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Alfalfa hay induced primary photosensitization in horses
Accepted Manuscript Title: Alfalfa hay induced primary photosensitization in horses Author: B. Puschner, X. Chen, D. Read, V.K. Affolter PII: DOI: Reference:
S1090-0233(16)00075-7 http://dx.doi.org/doi: 10.1016/j.tvjl.2016.03.004 YTVJL 4770
To appear in:
The Veterinary Journal
Accepted date:
5-3-2016
Please cite this article as: B. Puschner, X. Chen, D. Read, V.K. Affolter, Alfalfa hay induced primary photosensitization in horses, The Veterinary Journal (2016), http://dx.doi.org/doi: 10.1016/j.tvjl.2016.03.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Original Article Alfalfa Hay Induced Primary Photosensitization in Horses B. Puschner a,*, X. Chen a, D. Read b, V.K. Affolter c a
Department of Molecular Biosciences, School of Veterinary Medicine, University of California, 1089 Veterinary Medicine Drive, Davis, CA 95616, USA b California Animal Health and Food Safety Laboratory System, San Bernadino Branch, 105 W. Central Avenue, San Bernardino, CA, 92408, USA c Department of Pathology, Microbiology, Immunology, School of Veterinary Medicine, University of California, Davis, One Shields Avenue, Davis, CA 95616, USA * Corresponding author. Tel.: +1 530 7526285 E-mail address: [email protected] (B. Puschner).
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Highlights
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Alfalfa hay can cause primary photosensitization in horses.
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Chlorophyll a, chlorophyll b, and pheophorbide a are not the causative photodynamic
20 21 22 23 24
compounds.
Growing and climate conditions resulting in the presence of photodynamic compounds in alfalfa hay are unknown.
Abstract
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Photosensitization, also known as photodermatitis, occurs when phototoxic or
26
photoactive substances accumulate in the skin and interact with sunlight to result in an often
27
severe, crusting, itching or painful dermatitis in unpigmented and/or or lightly haired areas of
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the skin. Primary photosensitization, caused by direct ingestion of photosensitizing agents,
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has been reported anecdotally in horses after ingestion of alfalfa hay. Between 2004 and 2014,
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several large outbreaks of primary photosensitization in horses fed primarily alfalfa hay were
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investigated in California. Alfalfa hay samples were collected and carefully examined for the
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presence of known photosensitizing plants and pesticide residues but none were identified.
33
Select hay samples were evaluated for unusual fungal infestation and for phototoxicity assay
34
using a specific Candida albicans assay; results were negative. In the 2004 outbreak, a feeding
35
study was conducted with three horses exclusively fed alfalfa hay that was suspected to have
36
caused the outbreak. Two weeks after ingestion of alfalfa hay, two horses developed several
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lesions in non-pigmented skin characterized as chronic ulcerative and necrotizing dermatitis
38
with superficial vasculitis, which was consistent with photosensitization. In the 2014
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outbreak, seven different implicated alfalfa hay samples were analyzed for chlorophyll a and
40
b, and pheophorbide a. These compounds had been suspected to play a role in alfalfa-induced
41
primary photosensitization. The chlorophyll contents ranged from 0.90 - 2.30 mg/g in the
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alfalfa hay samples, compared to 1.37 and 2.94 mg/g in locally grown alfalfa and orchard
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grass hay. The pheophorbide a levels ranged from 3.36 - 89.87 µg/g in alfalfa samples
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compared to 81.39 and 42.33 µg/g in control alfalfa and orchard grass hay samples. These
45
findings eliminate chlorophyll a, chlorophyll b, and pheophorbide a as possible causes for
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alfalfa-hay induced primary photosensitization.
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Keywords: Chlorophyll, Horse, Pheophorbide a, Photodermatitis, Primary Photosensitization
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Introduction
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Photosensitization, also known as photodermatitis, occurs when phototoxic or
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photoactive substances accumulate in the skin, interact with sunlight and produce a severe
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dermatitis. There are several different types of photosensitization related to plant exposures
53
(Johnson, 1982). Type I is a result of the direct absorption of the photosensitizing agent into
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the blood, and distribution to the skin. Some of the more common pasture plants known to
55
cause Type I photosensitization include Hypericum perforatum (St. Johnswort), Fagopyrum
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esculentum (Buckwheat), Ammi majus (Bishop’s weed), Cymopterus spp. (spring parsley),
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and the Medicago spp. (clovers) (Campbell et al., 2010; Dollahite et al., 1978; Staker, 2014;
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Williams and Binns, 1968). Type I photosensitization is rarely reported in horses because
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exposure to these plants in pasture is unlikely. Type II photosensitization occurs subsequent to
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liver damage due to the insufficient excretion of phylloerythrin, a photoactive metabolite of
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chlorophyll (Campbell et al., 2010). Due to the horses’ extreme susceptibility to pyrrolizidine
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alkaloids (PAs), exposure to Senecio and Amsinckia sp. has resulted in hepatogenous
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photosensitization (Knight et al., 1984). Type II photosensitization has also been observed in
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cattle exposed to moldy alfalfa hay (Scruggs and Blue, 1994), and horses exposed to clover
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pastures that were infested with the fungus Cymodethea trifolii (Ames et al., 1994).
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Ingestion of alfalfa silage or Froelichia humboldtiana has resulted in primary
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photosensitization in cattle and horses (House et al., 1996; Ribeiro Knupp et al., 2014; Souza
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et al., 2012). Breakdown products of chlorophyll such as pheophorbide were suspected to be
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the responsible photoactive compounds in alfalfa silage (Takamiya et al., 2000). In the past
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ten years, several outbreaks of primary photosensitization in horses were observed after
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ingestion of alfalfa hay in California. To the authors’ knowledge, alfalfa hay-induced primary
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photosensitization in horses has not been confirmed based on a feeding study. Furthermore,
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no data has been established in previous investigations as to the possible role of pheophorbide
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a. Thus, the current case series describes seven outbreaks of primary photosensitization in
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horses between 2004 and 2014, the available epidemiological and clinical findings, the results
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of a feeding trial, and the concentrations of chlorophyll a, chlorophyll b, and pheophorbide a
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in feed samples associated with some of the outbreaks.
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Materials and methods
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Case series:
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Between 2004 and 2014, a total of seven outbreaks of photosensitization in horses in
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California were evaluated. For the 2004 and 2014 outbreaks detailed information was
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available, whereas for the outbreaks in 2008 (2), 2009 (2) and 2013, only limited
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epidemiological and diagnostic data exist. The first large outbreak occurred in San Joaquin
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and Kern Counties in July 2004. Approximately 70 horses housed in at least four different
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locations developed severe skin lesions (Plate 3 – a and d). Owners reported that skin lesions
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developed approximately 3 to 7 days after feeding a new shipment of alfalfa hay. Of the 70
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affected horses, one Quarter horse filly died and was examined post-mortem with a complete
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pathological and toxicological diagnostic work-up. Alfalfa hay samples were examined for
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the presence of potentially toxic weeds, herbicides, pesticides and mycotoxins. A
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phototoxicity assay and fungal identification were also performed on a hay sample collected
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from the site with the most severe presentation and the greatest number of affected horses.
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In 2008, two outbreaks were investigated in Fresno County, California. The first
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occurred in August 2008 and resulted in an unknown number of horses developing lesions in
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the non-pigmented skin areas. At one farm, three horses developed lesions on the muzzle and
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on their backs. The second outbreak occurred in September 2008 and the total number of
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affected horses was unknown, but was estimated to be more than ten. One severely affected
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horse, a pinto, developed severe skin lesions after being fed a diet of Alfalfa cubes that were
Comment [CX1]: Picture a and d are from 2004 outbreak. belong to the year 2004. Plate legends on page 21 and 22 have been updated accordingly.
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grown and made locally, and having been exclusively in the full sun for three weeks. The less
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severely affected horses had crusting and reddening of the non-pigmented skin on muzzle and
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face. In 2009, two outbreaks of photosensitization occurred in May in Madera and Kern
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County, California. Several horses developed skin lesions on the white areas of the muzzle
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and legs after a new batch of alfalfa hay was introduced several weeks earlier. In 2013, an
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outbreak occurred at a boarding stable in Santa Barbara County, California. Horses were
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turned out into a large dry pasture and fed alfalfa hay ad libitum. Approximately eight horses
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out of 116 developed skin lesions that consisted of dry, reddened thick folds on the muzzle,
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neck and tail approximately two weeks after a new shipment of alfalfa hay was introduced
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(Plate 2 – a, b, c, d). In addition to a change in feed, a one-time exposure to a fermenting pile
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of grass clippings consisting of annual meadow grass (Poa annua), bermuda grass (Cynodon
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dactylon) and perennial rye grass (Lolium perenne) occurred at about the same time as the hay
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change. The hay was evaluated for the presence of weed contamination.
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In July 2014, an outbreak of photosensitization occurred in Phelan, CA (Plate 3 – b
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and c). More than 12 horses developed skin lesions on the white areas of the muzzle and face
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after being offered a new shipment of alfalfa hay (http://www.vvng.com/phelan-horse-
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owners-concerned-about-tainted-hay/). Affected horses were examined by veterinarians. Hay
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samples were evaluated for poisonous weed contamination, chlorophyll and pheophorbide a
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analysis.
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Feeding Study:
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A feeding study was conducted over 21 days on the University of California, Davis
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campus at the Center for Equine Health facility and was approved by the Institutional Animal
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Care and Use Committee. Three horses with varying degrees of unpigmented skin were
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enrolled in the study. All horses underwent a complete physical examination prior to study
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begin. The horses were a 7-year old gray-colored Westphalian gelding, a 6-year old chestnut-
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colored Irish Sport gelding, and a 20-year old gray-colored Arabian cross gelding. Horses
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were housed together in a corral without protection from UV exposure, were each fed 6
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pounds of alfalfa hay twice per day, and offered water ad libitum. Horses were examined
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daily for development of skin abnormalities or other signs of illness, and blood samples were
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collected on days 2, 6, 9, 13, 21, and 27 of the study. Serum samples were evaluated for anion
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gap, sodium, potassium, chloride, CO2 total, calcium, phosphorus, creatinine, urea nitrogen,
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glucose, total protein, albumin, globulin, AST, creatinine kinase, ALP, GGT, SDH-37,
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bilirubin total, bilirubin direct, bilirubin indirect, iron, magnesium, zinc, and copper. Blood
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samples were evaluated for red blood cells, hemoglobin, hematocrit, MCV, MCH, MCHC,
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red cell distribution width, RBC morphology, neutrophils, lymphocytes, monocytes,
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eosinophils, basophils, platelets, plasma protein, plasma fibrinogen and selenium. All
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analyses were performed using standard operating procedures at the Veterinary Medical
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Teaching Hospital Laboratory and the California Animal Health and Food Safety Laboratory
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System of the University of California, Davis. A skin biopsy was taken from the 7-year old
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Westphalian gelding on day 13 of the feeding study and examined histologically. Alfalfa hay
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was discontinued after 22 days, and all three horses returned to the herd. Both horses that
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developed lesions during the feeding trial recovered uneventfully over the next 2 weeks.
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Chlorophyll a and b and pheophorbide a analysis:
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From the 2014 outbreak, a total of seven hay samples were analyzed for chlorophyll a
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and b, and pheophorbide a. Control hay samples were locally grown alfalfa and orchard grass
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hay that had been fed to horses in the Veterinary Medical Teaching Hospital for over four
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weeks. For chlorophyll a and b analysis, 0.15 g of hay were homogenized on ice with 2 mL of
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acetone: 0.1N NH4OH (9:1 vol/vol) using a homogenization tube and pestle for 30 seconds.
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After rinsing the pestle with 3 mL acetone: 0.1N NH4OH (9:1 vol/vol) to remove any plant
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residue, the tube was placed at 4 °C in the dark for two hours and then reground to extract any
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remaining chlorophyll. The extract was then transferred to a centrifuge tube. Five microlitres
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of 80% aqueous acetone were used to rinse the pestle and homogenization tube and added to
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the sample extract. Sample extracts were centrifuged for 20 min at 500 x g. The supernatant
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was transferred into a 10 mL graduated cylinder, the volume was brought to 10 mL with 80%
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aqueous acetone, and the sample was stored at -20 °C until analysis. Sample extracts were
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prepared in duplicate and analyzed for chlorophyll a and b with a spectrophotometer
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(SpectraMax M3， Molecular Devices, Sunnyvale, CA, USA) and absorption was measured
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at 645 nm and 663 nm once for each extract. The 80% aqueous acetone was used as a blank to
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zero the instrument initially and after every resetting of the wavelength. Chlorophyll a and b
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concentrations were determined according to previously developed formulas (Arnon, 1949;
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Porra, 2002; Warren, 2008):
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For pheophorbide a analysis, 0.2 g of hay were homogenized on ice with 4 mL of 80%
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aqueous acetone using a homogenization tube and pestle for 30 seconds. After rinsing the
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pestle with 2 mL 80% aqueous acetone the tube was placed at 4 °C in the dark for two hours
165
and then reground. The extract was then transferred to a centrifuge tube and centrifuged for
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20 min at 500 x g. After addition of 400 µL 80% aqueous acetone to 100 µL supernatant, the
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sample was store the at -20 °C until analysis. An Advance UHPLC high performance liquid
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chromatograph coupled to an EVOQ Elite MS/MS triple quadrupole mass spectrometer
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equipped with an atmospheric pressure chemical ionization (APCI) source was used for the
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pheophorbide a analysis (Bruker Corp, Freemont, CA, USA). The chromatograph was fitted
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with a 100 mm x 2.1 mm i.d. 1.7 µm ACQUITY BEH C18 column (Waters, Milford, MA).
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The mobile phases consisted of 0.1% formic acid in deionized water (channel A), and 0.1%
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formic acid in methanol (channel B). Starting conditions consisted of 90% B increasing to
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95% B over 2 minutes, then ramped up to 100% B over 1 min, which was held for 5 min and
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then returned to 90% B within 0.5 min and held for 1 min for re-equilibration. The column
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was maintained at 25 °C. Pheophorbide a eluted at approximately 4.2 min. Analytes were
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monitored by multiple reaction monitoring (MRM). The transitions for pheophorbide a were
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set as following: pheophorbide a m/z 593.2 m/z 533.6 with collision energy (CE) 45 eV.
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Nitrogen gas served as the nebulizer, dry and collision gas. Other source parameters were as
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follows: spray current 10 µA, cone temperature 350 °C, cone gas flow of 25 au, heated probe
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temperature 400 °C, probe gas flow of 35 au, nebulizer gas flow of 45 au. The injection
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volume was 10 µL. Bruker MSWS 8.1 software was used for all data acquisition and
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processing.
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Results
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Outbreaks:
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In the 2004 outbreak, the implicated alfalfa hay was purchased at various suppliers
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and presumably grown in Nevada and California. Veterinarians examining the horses
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confirmed photodermatitis; only unpigmented skin was damaged, and in severe cases there
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was crusting, itching and a painful dermatitis. Skin lesions were primarily in the face and on
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legs (Plate 3 – a and d). Of the affected horses, one a five month old Quarter horse filly died.
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The foal was initially presented with skin lesions (Plate 3 – d) and myositis. After initiation of
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supportive treatment, the foal improved over the next 24 hours before developing cool,
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discolored distal extremities. The foal progressed to recumbency, became unresponsive to
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fluids and oxygen, and died. A full post-mortem examination revealed subacute necrosis of
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non-pigmented and lightly pigmented skin of limbs and head and pansystemic bacterial
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embolism. Due to the lack of parenchymal or biliary liver lesions, hepatogenous
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photosensitization was ruled out. The portal of entry of the bacterial septicemia was
Comment [CX2]: Same corrections as above
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determined to be the necrotic skin.The liver contained acceptable concentrations of lead,
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manganese, cadmium, copper, iron, zinc, molybdenum, arsenic, mercury, selenium, and
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vitamin E. The brain was evaluated for acetylcholinesterase activity, which was found to be
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adequate for horses (4.9 µM/g/min; adequate for horses > 2.1 µM/g/min). Alfalfa hay samples
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implicated in this outbreak were examined visually. All hay samples consisted predominantly
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of alfalfa (Medicago sativa), but some samples had small amounts of Setaria pumila (yellow
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foxtail), Leptochloa uninervia (Mexican sprangletop), Cynodon dactylon (Bermuda grass),
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Eriochloa contracta (prairie cupgrass), and Sonchus oleraceus (common sowthistle). Some
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mold was identified on all alfalfa hay samples and was identified on direct tape mount as
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Cladosporium and Alternaria species. On potato flakes agar plates there was Mucor sp. and
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Cladosporium spp. growing. These findings are typical mold growth identified on most types
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of organic materials, such as alfalfa. A phototoxicity (Candida albicans) assay performed on
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individual fragments selected from various alfalfa hay samples was negative (Kavli and
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Volden, 1984). Hay samples contained non-toxic concentrations of heavy metals (lead,
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manganese, cadmium, copper, iron, zinc, molybdenum, arsenic, mercury). None of the listed
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carbamate insecticides (aldicarb, aldicarb sulfone, carbaryl, carbofuran, 3-hyroxycarbofuran,
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methiocarb, methomyl, mexacarbate, oxamyl, and propoxur), organophosphorus insecticides
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(43
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trichlorophenoxyacetic acid, and 2,4-DB), or mycotoxins (aflatoxin B1, ochratoxin A, T-2,
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DAS, and vomitoxin) were identified when analyzed by standard operating procedures.
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Follow-up with veterinarians and owners revealed that all affected horses, except for the foal
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that died, recovered with supportive care consisting of anti-inflammatory medications,
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protection from UV exposure, and change in feed.
different
compounds),
herbicides
(dicamba,
MCPA,
2,4-D,
silvex,
2,4,5-
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In all 2008 and 2009 outbreaks, horses were fed locally grown alfalfa hay or alfalfa
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cubes and had no elevations of liver enzymes when examined by the veterinarian. No deaths
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were reported and all horses recovered with supportive care consisting of change in feed,
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protection from UV exposure, and anti-inflammatories when indicated. In the 2013 outbreak
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with 8 affected horses, liver enzymes were evaluated in all affected horses and were within
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reference ranges. The alfalfa hay was examined carefully and although predominantly
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consisting of alfalfa, several plants contaminant were identified. Those included found to
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contain Bassia scoparia (Kochia, burning bush), Quercus lobata (Valley oak), Chenopodium
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murale (Nettleleaf goosefoot), Marrubium vulgare (Horehound), Rumex sp. (dock), Eurybia
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sp. (Aster), Lavandula angustifolia (Common lavender), Buxus microphylla japonica
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(Boxwood), Tagetes sp. (Marigold), and Festuca sp. (Fescue). Affected horses were removed
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from the sun, had a change in feed, and received local treatment of skin lesions with zinc
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oxide. There were no reports of deaths in these outbreaks.
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In the 2014 outbreak, none of the affected horses had elevated liver enzymes or other
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abnormalities when examined by their veterinarians. The alfalfa hay was suspected to have
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been grown in Southern California, and presumably fed shortly after harvest. A total of seven
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hay samples were carefully evaluated and confirmed to consist predominantly of clean, green
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alfalfa. Some of the alfalfa had some black spot mold, especially on older stems, and there
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were some water-soaked lesions on some of the larger leaves. While five of the hay samples
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contained no weed contamination, one of the hay samples contained four small fragments of
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puncture vine, Tribulus terrestris. Puncture vine has been associated with the development of
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liver disease and the development of secondary photosensitization.
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Feeding trial:
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All horses had good appetite and finished the provided alfalfa hay after each feeding.
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All serum and blood analytes were within reference ranges for horses at all assessed time
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points. Daily observations revealed no abnormalities until day 13 of the feeding trial. On day
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13, the 7-year old Westphalian gelding developed an erythematous, crusting lesion on the
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dorsum of his muzzle where white facial markings existed (Plate 1 – b). On day 19, the 6-year
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old chestnut-colored Irish Sport gelding developed two areas of red raw, peeling, and crusting
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skin on the medial aspects of the pastern and along cannon bone of the non-pigmented right
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hind leg (Plate 1 – a). The 20-year old Arabian cross developed no skin abnormalities
253
throughout the trial. This horse had no white or pink skin areas, and had a pigmented
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epidermis making him a valuable negative control horse of the feeding trial. A punch biopsy
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was collected from the 7-year old Westphalian gelding (Plate 1 – c). The hyperplastic
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epidermis was overlain by a prominent crusts composed of necrotic epidermal and dermal
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components, indicating a necrotizing process with re-epithelialization. Lymphocytes and
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plasma cells with rare neutrophils were surrounding dermal vessels, many of which had
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indistinct vascular walls with partial loss of endothelial cells (Plate 1 – d). Multiple
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microhemorrhages were present in the superficial dermis. Histologic features were consistent
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with a subacute stage of a vascular insult.
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Chlorophyll and Pheophorbide a analysis:
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The chlorophyll a concentrations in the 7 alfalfa hay samples from the 2014 outbreak
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were 1.44, 0.94, 1.20, 0.55, 1.37, 1.32, and 1.37 mg/g respectively, whereas the two control
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hay samples had 0.84 mg/g (alfalfa hay) and 1.77 mg/g (orchard grass hay). The chlorophyll b
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concentrations in the outbreak-associated alfalfa hay samples were 0.86, 0.59, 0.72, 0.35,
268
0.82, 0.79, and 0.81 mg/g, while two control hay samples had 0.53 and 1.17 mg/g. Thus, all
269
analyzed alfalfa hay samples implicated in the 2014 outbreak contained chlorophyll a and b in
270
concentrations similar to what was determined in control alfalfa and orchard grass hay
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samples that were not associated with any illness after long-term feeding to horses (Fig. 1).
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The pheophorbide a concentrations in the 7 alfalfa samples available from the 2014 outbreak
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were 45.75, 22.29, 89.87, 3.36, 15.90, 26.10, and 31.47 µg/g, while the alfalfa and orchard
274
grass hay control samples had 81.39 and 42.33 µg/g (Fig. 2).
275 276
Discussion
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This study confirms that alfalfa hay is able to induce primary photosensitization in
278
horses. The photosensitizing trigger remains unknown at this point, as chlorophyll a,
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chlorophyll b and pheophorbide a as the responsible photoactive compounds in alfalfa hay
280
have been ruled out. In all affected horses, liver function was not impaired when
281
photodermatitis was diagnosed confirming that there was direct exposure to a photosensitizer.
282
The only published report implicating alfalfa in an outbreak of primary photosensitization was
283
reported in dairy cattle from San Joaquin Valley, California (House et al., 1996). In this
284
outbreak, over 400 lactating dairy cows developed photosensitization after being fed alfalfa
285
silage. A feeding trial confirmed alfalfa silage to have caused the skin lesions. It was
286
suspected that breakdown products of chlorophyll, such as pheophorbide a, may have been the
287
cause of photosensitization. However, no testing for pheophorbide was done.
288
Photosensitizers are capable of absorbing UV radiation to form a reactive excited state
289
molecule that in turn transfers the energy to a surrounding molecule. Phototoxins are
290
commonly found in plants where they serve an important role for plant defense (Björn and
291
Huovinen, 2002). Common primary photosensitizing plants include Hypericum perforatum
292
(St. John’s wort), Fagopyrum esculentum (buckwheat) Ammi majus (Bishop’s weed),
293
Cymopterus (spring parsley), Heracleum sp. (cow parsnip, hogweed), Lomatium sp. (wild
294
parsley), Apium sp. (celery), Pimpinella sp. (burnet saxifrage), Ambrosia sp. (ragweed) and
295
the Medicago species (Downum, 1992; Ivens, 2011; Ivie, 1982; Johnson, 1982; Pathak, 1986).
296
None of these plants were identified in any of the outbreaks. In the 2004 outbreak, Setaria sp.
297
were identified in the hay. Setaria sp. have sharp and barbed bristles that are capable of
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penetrating the mucous membranes and may lead to erosions of the mouth. Setaria-induced
299
illnesses have been reported in horses; however, lesions are typically seen in the mouth and
300
horses are reluctant to eat. These clinical signs were not seen in the affected horses.
301
In addition, certain antibiotics and phenothiazine derivatives have been associated
302
with primary photosensitization in livestock. However, phenothiazine is primarily a problem
303
in ruminants due to the formation of the phototoxic compound by rumen microbes (Dirksen
304
and Tammen, 1964). While drug-induced photosensitization is a major health concern in
305
humans (Dawe and Ibbotson, 2014), documented reports in horses are missing. In all outbreak
306
investigations, owners and veterinarians were asked about use of medications in the affected
307
horses; none were reported.
308
The typical chemical structure of a photosensitizer is a polycyclic compound with
309
many double bonds (Björn and Huovinen, 2002). Chlorophylls are able to absorb light and act
310
as photosensitizers resulting in singlet oxygen and superoxide formation. Chlorophylls are
311
photosynthetic pigments that are widely distributed in nature and possess a basic skeleton
312
structure of porphyrin with a magnesium ion in the center and a long phytol group as the tail
313
(Huang et al., 2008). The major chlorophyll forms in plants are chlorophyll a and chlorophyll
314
b with the content of the former being about 3-fold higher than the latter. Under mild heating
315
conditions during the drying process of green plants, chlorophyll a is degraded to
316
chlorophyllide a through the activation of chlorophyllase and removal of phytol, which upon
317
activation of dechelatase can be further converted to pheophorbide a (Loh et al., 2012).
318
Pheophorbide a is structurally very similar to chlorophyll a with the exception of the loss of
319
Mg and the phytol group, and is photodynamic. Based on its phototoxic ability, pheophorbide
320
a is utilized in photodynamic therapy of cancer (Yoon et al., 2014). In addition, pheophorbide
321
a-induced phototoxicity has been reported in humans after ingestion algae-derived chlorella
322
tablets (Jitsukawa et al., 1984), that are sold as natural health supplement. Pickled vegetables
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have also caused phototoxicity in rats due to their substantial amounts of pheophorbide a
324
(Jitsukawa et al., 1984). Thus, our assessment of pheophorbide a levels in the various alfalfa
325
hay samples was warranted; however, results illustrated that pheophorbide a levels did not
326
differ between the hay samples causing primary photosensitization and the control hay.
327
Pheophorbide a, while one of the more in depth studied breakdown products, is not the only
328
photodynamic chlorophyll a metabolite, however. Pheophytin a, pyropheophytin a, and
329
chlorophyllide a (Lohrey et al., 1974; Takeda et al., 1989; Tapper et al., 1975) have been
330
identified and could conceivably play a role in these outbreaks. In addition, breakdown
331
products of chlorophyll b, even though present in much lower concentrations in plants, may
332
play a role.
333
Since the amount of chlorophyll, and most likely other potential photosensitizers, is
334
greater in green plant material compared to dry hay the risk for an animal to develop
335
photosensitization is considered greatest under grazing conditions (Quinn et al., 2014).
336
However, under certain condition, dried plant material can result in the formation of certain
337
phototoxic compounds that lead to disease. This was the case in cattle that were exposed to
338
dried leaves of Cooperia pedunculata in Texas and developed primary photosensitization
339
(Rowe et al., 1987). In another instance, specific conditions had to be present for disease to
340
develop; co-consumption of horse brush (Tetradymia spp.) and sage (Artemisia spp.)
341
appeared to be necessary to result in photosensitization in sheep (Johnson, 1974).
342
Sporadic outbreaks of primary photosensitization in horses are a great source of
343
frustration to owners, veterinarians, and growers. The motivation for this study was to
344
confirm that alfalfa hay can induce primary photosensitization in horses, and to determine
345
whether alfalfa hay, under certain conditions, can accumulate toxic levels of chlorophyll a,
346
chlorophyll b or pheophorbide a, all known photosensitizing agents. Even though we were
347
able to reproduce the primary photosensitization with a feeding trial, we were unable to
15
Page 15 of 23
348
determine the etiologic agent. Although the present study has yielded important findings, its
349
design is not without flaws. A number of caveats that are often encountered when relying on
350
the submission of case-based/outbreak material need to be noted: the quality of epidemiologic
351
information such as the number of affected horses, location of hay purchase, location of hay
352
grower, exact days of exposure provided by owners and veterinarians varied; the diagnostic
353
work-up varied by outbreak depending on availability of specimens and analytical tests;
354
analytical testing for chlorophyll a and b and pheophorbide a was only available for the 2014
355
investigation. In addition, as is true for any analysis of a feed sample, hay samples examined
356
and tested may not have been representative of the complete shipment.
357 358
Conclusions
359
To the authors’ knowledge, this is the first report of confirming that alfalfa hay can induce
360
primary photosensitization in horses. Horse owners as well as veterinarians should recognize
361
the possibility that alfalfa hay, on rare occasions, can induce skin lesions, and discontinue
362
feeding alfalfa as soon as signs are noted. In addition, rapid response and research efforts are
363
needed to identify the etiologic agent and the growing conditions for the toxin to be present as
364
the toxin may not be stable under long-term storage conditions. There are very few studies
365
that have evaluated the concentrations of photosensitizers in dried plant material implicated in
366
outbreaks of photosensitization. This is a critical step in not only the characterization of
367
photosensitizing compounds but in the development of an approach for prevention strategies.
368
In addition, outbreaks appear to occur sporadically. We have very limited information about
369
the growing and climate conditions when these disastrous events occur. Additional research is
370
needed to determine the nature of the photosensitizing compounds and the factors necessary
371
for production.
372
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373 374 375
Conflict of interest statement None of the authors has any financial or personal relationships that could inappropriately influence or bias the content of the paper.
376 377
Acknowledgements
378
The authors thank Dr. Asheesh Tiwary for his assistance during the 2004 outbreak
379
investigation and feeding study, Marcia Booth for her expertise in plant identifications, and
380
Drs. Phoebe Smith, Kristen Fosnaugh, Jeanette Mero, Robert Shaw, and Alisha Olmstadt for
381
their information on case management and outbreak epidemiology. The authors are also
382
grateful to Dr. John Reagor for performing the phototoxicity assay, and to Dr. Michael
383
Rinaldi for interpretation of fungal identification results. We also would like to thank Dr.
384
Sharon Spier for collection of a biopsy specimen, and Mike Davidson for the provision of
385
quarantined hay for the feeding study.
386 387 388 389
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Arnon, D.I., 1949. Copper enzymes in isolated chloroplasts. Polyphenoloxidase in beta
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Huang, S.C., Hung, C.F., Wu, W.B., Chen, B.H., 2008. Determination of chlorophylls and
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Ivie, G.W., 1982. Chemical and biochemical aspects of photo-sensitization in livestock and
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Jitsukawa, K., Suizu, R., Hidano, A., 1984. Chlorella photosensitization. New
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phytophotodermatosis. International Journal of Dermatology 23, 263-268.
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Johnson, A.E., 1974. Predisposing influence of range plants on tetradymia-related
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photosensitization in sheep: Work of drs. A. B. Clawson and w. T. Huffman. American
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Johnson, A.E., 1982. Toxicologic aspects of photo-sensitization in livestock. Journal of the
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Kavli, G., Volden, G., 1984. Phytophotodermatitis. Photo-Dermatology 1, 65-75.
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Knight, A.P., Kimberling, C.V., Stermitz, F.R., Roby, M.R., 1984. Cynoglossum officinale
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(hound's-tongue)--a cause of pyrrolizidine alkaloid poisoning in horses. Journal of the
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Loh, C.H., Inbaraj, B.S., Liu, M.H., Chen, B.H., 2012. Determination of chlorophylls in
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taraxacum formosanum by high-performance liquid chromatography-diode array detection-
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Lohrey, E., Tapper, B., Hove, E.L., 1974. Photosensitization of albino rats fed on lucerne-
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Pathak, M.A., 1986. Phytophotodermatitis. Clinics in dermatology 4, 102-121.
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Porra, R.J., 2002. The chequered history of the development and use of simultaneous
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equations for the accurate determination of chlorophylls a and b. Photosynthesis Research 73,
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Quinn, J.C., Kessell, A., Weston, L.A., 2014. Secondary plant products causing
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photosensitization in grazing herbivores: Their structure, activity and regulation. International
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Ribeiro Knupp, S.N., Borburema, C.C., de Oliveira Neto, T.S., de Medeiros, R., Knupp, L.S.,
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Riet-Correa, F., Lucena, R.B., 2014. Outbreaks of primary photosensitization in equidae
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caused by froelichia humboldtiana. Pesquisa Veterinaria Brasileira 34, 1191-1195.
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Rowe, L.D., Norman, J.O., Corrier, D.E., Casteel, S.W., Rector, B.S., Bailey, E.M., Schuster,
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J.L., Reagor, J.C., 1987. Photosensitization of cattle in southeast texas - identification of
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phototoxic activity associated with cooperia-pedunculata. American Journal of Veterinary
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Research 48, 1658-1661.
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Scruggs, D.W., Blue, G.K., 1994. Toxic hepatopathy and photosensitization in cattle fed
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moldy alfalfa hay. Journal of the American Veterinary Medical Association 204, 264-266.
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Souza, P.E.C., Oliveira, S.S., Aguiar-Filho, C.R., Cunha, A.L.B., Albuquerque, R.F.,
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Evencio-Neto, J., Riet-Correa, F., Mendonca, F.S., 2012. Primary photosensitization in cattle
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caused by froelichia humboldtiana. Research in Veterinary Science 93, 1337-1340.
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Staker, L., 2014. Macropod husbandry, healthcare and medicinals-volumes one and two.
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Lynda Staker, 336.
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Takamiya, K.-i., Tsuchiya, T., Ohta, H., 2000. Degradation pathway(s) of chlorophyll: What
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has gene cloning revealed? Trends in Plant Science 5, 426-431.
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Takeda, Y., Konuma, H., Uchiyama, S., Saito, Y., 1989. Microbial formation of pheophorbide
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a and pyropheophorbide a in takana during brining and fermentation. Journal of the Food
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Hygienic Society of Japan 30, 228-232.
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Tapper, B.A., Lohrey, E., Hove, E.L., Allison, R.M., 1975. Photosensitivity from chlorophyll-
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derived pigments. Journal of the Science of Food and Agriculture 26, 277-284.
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Warren, C.R., 2008. Rapid measurement of chlorophylls with a microplate reader. Journal of
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Plant Nutrition 31, 1321-1332.
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Williams, M.C., Binns, W., 1968. Experimental photosensitization by spring parsley
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(cymopterus watsonii) in chicks. American Journal of Veterinary Research 29, 111-115.
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Yoon, H.E., Oh, S.H., Kim, S.A., Yoon, J.H., Ahn, S.G., 2014. Pheophorbide a-mediated
469
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470
skin cancer cells. Oncology Reports 31, 137-144.
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471 472
Plate and Figure legends Plate 1: Skin lesions developing during feeding trial of provided alfalfa (2004)
473
a: Skin lesions observed on non-pigmented skin on distal lags of the 6-year old chestnut-
474
colored Irish Sport gelding on day 19: locally extensive oozing and crusting with ulcerations
475
b: Skin lesions observed on the non-pigmented portion of the muzzle of the 7-year old
476
Westphalian gelding on day 13: erythematous, crusting lesion on the white markings of the
477
muzzle. Note the abrupt demarcation of the lesions along the non-pigmented to pigmented
478
skin area.
479
c: Biopsy from the affected area of the muscle of the 7-year old Westphalian gelding: severe
480
serocellular crusting (C) composed of epidermal and dermal components are overlaying a
481
markedly hyperplastic newly formed epidermis (E). There is mild perivascular, mostly
482
mononuclear inflammation (I) in the dermis and small areas of microhemoprrhage (>).
483
d: Higher magnification of the biopsy shown in C: Multifocal microhemorrhages (H) are the
484
result of the damaged vascular walls (<), characterized by hyalinized vessel walls and loss of
485
endothelial cells.
486 487
Plate 2: 2013 outbreak of photodermatitis in horses. Severe erythema, erosions and
488
ulcerations (a-d) and crusting (a and c) are limited to non-pigmented areas of the skin. The
489
shoulder lesions and the lateral muzzle show abrupt demarcation of the lesions and the normal
490
appearing skin surface in the pigmented skin.
491 492
Plate 3: 2004 and 2014 outbreaks of photodermatitis in horses. In the 2004 outbreak, severe
493
skin lesions were primarily located in the face and on legs (a and d). One 5-month old Quarter
494
horse foal presented with swollen, red raw, peeling and crusty skin exclusively affecting non-
495
pigmented areas of rear pasterns (d). In the 2014 outbreak, erosion and ulcerations developed
496
on the muzzle (b and c).
21
Page 21 of 23
497
Fig. 1. Chlorophyll a and chlorophyll b concentrations (in mg/g) in seven hay samples from the 2014 outbreak. Control samples were alfalfa and
498
orchard grass hay. 3.50 chlorophyll b 3.00
concentration(mg/g)
chlorophyll a 2.50
2.00
1.50
1.00
0.50
0.00 Alfalfa #1 Alfalfa #2 Alfalfa #3 Alfalfa #4 Alfalfa #5 Alfalfa #6 Alfalfa #7 Sample ID
Control Alfalfa
Control Orchard grass
499 500 501
22
Page 22 of 23
502
Fig. 2. Pheophorbide a concentration (in µg/g) in seven hay samples from the 2014 outbreak. Control samples were alfalfa and orchard grass hay.
pheophorbaide a concentration (µg/g)
100.00
80.00
60.00
40.00
20.00
0.00 Alfalfa #1
Alfalfa #2
Alfalfa #3
Alfalfa #4
Alfalfa #5
Sample ID
Alfalfa #6
Alfalfa #7
Control Alfalfa
Control orchard grass
503 504
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Page 23 of 23
S1090-0233(16)00075-7 http://dx.doi.org/doi: 10.1016/j.tvjl.2016.03.004 YTVJL 4770
To appear in:
The Veterinary Journal
Accepted date:
5-3-2016
Please cite this article as: B. Puschner, X. Chen, D. Read, V.K. Affolter, Alfalfa hay induced primary photosensitization in horses, The Veterinary Journal (2016), http://dx.doi.org/doi: 10.1016/j.tvjl.2016.03.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Original Article Alfalfa Hay Induced Primary Photosensitization in Horses B. Puschner a,*, X. Chen a, D. Read b, V.K. Affolter c a
Department of Molecular Biosciences, School of Veterinary Medicine, University of California, 1089 Veterinary Medicine Drive, Davis, CA 95616, USA b California Animal Health and Food Safety Laboratory System, San Bernadino Branch, 105 W. Central Avenue, San Bernardino, CA, 92408, USA c Department of Pathology, Microbiology, Immunology, School of Veterinary Medicine, University of California, Davis, One Shields Avenue, Davis, CA 95616, USA * Corresponding author. Tel.: +1 530 7526285 E-mail address: [email protected] (B. Puschner).
16
1
Page 1 of 23
17
Highlights
18
Alfalfa hay can cause primary photosensitization in horses.
19
Chlorophyll a, chlorophyll b, and pheophorbide a are not the causative photodynamic
20 21 22 23 24
compounds.
Growing and climate conditions resulting in the presence of photodynamic compounds in alfalfa hay are unknown.
Abstract
25
Photosensitization, also known as photodermatitis, occurs when phototoxic or
26
photoactive substances accumulate in the skin and interact with sunlight to result in an often
27
severe, crusting, itching or painful dermatitis in unpigmented and/or or lightly haired areas of
28
the skin. Primary photosensitization, caused by direct ingestion of photosensitizing agents,
29
has been reported anecdotally in horses after ingestion of alfalfa hay. Between 2004 and 2014,
30
several large outbreaks of primary photosensitization in horses fed primarily alfalfa hay were
31
investigated in California. Alfalfa hay samples were collected and carefully examined for the
32
presence of known photosensitizing plants and pesticide residues but none were identified.
33
Select hay samples were evaluated for unusual fungal infestation and for phototoxicity assay
34
using a specific Candida albicans assay; results were negative. In the 2004 outbreak, a feeding
35
study was conducted with three horses exclusively fed alfalfa hay that was suspected to have
36
caused the outbreak. Two weeks after ingestion of alfalfa hay, two horses developed several
37
lesions in non-pigmented skin characterized as chronic ulcerative and necrotizing dermatitis
38
with superficial vasculitis, which was consistent with photosensitization. In the 2014
39
outbreak, seven different implicated alfalfa hay samples were analyzed for chlorophyll a and
40
b, and pheophorbide a. These compounds had been suspected to play a role in alfalfa-induced
41
primary photosensitization. The chlorophyll contents ranged from 0.90 - 2.30 mg/g in the
42
alfalfa hay samples, compared to 1.37 and 2.94 mg/g in locally grown alfalfa and orchard
2
Page 2 of 23
43
grass hay. The pheophorbide a levels ranged from 3.36 - 89.87 µg/g in alfalfa samples
44
compared to 81.39 and 42.33 µg/g in control alfalfa and orchard grass hay samples. These
45
findings eliminate chlorophyll a, chlorophyll b, and pheophorbide a as possible causes for
46
alfalfa-hay induced primary photosensitization.
47 48
Keywords: Chlorophyll, Horse, Pheophorbide a, Photodermatitis, Primary Photosensitization
3
Page 3 of 23
49
Introduction
50
Photosensitization, also known as photodermatitis, occurs when phototoxic or
51
photoactive substances accumulate in the skin, interact with sunlight and produce a severe
52
dermatitis. There are several different types of photosensitization related to plant exposures
53
(Johnson, 1982). Type I is a result of the direct absorption of the photosensitizing agent into
54
the blood, and distribution to the skin. Some of the more common pasture plants known to
55
cause Type I photosensitization include Hypericum perforatum (St. Johnswort), Fagopyrum
56
esculentum (Buckwheat), Ammi majus (Bishop’s weed), Cymopterus spp. (spring parsley),
57
and the Medicago spp. (clovers) (Campbell et al., 2010; Dollahite et al., 1978; Staker, 2014;
58
Williams and Binns, 1968). Type I photosensitization is rarely reported in horses because
59
exposure to these plants in pasture is unlikely. Type II photosensitization occurs subsequent to
60
liver damage due to the insufficient excretion of phylloerythrin, a photoactive metabolite of
61
chlorophyll (Campbell et al., 2010). Due to the horses’ extreme susceptibility to pyrrolizidine
62
alkaloids (PAs), exposure to Senecio and Amsinckia sp. has resulted in hepatogenous
63
photosensitization (Knight et al., 1984). Type II photosensitization has also been observed in
64
cattle exposed to moldy alfalfa hay (Scruggs and Blue, 1994), and horses exposed to clover
65
pastures that were infested with the fungus Cymodethea trifolii (Ames et al., 1994).
66
Ingestion of alfalfa silage or Froelichia humboldtiana has resulted in primary
67
photosensitization in cattle and horses (House et al., 1996; Ribeiro Knupp et al., 2014; Souza
68
et al., 2012). Breakdown products of chlorophyll such as pheophorbide were suspected to be
69
the responsible photoactive compounds in alfalfa silage (Takamiya et al., 2000). In the past
70
ten years, several outbreaks of primary photosensitization in horses were observed after
71
ingestion of alfalfa hay in California. To the authors’ knowledge, alfalfa hay-induced primary
72
photosensitization in horses has not been confirmed based on a feeding study. Furthermore,
73
no data has been established in previous investigations as to the possible role of pheophorbide
4
Page 4 of 23
74
a. Thus, the current case series describes seven outbreaks of primary photosensitization in
75
horses between 2004 and 2014, the available epidemiological and clinical findings, the results
76
of a feeding trial, and the concentrations of chlorophyll a, chlorophyll b, and pheophorbide a
77
in feed samples associated with some of the outbreaks.
78 79
Materials and methods
80
Case series:
81
Between 2004 and 2014, a total of seven outbreaks of photosensitization in horses in
82
California were evaluated. For the 2004 and 2014 outbreaks detailed information was
83
available, whereas for the outbreaks in 2008 (2), 2009 (2) and 2013, only limited
84
epidemiological and diagnostic data exist. The first large outbreak occurred in San Joaquin
85
and Kern Counties in July 2004. Approximately 70 horses housed in at least four different
86
locations developed severe skin lesions (Plate 3 – a and d). Owners reported that skin lesions
87
developed approximately 3 to 7 days after feeding a new shipment of alfalfa hay. Of the 70
88
affected horses, one Quarter horse filly died and was examined post-mortem with a complete
89
pathological and toxicological diagnostic work-up. Alfalfa hay samples were examined for
90
the presence of potentially toxic weeds, herbicides, pesticides and mycotoxins. A
91
phototoxicity assay and fungal identification were also performed on a hay sample collected
92
from the site with the most severe presentation and the greatest number of affected horses.
93
In 2008, two outbreaks were investigated in Fresno County, California. The first
94
occurred in August 2008 and resulted in an unknown number of horses developing lesions in
95
the non-pigmented skin areas. At one farm, three horses developed lesions on the muzzle and
96
on their backs. The second outbreak occurred in September 2008 and the total number of
97
affected horses was unknown, but was estimated to be more than ten. One severely affected
98
horse, a pinto, developed severe skin lesions after being fed a diet of Alfalfa cubes that were
Comment [CX1]: Picture a and d are from 2004 outbreak. belong to the year 2004. Plate legends on page 21 and 22 have been updated accordingly.
5
Page 5 of 23
99
grown and made locally, and having been exclusively in the full sun for three weeks. The less
100
severely affected horses had crusting and reddening of the non-pigmented skin on muzzle and
101
face. In 2009, two outbreaks of photosensitization occurred in May in Madera and Kern
102
County, California. Several horses developed skin lesions on the white areas of the muzzle
103
and legs after a new batch of alfalfa hay was introduced several weeks earlier. In 2013, an
104
outbreak occurred at a boarding stable in Santa Barbara County, California. Horses were
105
turned out into a large dry pasture and fed alfalfa hay ad libitum. Approximately eight horses
106
out of 116 developed skin lesions that consisted of dry, reddened thick folds on the muzzle,
107
neck and tail approximately two weeks after a new shipment of alfalfa hay was introduced
108
(Plate 2 – a, b, c, d). In addition to a change in feed, a one-time exposure to a fermenting pile
109
of grass clippings consisting of annual meadow grass (Poa annua), bermuda grass (Cynodon
110
dactylon) and perennial rye grass (Lolium perenne) occurred at about the same time as the hay
111
change. The hay was evaluated for the presence of weed contamination.
112
In July 2014, an outbreak of photosensitization occurred in Phelan, CA (Plate 3 – b
113
and c). More than 12 horses developed skin lesions on the white areas of the muzzle and face
114
after being offered a new shipment of alfalfa hay (http://www.vvng.com/phelan-horse-
115
owners-concerned-about-tainted-hay/). Affected horses were examined by veterinarians. Hay
116
samples were evaluated for poisonous weed contamination, chlorophyll and pheophorbide a
117
analysis.
118 119
Feeding Study:
120
A feeding study was conducted over 21 days on the University of California, Davis
121
campus at the Center for Equine Health facility and was approved by the Institutional Animal
122
Care and Use Committee. Three horses with varying degrees of unpigmented skin were
123
enrolled in the study. All horses underwent a complete physical examination prior to study
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Page 6 of 23
124
begin. The horses were a 7-year old gray-colored Westphalian gelding, a 6-year old chestnut-
125
colored Irish Sport gelding, and a 20-year old gray-colored Arabian cross gelding. Horses
126
were housed together in a corral without protection from UV exposure, were each fed 6
127
pounds of alfalfa hay twice per day, and offered water ad libitum. Horses were examined
128
daily for development of skin abnormalities or other signs of illness, and blood samples were
129
collected on days 2, 6, 9, 13, 21, and 27 of the study. Serum samples were evaluated for anion
130
gap, sodium, potassium, chloride, CO2 total, calcium, phosphorus, creatinine, urea nitrogen,
131
glucose, total protein, albumin, globulin, AST, creatinine kinase, ALP, GGT, SDH-37,
132
bilirubin total, bilirubin direct, bilirubin indirect, iron, magnesium, zinc, and copper. Blood
133
samples were evaluated for red blood cells, hemoglobin, hematocrit, MCV, MCH, MCHC,
134
red cell distribution width, RBC morphology, neutrophils, lymphocytes, monocytes,
135
eosinophils, basophils, platelets, plasma protein, plasma fibrinogen and selenium. All
136
analyses were performed using standard operating procedures at the Veterinary Medical
137
Teaching Hospital Laboratory and the California Animal Health and Food Safety Laboratory
138
System of the University of California, Davis. A skin biopsy was taken from the 7-year old
139
Westphalian gelding on day 13 of the feeding study and examined histologically. Alfalfa hay
140
was discontinued after 22 days, and all three horses returned to the herd. Both horses that
141
developed lesions during the feeding trial recovered uneventfully over the next 2 weeks.
142 143
Chlorophyll a and b and pheophorbide a analysis:
144
From the 2014 outbreak, a total of seven hay samples were analyzed for chlorophyll a
145
and b, and pheophorbide a. Control hay samples were locally grown alfalfa and orchard grass
146
hay that had been fed to horses in the Veterinary Medical Teaching Hospital for over four
147
weeks. For chlorophyll a and b analysis, 0.15 g of hay were homogenized on ice with 2 mL of
148
acetone: 0.1N NH4OH (9:1 vol/vol) using a homogenization tube and pestle for 30 seconds.
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Page 7 of 23
149
After rinsing the pestle with 3 mL acetone: 0.1N NH4OH (9:1 vol/vol) to remove any plant
150
residue, the tube was placed at 4 °C in the dark for two hours and then reground to extract any
151
remaining chlorophyll. The extract was then transferred to a centrifuge tube. Five microlitres
152
of 80% aqueous acetone were used to rinse the pestle and homogenization tube and added to
153
the sample extract. Sample extracts were centrifuged for 20 min at 500 x g. The supernatant
154
was transferred into a 10 mL graduated cylinder, the volume was brought to 10 mL with 80%
155
aqueous acetone, and the sample was stored at -20 °C until analysis. Sample extracts were
156
prepared in duplicate and analyzed for chlorophyll a and b with a spectrophotometer
157
(SpectraMax M3， Molecular Devices, Sunnyvale, CA, USA) and absorption was measured
158
at 645 nm and 663 nm once for each extract. The 80% aqueous acetone was used as a blank to
159
zero the instrument initially and after every resetting of the wavelength. Chlorophyll a and b
160
concentrations were determined according to previously developed formulas (Arnon, 1949;
161
Porra, 2002; Warren, 2008):
162
For pheophorbide a analysis, 0.2 g of hay were homogenized on ice with 4 mL of 80%
163
aqueous acetone using a homogenization tube and pestle for 30 seconds. After rinsing the
164
pestle with 2 mL 80% aqueous acetone the tube was placed at 4 °C in the dark for two hours
165
and then reground. The extract was then transferred to a centrifuge tube and centrifuged for
166
20 min at 500 x g. After addition of 400 µL 80% aqueous acetone to 100 µL supernatant, the
167
sample was store the at -20 °C until analysis. An Advance UHPLC high performance liquid
168
chromatograph coupled to an EVOQ Elite MS/MS triple quadrupole mass spectrometer
169
equipped with an atmospheric pressure chemical ionization (APCI) source was used for the
170
pheophorbide a analysis (Bruker Corp, Freemont, CA, USA). The chromatograph was fitted
171
with a 100 mm x 2.1 mm i.d. 1.7 µm ACQUITY BEH C18 column (Waters, Milford, MA).
172
The mobile phases consisted of 0.1% formic acid in deionized water (channel A), and 0.1%
173
formic acid in methanol (channel B). Starting conditions consisted of 90% B increasing to
8
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174
95% B over 2 minutes, then ramped up to 100% B over 1 min, which was held for 5 min and
175
then returned to 90% B within 0.5 min and held for 1 min for re-equilibration. The column
176
was maintained at 25 °C. Pheophorbide a eluted at approximately 4.2 min. Analytes were
177
monitored by multiple reaction monitoring (MRM). The transitions for pheophorbide a were
178
set as following: pheophorbide a m/z 593.2 m/z 533.6 with collision energy (CE) 45 eV.
179
Nitrogen gas served as the nebulizer, dry and collision gas. Other source parameters were as
180
follows: spray current 10 µA, cone temperature 350 °C, cone gas flow of 25 au, heated probe
181
temperature 400 °C, probe gas flow of 35 au, nebulizer gas flow of 45 au. The injection
182
volume was 10 µL. Bruker MSWS 8.1 software was used for all data acquisition and
183
processing.
184 185
Results
186
Outbreaks:
187
In the 2004 outbreak, the implicated alfalfa hay was purchased at various suppliers
188
and presumably grown in Nevada and California. Veterinarians examining the horses
189
confirmed photodermatitis; only unpigmented skin was damaged, and in severe cases there
190
was crusting, itching and a painful dermatitis. Skin lesions were primarily in the face and on
191
legs (Plate 3 – a and d). Of the affected horses, one a five month old Quarter horse filly died.
192
The foal was initially presented with skin lesions (Plate 3 – d) and myositis. After initiation of
193
supportive treatment, the foal improved over the next 24 hours before developing cool,
194
discolored distal extremities. The foal progressed to recumbency, became unresponsive to
195
fluids and oxygen, and died. A full post-mortem examination revealed subacute necrosis of
196
non-pigmented and lightly pigmented skin of limbs and head and pansystemic bacterial
197
embolism. Due to the lack of parenchymal or biliary liver lesions, hepatogenous
198
photosensitization was ruled out. The portal of entry of the bacterial septicemia was
Comment [CX2]: Same corrections as above
9
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199
determined to be the necrotic skin.The liver contained acceptable concentrations of lead,
200
manganese, cadmium, copper, iron, zinc, molybdenum, arsenic, mercury, selenium, and
201
vitamin E. The brain was evaluated for acetylcholinesterase activity, which was found to be
202
adequate for horses (4.9 µM/g/min; adequate for horses > 2.1 µM/g/min). Alfalfa hay samples
203
implicated in this outbreak were examined visually. All hay samples consisted predominantly
204
of alfalfa (Medicago sativa), but some samples had small amounts of Setaria pumila (yellow
205
foxtail), Leptochloa uninervia (Mexican sprangletop), Cynodon dactylon (Bermuda grass),
206
Eriochloa contracta (prairie cupgrass), and Sonchus oleraceus (common sowthistle). Some
207
mold was identified on all alfalfa hay samples and was identified on direct tape mount as
208
Cladosporium and Alternaria species. On potato flakes agar plates there was Mucor sp. and
209
Cladosporium spp. growing. These findings are typical mold growth identified on most types
210
of organic materials, such as alfalfa. A phototoxicity (Candida albicans) assay performed on
211
individual fragments selected from various alfalfa hay samples was negative (Kavli and
212
Volden, 1984). Hay samples contained non-toxic concentrations of heavy metals (lead,
213
manganese, cadmium, copper, iron, zinc, molybdenum, arsenic, mercury). None of the listed
214
carbamate insecticides (aldicarb, aldicarb sulfone, carbaryl, carbofuran, 3-hyroxycarbofuran,
215
methiocarb, methomyl, mexacarbate, oxamyl, and propoxur), organophosphorus insecticides
216
(43
217
trichlorophenoxyacetic acid, and 2,4-DB), or mycotoxins (aflatoxin B1, ochratoxin A, T-2,
218
DAS, and vomitoxin) were identified when analyzed by standard operating procedures.
219
Follow-up with veterinarians and owners revealed that all affected horses, except for the foal
220
that died, recovered with supportive care consisting of anti-inflammatory medications,
221
protection from UV exposure, and change in feed.
different
compounds),
herbicides
(dicamba,
MCPA,
2,4-D,
silvex,
2,4,5-
222
In all 2008 and 2009 outbreaks, horses were fed locally grown alfalfa hay or alfalfa
223
cubes and had no elevations of liver enzymes when examined by the veterinarian. No deaths
10
Page 10 of 23
224
were reported and all horses recovered with supportive care consisting of change in feed,
225
protection from UV exposure, and anti-inflammatories when indicated. In the 2013 outbreak
226
with 8 affected horses, liver enzymes were evaluated in all affected horses and were within
227
reference ranges. The alfalfa hay was examined carefully and although predominantly
228
consisting of alfalfa, several plants contaminant were identified. Those included found to
229
contain Bassia scoparia (Kochia, burning bush), Quercus lobata (Valley oak), Chenopodium
230
murale (Nettleleaf goosefoot), Marrubium vulgare (Horehound), Rumex sp. (dock), Eurybia
231
sp. (Aster), Lavandula angustifolia (Common lavender), Buxus microphylla japonica
232
(Boxwood), Tagetes sp. (Marigold), and Festuca sp. (Fescue). Affected horses were removed
233
from the sun, had a change in feed, and received local treatment of skin lesions with zinc
234
oxide. There were no reports of deaths in these outbreaks.
235
In the 2014 outbreak, none of the affected horses had elevated liver enzymes or other
236
abnormalities when examined by their veterinarians. The alfalfa hay was suspected to have
237
been grown in Southern California, and presumably fed shortly after harvest. A total of seven
238
hay samples were carefully evaluated and confirmed to consist predominantly of clean, green
239
alfalfa. Some of the alfalfa had some black spot mold, especially on older stems, and there
240
were some water-soaked lesions on some of the larger leaves. While five of the hay samples
241
contained no weed contamination, one of the hay samples contained four small fragments of
242
puncture vine, Tribulus terrestris. Puncture vine has been associated with the development of
243
liver disease and the development of secondary photosensitization.
244
Feeding trial:
245
All horses had good appetite and finished the provided alfalfa hay after each feeding.
246
All serum and blood analytes were within reference ranges for horses at all assessed time
247
points. Daily observations revealed no abnormalities until day 13 of the feeding trial. On day
248
13, the 7-year old Westphalian gelding developed an erythematous, crusting lesion on the
11
Page 11 of 23
249
dorsum of his muzzle where white facial markings existed (Plate 1 – b). On day 19, the 6-year
250
old chestnut-colored Irish Sport gelding developed two areas of red raw, peeling, and crusting
251
skin on the medial aspects of the pastern and along cannon bone of the non-pigmented right
252
hind leg (Plate 1 – a). The 20-year old Arabian cross developed no skin abnormalities
253
throughout the trial. This horse had no white or pink skin areas, and had a pigmented
254
epidermis making him a valuable negative control horse of the feeding trial. A punch biopsy
255
was collected from the 7-year old Westphalian gelding (Plate 1 – c). The hyperplastic
256
epidermis was overlain by a prominent crusts composed of necrotic epidermal and dermal
257
components, indicating a necrotizing process with re-epithelialization. Lymphocytes and
258
plasma cells with rare neutrophils were surrounding dermal vessels, many of which had
259
indistinct vascular walls with partial loss of endothelial cells (Plate 1 – d). Multiple
260
microhemorrhages were present in the superficial dermis. Histologic features were consistent
261
with a subacute stage of a vascular insult.
262 263
Chlorophyll and Pheophorbide a analysis:
264
The chlorophyll a concentrations in the 7 alfalfa hay samples from the 2014 outbreak
265
were 1.44, 0.94, 1.20, 0.55, 1.37, 1.32, and 1.37 mg/g respectively, whereas the two control
266
hay samples had 0.84 mg/g (alfalfa hay) and 1.77 mg/g (orchard grass hay). The chlorophyll b
267
concentrations in the outbreak-associated alfalfa hay samples were 0.86, 0.59, 0.72, 0.35,
268
0.82, 0.79, and 0.81 mg/g, while two control hay samples had 0.53 and 1.17 mg/g. Thus, all
269
analyzed alfalfa hay samples implicated in the 2014 outbreak contained chlorophyll a and b in
270
concentrations similar to what was determined in control alfalfa and orchard grass hay
271
samples that were not associated with any illness after long-term feeding to horses (Fig. 1).
272
The pheophorbide a concentrations in the 7 alfalfa samples available from the 2014 outbreak
12
Page 12 of 23
273
were 45.75, 22.29, 89.87, 3.36, 15.90, 26.10, and 31.47 µg/g, while the alfalfa and orchard
274
grass hay control samples had 81.39 and 42.33 µg/g (Fig. 2).
275 276
Discussion
277
This study confirms that alfalfa hay is able to induce primary photosensitization in
278
horses. The photosensitizing trigger remains unknown at this point, as chlorophyll a,
279
chlorophyll b and pheophorbide a as the responsible photoactive compounds in alfalfa hay
280
have been ruled out. In all affected horses, liver function was not impaired when
281
photodermatitis was diagnosed confirming that there was direct exposure to a photosensitizer.
282
The only published report implicating alfalfa in an outbreak of primary photosensitization was
283
reported in dairy cattle from San Joaquin Valley, California (House et al., 1996). In this
284
outbreak, over 400 lactating dairy cows developed photosensitization after being fed alfalfa
285
silage. A feeding trial confirmed alfalfa silage to have caused the skin lesions. It was
286
suspected that breakdown products of chlorophyll, such as pheophorbide a, may have been the
287
cause of photosensitization. However, no testing for pheophorbide was done.
288
Photosensitizers are capable of absorbing UV radiation to form a reactive excited state
289
molecule that in turn transfers the energy to a surrounding molecule. Phototoxins are
290
commonly found in plants where they serve an important role for plant defense (Björn and
291
Huovinen, 2002). Common primary photosensitizing plants include Hypericum perforatum
292
(St. John’s wort), Fagopyrum esculentum (buckwheat) Ammi majus (Bishop’s weed),
293
Cymopterus (spring parsley), Heracleum sp. (cow parsnip, hogweed), Lomatium sp. (wild
294
parsley), Apium sp. (celery), Pimpinella sp. (burnet saxifrage), Ambrosia sp. (ragweed) and
295
the Medicago species (Downum, 1992; Ivens, 2011; Ivie, 1982; Johnson, 1982; Pathak, 1986).
296
None of these plants were identified in any of the outbreaks. In the 2004 outbreak, Setaria sp.
297
were identified in the hay. Setaria sp. have sharp and barbed bristles that are capable of
13
Page 13 of 23
298
penetrating the mucous membranes and may lead to erosions of the mouth. Setaria-induced
299
illnesses have been reported in horses; however, lesions are typically seen in the mouth and
300
horses are reluctant to eat. These clinical signs were not seen in the affected horses.
301
In addition, certain antibiotics and phenothiazine derivatives have been associated
302
with primary photosensitization in livestock. However, phenothiazine is primarily a problem
303
in ruminants due to the formation of the phototoxic compound by rumen microbes (Dirksen
304
and Tammen, 1964). While drug-induced photosensitization is a major health concern in
305
humans (Dawe and Ibbotson, 2014), documented reports in horses are missing. In all outbreak
306
investigations, owners and veterinarians were asked about use of medications in the affected
307
horses; none were reported.
308
The typical chemical structure of a photosensitizer is a polycyclic compound with
309
many double bonds (Björn and Huovinen, 2002). Chlorophylls are able to absorb light and act
310
as photosensitizers resulting in singlet oxygen and superoxide formation. Chlorophylls are
311
photosynthetic pigments that are widely distributed in nature and possess a basic skeleton
312
structure of porphyrin with a magnesium ion in the center and a long phytol group as the tail
313
(Huang et al., 2008). The major chlorophyll forms in plants are chlorophyll a and chlorophyll
314
b with the content of the former being about 3-fold higher than the latter. Under mild heating
315
conditions during the drying process of green plants, chlorophyll a is degraded to
316
chlorophyllide a through the activation of chlorophyllase and removal of phytol, which upon
317
activation of dechelatase can be further converted to pheophorbide a (Loh et al., 2012).
318
Pheophorbide a is structurally very similar to chlorophyll a with the exception of the loss of
319
Mg and the phytol group, and is photodynamic. Based on its phototoxic ability, pheophorbide
320
a is utilized in photodynamic therapy of cancer (Yoon et al., 2014). In addition, pheophorbide
321
a-induced phototoxicity has been reported in humans after ingestion algae-derived chlorella
322
tablets (Jitsukawa et al., 1984), that are sold as natural health supplement. Pickled vegetables
14
Page 14 of 23
323
have also caused phototoxicity in rats due to their substantial amounts of pheophorbide a
324
(Jitsukawa et al., 1984). Thus, our assessment of pheophorbide a levels in the various alfalfa
325
hay samples was warranted; however, results illustrated that pheophorbide a levels did not
326
differ between the hay samples causing primary photosensitization and the control hay.
327
Pheophorbide a, while one of the more in depth studied breakdown products, is not the only
328
photodynamic chlorophyll a metabolite, however. Pheophytin a, pyropheophytin a, and
329
chlorophyllide a (Lohrey et al., 1974; Takeda et al., 1989; Tapper et al., 1975) have been
330
identified and could conceivably play a role in these outbreaks. In addition, breakdown
331
products of chlorophyll b, even though present in much lower concentrations in plants, may
332
play a role.
333
Since the amount of chlorophyll, and most likely other potential photosensitizers, is
334
greater in green plant material compared to dry hay the risk for an animal to develop
335
photosensitization is considered greatest under grazing conditions (Quinn et al., 2014).
336
However, under certain condition, dried plant material can result in the formation of certain
337
phototoxic compounds that lead to disease. This was the case in cattle that were exposed to
338
dried leaves of Cooperia pedunculata in Texas and developed primary photosensitization
339
(Rowe et al., 1987). In another instance, specific conditions had to be present for disease to
340
develop; co-consumption of horse brush (Tetradymia spp.) and sage (Artemisia spp.)
341
appeared to be necessary to result in photosensitization in sheep (Johnson, 1974).
342
Sporadic outbreaks of primary photosensitization in horses are a great source of
343
frustration to owners, veterinarians, and growers. The motivation for this study was to
344
confirm that alfalfa hay can induce primary photosensitization in horses, and to determine
345
whether alfalfa hay, under certain conditions, can accumulate toxic levels of chlorophyll a,
346
chlorophyll b or pheophorbide a, all known photosensitizing agents. Even though we were
347
able to reproduce the primary photosensitization with a feeding trial, we were unable to
15
Page 15 of 23
348
determine the etiologic agent. Although the present study has yielded important findings, its
349
design is not without flaws. A number of caveats that are often encountered when relying on
350
the submission of case-based/outbreak material need to be noted: the quality of epidemiologic
351
information such as the number of affected horses, location of hay purchase, location of hay
352
grower, exact days of exposure provided by owners and veterinarians varied; the diagnostic
353
work-up varied by outbreak depending on availability of specimens and analytical tests;
354
analytical testing for chlorophyll a and b and pheophorbide a was only available for the 2014
355
investigation. In addition, as is true for any analysis of a feed sample, hay samples examined
356
and tested may not have been representative of the complete shipment.
357 358
Conclusions
359
To the authors’ knowledge, this is the first report of confirming that alfalfa hay can induce
360
primary photosensitization in horses. Horse owners as well as veterinarians should recognize
361
the possibility that alfalfa hay, on rare occasions, can induce skin lesions, and discontinue
362
feeding alfalfa as soon as signs are noted. In addition, rapid response and research efforts are
363
needed to identify the etiologic agent and the growing conditions for the toxin to be present as
364
the toxin may not be stable under long-term storage conditions. There are very few studies
365
that have evaluated the concentrations of photosensitizers in dried plant material implicated in
366
outbreaks of photosensitization. This is a critical step in not only the characterization of
367
photosensitizing compounds but in the development of an approach for prevention strategies.
368
In addition, outbreaks appear to occur sporadically. We have very limited information about
369
the growing and climate conditions when these disastrous events occur. Additional research is
370
needed to determine the nature of the photosensitizing compounds and the factors necessary
371
for production.
372
16
Page 16 of 23
373 374 375
Conflict of interest statement None of the authors has any financial or personal relationships that could inappropriately influence or bias the content of the paper.
376 377
Acknowledgements
378
The authors thank Dr. Asheesh Tiwary for his assistance during the 2004 outbreak
379
investigation and feeding study, Marcia Booth for her expertise in plant identifications, and
380
Drs. Phoebe Smith, Kristen Fosnaugh, Jeanette Mero, Robert Shaw, and Alisha Olmstadt for
381
their information on case management and outbreak epidemiology. The authors are also
382
grateful to Dr. John Reagor for performing the phototoxicity assay, and to Dr. Michael
383
Rinaldi for interpretation of fungal identification results. We also would like to thank Dr.
384
Sharon Spier for collection of a biopsy specimen, and Mike Davidson for the provision of
385
quarantined hay for the feeding study.
386 387 388 389
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471 472
Plate and Figure legends Plate 1: Skin lesions developing during feeding trial of provided alfalfa (2004)
473
a: Skin lesions observed on non-pigmented skin on distal lags of the 6-year old chestnut-
474
colored Irish Sport gelding on day 19: locally extensive oozing and crusting with ulcerations
475
b: Skin lesions observed on the non-pigmented portion of the muzzle of the 7-year old
476
Westphalian gelding on day 13: erythematous, crusting lesion on the white markings of the
477
muzzle. Note the abrupt demarcation of the lesions along the non-pigmented to pigmented
478
skin area.
479
c: Biopsy from the affected area of the muscle of the 7-year old Westphalian gelding: severe
480
serocellular crusting (C) composed of epidermal and dermal components are overlaying a
481
markedly hyperplastic newly formed epidermis (E). There is mild perivascular, mostly
482
mononuclear inflammation (I) in the dermis and small areas of microhemoprrhage (>).
483
d: Higher magnification of the biopsy shown in C: Multifocal microhemorrhages (H) are the
484
result of the damaged vascular walls (<), characterized by hyalinized vessel walls and loss of
485
endothelial cells.
486 487
Plate 2: 2013 outbreak of photodermatitis in horses. Severe erythema, erosions and
488
ulcerations (a-d) and crusting (a and c) are limited to non-pigmented areas of the skin. The
489
shoulder lesions and the lateral muzzle show abrupt demarcation of the lesions and the normal
490
appearing skin surface in the pigmented skin.
491 492
Plate 3: 2004 and 2014 outbreaks of photodermatitis in horses. In the 2004 outbreak, severe
493
skin lesions were primarily located in the face and on legs (a and d). One 5-month old Quarter
494
horse foal presented with swollen, red raw, peeling and crusty skin exclusively affecting non-
495
pigmented areas of rear pasterns (d). In the 2014 outbreak, erosion and ulcerations developed
496
on the muzzle (b and c).
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497
Fig. 1. Chlorophyll a and chlorophyll b concentrations (in mg/g) in seven hay samples from the 2014 outbreak. Control samples were alfalfa and
498
orchard grass hay. 3.50 chlorophyll b 3.00
concentration(mg/g)
chlorophyll a 2.50
2.00
1.50
1.00
0.50
0.00 Alfalfa #1 Alfalfa #2 Alfalfa #3 Alfalfa #4 Alfalfa #5 Alfalfa #6 Alfalfa #7 Sample ID
Control Alfalfa
Control Orchard grass
499 500 501
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502
Fig. 2. Pheophorbide a concentration (in µg/g) in seven hay samples from the 2014 outbreak. Control samples were alfalfa and orchard grass hay.
pheophorbaide a concentration (µg/g)
100.00
80.00
60.00
40.00
20.00
0.00 Alfalfa #1
Alfalfa #2
Alfalfa #3
Alfalfa #4
Alfalfa #5
Sample ID
Alfalfa #6
Alfalfa #7
Control Alfalfa
Control orchard grass
503 504
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