AFRICAN WILD DOG (LYCAON PICTUS) BREEDING PROGRAM
Mkomazi Game Reserve, Tanzania
Report 1995-2001
Aart M. Visee, D.V.M.
AN ENDANGERED SPECIES PROGRAM UNDERTAKEN BY: THE WILDLIFE PRESERVATION TRUST FUND (TANZANIA) THE GEORGE ADAMSON WILDLIFE PRESERVATION TRUSTS (UK, USA, GER.) THE AFRICAN WILD DOG FOUNDATION (NETH.)
CONTENTS Page
Foreword 3 Introduction 4 Husbandry 5 Development of packs 5 One big pack 5 Lendenai pack 6 Sangito pack 7 Kisima pack 8 Ayubu pack 10 Dominance problems 11 Birth rate and pup survival 12 Restart/Kimondo pack 21 Success from tragedy 22 Zootechnique 26 Composition of packs 26 Identification 29 Population genetics 30 Housing 30 Nutrition 31 Bodyweight 32 Neck-size 33 Veterinary Work 34 Preventative Medicine 35 Canine Distemper Virus 35 Rabies 38 Parvoviral Disease 42 Parasites 42 Clinical Work-Diseases 44 Clinical Work 44 Anesthesia 45 Diseases 47 Canine Distemper 47 Pathology 50 Acknowledgements 54 Summery 54 References 55 Appendix I, bodyweight 57 Appendix II, distemper 59 Appendix III, rabies 62 Appendix IV, parvo 64 Genetic variation 66 Parenthood analysis 71 Distemper Outbreak and Its Effect on African Wild Dog Conservation attachment Distemper, Rabies and Parvovirus vaccinations in a captive breeding … attachment Plan for Re-introduction of the African Wild dog attachment
2
FOREWORD
With lots of enthusiasm and high hopes the African wild dog breeding program in Kisima camp, Mkomazi Game Reserve, Tanzania, was started in 1995. The future looked bright with 25 beautiful pups and we were determined to do something to preserve the African wild dog from extinction by way of breeding them and re-introducing them to the wild in places where they had disappeared or seemingly disappeared. The first 5 years of our breeding program were a success. We expected some difficulties, got them and were able to solve them. But we never expected to be confronted with a very serious canine distemper virus (CDV) outbreak that hit our African Wild Dog Program by the end of 2000. At that time the program contained 52 dogs. By the time the virus had had its way only three dogs were left. A real devastating experience, not only for the dogs but for the people involved as well. In fact, this outbreak symbolized the end of the first five years of our breeding program, which started with the arrival of 25 pups from the Masai Steppe in September of 1995. This period ended abruptly when the last of the 25 original dogs, present in Mkomazi, died on the fourth of August 2001, although two of the original dogs are still living at the Ol Jogi Ranch, Kenya. At about the same time, a new era began on the seventh of July 2001, symbolized by the birth of the first litter of pups to the dogs, which had survived the canine distemper outbreak. This report covers the years 1995 to 2001, i.e. concluded the era of the original 25 dogs and includes details of the fresh start.
The report is dedicated to the memory of the original dogs and their offspring that died during the canine distemper outbreak. The dogs did not like all aspects of the program, but accepted it, as being important for the welfare and the future of their species. We, as humans who carried out the program, are very grateful to have known them and their individual traits. They enriched our lives.
Aart Visee
Aart M. Visee. D.V.M. The African Wild Dog Foundation Gaspeldoornlaan 17. 3956DN Leersum The Netherlands Tel.: +31 343 769081 E-mail: [email protected]
3
INTRODUCTION
The George Adamson Wildlife Preservation Trusts, (joined in 2000 by the African Wild Dog Foundation), through their Field Director Tony Fitzjohn, were invited in July of 1988 by the Tanzanian Government, to establish a rehabilitation program for the African wild dog (Lycaon pictus) involving capture, captive breeding and re-introduction. The African wild dog is an extremely endangered species. Diseases, large predators (lions), and man, threaten their existence. In areas where game is scarce, wild dogs tend to get close to human settlements. As a consequence they are poisoned, and may come into close contact with domestic dogs. As the African wild dog is susceptible to diseases also found in domestic dogs (distemper, rabies and parvo virus), cross infection may occur--leading, in some cases, to high mortality. It took many years and much searching before dens were finally located from which pups could be collected. It was most important that the pups came from a non-conservation area (in this case the Masai Steppe), to prevent the unnecessary decline of numbers in conservation areas. And, it was thought likely that pastoralists would have poisoned them sooner or later anyway. Finally, the right moment arrived in early August of 1995. Twenty-five pups were lifted from three different dens in the Masai Steppe. At the time of lifting, they were not totally dependent on their mothers--they could eat solid food. They ranged in age, approximately, from three to five weeks. According to the location where they were found, they were called the Lendanai Group (sex 1m, 3f), the Llondirrigiss Group (sex 7m, 1f), and the Najo Group (sex 7m, 6f). The pups were flown to the Mkomazi Game Reserve (Kisima Camp) on September 3, 1995. For them, and the people involved, it represented a new start for the rescue of the genetically unique East African population of the African wild dog (Lycaon pictus)--considered by some researchers to be a distinct subspecies. With the arrival of the dogs, it was decided to collect as much data as possible without stressing the dogs too much, and with a minimum of interference in their lives. For these reasons, blood samples were not taken on all occasions from each of the dogs. Alpha females and alpha males were sedated as little as possible, to prevent any possible adverse effects on their social status. Pregnant females were not sedated for obvious reasons.
For 5 years the program developed well and prospered. By the end of 2000, there were a total of 52 dogs, and plans for re-introduction into the wild were seriously underway. However, just before Christmas 2000, fate struck. The breeding program was hit very hard with a canine distemper virus infection (CDV), despite the fact that the dogs had been vaccinated according to an intensive schedule. In the section 'Veterinary Work' this epidemic is fully reported and discussed, and includes the measures to be taken in the future.
This report is divided into two sections: Husbandry and Veterinary Work.
4
HUSBANDRY
Development of Packs A survey is given concerning the origin of the breeding packs and what happened within the various packs.
Zootechnique In this section, a report is drawn up about composition of packs, identification, housing, nutrition, bodyweight and neck-size.
Development of Packs
One big pack (1996)
On arrival at Kisima Camp the pups were kept separately, in their original litter formations. In December 1995, a transponder was inserted in the left side of the neck of each dog, so that it was possible to identify them at all times. On December 21, 1995, it was judged the right time to put the three litters together. They settled themselves into a new social order without much quarreling, and lived happily together. In December 1996 Llondirrigiss #297 came into season, which took us by surprise. Several of her brothers mated with her. The last male, who mated, was Najo #300. Separation of #297 was considered, but not effected as this might have caused big problems to reintroduce her in the pack.
On March 9th the annual vaccinations, blood sampling etc. took place. This occasion was at the same time used as an opportunity to establish the final breeding packs. The large original pack was split into three smaller packs. 'Kisima Pack' in boma I, 'Lendanai Pack' in boma II and 'Sangito Pack' in boma III. See chapter Composition of breeding packs. The alpha female #297 and male #300 with the pups were left together, in peace, in the original Kisima boma. Thus three breeding packs were composed, and the dogs were separated into three breeding compounds. In October of 1998, a fourth breeding pack was established (see Composition of packs).
In August of 1996, a request was received from the Kenya Wildlife Service (KWS) for four male dogs. KWS had recently captured a pack of four adult females with hunting experience, who had been stock raiding (sheep). Their intention was to add males to the pack, and reintroduce the whole pack in the Serengeti-Mara ecosystem. With the permission of the Tanzanian Wildlife Department and the Serengeti Wildlife Research Institute, four males were sent to Kenya: Llondirrigiss #299, Najo #273, #288 and #303. Eventually the dogs were released in Tsavo West. The project proved to be a partial success. Richard Kock, at that time head of the veterinary department of KWS, wrote a report about the release (available on request). However, two of the male dogs (#273 and #303) were fought out of the newly
5 composed pack during the introduction period in the Orphanage, Nairobi, and sent to the Ol Jogi ranch, Kenya.
From 1997 onward development of the different breeding packs will be given per pack.
Lendanai Pack 1997 At the end of June the Lendanai females showed signs of coming into season. Female #264 appeared to become the alpha female and Najo #274 the alpha male. Unfortunately #264 got a rectal prolapse and had to undergo surgery twice. After the second intervention, being separated for two days from the pack by a simple fence, she lost her leading position to female Lendanai #262. It was remarkable to note that in the original large pack she was the lowest in order. At the end of 1997 #262 showed signs of pregnancy. 1998 On 1 February 1998 alpha female #262 gave birth to 7 pups. Najo #274 was their father. The pups started off doing very well. However on 28 April one pup died and on 19 May another pup died. A third pup was noted being sick on 22 May. This pup fortunately recovered after medical treatment (see Clinical Work). After this bad episode, the remaining five pups grew up well without any problems. By the end of 1998 the total number of dogs was 10 and Lendanai #262 showed signs of pregnancy again. 1999 On the 6th of January, 1999, alpha female #262 gave birth. Najo #274 was still alpha male, and father of the newborn pups. It was the second litter of the pack. In the beginning, everything went normally, but after three weeks (on January 26th) a lot of noise was heard coming from the den. It was only possible to investigate the den the next day, while the mother was confined to the feeding area. Only one sick pup was left in the den, but the next day, it also died. For the rest of 1999, the alpha pair showed no signs of sexual activity. The pack began and finished the year with 10 dogs, with #262 and #274 being the alphas. 2000 Early in January 2000, the alpha female was pregnant and gave birth to seven pups on the 4th of April. The litter appeared to consist of a healthy set of pups; all pups survived, until the canine distemper virus outbreak--of which they were the first victims. The Lendanai pack was very balanced in their social interactions. The only aggression was displayed when the alpha female was eating (always with the alpha male), at which time she was fairly aggressive toward her sisters--but there was no fighting. Only one dog survived the canine distemper epidemic--a male, #325.
Lendanai pack established March1997
females males Lendanai Najo 261 262 264 274 300 alpha since alpha until alpha Oct.97 Oct.97
01-02-98 322 323 325 327 329
6 m f m f m
04-04-00 780 812 817 850 893 912 924 f f m m m f f
Sangito Pack 1997 After establishing the pack no particulars were observed in 1997 1998 By the end of 1997 the dogs had still not decided which dogs were going to be the alpha female and male. By early 1998 they had made up their minds. Alpha female was Najo #306 and alpha male Llondirrigiss #294. As a result, on 3 May Najo #306 gave birth to 8 pups. On 27 May one pup died; on 6 July two pups died and on 14 August a fourth pup died. After this nasty period the remaining four pups grew up well. What happened? Initially the alpha female and male looked after their pups very well. After a month the pups started to come to the top of the den to receive regurgitated food from their parents. This went well for a couple of days. Suddenly, one day, a fight broke out between the father, #294, and one of his brothers #291. Apparently #291 was after the mother #306. Fighting went on for a week. To make things even more complicated; mother #306 went off during this period with the third brother #289. Father #294 took control of looking after the pups. All pups were still healthy and well at this stage. Whilst he was caring for the pups, the fight between him and his brother #291 died down, but mother #306 and her new suitor #289 now started fighting him! Apparently, as a result, he did not feel confident enough, to leave the pups on their own and to come and take food from the feeding compound to feed him and the pups. This happened in the time that the two pups died (6 July). At that time feeding was started near the top of the den, exclusively for #294 and the pups. Fighting stopped and male #289 assumed alpha position. The pups grew up well, but one pup died on 14 August. At the end of 1998 the pack consisted of 11 dogs. 1999 By the end of 1998, finally the alpha-ship was firmly settled, with the alpha female being Najo #306, and alpha male Llondirrigiss #289. As a result, #306 gave birth to 10 pups on the 28th of February. Unfortunately, between the 12th and 25th of April, the pups died. On the 12th of September, #306 gave birth to another set of pups (her third), consisting of 12 pups. Once again, the pups started out well, but between October 22nd and December 12th, eight pups died. This time, an infestation of worms was suspected, and the pups were treated accordingly. As a result, three pups survived. By the end of 1999, the pack consisted of 14 dogs, with #306 and #289 being the alphas. 2000 The year 2000 was generally peaceful. The only exception involved two brothers, #289 (alpha) and #291, while the alpha female was 'in season'. It appeared that to retain his position, #289 had to fight his brothers. Also, when the alpha female (#306) was pregnant, she chased her sisters away from the food, and was generally aggressive toward them. On the 12th of June, the alpha female gave birth to 11 pups. Over the next four months the pups died sporadically. By the end of 2000, only 4 pups survived. Midway through February 2001, following the canine distemper epidemic, there was only one survivor: the alpha female #306.
7
Sangito pack established 1997
males females Llondirrigiss Najo 289 291 294 265 306 305 308 alpha 1999 alpha until alpha and onward 1999
03-05-98 352 355 358 359 m m m m
12-09-99 319 360 361 f m m
12-06-0 Pups Sangito III (3m,1f)
Kisima Pack
1997 At the end of January 1997 #297 showed signs of pregnancy, but was still mounted by the males. On the fourth of March 1997 she gave birth to 6 pups. About a week before giving birth she spent more and more time in the den, being guarded by male Najo #300. After birth it was clear that he was the alpha male. On April 8th 297 brought her pups out of the den for the first time. May 15th one female pup died. She was losing weight, while the others were prospering, no signs of disease. The 5 remainig pups matured without problems October 17th was a very sad day, the mother of the pups, Llondirrigiss #297, died the day after she was sedated. The pups were big enough (7 months) to be independent of their mother. However, it was heartwarming to see how the father of the pups (Najo #300) gave special care and attention to the pups. 1998 After the alpha female Llondirrigiss #297 died in October 1997, the alpha male, Najo #300 looked after the pups of the first litter. He did not bother to engage in the struggle for leadership, and perhaps withdrew as in the wild to allow one of his sons to establish himself as the alpha male. However, the sons were very young and in the interim period before they reached adult age, three males and two females struggled for alpha positions. (The Lendanai male #263 did not involve himself in the positioning). The males Londirrigiss #293, #296 and #298 went for alpha, but it was not clear which one was the real alpha. The same could be
8 applied to the struggle of the females, Najo #284 and #285. As a result, #284 gave birth on 9 May and #285 on 1 June. Apparently #285 took her pups down in the den of #284. Over the next two days there was a fight in the den. The first opportunity to look in the den was on 6 June but there were no signs of the pups of 285. Over the next three months #284 and #285 were continuously fighting. During this time, the litter of #284 did not once come up to the top of the den for food. This is highly unusual as pups usually come up once they are one month old for regurgitated food. Apparently the pups lived only on their mother's milk during these months, which is not enough and they slowly died one by one. One can only guess what the reasoning was for the pups refusal or inability to come out. However it is likely that the alpha position was not quite settled with both females and males and, as a consequence, neither father nor mother took full responsibility for the pups, in contrast to what happened in the Sangito pack. At the end of 1998 there were 9 dogs left in this pack 1999 The year 1999 started well. Alpha female Najo #284 gave birth to four pups on the 8th of January and the father was Llondirrigiss #298. The pups, 3m.1f, grew well, with the exception of the female pup. She lagged behind in growth. Being smaller than her brothers gave her a disadvantage at the food. As a result, her growth was retarded, and she died on the 5th of April. Her three brothers grew up well and without complications.
On June 22nd just over 12 months since her last litter was born and died, the beta female Najo #285 gave birth to six pups (4m.2f). The father was #296. The alpha female #284, who in 1998 had given birth about a month before her subordinate sister, but had subsequently lost her entire litter, began to harass her sister with the new pups. This harassment intensified in the following weeks, and the alpha female would chase #285 out of her den. By the end of July the alpha was pregnant again, which might have influenced her behavior toward her subordinate sister. As a result of the harassment, #285's pups suffered from malnutrition and died between July 27th and August 3rd. On the 16th of September, 15 months after her last litter, the alpha female #284 gave birth to four pups. The decision was taken to remove Najo #285 from the pack and introduce her into the Ayubu pack, which took place October 28th. At the end of 1999, the Kisima pack consisted of 14 dogs, with #284 and #298 being the alphas. 2000 The year 2000 was characterized with fights between the brothers #293, #296 and #298. Occasionally the younger two brothers in the pack, (#333 and #335) from the first Kisima litter, who were unrelated to either of the alpha pair, would join in. The alpha male, #298, was the main aggressor. It appeared that his alpha status was not well established in the pack. This may, in part, have been due to the behavior of the alpha female, #284, who appeared ambivalent in her choice of partner. The male aggression may also have been due to the attempt by the young males to displace the alpha, who they were not related to, with one of the younger males taking over as alpha. This behavior would be expected in a free-living pack, had the same situation arisen. In early February 2000, #284 mated with #298, and #276 mated with #296. However, both females miscarried during the next two months. During the fights between the males, alpha female #284 temporarily rejected #298, and consorted with #296. However, she finally returned to #298 and had her fourth set of pups, four in all, on the 22nd of September. The father was #298. For no obvious reasons, the pups deteriorated and died one-by-one by the end of November. This pack also had only one survivor, male #372, after the canine distemper epidemic.
9
Kisima pack established March 1997
males females Llondirrigiss Lendanai Najo Llondirrigiss Najo 293 296 298 263 300 297 276 284 285 alpha to Ayubu alpha alpha alpha to Ayubu 1998 and pack Oct.98 until Oct.97 until Oct.97 1998 and pack Nov. onward onward 99
04-03-97 333 335 336 337 348 m m f f f
to Ayubu pack Oct.98
08-01-99 366 369 372 m m m
16-09-99 312 344 349 m m m
Ayubu Pack 1998 In October the male Lendena1 #263 and the females Kisima #336, #337 and #339/348 were separated from the Kisima pack to form a new breeding pack. They were placed in the newly built Ayubu boma and settled down soon fairly well. At the end of 1998 the Ayubu pack consisted of these 4 dogs. 1999 In 1999 there were no indications that one of the females had become alpha. Peace reigned. To bring more activity in the group, it was decided to introduce Najo #285 to the group. On the 28th of October, she was sedated and put into the feeding compound of the Ayubu boma. After a complete recovery, the only male in the pack, Lendanai #263, was introduced to her the next day in the feeding area. The dogs showed no hostilities at all toward each other. Three days later, November 2nd, the three younger females were introduced, and once again no hostilities were shown--#285 was accepted. The only sign of stress #285 showed, was her calling every morning and night, for many weeks. This call is typically given by an individual dog, who has temporarily lost touch with its pack. The dogs in the Kisima pack responded to her calls. At the time, the alpha female (#284) in the Kisima boma had young pups, and there was a lot of activity there. This could be heard by
10 #285 in the Ayubu boma. She was likely responding to these activities in her former pack. It was remarkable that the introduction of #285 went so well, maybe due to the fact that #285 was not a strange dog, as they had all been members of the same pack until October 1998, when the Ayubu pack was established. The Ayubu pack consisted of five dogs by the end of 1999. The only male was #263, and there was no obvious alpha female. 2000 What we had hoped for, happened early on in 2000. The arrival of #285 in the pack stirred things up and it soon appeared that #337, one of the three Kisima sisters, had become the alpha female. On the 25th of May, she gave birth to three pups. The pups, the first dogs born to a captive Mkomazi-bred dog, grew well until the 19th of August. After having eaten well the night before, all that remained of two of the pups were their heads--their bodies were never found. The third pup grew up well, until her untimely death, when the whole pack perished due to canine distemper virus.
Ayubu pack established Oct.98 females male Kisima Najo Lendanai 336 337 348 285 263 alpha introduced alpha Nov.99
25-05-00 Pup Ayubu I f
Dominance Problems
It is possible that some of the dominance problems might be related back to the mixing of the three original litters, when the pups were approximately five months old. Until then, they lived in separate compounds, and each had separate male and female dominance hierarchies. The combined litter then lived together until February of 1997, when the individuals were approximately 19 months old. During this time, a new male and female dominance hierarchy was established--with Llondirrigiss #297, and Najo #300 in firm alpha positions.
Following the split in the three breeding packs, the newly formed packs, apart from the Kisima, which still contained #297 and #300, had to establish a new alpha pair. It took some time for the males in the Sangito compound to sort out a new dominance hierarchy in 1998. As the occurrences in the year 2000 show, the male alpha position was not as firmly established as we had assumed in 1999. After the death of Llondirrigiss #297 in 1997 in the Kisima compound, Najo #300 could no longer maintain his male alpha status. Thus, he became a subordinate male, who raised his own pups without assistance, once his mate had died. Najo #300 did not bother to fight for his alpha status at all. Subsequently, the remaining males and females had to establish a new dominance hierarchy, which caused the fighting between the females #284 and #285. The fighting stopped with the removal of #285 in 1999.
11 Due to the ongoing hostilities in 1999, and peaking in 2000, it was apparent that the male alpha status was never very firmly established.
Birth rate and pup survival
Introduction
This overview concerns pups born in the period 1995 to 2001 ending, for obvious reasons, with the canine distemper outbreak at the end of December 2000 (see Foreword). Of the 15 litters produced during this period, 13 were born to socially dominant (alpha) and two to subordinate (beta) females.
The pups were born in an artificial den (see Housing) and it was only possible to check the pups when the parents were safely locked up in the feeding area at feeding time. The date of birth of each litter was based on the observation that one day the pregnant dog had an enlarged belly, swollen mammary glands and a swollen vulva and the next day she appeared to be thin. During the first days following the pup's birth, one or both parents stayed at the den, thus making it impossible to check the pups. Also, during times of intensive vocalization it was impossible to check the pups as the dogs could not be removed from the area around the den. At other times it was thought wise not to look into the den to prevent causing unrest in the pack. As pups first emerge from the den at approximately 3-4 weeks of age, it was often difficult to determine what had happened during the first weeks of their lives, for the reasons mentioned above. Between four and six months of age, transponders were fitted. At the same time their vaccination program was started. This age was chosen to minimize the risk of sedation of the individual and to minimize possibly stressful interference in the pack whilst the pups were very young.
Litters (table 1)
The 15 litters may be divided according to the success in raising pups:
1. Litters produced but no pups were seen. 2. Litters where pups were seen, but the pups died before a transponder was fitted. 3. Litters in which pups received a transponder and vaccinations. Data on the last two litters to be born , Ayubu I 25-5-00 and Sangito III 12-6-00, are included here although these pups had not received their transponders. The transponders would have been fitted if distemper vaccine had been available in November 2000.
1. Litters where no pups were seen. This involved two litters: a. Litter born 1-6-98 in the Kisima pack. The mother (#285) was the beta female in the Kisima pack. The alpha female (#284) who already had pups interfered probably in an attempt to combine the subordinate female's (#285) new pups with her own older pups, resulting in the death of the second litter (see chapter Kisima pack). b. Litter born 6-1-99 in the Lendenai pack; mother #262. After three weeks the litter perished for unknown reasons (see chapter Lendenai pack).
2. Litters where pups were seen, but the pups died before a transponder could be
12 fitted. Four litters were involved: c. Litter born 9-5-98 in the Kisima pack, mother #284. Litter perished following aggressive disputes between the alpha and the beta female (see chapter Kisima pack). d. Litter born 28-2-99 in the Sangito pack, mother #306. At the age of 6-8 weeks the pups all died within 14 days. Their faeces consisted mainly of blood and the pups were thin. An infection is considered to have been the cause, i.e. worms, bacteria or virus. e. Litter born 22-6-99 in the Kisima pack. Once again the beta female #285 gave birth. Again there were (as in litter c.) problems with alpha female #284 (see chapter Kisima pack). f. Litter born 22-9-00 in the Kisima pack, mother #284. At the age of approximately eight weeks the pups died one by one for no obvious reasons, although it appeared that the mother did not show much interest in them anymore. This might have been normal as in free living packs the mother's interest in her pups declines rapidly during the short lactation period and most of the normal caring duties carried out by mothers in other species, apart from suckling the pups, are taken over by the rest of the pack particularly the yearlings (Roger Burrows, pers.com.).
3. Litters in which pups received a transponder and vaccinations. A total of 9 litters: g. Litter born 4-3-97 in the Kisima pack, mother #297; the first litter born in the breeding program. Litter size 6 pups, all but one pup matured without any problems. h. Litter born 1-2-98 in the Lendenai pack, mother #262. Litter size 8 pups, 5 pups matured; 3 pups died for unknown reasons. i. Litter born 3-5-98 in the Sangito pack, mother #306. Litter size 8, of which 4 pups survived. Malnutrition was suspected possibly due to hierarchy problems in the pack (see chapter Sangito pack). j. Litter born 8-1-99 in the Kisima pack, mother #284. Litter size 4 pups. One pup, the only female of the four, was doing less well from the start and being smaller than her brothers was possibly at a disadvantage at feeding time. Probably as a result of getting less food than her brothers her condition gradually deteriorated and she eventually died. k. Litter born 12-9-99 in the Sangito pack, mother #306. Litter size 11 pups. Eight pups died slowly, one at a time. An infestation of worms was diagnosed from the faeces samples collected. No behavioural problems were reported in the pack at that time. l. Litter born 16-9-99 in the Kisima pack, mother #284. Litter size 6 pups. Three pups matured, the other 3 pups died of unknown causes. m. Litter born 3-4-00 in the Lendenai pack, mother #262. Litter size 7 pups. All pups grew up without problems. n. Litter born 25-5-00 in the Ayubu pack, mother #337. Litter size 3 pups. Pups grew up well until the age of 12 weeks when two pups suddenly died. An encounter with a poisonous snake was suspected. o. Litter born 12-6-00 in the Sangito pack, mother #306. Litter size 12 pups. Between the age of 6-12 weeks 8 pups died. Worm infestation was suspected and the pups were treated accordingly.
13 Table 1 Litter composition, sex and death rate Name Litter Date of Number of Sex Matured Sex Died birth pups Kisima I ³ g 4-3-97 6 2.4 5 2.3 1 Lenjo I ³ h 1-2-98 8 3.2 (3 ¹) 5 3.2 3 Sangito I ³ i 3-5-98 8 4.0 (4 ¹) 4 4.0 4 Kisima ² c 9-5-98 6 (6 ¹) 0 6 Kisima ² a 1-6-98 ?* ?* 0 ?* Lenjo ² b 6-1-99 ?* ?* 0 ?* Kisima II ³ j 8-1-99 4 3.1 3 3.0 1 Sangito ² d 28-2-99 10 (10 ¹) 0 10 Kisima ² e 22-6-99 6 4.2 0 6 Sangito II ³ k 12-9-99 11 2.1 (8 ¹) 3 2.1 8 Kisima III ³ l 16-9-99 6 2.1 (3 ¹) 3 2.1 3 Lenjo II ³ m 3-4-00 7 3.4 7 3.4 0 Ayubu I ³ n 25-5-00 3 0.1 (2 ¹) 1 0.1 2 Sangito III ³ o 12-6-00 11 3.1 (7 ¹) 4 3.1 7 Kisima ² f 22-9-00 4 (4 ¹) 0 4 Total 90 26.17 35=38.9% 22.13 55=61.1% * number of pups not established ¹ sex not established ² litter received no Roman numeral as no pups survived ³ Roman numeral indicates the first, second or third litter that matured in the pack concerned
Table 1 shows that 38.9% of the pups matured and 61.1% died. From the 15 litters 35 pups matured which means an overall pup survival per litter of 2.3 pups.
Another way to divide the litters is shown in table 2.
Table 2 Pup survival per litter Number of litters % Survival 0 % 6 40 Survival <100 % >0 % 8 53.3 Survival 100 % 1 6.7 Total 15 100.0
Percentage of litters with one or more surviving pups was 60 %
Cause of death of pups (see table 3 and 4)
The cause of death of pups was difficult to determine for the following reasons:- a. It was seldom possible to retrieve the carcasses due to the fact that most pups died in the den. By the time it was possible to check the den either no remains were found or on some occasions, just the head. Apparently most pups that died were eaten by pack members as happens in free living packs (Roger Burrows, pers.com.). b. No pathologist was available, i.e. if the carcass was available the autopsy was performed by a layman. The only means for further investigation
14 available were from tissue samples preserved in formaldehyde 4%. As a result the cause of death most of the time was based only on observations of the physical condition of the pups and the examination of their faeces.
In cases were it is suggested that problems in the social hierarchy led to unrest and stress in the pack this may have led directly to some pup deaths. Also due to the pack unrest some pups may have died indirectly from the unrest and directly from malnutrition caused by the unrest, after which they were eaten by the adults.
Table 3 Cause of death per pup/litter Pup/Litter ¹ Date of Date of death Age in Cause of death birth days Kisima I g 4-3-97 15-5-97 72 unknown Lenjo I h 1-2-98 28-4-98 87 fleas + unknown Lenjo I h 1-2-98 19-5-98 108 fleas + unknown Lenjo I h 1-2-98 25-5-98 114 fleas + unknown Sangito I i 3-5-98 27-5-98 24 hierar. unrest + fleas Sangito I i 3-5-98 6-7-98 64 hierar. unrest + fleas Sangito I i 3-5-98 6-7-98 64 hierar. unrest + fleas Sangito I i 3-5-98 14-8-98 103 hierar. unrest + fleas Kisima 6 pups c 9-5-98 ??? <90 hierar. unrest Sangito 4 pups d 28-2-99 12-4-99 44 infection: par./bact./virus Sangito 2 pups d 28-2-99 20-4-99 52 infection: par./bact./virus Sangito 4 pups d 28-2-99 22 to 25 -4-99 54 - 57 infection: par./bact./virus Sangito II 8 pups k 12-9-99 12-10 to 12-12-99 30 - 90 endoparasites Kisima II j 8-1-99 5-4-99 87 failing to make physical progress ³ Kisima 6 pups e 22-6-99 22-7 to 3-8-99 35 - 42 hierarchical unrest Kisima III l 16-9-99 15-12-99 90 unknown Kisima III l 16-9-99 15-12-99 90 unknown Kisima III l 16-9-99 26-12-99 101 unknown Sangito III o 12-6-00 17-7-00 35 endoparasites ² Sangito III o 12-6-00 24-7-00 42 endoparasites ² Sangito III o 12-6-00 27-7-00 45 endoparasites ² Sangito III o 12-6-00 27-7-00 45 endoparasites ² Sangito III o 12-6-00 2-8-00 51 endoparasites ² Sangito III o 12-6-00 25-8-00 74 endoparasites ² Sangito III o 12-6-00 23-9-00 103 endoparasites ² Ayubu I 2 pups n 25-5-00 19-8-00 86 snake bite possible Kisima f 22-9-00 26-11-00 64 unknown Kisima f 22-9-00 28-11-00 66 unknown Kisima f 22-9-00 3-12-00 98 unknown Kisima f 22-9-00 11-12-00 106 unknown ¹ litter a and b excluded as number of pups unknown ² suspected ³ no real cause of death, in fact unknown
15 Of the 54 recorded pups in table 3 all were less than four months old at the time of their death, four months being the time that the pups start to get their permanent teeth. After having reached that age no more pups died. Not surprisingly, it appeared that events in the pack during the first four months of a pup’s life are the most crucial for their survival.
Table 4 Cause of death in % Cause of death Number of pups % Hierarchical unrest 16 29.1 Endoparasites 15 27.3 Infection: par./bact./virus ¹ 10 18.2 Unknown ² 14 25.4 Total 55 100.0 ¹ cause might be one or more of these ² including possible snakebite and failing to make physical progress
Most pup deaths (29.1%) appeared to be correlated with unrest in the social hierarchy (hierarchical unrest) closely followed by that associated with endoparasite infection (27.3%). Social hierarchy unrest was observed in the Kisima and Sangito pack. In the case of the Kisima pack this followed to the death of the alpha female October 1997. After her death it was impossible, due to their being confined in a boma, for the pack to follow the usual procedure (natural pack rule) i.e. that following the death of an alpha dog, the pack would split up into two single sex parties or groups. However, pup loss due to hierarchical unrest had, in the Kisima pack, probably mainly resulted from aggressive interactions between the alpha and the beta female. The beta female could not physically distance herself in the boma from the attentions of the alpha and this probably led to frequent aggressive interactions, which caused the perishing of litters a and c. With more space, as in free-living packs things might have turn out differently as suggested by the personal observations of Roger Burrows below :-
A subordinate (beta) female who dens alone some distance away from the alpha's den after the latter has pups will not be fed by the pack who concentrate on regurgitating food to the alpha female and her pups. After the beta female has given birth, due to interference by the alpha female and/or other pack members (i.e. a form of hierarchical unrest) the beta female is not fed and must hunt alone or suffer severe malnutrition. This will lead to a lack of milk and the subsequent starvation of all or most of the subordinate’s pups before they are old enough to emerge from the den. In other cases the alpha female physically carries the subordinate’s pups to her den. Sometimes, however, subordinate female’s pups may inadvertently die during the struggle to move the subordinate’s pups to the alpha's den. Sometimes the alpha female moves her older pups into the subordinate’s den.
Significantly after the merger of the two litters, the beta female (usually the alpha's sister) is not only allowed access to all the pups but she is fed by the pack and can suckle her own and sometimes also the older pups of the alpha female. So the often severe aggression shown by alpha females to subordinate females who attempt to produce a litter within the packs denning area, decreases dramatically once the alpha ceases to lactate and/or after she has successfully merged the subordinates litter with her own older pups.
16 After a successful litter merger the alpha females may even regurgitate food to the subordinate female who now has responsibility for looking after the merged litters when the rest of the pack, including the alpha female, goes hunting. Some pups from a subordinate’s litter therefore can and do survive in free-living packs and as yearlings help the alpha female raise her next litter.
Disturbances in the Sangito pack were probably mainly related to a male hierarchy problem. The problem arose in the composition of the original pack. Although the pack consisted of full brothers and full sisters of different packs, they were more or less picked at random from the litters they originated from. In the case of the sisters this worked well, in the case of the brothers not. Obviously the alpha male of the litter from which the brothers originated was not included, thus lacking a firm male alpha, which might have caused the disputes between the males.
Endoparasites, coincidence or not, the second most frequent cause of death (27.3%), occurred in the same two packs, Kisima and Sangito. There might be a connection between social hierarchy unrest leading to stress and so increasing susceptibility to endoparasite infection. This may have resulted from pups being less well-fed as a result of pack unrest and so less well able to cope with the infestation of endoparasites, than the pups from litters in more socially stable pack. Endoparasites are always a problem when animals are kept on soil without the possibility for the animals being able to move around freely. There are three ways to combat the problem: a. Not too many animals should be kept together to keep infection pressure as low as possible. b. A strict anthelmintic regime. c. Remove the top of the soil every couple of years and replace it with fresh soil. In this way most of the worm eggs in the boma will be removed. See also chapter Parasites.
One litter of 10 pups in the Sangito pack is listed as “Infection: par./bact./virus” The pups died within 14 days; all were listless, suffered loss of body-weight and had bloody faeces which might have resulted from any of the following:- endoparasites (Ancylostoma sp. and Toxascaris leonina), bacteria or virus.
A high percentage (14 pups or 25.4%), died from unknown causes.
Pup survival per pack and per alpha female (table 6 and 7)
A noticeable difference in pup survival per pack and survival of litters per pack is shown in table 6. The best performance was by the Lendenai pack with a pup survival rate of 80% and an average pup survival per litter of 6. Even with litter b. included the average pup survival rate per litter was still 5. The Sangito pack had the lowest pup survival rate, 27.5 %. Their average pup survival per litter was 2.7. The average pup survival per litter of the Kisima pack was the lowest 1.8. With litter a included in the figures of the Kisima pack the average survival rate per litter dropped to 1.6. Interestingly, the average litter size in the Sangito pack was the highest, 10.0, and in the Kisima pack the lowest, 5.3, with the Lendenai pack in between, 7.5.
17 Despite the differences (pack composition, hierarchical unrest, litter size, etc.) between the Kisima, Lendenai and Sangito pack the number of surviving pups was almost the same (11,12 and 11). The Ayubu pack is not discussed here as they had only one litter of three.
Table 6 Pup survival rate per pack Pack N. Pups Pups Pup Average Average pup litters born survived survival % litter size survival per litter Kisima* 6 32 11 34.4 5.3 1.8 Lendenai# 2 15 12 80.0 7.5 6.0 Sangito 4 40 11 27.5 10.0 2.7 Ayubu 1 3 1 33.3 3.0 1.0 Total 15 90 35 38.9 6.0 2.3 (total program) * exclusive of litter a # exclusive of litter b
The Lendenai pack was the only pack where the same alpha pair produced the litters, which resulted in the highest pup survival rate. In the Kisima pack after the first alpha female #297 died she was succeeded by #284. Pup survival rate of the new alpha female #284 was lower than that of the old alpha female #297 (see table 7). In free living packs once an alpha pair is established they often produce a litter each year with some pairs still doing so when 10 years old. The results from Mkomazi and those from free living packs in the Serengeti -Mara ecosystem (1985-91)(Fuller et al. 1992, Burrows et al 1994) suggest that there is a correlation between the longevity of the alpha pairs in a population resulting in high litter/pups survival in their pack and so enhancing the long term survival of the local wild dog population of which they form part.
Table 7 Pup survival rate per alpha female Alpha Nr. Pups Pups Pup Average Average pup litters born survived survival % litter size survival per litter 297 1 6 5 83.3 6.0 5.0 284 4 20 6 30.0 5.0 1.5 262* 2 15 12 80.0 7.5 6.0 306 4 40 11 27.5 10.0 2.7 337 1 3 1 33.3 3.0 1.0 * exclusive of litter b
Litter per alpha female and month of birth (table 5)
Few conclusions can be drawn from table 5. However, most births took place in the period of the long rains, which in the Tarangire - Masai Steppe area, where the original pups came from, is from March - June and is the period when the migrant prey species such as wildebeest and gazelles will be spread over the grassland of the Masai Steppe. This would be similar to the main birth period for packs formerly in Serengeti. The Lendenai alpha female #262 showed a tendency to give birth early in the year. The alpha females from the Kisima, Sangito and Ayubu pack showed a tendency to give birth in May, June and September.
18
Table 5 Litter per alpha female and month of birth 297(Kis.) 284(Kis.) 285(Kis.)# 262(Len.) 306(San.) 337(Ayu.) Total January * * 2 February * * 2 March * 1 April * 1 May * * * 3 June * * * 3 September * * * 3 Total 1 4 2 3 4 1 15 * one or more pups survived Kis.=Kisima * no pups survived Len.=Lendenai # beta female San.=Sangito Ayu.=Ayubu
Time interval between litters (Dates in red: no pup of that litter survived) Gestation period 68-70 days.
first second third fourth litter litter litter litter
#297 4-3-97
#262 1-2-98 6-1-99 3-4-00
11 months, 6 days 15 months
#306 3-5-98 28-2-99 12-9-99 12-6-00
10 months 6 months, 12 days 9 months
#284 9-5-98 8-1-99 16-9-99 22-9-00
8 months 8 months, 8 days 12 months, 6 days
#285 1-6-98 22-6-99
19
12 months, 22 days
The figures show #262 had one litter a year and the loss of the second litter apparently did not influence the interval before the next litter. For #306 the interval was eight to nine months; after the loss of her second litter it took her only six months to produce the next litter. Kisima #284 initially (on two occasions) had an eight months interval, which changed to a twelve months interval for the last litter. The beta female #285 had a one-year interval between her two litters. Average interval between litters was approximately 10 months.
Conclusions
Close observation of captive packs is essential for the welfare of the packs in the program but especially for identifying reasons for the loss of pups. The high number of 'cause unknown' emphasises the importance of developing ways to retrieve carcasses of dead pups. A strict anthelmintic program is essential in any captive breeding group.
For reasons given in the 'Introduction' and 'Cause of death of pups' sections, some of the data presented is not as complete as one could hope for. However data from the Lendenai pack, suggests that packs with no overt social hierarchy problems have a higher pup rate survival than packs with a lot of social unrest (e.g. Kisima and Sangito pack).
Based on even this limited data, if the known 'natural wild dog pack rules/protocols' are followed when captive breeding packs are initially artificially formed, this will diminish any potential social hierarchy stress leading to social instability and aggression.
It is likely therefore that if the natural pack rules/protocols that have been deduced from the long term study of free living packs are followed in captive breeding programs, the better the welfare of the captive groups and so the higher the rate of pup survival.
Restart/Kimondo Pack
After the devastating canine distemper period from December 2000 – February 2001, it was a real thrill for all the people and dogs involved to see the three survivors united on March 13th 2001. During the preceding weeks, they had been calling to each other every night.
20 For the occasion the two males, #325 and #372 were sedated and transferred to the feeding area of the Sangito boma. The female #306 was locked out of the feeding area. The moment she noticed the other dogs, still sedated, she started to whine softly. After waking up, the two males (seeing each other for the first time), showed no signs of hostility toward each other, although #325 took more time for a complete recovery. In the meantime, #306 tried to make contact through the wire, and started to roll over onto her back. After 2½ hours, #325 was completely recovered, and since no signs of hostilities were shown though the wire (in fact #306 and #372 were very interested in each other), the dogs were put together. Immediately #306 and #372 joined, and #306 was putting her nose under #372’s belly. #325 kept a little distance. The next day, the three of them were eating and sleeping together. Apparently at the introduction, it was already decided who was going to be alpha-male. In the second half of April, #306 and #372 mated, and on the 7th of July, #306 gave birth to eight pups (m5, f3). The pups flourished. Unfortunately, on the 4th of August, fate struck again. All of a sudden #306 showed signs of serious distress, and died within six hours (see Veterinary Work). That day, the pups were exactly four weeks of age, and old enough to live on solid food. Having a lot of experience in raising the original pups, it was decided to separate the pups from the males, to keep a close watch on them. The pups were transferred to the Kisima boma. Nevertheless, one female pup died on the 3rd of October (see Pathology). The remaining seven pups grew up well.
At the end of June, two female wild dogs showed up in the vicinity of our bomas. It was most likely that they were two sisters looking for an all-brother party to start a new pack. The females were very interested in the pups as well; they kept coming and going for the next couple of months. After the death of #306 they stayed even more closely to our bomas. Apparently, they had no intention of leaving, and the decision was made to try and introduce them to our males. At the end of August the door of one boma was kept open and the two females went in. Before the door could be properly shut one female escaped. However, she stayed close to the boma where her sister was kept. Toward the end of September she was still hanging around, and it was apparent that she had no intention of leaving. At that time the wildlife veterinarians from TAWIRI (Drs. Robert Fyumagwa and Harald Wiik) were in Mkomazi on other duties, and they decided to provide us with their professional assistance in order to transfer her safely into the boma--finally re-uniting her with her sister. Few hostilities took place when the four dogs were introduced to each other, and we expect that they will establish a new breeding pack soon (Kimondo pack).
In December 2001, it looked like #372 was the alpha male, and #040 the alpha female.
Success From Tragedy
Over the past three years Roger Burrows has visited our breeding program on several occasions. His visits were highly valued, as he offered his knowledge and expertise without reservations. It seemed only logical to compare the behavior of the captive Mkomazi wild dogs with the behavior found in free-living East African packs. We are fortunate and grateful that Roger wrote the next chapter in close collaboration with us.
21
Behavior
Despite the tragedy, the first six years of our African Wild Dog Breeding Program has produced some very positive results. The behavior (i.e. feeding and sexual hierarchy) of the original (wild-born) and captive-born dogs is comparable with the behavior found in East African free-living packs, studied in the Serengeti ecosystem between 1965-91 (FRAME et al 1979, BURROWS 1995).
1. Feeding Hierarchy
In Mkomazi the dogs are fed once a day. To ensure that each dog has access to the food at some time, the keeper developed, out of necessity, a highly artificial system. The system is subject to change, depending on the social interactions of the dogs at a particular time. Access to the food in the feeding enclosure is controlled by a couple of gates which makes it possible to rotate the dogs in the feeding area, ensuring that all dogs have the opportunity to feed. Different groups were given access for a few minutes at a time before they were removed and another group fed, with no group of individuals being given unlimited access to the food. To see if one of the captive packs would behave as if they were a free-living pack on a kill, a simple experiment was performed. During one evening feed in 2000, all of the individuals in a pack were allowed access to the food at the same time. For a better understanding of the experiment, the behavior of a pack in the wild at a kill is described first.
In the wild, when the pups are old enough to join the adults at a kill, the pups have feeding priority. The adult dogs, apart from the alpha female, immediately stand back and drive away would-be scavengers while the pups and the alpha female feed. The pups use behavioral postures and vocalizations to prevent the adults from coming back to the kill. Once the pups are satiated, they lose interest in the food and move away, allowing the yearlings (12-24 months old) to feed. Once the latter are satiated, the alpha male and older subordinate dogs of both sexes are the last to return to what remains of the kill. Some of these dogs may have made the kill before the rest of the pack arrived on the scene. Most of the pack now leaves the kill site with the pups, and move away to find shade or water. Consequently, a very small group of adults, or often a lone dog (particularly one of a subordinate pair), remain on the kill, and may get little time to feed before the remains are scavenged by spotted hyenas, vultures, eagles, jackals, and occasionally lions.
Experiment: In Mkomazi during an evening feed in 2000, all individuals of a pack were allowed access to the food at the same time. After a few seconds of chaos and much noise around the food, the pattern described above for the wild packs suddenly emerged, with the young pups left eating, and the adults standing around awaiting their turn. This admittedly very limited experimental evidence suggests that the feeding behavior in African wild dogs is not learned by example. Since none of the original 25 pups were old enough at the time of capture to have joined their parents in a hunt, the feeding behavior appears to be genetically determined. Released captive groups of wild dogs from Mkomazi into the wild, would in all probability, once a pack is formed, immediately show the same feeding hierarchy as that observed in free-living packs.
2. Sexual Hierarchy
22 a. Female dominance hierarchy In free-living groups of sisters, either within a pack or dispersing, one sister will be alpha. She will have assumed that position usually without overt aggression. The lower ranks in the female hierarchy can, and do, change; sometimes after serious fighting. In one Mkomazi pack, the Kisima, considerable female aggression was observed. In the Lendanai pack, the alpha female harassed, but did not fight, her lower ranking sisters.
In the wild, subordinate females are likewise harassed. If they produce pups, these pups may die either from starvation or be killed due to the aggressive interactions between the alpha and subordinate females. In the Kisima compound two litters born to a subordinate female died. It is interesting that, as in the wild, the beta litter of 1998 was born after the alpha had given birth. Any subordinate's pups are therefore younger than the alpha's; in the wild, sometimes by as much as two months.
In the wild however, sometimes a subordinate's pups do survive, but only after being taken over by the alpha. If the alpha loses her litter, she still may have the pups of the beta female to act as helpers in the following denning season. This way, the continuity of the pack is ensured until the alpha female is successful in raising her next litter.
In the Serengeti, the function of the sisters in a newly formed pack is to provide help in raising the first litter of the alpha. In the subsequent year(s), the subordinate sisters of the alpha leave the pack. This leaves the alpha female as the only adult female in the pack. The dispersing sisters can form new packs with groups of any unrelated males that they meet.
The losses of subordinate litters in Mkomazi, suggest that to prevent pack disruption and unnecessary mortality of pups, due to aggression between the adult females, only one adult breeding female should be kept in a captive pack. b. Male dominance hierarchy
Following the demise of most of the Mkomazi captive population in 2001, the two surviving males from different natal packs, both captive-born, were given access to the lone surviving mature wild-born female. A new pair bond was immediately established with the younger male as alpha. This behavior parallels the behavior found in free-living Serengeti packs.
Recently, observations of male hierarchies in South Africa found the same behavior as that observed in the Serengeti wild packs and the Mkomazi captive packs. When two hand-raised younger males and two free-born older males were introduced to a free-born female, the alpha male turned out to be one of the younger captive-raised males (HOFMEYR 2000).
The same thing happened in free-living Serengeti wild dog packs. When a dispersing group of related males of different ages left their natal pack, one of the younger males took over the alpha position. Similarly, when the last adult female in a pack died, a male from her last litter became the alpha in the now all male group. Such all male groups remain on the former packs' home range.
In October 1997, the alpha female (#297) in the Kisima pack died. One year later some dogs were removed from this pack to start the Ayubu pack. What remained of the Kisima pack contained two young males, sons of the deceased alpha female, their father (#300, former alpha male) and three males of the same age. The latter males were not related to the former
23 alpha male. A new alpha pair was formed, but neither was related to the sons of the former alpha. However, these young males were seen to join in disputes between the new alpha male and his two brothers, and in disputes between the adult females. It is likely that the younger males from the original alpha pair were, as the youngest males present, attempting to take over the alpha role, and would not accept an unrelated older male as the alpha in the compound.
Experiment: This time unplanned. At the end of June 2001, two wild females arrived at our bomas in Mkomazi. These females came and went. After the death of the captive alpha female on August 4th they stayed even closer to the boma, and eventually joined the program. This is the scenario that we hope will happen with future re-introductions: A group of captive- bred males await within a boma for the arrival of immigrant females. The captive-born males would then be allowed to join the females to form an instantaneous new pack.
Conclusions
The Mkomazi African Wild Dog Breeding Program has produced some extremely useful findings:
1. The social and feeding hierarchies of the captive Mkomazi packs parallel that of free-living African wild dogs in the Serengeti ecosystem.
In both the Mkomazi and the Madikwe captive packs, as in the free-living Serengeti population, in all-male groups of mixed age, a younger dog takes over the alpha position despite the presence of older, heavier, and more experienced males--including, if present, his father. This counter intuitive finding has considerable practical implications for any re- introduction or supplementation programs in East Africa, and in at least some southern Africa populations. Based on data from free-living Serengeti packs from 1965-91, and the captive packs in Mkomazi, a general protocol emerges: When in a pack, if either parent (one of the alpha pair) dies, a male from the youngest cohort in the pack becomes the alpha (see also FRAME et al., 1979, BURROWS 1995). Likewise, in an artificially constructed pack consisting of unrelated males, the alpha will be from the youngest cohort present-- irrespective of his relationship, relative strength, or hunting experience.
2. The behavioral similarities of captive and free-living packs suggest that captivity, in the short term at least, does not change basic behavior, although other studies show that hunting skills are acquired after release. For this reason, it is important that a combination of a wild- bred group and a captive-bred group of the opposite sex be released together.
3. It is unwise for pack stability to mix pups from different natal packs.
4. A 'nuclear pack' consisting of one breeding adult female and one unrelated male is all that is necessary to start a captive pack.
5. There is no reason to leave yearlings from a captive mating pair in the same enclosure with their parents once a new litter of pups is produced and raised. Such yearlings would be ideal subjects for translocation.
24
6. There is no evidence in captivity of aggression leading to severe physical injury resulting from hierarchy disputes in male African wild dogs. Conversely, in free-living packs, aggression between females for social rank can lead to severe injuries.
7. The arrival of the two females (June 2001) suggests that even in areas in which the African wild dog is described as 'absent' or 'vagrant', dispersing groups pass through, as they are known to do in the former Serengeti study areas. This also suggests that the technique proposed in the "Plan for the Re-introduction of the African Wild Dog" (VISEE et al., 2001) is realistic, and has a considerable chance of success.
Zoo technique
Composition of Packs
The original litters in 1995 had the following composition:
Lendanai: 261 F Llondirrigiss: 289 M Najo: 265 F 262 F 291 M 273 M 263 M 293 M 274 M 264 F 294 M 275 F 296 M 276 F 297 F 284 F 298 M 285 F 299 M 288 M 300 M
25 303 M 305 M 308 F 310 M
1.3 7.1 7.6
F= Female M= Male Najo 273, 288, 303 and Llondirrigiss 299 were sent to Kenya. Llondirrigiss 297 died in October of 1997.
The following points were taken in consideration for the composition of the breeding packs:
1. As little as possible genetic material should be lost. 2. No brother sister relationships. 3. Full sisters of a litter of one pack should form a new pack with the full brothers of a litter of another pack. 4. A breeding pack should consist of at least two females and two males. In case the alpha female or male dies, there is no need to introduce a new female or male, with all the risks of fighting and getting badly hurt.
Taking these guidelines into account, the following packs were composed in 1997:
Boma I: Kisima Pack: Najo x Llondirrigiss Males Females Males Females Llondirrigiss 293 Llondirrigiss 297 Kisima 333 Kisima 336 296 Najo 276 335 337 298 284 339/348* Lendanai 263 285 Najo 300 Born 4-March-97
* Transponder 339 was replaced by transponder 348
The composition of the Kisima Pack was not in agreement with the above-mentioned guidelines. It was more or less a leftover. Brother to sister relationship was possible. However, at the time of composing the packs female Llondirrigiss #297 and male Najo #300 already were alpha female and male and had their first litter (the Kisima I pups). Unfortunately female #297 died. At the end of 1997 the hierarchy of the alpha female and alpha male had not yet been established, it would be a Najo female anyway, because only Najo females were left. The establishing of the alpha’s caused a lot of trouble in the pack in the years to come.
Boma II: Lendanai Pack: Males Females Najo 274 Lendanai 261 310 262 264
26
Boma III: Sangito Pack: Males Females Llondirrigiss 289 Najo 265 291 275/306* 294 305 308
* Transponder #275 was replaced by transponder #306 The composition of the Lendanai and Sangito packs was according to the guidelines mentioned above. Nine dogs were left in the original boma.
In 1998 Boma IV was build and the Ayubu pack established:
Boma IV: Ayubu pack:
Male Females (Najo x Llondirrigiss) Lendenai 263 336 337 348
27 Below is an overview of each pack's composition at the time of the canine distemper virus (CDV) outbreak. Included are the dates of birth and death, and the origin of the dogs.
Boma I: Kisima Pack Identification Sex Date of birth Date of death Origin 293 M August 1995 05-02-2001 Llondirrigiss 296 M August 1995 01-02-2001 Llondirrigiss 298 alpha M August 1995 13-02-2001 Llondirrigiss 276 F August 1995 01-02-2001 Najo 284 alpha F August 1995 31-01-2001 Najo 300 M August 1995 26-01-2001 Najo Mother x Father 333 Kisima I M 04-03-1997 18-01-2001 Llondirrigiss x Najo 335 Kisima I M 04-03-1997 06-02-2001 Llondirrigiss x Najo 366 Kisima II M 08-01-1999 01-02-2001 Najo x Llondirrigiss 369 Kisima II M 08-01-1999 31-01-2001 Najo x Llondirrigiss 372 Kisima II M 08-01-1999 Alive Najo x Llondirrigiss 312 Kisima III M 16-09-1999 30-01-2001 Najo x Llondirrigiss 344 Kisima III F 16-09-1999 30-01-2001 Najo x Llondirrigiss 349 Kisima III F 16-09-1999 03-02-2001 Najo x Llondirrigiss
Number of dogs: 14, Sexes: 10,4 at the time of the CDV outbreak.
Boma II: Lendanai Pack Identification Sex Date of birth Date of death Origin 261 F August 1995 07-01-2001 c Lendanai 262 alpha F August 1995 09-01-2001 c Lendanai 264 F August 1995 07-01-2001 c Lendanai 274 alpha M August 1995 06-01-2001 c Najo 310 M August 1995 07-01-2001 c Najo Mother x Father 322 Lenjo I M 01-02-1998 11-01-2001 c Lendanai x Najo 323 Lenjo I F 01-02-1998 02-01-2001 Lendanai x Najo 325 Lenjo I M 01-02-1998 Alive Lendanai x Najo 327 Lenjo I F 01-02-1998 10-01-2001 c Lendanai x Najo 329 Lenjo I M 01-02-1998 29-12-2000 c Lendanai x Najo 780 Lenjo II F 03-04-2000 31-12-2000 Lendanai x Najo 812 Lenjo II F 03-04-2000 23-12-2000 Lendanai x Najo 817 Lenjo II M 03-04-2000 05-01-2001 c Lendanai x Najo 850 Lenjo II M 03-04-2000 05-01-2001 c Lendanai x Najo 893 Lenjo II M 03-04-2000 07-01-2001 c Lendanai x Najo 912 Lenjo II F 03-04-2000 21-12-2000 Lendanai x Najo 924 Lenjo II F 03-04-2000 29-12-2000 Lendanai x Najo c = day of death confirmed. The dates of death without a “c” means that the transponder was not checked at the time of death. The date given may not be exact. Number of dogs: 17, Sexes: 8,9 at the time of the CDV outbreak.
Boma III: Sangito Pack Identification Sex Date of birth Date of death Origin 289 alpha M August 1995 24-01-2001 Llondirrigiss 291 M August 1995 29-01-2001 Llondirrigiss 294 M August 1995 05-02-2001 Llondirrigiss 265 F August 1995 31-01-2001 Najo 306 alpha F August 1995 04-08-2001 Najo 305 F August 1995 29-01-2001 Najo 308 F August 1995 05-02-2001 Najo Mother x Father 352 Sangito I M 03-05-1998 14-07-2000* Najo x Llondirrigiss 355 Sangito I M 03-05-1998 29-01-2001 Najo x Llondirrigiss 358 Sangito I M 03-05-1998 14-07-2000* Najo x Llondirrigiss 359 Sangito I M 03-05-1998 24-11-2000* Najo x Llondirrigiss 319 Sangito II F 12-09-1999 27-01-2001 Najo x Llondirrigiss 360 Sangito II M 12-09-1999 25-01-2001 Najo x Llondirrigiss 361 Sangito II M 12-09-1999 05-02-2001 Najo x Llondirrigiss Pup Sangito III F 12-06-2000 18-01-2001 Najo x Llondirrigiss Pup Sangito III M 12-06-2000 18-01-2001 Najo x Llondirrigiss Pup Sangito III M 12-06-2000 29-01-2001 Najo x Llondirrigiss Pup Sangito III M 12-06-2000 31-01-2001 Najo x Llondirrigiss
Number of dogs: 15, Sexes 9,6 at the time of the CDV outbreak. * Died before the CDV outbreak.
Boma IV: Ayubu Pack Identification Sex Date of birth Date of death Origin 263 alpha M August 1995 03-02-2001 Lendanai Mother x Father 336 Kisima I F 04-03-1997 04-02-2001 Llondiirigiss x Najo 337 Kisima I alpha F 04-03-1997 29-01-2001 Llondirrigiss x Najo 348 Kisima I F 04-03-1997 11-02-2001 Llondirrigiss x Najo 285 F August 1995 06-02-2001 Najo Pup Ayubu I F 25-05-2000 25-01-2001 Llondirrigiss x Najo X Lendanai
Number of dogs: 6, Sexes 1,5 at the time of the CDV outbreak.
Identification
To run the breeding program properly it is necessary to be able identifying the dogs on all circumstances. Two ways were developed to achieve this goal. One way was to insert a transponder in each dog; the other way was making identification photographs of both sides of the dog in lateral recumbency. All transponders were placed subcutaneously on the left side of the neck at the first time of sedation, which happened to be when the dogs were still pups 4-6 months of age. They were inserted in a dorso-ventral direction. Before and after insertion the transponders were checked with the reader for proper action. All the numbers started with NLD 093500110, followed by 3 numbers to identify the individual dog. Datamars, Switzerland, manufactured the reader and the transponders.
29 Every time an animal was sedated, the transponder was checked. This happened to be the time of taking blood-samples most of the times. The transponders were on all occasions in good working order except on two occasions. March 1997 it appeared that the reader was not reading the transponder of Najo #275. It was not possible to locate the transponder manually, so it was decided to bring insert a new transponder #306. October 1998 the same applied to Kisima’s #339 transponder, she received a new transponder as well #348. Identification photographs were taken at the time of sedation. The first time at the first sedation, still being pups. When the dogs were full-grown new identification photographs were taken. No differences in the pattern of their coats were noticed. In the photograph the last three numbers of the transponder was included, which made it possible to recognize a dog. by number without sedation. This proved to be very helpful for observations especially for the keepers. In the two cases the transponder was not working identifying the dogs was simple due to the photographs
Population genetics.
In order to investigate the genetic variation within and between the three original African Wild Dog groups (Lendanai, Llondirrigiss and Najo) blood samples (EDTA blood) were collected in December 1995 and February 1996. The University of Groningen, Centre of Ecological and Evolutionary Studies, Population Genetics performed the investigations. In 1997 the results became available and showed that there was a considerable genetic variation. See attached report, Genetic Variation, from Dr. L. van de Zande. Female Llondirrigiss 297 gave birth to six pups on 4 March 1997. The behavior of the male Najo 300 was consistent with that of an alpha male. However it was not certain that he was the father of the pups as several males, brothers of 297, mated with the mother to be. EDTA blood samples were taken in October 1997 in order to investigate the fatherhood of the pups, which was important for breeding purposes. DNA fingerprinting made an end to speculations, Najo 300 proved to be the father of the pups. See attached report, Parenthood Analysis, from Dr. A.L. Kappe, Gendika B.V.. The combination of Najo and Llondirrigiss made these pups very suitable for a combination with Lendanai. Unfortunately it never came that far.
Housing.
The first and original enclosure (boma), the Kisima boma, was build in 1992 and 1993. It took two years before the first pups arrived in 1995. The Kisima boma was the largest boma; size approximately 90 m x 80 m. The set up of the boma was the same as described below for the Lendenai and Sangito boma, except the different parts of the boma were slightly larger than those of the other boma’s. In 1997 two new, identical, breeding boma’s (Lendenai and Sangito) were built, size 70 m x 40 m. Within these enclosures each contained two connected smaller enclosures, size 15 m x 5 m. These were used for feeding and separation if required. Connected to the last of the small enclosures was a 9 m long wired passageway, width 1 m, height 1 m. This passageway could be divided in 3 parts, 3 m long each, by means of wired slides. Each compartment gave sufficient space for 3 full grown dogs, to make it possible to dart them by blowpipe in order to vaccinate or sedate them. Every evening, after being fed, they left through this passageway, which made them grow accustomed to it. In the enclosure an artificial den was built with two opposite entrances. It was not possible for dog or men to look into the den from the
30 entrances. An artificial hill was provided as well. Natural shelter was provided by trees and thickets, artificial shelter was provided by a roof, 5 m x 2 m, constructed from palm leaves. The height of the fence was 2.25 m with an 80 cm extra overhang inside and outside. The fence was dug 60 cm into the soil and secured with heavy pieces of rock. The enclosures were situated in such a way, that the dogs from the different packs could hear each other, but could not see each other.
1 cm = 5 m A. Den, B. Hill, C. Passageway, D. Feeding area, E. Shelter for sick or injured dogs.
In 1998 the three boma’s of the dogs were not altered. A fourth boma was built to house the Ayubu pack. This boma was built exactly the same as the two boma’s in 1997.
As an extra precaution to keep small animals and people at a distance, a six foot high fence made of chicken wire was placed at a distance of approximately two meters from the outside of the perimeter fence of the Sangito and Kisima bomas in 2001. In the years to come a same fence will be placed around the Lendenai and Ayubu boma.
Nutrition.
The dogs were fed once a day. As a basis they received a commercial dog pellet, which was softened through soaking in water. Mixed with this dog food were pieces of cow meat. Once or twice a week one or two cows was bought and slaughtered, that is to say, as much meat as possible was taken from the cows and put into a freezer, operating on paraffin. As much as the freezer could contain was put in the freezer. The rest of the carcass was fed to the dogs. On the day they received the carcass, they did not receive the pellets. Pellets used were GILPA’s “Valu Complete”, a complete adult dog food. The moment the pups started to eat solid food, they received the same, softened, pellets with cow meat. The pups grew up with no, visible, bone abnormalities.
31
Bodyweight (See Appendix I)
It was only possible to measure the bodyweight of the dogs when they were sedated. They were weighed during each sedation.
The original dogs show the next, average, bodyweights in the course of time:
Bodyweight in Kilograms (kg)
Date Age (aprox.) Male Female 19-12-95 5 months 10.1 9.5 29-02-96 7 months 12.8 11.3 09-03-97 1 year 7 months 22.9 18.5 15-10-97 2 years 3 months 25.7 19.2 15-02-98 2 years 7 months 24.7 18.4 04-10-98 3 years 3 months 25.7 18.8 30-03-99 3 years 9 months 22.4 19.8 02-11-99 4 years 4 months 23.0 19.1
Although the number of dogs is not the same at every date of weighing, one might conclude that the average bodyweight for dogs above two years of age is 22-25 kg for males, and 18-19 kg for females.
The pups of the different litters showed the following development of, average, bodyweight:
Kisima I (04-03-1997) Date Age (aprox.) Male Female 15-10-97 6 months 17.4 15.4 15-02-98 1 year 22.3 20.1 04-10-98 1 year 7 months 23.0 21.0 02-11-99 2 years 8 months 23.8 20.3
It is interesting to note, that the Kisima I pups weigh far more than the average dog of the original group at approximately the same age (7 months). Males average 4.5 kg and females 4 kg more. One has to take into account, that the number of Kisima pups is only 5 against 25 of the original group. Also the original group of 25 animals was fed as a whole group with heavy competition at that time. The Kisima pups, at the time of weighing, were still fed separately, without any competition and they were fed partly commercial dog food, which did not happen with the original dogs at the same age. From the age of 1½ years onward these dogs have about the same average bodyweight as the original dogs , although the female Kisima dogs are slightly heavier (1 kg).
Lenjo I (01-02-1998) Date Age Male Female 04-10-98 9 months 16.6 16.8 30-03-99 1 year 2 months 18.8 20.2 02-11-99 1 year 9 months 19.7 19.8 30-03-00 2 years, 2 months 21.4 20.2
Sangito I (03-05-1998) Date Age Male 32 05-10-98 5 months 12.3 30-03-00 2 years, 1 month 21.7
Kisima II (08-01-1999) Date Age Male 02-11-99 10 months 18.4 08-11-00 1 year 10 months 20.3
Sangito II (12-09-1999) Date Age Male Female 08-11-00 1 year 2 months 17.8 15.6
Kisima III (16-09-1999) Date Age Male Female 08-11-00 1 year 2 months 19.9 15.4
Lenjo II (03-04-2000) Date Age Male Female 08-11-00 7 months 11.4 11.2
Neck-size (See Appendix I)
To get an impression what size of collar is needed for radio-collaring in the future, some dog’s neck-size was measured November 1999. This concerned 18 dogs, age 21 months or older, and 3 pups 11 months old. The measuring took place while the dogs were sedated for the annual blood-sampling with a tape measure in centimeters. The dogs older than 21 months had an average neck-size of 38 cm. Divided in females and males: the males had an average neck-size of 40 cm and the females 37 cm. The three pups had an average neck-size of 35 cm.
VETERINARY WORK
33
Preventative Medicine
Preventative medicine is the most important part of the work and takes the most time. It is far better to prevent diseases than to cure diseased animals, particularly in the given situation whereby many animals are kept in relatively close confinement and contagious diseases will spread easily.
The most dangerous diseases to the dogs are canine distemper, rabies and parvovirus infections. A program was developed in which the dogs were vaccinated against these diseases. In addition, their blood was taken on a regular basis to study the effect of the vaccinations, which made it possible to alter the vaccination schedules according to the results. Vaccination policy and results will be discussed in the chapter on Vaccines.
Another important part of preventative medicine is the prevention of parasites, which will be reported and discussed in the chapter on Parasites.
Clinical Work--Diseases
Our policy was, and remains, to interfere as little as possible in the breeding packs. Diseased dogs were only treated when it is possible to separate them, treat them and replace them immediately, or when pack treatment was required and possible. The canine distemper outbreak is described and discussed in detail.
Pathology
Unfortunately the alpha female #297 was lost in 1997. Pathology findings are reported Due to the canine distemper epidemic many necropsies could have been performed, but only a limited number were carried out for reasons described in the section on pathology. On two different occasions in 2001, not related to distemper, necropsy was also performed.
34 Preventative Medicine
Vaccines
The African wild dog appears to be extremely susceptible to diseases like canine distemper virus, parvoviral disease and rabies. Little is known about the effectiveness of vaccinating wild dogs against these diseases. The purpose of vaccinating our captive wild dogs is first, to attempt to protect them from the above-mentioned diseases, and secondly, to study the effectiveness of the vaccinations. As a common rule, only inactivated (i.e. killed) vaccines were used for two reasons: first, to prevent the introduction and spread of viruses in the environment, and secondly, to prevent any ill side effects on the dogs.
At the 40th International Symposium of Zoo-and Wildlife Diseases, Rotterdam, the Netherlands, a paper was presented by Aart Visee: Distemper, Rabies and Parvovirus vaccinations in a captive breeding programme for the African wild dog (Lycaon pictus) in Northe0rn Tanzania. (Verh. Erkrg. Zootiere (2001) 40, 243-250). This paper covers the results of the first 5 years of the vaccination program. Parts of this paper are quoted, as they give an excellent overview of the work accomplished. The paper is attached to this report.
Canine Distemper Virus (CDV) See Appendix II Vaccinations
No inactivated distemper vaccine is commercially available, however, the Institute of Virology, Erasmus University, Rotterdam, developed and kindly donated such a vaccine: CDV-ISCOM Vaccine. This vaccine must be kept frozen until used, i.e. in a cold chain. Individuals were vaccinated three times in the first year; with a 14-day interval between the first and second vaccination, and a 30-day interval between the second and third vaccination. The 3 vaccinations were followed by a single annual booster vaccination.
The Regime Adopted for the Original Dogs
1. All (N=25) pups were first vaccinated and blood-sampled following sedation on 19/12/1995 when approximately 5 months old. A second vaccination and a third vaccination was carried out on 03/01/1996 and 14/02/1996 by blowpipe without sedation and no blood-samples were taken. Blood sampling took place following sedation on 29/02/1996.
2. A fourth vaccination as the first annual booster was given by hand, following sedation on 09/03/1997. Blood-samples were taken also.
3. Blood-samples were taken but no vaccinations were given on 15/10/1997.
4. Blood-samples were taken on 15/02/1998 and a further vaccination by hand was carried out using a new batch of vaccines.
1. In 1998, the annual February booster vaccination was replaced by a similar vaccination in October (04/10/1998). This meant that the dogs were vaccinated twice in 1998, again using the new vaccine batch.
2. In March 1999, 5 dogs were tested.
35 3. In November 1999, the annual booster vaccination was given and blood-samples taken.
4. As there was no vaccine available (a new batch of vaccines was still in the safety-testing phase at the Erasmus University), the dogs were not vaccinated in November 2000. The dogs were supposed to receive their annual booster vaccination at that time.
The Regime Adopted for the Pups Born in the Breeding Program
1. The pups were vaccinated according the schedule described above. After finishing the initial course of three vaccinations in the first year of their lives, blood-samples were taken. Then the pups participated in the annual vaccination program. 2. Most pups were due to receive their annual booster vaccination November 2000. For reason described above, this did not happen.
Results
1. Pre-vaccination titers to CDV: Seventy-five percent (n=12) of the wild-born pups of 1995 were sero-positive for CDV when first vaccinated at approximately 5 months of age, on 19-12-95 (see Table 1). A group of (n=4) captive bred unvaccinated pups (Sangito Pack) were sero- positive for CDV on 04/10/1998 when first vaccinated with titer levels of 80 (1x3) and 20 (x1). 2. Post-vaccination titers from 29-2-96 to 30-03-2000(see Table 1). All dogs had completed the course of three vaccinations at the time of testing. One year after the annual booster vaccination of 1998, 82% of the tested dogs had antibody titers >20. In March 2000, 5 months after the booster vaccination of November 1999, 67% of the tested dogs had antibody-levels >20. 3. In November 2000, only one of the nine tested, vaccinated dogs had sufficient antibodies. These were all dogs that went through a complete course of vaccinations. The other 7 dogs (780-924) tested, had less than 20 neutralizing antibodies, but they were non-vaccinated pups at that time.
Table 1: Virus neutralizing antibody titers in African Wild Dogs during a vaccination trial with a CDV-ISCOM vaccine, after completing the three initial vaccinations followed by annual booster vaccination Number of dogs % Number of dogs % Total number of Date NT-titer >20 NT-titer <20 Dogs 19-12-1995* 9 75 3 25 12 29-02-1996 22 88 3 12 25 09-03-1997 5 26 14 74 19 15-10-1997** 0 0 22 100 22 15-02-1998*** 14 78 4 22 18 04-10-1998**** 29 100 0 0 29 30-03-1999 10 100 0 0 10 02-11-1999 14 82 3 18 17 30-03-2000 6 67 3 33 9 08-11-2000 1 11 8 89 9 13-03-2001 2 2 0 0 2 NT-titer >20 are considered protective. * pre-vaccination titer ** not vaccinated on this occasion *** a new vaccine batch was used in February 1998 ****in 1998 the annual booster vaccination was replaced from February to October, as a consequence the dogs were vaccinated twice that year
36 Table 1 and Figure 1 includes all dogs tested except seven non vaccinated dogs (Lenjo II) November 2000 and four non vaccinated dogs (Sangito I) October 1998.
Figure 1
120 100 Percentage dogs 80 with VNT titer 20 or more 60 Number dogs tested 40 20 0 19- 29- 09- 15- 15- 04- 30- 02- 30- 08- 13- 12- 02- 03- 10- 02- 10- 03- 11- 03- 11- 03- 95 96 97 97 98 98 99 99 00 00 01
Discussion
This is the first report of sero-positivity in unvaccinated African wild dog pups, both wild-caught and in captivity. As pups of 4-5 weeks of age are confined to the area of the den and exposure to CDV was confirmed in 75% of the pups from the three litters obtained from three different dens, it is likely that they carried maternal antibodies. This suggests that the adults in the three packs, from which the wild-captured pups were obtained, had been exposed to canine distemper virus (CDV) in the Masai Steppe. Strictly speaking CDV or morbillivirus. As CDV is a morbillivirus, it is theoretically possible that another morbillivirus instead of the CDV was involved. However, here it concerns land carnivores, and CDV is most likely.
The presence of CDV titers in the African wild dog population of the Masai Steppe is similar to the situation in the Selous (approximately 400 km south of the area from which the pups were obtained), where 59% of adults immobilized for radio-collaring were sero-positive. Here the evidence presented suggested they were not the result of surviving an epidemic (CREEL et al, 1997). Because of the absence of domestic dogs in both the Selous and Mkomazi, wildlife serving as a reservoir of CDV seems to be likely. The presence of antibodies in the captive-born pups, as with the wild-born pups, suggests maternal antibodies. In the case of the wild-born pups, a mild infection cannot be ruled out, as the titers of 1995 and early 1996 show a great variety (from <20 up to 540), and the maternal antibodies are believed to have disappeared after 2 months.
Although initially the titers achieved after three vaccinations appeared to be satisfactory, a year later (1997) the titers had declined below the protective level in the majority of the individuals. Failure of the CDV vaccinations was possibly due to the batch used. The way the vaccines are stored may also have had a negative influence; ideally, the vaccines should be stored in a freezer at minus 70 °C, but ‘in the bush’, it is impossible to reach that temperature in a freezer dependant mainly on solar energy. After a new batch was used (February 1998) results improved dramatically, although it remains inexplicable why October 1997 all dogs tested had titers <20 and four months later (February 1998) 13 out of 17 dogs tested showed a titer of 20 and only four dogs a titer <20 without a vaccination in the mean time.
37 Over the years most of the titers of the dogs tested were 20 or <20. Exceptions were dogs tested at 19-12-95, 29-2-96, 4-10-98 and 30-3-99. The results of the dogs tested at 19-12-95 and 29-2-96 are discussed above. However 11 of 33 dogs tested at 4-10-98 had a titer of 80, 12 a titer of 40 and 10 a titer of 20. Three of the dogs with a titer of 80 and one with a titer of 20, were not vaccinated at all. All 10 dogs tested at 30-3-99 had a titer of 80. These results are dramatically better than the average results. A mild infection with a morbillivirus is a possibility with or without combination with the new vaccine batch used. The bad results of November 2000, might be partially explained by the fact that the dogs received vaccines, stored over a longer period in the Mkomazi freezer, which might have reduced the activity of the vaccine; i.e. a breakdown in the cold-chain. All individuals (except #366, #369 and #372), were vaccinated with a freshly arrived batch of vaccine in November 1999. Next to the raised question of the vaccine batch used February 1997 and the breakdown in the cold- chain in the year 2000, one also might wonder if there is a three-year cycle (Diagram 1) at work (extremely low titers October 1997 and November 2000) like the distemper in the ringed seal and measles in humans. However October 1997 showed a good outcome, but November 2000 a disastrous one.
Conclusion
Vaccinating the dogs three times in the first year (14 days between the first and the second vaccination, and 30 days between the second and third vaccination), followed by a single annual booster vaccination appeared to be successful in providing a protective titer (>20). One year after the third vaccination, all dogs tested appeared to have (i.e. in 1998) antibody-titers, which are considered to be protective (OSTERHAUS pers.com.). However, much to our surprise and horror, in December 2000, a serious outbreak of canine distemper occurred. (See Clinical Work--Diseases).
Rabies See Appendix III
Vaccinations
In December 1995, at the age of approximately 5 months, the dogs received their first, single, rabies vaccination. However, due to lack of sero-conversion (see Results) in 1997, the vaccination schedule was changed and the dogs were vaccinated three times; a second and third vaccination was given after 1 and 5 months respectively, followed by an annual booster vaccination. Antibody testing was carried out at approximately 1 month, 5 months, and 12 months after the third vaccination and at the occasion of the annual booster vaccination. One month after the third vaccination (October 1997), antibodies were again measured in 22 dogs (see Table 3). Due to a shortage of vaccines, 9 dogs had received only 2 vaccinations; and 13 dogs received the full course of 3 vaccinations. March 1999 four dogs were due to receive their third vaccination but this slipped from our attention and was not carried out. As a result they were tested March 2000 17 months after their second vaccination. Another three dogs received only two vaccinations at the time of testing. The testing of these three dogs was performed because the dogs had to be sedated to apply a transponder anyway (November2000). In 1995 & 1997, Dohyrab® (Solvay Duphar) was given, but was replaced in 1998 by Rabdomun® (Schering-Plough). Both Dohyrab® and Rabdomun® are inactivated vaccines. According to Solvay Duphar and Schering-Plough, these vaccines protect domestic dogs against rabies for up to 3 years, provided the dog is more than 12 weeks of age at the time of vaccination. 38
Results (table 2)
1. Single vaccination: In February 1996, 2 ½ months after the single vaccination at the age of approximately 5 months, blood-samples from all dogs were collected. Not a single dog developed what are considered to be sufficient antibodies (>0.5 I.U./ml). However, for unaccountable reasons, one year later (15 months after the vaccination) seven of the 19 dogs tested showed a titer of >0.5 I.U.. 2. Two vaccinations: After two months half of the dogs tested showed a titer >0.5 I.U.. After 7 months five of the seven dogs tested showed a result >0.5, which dropped dramatically in the dogs tested after 17 months. None of these four dogs developed sufficient antibodies. 3. Three vaccinations: Results improved dramatically. Percentage of dogs tested with a titer of >0.5 I.U. varied from 80 to 92 % one, two and five months after the last vaccination. 75% of the tested dogs had sufficient antibodies (>0.5 I.U./ml) one year after the third vaccination (1998). One year after the annual booster vaccination (1999), the result dropped to 47 %; only half the number of dogs was tested compared to previous years. In between 8 dogs were tested 18 months after the third vaccination with a booster at twelve months; 50 % of them showed sufficient antibodies. Due to the distemper outbreak it was possible to test one dog only 30 months after the third vaccination with booster vaccinations at 12 and 24 months. That dog showed a titer >0.5 I.U..
An overview of the titers of all tests performed is presented in table 2.
Table 2: Rabies antibody titers in African Wild Dogs before and after vaccination with inactivated commercial rabies vaccines: Dohyrab® (Solvay Duphar) in 1995&1997 and Rabdomun® (Schering- Plough) from 1998 onward. Number of dogs % Number of dogs % Total number
39 >0.5 I.U./ml* <0.5 I.U./ml of dogs Not vaccinated 0 0 9 100 9 Months after one
vaccination 2 0 0 12 100 12 15 7 37 12 63 19 Months after two
rabies vaccination 2 5 50 5 50 10 5 5 71 2 29 7 17 0 0 4 100 4 Months after third
rabies vaccination 1 12 92 1 8 13 2 4 80 1 20 5 5 - 6 23 92 2 8 25 12 ¹ 21 75 7 25 28 18 ² 4 50 4 50 8 24 ³ 7 47 8 53 15 30 ³ 1 100 0 0 1 * a Rabies Specific VNA titer of >= 0.5 International Units (I.U./ml) is considered to be the minimum titer likely to provide protection against challenge. ¹ received booster vaccination at the time of blood sampling ² received booster vaccination 12 months after third vaccination ³ received booster vaccination 12 and 24 months after the third vaccination
Figure 2: Percentage of dogs with a rabies titer >0.5 I.U. after three vaccinations in the years 1996-2000
100 90 80 70 Percentage 60 >0.5 I.U. 50 40 30 20 10 0 1357911131517192123 Months after third vaccination
Table 3: Rabies antibody titers in African Wild Dogs before and after vaccination with inactivated commercial rabies vaccines: Dohyrab® (Solvay Duphar) in 1995&1997 and Rabdomun® (Schering-Plough) from 1998 onward after completing the three initial vaccinations followed by an annual booster vaccination in chronological order 40 Number of dogs % Number of dogs % Total number of Date NT-titer >0.5 I.U.* NT-titer <0.5 I.U.* Dogs 09-03-1997** 7 37 12 63 19 15-10-1997 14 82 3 18 17 15-02-1998 16 89 2 11 18 04-10-1998 21 84 4 16 25 30-03-1999 8 100 0 0 8 02-11-1999 7 39 11 61 18 30-03-2000 1 20 4 80 5 08-11-2000 4 67 2 33 6 13-03-2001 2 100 0 0 2 * a Rabies Specific VNA titer of >= 0.5 International Units (I.U./ml) is considered to be the minimum titer likely to provide protection against challenge. ** received one vaccination
Figure 3: Percentage of dogs tested with a rabies titer >0.5 I.U. in the years 1996-2000 and number of dogs tested.
120 100 Percentage of dogs 80 with a rabies titer >0.5 I.U. 60 Number of dogs 40 tested rabies 20 0 Percentage/number 19- 29- 09- 15- 15- 04- 30- 02- 30- 08- 13- 12- 02- 03- 10- 02- 10- 03- 11- 03- 11- 03- 95 96 97 97 98 98 99 99 00 00 01 Date of testing
Conclusion
From the data presented, it may be concluded that 3 vaccinations with an inactivated vaccine, according to the above-mentioned schedule, result in sufficient antibodies production in the majority of the dogs. Although the results tend to drop half a year and onward after the first annual booster vaccination (Figure 2). But, a single vaccination of such a vaccine, as recommended for domestic dogs, does not provide adequate titers. Two vaccinations (the second one month after the last vaccination) provide some adequate titers on the short term, but not on the long term.
Parvoviral Disease (CPV) See Appendix IV
Vaccination
41 The inactivated vaccine Dohyvac I-LP® (Solvay Duphar) for use against parvovirus and leptospirosis was used. In the first year, the dogs were vaccinated twice, with a one-month interval. This was followed by an annual booster vaccination. These vaccinations were following the manufacturer's protocol for treating domesticated dogs older than 12 weeks: 2 vaccinations with a 4-week interval followed by an annual, single booster vaccination.
In March 1997, antibody testing for parvovirus infection on an annual basis ceased, but the annual, single booster vaccination was continued. However, in 1999 it was impossible for us to acquire inactivated parvovirus vaccine. Instead, the dogs received the leptospirosis vaccination, Vanguard Lepto-CI® (Pfizer). In the year 2000, the parvovirus vaccination was resumed, with a double vaccination of Dohyvac I- LP® (Solvay Duphar).
Results
Fourteen days after the second vaccination (February 1996), all 25 dogs were well protected (Ig G >20). One year later (March 1997), the Ig G of all dogs tested (18) was still above 20. This excellent result was reason enough to stop antibody testing for parvovirus infection on an annual basis, but the annual vaccination was continued.
Conclusion
During the first year, two vaccinations at a 30-day interval, followed by one single booster vaccination proved to be successful: 100% of the tested dogs had sufficient antibodies one year after the second vaccination.
Parasites
Endoparasites
With the arrival of the pups in 1995 an anthelmintic regime was started. The drug of choice was Drontal® Dog*. Drontal® Dog* was chosen being a broad-spectrum anthelmintic, i.e. being active against roundworms, hookworms and tapeworms. Beside of that it is sufficient to administer the drog only once and can be simply added to the food, as there is no need to have an empty stomach for the dogs prior to administering the drug. The dosage used, was one tablet Drontal® Dog* per 10 kg bodyweight. The dogs were not individually treated, but as a pack as it is impossible to feed each dog individually. To prevent re-infection of the dogs, twice a day the faeces was removed from the boma’s. This way infestation of the soil of the boma’s with worm-eggs was prevented as much possible as well. Twice a year, five faeces samples were taken from each pack, and were preserved with a 5% formalin solution before sending the samples to a laboratory. In 1996 the dogs received Drontal® Dog* every month. Due to the negative results of the faeces samples the schedule was changed in the course of 1997 to once every three months. Newborn pups received their Drontal® Dog* still every month, as soon as they could eat solid food not regurgitated by their parents or other dogs. This schedule for the pups was followed until the age of 7-9 months as it was no longer possible to separate them any longer at feeding time. This schedule was not changed, as all samples were negative up until March 1999. However, in October 1999, the pups of the Sangito II litter started to deteriorate slowly and died one-by-one. Infection with worms was suspected, and Drontal® Dog* was administered three times to the pups at 10-day intervals. As a result, three pups survived. Faeces samples taken early in November, showed the following results: Lendanai and Ayubu pack negative, but in the Kisima pack a few Dipylidium caninum eggs and a few Ancylostoma sp. eggs, in the Sangito pack a few Ancylostoma 42 sp. eggs. However a faeces sample from a pup in the Sangito pack showed many Ancylostoma sp. eggs and many Toxascaris leonina eggs.
The following measures were taken or continued: 1. The removal of the faeces from the boma’s twice a day was continued in an even more scrutinized way. 2. Anthelmintic treatment of the Lendanai and Ayuba Packs was continued regularly--every three months. The dogs in the Sangito and Kisima boma received anthelmintic treatment every month for a period of six months. After six months, treatment was continued once every two months. 3. Females received anthelmintic treatment just after mating, and again 10 days later, to prevent the unborn pups from being infected in the uterus. As soon as they had given birth the females received the anthelmintic again, followed by a treatment 10 days later. 4. The pups received their first anthelmintic treatment as soon as they came to the surface of the dens and could take minced meat from a tray. This was followed by a second treatment 10 days later. Treatments took place when the pups were about four weeks old.
The faeces samples collected in March and November 2000, proved to be negative again. However, the pups of the litter born 7-7-2001 were treated in a different way. The moment they started to eat solid food, the Drontal® Dog* was provided in the food of the adult pack members; i.e. the regurgitated food contained the anthelmintic. In this way, the anthelmintic was administered to the pups at an age of three weeks instead of four weeks. Unfortunately, these pups had to be separated from the adult dogs at the age of 4 weeks. However, this made it possible to treat them in an even more secure way--by putting the anthelmintic directly into their food every fortnight until they were six months old.
*Drontal® Dog (praziquantel, pyrantelembonaat, febantel), Bayer.
Blood Parasites
In March of 2000, EDTA blood samples were taken from 5 different dogs to be tested for pathogens at the Department of Parasitology and Tropical Veterinary Medicine, Faculty of Veterinary Medicine, Utrecht University, the Netherlands. GUBBELS et al. (1999), describe the technique used. The advantage of the technique is that the parasite species present can be identified, and their numbers counted if specific probes are available. At the time of testing, probes were available for Babesia, Theilleria and Ehrlichia species. Fortunately, all of the samples were negative.
Ectoparasites
On each occasion of sedation, the skin of the dogs was checked for parasites. There appeared to be no signs of fungus or scabies. No fleas or ticks were found until September 1998, when there was a sudden outbreak of fleas in the Lendanai boma. This happened in the Sangito boma as well later on.
Initially the dogs were treated with Frontal*, by way of spraying them, while the dogs were walking through the passageway in the boma’s, but as expected this solved not the problem as it is almost impossible to spray them in a sufficient way. In October 1998 a regime was started to add Program** once a month to the food of the dogs, to prevent the fleas from multiplying. Every first of the month the dogs received one tablet of Program** 409 mg through their food in the way of pack treatment. The only way to kill fleas directly is either spray the dogs in a proper way or application of drops in the neck of the dog with a product like Advantage®***. Both ways the dogs have to be sedated to administer the insecticide properly. From October 1998 onward, at every occasion of sedation Advantage*** was applied on their neck 43 to kill the fleas. This happened to all the dogs in October 1998. After that the combination of Program®* in their food on the first day of each month and the application of Advantage®*** at every sedation appeared to be satisfactory in keeping the fleas to an acceptable level. As a result a few fleas were detected only occasionally on the dogs since then.
Also in October 1998 the first ticks were found. The ticks were preserved on a 96% alcohol solution and were sent for identifying to the University of Utrecht, Department Parasitology & Tropical Veterinary Medicine. The ticks were identified as nymphs of Haemaphysalis leachii, an important vector for pathogen protozoa. Unfortunately, the Program®** does not affect ticks. The Advantage®*** does kill ticks, but was not administered on each dog and the frequency of administering was so low, that destroying the ticks sufficiently was not possible this way. After the devastating canine distemper episode, the Kisima boma in particular was heavily infested with young ticks. To reduce the tick numbers, the grass around the bomas was burned in the spring of 2001. As a result, the dogs in the Kisima boma had only a few ticks when sedated at the end of that year.
* Frontal® (fipronil), Rhone Merieux. ** Program® (lufenuron), Novartis. *** Advantage® (imidacloprid), Bayer.
Clinical Work--Diseases
Clinical Work
Rectal Prolapse in Lendanai Dogs
Most unusual was the appearance and disappearance of rectal prolapse in the original Lendanai dogs. Over the years, three out of four Lendanai dogs occasionally showed a rectal prolapse. At the time of occurrence, they all held the alpha position, which makes it look like a stress-related condition. It may also be genetically related, as not a single dog from the Llondirrigiss or the Najo dogs showed this condition.
In 1997 the first dog, Lendanai #264, showed this condition. After achieving the position of alpha female in the Lendanai group and having mated and being probably pregnant, she showed a rectal prolapse in July. Initially the prolapse came and went but stayed eventually. Surgery took place, whereby the rectum was repositioned and colopexy performed, in which the colon was fixed to the abdominal wall 15 cm caudal from the navel with 3 stitches, to prevent recurrence of the prolapse.
Despite the fixation of the colon to the abdominal wall the rectal prolapse reoccurred in early October. This time a rectal amputation was performed, whereby the prolapsed part was resected 2 cm distal from the anus. On both occasions the dog recovered well with no complications, however after the last operation she lost her position as alpha female, being separated for 2 days from the pack, although she had contact with her pack mates through the fence. She lost that position to #262. In May of 2000, #262 also showed a rectal prolapse. The prolapse reverted in 10 days. After the male #263 was placed with the females of the first Kisima litter in the Ayubu boma in October 1998, and in a way being forced to be alpha (being the only male), every now and then he showed a rectal prolapse. The prolapse showed for 3-5 days and then disappeared.
44 Surgical intervention was never necessary. In both dogs, the prolapse was never larger than approximately 5 cm. As a means of precaution, both dogs were treated with Synulox®*. Since the prolapses disappeared in less than 10 days, except for the prolapse in #264, it seems wise, provided the prolapse is not too large (less than 5 cm), to be reticent with surgery.
Sick pups
Most of the time it was not possible to treat sick pups, but in 1998 a rare occasion presented itself. Two pups in the Lendanai compound were found dead, without signs of illness, in April and May. However another sick pup could be caught and manually treated. After consultation by phone it was decided to administer Synulox* and dexamethasone intramuscularly to the diseased pup. Next morning the pup showed improvement and subsequently recovered completely. All pups received Synulox* in their food for five days and the pups showed no signs of illness after that. A bacterial infection seems likely.
In the period October to December 1999 in the Sangito boma one pup after another fell ill. They could not be treated manually. Synulox* was added to there food, without result. An extra course of anthelmintic proved to be successful, supported by the results of the faeces samples of the pups.
Wounds
Because of the occasional fighting, some dogs (mainly males), showed some wounds. Most of the time it was not necessary to treat these wounds, with a few exceptions. In December 2000, the alpha male (#289) in the Sangito Pack, had a nasty gash just under his right lower eyelid. Male #291 had a bad hind leg, with a couple of wounds. He rarely used the leg for support. The dogs had been fighting each other. An oral course of Synulox®* was administered to both of them and was beneficial.
Synulox® (amoxicillin, clavulanic acid), Pfizer.
Anesthesia
Over the years, the combination of medetomidine (Domitor®*) and ketamine HCL (100 mg/ml), with atipamezole (Antisedan®**) as an antidote for the Domitor®*, proved to be a safe way of sedating the dogs. From 1995 to 2001 slightly more than 200 sedations (see table below) have been performed. Only one death (#297 in October 1997) occurred, which is likely to be related to the sedation (see below).
Table 4: Number sedations performed 1995-2001 Date Number sedations 19-12-95 25 29-2-96 25 Autumn 1996 Dogs to Kenya 4 9-3-97 19 Spring 1997 Start Sangito pack 7
45 15-10-97 18 15-2-98 18 4-10-98 33 30-3-99 9 2-11-99 19 30-3-00 9 8-11-00 16 13-3-01 2 13 occasions Total 204
The drugs were administered by blowpipe, intramuscularly, in the hindquarters. For that purpose, the dogs were enclosed in a passageway. The passageway was divided into small compartments, with 2 or 3 dogs per compartment, to avoid confusion during the darting process. Over the years, the adult dogs usually received 1.0 ml Domitor®* per 10 kg bodyweight, topped up with 0.1 ml ketamine HCL per dog--independent of their bodyweight. Apparently, pups and adolescent dogs need a larger dosage: 1.5 ml Domitor®* per 10 kg bodyweight, topped up with the ketamine HCL. Using the dosage of the adult dogs in the young ones meant that most of them needed an extra administration of Domitor®*. Induction time varied from 5 to 15 minutes. Some dogs were able to remove the syringe before it was completely emptied. Therefore, these, and other dogs who had received too little drug due to the mechanical failure of the syringe, received an extra 0.5 ml of Domitor®*.
This combination Domitor®* and ketamine HCL proved to be very satisfactory for purposes such as taking blood samples, applying transponders, measuring their bodyweight, etc.
The smaller dogs (<20 kg) received 1.5 ml Antisedan®**, and the larger dogs (>20kg) 2.0 ml Antisedan®**, immediately after the veterinary procedures involving them ended--on average 30 minutes after the administration of the Domitor®*. Recovery time was 3 to 15 minutes, with little side effects from the ketamine HCL. The very slight side effects, staring in the distance and ataxia, which were observed, disappeared usually within 30 minutes.
For her surgical intervention female Lendanai 264 received 2 ml Domitor* + 0.5 ml ketamine HCL on both occasions. This proved to be a good combination and dosage for this kind of surgery. Only on one occasion things went wrong. In October female Llondirrigiss 297 was routinely sedated according to the above-mentioned dosage. At the time of sedation she was not excited and looked normal, sedation went smoothly, blood samples etc. were taken. However after administering the Antisedan*, she hardly responded; only panted. After 15 minutes another dose of Antisedan* was administered, just in the unlikely event that the first syringe was not completely emptied in her body. Five minutes after the second dose of Antisedan she staggered around in a heavy ataxic way. Shock being suspected short acting dexamethason was administered intramuscularly. She went into a den and stayed there still heavily breathing. It was not possible to administer fluid by either subcutaneous or intraveneous way, as the dog would have to be sedated again, which would have caused her death for sure. The next morning she had expired. Autopsy revealed shock ( see Pathology). Although it was a very sad experience, nothing could have been done to prevent this, as everything had gone normally. One has to realize, that in more than 200 sedations an occurrence like this is likely to happen, how much sad it is.
*Domitor® (medetomidine hydrochloride 1 mg/ml), Pfizer. **Antisedan® (atipamezole hydrochloride 5 mg/ml), Pfizer.
46
Diseases
Canine Distemper
Introduction
“Canine distemper is a disease that primarily affects the lungs, intestinal tract, and nervous system of dogs. Among the virus-induced diseases in dogs, the mortality rate of distemper is second only to that of rabies. The virus is highly contagious and is passed directly from dog to dog by close contact. The virus is easily killed by detergents and heat. Most often young, unvaccinated dogs 3 to 6 months of age are infected with distemper. Nasal discharges containing virus are aerosolized by sneezing, thereby spreading the virus. The virus establishes itself in the nasal passages of a susceptible dog, multiplies, and spreads through the body. Dogs develop a fever a week after infection but this fever may not be noticed. Two weeks after infection, the virus produces severe damage to the cells of the nasal passages, eyes, lung, and intestinal tract. These damaged tissues commonly become secondarily infected with bacteria. This combined infection with virus and bacteria produces loss of appetite, fever, snotty nose, thick discharge from the eyes, pneumonia, and diarrhea. The virus also damages the immune system, thereby interfering with the body’s ability to fight off the infection. Half of the dogs with distemper develop neurological disease.”
From “Textbook of Veterinary Internal Medicine”; S.J.Ettinger, E.C.Feldman; page 1959.
Course of Infection
The first symptoms appeared in the Lendanai Pack on the 20th December, 2000. Two pups (#812 and #912), at 8 months old, showed no interest in food. One pup died overnight, the other 2 days later. The disease ended in the Lendanai Pack with the death of #322 on the 11th of January, 2001. Between 20-12-2001 and 11-01-2001, 15 of the 16 dogs died, leaving male #325 as the sole survivor. The infection lasted for exactly 21 days. Although it is very difficult to stop an infection like this, steps were taken to try to prevent spreading of the virus. These steps included a container with detergent posted at every boma entrance to disinfect footwear before entering and after leaving the boma, and a change of clothes and disinfecting utensils used in the boma. For the first five days, we had some hope that our efforts taken to stop the infection from spreading might succeed. However, this was not to be, and the next boma to be infected was that of the Sangito Pack. On January 16th two Sangito pups refused food, and the alpha male (#289) started to cough. Between 16-01-2001 and 05-02-2001, 14 of the 15 dogs died, leaving the alpha female (#306) as sole survivor. The infection lasted 16 days. At about the same time, the first symptoms of the disease appeared in the Kisima Pack, on January 18th and in the Ayubu Pack on January 24th. Between January 18th and February 13th, of 14 Kisima dogs--13 died, leaving the male (#372) as the only survivor. Between January 24th and February 11th all six dogs of the Ayubu Pack died. The duration of the infection in the Kisima Pack was 26 days, and in the Ayubu Pack it was 18 days. On February 13th 2001, the last death occurred. The alpha male of the Kisima Pack, #298, died after one week of fighting for his life. The epidemic lasted 56 days. By then, 49 of 52 animals had died of distemper.
Symptoms
47 The symptoms varied considerably and included: lack of appetite, listlessness, loss of condition, vomiting, diarrhea (greenish, watery, sometimes accompanied with blood), sneezing, coughing, and muco-purulent nasal and ocular discharge (nasal discharge more prominent than ocular discharge). Some dogs showed little symptoms. They were just listless and without appetite one day, and dead the next day. Other dogs showed all of the symptoms; although one symptom was more obvious than the others were. For example, in one dog coughing and nasal discharge was more prominent, while in another dog diarrhea was more prominent. The disease lasted in the individual dogs from one day, to as much as ten days. No neurological symptoms were observed.
Treatment
No antiviral drugs are available for the treatment of canine distemper. The presence of the virus presents a big opportunity for bacteria to invade the body. If a dog is lucky enough to survive the virus, it still runs the risk of dying from a secondary bacterial infection. Broad-spectrum antibiotics were used to control these infections. Since supportive therapy such as fluids and electrolytes are impossible to administer to African wild dogs without sedation, they were not used.
In all packs, antibiotics were put in the food and water. Only one dog (#289), was treated with injections of Synulox®* and Finadyne®*** (by blowpipe), for five consecutive days. He did not recover, and his misery was possibly prolonged. Antibiotics used were amoxicillin, Synulox®* and Baytril®**.
* Synulox® (amoxicillin, clavulanic acid), Pfizer ** Baytril® (enrofloxin), Bayer ***Fynadine® (flunixine meglumin), Schering-Plough
Discussion
The virus involved was Canine distemper virus (CDV)--(VAN DE BILDT et al., 2002). Most astonishing was that 49 out of the 52 dogs died. One might expect that some dogs die, but not in this horrifying quantity. The Mkomazi dogs lived in isolation, without direct contact with humans (and their domesticated dogs), and associated canid diseases. Consequently, the dogs had little opportunity, compared with their free-living relatives, of encountering naturally occurring canid viruses in the environment on a regular basis. Such exposure may have provided natural immunization, or at least boosted the dogs' immune system to combat such infectious diseases. Therefore, dogs kept in captive breeding programs such as the Mkomazi, appear to be more vulnerable to infectious diseases than free-living dogs, so their protection through vaccination is considered to be of paramount importance.
It is still unknown what the source of the canine distemper virus was. Direct contact with domesticated dogs can be ruled out. The nearest dogs are at a 28 km distance away, and have not been observed in the reserve for the last couple of years. Infected wild carnivores serving as reservoirs cannot be ruled out, although no diseased carnivores have been observed. Indirect contact is possible via humans or their equipment. In favor of the possibility of indirect transmission via humans, points to the fact that the outbreak started in the boma closest to the human lodgings. However it is not likely that the keepers of the dogs caused the transmission. As they enter all the bomas a few times every day, infection appearing more or less at the same time in every boma would have been more likely.
As Appendix II shows, we had good reason to believe that the dogs were well protected by the vaccinations. Although the results fluctuated somewhat; the majority of the dogs tested always had 48 a titer of 20 or more. Since the annual booster vaccinations were always given with a newly arrived batch of vaccine, any problems with the ‘cold chain’ can be dismissed. In November 2000, one month before the outbreak, the dogs were due to receive their annual booster. However, due to the fact that the safety procedures of the new batch of CDV-ISCOM vaccine had not been completed, no vaccine was available, and the dogs did not receive their annual booster vaccination. The annual booster was rescheduled for February 2001, but because this was (at the time of the outbreak) just one month overdue, it should not be the reason for the severity of the outbreak. However, of the nine dogs, that had completed the three initial vaccinations, tested November 2000 only one dog had a sufficient titer (20). Coincidence or not, that dog appeared to be one of the survivors. Also, the manner in which the vaccinations were applied should not have had an effect on the results. The dogs were either vaccinated by hand while sedated, or vaccinated by blowpipe and kept under strict observation (distance between blowpipe and target dog never more than one meter). The extremely high death rate might indicate a vaccination failure. It is possible that the vaccine used does not protect African wild dogs as well as it does the harbor seals (Phoca vitulina) and domesticated dogs. In addition, it is possible that the African wild dog differs in its antiviral immune response from that of the domesticated dog, although of the nine dogs tested November 2000, seven showed a sufficient titer for rabies, which contradicts the distemper results at that date in the same dogs. Very little or nothing is known about this subject.
Based on our experience, canine distemper appears to be a big problem in the captive African wild dog. It may be also a problem in free-living packs. For instance, 15 of the 16 Lendanai dogs died within 21 days; 14 of the 15 dogs withdrew into the den to die. If such an occurrence took place in the wild, it is quite likely that no dog carcasses would be found. It would appear that the pack had 'just disappeared'. Only one case of CDV in free-living wild dogs has been confirmed (ALEXANDER et al, 1996).
We shall continue the Mkomazi wild dog captive breeding program, although alterations have already been made, and others are still under consideration.
Steps Taken/Considered
1. New vaccination policy and study of effect of vaccinations: a. Change of vaccine. Instead of the CDV-ISCOM vaccine, Purevax™* (Merial) is going to be used. According to the manufacturer, Purevax™ is a lyophilized vaccine of a recombinant canarypox vector expressing the HA and F glycoproteins of canine distemper virus. More popular: a “canarypox-vectored canine distemper vaccine”; i.e. an inactivated distemper vaccine where genetic information of the distemper virus is built into the canarypox virus. The virus may multiply after injection and enhances the building up of immunity this way. After application in a mammal, the virus is unable to spread. The vaccine is developed for use in ferrets and tested in ferrets, domesticated dogs, and lesser pandas. Determination of the serum antibodies in response to the vaccination will be continued. b. Cell mediated immunity will be studied to see how the African wild dog responds on a cellular level to the vaccine virus, which is more important than the humoral immune response in the case of canine distemper.
Both a and b are developed in close cooperation with the Institute of Virology, Erasmus University Rotterdam, the Netherlands. This Institute will carry out all of the laboratory work as well. To enlarge the number of dogs participating in this vaccination program, i.e. to come to results earlier, African wild dog pups born in Artis, Amsterdam, and Safari Beekse Bergen, Hilvarenbeek (both in the Netherlands), will participate in this program.
49 2. As an extra precaution to keep small animals and people at a distance, a fence of six-foot high chicken wire was placed at a distance of approximately 2 meters from the outside perimeter fence of the Sangito and Kisima bomas. The moment the other bomas are in use again, the same provision will be installed.
3. A strict policy is established in which only people with clear business are allowed in the bomas. Before entering the boma, they must disinfect their shoes with the disinfectant supplied at the door of the boma.
4. At the time of the distemper outbreak, the number of dogs was relatively high, despite the fact that the dogs were divided into four packs. Three of the four packs contained dogs/pups of different litters. As the dogs matured, they had no opportunity to move on in a natural way. Two measures should be considered: a. As soon as pups have reached yearling age (18-24 months), and have assisted in raising the next litter, they should be removed from the pack. Accumulation of dogs is prevented that way. b. Relocating half of the breeding program. If a new outbreak occurs, only half of the program is affected. The larger the distance that separates the two groups the less the chance that the other group is affected. The fact that two of our original dogs (#273 and #303) are still alive and well in Kenya (Ol Jogi), emphasizes this. To prevent infection with the strain of canine distemper, circulating in East Africa, moving a pack abroad should be considered.
Pathology
Over the years only a few full-grown dogs died until the canine distemper outbreak; in fact four dogs. October 1997 alpha female #297 died, autopsy report below. In July 2000 two dogs of the Sangito I litter #352 and #358, followed by their brother #359 in November, died at the age of two years. #359 looked sickly, hair standing up and disappeared completely overnight. No sign of any remnants. After the distemper epidemic we had to mourn the loss of another two dogs in 2001. In August 2001, the remaining female #306, mother of the pups, died; and in October 2001 one of her pups. On both occasions the keeper of the dogs, who also preserved the tissue-samples in an excellent way, performed necropsy. The histology of these two dogs is reported in detail. Over the years pups died on different occasions. Most of the time only the head, or some other minor body-part or even nothing, of the deceased pup could be retrieved. Not enough to establish (for certain) the cause of death. Unsatisfactory, but there is nothing to do to prevent the adult dogs from scavenging the carcasses of the deceased pups.
As it is impossible to have pathological expertise at all times in Mkomazi, diagnoses have to be made on basis of histology only, most of the time. Not finding answers for all questions is a logical consequence.
Pathology report of #297
History See chapter Anestesia
Gross Examination of Organ Systems Good bodily condition. Mucous membranes pale. Lungs hyperaemia. Heart petechiae in heartmuscle. Liver, spleen and kidneys hyperaemia. Stomac haemorrhagic mucosa; duodenum,
50 jejunum and colon haemorrhagic mucosa and blood in lumen. Uterus, ovariae and bladder no abnormalities.
Histological Examination Lung: peri-brochial brown pigment in clusters, sometimes clearly containing crystals. Locally oedema. In some bronchiae repulsing of bronchial epithelia with sometimes some large macrophages. Paramyxo negative. Intestines: too autolytic for good judgement. Liver: in many blood vessels larger bacterial rods (bacteriae associated with decomposition ?), a fair number of lipocytes. Kidneys: hyperaemia in glomeruli and marrow: shock. Spleen: depleted, many macrophages and necrosis. PAP-Carré negative.
Conclusion: Shock, chronic bronchitis.
Macroscopic examination was performed on the spot in Kisima camp by Aart Visee. Microscopic examination was performed by University of Utrecht, the Netherlands, Faculty Veterinary Medicine, Department Pathology Exotic Animals. Parts of organs were preserved and transported in a 4% formaldehyde solution.
Pathology canine distemper victims
Only nine necropsies were performed out of the 49 dogs that died during the distemper outbreak. This low number is explained as follows: with the exception of three(performed by Aart Visee), all necropsies were performed by laymen, i.e. the keepers of the dogs: Sangito and Ayubu. Over the years a bond was established between the dogs and them. It was extremely hard on the keepers to fight for the life of a dog one day, and perform a necropsy the next day. Despite their emotional bond with the dogs, they performed extremely well; preserving tissue samples of the deceased dogs in a correct way. This is even more impressive when one realizes that they were never trained in this field. Fortunately, the diagnosis was made, and the virus isolated in an early stage of the epidemic, which made it less pressing to perform more necropsies.
VAN DE BILDT et al., (2002) describes the identification of the canine distemper virus (CDV) by means of a histological examination, virus isolation, reverse transcriptase-polymerase chain reaction analysis, and nucleotide sequencing. For results, the paper is attached to this report.
Pathology Report of #306 Date of Necropsy 04-08-2001
History Gave birth to eight pups July 7th 2001. Pups developed well. Saturday August 4th sudden onset of symptoms: lateral recumbency, increasing dyspnoea, gasping for breath, and some blood in the saliva. Died approximately 6 hours after onset of symptoms. To the day of her death she ate well, and water intake was normal. The last two days before her death, it was noticed that her belly was enlarged.
51 Gross Examination of Organ Systems No abnormalities noticed. Necropsy performed by the keeper of the dogs. Necropsy was primarily directed at collecting of tissue samples. No pus was observed in the abdominal cavity.
Histological Examination Brain: Multifocally in white and grey matter are small areas of haemorrhage. Heart: Multifocally in the epicardium, myocardium, and endocardium are small to medium-sized areas of hemorrhage. Spleen: The capsule is corrugated (spleen atrophy). There is marked depletion of lymphocytes in the white pulp. Liver: Except for a few small areas, the tissue is too autolysed for histology. There are abundant large bacilli (postmortem invaders) in the autolytic tissue. Lung: The tissue is slightly autolytic, with extensive sloughing of epithelium and leakage of serous fluid into the alveoli. No intracytoplasmic inclusion bodies or multinucleated giant cells are seen. Uterus: The lumen is distended and filled with pink amorphous material admixed with erythrocytes, inflammatory cells, and cell debris. Diffusely, the endometrium is infiltrated with moderate to many lymphocytes, plasma cells, and fewer macrophages, and neutrophils. Some macrophages are filled with amphophilic amorphous material. Multifocally the inflammatory infiltrate extends into the myometrium. There is multifocal hemorrhage in the uterine wall. No abnormalities found in the following tissues: kidney.
Final Diagnoses -Uterus: Endometrititis and myometritis, lympho-plasmacytic, diffuse, subacute, moderate. -Brain: haemorrhage, multifocal, acute, mild. -Heart: hemorrhage, multifocal, acute, moderate. -Spleen: lymphocytic depletion, marked.
Final Comments The primary lesion in this African wild dog was an inflammation of the uterus, (endo) metritis. In general, females are more likely to develop such an inflammation after parturition, as a result of bacterial infection. Based on the specimen submitted, it is not clear whether the endometritis would have been severe enough to cause death. It is possible that the lesion was more severe at other locations of the uterus. In principle, the hemorrhages observed in multiple organs fit with a toxemia due to endometritis.
Pathology Report of pup born 07-07-2001 Date of Necropsy 03-10-2001
History Stayed behind in growth. Water intake increased. A week prior to death drinking in recumbent position.
Gross Examination of Organ System Kidneys are markedly enlarged and pale. No other abnormalities noticed. Necropsy performed and tissue samples collected by keeper.
52 Histological Examination Kidney: Diffusely, the glomeruli show loss of capillaries and are thickened by amorphous pink material (fibrous connective tissue; glomerulosclerosis). Some Bowman’s capsules are filled with homogeneous light pink material (proteinaceous fluid). Diffusely, the epithelium of proximal tubuli, normally cuboidal, is squamous. Some tubuli are distended and filled with proteinaceous fluid. Multifocally in the interstitium of cortex are rare lymphocytes and plasma cells. No abnormalities noted in the following tissues: intestine
Final Diagnosis -Kidney: glomerulosclerosis, generalized, diffuse, marked, with tubular squamous degeneration, tubular cystic dilatation, and proteinaceous casts.
Final Comments The primary lesion in this animal was a severe and diffuse fibrosis of the renal glomeruli, resulting in renal failure, and no doubt leading to the animal’s death. The age in which this occurred and the virtual absence of inflammation or other lesions suggest that this is a genetic disease. For example, the histologic aspect resembles that of Samoyed hereditary glomerulopathy. Transmission electron microscopy of the glomerulus would be needed to further characterize the lesion.
Acknowledgments
My sincere gratitude for their help and support goes to:
Ministry of Natural Resources & Tourism, Government of the United Republic of Tanzania. Department of Wildlife, Tanzania. Tanzania Wildlife Research Institute (TAWIRI). Duncan Forbes, Ray Rowe Trust for Animals, for funding a big part of the program. Prof. A.D.M.E. Osterhaus, Marco van de Bildt and Thijs Kuiken, Institute of Virology, Erasmus University, Rotterdam, for supplying the CDV-ISCOM vaccines, performing the antibodies testing, isolating the canine distemper virus, performing histological research and most of all their invaluable advice. Roger Burrows, for giving us invaluable insights into the behavior of The African Wild Dog and his comments on this report. 53 Mark de Boer, Diergaarde Blijdorp, Rotterdam, the Netherlands for examining the faeces samples. Gilbertson & Page, England, for generously supplying dry dog food, GILPA “Value Complete”. Pfizer, the Netherlands for generously supplying Domitor®, Antisedan®, and Rabdomun®. Bayer, the Netherlands for generously supplying Drontal ® Dog and Advantage®. Novartis, the Netherlands for generously supplying Program®. Virbac, the Netherlands for generously supplying transponders. I.U.C.N. Canid Specialist Group. Tony Fitzjohn, field-director, for his invaluable support and help with the fieldwork Lucy Fitzjohn, for her support and preparing annually notes, which made Sangito’s notes accessible for me. Sangito Lema, keeper of the dogs, for his help with the fieldwork and his valuable notes Ayubu Mbise, assistant keeper of the dogs, for his help with the fieldwork. Dierenkliniek Visee, Netherlands, for generously supplying medical necessities.
Summary
In 1995, the George Adamson Wildlife Preservation Trusts, and The African Wild Dog Foundation, through the Ministry of Natural Resources and Tourism, and the Department of Wildlife, Tanzania, started a breeding program for the African Wild Dog in Mkomazi Game reserve, Tanzania.
In the first section, the development of the breeding packs is described, including the zoo technical aspects.
In order to protect the dogs from infectious diseases they were vaccinated against distemper, rabies and parvo-viral disease. To establish the effectiveness of the vaccinations, testing for antibodies was performed.
Distemper vaccination (CDV-ISCOM) according to the schedule three vaccinations the first year (14 days between the first and second vaccination, and 30 days between the second and third vaccination), followed by an annual booster proved to be satisfactory until December 2000. Despite the vaccinations, a serious outbreak of canine distemper occurred. This outbreak is extensively reported on and discussed.
Three rabies vaccinations in the first year--one month between the first and second, and five months between the second and the third, followed by an annual booster, gave a satisfactory quantity of antibodies. References
ALEXANDER, K.A., KAT, P.W., MUNSON, L.A., KALAKE, A. and M.J.G. APPEL (1996): Canine distemper-related mortality among wild dog (Lycaon pictus) in Chobe National Park, Botswana. Journal of Zoo and Wildlife medicine 27(3): 426-427. VAN DE BILDT, M.W.G., KUIKEN, T., VISEE, A.M., LEMA, S., FITZJOHN, A.R. and A.D.M.E. OSTERHAUS (2002): Distemper outbreak and its effect on African wild dog conservation. Emerging Infectious Diseases, 8, 2, 211-213. BURROWS, R. (1995): Demographic changes and social consequences in wild dogs 1964-1992. In: Serengeti II: research, management, and conservation of an ecosystem (Ed. A.R.E. Sinclair & P.Arcese), Chicago University Press.
54 BURROWS,R., HOFER.H. AND M.L. EAST (1994): Demography,extinction and intervention in a small population : the case of the Serengeti wild dogs (Lycaon pictus). Proceedings of the Royal Society of London B 256: 281-292.
CREEL, S., CREEL, N.M., MUNSON, L., SANDERLIN, D. and M.J.G. APPEL (1997): Serosurvey for selected viral diseases and demography of African wild dogs in Tanzania. Journal of Wildlife Diseases 33(4): 823-832. FRAME, L.H., MALCOLM, J.R., FRAME, G.W. and H. van LAWICK (1979): Social organization of African wild dogs (Lycaon pictus) on the Serengeti plains. Zeitschrift für Tierpsychologie: 50, 225-249. FULLER,T.K., KAT,P.W., BULGER,J.B., MADDOCK,A.H., GINSBERG,J.R., BURROWS,R., MCNUTT,J.W., AND M.G.L. MILLS (1992): Population dynamics of African wild dogs. Wildlife 2001:Populations (Ed.D.R.McCullough & H.Barrett), London: Elsevier Science Publishers GIRMAN, D.J., VILA, C., GEFFENS, E., CREEL, S., MILLS, M.G.L., McNUTT, J.W., GINSBERG, J., KAT, P.W., MAMIYA, K.H. and R.K. WAYNE (2001): Patterns of population subdivision, gene flow and genetic variability in the African wild dog (Lycaon pictus). Molecular Ecology: 10, 1703-1723. GUBBELS, J.M., DE VOS, A.P., VAN DER WEIDE, M., VISERAS, J., SCHOULS, L.M., de VRIES, E. and F.JONGEJAN (1999) : Simultaneous detection of bovine Theileria and Babesia species using reverse line blot hybridization. J. Clin. Microbiol. 37 :1782-1789. HOFMEYR, M., BINGHAM, J., LANE, E.P., IDE, A. and L. NEL (2000): Rabies in African wild dogs (Lycaon pictus) in the Madikwe Game Reserve, South Africa. Veterinary Record: 146, 50-52. VISEE, A.M., BURROWS, R., FITZJOHN, A.R. and L. FITZJOHN (2000): Plan for re- introduction of the African wild dog (Lycaon pictus). Unpublished. George Adamson Wildlife Preservation Trusts, The African Wild Dog Foundation. VISEE, A.M., (2001): Distemper, Rabies and Parvovirus vaccinations in a captive breeding programme for the African wild dog (Lycaon pictus) in Northern Tanzania. Verh. Erkrg. Zootiere: 40, 243-250. VAN DE ZANDE, L., (1997): Genetic variation within and between African Wild Dog (Lycaon pictus) groups from the Masaai Steppe, Tanzania. Unpublished, Population Genetics, Centre of Ecological and Evolutionary Studies, University of Groningen, the Netherlands. . 'Young Lycaon Male' is top dog:
55
Appendix I Bodyweight kilogrammes (kg) Neck-size centimeters (cm) Date 19-12-1995 29-2-1996 9-3-1997 15-10-1997 15-2-1998 4-10-1998 30-3-1999 2-11-1999 IdentificationSexof Birthkgkgkgkgkgkgkgkg Lendanai 261 F Aug.1995 12 13,1 20,5 19,6 19,7 Lendanai 262 F Aug.1995 11,1 12 19 19,6 18,8 Lendanai 263 M Aug.1995 13,3 14,5 23,3 25,5 25 23,3 23,1 Lendanai 264 F Aug.1995 12,4 14,1 21 21,2 20,7 Llondirrigiss 289 M Aug.1995 13,7 22 24,8 24 Llondirrigiss 291 M Aug.1995 9,2 11,9 20,2 26,5 22,7 20,3 Llondirrigiss 293 M Aug.1995 10 12,7 22 22,5 23,8 Llondirrigiss 294 M Aug.1995 10,5 14 25 28 27 25,6 23,6 Llondirrigiss 296 M Aug.1995 10 10,8 23,5 26,5 Llondirrigiss 297 F Aug.1995 9,1 13 22 died Llondirrigiss 298 M Aug.1995 11,1 15,5 25,5 27 25 24,9 Llondirrigiss 299 M Aug.1995 10,5 14,3 Kenya Najo 265 F Aug.1995 8,1 9 17,2 19,5 18,1 17,1 18,1 Najo 273 M Aug.1995 9,1 10,5 Kenya Najo 274 M Aug.1995 9,8 12,4 22,5 23,7 22,7 Najo 276 F Aug.1995 8,7 10,5 17,5 15 17,7 18 Najo 284 F Aug.1995 8,1 9,8 16,5 15,8 17,1 18 Najo 285 F Aug.1995 8 9,5 17 15,9 17,5 17,9 17,4 Najo 288 M Aug.1995 10,1 12 Kenya Najo 300 M Aug.1995 9,8 11,8 23 23,5 22 Najo 303 M Aug.1995 9,7 11,9 Kenya Najo 305 F Aug.1995 9,5 11,2 19 22,3 20,8 19,2 20,4 Najo 306 F Aug.1995 9,2 11,5 18,5 20,8 19,5 18,9 Najo 308 F Aug.1995 9,3 10,7 19 22,5 20,5 19,3 20,6 Najo 310 M Aug.1995 8,8 11,8 22 22,3 22 Kisima I 333 M 4-3-1997 17,8 21,9 24 24,4 Kisima I 335 M 4-3-1997 17 22,7 22,1 23,3 Kisima I 336 F 4-3-1997 14,5 19 20,6 20,6 Kisima I 337 F 4-3-1997 15,7 19 21,3 20 Kisima I 348 F 4-3-1997 16,1 22,3 21 20,4 Lenjo I 322 M 1-2-1998 14,7 17,8 19,7 Lenjo I 323 F 1-2-1998 16,5 19,9 19,1 Lenjo I 325 M 1-2-1998 17 19,9 Lenjo I 327 F 1-2-1998 17,2 20,6 20,6 Lenjo I 329 M 1-2-1998 18 Sangito I 352 M 3-5-1998 11,7 Sangito I 355 M 3-5-1998 12,7 Sangito I 358 M 3-5-1998 12,7 Sangito I 359 M 3-5-1998 12,2 Kisima II 366 M 8-1-1999 19,1 Kisima II 369 M 8-1-1999 18,3 Kisima II 372 M 8-1-1999 17,7 Sangito II 319 F 12-9-1999 Sangito II 360 M 12-9-1999 Sangito II 361 M 12-9-1999 Kisima III 312 M 16-9-1999 Kisima III 344 M 16-9-1999 Kisima III 349 F 16-9-1999 Lenjo II 780 F 3-4-2000 Lenjo II 812 F 3-4-2000 Lenjo II 817 M 3-4-2000 Lenjo II 850 M 3-4-2000 Lenjo II 893 M 3-4-2000 Lenjo II 912 F 3-4-2000 Lenjo II 924 F 3-4-2000 Ayuba I F 25-5-2000 Sangito III F/M 12-6-2000 Sangito III F/M 12-6-2000 Sangito III F/M 12-6-2000 Sangito III F/M 12-6-2000
56 Appendix I Bodyweight kilogrammes Neck-size centimeters Date 02-11--99 30-3-2000 8-11-2000 13-3-2001 Identification Sex of Birth neck-size kg kg kg Lendanai 261 F Aug.1995 36 Lendanai 262 F Aug.1995 Lendanai 263 M Aug.1995 38 Lendanai 264 F Aug.1995 Llondirrigiss 289 M Aug.1995 Llondirrigiss 291 M Aug.1995 39 Llondirrigiss 293 M Aug.1995 Llondirrigiss 294 M Aug.1995 40 Llondirrigiss 296 M Aug.1995 Llondirrigiss 297 F Aug.1995 Llondirrigiss 298 M Aug.1995 42 Llondirrigiss 299 M Aug.1995 Najo 265 F Aug.1995 36 Najo 273 M Aug.1995 Najo 274 M Aug.1995 Najo 276 F Aug.1995 Najo 284 F Aug.1995 Najo 285 F Aug.1995 37 Najo 288 M Aug.1995 Najo 300 M Aug.1995 Najo 303 M Aug.1995 Najo 305 F Aug.1995 38 Najo 306 F Aug.1995 Najo 308 F Aug.1995 38 Najo 310 M Aug.1995 39 Kisima I 333 M 4-3-1997 42 Kisima I 335 M 4-3-1997 41 Kisima I 336 F 4-3-1997 38 Kisima I 337 F 4-3-1997 36 Kisima I 348 F 4-3-1997 37 Lenjo I 322 M 1-2-1998 37 19,4 Lenjo I 323 F 1-2-1998 37 19,7 Lenjo I 325 M 1-2-1998 21,4 19,9 Lenjo I 327 F 1-2-1998 38 20,8 Lenjo I 329 M 1-2-1998 23,5 Sangito I 352 M 3-5-1998 21,7 Sangito I 355 M 3-5-1998 23,9 Sangito I 358 M 3-5-1998 21,5 Sangito I 359 M 3-5-1998 19,6 Kisima II 366 M 8-1-1999 36 20,7 Kisima II 369 M 8-1-1999 35 20,4 Kisima II 372 M 8-1-1999 35 19,9 20,5 Sangito II 319 F 12-9-1999 18,2 Sangito II 360 M 12-9-1999 15,6 Sangito II 361 M 12-9-1999 17,5 Kisima III 312 M 16-9-1999 19,9 Kisima III 344 M 16-9-1999 14,9 Kisima III 349 F 16-9-1999 15,9 Lenjo II 780 F 3-4-2000 12,2 Lenjo II 812 F 3-4-2000 10,4 Lenjo II 817 M 3-4-2000 10,7 Lenjo II 850 M 3-4-2000 12,1 Lenjo II 893 M 3-4-2000 11,5 Lenjo II 912 F 3-4-2000 9,8 Lenjo II 924 F 3-4-2000 12,3 Ayuba I F 25-5-2000 Sangito III F/M 12-6-2000 Sangito III F/M 12-6-2000 Sangito III F/M 12-6-2000 Sangito III F/M 12-6-2000
57 Appendix II Distemper antibodies levels (NVT) and vaccination dates Date 19-12-95 03-01-96 14-02-96 29-02-96 09-03-97 28-06-97 02-08-97 27-09-97 15-10-97 10-12-97 15-02-98 Identification Sex D-O NT-titer D-O NT-titer D-O NT-titer D-O NT-titer D-O NT-titer D-O NT-titer D-O NT-titer D-O NT-titer D-O NT-titer D-O NT-titer D-O NT-titer Lendanai 261 F V 540 V V 20 V 20 V Lendanai 262 F V 20 V V 60 V <20 V Lendanai 263 M V 540 V V 60 V <20 <20 V 20 Lendanai 264 F V <20 V V 60 V 20 <20 V Llondirrigiss 289 M V 20 V V <20 V <20 <20 V 20 Llondirrigiss 291 M V V V 180 V 20 <20 V 20 Llondirrigiss 293 M V <20 V V 60 V <20 <20 V Llondirrigiss 294 M V V V 20 V <20 <20 V 20 Llondirrigiss 296 M V V V 60 V 20 <20 V Llondirrigiss 297 F V V V <20 V died <20 Llondirrigiss 298 M V V V 20 V <20 <20 V 20 Llondirrigiss 299 M V 180 V V 60 Kenya Najo 265 F V 20 V V 20 V 20 <20 V 20 Najo 273 M V V V 60 Kenya Najo 274 M V V V <20 V <20 V Najo 276 F V V V 180 V <20 <20 V 20 Najo 284 F V V V 60 V <20 <20 V 20 Najo 285 F V V V 180 V <20 <20 V <20 Najo 288 M V V V 540 Kenya Najo 300 M V 60 V V 20 V <20 V 20 Najo 303 M V <20 V V 20 Kenya Najo 305 M V 20 V V 60 V <20 <20 V 20 Najo 306 F V 60 V V 60 V <20 <20 V 20 Najo 308 F V V V 20 V <20 <20 V 20 Najo 310 M V V V 60 V <20 V Kisima I 333 M V V V <20 V V <20 Kisima I 335 M V V V <20 V V 20 Kisima I 336 F V V V <20 V V <20 Kisima I 337 F V V V <20 V V 20 Kisima I 348 F V V V <20 V V <20
V = vaccination CDV-ISCOM Neutralizing antibodies were tested. 20 and up means well protected
Appendix II Distemper antibodies levels (NVT) and vaccinations dates Date 27-06-98 12-08-98 10-09-98 04-10-98 04-11-98 10-02-99 30-03-99 02-11-99 22-01-00 06-02-00 Identification Sex D-O NT-titer D-O NT-titer D-O NT-titer D-O NT-titer D-O NT-titer D-O NT-titer D-O NT-titer D-O NT-titer D-O NT-titer D-O NT-titer Lendanai 261 F V 40 80 V 60 Lendanai 262 F V 40 80 V Lendanai 263 M V 20 V 20 Lendanai 264 F V 40 80 V Llondirrigiss 289 M V V Llondirrigiss 291 M V 80 V 20 Llondirrigiss 293 M V 80 V Llondirrigiss 294 M V 20 V 60 Llondirrigiss 296 M V 40 V Llondirrigiss 297 F Llondirrigiss 298 M V 40 V 20 Llondirrigiss 299 M Najo 265 F V 80 V Najo 273 M Najo 274 M V 20 80 V Najo 276 F V 20 V Najo 284 F V 20 V Najo 285 F V 80 V <20 Najo 288 M
58
Appendix II Distemper antibodies levels (NVT) and vaccinations dates Date 27-06-98 12-08-98 10-09-98 04-10-98 04-11-98 10-02-99 30-03-99 02-11-99 22-01-00 06-02-00 Identification Sex D-O NT-titer D-O NT-titer D-O NT-titer D-O NT-titer D-O NT-titer D-O NT-titer D-O NT-titer D-O NT-titer D-O NT-titer D-O NT-titer Lendanai 261 F V 40 80 V 60 Lendanai 262 F V 40 80 V Lendanai 263 M V 20 V 20 Lendanai 264 F V 40 80 V Llondirrigiss 289 M V V Llondirrigiss 291 M V 80 V 20 Llondirrigiss 293 M V 80 V Llondirrigiss 294 M V 20 V 60 Llondirrigiss 296 M V 40 V Llondirrigiss 297 F Llondirrigiss 298 M V 40 V 20 Llondirrigiss 299 M Najo 265 F V 80 V Najo 273 M Najo 274 M V 20 80 V Najo 276 F V 20 V Najo 284 F V 20 V Najo 285 F V 80 V <20 Najo 288 M Najo 300 M V 80 V Najo 303 M Najo 305 M V 20 V 20 Najo 306 F V 80 V Najo 308 F V 20 V 60 Najo 310 M V 40 80 V 20 Kisima I 333 M V 80 V 20 Kisima I 335 M V 40 V 20 Kisima I 336 F V 40 V 20 Kisima I 337 F V 40 V 20 Kisima I 348 F V 20 V <20 Lenjo I322MVVVV40 80V20 Lenjo I323FVVVV20 80V<20 Lenjo I325MVVVV40 80V Lenjo I327FVVVV40 80V20 Lenjo I329MVVVV80 80V Sangito I 352 M V 80 V V V Sangito I 355 M V 80 V V V Sangito I 358 M V 80 V V V Sangito I 359 M V 20 V V V Kisima II 366 M V Kisima II 369 M V Kisima II 372 M V Sangito II 319 F VV Sangito II 360 M VV Sangito II 361 M VV Kisima III 312 M VV Kisima III 344 F VV Kisima III 349 F VV Lenjo II 780 F Lenjo II 812 F Lenjo II 817 M Lenjo II 850 M Lenjo II 893 M Lenjo II 912 F Lenjo II 924 F Ayubu I F Sangito III F/M Sangito III F/M Sangito III F/M Sangito III F/M
V = vaccination CDV-ISCOM Neutralizing antibodies were tested. 20 and up means well protected.
59
Appendix II Distemper antibodies levels (NVT) and vaccination dates Date 22-03-00 30-03-00 18-06-00 08-11-00 24-11-00 13-03-01 Identification Sex D-O NT-titer D-O NT-titer D-O NT-titer D-O NT-titer D-O NT-titer D-O NT-titer Lendanai 261 F Lendanai 262 F Lendanai 263 M Lendanai 264 F Llondirrigiss 289 M Llondirrigiss 291 M Llondirrigiss 293 M Llondirrigiss 294 M Llondirrigiss 296 M Llondirrigiss 297 F Llondirrigiss 298 M Llondirrigiss 299 M Najo 265 F Najo 273 M Najo 274 M Najo 276 F Najo 284 F Najo 285 F Najo 288 M Najo 300 M Najo 303 M Najo 305 M Najo 306 F Najo 308 F Najo 310 M Kisima I 333 M Kisima I 335 M Kisima I 336 F Kisima I 337 F Kisima I 348 F Lenjo I 322 M 20 Lenjo I 323 F 20 Lenjo I 325 M <20 V 320 Lenjo I 327 F 60 Lenjo I 329 M 20 Sangito I 352 M <20 Sangito I 355 M 20 Sangito I 358 M 20 Sangito I 359 M <20 Kisima II 366 M V V <20 Kisima II 369 M V V <20 Kisima II 372 M V V 20 V 80 Sangito II 319 F V <20 Sangito II 360 M V <20 Sangito II 361 M V <20 Kisima III 312 M V <20 Kisima III 344 F V <20 Kisima III 349 F V <20 Lenjo II 780 F V <20 V Lenjo II 812 F V <20 V Lenjo II 817 M V <20 V Lenjo II 850 M V <20 V Lenjo II 893 M V <20 V Lenjo II 912 F V <20 V Lenjo II 924 F V <20 V Ayubu I F Sangito III F/M Sangito III F/M Sangito III F/M Sangito III F/M
V = vaccination CDV_ISCOM Neutralizing antibodies were tested. 20 and up means well protected.
60 Appendix III Rabies antibody levels and vaccination dates Date 19-12-95 29-02-96 09-03-97 09-04-97 28-06-97 02-08-97 22-09-97 15-10-97 10-12-97 15-02-98 27-06-98 Identification Sex I.U. I.U. I.U. I.U. I.U I.U. I.U. I.U. I.U. I.U. I.U. Lendanai 261 F V 0 V <0.5 V V Lendanai 262 F V 0 V 0,5 V V Lendanai 263 M V 0 V >0.5 V V >0.5 >0.5 Lendanai 264 F V 0 V >0.5 V V >0.5 Llondirrigiss 289 M V 0 V <0.5 V V <0.5* >0.5 Llondirrigiss 291 M V V <0.5 V V >0.5* >0.5 Llondirrigiss 293 M V 0 V >0.5 V V >0.5 Llondirrigiss 294 M V V <0.5 V V <0.5* >0.5 Llondirrigiss 296 M V V <0.5 V V >0.5 Llondirrigiss 297 F V V V V <0.5 died Llondirrigiss 298 M V V <0.5 V V >0.5 >0.5 Llondirrigiss 299 M V 0 Kenya Najo 265 F V 0 V <0.5 V V >0.5 >0.5 Najo 273 M V Kenya Najo 274 M V V <0.5 V V Najo 276 F V V <0.5 V V >0.5 >0,5 Najo 284 F V V <0.5 V V >0.5 >0.5 Najo 285 F V V >0.5 V V >0.5 >0.5 Najo 288 M V Kenya Najo 300 M V 0 V V V >0.5 >0.5 Najo 303 M V 0 Kenya Najo 305 M V 0 V >0.5 V V >0.5* >0.5 Najo 306 F V 0 V <0.5 V V >0.5 >0.5 Najo 308 F V V <0.5 V V >0.5 >0.5 Najo 310 M V V >0.5 V V Kisima I 333 M V V <0.5* V <0.5 Kisima I 335 M V V <0.5* V >0.5 Kisima I 336 F V V <0.5* V >0.5 Kisima I 337 F V V <0.5* V >0.5 Kisima I 348 F V V <0.5* V >0.5 Lenjo I 322 M V Lenjo I 323 F V Lenjo I 325 M V Lenjo I 327 F V Lenjo I 329 M V Sangito I 352 M Sangito I 355 M Sangito I 358 M Sangito I 359 M Kisima II 366 M Kisima II 369 M Kisima II 372 M Sangito II 319 F Sangito II 360 M Sangito II 361 M Kisima III 312 M Kisima III 344 F Kisima III 349 F Lenjo II 780 F Lenjo II 812 F Lenjo II 817 M Lenjo II 850 M Lenjo II 893 M Lenjo II 912 F Lenjo II 924 F Ayubu I F Sangito III F/M Sangito III F/M Sangito III F/M Sangito III F/M
V = vaccination. Resistance against rabies is measured in International Units (I.U.) 0.5 I.U. and up means well protected. * Two times vaccinated at the time of testing (15-10-97).
61 Appendix III Rabies antibody levels and vaccination dates Date 12-08-98 04-10-98 04-11-98 30-03-99 02-11-9922-01-00 22-03-00 30-03-00 18-06-00 08-11-00 13-03-01 Identification Sex I.U. I.U. I.U. I.U. I.U. I.U. I.U. I.U. I.U. I.U. I.U. Lendanai 261 F V >0.5 >0.5 V <0.5 V Lendanai 262 F V >0.5 >0.5 V V Lendanai 263 M V >0.5 V <0.5 V Lendanai 264 F V >0.5 VV Llondirrigiss 289 M V >0.5 V V Llondirrigiss 291 M V <0.5 V >0.5 V Llondirrigiss 293 M V >0.5 V V Llondirrigiss 294 M V <0.5 V <0.5 V Llondirrigiss 296 M V >0.5 V V Llondirrigiss 297 F Llondirrigiss 298 M V >0.5 V <0.5 V Llondirrigiss 299 M Najo 265 F V >0.5 V >0.5 V Najo 273 M Najo 274 M V >0.5 >0.5 V V Najo 276 F V >0.5 V V Najo 284 F V >0.5 V V Najo 285 F V >0.5 V >0.5 V Najo 288 M Najo 300 M V <0.5 V V Najo 303 M Najo 305 M V >0.5 V >0.5 V Najo 306 F V >0.5 V V Najo 308 F V >0.5 V >0.5 V Najo 310 M V >0.5 V >0.5 V Kisima I 333 M V >0.5 V >0.5 V Kisima I 335 M V >0.5 V <0.5 V Kisima I 336 F V >0.5 V <0.5 V Kisima I 337 F V >0.5 V <0.5 V Kisima I 348 F V <0.5 V <0.5 V Lenjo I 322 M V V >0.5* >0.5 V <0.5 <0.5 V Lenjo I 323 F V V >0.5* >0.5 V <0.5 <0.5 V Lenjo I 325 M V V >0.5* >0.5 V >0.5 V V >0.5 Lenjo I 327 F V V >0.5* >0.5 V <0.5 <0.5 V Lenjo I 329 M V V >0.5* >0.5 V <0.5 V Sangito I 352 M V <0.5 V <0.5 V Sangito I 355 M V <0.5 V <0.5 V Sangito I 358 M V <0.5 V <0.5 V Sangito I 359 M V <0.5 V <0.5 V Kisima II 366 M V V V >0.5* Kisima II 369 M V V V >0.5* Kisima II 372 M V V V >0.5* V >0.5 Sangito II 319 F V V V V >0.5 Sangito II 360 M V V V V >0.5 Sangito II 361 M V V V V >0.5 Kisima III 312 M V V V V <0.5 Kisima III 344 F V V V V <0.5 Kisima III 349 F V V V V >0.5 Lenjo II 780 F V Lenjo II 812 F V<0.5 Lenjo II 817 M V<0.5 Lenjo II 850 M V<0.5 Lenjo II 893 M V<0.5 Lenjo II 912 F V Lenjo II 924 F V<0.5 Ayubu I F Sangito III F/M Sangito III F/M Sangito III F/M Sangito III F/M
V = vaccination. Resistance against rabies is measured in International Units (I.U.) 0.5 I.U. and up means well protected. * Two times vaccinated at the time of testing (15-10-97).
62 Appendix IV Parvo antibody level and vaccination dates Date 19-12-95 15-02-96 29-02-9609-03-97 28-06-97 02-08-97 15-02-98 27-06-98 12-08-98 04-10-98 Identification Sex Ig G Ig G Ig G Ig GIg GIg G Ig G Ig G Ig G Ig G Lendanai 261 F V V 13.500 V 100 V V Lendanai 262 F V V 1.500 V 90 V V Lendanai 263 M V V 4.500 V 150 V V Lendanai 264 F V V 1.500 V 270 V V Llondirrigiss 289 M V V 1.000 V 270 V V Llondirrigiss 291 M V V 13.500 V 270 V V Llondirrigiss 293 M V V 1.500 V 100 V Llondirrigiss 294 M V V 1.500 V 90 V V Llondirrigiss 296 M V V 500 V 90 V Llondirrigiss 297 F V V 4.500 died Llondirrigiss 298 M V V 1.500 V 100 V V Llondirrigiss 299 M V V 1.500 Kenya Najo 265 F V V 1.500 V 100 V V Najo 273 M V V 1.500Kenya V Najo 274 M V V 1.000 V 30 V V Najo 276 F V V 500 V 90 V Najo 284 F V V 500 V 80 V Najo 285 F V V 1.500 V 90 V Najo 288 M V V 1.500Kenya V Najo 300 M V V 1.500 V V Najo 303 M V V 13.500Kenya V Najo 305 M V V 1.500 V 90 V V Najo 306 F V V 1.500 V 90 V V Najo 308 F V V 4.500 V 90 V V Najo 310 M V V 100 V V V Kisima I 333 M V V V Kisima I 335 M V V V Kisima I 336 F V V V V Kisima I 337 F V V V V Kisima I 348 F V V V V Lenjo I 322 M VVV Lenjo I 323 F VVV Lenjo I 325 M VVV Lenjo I 327 F VVV Lenjo I 329 M VVV Sangito I 352 M V Sangito I 355 M V Sangito I 358 M V Sangito I 359 M V Kisima II 366 M Kisima II 369 M Kisima II 372 M Sangito II 319 F Sangito II 360 M Sangito II 361 M Kisima III 312 M Kisima III 344 F Kisima III 349 F Lenjo II 780 F Lenjo II 812 F Lenjo II 817 M Lenjo II 850 M Lenjo II 893 M Lenjo II 912 F Lenjo II 924 F Ayubu I F Sangito III F/M Sangito III F/M Sangito III F/M Sangito III F/M
63 Appendix IV Parvo antibody level and vaccination dates Date 04-11-98 30-03-99 30-03-00 18-06-0008-11-00 24-11-00 13-03-01 Identification Sex Ig G Ig G Ig G Ig G Ig G Ig G Ig G Lendanai 261 F V V Lendanai 262 F V V Lendanai 263 M V V Lendanai 264 F V V Llondirrigiss 289 M V V Llondirrigiss 291 M V V Llondirrigiss 293 M V V V Llondirrigiss 294 M V V Llondirrigiss 296 M V V V Llondirrigiss 297 F Llondirrigiss 298 M V V V Llondirrigiss 299 M Najo 265 F V V Najo 273 M Najo 274 M V V Najo 276 F V V V Najo 284 F V V V Najo 285 F V V V Najo 288 M Najo 300 M V V V Najo 303 M Najo 305 M V V Najo 306 F V V V Najo 308 F V V Najo 310 M V V Kisima I 333 M V V V Kisima I 335 M V V V Kisima I 336 F V V Kisima I 337 F V V Kisima I 348 F V V Lenjo I 322 M V V Lenjo I 323 F V V Lenjo I 325 M V V V Lenjo I 327 F V V Lenjo I 329 M V V Sangito I 352 M V V V Sangito I 355 M V V V Sangito I 358 M V V V Sangito I 359 M V V V Kisima II 366 M V V Kisima II 369 M V V Kisima II 372 M V V V Sangito II 319 F V V Sangito II 360 M V V Sangito II 361 M V V Kisima III 312 M V V Kisima III 344 F V V Kisima III 349 F V V Lenjo II 780 F V V Lenjo II 812 F V V Lenjo II 817 M V V Lenjo II 850 M V V Lenjo II 893 M V V Lenjo II 912 F V V Lenjo II 924 F V V Ayubu I F Sangito III F/M Sangito III F/M Sangito III F/M Sangito III F/M
64 GENETIC VARIATION WITHIN AND BETWEEN AFRICAN WILD DOG (LYCAON PICTUS) GROUPS FROM THE MAASAI STEPPE, TANZANIA.
Dr. L. Van de Zande
Population Genetics, Centre of Ecological and Evolutionary Studies, University of Groningen, The Netherlands. Biological Centre, P.O. Box 14, 9750 AA Haren, The Netherlands. Tel: +31 50 3632126, Fax +31 50 3632348; Email: [email protected]
Introduction
In order to investigate the genetic variation within and betweenthe African Wild Dog (Lycaon pictus) packs in the Mkomazi Game Reserve in Tanzania, a multilocus DNA fingerprint pattern of 25 individual Wild dog pups was made to serve as an initial survey. The Mkomazi Project concerns the capture and translocation of Wild dogs for captive breeding and reintroduction. The 25 individuals were captured in the Maasai Steppe, Tanzania. The pups can be divided into three groups: the Najo group (n=13), the Llondirrigiss group (n=8) and the Lendanai group (n=4). All pups within one group are full-sibs (relatedness r=0.5), while individuals belonging to different groups are considered to be unrelated.
Materials and methods
DNA extraction DNA was isolated from 5 ml whole blood collected in EDTA anticoagulant vacutainers. DNA was extracted by proteinase K digestion (100 g/ml) at 55oC overnight in lysis buffer (l0 mM Tris-HCI, pH 7.4,100 mM NaCI, 25 mM EDTA, pH 8.0, 0.5 per cent SDS (w/v), and purified by several extractions with phenol and chloroform:isoamyl-alcohol (24:1).
DNA fingerprinting Five micrograms of DNA were digested to completion with 15 unit's restriction enzyne Hinfl. DNA fragments were resolved on a 0.8 % agarose gel, without ethidium bromide, in TBE buffer (0.089 M Tris, 0.089 M boric acid, 0.002 M EDTA) for 60 h at 1.8 V/cm. After electrophoresis the DNA fragments were transferred to nylon membranes (Hybond-N, Amersham) by vacuum blotting and crosslinked by UV illumination. Minisatellite probe 33.15 (Jeffreys et al., 1985) was labeled with -32P-dCTP by random primed DNA labeling (Feinberg & Vogelstein; 1984). Filters were 0 prehybridized at 65 C in 0.263 M Na2HPO4, pH 7.2, 7% SDS (w/v), 1 mM EDTA, for 1 h. Hybridization was carried out overnight at 65oC in the same buffer. Filters were washed with 2xSSC, 0.1% SDS for 10 min at room temperature. The filters were exposed to Kodak X-OMAT X-ray film for 1-3 days at -700C using intensifying screens.
65 Data analysis
All clear bands in the range of 12-2 kb were scored manually. The coefficient of band-sharing between individuals was calculated as Sxy =2nxy/(nx + ny) where nx and ny are the number of bands present in individuals x and y, respectively, and nxy is the number of bands shared by x and y (Wetton et al., 1987) Mean similarity within a group was estimated by averaging the similarities of all pairs of individuals belonging to that group. Mean similarity between groups A and B was estimated by averaging the similarities of all pairs of individuals of which one belonged to group A and the other to group B. Due to the fact that in the field no direct freezing of the samples was possible and the samples were not frozen until they arrived in our lab, the condition of the DNA was not optimal, and some degeneration had occurred. Nonetheless, the quality of most DNA samples was sufficient to perform a multilocus DNA fingerprint analysis.
Results
In the first run 15 samples were randomly chosen from the collection and their fingerprint pattern was analyzed. Scorable bands were observed in the range from 11 - 2 kb (Pic. 1.) Eight of these samples were from Najo dogs, 5 were from Llondirrigiss dogs and 2 were from Lendanai dogs. Two samples failed to give an interpretable banding pattern (Najo 284 and Llondirrigiss 291). Analysis of the patterns yielded a mean similarity within the Najo group of 0.58 and within the Llondirrigiss group of 0.65. The mean similarity between the Najo and the Llondirrigiss group was 0.38 (Table 1.). The sample size of the Lendanai group was too small to yield reliable results. In the second run another 15 samples were fingerprinted: the 10 remaining samples plus 5 samples from run one, to serve either as cross reference or as second sampling. Seven of these samples were from Najo dogs, 5 from Llondirrigiss dogs and 3 from Lendanai dogs. One sample failed to give an interpretable banding pattern (Llondirrigiss 296). Unfortunately, only the high molecular weight bands (10 - 5 kb) could be analyzed as the lower molecular weight bands were obscured by aspecific hybridization (Pic. 2). Analysis of the patterns yielded a mean similarity within the Najo group of 0.51, within the Llondirrigiss group of 0.36 and within the Lendanai group of 0.52. The mean similarity between the Najo and the Llondirrigiss group was 0.23, between Najo and Lendanai 0.37 and between Llondirrigiss and Lendanai 0.22 (Table 1.).
66 Table 1. Mean similarity within and between three groups of wild dogs.
Conclusion
From the results can be concluded that there is considerable genetic variation in the wild dog population in the Maasai steppe in Tanzania. The most reliable results can be derived from run 1, where no aspecific hybridization has occurred. Full-sibs have a mean similarity ranging from 0.55 - 0.65 while unrelated animals have mean similarities of 0.38 and even lower. The observed similarity of approximately 0.6 for full-sibs is in accordance with parents with similarities in the order of 0.4. However; in order to obtain more reliable estimates of the degree of relatedness of individuals from different groups, reference data from animals of known relatedness are needed. Nevertheless, the social organization of the hunting dog population seems sufficiently structured to prevent high levels of inbreeding.
References
FEINBERG, A.P. AND VOGELSTEIN, B. 1983. A technique for radio labeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132, 6-13.
JEFFREYS, A.J., WILSON, V., AND THEIN, S.L. 1985. Hypervariable 'minisatellite' regions in human DNA. Nature, 314, 67-73.
WETTON, J.H., CARTER, R.E., PARKIN, D.T. AND WALTERS, D. 1987. Demographic study of a wild house sparrow population by DNA 'fingerprinting'. Nature, 327, 147-149.
67
Pic. 1. DNA fingerprint of 15 Lycaon pictus individuals. 1: Najo 285; 2: Najo 300; 3: Llondirrigiss 291; 4: Najo 310; 5: Najo 305; 6: Najo 275; 7: Najo 288; 8: Llondirrigiss 289; 9: Llondirrigiss 298; 10: Najo 273 11: Llondirrigiss 296; 12: Lendanai 263; 13: Llondirrigiss 299; 14: Najo 284; 15: Lendanai 261. DNA samples were digested with HinfI and hybridized with Jeffreys probe 33.15.
68
Pic. 2. DNA fingerprint of 15 Lycaon pictus individuals. 1: Najo 308; 2: Najo 303; 3: Llondirrigiss 298; 4: Llondirrigiss 297; 5: Llondirrigiss 296; 6: Llondirrigiss 294; 7: Llondirrigiss 293; 8: Najo 284; 9: Najo 276; 10: Najo 274; 11: Najo 273; 12: Najo 265; 13: Lendanai 264; 14: Lendanai 263; 15: Lendanai 262. DNA samples were digested with HinfI and hybridized with Jeffreys probe 33.15.
69 Parenthood analysis of the Afr ican Wild Dog from the Mkomazi Game Reserve in Tanzania
Dr. A.L. Kappe
Gendika B.V. Industrieweg 1 9641 HM Veendam The Netherlands
Tel: +31 598 619 343 / 616 306 Fax: +31 598 612 194 E-mail: [email protected]
70 Contents
Material P3
Method P3
1. DNA isolation P3 2. DNA fingerprinting P3 3. Data analysis P4
Results P5
Conclusion P6
71 Material
This report describes a DNA fingerprint analysis of seven blood samples of the African Wild Dog from the Mkomazi Game Reserve in Tanzania. The collected samples contained:
Code Identity
Llondirrigiss 297 mother Najo 300 presumable father Kisima 333 pup Kisima 335 pup Kisima 336 pup Kisima 337 pup Kisima 339 pup
Method
1. DNA isolation
DNA was isolated from 3 ml whole blood collected in EDTA anticoagulant vacutainer tubes. DNA was extracted by proteinase K digestion (100 g/ml) at 55º C overnight in lysisbuffer (10 mM Tris-HCl, pH 7.4, 100 mM NaCl, 25 mM EDTA, pH 8.0, 0.5% SDS (w/v)), and purified by several extractions with phenol and chloroform:isoamyl-alcohol (24:1).
2. DNA fingerprinting
Five micrograms of DNA were digested to completion with 15 units restriction enzyrn HinfI. DNA fragments were resolved on a 0.8% agarose gel, without ethidium bromide, in a TBE buffer (0.089 M Tris, 0.089 M boric acid, 0.002 M EDTA) for 60 h at 1.8 V/cm. After electrophoresis the DNA fragments were transferred to nylon membranes by vacuum blotting and cross linked by UV illumination. Minisatellite probe 33.15 (Jeffreys) was radioactive labeled with 32P by random- primed DNA labeling. The membrane was hybridized with the radioactive labeled probe and was carried out overnight at 65ºC in hybridization mix (0.263 M Na2HPO4, pH 7.2, 7% SDS (w/v), 1 mM EDTA). The membrane was exposed to X-ray film for 1 day at -70º C using intensifying screens.
72
DNA fingerprint of African Wild Dog. DNA was digested with restriction enzym HinfI and hybridized with minisatellite probe 33.15.
3. Data analysis
The resulting film with DNA fingerprinting data was analyzed with ImageMaster analyzing equipment. The data were analyzed with DNA Image analysis Elite - ID software.
73 Results
With the DNA Image analysis software a band matching was executed by computer. This means that bands, detected by the computer and assigned with a on this picture, that are situated on equal heights, are connected by a purple line.
The lines that are marked between lanes 1 and 10 (marker lanes) are reference lines used to match the other bands of the samples. The other purple lines are matching lines.
Lane 6 (blue) contained insufficient DNA to detect a signal, so no bands are visible.
Legends of the gel
1. marker 2. 297 (mother) 3. 300 father 4. 333 (pup) 5. 335 (pup) 6. 336 (pup) 7. 337 (pup) 8. 339 (pup) 9. negative control 10. marker
Analysis of the DNA fingerprint membrane with use of the matching program software in ImageElite.
74 Conclusion
From the data can be concluded that five bands assigned to the mother (dog 297) are independent of the three bands shown by the father (dog 300), there are no shared bands by mother and father. All descendants show bands that can be assigned to the father or the mother. All bands of the descendants can be assigned to father and mother. From the data can be concluded that dog 300, the presumed father, is the most probable father of the pups 333, 335, 337 and 339 (no results of dog 336 could be found). The possibility of fatherhood of one of the brothers of the mother, dogs 289, 291, 293, 294, 296 or 298, is very unlikely.
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© The African Wild Dog Foundation 2003