Ebola virus

1. Ebola Virus Antibody
Prevalence in Dogs
and Human Risk
Loïs Allela,* Olivier Bourry,* Régis Pouillot,† André Délicat,* Philippe Yaba,* Brice
Kumulungui,* Pierre Rouquet,* Jean-Paul Gonzalez,‡ and Eric M. Leroy *‡
Emerg Infect Dis. 2005 Mar; 11(3)

2. Background: Problem
Ebola virus-Z (Zaire) is one of the four Ebola virus species.
Occurs in central Africa and leads to 80% mortality in a few
days.
Five EB-Z outbreaks in three years in Gabon and Republic
of Congo (428 cases, 334 deaths)
It is a zoonotic disease: the index patient is often infected
by an animal source.

3. Background
Epidemic spreads in relation to close contact with EV-Z infected
animal carcasses.
Main sources of human cases: gorilla, chimpanzee and duiker
carcasses.
Some human cases in a recent outbreak (5-17%) were not
directly exposed to Ebola hemorrhagic fever patients or
infected carcasses. So,
Are there other routes for transmission?

4. Objective
1. To determine whether pet dogs can be asymptomatically infected and their potential as primary or
secondary sources of infection.
2. To analyze the Human risk of contact.
3. Canine infection by EB-V has never been documented before. Are they at risk?
4. To better define the routes of transmission of Ebola, Focusing on interspecies spread of the virus.
5. Study: large serologic survey on the prevalence of EB infection in pet dogs from an epidemic area
in Gabon.

5. Methods: Sample
102 France (negative controls)
159 Villages between Mekambo-Ekata-Mazingo (Gabon)
99 Mekambo city
50 Libreville
29 Port Gentil
A total of 439 dogs were sampled and divided into 4 groups.

6. Methods: Sampling was conducted in three ways
Dogs from: Sampled in Blood samples Serum
Libreville and Port
Gentil
veterinary clinic 5-ml dry Vacutainers by cetrifugation,
stored at -80ºC
Virus endemic area the villages 5-ml dry Vacutainers
and medetomidina
anesthesia
kept in liquid nitrogen
in 1-ml aliquots and
stored at -80ºC.
France Laboratoire des
Dosages Hormonaux
of the Ecole Nationale
Vétérinaire de Nantes,
France.

7. Methods: Laboratory Investigations:
Ebola virus–specific immunoglobulin (Ig) G was detected by using a standard enzyme-linked immunosorbent
assay (ELISA) method. Maxisorp plates were coated with Ebola virus–Z antigens diluted 1:1,000 in
phosphatebuffered saline (PBS), overnight at 4°C. Control plates were coated with uninfected Vero cell culture
antigens in the same conditions. Sera diluted 1:400 in 5% nonfat milk in PBS-Tween 20 (0.1%) were added to
the wells and incubated overnight at 4°C.
ELISA Maxisorp plates

8. Methods: Laboratory Investigations:
Trough using a peroxidase-labeled anti-dog IgG and the TMB detector system, IgG binding was
visualized. Optical density (OD) was measured at 450 nm with an ELISA plate reader. For each
sample we calculated the corrected OD. Samples were used for antigen detection and for viral
polymerase chain reaction (PCR) amplification.
For the detection of viral mRNA, total RNA was isolated from serum with the QIAmp viral RNA
kit (Qiagen, Courtaboeuf, France), and cDNA was synthesized from mRNA. We used two pairs
of degenerate primers corresponding to the L-gene of Ebola virus for 2 rounds of
amplification.

9. Methods: Statistical Methods
• Statistical Methods Confidence intervals for proportions were calculated by using the
Clopper and Pearson method.
• Statistical comparisons between seroprevalence rates were performed by using the
Fisher test.
• The Cochran-Armitage test was used as a trend test for proportions.
• All tests used a 0.05 significance level.
• Statistical analyses were performed by using R software.

10. Results:

11. Results:
• This indicates: true infection or simple antigenic stimulation.
• All tests: standardized at the 1:400 serum dilution.
• Most serum specimens had high OD values confirming the specificity of the reactions.
• These findings strongly suggest that dogs can be infected by Ebola virus.

12. Results:

13. Results:
The seroprevalence rate was
significantly lower in France (2%) than
in Gabon. It was significantly lower
compared to the 2 major towns, to
Mekambo, and to the Ebola epidemic
area.
This suggests that antigenic
stimulation in these towns occurred
despite they were not considered
endemic areas.
Major towns (Libreville
and Port Gentil)
P= 0.043
Mekambo P=0.001
Ebola virus-epidemic
área
P<0.001

14. Results:

15. Results:

16. Results:

17. Results: Problems finded:
1. Neither Ebola virus antigens nor nucleotide sequences were detected in any of the
positive or negative dog blood samples.
2. The authors also failed to isolate the virus from 3 positive and 3 negative samples on
VeroE6 cells.
3. No circulating Ebola antigens or viral DNA sequences were detected in either positive or
negative serum specimens, and attempts to isolate virus from these samples failed. These
findings indicate either old, transient Ebola infection of the tested dogs, or antigenic
simulation

18. Results-Discussion:
• Authors also investigated the potential involvement of domestic dogs in the
occurrence or disseminatcion of Ebola virus hemorrhagic fever in humans.
• Evidence that dogs can be infected by Ebola virus was found. This finding raises
important human health issues.
• Symptoms did not develop in any of these highly exposed animals during the
outbreak Antigenic stimulation
Asymptomatic
Mild Ebola virus infection
• Dogs: first animal shown to be naturally and asymptomatically infected by Ebola virus.
• They excrete infectious viral particles.

19. Results-Discussion:
• Potential risk factor for human infection and virus spread: the canine Ebola infection
• Close contact between humans and domestic dogs.
• Take into consideration domestic dogs during human Ebola outbreaks.
• Ebola virus reservoir species: unknown. Suggestion: bats and rodents living in central Africa.
• Ebola virus-positive pet dogs in undeclared affected áreas suggests these animals live in close
contact with Ebola virus reservoir.

20. Conclusions:
1. This study offers the first evidence that dogs might be asymptomatically infected by
Ebola virus in the wild.
2. This finding has potential implications for preventing and controlling human outbreaks.
3. The increasing canine seroprevalence gradient from low-risk to at-risk Ebola virus–
endemic areas indicates that this seroprevalence might be used as an epidemiologic
indicator of virus circulation.

21. Conclusions:
4. Asymptomatic Ebola infection in humans : very rare.
5. Canine Ebola infection: potential risk factor for human infection and virus spread.
6. Considerate dogs during the management of human Ebola outbreaks.