EEHV update from Europe2017-2018

Willem Schaftenaar, DVM

Rotterdam Zoo

Vet advisor European elephant TagEEHV Advisory Group MeetingAugust 12-14, 2018White Oak Conservation Center

1

2017-2018

• 2 Non-fatal EEHV-HD cases

• 3 Fatal EEHV-HD cases

2

Clinical EEHV-HD case 1

• Namsai – Kolmarden (Sweden): (March 2017, presented at London EEHV workshop 2017)

• EEHV1b, AHPA Weybridge• Monday-Saturday: slightly slower.• Saturday: bluish discoloration lateral on the tongue • Sunday: Oral Acyclovir and fluids rectally• Monday: frozen and fresh plasma, full blood,

dexamethason and hypertonic NaCl, antibiotics• Wednesday improvement was seen, also reflected in

slightly better blood parameters (raise in Leucocytes and thrombocytes, not too bad coagulation parameters) and a stagnating, then diminishing viral load.

3

Namsai - Kolmarden

4

• Tarli - Woburn (UK). December 2017• Weekly monitoring qPCR• EEHVB-1b viremia, APHA Weybridge• Ganciclovir, plasma, fuids• Prior to clinical signs:

– Drop heterophils / monocytes– Increase lymphocytes– Platelets 300 -> 100

• Dexamethason when clinical signs appeared (2x)– Increase monocytes– Increase platelets

Clinical EEHV-HD case 25

• Qijo – Planckendael (Belgium), May 2018

• 2 yrs-old

• EEHV1a, APHA Weybridge

• No monitoring, detected after clinical signs started

• Died within a few hours after symtoms were detected.

Fatal EEHV-HD case 16

• Kanja – Hamburg (Germany), † 6 June 2018

• 2.5 yrs-old

• EEHV1a, Free University Berlin & APHA Weybridge

• No routine monitoring

• Detected after clinical signs started

• No EEHV-HD treatment given

Fatal EEHV-HD case 27

1,0 Asian elephant (Elephas maximus) KANJA (*11.01.2016)

03.06.2018: calmer than usual; no feces over half a day; eating only dry/hard things (no fruits); in the evening 37,5°C ->Tried to give him paraffin + bran

04.06: in the morning better/pretty active; eating; feces normal; 37,1 °C; in the evening calmer; again very picky when eating; drinking a lot; 38,1 °C -> stays in his box overnight; only hay

05.06: behaves like having a colic; not eating at all; drinking a lot; no feces; tongue normal -> 15 ml Buscopan; 20 Penicillin/Streptomycin per blowpipe

06.06: poor general condition; abdominal cramping?/colic?; lies in the water; drinks a lot; definitely high temperature (not possible to come close to him)-> 25 ml Buscopan per blowpipe~12:00 walks a few steps out of the water and collapses; immediately dead

PCR-results:EDTA-blood (taken post mortem, 06.06.): qPCR EEHV-1: CT 22,8; positiveTissue (tongue): qPCR EEHV-1: CT 17,6; positive

8

• Anjuli – Hamburg (Germany), †13 June 2018

• 3 yrs-old

• EEHV1a, Free University Berlin & APHA Weybridge

• No monitoring

• Detected after onset of clinical signs– Treated with famciclovir, plasma, fluids, NSAID,

dexamethason

Fatal EEHV-HD case 39

0,1 Asian elephant (Elephas maximus) ANJULI (*13.07.2015)

08.06.2018: 15:15 temperature 38,5 °C; behavior totally “normal” -> (after getting the positive PCR-result) 15mg/kg Famciclovir TID (30 tablets 8:00/16:00/24:00h) p.o.; 2,5 mg/kg Enrofloxacin (25 ml Baytril 10%) hand-injected

09.06: normal behavior; takes all tablets Famciclovir; 2,5 mg/kg Enrofloxacin hand-injected (20ml) + per blowpipe (5ml); no more drinking than usual!; feces ok; in the afternoon 38,7 °C; tongue normal

10.06: normal behavior; doesn’t want to eat the tablets any more -> only 40 tablets Famciclovir over the whole day; 2,5 mg/kg Enrofloxacin per blowpipe; in the afternoon 37,9 °C; tongue normal

11:06: normal behavior; eating; feces ok; in the morning temperature 38,3°C; doesn’t want to take the tablets. Standing sedation: “Famciclovir-paste” (30 pulverized tablets with water) orally and rectally (30 tablets); Enrofloxacin i.m.; 0,2 mg/kg Meloxicam i.m.; blood sample (EDTA)

12.06: depressed; lies more than usual; eats a little (mainly willow); no feces -> standing sedation): “Famciclovir-paste” orally (60 tablets); Enrofloxacin i.m.; Emdocam i.m.; blood sample; 350 ml Plasma (donor 0,1 SHANDRA, tested EEHV-negative) i.v. (ear); water (several liters) rectally; temperature (rectum) 37,8°C

13.06: apathetic (first day we can manipulate her without sedation); edema on head/eyes and shoulder; tongue bluish; petechial bleeding in both eyes; small amount of blood in the rectum (after rectal manipulation) -> 0,07 mg/kg dexamethasone i.m.; every 2 hours enemas with water-hose; “Famciclovir-paste” orally (30 tablets); getting worse rapidly; ~11:00 collapses; can’t get up any more; no more treatment possible because the whole herd standing around her; ~11:30 dead

10

0,1 Asian elephant (Elephas maximus) ANJULI (*13.07.2015)

PCR-results– 06.06.(taken in the afternoon): Swab (trunk) qPCR EEHV-1:

negative!– 08.06.(taken in the morning): swab (trunk): qPCR EEHV-1:

CT 37; positive & Trunk-wash: qPCR EEHV-1: CT 37;positive– 11.06. (taken in the afternoon): blood: qPCR EEHV-1: CT

21; positive– 12.06. (taken in the evening): blood: qPCR EEHV-1: CT 20;

positive– 13./14.06. (taken post mortem): blood: qPCR EEHV-1: CT

22; positive & Tissues (tongue): qPCR EEHV-1: CT 20;

Cell count– 11.06.: thrombocytes 194 G/l (reference: 258-740)– 12.06.: thrombocytes 43 G/l

11

• APHA Wheybridge: Akbar Dastjerdi• IZW – Berlin:

– Sónia Fontes /Thomas Hildebrandt– Alex Greenwood

• Free University Berlin: Walid Azab• Dutch EEHV working group• PhD students:

– Kopenhagen: Kathryn Perrin– APHA Weybridge:

• Jonathan Haycock: second year• Hannah Davies : starts in October 2018

– Chester?

Research in Europe12

APHA Weybridge: Akbar Dastjerdi

• Weekly EEHV-1, 3 & 4 real time PCR testing.

Routinely: Twycross zoo and Woburn safari park

Occasionally: Blackpool and Dublin zoo

• Helped Chester zoo with training and purchase of reagents and equipment so that the zoo can perform the EEHV PCRs on site. This is now accomplished.

13

APHA Weybridge: Akbar Dastjerdi

• Emergency diagnostic and viral load measurement for Woburn Safari Park, UK (Tarli case: EEHV-1B) and Kolmarden, Sweden (Namsai case: EEHV-1B) to help assess their therapeutic interventions. Both elephant calves have survived the infection despite developing clinical signs.

• Emergency diagnostic services for two fatalities from KMDA, Belgium [Qiyo and Tarzen: EEHV-1A] and two fatalities from Tierpark zoo, Germany [Anjuli and Kanja: EEHV-1A].

• Almost finalized sequencing POL, gB, gH, gL, gO and thymidine Kinasegenes of EEHVs from most fatalities in European zoos including EEHVs detected from survival cases and latent infections. This work was done as part of an undergraduate placement project by Hannah Davies. The work was presented at the International Herpesvirus Workshop (IHW) -Vancouver in July 2018 as a poster.

• Purchased a software to manage its samples storage. All clinical elephant samples hold at the Weybridge are now lodged in this inventory which facilities their traceability.

14

APHA Weybridge: Akbar Dastjerdi

• Jonathan Haycock is approaching end of his 2nd year of PhD. He has been making progress assessing the use of elephant IFNs to control EEHV replication in the early stages of EEHV infection e.g. following a positive real time PCR.

• Hannah Davies will be starting her PhD in October. She will be working to identify glycoprotein/protein complexes which may play a role in EEHV attachment to the cells and hence targets for a vaccine.

PhD students:

15

IZW – Sónia Fontes & Thomas Hildebrandt

AIM: To find the factors that predict the likelihood of an elephant to die from EEHV-HD (management-related and genetics-related)

ACHIEVEMENTS/PROGRESS:- Assessing a survey on growth-process of the calves (7 breeding

facilities). - EEHV-HD epidemiology in Europe until 2015: 75% of EEHV-HD

victims between 1.4 and 3.2 years- The probability that a death in the population is caused by

EEHV is 28,8% (ranging from 18,8%-40,6%). - There is no gender correlation in this disease.

16

IZW – Sónia Fontes & Thomas Hildebrandt (cont.)

ACHIEVEMENTS/PROGRESS (cont.):

- Coagulation mutation Factor VII (Australia) checked in European population: 2 coagulation parameters (prothrombin time and activated partial thromboplastin time) using fresh blood in 58 animals, in 16 zoos

- Analysis of presence of the Factor VII (FVII) gene in frozen tissue (liver and myocardium) and blood for PCR and sequenciation (n=37) for detection of possible mutations in the FVII gene.

17

IZW – Sónia Fontes & Thomas Hildebrandt (cont.)

Evolutionary Genetics’ Department –Dr. Jörns Fickel:

• Successfully amplified and sequenced 7 out of 8 exons that constitute this gene

• 10 single nucleotide polymorphism (SNP) – mutation points found, in 4 exons.

• Primers for the missing exon were designed and are being processed now

• This will hopefully result in entire gene; check for the alteration this mutations might cause in the FVII shape and activity.

18

IZW – Alex Greenwood

• Large sequencing project of elephants, looking at immune gene diversity, including the data set of elephants with and without EEHV infections to see if there is an immune gene type correlation, sharing data with Paul Ling who can use the immune gene sequences to further develop his assays.

• The peptides are on ice…

19

Free University Berlin – Walid Azab

1. Analyzing now the two cases that died in Hamburg zoo last month; trying to isolate the virus (or to understand this virus).

2. Investigating whether the gB-antibodies from Petra van den Doel are reactive with EEHV in cells using immunofluorescence.

3. Diagnostics and routinely monitoring calves (qPCR):

20

Free University Berlin – Walid Azab

• Elephants samples (2-3 zoos routinely):

Samples Positive Negative

2017 226 10 (TW) 216

2018 198 4 (TW*/swab*)5 (Tissues/blood)

189

* 1 TW and 1 swab from EEHV-HD

21

Research in Europe

• Dutch EEHV working group– Artemis-OneHealth (Delft): Byron Martina (virus

reactivation, gN)– Veterinary faculty (Utrecht):

• Immunology (cytokines) : Victor Rutten• Pathology (IHC): Jooske IJzer

– Erasmus University Rotterdam: Petra van den Doel– University of Amsterdam: Timo Breit (student paper

on small RNA’s)– Kathryn Perrin (PhD-student) spent June-August 2018

at vet faculty Utrecht. Will probably come back later for cytikine and IHC studies

22

Research in Europe

• European EEHV steering group

– No meeting since May 2017

– Members are too busy with zoo work

23

Publications24

25

•In Switzerland, at least 3 fatal EEHV1a-HD cases•Trunk wash samples of 7 zoo elephants, 4 months•5 out of 7 were EEHV1b-shedders

2 shedded only once; 1 recent import shedded EEHV3/4 3 were multiple shedders (all closely related; 1 of these had been

in contact with the 3 HD-cases)

26

Acknowledgement

• APHA Weybridge: Akbar Dastjerdi• Free University Berlin: Walid Azab• IZW: Alex Greenwood, Sónia Fontes• Artemis-OneHealth: Byron Martina• Veterinary Faculty Utrecht: Victor Rutten, Peter van Kooten, Jooske

IJzer• Erasmus University Rotterdam: Petra van den Doel• University of Amsterdam: Timo Breit• Kolmarden Zoo: Louise Guevara DVM• Woburn Zoo: Nathalie Wissink-Argilaga DVM• Planckendael Zoo: Francis Vercammen DVM• Hamburg Zoo: Adriane Prahl DVM• Kathryn Perrin: Copenhagen Zoo

27

Thank you!