Final report for GNE17-145
- Problem Addressed/Solution Pursued:
The main goal of this project is to investigate the role of season and age of adult gastrointestinal nematodes of small ruminants, Haemonchus contortus, on the hatchability of their eggs and ability of the resulting infective L3 larvae to exsheath in vitro as well as in vivo within the rumen of four fistulated sheep.
Gastrointestinal nematode infections pose a major threat to the health of small ruminants, such as sheep and goats, leading to significant productivity and financial losses for producers. Small ruminants suffering from GIN infection may experience severe anemia, weight loss, diarrhea and in highly susceptible animals, death. The GIN species of greatest concern is H.contortus, also known as the barber pole worm. The barber pole worm is the most pathogenic GIN parasite accounting for the greatest portion of production losses. Young animals along with pregnant and lactating small ruminants are especially susceptible to infection due to their immature or suppressed immune system, respectively.
Ruminants have a four-chambered stomach that begins with the reticulum, and segments into the rumen, omasum and abomasum. Haemonchus contortus mature and reproduce in the abomasum where they feed on the host’s blood. Eggs are shed in feces, hatch out, go through the L1 and L2 stage and emerge from the fecal pellet as infective third stage larvae (L3). Animals on pasture ingest the L3 larvae that hatched out in the feces. The L3 parasite is encased in a protective sheath covering that must be shed in the rumen in order for the larvae to continue maturation into adult blood feeding worms. The process of shedding the sheath is termed ‘exsheathment’ and is critical for establishment of infection within the host. Although the details are not fully understood, it is believed that carbon dioxide (CO2) within the rumen environment plays a role in inducing exsheathment.
A rising issue with gastrointestinal nematodes (GIN) is the development of resistance to all three classes of commercially available anthelmintics (dewormers). This has created a need for research and development of anthelmintic alternatives. One promising area of research focuses on plants that contain condensed tannins and other plant secondary compounds. These compounds have been shown to have an anthelmintic effect against different life stages of GIN species as assessed primarily through in vitro assays. Using these assays, it has been observed that condensed tannins have an inhibitory effect on the hatchability of eggs produced as well as on the exsheathment process of L3 stage larvae in H.contortus.
Experimentally infected donor sheep and goats are routinely used to provide the fecal eggs and larvae that are used in these in vitro egg hatch as well as in vitro and in vivo exsheathment assays, on a year round basis. The effect of cold storage on experimentally obtained L3 is well known, with most published studies noting that larvae used in their studies had not been in refrigerated storage for longer than three months. It is also known that H.contortus larvae arrest their development within the host during the shorter and colder days of the winter months and transition into a hypobiotic state. While hypobiosis has been studied extensively, it is unknown how environmental factors such as season or the age of the adult H. contortus worm specifically affect the viability of the eggs deposited in the manure or the efficiency with which the resulting larvae exsheath in the rumen under experimental conditions.
We have observed unexplained variability in larval performance, particularly in their ability to exsheath using both in vitro as well as the less common in vivo assays. This variability has been noted in other laboratories, particularly in in vivo work related to time to complete exsheathment. This unexplained variability can impact the results/interpretation of the research. Understanding the factors that influence this variability can inform current recommendations for the control of these parasites.
- Research Approach:
This project was designed to run in a series of cycles according to the four different seasons (Fall, Winter, Spring, and Summer). Each cycle began with the infection of two donor lambs with 10,000 H. contortus L3. Fecal samples were collected from the donor animals once a month for eggs and larvae to be harvested from the feces. The eggs that were collected would be used in the in vitro egg hatch assay to assess the effect of season and age of adult worm on egg hatchability. The larvae that was collected was subjected to two different exsheathment assays (in vitro using CO2 and in vivo using rumen fistulated ewes). Both viability and exsheathment were assessed to determine any impact of season or age of adult worm. Due to observed variability in in vitro exsheathment data during the Fall cycle, a fifth cycle was added as a replicate of the Fall cycle. Data is currently being analyzed to determine how season and age of worm impact viability and exsheathment of H.contortus L3, as well as hatchability of eggs.
- Research conclusions:
Preliminary data analysis suggests that there is a combination effect of season and age of worm on both in vitro and in vivo larval exsheathment. These effects are not seen for the egg hatch life cycle stage. A pattern of variability in in vitro exsheathment was observed specifically for the Fall season, which was an unexpected outcome at the outset of this project. It has also been determined that when examining the effect of season and effect of age of worm separately, data between the in vitro and in vivo exsheathment assays differ within certain seasons and months, respectively.
This project was designed to further our understanding of how external factors, such as the environment and age of the adult worm, affect eggs and larvae produced by adult worms. By expanding our knowledge and having a better understanding of the life cycle of this economically important gastrointestinal nematode, researchers can improve practices, leading to better recommendations for farmers. The results of this project can have specific impacts on the agricultural community in the following ways: 1) Lead to the reduction of environmental and health risks in agriculture due to a clearer understanding of the impact of the environment on the adult parasite, leading to the development of targeted control strategies that will improve health and well being of small ruminants; 2) Can lead to improved productivity of sheep and goats through a reduction in the cost of raising sheep and goats and ultimately an increase in net farm income for small ruminant producers.
The main objective of this project is to elucidate factors affecting the hatchability of H. contortus eggs, as well as the viability and exsheathment of larvae from adult worms residing within the host animal (sheep and goats). This study can lead to new discoveries and broaden our understanding of the H. contortus life cycle in relation to sensitivity to environmental season and temperature, as well as length of infection within the host.
- To determine if age of the adult worm in the host animal affects the ability of the egg to hatch as well as viability and exsheathment of the resulting third stage H. contortus larvae.
- To determine the effect of season on the adult worm in the host animal and the ability of the eggs from these worms to hatch at varying times of year as well as viability and exsheathment of the resulting L3 H. contortus larvae.
- Correlate a common artificial in vitro exsheathment method using CO2 to an in vivo exsheathment method using ruminally fistulated ewes.
The overarching purpose of the proposed project is to expand knowledge on the life cycle of H. contortus and factors affecting infection development. Currently, there is limited work that has been conducted that looks specifically at both the effect of age of worm and season on the exsheathment stage (both in vitro and in vivo) and egg hatch stage of the H.contortus life cycle. Filling this gap in knowledge is crucial in order to refine methodology for how H.contortus infection is studied.
This study will further our understanding of the factors affecting various life stages of of H.contortus . GIN infection serves as a threat to animal health and welfare for small ruminants, which in turn has a major impact on small ruminant production operations, causing significant economic loss for farmers. Finding a sustainable solution for GIN control in small ruminants will improve animal health and welfare, as well as the economic viability of producers.
- (Educator and Researcher)
This project ran in a series of five cycles corresponding to the beginning of the four seasons of the year 1) Fall 2017 (autumnal equinox, September 22, 2017), 2) Winter 2017 (winter solstice, December 21, 2017), 3) Spring 2018 (vernal equinox, March 20, 2018), 4) Summer 2018 (summer solstice, June 21, 2018), and 5) Fall 2018 (autumnal equinox, September 22, 2018). Daily temperature, humidity and precipitation data were obtained from the weather station at the University of Rhode Island’s Peckham Farm (sponsored by NOAA). Each cycle began (time (t) = 0) with the experimental infection of two donor lambs with 10,000 H. contortus L3 larvae. Fecal egg counts (FEC) and packed cell volume (PCV) were conducted weekly at t = 0 and every week thereafter through 24 weeks of infection. Fecal samples were collected from each lamb for both egg and larval culture beginning at four weeks of infection, every four weeks, through 20 weeks of infection (6 collection times). Upon the collection of the fecal sample, eggs were extracted from the feces and used in the egg hatch assay to determine egg hatchability. The remainder of the fecal sample was used to prepare fecal cultures that yielded L3 larvae. The larvae was extracted from the culture samples and was used in an in vitro exsheathment assay using CO2 treatment, as well as in an in vivo exsheathment assay using 4 ruminally fistulated ewes. Results were statistically analyzed to determine the effect of adult worm age and season upon egg hatchability and exsheathment as well as agreement between in vivo versus in vitro exsheathment assays.
Lamb selection and infection:
Four sexually mature male lambs (born in the spring of 2017), 4 sexually mature female lambs (born in the fall of 2017) and two sexually mature male lambs (born in the spring of 2018) at the University of Rhode Island’s Peckham Farm, were experimentally infected with H. contortus for this project. These donor lambs provided the fecal samples needed for completion of the work outlined in this proposal. In addition, four fistulated ewes, housed at Peckham Farm, were used for the in vivo exsheathment assays. All procedures used in this study were approved by the University of Rhode Island’s Institutional Animal Care and Use Committee (IACUC).
Fecal Egg Count and Packed Cell Volume:
Blood samples were taken weekly from each lamb and processed to determine packed cell volumes (PCV) using the micro-hematocrit centrifuge method. Fecal samples were also taken on a weekly basis in order for fecal egg counts (FEC) to be determined for each lamb. Fecal egg counts were determined using the modified McMaster technique (Whitlock, 1948). All FEC are expressed in eggs per gram with each egg that is counted representing 50 eggs/gram of feces.
Egg Recovery and Egg Hatch Assay:
Upon collection of fecal samples from the two donor lambs during each season, approximately 10 grams of fecal matter was used for egg recovery. The 10 grams of feces was combined with water to create a slurry mixture. The slurry is poured over sieves of decreasing sizes (1 mm, 355, 150, 38 and 25 μm). Eggs are too large to pass through both the 38 and 25 μm sieves and were collected using a salt solution (Fecasol®, Vetoquinol U.S.A., Inc., Fort Worth, TX, USA). The egg-containing solution is centrifuged and eggs are collected using cover slips. The cover slips are rinsed using water and the final aqueous solution of eggs is used in the egg hatch assay that day. The egg hatch assay was conducted using established procedures (Assis et al. 2003; Marie-Magdeleine et al. 2009). Eggs were added (100 eggs in 100 μl of water per well) to a 24-well flat-bottomed microtitre plate (CorningTM, FalconTM, Polystyrene Microplates, Corning Life Sciences, Tewksbury, MA, USA). Water is added (1,900 μl) to each well to bring the final volume to 2 ml. A set of 10 wells will be run for each lamb. The well plates were incubated at 26°C for 24 hours and read under a microscope the following day to determine the the percent of eggs hatching.
After initial fecal sample collection, larvae was recovered using a modified version of the Baermann Technique (Zajac and Conboy, 2012). The fecal samples were maintained in a humid environment and incubated at 25°C for 1-2 weeks. The fecal samples rested on cheesecloth and were flooded with water to recover hatched larvae in a concentrated solution.
In vitro Exsheathment Assay:
In vitro CO2 Exsheathment Assay – Briefly, 2,000 L3 larvae were added to a polypropylene tube and Earle’s Balanced Salt Solution (EBSS, Sigma-Aldrich®, Inc., Natick, MA, USA) was added to bring the volume to one mL. An additional one mL of water was added to the tube to bring the final volume to two mL. Three tubes were prepared for each of the two fecal samples (three duplicates for each lamb). The tubes were exposed to CO2 treatment using a modified version of the technique proposed by Conder and Johnson (1996). The solutions were bubbled with CO2 for 15 minutes each, capped and then incubated at 37°C for 18 hours. After the incubation period, the larvae was observed for viability and presence of sheath. At least 100 live larvae were counted for each tube and percent viability as well as percent live exsheathment were calculated.
In Vivo Exsheathment Assay:
Prior to performing the in vivo assay, the L3 larvae that were briefly stored in the fridge were adjusted to room temperature. The exsheathment assay used for this project is a modified version of previously published procedures (Brunet, et al. 2007). Both concentration and viability were determined for each set of larvae. A total of 2,000 ensheathed L3 larvae were then added to a capsule composed of a piece Tygon® tubing (ID 9.5, OD 14.3 mm, Fisher Scientific, Hampton, NH) and two 8 μm NuncTM Cell Culture Inserts on either end of the tubing (#140629, Thermo Scientific, Waltham, MA). A total of 8 capsules were prepared—4 capsules per donor lamb. One capsule was placed in a heat-sealed concentrate bag (R510, ANKOM Technology, Macedon, NY), to prevent large particles in the rumen from clogging the insert membrane, and was tied to a 20 cm cord that was suspended in the rumen of the four fistulated ewes. The capsules were removed after an exposure period of 8 hours. The larvae in the capsules were observed for viability and exsheathment with approximately 150 live larvae being counted from each capsule.
For Egg Hatch, in vitro exsheathment and in vivo exsheathment assays:
Data was analyzed using the GLIMMIX procedure in SAS (SAS Institute Inc., Cary, NC).. The model included terms cycle (representing each seasonal cycle); worm age (age of infection); and cycle*worm age. A post hoc adjustment for multiple comparisons test was performed for simple effect comparisons of cycle*worm age least square means by cycle and by age. Significance of least square means was defined as p < 0.05.
Assis, L. M., C. M. L. Bevilaqua, S. M. Morais, L. S. Vieira, C. T. C. Costa, and J. A. L. Souza. 2003. Ovicidal and larvicidal activity in vitro of Spigelia anthelmia Linn. extracts on Haemonchus contortus. Vet. Parasitol. 117:43–49.
Brunet, S., J. Aufrere, F. El Babili, I. Fouraste, and H. Hoste. 2007. The kinetics of exsheathment of infective nematode larvae is disturbed in the presence of a tannin-rich plant extract (sainfoin) both in vitro and in vivo. Parasitology. 134:1253–1262.
Conder, G. A., and S. S. Johnson. 1996. Viability of infective larvae of Haemonchus contortus, Ostertagia ostertagi, and Trichostrongylus colubriformis following exsheathment by various techniques. J. Parasitol. 82:100–102.
Marie-Magdeleine, C., H. Hoste, M. Mahieu, H. Varo, and H. Archimede. 2009. In vitro effects of Cucurbita moschata seed extracts on Haemonchus contortus. Vet. Parasitol. 161:99–105.
Whitlock, H.V., 1948. Some modifications of the McMaster Helminth egg-counting
technique and apparatus. J. Counc. Sci. Res. 21, pp. 177–180.
Zajac, A.M., and G.A. Conboy. 2012. Veterinary clinical parasitology. 8th ed. John Wiley &Sons inc. Chichester, West Sussex, UK.
Preliminary data analysis has shown that there is a combination of season and age of worm effect on both the viability and exsheathment of H.contortus L3 both in vitro and in vivo. It has shown no evidence for an effect of season or age of worm on the egg hatch stage of the life cycle.
Preliminary data analysis has also detected differences in both % viability and % exsheathment between the two exsheathment methods (in vitro and in vivo). This may suggest that the two assays do not correlate accurately to one another.
Preliminary data analysis has also revealed an unexpected amount of variability in larval performance in the in vitro CO2 exsheathment assay as well as variability in the amount of time needed for larvae to reach the L3 stage prior to being used in assays.
The differences in development patterns between the seasonal cycles should be explored in more depth in future research.
Preliminarily, there appears to be an effect of environment as well as age of the adult worm that influences the ability of larvae to exsheath both in vitro and in vivo. Increasing our understanding of the impact of season and age of the worm on commonly used assays to study alternative methods of parasite control will increase the accuracy of future data and ultimately help shape recommendations for farmers.
Education & Outreach Activities and Participation Summary
In May of 2018, I conducted an educational workshop at the Compass School in South Kingstown, Rhode Island. The workshop focused on GIN infection, methods for detecting infection and general information about H. Contortus and how we study this parasite. The workshop was designed for elementary school children who were participating in a farming class at their school. The students participated in an interactive lecture, show and tell segment, as well as a hands-on field trip to Peckham Farm at the University of Rhode Island. During the field trip, students were able to interact with goats and learn how to perform FAMACHA© eye scoring and fecal floats. The teacher of the class is a farmer at the school and she also participated in all activities.
In July of 2018, I attended the annual meeting for the American Association of Veterinary Parasitologists. I presented a poster on the proposed project based on data gathered from September 2017-July 2018. Those attending the meeting included undergraduate/graduate students, ag service providers, and animal science/agriculture professors, as well as others interested in veterinary parasitology. A copy of the presentation abstract is attached to this report.
Once the results are finalized, they will be presented at farmer workshops and on the URI Small Ruminant website.
H. contortus is the most pathogenic GIN affecting small ruminant animals, which ultimately has a negative economic impact on producers. GIN infection can become fatal if not properly treated, which is a social concern for consumers in terms of animal health and welfare. Findings from this project will allow researchers in the field to better understand the life cycle and development of H.contortus, especially in a research setting, in terms of how larvae perform in commonly used assays. Having a clearer understanding of the life cycle of this parasite will ultimately lead to improved recommendations for small ruminant producers to control/manage infections. This can improve economic viability for producers and improve health and animal welfare for small ruminant animals.
This project has revealed more information than we had expected at the outset. While we were able to answer the questions asked in our objectives, we also uncovered trends and discrepancies that we did not have any knowledge of (i.e variability in in vitro data, varying culture periods, etc.). Having a more informed understanding of the factors that can produce variability in data has been one of the biggest sources of knowledge that my advisor and I have both gained from this experience. The results of this study have indicated that certain experimental methods need to be reevaluated, which has the potential to lead to skill development in learning new assays. Ultimately, we are now more informed as researchers in the field of sustainable agriculture and we hope to continue to explore factors that may influence our experiments. I hope to pursue a career in education as a teacher or lecturer in the field of Animal Science, Agriculture, Biology or a related field.