AN OUTBREAK OF FUNGAL DERMATITIS AND STOMATITIS
IN A WILD POPULATION OF PIGMY RATTLESNAKES,
SISTRURUS MILIARIUS BARBOURI, IN FLORIDA: DESCRIPTION, FACTORS,
CYCLICITY, AND PREVENTION
JOSEPH LATON CHEATWOOD
A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE
UNIVERSITY OF FLORIDA
For my wife, Amy Pazzalia Cheatwood. You have supported me unconditionally since
the first day. I am truly blessed. The best it yet to be.
I would like to acknowledge the many fine scientists that provided support for my
research. First, I would like to thank Dr. Elliott Jacobson, my major professor, without
whose sponsorship and input this work would have been impossible. Second, Dr. Peter
May and Dr. Terence Farrell, both from Stetson University in Deland, Florida, were the
keystones on which this study was constructed. Their long-time devotion to the
herpetological fauna of this great state has inspired many students and greatly enhanced
human knowledge of several key species.
I would like to thank Dr. James Kimbrough of the University of Florida,
Department of Plant Pathology, for lending his mycological expertise to the project. Dr.
Don Samuelson also deserves a great barrage of gratitude for his unending support of this
project and his histological expertise. A huge "thank you" is in order for Dr. Bruce
Homer, a pathologist at the University of Florida, College of Veterinary Medicine, for
helping prepare me to describe the lesions I found on snakes in the field and for his
microphotography advice. Finally, Dr. Jorge Hernandez deserves notice for his advice on
the analysis and interpretation of the data in Chapter 3. His help was indispensable.
Again, thank you all.
TABLE OF CONTENTS
A C K N O W L E D G M E N T S ................................................................................................. iii
LIST OF TABLES ........ ............ .......... ............. ......................... vi
AB STRA CT ............ ................... ........................ ........... ... ............. vii
1 REVIEW OF FUNGAL DISEASE AND METHODS LITERATURE..........................1
In tro d u ctio n ...................................... ...................................... ............... 1
Fungal Taxonom y ........................................... ... .... ..... ........... .. 2..
Review of Literature by Order of Reptiles ........................................................ 5
I. C helonia ................................. ......... ............... 5
II. Crocodilia .................... ... .. .... ........ .................. 7
III. S qu am ata : L acertilia ............................................................................................. 9
IV S qu am ata : S erp entes ........................................................................................... 10
2 DESCRIPTION OF DISEASE AND PATHOGENESIS........... ..............13
Intro du action ............ ... ...... .......................................................... ..... 13
M materials and M ethods................... .............................................. ........................... 14
R e su lts ......... .......................................................... .................................. ..... 1 8
D iscu ssion ......... .. ......... ................................................ ............... . ... 22
3 OUTBREAK SEASONALITY AND CYCLICITY ................................................ 30
Introduction................................ .......... .......... 30
M materials and M ethods......... ......... ....... ........... ........................... .............. 32
C ase D ata .............. ......... ............................................................... .... ... . 32
Environm mental D ata ....... .... ..... ..... ......................... ........ .. ............ .. 33
D ata A naly sis ..................................................................................... 34
D escriptiv e Statistics............................ ................ ... ............... ....... ................ 34
Regression Analyses ........ ... ..... ........ ..................... .......... .. .......... .. 34
T im e series .......................................................................................... . . 34
Results................................. .............. 36
D escriptiv e Statistics............................................................ ........... . ............ 36
R egression A analyses ....................................................... .. .......... .. 37
T im e S erie s ................................................................... 3 7
D iscu ssio n ..................................................... 4 0
4 HANDLING AND SAMPLING PROTOCOL FOR HERPETOLOGICAL
R E S E A R C H .....................................................................................................4 1
Introduction and B background ...................................................................... 41
Protocol for Safe Handling and Sampling of Reptiles .............................................. 46
Basic protocol ......................................... 46
Surgical P protocol ..................................................... 48
C o n c lu sio n .............................................................................. 5 0
A. HANDLING AND SAMPLING PROTOCOL SURVEY .......................................52
B SA M PL E D A T A SH E E T ....................................................................................... 56
LIST OF REFERENCES .................................................................. ...........57
BIOGRAPHICAL SKETCH ................................. ........................... ........66
LIST OF TABLES
1 Taxonomic Tree of Medically Important Fungi in Humans and Animals....................3
2 Incidence of disease by year (% affected).................................. ...................... ........... 19
3 G roups by yearly incidence...................................................................... ...................37
4 R results of regression analyses ................................... .... ............................ ...............37
5 Sum m ary of survey responses ................................................ .............................. 45
Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science
AN OUTBREAK OF FUNGAL DERMATITIS AND STOMATITIS
IN A WILD POPULATION OF PIGMY RATTLESNAKES,
SISTRURUS MILIARIUS BARBOURI, IN FLORIDA: DESCRIPTION, FACTORS,
CYCLICITY, AND PREVENTION
Joseph Laton Cheatwood
Chairman: Dr. Elliott R. Jacobson
Major Department: Veterinary Medicine
This study consists of three sections. First, a fungal disease in a wild population
of pigmy rattlesnakes, Sistrurus miliarius barbouri, was studied over a 20-month period
in 1998 and 1999. Weekly searches were conducted for infected animals in the study
population. Lesions found on infected snakes were biopsied and prepared for both
histology and fungal culture. Fungi successfully isolated from lesions included
Sporothrix schenkii (two snakes with severe facial lesions), an unidentified Paecilomyces
sp. (one snake with subdermal granulomas), Pestalotiapezizoides (one snake with
subdermal granulomas), and Geotrichum candidum (=Galactomyces geotrichum) (one
snake with subdermal granulomas). Fungi were also isolated from leather gloves used by
members of an ongoing ecological study in the population to restrain the snakes. Two
fungi were identified from the gloves: Cladosporium sphaerospermum and Pestalotia
pezizoides. Neither of these fungi has been previously identified as pathogenic organisms
in reptiles, though both are pathogens of plants.
Environmental data spanning the length of the ecological studies being conducted
by research group in this population were analyzed to determine which environmental
factors, if any, were correlated with an increase in the number of new cases of fungal
dermatitis and stomatitis. Factors analyzed included habitat water level, temperature, and
a calculated value used to represent the combined effect of the two. Simple and multiple
linear regressions did not indicate a statistically significant direct correlation between any
of the factors and the incidence of disease in the population at a given time.
Significant differences were shown to exist between the numbers of new cases
found per year. Years were placed into three groups based on the yearly incidence of
disease (new cases divided by total snakes captured). The eight years covered by the data
can be clearly divided into four groups: A, B, C, and D. The 1997-1998 outbreak is the
only member of group D, however, and is significantly different from all of the other
years covered by the study. Time series analyses show that there are significant seasonal
and cyclical patterns to the disease outbreaks. These repeating patterns could be due in
part to many factors including environmental conditions, even though a direct
relationship is not evident from the regression models.
While conducting the studies, concern arose about a possible anthropogenic
component of the disease. Though a relationship between research methods and disease
was never proven, the possibility prompted the construction of a pamphlet containing
recommended handling and sampling protocols for conducting research with wild
REVIEW OF FUNGAL DISEASE AND METHODS LITERATURE
Many different mycotic diseases have been reported in captive chelonians,
crocodilians, and squamates. No reports describing mycotic disease in the tuatara
(Sphenodon punctatus), a member of the order Rhynchocephalia, could be found in a
Medline search. Relatively few mycotic diseases have been seen in free ranging reptiles.
As in other vertebrates, fungal pathogens in reptiles may be primary or secondary
invaders. Mycotic disease may be associated with predisposing factors including high
humidity, overcrowding, and debris accumulation in the animal's environment. The
system affected may vary between the major groups of reptiles. For instance, whereas
mycotic pneumonia is uncommon in snakes, it is commonly seen in captive chelonians.
Compared to mammals, systemic mycotic diseases such as histoplasmosis,
coccidiodomycosis, and cryptococcosis are rarely seen in reptiles. Similarly, the
dermatophytes Trichophyton and Microsporum are rarely reported in reptiles. In contrast,
fungi that are seldom reported as significant problems in birds and mammals are common
in reptiles. Beginning with an overview of fungal taxonomy, this chapter will review the
available literature on fungal infections of reptiles.
Many different fungi have been identified in the tissues of humans (and other
mammals), reptiles, and birds. Almost any fungus can be a facultative pathogen, moving
into preexisting lesions and preying on soft tissues of immunocompromised patients.
However, some fungi are primary pathogens, causing damage to healthy tissue without
the aid of other organisms. These primary pathogens are considered medically important.
All fungi are members of the Kingdom Fungi (Myceteae). Most pathogenic fungi
are classified in the Division Amastigomycota. Within this division, there are seven
classes containing medically important fungi. Table 1 shows the taxonomic breakdown
in greater detail, listing genera in each family that are known human pathogens.
Hundreds of species of fungi have been shown to be of medical importance.
Since appropriate therapies differ for various pathogens, it is important to rapidly and
accurately identify fungi. Morphological characteristics, such as width of hyphae,
presence or absence of septae, type and size of reproductive structures, colony
morphology (including size, shape, rate of growth, and color), and optimum incubation
temperature are used to identify and classify fungi. These characteristics are explained in
greater detail in many medical and clinical mycology reference books (Kwon-Chung and
Bennett, 1992; Fisher and Cook, 1998). It is important to consult the most recently
published reference books to ensure that the most current techniques and products are
used for fungal identification and that the most widely accepted names are used.
Table 1 Taxonomic Tree of Medically Important Fungi in Humans and Animals
Kingdom Fungi (Myceteae)
Genus: Filobasidiella (Teleomorph of Cryptococcus)
Reprinted with permission from Jacobson et al., 2000.
Review of Literature by Order of Reptiles
Hyalohyphomycosis is a term that includes mycotic infections involving any
fungal agent with septate hyphae and non-pigmented (hyaline) walls in tissue. The term
encompasses a large number of fungi, some very different from each other, with one
common characteristic: septate hyaline hyphae. The term does not refer to a group of
common clinically recognizable symptoms (Fisher and Cook, 1998).
Fusarium solani has been reported as the cause of cutaneous mycosis in a
loggerhead sea turtle, Caretta caretta (Cabanes et al., 1997). The authors described the
fungal lesions on the turtle's skin as "white-scaled." The skin lesions from which the
fungus was isolated measured between 10mm and 35mm in diameter.
Paecilomyces lilicanus and Candida albicans were isolated from an Aldabra
tortoise, Geochelone gigantea (Heard et al., 1986). Paecilomyces lilicanus was isolated
from many macroscopic, firm yellow nodular lesions in the oral and gastric mucosas and
throughout the liver.
A case of systemic mycosis caused by Penicillium griseofulvum has been reported
in a Seychelles giant tortoise, Geochelone gigantea (Oros et al., 1996).
Aspergillosis is caused by members of the genus Aspergillus. Many Aspergillus
species are widespread saprobes (soil dwellers), breaking down plant materials for
nutrition (Fisher and Cook, 1998). A side-necked turtle, St. Hilaire's terrapin (Hydraspis
hilarii), died from a generalized aspergillosis (Hamerton, 1934). Mycotic granulomas
from the forefeet of a female musk turtle (S.i in,/lh'i //i1% odoratus) were found to contain
yeast-like organisms presumed to be an Aspergillus sp. (Frye and Dutra, 1974).
Mucormycosis is caused most frequently by members of the family Mucoraceae,
including Rhizopus, Absidia, Rhizomucor, Mucor, and Apophysomyces (Fisher and
Cook, 1998). In chelonians, mucormycosis has been reported in juvenile Florida
softshell turtles (Apaloneferox) that had necrotizing shell and skin lesions. Mucor was
isolated from the lesions (Jacobson et al., 1980). AMucor sp. was also isolated from
infected skin of wood turtles, Clemmys insculpta (Lappin and Dunstan, 1992).
Mixed mycoses (Sporotrichosis, Phaeohyphomycosis, Hyalohyphomycosis)
Mariculture-reared green sea turtles (Chelonia mydas) with mycotic pneumonia
were found upon necropsy to be infected with several different fungi including a
Sporothrix sp., a Cladosporium sp., and a Paecilomyces sp. (Jacobson et al., 1979). The
lesions were described as multifocal firm nodules that were more prominent in the right
lung. Histopathology of the granulomas showed that each contained numerous fungal
Superficial and deep mycoses (Hyalohyphomycosis, Aspergillosis, Beauveriosis,
One study isolated Fusarium solani from deep tissue mycoses in saltwater
crocodiles (Crocodylusporosus) and freshwater crocodiles (Crocodylusjohnstoni) on
farms in Australia. In the same study, Aspergillus niger, Penicllium oxalicum, and
Curvularia lunata varaeria coexisted in superficial lesions on the skin and gingiva. The
authors did not conclude which organisms were the causative agents (Buenviaje et al.,
1994). In the cases exhibiting deep tissue mycoses, lesions were found in the liver, lungs,
intestines, and stomach of affected animals. In a subsequent study, several additional
species of fungi were noted as pathogens on crocodile farms. These included Fusarium
sp. and Candida sp. (Buenviaje and Ladds, 1998).
A report was published of a case of a fatal Beauveria bassiana infection
(hyalohyphomycosis) in a captive American alligator, Alligator mississippiensis
(Fromtling et al., 1979). On necropsy, it was determined that only the lungs were
infected. The lungs were reportedly thickly covered with "mats" of fungal hyphae.
A hyaline Fusarium sp. and a yeast-like Trichosporon sp. were isolated from skin
lesions on two different caiman, Caiman crocodylus (Kuttin et al., 1978).
Mixed fungal pneumonias
There have been several unique reports of fungal pneumonia in crocodilians.
Candida albicans was identified as the causative agent of pneumonia in unspecified
species of crocodile and caiman (Zwart, 1968). A fatal, diffuse, granulomatous
pneumonia and accompanying necrotizing hepatitis was described in three six-month-old
Caiman sclerops (Trevino, 1972). In this paper, hyphae with terminal chlamydospores
morphologically consistent with Cephalosporium sp. were seen in tissue sections of the
lesions. In another paper, three species of crocodilians, including a Morelet's crocodile
(Crocodylus moreleti), an American crocodile (C. acutus), and a Nile crocodile (C.
niloticus), developed a fatal respiratory infection (Silberman et al., 1977). Lesions were
primarily confined to the lungs, from which a Mucor species was identified. Several
captive two to six-week-old American alligators (Alligator mississippiensis) were seen in
a separate study with pneumonic lesions from which Aspergillusfumigatus and A. ustus
were isolated (Jasmin et al., 1968).
Mixed necrotizing dermatitis (Aspergillosis, Mucormycosis)
Fungi from the genera Aspergillus, Mucor, and Rhizopus were isolated from
cutaneous lesions in a 100-year-old American crocodile (Crocodylus acutus) that was
infected with Erysipelothrix indiosa (Jasmin and Baucom, 1967). Interestingly, in the
same study, members of the genera Aspergillus, Rhizopus, and Penicillium were isolated
from seemingly normal skin and scales of an American alligator (Alligator
III. Squamata: Lacertilia
Cryptococcus neoformans, a yeast-like organism, has been isolated from a
subcutaneous lesion of an eastern water skink, Eulamprus quoyii (Hough, 1998). The
lesion was described as a "small, discrete swelling over the lower thoracic spine." Light
microscopic examination revealed numerous vacuoles containing the yeast-like cells.
A cutaneous fungal infection involving the Chrysosporium anamorph of
Nannizziopsis vriesii has been reported in chameleons (Pare et al., 1997). Chrysosporium
keratinophilum was seen in multifocal lung lesions and necrotic stomach lesions of two
green iguanas, Iguana iguana (Zwart et al., 1968).
Aspergillus terreus was isolated from two San Esteban chuckwallas, Sauromalus
various (Tappe et al. 1984). The lesions were described as edematous and necrotic.
Biopsies revealed the presence of numerous fungal hyphae.
A black-pointed teguexin (Tupinambis nigropunctatus) died following a
generalized mycosis caused by an unidentified species ofAspergillus sp. (Hamerton,
Mucor sp. was isolated from cutaneous lesions in a bearded dragon, Pogona
barbata (Frank, 1966).
Candida albicans has been isolated from multiple necrotic areas of the liver of a
two-banded chameleon, Chameleo bitaeniatus (Silberman et al., 1977), and from necrotic
esophageal lesions in a crocodile tegu, Crocodilurus lacertinus (Zwart et al., 1968).
One of the first papers reporting a fungal infection in a reptile described hyphae
that were seen in "tumours" of a green lizard, Lacerta viridis (Blanchard, 1890). The
hyphae were consistent with either a Fusarium sp. or a Selenosporium sp.
IV. Squamata: Serpentes
The only known causative agent of Trichosporonosis is Trichosporon beigeleii
(Fisher and Cook, 1998). This fungus was isolated from the liver and kidneys of several
captive banded rock rattlesnakes, Crotalus lepidus klauberi, but the authors were not
certain that this was also the yeast-like organism observed in tissue (Reddacliffe et al.,
Cladosporium sp. has been isolated from granulomatous lesions from the
mandible of an adult anaconda, Eunectes murinus (Marcus, 1971).
Chromoblastomycosis is defined as a superficial or subcutaneous mycosis
resulting from a fungus that produces round, non-budding forms called sclerotic bodies
(Fisher and Cook, 1998). A case of chromoblastomycosis has been reported in a
reticulated python, Python reticulates (Frank, 1970), with a severe ulcerative dermatitis
on the ventral scales. A similar fungus has been associated with skin lesions in a boa
constrictor, Constrictor constrictor (Frank, 1976).
Geotrichosis is an infection caused by Geotrichium candidum (Fisher and Cook,
1998). Mycotic dermatitis (Geotrichosis) due to G. candidum was documented in a
group of captive carpet pythons (Morelia spilotes variegata). Skin lesions were
prominent on the ventral scales (McKenzie and Green, 1976). Geotrichium candidum
was also seen in caseous subcutaneous nodules in a northern water snake, Nerodia
sipedon (Karstad, 1961).
An Aspergillus sp. was isolated from a puff adder (Bitis arietans) with peritonitis
Although fungal elements were seen on microscopic examination of histologic
sections taken from a mangrove snake (Boiga dendrophila), no fungi were isolated
(Jacobson, 1984). Similarly, fungal infections were diagnosed in a western Massasaugua
rattlesnake, Sistrurus catenatus (Williams et al., 1979), a red milksnake, Lampropeltis
triangulum syspila (Sindler et al., 1978), and an eastern indigo snake, Drymarchon corais
couperi (Werner et al., 1978), but an infectious agent was not isolated in any of these
cases. A zygomycete was observed in a captive gopher snake (Pituophis melanoleucos)
and a captive copperhead (Agkistrodon contortrix). Systemic disease resulted in the
deaths of these snakes (Jessup and Seely, 1981).
DESCRIPTION OF DISEASE AND PATHOGENESIS
In order to understand demographic and ecological aspects of dusky pigmy
rattlesnakes, Sistrurus miliarius barbouri, a study site was established in 1992 at Lake
Woodruff National Wildlife Refuge, Volusia County, Florida. This research provided
new information on the life history of this snake (Rabatsky and Farrell, 1996; Bishop et
al., 1996; May et al., 1996; Jemison et al., 1995; Farrell et al., 1995; Roth et al., 1999).
All pigmy rattlesnakes with a mass greater than 25g encountered were manually
restrained using leather welder's gloves and a passive integrated transponder tag (PIT-
tags) was inserted into the coelom using a modified hypodermic syringe (Jemison et al.,
1995). PIT-tags (AVID Marketing Inc., Norco, California) are small glass-encapsulated
microchips that contain unique identification numbers. These tags can be repeatedly
read with an external scanning device. This allows reliable, unique identification of
In the fall and winter of 1997, field studies were conducted and nine pigmy
rattlesnakes at this site were observed with severe skin, eye, and mouth lesions. Several
ribbon snakes, Thamnophis sauritis sauritis, and a garter snake, Thamnophis sirtalis
sirtalis, with similar lesions were also seen at the site during further surveys. Of these, a
few were either found dead in the field or were moribund. During the same period, other
pigmy rattlesnakes were seen with less severe multifocal subcutaneous masses or crusted
scutes. Skin lesions in snakes can be caused by a variety of pathogens including
bacteria, fungi, and parasites. Unfortunately, there is a paucity of information on wild
reptiles However, there is a variety of reports of fungal infection in captive reptiles. In
snakes, the integumentary system is commonly affected. The following fungi have been
identified in skin lesions of snakes: Geotrichium spp. (Karstad, 1961), Candida albicans
(Zwart, 1968), Penicillium spp. (Jacobson, 1980), and an unidentified Phycomycete
(Werner et al, 1978). Fungal skin and granulomatous disease have been seen in a variety
of species of captive snakes including an anaconda (Eunectes murinus) (Marcus, 1971),
reticulated pythons (Python reticulatus) (Frank, 1970), a boa constrictor (Constrictor
constrictor) (Frank, 1976), carpet pythons (Morelia spilotes variegata) (McKenzie and
Green, 1976), a northern water snake (Nerodia sipedon) (Karstad, 1961). In this chapter,
pathologic and microbiologic findings on snakes from the affected population are
Materials and Methods
Between September 1997 and March 1998, a survey of pigmy rattlesnakes at
Lake Woodruff National Wildlife Refuge (Deleon Springs, FL; 290 07' N, 810 22' 30"W)
revealed three pigmy rattlesnakes with severe eye, head, mouth, and multifocal skin
lesions (Figure 1A and Figure 1B). The snakes were transported to the University of
Florida where they were euthanitized with a concentrated barbiturate solution and
necropsied. The heads were removed and decalcified using a formic acid sodium citrate
decalcification solution (Luna, 1968). Samples of all major organ systems were collected
and placed in neutral buffered 10% formalin (NBF). For microbial isolation attempts,
samples of lesions were homogenized and streaked onto blood agar for bacterial isolation
and incubated at 360C. Samples were pressed into Sabouraud dextrose agar (SAB) and
mycobiotic agar for fungal isolation and incubated at 230C for 30 days.
Figure 1 Ventrodorsal (A) and lateral (B) views of a pigmy rattlesnake with
severe facial skin disease. The spectacle is cloudy and bulging beyond normal
limits. There are diffuse areas of epidermal necrosis with subcutaneous swelling
that distorts the appearance of the head.
After the initial snakes were found, weekly inspections of the field site were
conducted from September 1997 until November 1999. During these surveys, a ribbon
snake, Thamnophis sirtalis sirtalis, and a garter snake, Thamnophis sauritis sauritis, with
lesions similar to those seen in the pigmy rattlesnakes were found at the same study site.
Both of these snakes were euthanitized and necropsied. Samples were taken for histology
and fungal culture as described above.
Figure 2 Focal epidermal necrosis and subcutaneous masses in the
skin of a pigmy rattlesnake, Sistrurus miliarius barbouri.
During weekly surveys from January 1998 through December 1999, 22 total pigmy
rattlesnakes were seen at the study site with multifocal minimal to moderate necrotizing
skin lesions overlying subcutaneous masses (Figure 2). Some of these snakes were
captured multiple times over the course of their infection (and after their recovery) while
others were observed only once. Snakes were manually restrained using heavy leather
welding gloves and a ring block of 2% lidocaine (Butler Company, Columbus, Ohio,
USA) was used for local anesthesia. Field biopsies were obtained from six pigmy
rattlesnakes with subcutaneous masses only. Biopsies were taken aseptically by cleansing
the scales with Nolvasan (Fort Dodge, Fort Dodge, Iowa, USA) and immediately
removing the affected scales and subdermal masses with a sterile number-15 scalpel
blade. The incision site was then cleansed with an organic iodine solution (Betadine, Fort
Dodge, Fort Dodge, Iowa, USA) and the snakes were released. The tissues excised from
the snakes were divided in half and placed in either NBF or sterile water. Samples placed
in NBF were processed for histology. For isolation of fungi, biopsies in sterile water were
placed on SAB agar or mycobiotic agar and incubated at 230C.
All tissues in NBF were routinely processed, embedded in paraffin, and sectioned
at 5[im. Sections of each lesion were stained with hematoxylin and eosin, Periodic Acid
Schiff (PAS) stain, or Gomori's methenamine silver (GMS) stain and were evaluated by
Cultures for fungal isolation were observed for fungal growth over a 30-day
period. Fungi forming colonies on the plates were separated into pure culture on
additional SAB plates. Samples of pure fungal cultures were placed on malt extract agar
to encourage the production of reproductive structures. All fungi growing on plates were
identified by morphological characteristics and colony presentation using several
previously published keys (Fisher and Cook, 1998; Ellis, 1972; Ellis, 1976). Samples of
the mature cultures were placed on a slide in a drop of lactophenol cotton blue to help
delineate the morphological features of the fungi (Rippon, 1988). Measurements of fungi
in tissue were made with an optical micrometer.
Since a database was available that contained records of all captures since the
beginning of the ecological study, it was possible to conduct a retrospective evaluation to
determine the presence of lesions in previously captured snakes. Fields contained in the
database included mass, capture location, length, gravidity, gender, presence or absence
of prey, and any special comments necessary to describe the individual. In this database,
cutaneous masses were referred to as "lumps," "bumps," or "tumors" and snakes with
severe oro-facial lesions were described as having "mouth rot." Mild to moderate
multifocal skin lesions were often not recorded in the database. Querying the database
for these terms using the database software (Microsoft Access 97 for Microsoft Windows
platform) revealed how many snakes with each type of lesion had been seen during the
years of the study.
Between February 1992 and November 1999, a total of 10,727 dusky pigmy
rattlesnake captures and recaptures were made at the field site. This number represents
approximately 600 individual snakes (May et al., 1996). Since the initiation of the
ecological study, sixteen snakes have been seen with severe head and oral cavity lesions
(Table 2). Of the sixteen snakes with severe head and oral cavity lesions, six had been
previously captured and PIT-tagged during the course of the study.
During the same period, 48 individual snakes were found with small (3-5mm),
raised, firm, mild to moderate multifocal skin lesions scattered over the body surface.
Twenty-three (23) males and thirty-one (31) females were affected and ages of the snakes
ranged from less than one year old to greater than six years of age. Of the 45 snakes with
multifocal granulomatous lesions, 19 were seen with lesions for the first time during
weekly surveys for this study between January 1998 and November 1999.
Table 2 Incidence of disease by year (% affected)
6/912 6/800 10/1047 20/670 3/533 10/829 30/474 1/285
(0.66) (0.75) (0.96) (2.99) (0.56) (1.21) (6.32) (0.35)
By light microscopy, the severe facial and orbital lesions seen in the pigmy
rattlesnakes and several garter and ribbon snakes also found at the study site had similar
features. Affected skin, spectacles, and mucosa lining the oral cavity were diffusely
necrotic with either diffuse infiltrates of mixed inflammatory cells including heterophils,
small mononuclear cells, and macrophages (i.e. immature granulomas) or more
organized, mature granulomas. The mature granulomas observed in both the severe and
multifocal infections had a necrotic, deeply eosinophilic center with H&E staining
(Figure 3A). Using PAS and GMS stains, branching septate hyphae were seen (Figure
3B). Hyphae of several different widths (1.1 tm to 5.5[tm) and morphologies were
observed in tissues, suggesting that there were multiple fungal agents associated with the
granulomatous response in this population of snakes. Some hyphae in tissue branched
often (every 3-6[tm) and others had much longer regions between branches (20-30[tm).
Fungal hyphae were seen in all granulomas, both immature and mature. Evaluation of all
internal tissues did not reveal any fungal granulomas. The only lesion seen was a sperm
granuloma in the kidney of one pigmy rattlesnake.
Figure 3 -
A. Photomicrograph of the head of a pigmy rattlesnake showing necrotic epidermis
(E), subcutaneous granulomas (arrows), tooth (T), and oral cavity (0). H&E stain.
B. At a higher magnification, numerous hyphae can be seen in an area of epidermal
necrosis. PAS stain. 400x. Bar=10[m.
Biopsies of mild to moderate skin lesions showed epidermal hyperplasia, often
with ulceration, subtended by an edematous dermis and subdermis. In skin samples from
one affected pigmy rattlesnake, there was a severe epidermitis with focal to diffuse
coagulation necrosis of the epidermis and dermis. In the dermis there were multiple
mature granulomas with deeply eosinophilic centers clustered together (Figure 4).
Figure 4 Photomicrograph of a subcutaneous granuloma in a pigmy
rattlesnake with multifocal skin lesions. Hyphae can be seen within the
center of the granuloma. GMS stain. 400x.
Over the course of the study, biopsy samples were collected from nine snakes
with mild to moderate multifocal skin lesions. Fungi were isolated from five of the
samples. Based upon the morphology of conidia, spores, and other sexual structures, the
following fungi were identified on malt extract agar plates: from severe orofacial lesions,
Sporothrix schenkii (two snakes), a Paecilomyces sp. (one snake), Pestalotiapezizoides
(one snake) and Geotrichum candidum (=Galactomyces geotrichum) (one snake) were
isolated. The first three fungi were isolated from the initial cultures from the severely
affected snakes. Galactomyces geotrichum was also isolated from cultures of two
biopsies of granulomatous lesions from different snakes.
The samples taken from the gloves used to handle snakes while measurements
were made resulted in the isolation of an unidentified actinomycete and two species of
fungi: Pestalotia pezizoides and Cladosporium sphaerospermum.
In addition to the fungi, the following bacteria were isolated from the initial
severe oro-facial lesions of two snakes: a Xanthomonas sp., a Klebsiella-Enterobacter
sp., a Corynebacterium sp., and Bacillus spp.
The study site, Lake Woodruff National Wildlife Refuge, is within the floodplain
of the Saint Johns River in Volusia County, Florida. Habitats in the refuge include sandy
uplands, seasonally damp oak hammocks, pine flatlands, and areas of tall marsh grass. A
diverse reptile population is present in the refuge. During this study, we observed seven
species of turtles, one species of crocodilian, five species of lizards, and seventeen
species of snakes. Of these, pigmy rattlesnakes were the most plentiful reptile
encountered at the site. The most recent estimates of the size and density of the
population, published in 1996, indicated that there were approximately 600 individuals in
the research site, providing a density of greater than fifty pigmy rattlesnakes per hectare
(May et al., 1996). Density and population size fluctuate yearly, partially because the
number of juveniles born each year varies. One study published concerning the
reproduction strategy in the study population found that over a two-year period,
approximately 68% of the adult females were fertilized, with some snakes reproducing in
both years (Farrell et al., 1995). Each gravid female produces an average of
approximately six offspring. Neonate pigmy rattlesnakes typically have an average mass
of 4.79 grams (Farrell et al., 1995). The mean weight of adult pigmy rattlesnakes at the
site is approximately 47.5g, with a maximum weight in the population of 182.0 grams.
In the spring of 1998, surveys at the study site revealed pigmy rattlesnakes with
severe oral and integumentary lesions. Twenty-two of the eighty-five snakes (23%)
captured in the first three months of 1998 represented new cases of either mild to
moderate granulomatous lesions, crusted scutes, or a severe necrotizing fungal infection
involving the head. In the previous quarter, the winter months of 1997, five new cases
were observed in 92 snakes (5.4%). In a review of field records, similar appearing
lesions were recognized in pigmy rattlesnakes in this population sampled during previous
years. However, the number and frequency of the lesions was highest during the 1997-
1998 epidemic. Prior to the winter of 1997, 50 total cases had been documented. During
the sixth month period between October 1997 and March 1998, 27 new cases were
documented. Before this report, no previous histopathologic evaluation was undertaken
to determine the nature of these lesions.
In field studies on this population after the severe oral and integumentary lesions
were recognized, snakes with focal to multifocal less severe integumentary lesions also
were observed. In a review of field records, previous reports of similar lesions were
found. Calculated incidences based on the number of new cases and the numbers of
individual snakes captured per year (Table 2) indicate a high degree of variability in the
incidence of the disease in the population. The number of snakes captured each year
varied with a maximum of 1047 in 1994 and a minimum of 285 in 1999.
Lesions were not limited to pigmy rattlesnakes. Similar severe gross lesions were
observed in a ribbon snake and a garter snake at the site in the fall of 1997. Prior to 1997,
records indicated that only pigmy rattlesnakes were observed with these lesions. Similar
lesions were not recognized in any other reptiles at Lake Woodruff.
The severe facial and orbital lesions distorted the appearance of affected snakes.
Spectacles of severely affected snakes were edematous, white or clouded, and abnormally
bulged from the margins of the orbit. The oral cavity and surrounding tissues were
similarly edematous, thickened, and necrotic. The mild to moderate focal to multifocal
integumentary lesions were easily overlooked and only slightly elevated the overlying
epidermis, often resulting in a superficial necrosis. Histologic evaluation indicated that
the more severe lesions consisted of granulomatous inflammation intermixed with areas
of cellulitis and necrosis. The overlying mucosa and epidermis were often necrotic. The
mild to moderate integumentary lesions consisted of mature granulomas containing
eosinophilic centers when stained with H&E.
In reptiles, granulomatous inflammation can be caused by a wide variety of
pathogens including bacteria, fungi, and parasites. While several bacteria were isolated
from lesions in snakes in this study and were identified in tissue section using special
stains, in GMS and PAS stained tissue sections, fungal hyphae were consistently seen
within the center of organized granulomas, in areas of less organized granulomatous
inflammation, and also within necrotic tissue on the body surface. Similar appearing
integumentary lesions have been seen in captive snakes with fungal epidermitis and
dermatitis (Williams et al., 1979; Jacobson, 1984). One veterinarian has seen similar
fungal associated integumentary in other wild snakes in the southeast United States
including a corn snake (Elaphe guttata guttata), water snakes (Nerodia spp.), garter
snakes (Thamnophis spp.), and eastern indigo snakes (Drymarchon corais) (Jacobson,
pers comm.). Most of these lesions probably go unreported or unrecognized by
investigators working on snakes in the field.
Hyphae with several different morphologies were observed in the lesions. Fungi
seen in the tissues were irregularly branching, septate, hyaline hyphae ranging from 1 ltm
to 5[tm in width. It is often difficult to determine the identity of fungi in tissue without
special techniques such as immunoflourescent antibody assays, immunohistochemistry,
or molecular (i.e. polymerase chain reaction) assays (Fisher and Cook, 1998). In some
cases, these can be used to identify fungi to genus and species (Fisher and Cook, 1998;
Sandhu et al., 1995; Makimura et al., 1994). Identification via morphological
characteristics of cultures grown out on media is still considered the gold standard. Thus,
in this report, direct examination of paraffin embedded tissue sections by light
microscopy was not useful in identifying fungi to a generic and specific level.
Based on hyphae and spore characteristics (Kwon-Chung and Bennett, 1992;
Fisher and Cook, 1998), the following four species of fungi were isolated from snakes
with severe orofacial lesions: Sporothrix schenkii, Pestalotiapezizoides, Geotrichum
candidum (=Galactomyces geotrichum), and a Paecilomyces sp. Sporothrix schenkii is a
well-known pathogen. It has been reported to cause subcutaneous lesions in primates
(Costa et al., 1994; Vieira-Dias et al., 1997; Vismer and Hull, 1997; Conias and Wilson,
1998; Tomimori-Yamashita et al., 1998; Hajjeh et al., 1997; Kauffman, 1999; Werner
and Werner, 1994), ungulates (Irizarry-Rovira et al., 2000; Greydanus-van der Putten et
al., 1994), felids (Costa et al., 1994; Davies and Troy, 1996; Nakamura et al., 1996;
Marques et al., 1993; Reed et al., 1993), and armadillos (Wenker et al., 1998) that are
similar to those that we have observed in pigmy rattlesnakes. Previous isolation of S.
schenkii from reptiles has been limited to a group of mariculture-reared green sea turtles
(Chelonia mydas) with mixed mycotic pneumonia (Jacobson et al., 1979). A
Paecilomyces sp. was also isolated from the lungs of the same sea turtles. Only two
additional reports could be found indicating Paecilomyces as a pathogenic fungus in
reptiles. Infections in a captive Aldabra tortoise (Geochelone gigantea) that died with
macroscopic, firm yellow nodular lesions distributed across the oral surface, gastric
mucosa, and throughout the liver (Heard et al., 1986) and a systemic infection of a
captive crocodile (Crocodylusporosus) (Maslen et al., 1988) showed granulomatous
lesions of fungal origin in the liver, left lung, and spleen. Pestalotiapezizoides has never
been implicated as a pathogenic fungus, except in some plant species, and was probably a
contaminant in the tissue. However, it is possible that the organism was acting as a
falcultative pathogen because of the advanced necrotic condition of the lesions from
which it was isolated. Geotrichum candidum (Galactomyces geotrichum) has been
previously reported as a pathogen in a group of captive carpet pythons (Morelia spilotes
variegata), (McKenzie and Green, 1976) a northern water snake (Nerodia sipedon)
(Karstad, 1961) and most recently an unspecified garter snake (Thamnophis sp.)
(Vissiennon et al., 1999).
Based upon our findings, more than one fungus was probably involved. It is
possible, since we do not know the identities of all of the fungi seen in paraffin sections,
that other pathogenic or opportunistic fungi may have been present in the lesions but not
isolated in culture. It is also possible that some of the fungi that were isolated were
surface contaminants and not actually in the tissues. Transmission studies needed to
demonstrate a causal relationship were beyond the scope of this report.
The pathogenesis of these lesions, both the severe orofacial lesions and mild to
moderate integumentary lesions, is unclear. Penetrating wounds in the integument may
have resulted in infection and granuloma formation in the dermis in those snakes with
mild to moderate integumentary lesions. Though no ticks or other external parasites of
any kind were observed on pigmy rattlesnakes at the time of capture, this is also a
potential route of infection. This type of infection could explain the multifocal
distribution of the subcutaneous granulomas. Ticks do exist in the habitat, but their role,
if any, as a parasite of pigmy rattlesnakes is unknown. Another explanation could be the
association with subcutaneous parasites. Fungal granulomas associated with
subcutaneous pentastomid parasites in indigo snakes in Florida have been seen by a
specialist on diseases of reptiles, Dr. Elliott Jacobson (pers comm.). No pentastomes
were seen in any of the snakes in this study, but this does not exclude the possibility that
they may be in the population. Full necropsies on several snakes with severe lesions did
not indicate systemic disease. While this suggests that lesions commenced locally and
spread to surrounding tissues, it is still possible that the pathogen(s) spread via the
circulatory system, causing inflammation at the affected sites. Fungal hyphae may have
spread locally from granulomas to surrounding areas via the circulatory system. In
humans, the spread of S. schenkii and other systemic fungi has been shown to occur
through the circulatory system and lymphatics (Rippon, 1988).
Another possibility is showering of hyphae or spores from visceral structures.
Fungi have been previously cultured from kidney of apparently healthy snakes without
specific lesions seen in the kidney (Jacobson, pers comm.). It appears that reptiles may
harbor organisms in visceral structures that can cause disease when conditions allow the
organism to proliferate, stimulating an inflammatory response. A similar situation has
been described in amphibians where culture of the kidney of free ranging anurans in
Brazil indicated presence of multiple species of fungi (Mok et al., 1982).
An anthropogenic basis for the epidemic also was considered. One of the initial
concerns in this study was that fungi were transferred between snakes and entered tissues
due to handling and sampling techniques being used in the field. Due to their small size,
all pigmy rattlesnakes were manually restrained using a pair of leather welder's gloves.
The pair of gloves being used at the time of the 1997/1998 outbreak had been in use for a
period of approximately two years. Two fungi were identified in pure culture from
samples taken from the gloves: Cladosporium sphaerospermum and Pestalotia
pezizoides. No reports were found in the literature to indicate that C. sphaerospermum is
a primary pathogen of vertebrates. However, it was isolated from lesions in a lesser
octopus, Eledone cirrhosa, and transmission studies were conducted that confirmed the
pathogenic nature of the fungus in a marine environment (Polglase et al., 1984). It was
not isolated from any lesions in this study and probably did not play a role in the
1997/1998 outbreak. Though P. pezizoides, was isolated from both the severe
necrotizing lesions on one of the pigmy rattlesnakes and the gloves used for manual
restraint, it is unlikely that the fungus was a primary pathogen, since P. pezizoides has
never been reported as a pathogen of vertebrates. Thus, there is no evidence to suggest
that handling techniques were involved in the outbreak, other than the possible effects of
stress and abrasion of the skin, which could make infection more likely. Field records
indicated that twelve of the sixteen snakes with the severe oral and facial lesions
observed during the 1997-1998 epidemic had never been captured previously or handled
by the study team. Still, field equipment can represent important mechanisms for transfer
of pathogens between animals and should be disinfected as needed. This may necessitate
having more than one set of equipment available when handling snakes, especially if
snakes with lesions are encountered. A protocol for handling snakes in the field to reduce
the spread of transmission of pathogens between animals is needed and is currently being
developed by us.
OUTBREAK SEASONALITY AND CYCLICITY
This study was conducted to search for abiotic factors that could be related to
outbreaks of fungal dermatitis and stomatitis in a wild population of pigmy rattlesnakes
in Florida. Tests for seasonal and cyclical patterns in the incidence of the disease were
also conducted to better explain the occurrence and cyclicity of the outbreaks.
Temporal fluctuations have been described in a wide variety of biological
systems. Yearly changes in the sizes of mammal populations are the best studied, and
many have been shown to vary in a predictable way based on population and
environmental conditions (Oli and Dobson, 1999; Kendall et al., 1998; Seldal et al.,
1994). Within populations of wild and captive animals, the prevalence of diseases and
parasites has also been demonstrated to fluctuate temporally. The incidence of
salmonellosis in horses presented to a veterinary hospital in California, for example, was
shown to be strongly seasonal, occurring most frequently in June through September
(Carter et al., 1986). A study of rabies in Chile concluded that outbreaks follow a
seasonal trend, with cases increasing during November and December, and also exhibit a
cyclic behavior, repeating regularly every five years (Ernst and Fabrega, 1989).
Occurrence of human hemorrhagic septicemia has been shown to have a strong
seasonality as well, with outbreaks occurring regularly during the rainy season in several
areas of India (Dutta et al., 1990). A similar study on foot-and-mouth disease in India
also found a strong pattern of seasonality, again correlating occurrence of disease with
the rainy season (Sharma et al., 1991). Retrospective studies of data from historic
outbreaks of diseases in human populations have shown cyclic patterns. Analysis of the
widespread smallpox epidemics in Britain in the seventeenth and eighteenth centuries, for
example, show two distinctly different repeating patterns of occurrence based on the
number of people in an outbreak area (Duncan et al., 1994). Studies of epidemics of
whooping cough in London from 1701-1812 (Duncan et al., 1996a) and smallpox in
London from 1647-1893 (Duncan et al., 1996b) have determined the existence of similar
cyclical patterns of disease.
Outbreaks that occur seasonally during periods of increased rainfall (Sharma et
al., 1991; Dutta et al., 1990) provide support for the idea that disease incidence
fluctuations are sometimes due to changes in environmental conditions. Rainfall,
temperature, food availability, or any other stress factor that makes a population of
animals more susceptible to pathogens could all play roles of varying importance.
Studying these factors could conceivably help in the development of a model to predict
future outbreaks. Since these factors may be correlated to or dependent upon one another
(e.g. rainfall and availability of frogs as prey items for pigmy rattlesnakes), establishing a
linear relationship of one or more environmental variables and the incidence of a disease
can be difficult.
Materials and Methods
Cases were obtained by searching a database from an eight-year-long
mark/recapture study involving a population of pigmy rattlesnakes, Sistrurus miliarius
barbouri, in Florida. The database, obtained from researchers at Stetson University in
Deland, Florida, contains weekly entries that describe the number of snakes captured
during each sample date. These entries also contain notes on the condition of snakes.
During the study period, a subset of the study population was found with what have been
determined to be subdermal granulomas formed as a response to fungi in the tissue.
Snakes that were found with these lesions during the eight-year study were noted in the
database as having "bumps" or "lumps" on their skin. Snakes with a more severe fungal
infection involving the head were similarly designated in the database as having "head
rot" or "mouth rot." A search of the database for snakes described using these terms was
used to calculate the number of affected animals in the population during any given
period. Results returned by the queries of the database were carefully screened to be
certain that they were actual accounts of cases and not records describing snakes without
lesions of interest.
Monthly and quarterly incidences of disease were calculated for the study period
by querying the database for new cases and total snakes captured in a given month. The
number of new cases was subsequently divided by the total number of snakes captured in
a month, and the resulting figure was multiplied by 100 to yield an incidence per 100
snakes captured. The same process was repeated to determine quarterly incidences of
disease for the study period.
During weekly surveys at the study site, a water level reading was recorded for
the habitat. The water level value represents the depth of water covering the ground in
the habitat at a given point. A negative value reflects the depth at which wet soil can be
found. Readings are taken with the help of a permanently placed stake marked at 1cm
intervals. Using this method, the lowest measurable water depth is -10cm. Weekly
measurements were collected and averaged appropriately to yield mean monthly and
quarterly water level measurements for the habitat.
For the purpose of this study, this water level reading is more representative of the
conditions in the habitat than rainfall. The habitat for the study is part of a floodplain for
the Saint Johns River, which runs along the East coast of Florida. Because it is attached
to the river, the amount of water present in the habitat is dependent upon the water level
in the river. Since the river flows from South to North, emptying into the Atlantic Ocean
in Jacksonville, Florida, increased rain in South Florida or increased wind in Jacksonville
could theoretically have a large impact on water level in the habitat. This necessitates a
"bigger picture" measure of water present in the habitat than considering rainfall alone.
Thus, water level was chosen.
Daily minimum and maximum temperature measurements were obtained from a
local weather station for the duration of the study. Minimum and maximum temperatures
for each day were averaged to yield an average daily temperature.
The null hypotheses of this study were as follows:
1) There is no difference between annual incidence of disease in the population
2) There is no linear relationship between abiotic factors and disease
3) There is no increased "seasonal index" for any given month
4) There is no cyclical component of the disease outbreak
Incidence rates were calculated for each year by dividing the number of new cases
identified during the year by the total number of snakes captured. The X2 test (Rao,
1998) was used to compare annual incidence rates from 1992 to 1999.
Simple linear regressions and multiple linear regressions were performed with
Microsoft Excel and verified by SAS. In the simple linear regressions, incidence in a
given quarter was compared to either water level in the same quarter, mean temperature
for the quarter, or a combined factor representing the multiplicative effects of water level
and temperature. Multiple linear regressions were conducted using all three factors in the
Incidence rates were calculated for each month by dividing the number of new
cases identified during the month by the total number of snakes captured. The resulting
number was multiplied by 100 and expressed as a percentage. The time series model was
used to break the data into four components: trend, seasonal variation, cyclical variation,
and random variation as previously described by Carter et al (1986).
Previously calculated incidence rates were examined for seasonal patterns. This
was done by calculating a "seasonal index" for each calendar month. These indexes were
derived by dividing the previously calculated monthly incidence by the 12-month moving
average (12-month period centered around the month in question). The resulting value is
then added for each calendar month (e.g., all December values are added to get a
cumulative total for December). Dividing the cumulative index by the average index
value (obtained by adding all indexes together and dividing by 12) will allow the
expression of a monthly index value as a percentage of the mean. Months having index
values higher than 100 are months in which there is a seasonal increase in the incidence
In order to determine the long-term trend in incidence rates, a regression analysis
was performed using time in months (numbered consecutively starting at 1) as the
independent variable (x) and deseasonalized monthly incidence as the independent
variable. Deseasonalized data were calculated by dividing the monthly incidence rates by
the cumulative seasonal index for the corresponding month.
Data used to look for cyclical patterns in incidence were calculated by dividing
the deseasonalized incidence for each month by the corresponding trend value for the
month. Trend values are continuous, beginning with the y-intercept (m) calculated in the
above regression analysis and increasing in steps by the slope value (b) for the same
number of points as the deseasonalized data.
Figure 5 shows yearly incidence rates for all years in the study (1992-1999). The
year containing the majority of the outbreak addressed in Chapter 2 (1998) has a much
higher incidence than any other year in the study period. Groups developed based on
results of the X2 tests are shown in Table 3. The groups consist of years whose
incidences are not significantly different from one another (a>0.05). Some years are in
more than one group. The only year whose incidence is statistically different from all
others is 1998, the year of the outbreak that inspired this study.
1992 1993 1994 1995 1996 1997 1998 1999
1 Total Inc. 0.658 0.75 0.955 2.985 0.563 1.206 6.329 0.351
Figure 5 Yearly incidence of disease for all study years
Table 3 Groups by yearly incidence
1992 1993 1995 1998
1993 1994 1997
The results of regression analyses conducted to test for a linear relationship
between environmental conditions (habitat water level and temperature) and the
incidence of disease in the population are shown in Table 4.
Table 4 Results of regression analyses
Factor Intercept Coefficient P-value R2
Water level (W) 2.53 0.19 a=0.09 0.10
Temperature (T) 15.19 0.19 a=0.09 0.10
The raw monthly incidence rates, 12-month centered moving average, and the
trend are depicted in Figure 6.
Seasonal indices, calculated as previously described, are shown graphically in
Figure 7, below. Values greater than 100 indicate a month in which the incidence of
disease is consistently elevated above the mean incidence across the study period.
Indexes for December, January, March, and April are all greater than 100. All other
months are below 100.
The cyclical portion of this time series is found in Figure 8. Cycles seen
here represent patterns in the data not accounted for by the trend or seasonal variation.
Figure 6 -Monthly incidence, 12-month centered moving average, and trend of disease
4 4 1m-R 1 17AI1
S i en en 'I 'I n n k vO V N- N 0000 ON ON
N ON O ON O ON O ON O ON O ON O ON O O
I I I I I I I I I I I I I I I I
-1 lml I I
Figure 8 Cyclical component of time series
Figure 7 Seasonal Component of Disease Outbreaks
The regression analyses in Table 3 indicate that there is no statistically significant
linear relationship between the environmental factors of interest and the incidence of
disease. It is possible, however, that the relationships are not statistically significant
because the incidence of disease is profoundly affected by variables that were not
considered in this study. Some possible variables that could contribute to the
susceptibility of snakes to fungal disease are chemical pollutants, immune system
disease, prey abundance, or an unknown periodic physiological change in the snakes.
Table 4 demonstrates that incidence rates are different between the years
examined by the study. This indicates that the factors contributing to the outbreak,
whatever they may be, are not the same from year to year.
The seasonal effect noted in Figure 7 may be the result of changes in
environmental conditions in the habitat (i.e. water level, temperature, etc.) that occur in
the winter months, which encourage the growth of fungi. Likewise, it is also possible
that a repressive element exists in the summer months, helping to keep the occurrence of
Figure 8 indicates a clear cycle of disease after seasonal effects are removed. The
cycle repeats yearly, but does not reach the "outbreak" level in 1996 (i.e. does not pass
the "100%" mark). The peaks indicate that outbreaks occurred in six out of the seven
high cycles in the study. Even in years that never exceeded the outbreak classification
value of 100, the highest points in the cycles were seen in the same months as in years
that clearly exhibited outbreaks.
HANDLING AND SAMPLING PROTOCOL FOR HERPETOLOGICAL RESEARCH
Introduction and Background
Infectious diseases affecting reptiles are caused by many different pathogens.
Bacterial, viral, fungal, and parasitic infections have been documented in both wild and
captive reptiles (McLaughlin et al., 2000; Homer et al., 1998; Lackovich et al., 1999;
Lamirande et al., 1999; Graczyk and Cranfield, 2000; Jacobson et al., 2000; Mader,
1996). All genera of reptiles are susceptible to infection by pathogenic organisms.
Recently, several diseases have surfaced as causes of illness and mortality in wild
reptiles. Examples include fibropapillomatosis of marine turtles (Jacobson et al., 1989),
mycoplasma of tortoises (Brown et al., 1999), and, as reported in this thesis, fungal
infections of pigmy rattlesnakes. Outbreaks of disease have been studied in many
populations of captive reptiles, but few studies have been conducted with wild
populations (Jacobson et al., 2000). This is not to say that wild populations are less
susceptible to disease than captive animals. One reason for a relative lack of information
on diseases of wild populations of reptiles (compared to captive reptiles) is the simple
fact that they are monitored for disease much more infrequently than captive reptiles.
Most free-ranging populations of wild reptiles are not regularly studied by herpetologists.
In populations where studies are conducted, it is possible not to encounter affected
animals, not to recognize disease, or simply not to document cases. In captive
populations, however, caretakers and pet owners have regular and repeated opportunities
to inspect individual reptiles for signs of illness or disease and seek veterinary treatment.
This difference may account for the higher case report rate for captive reptiles than wild
reptiles. Also, when a reptile dies in the wild it is seldom found. The reason that disease
is reported more often for wild chelonians than other groups of reptiles is because the
shell of a dead chelonian will not decompose and will persist for a long period after
In captive reptiles, fungal and bacterial infections of the skin and integument
often result from predisposing factors like unsanitary living space or improper regulation
of environmental conditions (i.e. too wet, too cold, etc.). One recent study of the causes
of death of captive mammals, birds, and reptiles found that improper husbandry, such as
a poor diet, improper environmental conditions, etc., was responsible for a higher
percentage of animal deaths than infectious disease alone (Ferreira et al., 1999). In
addition, the condition of the reptile's immune system, like any other animal, is important
in the development of disease. A strong response can help keep the infection subclinical.
Conversely, a repressed immune system, whatever the cause, can predispose a reptile (or
any other animal) to infections that are less likely to develop in the presence of a normal
immune response. In the relatively close quarters of captivity, healthy reptiles may have
a higher rate of exposure to diseased individuals due to increased animal density. This
high rate of contact increases a reptile's chances of encountering and contracting
whatever pathogens may be in the collection. This type of horizontal transmission has
been documented in populations of captive animals, such as farm-reared broiler chicks
(Shanker et al., 1990) and sheep (Li et al., 2000).
Wild reptiles are made more or less vulnerable to infection by the same factors
that affect captive reptiles. Free-ranging reptiles, however, have a greater ability than
captives do to change their surroundings. Their chances of developing disease are
increased by the same variables, but wild reptiles can relocate to conditions that are more
satisfactory with greater ease. In addition, healthy free-ranging reptiles may have less
exposure to diseased individuals, reducing the spread of disease. This is also highly
density dependent and populations that are steadily increasing in density may be
increasingly at risk. This could help explain the fact that large-scale outbreaks of disease
are rarely documented in free-ranging populations. The more obvious side affect of
disease in a population is a reduction of population size.
When herpetologists study populations of free-ranging reptiles, the possibility
exists of that whatever pathogens may be in the population can be transferred from
animal to animal via contaminated equipment. Thus, there is a chance of unwittingly
facilitating the spread of an infectious agent. Pathogens can be spread through a
population simply by touching an uninfected reptile after handling a diseased animal with
hands or capture and restraint tools. Unfortunately, it is often difficult to determine the
status of a free-ranging reptile before it is captured. This fact necessitates the
development of a standardized set of common, safe, and widely accepted handling and
sampling protocols so that researchers can avoid spreading potentially pathogenic
organisms from infected to healthy reptiles.
Preventing the spread of diseases between animals is the goal of preventative
medicine programs in both veterinary medicine and the human medical profession.
Practices designed to reduce the chances of spreading infections between patients have
become compulsory in hospitals around the world. Sets of guidelines designed to
encourage practitioners to thoroughly cleanse equipment and avoid spreading diseases
have been widely distributed. The recommended practices include hand washing,
wearing latex gloves, avoiding mixing of healthy and ill patients, proper methods of
disinfection, and sterilization of appropriate equipment (Anonymous, 2000).
Proper maintenance of instruments used to examine or manipulate reptile patients
is also important. Much of the equipment used in modem human and veterinary hospitals
is disposable. This equipment, such as syringes, are used once and then disposed of.
When instruments designed for multiple uses are used for invasive procedures, they are
routinely disinfected or sterilized to prevent the spread of infectious agents. Chemicals
used frequently to disinfect equipment between uses include glutaraldehyde, hydrogen
peroxide, peracetic acid, sodium hypochlorite, alcohol, iodophors, phenolics, and
quaternary ammonium compounds (Rutala and Weber, 1999). However, gas or heat
sterilization are the methods of choice.
In the spring and summer of 1999, a survey was conducted through an interest
site at the University of Florida College of Veterinary Medicine to determine the
handling and sampling protocols of herpetologists from around the United States and the
world (Appendix A). Over 40 invitations to complete the survey were distributed via
Email and 14 herpetologists from the United States, Europe, and Australia responded.
All 14 responders (100%) reported conducting invasive procedures on free-ranging
animals as part of their research, either in the field or in the lab. Of these 14, only six
(42%) reported having received some kind of formal training on surgical and biomedical
sampling procedures. Seven responders (50%) stated that they clean and sterilize their
non-surgical equipment (tongs, restraint tubes, etc.) at least periodically, if not between
animals. Complete survey results are presented in Table 5.
Table 5 Summary of survey responses
iQ sioI. IIR
Invasive procedures used
Assess gender with a probe 13/14 (93%)
Implant radio transmitters 9/14 (64%)
Insert ID microchips (PIT-tags) 9/14 (64%)
Sterile/aseptic techniques training 6/14 (43%)
Use anesthesia for surgery 10/14 (71%)
Sterilize non-surgical tools periodically 9/14 (64%)
Sterilize non-surg. tools after each use 0/14 (0%)
Sterilize surgical tools periodically 14/14 (100%)
Sterilize surgical tools after each use 6/14 (43%)
Observed health problems in study pop. 8/14 (57%)
Protocol for Safe Handling and Sampling of Reptiles
For mark-recapture studies and surveys, it is important to try to avoid contributing
the spread of infectious agents through the study population. Following a basic protocol
for safe handling and sampling can help reduce the chances of spreading pathogens
between study animals. First, it is important to regularly clean and disinfect capture and
restraint equipment to the highest possible level. Tongs can be washed with a sanitizing
solution like Nolvasan or a diluted bleach solution. Since snake bags and containers are
frequently soiled by specimens, they should be washed in a bleach solution after each
time they are used to hold an animal.
Small instruments, like gender (sexing) probes, can be autoclaved between uses or
trips to the study site. Gender probes are an area of special concern since they are tools
used for an invasive procedure. Carrying several autoclaved probes can allow the gender
of several snakes to be identified, should the need arise, so that no instruments are reused.
Minimally, tools should be cleansed with a glutaraldehyde, bleach, or a Nolvasan
solution and rinsed with sterile water between animals. Pathogen transmission via body
fluids has been documented for several pathogens including the transmission of bacteria
and viruses through human urine, though no references to vertical transmission in reptiles
via cloacal contact could be found in searches of the primary literature (Knutsson and
Kidd-Ljunggren, 2000). There are reports in the literature of horizontal cloaca-to-cloaca
transfer of sexually transmitted diseases in avians (Westneat and Rambo, 2000). Since
this is also a possible risk for reptiles, gender-probing bears further investigation to
determine the risk of transmitting pathogens between animals.
Second, it is important to handle reptiles as gently as possible during capture and
data recording to avoid abrading the skin or scales. It has been shown conclusively that
skin abrasion can predispose reptiles to fungal or bacterial infections (Lillywhite, 1996).
Because of this, every effort should be made to maintain the integrity of the skin.
Complete instructions on handling many types of reptiles in a way that is safe for the
animal and the herpetologist can be found in several books (Jenkins, 1996; Cunningham
and Gili, 1994; Barnard, 1996).
Thirdly, try to handle individuals that have obvious signs of disease with different
tools than healthy animals. These animals frequently exhibit brown or crusted scales or
scutes, open sores, infections of the structures of the head, or subdermal masses. The best
way to accomplish this is to carry two sets of capture and restraint tools, especially those
integral to restraining the infected animal (i.e. tongs). Alternately, a disinfectant solution
may be used in the field to cleanse tools that must be reused for the capture and restraint
of healthy reptiles. If hand capture is used, carrying several sets of disposable latex-type
gloves for use with obviously infected individuals is recommended. It has long been
accepted that washing hands with soap and water helps reduce the likelihood of spreading
pathogens between animals. Hand washing has also been shown to be protective against
the transmission of Salmonella enteritidis, a common cause of salmonellosis, from
reptiles to humans (Friedman et al., 1998). Minimally, three minutes of hand washing is
necessary for proper cleansing.
When reptiles with signs of disease are encountered, a report sheet specially
designed for that species should be completed. This report sheet, often referred to as a
health sheet, can be used to document the location, size, color, shape, and other physical
characteristics of any lesions that may be found on the specimen. The sheet may also be
used to record information such as location, behavior, breeding status, gender, mass,
length, and any distinguishing marks that the subject may have. An example health sheet
for use in documenting diseases in reptile populations can be found in Appendix B.
Many herpetologists who are now performing surgical procedures are doing so
under the direct supervision of a veterinarian. This is advisable, whenever possible, to
ensure the health and safety of the animals. The guidelines provided here are a basic
outline of the principles needed to reduce the risk of disease transmission between
reptiles and the risk of postoperative infection.
Surgeries that are conducted in the field setting should be performed with the
utmost attention to aseptic techniques in order to avoid spreading pathogens or
encouraging post-operative infection. First, cleansing the skin and scales with an
antimicrobial solution prior to starting surgery will help reduce the number of organisms
on the surface of the skin and can help prevent postoperative infections. Commonly used
solutions for surgery on reptile patients include povidone-iodine and chlorhexidine
(Bennett and Mader, 1996).
Second, it is important to use an anesthetic when any type of surgery is conducted
on a reptile. The anesthetic will reduce the amount of pain that the subject feels and,
therefore, help keep the subject from writhing around during the procedure. In the field,
for minimally invasive surgeries, a local anesthetic is usually preferred because of the
ease with which they can be administered and the reduced necessity for post-operative
recovery time and observation. This method is especially useful when biopsying skin or
subcutaneous masses. A lidocaine ring block can be performed by injecting an
appropriate quantity a 2% solution in a circle (ring-block) around the biopsy or incision
site. When surgeries are conducted in a laboratory or operating room, general anesthetics
may be used, per a veterinarian's advice. Common injectable anesthetics used for
reptiles include sodium pentobarbital, methohexital, ketamine, and telazol. Isoflurane is
the preferred gas anesthetic for reptiles (Bennett, 1996). Consultation of a contemporary
text on reptile anesthesia is recommended to help determine which protocols should be
Thirdly, it is imperative that sterile surgical tools be used for each subject as
previously discussed. For transmitter implants or biological sampling, sterile scalpels
should be used for only one surgery and then replaced. For PIT-tag implants, a sterile
insertion needle should be used for each individual. This level of sterility can be
achieved by carrying several sterile surgical packs into the field when transmitter
implants or biological sampling are anticipated.
All surgical wounds, especially when large, should be closed using either surgical
glue or sterile suture material and the animal treated with an appropriate antimicrobial
agent, usually an iodine solution (e.g. Betadine). Commonly used suture materials
include nylon or polypropylene sutures. Skin staples designed for human surgery can
also be used to close wounds in reptiles (Bennett and Mader, 1996). This is a concern for
animals that will be released immediately after surgery, however, since most suture
materials require removal. For field surgeries, therefore, a skin glue product specifically
designed for postoperative wound closure may be more appropriate.
If animals are kept for observation after surgery or are part of a collection, closely
monitor the wound for signs of infection after the surgery is complete. If the subjects are
released after surgery, pay close attention to the surgical wound at the time of any
subsequent recaptures and beware of possible infections. If any infections are noticed,
contact a veterinarian immediately for instructions on treatment.
By applying the techniques described in this paper, researchers can reduce the
chances of causing disease outbreaks by spreading pathogens between individuals and
may lower postoperative infection rates. These simple steps can help insure the health of
reptile populations. The guidelines stated here are basic approaches to handling and
sampling reptiles in the field and in the lab. These simple rules can be applied to many
research protocols to help protect the health of wild and captive reptile populations.
Using the safest techniques possible will help reduce the risk of transmitting potentially
pathogenic organisms between animals, thereby helping keep wild and captive study
HANDLING AND SAMPLING PROTOCOL SURVEY
Thank you very much for taking time out of your busy schedule to help us. This questionnaire was developed
to document the handling and sampling methods of snake researchers worldwide. Within the last year,
individuals in a Florida population of pigmy rattlesnakes, Sistrurus miliarius barbouri, developed severe
lesions of the orbital, facial, and oral regions of the head. Several species of fungi have been isolated from
these lesions and may be the causative agentss.
The investigation into the cause of these lesions leads us to the question: "Is there some way that human
interaction with this population could have induced the appearance of the lesions?" To answer this question,
we must compare what is known of the handling and sampling protocol used in the study of the Florida
population to that of studies in populations across the world. Please take the time now to complete the
questionnaire below. Your input is invaluable to us. If you have any questions or comments about the nature,
structure, or intent of this questionnaire, please feel free to send those concerns via email to
email@example.com. Thank you.
Please enter your name:
Please enter your email address:
1) Which snakes (genus and species) do you study?
2) How long have you been studying each of the above mentioned snakes?
3) Have you published one or more papers about the handling and sampling methods you use?
(If "yes ", please include pertinent references below.)
4) What type(s) of data do you collect when studying your snakes?
Dimensions 0 Mass 0 Gender 0 Geographic location [
Parasites 0 Prey 0 Fecundity O Disease 0
Air temperature O Ground temperature O Body temperature O Feeding status 0
Shed status O Skin abnormalities 0 Other O
(If "other", please elaborate below.)
5) Do any of your methods require perforation of the skin or other invasive procedures?
(If "yes ", please specify which data you are collecting invasively.)
6) Do you use radio telemetry or inject a microchip into any of your research animals?
Yes transmitter No transmitter O
Yes microchip O No microchip 0
7) Have you had any formal training in aseptic surgical techniques and biomedical sampling?
8) Do you use anesthesia in any of your procedures?
(If "yes", please specify which anesthetics you use.)
9) Are your snakes considered venomous?
10) What techniques do you use for capturing and restraining snakes if samples and data are to be collected?
11) How often do you sterilize your data collection tools and precautionary tools?
A) After each use O
B) After several uses 0
C) Weekly (without regard to number of uses) O
D) Monthly (without regard to number of uses)O
E) Irregularly O
F) Rarely O
G) Never O
12) How often do you sterilize any surgical tools you are using?
(Including materials usedfor injections of any kind)
A) After each use O
B) After several uses O
C) Weekly (without regard to number of uses) O
D) Monthly (without regard to number of uses)O
E) Irregularly O
F) Rarely 0
G) Never 0
13) Have you noticed any health problems in any of your research snakes?
(If "yes", please specify which health problems you have noticed)
14) Have you conducted any studies specifically designed to test the safety of your data collection methods or surgical
15) Do you have any questions, comments, or additional information about your research that you would like to share
Please click on the button below when you have completed answering all of the questions.
This page constructed by Joseph L. Cheatwood (cheatwoodufl.edu).
Last updated: 10/14/99
SAMPLE DATA SHEET
Snake Observation Data Sheet
Sex: (circle one) M
Nasal exudate: Y N
Nares crusted: Y N
Lesion/lumps: Y N
Eyes cloudy: Y N
Eyes swollen: Y N
Description of lesions:
Ambient temp. (C):
Photographs Taken?: Y N
Draw any lesions on diagrams below:
Alert & responsive:
Defensive behavior obs.:
Coiled when found:
Moving when found:
Normal elasticity: Y N
Near/in ecdysis: Y N
Abnormal shed: Y N
Lesions/lumps: Y N
Normal muscle tone: Y N
Normal vertebral column: Y N
Description of lumps/lesions:
Draw any lesions on diagrams below:
Blood: Y N Swabs: Y N
Tissue: Y N Feces: Y N
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Joseph Laton Cheatwood was born in Lakeland, Florida, in 1976. He graduated
from Lake Gibson High School (Lakeland, Florida) in 1994. He received a Bachelor of
Science degree from Stetson University (Deland, Florida) in May, 1998. He entered the
graduate degree program at the University of Florida in May, 1998, to pursue a Master of
Science under Dr. Elliott Jacobson at the University of Florida College of Veterinary
Medicine. Upon completion of this work he will continue his studies in a Ph.D. program
at the University of Florida.