BLOOD PARASITES OF TWO OF RAT SNAKE (ELAPHE SPP.) FROM

SOUTHEASTERN NEBRASKA AND NORTHEASTERN KANSAS

A Thesis

Presented to the

Department of Biology

and the

Faculty of the Graduate College

University of Nebraska

in Partial Fulfillment

of the Requirements for the Degree

Master of Arts

University of Nebraska at Omaha

by

Maggie Lynn Lalley

May 2004 THESIS ACCEPTANCE

Acceptance for the faculty of the Graduate College,

University of Nebraska, in partial fulfillment of the

requirements for the degree Master of Arts,

University of Nebraska at Omaha.

Committee

Chairperson ] ✓ Date ,z ABSTRACT

Blood Parasites of Two Species of Rat SnakeElaphe ( spp.) from Southeastern Nebraska and Northeastern Kansas

Maggie Lynn Lalley

University of Nebraska at Omaha, 2004

Advisor: Dr. James Fawcett

Thirty Elaphe obsoleta obsoleta and 14 Elaphe guttata emoryi were collected from Gage County, Nebraska and Atchison, Marshall, and Washington counties in

Kansas between April and October 2000 and 2001. Blood was collected and then examined with light microscopy for presence of parasites. The size of 30 infected red blood cells (RBCs), 30 uninfected RBCs, and 30 parasites was measured from each infected snake. The proportion of infected RBCs per 10,000 uninfected RBCs was calculated for each infected snake. The proportion of white blood cells per 1,000 RBCs was also calculated.

Representatives of the were the only parasites recovered from the blood. Prevalence ofHepatozoon species in E. o. obsoleta was 40.0% (12 infected of

30 total snakes), whereas Hepatozoon species were found in only 7.1% (one infected of

14 total snakes) of E. g. emoryi. Due to the low frequency of infection,E. g. emoryi were removed from all other analyses.Elaphe o. obsoleta with snout-vent lengths larger than

115.0 cm were all (n = 11) infected with Hepatozoon species, while all but one snake

(106.1 cm) smaller than 115.0 cm (n = 19) were not infected. These differences in prevalence may be due to differences in habitats and diets of the snakes. Infected RBCs were found to be significantly larger than uninfected RBCs, possibly due to the presence iv

of parasitic gamonts. Infected snakes had significantly fewer white blood cells than uninfected snakes, but there is not enough information available about snake immune systems to form a plausible explanation. V

ACKNOWLEDGMENTS

I would like to thank the members of my committee, Dr. James Fawcett, Dr,

Richard Stasiak, and Dr. Daniel Sullivan. These men not only helped me complete my master's degree, but they were also instrumental in the completion of my bachelor's

degree. Without their patience, guidance, and support, I would not have applied for graduate school.

I would like to thank a few individuals who were a tremendous help to me during this study. Dan Fogell transported me to and from my field site, helped me wrangle my

snakes out of trees and cliffs, and most importantly kept me out of harm's way. I could not have done this study without him. Ted Leonard and Dena Quigley were also faithful

assistants, both in the field and in the lab. I would like to give special thanks to Dr.

Michael Barger who helped me with my statistical analyses.

I received financial support from the Department of Biology at the University of

Nebraska at Omaha and the University Committee on Research. Snakes were collected under Institutional Animal Care and Use Committee protocol number 00-051-04 and under Kansas Wildlife and Parks permit SC-061-2001.

Last and most importantly, I would like to thank my family. My mother and father have always supported me in whatever challenges I have taken on, and I am eternally grateful to them. And then there's Scott. I truly would not be receiving this degree without him. He is the most patient, honest, and gentle man I have ever known.

Thank you. TABLE OF CONTENTS

PAGE

THESIS ACCEPTANCE ii

ABSTRACT iii

ACKNOWLEDGEMENTS v

TABLE OF CONTENTS vi

LIST OF FIGURES vii

INTRODUCTION 1

MATERIALS AND METHODS 4

Field Techniques 4

Laboratory Techniques 5

Statistics 6

RESULTS 7

DISSCUSSION 13

Parasite Identification 13

Prevalence 16

Host Blood Cells 19

Conclusion 22

LITERATURE CITED 23 vii

LIST OF FIGURES

PAGE

Figure 1. Prevalence (number infected) of Hepatozoon species in Elaphe 8 obsoleta obsoleta and Elaphe guttata emoryi.

Figure 2. Snout-vent length (SVL) of uninfected (A) and infected (□) 10 Elaphe obsoleta obsoleta.

Figure 3. Red blood cell infected with Hepatozoon species gamont. 11

Figure 4. Mean number of white blood cells (WBCs) per 1,000 red blood 12 cells (RBCs) in uninfected (n=12) and infected (n=13) Elaphe obsoleta obsoleta.

Figure 5. Generalized life cycle ofHepatozoon species of snakes. 15 1

INTRODUCTION

A parasite is a symbiont that harms its host, and there is a far greater number of

parasitic organisms than nonparasitic organisms in the world (Roberts and Janovy, 2000).

Parasites are distributed globally and consist of both protozoans and metazoans (Noble

and Noble, 1976). Every species of animal that has been examined has been shown to be

host to at least one species of parasite, and often animals will have on or in their bodies

hundreds or even millions of individual parasites (Noble and Noble, 1976).

All classes of have parasites, including reptiles. Those that live in the

blood of a host are called blood parasites or hemoparasites, and are usually transmitted by

hematophagous invertebrates such as arthropods or leeches. Blood parasites can live

freely in the blood plasma of the host or inside host blood cells, either red blood cells

(RBCs) or white blood cells (WBCs). Some of the common genera of parasitic protozoans found in reptilian blood areFallisia, Hemogregarina, ,

Hepatozoon, , , and Trypanosoma (Roudabush and Coatney,

1937; Thompson and Huff, 1944; Jordan and Friend, 1971; Nickerson and Ayala, 1982;

Lowichik et al., 1988; Siddall and Desser, 1991; Smith and Desser, 1997; Telford, 1998;

Smallridge and Bull, 1999).

There is a great deal of information available documenting the effects of virulent parasites on humans and domestic animals, but very little information is known about parasites in the wild and their effects on natural hosts (Smallridge and Bull, 2000).

Persistent and endemic species of parasites that occur in natural populations have not 2

been studied as extensively (Anderson, 1995) so data on the abundance of a parasite

species necessary to gain an understanding of host population dynamics and ecology is

lacking (Smallridge and Bull, 2000). Prevalence, the percentage of suitable hosts

infected, and intensity or parasitemia, the number of parasites infecting an individual

host, are the measurements used to define abundance, and are the most basic of data

collected in a study on the ecology of any species (Perkinset al., 1998). There are

several studies that focus on the prevalence and intensity of parasite species in a limited

number of host species (e.g., Jordan and Friend, 1971; Schall, 1986; Mahrt, 1989; Staats

and Schall, 1996; Smallridge and Bull, 2000; Clark et a l, 2003), including the current

study.

Parasites that live in the blood of a host often have detrimental effects on host

physiology, reproduction, and ability to escape predation and capture prey (Schall, 1983;

Daly, 1984; Schall and Dearing, 1987; Oppliger et a l, 1996). An exhaustive examination

of these factors was beyond the scope of the current study, largely because the presence

of blood parasites in snakes from Kansas and Nebraska has never been reported. Studies

that determine the impact of a parasite on its host cannot be performed until the presence

of the parasite is established. The species of parasite also needs to be identified and its

abundance within a certain population needs to be determined. The current study examined and compared the prevalence and intensity of blood parasites in two species of

sympatric snakes in southeastern Nebraska and northeastern Kansas. The study also examined blood cell morphology and cellular immune response in parasitized snakes. 3

The Black Rat Snake, Elaphe obsoleta obsoleta, is the largest snake species in

Nebraska and Kansas (Collins, 1993), with adults ranging from 106.7 cm to 183.0 cm snout-vent length (SVL). These snakes are mostly diurnal in the spring and fall but become more nocturnal in the warm summer months. They inhabit riparian forests, rocky hillsides and open woodlands and spend the winter in hillside dens. Elaphe o. obsoleta are excellent climbers and feed opportunistically on frogs, , snakes, , eggs, and mammals. In contrast, adult Great Plains Rat Snakes, Elaphe guttata emoryi, range in SVL from 61.0 cm to 122.0 cm. They are strictly terrestrial and primarily nocturnal, hiding under rocks during the day. They inhabit hillsides, canyons, and caves when available, and roam open woods or woodland edges, avoiding thick forests. Elaphe g. emoryi feed mainly on small birds and mammals (Collins, 1993).

In southeastern Nebraska and northeastern Kansas E. o. obsoleta and E. g. emoryi live sympatrically in the wooded areas along the Big Blue River drainage system

(Collins, 1993; Conant and Collins, 1998). These species have probably evolved together, which may explain why their habitats and diets are so different (Vitt, 1987;

Luiselli and Rugiero, 1993). The Big Blue River drainage was chosen for the current study in order to compare the prevalence and intensity of blood parasites between two species of snake belonging to the same genus. The two populations of snakes in the study inhabit the same ecological community, which means individuals from both species could encounter the same potential vectors and intermediate hosts for any parasites that are also present in the community. 4

MATERIALS AND METHODS

Field Techniques

Thirty Elaphe obsoleta obsoleta and 14 Elaphe guttata emoryi were collected from Gage County, Nebraska and Atchison, Marshall, and Washington counties in

Kansas. Snakes were collected between April and October 2000 and 2001 near known hibemacula and large trees, and often found under cover boards or large flat rocks.

Snakes were identified and the following parametric data were recorded for each: tail length, SVL, mass, and sex. Tail length and SVL were measured in centimeters with a flexible measuring tape. Mass in grams was measured by placing the snake in a cloth bag of known mass, attaching the bag to a spring scale, and subtracting the mass of the bag from the total mass. Snake sex was determined with the use of a lubricated probe according to Kams (1986) and each snake was assigned a unique subcaudal scale clip for future identification.

Several thin blood smears were made (Pritchard and Kruse, 1982) from each snake by collecting between 0.05-0.20 pi of blood from the subcaudal vein (Frye, 1991) with a 25 gauge needle and syringe. The blood was smeared onto microscope slides, which were labeled and allowed to air dry. Snakes were released near the site of capture and the blood smears were taken to the laboratory for processing and examination. 5

Laboratory Techniques

Blood smears were stained with Price’s Giemsa (Luna, 1968) and covered with

Damar balsam and cover slips. At least one slide from each snake was examined in its

entirety under a compound microscope to determine the presence of any parasites. If

parasites were present, six measurements were recorded from 30 infected RBCs and the

intraerythrocytic parasites. The six measurements were recorded in micrometers at

lOOOx magnification and are as follows: length of parasite, width of parasite, length of

parasite nucleus, width of parasite nucleus, length of host cell, and width of host cell.

Additionally, the length and width of 30 uninfected RBCs were measured for each

infected snake.

Digital images were used to calculate parasitemia, a measure of the intensity of a

parasitic infection in a single host. Images of blood smears from infected snakes were

taken at 400x magnification with an Olympus DP 11 digital camera mounted on an

Olympus BX41 compound microscope. These images were transferred to a computer,

image quality and size were adjusted using Adobe Photoshop®, and color images were

printed onto 21.8 x 28.2 cm paper. The printed images were used to determine the

number of infected RBCs out of 10,000 uninfected RBCs by marking each cell as it was counted.

The proportion of WBCs present in infected and uninfected snakes was determined by calculating the number of WBCs present per 1,000 RBCs. Images of blood smears from both infected and uninfected snakes were taken with a microscope- mounted digital camera, printed, and counted for number of RBCs and WBCs, as above. 6

Statistics

All of the statistics in the current study were performed according to Zar (1996).

The level of significance for all statistics was determined by p < 0.05. 7

RESULTS

Representatives of the genus Hepatozoon were the only parasites recovered from the blood ofElaphe obsoleta obsoleta and Elaphe guttata emoryi. These parasites are intracellular hemogregarines circumscribed within the phylum and are represented in the current study by one to three different species (Dr. Todd G. Smith, pers. comm., Acadia University, Nova Scotia, Canada).

Prevalence of Hepatozoon species in E. o. obsoleta was 40.0% (12 infected of 30 total snakes), whereas Hepatozoon species were found in only 7.1% (one infected of 14 total snakes) ofE. g. emoryi (Figure 1). Statistical comparison of these values using a

Fisher’s Exact test revealed E. o. obsoleta to have a significantly higher rate of infection than E. g. emoryi (p <0.05). Due to the low frequency of infection,E. g. emoryi were removed from all other analyses.

Male and female snakes were collected in both spring and autumn. To examine the effects of sex and season on prevalence, a three-dimensional contingency table was performed (Zar, 1996). This test revealed that there were no significant effects of sex or season on prevalence (%2= 7.54, p > 0.05).

The influence of snake size on prevalence was also examined. Infected snakes had an average SVL of 128.7 cm, while uninfected snakes averaged 89.7 cm SVL. A t- test demonstrated that infected snakes were significantly larger than uninfected snakes

(p < 0.05). Snakes with SVLs larger than 115.0 cm were all (n = 11) infected with 8

35 -

E.o.o. E.g.e. n Infected ■ Uninfected

Figure 1. Prevalence (number infected) of Hepatozoon species in Elaphe obsoleta obsoleta and Elaphe guttata emoryi. 9

Hepatozoon species, while all but one snake (106.1 cm) smaller than 115.0 cm (n = 19) were not infected (Figure 2).

In order to examine the relationship between snake size and parasitemia, parasitemia data were transformed using a modified Freeman and Tukey arcsine transformation to normalize their distribution (Zar, 1996). A correlation coefficient revealed that there was no significant relationship between snake size and parasitemia

(r = 0.178, p > 0.05).

The average length and width of infected RBCs (19.9 x 11.6 pm) and uninfected

RBCs (18.3 x 10.0 pm) were calculated in order to determine any influence of parasitic infection on RBC size. Two f-tests revealed that infected RBCs were significantly longer

(p < 0.05) and wider (p < 0.05) than uninfected RBCs (Figure 3).

The number of WBCs per 1,000 RBCs was calculated for both infected and unintected snakes. Infected snakes averaged 2.0 WBCs/1,000 RBCs, while uninfected snakes averaged 3.8 WBCs/1,000 RBCs (Figure 4). A r-test revealed that infected snakes had significantly fewer WBCs than uninfected snakes (p < 0.05). The relationship between number of WBCs per 1,000 RBCs and parasitemia was examined by calculating a correlation coefficient (r = 0.605,p < 0.05) with parasitemia data that were normalized using a modified Freeman and Tukey arcsine transformation (Zar, 1996). This analysis revealed an inverse relationship between parasitemia and number of WBCs in the host. 10

160

□ 140 n

cP 120 □ □ E o 100 > CO 80

60

40 40 60 80 100 120 140 160 SVL (cm) Figure 2. Snout-vent length (SVL) of uninfected (A) and infected (□ ) Elaphe obsoleta obsoleta. 11

♦ •

r a# *

Figure 3. Red blood cell infected with Hepatozoon species gamont. Scale bar = 10 pm. Figure 4. Mean number of white blood cells (WBCs) per 1,000 red blood cells (RBCs) in error. (RBCs) standard cells blood red 1,000 per (WBCs) cells blood white of number Mean 4. Figure uninfected (n=12) and infected (n=13) (n=13) infected and (n=12) uninfected

Mean#WBCs/ 1,000 RBCs 4.5 4.5 0.5 - 2.5 . - 3.5 . - 1.5 0 2 3 - 4 i 5 1 - Infected Elaphe obsoleta obsoleta. obsoleta Elaphe \ y ■ m : — — Uninfected ...... •* ; ;•;* -. • Error bars represent represent bars Error .' ' ' . . : 2 1 13

DISCUSSION

Parasite Identification

Membeis of the genus Hepatozoon were the only hemoparasites found

among the forty-four snakes examined in this study. Only one other study has sought to examine blood parasites of reptiles in Nebraska or Kansas (Greiner and Daggett, 1973).

These authors examined 20 eastern fence lizards ( Sceloporus undulatus) from the

Sandhills of Nebraska for species ofPlasmodium but all of the lizards were negative.

The present study is the first account of reptilian Hepatozoon species in Nebraska or

Kansas.

The number of species ofHepatozoon in snakes from the current study was between one and three, according to Dr. Todd G. Smith (pers. comm., Acadia University,

Nova Scotia, Canada), but species identification was not possible. The morphology of the intraerythrocytic stages ofHepatozoon is highly variable among species, and species determination cannot be made based solely on parasite ontogenetic stages viewed in blood smears (Ball et al., 1967; Wozniak and Telford, 1991; Desseret al., 1995; Siddall,

1995). Species identification requires an examination of parasites found in the invertebrate vector (Nadler and Miller, 1984; Wozniak and Telford, 1991; Desser et al.,

1995) in addition to data collected from parasites in the hosts (Telfordet al.,

2001). At the present time, the complete life cycles of only five species of snake

Hepatozoon have been elucidated (Ball et al., 1967; Tandan et al., 1972; Bashtaret al.,

1984; Nadler and Miller, 1984; Smith et al., 1994). 14

The typical life cycle of Hepatozoona species in a snake, as described by Smith

(1996), is as follows: Gamonts, inside the snake's erythrocytes, are ingested by a

mosquito during a blood meal. Micro- and macrogamonts pass through the gut wall of

the mosquito into fat body cells where they associate in syzygy and undergo

gametogenesis. Microgametes fertilize the macrogamete and the resulting zygote

expands into an immature oocyst. After nuclear segmentation and sporoblast formation,

the mature oocyst releases numerous sporocysts, each containing numerous sporozoites,

into the gut of whatever vertebrate intermediate host ingests the infected mosquito. Cysts

that contain two cystozoites each form from sporozoites in the vertebrate's hepatocytes.

A snake then ingests the vertebrate intermediate host and the cystozoites are released into

the gut of the snake. Macromeronts, each containing numerous macromerozoites, form

in the snake's liver, lungs, and other visceral organs. After two rounds of merogony take

place, micromerozoites are released into the snake's bloodstream where they invade

erythrocytes and form gamonts (Figure 5).

Although the current study did not examine any possible arthropod vectors,

researchers who have described the life cycles of snakeHepatozoon species employed

mosquitoes representing the generaCulex and Aedes to infect snakes (Ball et al., 1967;

Bashtar et al., 1984; Nadler and Miller, 1984; Smith et al., 1994). There are six species

of Culex and 18 species of Aedes in the geographic region encompassing the study site

(Lunt and Rapp, 1981), and a total of approximately 50 different species of mosquito in

this area (Moore, 2001). This large number of potential vectors makes vector identification in the field difficult and the low prevalence of blood parasites in mosquito mosquito

or frog

Figure 5. Generalized life cycle ofHepatozpon species of snakes, a. Gamont in the red blood cell of a snake, b-e. The gamont is ingested by a mosquito and undergoes sexual and asexual reproduction, f. Sporozoites are ingested by a lizard or frog intermediate host when the intermediate host eats a mosquito, g-h. Sporozoites infect intestinal cells of the lizard or frog intermediate host. «. Cystozoites are released from the intestine of the lizard or frog intermediate host when that animal is ingested by a snake, j-m. Micromerozoites are produced by asexual reproduction in the cells of snake visceral organs and are released to invade red blood cells and develop into gamonts (1). From Smith (1996). 16

vectors, in general, makes such a field-based effort prohibitive. For example, during a

recent outbreak ofPlasmodium vivax in humans in northern Queensland,

Australia, none of the 706Anopheles farauti collected was infected with P. vivax, even

though this species of mosquito is known to vector this parasite (Hannaet al., 2004). Not

only are laboratory experiments necessary to identify the vectors, but vertebrate

intermediate hosts cannot be identified without conducting experimental transmission

studies in a laboratory (Smithet al. 1994).

Prevalence

Elaphe obsoleta obsoleta were infected with species of Hepatozoon at a

significantly higher rate than Elaphe guttata emoryi, with 12 out of 30 E. o. obsoleta

infected and only one out of 14 E. g. emoryi infected. The difference between infection

rates may be related to snake diet. Elaphe. g. emoryi eat only birds and mammals,

whereas E. o. obsoleta eat birds, mammals, reptiles, and amphibians (Collins, 1993). It has been noted that sympatric species can live together successfully if they partition their prey resources (Vitt, 1987), especially if one of the species is terrestrial and the other is

arboreal (Shine, 1983; Luiselli and Rugiero, 1993). Species ofHepatozoon are transmitted to snakes by the ingestion of infected prey items, that in turn have ingested infected mosquitoes (Smith, 1996). Therefore, diet is a major componentHepatozoon of species transmission to snakes, and differences in snake diets may explain why E. o. obsoleta had a much higher prevalence of Hepatozoon infection than E. g. emoryi. 17

Juvenile and sub-adult E. o. obsoleta are exclusively terrestrial whereas sexually mature adults exhibit arboreal behavior (pers. comm., Daniel D. Fogell, University of

Nebraska at Omaha, Nebraska). In this study, infected snakes averaged 128.7 cm SVL and were significantly larger than uninfected snakes, which averaged 89.7 cm SVL. The

SVL of infected snakes ranged from 106.1 cm to 153.0 cm. The SVL of uninfected snakes ranged from 60.0 cm to 111.0 cm and all but one of the uninfected snakes had a

SVL less than 115.0 cm (Figure 2). According to Blouin-Demers et al. (2002), Elaphe obsoleta in Ontario do not mature sexually until they reach a minimum SVL of 105.0 cm.

Sexual maturity in E. o. obsoleta coincides with arboreal behavior and a possible shift in diet. In the current study, the size of snakes infected with species of Hepatozoon exceeded the size corresponding with sexual maturity and the size of the snakes that were negative for Hepatozoon species fall below this size threshold.

Sexually mature and immature snakes may display different feeding behaviors.

For example, Weatherheadet al. (2003), found that E. o. obsoleta exhibit an ontogenetic change in diet as their size increases. According to Dr. Gabriel Blouin-Demers (pers. comm., University of Ottawa, Ontario, Canada), terrestrialE. o. obsoleta (juveniles and sub-adults) most likely prey on small reptiles and amphibians, and small, terrestrial mammals, whereas the arboreal adults feed on larger prey items, including insectivorous birds and bats.

Few life cycles have been completed for species of snakeHepatozoon (Ball et al.,

1967; Landau et al., 1972; Bashtaret al., 1984; Nadler and Miller, 1984; Smith et al.,

1994) and the only intermediate hosts described in these life cycles are frogs and lizards. 1 8

Smith (1996) notes that: . .other insectivorous vertebrates that are dietary items of a specific snake are potentially capable of serving as an intermediate vertebrate host for species ofHepatozoon." Drs. Todd G. Smith and Gabriel Blouin-Demers suggested during independent personal communications that bats could be serving as potential intermediate hosts for Hepatozoon species in snakes by ingesting infected mosquitoes and in turn being ingested by the snakes. Thus, the shift in habitat and diet exhibited by adult E. o. obsoleta may allow them to become infected withHepatozoon species while juveniles and sub-adults remain uninfected.

The notion that insectivorous birds and/or bats transmitHepatozoon species to snakes is also supported by the difference in prevalence of Hepatozoon species between

E. o. obsoleta and E. g. emoryi. As mentioned previously, species ofHepatozoon are transmitted to snakes by the ingestion of infected prey items (Smith, 1996). Species of snake Hepatozoon are transmitted principally by mosquitoes (Ballet ah, 1967; 1969;

Chao and Ball, 1969; Nadler and Miller, 1984; Lowichik et al., 1993; Smith et al., 1994) making essential the ingestion of a mosquito by an insectivorous intermediate host.

There are several species of bats, such asEptesicus juscus, Lasionycteris noctivigans,

Lasiurus cinereus, Myotis lucifugus, and Pipistrellus subflavus, within the study site that eat mosquitoes (Whitaker, 1972; Anthony and Kunz, 1977; Barclay, 1985; Whitaker and

Lawhead, 1992; Whitaker, 1995). Both species of snake in the current study eat birds and mammals, but E. g. emoryi are strictly terrestrial and feed mostly on birds and terrestrial mammals that would not likely ingest mosquitoes (Joneset al, 1985). Adult E. o. obsoleta are arboreal, and have access to insectivorous birds and bats (Barr and 19

Norton, 1963; Fitch, 1963; Jackson, 1970; Plummer, 1977; Mullin and Cooper, 1998;

Thompsonet al., 1999).

If species ofHepatozoon in the current study were transmitted by a

hematophagous arthropod other than a mosquito, it would be expected E.that g. emoryi

and juvenile and sub-adult E. o. obsoleta would have higher parasite prevalence than

observed. Other hematophagous arthropods, such as ticks, mites, and fleas, are

ectoparasites and could be found living on all of the vertebrates that inhabit the present

study site (Jones et al., 1985; Frye, 1991; Janovy, 1997). All of these ectoparasites are

potential intermediate hosts for species ofHepatozoon (Smith, 1996). However, a study published by Wozniak and Telford (1991) demonstrated that some species of ticks are incapable of transmitting Hepatozoon species to snakes. Therefore, it is likely that the species ofHepatozoon examined in the current study use mosquitoes as vectors, which supports the notion that the intermediate host must be a mosquito-eating insectivore.

Host Blood Cells

Snake RBCs, when uninfected, are ovoid in shape and each cell contains a single, centrally located ovoid nucleus. The RBCs of snakes infected with Hepatozoon species are invaded by gamonts (Smith, 1996). The gamonts, which have been described as

“sausage-shaped” (Levine and Wacha, 1983; Ahmed, 1998), are positioned alongside the host cell nucleus, often causing the nucleus to become displaced laterally (Bashtar et al.,

1984; Desser et al., 1995; Saoudet al., 1996; Desser, 1997). Cell distortion and hypertrophy of host RBCs are also common effects of parasitic infections (Ballet al., 2 0

1969; Daly, 1984; Nadler and Miller, 1984; Telfordet al., 2001). In the current study, infected RBCs were found to be significantly larger than uninfected RBCs. Uninfected

RBCs had an average length of 18.3 jam and an average width of 10.0 jam; infected RBCs averaged 19.9 x 11.6 jam for length and width. The average lengths and widths of gamonts in this study were 15.3 x 4.0 jam. The difference in size between infected and uninfected RBCs may be due to the presence of large parasitic inclusions inside the infected RBCs. Nadler and Miller (1984) hypothesized that hypertrophy of infected

RBCs was either caused by the added volume inside each RBC due to the presence of gamonts, or an adaptation of the RBCs in response to parasitic infections.

In addition to examining the response of RBCs to a parasitic infection, the number of WBCs per 1,000 RBCs was calculated for infected and uninfected E. o. obsoleta to determine if a parasitic infection would influence host immune response, as measured by

WBC count. Elevated levels of WBCs have been demonstrated in birds infected with blood parasites (Ots and Horak, 1998), but very little information is available regarding cell-mediated immunity to parasites in reptiles. Nadler and Miller (1985) described potentially antigenic ultrastructural alterations ofHepatozoon -infected RBCs in snakes, but stage specific antibodies have not yet been reported (Wozniaket al., 1996).

The current study did not include any adult E. o. obsoleta that were not infected with Hepatozoon species, so the comparison between infected and uninfected snakes is also a comparison between juveniles/sub-adults and adults. Infected snakes in the current study had a significantly lower average proportion of WBCs than uninfected snakes.

These results are contrary to a study published by Watkins and Nowell (2003) which 2 1

demonstrated an increase of WBCs in individuals infected with Hepatozoon species, but

the individuals examined were Grey Squirrels (Sciurus carolinensis), not snakes.

Hepatozoon species in mammals invade host WBCs, not RBCs (Britt and Molyneaux,

1979), so it is difficult to compare the immune response of a mammal with the immune response of a reptile. . Moreover, a recent cladistic analysis suggested thatHepatozoon

species of reptiles are evolutionarily distinct fromHepatozoon species of mammals

(Jakes et al., 2003), thus, these distantly related parasites may illicit different immunological responses.

Alternatively, the transition from sexual immaturity to sexual maturity may trigger a change in the number of circulating WBCs in E. o. obsoleta. This possibility could not be tested in the present study because all of the adult snakes were infected and a representative number of WBCs for uninfected adults could not be determined. If uninfected adult snakes could be found it would be relatively easy to determine if infection status or ontogenetic shift is important in the number of circulating WBCs.

The effects ofHepatozoon species infections on host RBCs are well documented, but the effects of such an infection on a host immune system is far from being understood due to a lack of information available about the immune system of reptiles. The ability to

“domesticate” the life cycles of these parasites would also allow for more studies on the physiology and pathology of gamonts inside host RBCs and the effects of gamonts on host WBCs. 2 2

Conclusion

Blood parasites infecting E. o. obsoleta and E. g. emoryi in the current study are

members of the genus Hepatozoon. Specific identification of the parasites cannot be

determined without knowledge and examination of the vertebrate intermediate host and the arthropod vector. Many suggestions have been made as to what organisms may act as vertebrate intermediate hosts for this population of snakes. Based on prevalence data and life histories of both species of snake, which include habitat and diet, it is suggested here that bats are most likely the intermediate hosts responsible for transmittingHepatozoon species to adult E. o. obsoleta in Nebraska and Kansas. Elucidation of the life cycles of

Hepatozoon species fromE. o. obsoleta in Nebraska and Kansas minimally would require experimental exposures of snakes, mosquitoes, and locally abundant bats to infective stages ofHepatozoon species. Completion of the life cycle would allow for identification of the Hepatozoon species involved and would set the stage for controlled experiments to examine the effects of snake age and diet on the transmission of these parasites. 23

LITERATURE CITED

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