The main goal of this project is to investigate the role of season and age of adult gastrointestinal nematode of small ruminants, Haemonchus contortus, on the hatchability of their eggs and ability of the resulting infective L3 larvae to exsheath in vitro and 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 highest 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 refrigerated storage on experimentally obtained L3 is well known, with most published studies noting that larvae used in their studies had not been in storage for more than three months. Although it is known that H.contortus larvae arrest their development within the host during the shorter and colder days of the winter months, it is unknown how the season or the age of the adult H. contortus worm affects 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 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 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.
This project is 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. This proposal has direct links to agricultural sustainability in the following areas: 1) the reduction of environmental and health risks in agriculture by 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) 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 viability of H. contortus eggs and larvae from adult worms residing within the host animal (sheep and goats). I hypothesize that both larval viability and larval exsheathment will be significantly lower when infection is developing in colder versus hotter temperatures. I also hypothesize that no significant differences will be observed for egg hatchability according to age of infection. This study can lead to new discoveries and broaden our understanding of 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 in vivo exsheathment using ruminally fistulated ewes.
The over-arching purpose of the proposed project is to further expand knowledge on the topic of the H. contortus life cycle and how this pertains to infection development and how researchers study it. This increase in knowledge will ultimately inform future research and allow for more educated and efficient decisions to be made in important studies. 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) of H.contortus L3. Filling this gap in knowledge is crucial in order to refine methodology for how H.contortus infection is studied.
By improving the quality of H.contortus research– commonly studied in association with anthelmintics– there is an increased chance in advancing efforts in finding alternative anthelmintic methods to combat GIN infection. As mentioned above, GIN infection 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.
This project will be run 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 (autmnal equinox, September 22, 2018). Daily temperature, humidity and precipitation will be obtained from the weather station at the University of Rhode Island’s Agronomy Farm. Each cycle will begin (time (t) = 0) with the experimental infection of two male donor lambs with 10, 000 H. contortus L3 larvae. Fecal egg counts (FEC) and packed cell volume (PCV) will be conducted weekly at t = 0 and every week thereafter through 20 weeks of infection. Fecal samples will be collected from each ram 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 will be extracted from the feces and used in the egg hatch assay to determine egg hatchability. The remainder of the fecal sample will be used to prepare fecal cultures that will yield L3 larvae. The larvae will be extracted from the culture samples and will be used in two different artificial in vitro exsheathment assays—bleach treatment and CO2 treatment—as well as in an in vivo exsheathment assay using 4 ruminally fistulated ewes. Results will be 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, will be experimentally infected with H. contortus for this project. These donor lambs will provide the fecal samples needed for completion of the work outlined in this proposal. In addition, four fistulated ewes, housed at Peckham Farm, will be available to use for the in vivo exsheathment assays. All procedures used in this study will be approved by the University of Rhode Island’s Institutional Animal Care and Use Committee (IACUC).
Fecal Egg Count and Packed Cell Volume:
Blood samples will be taken weekly from each lamb and processed to determine packed cell volumes (PCV) using the micro-hematocrit centrifuge method. Fecal samples will also be taken on a weekly basis in order for fecal egg counts (FEC) to be determined for each lamb. Fecal egg counts will be determined using the modified McMaster technique (Whitlock, 1948). Briefly, two grams of feces will be combined with sodium nitrate flotation solution (Fecasol®, Vetoquinol U.S.A., Inc., Fort Worth, TX, USA) and mashed into a slurry, then poured over cheese cloth to yield an aqueous egg solution that is then read under a microscope. All FEC are expressed in eggs per gram with each egg that is counted representing 50 eggs/gram of feces and are indicative of worm burden.
Egg Recovery and Egg Hatch Assay:
Upon collection of fecal samples from the two donor rams, approximately 10 grams of fecal matter will be used for egg recovery. The 10 grams of feces will be combined with water to create a slurry mixture. The slurry will be 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 will be collected using a salt solution (Fecasol®, Vetoquinol U.S.A., Inc., Fort Worth, TX, USA). The egg-containing solution is centrifuged and eggs will be collected using cover slips. The cover slips are rinsed using water and the final aqueous solution of eggs will be used in the egg hatch assay that day. The egg hatch assay will be conducted using established procedures (Assis et al. 2003; Marie-Magdeleine et al. 2009). Eggs will be added (100 eggs in 100 μl of water per well) to a 24-well flat-bottomed microtitre plates (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 4 wells will be run for each ram. The wells will be incubated at 26°C for 24 hours and read under a microscope the following day to determine number of eggs and number of larvae in order to determine percent egg hatchability.
After initial fecal sample collection, larvae will be recovered using a modified version of the Baermann Technique (Todd et. al, 1970). 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. The flooded cultures sat for 6-12 hours in order for larvae to settle within the water solution. Excess water was siphoned off the top so that a concentrated larvae solution was obtained.
In vitro Exsheathment Assays:
In vitro CO2 Exsheathment Assay – Briefly, 2,000 L3 larvae will be added to a polypropylene tube and Earle’s Balanced Salt Solution (EBSS, Sigma-Aldrich®, Inc., Natick, MA, USA) will be added to bring the volume to one mL. An additional one mL of water will be added to the tube to bring the final volume to two mL. Two tubes will be prepared for each of the two fecal samples (two duplicates for each ram). The tubes will be exposed to CO2 treatment using a modified version of the technique proposed by Conder and Johnson (1996). The four tubes will first be covered using Parafilm M® (Parafilm M®, Bemis Company, Inc., Neenah, WI). A glass pipet tip, connected to CO2, will be pushed through the Parafilm M® covering so that it is suspended in the aqueous larvae solution. The solutions will be bubbled with CO2 for 15 minutes each and then resealed using Parafilm M® before being capped. The capped tubes will then be incubated at 37°C for 18 hours. After the incubation period, the larvae will be observed for viability and presence of sheath. At least 100 live larvae will be counted for each tube and percent viability as well as percent live exsheathment will be calculated.
In Vivo Exsheathment Assay:
Prior to performing the in vivo assay, the L3 larvae that were briefly stored in the fridge will be adjusted to room temperature. The exsheathment assay used for this project was developed by a graduate student in our lab (Lonngren et al., 2017) as a modification of a previously published procedure (Hertzberg et al. 2002). Both concentration and viability will be determined for each set of larvae. A total of 2,000 ensheathed L3 larvae will then be added to a capsule that is composed of a piece Tygon® tubing (ID 9.5, OD 14.3 mm, Fisher Scientific, Hampton, NH) and an 8 ?m NuncTM Cell Culture Insert (#140629, Thermo Scientific, Waltham, MA). A total of 8 capsules will be prepared—4 capsules per donor ram. One capsule will each be placed in a heat-sealed concentrate bag (R510, ANKOM Technology, Macedon, NY), to prevent large particles in the rumen from clogging the insert membrane, which will be tied to a 20 cm cord that is suspended in the rumen of the four fistulated ewes. The capsules will be removed after an exposure period of 8 hours. The larvae in the capsules will be observed for viability and exsheathment with approximately 150 live larvae being counted from each capsule.
Statistical analysis of data is still pending, therefore there are no definitive results to present at this time. Preliminary data analysis has shown that there is a potential combination of season and age of worm effect on ability of larvae to exsheath in the in vitro CO2 assay. This trend was not seen for the in vivo assay data in preliminary analysis, which may suggest that the two exsheathment assays do not correlate accurately.
Preliminary data analysis also revealed an unexpected amount of variability in larval performance in the CO2 exsheathment assay specifically during the fall cycle. This variability led to a repetition of the entire fall cycle, which is currently being conducted. The data from the second fall cycle will be compared to the first fall cycle in order to determine the legitimacy of attributing the observed variability to season or age of larvae.
At this point in time, data has been collected for the the following cycles: Fall 2017, Winter 2017, Spring 2018, and Summer 2018. Data collection for the Fall 2018 cycle is ongoing. Upon the completion of data collection, the data will be statistically analyzed.
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, and animal science/agriculture professors, as well as others interested in veterinary parasitology. A copy of the presentation abstract is attached to this report.
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. As H. contortus is the most pathogenic GIN affecting small ruminant animals, 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.
Once data has been fully analyzed, this project has the potential to increase our knowledge about external factors that influence the life cycle of H. contortus. The main goal is to uncover knowledge that will guide future research directions and allow members of this research group to develop improved methods for studying H.contortus. I hope to pursue a PhD in a related field and ultimately become a lecturer or professor in the field on Animal Sciences and/or Sustainable Agriculture.