1. VERTICAL MIGRATION OF HAEMONCHUS CONTORTUS INFECTIVE LARVAE ON CYNODON DACTYLON AND PASPALUM NOTATUM PASTURES ABSTRACT OVERVIEW: Observations were made on vertical migration patterns of Haemonchus contortus infective larvae on Cynodon dactylon (bermudagrass) and Paspalum notatum (bahiagrass) pastures under summer climatic conditions typical of East Texas. OBJECTIVES: 1. To determine the limits of vertical migration if the L 3 stage of H. contortus in pasture swards of bermudagrass and bahiagrass, 2. Which climatic conditions have the strongest influence on nematode vertical migration, and 3. Whether grass species influenced larval migration. METHODS: Ten thousand H. contortus infective larvae (L 3 ) were introduced to 100 cm 2 subplots of each pasture species within a plot area of 1 m 2. Herbage from the inoculated areas was harvested on five sampling days over a span of 21 days. L 3 recoveries were observed and recorded each day on four herbage strata viz. 0 - 5, 5 - 10, 10 - 20 and > 20 cm from ground level. The log transformed larval recovery data were analyzed for effect of day, stratum, and day X stratum interaction for each grass species during two separate experimental periods. Precipitation, relative humidity and temperature during the study were subjected to correlation and multiple regression analyses with the larval counts. RESULTS: Significant (p ≤ 0.0017) differences were found for the effect of day on larval recoveries. Stratum effect, though not significant, provided a strong indication of influencing larval recovery. A high positive correlation (≥ 0.93) between rainfall and total average daily larval counts was apparent. CONCLUSIONS: The H. contortus infective larvae can survive beyond 21 days in the soil and infest pasture grasses when climatic conditions are favorable. Avoiding use of H. contortus contaminated pasturelands in summer at the onset of rainfall following a dry spell may effectively reduce worm loads in susceptible farm animals. Total larval recovery of H. contortus in this study was greater in bahiagrass than bermudagrass. While the design of this study did not allow statistical comparison between the two pasture species, studies with potted plants with a proper experimental design would allow for valid comparisons. Robert A. Lane 1 and Bimal S. Amaradasa 2 1 Professor of Agronomy, Sam Houston State University; 2 Ph.D. Student, Virginia Tech Culture of L 3 (free living) infective larvae –Fresh goat feces were placed in a gauze bag and suspended in a humid chamber for 7-9 days to initiate hatching of larval eggs and development to the L 3 infective stage –Feces bags were run overnight through the Baermann apparatus filled with water between 30º - 35º C –Larval-containing medium (bottom 5-8 ml) in the Baermann apparatus transferred to centrifuge tube Larval identification and enumeration –A key adapted from the Manual of Veterinary Parasitological Laboratory Techniques (1971) used –The nematodes from the centrifuge tubes were decanted into a Petri dish using 10 ml of tap water and diluted further for enumeration. –Petri dish diameter: 14 cm ; Grid size ; 2 cm 2 –Enumeration with aid of a dissecting microscope Storage of larvae –Larvae were stored in quantities of 10,000 per container in 150 cm 2 cell culture flasks Field Activities –Conducted at SHSU Gibbs Ranch, Huntsville, TX from June 20 - July 12 and July 30 - August 21, 2006 –Field sites of bermudagrass (Cynodon dactylon) var. Jiggs and bahiagrass (Paspalum notatum) had notbeen grazed by ruminants for at least 12 months prior –Each experimental site was divided into 30 equal plots, each having an area of 1 m 2 with a buffer zone of 0.5 m –10,000 larvae inoculated to the soil or grass mat within a marked 100 cm 2 area using a fine glass pipette –Vegetation within the marked areas was harvested beginning at 8.00 a.m. each day of harvest –Harvest days were randomly assigned to sub-plots & the time intervals were as follows: D1 = 1.5 days after inoculation D2 = 3.5 days after inoculation D3 = 7.5 days after inoculation D4 = 14.5 days after inoculation D5 = 21.5 days after inoculation –The harvested grass samples were straightened and clipped into four strata for larval recovery –Herbage strata used for larval recovery Soil level to 5 cm 5-10 cm height 10-20 cm height Above 20 cm from the ground Statistical Analysis –The log transformed numbers of larvae recovered per kg of dry mater were analyzed with ANOVA –The analysis for the two pasture species was done separately –The treatment means were separated using Fisher’s protected t-test least significant difference (LSD) –Larval counts & climatic data were analyzed by correlation & multiple linear regression Extracting L 3 larvae from herbage –The Baermann apparatus technique used again to extract L 3 larvae from herbage –A second inoculation was carried out on same bahiagrass site. The original bermudagrass plots lodged second inoculation had to move to another site having var. Cheyenne RESULTS CONCLUSIONS Though not statistically significant, this study gave a strong indication of strata effect on larval recovery. A number of larvae were recovered from the 10-20 cm strata. Precipitation and humidity are the predominant controlling factors affecting vertical migration of L 3 larvae during the summer months. Further study is needed to identify plant morphological features that favor or impede vertical larval nematode migration. The avoidance of grazing susceptible farm animals on H. contortus contaminated pasturelands at the onset of rainfall following a dry spell may effectively reduce heavy intake of worm loads. DISCUSSION  Past studies showed precipitation played a significant role in larval migration to herbage under both field and laboratory conditions (Fakae & Chiejina, 1988; Misra & Ruprah, 1972; Okon & Enyenihi, 1977; Silangwa & Todd, 1964). Our results support these findings.  Only BH1 had a significant stratum effect on larval counts. There was a strong trend for stratum effect in BH2 (P=0.13) and BM2 (P=0.09). A few larvae were observed in the >20cm stratum.  The larval recovery percentages for the four experiments ranged from 0.05 to 1 percent. Misra & Ruprah (1972) recorded larval recovery of 35.2, 27.8, 28.6 and 9.3 percent in autumn winter, spring and summer respectively in bermudagrass. The laboratory study carried out by Silangwa and Todd (1964) found only 2 to 3 percent of Trichostrongylid L 3 larvae on the grass blades.  Larval migration is greatly dependent on prevailing micro- and macro-climatic factors as well as plant features. More successful migration of L 3 onto the bahiagrass compared to Bermudagrass may be due to coarser leaf blade texture, more pubescence and a dense cluster of stems, possibly aiding in retention of a water film.  Lyaku, Monrad, & Kassaku (1988) recovered Haemonchus L 3 from soil samples artificially infected and studied under laboratory conditions for three weeks only. The field study conducted by Krecek et al. (1991) observed larval herbage recoveries were very low or negative after approximately 3 weeks from first migration. This study proved H. contortus could survive beyond 21 days under the environmental conditions prevalent in E. Texas. Source: Texas Agricultural Extension Service pamphlet L-5095 Symptoms & Economic losses - Chronic blood loss/ goats do not thrive/grow well - Reduced milk production; Hypoproteinemia (deficiency of blood proteins); edema (bottle jaw) - Anemia ; pale membranes of the eyes and gums and lethargy; Diarrhea (scours) common BACKGROUND Internal parasite management, especially of Haemonchus contortus (barberpole worm, stomach worm), is a primary concern for the majority of sheep and goat producers. Animal mortality levels from infection with this nematode species results in millions of dollars in losses to producers annually. These parasites have become increasingly difficult to manage due to apparent resistance to nearly all available dewormers. MATERIALS AND METHODS http://ucdnema.ucdavis.edu/imagemap/nemmap/Ent156html/upenn/hpl acF.gif Source: www.attra.ncat.org Adult H. contortus in goat abomasum Typical appearance of goat severely infected with H. contortus Baermann apparatus, used to collect L 3 larvae of H. contortus from fecal pellets L 3 larvae of H. Contortus (100X) No H. contortus L 3 larvae were recovered from herbage samples from control plots Larval recoveries from both grasses differed significantly for day (P≤ 0.0017) Interactions of stratum with day were not significant for either grass species There were no significant stratum effects on larval count except in bahiagrass - experiment 1 Observations of high L 3 on both grasses corresponded with occurrence of a rainy days For experiment 1, days four and five were significantly different from the other three days In experiment 2 significantly higher larval counts were recorded for day three with 19.3 mm of rainfall Neither of the two grasses tested appear suited to rapid or easy vertical migration by H. contortus larvae under the environmental conditions experienced during this study It seems that larvae are more successful in upward movement on bahiagrass than bermudagrass Very high positive correlation coefficients (R) for rainfall and total average daily larval counts were apparent Multiple regression analysis was not significant for climatic variables (temperature, relative humidity & rainfall) and larval counts Larval counts showed H. contortus larvae could survive dry periods exceeding 21 days after inoculation under summer weather conditions prevailing at Huntsville, Texas