Trichostrongylus colubriformis infection: Impact on digesta passage rate and lamb performance.

In this study we aimed to evaluate the effect of Trichostrongylus colubriformis infection on digesta passage rate, rumen fermentation and lamb performance. Eighteen three-month-old Santa Ines castrated male lambs (16.9 ±â€¯1.43 kg of body weight) were randomly distributed in two experimental treatments: Infected with T. colubriformis (I, n = 9) and Uninfected (U, n = 9). The I lambs received a total of 45,000 L3 larvae of T. colubriformis (5,000 infective larvae, three times per week for three weeks). Daily feed intake was assessed using the I lambs as a reference for their respective pairs on the U group (pair-fed). Weight, body condition score and faeces (stool) samples were obtained every 15 days for 75 days. In both treatments, faecal egg count (FEC), digesta passage rate, rumen fermentation parameters, protozoa count and short chain fatty acids (SCFA) were evaluated. The lambs presented moderate infection (FEC = 620). The retention time of the digesta in the rumen-reticulum segment was lower (P < 0.05) in I lambs. The I lambs presented no inappetence, however, lower concentrations of total SCFA and butyrate, while higher acetate concentration were observed in these lambs (P < 0.05). The present findings highlight that T. colubriformis infection decreased the retention time (solid and liquid content) of the digesta in the rumen-reticulum, as well as negatively affected lamb growth.

Four clusters of Trichostrongylus infection diagnosed in a single center, in Italy.

Trichostrongylus spp. are parasites that are seldom recognized as a cause of eosinophilia and gastroenteric symptoms in industrialized countries. The index of suspicion raises when several members of a same household present eosinophilia. We report four clusters of Trichostrongylus infection diagnosed in a single center, in northern Italy. Patients came from four different provinces of three Italian Regions. Some patients presented symptoms (abdominal pain and diarrhea were the most frequent ones, reported by 67 and 42% of our patients, respectively), while other were asymptomatic. All of them presented eosinophilia, that was severe (>5000 eosinophils/mmc) in 58% cases. Obtaining an accurate history from patients, investigating possible ingestion of vegetables contaminated by organic manure or sheep dejections, is particularly important to achieve diagnosis, also in light of the low sensitivity of parasitological tests.

Efferent intestinal lymph protein responses in nematode-resistant, -resilient and -susceptible lambs under challenge with Trichostrongylus colubriformis.

The mechanisms underlying resistance to challenge by gastrointestinal nematode parasites in sheep are complex. Using DIGE, we profiled ovine lymph proteins in lambs with host resistance (R), resilience (Ri) or susceptibility (S) to a daily trickle challenge with the nematode Trichostrongylus colubriformis. Efferent intestinal lymph was collected prior to infection (day 1) and on days 5 and 10 post-infection. Eight proteins identified by LC-MS/MS, showed differences relating to host genotype. Of these, Serpin A3-3 and Serpin A3-7 have not been reported previously in the lymph proteome. Three acute phase proteins showed significant differences relating to interactions between breeding line and parasite challenge, including complement C3ß, C3α and haptoglobin (Hp) ß. In the R lambs C3α was significantly up regulated (P<0.05) on day 10, while in the Ri lambs Hp ß was significantly down regulated (P<0.05). In the S lambs, levels of C3ß were up regulated and levels of Hp ß down regulated (both P<0.05) on day 10. Hence we demonstrate that acute phase inflammation proteins contribute to differences in the innate immune response of sheep to challenge by T. colubriformis. The findings may lead to the development of new approaches to combat nematode infestations in sheep production systems. BIOLOGICAL SIGNIFICANCE: Breeding lines of sheep with resistance (R), resilience (Ri) or susceptibility (S) to nematode infections provide an experimental model to examine the biological mechanisms underlying the ability of some sheep to expel worms and remain healthy without the use of an anthelmintic. Using proteomics we identified differences in the expression of acute phase lymph proteins in the R, Ri and S lambs. The results will assist the development of alternative control strategies to manage nematode infections in livestock.

Experimentally elevated levels of testosterone at independence reduce fitness in a territorial bird.

Environmental conditions and individual strategies in early life may have a profound effect on fitness. A critical moment in the life of an organism occurs when an individual reaches independence and stops receiving benefits from its relatives. Understanding the consequences of individual strategies at the time of independence requires quantification of their fitness effects. We explored this period in the Red Grouse (Lagopus lagopus scoticus). In this system, testosterone and parasite (Trichostrongylus tenuis) levels are known to influence survival and reproduction, the two key components of individual fitness. We experimentally and simultaneously manipulated testosterone and parasites at three levels (high, intermediate, and control levels for both factors) in 195 young males in five populations using a factorial experimental design. We explored the effects of our treatments on fitness by monitoring reproduction and survival throughout the life of all males and estimating lambda(ind), a rate-sensitive index of fitness. Parasite challenges increased the number of worms with a time lag, as previously found. However, we did not find significant effects of parasite manipulations on fitness, possibly because parasite abundance did not increase to harmful levels. Our hormone manipulation was successful at increasing testosterone at three different levels. Such increases in hormone levels decreased overall fitness. This was caused by reduced offspring production in the first breeding attempt rather than by any effect of the treatment on bird survival. Our results highlight that investing in high testosterone levels at independence, a strategy that might enhance short-term recruitment probability in territorial species such as Red Grouse, has a fitness cost, and can influence the resolution of the trade-off between reproduction and survival later in life.

Prevalence and clinical aspects of human Trichostrongylus colubriformis infection in Lao PDR.

There have been few studies on human trichostrongyliasis in Southeast Asia, information on its clinical manifestations is also sparse. Trichostrongyliasis occurs predominantly in areas where poor hygiene is common especially where human/animal feces are used as a fertilizer, thereby contaminating vegetables and stream water. The intimate coexistence of domestic animals and humans explains the prevalence of Trichostrongylus infection in such areas. The goal of the current study was to determine the prevalence of trichostrongyliasis among villagers in Thakamrien village, Sonkon district, Savannakhet province, Laos, and to investigate potential relationships between clinical features, laboratory data, and severity of infection. Of 272 villagers examined, 160 (58.8%) were determined positive for helminthic infections by fecal examination, and 59 (36.9%) of these were infected with Trichostrongylus. Only 58 cases were in the inclusion criteria of the study and then underwent further assessment, including a questionnaire on personal behaviors, physical examination, and laboratory tests. Villagers in the trichostrongyliasis group were more likely than the control group to have consumed fresh vegetables, not washed their hands before meals or after using the toilet, and to have had close contact with herbivorous animals (goats and cows). Similarly, villagers in the trichostrongyliasis group were more likely than the control group to have a history of loose feces, rash, or abdominal pain; however, no obvious clinical symptoms were observed during physical examination of the trichostrongyliasis patients. The degree of infection was determined by both fecal egg counts and quantification of adult worms after deworming. Laboratory data were evaluated for any relationship with severity of infection. No significant differences were found in laboratory values between the trichostrongyliasis and control groups, with most values being within normal limits; however, both groups had high eosinophil counts. This study demonstrated that the useful clinical characteristics of trichostrongyliasis patients include history of loose feces, rashes, and abdominal pain, as well as in personal behaviors, such as the regular consumption of fresh vegetables, lack of hand washing, and close contact with cattle.

Histopathological, histochemical and immunohistochemical findings of the small intestine in goats naturally infected by Trichostrongylus colubriformis.

Gastrointestinal (GI) strongyle infection remains one of the main constraints to goat production worldwide. Samples of small intestine from 15 Syrian goats naturally infected with Trichostrongylus colubriformis were examined by routine histology, histochemistry and immunohistochemistry to describe the histological changes and the phenotypes of inflammatory cellular components of the mucosa. Results indicated that the immune response to infection by T. colubriformis was characterized by an increased rate of the severity of the histologic lesions, an increase rate of T cell lymphocytes recruitment to the intestinal mucosa and quantitative and qualitative changes in the histochemical composition of mucin in goblet cells.

Trichostrongylus colubriformis larvae induce necrosis and release of IL33 from intestinal epithelial cells in vitro: implications for gastrointestinal nematode vaccine design.

Gastrointestinal nematodes represent a major production problem for ruminant livestock. Enhancing immunity to gastrointestinal nematodes through vaccination is desirable but mechanistic understanding of initial host responses that facilitate gastrointestinal nematode protective immunity is limited. We hypothesise that gastrointestinal nematode invasion induces mucosal epithelium damage and alarmin (e.g. IL33) release, thereby contributing to initiation of protective gastrointestinal nematode immunity. To test this, an in vitro air-liquid interface human HT-29 epithelial cell-Trichostrongylus colubriformis co-culture system was developed. Exsheathed L3 T. colubriformis exhibited both sinusoidal and burrowing motions in the co-culture system. Burrowing parasites, but not ivermectin-paralysed larvae, induced necrotic death of epithelial cells (annexin V(+)/propidium iodide(+)/caspase 3/7(-)). Microscopy confirmed that larvae consumed labelled necrotic epithelial cell contents. Trichostrongylus colubriformis larvae and their post-exsheathment antigens (excretory/secretory products) significantly induced IL33 mRNA expression in the epithelial cells. Immunoblot confirmed that IL33 was released from epithelial cells due to the damage caused by motile larvae. Exposure of HT-29 cells to alum or Sigma proprietary adjuvants induced significant epithelial cell IL33 mRNA expression without inducing cellular necrosis. Hence, the intracellular contents were not released externally where they might exert alarmin activity and this may limit their ability to trigger a protective anti-gastrointestinal nematode response. We conclude that T. colubriformis motion at the infection site induces intestinal epithelial cell necrosis which facilitates the release of intracellular contents, including IL33, and may be fundamental to the initiation of an appropriate host response to gastrointestinal nematodes. Our co-culture model is useful for studying initial epithelial cell-parasite interactions without conducting expensive animal trials.

Responses of the humid zone ecotype of the Nigerian West African Dwarf sheep to mixed infections with Haemonchus contortus and Trichostrongylus colubriformis.

The responses of the Nigerian West African Dwarf sheep to experimental infections with two of its most important gastrointestinal (GI) nematodes, namely, Haemonchus contortus and Trichostrongylus colubriformis were studied by means of two measures of parasitological response, namely, faecal egg count (FEC) and worm burden (Wb), and three measures of host pathology, namely, packed cell volume (PCV), body weight (Bwt) and body condition score (BCS). Following exposure to weekly escalating infections (60% H. contortus and 40% T. colubriformis) starting with single doses of 500 infective larvae (L(3)) at week 1, 1,000 L(3) at week 2, 2,000 L(3) at week 3 and 4,000 L(3) at week 4, two distinctive worm burden response phenotypes were readily recognisable, namely, low Wb (LWb) and high Wb (HWb) phenotypes. The percentage of inoculums (adults and immature stages) recovered at necropsy were 3.75% and 33.08% respectively for H. contortus and T. colubriformis. The results showed that the FECs of the lambs belonging to the LWb phenotype were significantly lower than the FECs of their HWb counterpart. Among the measures of host pathology tested, the LWb phenotype sheep had similar Bwt, PCV and BCS to the uninfected control sheep, whereas the HWb phenotype sheep had significantly lower values than their LWb counterparts and the control sheep towards the end of the experiment. There was a highly significant positive correlation between worm burden transformed as log(10) (Wb + 10) and the average of D56 and D59 FECs. A highly significant negative correlation also was obtained between log(10) (Wb + 10) and the PCV, Bwt and BCS. The low percentage of H. contortus inoculums recovered at necropsy suggested that the sheep may be resistant to their most important GI nematode, H. contortus but less so to T. colubriformis.

The ornament-condition relationship varies with parasite abundance at population level in a female bird.

Environmental heterogeneity is expected to create variation in the ornament-condition relationship. This topic has been studied in males with less attention being given to females. Here, we explore inter-population variation in the relationship between the size of a male-like trait, supra-orbital combs, and body mass in female red grouse Lagopus lagopus scoticus. We used the abundance of the nematode Trichostrongylus tenuis, a parasite with strong negative effects on this species, as a proxy of environmental conditions. We studied six populations over 5 years and showed that the comb size-body mass relationship varied with mean parasite abundance, with stronger ornament-condition relationships in populations with higher parasite infection levels. Our study supports the idea that environmental conditions, and in particular parasite infection levels, may affect the reliability of female ornaments as condition indicators.

The experimental establishment of ruminant nematodes in European hares (Lepus europaeus).

The factors that control the demography of European hare Lepus europaeus populations are poorly understood, but it has been recognized that the decline of hares in Europe is associated with an increasing intensity of agricultural activity. Many mechanisms have been suggested. We propose another mechanism; a negative impact arising from ingestion of the infective larvae of ruminant livestock. We dosed juvenile hares from a worm-free colony with a conservative dose from a mixed culture of infective larvae of the nematode parasites of sheep and cattle. We examined the hares post-mortem for the establishment of those ruminant nematodes, differences in weight changes, and the shedding of eggs. We found that under the circumstances of our trial, Trichostrongylus colubriformis and to a lesser extent T. rugatus, T. vitrinus, and Teladorsagia circumcincta were able to establish as adults in the dosed animals. We found strongyle eggs in the faeces of the dosed hares, and were able to culture larvae from those eggs. However, the ecological significance of our findings, if any, remains to be elucidated. Because of their mobility, hares may transmit resistant strains of parasites between grazing properties.

The effects of concurrent experimental infections of sheep with Trichostrongylus colubriformis and T. vitrinus on nematode distributions, numbers and on pathological changes.

Simultaneous infections of Trichostrongylus colubriformis and T. vitrinus in the small intestine of the sheep were examined by comparing the numbers of worms which established and their distribution within the intestine in both monospecific infections and mixed infections. The results differed depending upon the species and number of parasites. The establishment of T. colubriformis was reduced and the distribution of the nematode population was displaced posteriorly within the intestine when 30,000 larvae of both species were administered, compared with pure infections of T. colubriformis. The reduced establishment was less marked with infections of 15,000 larvae of both species and there was only a slight posterior displacement of T. colubriformis. Neither effect was evident with infections of 7,500 larvae of both species. The rate of establishment and distribution of T. vitrinus were unaffected by the presence of T. colubriformis at all three rates of infection. Atrophy of villi and hypertrophy of crypts occurred at the main site of infection in the anterior duodenum. The severity of villus atrophy was related to the number of infective larvae administered and/or the worm burden. In the ileum, beyond the main site of infection, hypertrophy of villi was only found in sheep receiving the greatest number of infective larvae.

Intestinal migrations of Trichostrongylus retortaeformis (Trichostrongylina, Trichostrongylidae) in the rabbit.

Observations were made on histological sections of the stomach and small intestine of seven rabbits infected with Trichostrongylus retortaeformis and from one uninfected control rabbit. At 12h post-infection, larvae were found in the small intestine. At first, only a few larvae were observed entering the mucosa through capillaries of the stroma of villi; the majority of larvae remained in the intestinal lumen, within mucus of the crypts. We consider that the presence of the worms in the stroma is the result of a larval migration. From a phyletic point of view, this migration is interpreted as an ancestral memory of the pulmonary migration seen in the primitive Strongylida.

Osteopenia and reduced serum alkaline phosphatase activity in grazing lambs naturally infected with gastrointestinal nematodes.

The effect of gastrointestinal nematode infections on bone development was investigated in growing sheep on pasture. Forty-five weaned lambs from six groups in a two-factorial design incorporating stocking rate (SR; low, medium and high) and presence or absence of infection on pasture were sampled in the late grazing season. Worm counts were performed at slaughter, and the left metacarpal bones were excised for bone assessment. Faecal egg counts and worm burdens, primarily of Ostertagia circumcincta and Trichostrongylus vitrinus, were considerably higher in the high SR infected group ("I-High") than in comparable animals at low or medium SRs, whereas uninfected groups showed negligible egg excretion. Clinical biochemistry revealed significantly reduced serum concentrations of albumin, calcium and alkaline phosphatase in infected lambs. Nematode infections were associated with significant reductions in bone mineral density (30% at high SR), measured by dual energy X-ray absorptiometry, and in bone size (9%). Histomorphometry indicated thinning of the trabecular structure and reduced bone formation in the infected groups, particularly the I-High group. Bone mineral density, bone tissue volume and structural changes were strongly associated with log-transformed worm counts. The study showed that lambs suffering from moderate to heavy degrees of naturally acquired gastrointestinal nematode infection developed marked osteopenia after weaning, i.eduring the later part of the grazing season.

Quantitative and qualitative changes in intestinal goblet cells during primary infection of Trichostrongylus colubriformis high and low responder guinea pigs.

Small intestine goblet cell numbers and the composition of their mucus were compared in guinea pigs with genetically determined differences in responsiveness to Trichostrongylus colubriformis infection. Prior to infection, no differences between high responder and low responder animals were detected. However, following primary infection with T. colubriformis, pronounced goblet cell hyperplasia developed and the proportion of sulphomucin in these cells increased. Both changes developed significantly earlier in high responder animals.

In vivo depletion of T-cells and cytokines during primary exposure of sheep to parasites.

This study examined the role of CD8+ and WC1+ T-cells and of interferon (IFN)-gamma in the development of protective immunity against infection with the enteric nematode parasite Trichostrongylus colubriformis in sheep. Monoclonal antibodies (mAb) were administered during induction of the immune response to deplete or neutralise these components. Protection against the primary and challenge infections was assessed by faecal egg count and total worm count. Prolonged administration of mAb recognising IFN-gamma and CD8 resulted in significantly increased protection during the 6 week primary infection and following challenge. CD8+ cells were depleted from blood but not from intestinal mucosa. After injection of mAb (CC15) recognising the surface antigen WC1, WC1+ and Tcr gamma delta + cells were depleted from blood but not markedly from enteric mucosa, and protection against challenge, although variable, was increased by up to 88%. It appears that CD8+ and WC1+/gamma delta+ cells and IFN-gamma all retard the potential development of naturally acquired immunity against the parasite.

Development of morphological changes and ileal glucagon gene expression in the small intestine of lambs infected with Trichostrongylus colubriformis.

To investigate the consequences of subclinical Trichostrongylus colubriformis infection on the intestinal mucosa and the associated changes in entero-glucagon gene expression, sheep were infected with 30000 larvae and killed 5, 10, 15 or 20 days after infection. Histological and cytological changes were examined. In the main site of infection, the upper duodenum, villous atrophy associated with crypt hyperplasia developed gradually. Cytological changes in the enterocytes appeared concurrently, characterized by a progressive reduction in brush border and in the number of ribosomes in the cytoplasm, changes in the internal structure of mitochondria, and enlargement of the intercellular spaces. Neither histological nor cytological modifications were found before day 15. At the same time, villous hypertrophy developed distally, beyond the main site of infection; this was interpreted as an adaptive response to parasitism. Enteroglucagon gene expression in the ileum was measured in parallel with the mucosal changes but did not reveal any difference between infected and control sheep. The results indicate that this gastrointestinal hormone does not have a major role in the response to nematode parasitism.

The immune response of sheep surgically modified with intestinal loops to challenge with Trichostrongylus colubriformis.

Aspects of the local immune response to nematode challenge were investigated in vivo in isolated loops of the upper small intestine of mature sheep that were immunised by repeated infections with Trichostrongylus colubriformis infective larvae (L3). Groups of 3 sheep were challenged either through the loop (Group 1) or orally (Group 2) with T. colubriformis L3, the third group served as unchallenged controls (Group 3). Nematode specific antibody levels, mast cell proteinase levels (SMCP) and larval migration inhibition (LMI) activity were determined in loop secretions for 4 weeks after challenge. The intestinal loops remained functional throughout the experiment. Groups 1 and 2 were re-challenged 2 weeks after the first challenge, and all 3 Groups were slaughtered 2 weeks later. Histopathological examination showed elevated numbers of globule leukocytes (GL) in both the nematode-challenged loop and unchallenged small intestine of Group 1 and small intestine of Group 2 indicating that nematode infections induce the local appearance of large numbers of GL. Oral, but not loop challenge caused increased antibody levels in loop secretions when compared to unchallenged controls. Only loop-challenged sheep showed a peak in loop fluid SMCP levels 10-13 days after the first challenge which coincided with a peak in numbers of mucosal GL. The isolated loops of all 3 groups showed highly elevated numbers of eosinophils when compared to the intact small intestine. Loop fluid of all 3 groups showed a high level of LMI activity reflecting the high level of nematode-resistance induced by the immunisation procedures. Sheep in Groups 1 and 2 were both able to expel challenge infections, and when compared to Group 3, showed higher blastogenic activity of unstimulated cells derived from a mesenteric lymph node in the region of the challenged part of the intestine. The present experiment showed that surgically constructed intestinal loops provided a model system by which the substantial changes associated with the local intestinal immune response to challenge with T. colubriformis could be investigated.

Measurement of an eosinophil differentiation factor in sheep before and after infection with Trichostrongylus colubriformis.

A bioassay for the measurement of an ovine eosinophil differentiation factor (EDF) was developed. Recombinant murine interleukin-5 (rmIL-5) and supernatant from concanavalin A stimulated lymphocytes induced eosinophil differentiation and proliferation in ovine bone marrow cell culture, which was determined by an eosinophil peroxidase assay (EPO). The assay was used to measure the production of an ovine eosinophil differentiation factor by lymphocytes following experimental infection of sheep with Trichostrongylus colubriformis. Sixteen sheep were immunised and challenged with T. colubriformis and grouped into responders (n = 10) and non-responders (n = 6) based on the lower 99% confidence limit of the worm burden of the unimmunised control group (n = 10). Total worm burdens of all animals were established 10 days after initial challenge. Peripheral blood lymphocytes at Day 0 (pre-challenge values) and Day 9 (post-challenge values) from all sheep and lymphocytes of mesenteric lymph nodes from eight sheep were stimulated with third stage larval antigen of T. colubriformis. The resulting lymphocyte conditioned medium (LyCM) was tested for EDF by its ability to induce growth and differentiation of eosinophils in liquid bone marrow cell cultures. No significant difference was found between the animal groups. No correlation was found between EDF activity and total worm numbers.

Trichostrongylus colubriformis infection in rabbits: persistence of the distal adaptive response to parasitism after anthelmintic treatment.

Mucosal changes associated with anthelmintic treatment in Trichostrongylus colubriformis-infected rabbits were assessed along the entire length of the small intestine. The following groups, each of five rabbits, were compared: infected (group I); infected and treated on day 21 with fenbendazole (group IT); uninfected but given fenbendazole on day 21 (group C, controls). All animals were killed on day 28. In the proximal part of the small intestine of group I rabbits, the worms were associated with shortening of the villi and a significant depletion in alkaline phosphatase activity, which differed significantly from the findings in groups C and IT. In the same region, no difference was found between groups C and IT. In the distal small intestine, hypertrophy of villi and crypts (an adaptive response to the infection), coupled with an increase in enzymic activity, were present in both groups I and IT, in contrast to group C. These results suggest that a complete mucosal restoration occurred within 7 days of anthelmintic treatment in the parasitized part of the intestine. In contrast, the adaptive response observed beyond the main site of parasitism was not abolished by treatment. The functional significance of these findings is discussed in relation to the compensatory growth commonly observed after anthelmintic treatment in ruminants.