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INVESTIGATIONS INTO THE PHYSIOLOGICAL AND BIOMECHANICAL BASIS OF DIFFERENTIAL SUCCESS IN ORAL VACCINATION BETWEEN SKUNKS ( MEPHITIS) AND ( LOTOR)

A Thesis Presented to the Honors Tutorial College Ohio University

In Partial Fulfillment of the Requirements for Graduation from the Honors Tutorial College with the degree of

Bachelor of Science in Biological Sciences

by

Charlotte M. Klimovich

August 2017

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Table of Contents INTRODUCTION ...... 3 EXECUTION OF ORAL RABIES VACCINATION PROGRAMS ...... 4 HISTORY OF DISEASE CONTROL PROGRAMS ...... 5 A NEW ERA OF DISEASE CONTROL ...... 7 OPTIMIZATION OF ORAL RABIES VACCINE PROGRAMS ...... 9 PROJECT HYPOTHESES AND OBJECTIVES ...... 12 AIM 1: FACTORS INFLUENCING BITE FORCE PRODUCTION CAPABILITIES AT DIFFERENT GAPES USING BIOMECHANICAL MODELS INFORMED BY JAW MUSCLE AND SKELETAL STRUCTURE ...... 19 Background and Hypotheses ...... 19 Materials and Methods ...... 26 Results ...... 32 Discussion ...... 37 AIM 2: COMPARE NATURAL BITE FORCES AT DIFFERENT GAPES IN LIVE .. 40 Background and Hypotheses ...... 40 Materials and Methods ...... 41 Results ...... 43 Discussion ...... 45 AIM 3: EVALUATE DIFFERENCES IN ORAL HANDLING, MASTICATION, AND SWALLOWING OF THE SACHETS USING BIPLANAR FLUOROSCOPY ...... 47 Background and Hypotheses ...... 47 Materials and Methods ...... 48 Results ...... 50 Discussion ...... 55 SYNTHESIS ...... 57 REFERENCES ...... 62 APPENDIX 1...... 69

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INTRODUCTION

A rabies variant (RRV) epidemic has been raging on the east coast of the

United States since the 1970s (Anonymous 2000). Over 90% of all rabid cases reported to the Centers for Disease Control and Prevention (CDC) in modern times occur in wild animals.

Rabies used to be rare outside of the Southern United States, but uncontrolled outbreaks in wildlife are now placing unprecedented numbers of citizens at risk of exposure. An increase in prophylactic rabies treatments given due to possible exposures indicates the seriousness of this issue; while fewer than one hundred treatments were given in the entire state of New York before 1990, more than 10,000 are now given annually. The epidemic had spread from Florida to West Virginia by 1984, when the first oral vaccine (V-RG, now called RABORAL V-RG) was created (USDA 1995).

The CDC spends more than $300 million a year attempting to contain RRV and the other rabies variants found around the United States (Centers for Disease Control and

Prevention. 2015). Oral rabies vaccination (ORV) programs have been shown to be effective in many species, with reported cases decreasing in all animal species measured during 2010 (Blanton et al. 2011). However, the CDC must constantly develop and implement new techniques to keep up with the changes in both the disease itself and the way it is transmitted to ensure the numbers of rabies cases found annually continue to decrease.

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EXECUTION OF ORAL RABIES VACCINATION PROGRAMS

The RRV ORV program utilizes sachets of liquid RRV vaccine covered by an

attractant such as fishmeal (Anonymous 2000). In the United States, these sachets are

produced by Merial (Duluth, Georgia). They are distributed by plane or vehicle,

depending on the density of humans in the area. When animals find the vaccine bait, they

eat it and ingest liquid vaccine. Virus in the liquid absorbed through the oropharyngeal

mucosa will penetrate the lymphoid tissue, including the lingual and palatine tonsils,

lining the opening of the Figure 1. Example of a fishmeal polymer oropharynx and induce the Raboral V-RG bait.

production of rabies antibodies

that enable the animal to mount an immune response in case of exposure (Brown et al.

2014).

Raccoon (Procyon lotor) populations have satisfactory rates of immunization

from oral vaccination using the sachets (Roscoe et al. 1998). It is commonly accepted that

around 70% of animals in a population should be immunized to effectively control

transmission of a disease, with evidence from both practice and theory supporting this

percentage (Zalma, Older, and Brooks 1970; Simonsen et al. 1987). However, oral rabies

vaccination of raccoons is not infallible. For example, in 2004, a raccoon was found in

Lake County (Ohio) with rabies strain later shown to have originated from within the

'rabies-safe' zone created by the ORV program (Henderson et al. 2008). This means that enough rabid raccoons had survived within the zone to create a distinct strain of rabies virus - quite the opposite of what the CDC had intended (Klimovich 2013, unpublished data).

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In contrast, 2010 census data from the Centers for Disease Control and Prevention

show that skunks (Mephitis mephitis) are unresponsive to ORV programs (see

(http://www.cdc.gov/rabies/location/usa/surveillance/wild_animals.html). The striped

skunk is second only to the raccoon in number of rabies cases reported and there is

evidence that RRV (usually only found within the raccoon population) is now endemic in

the skunk population (Guerra et al. 2003). Host shifts are associated with the emergence

of some of the most virulent infectious diseases in humans including AIDS, SARS, swine

flu, and Ebola, so it is imperative to explore new methods of control before a large-scale public health threat emerges from the disease running rampant in the skunk population.

It remains unclear why the response to oral vaccination differs between skunks

and raccoons. Other studies have analyzed bait distribution strategies, bait campaign

timing, natural barriers, gender, age, population density, eating behaviors, bait color, bait

odor, and bait texture to offer suggestions about ways to improve efficacy (Robinson,

Jojola, and VerCauteren 2004; Jojola, Robinson, and VerCauteren 2007; Feldhamer,

Thompson, and Chapman 2003; Greenwood 1980). Even using improved baits, however,

immunization rates in skunks still remain low (Fehlner-Gardiner et al. 2012; Mainguy et

al. 2012; Tolson et al. 1987).

HISTORY OF DISEASE CONTROL PROGRAMS

In the process of exploring options to vaccinate skunks, it is beneficial to

understand the evolution of oral rabies immunization programs in raccoons over time in

order to inform possible skunk ORV development. Prior to 1950, raccoon rabies was not

reported as a problem in the United States. The first increase in numbers of rabid

raccoons occurred between 1950 and 1970, mostly in Florida and Georgia. The rabies

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variant implicated in the current outbreak was detected in 1977 in Virginia and West

Virginia, marking it as separate from the rabies strain observed in Florida and Georgia,

although they eventually converged into one rabies strain (Anonymous 2000). There is

evidence that this 1977 rabies variant appeared due to translocation of an infected raccoon from the southeastern United States to the region (Jenkins and Winkler 1987;

Nettles et al. 1979; J. S. Smith et al. 1984).

There were many possible options to control the spread of this disease. One common historical technique to control rabies was population reduction, as it was

believed that a certain number of animals are required to effectively pass rabies through

the group (Debbie 1991). This approach has not been popular due to impractical cost and

predicted impacts on nontarget species due to the techniques employed (such as ,

poison baits, gassing, and trapping) (Debbie 1991; MacDonald and Research 1980).

Habitat modification is a similar technique that is often used in site specific cases (i.e.

removing garbage from specific public spaces where animals may come in contact with

humans and domestic animals) (Wobeser 2002).

Immunization techniques were also explored. Trap-Vaccinate-Release programs

represent a more practical option than these others already described due to factors of

cost, scale, and past observed effectiveness in a wide range of environments. Indeed,

these programs have seen success when used on an interim basis. One example is the

intramuscular injection of inactivated rabies virus into live-trapped animals in Toronto;

while costs were high, fewer people were exposed to rabies (as measured by the number

of people undergoing post-exposure prophylaxis, or PEP) (R. C. Rosatte 1987; R. C.

Rosatte et al. 1992). Another problem was the projected number of animals that needed to

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be effectively immunized to create a zone of herd immunity. One of the earliest estimated

herd immunity thresholds for any disease was proposed by Godfrey in 1933. He predicted

that vaccination of 30% of children 0 to 4 years old and 50% of children 5-14 years old

would be sufficient to eliminate the problem of diptheria (Godfrey 1933). This figure was

later revised to be 70-90% when practical experience was applied, while simple theory

dictates it should be around 85% (Zalma, Older, and Brooks 1970; Simonsen et al. 1987;

R. M. Anderson 1992). This value was difficult to reach in Trap-Vaccinate-Release programs due to their labor and monetary costs (while lower than that of other techniques, they are still significant).

A NEW ERA OF DISEASE CONTROL

These techniques have been impractical on a large scale due to reasons already

discussed. During the 1960s, researchers at the CDC proposed the idea of an oral

immunization program for wild animals. To test this concept, red were given

attenuated Evelyn-Rokitnicke-Abelseth vaccine via the oral route in the early 1970s and

the practicality of the idea was demonstrated through their effective immunization (Baer,

Abelseth, and Debbie 1971). Inspired by this first attempt, the World Health Organization

(WHO) encouraged development of oral rabies immunization programs for foxes in

Europe (Steck et al. 1982). The first country to use ORV in wildlife species starting in

1978, Switzerland, was declared free of rabies in 1999 after a long enzootic for rabies in

red foxes.

In 1989, southern Ontario began to pilot a similar ORV program in North

America, which is continuing to this day and which has been successful in reducing the

incidence of rabies in arctic foxes. After seeing the success of this program, the United

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States developed a vaccinia-rabies glycoprotein (V-RG) recombinant virus vaccine for use in controlling the RRV outbreak (Kieny, Lathe, and Drillien 1984; Wiktor et al. 1984,

1985; C E Rupprecht et al. 1986; Charles E. Rupprecht et al. 1987, 1988). The US

version of the ORV program utilizes plastic sachets of liquid Raboral V-RG vaccine covered by an attractant such as fishmeal. They are distributed by plane or vehicle, depending on the density of humans in the area. This vaccine was fully licensed after

field testing in 1997 for use by the state or federal government in programs targeting

raccoons (USDA 1995). It first demonstrated success in preventing the spread of disease

in New Jersey at the Cape May peninsula (Roscoe et al. 1998).

A National Rabies Management Team was formed in 1998 by the U.S.

Department of Agriculture, Animal and Plant Health Inspection Service, Wildlife

Services (APHIS-WS) in order to comply with their federal appropriation to cooperate in

existing ORV programs. This team worked to expand ORV to include 16 states and to

take into consideration geographic features when dropping baits such as lakes, rivers, and

quality. Unfortunately, in recent years, unexpected problems have come up that

retarded their progress in eradicating rabies in the United States. The most serious of

these is the fact that Raboral V-RG (the only oral rabies vaccine licensed for use in the

US) does not effectively immunize skunks (Tolson et al. 1987; Vos et al. 2017). A

significant amount of RRV cases (30%) have implicated skunks, pointing to the existence

of independent transmission of the virus within their population and thus necessitating

targeting their populations specifically (Guerra et al. 2003; Klimovich, unpublished data).

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OPTIMIZATION OF ORAL RABIES VACCINE PROGRAMS

A number of studies have been conducted that focus on factors contributing to

ORV success. Robbins et al. (1998) tested different bait distribution strategies (high-

density with uniform distribution, low-density with additional targeted bait distribution,

and high-density with additional bait distribution) to determine the most optimal one,

weighing cost against effectiveness. Sattler et al. conducted a similar study in 2009 to determine any associations between bait distribution and demographic factors. Variables such as gender, age, and macro-habitat were considered, but found to be non-significant.

Sattler et al. (2009) also considered the bait campaign timing (semi-annual, annual, etc.)

to determine the optimal frequency with which to bait. This is important to consider due

to the presence of naive young animals in the population at certain times of the year. Côté

et al. (2012) studied how natural barriers informed the movement of hosts and thus the

transmission of disease. They found that variation in disease progression may be

explained by the presence of natural barriers such as rivers, mountains, or highways,

whose characteristics may change as the seasons progress (Childs et al. 2000; Russell,

Real, and Smith 2006; Blanchong et al. 2008; Neaves et al. 2009). Côté et al. (2012) also

suggest that sex-specific characteristics be considered because males and juveniles in

most mammalian species like raccoons are more likely to disperse than adult females due

to their mating systems (Greenwood 1980). Wallace et al. (2014) expanded on these thoughts in 2014 in their “20-Year Review of Rabies Virus Cross Species Transmission among Terrestrial in the United States." Raccoons appeared to be four times more likely than skunks to participate in cross-species transmission of rabies, and this may be due to their high population density (1-250/km2, with highest densities observed

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in urban ) and high frequency of interspecies interactions (H. T. Smith and

Engeman, n.d.; R. C. Rosatte et al. 1992; Riley, Hadidian, and Manski 1998; Prange,

Gehrt, and Wiggers 2003; Broadfoot, Rosatte, and O’Leary 2001; Fascione et al. 2004;

Hirsch et al. 2013). For comparison, skunks were found to have a population density of

only 3-6/km2 (with no data reported on the effects of urban environments) (Feldhamer,

Thompson, and Chapman 2003). Raccoons are also highly curious, which may entice

them to confront rabid animals acting strangely rather than avoiding them, increasing

their chance of exposure (Feldhamer, Thompson, and Chapman 2003). Skunks, in

contrast, employ techniques to drive away other animals such as their characteristic

spray, which may lead to decreased risk of exposure (Totton et al. 2002; Breed and

Moore 2015). Additionally, raccoons use latrine sites that other animals may come into

contact with, facilitating disease transmission (Page, Swihart, and Kazacos 1999). Lastly,

raccoons will share den sites, sometimes with other species such as skunks, and feeding

sites with many different species, also facilitating transmission of disease to other

animals (Shirer and Fitch 1970; Rivest and Bergeron 1981). Wallace et al. (2014) also stressed the importance of considering the possibility of the virus undergoing a host shift in the skunk population, leading to differences in transmission.

Some researchers have studied skunks specifically in an effort to understand why they cannot be effectively immunized. For example, Robinson et al. (2004) explored the role of bait manipulation in the delivery of the vaccine to skunks. Several rabies vectors' eating behaviors were evaluated and compared, including skunks, , and raccoons.

The team also evaluated the direction from which a skunk approached the plate of offered baits and also the baits' odor, color, shape, texture, and size and observed that skunks

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typically held the larger baits vertically to eat them from the outer edges (Jojola,

Robinson, and VerCauteren 2007). Depending on the shape, the bait would either be consumed starting from the corner or the whole bait would be placed in the mouth.

Because of this behavior, the vaccine sachet would sometimes become separated from the attractant and fall to the ground. The teeth the skunks used to bite at the baits were also noted, along with their reaction to the taste and consistency of the bait. Most

interestingly, Brown et al. (2014) conducted a study comparing oral immunization success between skunks who had eaten baits versus skunks given an oral inoculation of the vaccine. All skunks administered vaccine via direct instillation into the oral cavity survived the rabies challenge and produced rabies antibodies. This proves that the vaccine is effective in skunks via the oral route and something about the administration of the vaccine bait is preventing effective oral immunization.

A new kind of vaccine called ONRAB is being produced in Canada as an alternative to Raboral V-RG that takes many of these studies' findings into account.

ONRAB is a live, recombinant human adenovirus (AdRG1.3) rabies virus glycoprotein vaccine in a polyvinyl chloride (PVC) blister pack coated directly with a sweet attractant.

Some trials conducted on skunks using this bait either did not capture enough skunks in order to draw a conclusion about efficacy (Slate et al. 2014) or found no significant difference between vaccination rates of the on either ONRAB or Raboral

V-RG (Fehlner-Gardiner et al. 2012). However, Mainguy et al. (2012) reported percentages of antibody-positive skunks found after baiting to be between 11 to 17%, which is better than the negligible percentages of antibody-positive skunks reported in previous studies. Still, this is nowhere near the required 70% of animals needed for

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effective herd immunity (Zalma, Older, and Brooks 1970; Simonsen et al. 1987; R. M.

Anderson 1992).

PROJECT HYPOTHESES AND OBJECTIVES

The overarching hypothesis I have developed for this project is that there are biomechanical and/or physiological differences between skunks and raccoons that affect their ability to penetrate the vaccine capsule or the manner in which the vaccine liquid is handled intra-orally. This is indirectly supported by a recent study demonstrating that skunks are immunized effectively through direct oral inoculation (Brown et al. 2014). In this study, the same oral vaccine that is effective in raccoons was used, suggesting that the vaccine formula is not the cause of the lower vaccination rates in this species. Thus there may be differences in how much the vaccine coats the oropharyngeal mucosa.

Indeed, this is alluded to by Vos et al. (2017) in a study comparing uptake of the rabies

vaccine in the lymphoid tissue of skunks and other species in rabies vaccination

programs.

The overall objective of the proposed research is to elucidate the relevant

biomechanical and physiological differences between skunks and raccoons that may

contribute to differences in oral immunization success. The present project provides a new perspective and data that could inform the development of new vaccine baits specifically for skunks. Studies to date have failed to consider cranial morphology and oral behaviors as an explanation for differences in vaccine effectiveness. With this in mind, the following three aims will be completed: (1) characterize and compare factors relating to the mechanics of bite force production using biomechanical models of jaw muscle and skeletal structure; (2) compare natural bite forces at different gapes in live

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animals; and (3) evaluate differences in oral handling, mastication and swallowing of the sachets using biplanar fluoroscopy (i.e., X-ray video).

While these approaches have never been applied to the study of oral immunization success, there is evidence that the techniques will produce data relevant to the questions being asked. All of these approaches are rooted in the field of comparative biomechanics and functional morphology, and have been utilized to study differences in cranial form and function in mammals. However, these studies have typically focused on evolutionary, behavioral and ecological questions aimed at understanding why there are interspecific differences in feeding behaviors and performance. To my knowledge, this is the first application of these approaches for understanding differences in inoculation rates in wild animals, with the ultimate goal of yielding data that are informative for developing new prevention strategies. A brief background on the relevance and application of the approaches utilized in the present study is provided.

In regards to the first aim of comparing factors relating to the mechanics of bite force production, I evaluated several different parameters that either impact the ability of an animal to bite or the resistance to elevated skeletal loading due to habitually higher bite forces. Inferring relative biting performance by modelling bite force capabilities, or factors that influence bite performance, from musculoskeletal morphology is pervasive in the literature on skull morphology and evolution in mammals and other vertebrates (e.g.,

Christiansen and Adolfssen 2005; Christiansen and Wroe 2007; Davis et al. 2010;

Vinyard et al. 2003; Williams et al. 2002; Wroe, McHenry, and Thomason 2005). This is because maximum bite force is linked to ecologically and evolutionarily relevant behaviors including food acquisition, dietary niche, mate selection, and defense in

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vertebrate (R. A. Anderson, McBrayer, and Herrel 2008). Thus by characterizing

musculoskeletal factors that influence bite forces in skunks and raccoons we can evaluate

anatomical differences that may contribute to difference in accessing the bait packets or

sachets between skunks and raccoons. There are myriad ways in which anatomy can

affect bite force production, and the research presented here focuses on a few key

parameters, including lever arms and mechanical advantage of the jaw muscles and jaw

muscle physiological cross-sectional area.

At the same time, if an animal is habitually producing higher bite forces, perhaps due to evolutionary changes in musculoskeletal anatomy, it has also been hypothesized that the jaw must be better equipped to resist the loads that are incurred due to reaction forces at the bite point and the temporomandibular joint during biting (e.g., Hylander,

1985). Indeed, a number of studies on cranial morphology in mammals have focused on

comparing relative size and shape of the jaw as a way of inferring whether animals

habitually produce (and thus resist) elevated loads during biting (e.g., Biknevicius and

Ruff 1992; Williams et al., 2002). This general hypothesis about the link between force production during biting and skeletal morphology is based on in vivo studies in which strains along the jaw, representing deformation of the local bone, are recorded during biting on a bite force transducer measuring bite force. These studies demonstrate that the jaw incurs higher stresses and strains when animals bite harder (William L. Hylander

1977; William L Hylander 1979; William L. Hylander 1985, 1979; W. L. Hylander and

Bays 1979). This hypothesis is also based on studies in which animals are fed hard and soft diets over the same duration, and the hard-diet animals demonstrate increased modelling and remodeling of the jaw as well as generally more robust jaws (e.g.,

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Ciochon, Nisbett, and Corruccini 1997; Marianne Bouvier 1986a, 1986b; Marianne

Bouvier and Hylander 1981; M Bouvier and Hylander 1982; Marianne Bouvier and

Hylander 1984). Thus we might expect differences between skunks and raccoons in the relative size of different parts of the jaw relevant for resisting loads, if one animal habitually bites harder than the other.

Finally, because this project is interested in a particular kind of biting – biting on the rabies sachets – I also evaluated how musculoskeletal anatomy might limit or enhance bite force production during biting at the different gapes that would be utilized to penetrate the different baits. For this work, I drew on work by Herring and Herring’s

(1974) model that evaluates the impact of gape on muscle stretch as well as a basic principle of muscle biology relating to muscle architecture that related to muscle stretch and force production. This principle is the length-tension relationship of muscle which describes the amount of isometric force that can be produced by a muscle at a given length (Gordon, Huxley, and Julian 1966). The active part of this relationship stems from the basic unit of muscle, the sarcomere, being made up of actin and myosin elements that interact to allow the muscle to produce force. This interaction works best at a certain fiber length, with shorter or longer fiber lengths leading to suboptimal force production. When a muscle is stretched, it may be producing force in a less advantageous region of the length-tension curve. Thus factors that limit stretch, including relatively longer fiber lengths, may help to maintain or even increase force at wider gapes. Again, a number of studies of mammalian jaw muscles have looked at the effect of gape on muscle stretch including in (e.g., Williams, Peiffer, and Ford 2009), primates (e.g., (Eng et al.

2009; Vinyard et al. 2003; Taylor and Vinyard 2009), and bats (Santana 2015).

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Fewer studies have looked at the relationship between muscle architecture, gape

and bite force production, but a notable series of studies in this realm compare tree-

gouging marmosets (common marmosets, Callithrix jacchus) to the non-gouging cotton-

top tamarins (Saguinus oedipus). Common marmosets use wide gapes to gouge trees to

obtain exudates, and this is their primary mode of food acquisition all year. Thus they

must be able to produce large bite forces at wide gapes. In comparing their muscle

architecture, Eng et al. (2009) demonstrated that common marmosets have a muscle configuration (longer muscle fibers and smaller muscle excursion) allowing them to act on a more favorable portion of the length-tension curve at larger gapes (Eng et al. 2009).

It might be expected that we find these same characteristics in raccoons as compared to skunks, due to their greater observed success with puncturing the vaccine baits.

Taylor and Vinyard's (2009) study had similar aims, but evaluated biting at different gapes in different ways. Cebus apella (a tufted capuchin known for its capacity to exploit hard and tough food items) was compared to two species of "untufted" capuchin (C. capucinus and C. albifrons) that do not have such a varied diet. Taylor and

Vinyard (2009) evaluated physiologic cross-sectional area (PCSA), pinnation, and ratio of mass to tetanic tension for all species in order to see which characteristics may be an adaptation. These are the methods I will use in my study as they incorporate the study of muscle fibers and excursions as Eng et al. (2009) did while going even more into depth about the cause of the differences. Taylor and Vinyard (2009) found that tufted capuchins have enlarged their jaw muscle mass to increase PCSA, allowing them to produce larger bite forces without compromising muscle excursion and contraction velocity (in such situations as large gapes). Again, we might expect that similar trends will be found in

17 raccoons, which have been observed to be more effective in biting the vaccine bait.

The second aim is to measure bite forces directly in skunks and raccoons. These data are critical for characterizing actual biting performance in these species. Whereas some researchers have estimated bite force from mechanical models in skunks and raccoons (e.g., Christiansen and Adolfssen 2005; Christiansen and Wroe 2007), obtaining in vivo bite forces is a rather simple procedure that has significant value for interpreting cranial morphology and function. Thus this gives us the opportunity to validate or reject the hypotheses for maximum bite forces developed from musculoskeletal modelling.

Specific methods to measure bite force in live animals are further explained in Williams,

Peiffer, and Ford (2009). A custom made bite force transducer that can be adjusted to the gapes of interest will be used to collect bite force data. This will allow us to determine optimal gape as it relates to bite force for the animals. They also calculated stretch factors of the mastication muscles (describing how far past optimum it was stretched) to back up these findings. I will follow the same methods in my study. Based on their results, it would be expected that raccoons' optimal gape would be closer to the gape needed to bite the vaccine bait than skunks. Skunks may be experiencing more muscle stretch (i.e. have a greater stretch factor) that is negatively impacting their ability to produce large bite forces needed to puncture the vaccine sachet.

Lastly, biplanar fluoroscopy will be used to evaluate oral handling and swallowing behaviors animals perform when chewing the baits. The project mentor, Dr.

Susan Williams, has extensive experience utilizing biplanar fluoroscopy in the context of the XROMM workflow to study mastication in a variety of species (Brainerd et al. 2010).

There is also precedent to study swallowing behavior using fluoroscopes and radiopaque

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solutions like barium (Logemann 1993; Rasley et al. 1993). Barium studies are a

common clinical tool to identify problems with the pharynx that contribute to difficulty

swallowing. All of these studies rely on qualitative assessment of the x ray videos for

characteristics related to the behavior of interest. Thus, it is difficult to draw many

connections between the methodology of these studies and my own due to the differences

in our specific aims. A protocol detailing which specific behaviors related to swallowing are of interest was developed specifically for my project. These include oropharynx

transit time and numbers of swallows per bolus based on preliminary literature review

(Lever et al. 2015), but experiments will show if any other interesting trends develop.

The aims, ranging from simple force modeling to complex behavior

quantification, should be completed in this order to ease interpretation of data. For

example, if simple models dictate that skunks should be able to bite harder than raccoons

based on muscle architecture, but the natural bite force studies show that raccoons are

actually biting harder, we can conclude that some other factor besides simple anatomy is dictating force production in skunks.

In conclusion, my study can be clearly seen to bridge the gap between the fields of functional morphology and public health. Because of this design, I have made connections either with studies with methodological similarities to my own or similar theoretical ideas. This study will be the first of its kind, to my knowledge, to apply anatomical methodology to the problem of vaccine efficacy.

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AIM 1: FACTORS INFLUENCING BITE FORCE PRODUCTION CAPABILITIES AT DIFFERENT GAPES USING BIOMECHANICAL MODELS INFORMED BY JAW MUSCLE AND SKELETAL STRUCTURE

Background and Hypotheses

The main structures involved in chewing and biting are the mandible, teeth,

temporomandibular joint and muscles of mastication. The mandible is simply the lower

jaw bone. The temporomandibular joint (TMJ) is the joint where the mandible meets the

skull. The skeletal components of the TMJ are the condyle, a rounded structure at the

back of the mandible, and the glenoid , a depression on the base of the skull. The

condyle sits in the glenoid fossa to form the TMJ.

Two major muscles involved in mastication that will be the focus of this study are

the temporalis and the masseter. As seen in the Figure 2, the temporalis (Figure 2 left) is

the fan-shaped muscle found on the side of the head that has an effect in the closing of

the jaw when it contracts. The masseter (Figure 2 right), seen stretched over the rear

section of the jaw bone below the TMJ, is one of the strongest muscles involved in mastication. Therefore, it is an important contributor to the overall maximal force the animal is able to generate when closing the jaw.

Bite force is an important measure of performance of the mammalian masticatory system. This is particularly true of predatory animals that must capture live prey and/or engage in inter- or intraspecific antagonistic behaviors

(R. A. Anderson, Figure 2. Major jaw adductors in raccoons. Left, the temporalis McBrayer, and Herrel muscle. Right, the masseter muscle. Not shown is the medial pterygoid.

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2008). Historically, bite force has been modelled in a number of different ways, from simplistic 2-dimensional lever mechanics based on the cranial skeleton (e.g., Christiansen and Adolfssen 2005) to more complex 3-dimensional modelling using information from both the muscles and bones of the masticatory system (e.g., Santana 2015). In all of these models, there are a number of factors that influence biting mechanics and the ability of the masticatory system to produce force during isometric biting (i.e., when the jaw adductor muscles are not shortening). These factors include not only the size and architecture of the jaw muscles, but also the relative positions of the jaw muscles, the jaw joint and the teeth on which the bites are produced. Models that take into account all of these factors offer more accurate approximations of bite performance, however, in most cases a number of these parameters are not known, particularly in the case of the soft tissues (i.e., the jaw muscles). Nevertheless, the absence of these data are not critical as relative estimations for inter- or intraspecific comparisons can often address ecologically- and biologically-relevant questions. In the context of the present project, it is these relative differences that are of interest. Specifically, given the differences in rabies vaccination rates between skunks and raccoons, the goal of the present study is to determine factors influencing biting mechanics between skunks and raccoons.

One of the most utilized models of factors influencing bite force mechanics was by Walter Greaves in 1978. The model predicts the location where maximum bite forces can be generated given a hypothetical muscle force by considering a number of variables relating to the configuration of the muscle resultant, bite point, and interglenoid width, as illustrated in Figure 3A. The model assumes a “triangle of support” formed by lines

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Figure 3. The constrained model of Greaves (1978). A) Ventral view of a skull showing the relevant parameters described in the text. Reg I = Region I; Reg II = Region II. The blue triangle is the triangle of support when working and balancing side muscle.

between the two temporomandibular joints (TMJs) (i.e., the baseline axis; indicated by

the yellow line in Figure 3) and the bite point (a tooth, indicated with a green star). In the model, a resultant muscle force (the sum of the working and balancing side muscle forces; purple circle in Figure 3) must pass through the triangle of support to ensure that the mandible does not rotate and produce tensile forces within the TMJ on either the working or balancing side (Spencer and Demes 1993). Tensile forces in the TMJ negatively affect the stability of the jaw joint as they cause the condyle to pull away from the glenoid fossa. When jaw muscles in the working and balancing sides are equally active, the muscle force resultant lies directly between the working and balancing side glenoids. A line drawn through the balancing side TMJ and the midline muscle resultant

(pink line in Figure 3) will intersect the boundary of what is termed Regions I and II on the working side teeth (indicated by green star in Figure 3).

According to the constrained model of Greaves (1978), maximal bite force magnitudes must be calculated using different mechanical rules depending on what

22 region they are found in. For teeth in Region I, jaw muscles can contract maximally on both sides and the muscle resultant will still lie within the triangle of support causing no negative forces at the TMJ (Figure 3C). Additionally, in Region I, bite force maxima increase at the teeth closer to the Region I-II boundary. This can be understood using simple lever mechanics, as the bite force can be estimated as the ratio of the in-lever to the out-lever. The in-lever is the distance from a line connecting the baseline axis to the muscle resultant (shown as the red line in Figure 3B) and the out-lever is the distance from the baseline axis to the bite point (a tooth; shown as the black line in Figure 3B).

Therefore, in Region I, calculating the ratio of the in-lever to the out-lever will reveal an increase in bite force posteriorly along the tooth row because the out-lever decreases posteriorly (e.g., from the incisor to the premolars).

In contrast, in Region II, when the working and balancing side muscle forces are equally and maximally active, the midline muscle resultant will be found outside the triangle of support resulting in tensile forces at the TMJs, for which is not well-suited to resist. Therefore, in order to prevent damage to the TMJs, the balancing side muscles must decrease their contractile force. This shifts the muscle resultant back into the triangle of support and results in a decrease in bite force posteriorly along the tooth row in Region II (Figure 3D).

Animals that differ in skull shape will have differences in the size and shape of the triangle of support. Thus with this investigation, I attempted to quantify the differences between skunks and raccoons, and between the genders when applicable, in order to understand how their skull shape affects biting mechanics. In addition to differences in overall size, skunks and raccoons have qualitatively different head shapes,

23 with skunks having longer narrow heads with a shortened tooth row and a longer cranium as compared to raccoons. In addition, female skunks are on average smaller than male skunks. These differences would not only impact the location of the bite point relative to the jaw adductor muscles, but also the lever mechanics through changes in palate and interglenoid width.

The next study aimed to investigate the effect that opening the jaw (i.e., gape) has on the production of bite forces for each species because of its impact on muscle stretch.

In order to bite the vaccine baits, the jaws must be open wide enough for the bait to be positioned between an upper and lower tooth (or teeth). However, maximum isometric bite forces are thought to be produced at or near occlusion, when the jaw muscles such as the masseter and temporalis are not significantly stretched (Nordstrom and Yemm

1972).Thus, when these muscles are stretched beyond resting length, bite force should decrease. It has historically been thought decreasing the size of the bait may increase efficacy, as skunks may have to open their jaws so wide to bite the current baits that they are unable to produce enough force to puncture it (Jojola, Robinson, and VerCauteren

2007).

Herring and Herring (1974) developed a biomechanical model to compare the effect of gape on masseter and temporalis muscle stretch, and by extension bite force production. Using this model, I considered the following questions:

1. Do skunks and raccoons differ in masseter and temporalis orientation? If so, what effect does this have on muscle stretch when the jaw is opened?

2. How do different bait sizes impact muscle stretch in skunks and raccoons?

24

Herring and Herring (1974) mathematical model first considers the length of the origin and insertion of the jaw muscles and the angle in between when the teeth are in occlusion. From this, the amount of muscle stretch (i.e., the stretch factor, or L/l) can be determined for a given amount of angular mandibular rotation when the jaw is opened to a certain gape. High or low stretch factors (i.e., greater than or less than 1) indicate that the muscle is stretched beyond its resting length, which could negatively impact bite force. Changing the angle between the muscle origin and insertion and/or the origin- insertion ratio will impact muscle stretch. Mechanisms to maintain high jaw adducting force when muscles are stretched beyond their resting length involve reducing the stretch factor. This can be achieved by increasing the angle between the origin and insertion or by having high or low origin/insertion ratios. Finally, this model assumes that there are no changes in the internal architecture of the muscles that would impact the operating range of the muscle (e.g., fiber length) or force production (e.g., physiological cross-

Figure 4. Skull measurements and the equation used to calculate adductor stretch factors (Herring & Herring 1974), where t = temporalis, m = masseter, a = the distance between the temporomandibular joint (TMJ) and the origin of the muscle, b = the distance between the TMJ and the insertion of the muscle, c = the distance between the origin and insertion of the muscle, Ф = the angle between a and b in close jaw position, Ѳ = jaw opening angle, l = the length of muscle, and L = the length of stretched muscle (L/l being the stretch factor).

25

sectional area). Measurements taken can be seen in Figure 4.

Muscle Measurements

The next study aimed to quantify any differences in muscle fiber architecture that

could be contributing to differences in function. Muscles have a number of different

physical properties that contribute to their function. The sarcomere is the basic unit of a

muscle that allows it to contract; muscle cells have many repeating sections of

sarcomeres along their length. The muscle body is made up of many muscle cells bound

in fascicles (bundles of muscle cells) which in turn are bound together to produce

numerous muscle fibers. Physiological cross sectional area (PCSA) is an estimate of the total cross sectional area of the muscle fibers. It is a proxy for peak isometric force production, or the maximum force a muscle can produce when exerting a constant force

that does not change its length (as might be observed when holding up an object in a set

position).

Pinnation of a muscle is another way that muscle strength can be affected by

structure. A pinnate muscle is one with fascicles that attach obliquely instead of parallel

to the tendon. Muscles may be bipinnate, like the temporalis, or multipinnate, like the

masseter. The more pinnation a muscle displays, the stronger it will be, as pinnation

allows for many fibers to be packed into a small space (Gans 1982). Multipinnate

muscles typically have multiple myotendinous junctions, or the site of muscle tendon

interface.

Variation in muscle architecture can contribute to variation in muscle function

under different circumstances. Taylor and Vinyard (2004) demonstrated that long muscle

fiber length seems to allow for increased gape, while large PCSA and greater angle of

26

pinnation enable greater force production capability. Due to the raccoon’s observed

greater success in immunization, it would be expected that their muscles would display

these muscle architecture parameters suited to gape and force production.

Materials and Methods

Skull Measurements

A sample size of 16 raccoon and 15 skunk skulls were used in this investigation, consisting of 8 female/8 male raccoons and 7 female/8 male skunks to account for sexual dimorphism (e.g., differences in body size between males and females). All specimens

were dentally mature. Specimens were obtained on loan from the United States Museum

of Natural History as well as from the Ohio University Vertebrate Collection (Appendix

1).

Measurements were taken with digital calipers directly on the skulls to the nearest

0.01 mm (Figure 5, Appendix 1). To account for size differences between individuals and species, we created a biomechanical shape ratio by dividing each variable by jaw length (with the exception of jaw length). Jaw length was used because it represents the load arm during chewing and biting (William L. Hylander 1985). For jaw length, however, a skull shape ratio was created using the geometric mean of several variables

(see Figure 5) following Darroch and Mosimann (1985) and Jungers, Falsetti, and Wall

(1995) .

Ln-transformed shape ratios were compared using a one-tailed Mann Whitney U test in SPSS 13.0. We first compared males and females within each species. We then compared males and females between species. In light of the number of statistical

27 comparisons used in this study, a Bonferroni adjusted α (p=0.05) was used to reduce spurious type I errors (Rice 1989).

Calculation of Stretch Factors

For this study, we utilized a sample of 6 raccoons (4 male; 2 female) and 6 skunks

(3 male; 3 female) obtained locally from a wildlife control service, from the Smithsonian

National Museum of Natural History collections, or from the Ohio University

Comparative Vertebrate collections. Measurements were obtained from either cadaveric or osteological specimens. In the case of cadaveric specimens, the muscles were still

28

Skunk (♂)

Raccoon (♂)

Figure 5. Male skunk (top) and raccoon (bottom) crania and jaws showing the measurements used in this study. Scale bars represent 1 cm. The skunk specimens have been scaled to match the raccoons for visualization purposes. Measurements in green are used to calculate an overall skull size estimate for the geometric mean. Measurements in red are the jaw adductor moment arms and jaw length, the ratio of which provides an indication of leverage for each muscle. Measurements in blue provide an indication of load resistance capabilities at the mandibular condyle, corpus, and symphysis. See Taylor and Vinyard (2004).

attached so the specimens were CT scanned at the OU microCT. The skulls were reconstructed in Avizo then imported into Geomagic for measurement. For osteological specimens, measurements were taken directly on the skulls using digital calipers to the nearest 0.01 mm (Figure 5). For all measurements, the distance between the TMJ and the

29

origin and insertion of the temporalis and masseters muscles were determined using

osteological landmarks based on dissections.

Angles were initially calculated using the law of cosines for Ф in the neutral jaw

position. For Ѳ, angles were again calculated using the law of cosines when 1 cm and 2

cm blocks, representing the 2 different baits, were placed at the canines and first molar in

raccoons and the canine and carnassial in skunks. For visualization, this was done

digitally in Geomagic on the scanned specimens (Figure 6). Males and females were considered separately because of potential differences in skull size and shape due to sexual dimorphism.

Raccoons were predicted to have higher angles between the origin and insertion of the masseter and temporalis. It was also expected that raccoons will have higher

Figure 6. Skunk skulls showing the various jaw angles considered in this study.

30

origin/insertion ratios than skunks. Both of these would have the effect reducing muscle

stretch (i.e., lower stretch factors) during jaw opening around the two baits, which would

be advantageous for biting through the baits.

Muscle Measurements

All methods and measurements for this study follow those utilized by Taylor and

Vinyard (2004). Heads of fresh cadaveric specimens (3 female and 7 male raccoons; 3

female and 4 male skunks) obtained locally were removed and skinned. They were then

fixed (preserved) in 10% neutral buffered formalin in preparation for dissection. Care

was taken to ensure that the jaws were fixed in a closed position to avoid muscle stretch.

Once fixed, the masseter and temporalis muscles were carefully removed. Fascia was removed from each muscle, and then they were weighed using an analytical balance to the nearest 0.1 g to determine muscle mass. Each muscle was sectioned to identify myotendinous junctions following the methods of Taylor and Vinyard (2004). The proximal and distal tendinous attachments of six individual muscle fibers from each muscle or muscle section were then visualized under a dissecting microscope.

Photographs were taken and the relevant measurements made using ImageJ. The average

of the distance between the proximal and distal attachments was considered the muscle

fiber length.

31

Figure 7. Cross- section through a muscle showing how pinnation angle is determined. MTJ = myotendinous junction. Based on Taylor and Vinyard (2004).

To calculate pinnation angle, a simple geometric method is used that is based on a

triangle formed by the following “sides”, as shown in Figure 7: 1) the perpendicular distance from the proximal myotendinous junction to the tendon of distal muscle attachment (green line); 2) the individual fiber lengths running from the central tendon to the distal tendon (red line), and 3) the myotendinous junction (purple line). The angle of pinnation (cosθ; yellow arc) is determined geometrically using the law of cosines as the angle between the sides formed by the fiber length and myotendinous junction.

The calculation of PCSA (physiological cross-sectional area), an estimate of the maximum force production capabilities of a muscle, relies on the above measurements of muscle mass, pinnation angle and fiber length. The formula is given below:

( ) × ( ) = ( ) × 1.0564 ( ) 2 𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚 𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚 𝑔𝑔𝑔𝑔 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃 𝑐𝑐𝑐𝑐 𝑔𝑔𝑔𝑔 𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓 𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙ℎ 𝑐𝑐𝑐𝑐 3 𝑐𝑐𝑐𝑐

32

The value of 1.0564 is the specific density of muscle (Mendez and Keys 1960).

Mean PCSA for males and females within each species was calculated. Statistical

comparisons were made between males and females within and across species to

determine whether 1) there are sex-related differences in the maximum muscle force that can be produced within species that might be important for understanding the distribution of effective oral vaccines within a population and 2) whether skunks consistently have less favorable masticatory configurations for producing maximum muscle forces than

raccoons.

Shape ratios relative to biomechanical standards were created by dividing

fiber length and PCSA against jaw length (in this case from the back of the condyle to

infradental). One-tailed Mann-Whitney U tests were then used to determine if fiber

lengths and PCSAs for all muscle groups (for fiber lengths, whole masseter complex,

outer temporalis portion, and inner temporalis portion; for PCSA, whole masseter

complex along with its components of zygomaticomandibularis and masseter, plus outer

temporalis portion and inner temporalis portion) were significantly different. These tests

were performed in SPSS version 23.

Results

Skull Measurements

Compared to males, female skunks have relatively shorter jaws and medial

pterygoid moment arms. The mechanical advantage of the medial pterygoid is also

significantly reduced in females (Table 1a).

33

This indicates that the anatomical configuration of the medial pterygoid in males

is in a more favorable position to produce bite forces even with a relatively longer jaw. In

contrast, male and female raccoons did not differ in any of the variables examined (Table

1b). Compared to skunks, raccoon males and females have relatively shorter jaws but relatively longer moment arms for all of the jaw muscles resulting in significantly higher mechanical advantages for all of the jaw adductors (Table 1c). This suggests that male and female skunks are at a relative disadvantage for producing bite forces to break open the packets. Load resistance capabilities in raccoons and skunks also differ, with raccoons having relatively more robust mandibular condyles, corpora and symphyses. Results suggest that raccoons must resist relatively higher forces due to the production of relatively higher bite forces.

Raccoon ♀ Raccoon ♂ Skunk ♀ Skunk ♂ Variable A Mean S.D. Mean S.D. Mean S.D. Mean S.D. Jaw Length 1.075 0.017 1.074 0.007 1.091 0.009 1.103 0.036 Masseter Moment Arm 3.478 0.061 3.464 0.034 3.089 0.058 3.156 0.065 Temporalis Moment Arm 3.270 0.133 3.288 0.057 2.767 0.060 2.816 0.093 Medial Pterygoid Moment Arm 3.110 0.092 3.147 0.084 2.762 0.074 2.843 0.058 Masseter Mech Adv 0.818 0.014 0.814 0.008 0.726 0.014 0.742 0.015 Temporalis Mech Adv 0.769 0.031 0.773 0.013 0.650 0.014 0.662 0.022 Medial Pterygoid Mech Adv 0.731 0.022 0.740 0.020 0.649 0.017 0.668 0.014 Anteroposterior Condyle Length 1.174 0.064 1.194 0.059 0.760 0.065 0.768 0.075 Medioloateral CondyleWidth 1.894 0.059 1.928 0.055 1.673 0.046 1.692 0.035 Corpus Width at M1 1.694 0.048 1.696 0.042 1.293 0.018 1.340 0.081 Symphysis Length 1.486 0.062 1.464 0.039 1.071 0.085 1.069 0.068 Symphysis Height 1.814 0.071 1.855 0.076 1.613 0.047 1.660 0.086

Variable B Raccoon Skunk Variable C Raccoon vs Skunk ♂vs♀ ♂vs♀ ♂vs♂ ♀vs♀ ♂vs♀ ♀vs♂ Jaw Length NS 0.021 Jaw Length 0.021 0.04 0.002 0.065 Masseter Moment Arm NS NS Masseter Moment Arm <0.001 <0.001 <0.001 <0.001 Temporalis Moment Arm NS NS Temporalis Moment Arm <0.001 <0.001 <0.001 <0.001 Medial Pterygoid Moment Arm NS 0.037 Medial Pterygoid Moment Arm <0.001 <0.001 <0.001 <0.001 Masseter Mech Adv NS NS Masseter Mech Adv <0.001 <0.001 <0.001 <0.001 Temporalis Mech Adv NS NS Temporalis Mech Adv <0.001 <0.001 <0.001 <0.001 Medial Pterygoid Mech Adv NS 0.037 Medial Pterygoid Mech Adv <0.001 <0.001 <0.001 <0.001 Anteroposterior Condyle Length NS NS Anteroposterior Condyle Length <0.001 <0.001 <0.001 <0.001 Medioloateral CondyleWidth NS NS Medioloateral CondyleWidth <0.001 <0.001 <0.001 <0.001 Corpus Height at M1 NS NS Corpus Height at M1 <0.001 NS <0.001 <0.001 Symphysis Length NS NS Symphysis Length <0.001 <0.001 <0.001 <0.001 Symphysis Height NS NS Symphysis Height 0.002 <0.001 <0.001 0.005 Table 1. (a) Means and standard deviations (S.D.) for ln-transformed shape ratios by species and sex. (b) Results of comparisons between males and females of each species. (c) Results of comparisons between species.

34

Calculation of Stretch Factors

Mephitis mephitis Procyon lotor Origin-insertion ratios and ɸ Male Female Male Female when jaws are closed (n=3) (n=3) (n=4) (n=2) Masseter Origin-insertion 2.75 2.59 2.31 2.41 ratio Masseter ɸ (°) 87.65 81.98 104.46 107.63 Temporalis Origin-insertion 1.63 1.68 1.48 1.53 ratio Temporalis ɸ (°) 91.95 93.67 85.29 91.51

Table 2. Comparison of Origin-Insertion ratios and Φ angles for the masseter and temporalis muscles between species.

Counter to our prediction, male and female raccoons have lower masseter and temporalis origin-insertion ratios than male and female skunks. While ɸ is higher in raccoons than skunks for the masseter, ɸ for the temporalis is comparable in both species

(Table 2). Statistical tests for significance were unable to be conducted due to low

sample size. This issue will be resolved for final publication.

Stretch factors for the masseter are higher in skunks than raccoons for both males

and females at all tooth positions and for each bait size (Figure 10). Thus, even though

origin-insertion ratios for the skunks are higher than raccoons, the skunk masseter is

stretched more than in raccoons. This likely has to do with the gross size differences

between the species, which require larger absolute gapes in skunks for the two bait sizes.

Stretch factors in raccoons may also be reduced by their higher ɸ. The temporalis stretch

factor is much higher in raccoons. This reflects the low origin-insertion ratios and

comparable ɸ for this muscle. Thus, even though raccoons are bigger than skunks, they

35

are at a disadvantage for producing bite force with the temporalis when biting the two

bait sizes. Sex differences within species are relatively minor except for the temporalis stretch factors in skunks, which are much lower in females. Stretch of the temporalis muscle in female skunks may have less impact on bite force production compared to males, all else being equal.

Muscle Measurements

A Mann-Whitney U test comparing skunk and raccoon muscle fiber

lengths showed that all muscles tested (the masseter and the inner/outer temporalis

portions) have significantly different (p value < 0.05) fiber lengths between the two

species (Figure 11). Specifically, skunks were shown to have significantly longer muscle

fibers, when scaled to jaw length, than raccoons. Thus skunks have relatively longer

muscle fibers than raccoons.

Figure 10. Graph comparing stretch factors when bait is placed in the mouth (1 and 2 cm baits, placed either at the canines or carnassial teeth) between species.

36

Figure 11. Boxplots showing results of a Mann-Whitney U Test comparing skunk and raccoon muscle fiber lengths. Mass=Masseter, TempO=Outer Temporalis, TempI=Inner Temporalis.

Comparisons also showed that relative to jaw length, raccoons have significantly larger PCSAs for most of the muscle or muscle groups: the whole masseter complex

(U=0.000, p value=0.001); the zygomaticomandibularis (U=0.000, p=0.001), the deep/superficial masseter (U=9.000, p=0.011), the inner temporalis (U=18.000, p=0.097).

The one exception was the inner temporalis which did not differ significantly between the two species (U=8.000, p=0.008).

Next, males and females were compared to each other within the species.

For raccoons, no fiber lengths were significantly different between males and females, and only the PCSAs of the zygomaticomandibularis and inner temporalis were significantly different (respectively, U=0.000, p=0.017 and U=1.000, p=0.030).

Specifically, male raccoons have significantly larger PCSAs in those two muscle groups

37 than females.

A similar trend was observed within the skunk species. For fiber lengths, only the fibers of the outer temporalis muscle group were significantly different between the two sexes (U=0.000, p=0.034), with the females having longer muscle fibers than the males.

For PCSAs, only the inner temporalis muscle group was significantly different between the genders (U=0.000, p=0.034). Again, females had larger PCSAs in this muscle group than males. It is surprising that there were so few sex differences observed, as in most of the other investigations completed, female and male skunks have tended to have easily observable sex differences.

Discussion

The skeletal measurements, performed first, seemed overall to indicate that skunks were at a relative and absolute disadvantage for producing bite forces as compared to raccoons. Also, while female and male raccoons were not significantly different in their skeletal anatomy, female skunks were at a disadvantage to male skunks when producing bite forces. All of this was consistent with the commonly held belief that skunks may not be getting immunized due to their inability to puncture the vaccine bait, possibly due to their small size compared to raccoons.

However, the calculation of stretch factors complicated that prediction.

While stretch factors at the masseter muscle were higher in skunks than in raccoons, stretch factors at the temporalis muscle were actually lower in skunks. The temporalis muscle is the major force production muscle in these species, so skunks may have found a way to compensate for their small size necessitating the production of larger gapes and

38 not be at a disadvantage to raccoons when it comes to producing bite forces. Female skunks also had low temporalis stretch factors compared to male skunks, which may also mean that they are not actually at a disadvantage to male skunks when it comes to producing bite forces.

Consistent with these conclusions, the results of the muscle dissections reveal that skunks have significantly longer fiber lengths than raccoons, meaning they are more suited to producing bite forces at larger gapes. This again displays how they may be compensating for their small size. Raccoons had larger PCSAs than skunks, meaning they are more suited to producing large bite forces, which is more consistent with original predictions. In contrast to the other studies, sex differences were minimal. Male raccoons had larger PCSAs and thus larger force production abilities in two muscle groups, with no other significant differences. Female skunks had longer muscle fiber lengths in one of the muscle groups and larger PCSAs in another one of the groups. These are most consistent with our overall conclusions, but we may find more significant differences between male and female skunks if sample size was increased.

Overall, these studies together seem to indicate that the simple conclusion that predominates in the literature about skunks simply being smaller and less able to bite than raccoons may be incorrect. At several points in these studies, there was evidence that skunks were compensating for their smaller size by optimizing their muscles in various ways and may not actually be at a disadvantage to raccoons for producing bite forces.

However, all of these studies rely heavily on theoretical modeling, which necessarily misses some of the actual complexity found in nature. Also, some of the investigations suffered from small sample sizes due to low availability, which should be corrected

39 before publication to ensure all conclusions are not being skewed. This may help increase statistical significance as well.

40

AIM 2: COMPARE NATURAL BITE FORCES AT DIFFERENT GAPES IN

LIVE ANIMALS

Background and Hypotheses

In this study, I investigated the effect that opening the jaw (i.e. to bite a vaccine

bait) would have on the production of bite forces for each species. Maximum isometric

bite forces are thought to be produced at or near occlusion, when the jaw muscles such as the masseter and temporalis are not stretched (Nordstrom and Yemm 1972). Thus, when these muscles are stretched beyond resting length, bite force should decrease. Previously,

Herring and Herring (1974)’s “stretch factor” model was used to determine how much the jaw muscles are stretched when a 1 cm or 2 cm bait is placed on the canines or carnassials (Figure 6). Assuming that the jaw adductors are near the peak of their length–

tension curves at rest, muscle force is reduced when muscles are stretched at wide gapes

(Nordstrom and Yemm 1972).

Because the skulls of skunks are smaller on average than raccoons, we would

predict skunks to be less able to produce bite forces at the different gapes as their jaws

would need to stretch more to produce the same gape. However, the results of the

theoretical model show that skunks have higher masseter but low temporalis stretch

factors at all tooth positions for both bait sizes, meaning that the skunk masseter

undergoes significant stretching when the jaws are open whereas the skunk temporalis is

stretched less compared to the raccoon. Because the temporalis is the dominant muscle in both species, skunks may not actually be at a significant disadvantage for producing bite forces at each gape and tooth location, beyond that due to their overall smaller body size.

41

We aimed to validate the results of the model by measuring actual bite forces in live

animals at different gapes.

Materials and Methods

Natural bite forces were measured in 2 skunks and 2 raccoons using a custom bite

force strain-gauge based transducer designed after (Dechow and Carlson 1983) (Figure

12). All methods involving live animals were approved by the IACUC under protocol 16-

U-003. Two aluminum beams, each with two single-element strain gauges (TML UFLA-

1-350-11-3LT), were used as the bite plates and mounted parallel to each other using adjustable screws. Due to this design, the beams of the transducer could be adjusted from

1 to 2 cm (corresponding to the sizes of the rabies baits) so that bite force data could be collected at both gapes. One end of each beam was covered with a rubber coating to protect the animals’ teeth from injury. The four strain gauges were connected in a

Wheatstone bridge. Deformation of the beams by biting results in a proportional change in resistance in the gauges and thus produces a change in voltage output by the

Wheatstone bridge.

The voltage output was conditioned and amplified with a Vishay Micro-

Measurements 2120 System and recorded using Qualysis software along with synchronized digital video to record bite location (Figure 12). Prior to each recording session, the bite force transducer was calibrated by determining the relationship between the voltage output and calibration weights suspended from the bite plate. Calibration regressions consistently had high r2 values (all > 0.92) indicating a strong linear

42

Figure 12. Image of an adjustable bite force transducer (scale bar = 1 cm) from Williams et al. (2009) and image from Qualysis of a raccoon biting the force transducer. relationship between the calibration weights and voltage output. For each bite, the peak voltage output was extracted and converted to grams using the calibration equation for the relationship between the calibration weight. Data were then converted to Newtons, as is the standard for bite force data. Because we are interested in maximum performance, we analyzed the top 10 bites at each bite location and gape for each species.

Due to their smaller body size, we would expect that skunks would prefer to bite at the canine position to reduce the effect of muscle stretch on force production and to have smaller observed bite forces overall than raccoons. Also due to muscle stretch, both species are expected to produce smaller bite forces at the larger 2 cm gape than at the 1 cm. For raccoons, measured bite force would be expected to increase caudally along the toothrow following simple lever mechanics. Unfortunately, no data were able to be collected at the 2 cm molar/carnassial position in skunks due to gape limitations.

43

Results

Figure 13. Results of χ2 tests and graphs of biting frequency by location. Skunks were unable to bite at the molar/carnassial at the 2 cm gape due to gape limitations.

A χ2 test for expected equal frequencies at each bite location showed no significant location preference at the 1 cm gape for skunks (Figure 13). At the 2 cm gape, however, skunks preferred premolar biting significantly more than canine biting. Both of these results were counter to our original predictions. Raccoons, in contrast, showed a significant preference for biting in the molar region regardless of gape.

Unexpectedly, a 1-tailed t-test showed skunks produced significantly higher bite forces Figure 14. A bar graph showing force magnitude as it varies by bite location. Error bars show the at the canines than raccoons. At standard error.

44 the other biting locations, there was no significant difference between the bite forces produced by the two species (Figure 14). Both of these results were contrary to the predicted.

Also counter to our predictions, a 1-tailed t-test 250 1 cm gape 2 cm gape showed that skunk bite 200 forces at the smaller 1 cm 150 gape were not significantly 100 Bite Force (N) Force Bite larger than bite forces at 50 the 2 cm gape (Figure 15). 0 However, bite forces were Canine Premolar Carn/Molar Figure 15. Bar graph showing skunk bite shown to increase caudally forces as they vary by bite location and gape. Error bars show the standard error. (from the canine to carnassial/molar at the 1 cm gape and from the canine to premolar at the 2 cm gape) at both gapes as anticipated.

The results of a 1-tailed t-test showed that only the raccoon carnassial/molar bite forces at 2 cm gape were significantly lower than bites on the same tooth at 1 cm gape

(the rest of the forces were not significantly different) (Figure 16). Bite forces were shown to increase caudally as expected at the 1 cm gape, but contrary to our prediction, at the 2 cm gape bite forces were highest at the premolar location.

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Figure 16. Bar graph showing raccoon bite forces as they vary by bite location and gape. Error bars show the standard error.

Discussion

In conclusion, because the force produced by skunks at the premolar position at a

2 cm gape was higher than at the 1 cm gape, it would seem that they have a fiber

architecture and length that helps them maintain bite force at wider gapes. In contrast,

raccoons had lower bite forces at the carnassial/molar at the 2 cm gape compared with the

1 cm. This means that despite being larger than skunks, muscle stretch is affecting their

ability to bite posteriorly. The two species did not significantly differ in overall observed

force production, except that skunks had significantly higher bite forces at the canines.

This indicates that the animals’ ability to puncture vaccine sachets is likely not the cause

of differential immunization success as was previously thought and factors related to

chewing and swallowing need to be considered in future experiments.

Although there are no other natural bite force studies for these species existing in the literature, there have been several published studies estimating maximal bite forces in

46 raccoons and skunks. In Christiansen and Adolfssen (2005), maximal bite forces for raccoons were estimated at the canines to be 119.5 N and at the carnassial to be 176.4 N.

In Christiansen and Wroe (2007), raccoon maximal bite forces were estimated as 121.4 N at the canines and 176.7 N at the carnassial. Interestingly, in our study raccoon maximal bite forces were recorded at the canines to be 82.4 N and at the carnassial to be 259.5 N.

Only Christiansen and Wroe (2007) calculated theoretical skunk maximal bite force values of 73.3 N at the canines and 99.9 N at the carnassial. Our study recorded forces of

87.6 N at the canines and 226.5 N at the carnassial. The lower value at the canines may be due to the animals’ unwillingness to bite a hard surface like the transducer, although the values may be so close that the difference is negligible. The higher observed values at the carnassial is likely due to their model not taking into account all of the complexity in actual animals.

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AIM 3: EVALUATE DIFFERENCES IN ORAL HANDLING, MASTICATION, AND SWALLOWING OF THE SACHETS USING BIPLANAR FLUOROSCOPY

Background and Hypotheses

The behavior the animal displays when confronted with the bait is vitally important to ensuring successful delivery of the oral rabies vaccine. While most studies have focused on external aspects of behavior that can be easily observed (Jojola,

Robinson, and VerCauteren 2007; Robinson, Jojola, and VerCauteren 2004), it is highly likely that other behaviors occurring inside the oral cavity also contribute to immunization success. High speed digital and fluoroscopy videos of animals drinking liquid and also puncturing and ingesting a sachet filled with radiopaque fluid were collected in order to determine if the sachet handling behavior, both extraorally and intraorally differed between the two species, and if so, whether the manner in which one species handles liquids and the baits is more optimal to induce production of antibodies.

The hypothesis is that, compared to skunks, oral behaviors in raccoons are more likely to result in greater consumption of the rabies fluid within the bait or result in better coating of the oropharyngeal mucosa.

The project mentor, Dr. Susan Williams, has extensive experience utilizing biplanar fluoroscopy in the context of the XROMM workflow (Brainerd et al. 2010) to study feeding in a variety of species. There is also precedent to study swallowing behavior using fluoroscopes and radio opaque solutions like barium (Logemann 1993;

Rasley et al. 1993). Barium studies are a common clinical tool to identify problems with the pharynx that contribute to difficulty swallowing. Combining high speed video and fluoroscopy is ideal for assessing whether there are factors that may contribute to

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Figure 17. A screenshot of Qualysis user interface showing a skunk in the restraint system in front of the two fluoroscopes (top) and the external webcam (bottom).

differences in the amount of coverage on the mucosa-associated lymphoid tissue

(MALT), known to be important for inducing an immune response (Vos et al. 2017).

Materials and Methods

Two synchronized fluoroscopes (OEC-9000) fitted with 30 cm diameter image intensifiers were used to monitor chewing, licking drinking and swallowing behavior as well as bait manipulation. They were placed so that dorsal and lateral views of the cranium can be taken simultaneously. Fluoroscope intensity was set to 70-90 kVp and

3.5-4.0 mA, depending on the specimen, for recording. High-speed Oqus 310 digital video-cameras mounted to the output ports of the fluoroscopes recorded the x-ray movies as well as a synchronized video from a Logitech webcam. Videos were recorded at 250 frames per second, and lasted up to 33.95 seconds using Qualysis Track Manager 2.6 software (Sweden) (Figure 17).

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Animals were placed in custom restraints that allow mobility of the head and

body but prevent removal of recording devices or movement out of the field of view.

They were allowed to acclimate to this restraint system and the experiment room until

calm before the test to reduce stress. Clicker training and positive reinforcement were

used when necessary to ensure their cooperation with the test. All methods involving live

animals are approved by the IACUC under protocol 16-U-003.

ORV coated sachets filled with sterile saline instead of active vaccine were obtained from Merial. The approximately 2 ml of saline was extracted via syringe, and replaced via syringe with a barium solution made with E-Z-Paque Barium Sulfate suspension (E-Z EM Inc.) reconstituted with water or apple juice. Prior to resealing the packet, it was pressurized, also via syringe, by inflating it with a can of compressed air. It was then sealed by melting the edges of the sachet punctured by the syringes with an electric wire stripper. Apple juice barium mix was used for some raccoon tests to encourage participation.

Animals were first given the baits and filmed to record their behavior in response to the bait as a novel object. This was done with high speed video cameras only without the fluoroscopes to reduce the exposure to the animals and operators. A number of tests in front of the fluoroscope were then performed in the restraint system to gather data about what happens to the bait once it is punctured and the liquid ingested. Lastly, a few tests using pure barium solution made with water and E-Z-Paque Barium Sulfate suspension (E-Z EM Inc.) powder in a bowl were conducted to observe more instances of each animal swallowing.

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After recording, high speed digital videos (both standard and fluoroscopy) were

watched in slow motion in Qualysis Track Manager software. From these videos, I made subjective observations on extra- and intraoral liquid and sachet handling behaviors. I

also extracted the following variables: the time it took the animal to puncture the bait, the

total number of swallows, the amount of time over which swallows occurred, how many

transports (licks) occurred per swallow, the time of each swallow, and the degree of bolus

clearance. These data were tested to see if they met the criteria for parametric statistical

tests. As they did not, I compared Time to Puncture, Number of Swallows per sequence,

Number of Swallows/Length of Time the packet was open, and Transports

(licks)/Swallow the data using a Mann-Whitney U-Test in SPSS (version 23.0).

Results

Both skunks and raccoons readily consumed the barium solution, and observation

of swallowing behaviors revealed interesting differences about oral handling of liquid

during drinking. Most notably, skunks were observed to create less of a bolus in the vallecula, the space at the back of the tongue in front of the epiglottis, than raccoons and as a result had faster swallows. This appeared to be primarily due to the fact that skunks tended to swallow every time they licked the barium solution. Raccoons appeared to collect fluid in the vallecula resulting in a larger bolus in general but fewer individual swallows.

For bolus clearance, there also were few clear results, again probably due to small sample size. When comparing the female skunk to the male, she had seemingly many more swallows where there was not complete clearance (64% as compared to 22 %).

When compared as a species to raccoons, raccoons appeared to have even fewer

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swallows with complete bolus clearance than skunks (75% as compared to 49%).

However, because raccoons seemed to have fewer swallows with greater volume than

skunks, we were able to observe fewer swallows over the course of the experiment

making these results somewhat suspect. Bolus clearance appeared to be very related to

the animal’s position in the restraint system. The female skunk laid low to the ground for

most of the tests, for example, creating a kink in her esophagus where liquid would get

stuck (Figure 18).

Figure 18. Screenshots from Qualysis showing the female skunk on the left (with incomplete clearance of liquid indicated by arrow) and male skunk on the right for comparison.

In terms of bait access behavior the male grasped baits firmly between both front

paws, turned his head to the side, and bit with teeth in the molar region towards the throat

(Figure 19). He would also pin the bait to the ground with a paw and lick it at several points after the initial puncture.

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Figure 19. Male skunk displayed the same behavior as skunks reported in Jojola et al. (2007).

This behavior held constant from the novel bait introduction through all the

experiments, and even when he was offered normal foods (Figure 20). This appears to be an optimal position to bite the bait to facilitate lymphoid tissue absorption, as it is very close to the back of the throat.

Figure 20. Male skunk eating a sweet potato and pea.

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In contrast, the female skunk rarely adopted the position that was utilized by the

male for accessing the baits. In nearly all tests, she began by pinning the bait to the

ground and nibbling at it with her canine or premolar teeth (Figure 21). This meant that she needed more trials in the experiment box to get the bait open, as even sometimes after we had observed her biting it, she had not punctured it.

Figure 21. Fluoroscope image of the female skunk biting the bait on the bottom of the test cage.

This difference in behavior may be due to individual variation, as we only had

one individual of each species to compare to. It may also be related to sex differences;

female skunks are much smaller than male skunks, and as our previous experiments have

shown, skunks have many physical sex differences. Either way, this behavior is a novel

bait opening technique never before reported in the literature. This study is also the first

to our knowledge to report sex as it relates to bait opening behavior.

Raccoons first respond to a bait by feeling all over it with their paws. Once they

have decided to eat it, they hold the bait between their paws, bite with their canine teeth,

and pull to puncture it (Figure 22). On occasion, the bait will be inserted into the mouth

and chewed vigorously to release the liquid within.

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Figure 22. Image from Qualysis showing usual raccoon bait opening behavior.

Raccoons were much more energetic during the tests than skunks, and thus seemed to treat the baits much more roughly, which would seem consistent with their higher immunization rate since that usually led to bait puncture. However, they were also much more discerning about eating the baits; after a few trials with the baits, they lost interest and had to be given baits filled with apple juice barium solution to participate in the trials. The skunks mostly continued to be interested in baits as long as experiments were being conducted.

All tests comparing the two species were not significant (LicksPerSwallow

U=1037.5, p=0.127; TimeForSwallows U=45.0, p=0.248; NumOfSwallows U=43.0, p=0.293; TimeToPuncture U=24.0, p=0.155). This is likely due to low sample size; only two individuals of each species were used. For the same reason, we were unable to compare between the sexes, although it seems likely there might be differences between the two based on our other results.

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Discussion

First and foremost, future trials using the same methodology should be conducted

to increase sample size and control for any individual variation in the species. The small

sample size of individuals may account for the lack of statistical difference in the

quantitative variables. However, some important information can still be gathered by the

qualitative observations.

The importance of recording demographic information like sex when doing

studies on behavior becomes vitally important in this context. It is difficult to compare

our results to others (which might help increase confidence in them with the current low

sample size) because this information is not known. At least in the case of skunks, sex

appears to have a large effect on both behavior and physiology. For example, female

skunks are known to have significantly smaller home ranges than male skunks (Talbot et

al. 2012). This would be important information when conducting ORV trials, as there is always a question of how to target the animals that are most likely to pass along disease or be successfully immunized. Our results also reveal that there may be a sex difference in skunk bait access behavior, with the female oral behavior being less conducive to successful immunization than male oral behavior. Female skunks in particular should be

further studying to elucidate any behavior differences in bait access and explore options

to resolving the issue. This is a particularly interesting given that male and female skunks

show some sexual dimorphism in body size with males being larger than females.

However, as this was not reflected in the small sample of bite force measurements (i.e.,

the female had a larger bite force than the male), this suggests that the different in accessing the baits may be related to gape.

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This study is also the first to our knowledge to test the animals’ overall interest in

the baits, and the first observations of bait opening behavior in raccoons. Surprisingly, the

raccoons got tired of the baits much more quickly than the skunks did. This is unexpected

considering the attractant (fishmeal) is optimized for raccoons and not skunks (Hanlon et

al. 1989). Clearly, this is relevant when deciding how often to bait. An interesting test to

conduct in the future would be to test animals with larger time periods in between to see

if their previous knowledge of the rabies baits would affect their later behavior when they

encounter one.

Lastly, this study provides a first look at what happens inside the mouth and

oropharynx once the animals ingest the baits. The extremely fast swallow speeds of

skunks versus the longer, larger swallows of raccoons are a difference in the way the

species respond to the baits that could not have been known any either way but has

serious implications for oral immunization due to possible insufficient lymphoid tissue

coverage. Because oral inoculation is known to be effective for skunks with current

vaccine formulas (Brown et al. 2014), one possible way to overcome this behavioral difference is to put more vaccine than 2 ml into the vaccine sachets. Another possible strategy, of course, would be to make an alternate vaccine formula optimized for quick uptake in skunks. A final option might be to increase the viscosity of the vaccine medium so that it coats the oropharyngeal mucosa more or slows the speed of the swallow. Vos et al. (2017) recently published a study demonstrated that striped skunks may have a less

efficient uptake of vaccine virus leading to decreased immunization success. Further

study of that nature about what causes that resistance or what methods may be able to

overcome it should be completed.

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SYNTHESIS

At the beginning of the present study, skeletal measurements were completed.

These indicated the presence of a sex difference between male and female skunks (with female skunks being at a disadvantage to biting compared to male skunks) and the lack of a sex difference between male and female raccoons. Raccoons were also found to have significantly higher mechanical advantages for all jaw adductors and significantly more robust mandibular condyles, corpora, and symphyses. This suggests they are at an advantage for both biting and for resisting the production of higher bite forces than skunks. Because these disadvantages for skunks were both relative and absolute, this suggested that proportionate size reductions in the baits may not be enough to achieve higher rates of immunization in skunks.

Next, Herring and Herring’s (1974) model was used to evaluate the effect of gape and muscle stretch on biting mechanics. Sex differences were minor, except for female skunks having a lower temporalis stretch factor compared to male skunks, suggesting that temporalis muscle stretch may have less impact on bite force production in female skunks. Due to their small size compared to male skunks, this may allow them to compensate and still produce comparable forces. Stretch factors for skunks in the masseter muscle were higher than in raccoons, suggesting that the masseter is negatively affected by stretch, but actually had lower temporalis stretch factors than raccoons.

Because the temporalis muscle is the dominant force production muscle in these species, these results suggest that skunks may not actually be at a disadvantage when it comes to overall force production capability.

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Muscle measurements were then taken to complement these models. Sex differences were minimal. For raccoons, male raccoons had significantly larger PCSAs and thus larger bite force production capability. For skunks, female skunks had significantly longer muscle fibers in one of the muscle groups (indicating maintenance of force production ability during muscle stretch) and larger PCSAs in another one of the groups (corresponding to higher force production capability). Skunks were shown to have significantly longer fiber lengths than raccoons, suggesting that they are more suited to producing high bite forces at large gapes than raccoons. Raccoons had significantly larger

PCSAs than skunks, a measurement which indicates they are at an advantage to producing large bite forces. This again seems to indicate skunks are compensating for their small size and may still be able to compete with raccoons in bite force production capability.

Natural bite forces were then measured on live animals to validate the results of the models. Bite forces were measured at the canines, premolar, and carnassial, at a 1 cm gape and then a 2 cm gape. Contrary to our hypothesis, skunks were able to produce absolutely higher bite forces at the canines than raccoons (with all other tooth regions being nonsignificant). Skunks also were able to bite harder at the 1 cm gape when compared to the 2 cm gape at the premolar tooth position, showing their ability to still produce large bite forces at large gapes. Raccoons, in contrast, had lower bite forces at the carnassial/molar at the 2 cm gape when compared with the 1 cm, as was expected, showing that muscle stretch was affecting their bite force production. Contrary to popular belief, these results indicate that production of bite forces sufficient to break the plastic

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sachet inside the baits is not a problem for skunks. Other explanations thus needed to be

considered.

To further explore explanations for differences in vaccine efficacy, live animals

were given baits filled with a barium solution in front of two fluoroscopes. The raccoons

tired more quickly than the skunks of eating the baits, but were much rougher about

puncturing them (possibly indicating a greater rate of success ingesting vaccine despite

fewer attempts). They were very interested in feeling the baits with their hands and then

biting and pulling to puncture the baits. The male skunk usually held the bait between his

paws and bit into it with his rear teeth, which is the skunk bait consumption behavior

reported in the literature. It would seem to facilitate antibody production, as vaccine

should enter the mouth near the throat. The female skunk, in contrast, performed a bait access behavior not reported in the literature; she held the bait to the ground and took small bites at it with her canines. She seemed to have the most problems puncturing the bait with this strategy out of all the animals. Once the baits were punctured, skunks were shown to swallow much more often and thus create smaller boluses than raccoons. This could possibly contribute to their poor immunization rates, as the vaccine may not have sufficient contact with the lymphoid tissue to induce antibody production.

These studies together seem to indicate a more complicated explanation than

those that have been reported before, such as suggestions that the bait is too large for

skunks to bite with enough force to puncture the plastic sachet. However, we found no

significant differences in force production except that skunks were actually able to bite

harder than raccoons at the canine position. All evidence suggests that skunks have a

fiber architecture conducive to the production of bite forces at large gapes, allowing them

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to out compete raccoons in bite performance. A possible alternative explanation is the

one suggested by the fluoroscopy study, which is that the way skunks swallow does not

allow time for proper lymphoid tissue coverage.

Unfortunately, not much is known about the entry of the rabies virus vaccines in

the lymphoid tissues. A recent study by Vos et al. (2017) begins to investigate this problem by comparing the dissemination of the vaccine virus construct in the lymphoid tissues in the red and the striped skunk. Vaccine absorption in the oral cavity was shown to be much less pronounced in the striped skunk, but for unknown reasons. They discuss that uptake may be influenced by the duration of exposure in the oral cavity, as might be affected by high saliva excretion (Vos et al. 2017). They also mention that the anatomical configuration of the tissue (a Waldeyer’s tonsillar ring with a dominating palatine tonsil) is likely not the issue as red foxes, a highly compliant species, have the same crypt-free tonsil covered within its mucosal fossa. This study complements these results by suggesting that this low uptake may be due to nonoptimal swallowing behavior. This situation will need to be approached with strategies different than simply shrinking bait size.

One possible solution that could be tried is to increase the amount of vaccine included in the sachets; right now, it is only about 2 ml. The research that Vos et al.

(2017) has done could be continued to research why skunk lymphoid tissue is resistant to uptake and test new vaccine formulas to improve uptake. They do mention that all current oral rabies vaccine are based on viruses that require primary virus replication in the body, so perhaps different strategies to make an effective virus should be explored.

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Another factor that has been made apparent through these studies is the importance of studying female skunks as compared to male skunks in these investigations. While their size difference has been well documented, there are many less obvious physical differences (such as muscle architecture) that are critically important to vaccine effectiveness. Behavior differences, also, have not been addressed. Female skunk bait access behavior appears to be less optimal than male skunks, but only the behavior seen in the male skunk has been previously reported in the literature. Female skunks are also known to be philopatric, while male skunks disperse greater distances (Talbot et al.

2012). How this affects oral rabies immunization has not been much discussed in the literature. The consensus mostly seems to be that male skunks should be the focus of

ORV interventions, as they would be the most active sources of .

However, being that in past research raccoon den sites have been a source of concern for their role in disease transmission, along with mother-offspring rabies transmission being considered the cause of juvenile rabies infections peaking in early fall (Hirsch et al. 2013;

R. Rosatte et al. 2006), it seems likely that skunk females probably play a significant role in rabies transmission. Now that skunks are a major rabies vector, future research should address such demographic factors.

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APPENDIX 1 Specimen List

Genus Species Specimen # Sex (F=1, M=2) Procyon lotor 98595 1

Procyon lotor 187894 1

Procyon lotor 275291 1

Procyon lotor 275310 1

Procyon lotor 283195 1

Procyon lotor 286012 1

Procyon lotor 286558 1

Procyon lotor 287001 1

Procyon lotor 81816 2

Procyon lotor 91744 2

Procyon lotor 187893 2

Procyon lotor 265613 2

Procyon lotor 275311 2

Procyon lotor 277762 2

Procyon lotor 286986 2

Procyon lotor 287017 2

Mephitis mephitis 51439 1

Mephitis mephitis 56301 1

Mephitis mephitis 56568 1

Mephitis mephitis 81820 1

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Mephitis mephitis 81822 1

Mephitis mephitis 116479 1

Mephitis mephitis 241175 1

Mephitis mephitis 61768 2

Mephitis mephitis 87236 2

Mephitis mephitis 87237 2

Mephitis mephitis 91727 2

Mephitis mephitis 91749 2

Mephitis mephitis 135424 2

Mephitis mephitis 188534 2

Mephitis mephitis 188537 2