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)

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.

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?

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.

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.

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.

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

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.

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.

Results:

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.

Results:

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

Results:

Results:

Results:

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

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.

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.

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.

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.