Serologic Surveillance for West Nile Virus in Dogs, Africa

To the Editor: West Nile fever is caused by the West Nile virus (WNV), a mosquito-borne member of the genus Flavivirus. Birds are the natural reservoir of the virus, which is maintained in nature in a mosquito–bird–mosquito transmission cycle. WNV has been detected in many regions worldwide, including North America, Europe, Africa, the Near East, and Asia (1). WNV has been shown to cause meningoencephalitis in humans and horses. In the United States, seroconversion in dogs was detected 6 weeks before a human case was reported (2). Thus, dogs could be considered as sentinels for WNV infection, but their role as reservoir is unlikely because of short-term and low levels of viremia (3). In this study, we determined the seroprevalence of WNV in dogs living close to humans in different environments to assess their role as sentinels of this potentially severe zoonosis. 
 
During 2003–2012, blood samples were collected from 753 adult dogs from France and 6 countries in Africa (Table). Samples were centrifuged within 24 h after collection, separated, frozen at –20°C, and sent to the virology laboratory of the Institut de Recherche Biomedicale des Armees (Marseille, France). Each sample was systematically tested for IgG against WNV by using an in-house ELISA with inactivated WNV as antigen. Serum samples were considered positive if the optical density at 450 nm was >3-fold the mean of that for negative antigen. Because of the antigenic cross-reactivity among flaviviruses, all positive samples were further tested by Western blot for WNV-specific antibodies (4); seroprevalence was calculated on the basis of Western blot–confirmed cases only. For the statistical analysis, we used the exact binomial method to calculate 95% CIs of the proportions and the Fisher exact test to calculate p values and compare the seroprevalence rates between countries; significance was set at p<0.05. 
 
 
 
Table 
 
Prevalence of West Nile virus antibodies in dog populations, France and Africa, 2003–2012 
 
 
 
Seropositive dogs were found in all portions of Africa and France surveyed except northeastern Gabon and Corsica (Table). Seroprevalence of WNV in native dogs was significantly higher in Chad than in the Democratic Republic of the Congo (DRC) (p<0.001), Senegal (p<0.00001), Cote d’Ivoire (p<0.000001), and Gabon (p<0.000001). Seroprevalence was low in Kinshasa, DRC (12.5%), and Dakar, Senegal (11.1%), but in N’Djamena, Chad, all 5 native dogs tested had specific antibodies against WNV. 
 
As part of the study, we tested 50 military dogs from France twice, before and after a 4-month mission in Chad; 12 (24.0%) became seropositive after the stay. In addition, 12.5% of military working dogs in France imported from Hungary were seropositive on initial testing. We also found that, in France, dogs are the sentinels of WNV circulation in the Var (12.0%) and Gard (9.1%) departments. All dogs we tested that were positive for IgG were negative for IgM, a finding that indicates infection by the virus did not occur recently. 
 
The results and the statistical analysis reveal notable differences in the seroprevalence rates, according to the geographic area. N’Djamena, Chad, where all native dogs tested positive for WNV, is located at the confluence of the Chari and Logone Rivers and is an area with high densities of residential and migratory birds. In contrast, the northeastern region (Haut-Ogooue) of Gabon, where no native dogs tested positive for WNV, is an ecosystem of wet forests without migratory birds, unfavorable to virus circulation. In Dakar, 18.7% of native dogs were seropositive. In these central parts of Senegal, characterized by a semi-arid climate and vegetation composed of steppe plants and bluegrass, several WNV strains have been isolated from birds and mosquitoes. The seroprevalence was lower (0%–12.5%) in the sub-Saharan area, including Cote d’Ivoire, Gabon, DRC, and Senegal (Sine-Saloum and Casamance), where the humid or semihumid climate is linked with tropical rain forests or woodland savannah known to favor sedentary birds (5). 
 
In a large proportion of the human and animal population of Africa, immunity to WNV has developed (1). A serologic survey of dogs from the Highveld region of South Africa showed that 37% (138/377) had neutralizing antibodies against WNV (6). Similarly, seroprevalence of antibodies against WNV is high among dogs in the United States, for example, 55.9% (218/390) in the Gulf Coast region (7). In Turkey, an area where many birds stop over during migration, seroprevalence among dogs was high (37.7%, 43/114) (8). 
 
Our study highlights the role of dogs as sentinels for WNV circulation, particularly in southeastern France (Gard and Var departments), where WNV epidemics and epizootics occurred in 2000 and 2003. In addition, we observed that military working dogs purchased from Hungary, where WNV infection is common (9), may be seropositive. Seroconversion in dogs returning from short missions in WNV-endemic countries such as Chad was also observed. Therefore, our data emphasize the usefulness and convenience of WNV seroprevalence surveys in dogs for studying WNV epidemiology and circulation. It is possible that dogs living close to humans could attract infected mosquitoes, thereby reducing human infection.

Although only X. cheopis and S. fonqueniei fleas had previously been described as plague vectors in Madagascar, P. irritans fleas were most commonly collected during this field study; engorged and unfed male and female P. irritans fleas carried Y. pestis. Other studies have found P. irritans fleas in the plague risk area in other countries in Africa (5,6); one study found that P. irritans fleas may play a role in plague epidemiology in Tanzania (5). Data on P. irritans fleas in rats make it unlikely that these fleas are involved in rat-to-human transmission of Y. pestis in Madagascar. During 1922-1995, a total of 118,608 rats were caught and examined in Madagascar, but only 148 P. irritans fleas were identified, and none have been found on rats since 1996 (http://www. pasteur.mg/spip.php?rubrique124 To the Editor: West Nile fever is caused by the West Nile virus (WNV), a mosquito-borne member of the genus Flavivirus. Birds are the natural reservoir of the virus, which is maintained in nature in a mosquito-bird-mosquito transmission cycle. WNV has been detected in many regions worldwide, including North America, Europe, Africa, the Near East, and Asia (1). WNV has been shown to cause meningoencephalitis in humans and horses. In the United States, seroconversion in dogs was detected 6 weeks before a human case was reported (2). Thus, dogs could be considered as sentinels for WNV infection, but their role as reservoir is unlikely because of shortterm and low levels of viremia (3). In this study, we determined the seroprevalence of WNV in dogs living close to humans in different environments to assess their role as sentinels of this potentially severe zoonosis.
During 2003-2012, blood samples were collected from 753 adult dogs from France and 6 countries in Africa (Table). Samples were centrifuged within 24 h after collection, separated, frozen at -20°C, and sent to the virology laboratory of the Institut de Recherche Biomédicale des Armées (Marseille, France). Each sample was systematically tested for IgG against WNV by using an inhouse ELISA with inactivated WNV as antigen. Serum samples were considered positive if the optical density at 450 nm was >3-fold the mean of that for negative antigen. Because of the antigenic cross-reactivity among flaviviruses, all positive samples were further tested by Western blot for WNV-specific antibodies (4); seroprevalence was calculated on the basis of Western blot-confirmed cases only.

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For the statistical analysis, we used the exact binomial method to calculate 95% CIs of the proportions and the Fisher exact test to calculate p values and compare the seroprevalence rates between countries; significance was set at p<0.05.
Seropositive dogs were found in all portions of Africa and France surveyed except northeastern Gabon and Corsica (Table). Seroprevalence of WNV in native dogs was significantly higher in Chad than in the Democratic Republic of the Congo (DRC) (p<0.001), Senegal (p<0.00001), Côte d'Ivoire (p<0.000001), and Gabon (p<0.000001). Seroprevalence was low in Kinshasa, DRC (12.5%), and Dakar, Senegal (11.1%), but in N'Djamena, Chad, all 5 native dogs tested had specific antibodies against WNV.
As part of the study, we tested 50 military dogs from France twice, before and after a 4-month mission in Chad; 12 (24.0%) became seropositive after the stay. In addition, 12.5% of military working dogs in France imported from Hungary were seropositive on initial testing. We also found that, in France, dogs are the sentinels of WNV circulation in the Var (12.0%) and Gard (9.1%) departments. All dogs we tested that were positive for IgG were negative for IgM, a finding that indicates infection by the virus did not occur recently.
The results and the statistical analysis reveal notable differences in the seroprevalence rates, according to the geographic area. N'Djamena, Chad, where all native dogs tested positive for WNV, is located at the confluence of the Chari and Logone Rivers and is an area with high densities of residential and migratory birds. In contrast, the northeastern region (Haut-Ogooué) of Gabon, where no native dogs tested positive for WNV, is an ecosystem of wet forests without migratory birds, unfavorable to virus circulation. In Dakar, 18.7% of native dogs were seropositive. In these central parts of Senegal, characterized by a semi-arid climate and vegetation composed of steppe plants and bluegrass, several WNV strains have been isolated from birds and mosquitoes. The seroprevalence was lower (0%-12.5%) in the sub-Saharan area, including Côte d'Ivoire, Gabon, DRC, and Senegal (Siné-Saloum and Casamance), where the humid or semihumid climate is linked with tropical rain forests or woodland savannah known to favor sedentary birds (5).
In a large proportion of the human and animal population of Africa, immunity to WNV has developed (1). A serologic survey of dogs from the Highveld region of South Africa showed that 37% (138/377) had neutralizing antibodies against WNV (6). Similarly, seroprevalence of antibodies against WNV is high among dogs in the United States, for example, 55.9% (218/390) in the Gulf Coast region (7). In Turkey, an area where many birds stop over during migration, seroprevalence among dogs was high (37.7%, 43/114) (8).
Our study highlights the role of dogs as sentinels for WNV circulation, particularly in southeastern France (Gard and Var departments), where WNV epidemics and epizootics occurred in 2000 and 2003. In addition, we observed that military working dogs purchased from Hungary, where WNV infection is common (9), may be seropositive. Seroconversion in dogs returning from short missions in WNV-endemic countries such as Chad was also observed. Therefore, our data emphasize the usefulness and convenience of WNV seroprevalence surveys in dogs for studying WNV epidemiology and circulation. It is possible that dogs living close to humans could attract infected mosquitoes, thereby reducing human infection.