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Ebola virus antibody prevalence in dogs. elena ortiz (1)
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.
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.
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
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.
Editor's Notes
Good morning, my name is Elena Ortiz. I would like to thank Dr Miller for his kind invitation to this meeting and to all of my colleagues for attending to my presentation. I will speak about Ebola virus infection in dogs, an important issue regarding Ebola epidemiology.
Ebola hemorrhagic fever is a very severe disease caused by three of the four Ebola virus species. It affects humans and primates, and it is associated with an 80% mortality. Epidemics are uncommon and the source of infection is usually an infected animal; this makes Ebola a zoonotic disease. In the past three years, outbreaks caused by Ebola virus-Z (Z from Zaire) have affected the region of Central Africa, including Gabon and the Republic of Congo.
Epidemiologic and genetic analyses identified gorilla, chimpanzee, and duiker carcasses as the main sources of human cases. Once the first human case is confirmed, the disease spreads among humans by direct physical contact.
Ebola hemorrhagic-fever outbreaks occur in villages where people normally keep domestic animals, including dogs. Although canine infection by Ebola virus has never been documented, domestic dogs’ behavior and diet place them at risk.
It is important to remember that the main Ebola infection symptoms are: fever, headache, muscle and stomach pain, fatigue, diarrhea, vomiting, and unexplained hemorrhage (bleeding or bruising). Symptoms may appear between day 1 and 21 after exposure to the virus, with an average of 8 to 10 days. In order to survive the infection, a good supportive clinical care is required. Also, the patient’s immune response is important: people who recover from Ebola infection develop protective antibodies that last more than 10 years. (No está mal comentar algo sobre la epidemiología general y la clínica del virus, siempre y cuando no te pases del tiempo…)
The aim of this investigation is to determine whether pet dogs can be infected and their potential as primary or secondary sources of human infection.
The second objective is to analyze the risk of infection between domestic dogs and humans, easily occurring by licking, biting or grooming.
Also, to better define the routes of transmission, focusing on interspecies spread of the virus, and to determine the prevalence of Ebola virus in dogs from several African regions.
A total of 439 dogs were selected and divided into 4 groups. The first group comprised 102 dogs from France (which were the negative controls). A second group comprised 258 dogs from Gabon (from an outbreak in 2001-2002), that was subdivided into 2 clusters: 159 dogs from villages located between Mekambo and Ekata and between Mekambo and Mazingo, and 99 dogs from Mekambo city, where human cases were also reported. The last group comprised 50 dogs from Libreville, the capital of Gabon, and 29 dogs from Port Gentil.
Sampling was conducted in three ways:
Dogs in Libreville and Port Gentil were sampled in a veterinary clinic. Blood was collected in 5-ml dry Vacutainers and serum was prepared by centrifugation. Serum specimens were stored at -80ºC until testing.
Dogs from the Ebola virus endemic area were sampled in the villages. An experienced veterinary team was located at Mekambo. Blood samples were daily collected in the vicinity of the village by using 5-ml dry Vacutainers and medetomidina anesthesia. Serum samples were kept in liquid nitrogen in 1-ml aliquots and stored at -80ºC until serologic testing, antigen detection, and RNA amplification were carried out.
Dogs in France were sampled in the Laboratoire des Dosages Hormonaux of the Ecole Nationale Vétérinaire de Nantes, France.
Dog owners were interviewed on their pets’ activities, such us participation in hunting, and health history. The focus of the interviews was on potential Ebola virus exposure events, including human cases that occurred in the village and among dog owners.
Ebola virus–specific immunoglobulin (Ig) G was detected by using a standard enzyme-linked immunosorbent assay (ELISA) method. Sera was diluted 1:400 and incubated at 4ºC.
This is the specific laboratory methodology used for virus infection confirmation: IgG binding, ELISA test, PCR amplification and viral mRNA detection…)
The authors used specific statistical tests for comparison of variables with a 0.05 significance level, with the aid of the appropriate licensed software.
All 439 blood samples from dogs were screened for Ebola virus–specific IgG. Two of the 102 blood samples from the dogs living in France had detectable Ebola virus–reactive IgG. Seven of the 79 dogs sampled in Libreville and Port Gentil, 15 of the 99 dogs sampled in Mekambo, and 40 of the 159 dogs sampled in villages located within the Ebola virus–epidemic area had detectable IgG to Ebola virus antigens.
This indicates either true infection or simple antigenic stimulation. All the tests were standardized at the 1:400 serum dilution, and most serum specimens had high OD values even at higher dilutions, confirming the specificity of the reactions.
These findings strongly suggest that dogs can be infected by Ebola virus. The prevalence of Ebola virus–reactive IgG among dogs from the villages where humans cases occurred was 27.2%, compared to 22.4% among dogs from villages where no human cases were noted.
Understandably, the seroprevalence rate was significantly lower in France than in Gabon. It was significantly lower compared to the 2 major towns, to Mekambo, and to the Ebola epidemic area.
In particular, it was lower than in the 2 major towns (p = 0.043), in Mekambo (p = 0.001), and in the Ebola virus–epidemic area (p &lt; 0.001).
This suggests that antigenic stimulation in these towns occurred despite they were not considered endemic areas.
The seroprevalence rates in dogs increased linearly as the sampling area approached the sites of human cases, as confirmed by the highly significant Cochran-Armitage test for trends in proportions.
In parallel, the seroprevalence rates in dogs increased linearly as the sampling area approached animal sources, also confirmed by the statistical test. However, epidemiologic investigations showed that most seropositive dogs in Libreville and Port Gentil had probably never been in contact with an infected source. They may therefore have been in contact with free viral antigens, transmitted by aerosol or experienced conjunctival exposure to virus droplets of urine, feces or blood.
One striking result of this study is the significant increasing gradient of canine seroprevalence from France to the Ebola virus–epidemic area, including from villages with and without human cases in the area.
The virus appears to jump from its natural host to humans only in specific, but unknown, conditions. The authors suggest that the seroprevalence rates in dogs may serve as an indicator for Ebola virus in regions in which no animal deaths or human cases have been observed in a given moment
It is important to mention that neither Ebola virus antigens nor nucleotide sequences were detected in any of the positive or negative dog blood samples. The authors also failed to isolate the virus from 3 positive and 3 negative samples on VeroE6 cells.
The authors also investigated the potential role of domestic dogs in the occurrence or dissemination of Ebola virus hemorrhagic fever in humans. Based on a serologic survey of dogs in the 2001–2002 Ebola outbreak area in Gabon, evidence was found that dogs can be infected by Ebola virus. However, symptoms did not develop in any of these highly exposed animals during the outbreak, a finding that means antigenic stimulation, asymptomatic, or very mild Ebola virus infection. Dogs appear to be the first animal species shown to be naturally and asymptomatically infect by Ebola virus. However, they may excrete infectious viral particles in urine, feces and saliva. Asymptomatic Ebola infection in humans has also been observed during outbreaks but it is very rare.
Due to the close contact between humans and domestic dogs, canine Ebola infection must be considered as a potential risk factor for human infection and virus spread. The results suggest that dogs should be taken into consideration during the management of human Ebola outbreaks. The Ebola virus reservoir species are currently unknown but epidemiologic observations suggest that bats and rodents living in central Africa are the major candidates. The discovery of Ebola virus-positive pet dogs in undeclared affected areas suggests that these animals live in close contact with the Ebola virus reservoir, and this finding may help to identify the true reservoir.
This study offers the first evidence that dogs might be asymptomatically infected by Ebola virus in the wild.
This finding has potential implications for preventing and controlling human outbreaks.
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 in regions where no other means of virus detection are available.
