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The effectiveness of a single dose of doramectin pour-on in the control of gastrointestinal nematodes in yearling stocker cattle.

Three field studies were conducted to determine the efficacy of a single dose of doramectin pour-on in the control of gastrointestinal nematode infections in yearling stocker calves on pasture. These 140-day studies were carried out between October 1995 and March 1996 in Tennessee (TN), between January and June 1997 in Louisiana (LA), and between May and September 1997 in Wisconsin (WI). Calves with patent nematode infections were equally allocated to treatments (doramectin pour-on, at 500 microg/kg body weight or untreated control) and pastures as randomized complete-block designs (LA and TN studies) or completely at random (WI study). There were six pasture replicates per treatment at each site, with each pasture replicate accommodating six calves at the TN site (36 calves per treatment), five calves at the LA site (30 calves per treatment), and seven calves at the WI site (42 calves per treatment). Fecal samples for nematode egg counts were collected on Day 0, and at 28-day intervals thereafter. Body weights were recorded on Day 0 and at 28-day intervals until study termination. Nematode egg output of the doramectin-treated groups was reduced over the entire grazing period compared to those in the untreated control groups, resulting in average daily weight-gain advantages of 0.055 kg (p < or = 0.05) for the TN study, 0.208 kg (p < or = 0.05) for the LA study, and 0.116 kg (p < or = 0.05) for the WI study.

Administration, Topical↗

Antigen-presenting cells recruited by Brugia malayi induce Th2 differentiation of naïve CD4(+) T cells.

A key feature of nematode infection is a bias towards a type 2 immune response. To investigate the role that antigen-presenting cells (APC) may play in promoting this bias, we used adherent peritoneal exudate cells (PEC) recruited in response to the filarial nematode Brugia malayi, to stimulate naïve T cells from pigeon cytochrome c (PCC)-specific TCR transgenic (PCC-tg) mice. Although the proliferation of PCC-tg T cells was inhibited by parasite- induced PEC during primary stimulation, they proliferated normally upon secondary stimulation and were not rendered anergic. However, PCC-tg T cells primed by suppressive APC differentiated into IL-4-producing Th2 cells upon secondary stimulation instead of IFN-gamma-producing Th1 cells, as has been previously described. Studies with carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled cells indicated that Th2 differentiation was associated with the inhibition of (or failure to stimulate) IFN-gamma production during primary stimulation. Interestingly, blocking antibodies against TGF-beta (but not IL-10) restored the differentiation of IFN-gamma-producing Th1 cells. Identical results with CFSE-labeled cells were obtained using purified IL-4-dependent F4/80(+) macrophages. These data indicate that T cells exposed to parasite-induced alternatively activated macrophages are driven towards Th2 differentiation. This may be an important factor in the Th2 bias that accompanies nematode infection.

Animals↗

Haemonchus contortus GA1 antigens: related, phospholipase C-sensitive, apical gut membrane proteins encoded as a polyprotein and released from the nematode during infection.

It was previously shown that the Haemonchus contortus apical gut surface proteins p46, p52, and p100 induced protective immunity to challenge infections in goats. Here, it is shown that the three proteins are all encoded by a single gene (GA1) and initially expressed in adult parasites as a polyprotein (p100GA1). p46GA1 and p52GA1 are related proteins with 47% sequence identity, including a cysteine-containing region, which appears to confer secondary structure to these proteins, and a region with sequence similarity to bacterial Tolb proteins. GA1 protein expression is regulated during the life cycle at the level of transcript abundance. Only p52GA1 has characteristics of a glycosylinositolphospholipid membrane-anchored protein. However, both p46GA1 and p52GA1 were released from the gut membrane by phosphatidylinositol specific-phospholipase C, suggesting that p46GA1 membrane association depends on interactions with a glycosylinositolphospholipid gut membrane protein. Finally, GA1 proteins occur in abomasal mucus of infected lambs, demonstrating possible presentation to the host immune system during H. contortus infection. The results identify multiple characteristics of the GA1 proteins that should be considered for design of recombinant antigens for vaccine trials and that implicate a series of cellular processes leading to modification and expression of GA1 proteins at the nematode apical gut surface.

Amino Acid Sequence↗

Effects of the repeated distribution of sainfoin hay on the resistance and the resilience of goats naturally infected with gastrointestinal nematodes.

Due to the high prevalence of anthelmintic resistance in goats, the need to explore novel approaches to control nematodes and to reduce the exclusive reliance on chemotherapy is strongly demanded in this host species. In sheep, several studies have shown that the consumption of tannin-rich legume forages was associated with positive effects on host resilience and resistance to parasite infection. In goats, studies on such interactions between tanniferous plants and nematode infections remain few. The objectives of the current study were to examine under natural conditions the effects of consumption of sainfoin hay by goats on the parasite populations and on host resilience. Eighteen adult cull goats naturally infected with Haemonchus contortus, Teladorsagia circumcincta and Trichostrongylus colubriformis were used in the study. At the start of the assay, the goats were allocated into two groups, balanced according to weight and the levels of egg excretion. The two groups grazed separate pastures for 3 months with similar stocking rates. Goats from group S received each month indoors, for 7 days, sainfoin hay and control goats (group C) received hay of ryegrass. The diets in both groups were made isoenergetic and isoproteic and the refusals measured. Individual parasitological and pathophysiological measurements were performed fortnightly in order to compare host resistance and resilience. At the end of the study, five goats per group were necropsied. The distribution of sainfoin was associated with: (1) a higher consumption of hay; (2) significant, lower levels of nematode egg excretion which was associated with a decrease in worm fertility but no change in worm population; however, the number of intestinal worms was reduced by 50% in group S; (3) a better host resilience. In particular, after 2 months of grazing, two control goats died and half of the remaining animals needed to be treated whereas this was not the case in group S. These differences were related to significant changes in pepsinogen and phosphate values (PCV) but not in pepsinogen and phosphate concentrations. These results demonstrate that a repeated distribution of sainfoin hay to grazing goats might be beneficial in regard of pasture contamination and host resilience. They suggest that administration of sainfoin hay might represent a valuable alternative and adjunct to reduce nematode infections in dairy goat flock.

Animal Feed↗

Energy deficits suppress both systemic and gut immunity during infection.

Protein-energy malnutrition and gastrointestinal nematode infections widely coexist in developing countries. Evidence is provided demonstrating the profound impact of dietary energy deficiency on immune function. Energy-restricted (ER) mice infected with a gastrointestinal nematode showed impaired lymphocyte proliferation and reduced production of Th2 cytokines and lower levels of IgE, parasite-specific IgG1, and eosinophils, which led to higher worm burdens and fecundity. We conclude that mild ER, without concurrent protein malnutrition, can modulate protective immunity from (a) activation early during a primary infection to (b) the expression of acquired immunity during reinfection in both systemic and gut-associated lymphoid tissues.

Animals↗

Differences between IL-4R alpha-deficient and IL-4-deficient mice reveal a role for IL-13 in the regulation of Th2 responses.

Allergens and infections with parasitic helminths preferentially induced Th2 immune responses associated with elevated levels of serum immunoglobulin E (IgE) and expansion of eosinophils and mast cells. Interleukin-4 (IL-4) is a key cytokine in the differentiation of naive CD4+ T cells into Th2 cells, which produce a panel of cytokines including IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13 [1] and have been shown to trigger recovery from gastrointestinal nematodes [2]. Nonetheless, mice deficient for IL-4 have been shown to develop residual Th2 responses [3-5] and can expel the nematode Nippostrongylus brasiliensis [6], suggesting that there is a functional equivalent of IL-4 in these processes. IL-13 is a cytokine that shares some, but not all, biological activities with IL-4 [7,8]. There is now compelling evidence that IL-4 and IL-13 share receptor components, including IL-4R alpha and IL-13R alpha 1 [9]. In order to dissect the roles of IL-4 and IL-13 in the regulation of Th2 cells and in the response to nematode infections, we looked for differences between mice deficient for either the IL-4 gene or the IL-4R alpha gene. Unlike IL-4, IL-4R alpha was required for control of N. brasiliensis, and Th2 development during infection--as characterized by cytokine production, GATA-3 and surface CD30 expression--was more severely affected in IL-4R alpha-/- mice than in IL-4-/- mice. Injection of recombinant IL-13 induced worm expulsion in otherwise incompetent RAG2-/- mice. Our results suggest that IL-13 regulates Th2 responses to nematode infection and requires IL-4R alpha.

Animals↗

Feasibility of genus-specific real-time PCR for the differentiation of larvae from gastrointestinal nematodes of naturally infected sheep.

Results of real-time PCR analysis of coproculture third stage larvae (L3) using genus specific TaqMan minor groove binder probes were compared with the results of morphological differentiation of L3 after coprocultured and direct morphological worm differentiation from gastrointestinal samples of eight sheep with naturally acquired nematodes infections. Faecal egg counts prior to postmortem confirmed infections with trichostrongyles with a geometric mean count of 4828 eggs per gram for all sheep. Individual egg counts correlated positively with total worm counts (correlation coefficient 0.794). Five different nematode species and one genus were found in the abomasi and small intestines: Cooperia curticei, Haemonchus contortus, Nematodirus spp., Teladorsagia (Ostertagia) circumcincta, Trichostrongylus axei and Trichostrongylus colubriformis. Coproculture of faecal eggs yielded five of these, Cooperia spp., Haemonchus spp., Ostertagia/Teladorsagia spp. and Trichostrongylus spp. Comparison between morphological L3 and worm differentiation data showed high congruence (94%). The agreement between PCR analysis of L3 after coproculture and direct morphological worm differentiation was 84%. Thus, real-time PCR was found to be suitable as a speedy and reliable diagnostic tool for the assessment of gastrointestinal nematode infections of ruminants in the field.

Animals↗

Health status of primary schoolchildren in Sri Lanka.

OBJECTIVE: To assess health status of 9-10-year old school children in Sri Lanka. DESIGN: A cross-sectional, descriptive study. Schools were selected to obtain a sample representative at national and provincial levels and 20 children were randomly selected from Grade 5 classes in each school. MEASUREMENTS: Children were examined for Bitot's spots and goitre. Height, weight, and visual acuity were measured according to standard procedures. Haemoglobin level was measured using finger-prick blood and a HemoCue meter. Geohelminth infections were quantified by faecal examination using the modified Kato-Katz technique. Height for age Z-scores (HAZ) and body mass index (BMI) were calculated as indicators of nutritional status. RESULTS: Two thousand five hundred and twenty eight children (1351 boys) from 144 schools (140 state schools and four private schools) were examined. Nationally, 15.5% of children were stunted (HAZ lower than -2.0 SD); 52.6% were thin (BMI < 5th centile of age- and sex-matched reference population); 3.1% were overweight (BMI > 85th centile); 12.1% were anaemic; 0.3% had Bitot's spots; 3% had a visible or palpable goitre; 4.6% were shortsighted; and 6.9% had one or more soil-transmitted nematode infection. Among children on whom anthropometry, haemoglobin and faecal examinations were all done, 64.6% (1332/2063) were thin, stunted, anaemic or infected with worms. A much higher proportion of children in the Northern and Eastern provinces had health problems when compared to the other provinces. CONCLUSIONS: The majority of older primary schoolchildren in Sri Lanka are undernourished. Anaemia, vitamin A deficiency, iodine deficiency and soil-transmitted nematode infections affect a much smaller proportion of them.

Child↗

Cytokine and antibody subclass responses in the intestinal lymph of sheep during repeated experimental infections with the nematode parasite Trichostrongylus colubriformis.

The expression of interleukin (IL)-4, IL-5, IL-10, IL-13, TNF-alpha and IFN-gamma genes, and parasite-specific IgM, IgG1, IgG2, IgA and total IgE levels, were monitored daily in intestinal lymph of sheep infected repeatedly with the nematode parasite Trichostrongylus colubriformis. Host genotype had a significant influence on IL-13 gene activity, with resistant-line (R) sheep consistently expressing higher levels of mRNA than susceptible-line (S) sheep. Mean gene expression of IL-13, IL-4 and IFN-gamma did not differ significantly between the first and second nematode challenge. Field-primed R and S as well as field-primed R and naïve S sheep had lower mean gene expression of IL-5 and IL-10, respectively, during the second when compared to primary challenge. Genes for IL-13 and IL-5 were transiently and strongly up-regulated after nematode infection, particularly in animals with previous exposure to nematodes. Genes for TNF-alpha and IFN-gamma were also transiently up-regulated, but to a lesser extent and more typically after primary challenge. Naïve sheep of both genotypes produced relatively little antibody response after primary challenge. A second nematode challenge resulted in large increases in the lymphatic levels of all antibody sub-classes which were significant for adult antigen-specific IgA and larval antigen-specific IgG1. In naïve S line sheep, the larval-specific IgA and IgG2 response appeared delayed when compared to the R line animals. Field-primed R and S line sheep had relatively high lymphatic IgG1 levels prior to experimental infection and these did not change significantly afterwards. These results demonstrate that during nematode infections, the intestinal micro-environment of sheep is transiently skewed towards Th2 cytokine dominance, although IFN-gamma gene expression continues. This response is accompanied by increases of nematode-specific IgG1, IgA, IgG2 and IgM, as well as of total IgE in lymph plasma.

Animals↗

Functional relationship between mast cells and C-sensitive nerve fibres evidenced by histamine H3-receptor modulation in rat lung and spleen.

1. Mast cell populations in rat lung and spleen were characterized by the presence of two specific protease markers, rat mast cell protease I and II, using both histochemical and radioimmunoassay techniques. Three mast cell populations with different size, morphology, and localization were found in lung and spleen and were identified according to the expression of rat mast cell protease I (RMCPI+) or rat mast cell protease II (RMCPII+) or of both proteases (RMCPI/II+). 2. All three mast cell types were in the vicinity of calcitonin-gene-related-peptide-immunoreactive (CGRP+) nerve fibres in controls as well as in rats infected by Nippostrongylus brasiliensis in which a large increase in the number of both RMCPII+ and RMCPI/II+ mast cells was found. Ablation of the CGRP+ fibres by neonatal treatment with capsaicin resulted in a marked increase in the number of RMCPII+ and RMCPI/II+ cells in lung and, even more, in spleen of adult rats. 3. The interaction of mast cells with CGRP+ C-fibres was assessed pharmacologically by evaluation of the effects of histamine H3-receptor ligands known to act on various types of nerve endings, including those of C-fibres. The effects of H3-receptor ligands were assessed in controls, nematode-infected rats and neonatally capsaicinized rats. Mast cell activity was evaluated by measurement of [3H]histamine synthesis from [3H]histidine. In control rats, administration of the H3-receptor agonist (R)-alpha-methylhistamine and antagonist thioperamide, decreased and enhanced respectively [3H]histamine synthesis in lung and spleen, indicating a tonic control of mast cell activity by histamine via H3-receptors. Such effects were not found in the jejunum, although RMCPII+ mast cells are in close apposition with neuropeptide-containing fibres. The effects of the H3-receptor agents were maintained in lung and spleen of nematode-infected rats, but were almost suppressed in capsaicinized rats. 4. It is concluded that the control of mast cells by histamine acting at H3-receptors involves neuropeptide-containing nerves and presumably reflects the operation of a local neuron-mast cell feedback loop controlling processes such as 'neurogenic inflammation'. This loop still functions when mast cells proliferate in an inflammatory condition. These observations suggest that the use of histamine H3-receptor agonists may constitute a novel therapeutic approach to limit excessive inflammatory responses resulting from dysregulation of this feedback loop.

Animals↗

The generation of ovine bone marrow-derived mast cells in culture.

Ovine mast cells generated in vitro are morphologically and biochemically similar to mast cells localized to the gastrointestinal tract in vivo. Bone marrow (BM)-derived haemopoietic precursor cells were cultured in the presence of conditioned medium (CM) prepared from in vitro-activated lymphocytes from nematode-infected sheep. Mast cells developed rapidly after 2 weeks of culture and could persist for 2-3 months. The presence of sheep mast cell proteinase (SMCP) in the cultured cells correlated with the number of morphologically recognizable mast cells by Leishman-Giemsa staining and is definitive as, in vivo, this enzyme is unique to mast cells. The content of SMCP per cell increased as the cultures developed. In vivo, increased serum concentrations of SMCP are associated with mast cell function at the site of nematode infection. The availability of the cultured cells should now facilitate mast cell biochemical and functional studies.

Animals↗

Induction of enhanced immunity to intestinal nematodes using IL-9-producing dendritic cells.

Dendritic cells can be considered natural adjuvants and are able to act as cellular vaccines to protect against disease. Adoptive transfer of Ag-pulsed bone marrow-derived dendritic cells (BMDCs) enhanced expulsion of the intestinal nematode, Trichinella spiralis, from the small intestine. IL 9 is a critical cytokine in protective immunity to intestinal nematode infection and is believed to enhance Th2 immune responses. Deriving dendritic cells from an IL-9 transgenic (IL-9t) mouse has enabled a detailed investigation of the importance of IL-9 during Ag presentation. Indeed, IL-9t dendritic cells significantly enhanced T cell proliferation and Th2 responses and, after adoptive transfer, enhanced parasite-specific IgG1 and intestinal mastocytosis in vivo, leading to accelerated expulsion of adult worms from the intestine. Overall, this paper demonstrates that dendritic cell vaccination can be used to successfully protect the host against intestinal nematode infection and suggests that IL-9 can act as a potent type 2 adjuvant during Ag presentation and the early stages of Th2 activation.

Animals↗

Hyporesponsiveness of murine B lymphocytes exposed to the filarial nematode secreted product ES-62 in vivo.

ES-62 is a phosphorylcholine (PC)-containing glycoprotein secreted by filarial nematodes, parasites of vertebrates including humans. We have previously demonstrated that pre-exposure to this molecule in vitro interferes with subsequent B-cell receptor (BCR)-dependent activation of murine splenic B lymphocytes. To investigate the significance of this during filarial nematode infection, we now employ mice exposed to ES-62, at concentrations equivalent to those found for PC-containing molecules in the bloodstream of parasitized humans, via release from implanted osmotic pumps. Using this approach, we reveal that splenic and lymph node mononuclear cells, and also purified splenic B cells recovered from these mice have reduced ability ex vivo to proliferate in response to BCR ligation. The effect on BCR-induced proliferation was further investigated with respect to elucidating the mechanism of action of the parasite product and was shown to be associated with impaired signal transduction affecting the ErkMAPkinase pathway. Also, it was found that ES-62 did not act by promoting apoptosis or by priming for apoptosis following subsequent stimulation, but rather, appeared to render cells hyporesponsive to stimulation. ES-62 is thus shown for the first time to be a potent modulator of B lymphocyte function in vivo at a concentration relevant to natural filarial nematode infection. This finding considerably strengthens the idea that ES-62 plays a role in evasion of the immune response during parasitism.

Animals↗

In vivo activation of murine peritoneal B1 cells by the filarial nematode phosphorylcholine-containing glycoprotein ES-62.

Mice were subcutaneously implanted with osmotic pumps loaded with ES-62, an immunomodulatory phosphorylcholine (PC)-containing glycoprotein secreted by filarial nematodes. The concentration of ES-62 was set to give a serum level within the range found for PC-containing molecules during natural filarial nematode infection of humans. Peritoneal B1 cells were recovered from the mice and the effect of exposure to ES-62 on a number of parameters determined ex vivo. B1 cells exposed to ES-62 showed an increase in spontaneous proliferation that was enhanced by ex vivo exposure to F(ab')(2) fragments of anti-IgM antibodies (anti-IgM), to activate via the antigen receptor, or LPS. Consistent with this, cell-cycle analysis indicated that cells pre-exposed to ES-62 showed increased cell-cycle progression following stimulation with anti-IgM. Pre-exposed cells also showed an increase in both spontaneous and anti-IgM induced IL-10 secretion. Taken together, these data indicate that ES-62 activates murine B1 cells in vivo. Conversely, we have previously shown conventional (B2) B cells to be rendered hypo-responsive by in vivo exposure to ES-62 and the different effect on the two cell types is discussed in relation to the nature of the antibody response arising during filarial nematode infection.

Animals↗

Diarrhoea in merino ewes during winter: association with trichostrongylid larvae.

A study on 3 farms, each having a preventive strategy for the control of nematode infections, determined the risk factors associated with diarrhoea among Merino ewes in winter. Comparisons were made among groups of mated and unmated ewes with and without treatment with controlled-release capsules containing albendazole. Diarrhoea was assessed from the accumulations of faeces around the breech (dag score) and related to worm egg counts, total worm counts and histopathological findings. The overall prevalence of severe dag in ewes not treated with a capsule was 26% (221/844). In comparison, only 2.7% of treated ewes had severe dag. The adjusted odds ratio on each farm indicated that untreated ewes were between 12 and 16 times more likely to be affected with severe dag than ewes treated with a capsule. The effect of lactation was significant on only one farm whereas initial body weight had no significant effect. There was no significant association between worm egg counts and the occurrence of severe dag. There were significantly more eosinophils in the small intestine of ewes affected with severe dag compared with unaffected ewes. There were no significant differences in mast cell and globule leucocyte numbers between affected and unaffected ewes. This study provides strong evidence that the main cause of diarrhoea among Merino ewes grazing winter and early spring pastures is the ingestion of trichostrongylid larvae even by sheep that have a well-developed protective immune response to these parasites. New strategies for the control of nematode infections are needed to prevent diarrhoea and dag formation in adult sheep.

Animals↗

Administration of Duddingtonia flagrans chlamydospores to goats to control gastro-intestinal nematodes: dose trials.

The ability of the nematophagous fungus Duddingtonia flagrans to reduce the number of infective nematode larvae in coproculture was investigated in goats using different doses of chlamydospores (0, 1.25 x 10(5), 2.5 x 10(5), 5 x 10(5) chlamydospores/kg BW/day) given by oral administration or by voluntary consumption in feed during natural or experimental infections with nematodes. The kinetics of excretion of D. flagrans chlamydospores in the faeces was also determined using a dose of 5 x 10(5) chlamydospores/kg BW/day for five days. For all the trials, the faecal nematode egg outputs were determined by a modified McMaster method and standard coprocultures were set up (14 days, 25 degrees C) to determine the number of larvae emerging from culture in fungus treated and control faeces. When chlamydospores were orally administered, the number of larvae were reduced by 50 to 97% when compared to control cultures. No difference in the level of larval emergence from the culture was seen for experimental or natural infections at the different chlamydospore dose rates. In contrast, when chlamydospores were distributed in the feed, a dose-dependent relationship was observed 10 days after the start of administration, the larval development being 2.0%, 14.0% and 86.9% for 5 x 10(5), 2.5 x 10(5) and 0 spores/kg BW/day, respectively. In addition, the kinetic study showed that the larval emergence from coproculture in the fungus group was statistically lower than in the control group from the second day of administration of the chlamydospores and remained lower until the second day after the last administration (p < 0.05). The results indicate that, for goats in farm conditions, a minimum daily dose of 5 x 10(5) chlamydospores/kg BW must be used to ensure a high treatment efficacy and that daily administration is preferable for maintenance of efficacy over time.

Animals↗

Persistent efficacy of topical eprinomectin against nematode parasites in cattle.

Six studies were conducted to evaluate the persistent efficacy of eprinomectin pour-on against experimental challenges with infective nematode larvae in calves. In each study, calves were randomly assigned to one untreated group and up to four test groups, which were treated with eprinomectin at 500 microg/kg body weight at weekly intervals before single bolus challenge. The calves were necropsied approximately 4 weeks after challenge infection for nematode recovery. Eprinomectin pour-on provided > or =90% efficacy against challenge with Haemonchus placei, Trichostrongylus axei and T. colubriformis at 21 days after treatment and against Cooperia oncophora, C. punctata, C. surnabada, Dictyocaulus viviparus, Nematodirus helvetianus, Oesophagostomum radiatum and Ostertagia ostertagi at 28 days after treatment.

Administration, Topical↗

Selective loss of gastrointestinal mast cells and impaired immunity in PI3K-deficient mice.

Mice that lack the p85alpha regulatory subunit of phosphatidylinositol-3 kinase (PI3K) are deficient in gastrointestinal and peritoneal mast cells but have dermal mast cells. Accordingly, these mice show impaired bacterial clearance in response to acute septic peritonitis and are highly susceptible to infection by the intestinal nematode Strongyloides venezuelensis. Systemic anaphylactic shock responses, however, are intact. We found that although reconstitution of PI3Kminus sign/minus sign mice with bone marrow--derived mast cells (BMMCs) restored anti-bacterial immunity, only T helper type 2 (TH2)-conditioned BMMCs, not "standard" BMMCs, were able to restore anti-nematode immunity. This finding highlights the importance of the TH2 response in the control of nematode infection. Thus, PI3K likely plays an essential role in host immune responses by regulating both the development and induction of mast cells.

Animals↗