Dissecting the impact of protein versus energy host nutrition on the expression of immunity to gastrointestinal parasites during lactation

Many mammals exhibit a periparturient relaxation of previously established immune responses (PPRI) to gastrointestinal nematodes culminating in increased worm burdens. It has been suggested that the extent of PPRI may have a nutritional basis as it is considerably augmented when protein supply is scarce. Subsequent studies have shown that increased dietary protein intake can ameliorate this phenomenon. However, this effect is often confounded with increased food intake and thus increased energy levels. Herein, we aimed to dissect the effects of protein and energy nutrition on the immune status and resistance to re-infection with gastrointestinal nematodes in the periparturient host. The lactating, Nippostrongylus brasiliensis re-infected rat was utilised as an established model for mammalian PPRI. Experimental animals were assigned to restricted feeding regimens designed to achieve four pre-determined levels of crude protein (CP) at one of two levels of metabolisable energy (ME) and parasitological and immunological measurements taken at either day 6 or day 9 post re-infection. We clearly show that increased supply of dietary CP, but not increased dietary ME, significantly reduced worm burdens. The increased magnitude of worm expulsion with increased dietary CP supply strongly correlated with mucosal mast cell accumulation in the small intestine. In addition, increased CP and not ME supply increased mucosal eosinophil numbers. Furthermore, increased CP led to higher levels of total IgG at high ME only and there were interactive effects of CP and ME on serum levels of IgG1 and IgG2a. Perhaps surprisingly, CP nutrition did not affect expression of either Th1 (IFN-γ) or Th2 (IL-4, IL-13) cytokines in the mesenteric lymph nodes. These data emphasise the role of immunonutrition, and particularly dietary protein, in combating infectious disease such as gastrointestinal parasitism.     

The effect of vaccinating infection during pregnancy and dietary protein supply on the peri-parturient immune response of sheep to infection with Teladorsagia circumcincta and Trichostrongylus colubriformis larvae

It is well established that dietary protein supply can influence the peri-parturient breakdown of immunity to nematode parasites but there is no information on the importance of exposure to nematode larvae during pregnancy for this response. We investigated this by exposing housed pregnant sheep, scanned as carrying two lambs, to a vaccinating infection with a trickle mixed infection of Teladorsagia circumcincta and Trichostrongylus colubriformis larvae (L3) or to no infection during weeks − 9 to − 4 relative to parturition. At the beginning of week − 3 all sheep were treated with anthelmintic to remove any vaccinating worm burden and from week − 2 to week +6 received a trickle challenge infection with the same nematodes. Within each vaccinating treatment there were two nutritional treatments (no. = 20 per subgroup) designed to provide 1.5 or 1.0 and 1.3 or 0.8 of metabolisable protein (MP) requirement during pregnancy and lactation, respectively. Five ewes were necropsied during weeks +1 and +3 to measure worm burdens and mucosal inflammatory cells and the remainder maintained until week +6. Serum levels of total, IgA and IgE antibodies against L3 antigen of each nematode were measured.Scanning errors and lamb losses resulted in some ewes carrying and/or rearing only one lamb. Numbers of lambs reared was therefore introduced as a treatment effect. Vaccinating infection delayed the peri-parturient rise in faecal egg count (FEC) by an average of 2 weeks but its effect on FEC during the first 6 weeks of lactation was smaller and less persistent than that of dietary MP supply and single- v. twin-suckling.Populations of both nematodes were lower in association with high MP supply, vaccination and single suckling. These changes were associated with increases in numbers of mucosal mast cells (MMC) as a result of both increased MP supply and vaccination. Evidence for a more rapid return of host ability to limit populations of the abdominal nematode T. circumcincta than of the intestinal nematode T. colubriformis was associated with fewer eosinophils and more globule leucocytes (GL) in abomasal than in intestinal tissue.None of the serum antibody isotypes was affected by dietary protein supply. Total and IgA antibodies were maintained by a current larval (vaccinating) intake. IgA titres, however, increased progressively during pregnancy, especially in twin-bearing ewes. IgE titres appeared to be sensitive primarily to the reproductive cycle itself, peaking around parturition.This work supports the conclusion that availability of MP supply influences the recruitment and activity of cells of the immune armoury of the gastro-intestinal tract to nematode parasites. The precise outcome may differ with site and/or nematode species.     

Preferential infectivity of entomopathogenic nematodes in an envenomed host

Entomopathogenic nematodes and parasitoid wasps are used as biological control agents for management of insect pests such as the Indian meal moth, Plodia interpunctella. The parasitoid wasp Habrobracon hebetor injects a paralytic venom into P. interpunctella larvae before laying eggs. A previous study reported that the entomopathogenic nematode Heterorhabditis indica preferentially infects P. interpunctella that have been envenomed by H. hebetor while results in this study showed a similar preference by the entomopathogenic nematode, Steinernema glaseri. We therefore tested four hypotheses for why nematode infection rates are higher in envenomed hosts: (1) elevated CO₂ emission from envenomed hosts attracts nematodes, (2) paralysis prevents hosts from escaping nematodes, (3) volatile chemicals emitted from envenomed hosts attract nematodes and increase infection, and (4) reduced immune defenses in envenomed hosts increase nematode survival. Results showed that envenomed P. interpunctella larvae emitted lower amounts of CO₂ than non-envenomed larvae. Physical immobilization of P. interpunctella larvae did not increase infection rates by S. glaseri but did increase infection rates by H. indica. Emissions from envenomed hosts were collected and analyzed by thermal desorption gas chromatography/mass spectrometry. The most abundant compound, 3-methyl-3-buten-1-ol, was found to be an effective cue for S. glaseri attraction and infection but was not an effective stimulus for H. indica. Envenomed P. interpunctella exhibited a stronger immune response toward nematodes than non-envenomed hosts. Altogether, we conclude that different mechanisms underlie preferential infection in the two nematode species: host immobilization for H. indica and chemical cues for S. glaseri.     

Intraspecific Epitopic Variation in a Carbohydrate Antigen Exposed on the Surface of Trichostrongylus colubriformis Infective L3 Larvae

The carbohydrate larval antigen, CarLA, is present on the exposed surface of all strongylid nematode infective L3 larvae tested, and antibodies against CarLA can promote rapid immune rejection of incoming Trichostrongylus colubriformis larvae in sheep. A library of ovine recombinant single chain Fv (scFv) antibody fragments, displayed on phage, was prepared from B cell mRNA of field-immune sheep. Phage displaying scFvs that bind to the surface of living exsheathed T. colubriformis L3 larvae were identified, and the majority of worm-binding scFvs recognized CarLA. Characterization of greater than 500 worm surface binding phage resulted in the identification of nine different anti-CarLA scFvs that recognized three distinct T. colubriformis CarLA epitopes based on blocking and additive ELISA. All anti-CarLA scFvs were specific to the T. colubriformis species of nematode. Each of the three scFv epitope classes displayed identical Western blot recognition patterns and recognized the exposed surface of living T. colubriformis exsheathed L3 larvae. Surprisingly, each of the anti-CarLA scFvs was able to bind to only a subset of worms. Double-labelling indirect immunofluorescence revealed that the three classes of anti-CarLA scFvs recognize distinct, non-overlapping, T. colubriformis sub-populations. These results demonstrate that individual T. colubriformis L3 larvae display only one of at least three distinct antigenic forms of CarLA on their surface at any given time, and suggest that antigenic variation within CarLA is likely a mechanism of immune evasion in strongylid nematodes.     

Effect of nematode Panagrellus redivivus density on growth, survival, feed consumption and carcass composition of bighead carp Aristichthys nobilis (Richardson) larvae

The study aimed to determine the optimum density of free‐living nematodes in feeding bighead carp, Aristichthys nobilis, larvae. In the first experiment, carp stocked at 25 larvae L^?1 were fed varying levels of nematodes (50, 75, 100, 125 and 150 per ml) twice a day for 21 days from the start of exogenous feeding. Final body weight was significantly higher (P < 0.05) in larvae fed 125 and 150 nematodes per ml than in those fed 50 and 75 per ml, but survival was low (61.8 and 63.6%, respectively). Survival rate was highest in larvae fed 100 nematodes ml^?1 (81.3%). Carcass analysis showed that larvae fed 125 and 150 nematodes ml^?1 had significantly lower body protein and higher body lipid than those fed other nematode densities. Carcass ash was similar for larvae fed 50–100 nematodes ml^?1 but it decreased significantly at the higher nematode densities. Carp larvae in a subsequent experiment were given 50, 75 and 100 nematodes ml^?1 per feeding. Newly hatched Artemia was the control feed. Nematode consumption and growth of the larvae were determined. Larvae were sampled at intervals of 2–4 days and the nematodes in the gut were counted and measured. At each nematode density, the number of nematodes present in the gut of the larvae increased significantly with time. At each sampling day, the number of nematodes in the gut did not differ significantly among treatments (P > 0.05) although it tended to increase with nematode density at day 2 and day 4 but decrease at day 7 onward. The carp larvae consumed significantly shorter nematodes on day 2 and day 4 than on the succeeding sampling days regardless of nematode density. However, the length of nematodes in the gut of the larvae did not differ significantly among the nematode densities. The final body weight of larvae increased with increasing nematode density. The body weight of larvae fed 100 nematodes ml^?1 did not differ significantly from that of larvae given Artemia nauplii. Results show that bighead carp larvae should be fed 100 free‐living nematodes per ml at each feeding time.     

Serine Protease(s) Secreted by the Nematode Trichuris muris Degrade the Mucus Barrier

The polymeric mucin component of the intestinal mucus barrier changes during nematode infection to provide not only physical protection but also to directly affect pathogenic nematodes and aid expulsion. Despite this, the direct interaction of the nematodes with the mucins and the mucus barrier has not previously been addressed. We used the well-established Trichuris muris nematode model to investigate the effect on mucins of the complex mixture of immunogenic proteins secreted by the nematode called excretory/secretory products (ESPs). Different regimes of T. muris infection were used to simulate chronic (low dose) or acute (high dose) infection. Mucus/mucins isolated from mice and from the human intestinal cell line, LS174T, were treated with ESPs. We demonstrate that serine protease(s) secreted by the nematode have the ability to change the properties of the mucus barrier, making it more porous by degrading the mucin component of the mucus gel. Specifically, the serine protease(s) acted on the N-terminal polymerising domain of the major intestinal mucin Muc2, resulting in depolymerisation of Muc2 polymers. Importantly, the respiratory/gastric mucin Muc5ac, which is induced in the intestine and is critical for worm expulsion, was protected from the depolymerising effect exerted by ESPs. Furthermore, serine protease inhibitors (Serpins) which may protect the mucins, in particular Muc2, from depolymerisation, were highly expressed in mice resistant to chronic infection. Thus, we demonstrate that nematodes secrete serine protease(s) to degrade mucins within the mucus barrier, which may modify the niche of the parasite to prevent clearance from the host or facilitate efficient mating and egg laying from the posterior end of the parasite that is in intimate contact with the mucus barrier. However, during a T_H2-mediated worm expulsion response, serpins, Muc5ac and increased levels of Muc2 protect the barrier from degradation by the nematode secreted protease(s).     

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.     

Assaying Predatory Feeding Behaviors in Pristionchus and Other Nematodes

This protocol provides multiple methods for the analysis and quantification of predatory feeding behaviors in nematodes. Many nematode species including Pristionchus pacificus display complex behaviors, the most striking of which is the predation of other nematode larvae. However, as these behaviors are absent in the model organism Caenorhabditis elegans, they have thus far only recently been described in detail along with the development of reliable behavioral assays ¹. These predatory behaviors are dependent upon phenotypically plastic but fixed mouth morphs making the correct identification and categorization of these animals essential. In P. pacificus there are two mouth types, the stenostomatous and eurystomatous morphs ², with only the wide mouthed eurystomatous containing an extra tooth and being capable of killing other nematode larvae. Through the isolation of an abundance of size matched prey larvae and subsequent exposure to predatory nematodes, assays including both "corpse assays" and "bite assays" on correctly identified mouth morph nematodes are possible. These assays provide a means to rapidly quantify predation success rates and provide a detailed behavioral analysis of individual nematodes engaged in predatory feeding activities. In addition, with the use of a high-speed camera, visualization of changes in pharyngeal activity including tooth and pumping dynamics are also possible.     

Susceptibility of early larval stages of Pseudaletia unipuncta and Spodoptera exigua (Lepidoptera: Noctuidae) to the entomogenous nematode Steinernema feltiae (Rhabditida: Steinernematidae)

Early stages (neonate to 7- or 8-day-old larvae) of Spodoptera exigua and Pseudaletia unipuncta were exposed to the entomogenous nematode, Steinernema feltiae, at concentrations of 0, 10, 25, 60, 100, or 200 nematodes per larva. Larvae of both species were susceptible to nematode infections. However, neonate larvae of S. exigua were significantly less susceptible to nematode infection than 3- or 8-day-old larvae at or above 50 nematodes per larva. Mortalities of neonate larvae exposed to 50 or more nematodes ranged from 68 to 74% while mortalities of 3- and 8-day-old larvae ranged from 91 to 100%. The results with P. unipuncta showed similar trends as described for S. exigua, albeit at a lower mortality level and usually with no statistical differences. Mortalities of neonate larvae exposed to 50 or more nematodes ranged from 34 to 44% while mortalities of 7-day-old larvae ranged from 32 to 91%.     

For the Insect Pathogen Photorhabdus luminescens, Which End of a Nematode Is Out?

The nematode Heterorhabditis bacteriophora is the vector for transmitting the entomopathogenic bacterium Photorhabdus luminescens between insect larvae. The dauer juvenile (DJ) stage nematode selectively retains P. luminescens in its intestine until it releases the bacteria into the hemocoel of an insect host. We report the results of studying the transmission of the bacteria by its nematode vector. Cells of P. luminescens labeled with green fluorescent protein preferentially colonized a region of the DJ intestine immediately behind the basal bulb, extending for various distances toward the anus. Incubation of DJ nematodes in vitro in insect hemolymph induced regurgitation of the bacteria. Following a 30-min lag, the bacteria migrated in a gradual and staggered movement toward and ultimately exited the mouth. This regurgitation reaction was induced by a low-molecular-weight, heat- and protease-stable, anionic component present in arthropod hemolymph and in supernatants from insect cell cultures. Nematodes anesthetized with levamisole or treated with the antihelmenthic agent ivermectin did not release their bacteria into hemolymph. The ability to visualize P. luminescens in the DJ nematode intestine provides the first clues to the mechanism of release of the bacteria during infection of insect larvae. This and the partial characterization of a component of hemolymph triggering release of the bacteria render this fascinating example of both a mutualistic symbiosis and disease transmission amenable to future genetic and molecular study.     

Nippostrongylus brasiliensis and Haemonchus contortus: Function of the excretory ampulla of the third-stage larva

An improved technique for measuring the water content of nematodes is described using an electronic interferometer. Changes in phase of a laser beam passing through a known pathlength of the nematode have been used to measure the refractive index and hence the water content and relative volume of the animal. Third-stage larvae of Nippostrongylus brasiliensis and Haemonchus contortus which possess an excretory ampulla, differed from second-stage larvae lacking this ampulla in requiring a greater fall in the osmolarity of artificial tap water before there was a significant increase in their water content. Increases in the pulsation frequency of the ampulla also occurred in less hypotonic solutions than those required to increase the water content of the third-stage larvae. The ampulla pulsation frequency of third-stage larvae of N. brasiliensis increased after locomotor activity in hypotonic tap water and locomotory wave frequency of third-stage larvae of N. brasiliensis was independent of the extent of hypotonicity for a range of solutions that reduced wave propogation by its second-stage larva. The results suggest that the ampulla is an adaptation to hypotonic conditions favouring a volume homeostasis that is required for optimal locomotor activity of the third-stage infective larvae of these nematodes.     

Biological Control of the Leafminer Liriomyza trifolii (Burgess): Implications for Intraguild Predation between Diglyphus begini Ashmead and Steinernema carpocapsae (Weiser)

Studies were conducted on the combined use of the eulophid parasitoid wasp Diglyphus begini Ashmead and the entomopathogenic nematode Steinernema carpocapsae (Weiser) for control of the leafminer Liriomyza trifolii (Burgess) on chrysanthemums. Several factors indicated that these two agents were suitable for combined use: adult D. begini were not susceptible to nematode infection, leafminer larvae parasitized by the wasp were less susceptible to nematode infection, adult wasps detected and tended to avoid ovipositing on nematode-infected leafminer larvae, nematode-infected larvae served as host-feeding sources for the adult wasps, and nematodes showed equal orientation toward paralyzed/parasitized leafminer larvae and healthy leafminer larvae. However, interspecific interference and intraguild predation (IGP) between the agents were found. Infection of D. begini larval stages by nematodes was seen in petri dishes and in intact leaf mines. The presence of nematodes in mines with wasp eggs decreased the chance of wasp survival to adulthood. IGP may be minimized through proper timing of natural enemy releases.     

Effect of the Entomogenous Nematode Nemplectana carpocapsae on the Tachinid Parasite Compsilura concinnata (Diptera: Tachinidae)

The entomogenous nematode Neoaplectana carpocapsae and its associated bacterium, Xenorhabdus nematophilus, could not infect the pupal stage of the tachinid Compsilura concinnata through the puparium. N. carpocapsae had an adverse effect on 1-, 2- and 3-day-old C. concinnata larvae within the armyworm host in petri dish tests. All 1-day-old larvae treated with nematodes died in their hosts, whereas 61% and 69% of 2- and 3-day-old larvae treated with nematodes, respectively, died. However, the survivors developed to adults. Nine to thirty-seven percent of adult tachinids which emerged from nematode-treated soil (50 nematodes/cm²) were infected with N. carpocapsae. The nematode adversely affects C. concinnata directly by the frank infection of the tachinid and indirectly by causing the premature death of the host which results in tachinid death.     

Manipulation of environment to facilitate use of nematodes in biocontrol of insects

The pre- and postparasitic phases of entomophilic nematodes are exposed to different microenvironments which should be changed in order to facilitate the use of nematodes as biological control agents of insects. The mixing of “thick water” with nematodes prior to spraying on exposed surfaces retards nematode desiccation and so increases the mortality of forest and agricultural insect pests. Aquatic larvae of Simulium vittatum and of Culiseta inornata feed fastest at 20 and 25 C, respectively, and so engulf more infective larvae of Neoaplectana carpocapsae (DD-136). When exposed to N. carpocapsae (DD-136) on potato foliage, the percentage of encapsulated nematodes in the haemocoel of adult Leptinotarsa decemlineata was less than in the third stage nymphs. The percentage of unencapsulated, dead nematodes was greatest in the nymphal stages. The value of baits and phagostimulants to increase the insects' oral uptake of infective nematodes and of the manipulation of the insect's physiological processes so as to modify its susceptibility to nematodes is discussed.     

A gastrointestinal nematode in pregnant and lactating mice alters maternal and neonatal microbiomes

The maternal microbiome is understood to be the principal source of the neonatal microbiome but the consequences of intestinal nematodes on pregnant and lactating mothers and implications for the neonatal microbiome are unknown. Using pregnant CD1 mice infected with Heligmosomoides bakeri, we investigated the microbiomes in maternal tissues (intestine, vagina, and milk) and in the neonatal stomach using MiSeq sequencing of bacterial 16S rRNA genes. Our first hypothesis was that maternal nematode infection altered the maternal intestinal, vaginal, and milk microbiomes and associated metabolic pathways. Maternal nematode infection was associated with increased beta-diversity and abundance of fermenting bacteria as well as Lactobacillus in the maternal caecum 2 days after parturition, together with down-regulated carbohydrate, amino acid and vitamin biosynthesis pathways. Maternal nematode infection did not alter the vaginal or milk microbiomes. Our second hypothesis was that maternal infection would shape colonization of the neonatal microbiome. Although the pup stomach microbiome was similar to that of the maternal vaginal microbiome, pups of infected dams had higher beta-diversity at day 2, and a dramatic expansion in the abundance of Lactobacillus between days 2 and 7 compared with pups nursing uninfected dams. Our third hypothesis that maternal nematode infection altered the composition of neonatal microbiomes was confirmed as we observed up-regulation of several putatively beneficial microbial pathways associated with synthesis of essential and branched-chain amino acids, vitamins, and short-chain fatty acids. We believe this is the first study to show that a nematode living in the maternal intestine is associated with altered composition and function of the neonatal microbiome.     

Neutrophils clear bacteria associated with parasitic nematodes augmenting the development of an effective Th2-type response

Infection with the parasitic nematode Nippostrongylus brasiliensis induces a potent Th2 response; however, little is known about early stages of the innate response that may contribute to protective immunity. To examine early events in this response, chemokine expression in the draining lymph node was examined after N. brasiliensis inoculation. Pronounced increases of several chemokines, including CCL2, were observed. Compared with wild-type mice, elevations in a Gr-1bright population in the draining lymph node was significantly decreased in CCL2-/- mice after N. brasiliensis inoculation. Further flow cytometric and immunofluorescent analysis showed that in wild-type mice, Gr-1+ cells transiently entered and exited the draining lymph node shortly after N. brasiliensis inoculation. The Gr-1bright population was comprised of neutrophils expressing TGF-beta and TNF-alpha. Following Gr-1+ cell depletion, N. brasiliensis infection resulted in transient, but significantly increased levels of IFN-gamma, increased serum IgG2a, reduced Th2 cytokines and serum IgE, greatly increased mortality, and delayed worm expulsion. Furthermore, bacteria were readily detected in vital organs. Infection of Gr-1+ cell-depleted mice with N. brasiliensis larvae that were pretreated with antibiotics prevented bacterial dissemination, Th1 inflammatory responses, and decreases in host survival. This study indicates that parasitic nematodes can be an important vector of potentially harmful bacteria, which is typically controlled by CCL2-dependent neutrophils that ensure the optimal development of Th2 immune responses and parasite resistance.