Tetracycline Labeled Bone Content Analysis of Ancient Nubian Remains from Kulubnarti

TETRACYCLINE LABELED BONE CONTENT ANALYSIS OF ANCIENT NUBIAN REMAINS FROM KULUBNARTI

THESIS

Presented in Partial Fulfillment of the Requirements for the Degree Master of Arts in the Graduate School of The Ohio State University

Julie Anna Margolis

Graduate Program in Anthropology

The Ohio State University

2015

Master's Examination Committee:

Dr. Clark Spencer Larsen, co-advisor

Dr. Samuel D. Stout, co-advisor

Dr. Douglas E. Crews

Copyrighted by

Julie Anna Margolis

2015

ABSTRACT

Armelagos and colleagues (2001) have hypothesized that beer is a conduit for in vivo tetracycline consumption by ancient Nubians. Streptomycetes bacteria has a high prevalence in Sudanese-Nubian soil (60 -70%) and secretes the antibiotic under harsh conditions such as fermentation. At the site of Kulubnarti, 21-S-46 cemetery (716 CE) skeletons likely represent a working underclass contemporaneous with the 21-R-2 cemetery (752 CE) containing the remains of a land-owning class. Interpretations of archaeological and osteological evidence suggest that poorer health and higher mortality occurred in the S population. To test whether an anticipated difference in tetracycline ingestion between S and R cemetery populations existed, the amount of tetracycline- labeled bone was quantified under ultra violet light using image analysis software.

Amount of tetracycline labeling was expressed in terms of the total area of labeled bone tissue in square micrometers, number of labeled osteons, and number of grid intersections over labeled bone. No significant differences in percent tetracycline-labeled bone tissue, or percent labeled osteons was observed between cemeteries. These results suggest that tetracycline ingestion was similar for S and R group members, class differences were not mediating tetracycline ingestion, and both sub-groups had equal access to beer.

Dedicated to my mentor, George J. Armelagos.

iii

ACKNOWLEDGMENTS

This thesis would not have been possible without support from my parents, friends, and mentors. I would like to thank my committee co-chairs Dr. Samuel D. Stout and Dr. Clark S. Larsen, committee member Dr. Douglas E. Crews, and Dr. Mark Hubbe for their guidance and helpful comments regarding my writing and presentation of statistics. I am grateful to my friend Sara Becker (MSc. Maastricht University) for translating Pipenbrink’s 1983 work from German to English for me. I extend thanks to the Department of Anthropology at Emory University for allowing me to use their facilities and resources for data collection. I thank Nicole L. Henderson for having been an incredible lab assistant and for helping me test my proposed technique of image analysis by re-measuring several samples as part of a preliminary inter-observer study.

Most of all, I would like to thank Dr. George J. Armelagos and Dr. Dennis P. Van

Gerven, as without them this project would not have been possible. I am extremely grateful to Dennis for providing the Kulubnarti rib cross-sections for me to work with in the first place, helping me work through and understand my statistics, and answering a vast array of questions on his previous studies of ancient Nubia. As for George, I cannot express how thankful and appreciative I am for everything he did for me. George gave me this project to work on originally, gave me access to all of his equipment and resources (including funding for materials), answered my endless questions, acted as a

iv sounding board for me to develop further research questions, and provided guidance as only a friend and mentor can in their ideal form. George prepared me for graduate school and a career in anthropology, and without him I would not be where I am today. I am also thankful that, while George did not see the final written version of this thesis before his death (only earlier drafts), he was able to see this project of ours through to the end of my analysis and the future directions of my research. I will be forever grateful to George

Armelagos, for everything.

VITA

2008...... Diploma, Libertyville High School

2010...... A.A. Anthropology, Oxford College of Emory University

2012...... B.A. Anthropology, Emory University

2012 to present ...... Graduate Student, Department of

Anthropology, The Ohio State University

Publications

Margolis JA, Van Gerven DP, and Armelagos GJ. 2013. Tetracycline labeling in early

Christian burials from Kulubnarti, Nubia: Measure of class differences. American Journal of Physical Anthropology Supplement 56, 2013 (Annual Meeting Issue):189.

Fields of Study

Major Field: Anthropology

Table of Contents

ABSTRACT ...... ii

ACKNOWLEDGMENTS ...... iv

VITA ...... vi

Publications ...... vi

Fields of Study ...... vi

Table of Contents ...... vii

List of Tables ...... ix

List of Figures ...... x

CHAPTER 1: INTRODUCTION ...... 1

Purpose ...... 1

Background of Kulubnarti ...... 2

Health of the Kulubnarti Populace ...... 5

Nubian Antibiotics ...... 7

Why Study Tetracycline Consumption at Kulubnarti? ...... 10

CHAPTER 2: MATERIALS AND METHODS ...... 12

vii

Materials ...... 12

Methods ...... 14

New Technique of Image Analysis Method ...... 14

Osteon Count Method ...... 17

Point-Count Method ...... 17

CHAPTER 3: RESULTS ...... 19

CHAPTER 4: DISCUSSION ...... 24

CHAPTER 5: CONCLUSIONS ...... 31

REFERENCES ...... 33

APPENDIX A: RAW DATA ...... 37

APPENDIX B: RIB CROSS-SECTION PHOTOS ...... 39

viii

List of Tables

Table 1: Sample specimens and associated sex and age estimations from Kulubnarti .... 13

Table 2: Percent averages of tetracycline-labeled bone for all ages at Kulubnarti ...... 20

Table 3: Percent averages of tetracycline-labeled bone among those aged 12 and older at

Kulubnarti ...... 23

Table 4: Kulubnarti cemetery 21-S-46 (S Cemetery) raw data ...... 37

Table 5: Kulubnarti cemetery 21-R-2 (R Cemetery) raw data…………………..………38

List of Figures

Figure 1: Map of Nubia (Turner et al. 2007) ...... 3

Figure 2: Tetracycline molecule ...... 8

Figure 3: Rib cross-section from specimen S48 before digital cleaning ...... 15

Figure 4: Rib cross-section from specimen S48 after digital cleaning ...... 16

Figure 5: Masks used for measurement of total bone area in microns (left) and tetracycline-labeled bone area in microns (right) for specimen S48 ...... 17

Figure 6: Frequency of tetracycline-labeled bone area (in microns) by age for both cemeteries at Kulubnarti (loess line of fit included) ...... 21

CHAPTER 1: INTRODUCTION

Purpose

Ancient Nubians were ingesting the antibiotic, tetracycline, likely through beer

(Bassett et al. 1980; Hummert and Van Gerven 1982; Keith and Armelagos 1988;

Armelagos et al. 2001). If tetracycline was ingested through a culturally influenced activity as part of the daily lives of the ancient Nubians, then the consumption of tetracycline should vary with cultural-behavioral differences. To test this hypothesis, amounts of tetracycline labeling of skeletal remains from two contemporaneous cemeteries were compared. At the site of Kulubnarti, 21-S-46 cemetery (716 CE) skeletons likely represent a working underclass, and the 21-R-2 cemetery (752 CE) contains the remains of a land-owning class (Adams and Adams 2007). Interpretations of archaeological and osteological evidence also suggest that poorer health and higher mortality occurred in the S population (Van Gerven et al. 1995; Adams and Adams

2007).

Past studies measuring amounts of tetracycline-labeled bone in ancient Nubians, such as those conducted by and Bassett et al. (1980) and Hummert and Van Gerven

(1982), were limited by available technology and methods of the time. Results of the current study highlight the merit of reexamination with newer methods; and demonstrate

1 how skeletal biology and bioarchaeology can augment our understanding of biocultural interactions in the past.

Background of Kulubnarti

The site of Kulubnarti contains two, cotemporaneous, Christian cemeteries dated to AD 550-800 (Turner et al. 2007) located within the Batn el Hajar (or “belly of rock”) region of Upper Nubia (Fig. 1). This "barren region of rocks and rapids" extends for 150 miles along the Nile, from the Second Cataract to the Dal Cataract (Van Gerven et al.

1981). Kulubnarti is a penninsula of tall granite outcrops, connected to the west riverbank of the Nile. In modern times, an island was formed when the peninsula separated from the mainland by flooding, resulting from construction of the Aswan Dam (Van Gerven et al. 1981; Campbell Hibbs 2010). Beyond the Nile in this region, the desert is "a virtually lifeless expanse of rocky jebels (large outcroppings of rock) interspersed with pockets of sandy wadi" (Van Gerven et al. 1995: 468).

Regional subsistence patterns have changed relatively little over the course of

5,000 years, consisting predominately of small-scale farming villages, which were marginal at best. Villages depended on retaining walls in order to protect alluvial soils necessary for agriculture. These soils existed only in pockets and coves, and not as a continuous floodplain. Crops were irrigated by means of a saqia water wheel (Van

Gerven et al. 1995) and annual flooding (Campbell Hibbs 2010). Staple crops consisted of beans, barely, lentils, peas, sorghum, millet, dates, and wheat. Domestication of a few

2 animals such as cattle, sheep, and pigs was employed, but animal products appeared to have been a minor part of the diet (Van Gerven et al. 1995).

Figure 1: Map of Nubia (Turner et al. 2007)

Trace element and isotope analyses, coprolite analyses, and skeletal indicators of stress suggest the diet at Kulubnarti was reflective of poor agricultural yields (Martin et 3 al. 1989; Sheridan 1992; White et al. 2004; Turner et al. 2007). Meager harvests likely were due to the inhospitable nature of the Batn el Hajar, low water levels in the Nile, and general isolation and poverty of the people inhabiting Kulubnarti. This diet has been estimated to be deficient in vitamins C, B6, B12, folacin, and protein; but high in phytates, fiber, tannins, and phosphorous, which all inhibit iron uptake (Carlson et al. 1974;

Sheridan 1992; Van Gerven et al. 1995; Turner et al. 2007). Most dietary sources of protein came from beans and legumes, which are low in iron content in comparison to animal products. Due to this diet, in conjunction with the high likelihood of parasites, such as hookworm, which may cause intestinal bleeding, it is probable that the population suffered from iron-deficiency anemia (Martin et al. 1989; Sheridan 1992; Mittler and Van

Gerven 1994). However, despite widespread poverty and malnutrition of the Kulubnarti populace, class divisions may still have been present (Van Gerven et al. 1995; Adams

1999; Adams and Adams 2007; Turner et al. 2007).

Ethnographic and historical evidence indicates that within Nubian society are social distinctions between land-owners and itinerate farmers working the land (Adams and Adams 2007). Interpretations that the Kulubnarti R and S cemeteries represent communities of each respective class are supported by archaeological and paleopathological evidence (Van Gerven et al. 1995; Adams 1999; Adams and Adams

2007; Turner et al. 2007). Archaeological evidence consists primarily of differences in architecture and burial goods associated with wealth, such as more elaborate textiles

(Adams and Adams 2007; Turner et al. 2007). Higher frequencies of material remains associated with greater wealth (according to estimated production efforts of particular

4 item qualities) are found at the R cemetery site, and higher frequencies of structures and lower quality goods, associated with lesser wealth, are found at the S cemetery site

(Adams 1999; Adams and Adams 2007). All skeletal markers of stress indicate that the remains interred in the S cemetery suffered poorer health than in the R cemetery community, particularly among sub-adults (Martin et al. 1984; Martin et al. 1989; Mittler and Van Gerven 1994; Van Gerven et al. 1995). Higher sub-adult mortality rates and growth retardation, cribra orbitalia lesion frequencies, and distributions of enamel hypoplasia frequencies have been reported in the S cemetery group compared to the R

(Van Gerven et al. 1981; Martin et al. 1984; Martin et al. 1989; Van Gerven et al. 1990;

Mittler and Van Gerven 1994; Van Gerven et al. 1995).

Health of the Kulubnarti Populace

Cribra orbitalia, a form of porotic hyperostosis associated with childhood nutritional stress (Martin et al. 1989; Van Gerven et al. 1995), is highly prevalent in remains from both the R and S cemeteries of Kulubnarti; with more than 80% of individuals from the Kulubnarti cemeteries exhibiting lesions characteristic of cribra orbitalia (Mittler and Van Gerven 1994; Van Gerven et al. 1995). This condition is characterized by porous, pathological lesions in the bones of the orbit which form through the expansion of bone marrow as the body attempts to compensate for the inadequate production of red blood cells associated with various forms of anemia, suffered during childhood stages of growth and development (Lallo et al. 1977; Sheridan

1992; Mittler and Van Gerven 1994; Van Gerven et al. 1995; Wapler et al. 2004; Walker et al. 2009; Oxenham et al. 2010). Traditionally, the pathology has been associated with iron-deficiency anemia (Carlson et al. 1974; Lallo et al. 1977; Mittler and Van Gerven

1994; Van Gerven et al. 1995; Oxenham et al. 2010), though arguments have been made for its attribution to other forms of anemia as well, such as hemolytic or megaloblastic anemias (Wapler et al. 2004; Walker et al. 2009). These other types of anemia are also associated with nutritional deficiencies and are able to produce the sort of marrow hypertrophy characteristic of porotic hyperostosis (Walker et al. 2009). However, regardless of the type of anemia, the presence of cribra orbitalia is associated with childhood anemias and malnutrition; from which the Kulubnarti populations likely suffered (Sheridan 1992; Mittler and Van Gerven 1994; Van Gerven et al. 1995; Turner et al. 2007).

Further indicators of dietary stress found at Kulubnarti include enamel hypoplasias and osteoporosis. Enamel hypoplasias represent episodes of childhood nutritional stress resulting in the disruption of ameloblast activity causing zones of enamel to be reduced in thickness (Van Gerven et al. 1995). Every individual at

Kulubnarti exhibited evidence of at least one hypoplasmic event. In addition, the dental age in 70.5% of individuals at Kulubnarti exceeded their estimated age as reflected by other skeletal markers, suggesting that there had been significant skeletal growth retardation during development (Van Gerven et al. 1995). It has also been suggested that the Kulubnarti diet may have been deficient in calcium (Sheridan 1992). While calcium deficiency may have impacted health aspects such as bone tissue production and

6 reproductive capabilities, it would not affect percentages of bone labeled with tetracycline.

However, several cultural factors may have actually served as buffers to the morbidity of the Kulubnarti populations. For instance, Campbell Hibbs (2010) suggests that irrigation practices may have buffered infection rates of schistosomiasis at

Kulubnarti. During the Christian period, it is likely that most irrigation at Kulubnarti was a result of annual flooding, rather than irrigation canals. While saqia water wheels were used by the end of the Christian period, there is little to no evidence of their usage before this time. Lack of irrigation canals and standing water therefore reduced the chance of exposure to water-born parasites - especially schistosomiasis (a deadly parasite carried by host snails) - at Kulubnarti, compared to other ancient Nubian sites. Campbell Hibbs

(2010) detected significantly fewer antigens for Schistosoma mansoni in desiccated tissue samples from Kulubnarti than in Wadi Halfa samples. Infection from schistosomiasis would have further compounded the effects of malnutrition causing additional diarrhea, growth retardation, more severe symptoms of anemia, and a variety of other disabling factors (Campbell Hibbs 2010).

Nubian Antibiotics

Evidence of consumption of the broad spectrum antibiotic, tetracycline, has been observed in Nubian remains from the sites of Kulubnarti and Wadi Halfa (Bassett et al.

1980; Hummert and Van Gerven 1982; Armelagos et al. 2001). "Rediscovered" in the

7 modern era, tetracycline molecules (Fig. 2) are composed of four six-membered rings fused linearly - to which a variety of functional groups attach – with characteristic double bond arrangements (Frost et al. 1961; Dürckheimer 1975; Chopra and Roberts 2001)

Tetracycline binds to calcium when present and therefore labels bone undergoing mineralization at the time of ingestion. This labeling appears as a yellow-green fluorescence when thin sections of bone are viewed microscopically under an ultraviolet light at 490 nanometers (Bassett et al. 1980; Armelagos et al. 2001). Since fluorescence patterns indicate that osteons were labeled at various times during their formation, and the remains do not show any signs of post-mortem mold infestation, it is likely that tetracycline was ingested in vivo (Keith and Armelagos 1988).

H N(CH3)2 CH3 OH H H

CONH2

OH OH O OH O

Figure 2: Tetracycline molecule

The bacterium which produces tetracycline, Streptomycetes, flourishes in warm, dry, and alkaline environments. Therefore, mud pots in which ancient Nubians stored grain for brewing beer, produced a perfect environment for cultivating Streptomycetes. In addition, Streptomycetes comprises 60 to 70% of soil bacteria in Sudanese Nubia.

Consumption of beer seems the ideal conduit for tetracycline ingestion and is favored over an alternative hypothesis of eating bread baked with Streptomycetes contaminated grain during a food shortage. This suggestion is based on the nature of tetracycline with regard to its production and how it labels bone. Streptomycetes will only secrete tetracycline under harsh environmental conditions, such as those created by fermentation processes (Armelagos et al. 2001). Generally, 250 mg of tetracycline is considered a safe therapeutic dosage in modern medicine (Frost et al. 1961; Delaney et al. 1974; Sauer

1976; Basset et al. 1980; Martin et al. 1989); and only 1 to 2 grams of tetracycline per day are required to produce fluorescence in human bones (Armelagos et al. 2001; Fabsits

2008). Any observable labeling in human bone must therefore result from ingesting tetracycline at therapeutic dosages. Thus, the amount of bone labeled by tetracycline at

Kulubnarti appears to be a level of intake too high to have occurred from chance exposure, but rather appears to arise from a culturally sanctioned activity (Bassett et al.

1980).

Why Study Tetracycline Consumption at Kulubnarti?

For years bioarchaeologists and paleopathologists in particular, have studied health and its cultural implications in ancient populations from patterns of skeletal morphologies indicative of nutritional status, activity levels, morbidity and mortality

(Larsen 1987; Goodman 1993; Armelagos 2003; Armelagos and Van Gerven 2003).

However, information which can be obtained through analyses of skeletal indicators of health is often complicated and limited. These skeletal markers are often only present in individuals who survived a disease or period of nutritional stress long enough for the stressor to leave its mark in bone (Goodman 1993; Ortner 2003). Bioarchaeologists, therefore, must rely on the general patterns of health indicators which emerge from the skeletal analysis of a variety of morphological and histological features observed within an archaeological population in order to draw conclusions (Goodman 1993; Armelagos

2003; Armelagos and Van Gerven 2003).

The presence of tetracycline labeled bone provides a unique opportunity for studying health in ancient Nubian populations. Since tetracycline and its effects on the body are able to be studied in modern clinical settings, any influence tetracycline has on health in modern populations can be extrapolated to ancient populations, even when physical evidence is limited. Such analogies are possible provided the environments in which effects are produced are similar. However, when used with other skeletal indicators relating to health and biocultural interactions, the environmental context in which the population lived can be reconstructed (Goodman 1993; Armelagos 2003;

Armelagos and Van Gerven 2003; Larsen 2015). Understanding past biocultural

10 interactions in particular ecological contexts can then be applied to understand biocultural interactions in living populations. Results from this project may also be used in future research to further understand how tetracycline affects human health in nutritionally challenging environments.

Summary

The ancient Nubians of Kulubnarti were a marginal farming community of the inhospitable Batn el Hajar region. Freehold farmers and itinerate sharecroppers were buried with their peers in two separate, Christian cemeteries in association with material remains reflective of their status. Despite the generally poor diet of this peasant community, there is skeletal evidence indicating that the sharecroppers were even more nutritionally stressed than their land-owning neighbors. Additionally, both cohorts exhibit evidence of having ingested the antibiotic tetracycline – likely through their dietary staple, beer. Whether or not social status influenced access to tetracycline is under investigation in this study.

CHAPTER 2: MATERIALS AND METHODS

Materials

Thirty-eight thin rib cross-sections from Kulubnarti were analyzed. Previously, rib samples were embedded in epoxy resin and ground down to a thickness approximating 100 micrometers. S and R cemetery sub-group samples consisted of 19 single burials (n = 38), matched for sex and age as closely as possible using available slides and data (Table 1). Aided by the remarkable preservation and often natural mummification of the remains, age at death and sex were estimated utilizing several methods, detailed by Van Gerven et al. (1981). These methods included visual examination of preserved external genitalia, pelvic morphologies, epiphyseal unions, dental eruptions, and changes in the os pubis. Eleven sub-adults age 16 or younger and eight adults were selected from each cemetery. Sub-adult females were under-represented in the sample. This under-representation likely stems from difficulties estimating the sex for pre-pubescent skeletal remains as noted by Phenice (1967) and varying degrees of natural mummification. Comparative analysis of the sub-adult populations by sex is therefore not presented. Sample size was limited to n=38 by access to materials and funding.

Table 1: Sample specimens and associated sex and age estimations from Kulubnarti

Specimens Specimens by Burial Sex Age by Burial Sex Age Number Number S13 male 12 years R68 male 12 years S62 male 14 years R146 male 15 years S68 a male 4 years R147 male 16 years S236 male 16 years R196 male 1.5 years S36 ? 13 years R18 female 15 years S23 ? 5 years R84 female 2 years S28 ? 7 years R192 female 16 years S37 ? 1 year R21 ? 6 years S41 ? 15 years R142 ? 9 years S47 ? 8 years R179 ? 14 years S48 ? 11 years R201 ? 7 years S16 male 27 years R15 male 22 years S99 male 39 years R34 male 31 years S173 male 42 years R158 male 19 years S206 male 37 years R163 male 21 years S1 female 42 years R72 female 38 years S109 female 31 years R107 female 51+ years S186 female 31 years R122 female 27 years S207 female 49 years R144 female 34 years

Methods

Thin sections were viewed under an ultraviolet light microscope at 490 nm. At this intensity, fluorophors in bone labeled with tetracycline fluoresce yellow-green (Frost

1969; Bassett et al. 1980; Armelagos et al. 2001). Amount of tetracycline-labeled bone was quantified by area and labeled osteon frequencies using three measurement methods.

Labeled-bone area was determined through application of a new image analysis technique and point-count methods. Then labeled osteon frequencies were determined by manually counting fluorescing osteons – similar to the osteon count methods employed in prior studies of tetracycline labeling in Nubian populations (Bassett et al. 1980; Hummert and Van Gerven 1982; Fabsits 2008). However, this technique does not count lamellar or trabecular tetracycline-labeled bone tissues or partially resorbed labeled osteons.

Therefore, a new technique of image analysis - described below - was developed to directly measure labeled bone area. Additionally, tetracycline-labeled bone area was estimated using a point-count method, commonly used to study bone histology (Frost

1969).

New Technique of Image Analysis Method

A video motion camera was mounted to a UV light microscope with a 0.63x lens attached. Photographs were streamed from the microscope camera to a computer using

Image-Pro Plus 7.0. Then a series of images spanning entire rib cross-sections under the

5x optical lens were photographed. Image brightness and contrast were set to 33, default enhancements; maintaining section photograph qualities were as similar to their actual

14 appearance (observed directly through the microscope) as possible and contained minimal background noise.

Photographs were imported into Microsoft Image Composite Editor (ICE) and each series of images stitched together to produce a single composite image of each rib cross-section (Fig. 3). Adobe Photoshop was used to “digitally clean” these images of soft tissue attached to the bone and background colorations (as much as possible) to minimize interference during image analysis (Fig. 4). Cross-section photographs were archived before and after digital cleaning.

Figure 3: Rib cross-section from specimen S48 before digital cleaning

Figure 4: Rib cross-section from specimen S48 after digital cleaning

To distinguish tetracycline-labeled bone tissue, two masks of the cross-section were created for each slide. One mask selected all pixels of bone tissue, the second only pixels of bone tissue labeled with tetracycline (Fig. 5). Tissue differentiation was accomplished using color bitmap manipulation in which ranges of green intensity levels were selected for mask production. Red and blue intensity levels were set to zero. Since each bone cross-section and image differed in preservation quality and UV light exposure time - which affected the intensity of yellow-green tetracycline fluorescence (Keith and

Armelagos 1988; Maggiano et al. 2006) - unique green color ranges had to be manually set to select the correct tissue areas for each mask. The total mask area in micrometers was then measured using Image-Pro. Total bone and total tetracycline-labeled bone areas for each cross-section were obtained, and percentages of tetracycline-labeled bone were calculated. 16

Figure 5: Masks used for measurement of total bone area in microns (left) and tetracycline-labeled bone area in microns (right) for specimen S48

Osteon Count Method

Digitally cleaned images were then reopened in Adobe Photoshop, and its counting tool was used to track manual osteon selections. All osteons containing a

Haversian canal were counted. Osteons visually determined to be over 50% labeled were then counted separately. Most tetracycline-labeled osteons were not homogenously labeled. However, this pattern is expected if tetracycline consumption was not restricted to the average osteon formation period of 80 days. Therefore, it was not considered necessary to count only fully labeled osteons. Osteon counts were recorded and labeled- osteon percentages calculated.

Point-Count Method

Image-Pro was used to create a grid overlay containing at least 100 intersections sized to each individual cross-section image. The Image-Pro counting tool was then used 17 to track all grid intersections over bone and then over tetracycline-labeled bone tissue.

Resulting counts, grid sizes, and percent intersections over labeled bone calculations for individual cross-sections were also recorded in Excel.

Descriptive statistics of results for key variables for each method of measurement were determined (Table 2). Visual examinations suggested that variables are not distributed normally and therefore require the use of non-parametric statistical tests. The statistical significance of differences in tetracycline-labeled bone content between the cemeteries were evaluated using the non-parametric Mann-Whitney test and a 95% confidence level (p ≤ 0.05).

Summary

For 38 individuals from both Kulubnarti cemeteries, three measurements were taken under UV light from thin rib cross-sections to quantify amounts of bone labeled with tetracycline. A new technique was developed to measure labeling directly by area and two established techniques - counting osteons and labeled points - were also employed for comparison. Mann-Whitney U tests were used to compare amounts of tetracycline-labeled bone between the cemeteries at the 95% confidence interval (p ≤

0.05) for each method of measurement.

CHAPTER 3: RESULTS

In all cases, regardless of whether tetracycline-labeled bone was quantified via area or osteon frequencies, no significant differences in percentages of tetracycline- labeled bone tissue were observed between S and R cemeteries (U: p ≤ 0.05) (Table 2). In addition, results obtained using the new technique of image analysis to measure total area of tetracycline-labeled bone were similar those obtained using previously established methods. However, when percent area of labeled bone tissue was graphed against estimated age at death, a different pattern for percent labeled bone tissue between the two cemeteries - especially for sub-adults - was revealed (Fig. 6). The amount of labeled bone tissue for sub-adults increased with age in the R cemetery, while it decreased in individuals from the S cemetery. After the age of approximately 15 years, the age- associated patterns for tetracycline labeling for both cemetery samples begin to follow a similar pattern of relatively gradual increase with age.

Table 2: Percent averages of tetracycline-labeled bone for all ages at Kulubnarti

Std. Measurement Cemetery n Median Mean U z p* Dev. % Labeled S 19 21.595 26.494 14.930 Bone Area

% Labeled R 19 27.669 26.599 19.044 178.00 - 0.073 0.954 Bone Area

% Labeled Combined 38 23.660 26.547 16.878 Bone Area

% Labeled S 19 21.693 26.730 14.549 Osteons

% Labeled R 19 27.826 25.103 15.445 162.00 - 0.540 0.603 Osteons

% Labeled Combined 38 25.741 25.917 14.822 Osteons % Labeled Grid S 19 24.490 25.848 14.095 Intersections % Labeled Grid R 19 23.423 24.046 16.169 166.00 -0.423 0.686 Intersections % Labeled Grid Combined 38 23.957 24.947 14.989 Intersections * Mann-Whitney test statistic for cemetery comparison, analyzed using a 95% confidence interval

Figure 6: Frequency of tetracycline-labeled bone area (in microns) by age for both cemeteries at Kulubnarti (loess line of fit included)

Observed differences in the patterns for tetracycline labeling between the cemeteries for sub-adults may reveal differences in diet, especially breast feeding behavior. Tetracycline can be passed from mother to child through breast-feeding (Chopra and Roberts 2001;

Sánchez et al. 2004). To control for possible effects of mother-offspring transmission of tetracycline on the total amount of labeled bone, and account for differences in tetracycline-labeled bone vary between cemeteries, Mann-Whitney tests were applied to

21 samples of only individuals age 12 years and older. By age 12, bone turnover should completely replace any bone formed during breast-feeding (Stout p.c. 2014). Again, no significant differences (U: p ≤ 0.05) in tetracycline-labeled bone content between cemeteries were observed (Table 3). Small sample size precluded testing only individuals estimated to be younger than age 12 at death for differences in tetracycline-labeled bone between the cemeteries.

Summary

Regardless of which measurement technique was used, there are no significant differences in the amounts of tetracycline-labeled bone between the cemeteries at the

95% confidence level. Whether there is a difference between sub-adults and adults within cemeteries, or sub-adults between the cemeteries, requires an expansion of the sample to test. However, it does not appear that there is a significant difference in tetracycline consumption between the adults of each cemetery.

Table 3: Percent averages of tetracycline-labeled bone among those aged 12 and older at Kulubnarti

Std. Measurement Cemetery n Median Mean U z p* Dev. % Labeled S 13 18.016 22.225 14.891 Bone Area % Labeled R 14 29.182 29.126 18.749 72.00 - 0.922 0.375 Bone Area % Labeled Combined 27 20.248 25.803 17.043 Bone Area

% Labeled S 13 17.526 21.985 13.132 Osteons % Labeled R 14 27.078 26.512 14.949 81.00 - 0.485 0.650 Osteons % Labeled Combined 27 21.004 24.332 14.022 Osteons % Labeled Grid S 13 18.452 21.494 12.961 Intersections % Labeled Grid R 14 25.448 25.779 16.054 79.00 - 0.582 0.583 Intersections % Labeled Grid Combined 27 21.324 23.716 14.531 Intersections * Mann-Whitney test statistic for cemetery comparison, analyzed using a 95% confidence interval

CHAPTER 4: DISCUSSION

The use of image analysis technology to measure tetracycline labeling in terms of labeled bone area proved to be appropriate, and comparable to other methods. Observed small differences of 1-2 percentage points between the three measurement techniques

(Table 2) likely result from what is actually being measured by each method. Where the proposed image analysis technique and point count methods measures area of tetracycline-labeled bone, the osteon count method only provides a frequency of labeled osteons; and does not include other types of bone tissue, such as primary lamellar bone, or partially resorbed secondary bone of remodeled fragmentary osteons. Results of this study also suggest that the point count method can serve as a substitute for area measurements, when technology does not make direct tetracycline-labeled bone area measurements feasible.

Means for tetracycline-labeled bone for each method of measurement fall within the range of 24-27% of bone tissue being labeled (Table 2). The amount of bone labeled with tetracycline in the Kulubnarti Nubian populations supports the assumption that tetracycline ingestion by means of a common, characteristic activity, exceeding a therapeutic threshold. Beer consumption (a culturally influenced activity) continues to be the most probable source of tetracycline for ancient Nubian populations (Bassett et al.

1980; Armelagos et al. 2001). Because beer was a staple of the Nubian diet (Bassett et al.

1980), it follows that all members of society, regardless of class, would have access to beer. Unless beer was used in a ritual context beyond nutritional sustenance, one would expect access to beer to be mostly uniform across classes. Thus, if tetracycline was consumed through beer, the amount of labeled bone should be homogeneous across groups, which is the case at Kulubnarti. Despite the S cemetery exhibiting poorer health and less material wealth than the R cemetery (according to archaeological and paleopathological findings (Sheridan 1992; Mittler and Van Gerven 1994; Van Gerven et al. 1995; Adams and Adams 2007; Turner et al. 2007)), there are no significant differences in tetracycline consumption. Therefore, the classed communities (represented by the S and R cemeteries at Kulubnarti) likely had equal access to beer. Future sample expansions are needed to further investigate age trends in tetracycline consumption.

Other hypotheses have been posed regarding how tetracycline was ingested or whether the fluorescence observed in Nubian remains is from tetracycline in the first place. Pipenbrink (1983, 1986) suggests that mold infestation during diagenesis could be responsible for the yellow-green fluorescence observed in Nubian samples. However, characteristic tunneling, cuffing, and fluorescence in wake of this microbial destruction

(Pipenbrink 1986) are not observed in the Kulubnarti sample; nor in the ancient Nubian

NAX population from Wadi Halfa (Bassett et al. 1980; Keith and Armelagos 1988;

Nelson et al. 2010). Observed osteon fluorescence appears as more discrete patterns rather than diffused (a common pattern of fluorescence in skeletal remains when caused by mold infestation (Pipenbrink 1986; Keith and Armelagos 1988)). In addition, labeling

25 does not only appear as surface labeling or staining, but embedded internal microstructures of bone are labeled as well. Surface-only labeling is reflective of in vitro labeling (Pipenbrink 1986; Keith and Armelagos 1988) and this pattern is not present in the Kulubnarti sample. Pipenbrink (1983, 1986) was not convinced that published photographs of fluorescing osteons from Nubian sites were not actually surface labels.

This skepticism may be due in part to technology available at the time, preventing photography and publication of the entire bone cross-section (Bassett et al. 1980; Keith and Armelagos 1988). By incorporating new technology in developing updated methods, it is clear that bone material is labeled throughout the bone, rather than just the surface areas (Figs. 1-2). However, successful extraction of tetracycline from the NAX population is the most conclusive evidence that fluorescence in ancient Nubian skeletal remains is from tetracycline consumed in vivo (Nelson et al. 2010).

Tetracycline was also not likely consumed by eating mold-infested or

Streptomycetes contaminated grains (made into foodstuffs such as bread) during a food shortage. First of all, tetracycline is only secreted under harsh conditions, such as fermentation (Armelagos et al. 2001; Maggiano et al. 2003; Nelson et al. 2010). Thus, foodstuffs would not have regularly produced the necessary environment for secretion of this antibiotic. Secondly, even if tetracycline secretion had been managed, eating contaminated grains during a food shortage would have produced sporadic labeling.

Observed patterns of tetracycline-labeled bone fluorescence indicate that the drug was ingested on a regular basis (Bassett et al. 1980; Keith and Armelagos 1988; Armelagos et al. 2001; Nelson et al. 2010). High percentages of tetracycline-labeled bone content

26 resulting from this project further support this conclusion suggesting that tetracycline was ingested often. Maggiano et al. (2003) also reached the conclusion that tetracycline was ingested in vivo near the Dakhleh Oasis, although the authors argued that contaminated foodstuffs were the source of tetracycline. Their main argument was that contaminated and spoiling grains were the source of tetracycline in contrast to ale. This conclusion centers mainly on their assumption that without controlling the amount of Streptomycetes present during fermentation, the resulting beverage would be “foul-tasting” (Maggiano et al. 2003: 341). Taste is however, a subjective quality that may be influenced by cultural preferences. Without having an exact recipe used for ancient Nubian beer brewing or knowledge of cultural notions regarding food, such an argument does not adequately counter the apparent regularity of tetracycline consumption; which is greater than would be expected if spoiling food had been the source.

Tetracycline-labeled bone percentages resulting from this study (Table 2) are similar to previous findings of Bassett et al. (1980), where about 30% of all observed osteons from the ancient Sudanese-Nubian X-Group population exhibited some level of fluorescence from tetracycline labeling. However, the results differ about eightfold from those reported by Hummert and Van Gerven (1982) for the Kulubnarti population (3.6% of osteons were labeled with tetracycline). These discrepancies are likely explained by the prior study using thin bone sections from metacarpals and femurs rather than ribs and differences in slide preparation techniques. Their slides were prepared by a different technician using currently available, but different technologies and measurement methods. The technology available in 1982 also limited the scope of measurements,

27 making sub-sampling cross-sections the only practical means of measurement. By sub- sampling larger bones, the portions of cross-sections measured for labeled osteon frequencies may have simply had fewer osteons than elsewhere on the same cross-section and/or what would be expected in a comparable sub-sample from ribs. Interestingly,

Hummert and Van Gerven (1982) found that only 63% of their sample contained fluorophors (41 of the 110 samples contained no tetracycline labeling) as opposed to the

100% reported by Bassett et al. (1980) for the X-Group. In preparation for conducting this study on the Kulubnarti populations, of 182 individuals from which rib slides were created (and from which samples were used for this study), only six had little to no tetracycline labeling. However, despite discrepancies in resulting means of tetracycline- labeled bone between studies, Hummert and Van Gerven (1982) also found there was not a significant difference in amount of labeled bone between the S and R cemeteries.

The results also differ from those that were previously reported (Margolis et al.

2013) for this sample. After reexamination of data and materials, it became apparent that the standard UV light alignment settings had slipped during cross-section photography.

Which cross-section photos were affected by this setting change were determined based on visual appearance of photographs in conjunction with the timing of the original settings slip and order in which photographs were stitched together (as were recorded in laboratory notes). All cross-section photographs affected by the change of settings were reproduced using proper UV light alignment settings and data re-analyzed. In addition, the hit-and-miss method and more appropriate, non-parametric statistical testing have been employed in this updated analysis.

Since the sample size was relatively small, a future expansion of the sample may provide a more accurate estimate of tetracycline consumption. To further investigate why there are opposing trends in accumulation of tetracycline labeling between the cemetery communities, sample size expansion would be particularly important. Sample expansion would help determine if the observed trend opposition resulted from small sample size or is real. Additional research on tetracycline-labeled bone accumulation trends in the

Kulubnarti cemeteries will also aid in examination of whether beer (as the conduit of tetracycline consumption) was a dietary staple or was used in different contexts according to class. Data obtained from this thesis however, may be used to better understand health in the Kulubnarti, Nubian population and why certain skeletal pathology trends, such as infection rates, are observed.

Summary

Resulting means of tetracycline-labeled bone in the Kulubnarti cemeteries are similar to those reported for the X-Group at Wadi Halfa, but differ greatly from those previously reported for Kulubnarti (Bassett et al. 1980; Hummert and Van Gerven 1982).

These discrepancies are likely explained by differences in measurement methods as constrained by available technology. However, as was previously concluded by Hummert and Van Gerven (1982), there are no significant differences in tetracycline consumption between the cemeteries at Kulubnarti, despite marked differences in skeletal indicators of stress and status. Due to labeling patterns, the high percentages of labeled-bone, and

29 nature of tetracycline production, it is likely that the ancient Nubians were ingesting tetracycline through culturally characteristic beer drinking.

CHAPTER 5: CONCLUSIONS

High percentages of bone labeled with tetracycline in the Kulubnarti Nubian populations offers more support to the hypothesis that tetracycline was ingested in therapeutic doses in vivo by means of a common, culturally characteristic activity. The hypothesized means of ingestion for the antibiotic, as a culturally influenced activity, through consumption of beer (Bassett et al. 1980; Armelagos et al. 2001), continues to be the most probable source of tetracycline for the ancient Nubian populations. However, the results from this study are surprising as the skeletal indicators of health and archaeological evidence between the two cemeteries are markedly different (Sheridan

1992; Mittler and Van Gerven 1994; Van Gerven et al. 1995; Adams and Adams 2007;

Turner et al. 2007). Despite the S cemetery exhibiting poorer health and less material wealth than the R cemetery, the two groups appear to have been equal in regards to their tetracycline consumption. While it is still likely that the S cemetery represents an underclass to those interred in the R cemetery (Adams and Adams 2007), it can be concluded that class differences were not mediating access to tetracycline, and in turn, beer. In addition, the new method of measuring tetracycline-labeled bone area using image analysis (as presented in this thesis) provides an efficient and comprehensive procedure for determining tetracycline-labeled bone content. This methodology will be

31 beneficial not only for bioarchaeological studies on health in ancient populations, but for any skeletal histological research incorporating use of tetracycline as a marker of bone growth and resorption, and clinical studies on the physiological effects of this antibiotic.

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APPENDIX A: RAW DATA Table 4: Kulubnarti cemetery 21-S-46 (S Cemetery) raw data

Green Total Green Total # of color range Area of Grid Used Specimens color range Area of % Bone # of Mostly % Osteons Total Hits Labeled % Labeled Sex Age (Total Labeled (in by Burial # (Total bone Bone (in Labeled Osteons Labeled Labeled and Misses Hits Hits labeled Bone (in microns) area) microns) Osteons bone) microns)

S13 male 12 years 62-255 15140824 112-255 5334595.5 35.233191 208 92 44.23077 103 39 37.86408 400x400 S62 male 14 years 33-255 23732366 76-255 941806.31 3.9684468 183 11 6.010929 101 6 5.940594 500x500 S68 a male 4 years 47-255 6590091.5 77-255 1423142.6 21.595187 46 9 19.56522 100 21 21 275x275 S236 male 16 years 48-255 29471674 81-255 5309460 18.015468 189 41 21.69312 117 23 19.65812 500x500

37 S36 ? 13 years 50-255 17232128 82-255 2823355.8 16.384255 73 22 30.13699 111 15 13.51351 400x400 S23 ? 5 years 65-255 10136109 10-255 4464284 44.04337 99 47 47.47475 112 48 42.85714 300x300 S28 ? 7 years 55-255 15105379 98-255 4027931.8 26.665546 81 23 28.39506 98 24 24.4898 425x425 S37 ? 1 year 51-255 6146576.5 92-255 2662261.3 43.312912 71 32 45.07042 107 50 46.72897 250x250 S41 ? 15 years 45-255 24893706 80-255 2721774.3 10.933584 194 34 17.52577 133 20 15.03759 450x450 S47 ? 8 years 86-255 18928696 147-255 5744530 30.348261 190 57 30 96 26 27.08333 450x450 S48 ? 11 years 96-255 23435542 158-255 11367031 48.503384 192 99 51.5625 109 54 49.54128 475x475 S16 male 27 years 60-255 19538928 98-255 3283693.8 16.805906 263 36 13.68821 115 16 13.91304 425x425 S99 male 39 years 74-255 30002128 128-255 2405812.5 8.0188062 304 31 10.19737 129 6 4.651163 500x500 S173 male 42 years 67-255 28764506 103-255 7399316.5 25.723774 314 52 16.56051 129 33 25.5814 500x500 S206 male 37 years 73-255 13945925 129-255 2879463.5 20.647347 87 14 16.09195 166 43 25.90361 300x300 S1 female 42 years 83-255 31431152 150-255 17668838 56.214414 290 105 36.2069 129 59 45.73643 500x500 S109 female 31 years 68-255 18781614 114-255 8293123.5 44.155542 157 71 45.22293 97 39 40.20619 450x450 S186 female 31 years 65-255 19293838 102-255 2425776.8 12.572806 235 17 7.234043 108 14 12.96296 450x450 S207 female 49 years 95-255 18376276 151-255 3720810.8 20.247904 219 46 21.00457 168 31 18.45238 350x350 37

APPENDIX A: RAW DATA Table 5: Kulubnarti cemetery 21-R-2 (R Cemetery) raw data

R68 male 12 years 86-255 20606906 123-255 7524867 36.516239 165 72 43.63636 111 26 23.42342 450x450 R146 male 15 years 89-255 37938160 160-255 23443904 61.795048 207 101 48.79227 196 108 55.10204 450x450 R147 male 16 years 96-255 38324144 165-255 19131964 49.921439 367 159 43.32425 208 79 37.98077 450x450 R196 male 1.5 years 66-255 11313690 140-255 149101.77 1.3178881 315 13 4.126984 104 4 3.846154 350x350

R18 female 15 years 93-255 26986584 153-255 11987440 44.419998 267 96 35.95506 229 91 39.73799 350x350 38 R84 female 2 years 60-255 12388930 139-255 217328.91 1.7542186 149 0 0 102 3 2.941176 350x350 R192 female 16 years 76-255 36035896 128-255 19281566 53.506554 361 159 44.04432 152 73 48.02632 500x500 R21 ? 6 years 69-255 14038496 140-255 2150403.5 15.317905 115 32 27.82609 119 22 18.48739 350x350 R142 ? 9 years 87-255 20773254 155-255 10007632 48.175563 186 66 35.48387 103 46 44.66019 450x450 R179 ? 14 years 72-255 31230090 141-255 2111030.8 6.7596052 219 24 10.9589 133 7 5.263158 500x500 R201 ? 7 years 60-255 10227909 105-255 3176060.8 31.052885 73 28 38.35616 96 25 26.04167 350x350 R15 male 22 years 46-255 35485164 79-255 5858308 16.509175 322 49 15.21739 142 14 9.859155 500x500 R34 male 31 years 28-255 25347862 70-255 1045681.8 4.1253254 236 23 9.745763 137 9 6.569343 450x450 R158 male 19 years 28-255 21047066 54-255 2885944 13.711859 125 7 5.6 106 10 9.433962 450x450 R163 male 21 years 91-255 32027640 148-255 6435213 20.092686 186 44 23.65591 136 29 21.32353 500x500 R72 female 38 years 82-255 18889880 129-255 6794993 35.971605 200 61 30.5 100 36 36 450x450 R107 female 51+ years 58-255 25045434 115-255 6929769.5 27.668794 395 54 13.67089 131 38 29.00763 450x450 R122 female 27 years 73-255 15637168 141-255 4799962.5 30.695856 160 50 31.25 91 25 27.47253 450x450 R144 female 34 years 26-255 16346545 66-255 991312.5 6.0643549 162 24 14.81481 94 11 11.70213 450x450 38

APPENDIX B: RIB CROSS-SECTION PHOTOS

Specimen Cross-Section After Stitched Cross-Section Total Bone Mask Labeled Bone Mask Burial # Digital Clean-Up

S13 39

S16

S23

S28

APPENDIX B: RIB CROSS-SECTION PHOTOS

Specimen Cross-Section After Stitched Cross-Section Total Bone Mask Labeled Bone Mask Burial # Digital Clean-Up

S36

S37

S41

S47

S48

APPENDIX B: RIB CROSS-SECTION PHOTOS

Specimen Cross-Section After Stitched Cross-Section Total Bone Mask Labeled Bone Mask Burial # Digital Clean-Up

S62

S68a

S99

S109

S173

APPENDIX B: RIB CROSS-SECTION PHOTOS

Specimen Cross-Section After Stitched Cross-Section Total Bone Mask Labeled Bone Mask Burial # Digital Clean-Up

S186

S206

S207

S236

APPENDIX B: RIB CROSS-SECTION PHOTOS

Specimen Cross-Section After Stitched Cross-Section Total Bone Mask Labeled Bone Mask Burial # Digital Clean-Up

R15

R18

R21

R34

R68

APPENDIX B: RIB CROSS-SECTION PHOTOS

Specimen Cross-Section After Stitched Cross-Section Total Bone Mask Labeled Bone Mask Burial # Digital Clean-Up

R72

R84

R107

R122

R142

APPENDIX B: RIB CROSS-SECTION PHOTOS

Specimen Cross-Section After Stitched Cross-Section Total Bone Mask Labeled Bone Mask Burial # Digital Clean-Up

R144

R146

R147

R158

R163

APPENDIX B: RIB CROSS-SECTION PHOTOS

Specimen Cross-Section After Stitched Cross-Section Total Bone Mask Labeled Bone Mask Burial # Digital Clean-Up

R179

R192

R196

R201