Radiographic Assessment of Dental Pathology and

Radiographic Assessment of Dental Pathology and Abnormalities in Dolphins Author(s): Carolina Loch, Liliane J. Grando, Maria I. Meurer, Michella Zastrow, Angela Fernandes and Paulo C. Simões-Lopes Source: Zoological Science, 34(4):295-299. Published By: Zoological Society of Japan https://doi.org/10.2108/zs160151 URL: http://www.bioone.org/doi/full/10.2108/zs160151

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ZOOLOGICAL SCIENCE 34: 295–299 (2017)

X-rays dolphin teeth

© 2017 Zoological Society of Japan 295

Radiographic Assessment of Dental Pathology and Abnormalities in Dolphins Carolina Loch1,2*, Liliane J. Grando3, Maria I. Meurer3, Michella Zastrow4, Angela Fernandes5, and Paulo C. Simões-Lopes2 1

Sir John Walsh Research Institute, Faculty of Dentistry, University of Otago, Dunedin, New Zealand 2 Departamento de Ecologia e Zoologia, Laboratório de Mamíferos Aquáticos, Universidade Federal de Santa Catarina. Florianópolis, Santa Catarina, Brazil 3 Departamento de Patologia, Centro de Ciências da Saúde, Universidade Federal de Santa Catarina. Florianópolis, Santa Catarina, Brazil 4 RadImagem Digital. Florianópolis, Santa Catarina, Brazil 5 Departamento de Estomatologia, Setor de Ciências da Saúde, Universidade Federal Do Paraná. Curitiba, Paraná, Brazil

This study proposes a simple standardized method for the production of analog X-ray images of dolphin teeth, and to explore its potential use as a complementary technique in the evaluation of dental pathology in small cetaceans. We investigated exposure times that produced the best results, and whether radiographs helped in the diagnosis of macroscopic abnormalities. Teeth of six species of dolphins (Delphinidae: Tursiops truncatus, Steno bredanensis, Sotalia guianensis, Delphinus sp., Stenella coeruleoalba, and Stenella frontalis) were X-rayed in an analog dental X-ray machine operating at 70 kVp and 7 mA. Intraoral size 2 standard films were used, and the focus–film distance was standardised at 35 cm. Those species with smaller teeth (total length 12–20 mm) had the best results when exposed for 0.3 seconds, while species with larger teeth (30–45 mm) had to be exposed for 0.4 seconds for their best result. Three independent examiners analysed all the images taken. The average pairwise percent agreement was 73% (Fleiss’ Kappa = 0.229), suggesting fair agreement between examiners. Analog X-ray images produced were useful in complementing the diagnosis of dental pathology and abnormalities in dolphins, in addition to allowing the observation of internal details and lesion depths, which would not be possible with conventional macroscopic methods. The use of analog X-ray imaging is easily applicable to the study of dolphin teeth, with low operating costs and simple logistics compared to other non-destructive analytical approaches such as Micro-CT. Key words: Cetacea, X-rays, teeth, pulp stones, dental wear

INTRODUCTION Cetaceans—dolphins and whales—have simplified teeth in comparison to most mammals. Dolphins have a single set of teeth that remain in place throughout their lives (monophyodonty), the teeth are undifferentiated and simplified in shape (homodonty), and the number of teeth is muchincreased (polydonty) (Myrick, 1991; Ungar, 2010). Similar to humans and domestic mammals, a range of developmental and pathological conditions may affect the dentition of dolphins. These include disturbances in the size and structure of teeth, physiological tooth wear, and bacterial and non-bacterial tooth structure loss (Loch et al., 2011; Loch and Simões-Lopes, 2013). While the majority of studies on dental pathology in humans and other mammals have employed a range of analytical methods and techniques such as histology, molecular biology and X-ray imaging, the * Corresponding author. E-mail: [email protected]. doi:10.2108/zs160151

same is not true for studies on dental pathology and anomalies in dolphins. Radiographs obtained from specific body parts or organs of deceased-stranded animals are valuable tools in the development of standardized techniques for veterinary clinical practice and also for comparative anatomy studies (Van Bonn and Brook, 2001). Standardized methods of image capture, display, and interpretation, produce consistent radiographic results and thus should be encouraged in both clinical and research settings (Van Bonn et al., 2001). Radiographs have been used in the past to study normal osteological features, including skeletal maturity (Lee, 1978; Stockin et al., 2008), as well as bone lesions in cetaceans and pinnipeds (Alexander et al., 1989; Van Bonn et al., 2001; Bonar et al., 2007). However, the same cannot be said for other hard tissues, such as teeth. Most previous studies on dental pathology and anomalies relied on macroscopic assessment and comparison with published accounts for other species (Ness, 1966; Brooks and Anderson, 1998; Loch et al., 2011, 2013a; Loch and Simões-Lopes, 2013);

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however, a few of these studies employed complementary analytical methods (Loch et al., 2013a, b). Dolphin teeth have been radiographed to characterize the mineral density of growth layer groups (GLG) in dentine and cementum of bottlenose dolphins (Hohn, 1980). Radiographs were also used in age estimation studies with teeth of harbor seals (Norgaard and Larsen, 1991). The use of radiographic methods beyond age determination studies is still very restricted in dolphin teeth. This study aims to propose a simple standardized method for the production of analog X-ray images of dolphin teeth, to investigate the exposure times that produce the best results, and to explore its potential use as a complementary technique in the evaluation of dental pathology in cetacean museum specimens. MATERIALS AND METHODS Specimens Six species of small delphinids were studied: bottlenose dolphin

Table 1. Species, cases of dental anomalies and number of specimens and teeth analyzed via X-rays in this study. Species

Sotalia guianensis (n = 72)

Delphinus sp. (n = 7)

Stenella frontalis (n = 13) Stenella coeruleoalba (n =1)

Steno bredanensis (n = 9)

Tursiops truncatus (n = 22)

TOTAL

Anomaly/pathology

Number of Number specimens of teeth

Mechanical dental wear

30

90

Calculus

13

39

Erosion

9

27

Geminated or fused teeth

7

7

Caries-like lesions

4

4

Root resorption Dental fractures

4 5

12 8

6

18

1

1


6

18

Caries-like lesions Root resorption

5 2

5 6

Dental fractures

1

2


4

12

Caries-like lesions

1

1

Calculus

1

3

Enamel hypoplasia Erosion

2 1

6 3


7

21

Caries-like lesions

3

3

Root resorption

3

9

Calculus

1

3

Enamel hypoplasia

5

12

Dental fractures Geminated or fused teeth

2

4

1

1

124

315

Mechanical dental wear Root resorption

Tursiops truncatus, rough-toothed dolphin Steno bredanensis, Guiana dolphin Sotalia guianensis, common dolphin Delphinus sp., Atlantic spotted dolphin Stenella frontalis, and striped dolphin Stenella coeruleoalba (Table 1). Animals of all age ranges were evaluated. Specimens were accessed in three scientific collections from southern Brazil: Instituto de Pesquisas Cananéia, Paraná (acronym IPeC); Museu de Ciências Naturais UFPR, Paraná (MCN) and Departamento de Ecologia e Zoologia UFSC, Santa Catarina (UFSC). The same specimens had been investigated in three previous studies on the prevalence, clinical characteristics and functional implications of dental wear and dental pathology in dolphins (Loch et al., 2011, 2013 and Loch and Simões-Lopes, 2013). Radiographed specimens consisted of dried loose teeth preserved in alcohol in storage containers following preparation via water maceration and mechanical cleaning. Teeth were selected due to the presence of pathology and/or abnormalities observed macroscopically in the crown or roots of the teeth (following Loch et al., 2011 and Loch and Simões-Lopes, 2013). These include pathological processes such as caries-like lesions, root resorption, and tooth erosion; developmental and/or normal physiological processes such as dental fusion and gemination, enamel hypoplasia, calculus deposits and physical dental wear; and traumatic processes such as dental fractures (Table 2). No skulls or jaws were radiographed. An average of 30% of the specimens with macroscopic abnormalities was radiographed. In cases of pathologies and anomalies restricted to a small number of teeth, the teeth affected were

Table 2. Description of cases of dental pathology and/or abnormalities investigated via analog radiography. Definitions follow Goga et al. (2008), Loch et al. (2011), Loch and Simões-Lopes (2013). Pathology/Abnormality

Description

Pathological processes Caries-like lesions

Cavity formed by destruction and loss of dental hard tissues due to presumed bacterial fermentation.

Root resorption

Resorption and loss of root tissue due to trauma and excessive stress. Chemical wear of dental hard tissues, resulting in shallow defects in enamel and/or cupping of the dentine.

Erosion

Developmental/Physiological processes Mechanical dental wear

Mechanical loss of dental hard tissues caused by attrition and/ or abrasion.

Geminated or fused teeth

Teeth with bifid crowns and single roots.

Pulp stones

Discrete calcifications embedded within the dentine.

Enamel hypoplasia

Pits, horizontal lines, grooves or missing areas of enamel. Adherent mineral substance firmly attached to the tooth surface.

Calculus

Traumatic processes Dental fractures

Loss of portions of the crown and/or cervix due to trauma.


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selected for X-ray analyses. In cases of abnormalities with high prevalence among individuals, such as in the cases of dental wear, two or three representative teeth per specimen were selected for analysis. Three examiners analyzed the images independently and inter-rater reliability was calculated. Radiography standardization Radiographs were taken in a wallmounted Spectro 1070 analog dental X-ray machine (Dabi Atlante, Ribeirão Fig. 1. (A) Overview of equipment set up for X-ray imaging of isolated dolphin teeth. (B) Detail of Preto, Brazil), operating at 70 kVp and 7 bottlenose dolphin tooth placed over intra-oral film, with a lead number label on the lower left mA (Fig. 1A). Intraoral size 2 (31 × 41 corner. mm) standard films were used (Kodak E-Speed) and each radiograph was identified with a small numbered lead plate (Fig. 1B). Selected teeth were positioned on individual films with either the mesial or distal faces facing upwards; however, sometimes either the lingual or buccal faces faced upwards depending on the location of the pathology/alteration. When needed, a small amount of dental wax was used to secure the specimens in place. The X-ray source was always located directly above the specimen and film. The distance from the margin of Fig. 2. Standardization of the X-ray exposure times for teeth of the bottlenose dolphin (T. truncatus). the tubehead to the film was stan(A) 0.1 sec. (B) 0.2 sec. (C) 0.3 sec. (D) 0.4 sec. Scale bar = 1 cm. dardised at 20 cm, resulting in a focusfilm distance (FFD) of 35 cm. In order to standardize the appropriate exposure times, two different groups were produced: 1) species with larger teeth (T. truncatus and S. bredanensis, tooth length 30 to 45 mm), and 2) species with smaller teeth (S. guianensis, S. frontalis, S. coeruleoalba and D. capensis, tooth length 12 to 20 mm). Exposure times tested for both groups were 0.1 sec., 0.2 sec., 0.3 sec., 0.4 sec. Radiographs were manually processed in a dark room using the time-temperature developing method. This method establishes that if a developing solution of fixed temperature is used, the films are immersed in that solution for a specific length of time (Whaites and Drage, 2003). Intraoral standard films used were immersed for four minutes in the developing solution and 10 minutes in the fixing solution, both at 20°C. Developed films were then washed in clean tap water and dried at room temperature for 12 hours.

RESULTS Method standardization The exposure times that generated the best results were 0.3 sec for species with smaller teeth (S. guianensis, S. frontalis, S. coeruleoalba and Delphinus sp.) and 0.4 sec for species with larger teeth (T. truncatus and S. bredanensis). For both groups, shorter exposure times produced lighter images and longer exposure times resulted in denser images. Overexposed teeth resulted in images that were considerably darker (Fig. 2). X-ray images Three independent examiners analysed all the images taken. The average pairwise percent agreement was 73%

Fig. 3. (A) Calculus deposits (arrows) in teeth of the bottlenose dolphin T. truncatus (UFSC 1317). (B) Loss of dental tissue due to erosion (arrows) in teeth of the rough toothed dolphin S. bredanensis (IPeC 015). Scale bar = 1 cm.

(Fleiss’ Kappa = 0.229), suggesting fair agreement between examiners. Anomalies located at the crown and cingulum of the teeth, in addition to root and pulp anomalies not visible through macroscopic examination, were assessed. X-ray images enhanced the diagnostic accuracy of abnormalities, resulting in improvement in detecting anomalies and pathol-

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ogies with radiographs, rather than with visual examination alone. For the majority of the cases, the radiographic assessment confirmed the macroscopic diagnosis of dental abnormalities and allowed the estimation of lesion depths, coverage and thickness (Fig. 3A, B). X-ray images also showed evidence of pulp stones, root resorptions, partial or total closure of root apices and pulp cavity exposure due to excessive mechanical dental wear, which would not be possible with conventional macroscopic methods (Fig. 4A, B). Abnormal root spaces associated with pulp exposure and excessive mechanical wear were observed in a few specimens, while others with less prominent wear showed a different obliteration pattern (Fig. 4B). Cases of dental fusion and gemination were also observed. X-ray images allowed for appropriate differential diagnosis of these abnormalities. In particular, cases of dental fusion presented a doubled/expanded pulp space while geminated teeth had a similar pulp space to normal teeth (Fig. 5). DISCUSSION In most analog dental X-ray machines which operate with constant values for kilovoltage (kV) and Milliamperage (mA), the standardization of exposure times and control of film development variables through the time-temperature method are the main alternatives to regulate and standardize the density of radiographs (Whaites and Drage, 2003). In

Fig. 4. (A) Pulp stone in a tooth of the bottlenose dolphin T. truncatus (UFSC 1209). (B) Mechanical dental wear in teeth of the Guiana dolphin, S. guianensis (IPeC 199). The specimen at the right has an exposed pulp cavity in association with the wear in the crown. (C) Root resorption in a tooth of the Guiana dolphin S. guianensis (IPeC 225). Scale bar = 1 cm.

Fig. 5. (A) Normal (left) and fused tooth (right) in the Guiana dolphin S. guianensis (MCN 168). (B) Normal (left) and geminated tooth (right) in the Guiana dolphin S. guianensis (MCN 28). Scale bar = 1 cm.

our study, the standardization of exposure times based on the size of the teeth, in addition to control of time and temperature during film development, were fundamental steps to produce useful X-ray images with good quality. In this investigation, the use of analog intra-oral radiography was a viable technique to complement the diagnosis of dental pathology and abnormalities in dolphins. The methodology was easily applicable and the operating costs were low. X-ray imaging might be a useful technique for multidisciplinary research centers which already have operating X-ray equipment, or for researchers who can access those services at dental X-ray clinics. With the increased replacement of analog by digital X-ray machines, it is possible that more analog equipment will be decommissioned from private practices, thus potentially making it available for research use. Another technique available for imaging of internal morphological structures of teeth is Micro-Computed Tomography (Micro-CT). Micro-CT is a non-invasive and non-destructive technique that allows 3-dimensional (3-D) study of mineralized tissues and their physical properties, and its use has been trialed for fossil and modern dolphin teeth (Loch et al., 2013b). Despite its advantages, Micro-CT is still mostly avoided for mainstream research due to high equipment costs, the length of time required for scanning, specimen size requirements and high computing power needed to process the scans. The results obtained in this study showed that analog X-ray images are readily applicable to the complementary diagnosis of pathology and abnormality in dolphin teeth. In addition to the complementary diagnosis, images also assisted in revealing alterations and anomalies in the pulp region and within the dentine layer, which would otherwise not be observed through conventional macroscopic inspection. Developmental/physiological abnormalities such as pulp stones have already been detected and characterized in previous histological investigations (Perrin and Myrick, 1980; Goga et al., 2008; Luque et al., 2013). However, abnormalities in the internal structure of the pulp cavity, particularly those associated with cases of extreme wear and erosion, have not been appropriately documented before. Common age determination methods use methodologies that involve mechanical grinding and decalcification of hard tissues (Hohn, 1990), which could alter and hamper the identification of anomalies in hard tissues. On the other hand, X-ray imaging is non-destructive and logistically easy, since no sectioning or chemical treatment is needed, preserving the physical integrity of the specimens. X-ray images also allowed for the differential diagnosis of fusion and gemination cases due to the increased size of the pulp space in fusion cases. As most cetaceans have numerous homodont teeth and dental formulas are widely variable, the number of teeth is not a reliable factor in the diagnosis of fusion and gemination cases, as it is for human teeth. In humans, the observation of a lower tooth count would be consistent with fusion cases, while cases of gemination do not alter the final tooth count (Schuurs and van Loveren, 2000). Other potential research applications of X-ray imaging of cetacean teeth include the possibility of performing basic linear measurements of internal structures such as dimensions of the pulp cavity, provided that the


images have been taken consistently and the magnification factors are known. ACKNOWLEDGMENTS We sincerely acknowledge the curators of the scientific collections (Emygdio Monteiro-Filho, IpeC; Fernando Sedor, MCN) for allowing us to access specimens under their care. Thanks are also extended to the staff at the Laboratório de Ensino e Pesquisa de Imaginologia (UFPR, Curitiba, Brazil) and RADImagem Digital (Florianopolis, Brazil) for their valuable logistic support. Karla Rovaris (UEPB, Brazil) kindly reviewed early drafts of this manuscript and Glynny Kieser helped with proofreading and editorial review. We are indebted to two anonymous reviewers whose suggestions greatly improved this paper. Cetacean Society International through Bill Rossiter provided financial support for this research. Carolina Loch acknowledges Conselho Nacional de Desenvolvimento Científico e Tecnológico-CNPq for a MSc. Scholarship (process number 132356/2007-4) and Programa de Pós-graduação em Ciências Biológicas–Zoologia/UFPR for institutional support. Current support for Carolina Loch is provided by a University of Otago Research Fellowship.

COMPETING INTERESTS The authors have no competing interests to declare.

AUTHOR CONTRIBUTIONS CL, LJG, PCSL conceived and designed the study; MIM, MZ, AF contributed with analytical tools; CL performed the experiments; CL, LJG, PCSL, MIM, MZ, AF wrote the paper.

REFERENCES Alexander JW, Solangi MA, Riegel LS (1989) Vertebral osteomyelitis and suspected diskospondylitis in an Atlantic bottlenose dolphin (Tursiops truncatus). J Wildl Dis 25: 118–121 Bonar CJ, Boede EO, Hartmann MG, Lowenstein-Whaley J, Mujica-Jorquera E, Parish SV, et al. (2007) A retrospective study of pathologic findings in the Amazon and Orinoco river dolphin (Inia geoffrensis) in captivity. J Zoo Wildl Med 38: 177–191 Brooks L, Anderson HF (1998) Dental anomalies in Bottlenose Dolphins, Tursiops truncatus, from the west coast of Florida. Mar Mamm Sci 14: 849–853 Goga R, Chandler NP, Oginni AO (2008) Pulp stones: a review. Int Endod J 41: 457–468 Hohn A (1980) Analysis of growth layers in the teeth of Tursiops truncatus, using light microscopy, microradiography and SEM. Rep Int Whal Comm 3: 155–160 Hohn A (1990) Reading between the lines: analysis of age estimation in dolphins. In “The Bottlenose dolphin” Ed by Leatherwood S, Reeves R, Academic Press, San Diego, pp 575–586

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Lee KE (1978) Radiographic anatomy and development of the cetacean flipper. DM Thesis, Yale University School of Medicine, New Haven Loch C, Simões-Lopes PC (2013) Dental wear in dolphins (Cetacea: Delphinidae) from southern Brazil. Arch Oral Biol 58: 134–141 Loch C, Grando LJ, Kieser JA, Simões-Lopes PC (2011) Dental pathology in dolphins (Cetacea: Delphinidae) from the southern coast of Brazil. Dis Aquat Org 94: 225–234 Loch C, Grando LJ, Schwass DR, Kieser JA, Fordyce RE, Simões-Lopes PC (2013a) Dental erosion in South Atlantic dolphins (Cetacea: Delphinidae): a macro and microscopic approach. Mar Mamm Sci 29: 338–347 Loch C, Schwass DR, Kieser JA, Fordyce RE (2013b) Use of Microcomputed tomography for dental studies in modern and fossil odontocetes: potential applications and limitations. NAMMCO Scientific Publications 10, doi: http://dx.doi.org/10.7557/3.2616 Luque PL, Pierce GJ, Learmonth JA, Ieno E, Santos B, Lopez A, Lockyer CH (2013) Are mineralization anomalies in common dolphin teeth associated with life-history events and/or the exposure to anthropogenic pollutants? J Zool 291: 194–204 Myrick AC Jr (1991) Some new and potential uses of dental layers in studying delphinid populations. In “Dolphin Societies: Discoveries and Puzzles” Ed by K Pryor, KS Norris, University of California Press, Los Angeles, pp 251–279 Ness AR (1966) Dental caries in the platanistid whale Inia geoffrensis. J Comp Pathol 76: 271–279 Norgaard N, Larsen BH (1991) Age determination of harbour seals, Phoca vitulina, by cementum growth layers, x-ray of teeth and body length. Dan Rev Game Biol 14: 17–32 Perrin WF, Myrick AC (1980) Report of the workshop. Rep Int Whal Comm (Special Issue) 3: 1–50 Schuurs AHB, Van Loveren C (2000) Double teeth: review of the literature. J Dent Child 67: 313–325 Stockin KA, Wiseman N, Hartman A, Moffat N, Roe WD (2008) Use of radiography to determine age class and assist with the post-mortem diagnostics of a Bryde’s whale (Balaenoptera brydei). New Zeal J Mar Fresh 42: 307–313 Ungar P (2010) Mammal teeth: origin, evolution and diversity. The Johns Hopkins University Press, Baltimore Van Bonn W, Brook F (2000) Overview of diagnostic imaging. In “CRC Handbook of marine mammal medicine” Ed Dierauf LA, Gulland FMD, CRC Press, Florida, pp 551–556 Van Bonn W, Jensen ED, Brook F (2001) Radiology, computed tomography and magnetic resonance imaging. In “CRC Handbook of marine mammal medicine” Ed by LA Dierauf, FMD Gulland, CRC Press, Florida, pp 557–592 Whaites E, Drage N (2013) Essentials of dental radiography and radiology. Elsevier Health Sciences, Edinburgh (Received September 11, 2016 / Accepted March 12, 2017)