L3 Recovery Research Articles

Comparative development of human filariae Loa loa, Onchocerca volvulus and Mansonella perstans in immunocompromised mouse strains.

Mouse models of human filarial infections are not only urgently needed to investigate the biology of the nematodes and their modulation of the host's immunity, but will also provide a platform to screen and test novel anti-filarial drugs. Recently, murine Loa loa infection models have been stablished using immunocompromised mouse strains, whereas murine Mansonella perstans infections have not been implemented until now. Therefore, we aim to establish experimental M. perstans infections using the immunocompromised mouse strains RAG2IL-2Rγ-/- (lack B, T and natural killer cells), IL-4Rα/IL-5-/- (impaired IL-4/5 signalling and eosinophil activation) and NOD.Cg-PrkdcscidIl2rgtm1Wj l/SzJ (NOD scid gamma, NSG) BALB/c mice (lack mature lymphocytes) through subcutaneous (s.c.) or intraperitoneal (i.p.) inoculation of infective stage 3 larvae (L3) isolated from engorged vectors. In total, 145 immunocompromised mice have been inoculated with 3,250 M. perstans, 3,337 O. volvulus, and 2,720 Loa loa L3 to comparatively analyse which immunocompromised mouse strain is susceptible to human filarial infections. Whereas, no M. perstans and O. volvulus L3 could be recovered upon 2-63 days post-inoculation, a 62-66% Loa loa L3 recovery rate could be achieved in the different mouse strains. Gender of mice, type of inoculation (s.c. or i.p.) or time point of analysis (2-63 days post inoculation) did not interfere with the success of L3 recovery. In addition, administration of the immune suppressants hydrocortisone, prednisolone and cyclophosphamide did not restore M. perstans L3 recovery rates. These findings show that RAG2IL-2Rg-/-BALB/c and C57BL/6, IL-4Rα/IL-5-/- BALB/c and NSG mice were not susceptible to M. perstans and O. volvulus L3 inoculation using the applied methods, whereas Loa loa infection could be maintained. Further studies should investigate if humanized immunocompromised mice might be susceptible to M. perstans. and O. volvulus.

61 Determining in vivo and in vitro predatory activity of Duddingtonia flagrans and interaction with dietary coccidiostat

Abstract A nematode trapping fungus, Duddingtonia flagrans (Df), fed to sheep is useful for biocontrol of gastrointestinal nematodes on pasture. The objectives were: 1) validate methods to determine viability of a commercial source of Df before beginning research studies, and 2) examine the interaction between Df and dietary coccidiostat (as decoquinate). Methods: Study 1: In Jan 2023, weaned Katahdin lambs (97.3 ± 1.2 d of age; 19.5 ± 1.3 kg BW; packed cell volume of 26.5 ± 1.4 %), naturally infected with predominantly Haemonchus contortus (89%) and mean fecal egg count (FEC) of 6,860 ± 1,535 eggs/g, were randomly assigned to: 1) no Df (-g)/(FEC-g)×100]. Study 2: In Aug 2023, 7 mo old anthelmintic-treated ram lambs (FEC < 50 egg/s; 51.6 ± 3.9 kg in BW) were fed grain supplement daily with decoquinate (Deccox; +C) or without (-C; n = 3/diet) on d -7 to 1, and, within diet, administered 3 g BioWorma/d in 6 gel capsules (+DF; n = 2/diet) or not (-DF; n = 1) on d -1 and -2. Fecal samples were collected on d 0 (2 g/plate + 300 L3 of H. contortus) and d 1 (6 g/plate + 1000 L3) and placed on water agar plates (n = 3/lamb) at 25°C. Trapping structures were visually assessed after 24, 48, and 144 h (6 d), and cultures Baermannized on d 7 for L3 recovery. Relative reduction was (x̅-DF-C-x̅+DF+C/ x̅-DF-C)×100%. Data for variables collected one time were analyzed by Proc GLM (SAS), and for those collected serially, a Proc Mixed with repeated measures. A 2×2 factorial was used in Study 2 and included treatments, day and interaction in model. Results: Study 1: As expected, L3 recovery was reduced (P = 0.005) in DF lambs by 67-78% relative to control. Study 2: DF formed loops and traps around L3 by d 6 and reduced L3 recovery on both days (P < 0.001) without an interaction by decoquinate (P = 0.69). The L3 reduction relative to -DF-C was between 60.2-73.5%. The coccidiostat, decoquinate, can be added to the diet in the presence of BioWorma without antagonism. In conclusion, the in vitro studies allow detection of adverse issues with viability of Df and can reduce the number of animals needed for research studies.

Ecology of the free-living stages of cattle nematodes in the dry season in the Lerma Valley, Salta province, Argentina

The objective of this work was to describe the dynamics of development and survival of the free-living stages of cattle gastrointestinal nematodes (GIN) in fecal matter (FM) and pasture during the dry season in the Lerma Valley, Salta province, northwestern Argentina (NWA) to contribute to GIN management. The climate in the region is characterized by a rainy summer followed by a dry season from middle autumn to early spring. Fecal matter from calves naturally infected with GIN was deposited on three experimental field plots in April, July and October 2019, corresponding to the beginning, middle and end of the dry season, respectively. Each experimental unit consisted of 7 stools of about 800 g and had four repetitions. To determine the development from egg to infective larvae (L3), the first sampling (5 g fecal matter) was performed from the 10th day post-contamination and continued every 3 days until L3 were found. Subsequently, a monthly sampling was made until two consecutive negative results were obtained. Sampling of pasture began three days after the L3 recovery from FM, and continued monthly until two negative results were obtained. The following parameters were evaluated: development time and development rate from egg to L3; permanence time of L3 in feces; time of appearance on pasture; migration rate; and permanence time of L3 on pasture. The main genera of parasites present were Cooperia and Haemonchus. Significant differences were observed in the development time among contamination months (p < 0.001); development time was highest in the July contamination (28 days), with October and April contamination averaging 9 and 10 days, respectively. Development time also showed significant differences (p < 0.01) among contamination months, being highest in October (31.48%). The highest permanence time in fecal matter values were recorded in the July contamination (183 days) and migration rate was highest in the October contamination (42.49%). The highest time of appearance on pasture value was recorded in the July contamination (117 days). Finally, the highest permanence time of L3 in feces values were detected in the October contamination (148 days). The results of this work show that fecal contamination in the NWA region in the dry season would play an epidemiological role in the GIN cycle as a source of infection for the next productive cycle in the rainy season.

Monacrosporium sinense and Pochonia chlamydosporia for the biological control of bovine infective larvae in Brachiaria brizantha pasture

Ecologically correct solutions are necessary to minimize the use of anthelmintics in the control of cattle parasitic diseases. This study evaluated the effects of oral administration pellets containing the fungal combination Monacrosporium sinense and Pochonia chlamydosporia in the biological control of infective larvae (L3) of parasitic gastrointestinal nematodes of cattle grazed in pasture. Twelve crossbred bovines (Holstein × Zebu), seven to nine years old, after dewormed, were randomly divided into two groups (group treated with the combination of fungi and control group) and placed in separate paddocks of Brachiaria brizantha that were naturally infested with L3. Feces samples were collected from the animals to determine the parasitic load and pasture samples were collected from the soil to determine the L3 infestation. The recovery of L3 from the pasture of the treated group was 91.7% and 86.4% less than in the pasture of the control group, respectively for samples collected at 20 cm and 40 cm from the faecal pats. The values of eggs per gram of feces in the months of May 2019 and January 2020 were lower (p ≤ 0.05) in the treated group with the fungal combination. The combined administration of M. sinense and P. chlamydosporia reduced the L3 infestation on pasture and in animal parasitic load, making it a tool for controlling bovine gastrointestinal parasitic nematodes.

Predatory activity of nematophagus fungus Duddingtonia flagrans in infective larvae after gastrointestinal transit: biological control in pasture areas and in vitro.

Biological control is a strategy to decrease parasitic populations, and the action takes place through natural antagonists in the environment. We studied the predatory activity of the fungus Duddingtonia flagrans in infective larvae (L3) of gastrointestinal nematodes after gastrointestinal transit. Ten heifers were divided into two groups: treated (animals received pellets containing fungus) and control (animals received pellets without fungus). Twelve hours after administration, faeces samples were collected for in vitro efficacy tests. The animals then remained for 7 h in the experimental pasture area. At the end of this period, 20 faecal pads (ten treated and ten control) were selected at random. Pasture, faecal pad and soil collections occurred with an interval of 7 days, totalling four assessments. In vitro activity demonstrated that fungi effectively preyed on L3, achieving a reduction percentage of 88%. In the faecal pad of the pasture area, there was a difference (P < 0.05) between collections 3 and 4 for both groups; in the treated group a reduction of 65% was obtained, while in the control group there was an increase of 217% in the number of L3. The recovery of L3 in the soil and in the pasture was similar in both groups. There was no influence (P = 0.87) of the passage time on the fungus predatory activity. Duddingtonia flagrans demonstrated the ability to survive gastrointestinal transit in the animals, reducing the number of L3 in the faeces, indicating that this biological control has great potential in the control of worm infections.

Generation of Loa loa infective larvae by experimental infection of the vector, Chrysops silacea

Basic and translational research on loiasis, a filarial nematode infection of medical importance, is impeded by a lack of suitable Loa loa infection models and techniques of obtaining and culturing life cycle stages. We describe the development of a new method for routine production of infective third-stage larvae (L3) of L. loa from the natural intermediate arthropod vector host, Chrysops silacea, following experimental infection with purified microfilariae. At 14-days post-infection of C. silacea, the fly survival rate was 43%. Survival was significantly higher in flies injected with 50 mf (55.2%) than those that received 100 mf (31.0%). However, yield per surviving fly and total yield of L3 was markedly higher in the group of flies inoculated with 100 mf (3474 vs 2462 L3 produced). The abdominal segment hosted the highest percentage recovery of L3 (47.7%) followed by head (34.5%) and thorax (17.9%). L. loa larval survival was higher than 90% after 30 days of in vitro culture. The in vitro moulting success rate to the L4 larval stage was 59.1%. After experimental infection of RAG2-/-IL-2γc-/-mice, the average L. loa juvenile adult worm recovery rate was 10.5% at 62 dpi. More than 87% of the worms were recovered from the muscles and subcutaneous tissues. Worms recovered measured an average 24.3 mm and 11.4 mm in length for females (n = 5) and males (n = 5), respectively. In conclusion, L. loa mf injected into C. silacea intrathoracically develop into infective larvae that remain viable and infective comparable to L3 obtained through natural feeding on the human host. This technique further advances the development of a full laboratory life cycle of L. loa where mf derived from experimentally-infected animals may be utilized to passage life cycle generations via intrathoracic injections of wild-caught vector hosts.

Control of sheep gastrointestinal nematodes using the combination of Duddingtonia flagrans and Levamisole Hydrochloride 5.

The objective was to evaluate the action of D. flagrans pellets in association with Levamisole Hydrochloride 5% for controlling sheep gastrointestinal nematodes in the northeastern Brazil. Three groups of six sheep each were formed: group 1 received 3 g of the pellets (0.6 g of D. flagrans mycelium) for each 10 kg b.w., twice a week for six months, and deworming with Levamisole Hydrochloride 5% when EPG ≥ 1500; group 2 received a dosage of Levamisole Hydrochloride 5% when EPG ≥ 1500; and group 3 received 3 g of pellets without fungi for each 10 kg b.w., twice a week for six months. EPG counts, larval cultures, packed cell volume (PCV) and weighing were performed every 15 days; monthly, samples of grass from each paddock were collected. The mean EPG of the groups began to statistically differ from day 30 (p < 0.05). Group 1 required less deworming with Levamisole Hydrochloride 5% and showed superiority of PCV values ​​throughout the experiment (p < 0.05). There was a significant reduction (p < 0.05) in L3 recovery in the group 1 paddock from day 30 onwards. The use of D. flagrans pellets in association with Levamisole Hydrochloride 5% was effective for controlling gastrointestinal nematodes.

Effects of commonly used chemical fertilizers on development of free-living stages of Haemonchus contortus in experimentally infected pasture.

Aim:The effects of N-P-K fertilizers in the form of urea, single super phosphate and muriate of potash on development of free-living stages of Haemonchus contortus were studied.Materials and Methods:Five parasite free experimental plots of 1 m×1 m area, each of paddy leaves (15-day-old) and an equal number of Cynodon dactylon grass were infested with about 10×104 eggs/ml phosphate buffer saline along with the application of the calculated amount of fertilizers solution. On the 10th day of posttreatment, the pasture was cut, processed, larvae recovered by Baermann method and counted, which was expressed as number of L3 per kg dry matter (DM) of pasture.Results:The average recovered population of L3 of H. contortus per kg DM varied significantly (p<0.05) between the paddy leaves (5933.57±22.718) and Cynodon grass (4861.00±22.718). When different doses of chemical fertilizer and their impact on different pasture were analyzed for control (T-1, 0-0-0 kg/ha N-P-K), the mean L3 recovery per kg DM of paddy (19512.7±50.80) was more than that of Cynodon grass (16540.9±50.80). Larvae recovery per kg DM for different pastures under treatment were in decreasing order as follows: T-2 of paddy (6981.33±50.80, 35.77%), T-2 of Cynodon (5545.38±50.80, 33.52%), T-3 of paddy (317378±50.80, 16.26%), and T-3 of Cynodon (2218.72±50.80, 13.41%) which showed significant difference (p<0.05) among the treatments. In T-4 (paddy) and T-5 (Cynodon), the average number of recovery of larvae was nil implying no significant variation (p>0.05).Conclusion:This study shown that when N-P-K fertilizers administered at recommended level, significantly reduced larval translation of H. contortus minimizing pasture infectivity for the free range grazing animals.

Susceptibility of Biomphalaria glabrata submitted to concomitant infection with Angiostrongylus costaricensis and Schistosoma mansoni.

The easy adaptation of Angiostrongylus costaricensis, nematode responsible for abdominal angiostrongyliasis to several species of terrestrial and freshwater molluscs and the differences observed in the interactions of trematodes with their intermediate hosts have induced us to study the concomitant infection of Biomphalaria glabrata with Schistosoma mansoni and A. costaricensis. Prior exposure of B. glabrata to A. costaricensis (with an interval of 48 hours), favored the development of S. mansoni, observing higher infection rate, increased release of cercariae and increased survival of molluscs, when compared to molluscs exposed only to S. mansoni. Prior exposure of B. glabrata to A. costaricensis and then to S. mansoni also enabled the development of A. costaricensis since in the ninth week of infection, higher amount of A. costaricensis L3 larvae was recovered (12 larvae / mollusc) while for molluscs exposed only to A. costaricensis, the number of larvae recovered was lower (8 larvae / mollusc). However, pre-exposure of B. glabrata to S. mansoni (with an interval of 24 hours), and subsequently exposure to A. costaricensis proved to be very harmful to B. glabrata, causing extensive mortality of molluscs, reduced pre-patent period to release cercariae and greater recovery of L3 A. costaricensis larvae.

Distribution of infective gastrointestinal helminth larvae in tropical erect grass under different feeding systems for lambs.

This study examined tropical pasture contamination dynamics under different feeding systems for finishing lambs. The experiment aimed to evaluate the vertical distribution of gastrointestinal helminth infective larvae (L3) in erect grass subjected to grazing and to assess the parasite load and its impact on lamb performance in three production systems. Three treatments based on Aruana grass (Panicum maximum cv. IZ-5) were as follows: T1, grass only; T2, grass with 1.5% of body weight (BW) nutrient concentrate supplementation; and T3, grass with 2.5% BW concentrate supplementation. The randomized block design had three replicates of three treatments, with six lambs per replicate. L3 were recovered from three pasture strata (upper, middle, and bottom), each representing one third of the sward height, and correlated with microclimatic data. Significant differences (P < 0.05) were observed among treatments in the L3 recovery. Despite different grass heights between treatments and microclimates within the sward, the L3 concentration generally did not differ significantly among the three strata within a treatment (P > 0.05). Pasture microclimate did not correlate with larval recovery. At the end of the experiment, the animal fecal egg count was similar among treatments (P > 0.05). The results indicated that different lamb feeding systems in a tropical erect grassland caused differences in grass height but did not affect the distribution of infective larvae among strata. Larvae were found from the base to the top of the grass sward.

Rapid method for recovery of strongylid third stage larvae of parasitic nematodes from small soil samples

Livestock with access to pasture is generally exposed to infections with parasitic nematode species by uptake of infective third stage larvae (L3) with the grass. L3 can survive on pasture and particularly also in the soil up to several months and sometimes even longer, depending on temperature and humidity. As indicators for health and productivity of grazing animals it is important to determine the intensity and species spectrum of parasitic nematode larvae by analysing grass as well as soil samples. A rapid method for the recovery of L3 using a centrifugal-flotation technique from soil samples of 50–500g was developed. The method takes advantage of the low specific weight of larvae to separate them from equal sized soil and debris particles by centrifuging them in a saturated sugar solution. A stack of differently sized sieves is used to achieve elimination of larger particles, dust and sugar from the sample to enable easy counting of larvae. Independent of the number of larvae used for inoculation of the samples a mean recovery of 75.3% was obtained. The recovery rates obtained ranged between 60.8% and 88.0% which demonstrates a considerably lower variability compared to earlier approaches and therefore a more precise estimation of the actual numbers of parasite larvae in soil is achieved. Further advantages over already developed methods are the use of easy, affordable and eco-friendly materials, the simplicity of the procedure and a faster processing time with the possibility to examine up to 20 samples per day.

Biochemical and histopathological alterations in Biomphalaria glabrata due to co-infection by Angiostrongylus cantonensis and Echinostoma paraensei

The effect of concurrent infection by Echinostoma paraensei and Angiostrongylus cantonensis on the activity of the enzymes alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and the concentration of total proteins, uric acid and urea in the hemolymph of Biomphalaria glabrata were investigated. Additionally, histopathological studies were conducted to better understand the dynamics of ontogenic development of both helminths in the host and the possible biochemical effects. Co-infections by helminths and other parasites often occur due to the wide distribution of helminths and the chronic nature of the infection. The biochemical parameters were measured at the end of the seventh week after exposure. The co-infection resulted in a significant decrease in the total proteins concentration in the hemolymph of snails as well as an increase in the nitrogen excretion products, these results showed that the infection leads to exhaustion of free circulating and stored carbohydrates and the infected snails make use alternative substrates, such as free amino acids. So, the protein degradation to release free amino acids causes a decrease in the content of total proteins in the snail host and an amino acids deamination process, increasing the content of ammonium, which needs to be detoxified. This occurs by increasing the urea and uric acid contents. This observation is corroborated by the increase of ALT and AST activities, enzymes directly related to amino group from an amino acid to an α-ketoacid an important step to generate new carbon skeleton for glucose synthesis de novo, as well as new intermediates to the Krebs cycle. Additionally, reduction in the recovery of L3 from the co-infected group (A+E) was observed, since in this association the burden was higher than in the other. Histopathological results showed a change in the distribution of A. cantonensis in the presence of E. paraensei, indicating that the presence of this trematode impairs the dynamic transmission of A. cantonensis.

Soil moisture modulates the effects of the timing and amount of rainfall on faecal moisture and development of Haemonchus contortus and Trichostrongylus colubriformis to infective third stage larvae

Recent experiments on the effects of rainfall and/or soil moisture (SM) on development of sheep gastro-intestinal nematodes to infective L3 stage have used soil of relatively low moisture content in small experimental samples that dry out faster than field soil. To determine whether higher and more sustained SM content modulates the effects of rainfall amount and timing on faecal moisture (FM) and development of H. contortus and T. colubriformis to infective third stage larvae (L3), a climate-controlled chamber experiment was conducted. It was designed to test the effects of rainfall amount (0, 12 and 24mm), rainfall timing (days −1, 0 and 3 relative to faecal deposition) and soil moisture maintained at 10, 20 and 30% on these variables. Total recovery of L3 14 days after faecal deposition was significantly affected by SM, rainfall timing and their interaction (P<0.01), but not by rainfall amount or species or other two-way interactions. Recovery of L3 was maximal (28%) with a SM treatment of 30% and simulated rainfall on day 3. Faecal moisture was significantly affected by collection day, SM treatment, rainfall amount and rainfall timing with significant interaction between many of these effects (P<0.05). A positive linear association between FM and total L3 recovery was strongest on day 4 after faecal deposition (R2=0.64, P<0.001) for H. contortus and day 6 (R2=0.78, P<0.001) for T. colubriformis. Overall the results show that SM is able to modulate the effects of rainfall timing and amount with increased SM acting to broaden the window of opportunity for the free-living stages to respond to post deposition rainfall to complete development to L3. If SM is maintained in the range 10–30%, the reported benefits of early rainfall (days −1 and 0) of up to 24mm appear to be negated with later rainfall (day 3) proving more beneficial. These results require field confirmation.

Soil moisture influences the development of Haemonchus contortus and Trichostrongylus colubriformis to third stage larvae

Two climate chamber experiments were conducted to determine the effect of varying initial soil moisture (0, 10 and 15%), simulated rainfall amount (0, 12 and 24mm) and simulated rainfall timing (days −1, 0 and 3 relative to faecal deposition) on development (day 14) of Haemonchus contortus and Trichostrongylus colubriformis to the third stage larvae (L3) and faecal moisture (FM). Increasing initial soil moisture content from 0 to 10 or 15% led to higher recovery of total L3 (P<0.001). Total L3 recovery increased with each level of simulated rainfall (P<0.001) in the ascending order of 0, 12 and 24mm. There was an interaction between the effects of initial soil moisture and simulated rainfall amount on the recovery of total L3, showing that the benefit of increased simulated rainfall lessened with increasing soil moisture. Simulated rainfall on the day of deposition resulted in higher recovery of L3 (P<0.001) than simulated rainfall on other days. FM on day 3 relative to faecal deposition was best associated with recovery of total H. contortus and T. colubriformis L3 (R2=0.32–0.46), reinforcing the importance of sufficient moisture soon after faecal deposition. The effects of initial soil moisture, and the amount and timing of simulated rainfall on development to L3 were largely explained by changes to FM and soil moisture values within 4 days relative to faecal deposition. These results highlight the influence of soil moisture and its interaction with rainfall on development of H. contortus and T. colubriformis to L3. Consequently we recommend that soil moisture be given greater importance and definition in the conduct of ecological studies of parasitic nematodes, in order to improve predictions of development to L3.

Effect of simulated rainfall timing on faecal moisture and development of Haemonchus contortus and Trichostrongylus colubriformis eggs to infective larvae

Three climate-controlled chamber experiments were conducted to determine the effect of 32mm simulated rainfall applied prior to (days −4 to −1) or after (days 0–7) faecal deposition and as a single (32mm) or split (2×16mm) application on faecal moisture (FM) and development of H. contortus and T. colubriformis to third stage infective larvae (L3). The timing of simulated rainfall regulated extra-pellet L3 recovery for H. contortus (P<0.05) but not T. colubriformis. Recovery of L3 was highest (P<0.05) when simulated rainfall was applied on the day of deposition followed by days −1, 1 and 2, which resulted in similar but lower development success rates. Recovery of intra-pellet T. colubriformis L3 was two-fold greater (P=0.008) than for H. contortus and was higher (P=0.007) following simulated rainfall on days 0 and 1 than on other days. There was a positive association between FM and total L3 recovery indicating the importance of FM in the period 48–72h (H. contortus) and 72–96h (T. colubriformis) after deposition. Simulated rainfall on the day prior to deposition was as effective in supporting total L3 recovery as application on days 1 or 2 and this effect could be predicted through FM. This highlights the importance of soil in transferring moisture to the faecal pellet. The importance of precedent rainfall and soil moisture in determining the development success of H. contortus and T. colubriformis, in addition to the general effects of the timing of simulated rainfall, need to be accommodated in grazing management programs to combat these species.

Environmental factors influencing the transmission of Haemonchus contortus

Infection with the gastrointestinal nematode Haemonchus contortus causes considerable losses in the sheep industry. In this study, we evaluated the effect that climate has on third-stage larvae (L3) of H. contortus in terms of their migration from sheep feces to Brachiaria decumbens grass, as well as their distribution among the forage plants. Fecal samples containing H. contortus L3 was deposited on the soil among the herbage at an initial height of 30cm. Sample collection began 24h after contamination and was performed on alternate days over 13 days. The L3 were recovered and quantified in three strata (heights) of grass (0–10cm, 10–20cm and >20cm) as well as in the remaining feces and a superficial layer of soil, collected from beneath the feces. In order to obtain results under different environmental conditions, fecal samples containing H. contortus L3 were deposited on pasture in January (summer), in April (autumn), and July (winter). In all of the periods, the L3 were able to migrate from the feces to the herbage. However, rains, accompanied by high relative humidity and high temperatures, apparently favored migration. The highest L3 recovery rate in the pasture was in the summer observation period, which had the highest number of days with measurable precipitation, high relative humidity (>68.2%), and the highest temperatures at the soil level (minimum and maximum means of 19°C and 42°C, respectively). Under those conditions, larvae began to reach the upper stratum of the grass (>20cm) by 24h after the deposition of fecal matter, the number of larvae having reached that stratum peaking at seven days after deposition. In the autumn observation period, there was no rainfall in the first five days post-contamination. During that period, high numbers of larvae were found in the fecal samples demonstrating that feces can act as a reservoir of larvae in the absence of rain. Except for two days in the summer observation period, when most of the L3 were recovered from the tops of blades of grass, L3 where located predominantly at the base of the herbage. In conclusion, rainfall favors the migration of L3 from feces to herbage. In addition, larval migration up and along blades of grass can occur relatively rapidly when the temperature is high.

Dynamics of the free-living stages of sheep intestinal parasites on pasture in the North Island of New Zealand. 2. Weather variables associated with development

AIM: To identify weather variables associated with the development of eggs of Teladorsagia (=Ostertagia) circumcincta, Trichostrongylus colubriformis and Haemonchus contortus to third-stage infective larvae (L3) under a range of climatic conditions on pasture in the North Island of New Zealand. METHODS: Sheep faeces containing known numbers of eggs of all three nematode species were deposited on, or buried in, pasture plots at three sites, viz coastal Manawatu, Upper Hutt Valley, and East Cape hill country. Development was measured by recovering L3 from faeces, herbage and soil 28–31 days after deposition, on 13–18 occasions between January 2005 until July 2006. Weather data were recorded at each site, and the association between weather variables and number of L3 recovered was analysed using subsets regression to select best-fitting models from several candidate variables, after adjustment for efficiency of recovery of L3. A multiple linear regression model was then developed for each species, to select weather variables that had both significant and substantive effects on the number of L3 recovered. RESULTS: For all species, mean daily temperature was the bestpredictor of the number of L3 recovered (p=0.001). For T.circumcincta the final model included mean daily temperature and soil temperature (R2=51%), and for T. colubriformis the model only included mean daily temperature (R2=55%). For development of H. contortus, mean daily temperature was the most significant variable, but moisture in the form of rainfall entropy, i.e. the temporal distribution of rainfall, overthe first 14 days was also significant in the final model (R2=34%). CONCLUSIONS: Temperature was the most important determinant of developmental success of free-living nematodes on pasture at the study sites, and probably also for other parts ofNew Zealand with similar climates. Moisture was not significant in the development of T. circumcincta or T.colubriformis, implying that under the generally moist temperate climate in New Zealand, moisture is seldom limiting for development of these species. Haemonchus contortusappeared to be sensitive to moisture availability and precipitation in the first 14 days after deposition of faeces. The results of this study will be further developed as part of species-specific climate-driven models of parasite development in New Zealand.

Desenvolvimento e migraÇão de larvas infectantes de ciatostomíneos (Nematoda: Cyathostominae) em gramínea coast cross (Cynodon dactylon) em clima tropical, na Baixada Fluminense, RJ, Brasil

A study following the development and migration of Cyathostominae infective larvae was conducted from July 2003 to November 2004 in tropical climate, Baixada Fluminense, RJ, Brazil. Samples of naturally infected feces were placed on 12 m2 plot each month on a cyathostomin-free "Bermuda grass" pasture (Cynodon dactylon). After Seven days, samples of feces and grass were collected every week at 8 a.m, 1 and 5 p.m., weighed and processed by Baermann technique. Higher survival of L3 was found at dry season, 15 and 12 weeks on feces and sward respectively, at rainy season the survival was smaller. The multivariable analysis of main components was evident the influence of time and environment variables on L3 recovery from feces and grass. Close relationship between time and the number of L3 in feces could be noted, in contrast with L3 in sward. The climatic conditions influenced directly the number of infective larvae. The infective larvae were recovered during three times and the Kruskal-Wallis test did not present significance among them.

Recuperação de larvas infectantes de Trichostrongylus colubriformis em três espécies de gramíneas contaminadas no verão

O experimento teve como objetivo avaliar a recuperação de larvas infectantes (L3) de Trichostrongylus colubriformis em Brachiaria decumbens cv. Australiana, Cynodon dactilon cv. Coast-cross e Panicum maximum cv. Aruana. Foram utilizados módulos experimentais constituídos por seis canteiros de 32,4 m² cada, perfazendo dois canteiros por espécie forrageira. Cada canteiro foi dividido em 36 parcelas, de 30 x 30 cm, de forma a permitir seis repetições por espécie e por altura da forragem em cada semana de colheita de material. A sobrevivência larval foi avaliada do meio do verão e até meados do outono, sob o efeito de duas alturas de poda das forragens: baixa, 5 cm e alta, 30 cm. A poda foi realizada imediatamente antes da deposição das fezes contaminadas com ovos de T. colubriformis, obtidos de ovinos, que ocorreu no dia 05/02/ 2004. A colheita das fezes e da forragem foi realizada uma, duas, quatro, oito, 12 e 16 semanas após a deposição das fezes nos canteiros experimentais. A altura da forragem foi medida em cada uma das subdivisões imediatamente antes da colheita. A forragem foi cortada rente ao solo, de uma área delimitada com o auxílio de um círculo de 10 cm de raio. As fezes foram recolhidas manualmente dos canteiros. O número de larvas infectantes recuperado foi muito pequeno em comparação com a quantidade de larvas produzidas nas culturas controle, mantidas no laboratório (máximo de 6,7% no capim Aruana com 30 cm de altura). Arecuperação de L3 das amostras fecais foi maior quando as fezes foram depositadas em meio ao capim alto (com 30 cm - P&lt;0,05). Porém, a recuperação de L3 das forragens foi similar em ambos os cortes. Em relação à concentração de L3 (número de L3/kg de matéria seca), não houve diferença entre o corte baixo e alto (P&gt;0,05) em nenhuma das semanas experimentais. Dentre as espécies forrageiras, o capim aruana foi o que, no geral, apresentou maiores concentrações de L3 de T. colubriformis.

Effect of plant trichomes on the vertical migration of Haemonchus contortus infective larvae on five tropical forages

The influence of trichomes on vertical migration and survival of Haemonchus contortus infective larvae (L3) on different forages was investigated. Four different forages showing different distributions of trichomes (Brachiaria brizantha cv. Marandu, Brachiaria brizantha cv. Xaraes, Andropogon gayanus, and Stylosanthes spp.), and one forage species without trichomes (Panicum maximum cv. Tanzania), were used. Forages cut at the post-grazing height were contaminated with faeces containing L3. Samples of different grass strata (0-10, 10-20, >20 cm) and faeces were collected for L3 quantification once per week over four weeks. In all forages studied, the highest L3 recovery occurred seven days after contamination, with the lowest recovery on A. gayanus. In general, larvae were found on all forages' strata. However, most of the larvae were at the lower stratum. There was no influence of trichomes on migration and survival of H. contortus L3 on the forages.