brasiliensis.     

Host influences on reproduction and establishment of mouse-adapted Nippostrongylus brasiliensis (Nematoda)

Various influences of the host altered the establishment and reproduction of mouse-adapted Nippostrongylus brasiliensis. Establishment and egg production were greater in mature male than in female mice. Worm fecundity increased similarly in both sexes of mice prior to 41 days of host age. Male mice at 62 days of age or older harbored more N. brasiliensis than did females, but the number of nematodes was similar in younger hosts of both sexes. Crowding of male mice significantly increased the establishment of helminths, but not the production of eggs by N. brasiliensis. Pregnancy of the host significantly increased both establishment of and reproduction by N. brasiliensis. Ligation of the bile duct of the mouse reduced both establishment and fecundity of the nematode after subcutaneous or surgical infection with larvae and adults, respectively.     

Impaired protection against Trichinella spiralis in mice with high levels of IgE

Helminth infection induces production of a large amount of immunoglobulin E (IgE) to nonhelminth antigens. Although such “irrelevant” IgE is a major proportion of total IgE in the host, its biological significance remains unclear. Therefore, I examined protective activity against Trichinella spiralis in mice with high levels of IgE by repeated injections of anti-dansyl IgE monoclonal antibody or Nippostrongylus brasiliensis infection. Injected anti-dansyl IgE occupied IgE receptors on mast cells in naive mice. Protective activity against T. spiralis, determined with number of muscle larvae 5 weeks after infection, was impaired in mice treated with anti-dansyl IgE. The impaired protection was found in mice treated with anti-dansy IgE 7 and 14 days after infection, but not 21 and 28 days after infection, indicating that IgE-dependent protection operates at an early stage after infection. In the next experiments, mice were infected with N. brasiliensis 4 weeks before T. spiralis infection to obtain high levels of IgE. The protective activity against T. spiralis was decreased by N. brasiliensis infection. On the other hand, protection against T. spiralis was comparable in IgE-deficient SJA/9 mice and in anti-IgE-treated BALB/c mice with or without N. brasiliensis infection, suggesting that impairment of protection is dependent on IgE. These results indicate that the high levels of irrelevant IgE are beneficial for helminths and, alternatively, that anti-helminth IgE antibodies are protective for hosts. In addition, the impaired protection was found in IgE high-responder mice but not in low-responder mice, suggesting that protection against T. spiralis is controlled by IgE responsiveness in the host.     

Influence of the intestinal anticoagulant in the feeding performance of triatomine bugs (Hemiptera; Reduviidae)

Triatomines are haematophagous insects in all post-embryonic life stages. They are vectors of Trypanosoma cruzi, the causative agent of Chagas disease. Their vectorial ability is influenced by their feeding performance, which varies greatly amongst species. Recent work showed that inhibition of the coagulation process in the anterior midgut (crop) environment considerably influences the blood meal size. In this work, we performed a comparative study of the level of anticoagulant activity in the saliva and crop contents of three triatomine species - Triatoma infestans, Triatoma brasiliensis and Rhodnius prolixus - and correlated this with their feeding performance on live hosts. Moreover, the feeding parameters on a large diameter vessel influenced by the crop anticoagulants were evaluated in detail. The anticoagulant activity was significantly higher in the crop contents than in salivary glands, varying from 1.6-fold higher for R. prolixus to 70-fold higher for T. brasiliensis. Amongst the species, T. brasiliensis had the lowest crop anticoagulant activity, the lowest concentration of thrombin inhibitor, and took the longest to feed. Triatoma brasiliensis nymphs that had their intestinal anticoagulant (brasiliensin) knocked down by RNA interference had the lowest capacity to maintain cibarial pump frequency at higher levels throughout the feeding process and consequently a lower ingestion rate (mg/min), even when fed under favourable conditions (large diameter vessel). However, the feeding difficulty for brasiliensin knockdown T. brasiliensis nymphs was reversed by treating the host mice with heparin (a potent systemic anticoagulant) before blood feeding. The results indicate that crop anticoagulant activity influences modulation of the blood-pumping frequency to the intestine and significantly affects the feeding efficiency of triatomine spp. on live hosts.     

Feeding damage by Bagrada hilaris (Hemiptera: Pentatomidae) and impact on growth and chlorophyll content of Brassicaceous plant species

Bagrada hilaris Burmeister is an invasive species native to the old world and is currently threatening commercial vegetable production in the southwestern USA. A series of no-choice experiments were conducted to investigate multiple plant responses in six Brassica crops to feeding by B. hilaris. Varying numbers of adults were caged onto cotyledon, 2-true leaf, and 4-true leaf-stage plants of broccoli, green cabbage, red cabbage, cauliflower, kale, and radish for a 48-h infestation period. Feeding damage on leaf surfaces, total leaf area, and relative chlorophyll content on plants of each crop were measured before and after the 48-h infestation period. In addition, dry weights and total leaf area for the 4-leaf-stage plants were measured at 21 days post-infestation to estimate the residual impacts on older plants. In all crops tested, feeding damage increased with greater numbers of B. hilaris adults caged on cotyledon and 2-leaf-stage plants. Significantly more feeding damage occurred on the upper (younger) two leaves than on the lower (older) two leaves of the 4-true leaf plants for all host plants suggesting that B. hilaris feeds preferentially on newer leaf tissue. Significant reductions in leaf area, relative chlorophyll content, and dry weight in all crops indicated negative impacts on plant growth by B. hilaris. Moreover, cotyledon and 2-leaf plants were more severely impacted by B. hilaris-induced injury than the 4-leaf plants, and kale appeared to be less sensitive to B. hilaris feeding than the other five Brassicaceous hosts.     

The location of parasites within their hosts: The influence of host feeding and diet on the dispersion of adults of Nippostrongylus brasiliensis in the intestine of the rat

This paper is the first in a series to experimentally examine factors which determine the location of parasites within their hosts. Nippostrongylus brasiliensis is typically found one third of the way along the small intestine of the rat between days 7 and 12 of a primary infection. When rats were fasted for 2 days, N. brasiliensis became evenly distributed along the intestine and many individuals, mostly females, entered the caecum. Upon being fed again, the parasites were at their ‘normal site’ within 24 h. Experimental diets of α-cellulose, or simple dietary compounds caused the same re-distribution as did normal food. By sectioning the rat intestine when it was full of food and relating the dispersion of worms to the presence of intestinal contents, it was confirmed that N. brasiliensis is found amongst the intestinal contents of its host. The notion of a ‘site’ is critically discussed and the merit of ‘habitat’ is argued because the ‘site’ of N. brasiliensis would appear to be less a ‘place’ but rather a combination of environmental factors.     

The location of parasites within their hosts: the behaviour of Nippostrongylus brasiliensis in the anaesthetised rat

N. brasiliensis have been observed in vivo through the exposed wall of the small intestine of anaesthetised rats. Closed circuit television has been used to record the types of movement and analyses of the images indicate that differences occur. It would appear that these differences are correlated with the feeding regime of the host and could thus lead to habitat selection. A constantly changing picture of behavioural events emerges.     

Surfactant Protein-D Is Essential for Immunity to Helminth Infection

Pulmonary epithelial cell responses can enhance type 2 immunity and contribute to control of nematode infections. An important epithelial product is the collectin Surfactant Protein D (SP-D). We found that SP-D concentrations increased in the lung following Nippostrongylus brasiliensis infection; this increase was dependent on key components of the type 2 immune response. We carried out loss and gain of function studies of SP-D to establish if SP-D was required for optimal immunity to the parasite. N. brasiliensis infection of SP-D-/- mice resulted in profound impairment of host innate immunity and ability to resolve infection. Raising pulmonary SP-D levels prior to infection enhanced parasite expulsion and type 2 immune responses, including increased numbers of IL-13 producing type 2 innate lymphoid cells (ILC2), elevated expression of markers of alternative activation by alveolar macrophages (alvM) and increased production of the type 2 cytokines IL-4 and IL-13. Adoptive transfer of alvM from SP-D-treated parasite infected mice into naïve recipients enhanced immunity to N. brasiliensis. Protection was associated with selective binding by the SP-D carbohydrate recognition domain (CRD) to L4 parasites to enhance their killing by alvM. These findings are the first demonstration that the collectin SP-D is an essential component of host innate immunity to helminths.     

Nippostrongylus brasiliensis and Nematodirus battus: Changes in numbers and weight during the course of infection

Nematodirus battus has been shown to be subject to a “self-cure” mechanism when lambs 6 weeks of age are infected with 60,000 larvae. It is proposed that this self-cure phenomenon is an immune response similar to that which occurs in rats infected with Nippostrongylus brasiliensis. Changes in the weight of adult Nematodirus sp. and Nippostrongylus sp. occur over the period of an infection in their hosts. Females of both species from high-dose infections increased in weight up to that time postinfection where most adult worms were present, then decreased in weight with the onset of rejection from the host. Male Nematodirus sp. showed a similar pattern of weight change to that of females but male Nippostrongylus sp. maintained a steady weight throughout the infection. The consequences of these changes in weight are discussed with relevance to expression of enzyme activities of the nematodes on a weight of individual nematode basis.     

Neutral lipids of Aedes aegypti and Aedes albopictus cells cultured in vitro.

Aedes aegypti feeding on chickens infected with Plasmodium gallinaceum take less blood and lay fewer eggs than those feeding on uninfected hosts. Both activities show an inverse correlation with the degree of parasitemia. Mosquitoes feeding on infected chickens ingest blood in amounts directly proportional to the length of time spent on the hosts, whereas there is no relationship between host contact and blood meal size for mosquitoes feeding on uninfected hosts. Feeding and probing choice experiments demonstrate that infected chickens are less attractive to Aedes aegypti than uninfected chickens.     

Eimeria nieschulzi: glucose absorption in infected rats

Liquid scintillation spectrophotometry was employed to determine absorption of ³H-glucose by rats infected with the coccidium Eimeria nieschulzi. In vivo studies showed increased uptake of label into small intestinal tissue and hepatic portal plasma at 3 days postinoculation and decreased absorption at 8 days postinfection compared to uninfected control animals. Observations of tissues incubated in labeled glucose in vitro confirm in vivo findings of increased uptake early in infection and malabsorption coinciding with the observation of clinical disease symptoms.     

Effect of intestinal erythrocyte agglutination on the feeding performance of Triatoma brasiliensis (Hemiptera: Reduviidae)

Triatoma brasiliensis is an important vector of Trypanosoma cruzi in Brazil. The feeding efficiency on its hosts depends on several parameters including the maintenance of the ingested blood at low viscosity, which could be modulated by the anterior midgut (crop) anticoagulant and haemagglutinant activities. In the present study, we characterized T. brasiliensis crop haemagglutination activity and evaluated its importance in the feeding process. Soluble crop contents (SCC) of T. brasiliensis were able to agglutinate rat, mouse and rabbit eryhtrocytes, but had no activity on cattle and Thrichomys apereoides, a rodent species commonly associated with T. brasiliensis in the wild. The haemagglutination was characterized by the immediate formation of several clusters of erythrocytes connected by flexible elastic-like fibers. The feeding efficiency of T. brasiliensis on rat (agglutinated by SCC) was almost double that from T. apereoides (not agglutinated by SCC). The influence of haemagglutination on feeding was confirmed by artificially feeding bugs on a diet composed of cattle or rat erythrocytes. The bugs fed on cattle erythrocytes had lower ingestion rates in comparison to those fed on rats. The results indicate that, in addition to other parameters, haemagglutination brought about by SCC has an important role in the feeding efficiency of T. brasiliensis.     

Immunity against trichostrongylus colubriformis infection in guinea-pigs and sheep: Some comparisons with Nippostrongylus brasiliensis infection in the rat

In Nippostrongylus brasiliensis infection in rats there is evidence that antibodies ‘damage’ the worms rendering them susceptible to other components of the host immune response. Some of the experiments from which this evidence was obtained were repeated in Trichostrongylus colubri-formis infections in guinea-pigs and sheep.Lesions similar to those described in ‘damaged’ N. brasiliensis were present in T. colubriformis, but unlike damaged N. brasiliensis, damaged T. colubriformis were not rapidly expelled from the intestine of either normal or adoptively immunized hosts. Other differences between the two infections included the failure of serum from T. colubriformis-immune donors either to regularly transfer immunity against infection to non-immune recipients or to endow additional immunity on recipients of lymphoid cells from immune donors.It is suggested that the results might reflect differences between the manner in which guinea-pigs and rats achieve immune expulsion of gastro-intestinal nematode parasites.     

INSEMINATION AND EGG PRODUCTION OF MOUSE-ADAPTED NIPPOSTRONGYLUS BRASILIENSIS (NEMATODA)

Average egg output per individual female Nippostrongylus was increased as the helminth density approached 148 worms per host. After this number, the individual production by females decreased, but was compensated for by the increased helminth density. Egg release, based on fecal recovery, began at 5 days postinfection. The period of maximum egg production occurred earlier with increased numbers of intestinal adults during the preimmune period in the host. A male ratio of 9.1% yielded fecal egg values that were equivalent to the normal levels with 31–44% males in a natural laboratory infection. Immunosuppression of the mouse host revealed cyclic peaks in fecal egg production. The peak intervals were dependent on helminth density, based on worm recovery at 29 days postinfection.

At 105 h after subcutaneous inoculation, 17% of the female N. brasiliensis were inseminated while 81% were mated by 110 h. Intestinal temperature showed no significant effect on female insemination. Examination of intestinal sections also revealed no distinct influence on the time that was required for a 50% insemination level. Observation of egg production by females in vitro showed that 2.2 eggs were released in a ½-h period.     

Contrasting patterns of ovarian development and oogenesis in two sympatric host-feeding parasitoids,Diglyphus isaea and Neochrysocharis formosa (Hymenoptera: Eulophidae)

Diglyphus isaea (Walker) and Neochrysocharis formosa (Westwood) (Hymenoptera: Eulophidae) are common idiobiont parasitoids of leafminers attacking vegetable crops. They exhibit differing levels of synovigeny, and host feeding enhances their fecundity and longevity. The reproductive systems of these two parasitoids are typical of hymenopteran eulophids, consisting of two ovaries, each usually comprising three polytrophic meroistic ovarioles. Diglyphus isaea possesses two obvious oviduct accessory glands, which are absent in N. formosa. Both parasitoids underwent oosorption when starved, while feeding on host larvae promoted oogenesis and egg maturation. In both, oogenesis and vitellogenesis commenced on the first day of the pupal stage rather than after eclosion. Formation of ovarioles in D. isaea commenced 1 day earlier than in N. formosa. Mature eggs were rarely observed in ovaries of newly emerged D. isaea, but usually a few were present in N. formosa. When hosts (second–third instar Liriomyza sativae larvae) were provided, the number of mature eggs in D. isaea ovaries initially increased and then stabilized, while in N. formosa, the number first increased and then decreased. Diglyphus isaea had fewer but larger eggs than N. formosa did. Thus, synovigenic divergence begins at the pupal stage and may result in different life-history traits of adults.     

Amblyomma americanum (L): Tick macrophage migration inhibitory factor peptide immunization lengthens lone star tick feeding intervals in vivo

Immunizations of New Zealand White rabbits with specific macrophage migration inhibitory factor (MIF) tick peptide (PEP) produced circulating anti-tick PEP antibodies in the hosts. Antibody titers of greater than 1:5000 to tick MIF peptide were observed for crude sera from PEP-immunized rabbits. PEP- and BSA-vaccinated rabbits were infested with Amblyomma americanum adults. Feeding intervals, female weights, egg masses and percent egg hatch were measured for ticks feeding on control and immunized hosts. Feeding intervals were significantly lengthened to 13.3 days for PEP-vaccinated hosts compared to BSA-vaccinated controls at 12.4 days, while female engorgement weights and egg masses were unchanged. By immunizing hosts using specific tick PEP, we were able to alter the length of time the ticks fed on their hosts.     

Adult diet affects the life history and host-killing behavior of a host-feeding parasitoid

Neochrysocharis formosa (Westwood), an important biocontrol agent of agromyzid leafminers worldwide, is a host-feeding, idiobiont parasitoid. Female wasps have three types of host-killing behaviors: reproductive (parasitism), non-reproductive host feeding (host feeding), and host stinging without oviposition or feeding (host stinging). In this study, we compared the life history and host-killing behaviors of female parasitoids under four adult diets: starvation, hosts only, hosts plus honey (10% w/v honey solution), and honey only. Furthermore, we analyzed the host-feeding and oviposition preferences of adult females in the hosts-only and hosts-plus-honey treatments. Female parasitoids feeding on hosts had significantly increased longevity, higher fecundity, more host-stinging events, and caused a higher total host mortality than parasitoids in the starvation treatment. The honey supplement significantly increased longevity, fecundity, host-stinging events, and total host mortality, as well as average daily fecundity, but did not alter host-feeding events, host-stinging events, or daily total host mortality. However, the honey supplement did reduce the number of daily host-feeding events and induced a shift toward oviposition. Finally, we found that the non-reproductive host killing caused by host feeding and host stinging enhanced the control potential of N. formosa. These results should contribute to a better understanding of the biocontrol efficiency of destructive host feeders.     

Neutrophil Extracellular Traps Identification in Tegumentary Lesions of Patients with Paracoccidioidomycosis and Different Patterns of NETs Generation In Vitro

Paracoccidioidomycosis (PCM) is a systemic mycosis, endemic in most Latin American countries, especially in Brazil. It is caused by the thermo-dimorphic fungus of the genus Paracoccidioides (Paracoccidioides brasiliensis and Paracoccidioides lutzii). Innate immune response plays a crucial role in host defense against fungal infections, and neutrophils (PMNs) are able to combat microorganisms with three different mechanisms: phagocytosis, secretion of granular proteins, which have antimicrobial properties, and the most recent described mechanism called NETosis. This new process is characterized by the release of net-like structures called Neutrophil Extracellular Traps (NETs), which is composed of nuclear (decondensed DNA and histones) and granular material such as elastase. Several microorganisms have the ability of inducing NETs formation, including gram-positive and gram-negative bacteria, viruses and some fungi. We proposed to identify NETs in tegumentary lesions of patients with PCM and to analyze the interaction between two strains of P. brasiliensis and human PMNs by NETs formation in vitro. In this context, the presence of NETs in vivo was evidenced in tegumentary lesions of patients with PCM by confocal spectrum analyzer. Furthermore, we showed that the high virulent P. brasiliensis strain 18 (Pb18) and the lower virulent strain Pb265 are able to induce different patterns of NETs formation in vitro. The quantification of extracellular DNA corroborates the idea of the ability of P. brasiliensis in inducing NETs release. In conclusion, our data show for the first time the identification of NETs in lesions of patients with PCM and demonstrate distinct patterns of NETs in cultures challenged with fungi in vitro. The presence of NETs components both in vivo and in vitro open new possibilities for the detailed investigation of immunity in PCM.