AFRICAN WILD DOG (LYCAON PICTUS) BREEDING PROGRAM Mkomazi Game Reserve, Tanzania Report 2001-2005

Aart Visee, D.V.M.

AN ENDANGERED SPECIES PROGRAM UNDERTAKEN BY: THE WILDLIFE PRESERVATION TRUST FUND (TANZANIA) THE GEORGE ADAMSON WILDLIFE PRESERVATION TRUSTS (UK,USA, NETH., GER., KEN.) and THE AFRICAN WILD DOG FOUNDATION (NETH.) CONTENTS Page

Foreword 3 Introduction 4 Husbandry 5 Pack composition, development and behaviour 5 Introduction 5 2001: Restart / Malaika pack 5 2002: Kimondo pack / Start Zawadi pack 7 2003 9 2004 10 2005 14 Zootechnique 18 Identification 18 DNA 20 Housing 21 Nutrition 21 Bodyweight and Neck size 22 Veterinary 25 Preventative Medicine 26 Canine Distemper Virus 26 Rabies 29 Parvovirus 31 Parasites 31 Clinical Work-Diseases 33 Clinical Work 33 Anaesthesia 34 Pathology 35 Acknowledgements 36 Summery 37 References 38 Appendix I, Bodyweight and neck size 40 Appendix II, Distemper VNT 41 Appendix III, Distemper Elisa 42 Appendix IV, Rabies 43 Appendix V, Pack composition December 2005 44 Appendix VI, Breeding results 2001-2005 45 Appendix VII, Breeding results 2001-2005 including similarity 46 Genetic Analysis 47

2 Foreword.

With lots of enthusiasm and high hopes, we started the African wild dog breeding program in Kisima camp, Mkomazi Game Reserve, Tanzania, 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 reintroducing 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 really 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. This period ended abruptly, and completely when the last of the 25 original dogs died August 2001. 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.

The years 1995 to 2001, including the canine distemper epidemic, were extensively reported in the Report 1995-2001. Since then no new report appeared, as there was not much to report, being quite and thus nice years for a change. The establishing of new breeding packs with subsequently the birth of pups, and the progress made in the canine distemper vaccination research made it worthwhile to come out with a new report covering 2001-2005.

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 reintroduction.

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 parvovirus), 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.

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. The Mkomazi African wild dog population was almost completely wiped out, and in 2001 it was needed to start all over again with the three survivors as a basis for a new population. Soon two “volunteers” dogs would join them. Together with one of the distemper survivors, and the first litter born out of the distemper survivors, they would form the nucleus of our new breeding program.

This report is divided into two parts, Husbandry and Veterinary.

4 HUSBANDRY

Pack composition, development and behaviour. This report includes how the new breeding packs were composed and developed. Where necessary behaviour is reported and commented on.

Zoo technique. In this section, a report is drawn up about identification, DNA, housing, nutrition, bodyweight and neck size.

Pack composition, development and behaviour (see appendix VI)

Introduction

The key to understanding how new free living wild dog packs form and persist, despite major changes in their composition in subsequent years, is the existence of a basic protocol in wild dog behaviour, that was first identified in the East African wild dog population to which the Mkomazi captive breeding group belong.

In both the breeding and feeding hierarchies older dogs of both sexes (apart from the alpha [i.e. main breeding female - the ‘Selfish Matriarch’ (Burrows 1992)] give priority to both related and unrelated younger dogs in the pack (Malcolm & Marten 1982, Burrows 1995 & 2004, McNutt, 1996, Visee et al 2001). In this report the term ‘natural rules’ is applied to any behaviour observed in the Mkomazi captive packs that is known to be typical of the behaviour of free living dogs.

2001: Restart / Malaika 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.

For the occasion the two males, #325 (born 4-9-97) and #372 (born 8-1-99), 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

5 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 through the wire (in fact #306 and #372 were very interested in each other), the dogs were put together and the Malaika pack was established.

Immediately female #306 and the younger male #372 joined, and #306 was putting her nose under #372’s belly. #325 kept a little distance. It appeared that the female chose the younger male.

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, #372, being the youngest of the two males. 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.

On the 4th of August, fate struck again when suddenly female #306 showed signs of serious distress and died within six hours (see Report 1995-2001). 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 Report 1995-2001). The remaining seven pups grew up well.

The Malaika pack was short lived but left 7 beautiful pups as a strong basis for the breeding program.

At the end of June, two female wild dogs showed up in the vicinity of our boma’s. It was most likely that they were two young sisters (18-24 months old) who had emigrated from their free living natal pack and were 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 boma’s. 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

6 the boma; finally reuniting her with her sister. Few hostilities took place when the four dogs were introduced to each other.

In December 2001, it was apparent that #372 was the alpha male, and #040 the alpha female. Normally in a free living pack which contained younger males it might have been expected that after the death of the alpha female (#306) the alpha male would also have lost his position. As in this case there was no younger male, only his older male pack member, he kept his alpha position. If there had been a younger male, #372 might have lost his alpha position (based on the ‘young male first’ protocol in wild dog society) when new packs are formed.

Pack composition December 2001:

KISIMA boma

Females Males 040 alpha, Kimondo 372 alpha, Chupu chupu 006, Zawadi 325, Bahati

SANGITO boma

pups born 07-07-01 (306 x 372)

Females Males 448 202 464 251 269 368 389

2002: Kimondo pack / Start Zawadi pack

January 2002 mating was observed between #040 (Kimondo) and #372 (Chupu chupu), giving evidence that they were indeed the alpha’s. #325 (Bahati), the other distemper male survivor, was subordinate to #372 from the moment these two were put together in the same boma in March 2001. While #325 was fast asleep he was bitten in the neck many times by #372. The fact that they originated from different packs might have contributed to this kind of behaviour.

March 20th #325 the subordinate male died suddenly 5 minutes after eating from, what appeared to be, heart failure.

7 Kimondo (#040) gave birth 18 April. Some weeks later it appeared to be a litter of 4 pups. Four days later(22 April) Zawadi (#006) gave birth to four pups as inspection of the den showed a couple of days later. It was observed that both females had mated with Chupu chupu (#372).

In the following days Kimondo (#040) went in Zawadi’s den on a number of different occasions. Inspection of the den on 29 April revealed no signs of pups. Thereafter Zawadi assisted in raising and feeding Kimondo’s pups, being an integral part of the upbringing of the pups as it should be. When the pups were about 6 months old, Kimondo and Chupu chupu started to chase Zawadi off and as a result she kept herself more and more on the side. Kimondo rarely bit her sister but just chased her off until she stayed away.

In free living packs, subordinate sisters of the alpha female often leave (re-emigrate from) their alpha sister’s pack, having assisted in raising a new packs first litter, and attempt to locate new males to form a new pack.

Following these free living pack procedures, Zawadi was removed on December 8th and moved to the Lendanai boma to start a new pack with the brothers #202 and #251 from the Malaika litter in the Sangito boma. In this way a new pack, Zawadi pack, was established. To prevent brother sister relationships the other 3 males from the Sangito boma, #269, #368 and #389 were moved to the Ayubu boma.

Pack composition December 2002:

KISIMA boma

Kimondo pack Females Males 040 alpha, Kimondo 372 alpha, Chupu chupu

Pups born 18-04-02 104 651 348 730

LENDANAI boma

Zawadi pack Females Males 006, Zawadi 202 251

SANGITO boma

Females 448 464

8

AYUBU boma Males 269 368 389

2003

Kimondo pack: May 31st Kimondo (#040) gave birth to six pups; four males and two females. There was a lot of excitement from the first litter of pups (now 14 months old yearlings) and they all assisted in feeding and raising the new pups. At the time the new pups started to eat non-regurgitated food in the feeding area, the yearlings hung back and the alpha male (#372), chased them back as well, just to make sure.

An experiment in 1998 in Mkomazi, to test whether the ‘natural rules’ observed in the wild applied equally in captivity, showed that the order of feeding and the time spent feeding at the food provided (i.e. as at the kill of a free living pack) were very similar. In both cases pups monopolize food often with the alpha female (the ‘Selfish Matriarch’) and defended the food/kill from the other adults and only after the pups are satiated do the older dogs return to the food with older subordinate dogs the last to feed.

The time spent by the Mkomazi captive pack feeding on the food provided was 40 minutes at one feed and 50 minutes at another with the pups (4) monopolizing the food for 35 of the 50 minute feeding session. This is similar to the time taken by free living Serengeti packs with pups and/or yearlings spent feeding (‘Access Time’) at a kill (Schaller 1972, Malcolm 1979, Fanshawe et al 1993). There is no ‘scramble competition’ for food (contra Carbone et al 1997) and dogs of different ages/cohorts do not feed equally at any one feed/kill. This ‘natural rule’ (the ‘feeding’ or ‘access’ hierarchy’ protocol) is now known to apply to both captive and free living packs. This is important as we know that the ‘natural rules’ will be followed by Mkomazi bred dogs post release.

When the new pups were approximately six months old the two male yearlings started to snap at their father, #372, at feeding time. They attacked him together from each side. As a result #372 made sure that he quickly was in the feeding area first, had a quick bite and hung back to allow the pups and the yearlings to eat. Kimondo did not have any real problems with any of her pups or yearlings. She drove them away if they attempted to stop her feeding with them.

Zawadi pack: The initial introduction of the dogs to each other was peaceful, although the dogs were periodically somewhat exited. They settled down in their own way, i.e. Zawadi (#006), was much shyer and as a consequence more nervous and remained most of the time on the periphery. Until October both males, #202 and #251, chased and snapped at Zawadi occasionally, although #202 tried to get closer to her and sometimes even played with her.

9 Most of the times, however, #251 interfered and chased Zawadi away. At this time it was difficult to tell who was the alpha male. Originally, in the litter, #202 appeared to be alpha. As a consequence Zawadi had to be feed separately to make sure that she got enough food. On the rare occasions that they played together Zawadi was always at the back, nervous and never really involved.

By October #251 started to come closer to Zawadi, however #202 started to chase her off when she came too close to #251.

Sangito boma: The only two dogs in this compound, the two sisters, #448 and # 464, had an uneventful year. In 2002 it looked like #464 was alpha, but in the course of 2003 it showed that #448 was alpha. At feeding time she chased #464 off but outside feeding time they were very close.

Ayubu boma: Over the year the positions of the three brothers in this boma were very clear; #368 is the alpha, being very fierce and displays it to his brothers. #269 is the most subordinate in the pack. He did not display much physical strength but made the most noise (typical for submission): he was the one who started to “bark” when people or some animal approached the boma. As a result of their clear dominant/subordinate relationship they did not fight each other.

Pack composition December 2003:

KISIMA boma

Kimondo pack Females Males 040 alpha, Kimondo 372 alpha, Chupu chupu

Pups born 18-04-02 104 651 348 730 Pups born 31-05-03 001 092 031 187 212 393

Lendanai, Sangito and Ayubu boma as December 2002

2004

Kimondo pack: In June Kimondo, #040, again came in season and on August 29th gave birth to her third litter that appeared to contain six pups. When they were old enough to come out of the den it appeared as if they were going to be fed by quite a number of dogs.

However, already during pregnancy Kimondo did not look well and after coming out of the den the pups remained very tiny and did not increase in weight or size. The pups of Kimondo II litter (now 14 months old yearlings) hassled them. Although the yearlings fed them they

10 also picked them up by the ears or the middle of the back, not by the neck and were throwing them around. Also, while trotting after their parents along the fence, they were trampled upon by the yearlings. The first three pups died 11 October and the last three died on October 23rd, i.e. they disappeared and no remnants were found.

The intriguing facts here are that the dogs of the first born litter, Kimondo I (now 28 months old), ignored the small pups and the dogs of the second born litter, Kimondo II (now 16 months old), hassled them in a rougher way than we observed in other equal events. If there was going to be any trouble it would have been expected from the dogs of Kimondo I litter. The removal of these four dogs of the first litter was delayed as it was not advisable at that time to start a third and fourth breeding pack for which the dogs were intended. The yearlings of Kimondo II litter were underweight and did not have the body size expected for their age.

It is possible therefore that the apparently aggressive behaviour of the yearlings toward the new pups was due to lack of food but this is unlikely as they did feed the pups. Also as their mother appeared unwell during her pregnancy and the pups failed to grow as expected perhaps the pups were ill.

Yearlings are fascinated by new pups and attempt to interact with them as much as possible, so there are many accounts of free living yearlings appearing to hassle new pups. If the pups are healthy and well fed they just retreat into the den and survive despite the rough play of the yearlings.

There is just a single case reported in a free living pack when yearlings reportedly did not feed young pups at a den and even stole food regurgitated to the pups by other dogs. This was a pack watched in the dry season in Serengeti when prey was scarce and the pups were obviously both starving and in very poor health (Frame & Frame 1981). This was a time in Serengeti when canine distemper was suspected as the cause of high mortality in pups and perhaps in some young adults (Frame et al 1979). It may be that these free living pups were not behaving ‘normally’ (as perhaps were the pups in the Kimondo pack) and had no appetite when fed regurgitated food by the adults, so the yearlings took any uneaten food.

Not removing these four dogs meant that natural pack rules were not followed and might have caused trouble, which did not happen. That leaves the abnormal behaviour of the dogs, yearlings, of the Kimondo II litter. Looking at the dogs of the Kimondo II litter, underweight and not a body-size according to their age, it still might have been possible that their behaviour towards the newborn pups was instigated by lack of food. Not being full-grown yet and receiving not enough food for their own growth the newborn pups were a direct threat to their own existence.

11 Early December the four dogs of the Kimondo I litter were moved from the Kimondo pack to the Sangito and Ayubu boma.

Zawadi pack: Between late December 2003 to January 2004 #251 stayed close to Zawadi. #202 still chased her at feeding time but normally they all slept near each other and were getting along well in a more normal way. However, due to her nervousness, if one of the males woke up, Zawadi immediately followed him. Early in February it was observed that the three dogs were running together more often and by the end of the month Zawadi came in season. She started to pursue #251 and #251 also pursued her. Both chased off #202, with the male #251 the most vigilant and active chaser of #202. The alpha pair, #006 and #251 showed all the behaviour one might expect from an alpha pair, i.e. joint urine marking with raised leg posture, dominance display with #202 performing appeasement rolling, cringing and whining with ears back and tail between legs.

On May 11th Zawadi gave birth to nine pups, very unusually all were males.

Towards the end of the year the beta male, #202, was chased away at feeding time. However #202 had enough confidence in the keeper to come in and have some food before the other dogs were allowed to come into the feeding area.

Sangito boma: On the third of December the males, #651 and #730, from Kimondo’s firstborn litter were moved from the Kisima boma to the Sangito boma. The dogs were sedated and woke up in the feeding area of the Sangito boma. The resident females were locked out of the feeding area. Immediately the dogs showed an interest in each other but with some reservation in their behaviour. In the morning of 5 December, the day after the transfer of the male dogs, the dogs were allowed to join each other. No hostilities whatsoever took place and at feeding time all the dogs fed together. From the beginning, male #651 and female #464 took up alpha position. The relationship between the males and the females was pretty relaxed from the beginning. The alpha pair started mating within a couple of weeks.

Ayubu boma: On the third of December the females, #104 and #348, from Kimondo’s firstborn litter were moved from the Kisima boma to the Ayubu boma. The dogs were sedated and woke up after transport in the Ayubu boma outside the feeding area. The resident males were locked up in the feeding area. In this way the females had the opportunity to inspect and get familiar with their new housing arrangements should the males be aggressively interested in them. The opposite happened. The females showed more interest in the males than their brothers did in the females in the Sangito boma. The females slept and rested against

12 the fence of the feeding area to be as close as possible to the males. In the morning of 5 December the dogs were allowed to join. Here too no hostilities took place and the dogs immediately started to act as a pack. The male #368 behaved as the alpha male from the start and took great interest in the females, following them around. The female #104 appeared to be the alpha female from the start. The other two males had to keep a subordinate position which meant that they had to eat in a secondary pattern to the alpha pair. The alpha pair has been seen mating within a couple of weeks.

Pack composition December 2004:

KISIMA boma

Kimondo pack Females Males 040 alpha, Kimondo 372 alpha, Chupu chupu

Pups born 31-05-03 001 092 031 187 212 393

LENDANAI boma

Zawadi pack Females Males 006 alpha, Zawadi 202 251 alpha

Pups born 11-05-04 666 671 732 853 860 887 926 939 949

SANGITO boma

Females Males 448 alpha 651 464 730 alpha

AYUBU boma

Females Males 104 alpha, Mrefu 269 348 beta, Regina 368 alpha 89

13 2005

Kimondo pack: After the removal of the members of the Kimondo I litter the stress during feeding time completely died down and mid January it was observed that the alpha pair was mating again. During pregnancy Kimondo was very vocal and at feeding time she whined endlessly to ensure that the other dogs regurgitated their food for her. She gave birth 10th April to six pups, two males and four females. From the moment the pups emerged from the den a female pup appeared to be smaller and weaker than her littermates. Lacking in physical strength at feeding time she had to struggle to get some food and as a consequence was steadily falling back in strength and size in comparison to her littermates. July 17th she gave in and died. The other five pups grew up well and behaved in a normal way, thus taking and being allowed first place at feeding time.

On 26th November the four males of the Kimondo II litter were removed from the pack and transferred to the Kilo Sierra compound in the rhino sanctuary, to be the first dogs to be released.

Zawadi pack: Mid February Zawadi #006 and #251, the alpha pair, was mating again. Zawadi too was very vocal during pregnancy and also tried to encourage the other dogs to regurgitate food for her. The subordinate male #202 was the only one to do so. She gave birth 22nd May to five pups; once again all males. After the pups emerged from the den the nine pups from the older litter assisted in feeding them as it should be.

On 25th November the nine male pups, Zawadi I litter, were transferred to a newly build boma as they had reached an age that usually free living yearlings leave their natal pack. They settled well with hardly any calling at night.

Sangito boma: Despite the observed mating in December 2004 and the display of aggressive behaviour by the alpha male #730 towards his brother and the alpha female chasing off her sister it appeared that the alpha female #448 was not pregnant.

The feeding system had to be manipulated because the alpha pair would otherwise leave the beta dogs with very little food.

The following interactions were observed: At the time it was presumed that the alpha female was pregnant, she chased her sister out of the group. However, as the months passed, the beta sister returned slowly to the group, but still kept some distance. When the dogs were trotting through the compound, the alpha pair was first in line, followed by the beta male, and slightly behind the beta female. Also, when they slept, the alpha pair and the beta male slept together, and the beta female slept apart.

14 Ayubu boma: Since the alpha female #104 became pregnant December 2004 the other two subordinate males in the compound (#269, #389) had been experiencing problems. The alpha male continually chased them off during eating and they had to be fed separately. However, the subordinate female #348 was chased off by the alpha female for the first couple of weeks of pregnancy, followed by a couple of weeks that she was accepted again and the last couple of weeks of her pregnancy #348 was chased off again.

March 27th #104 gave birth to 7 pups. As in Kimondo’s litter one pup was half the size of the others at the first sighting and died 31st May.

Of the six left, three were male and three were female. Another female pup appeared to be retarded in growth and died July 17th.

Mid June mating was observed by the alpha male #368 and beta female #348. From that moment on the alpha female #104 chased her subordinate sister off.

The subordinate beta female #348 gave birth on 6th August 2005. The pups were for the first time sighted on 19th September and appeared to be seven pups in all. When #348 was pregnant, the alpha female chased her off at feeding time and tried to keep the alpha male away from her. But since she gave birth, the alpha female allowed her to feed normally, i.e. allowed her to come to the food in the feeding area. Then #348 went around all the adult males whining and succeeded in getting them to regurgitate for her. She ate that food as well and then regurgitated for her pups.

Meanwhile the alpha female, #104, looked after her own pups without getting any assistance from the adult males in the compound. The alpha male too assisted in feeding the pups of the beta female.

The pups from the alpha female were very curious to see what was inside the den of the beta female, as they could hear the noises and cries. They often went down into the den and came out the other side, as the new pups (beta) were hidden in a channel off the main den.

The alpha male was fierce in protecting both litters of pups but spent most of his time with the beta female #348.

On 26th November a male pup of the litter of the beta female died.

15 Pack composition December 2005 (see also appendix V):

KISIMA boma

Kimondo pack Females Males 040 alpha, Kimondo 372 alpha, Chupu chupu

Pups born 31-05-03 001 031

Pups born 10-04-05 039 043 2040 073 046

LENDANAI boma

Zawadi pack Females Males 006 alpha, Zawadi 202 251 alpha

Pups born 11-05-04 666 671 732 853 860 887 926 939 949

Pups born 22-05-05 519 521 560 567 572

SANGITO boma

Females Males 448 alpha 651 464 730 alpha

16 AYUBU boma

Females Males 104 alpha, Mrefu 269 348 beta, Regina 368 alpha 389

Pups born 27-03-05 444 557 481 586 643

Pups born 06-08-05 000 232 6372 296 0348 2368

17 Zootechnique

Identification All the dogs when first sedated were fitted with a transponder. The transponders were inserted subcutaneously on the left side of the neck. The transponders were checked on every occasion the individuals were sedated. All appeared to be in good order. The dogs received transponders starting with the same number 528210000 for every dog, followed by a different six number identity for each dog. Figure 1 shows the code for each dog. The only exception is #372, the last living survivor of the canine distemper epidemic. His complete number is: 981093500110372.

To make the numbers more workable only the last three numbers are used on daily basis.

As an extra precaution photographs were taken from both sides of the dogs in lateral recumbency.

18

Table 1: Transponders Number ID number Litter of origin Sex Date of Birth Date transponder Applied 181006 006 F 1999 20-11-01 181040 040 F 1999 20-11-01 180202 202 Malaika M 07-07-01 22-11-01 180251 251 Malaika M 07-07-01 22-11-01 180269 269 Malaika M 07-07-01 22-11-01 181368 368 Malaika M 07-07-01 22-11-01 180389 389 Malaika M 07-07-01 22-11-01 180448 448 Malaika F 07-07-01 22-11-01 180464 464 Malaika F 07-07-01 22-11-01 181104 104 Kimondo I F 18-04-02 05-12-02 180348 348 Kimondo I F 18-04-02 05-12-02 179651 651 Kimondo I M 18-04-02 05-12-02 179730 730 Kimondo I M 18-04-02 05-12-02 268001 001 Kimondo II F 31-05-03 04-12-03 269031 031 Kimondo II F 31-05-03 04-12-03 273092 092 Kimondo II M 31-05-03 04-12-03 269187 187 Kimondo II M 31-05-03 04-12-03 268212 212 Kimondo II M 31-05-03 04-12-03 266393 393 Kimondo II M 31-05-03 04-12-03 269666 666 Zawadi I M 11-05-04 04-12-04 266671 671 Zawadi I M 11-05-04 04-12-04 265732 732 Zawadi I M 11-05-04 04-12-04 266853 853 Zawadi I M 11-05-04 04-12-04 266860 860 Zawadi I M 11-05-04 04-12-04 268887 887 Zawadi I M 11-05-04 04-12-04 267926 926 Zawadi I M 11-05-04 04-12-04 264939 939 Zawadi I M 11-05-04 04-12-04 272949 949 Zawadi I M 11-05-04 04-12-04 268444 444 Mrefu I F 27-03-05 24-11-05 268481 481 Mrefu I F 27-03-05 24-11-05 272557 557 Mrefu I M 27-03-05 24-11-05 269586 586 Mrefu I M 27-03-05 24-11-05 269643 643 Mrefu I M 27-03-05 24-11-05 788039 039 Kimondo IV F 10-04-05 26-11-05 802040 2040 Kimondo IV F 10-04-05 26-11-05 788043 046 Kimondo IV F 10-04-05 26-11-05 794046 043 Kimondo IV M 10-04-05 26-11-05 805073 073 Kimondo IV M 10-04-05 26-11-05 789519 519 Zawadi II M 22-05-05 25-11-05 796521 521 Zawadi II M 22-05-05 25-11-05 795560 560 Zawadi II M 22-05-05 25-11-05 786567 567 Zawadi II M 22-05-05 25-11-05 792572 572 Zawadi II M 22-05-05 25-11-05 Number all transponders start with 528210000 followed by the number in the first column

19

DNA (see also appendix VII)

November 2002 blood was collected and DNA isolated from the following dogs:

Malaika litter, born 07-07-01, parents 306 (F) and 372 (M): 464, 448, 251, 368, 269, 202, 389

Wild caught: 006 and 040

Bred in Mkomazi: 372, parents 284 (F) and 298 (M) 325, parents 262 (F) and 274 (M), died March 2002, no off-spring

December 2004 blood was collected and DNA isolated from the following dogs:

Kimondo I litter, born 18-4-02, parents 040 (F) and 372 (M): 104, 348, 651, 730

Zawadi I litter, born 11-05-04, parents 006 (F) and 251 (M): 666, 671, 732, 853, 860, 887, 926, 939, 949

The DNA was isolated by Gendika BV, Veendam, the Netherlands. Gendika BV performed DNA fingerprinting on the DNA of the above-mentioned dogs (see attached report). From the results could be concluded that there is still a considerable genetic variation in the African wild dog population in Mkomazi. Answers were given to the following questions:

a. It was assumed that the new dogs #006 and #040 as they arrived together were sisters. The similarity between these two dogs proved to be 0.78, which is quite high and makes it most likely that they are sisters indeed. b. What is the genetic relationship between the new dogs, #006 and #040, and the original dogs (v.d.Zande1997)? Apparently the relationship is not high and these females could have bred very well with the original dogs especially dogs from the Lendanai group. Unfortunately all the original dogs, except three, died in the distemper outbreak. Of these three dogs only male #325 descended from the Lendanai but died one year after the outbreak bringing the Lendanai line to an end. c. What is the genetic relationship between the new dogs, #006 and #040, and the last living off-spring, #372, of the original dogs (being father of the Malaika litter and Kimondo I litter)? The similarity proved to be rather high, higher than the similarity between the three groups of original dogs in 1995. d. Is it acceptable to combine pups born out of the combination #372 x #306 with pups born out of the combination #372 x #040? It appeared to be that all combinations of different nests is possible as all combinations show lower similarity with other combinations than the similarity calculated from within nests. e. Would it be genetically acceptable for re-introduction purposes to mix dogs born out of the combination mentioned under d with pups born out of the combination #006

20 and #251? This question is already answered under d, although there is a preference to combine pups out of combination #006 x #251 with pups out of the combination #040 x #372. f. Are there two sub species of African wild dogs as researchers originally claimed (Girman et al 1993) but later on further investigation changed their minds (Girman et al 1997)? The similarity between the two groups (Mkomazi dogs and Amsterdam Z00- Artis- dogs) proved to be 0.26 and the conclusion might be justified that there is a different genetic basis in the Artis wild dogs (Southern Africa originated) compared to the Mkomazi wild dogs (Eastern Africa originated) but not sufficient to conclude that there are two subspecies.

Housing

The original four boma’s of the dogs were not altered. (For details see Report 1997- 2001). The Lendanai boma was completely stripped for considerable maintenance in 2002.

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 boma’s in 2001, the Lendanai boma in 2002 and the Ayubu boma in 2003.

Two new boma’s were built by the end of 2005 to provide accommodation for the yearlings that had to be separated from their natal pack due to following natural pack rules. The new boma’s were constructed in the same way as the other boma’s and have the same size, 70 x 40 meters.

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 every fortnight two cows were bought at the local market 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. The rest of the carcass was fed to the dogs. On the day they received the carcass, they did not receive the pellets.

21 Due to the fact that the dogs were rather lean December 2004 the temporary measure was taken to buy two cows every week and thus the quantity meat they received was increased. In November 2005 the pups born in 2005 showed once again a disappointing bodyweight as well as the dogs of the Zawadi I litter, born in 2004. As the number of dogs had grown in the course of 2005, the quantity of cows was increased to three cows a week.

Pellets used were initially 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 with no obvious bone abnormalities.

November 2002 the commercial dog food pellet brand had to be changed due to the impossibility of getting an export license in the UK for Tanzania. After an intensive search “Bobtail Classic” [Foodcorp (Pty) Ltd, SA] was selected, having the best quality price ratio, to replace “Valu Complete”.

The dogs were more eager to eat the new pellets than the old ones.

Bodyweight and Neck size (See appendix I)

It was our policy to take the bodyweight and the neck size of a dog every time the dog was sedated for whatever reason. The table below gives the average bodyweights and neck sizes of the dogs. The purpose of the neck sizes is to get an impression of size of collar, if radio-collaring in the future is needed and desirable. kg = kilogram cm = centimetre

Adult dogs*:

Dog Sex Number of times Average Average bodyweight taken bodyweight neck size in kg. in cm. Zawadi #006 F 4 20,2 36 Kimondo #040 F 2 19,6 35 Chupu Chupu #372 M 3 24,0 39 Bahati #325 M 2 24,6 39 * The dogs involved were weighed in the period 23-11-01 to 06-12-02 but not subsequently.

The average bodyweight of the adult dogs is in agreement with the range of bodyweight range of adult dogs in the period 1995-2001, i.e. males 22-25 kg, females 18-21 kg.

22 The average neck size of the adult dogs, 39 cm for the males and 36 cm for the females, is one cm less than the adult dogs in 1999 i.e. 40 cm for the males and 37 cm for the females.

Litters:

Malaika litter (born 07-07-01) Date Age in Male Female months Average Average Average Average bodyweight neck size bodyweight neck size in kg. in cm. in kg. in cm. 23-11-01 4 9,9 9,3 15-12-01 5 13,0 30 11,9 28 15-03-02 8 17,0 34 16,0 33 06-12-02 17 22,1 37 19,2 34 05-12-03 29 21,4 36 19,2 34 03-12-04 41 19,9 36 17,7 35

December 2004 the average bodyweight of the males was approximately 3 kg, and for the females 2 kg, too low. At all other dates the dogs of this litter had average bodyweights based on the bodyweights of the dogs in the period 1995-2001 at the same age which are taken as the normal value.

The Malaika litter is used as point of reference for all the litters below. As diagram 1 shows these pups grew up in a consistent way due the fact that they were on their own after the mother died and thus not living in a pack. In this way this litter is a good point of reference to compare other litters but one has to keep in mind that pups being raised in a pack might deviate in bodyweight depending on the composition of the pack and the social behaviour within the pack. However, this deviation should not be more than a couple of kilograms and should be made up at the time the dogs are 17-20 months old when they would normally have been expected to have reached average adult bodyweight.

Diagram 1: Growth rate Malaika litter

35 30 25 20 Bodyweight in kg 15 Male Malaika litter 10

5 Female Malaika litter 0 4 5 8 17 29 Lineair (Male Malaika litter) age in months Lineair (Female Malaika litter)

23 Kimondo I litter (born 18-04-02) Date Age in Male Female months Average Average Average Average bodyweight neck size bodyweight neck size in kg. in cm. in kg. in cm. 06-12-02 9 15,2 30 15,8 30 05-12-03 21 16,4 32 15,9 32 03-12-04 33 14,5 31 15,3 34

December 2003 and 2004 the average bodyweight for the males was 6-8 kg and for the females 4 kg too low.

Kimondo II litter (born 31-05-03) Date Age in Male Female months Average Average Average Average bodyweight neck size bodyweight neck size in kg. in cm. in kg. in cm. 05-12-03 6 9,4 25 8,4 25 25-11-05 30 18,5 36

Average bodyweight approximately 4 kg too low for males and females.

Kimondo IV litter (born 10-04-05) Date Age in Male Female months Average Average Average Average bodyweight neck size bodyweight neck size in kg. in cm in kg. in cm. 25-11-05 7 10,5 27 9,5 24

Zawadi I litter (born 11-05-04) Date Age in Male Female months Average Average Average Average bodyweight neck size bodyweight neck size in kg. in cm. in kg. in cm. 04-12-04 7 7,4 24 25-11-05 19 14,5 33

Compared with the Malaika litter, average bodyweight for the males at 5 months 13 kg, the average bodyweight of the pups of the Zawadi litter was far too low December 2004. One year later the dogs still had not made up arrears.

Zawadi II litter (born 22-05-05) Date Age in Male Female months Average Average Average Average bodyweight neck size bodyweight neck size in kg. in cm. in kg. in cm. 25-11-05 6 11,1* 28* * Only four out of five pups were measured as one pup was insufficiently sedated.

Mrefu I litter (born 27-03-05) Date Age in Male Female months Average Average Average Average bodyweight neck size bodyweight neck size in kg. in cm. in kg. in cm. 25-11-05 8 12,9 30 13,2 31

24 In general one might conclude that in December 2004 the bodyweights were too low. However, most dogs did not appear to be underweight. It was observed that some subordinates dogs did not look thinner than others of higher rank. This might have caused the people in charge to miss the physical signs/symptoms that all the dogs were underweight, most likely as a result of underfeeding. Measures were taken immediately as described in chapter Nutrition.

VETERINARY

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. Sadly enough the importance of the preventative medicine was heavily emphasized by the canine distemper outbreak December 2000. As it was impossible to combat the disease successfully with medications available nowadays, the only option left was the prevention of the disease by vaccination to protect the dogs in our breeding program.

Next to keeping the dogs in the best possible physical shape, the prevention of canine distemper, rabies and parvovirus infections is most important. These infections being the most threatening infectious diseases. In the past 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. As the results for rabies and parvovirus vaccinations were satisfying in the past, the same vaccination schedules for these diseases were continued. The canine distemper was a completely different story; a new vaccine and schedule was used.

Vaccination policy for the different diseases and results will be presented and 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.

25 Clinical Work--Diseases

Our policy was, and remains, to interfere as little as possible in the breeding packs. Diseased dogs were treated only when it was possible to separate them, treat them and replace them immediately, or when pack treatment was required and possible.

Pathology

Only a few dogs died. These deaths are reported and discussed in the chapter on pathology.

Preventative Medicine

Vaccinations.

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 firstly 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.

Canine Distemper Virus (CDV) – (See Appendix II and III)

Introduction

From 1995 to 2001 the CDV-Iscom vaccine, developed and donated by the Institute of Virology, Erasmus University, Rotterdam, was used. At the first real challenge however it appeared that this vaccine did not provided enough resistance. As announced in the 1995-2001 report, (chapter Steps Taken/Considered), the choice fell upon Purevax™ (Merial). According to the manufacturer, Purevax™ is a lyophilized vaccine of a recombinant canarypox vector expressing the HA and F glycoprotein’s of canine distemper virus. More popular: a “canarypox-vectored canine distemper vaccine”; i.e. a 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. 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. However, it is a live attenuated vaccine, not an

26 inactivated one, which is in contradiction with our vaccination policy not to use live attenuated vaccines. But the vaccine-virus multiplies in the cell it enters only. It is not excreted from that cell and is not passed on to the next cell. This way it is impossible for the vaccine-virus to spread itself in the environment.

Immunity is developed in two different ways. There is humoral immunity and cellular immunity. The humoral immunity is the response of B-lymphocytes to an invading infectious agent in the form of antibodies that are produced by plasma cells that originate from the B- lymphocytes. The cellular immunity is not the response in the form of antibodies but the effects or reactions of different cells itself. It is the response in the form of T-lymphocytes.

What has been done in the past in the Mkomazi dogs was the study of humoral immunity, i.e. the study of the antibodies. However, in some cases the study of cellular immunity is more important than the study of humoral immunity as some intracellular parasites like the intracellular life because of the protection it offers. In the cell it is impossible for humoral antibodies to reach them (ROITT & DELVES, 2001). One of these cases is the group of morbilli viruses and canine distemper virus is a morbilli virus.

Therefore, next to the study of humoral immunity, a study of cellular immunity (cell mediated immunity) had to be set up. We are very happy that this will be undertaken at the Institute of Virology, Erasmus University, Rotterdam, as well. To increase the number of pups participating in the cell mediated immunity research, i.e. to come to results more quickly, we were very happy that Artis, Amsterdam Zoo, the Netherlands, agreed to have their pups participating in the research program. This happened with two litters. One litter in 2001 eight pups and the other litter in 2002 twelve pups. Also a comparison was made between pups vaccinated with Purevax™ and pups vaccinated with CDV-Iscom. Separately will be reported over the results of the Artis pups.

However, due to the unavailability of the Purevax™ for us, we had to decide December 2004 either to continue with the CDV- Iscom vaccine or stop vaccinating against CDV until the Purevax™ would be available for us again. As, after vaccinating with CDV-Iscom vaccine, the canine distemper outbreak in December 2000 ran a devastating course it was decided to stop vaccinating against CDV temporarily.

Method

The following vaccination schedule was adopted for the use of Purevax™: The first year two vaccinations with one month in between followed by annual booster vaccinations.

For the purpose of antibody testing (humoral immunity) and cell mediated immunity study EDTA blood had to be collected at the time of the first vaccination, at the time of the second vaccination (one month after the first vaccination) and three months and one year after the second vaccination. For the cell-mediated immunity study EDTA blood should preferably not be older than 72 hours at the time of arrival at the University. Thanks to a good cooperation of all the people involved, in Mkomazi as well as in Rotterdam, this succeeded on every

27 occasion. This procedure was undertaken for the first litter of pups born after the distemper disaster (Malaika litter 07-07-2001), seven pups in all. The same happened to the new dogs #006 and #040 and the last two distemper epidemic survivors #372 and #325. Only #006 followed the whole procedure but quitted the program after her transfer to the Lendanai boma to start her own pack, i.e. she received her vaccinations but was not sedated anymore for blood samples. The same happened to #040 March 2002 as she was pregnant at that time and the same applies to her mate Alpha male #372. Unfortunately #325 died March 2002, so we could not follow up his antibody history anymore.

The pups of the second litter born after the distemper epidemic, Kimondo I litter, were vaccinated according a slightly different schedule. They were vaccinated three times with three weeks between the first and second and between the second and third vaccination of Purevax™. Blood samples were taken only at the time of the first vaccination and one year later.

The next litter, Kimondo II litter, was not vaccinated the first year to act as a control group.

Results humoral immunity.

1. Pre-vaccination titres to CDV: Only three dogs had a pre- vaccination titre, #104 a titre level of 80 and #092 and #853 a titre level of 20. All three were the only dog in their litter with a titre. 2. Post-vaccination titres: Malaika litter: One month after the first vaccination there were no titres, 3 months after the second vaccination four of the seven dogs had a titre and after one year all but one dog had a titre. Two years after the second vaccination, with an annual booster after one year, once again only one dog had no titre. Three years after the second vaccination with an annual booster after one and two years all seven dogs had a titre. Kimondo I litter: One year after three vaccinations all four dogs had a titre. After two years with an annual booster after one year, both dogs tested had a titre. 3. The two new, adult, arrivals #006 and #040 had no titre at the time of their first vaccination and again no titre one month after the first vaccination. Three months after the second vaccination and one year after the second vaccination #006 still had no titer. 4. In the control, non vaccinated, group ( Kimondo litter II) 7 months after birth one dog showed a titre of 20. The other dogs showed no antibodies.

Table 2: Virus Neutralizing antibody Titres in African Wild Dogs during a vaccination trial with Purevax™ vaccine Date Time-lapse between Number of % Number of % Total 2nd or 3rd vaccination dogs VNT- dogs VNT- number of And date of testing titre >20 titre <20 dogs 15-03-02* 3 months 4 50 4 50 8 06-12-02* 1 year 6 75 2 25 8 05-12-03* 2 years 6 85 1 15 7 05-12-03** 1 year 4 100 0 0 4 03-12-04* 3 years 7 100 0 0 7 03-12-04** 2 years 2 100 0 0 2

28 * Completed 2 initial vaccinations **Completed 3 initial vaccinations Results cellular immunity

Up until now (November 2004), there has been a technical problem at the University with the growth of white blood cells, lymphocytes, required for the study of cell mediated immunity. This problem has to be solved first before it is possible to get results. We hope that the cell mediated immunity study will provide an answer to the question why the canine distemper epidemic ran such a disastrous course.

Discussion/Conclusion

The number of dogs in the study is low. It appeared that dogs initially vaccinated three times, with three weeks between vaccinations, show a slightly better response than dogs that started initially with two vaccinations: 100% as against 75%. However, after two and three years respectively there was no difference, 100% both.

As appendix II shows titres in the years 2002-2004 are considerably higher than titres in the years 1995- 2001 after vaccination with CDV-Iscom (VISEE et al, 2001).The titre levels ranged from <20 to 80 in the years 1995-2001, compared with a range of titres from <20 to 640 in the years 2002-2004.

Although CDV-Iscom vaccine is known for its low titres, >=20, but to provide good immunity in seals (Van de Bildt, pers.com.), the vaccine obviously failed to provide sufficient immunity in our African wild dogs in 2000 (VISEE et al, 2001). The level of antibodies, after vaccination with Purevax™, suggests that vaccination with Purevax™ provides higher antibody titres and thus may provide a better protection against canine distemper virus.

At this moment to protect our African wild dogs, we prefer to use Purevax™ vaccine following a vaccination schedule which provides three initial vaccinations with three weeks in between followed by an annual booster vaccination. This schedule is advised for ferrets by the manufacturer Merial.

Rabies (See Appendix IV).

Introduction/Method

Initially, in 1995, rabies vaccination was started with a single vaccination. Due to a complete lack of sero-conversion the vaccination schedule was changed. From 1996 onward the dogs received three vaccinations in the first year: a second and a third vaccination was given one and five months after the first vaccination respectively, followed by an annual booster vaccination. This schedule proved to be successful as reported in the 1995-2001 report. It was only natural to carry on according this schedule. Vaccine used in the year 2001-2005 was Vanguard® *.

29 Results

1. Single vaccination: December 15th 2001 three weeks after a single vaccination only one dog of the Malaika litter (#269) developed what is considered to be sufficient antibodies: >0.5 I.U./ml. 2. Two vaccinations: March 15th 2002 four months after the first vaccination not a single dog of the Malaika litter showed sufficient antibodies. 3. Three vaccinations: One year after the first vaccination only one dog of the Malaika litter showed sufficient antibodies, once again #269. However all four dogs of the Kimondo I litter showed sufficient antibodies. 4. Three vaccination + a single annual booster vaccination: Four of the seven dogs of the Malaika litter showed sufficient antibodies. 5. Three vaccination + a single annual booster vaccination after one and two years: Six of the seven dogs of the Malaika litter showed sufficient antibodies. 6. The two new, adult, arrivals #006 and #040 showed no sufficient antibodies independent of the number vaccinations.

Table 3: Rabies antibody titres in African wild dogs after vaccination with inactivated commercial rabies vaccine: Vanguard® R (Pfizer) in the period 2001-2005. Months after Number of dogs % Number of dogs % Total number of third rabies >0.5 I.U./ml* <0.5 I.U./ml* dogs vaccination 5-6 11 90,9 1 9,1 4 9 1 14 7 86 8 18 2 100 0 0 2 21** 4 57 3 43 7 33*** 6 86 1 14 7 * a Rabies specific VNA titre of >0.5 International Units (I.U./ml) is considered to be the minimum titre likely to provide protection against challenge. ** received booster vaccination 9 months after third vaccination. *** received booster vaccination 9 and 21 months after third vaccination.

Table 4: Rabies antibody titres in African wild dogs after vaccination with inactivated commercial rabies vaccines: Dohyrab® (Solvay Duphar) in 1995&1997 and Rabdomun® (Schering-Plough) from1998 to 2001 (from VISEE et al, 2001) in the period 1997-2001. Months after Number of dogs % Number of dogs % Total number of third rabies >0.5 I.U./ml* <0.5 I.U./ml* dogs vaccination 1 12 92 1 8 13 5 13 100 0 0 13 12** 17 85 3 15 20 24 6 60 4 40 10 * a Rabies specific VNA titre of >0.5 International Units (I.U./ml) is considered to be the minimum titre likely to provide protection against challenge. ** received booster vaccination at the time of blood sampling.

Discussion/Conclusion

Although numbers of dogs are low, it is interesting to compare the results of the titres after three vaccinations with the results over the period 1995-2001 (Visee et al, 2001).

30 In that period 5 months after the third vaccination 100% of the dogs tested had a titre >0.5 I.U. (table 4). In the period 2001-2005 90,9% of the dogs tested had a titre >0,5 I.U. (table 3). However, in the latter case it concerned 11 dogs against 4 dogs in the period 1997-2001.

Also, 21 – 24 months after the third vaccination, with an annual booster vaccination in between, percentages are in the same order: 60% titre >0.5 I.U. in 1997-2001 and 57% in the period 2001-2005.

Not in accordance with these results were the titres 9-12 months after the last vaccination. In the period 1997-2001 85% of the dogs had a titre >0.5 I.U., in the period 2001-2005 14%, i.e. one dog had a titre >0.5 I.U... This concerned the Malaika litter. One year and one booster vaccination later the same dogs fitted into the expected range. We have no explanation for these, temporarily, abnormal titres.

After two annual booster vaccinations 86% of the same dogs in the Malaika litter had a titre >0.5 I.U. which indicates that following the vaccination regime used a satisfactory titre was eventually achieved.

All in all it may be concluded that three vaccinations with an inactivated vaccine, according to the above mentioned schedule, results in production of sufficient antibodies.

* Vanguard® Pfizer Animal Health B.V.

Parvovirus (CPV)

For vaccination the inactivated vaccine Dohyvac I-LP® (Solvay Duphar) was used for protection against parvovirus and leptospirosis. The vaccination protocol of the manufacturer for domesticated dogs older than 12 weeks was adopted: two vaccinations with a four-week interval followed by an annual, single booster vaccination. No antibody testing was performed due to positive results in the past. See Report 1995-2001.

In 2005 inactivated parvo virus vaccine was not available any more. It was decided to follow our policy of using inactivated vaccines only. Thus the vaccination against parvo virus was temporarily suspended except for the dogs that are not going to be used for re-introduction. They were vaccinated with Fel-O-Vax®* i-CHP (Fort Dodge), an inactivated feline enteritis vaccine. * Fel-O-Vax®* i-CHP, Fort Dodge.

31 Parasites

Endoparasites

To keep healthy dogs in a breeding program in captivity an anthelmintic regime is obligatory. Preventing worm-infection is preferable to combating the problem. Therefore twice a day the faeces were removed from the boma’s in a very diligent way, to prevent the soil being infested with worm-eggs as much as possible.

To check the anthelmintic status of the dogs faeces samples were taken; once or twice a year two to five samples per boma depending on the number of dogs in the boma. These samples were preserved with a 5% formalin solution before sending the samples to a laboratory.

To complete the regime the dogs received an anthelmintic, Drontal® Dog*, on a regular basis, in a dosage as prescribed by the manufacturer for domesticated dogs, i.e. one tablet per 10 kg bodyweight. Drontal® Dog was chosen as the drug of choice as it is a broad-spectrum anthelmintic (effective against round-, tape-, and hookworms) that needs to be administered only once and no empty stomach required, which makes it possible to mix the tablets with the food. As no side effects are known it may be administered to pregnant females and pups as well.

Every two months the adult dogs received a dosage Drontal® Dog, i.e. 15 mg febantel, 14,4 mg pyrantel and 5 mg praziquantel per kg bodyweight. Pregnant females received treatment just after mating, again 10 days later to prevent the unborn pups from being infected in the uterus and also 10 days after giving birth. The pups received the first treatment the moment they came out of the den and started to eat solid food, regurgitated by the pack members. At these occasions the tablets were put in the food of the adult dogs, which made it possible to administer an anthelmintic at an earlier age. In this way the adult dogs received some extra treatments as well, which causes less secretion of worm eggs by the adult dogs when the pups were at the smallest and most vulnerable.

As a result all the faeces samples proved to be negative in the years 2001-2004, which does not mean that we exterminated all the worms but kept them at a low, apparently acceptable, level. November 2005 a few Ancylostoma sp. eggs were found in the faeces of the dogs in the Kisima boma.

From December 2005 onward a change was made in the anthelmintic used. Instead of the Drontal® Dog*, Program Plus®** is being used. It might be beneficial to change anthelmintic every now and then to prevent the parasites getting used to the drugs. Another advantage was that by using Program Plus®** the quantity of tablets that have to be administered to the dogs can be reduced considerably as Program Plus®** is a combination of an anthelimintic (milbemycine)

32 and an Insect Development Inhibitor (lufenuron). Thus, a tablet has to be administered only once a month rather than twice. Dosage used as prescribed by the manufacturer for domesticated dogs, i.e. 0.5 mg milbemycine and 10 mg per kg bodyweight.

Since December 2003 at every occasion of sedation Stronghold®*** drops were applied onto the skin of neck of the dog in a dosage according to their bodyweight as prescribed by the manufacturer for domesticated dogs. Stronghold® is effective against round- and tapeworms and fleas.

*Drontal® Dog (praziquantel, pyrantelembonaat, febantel), Bayer B.V. **Program Plus®** (milbemycine, lufenuron), Novartis Consumer Health B.V./Animal Health *** Stronghold® (selamectin), Pfizer Animal Health B.V.

Ectoparasites

Just as in the case of the endoparasites, keeping dogs in relatively close confinement, is bound to be a good nurture ground for fleas and ticks. As a consequence preventive treatment has to be enforced to keep the levels of the ectoparasites as low as possible.

On each occasion when a dog was sedated, the skin was carefully checked for parasites. There were no signs of fungus infection or scabies. Fleas were detected occasionally. Ticks were noticed to a somewhat bigger extent. These satisfying results are due to the following anti flea and tick protocol.

To prevent fleas from multiplying the dogs receive every month Program®* in their food. As Program® is not an insecticide but reduces the number of larvae by inhibiting the development of the flea eggs, an insecticide has to be used to combat fleas. Therefore at every sedation Advantage®*** was applied onto the neck of the dog to top up the monthly Program® administration. December 2003 Advantage®*** was replaced by Stronghold® and December 2005 Program® was replaced by Program Plus®** as explained in chapter on endoparasites.

Unfortunately Program® does not affect ticks. To reduce the breeding environment for the ticks grass in and around the boma’s is kept low with satisfactorily results up until now.

*Program® (lufenuron), Novartis Consumer Health B.V./Animal Health **Program Plus®** (milbemycine, lufenuron), Novartis Consumer Health B.V./Animal Health ***Advantage® (imidacloprid), Bayer B.V.

33 Clinical Work-Diseases

Clinical work

Just one exception a diseased dog had to be treated the past years. Halfway through March 2002 one of the pups of the Malaika litter, #202, was behaving in a rather sluggish way: after resting he was the last to get up and was the first to lie down; he behaved very lethargically and showed little interest in eating. As this coincided with the time when blood samples were taken an examination of his blood was carried out, but this showed no abnormalities. The dog was put on an oral course of Synulox®* and recovered quickly.

Small physical abnormalities were noticed in two dogs while sedated. The left testicle of #269, male, was not completely descended. The testicle appeared to be just in front of the scrotum.

The right canine tooth, lower jaw, of the female #348, appeared to protrude laterally rather than upward.

* Synulox® (amoxicillin, clavulanic acid), Pfizer Animal Health B.V.

Anaesthesia

Over the years, the combination of medetomidine (Domitor®*) and ketamine HCL (100 mg/ml), with atipamezole (Antisedan®**) as an antidote for Domitor®*, proved to be a safe way of sedating the dogs. From 2001 to 2005 110 sedations (see table below) have been performed.

Table Number sedations 2001-2005 Date Number sedations 13-03-01 2 23-11-01 11 15-12-01 11 15-03-02 8 06-12-02 13 05-12-03 17 03-12-04 20 25-11-05 28 7 occasions Total 110

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.

Next quote from the Report 1995-2001 still

34 applies except for a small change. “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. Pups and adolescent dogs needed 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®*.”

From December 2002 onward the dosage of ketamine HCL was raised to 0.2 ml per dog independent of their bodyweight. As a result no more dogs needed a ‘top up’ with Domitor® except for dogs described in the next paragraph.

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 raising of the dosage of ketamine HCL did not increase the side effects. The very slight side effects observed were: staring into the distance and ataxia, which disappeared usually within 30 minutes and on four occasions a small seizure lasting less than a minute.

* Domitor® (medetomidine hydrochloride 1 mg/ml), Pfizer Animal Health B.V. ** Antisedan® (atipamezole hydrochloride 5 mg/ml), Pfizer Animal Health B.V.

Pathology

In the period 2001-2004 just three dogs died; two adults, #306 and # 325, and one pup. Information on #306 and the pup is extensively reported in Report 1995-2001 (VISEE et al, 2001). The dogs #306 and #325 were two of the three survivors of the canine distemper epidemic, which leaves #372 as the only living survivor today. #325 suddenly collapsed whilst eating on the night of 20 March 2002 and died within five minutes. The manager of the dogs, Sangito, was alone and too distressed to perform an autopsy. However, based on the reported symptoms, gasping with wide-open mouth prior to collapsing, it is not unreasonable to assume that #325 died of a heart failure, perhaps in part induced by his earlier traumatic experiences. Sadly with him the last blood of the original Lendanai dogs disappeared.

35 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). George and Natasha Duffield for funding part of the program. Duncan Forbes, Ray Rowe Trust for Animals, for funding part of the program. Prof. A.D.M.E. Osterhaus, Marco van de Bildt and Joost Philippa, Institute of Virology, Erasmus University, Rotterdam, for performing the antibodies testing and most of all their invaluable advice. Roger Burrows, for giving us invaluable insights into the behaviour of The African Wild Dog and his comments on this report. 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®, Vanguard®, Synulox® and Stronghold®. 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. Aart Visee, for supplying the photos used in this report. Sylvia Kappe†, for the final layout.

36 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. This Report reports over the years 2001-2004.

In the first section, the development of the breeding packs is described, including the zoo technical aspects. The second section deals with the veterinary 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.

Two different litters of dogs were vaccinated against distemper (Purevax®) according to two, different vaccination schedules. One litter received two vaccinations (one month in between) followed by an annual booster vaccination. The other litter received three vaccinations (three weeks between vaccinations) followed by an annual booster vaccination. In both cases satisfactory levels of antibodies were produced

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 satisfactory levels of antibodies.

37 References

BURROWS, R. (1992): Wild Dogs of the Serengeti Part 2. Miombo, 8, 4-5. 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 :Chicago University Press. BURROWS, R., HOFER, H. & M.L. EAST (1995): Population dynamics, intervention and survival in African wild dogs (Lycaon pictus). Proc. R. Soc. Lond. B 262, 235-245. BURROWS, R. (2004): Pack cohesion in the African wild dog (Lycaon pictus) and a "young- male first" protocol in the acquisition of dominance. In : Supplements to Ethology; Advances in Ethology 38 :124. CARBONE, C., DU TOIT, J.T. and I.J. Gordon (1997): Feeding success in African wild dogs: Does kleptoparasitism by spotted hyaenas influence hunting-group size? J.Anim.Ecol. 66 : 318-326. FANSHAWE, J.H. and C.D. Fitzgibbon (1993): Factors influencing the hunting success of an African wild dog pack. Animal Behaviour 45, 479-490. 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. FRAME, G.W. and L.H. FRAME (1981): Swift and Enduring: Cheetahs and Wild Dogs of the Serengeti. New York: E.P. Dutton. GIRMAN 1993. Molecular Genetic and Morphological Analyses of the African wild dog (Lycaon pictus). Journal of Heredity. 84, 450-459. GIRMAN, D.J., MILLS, M.G.L., GEFFEN, E. & R.K. WAYNE (1997): A molecular genetic analysis of social structure, dispersal, and interpack relationships of the African wild dog (Lycaon pictus). Behav. Ecol. Sociobiol. 40, 187-198. MACNUTT, J.W. (1996) : Adoption in African wild dogs, Lycaon pictus. J. Zool. Lond. 240, 163-173. MALCOLM, J.R. (1979): Social organisation and communal rearing in African wild dogs. PhD Thesis, Harvard University. MALCOLM, J.R.and K. Marten (1982): Natural selection and the communal rearing of pups in African wild dogs (Lycon pictus). Behavioural Ecology & Sociobiology 10: 1-13. ROITT, I.M. and P.J. DELVES (2001): Roitt’s Essential Immunology. Blackwell Science Ltd.. SCHALLER, G. B. (1972): The Serengeti Lion. The Chicago University Press. VISEE, A.M., FITZJOHN, A.R., FITZJOHN, L. and S. LEMA (2001): African Wild Dog (Lycaon pictus) Breeding program, Mkomazi Game Reserve, Tanzania, Report 1995-2001. Unpublished. George Adamson Wildlife Preservation Trusts, The African Wild Dog Foundation.

38 litter I Mrefu litter IV Kimondo litter II Zawadi litter I Zawadi litter II Kimondo litter I Kimondo litter Malaika Chupu Chupu Bahati Kimondo Zawadi Identification I Appendix 2040 643 586 557 481 444 073 046 043 572 567 560 521 519 949 939 926 887 860 853 732 671 666 393 212 187 092 031 001 730 651 348 104 389 368 269 251 202 464 448 372 325 040 006 Sex M M M M F M F M F M M M M M M M M M M M M M M M M M M F F M M F F M M M M M F F M M F F of Birth of Date 27-03-05 27-03-05 27-03-05 27-03-05 27-03-05 10-04-05 10-04-05 10-04-05 10-04-05 22-05-05 22-05-05 22-05-05 22-05-05 22-05-05 11-05-04 11-05-04 11-05-04 11-05-04 11-05-04 11-05-04 11-05-04 11-05-04 11-05-04 31-05-03 31-05-03 31-05-03 31-05-03 31-05-03 31-05-03 18-04-02 18-04-02 18-04-02 18-04-02 07-07-01 07-07-01 07-07-01 07-07-01 07-07-01 07-07-01 07-07-01 08-01-99 04-09-97 23-11-01 (ns) centimeters in Necksize (kg) kilograms in Bodyweight 10,4 10,8 10,0 23,0 24,3 20,0 20,6 9,1 9,0 9,3 9,4 kg 38 39 35 38 ns 15-12-01 13,7 13,0 13,3 11,9 11,7 12,1 24,3 24,9 19,3 20,1 kg 31 32 31 29 26 28 28 39 39 35 37 ns 15-03-02 17,6 17,9 18,0 16,5 14,7 16,2 15,8 20,2 kg 34 35 34 34 31 34 32 35 ns 06-12-02 16,2 14,1 14,5 17,1 21,8 24,4 21,6 21,6 21,0 19,3 19,1 24,8 19,8 kg 32 29 30 31 36 39 36 37 37 35 34 40 35 ns 05-12-03 10,8 17,3 15,5 14,7 17,0 20,6 21,8 20,2 22,3 22,0 19,7 18,6 8,0 9,1 9,7 8,4 8,5 kg 24 26 24 27 25 25 34 31 31 32 35 37 35 37 36 35 34 ns 03-12-04 14,8 14,1 14,4 16,2 19,9 18,4 7,0 7,0 8,6 8,0 7,4 7,9 6,5 7,3 6,8 kg 23 25 25 25 25 24 22 25 24 32 30 32 33 36 35 ns 25-11-05 11,5 13,9 13,6 12,8 13,2 11,4 10,4 11,1 10,5 10,4 12,3 14,5 13,7 15,9 14,7 15,8 14,8 12,9 14,2 14,2 17,0 18,6 19,4 18,9 9,5 8,8 kg 29 31 31 31 32 26 26 29 28 28 28 28 29 33 32 33 32 34 32 32 34 32 35 36 36 38 ns

39 Appendix II Distemper antibody levels (VNT) + Vaccination dates Mkomazi Date 23-11-01 15-12-01 15-03-02 06-12-02 29-12-02 21-01-03 05-12-03 03-12-04 25-11-05 Identification Sex VNT titer VNT titer VNT titer VNT titer VNT titer VNT titer VNT titer VNT titer VNT titer Zawadi 006 F V <20 V <10 <10 V T10 V Kimondo 040 F V <20 V <10 V V Bahati 325 M V 80 V 20 died 20-03-02 Chupu Chupu 372 M V 160 V 80 V 160 V Malaika 448 F V <20 V <10 20 V 20 V 320 1280 litter 464 F V <20 V <10 40 V T10 V 160 640 202 M V <20 V <10 T10 V 20 V T10 160 251 M V <20 V <10 80 V 40 V 40 640 269 M V <20 V <10 T20 V 40 V 40 320 368 M V <20 V <10 160 V 80 V 160 1280 389 M V <20 V <10 T20 V 40 V 640 1280 Kimondo I 104 F V 80 V V V 160 1280 litter 348 F V T10 V V V 80 160 651 M V T20 V V V 20 730 M V T10 V V V 160 Kimondo II 001 F T10 litter 031 F <10 092 M 20 <10 187 M <10 <10 212 M T10 <10 393 M T10 Zawadi I 666 M <10 litter 671 M 10 732 M <`10 853 M 20 860 M <10 887 M T 926 M <10 939 M <10 949 M <10 Zawadi II 519 M <10 litter 521 M 560 M <10 567 M <10 572 M <10 Kimondo IV 039 F litter 2040 F <10 043 M 046 F <10 073 M <10 Mrefu I 444 F <10 litter 481 M <10 557 M <10 586 M <10 643 M <10 V=Vaccinated T=Toxid

40 Appendix III Distemper antibody levels (Elisa) + Vaccination dates Mkomazi Date 23-11-01 15-12-01 15-03-02 06-12-02 29-12-02 21-01-03 05-12-03 03-12-04 25-11-05 Identification Sex Elisa titer Elisa titer Elisa titer Elisa titer Elisa titer Elisa titer Elisa titer Elisa titer Elisa titer Zawadi 006 F V <10 V <10 <10 V 20 V Kimondo 040 F V <10 V <10 V V Bahati 325 M V V >1280 died 20-03-02 Chupu Chupu 372 M V V 640 V 160 V Malaika 448 F V <10 V <10 80 V 160 V 640 # litter 464 F V <10 V <10 80 V 80 V 80 # 202 M V <10 V <10 40 V 80 V <10 # 251 M V <10 V <10 40 V 80 V 20 # 269 M V <10 V <10 <10 V 160 V 40 # 368 M V <10 V <10 160 V 160 V 40 # 389 M V <10 V <10 <10 V 80 V 640 # Kimondo I 104 F V 80 V V V 40 # litter 348 F V <10 V V V 20 # 651 M V 20 V V V 320 730 M V <10 V V V 40 Kimondo II 001 F V <10 litter 031 F V <10 092 M V 20 <10 187 M V <10 <10 212 M V <10 <10 393 M V <10 Zawadi I 666 M <10 litter 671 M 20 732 M <10 853 M 20 860 M <10 887 M 40 926 M <10 939 M <10 949 M <10 Zawadi II 519 M <10 litter 521 M 560 M <10 567 M <10 572 M <10 Kimondo IV 039 F litter 2040 F <10 043 M 046 F <10 073 M <10 Mrefu I 444 F <10 litter 481 M <10 557 M <10 586 M <10 643 M <10 #= results not received V= Vaccination Purevax

41 Appendix IV Rabies antibody levels + Vaccination dates Mkomazi Date 23-11-01 15-12-01 15-03-02 06-12-02 29-12-02 21-01-03 12-06-03 05-12-03 03-12-04 07-01-05 28-06-05 25-11-05 Identification Sex I.U. I.U. I.U. I.U. I.U. I.U. I.U. Zawadi 006 F V <0.5 V <0.5 V <0.5 V <0.5 V V Kimondo 040 F V <0.5 V <0.5 V V V V V Bahati 325 M V >0.5 V >0.5 died 20-03-02 Chupu Chupu 372 M V <0.5 V <0.5 V >0.5 V V V Malaika 448 F V <0.5 V <0.5 V <0.5 V <0.5 V <0.5 V >0.5 V litter 464 F V <0.5 V <0.5 V <0.5 V <0.5 V >0.5 V >0.5 V 202 M V <0.5 V <0.5 V <0.5 V <0.5 V 0.5 V <0.5 V 251 M V <0.5 V <0.5 V <0.5 V <0.5 V <0.5 V >0.5 V 269 M V <0.5 V >0.5 V <0.5 V >0.5 V >0.5 V >0.5 V 368 M V <0.5 V <0.5 V <0.5 V <0.5 V >0.5 V >0.5 V 389 M V <0.5 V <0.5 V <0.5 V <0.5 V >0.5 V >0.5 V Kimondo I 104 F V <0.5 V V V >0.5 V >0.5 V litter 348 F V <0.5 V V V >0.5 V >0.5 V 651 M V <0.5 V V V >0.5 V V 730 M V <0.5 V V V >0.5 V V Kimondo II 001 F <0.5 V V V litter 031 F <0.5 V V V 092 M <0.5 V V V >0,5 187 M <0.5 V V V >0,5 212 M <0.5 V V V >0,5 393 M <0.5 V V V Zawadi I 666 M V V V V >0,5 litter 671 M V V V V >0,5 732 M V V V V 853 M V V V V >0,5 860 M V V V V >0,5 887 M V V V V >0,5 926 M V V V V <0,5 939 M V V V V >0,5 949 M V V V V Zawadi II 519 M V <0,5 litter 521 M V 560 M V <0,5 567 M V <0,5 572 M V <0,5 Kimondo IV 039 F V litter 2040 F V <0,5 043 M V 046 F V <0,5 073 M V <0,5 Mrefu litter I 444 F V <0,5 481 M V <0,5 557 M V <0,5 586 M V <0,5 643 M V <0,5 V = Vaccination

42

05 α β

-

08 -

α 06

F 104 F 348 2368 M Born 730 M 730 M 651

05 05 α - AYUBU

α SANGITO 03 - 27 464 F 464 F 448 389 M 389 368 M 368 M 269 444 F 444 F 000 F 481 F 6372 F M 557 232 M M 586 296 M M 643 0348 M Born

Game Reserve December 2005

α 05 05

Mkomazi -

05 - dogs

α 22 wild 202 M 202 M251

Born African M372

031 F 046 F composition 2040 F 2040 073 M F 04 04

- α

05 Pack Pack 03001 F 05039 M F 043 - LENDANAI - -

KISIMA 11 05 04 α - -

31 10 006 F 006 666 M M 887 M 666 519 M M 926 M 671 521 M M 939 M 732 560 M M 949 M 853 567 M M 860 572 M Born Appendix V Appendix Born Born F 040

43

05 - 04 - IV 10 IV 039 F 039 F 046 043 M 043 M 073 2040 F 2040 survivors program a introduction - outbreak Kimondo re introduced - CD 2005 - For Re Breeding 03 - = = = 306 and 372 372 and 306 05 - Kimondo 2001 040 F F 040 031 F 031 092 M 092 M 187 M 212 M 393 0 01 F 01 0 II 31 II results 05 - Kimondo 08 x - Breeding I 06 I 02 F 000 232 M 232 M 296 6372 F 6372 - 0348 M 0348 M 2368 a ß 04 a - Regina 18 Chupu 651 M 651 M 730 X M M litter 104 F F 104 F 348 I 372 Chupu 05 Kimondo - Mkomazi Game Reserve. Reserve. Game Mkomazi X 03 - x dogs I 27 I 444 F 444 481 M 481 M 557 M 586 M 643 01 wild wild - Mrefu 07 - a a 07 M F F African litter 464 F 464 F 448 202 M 202 M 251 M 389 269 Malaika 368 M M 368 306 Malaika program program x Breeding 05 a - 04 - 05 - 05 - II 22 II Zawadi I 11 I 006 006 521 M 521 M 560 M 567 M 572 519 M 519 666 M 666 M 671 M 732 M 853 M 860 M 887 M 926 M 939 M 949 Appendix VI : VI Appendix Zawadi Zawadi

44

05 - 04 -

10 IV 039 F 039 F 046 043 M 043 M 073 F 2040 program

α 2005 + 2005 Similarity + introduction - - Kimondo re introduced -

2001 For Re Breeding 03 - = = =

= Similarity 05 - Kimondo

F 040 results 001 F 001 031 F 031 092 M 092 M 187 M 212 M 393 31 II

05 - Breeding Kimondo 08 0,50 x -

I 06 I

296 373 0348 2368 6372 4448 02 - 04 α α β - Regina

18 Chupu

651 M 730 M 104 F 348 F litter

I

M 372 X Mkomazi Game Reserve. Mkomazi Chupu 05 Kimondo - dogs X 0,72 03 - x 0,56 0,57 wild wild I 27 444 F 444 M 481 M 557 M 586 M 643

01 - Mrefu 07 - African

α α α 07

litter 464 F 448 F 202 M 269 M 389 M 251 M 251 M 368 M 368 M Malaika program 306 F 306

0,53 Malaika x

Breeding 0,69 0,56

0,62

0,73

05 α - 04 - 05 - 05 - II 22 II Zawadi I 11 I

006 666 M 666 M 671 M 732 M 853 M 860 M 887 M 926 M 939 M 949 M 519 M 521 M 560 M 567 M 572

Zawadi

Zawadi Appendix VII Appendix

45 Genetic Analysis of African Wild Dogs (Lycaon pictus)

from the Mkomazi Project (1995-2004)

For: The the African Wild Dog Foundation

Att: Drs. A. Visee

By: Dr. Ir. A. Kappe

Gendika

Industrieweg 1

9641 HM Veendam

Date: 1 november 2005 (toevoeging januari 2007)

46 Introduction

The African wild dog (Lycaon pictus) is an extremely endangered species. In order to prevent the species for extinction a rehabilitation program was established by the George Adamson Wildlife Preservation Trusts and the African Wild Dog Foundation involving capture, captive breeding and re-introduction. In 1995 a captive breeding program was started by 25 wild dog pups from three locations in the Masai Steppe, Tanzania, forming three family-groups; Najo- group (n=13), Llonddirrigiss group (n=8) and Lendanai group (n=4). In this report a genetic analysis of the dogs involved in the Mkomazi Project will be presented.

Materials and methods

Genetic samples

Samples were collected from 58 African wild dogs. Blood-samples from the original wild dogs were collected in 1995 and DNA was isolated by Dr. L. van de Zande, University of Groningen, the Netherlands (n=23). Blood-samples from pups born within the Mkomazi Project were collected per year and stored for analysis (n=18). In addition, 2 samples from wild caught animals were collected. See also Appendix 1 for a complete list of samples.

DNA isolation

DNA was isolated from 5 ml whole blood collected in EDTA anticoagulant vacutainers. DNA was extracted by proteinase K digestion (100 μg/ml) and DNA was isolated and purified by using Standard Gendika protocol GDKiso06.

Identification of microsatellite alleles

Wild dog samples were screened by the microsatellite technique. In the microsatellite technique highly variable microsatellites are used as genetic markers. Microsatellites, also known as simple sequence repeats or short tandem repeats, are genomic sequences that consist of di-nucleotide motif repeated in multiple tandem copies, in mammals CA-repeats are most common. The variation within the microsatellite loci has been shown to arise from variation in the number of repeat units (the number of CA-repeats). The variable appearances of a microsatellite are called alleles. Alleles present for a microsatellite are generated by PCR amplification. The lenght of the various alleles can be detected by cappilary gel electrophoresis in a Genetic Analyzer. By examination of a set of microsatellites a specific pattern of alleles will be produced for each individual. This specific pattern of alleles is also known as a DNA fingerprint. Wild dog samples were screened for variation in 10 CA(n) microsatellite loci, originally isolated from a domestic dog library, combined in a Canine 10 Plex Kit (Applied Biosystems). Nine microsatellites were identified that consistently gave PCR product, were polymorphic in wild dogs (see Appendix 2).

47 Detection of microsatellite alleles from DNA was achieved by performing 35 cycles of PCR amplification in a 10 μl reaction volume using 10 ng of target DNA and 0.36 units of Taq DNA polymerase (Applied Biosystems). Alleles were detected by a Genetic Analyzer.

Data analysis

The similarity, which is the average of shared alleles between two animals, was calculated as

Sxy= 2nxy / (nx + ny), where nx and ny are the number of microsatellite alleles present in individual x and indiviual y, respectively, and nxy is the number of alleles shared by individuals x and y (Wetton et al., 1987). The similarity between animals is presented as a percentage.

With this percentage the amount of Mean similarity within a group was estimated by averaging the similarities of all pairs of individuals that belong to the 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.

Results

For all individual African wild dogs a pattern of microsatellite alleles could be scored and individual specific genotypes were identified, which means that for all animals a specific DNA fingerprint could be produced.

The patterns of alleles for the nine microsatellites of all individual animals were compared and the percentage of shared alleles, the similarity Sxy, was calculated between animals, see Appendix 3. The calculated similarity Sxy is a measure for the relationship between animals. Parents share at least 50% of the alleles with their offspring, which means that the similarity between parents and offspring will be on average 0.5 or higher. Between siblings the similarity will be higher, than the similarity between unrelated animals, normally a similarity of 0.4 - 0.6 is found between siblings and a similarity of 0.0 – 0.3 is found in unrelated animals. In small populations all similarities will be higher, more alleles are shared between the animals because of the appearance of inbreeding within small populations.

Similarity in original dogs

The relationship within the three groups of original African wild dogs, Najo, Llondirrigiss and Lendanai, was in this project re-calculated and analysis of patterns yielded mean similarities of 0.55 - 0.58, which were comparable to the values found by van de Zande, 1997 (see Table 1).

48 Table 1: Similarity of alleles, Sxy (%) within and between original groups of African wild dogs

Similarity

Najo (n=13) Within group 0.58 Between groups N-Llondirrigiss 0.45 N-Lendanai 0.39

Llondirrigiss (n=6) Within group 0.58 Between groups Ll-Najo 0.45 Ll-Lendanai 0.37

Lendanai (n=4) Within group 0.55 Between groups Le-Najo 0.39 Le-Llondirrigiss 0.37

The values of mean similarity between the three groups were considerably lower than the values within the groups, which was also consistent with the data found by van de Zande.

Similarity in new dogs

In this analysis two newly introduced dogs, wild caught, were taken in to the research. Between the two new animals, 006 and 040, a high mean similarity of 0.78 was calculated (see Table 2). Also a mean similarity between the new animals and the three original groups was calculated. These values were comparable to the values of similarity calculated between the three original groups. The value between animal 006 and the Londirrigiss group is 0.62, which is considered to be quite high for unrelated animals.

Table 2: Similarity of alleles, Sxy (%) between new dogs and original groups of African wild dogs

Similarity

New dogs

006 040

006 - 0.78

040 0.78 -

Najo 0.53 0.48

Llondirrigiss 0.62 0.51

Lendanai 0.29 0.33

49

Similarity in various litters

In order to investigate the possibilities for the breeding program to combine pups from various litters the mean similarity of alleles between pups born within the Mkomazi Project was calculated, see table 3. In table 3a the similarity of alleles within the various litters is explained. In Table 3b the similarity between the various litters of variable years is indicated. And in this table also a comparison of animals in the litter of 2004 with newly formed packs of animals, called Sangito and Ayubu, is included. In both tables the litters are indicated by the combination of parents and the year the litter was born. See Appendix 1 for a complete list of animals.

Table 3a: Similarity of alleles, Sxy (%) within litters of African wild dog pups

Parents (female x male)

297 x 300 262 x 274 306 x 372 040 x 372 006 x 251

1997 1999 2001 2002 2004

S xy 0.63 0.72 0.69 0.72 0.73 Within litter (n=3) (n=2) (n=7) (n=4) (n=9)

Similarity between siblings is normally estimated between 0.4 – 0.6, in table 3a is quite evident that the similarity within the litter in the Mkomazi Project this similarity is slightly higher. Similarities calculated for the various litters is comparable to each other.

Table 3b: Similarity of alleles, Sxy (%) between litters of African wild dog pups

297 x 300 262 x 274 306 x 372 040 x 372 006 x 251

1997 1999 2001 2002 2004 S xy Between litters

1997 - 0.48 0.43 0.45 0.47 1999 - 0.38 0.50 0.33 2001 - 0.57 0.62 2002 - 0.53 2004 -

S xy Between litter and packs

Sangito 0.60 Ayubu 0.57

50 The similarity between the litters is considerably lower than the calculated similarity within the litters. All similarities between the litters are in the same order as the similariy calculated within the litters of the original dogs. The comparison of the litter of 2001 (306 (F) x 372 (M)) and the litters of 2002 (040 (F) x 372 (M)) and of 2004 (006 (F) x 251 (M)) show a slightly higher similarity.

The newly formed packs are animals which should form a new breeding group. Two packs of animals were formed; Sangito (animal 448, 464, 651and 730) and Ayubu (animal 269, 368, 389, 104, 348). Similarity between the newly formed packs and the litter from 2004 (006 (F) x 251 (M)) was calculated as 0.60 for the Sangito pack and 0.57 for the Ayubu pack, respectively, which is slightly higher than the similarity between the other litters, but in the same order as the comparison of the 2004 litter and the litter of 2001, 0.62. This comparison gives also a considerable lower similarity than within litter comparison of the 2004 litter, which was 0.73.

51 Conclusion

General

From the results of the genetic analysis by DNA microsatellites can be concluded that there is considerable genetic variation in the African wild dog population in the Mkomazi project. As van der Zande (1997) also concluded the genetic variation in the original African wild dog population, separated in three groups, was considerable, with a mean similar ranging from 0.55 - 0.58 for related animals and a mean similarity for unrelated animals ranging from 0.37 - 0.45. In literature a similarity of 0.0 – 0.3 is described in unrelated animals, a similarity of 0.5 or higher is found for parents and offspring and a similarity of 0.4 - 0.6 is found between siblings (Avise. 1994). Girman et al. 2001, reported quite high genetic variation in other wild dog populations in Africa as well.

The “new” dogs

The similarity between two new dogs 006 and 040, result of wild caught, is quite high 0.78. As mentioned above the similarity between siblings is in the order of 0.4 – 0.6. From this result the conclusion can be that the two animals are most likely close related, and probably these animals are sisters.

The observed similarity between these two new dogs and the original dogs in the Mkomazi project is ranging 0.29-0.33 for the Lendanai group, 0.48-0.53 for the Najo group and up to 0.51-0.62 with the Llondirrigiss group, which is a bit lower compared to the similarities within these groups. This implies that the “new” animals, 006 and 040, can be well bred into the original population of wild dogs from the Masai Steppe. Considering the results it is therefore recommendable to combine the two females with animals originated from or related to the Lendanai group, which shows very low similarity with both new dogs. But unfortunately these dogs did not survive the Canine Distemper-virus outbreak.

The female dog 040 was bred with a male dog 372, offspring of the original dog 284 (F) from the Najo group and original dog 298 (M) from the Llondirrigiss group. The observed similarity between these two dogs is 0.50, which is high for unrelated animals. Especially when the similarity is compared to the similarities between the three original groups. A higher degree of similarity can be expected in pups of this combination. This result is showed in the comparisons of the various litters, see table 3b and also the following part.

52 Future breeding

A measure of relatedness in pups born from variable combinations was made by calculating the similarity within the litters of pups born in the Mkomazi Project. With the results from these calculations a status can be revealed of the genetic variation in the pups. All full-sibs combinations were calculated and resulted in a similarity of approximately 0.70, which is higher as found in the original groups of dogs, the similarity at that point was 0.55 – 0.58. Between the litters we see a considerable amount of variation, which is shown by considerable lower similarities. Therefore concluded can be that there are good possibilities to combine pups from different litter (combinations) for future breeding.

Preferable combinations for breeding are; a: pups born out of combination 262 x 274 (litter 1999) and combination 297x300 (litter 1997) can be bred with pups from all other litters, depending on the male/female ratio b: pups born out of combination 006 x 251 (litter 2004) can be bred with pups born out of combination 040 x 372 (litter 2002). For practical reasons, pups out of the litter 2002 are used in the breeding program.

All other combinations of different litters show lower similarity with other combinations, than the similarity calculated from within litters (table 3b), which implies that the average similarity will not increase by combining the pups of various combinations/litters. Inbreeding needs to be taken into account, so combinations of pups with the same parents or grandparents needs to be avoided.

Reintroduction

For breeding purposes two newly fromed packs of wild dogs Sangito and Ayubu were screened on relatedness within the packs by calculating similarity within the animals in the two packs. Both formed packs showed a similarity of approximately 0.60. This similarity is lower than calculated for pups born in the various combinations, concluded can be that the Sangito and Ayubu packs can be a good start for future breeding between the offspring of various litters.

The combination of pups from the litter of 006 x 251 born in 2004 will give a comparable similarity when these pups are compared with animals in the two packs and therefore these pups can be a good addition to the formed packs of wild dogs.

The observed similarities in the African wild dogs within the Mkomazi project are still at a very acceptable level, when all animals are taken into account a similarity of 0.46 for all related and unrelated dogs together can be calculated. The results in this analysis of the African Wild dog from the Mkomazi Project show very good genetic opportunities for this species.

53 Literature

Avise, J.C. 1994. Molecular markers, natural history and evolution. Chapman and Hall, Inc

Girman, D.J., Vila, C., Geffen, E., Creel, S., Mills, M.G.L., McNutt, J.W., Ginsberg, J., Kat, P.W., Mamiya, K.H. and Wayne, R.K. 2001. Patterns of population subdivision, gene flow and genetic variablility in the African wild dog (Lycaon pictus). Molecular Ecology, 1703- 1723.

Van de Zande, L. 1997. Genetic variation within and between African Wild dog (Lycaon Pictus) groups from the Maasai steppe, Tanzania. Mkomazi project.

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.

54

55

1 2

© The African Wild Dog Foundation 2012

3