**Doppler Ultrasonography for Evaluating Vascular Responses to Ergopeptine Alkaloids in Livestock**

G.E. Aiken and J.R. Strickland *USDA-Agricultural Research Service, Forage-Animal Production Research Unit, Lexington, KY, USA* 

#### **1. Introduction**

566 A Bird's-Eye View of Veterinary Medicine

Ostrowski, S.; Dorrestein, G.M.; Ancrenas, M. & Saint-Jalme, M. Debilitanting cutaneous

PurcelL, D.A.; Clarke, J.K.; McFerran, B.J. & Hughes, D.A. The morphogenesis of pigeonpox

Reinolds, E.S. The use of lead citrate at high pH as an electron-opaque stain in electron

Ritchie, B.W. & Carter, K. Avian viruses: Function and control. Lake Worth, Florida, Ed.

Ritchie, B.W.; Harrison, G.J. & Harrison, L.R. Avian Medicine: Principles and application.

Sadosiv, E.C.; Chang, P.W. & Gulka, G. Morphogenesis of canary poxvirus and its entrance

Shivaprasad, H.L.; Kim, T.; Tripathy, D.; Woolcook, P.R.; Uzal, F.; Unusual pathology of

Smits, J.E.; Tella, J.L.; Carrete, M.; Serrano, D. & López, G. Na epizootic of avian pox in

Thiele, J.; Kiel, H. & Adolphs, H.D. Avien pox virus. An ultrastructural study on a cherrug

Trapp, J.L. Avian pox in the gray-crowned rosy finck in Alaska. North Am.Birds Bander., 5

Tripathy, D.N.; Schnitzlein, W.M.; Morris, P.J.; Janssen, D.L.; Zuba, J.K.; Massey, G.;

Tripathy, D.N., and W.M. Reed. 2003. Pox. In *Diseases of Poultry* , 11th Ed., Y.M. Saif, H.J.

Van Riper III, C.V.; Riper, S.G.V. & Hansen, W.R. Epizootiology and effect of avian pox on

Van Riper III, C. & D. J. Forrester. *Avian pox.* In: Infectious Diseases of Wild Birds. Ames, IA,

Watson, M. L. *Staining of tissue sections for electron microscopy with heavy metals. J. Biophyis.* 

Weli, S.C.; Okeke, M.I.; Tryland, M.; Nilssen, O. & Traavik, T. Characterization of avipoxviruses from wild birds in Norway. Can. J. Vet. Res., 68: 140-5, 2004. Weli, S. & Tryland, M. Avipoxviruses: infection, biology and their use as vaccine vectors.

Atkinson, C.T. Characterization of poxviruses from forest birds in Hawaii. *J. Wildl.* 

Barnes, J.R. Glisson, A.M. Fadly, and L.R. McDougald (eds.). Iowa State University

breeder canaries (Serinus canaria). Avian Pathol., 38(4):311-6, 2009.

berthelotti) in the Canary Island, Spain. Vet. Pathol., 42: 59-65, 2005. Terregino, C.; Catelli, E.; Delogu, M.; Capua, I. & Tonelli, A. Poxvirus infection in a blue

canary poxvirus infection associated with high mortality in young and adult

endemic short-toed larks (Calandrella rufescens) and berthelot's pipits (Anthus

Dis., 39(4): 904-11, 1995.

(2): 146-7, 1980.

*Dis., 36:*225-30, 2000.

virus. J. Gen. Virol., 15: 79-83, 1972.

microscopy. J. Cell. Biol., 17:208-12, 1963.

Publishing Incorporated, 1995. pp.285-311.

Florida, Ed. Wingers Publishing Inc., 1994. pp.865-74.

into inclusion bodies. Am. J. Vet. Res., 46 (2): 529-35, 1985.

bonnet owls from Florida. J. Wildl. Dis., 145 (9): 264, 1999.

falcon. Brief report. Arch. Virol., 62 (1): 77-82, 1979.

havaiian forest birds. *The Auk., 119(4):* 929-42, 2002.

Press, Ames, Iowa, U.S.A., pp. 253–269.

Ed. Blackwell Publishing, 2007. pp.131–176.

*Biochem. Cytol.,* 4:475-8, 1958.

*Virol. J*., 8(49):1-15, 2011.

poxvirus lesion on two captive houbara bustards (Chlamydotis undulata). Avian

Ergot alkaloids are produced by non-spore producing fungal endophytes that infect certain species of grasses, most notably tall fescue [*Lolium arundinaceum* (Schreb.) Darbysh.] and perennial ryegrass (*Lolium perenne* L.), and the spore producing *Claviceps* spp. that infect seed heads of certain grasses and particularly the cereal grains [rye (*Secale cereal* L.), barley (*Hordeum vulgare* L.), wheat (*Triticum aestivum* L.), , and oats (*Avena sativa* L.)] (Strickland et al., 2011). Ergot alkaloids induce a toxicosis in grazing livestock, with symptoms in cattle that include rough hair coats during the warm season, severe heat stress in warm and humid temperatures, reduced dry matter intake, agalactia, and poor reproductive and weight gain performance (Porter & Thompson, 1992; Paterson et al., 1995). Sheep grazing endophyte-infected fescue also can have elevated core body temperatures in warm and humid environments (Zanzalari et al., 1989; Hanna et al., 1990), and reduced dry matter intakes (Chestnut et al., 1992; Aldrich et al., 1993). The most pronounced effect on horses is observed with broodmares, which can exhibit prolonged gestation and agalactia (Cross et al., 1995). Symptomatology of the malady are reflective of alterations in hormone profiles (Porter & Thomson, 1992; Browning et al., 1997, 1998) and reductions in blood flow to peripheral tissues caused by interactions of ergopeptine ergot alkaloids with biogenic amine receptors in the vasculature (Oliver et al., 1998) to induce persistent vasoconstriction and restrict regulation of core body temperature by the sympathetic nervous system (Oliver, 1997). Consequently, lack of thermoregulation by livestock exposed to ergopeptines are extremely vulnerable to heat and cold stresses (Hemken et al., 1981; Al-Haidary et al., 2001).

Ergot alkaloids contain a common tetracycline ergoline ring structure and there are 3 different classes: 1) clavine alkaloids, 2) lysergic acid and its derivatives, and 3) ergopeptines (Lyons et al., 1986; Bush and Fannin, 2009; Strickland et al. 2011). Ergopeptines exert the greatest influence on the vasculature, with ergovaline being the ergopeptine of highest concentration in tall fescue (Lyons et al., 1986) and with a demonstrated high potency as a vasoconstrictor (Klotz et al., 2006).

Doppler Ultrasonography

for Evaluating Vascular Responses to Ergopeptine Alkaloids in Livestock 569

environments by endogenous biogenic amines: primarily serotonin, norepinephrine, and

Ergopeptines are ergot alkaoids that are produced by an endophyte (*Neotyphodium coenphialum*) that infects tall fescue plants (Bacon, 1995), and by the *N. lolii* endophyte that infects perennial ryegrass (Easton & Tapper, 2005). Although the endophyte that infects perennial ryegrass produces small amounts of ergot alkaloids that can induce vasoconstriction (Aiken et al., 2011), it is its production of lolitrem B that causes ryegrass staggers cattle and sheep which is of greater concern (Fletcher and Harvey, 1981). Livestock exhibiting staggers are incapacitated due to tremors, but the malady also affects animal performance (Siegel et al., 1985). All plant parts of tall fescue and perennial ryegrass contain ergot alkaloids, but alkaloid concentrations differ among plant parts. Rottinghaus et al. (1991) determined the ranking of plant parts from highest to lowest ergot alkaloid concentrations is seed, stem, leaf sheath, and leaf blade. There is a distinction between the mutual relationships between the wild-type, toxic endophytes that infect naturalized populations of tall fescue and perennial ryegrass and non-ergot alkaloid producing novel endophytes that are artificially infected into commercially released cultivars of each grass (Bouton et al., 2002). Claviceps spp. also produce ergot alkaloids, but fungal colonization and alkaloid concentrations are restricted to the seed or grain (Bandyopadhyay et al., 1998). Ergovaline has been proposed as the likely causal agent in the fescue toxicosis syndrome (Lyons et al, 1986). In vitro electromyograph studies have reported ergovaline to cause contractile responses of bovine uterine and umbilical arteries (Dyer, 1993), rat tail and guinea pig iliac arteries (Schoning, et al., 2001), and lateral saphenous weins of cattle (Klotz et al., 2007). Klotz et al. (2007) reported similar contractile responses between ergovaline and ergotamine, with contractile responses being initiated at 1 x 10-8 M concentrations for both ergopeptines. An earlier experiment by Klotz et al. (2006) determined a weak in vitro contractile response of the lateral saphenous vein to lysergic acid (a structurally simpler ergot alkaloid (reviewed by Strickland et al., 2011) that did not mediate contraction until concentrations reached supraphysiological levels (1 x 10-4 M). Dyer (1993) and Schoning et al. (2001) both showed that ergovaline elicited its contractile effects through activation of 5HT2A serotonergic receptors. However, in contrast to Dyer (1993) who showed that the 1-adrenergic receptor was not important in the contractile effects of ergovaline on the bovine uterine and umbilical arteries; Schoning et al. (2001) clearly demonstrated that the 1-adrenergic receptors were important to vascular regulation by ergovaline in their blood vessel models. Similar findings have been noted for other ergot alkaloids produced by both Neotyphodium and Claviceps spp. (reviewed

Although in vitro models are useful tools for investigating and identifying the modes by which the ergot alkaloids may effect vascular dysfunction, the data from these models must be interpreted with care until fully validated by in vivo models. Partial validation is provided by the results of earlier in vivo studies. Lewis and Gelfand (1935) demonstrated that ergotamine treatment of chickens resulted in cessation of blood flow to the comb and subsequent gangrene. They postulated that the gangrene developed as a result of endothelia damage. Shappell (2003) demonstrated that ergovaline was, in fact, directly cytotoxic to a

epinephrine (Johnson and Proppe, 1996; Strickland et al., 2011).

by Strickland et al., 2009a, b; Strickland et al., 2011).

**3. Ergopeptnes and their effects on vascular circulation** 

Reduced blood flows in cattle and sheep consuming endophyte-infected tall fescue was first reported by Rhodes et al. (1991) who used radioisotope labeled microspheres to estimate flow rates between those consuming endophyte-infected and endphyte-free tall fescue. Vascular dysfunction is also evidenced by in vitro findings of ergovaline induced constriction of bovine lateral saphenous veins (Klotz et al., 2006, 2007), uterine and umbilical arteries (Dyer, 1993), and rat tail and guinea pig iliac arteries (Schoning, et al., 2001). *Caviceps* spp. do not produce ergovaline, but can produce high quantities of ergotamine. Blaney et al. (2009) indentified ergotamine as the dominant ergopeptine produced by *Claviceps purpurea* sclerotia, which is the ergot that infects Australian rye. Blaney et al. (2011) reported severe hyperthermia in steers fed feedyard rations containing sorghum infected with Claviceps Africana, as compared to steers consuming non-infected sorghum.

Color Doppler ultrasonography has been used with humans as a noninvasive technique for real time diagnoses of aberrant blood flow (Someda et al, 1995; Whelan and Barry, 1992), stenosis (Hatle et al., 1980; Olin et al., 1995; Schmidt et al., 1997), and occlusions (Moneta et al., 1992; Müller et al., 1995). It also has been used to determine vasoconstrictive and blood flow responses in cattle (Aiken et al., 2007; 2009b) and sheep (Aiken et al., 2011) to ergot alkaloids, and was performed with steers to quantify artery lumen area and blood flow responses to heat and cold challenges (Kirch et al., 2008). Color doppler ultrasonography has potential use as a diagnostic or research tool in identifying and quantifying aberrant constriction caused by ergot alkaloids or other toxicants. This chapter will discuss: 1) blood flow aspects of thermoregulation in livestock, 2) effects of ergot alkaloids on blood flow and thermoregulation and, 3) procedures and sources of error in using Doppler ultrasonography as a research tool in evaluating vasoconstrictive responses in livestock exposed to ergot alkaloids.
