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# Bayesian modeling of post-vaccination serological data suggests that yearly vaccination of dog aged

Heidi Auerswald, Julia Guillebaud, Benoit Durand, Mathilde Le Vu, Sopheak

Sorn, Saraden In, Vutha Pov, Holl Davun, Veasna Duong, Sowath Ly, et al.

► To cite this version:

Heidi Auerswald, Julia Guillebaud, Benoit Durand, Mathilde Le Vu, Sopheak Sorn, et al. Bayesian modeling of post-vaccination serological data suggests that yearly vaccination of dog aged

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RESEARCH ARTICLE

# Bayesian modeling of post-vaccination serological data suggests that yearly vaccination of dog aged <2 years old is efficient to stop rabies circulation in Cambodia

Heidi Auerswald<sup>1,*</sup>, Julia Guillebaud<sup>1,*</sup>, Benoit Durand<sup>2,*</sup>, Mathilde Le Vu<sup>3,*,*</sup>, Sopheak Sorn<sup>3</sup>, Saraden In<sup>1</sup>, Vutha Pov<sup>3</sup>, Holl Davun<sup>4†</sup>, Veasna Duong<sup>1</sup>, Sowath Ly<sup>3</sup>, Philippe Dussart<sup>1,‡b</sup>, Véronique Chevalier<sup>3,5,6,‡b*</sup>

1 Virology Unit, Institut Pasteur du Cambodge, Pasteur Network, Phnom Penh, Cambodia, 2 Epidemiology Unit, Laboratory for Animal Health, French Agency for Food, Environmental and Occupational Health and Safety (ANSES), University Paris-Est, Maisons-Alfort, France, 3 Epidemiology and Public Health Unit, Institut Pasteur du Cambodge, Pasteur Network, Phnom Penh, Cambodia, 4 General Directorate of Animal Health and Production, Ministry of Agriculture, Forestry and Fisheries, Phnom Penh, Cambodia, 5 Centre de Coopération Internationale en Recherche Agronomique pour le Développement (CIRAD), Unité Mixte de Recherche ASTRE, Montpellier, France, 6 ASTRE, Université de Montpellier, CIRAD, INRAE, Montpellier, France

a These authors contributed equally to this work.

† Deceased.

¤a Current address: Institute of Evolutionary Medicine, University of Zurich, Zurich, Switzerland

¤b Current address: Institut Pasteur de Madagascar, Pasteur Network, Antananarivo, Madagascar

* veronique.chevalier@cirad.fr

<figure><img src="image_2.png" /></figure>

OPEN ACCESS

**Citation:** Auerswald H, Guillebaud J, Durand B, Vu ML, Sorn S, In S, et al. (2024) Bayesian modeling of post-vaccination serological data suggests that yearly vaccination of dog aged <2 years old is efficient to stop rabies circulation in Cambodia. PLoS Negl Trop Dis 18(4): e0012089. https://doi.org/10.1371/journal.pntd.0012089

**Editor:** Victoria J. Brookes, The University of Sydney School of Veterinary Science, AUSTRALIA

**Received:** November 30, 2023

**Accepted:** March 21, 2024

**Published:** April 18, 2024

**Copyright:** © 2024 Auerswald et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

**Data Availability Statement:** All relevant data are in the manuscript and it supporting information files. Data underlying the findings were anonymised.

**Funding:** This study was financially supported by Région Occitanie, Swedish Research Council and the French Embassy in Cambodia to VC; by the German Centre for International Migration and Development to HA; by International Direction of Pasteur Institute through Fondation Pierre Ledoux

## Abstract

Rabies control remains challenging in low and middle-income countries, mostly due to lack of financial resources, rapid turnover of dog populations and poor accessibility to dogs. Rabies is endemic in Cambodia, where no national rabies vaccination program is implemented. The objective of this study was to assess the short and long-term vaccination-induced immunity in Cambodian dogs under field conditions, and to propose optimized vaccination strategies. A cohort of 351 dogs was followed at regular time points following primary vaccination only (PV) or PV plus single booster (BV). Fluorescent antibody virus neutralization test (FAVNT) was implemented to determine the neutralizing antibody titer against rabies and an individual titer ≥0.5 IU/mL indicated protection. Bayesian modeling was used to evaluate the individual duration of protection against rabies and the efficacy of two different vaccination strategies. Overall, 61% of dogs had a protective immunity one year after PV. In dogs receiving a BV, this protective immunity remained for up to one year after the BV in 95% of dogs. According to the best Bayesian model, a PV conferred a protective immunity in 82% of dogs (95% CI: 75–91%) for a mean duration of 4.7 years, and BV induced a lifelong protective immunity. Annual PV of dogs less than one year old and systematic BV solely of dogs vaccinated the year before would allow to achieve the 70% World Health Organization recommended threshold to control rabies circulation in a dog
RESEARCH ARTICLE

# Bayesian modeling of post-vaccination serological data suggests that yearly vaccination of dog aged <2 years old is efficient to stop rabies circulation in Cambodia

<figure><img src="image_3.png" /></figure>

Heidi Auerswald<sup>1,*</sup>, Julia Guillebaud<sup>1,*</sup>, Benoit Durand<sup>2,*</sup>, Mathilde Le Vu<sup>3,*,*</sup>, Sopheak Sorn<sup>3</sup>, Saraden In<sup>1</sup>, Vutha Pov<sup>3</sup>, Holl Davun<sup>4†</sup>, Veasna Duong<sup>1</sup>, Sowath Ly<sup>3</sup>, Philippe Dussart<sup>1,‡b</sup>, Véronique Chevalier<sup>3,5,6,‡b*</sup>

1 Virology Unit, Institut Pasteur du Cambodge, Pasteur Network, Phnom Penh, Cambodia, 2 Epidemiology Unit, Laboratory for Animal Health, French Agency for Food, Environmental and Occupational Health and Safety (ANSES), University Paris-Est, Maisons-Alfort, France, 3 Epidemiology and Public Health Unit, Institut Pasteur du Cambodge, Pasteur Network, Phnom Penh, Cambodia, 4 General Directorate of Animal Health and Production, Ministry of Agriculture, Forestry and Fisheries, Phnom Penh, Cambodia, 5 Centre de Coopération Internationale en Recherche Agronomique pour le Développement (CIRAD), Unité Mixte de Recherche ASTRE, Montpellier, France, 6 ASTRE, Université de Montpellier, CIRAD, INRAE, Montpellier, France

These authors contributed equally to this work.

† Deceased.

a Current address: Institute of Evolutionary Medicine, University of Zurich, Zurich, Switzerland

b Current address: Institut Pasteur de Madagascar, Pasteur Network, Antananarivo, Madagascar

* veronique.chevalier@cirad.fr

<figure><img src="image_4.png" /></figure>

OPEN ACCESS

**Citation:** Auerswald H, Guillebaud J, Durand B, Vu ML, Sorn S, In S, et al. (2024) Bayesian modeling of post-vaccination serological data suggests that yearly vaccination of dog aged <2 years old is efficient to stop rabies circulation in Cambodia. PLoS Negl Trop Dis 18(4): e0012089. https://doi.org/10.1371/journal.pntd.0012089

**Editor:** Victoria J. Brookes, The University of Sydney School of Veterinary Science, AUSTRALIA

**Received:** November 30, 2023

**Accepted:** March 21, 2024

**Published:** April 18, 2024

**Copyright:** © 2024 Auerswald et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

**Data Availability Statement:** All relevant data are in the manuscript and it supporting information files. Data underlying the findings were anonymised.

**Funding:** This study was financially supported by Région Occitanie, Swedish Research Council and the French Embassy in Cambodia to VC; by the German Centre for International Migration and Development to HA; by International Direction of Pasteur Institute through Fondation Pierre Ledoux

## Abstract

Rabies control remains challenging in low and middle-income countries, mostly due to lack of financial resources, rapid turnover of dog populations and poor accessibility to dogs. Rabies is endemic in Cambodia, where no national rabies vaccination program is implemented. The objective of this study was to assess the short and long-term vaccination-induced immunity in Cambodian dogs under field conditions, and to propose optimized vaccination strategies. A cohort of 351 dogs was followed at regular time points following primary vaccination only (PV) or PV plus single booster (BV). Fluorescent antibody virus neutralization test (FAVNT) was implemented to determine the neutralizing antibody titer against rabies and an individual titer ≥0.5 IU/mL indicated protection. Bayesian modeling was used to evaluate the individual duration of protection against rabies and the efficacy of two different vaccination strategies. Overall, 61% of dogs had a protective immunity one year after PV. In dogs receiving a BV, this protective immunity remained for up to one year after the BV in 95% of dogs. According to the best Bayesian model, a PV conferred a protective immunity in 82% of dogs (95% CI: 75–91%) for a mean duration of 4.7 years, and BV induced a lifelong protective immunity. Annual PV of dogs less than one year old and systematic BV solely of dogs vaccinated the year before would allow to achieve the 70% World Health Organization recommended threshold to control rabies circulation in a dog
grant of the Fondation de France to MLV; and by
the FSPI project CORAMAD funded by the French
Embassy in Madagascar. The funders had no role
in study design, data collection and analysis,
decision to publish, or preparation of the
manuscript.

**Competing interests:** The authors have declared
that no competing interests exist.

population in three to five years of implementation depending on dog population dynamics.
This vaccination strategy would save up to about a third of vaccine doses, reducing cost and
time efforts of mass dog vaccination campaigns. These results can contribute to optimize
rabies control measures in Cambodia moving towards the global goal of ending human
death from dog-mediated rabies by 2030.

Author summary

Rabies is a fatal zoonotic viral disease. Dog vaccination is recognized as the most cost-effective and sustainable solution to rabies prevention. Cambodia is endemic with canine mediated rabies, that would cause to around 800 human death each year. This country also has a large owned but mostly free-roaming dog population that makes efficient massive vaccination hard to achieve. In this study, we vaccinated 351 dogs with or without booster one year later. We then used serological assessed the short and long-term vaccination-induced immunity in these dogs, and Bayesian modeling to identify a vaccination strategy adapted to the Cambodian dog population characteristics. Yearly primary vaccinations for young dogs, supplemented with a single booster injection of these dogs promises to achieve 70% dog vaccination coverage effectively and efficiently, and control rabies circulation in dog populations. Applying this new strategy running for five more years after the vaccination coverage of at least 70% is achieved, this implies per 1,000 dogs between 2000 and 5 000 saved vaccine doses depending on dog population characteristics. Our results could help controlling rabies in Cambodia and potentially be adopted by other countries facing similar challenges.

**Introduction**

Human infection with rabies virus (RABV) causes almost always fatal encephalitis when not treated by timely administration of the vaccine before the onset of symptoms. It still affects more than 150 countries leading annually to estimated 59,000 deaths, half of them occurring in Asia ([1]. Up to 99% of human rabies cases are acquired from bites of infected dogs. Since dogs are the main reservoir and source of infection for humans, dog vaccination is recognized as the most cost-effective and sustainable solution to rabies prevention [2]. Elimination of rabies in domestic dog populations can be achieved with ≥70% dog vaccination coverage each year for at least five years [3]. Annual vaccination using injectable inactivated virus vaccines is recommended for all dogs irrespective of age and health status. However, vaccination coverage may be limited through insufficient vaccine delivery during vaccination campaigns, the disappearance of vaccinated dogs and/or the introduction of unvaccinated, susceptible dogs through dog movements, trade and demographic processes [4]. The vaccine-induced protection can be compromised by immuno-suppression through malnutrition, infection and other stressors [4,5]. Dog vaccination has been successfully used in many industrialized countries to prevent dog-mediated rabies [3]. However, in low- and middle-income countries (LMICs), controlling and eventually eliminating rabies is more challenging due to the lack of financial resources to sustain vaccination and surveillance over longer time, as well as lack of accessibility to human treatment and dog vaccination especially in rural communities where most rabies cases occur.
grant of the Fondation de France to MLV; and by
the FSPI project CORAMAD funded by the French
Embassy in Madagascar. The funders had no role
in study design, data collection and analysis,
decision to publish, or preparation of the
manuscript.

**Competing interests:** The authors have declared
that no competing interests exist.

population in three to five years of implementation depending on dog population dynamics.
This vaccination strategy would save up to about a third of vaccine doses, reducing cost and
time efforts of mass dog vaccination campaigns. These results can contribute to optimize
rabies control measures in Cambodia moving towards the global goal of ending human
death from dog-mediated rabies by 2030.

Author summary

Rabies is a fatal zoonotic viral disease. Dog vaccination is recognized as the most cost-effective and sustainable solution to rabies prevention. Cambodia is endemic with canine mediated rabies, that would cause to around 800 human death each year. This country also has a large owned but mostly free-roaming dog population that makes efficient massive vaccination hard to achieve. In this study, we vaccinated 351 dogs with or without booster one year later. We then used serological assessed the short and long-term vaccination-induced immunity in these dogs, and Bayesian modeling to identify a vaccination strategy adapted to the Cambodian dog population characteristics. Yearly primary vaccinations for young dogs, supplemented with a single booster injection of these dogs promises to achieve 70% dog vaccination coverage effectively and efficiently, and control rabies circulation in dog populations. Applying this new strategy running for five more years after the vaccination coverage of at least 70% is achieved, this implies per 1,000 dogs between 2000 and 5 000 saved vaccine doses depending on dog population characteristics. Our results could help controlling rabies in Cambodia and potentially be adopted by other countries facing similar challenges.

**Introduction**

Human infection with rabies virus (RABV) causes almost always fatal encephalitis when not treated by timely administration of the vaccine before the onset of symptoms. It still affects more than 150 countries leading annually to estimated 59,000 deaths, half of them occurring in Asia ([1]. Up to 99% of human rabies cases are acquired from bites of infected dogs. Since dogs are the main reservoir and source of infection for humans, dog vaccination is recognized as the most cost-effective and sustainable solution to rabies prevention [2]. Elimination of rabies in domestic dog populations can be achieved with ≥70% dog vaccination coverage each year for at least five years [3]. Annual vaccination using injectable inactivated virus vaccines is recommended for all dogs irrespective of age and health status. However, vaccination coverage may be limited through insufficient vaccine delivery during vaccination campaigns, the disappearance of vaccinated dogs and/or the introduction of unvaccinated, susceptible dogs through dog movements, trade and demographic processes [4]. The vaccine-induced protection can be compromised by immuno-suppression through malnutrition, infection and other stressors [4,5]. Dog vaccination has been successfully used in many industrialized countries to prevent dog-mediated rabies [3]. However, in low- and middle-income countries (LMICs), controlling and eventually eliminating rabies is more challenging due to the lack of financial resources to sustain vaccination and surveillance over longer time, as well as lack of accessibility to human treatment and dog vaccination especially in rural communities where most rabies cases occur.
Several inactivated canine vaccines are licensed in the EU and US. Most vaccines, available in different formulations, are intended to induce immunity for one to three years [6]. The individual immunization rate induced by vaccination can be monitored by measuring neutralizing antibodies (nAb) as they are a proxy for protection [5]. These antibodies can be measured by virus neutralization assays such as rapid fluorescent focus inhibition test (RFFIT) or fluorescent antibody virus neutralization test (FAVNT) [7]. A nAb titer of ≥ 0·5 IU/mL is considered protective in humans [3] and dogs [7]. The amount of nAbs produced after vaccination can be influenced by individual dog characteristics like age [5,8–10], health status [4,5], sex and neutering status [4,8,10] as well as size and breed [8,9,11,12]. Even vaccination studies of laboratory bred dogs of the same age, breed and health status showed high individual variability of immune reaction and nAb production [5,13].

The factors influencing vaccine-induced protection are complex, and serological profiles after vaccination can oscillate around the binary threshold of nAb titer, often due to inter-assay variability [14–17]. Traditional statistical approaches might not fully capture the variability and interdependencies among dog demographics, vaccination history, and serological responses. The application of latent class models (LCMs) in a Bayesian framework is a strategic choice to assess test sensitivity and specificity in the absence of a gold standard, and Bayesian methods are commonly used to fit this type of modelling [18]. Additionally, these models are well-suited for heterogenous populations where individuals can be categorized into latent groups based on unobservable characteristics. This approach allows us to model the actual immune states of individuals as “latent” states, providing a more nuanced understanding of the underlying serological dynamics.

Rabies is endemic in Cambodia, a Southeast Asian country where around 75% of the people live in rural areas [19]. The dog population in Cambodia is extremely dense, with an estimated dog-to-human ratio of 1:3–4 [20,21]. There is no national rabies vaccination program nor canine population management. It is estimated that annually over 800 people die from rabies but this is likely an underestimation as this calculation is based on data available only for the capital city Phnom Penh and surrounding regions [22]. Like in many endemic countries, financial constraints, the possibility of handling aggressive dogs, and their low survival rate [21], raise questions about the best strategy for feasible and cost-effective annual mass dog vaccinations (MDV).

A multi-partner rabies control program started in 2017 to reduce human rabies cases in the Cambodian provinces Kandal and Battambang. The first achievements of this program were an estimation of demographic parameters of the dog populations, a deep understanding of dog-human relationships and dog management practices, as well as the estimation of the annual bite incidence rate and associated risk factors [21]. Furthermore, this program aimed to design a dog vaccination strategy accounting for the dog demography in Cambodia and the vaccination-induced immunity of these dogs. To achieve this goal, we used post-vaccination serological follow-up accompanying the above-mentioned dog demography survey, and applied Bayesian modeling to evaluate the individual duration of protection against rabies and to compare two different alternative vaccination strategies: annual primary vaccination of young dogs (<1 year old) vs annual primary vaccination of young dogs (<1 year old) with systematic booster vaccination solely of dogs that have been vaccinated the year before.

## Materials and methods

### Ethics statement

We followed WOAH guiding principles on animal welfare [23]. The protocol of the survey has been approved by General Directorate of Animal Health and Production of Cambodia
Several inactivated canine vaccines are licensed in the EU and US. Most vaccines, available in different formulations, are intended to induce immunity for one to three years [6]. The individual immunization rate induced by vaccination can be monitored by measuring neutralizing antibodies (nAb) as they are a proxy for protection [5]. These antibodies can be measured by virus neutralization assays such as rapid fluorescent focus inhibition test (RFFIT) or fluorescent antibody virus neutralization test (FAVNT) [7]. A nAb titer of ≥ 0·5 IU/mL is considered protective in humans [3] and dogs [7]. The amount of nAbs produced after vaccination can be influenced by individual dog characteristics like age [5,8–10], health status [4,5], sex and neutering status [4,8,10] as well as size and breed [8,9,11,12]. Even vaccination studies of laboratory bred dogs of the same age, breed and health status showed high individual variability of immune reaction and nAb production [5,13].

The factors influencing vaccine-induced protection are complex, and serological profiles after vaccination can oscillate around the binary threshold of nAb titer, often due to inter-assay variability [14–17]. Traditional statistical approaches might not fully capture the variability and interdependencies among dog demographics, vaccination history, and serological responses. The application of latent class models (LCMs) in a Bayesian framework is a strategic choice to assess test sensitivity and specificity in the absence of a gold standard, and Bayesian methods are commonly used to fit this type of modelling [18]. Additionally, these models are well-suited for heterogenous populations where individuals can be categorized into latent groups based on unobservable characteristics. This approach allows us to model the actual immune states of individuals as “latent” states, providing a more nuanced understanding of the underlying serological dynamics.

Rabies is endemic in Cambodia, a Southeast Asian country where around 75% of the people live in rural areas [19]. The dog population in Cambodia is extremely dense, with an estimated dog-to-human ratio of 1:3–4 [20,21]. There is no national rabies vaccination program nor canine population management. It is estimated that annually over 800 people die from rabies but this is likely an underestimation as this calculation is based on data available only for the capital city Phnom Penh and surrounding regions [22]. Like in many endemic countries, financial constraints, the possibility of handling aggressive dogs, and their low survival rate [21], raise questions about the best strategy for feasible and cost-effective annual mass dog vaccinations (MDV).

A multi-partner rabies control program started in 2017 to reduce human rabies cases in the Cambodian provinces Kandal and Battambang. The first achievements of this program were an estimation of demographic parameters of the dog populations, a deep understanding of dog-human relationships and dog management practices, as well as the estimation of the annual bite incidence rate and associated risk factors [21]. Furthermore, this program aimed to design a dog vaccination strategy accounting for the dog demography in Cambodia and the vaccination-induced immunity of these dogs. To achieve this goal, we used post-vaccination serological follow-up accompanying the above-mentioned dog demography survey, and applied Bayesian modeling to evaluate the individual duration of protection against rabies and to compare two different alternative vaccination strategies: annual primary vaccination of young dogs (<1 year old) vs annual primary vaccination of young dogs (<1 year old) with systematic booster vaccination solely of dogs that have been vaccinated the year before.

**Materials and methods**

**Ethics statement**

We followed WOAH guiding principles on animal welfare [23]. The protocol of the survey has been approved by General Directorate of Animal Health and Production of Cambodia
(GDAHP). All sampling sessions and interviews were implemented by trained Institut Pasteur du Cambodge officers with supervision of GDAHP agents, local veterinary services and local village authorities. All data were anonymized.

## Vaccination and serological follow-up

The details of the demographic survey performed in Kandal and Battambang provinces are provided in Chevalier *et al.* [21]. During this survey, at enrollment (T0), amenable dogs were vaccinated against rabies via subcutaneous injection of Rabisin (Merial, Lyon, France) provided by the World Organization for Animal Health (WOAH) through the General Directorate of Animal Health and Production of Cambodia (GDAHP). Before, venous blood was collected either from jugular or brachial vein, serum was separated from the clot by centrifugation and then was stored at -20°C until serological analysis.

The study included two groups of dogs (Fig 1). Group 1 (n = 221) relates to dogs with an individual blood sample at enrollment (T0) before these dogs received their primary vaccination. These dogs were formally re-identified with their owners, and re-sampled approximately every 6 months for up to 1.5 years (18 months, T18). Dogs of group 2 (n = 130) were primary vaccinated (T0) without a prior blood collection. These dogs were re-captured and re-identified one year after their primary vaccination, a blood sample was collected and they received a booster vaccination (T12). An additional follow-up blood collection of group 2 dogs was performed 14 months (T26, n = 130), and 22 months (T34, n = 22) after booster vaccination.

<figure><img src="image_3.png" /><figcaption>**Fig 1.** Vaccination and follow-up schedule for study groups (created by authors using the licensed program BioRender (http://www.biorender.com). Dogs without documented previous rabies vaccination (grey) received their primary vaccination (T0, grey). For group 1 follow-up blood samples were taken seven months (T7), around one year (T12-14) and 1.5 years (T18) after primary vaccination. Due to logistic restraints the follow-up sequence differs (blue) within this group between dogs from Kandal (n = 16) and dogs from Battambang (n = 205). From dogs of group 2, a blood sample (T12) was collected one year after their primary immunization to document the immune response of this primary immunization. Afterwards these dogs received a booster vaccination (green), and additional samples were collected (green) more than one year (T26) and three years (T34) after the booster vaccination to monitor its effect on the immune response.</figcaption></figure>

https://doi.org/10.1371/journal.pntd.0012089.g001
(GDAHP). All sampling sessions and interviews were implemented by trained Institut Pasteur du Cambodge officers with supervision of GDAHP agents, local veterinary services and local village authorities. All data were anonymized.

## Vaccination and serological follow-up

The details of the demographic survey performed in Kandal and Battambang provinces are provided in Chevalier *et al.* [21]. During this survey, at enrollment (T0), amenable dogs were vaccinated against rabies via subcutaneous injection of Rabisin (Merial, Lyon, France) provided by the World Organization for Animal Health (WOAH) through the General Directorate of Animal Health and Production of Cambodia (GDAHP). Before, venous blood was collected either from jugular or brachial vein, serum was separated from the clot by centrifugation and then was stored at -20°C until serological analysis.

The study included two groups of dogs (Fig 1). Group 1 (n = 221) relates to dogs with an individual blood sample at enrollment (T0) before these dogs received their primary vaccination. These dogs were formally re-identified with their owners, and re-sampled approximately every 6 months for up to 1.5 years (18 months, T18). Dogs of group 2 (n = 130) were primary vaccinated (T0) without a prior blood collection. These dogs were re-captured and re-identified one year after their primary vaccination, a blood sample was collected and they received a booster vaccination (T12). An additional follow-up blood collection of group 2 dogs was performed 14 months (T26, n = 130), and 22 months (T34, n = 22) after booster vaccination.

<figure><img src="image_5.png" /><figcaption>**Fig 1.** Vaccination and follow-up schedule for study groups (created by authors using the licensed program BioRender (http://www.biorender.com). Dogs without documented previous rabies vaccination (grey) received their primary vaccination (T0, grey). For group 1 follow-up blood samples were taken seven months (T7), around one year (T12-14) and 1.5 years (T18) after primary vaccination. Due to logistic restrains the follow-up sequence differs (blue) within this group between dogs from Kandal (n = 16) and dogs from Battambang (n = 205). From dogs of group 2, a blood sample (T12) was collected one year after their primary immunization to document the immune response of this primary immunization. Afterwards these dogs received a booster vaccination (green), and additional samples were collected (green) more than one year (T26) and three years (T34) after the booster vaccination to monitor its effect on the immune response.</figcaption></figure>

https://doi.org/10.1371/journal.pntd.0012089.g001
## Fluorescent antibody virus neutralization test (FAVNT)

FAVNT was used to determine RABV nAb titer in dogs at T0, T6, T12, T18, T24 and T36. The assay was performed following WOHA guidelines [7,24]. Antibody titers of ≥ 0·5 IU/mL were considered positive [25].

## Statistical model

Age of the dogs at primary vaccination was categorized in four groups: <12 months, 12–24 months, 24–48 months and >48 months old based on the 25th, 50th and 75th percentiles of the age distribution. The body condition score (BCS) was categorized as “skinny” for BCS<4, “normal weight” for BCS 4–6, and “overweight” for BCS>6.

Subsequently, the term of ‘protective immunity’ is used to refer to an immune state indicated by a positive FAVNT result of ≥0.5 IU/mL as this is indicated by the World Organization for Animal Health as a reasonable level of seroconversion after rabies vaccination [26]. We modeled the immune state of each dog in the study cohorts using three states: susceptible (denoted S), having acquired a protective immunity after a primary vaccination (denoted V1), or after a booster vaccination (denoted V2). As RABV is endemic in Cambodia, some dogs could have been naturally exposed to RABV prior to, or during the study, and therefore have developed a non-lethal infection and subsequent immunity. This may bias the analysis. To control for this bias, we explicitly represented natural exposure to RABV in the model, and added a 4th immune state for dogs that acquired a protective immunity after a natural infection (denoted R). In order to create a usable, simplified model we assumed that in this latter case, the protective immunity was lifelong. Transitions between immune states are described in Fig 2. Vaccination of a susceptible dog (naïve, or with lost protective immunity after primary or booster vaccination) induced a protective immunity (state V1) with a probability q. The protective immunity is subsequently lost with a rate ρ1. We assume that the dogs experience a constant rate of immunity loss post-vaccination, suggesting that the duration of immunity follows an exponential distribution. This hypothesis is particularly relevant in the Cambodian context, given the short life expectancy of dogs there. We assumed that dogs that received a booster vaccination (state V2), while already protected since primary vaccination, lost their protective immunity with a different rate (ρ2). All dogs were assumed exposed to RABV via natural infection (or to a rabies-like lyssavirus inducing a cross-protection) with a force of infection λ. When exposed, susceptible dogs may develop a non-lethal form with a probability π (or died otherwise with a probability of 1-π). They then acquired lifelong protective immunity (R state). We assumed that FAVNT allowed the detection of protective immunity (nAb titer ≥ 0·5 IU/mL) in vaccinated dogs (states V1 and V2) with a probability p, and that dogs that acquired a natural immunity (R state) were always positive. The specificity of the FAVNT was assumed perfect.

The dynamics of the different immune states in a cohort of dogs receiving a primary vaccination at t = PV, and a booster vaccination at t = BV, is described by the following equations (with 1_b the dummy function: 1_b = 1 if b is true, and 0 otherwise):

$$ \frac{dS}{dt} = -\lambda S - 1_{(t=PV)or(t=BV)} qS + \rho_1 V_1 + \rho_2 V_2 $$

$$ \frac{dV_1}{dt} = 1_{(t=PV)or(t=BV)} qS - V_1(\rho_1 + \lambda) - 1_{t=BV} V_1 $$
## Fluorescent antibody virus neutralization test (FAVNT)

FAVNT was used to determine RABV nAb titer in dogs at T0, T6, T12, T18, T24 and T36. The assay was performed following WOHA guidelines [7,24]. Antibody titers of ≥ 0·5 IU/mL were considered positive [25].

## Statistical model

Age of the dogs at primary vaccination was categorized in four groups: <12 months, 12–24 months, 24–48 months and >48 months old based on the 25th, 50th and 75th percentiles of the age distribution. The body condition score (BCS) was categorized as “skinny” for BCS<4, “normal weight” for BCS 4–6, and “overweight” for BCS>6.

Subsequently, the term of ‘protective immunity’ is used to refer to an immune state indicated by a positive FAVNT result of ≥0.5 IU/mL as this is indicated by the World Organization for Animal Health as a reasonable level of seroconversion after rabies vaccination [26]. We modeled the immune state of each dog in the study cohorts using three states: susceptible (denoted S), having acquired a protective immunity after a primary vaccination (denoted V1), or after a booster vaccination (denoted V2). As RABV is endemic in Cambodia, some dogs could have been naturally exposed to RABV prior to, or during the study, and therefore have developed a non-lethal infection and subsequent immunity. This may bias the analysis. To control for this bias, we explicitly represented natural exposure to RABV in the model, and added a 4th immune state for dogs that acquired a protective immunity after a natural infection (denoted R). In order to create a usable, simplified model we assumed that in this latter case, the protective immunity was lifelong. Transitions between immune states are described in Fig 2. Vaccination of a susceptible dog (naïve, or with lost protective immunity after primary or booster vaccination) induced a protective immunity (state V1) with a probability q. The protective immunity is subsequently lost with a rate ρ1. We assume that the dogs experience a constant rate of immunity loss post-vaccination, suggesting that the duration of immunity follows an exponential distribution. This hypothesis is particularly relevant in the Cambodian context, given the short life expectancy of dogs there. We assumed that dogs that received a booster vaccination (state V2), while already protected since primary vaccination, lost their protective immunity with a different rate (ρ2). All dogs were assumed exposed to RABV via natural infection (or to a rabies-like lyssavirus inducing a cross-protection) with a force of infection λ. When exposed, susceptible dogs may develop a non-lethal form with a probability π (or died otherwise with a probability of 1-π). They then acquired lifelong protective immunity (R state). We assumed that FAVNT allowed the detection of protective immunity (nAb titer ≥ 0·5 IU/mL) in vaccinated dogs (states V1 and V2) with a probability p, and that dogs that acquired a natural immunity (R state) were always positive. The specificity of the FAVNT was assumed perfect.

The dynamics of the different immune states in a cohort of dogs receiving a primary vaccination at t = PV, and a booster vaccination at t = BV, is described by the following equations (with 1_b the dummy function: 1_b = 1 if b is true, and 0 otherwise):

$$ \frac{dS}{dt} = -\lambda S - 1_{(t=PV)or(t=BV)} qS + \rho_1 V_1 + \rho_2 V_2 $$

$$ \frac{dV_1}{dt} = 1_{(t=PV)or(t=BV)} qS - V_1(\rho_1 + \lambda) - 1_{t=BV} V_1 $$
<figure><img src="image_4.png" /><figcaption>**Fig 2. Probabilistic model of the evolution of dog rabies and serological status during the study period.** The whole dog population is separated in 4 states (indicated by squares): S susceptible dog; V₁ dog with protective immunity induced by primary vaccination; V₂ dog with protective immunity induced by primary and subsequent booster vaccination; R dog with protective immunity induced by a non-lethal infection. The serological results (by FAVNT) are indicated by circles: N negative FAVNT (<0.5 IU/mL); P positive FAVNT (≥0.5 IU/mL). The parameters describing the model dynamics are: λ average force of infection; π probability of non-lethal infection; q probability of acquiring protective immunity after vaccination; ρ₁ rate of loss of protective immunity induced by primary vaccination; ρ₂ rate of loss of protective immunity induced by primary and subsequent booster vaccination; p sensitivity of the serological test for dogs in V₁ or V₂ status. (*) Sensitivity is assumed perfect for animals in the R state. (**) Specificity is assumed perfect. (***) Dogs in the V₁ state are assumed immuno-competent (since they have acquired a protective immunity after primary vaccination), therefore, all are assumed to enter the V₂ state after the booster vaccination.</figcaption></figure>

https://doi.org/10.1371/journal.pntd.0012089.g002

$$ \frac{dV_2}{dt} = 1_{t=\text{BV}}V_1 - V_2(\rho_2 + \lambda) $$

$$ \frac{dR}{dt} = \lambda(\pi S + V_1 + V_2) $$

The model was not identifiable since λ and π always appeared in a multiplicative way. We therefore defined three scenarios for π, the probability to acquire/develop a non-lethal infection: the estimate of 0.51 proposed by Hampson *et al.* [27], and two arbitrary extreme values of 0.05 and 0.95. The five remaining parameters of the model (q, λ, ρ₁, ρ₂ and p) could then be estimated for each scenario. We analyzed the influence of three individual dog characteristics (age group, BCS, and sex) on the five model parameters. For a given dog characteristic and a given parameter, we compared the model in which the parameter changed according to the dog characteristic, with the reference model in which the respective parameter value was the same for all dogs. We thus successively compared 15 alternative models (five parameters × three individual characteristics) with the reference model, using the Bayes' factor,
<figure><img src="image_6.png" /><figcaption>**Fig 2. Probabilistic model of the evolution of dog rabies and serological status during the study period.** The whole dog population is separated in 4 states (indicated by squares): S susceptible dog; V₁ dog with protective immunity induced by primary vaccination; V₂ dog with protective immunity induced by primary and subsequent booster vaccination; R dog with protective immunity induced by a non-lethal infection. The serological results (by FAVNT) are indicated by circles: N negative FAVNT (<0.5 IU/mL); P positive FAVNT (≥0.5 IU/mL). The parameters describing the model dynamics are: λ average force of infection; π probability of non-lethal infection; q probability of acquiring protective immunity after vaccination; ρ₁ rate of loss of protective immunity induced by primary vaccination; ρ₂ rate of loss of protective immunity induced by primary and subsequent booster vaccination; p sensitivity of the serological test for dogs in V₁ or V₂ status. (*) Sensitivity is assumed perfect for animals in the R state. (**) Specificity is assumed perfect. (***) Dogs in the V₁ state are assumed immuno-competent (since they have acquired a protective immunity after primary vaccination), therefore, all are assumed to enter the V₂ state after the booster vaccination.</figcaption></figure>

https://doi.org/10.1371/journal.pntd.0012089.g002

$$ \frac{dV_2}{dt} = 1_{t=\text{BV}}V_1 - V_2(\rho_2 + \lambda) $$

$$ \frac{dR}{dt} = \lambda(\pi S + V_1 + V_2) $$

The model was not identifiable since λ and π always appeared in a multiplicative way. We therefore defined three scenarios for π, the probability to acquire/develop a non-lethal infection: the estimate of 0.51 proposed by Hampson et al. [27], and two arbitrary extreme values of 0.05 and 0.95. The five remaining parameters of the model (q, λ, ρ₁, ρ₂ and p) could then be estimated for each scenario. We analyzed the influence of three individual dog characteristics (age group, BCS, and sex) on the five model parameters. For a given dog characteristic and a given parameter, we compared the model in which the parameter changed according to the dog characteristic, with the reference model in which the respective parameter value was the same for all dogs. We thus successively compared 15 alternative models (five parameters × three individual characteristics) with the reference model, using the Bayes' factor,
interpreted according to the usual rules: the strength of evidence in favor of the alternative model (relative to the reference model) was considered substantial if $3<\text{BF}<20$, strong if $20<\text{BF}<150$, and very strong if $\text{BF}>150$. Models were fitted in a Bayesian Markov Chain Monte Carlo (MCMC) framework. Priors were non-informative. We used R version 4.2.1 [28] and RStan version 2.21.5 [29]. R and Stancodes, and raw data- group1a (n = 205), group1b (n = 16) and group2 (n = 130) are provided in S1 Text.

## Evaluation of vaccination strategies

We used the best model estimates to compare two vaccination strategies: (A) annual primary vaccination of dogs <1 year old; (B) annual primary vaccination of dogs <1 year old with systematic booster vaccination of dogs that have been vaccinated the year before. For each of the strategies, we calculated the percentage of the protected population N years after the strategy started. In this calculation, we considered only vaccine-induced immunity, and neglected natural immunity (induced by non-fatal infection or cross-protection due to infection with rabies-like lyssavirus). Mortality rates for each dog population was assumed constant for this preliminary analysis. Details on calculations are provided in S2 Text.

We calculated the proportion of the dogs with vaccine-induced protective immunity each year during the first ten years, and after 20 years of implementation (i.e. after complete renewal of the dog population). Two different Cambodian dog populations were considered: Kandal province with a 24-months survival rate of 52%, and Battambang province with 34% as described previously [21]. Confidence intervals (CIs) were calculated using the lower limit of the CI of *q* and the upper limit of that of $\rho_1$ and $\rho_2$, for the lower bound, and the upper limit of the CI of *q* and the lower CI limit of that of $\rho_1$ and $\rho_2$ for the upper bound.

## Results

### Cohort characteristics

In total, we included 351 dogs, where 221 dogs received a primary vaccination (group 1), and 130 dogs received an additional booster vaccination (group 2). The median age of dogs at inclusion was 24 months (min = 1 months; max = 17 years). Overall, 78% (273/351) of the dogs had a medium BCS, corresponding to normal weight. The male-to-female ratio was 1·26.

### Serological results

Serological results were used to categorize each dog into different trajectory groups regarding their serological status dynamics (Table 1).

Overall, 61% of the dogs had a protective immunity one year after their primary vaccination. In the dogs receiving a booster vaccination, the rate of protected dogs rose to 95% measured one year after booster vaccination. Seven dogs presented positive nAb titers (≥ 0·5 IU/mL) before they received the rabies vaccination. Overall, 29% (n = 59) remained seronegative after primary vaccination. Eighty-five dogs (41%) remained seropositive through T6 and T18, whereas 9% (n = 19) were positive at T6 but became negative at T18, and 7% (n = 14) were positive at T6 but negative at T12. Nineteen dogs were negative at T6, but became positive at T12 (n = 14) or T18 (n = 5) without being re-vaccinated. For the dogs that received a booster vaccination (group 2, n = 130) and were already seropositive T12 (n = 80), one turned negative at T24, and 67 (84%) remained positive at T24. Among dogs that were negative at T12 (n = 50), 6 were still negative at T24. In the small cohort of dogs (n = 22) that were followed up until two years after their booster vaccination (three years after primary vaccination, T36) 100% of dogs remained positive over the whole study period.
interpreted according to the usual rules: the strength of evidence in favor of the alternative model (relative to the reference model) was considered substantial if $3<\text{BF}<20$, strong if $20<\text{BF}<150$, and very strong if $\text{BF}>150$. Models were fitted in a Bayesian Markov Chain Monte Carlo (MCMC) framework. Priors were non-informative. We used R version 4.2.1 [28] and RStan version 2.21.5 [29]. R and Stancodes, and raw data- group1a (n = 205), group1b (n = 16) and group2 (n = 130) are provided in S1 Text.

## Evaluation of vaccination strategies

We used the best model estimates to compare two vaccination strategies: (A) annual primary vaccination of dogs <1 year old; (B) annual primary vaccination of dogs <1 year old with systematic booster vaccination of dogs that have been vaccinated the year before. For each of the strategies, we calculated the percentage of the protected population N years after the strategy started. In this calculation, we considered only vaccine-induced immunity, and neglected natural immunity (induced by non-fatal infection or cross-protection due to infection with rabies-like lyssavirus). Mortality rates for each dog population was assumed constant for this preliminary analysis. Details on calculations are provided in S2 Text.

We calculated the proportion of the dogs with vaccine-induced protective immunity each year during the first ten years, and after 20 years of implementation (i.e. after complete renewal of the dog population). Two different Cambodian dog populations were considered: Kandal province with a 24-months survival rate of 52%, and Battambang province with 34% as described previously [21]. Confidence intervals (CIs) were calculated using the lower limit of the CI of *q* and the upper limit of that of $\rho_1$ and $\rho_2$, for the lower bound, and the upper limit of the CI of *q* and the lower CI limit of that of $\rho_1$ and $\rho_2$ for the upper bound.

## Results

### Cohort characteristics

In total, we included 351 dogs, where 221 dogs received a primary vaccination (group 1), and 130 dogs received an additional booster vaccination (group 2). The median age of dogs at inclusion was 24 months (min = 1 months; max = 17 years). Overall, 78% (273/351) of the dogs had a medium BCS, corresponding to normal weight. The male-to-female ratio was 1·26.

### Serological results

Serological results were used to categorize each dog into different trajectory groups regarding their serological status dynamics (Table 1).

Overall, 61% of the dogs had a protective immunity one year after their primary vaccination. In the dogs receiving a booster vaccination, the rate of protected dogs rose to 95% measured one year after booster vaccination. Seven dogs presented positive nAb titers (≥ 0·5 IU/mL) before they received the rabies vaccination. Overall, 29% (n = 59) remained seronegative after primary vaccination. Eighty-five dogs (41%) remained seropositive through T6 and T18, whereas 9% (n = 19) were positive at T6 but became negative at T18, and 7% (n = 14) were positive at T6 but negative at T12. Nineteen dogs were negative at T6, but became positive at T12 (n = 14) or T18 (n = 5) without being re-vaccinated. For the dogs that received a booster vaccination (group 2, n = 130) and were already seropositive T12 (n = 80), one turned negative at T24, and 67 (84%) remained positive at T24. Among dogs that were negative at T12 (n = 50), 6 were still negative at T24. In the small cohort of dogs (n = 22) that were followed up until two years after their booster vaccination (three years after primary vaccination, T36) 100% of dogs remained positive over the whole study period.
<table><caption>Table 1. Dynamics of serological status.</caption><thead><tr><th rowspan="2">Time points</th><th colspan="6">Primary vaccination*</th><th rowspan="2">Number of dogs</th></tr><tr><th>T0</th><th>T7</th><th>T12-T14</th><th>T18</th><th>T26</th><th>T34</th></tr></thead><tbody><tr><td rowspan="10">Trajectories of serological status</td><td>N</td><td>N</td><td>N</td><td>P</td><td>n/a</td><td>n/a</td><td>5</td></tr><tr><td>N</td><td>N</td><td>P</td><td>N</td><td>n/a</td><td>n/a</td><td>7</td></tr><tr><td>N</td><td>N</td><td>P</td><td>P</td><td>n/a</td><td>n/a</td><td>7</td></tr><tr><td>N</td><td>P</td><td>N</td><td>N</td><td>n/a</td><td>n/a</td><td>14</td></tr><tr><td>N</td><td>P</td><td>N</td><td>P</td><td>n/a</td><td>n/a</td><td>4</td></tr><tr><td>N</td><td>P</td><td>P</td><td>N</td><td>n/a</td><td>n/a</td><td>19</td></tr><tr><td>N</td><td>P</td><td>P</td><td>P</td><td>n/a</td><td>n/a</td><td>85</td></tr><tr><td>P</td><td>P</td><td>P</td><td>P</td><td>n/a</td><td>n/a</td><td>5</td></tr><tr><td>N</td><td>n/a</td><td>N</td><td>n/a</td><td>n/a</td><td>n/a</td><td>4</td></tr><tr><td>N</td><td>n/a</td><td>P</td><td>n/a</td><td>n/a</td><td>n/a</td><td>10</td></tr><tr><td>P</td><td>n/a</td><td>P</td><td>n/a</td><td>n/a</td><td>n/a</td><td>2</td></tr><tr><td>Seropositive dogs</td><td>3.3% (7/211)</td><td>62.0% (127/205)</td><td>61.1% (135/221)</td><td>51.7% (106/205)</td><td>n/a</td><td>n/a</td><td></td></tr></tbody><thead><tr><th rowspan="2">Time points</th><th colspan="6">Booster vaccination**</th><th rowspan="2">Number of dogs</th></tr><tr><th>T0</th><th>T7</th><th>T12-T14</th><th>T18</th><th>T26</th><th>T34</th></tr></thead><tbody><tr><td rowspan="6">Trajectories of serological status</td><td>n/a</td><td>n/a</td><td>N</td><td>n/a</td><td>N</td><td>n/a</td><td>6</td></tr><tr><td>n/a</td><td>n/a</td><td>N</td><td>n/a</td><td>P</td><td>n/a</td><td>34</td></tr><tr><td>n/a</td><td>n/a</td><td>N</td><td>n/a</td><td>P</td><td>P</td><td>10</td></tr><tr><td>n/a</td><td>n/a</td><td>P</td><td>n/a</td><td>N</td><td>n/a</td><td>1</td></tr><tr><td>n/a</td><td>n/a</td><td>P</td><td>n/a</td><td>P</td><td>n/a</td><td>67</td></tr><tr><td>n/a</td><td>n/a</td><td>P</td><td>n/a</td><td>P</td><td>P</td><td>12</td></tr><tr><td>Seropositive dogs</td><td>n/a</td><td>n/a</td><td>61.5% (80/130)</td><td>n/a</td><td>94.6% (123/130)</td><td>100.0% (22/22)</td><td></td></tr></tbody></table>

## Modeling of transmission and immunity dynamics

Estimated Bayes' factor in favor of the alternative models (in which one of the four estimated parameters varies with one of the three dog characteristics) versus the reference model (with all parameters having the same value for all dogs) was always <3 (Table 2). We thus selected the reference model for further analysis.

According to the selected model, primary vaccination conferred a protective immunity in 82% of dogs (95% CI: 75–91%), for a mean duration of $1/\rho1 = 4.7$ years (95% CI: $3.1–8.2$ years). Booster vaccination induced a lifelong protective immunity (the lower bound of the credibility interval was 21 years). Both results were only marginally affected by changes of the value for the proportion of non-lethal infections (Table 3). As expected, the proportion of non-lethal infections affected the estimated force of infection.

## Evaluation of vaccination strategies

Following vaccination strategy A, 20 years after its implementation with an annual primary vaccination of 100% of the dogs <1 year old, the proportion of protected dogs was 55% (95% CI: 44–70) and 65% (95% CI: 64–78) in Kandal and Battambang, respectively (Fig 3). With vaccination strategy B (annual primary vaccination of <1 year old dogs with a systematic booster vaccination of dogs that had been vaccinated the year before), the proportion of dogs with protective immunity would reach 83% in Kandal (95% CI: 70–93%) and 85% in

<footnote>* Primary rabies vaccination on T0</footnote>

<footnote>** primary rabies vaccination on T0 and booster vaccination on T12. N negative FAVNT (<0.5 IU/mL); P positive FAVNT (≥0.5 IU/mL). n/a indicates not applicable as no sample collection was performed on the respective time point</footnote>
<table><caption>Table 1. Dynamics of serological status.</caption><thead><tr><th rowspan="2">Time points</th><th colspan="6">Primary vaccination*</th><th rowspan="2">Number of dogs</th></tr><tr><th>T0</th><th>T7</th><th>T12-T14</th><th>T18</th><th>T26</th><th>T34</th></tr></thead><tbody><tr><td rowspan="10">Trajectories of serological status</td><td>N</td><td>N</td><td>N</td><td>P</td><td>n/a</td><td>n/a</td><td>5</td></tr><tr><td>N</td><td>N</td><td>P</td><td>N</td><td>n/a</td><td>n/a</td><td>7</td></tr><tr><td>N</td><td>N</td><td>P</td><td>P</td><td>n/a</td><td>n/a</td><td>7</td></tr><tr><td>N</td><td>P</td><td>N</td><td>N</td><td>n/a</td><td>n/a</td><td>14</td></tr><tr><td>N</td><td>P</td><td>N</td><td>P</td><td>n/a</td><td>n/a</td><td>4</td></tr><tr><td>N</td><td>P</td><td>P</td><td>N</td><td>n/a</td><td>n/a</td><td>19</td></tr><tr><td>N</td><td>P</td><td>P</td><td>P</td><td>n/a</td><td>n/a</td><td>85</td></tr><tr><td>P</td><td>P</td><td>P</td><td>P</td><td>n/a</td><td>n/a</td><td>5</td></tr><tr><td>N</td><td>n/a</td><td>N</td><td>n/a</td><td>n/a</td><td>n/a</td><td>4</td></tr><tr><td>N</td><td>n/a</td><td>P</td><td>n/a</td><td>n/a</td><td>n/a</td><td>10</td></tr><tr><td>P</td><td>n/a</td><td>P</td><td>n/a</td><td>n/a</td><td>n/a</td><td>2</td></tr><tr><td>Seropositive dogs</td><td>3.3% (7/211)</td><td>62.0% (127/205)</td><td>61.1% (135/221)</td><td>51.7% (106/205)</td><td>n/a</td><td>n/a</td><td></td></tr></tbody><thead><tr><th rowspan="2">Time points</th><th colspan="6">Booster vaccination**</th><th rowspan="2">Number of dogs</th></tr><tr><th>T0</th><th>T7</th><th>T12-T14</th><th>T18</th><th>T26</th><th>T34</th></tr></thead><tbody><tr><td rowspan="6">Trajectories of serological status</td><td>n/a</td><td>n/a</td><td>N</td><td>n/a</td><td>N</td><td>n/a</td><td>6</td></tr><tr><td>n/a</td><td>n/a</td><td>N</td><td>n/a</td><td>P</td><td>n/a</td><td>34</td></tr><tr><td>n/a</td><td>n/a</td><td>N</td><td>n/a</td><td>P</td><td>P</td><td>10</td></tr><tr><td>n/a</td><td>n/a</td><td>P</td><td>n/a</td><td>N</td><td>n/a</td><td>1</td></tr><tr><td>n/a</td><td>n/a</td><td>P</td><td>n/a</td><td>P</td><td>n/a</td><td>67</td></tr><tr><td>n/a</td><td>n/a</td><td>P</td><td>n/a</td><td>P</td><td>P</td><td>12</td></tr><tr><td>Seropositive dogs</td><td>n/a</td><td>n/a</td><td>61.5% (80/130)</td><td>n/a</td><td>94.6% (123/130)</td><td>100.0% (22/22)</td><td></td></tr></tbody></table>

## Modeling of transmission and immunity dynamics

Estimated Bayes' factor in favor of the alternative models (in which one of the four estimated parameters varies with one of the three dog characteristics) versus the reference model (with all parameters having the same value for all dogs) was always <3 (Table 2). We thus selected the reference model for further analysis.

According to the selected model, primary vaccination conferred a protective immunity in 82% of dogs (95% CI: 75–91%), for a mean duration of 1/ρ1 = 4.7 years (95% CI: 3.1–8.2 years). Booster vaccination induced a lifelong protective immunity (the lower bound of the credibility interval was 21 years). Both results were only marginally affected by changes of the value for the proportion of non-lethal infections (Table 3). As expected, the proportion of non-lethal infections affected the estimated force of infection.

## Evaluation of vaccination strategies

Following vaccination strategy A, 20 years after its implementation with an annual primary vaccination of 100% of the dogs <1 year old, the proportion of protected dogs was 55% (95% CI: 44–70) and 65% (95% CI: 64–78) in Kandal and Battambang, respectively (Fig 3). With vaccination strategy B (annual primary vaccination of <1 year old dogs with a systematic booster vaccination of dogs that had been vaccinated the year before), the proportion of dogs with protective immunity would reach 83% in Kandal (95% CI: 70–93%) and 85% in

<footnote>* Primary rabies vaccination on T0</footnote>

<footnote>** primary rabies vaccination on T0 and booster vaccination on T12. N negative FAVNT (<0.5 IU/mL); P positive FAVNT (≥0.5 IU/mL). n/a indicates not applicable as no sample collection was performed on the respective time point</footnote>
<table><caption>Table 2. Bayes' factor based comparison of 15 alternative models under three scenarios of non-lethal infection probability upon exposure to RABV.</caption><thead><tr><th rowspan="2">Estimated parameter</th><th rowspan="2">Dog characteristic</th><th colspan="3">Proportion of non-lethal infections</th></tr><tr><th>π = 0.51</th><th>π = 0.05</th><th>π = 0.95</th></tr></thead><tbody><tr><td rowspan="3">q</td><td>Age group<sup>a</sup></td><td>0.01</td><td>0.02</td><td>0.01</td></tr><tr><td>Body condition<sup>b</sup></td><td>0.20</td><td>0.22</td><td>0.20</td></tr><tr><td>Sex</td><td>0.30</td><td>0.35</td><td>0.30</td></tr><tr><td rowspan="3">λ</td><td>Age group<sup>a</sup></td><td>0.07</td><td>&lt;0.01</td><td>&lt;0.01</td></tr><tr><td>Body condition<sup>b</sup></td><td>&lt;0.01</td><td>&lt;0.01</td><td>&lt;0.01</td></tr><tr><td>Sex</td><td>&lt;0.01</td><td>0.01</td><td>&lt;0.01</td></tr><tr><td rowspan="3">ρ<sub>1</sub></td><td>Age group<sup>a</sup></td><td>&lt;0.01</td><td>&lt;0.01</td><td>&lt;0.01</td></tr><tr><td>Body condition<sup>b</sup></td><td>&lt;0.01</td><td>&lt;0.01</td><td>&lt;0.01</td></tr><tr><td>Sex</td><td>0.16</td><td>0.21</td><td>0.16</td></tr><tr><td rowspan="3">ρ<sub>2</sub></td><td>Age group<sup>a</sup></td><td>&lt;0.01</td><td>&lt;0.01</td><td>&lt;0.01</td></tr><tr><td>Body condition<sup>b</sup></td><td>&lt;0.01</td><td>&lt;0.01</td><td>&lt;0.01</td></tr><tr><td>Sex</td><td>&lt;0.01</td><td>&lt;0.01</td><td>&lt;0.01</td></tr><tr><td rowspan="3">p</td><td>Age group<sup>a</sup></td><td>&lt;0.01</td><td>&lt;0.01</td><td>&lt;0.01</td></tr><tr><td>Body condition<sup>b</sup></td><td>0.03</td><td>0.05</td><td>0.03</td></tr><tr><td>Sex</td><td>0.11</td><td>0.14</td><td>0.11</td></tr></tbody></table>

π probability of non-lethal infection upon exposure; q probability of acquiring protective immunity after vaccination; λ average force of infection exerted on dogs before and during the study period; ρ₁ rate of loss of the protective immunity induced by primary vaccination; ρ₂ rate of loss of the protective immunity induced by primary and booster vaccination; p sensitivity of the serological test for dogs in the V₁ and V₂ states. a Four age groups based on quantiles of age distribution: age < 12 months, 12–23 months, 24–48 months. b Three classes of body condition score (BCS): skinny (BCS ≤3), normal (BCS 4–6) and overweight (BCS >6). In the reference model, parameter values do not depend on dog characteristics. Bayes' factor (BF) was interpreted according to the usual rules: the strength of evidence in favor of the alternative model (relative to the reference model) is substantial if 3< BF <20, strong if 20< BF <150, and very strong if BF >150

https://doi.org/10.1371/journal.pntd.0012089.t002

Battambang (95% CI: 75–94%). The recommended 70% vaccination coverage would be achieved in Battambang three years after implementation of this vaccination strategy, and in Kandal after 5 years (Fig 3).

To calculate the vaccine doses that could be saved when our proposed second strategy would be applied instead of annual vaccination of all dogs, we assumed an exponentially distributed life expectancy. For a vaccination campaign applying this new strategy running for five more years after the vaccination coverage of at least 70% is achieved, this implies per 1,000 dogs an amount of 5,200 saved vaccine doses for the Kandal dog population and 2,720 doses when applied in Battambang province (Table 4).

<table><caption>Table 3. Estimated parameter values obtained using the selected model for three scenarios of the probability of non-lethal infection upon exposure to RABV.</caption><thead><tr><th rowspan="2">Parameter</th><th rowspan="2">Unit</th><th colspan="3">Proportion of non-lethal infections</th></tr><tr><th>π = 0.51</th><th>π = 0.05</th><th>π = 0.95</th></tr></thead><tbody><tr><td>q</td><td>Probability</td><td>0.82<sup>a</sup> (0.75–0.91)<sup>b</sup></td><td>0.84 (0.75–0.94)</td><td>0.83 (0.75–0.91)</td></tr><tr><td>λ</td><td>month<sup>-1</sup></td><td>0.003 (0.001–0.004)</td><td>0.016 (0.010–0.022)</td><td>0.002 (0.001–0.003)</td></tr><tr><td>ρ<sub>1</sub></td><td>month<sup>-1</sup></td><td>0.018 (0.010–0.027)</td><td>0.022 (0.013–0.035)</td><td>0.017 (0.010–0.026)</td></tr><tr><td>ρ<sub>2</sub></td><td>month<sup>-1</sup></td><td>0.003 (0.001–0.005)</td><td>0.002 (0.000–0.006)</td><td>0.001 (0.000–0.004)</td></tr><tr><td>p</td><td>Probability</td><td>0.90 (0.79–0.97)</td><td>0.87 (0.82–0.91)</td><td>0.91 (0.87–0.94)</td></tr></tbody></table>

π probability of non-lethal infection upon exposure; q probability of acquiring protective immunity after vaccination; λ average force of infection exerted on dogs before and during the study period; ρ₁ rate of loss of the protective immunity induced by primary vaccination; ρ₂ rate of loss of the protective immunity induced by primary and booster vaccination; p sensitivity of the serological test for dogs in the V₁ and V₂ states. a Mean of the posterior distribution. b 95% credibility interval.

https://doi.org/10.1371/journal.pntd.0012089.t003
<table><caption>Table 2. Bayes' factor based comparison of 15 alternative models under three scenarios of non-lethal infection probability upon exposure to RABV.</caption><thead><tr><th rowspan="2">Estimated parameter</th><th rowspan="2">Dog characteristic</th><th colspan="3">Proportion of non-lethal infections</th></tr><tr><th>π = 0.51</th><th>π = 0.05</th><th>π = 0.95</th></tr></thead><tbody><tr><td rowspan="3">q</td><td>Age group<sup>a</sup></td><td>0.01</td><td>0.02</td><td>0.01</td></tr><tr><td>Body condition<sup>b</sup></td><td>0.20</td><td>0.22</td><td>0.20</td></tr><tr><td>Sex</td><td>0.30</td><td>0.35</td><td>0.30</td></tr><tr><td rowspan="3">λ</td><td>Age group<sup>a</sup></td><td>0.07</td><td>&lt;0.01</td><td>&lt;0.01</td></tr><tr><td>Body condition<sup>b</sup></td><td>&lt;0.01</td><td>&lt;0.01</td><td>&lt;0.01</td></tr><tr><td>Sex</td><td>&lt;0.01</td><td>0.01</td><td>&lt;0.01</td></tr><tr><td rowspan="3">ρ<sub>1</sub></td><td>Age group<sup>a</sup></td><td>&lt;0.01</td><td>&lt;0.01</td><td>&lt;0.01</td></tr><tr><td>Body condition<sup>b</sup></td><td>&lt;0.01</td><td>&lt;0.01</td><td>&lt;0.01</td></tr><tr><td>Sex</td><td>0.16</td><td>0.21</td><td>0.16</td></tr><tr><td rowspan="3">ρ<sub>2</sub></td><td>Age group<sup>a</sup></td><td>&lt;0.01</td><td>&lt;0.01</td><td>&lt;0.01</td></tr><tr><td>Body condition<sup>b</sup></td><td>&lt;0.01</td><td>&lt;0.01</td><td>&lt;0.01</td></tr><tr><td>Sex</td><td>&lt;0.01</td><td>&lt;0.01</td><td>&lt;0.01</td></tr><tr><td rowspan="3">p</td><td>Age group<sup>a</sup></td><td>&lt;0.01</td><td>&lt;0.01</td><td>&lt;0.01</td></tr><tr><td>Body condition<sup>b</sup></td><td>0.03</td><td>0.05</td><td>0.03</td></tr><tr><td>Sex</td><td>0.11</td><td>0.14</td><td>0.11</td></tr></tbody></table>

π probability of non-lethal infection upon exposure; q probability of acquiring protective immunity after vaccination; λ average force of infection exerted on dogs before and during the study period; ρ₁ rate of loss of the protective immunity induced by primary vaccination; ρ₂ rate of loss of the protective immunity induced by primary and booster vaccination; p sensitivity of the serological test for dogs in the V₁ and V₂ states. a Four age groups based on quantiles of age distribution: age < 12 months, 12–23 months, 24–48 months. b Three classes of body condition score (BCS): skinny (BCS ≤3), normal (BCS 4–6) and overweight (BCS >6). In the reference model, parameter values do not depend on dog characteristics. Bayes' factor (BF) was interpreted according to the usual rules: the strength of evidence in favor of the alternative model (relative to the reference model) is substantial if 3< BF <20, strong if 20< BF <150, and very strong if BF >150

https://doi.org/10.1371/journal.pntd.0012089.t002

Battambang (95% CI: 75–94%). The recommended 70% vaccination coverage would be achieved in Battambang three years after implementation of this vaccination strategy, and in Kandal after 5 years (Fig 3).

To calculate the vaccine doses that could be saved when our proposed second strategy would be applied instead of annual vaccination of all dogs, we assumed an exponentially distributed life expectancy. For a vaccination campaign applying this new strategy running for five more years after the vaccination coverage of at least 70% is achieved, this implies per 1,000 dogs an amount of 5,200 saved vaccine doses for the Kandal dog population and 2,720 doses when applied in Battambang province (Table 4).

<table><caption>Table 3. Estimated parameter values obtained using the selected model for three scenarios of the probability of non-lethal infection upon exposure to RABV.</caption><thead><tr><th rowspan="2">Parameter</th><th rowspan="2">Unit</th><th colspan="3">Proportion of non-lethal infections</th></tr><tr><th>π = 0.51</th><th>π = 0.05</th><th>π = 0.95</th></tr></thead><tbody><tr><td>q</td><td>Probability</td><td>0.82<sup>a</sup> (0.75–0.91)<sup>b</sup></td><td>0.84 (0.75–0.94)</td><td>0.83 (0.75–0.91)</td></tr><tr><td>λ</td><td>month<sup>-1</sup></td><td>0.003 (0.001–0.004)</td><td>0.016 (0.010–0.022)</td><td>0.002 (0.001–0.003)</td></tr><tr><td>ρ<sub>1</sub></td><td>month<sup>-1</sup></td><td>0.018 (0.010–0.027)</td><td>0.022 (0.013–0.035)</td><td>0.017 (0.010–0.026)</td></tr><tr><td>ρ<sub>2</sub></td><td>month<sup>-1</sup></td><td>0.003 (0.001–0.005)</td><td>0.002 (0.000–0.006)</td><td>0.001 (0.000–0.004)</td></tr><tr><td>p</td><td>Probability</td><td>0.90 (0.79–0.97)</td><td>0.87 (0.82–0.91)</td><td>0.91 (0.87–0.94)</td></tr></tbody></table>

π probability of non-lethal infection upon exposure; q probability of acquiring protective immunity after vaccination; λ average force of infection exerted on dogs before and during the study period; ρ₁ rate of loss of the protective immunity induced by primary vaccination; ρ₂ rate of loss of the protective immunity induced by primary and booster vaccination; p sensitivity of the serological test for dogs in the V₁ and V₂ states. a Mean of the posterior distribution. b 95% credibility interval.

https://doi.org/10.1371/journal.pntd.0012089.t003
<figure><img src="image_5.png" /><figcaption>**Fig 3.** Evolution of the proportion of protected dogs in two Cambodian provinces. For the dog population of the Cambodian provinces of Kandal and Battambang, the proportion of protected dogs were modeled for two vaccination strategies: (A) annual primary vaccination of animals <1 year old and, (B) annual primary vaccination of animals <1 year old and systematic booster vaccination of dogs that have been vaccinated the year before. Plain lines indicate the mean value and grey areas show the 95% confidence intervals. Horizontal dashed lines mark the threshold of 70% vaccination rate recommended by WOAH to control rabies circulation in a dog population, and the vertical dashed lines indicate the year at which this threshold is achieved after implementation of the strategy.</figcaption></figure>

https://doi.org/10.1371/journal.pntd.0012089.g003

Discussion

We used a post-vaccination serological follow-up to investigate the development and persistence of RABV nAbs in 351 dogs under field conditions in Cambodia after a single Rabisin vaccination, with or without booster injection one year after primary vaccination. We then

<table><caption>**Table 4.** Estimation of vaccine doses for different vaccination strategies in Kandal and Battambang province.</caption><thead><tr><th></th><th>Kandal</th><th>Battambang</th></tr></thead><tbody><tr><td>Duration (years) of strategy B*</td><td>10</td><td>8</td></tr><tr><td>Necessary vaccine doses per 1,000 dogs</td><td></td><td></td></tr><tr><td>Strategy B</td><td>4,800</td><td>5,280</td></tr><tr><td>Annual vaccination</td><td>10,000</td><td>8,000</td></tr><tr><td>Saved doses</td><td>5,200</td><td>2,720</td></tr></tbody></table>

https://doi.org/10.1371/journal.pntd.0012089.t004

<footnote>* Vaccination program for five more years after at least 70% vaccination coverage was reached</footnote>
<figure><img src="image_7.png" /><figcaption>**Fig 3.** Evolution of the proportion of protected dogs in two Cambodian provinces. For the dog population of the Cambodian provinces of Kandal and Battambang, the proportion of protected dogs were modeled for two vaccination strategies: (A) annual primary vaccination of animals <1 year old and, (B) annual primary vaccination of animals <1 year old and systematic booster vaccination of dogs that have been vaccinated the year before. Plain lines indicate the mean value and grey areas show the 95% confidence intervals. Horizontal dashed lines mark the threshold of 70% vaccination rate recommended by WOAH to control rabies circulation in a dog population, and the vertical dashed lines indicate the year at which this threshold is achieved after implementation of the strategy.</figcaption></figure>

https://doi.org/10.1371/journal.pntd.0012089.g003

Discussion

We used a post-vaccination serological follow-up to investigate the development and persistence of RABV nAbs in 351 dogs under field conditions in Cambodia after a single Rabisin vaccination, with or without booster injection one year after primary vaccination. We then

<table><caption>**Table 4.** Estimation of vaccine doses for different vaccination strategies in Kandal and Battambang province.</caption><thead><tr><th></th><th>Kandal</th><th>Battambang</th></tr></thead><tbody><tr><td>Duration (years) of strategy B*</td><td>10</td><td>8</td></tr><tr><td colspan="3">Necessary vaccine doses per 1,000 dogs</td></tr><tr><td>Strategy B</td><td>4,800</td><td>5,280</td></tr><tr><td>Annual vaccination</td><td>10,000</td><td>8,000</td></tr><tr><td>Saved doses</td><td>5,200</td><td>2,720</td></tr></tbody></table>

https://doi.org/10.1371/journal.pntd.0012089.t004

<footnote>* Vaccination program for five more years after at least 70% vaccination coverage was reached</footnote>
<table><caption>Table 5. Studies on the immunogenicity of rabies field primary vaccinations.</caption><thead><tr><td>Study site</td><td>Vaccine</td><td>Method used for serology</td><td>Number of tested dogs</td><td>Duration of study</td><td>Observed seroprevalence</td><td>Reference</td></tr></thead><tbody><tr><td>Peru</td><td>Rabisin</td><td>RFFIT</td><td>198</td><td>1 year</td><td>97%</td><td>[49]</td></tr><tr><td>Finland</td><td>Madivak</td><td>RFFIT</td><td>47</td><td>1 year</td><td>91%</td><td>[50]</td></tr><tr><td>Finland</td><td>Rabisin</td><td>RFFIT</td><td>85</td><td>1 year</td><td>84%</td><td>[50]</td></tr><tr><td>Tunisia</td><td>Rabisin</td><td>RFFIT</td><td>200</td><td>1 year</td><td>73%</td><td>[51]</td></tr><tr><td>Thailand</td><td>Rabdomun</td><td>RFFIT</td><td>31</td><td>1 year</td><td>72%</td><td>[52]</td></tr><tr><td>Cambodia</td><td>Rabisin</td><td>FAVNT</td><td>221</td><td>1 year</td><td>61%</td><td>this study</td></tr><tr><td>Japan</td><td>Various*</td><td>RFFIT</td><td>92</td><td>1 year</td><td>51%</td><td>[53]</td></tr><tr><td>Sri Lanka</td><td>Nobivac</td><td>RFFIT</td><td>110</td><td>1 year</td><td>50%</td><td>[54]</td></tr><tr><td>Indonesia</td><td>Rabisin</td><td>ELISA (Pusvetma, Indonesia)</td><td>171</td><td>90 days</td><td></td><td>[55]</td></tr></tbody></table>

used Bayesian modelling to compare immunization coverage obtained using two different vaccination strategies: (A) annual primary vaccination of animals <1 year old and, (B) annual primary vaccination of animals <1 year old with a systematic booster vaccination of dogs that have been vaccinated the year before.

The peak of antibody response after primo-vaccination is expected 2–3 weeks after vaccination [5,30,31]. Due to logistical constraints, this recommended sampling could not be achieved. However, our results are in concurrence with other studies even if findings of other studies vary greatly (Table 5). One year after the primary vaccination we observed a protective nAb titer in 61% of the dogs. Failure to induce protective immunity with single injection in our study appeared high, with 29% (n = 59) of dogs that received primary vaccination remained seronegative until T18. However, loss of immunity is probably overestimated as dogs were sampled the earliest six months after vaccination and therefore the seroconversion rate usually determined 2–3 weeks after vaccination could not be measured timely. Additionally, vaccinated dogs with a low or negative nAb level may still be protected because of cellular immunity triggered by primary vaccination [5,32].

Measuring rabies nAbs is only a proxy for protection and might not reflect the whole spectrum of the immune response and protection upon a rabies vaccination. Besides humoral immunity, vaccination induces immunologic memory even in dogs with a nAb titer < 0.1 IU/mL as seen in laboratory RABV challenge [32].

In our study, seven dogs presented positive nAb levels (≥ 0.5 IU/mL) before they received RABV vaccination. Moreover, 19 dogs were seronegative at T6, and became positive at T12 (n = 14) or T18 (n = 5) without being re-vaccinated. Vaccine-unrelated rise in nAb titers or the presence of nAb before primary vaccination is well documented [30,33–36] and can be explained either by (i) maternal immunity, (ii) non-lethal exposure and/or (iii) cross-reaction due to exposure with related lyssaviruses. The presence of maternal nAbs is highly unlikely in our study due to the age of most of the included dogs (>3 months) and the fact that rabies animal vaccination is not accessible in rural Cambodia. Maternal antibodies are unlikely to protect the pups longer than six weeks after birth [37]. Several RABV challenge trials had the issue of insufficiently virulent virus strains [32,38], building evidence for non-lethal RABV exposure. Our dogs with nAb prior to rabies vaccination could also have been exposed to a non-rabies bat lyssavirus, widely distributed in South-East Asia [39–41], as some of them are known to cross-neutralize canine RABV [42,43].

Several dogs presented unexpected serological profiles oscillating between seropositive and-negative results. Several explanations can be put forward (vaccination failure, non-lethal

<footnote>* 6 different commercially available rabies vaccines, all derived from the RC-HL strain</footnote>
<table><caption>Table 5. Studies on the immunogenicity of rabies field primary vaccinations.</caption><thead><tr><td>Study site</td><td>Vaccine</td><td>Method used for serology</td><td>Number of tested dogs</td><td>Duration of study</td><td>Observed seroprevalence</td><td>Reference</td></tr></thead><tbody><tr><td>Peru</td><td>Rabisin</td><td>RFFIT</td><td>198</td><td>1 year</td><td>97%</td><td>[49]</td></tr><tr><td>Finland</td><td>Madivak</td><td>RFFIT</td><td>47</td><td>1 year</td><td>91%</td><td>[50]</td></tr><tr><td>Finland</td><td>Rabisin</td><td>RFFIT</td><td>85</td><td>1 year</td><td>84%</td><td>[50]</td></tr><tr><td>Tunisia</td><td>Rabisin</td><td>RFFIT</td><td>200</td><td>1 year</td><td>73%</td><td>[51]</td></tr><tr><td>Thailand</td><td>Rabdomun</td><td>RFFIT</td><td>31</td><td>1 year</td><td>72%</td><td>[52]</td></tr><tr><td>Cambodia</td><td>Rabisin</td><td>FAVNT</td><td>221</td><td>1 year</td><td>61%</td><td>this study</td></tr><tr><td>Japan</td><td>Various*</td><td>RFFIT</td><td>92</td><td>1 year</td><td>51%</td><td>[53]</td></tr><tr><td>Sri Lanka</td><td>Nobivac</td><td>RFFIT</td><td>110</td><td>1 year</td><td>50%</td><td>[54]</td></tr><tr><td>Indonesia</td><td>Rabisin</td><td>ELISA (Pusvetma, Indonesia)</td><td>171</td><td>90 days</td><td></td><td>[55]</td></tr></tbody></table>

used Bayesian modelling to compare immunization coverage obtained using two different vaccination strategies: (A) annual primary vaccination of animals <1 year old and, (B) annual primary vaccination of animals <1 year old with a systematic booster vaccination of dogs that have been vaccinated the year before.

The peak of antibody response after primo-vaccination is expected 2–3 weeks after vaccination [5,30,31]. Due to logistical constraints, this recommended sampling could not be achieved. However, our results are in concurrence with other studies even if findings of other studies vary greatly (Table 5). One year after the primary vaccination we observed a protective nAb titer in 61% of the dogs. Failure to induce protective immunity with single injection in our study appeared high, with 29% (n = 59) of dogs that received primary vaccination remained seronegative until T18. However, loss of immunity is probably overestimated as dogs were sampled the earliest six months after vaccination and therefore the seroconversion rate usually determined 2–3 weeks after vaccination could not be measured timely. Additionally, vaccinated dogs with a low or negative nAb level may still be protected because of cellular immunity triggered by primary vaccination [5,32].

Measuring rabies nAbs is only a proxy for protection and might not reflect the whole spectrum of the immune response and protection upon a rabies vaccination. Besides humoral immunity, vaccination induces immunologic memory even in dogs with a nAb titer < 0.1 IU/mL as seen in laboratory RABV challenge [32].

In our study, seven dogs presented positive nAb levels (≥ 0.5 IU/mL) before they received RABV vaccination. Moreover, 19 dogs were seronegative at T6, and became positive at T12 (n = 14) or T18 (n = 5) without being re-vaccinated. Vaccine-unrelated rise in nAb titers or the presence of nAb before primary vaccination is well documented [30,33–36] and can be explained either by (i) maternal immunity, (ii) non-lethal exposure and/or (iii) cross-reaction due to exposure with related lyssaviruses. The presence of maternal nAbs is highly unlikely in our study due to the age of most of the included dogs (>3 months) and the fact that rabies animal vaccination is not accessible in rural Cambodia. Maternal antibodies are unlikely to protect the pups longer than six weeks after birth [37]. Several RABV challenge trials had the issue of insufficiently virulent virus strains [32,38], building evidence for non-lethal RABV exposure. Our dogs with nAb prior to rabies vaccination could also have been exposed to a non-rabies bat lyssavirus, widely distributed in South-East Asia [39–41], as some of them are known to cross-neutralize canine RABV [42,43].

Several dogs presented unexpected serological profiles oscillating between seropositive and-negative results. Several explanations can be put forward (vaccination failure, non-lethal

<footnote>* 6 different commercially available rabies vaccines, all derived from the RC-HL strain</footnote>
infection, lack of sensitivity of the test) but confirm again that a positive result allows to iden-
tify a protected dog but that a negative result does not allow to conclude that the dog is suscep-
tible. The fact that the serological test does not allow to detect 100% of protected dogs was
considered a lack of sensitivity in the Bayesian model. This model therefore allowed (i) to
explicitly represent the non-observable biological status of dogs and (ii) to evaluate the sensi-
tivity of serological testing. According to the model, the mean duration of immunity conferred
by primary vaccination was 4·7 years for 82% of vaccinated dogs. A booster injection one year
later allowed to induce a protective immunity when the previous injection failed, and to induce
a lifelong protective immunity in dogs already seropositive after primary vaccination. Our
models' predictions extend beyond the observed data timeframe. The short life expectancy of
dogs in Cambodia justifies our assumption of a constant rate of immunity loss post-vaccina-
tion, leading to an exponential distribution for the immunity duration. This assumption might
not hold in other contexts where dogs experience longer life expectancies. Therefore, validat-
ing this hypothesis with more extended follow-up studies is crucial for broader application.
However, the estimated duration of immunity in our study aligns with findings from previous
studies. For instance, a challenge trial showed that 80% of dogs survived more than 6 years
post-primary vaccination [32]. Furthermore, it was observed that dogs with expired vaccina-
tion status exhibited an antibody response to booster rabies vaccination comparable to that of
dogs with up-to-date vaccination status [44]. Given the fast turnover of dog populations in
rabies-endemic countries, this generic approach could be adaptable for use in these regions.
Our assumption of constant mortality rate may lead to overestimate the proportion of dogs
with protective immunity. While the current approach, which overlooks age-related variations
in mortality rates and rates of loss of protective immunity, is preliminary and in need of refine-
ment through extended follow-up, its genericity makes it potentially applicable to other rabies-
endemic countries with rapid dog population turnover.

Based on the declining seropositivity rate after one year, booster vaccinations of dogs are recommended [13]. Dogs that are primary vaccinated when < six months should receive a booster dose not later than one year following administration of the first dose [6]. Annual parenteral vaccination of ≥70% of the dog population is effective to control rabies but difficult to achieve in LMICs due to lack of adequate resources, and aggressive and/or free-roaming dogs. Several surveys from focal point vaccination campaigns showed that 15-40% of adult dogs were not brought to veterinarians because they were hard to handle [45-47]. The above-mentioned constraints may result in a declined vaccination coverage to an insufficient 20-45% [27]. Very few surveys have been carried out under field conditions, based on immunity duration and taking into account dog population dynamic to propose pragmatic vaccination strategies. A study in Kinshasa, where the dog population characteristics are close Cambodia's dog population, demonstrated that systematic vaccination of puppies as well as annual vaccination of dogs aged between 3 and 15 months would be an efficient alternative to annual MDV [48]. In Cambodia, most dogs are owned but are also free roaming and are therefore often hard to handle. In contrast, younger dogs are more reachable since they mostly stay around their owner's houses and are easier to handle. Here, we show that the proportion of protected dogs would reach 83% in Kandal and 85% in Battambang with an annual vaccination of <1 year old dogs, and a systematic booster injection of dogs that had been vaccinated the year before. This strategy would allow controlling rabies transmission in both investigated dog populations within 3-5 years. We estimated 14-34% of vaccine doses required for annual vaccination of all dogs could be saved by implementation of strategy B. Furthermore, not only vaccine doses would be saved but also personnel costs would be reduced with strategy B as less dogs need to be vaccinated and younger dogs (<12 months) are usually easier to handle than adult dogs.
infection, lack of sensitivity of the test) but confirm again that a positive result allows to iden-
tify a protected dog but that a negative result does not allow to conclude that the dog is suscep-
tible. The fact that the serological test does not allow to detect 100% of protected dogs was
considered a lack of sensitivity in the Bayesian model. This model therefore allowed (i) to
explicitly represent the non-observable biological status of dogs and (ii) to evaluate the sensi-
tivity of serological testing. According to the model, the mean duration of immunity conferred
by primary vaccination was 4·7 years for 82% of vaccinated dogs. A booster injection one year
later allowed to induce a protective immunity when the previous injection failed, and to induce
a lifelong protective immunity in dogs already seropositive after primary vaccination. Our
models' predictions extend beyond the observed data timeframe. The short life expectancy of
dogs in Cambodia justifies our assumption of a constant rate of immunity loss post-vaccina-
tion, leading to an exponential distribution for the immunity duration. This assumption might
not hold in other contexts where dogs experience longer life expectancies. Therefore, validat-
ing this hypothesis with more extended follow-up studies is crucial for broader application.
However, the estimated duration of immunity in our study aligns with findings from previous
studies. For instance, a challenge trial showed that 80% of dogs survived more than 6 years
post-primary vaccination [32]. Furthermore, it was observed that dogs with expired vaccina-
tion status exhibited an antibody response to booster rabies vaccination comparable to that of
dogs with up-to-date vaccination status [44]. Given the fast turnover of dog populations in
rabies-endemic countries, this generic approach could be adaptable for use in these regions.
Our assumption of constant mortality rate may lead to overestimate the proportion of dogs
with protective immunity. While the current approach, which overlooks age-related variations
in mortality rates and rates of loss of protective immunity, is preliminary and in need of refine-
ment through extended follow-up, its genericity makes it potentially applicable to other rabies-
endemic countries with rapid dog population turnover.

Based on the declining seropositivity rate after one year, booster vaccinations of dogs are
recommended [13]. Dogs that are primary vaccinated when < six months should receive a
booster dose not later than one year following administration of the first dose [6]. Annual par-
enteral vaccination of ≥70% of the dog population is effective to control rabies but difficult to
achieve in LMICs due to lack of adequate resources, and aggressive and/or free-roaming dogs.
Several surveys from focal point vaccination campaigns showed that 15-40% of adult dogs
were not brought to veterinarians because they were hard to handle [45-47]. The above-men-
tioned constraints may result in a declined vaccination coverage to an insufficient 20-45%
[27]. Very few surveys have been carried out under field conditions, based on immunity dura-
tion and taking into account dog population dynamic to propose pragmatic vaccination strate-
gies. A study in Kinshasa, where the dog population characteristics are close Cambodia's dog
population, demonstrated that systematic vaccination of puppies as well as annual vaccina-
tion of dogs aged between 3 and 15 months would be an efficient alternative to annual
MDV [48]. In Cambodia, most dogs are owned but are also free roaming and are therefore
often hard to handle. In contrast, younger dogs are more reachable since they mostly stay
around their owner's houses and are easier to handle. Here, we show that the proportion of
protected dogs would reach 83% in Kandal and 85% in Battambang with an annual vaccina-
tion of <1 year old dogs, and a systematic booster injection of dogs that had been vaccinated
the year before. This strategy would allow controlling rabies transmission in both investi-
gated dog populations within 3-5 years. We estimated 14-34% of vaccine doses required for
annual vaccination of all dogs could be saved by implementation of strategy B. Furthermore,
not only vaccine doses would be saved but also personnel costs would be reduced with strat-
egy B as less dogs need to be vaccinated and younger dogs (<12 months) are usually easier
to handle than adult dogs.
The here described model is the first one outlining the life-long immunity induced in dogs after only two vaccinations and its implications for rabies control. These results can contribute to adapt control measures in Cambodia, but also in other countries with similar dog population characteristics moving towards the global goal of ending human deaths from dog-mediated rabies by 2030.

**Supporting information**

**S1 Text. R and Stan codes, and anonymized raw data.** "dogs_model" was used to estimate parameters and compare different vaccination strategies, using the following stan programs: "dogs_H0.stan" file to test the null hypothesis of an absence of link between the model parameters and individual covariables, and "dogs_H1_pSe.stan", "dogs_H1_pSV.stan", "dogs_H1_rBS.stan", "dogs_H1_rVS.stan", "dogs_H1_rFOI.stan" and "dogs_H1_rFOI_cage.stan" files to test alternative assumptions.
(ZIP)

**S2 Text. Evaluation of vaccination strategies—Calculations.**
(DOCX)

**Acknowledgments**

The authors thank all dog owners who participated in the study and allowed the sampling of their animals. We also like to thank the village chiefs for the support of this study. We are grateful for the dedication of the field teams and all people involved in the organization of this study. We thank Nancy Laboratory for Rabies and Wildlife and in particular Marine Wasniewski for all the support in the implementation and maintenance of the FAVN test. We also like to thank the General Directorate of Animal Health and Production (GDAHP) of Ministry of Agriculture, Forestry and Fisheries (MAFF) for supporting this study. We would like to pay special tribute to late Dr Davun for his contribution to this work.

**Author Contributions**

**Conceptualization:** Benoit Durand, Philippe Dussart, Véronique Chevalier.

**Data curation:** Sopheak Sorn.

**Formal analysis:** Heidi Auerswald, Julia Guillebaud, Benoit Durand, Mathilde Le Vu, Saraden In.

**Funding acquisition:** Véronique Chevalier.

**Investigation:** Sopheak Sorn, Vutha Pov, Holl Davun, Véronique Chevalier.

**Methodology:** Heidi Auerswald, Julia Guillebaud, Benoit Durand, Véronique Chevalier.

**Project administration:** Véronique Chevalier.

**Supervision:** Heidi Auerswald, Julia Guillebaud, Holl Davun, Véronique Chevalier.

**Validation:** Benoit Durand, Véronique Chevalier.

**Visualization:** Heidi Auerswald.

**Writing – original draft:** Heidi Auerswald, Julia Guillebaud, Benoit Durand.
The here described model is the first one outlining the life-long immunity induced in dogs after only two vaccinations and its implications for rabies control. These results can contribute to adapt control measures in Cambodia, but also in other countries with similar dog population characteristics moving towards the global goal of ending human deaths from dog-mediated rabies by 2030.

**Supporting information**

**S1 Text. R and Stan codes, and anonymized raw data.** "dogs_model" was used to estimate parameters and compare different vaccination strategies, using the following stan programs: "dogs_H0.stan" file to test the null hypothesis of an absence of link between the model parameters and individual covariables, and "dogs_H1_pSe.stan", "dogs_H1_pSV.stan", "dogs_H1_rBS.stan", "dogs_H1_rVS.stan", "dogs_H1_rFOI.stan" and "dogs_H1_rFOI_cage.stan" files to test alternative assumptions.
(ZIP)

**S2 Text. Evaluation of vaccination strategies—Calculations.**
(DOCX)

**Acknowledgments**

The authors thank all dog owners who participated in the study and allowed the sampling of their animals. We also like to thank the village chiefs for the support of this study. We are grateful for the dedication of the field teams and all people involved in the organization of this study. We thank Nancy Laboratory for Rabies and Wildlife and in particular Marine Wasniewski for all the support in the implementation and maintenance of the FAVN test. We also like to thank the General Directorate of Animal Health and Production (GDAHP) of Ministry of Agriculture, Forestry and Fisheries (MAFF) for supporting this study. We would like to pay special tribute to late Dr Davun for his contribution to this work.

**Author Contributions**

**Conceptualization:** Benoit Durand, Philippe Dussart, Véronique Chevalier.

**Data curation:** Sopheak Sorn.

**Formal analysis:** Heidi Auerswald, Julia Guillebaud, Benoit Durand, Mathilde Le Vu, Saraden In.

**Funding acquisition:** Véronique Chevalier.

**Investigation:** Sopheak Sorn, Vutha Pov, Holl Davun, Véronique Chevalier.

**Methodology:** Heidi Auerswald, Julia Guillebaud, Benoit Durand, Véronique Chevalier.

**Project administration:** Véronique Chevalier.

**Supervision:** Heidi Auerswald, Julia Guillebaud, Holl Davun, Véronique Chevalier.

**Validation:** Benoit Durand, Véronique Chevalier.

**Visualization:** Heidi Auerswald.

**Writing – original draft:** Heidi Auerswald, Julia Guillebaud, Benoit Durand.
**Writing - review & editing:** Heidi Auerswald, Julia Guillebaud, Benoit Durand, Mathilde Le Vu, Sopheak Sorn, Saraden In, Vutha Pov, Veasna Duong, Sowath Ly, Philippe Dussart, Véronique Chevalier.

References

1. Hampson K, Coudeville L, Lembo T, Sambo M, Kieffer A, Attlan M, et al. Estimating the global burden of endemic canine rabies. PLOS Negl Trop Dis (2015) 9:e0003709. https://doi.org/10.1371/journal.pntd.0003709 PMID: 25881058

2. Wallace RM, Undurraga EA, Gibson A, Boone J, Pieracci EG, Gamble L, et al. Estimating the effectiveness of vaccine programs in dog populations. Epidemiol Infect (2019) 147:e247. https://doi.org/10.1017/S0950268819001158 PMID: 31364582

3. World Health Organization. WHO expert consultation on rabies: third report. (2018). https://apps.who.int/iris/handle/10665/272364 [Accessed March 22, 2021]

4. Morters MK, McKinley TJ, Horton DL, Cleaveland S, Schoeman JP, Restif O, et al. Achieving Population-Level Immunity to Rabies in Free-Roaming Dogs in Africa and Asia. PLOS Negl Trop Dis (2014) 8:e3160. https://doi.org/10.1371/journal.pntd.0003160 PMID: 25393023

5. Aubert M. Practical significance of rabies antibodies in cats and dogs. Rev Sci Tech OIE (1992) 11:735-760. https://doi.org/10.20506/rst.11.3.622 PMID: 1472723

6. Brown CM, Slavinski S, Ettestad P, Sidwa TJ, Sorhage FE. Compendium of Animal Rabies Prevention and Control, 2016. Journal of the American Veterinary Medical Association (2016) 248:505-517. https://doi.org/10.2460/javma.248.5.505 PMID: 26885593

7. World Organisation for Animal Health (OIE). "Rabies (Chapter 2.1.13).," *Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (Terrestrial Manual)*. (2008). p. 304–322

8. Kennedy LJ, Lunt M, Barnes A, McElhinney L, Fooks AR, Baxter DN, et al. Factors influencing the antibody response of dogs vaccinated against rabies. Vaccine (2007) 25:8500–8507. https://doi.org/10.1016/j.vaccine.2007.10.015 PMID: 18006120

9. Wallace RM, Pees A, Blanton JB, Moore SM. Risk factors for inadequate antibody response to primary rabies vaccination in dogs under one year of age. PLOS Negl Trop Dis (2017) 11:e0005761. https://doi.org/10.1371/journal.pntd.0005761 PMID: 28759602

10. Mansfield KL, Sayers R, Fooks AR, Burr PD, Snodgrass D. Factors affecting the serological response of dogs and cats to rabies vaccination. Veterinary Record (2004) 154:423-426. https://doi.org/10.1136/vr.154.14.423 PMID: 15119893

11. Nokireki T, Jakava-Viljanen M, Virtala A-M, Sihvonen L. Efficacy of rabies vaccines in dogs and cats and protection in a mouse model against European bat lyssavirus type 2. Acta Vet Scand (2017) 59:64. https://doi.org/10.1186/s13028-017-0332-x PMID: 28969696

12. Berndtsson LT, Nyman A-KJ, Rivera E, Klingeborn B. Factors associated with the success of rabies vaccination of dogs in Sweden. Acta Vet Scand (2011) 53:22. https://doi.org/10.1186/1751-0147-53-22 PMID: 21439029

13. Cliquet F, Verdier Y, Sagne L, Aubert M, Schereffer JL, Selve M, et al. Neutralising antibody titration in 25,000 sera of dogs and cats vaccinated against rabies in France, in the framework of the new regulations that offer an alternative to quarantine: -EN- -FR- -ES-. Rev Sci Tech OIE (2003) 22:857-866. https://doi.org/10.20506/rst.22.3.1437 PMID: 15005543

14. Bahloul C, Taieb D, Kaabi B, Diouani MF, Ben Hadjahmed S, Chtourou Y, et al. Comparative evaluation of specific ELISA and RFFIT antibody assays in the assessment of dog immunity against rabies. Epidemiol Infect (2005) 133:749-757. https://doi.org/10.1017/s095026880500381x PMID: 16050522

15. Servat A, Cliquet F, OIE Reference Laboratory for Rabies, WHO. Collaborative study to evaluate a new ELISA test to monitor the effectiveness of rabies vaccination in domestic carnivores. Virus Res (2006) 120:17-27. https://doi.org/10.1016/j.virusres.2006.02.011 PMID: 16603271

16. Smith TG, Gilbert AT. Comparison of a Micro-Neutralization Test with the Rapid Fluorescent Focus Inhibition Test for Measuring Rabies Virus Neutralizing Antibodies. Trop Med Infect Dis (2017) 2:24. https://doi.org/10.3390/tropicalmed2030024 PMID: 28845465

17. Moore SM. Challenges of Rabies Serology: Defining Context of Interpretation. Viruses (2021) 13:1516. https://doi.org/10.3390/v13081516 PMID: 34452381

18. Keddie SH, Baerenbold O, Keogh RH, Bradley J. Estimating sensitivity and specificity of diagnostic tests using latent class models that account for conditional dependence between tests: a simulation study. BMC Medical Research Methodology (2023) 23:58. https://doi.org/10.1186/s12874-023-01873-0 PMID: 36894883
**Writing - review & editing:** Heidi Auerswald, Julia Guillebaud, Benoit Durand, Mathilde Le Vu, Sopheak Sorn, Saraden In, Vutha Pov, Veasna Duong, Sowath Ly, Philippe Dussart, Véronique Chevalier.

References

1. Hampson K, Coudeville L, Lembo T, Sambo M, Kieffer A, Attlan M, et al. Estimating the global burden of endemic canine rabies. PLOS Negl Trop Dis (2015) 9:e0003709. https://doi.org/10.1371/journal.pntd.0003709 PMID: 25881058

2. Wallace RM, Undurraga EA, Gibson A, Boone J, Pieracci EG, Gamble L, et al. Estimating the effectiveness of vaccine programs in dog populations. Epidemiol Infect (2019) 147:e247. https://doi.org/10.1017/S0950268819001158 PMID: 31364582

3. World Health Organization. WHO expert consultation on rabies: third report. (2018). https://apps.who.int/iris/handle/10665/272364 [Accessed March 22, 2021]

4. Morters MK, McKinley TJ, Horton DL, Cleaveland S, Schoeman JP, Restif O, et al. Achieving Population-Level Immunity to Rabies in Free-Roaming Dogs in Africa and Asia. PLOS Negl Trop Dis (2014) 8:e3160. https://doi.org/10.1371/journal.pntd.0003160 PMID: 25393023

5. Aubert M. Practical significance of rabies antibodies in cats and dogs. Rev Sci Tech OIE (1992) 11:735-760. https://doi.org/10.20506/rst.11.3.622 PMID: 1472723

6. Brown CM, Slavinski S, Ettestad P, Sidwa TJ, Sorhage FE. Compendium of Animal Rabies Prevention and Control, 2016. Journal of the American Veterinary Medical Association (2016) 248:505-517. https://doi.org/10.2460/javma.248.5.505 PMID: 26885593

7. World Organisation for Animal Health (OIE). "Rabies (Chapter 2.1.13).," *Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (Terrestrial Manual)*. (2008). p. 304–322

8. Kennedy LJ, Lunt M, Barnes A, McElhinney L, Fooks AR, Baxter DN, et al. Factors influencing the antibody response of dogs vaccinated against rabies. Vaccine (2007) 25:8500–8507. https://doi.org/10.1016/j.vaccine.2007.10.015 PMID: 18006120

9. Wallace RM, Pees A, Blanton JB, Moore SM. Risk factors for inadequate antibody response to primary rabies vaccination in dogs under one year of age. PLOS Negl Trop Dis (2017) 11:e0005761. https://doi.org/10.1371/journal.pntd.0005761 PMID: 28759602

10. Mansfield KL, Sayers R, Fooks AR, Burr PD, Snodgrass D. Factors affecting the serological response of dogs and cats to rabies vaccination. Veterinary Record (2004) 154:423-426. https://doi.org/10.1136/vr.154.14.423 PMID: 15119893

11. Nokireki T, Jakava-Viljanen M, Virtala A-M, Sihvonen L. Efficacy of rabies vaccines in dogs and cats and protection in a mouse model against European bat lyssavirus type 2. Acta Vet Scand (2017) 59:64. https://doi.org/10.1186/s13028-017-0332-x PMID: 28969696

12. Berndtsson LT, Nyman A-KJ, Rivera E, Klingeborn B. Factors associated with the success of rabies vaccination of dogs in Sweden. Acta Vet Scand (2011) 53:22. https://doi.org/10.1186/1751-0147-53-22 PMID: 21439029

13. Cliquet F, Verdier Y, Sagne L, Aubert M, Schereffer JL, Selve M, et al. Neutralising antibody titration in 25,000 sera of dogs and cats vaccinated against rabies in France, in the framework of the new regulations that offer an alternative to quarantine: -EN- -FR- -ES-. Rev Sci Tech OIE (2003) 22:857-866. https://doi.org/10.20506/rst.22.3.1437 PMID: 15005543

14. Bahloul C, Taieb D, Kaabi B, Diouani MF, Ben Hadjahmed S, Chtourou Y, et al. Comparative evaluation of specific ELISA and RFFIT antibody assays in the assessment of dog immunity against rabies. Epidemiol Infect (2005) 133:749-757. https://doi.org/10.1017/s095026880500381x PMID: 16050522

15. Servat A, Cliquet F, OIE Reference Laboratory for Rabies, WHO. Collaborative study to evaluate a new ELISA test to monitor the effectiveness of rabies vaccination in domestic carnivores. Virus Res (2006) 120:17-27. https://doi.org/10.1016/j.virusres.2006.02.011 PMID: 16603271

16. Smith TG, Gilbert AT. Comparison of a Micro-Neutralization Test with the Rapid Fluorescent Focus Inhibition Test for Measuring Rabies Virus Neutralizing Antibodies. Trop Med Infect Dis (2017) 2:24. https://doi.org/10.3390/tropicalmed2030024 PMID: 28845465

17. Moore SM. Challenges of Rabies Serology: Defining Context of Interpretation. Viruses (2021) 13:1516. https://doi.org/10.3390/v13081516 PMID: 34452381

18. Keddie SH, Baerenbold O, Keogh RH, Bradley J. Estimating sensitivity and specificity of diagnostic tests using latent class models that account for conditional dependence between tests: a simulation study. BMC Medical Research Methodology (2023) 23:58. https://doi.org/10.1186/s12874-023-01873-0 PMID: 36894883
19. Bank World. Rural population (% of total population)—Cambodia. Rural population (% of total population)—Cambodia (2020) https://data.worldbank.org/indicator/SP.RUR.TOTL.ZS?end=2020&locations=KH&start=1960&view=chart [Accessed November 4, 2021]

20. Sor S, Higuchi M, Sarker MAB, Hamajima N. Knowledge of rabies and dog-related behaviors among people in Siem Reap Province, Cambodia. *Trop Med Health* (2018) 46:20. https://doi.org/10.1186/s41182-018-0102-0 PMID: 29930480

21. Chevalier V, Davun H, Sorn S, Ly P, Pov V, Ly S. Large scale dog population demography, dog management and bite risk factors analysis: A crucial step towards rabies control in Cambodia. PLOS ONE (2021) 16:e0254192. https://doi.org/10.1371/journal.pone.0254192 PMID: 34237103

22. Ly S, Buchy P, Heng NY, Ong S, Chhor N, Bourhy H, et al. Rabies situation in Cambodia. PLOS Negl Trop Dis (2009) 3:e511. https://doi.org/10.1371/journal.pntd.0000511 PMID: 19907631

23. World Organisation for Animal Health. "Use of Animals in Research and Education." *Terrestrial Code Online Access*. (2021) https://www.oie.int/en/what-we-do/standards/codes-and-manuals/terrestrial-code-online-access/ [Accessed November 5, 2021]

24. Cliquet F, Aubert M, Sagné L. Development of a fluorescent antibody virus neutralisation test (FAVN test) for the quantitation of rabies-neutralising antibody. *Journal of Immunological Methods* (1998) 212:79-87. https://doi.org/10.1016/s0022-1759(97)00212-3 PMID: 9671155

25. Moore SM, Hanlon CA. Rabies-Specific Antibodies: Measuring Surrogates of Protection against a Fatal Disease. PLOS Negl Trop Dis (2010) 4:e595. https://doi.org/10.1371/journal.pntd.0000595 PMID: 20231877

26. World Organisation for Animal Health. "Chapter 3.1.18 Rabies (Infection with Rabies Virus and Other Lyssaviruses).," *Manual of Diagnostic Tests and Vaccines for Terrestrial Animals*. (2023)

27. Hampson K, Dushoff J, Cleaveland S, Haydon DT, Kaare M, Packer C, et. Transmission dynamics and prospects for the elimination of canine rabies. PLOS Biol (2009) 7:e53. https://doi.org/10.1371/journal.pbio.1000053 PMID: 19278295

28. R Core Team. R: A language and environment for statistical computing. (2021) https://www.R-project.org/

29. Stan Development Team. RStan: the R interface to Stan. (2022) https://mc-stan.org/

30. Darkaoui S, Fassi Fihri O, Schereffer JL, Aboulfidaa N, Wasniewski M, Zouine K, et al. Immunogenicity and efficacy of Rabivac vaccine for animal rabies control in Morocco. Clin Exp Vaccine Res (2016) 5:60. https://doi.org/10.7774/cevr.2016.5.1.60 PMID: 26866025

31. Minke JM, Bouvet J, Cliquet F, Wasniewski M, Guiot AL, Lemaitre L, et al. Comparison of antibody responses after vaccination with two inactivated rabies vaccines. Veterinary Microbiology (2009) 133:283-286. https://doi.org/10.1016/j.vetmic.2008.06.024 PMID: 18757142

32. Dodds WJ, Larson LJ, Christine KL, Schultz RD. Duration of immunity after rabies vaccination in dogs: The Rabies Challenge Fund research study. Can J Vet Res (2020) 84:153-158. PMID: 32255911

33. Gold S, Donnelly CA, Nouvellet P, Woodroffe R. Rabies virus-neutralising antibodies in healthy, unvaccinated individuals: What do they mean for rabies epidemiology? PLOS Negl Trop Dis (2020) 14: e0007933. https://doi.org/10.1371/journal.pntd.0007933 PMID: 32053628

34. Kitala P, McDermott J, Kyule M, Gathuma J, Perry B, Wandeler A. Dog ecology and demography information to support the planning of rabies control in Machakos District, Kenya. Acta Tropica (2001) 78:217-230. https://doi.org/10.1016/S0001-706X(01)00082-1 PMID: 11311185

35. Smith TG, Millien M, Vos A, Fracciterne FA, Crowdis K, Chirodea C, et al. Evaluation of immune responses in dogs to oral rabies vaccine under field conditions. Vaccine (2019) 37:4743-4749. https://doi.org/10.1016/j.vaccine.2017.09.096 PMID: 29054727

36. Mauti S, Traoré A, Hattendorf J, Schelling E, Wasniewski M, Schereffer JL, et al. Factors associated with dog rabies immunisation status in Bamako, Mali. Acta Tropica (2017) 165:194-202. https://doi.org/10.1016/j.actatropica.2015.10.016 PMID: 26691990

37. Arega S, Conan A, Sabeta CT, Crafford JE, Wentzel J, Reininghaus B, et al. Rabies Vaccination of 6-Week-Old Puppies Born to Immunized Mothers: A Randomized Controlled Trial in a High-Mortality Population of Owned, Free-Roaming Dogs. TropicalMed (2020) 5:45. https://doi.org/10.3390/tropicalmed5010045 PMID: 32178448

38. Troupin C, Dacheux L, Tanguy M, Sabeta C, Blanc H, Bouchier C, et al. Large-Scale Phylogenomic Analysis Reveals the Complex Evolutionary History of Rabies Virus in Multiple Carnivore Hosts. PLoS Pathog (2016) 12:e1006041. https://doi.org/10.1371/journal.ppat.1006041 PMID: 27977811

39. Reynes J-M, Molia S, Audry L, Hout S, Ngin S, Walston J, et al. Serologic evidence of lyssavirus infection in bats, Cambodia. Emerg Infect Dis (2004) 10:2231-2234. https://doi.org/10.3201/eid1012.040459 PMID: 15663870
19. Bank World. Rural population (% of total population)—Cambodia. Rural population (% of total population)—Cambodia (2020) https://data.worldbank.org/indicator/SP.RUR.TOTL.ZS?end=2020&locations=KH&start=1960&view=chart [Accessed November 4, 2021]

20. Sor S, Higuchi M, Sarker MAB, Hamajima N. Knowledge of rabies and dog-related behaviors among people in Siem Reap Province, Cambodia. *Trop Med Health* (2018) 46:20. https://doi.org/10.1186/s41182-018-0102-0 PMID: 29930480

21. Chevalier V, Davun H, Sorn S, Ly P, Pov V, Ly S. Large scale dog population demography, dog management and bite risk factors analysis: A crucial step towards rabies control in Cambodia. PLOS ONE (2021) 16:e0254192. https://doi.org/10.1371/journal.pone.0254192 PMID: 34237103

22. Ly S, Buchy P, Heng NY, Ong S, Chhor N, Bourhy H, et al. Rabies situation in Cambodia. PLOS Negl Trop Dis (2009) 3:e511. https://doi.org/10.1371/journal.pntd.0000511 PMID: 19907631

23. World Organisation for Animal Health. "Use of Animals in Research and Education." *Terrestrial Code Online Access*. (2021) https://www.oie.int/en/what-we-do/standards/codes-and-manuals/terrestrial-code-online-access/ [Accessed November 5, 2021]

24. Cliquet F, Aubert M, Sagné L. Development of a fluorescent antibody virus neutralisation test (FAVN test) for the quantitation of rabies-neutralising antibody. *Journal of Immunological Methods* (1998) 212:79-87. https://doi.org/10.1016/s0022-1759(97)00212-3 PMID: 9671155

25. Moore SM, Hanlon CA. Rabies-Specific Antibodies: Measuring Surrogates of Protection against a Fatal Disease. PLOS Negl Trop Dis (2010) 4:e595. https://doi.org/10.1371/journal.pntd.0000595 PMID: 20231877

26. World Organisation for Animal Health. "Chapter 3.1.18 Rabies (Infection with Rabies Virus and Other Lyssaviruses).," *Manual of Diagnostic Tests and Vaccines for Terrestrial Animals*. (2023)

27. Hampson K, Dushoff J, Cleaveland S, Haydon DT, Kaare M, Packer C, et. Transmission dynamics and prospects for the elimination of canine rabies. PLOS Biol (2009) 7:e53. https://doi.org/10.1371/journal.pbio.1000053 PMID: 19278295

28. R Core Team. R: A language and environment for statistical computing. (2021) https://www.R-project.org/

29. Stan Development Team. RStan: the R interface to Stan. (2022) https://mc-stan.org/

30. Darkaoui S, Fassi Fihri O, Schereffer JL, Aboulfidaa N, Wasniewski M, Zouine K, et al. Immunogenicity and efficacy of Rabivac vaccine for animal rabies control in Morocco. Clin Exp Vaccine Res (2016) 5:60. https://doi.org/10.7774/cevr.2016.5.1.60 PMID: 26866025

31. Minke JM, Bouvet J, Cliquet F, Wasniewski M, Guiot AL, Lemaitre L, et al. Comparison of antibody responses after vaccination with two inactivated rabies vaccines. Veterinary Microbiology (2009) 133:283-286. https://doi.org/10.1016/j.vetmic.2008.06.024 PMID: 18757142

32. Dodds WJ, Larson LJ, Christine KL, Schultz RD. Duration of immunity after rabies vaccination in dogs: The Rabies Challenge Fund research study. Can J Vet Res (2020) 84:153-158. PMID: 32255911

33. Gold S, Donnelly CA, Nouvellet P, Woodroffe R. Rabies virus-neutralising antibodies in healthy, unvaccinated individuals: What do they mean for rabies epidemiology? PLOS Negl Trop Dis (2020) 14: e0007933. https://doi.org/10.1371/journal.pntd.0007933 PMID: 32053628

34. Kitala P, McDermott J, Kyule M, Gathuma J, Perry B, Wandeler A. Dog ecology and demography information to support the planning of rabies control in Machakos District, Kenya. Acta Tropica (2001) 78:217-230. https://doi.org/10.1016/S0001-706X(01)00082-1 PMID: 11311185

35. Smith TG, Millien M, Vos A, Fracciterne FA, Crowdis K, Chirodea C, et al. Evaluation of immune responses in dogs to oral rabies vaccine under field conditions. Vaccine (2019) 37:4743-4749. https://doi.org/10.1016/j.vaccine.2017.09.096 PMID: 29054727

36. Mauti S, Traoré A, Hattendorf J, Schelling E, Wasniewski M, Schereffer JL, et al. Factors associated with dog rabies immunisation status in Bamako, Mali. Acta Tropica (2017) 165:194-202. https://doi.org/10.1016/j.actatropica.2015.10.016 PMID: 26691990

37. Arega S, Conan A, Sabeta CT, Crafford JE, Wentzel J, Reininghaus B, et al. Rabies Vaccination of 6-Week-Old Puppies Born to Immunized Mothers: A Randomized Controlled Trial in a High-Mortality Population of Owned, Free-Roaming Dogs. TropicalMed (2020) 5:45. https://doi.org/10.3390/tropicalmed5010045 PMID: 32178448

38. Troupin C, Dacheux L, Tanguy M, Sabeta C, Blanc H, Bouchier C, et al. Large-Scale Phylogenomic Analysis Reveals the Complex Evolutionary History of Rabies Virus in Multiple Carnivore Hosts. PLoS Pathog (2016) 12:e1006041. https://doi.org/10.1371/journal.ppat.1006041 PMID: 27977811

39. Reynes J-M, Molia S, Audry L, Hout S, Ngin S, Walston J, et al. Serologic evidence of lyssavirus infection in bats, Cambodia. Emerg Infect Dis (2004) 10:2231-2234. https://doi.org/10.3201/eid1012.040459 PMID: 15663870
40. Lumlertdacha B, Boongird K, Wanghongsa S, Wacharapluesadee S, Chanhome L, Khawplod P, et al. Survey for bat lyssaviruses, Thailand. Emerg Infect Dis (2005) 11:232–236. https://doi.org/10.3201/eid1102.040691 PMID: 15752440

41. Nguyen ATK, Nguyen TT, Noguchi A, Nguyen DV, Ngo GC, Thong VD, et al. Bat lyssaviruses, northern Vietnam. Emerg Infect Dis (2014) 20:161–163. https://doi.org/10.3201/eid2001.130813 PMID: 24377728

42. Badrane H, Bahloul C, Perrin P, Tordo N. Evidence of Two Lyssavirus Phylogroups with Distinct Pathogenicity and Immunogenicity. J Virol (2001) 75:3268–3276. https://doi.org/10.1128/JVI.75.7.3268-3276.2001 PMID: 11238853

43. Servat A, Wasniewski M, Cliquet F. Cross-Protection of Inactivated Rabies Vaccines for Veterinary Use against Bat Lyssaviruses Occurring in Europe. Viruses (2019) 11: https://doi.org/10.3390/v11100936 PMID: 31614675

44. Moore MC, Davis RD, Kang Q, Vahl CI, Wallace RM, Hanlon CA, et al. Comparison of anamnestic responses to rabies vaccination in dogs and cats with current and out-of-date vaccination status. J Am Vet Med Assoc (2015) 246:205–211. https://doi.org/10.2460/javma.246.2.205 PMID: 25554936

45. Minyoo AB, Steinmetz M, Czupryna A, Bigambo M, Mzimbiri I, Powell G, et al. Incentives Increase Participation in Mass Dog Rabies Vaccination Clinics and Methods of Coverage Estimation Are Assessed to Be Accurate. PLOS Negl Trop Dis (2015) 9:e0004221. https://doi.org/10.1371/journal.pntd.0004221 PMID: 26633821

46. Kazadi Kawaya E, Marcotty T, Mulumba Mfumu-Kazadi L, Van Gucht S, Kirschvink N. Factors of maintenance of rabies transmission in dogs in Kinshasa, Democratic Republic of the Congo. Prev Vet Med (2020) 176:104928. https://doi.org/10.1016/j.prevetmed.2020.104928 PMID: 32113177

47. Muthiani Y, Traoré A, Mauti S, Zinsstag J, Hattendorf J. Low coverage of central point vaccination against dog rabies in Bamako, Mali. Preventive Veterinary Medicine (2015) 120:203–209. https://doi.org/10.1016/j.prevetmed.2015.04.007 PMID: 25953653

48. Kazadi Kawaya E, Marcotty T, Mfumu-Kazadi LM, Marcotty D, Van Gucht S, Kirschvink N. Evaluation of dog vaccination schemes against rabies in Kinshasa, Democratic Republic of the Congo. Prev Vet Med (2022) 198:105531. https://doi.org/10.1016/j.prevetmed.2021.105531 PMID: 34773832

49. Chomel B, Chappuis G, Bullon F, Cardenas E, de Beublain TD, Lombard M, Giambruno E. Mass Vaccination Campaign Against Rabies: Are Dogs Correctly Protected? The Peruvian Experience. Clinical Infectious Diseases (1988) 10:S697–S702. https://doi.org/10.1093/clinids/10.supplement_4.s697 PMID: 3206083

50. Sihvonen L, Kulonen K, Neuvonen E, Pekkanen K. Rabies antibodies in vaccinated dogs. Acta Vet Scand (1995) 36:87–91. https://doi.org/10.1186/BF03547705 PMID: 7572461

51. Bahloul C, Taieb D, Diouani M, Ahmed S, Chtourou Y, Bchir B, Kharmachi H, Dellagi K. Field trials of a very potent rabies DNA vaccine which induced long lasting virus neutralizing antibodies and protection in dogs in experimental conditions. Vaccine (2006) 24:1063–1072. https://doi.org/10.1016/j.vaccine.2005.09.016 PMID: 16213633

52. Tepsumethanon W, Polsuwan C, Lumlertdaecha B, Khawplod P, Hemachudha T, Chutivongse S, Wilde H, Chiewbamrungkiat M, Phanuphak P. Immune response to rabies vaccine in Thai dogs: A preliminary report. Vaccine (1991) 9:627–630. https://doi.org/10.1016/0264-410x(91)90186-a PMID: 1950096

53. Watanabe I, Yamada K, Aso A, Suda O, Matsumoto T, Yahiro T, Ahmed K, Nishizono A. Relationship between Virus-Neutralizing Antibody Levels and the Number of Rabies Vaccinations: a Prospective Study of Dogs in Japan. Jpn J Infect Dis (2013) 66:17–21. https://doi.org/10.7883/yoken.66.17 PMID: 23429079

54. Pimburage RMS, Gunatilake M, Wimalaratne O, Balasuriya A, Perera KADN. Sero-prevalence of virus neutralizing antibodies for rabies in different groups of dogs following vaccination. BMC Vet Res (2017) 13:133. https://doi.org/10.1186/s12917-017-1038-z PMID: 28521804

55. Wera E, Warembourg C, Bulu PM, Siko MM, Dürr S. Loss of binding antibodies against rabies in a vaccinated dog population in Flores Island, Indonesia. PLOS Neglected Tropical Diseases (2021) 15: e0009688. https://doi.org/10.1371/journal.pntd.0009688 PMID: 34492033
40. Lumlertdacha B, Boongird K, Wanghongsa S, Wacharapluesadee S, Chanhome L, Khawplod P, et al. Survey for bat lyssaviruses, Thailand. Emerg Infect Dis (2005) 11:232–236. https://doi.org/10.3201/eid1102.040691 PMID: 15752440

41. Nguyen ATK, Nguyen TT, Noguchi A, Nguyen DV, Ngo GC, Thong VD, et al. Bat lyssaviruses, northern Vietnam. Emerg Infect Dis (2014) 20:161–163. https://doi.org/10.3201/eid2001.130813 PMID: 24377728

42. Badrane H, Bahloul C, Perrin P, Tordo N. Evidence of Two Lyssavirus Phylogroups with Distinct Pathogenicity and Immunogenicity. J Virol (2001) 75:3268–3276. https://doi.org/10.1128/JVI.75.7.3268-3276.2001 PMID: 11238853

43. Servat A, Wasniewski M, Cliquet F. Cross-Protection of Inactivated Rabies Vaccines for Veterinary Use against Bat Lyssaviruses Occurring in Europe. Viruses (2019) 11: https://doi.org/10.3390/v11100936 PMID: 31614675

44. Moore MC, Davis RD, Kang Q, Vahl CI, Wallace RM, Hanlon CA, et al. Comparison of anamnestic responses to rabies vaccination in dogs and cats with current and out-of-date vaccination status. J Am Vet Med Assoc (2015) 246:205–211. https://doi.org/10.2460/javma.246.2.205 PMID: 25554936

45. Minyoo AB, Steinmetz M, Czupryna A, Bigambo M, Mzimbiri I, Powell G, et al. Incentives Increase Participation in Mass Dog Rabies Vaccination Clinics and Methods of Coverage Estimation Are Assessed to Be Accurate. PLOS Negl Trop Dis (2015) 9:e0004221. https://doi.org/10.1371/journal.pntd.0004221 PMID: 26633821

46. Kazadi Kawaya E, Marcotty T, Mulumba Mfumu-Kazadi L, Van Gucht S, Kirschvink N. Factors of maintenance of rabies transmission in dogs in Kinshasa, Democratic Republic of the Congo. Prev Vet Med (2020) 176:104928. https://doi.org/10.1016/j.prevetmed.2020.104928 PMID: 32113177

47. Muthiani Y, Traoré A, Mauti S, Zinsstag J, Hattendorf J. Low coverage of central point vaccination against dog rabies in Bamako, Mali. Preventive Veterinary Medicine (2015) 120:203–209. https://doi.org/10.1016/j.prevetmed.2015.04.007 PMID: 25953653

48. Kazadi Kawaya E, Marcotty T, Mfumu-Kazadi LM, Marcotty D, Van Gucht S, Kirschvink N. Evaluation of dog vaccination schemes against rabies in Kinshasa, Democratic Republic of the Congo. Prev Vet Med (2022) 198:105531. https://doi.org/10.1016/j.prevetmed.2021.105531 PMID: 34773832

49. Chomel B, Chappuis G, Bullon F, Cardenas E, de Beublain TD, Lombard M, Giambruno E. Mass Vaccination Campaign Against Rabies: Are Dogs Correctly Protected? The Peruvian Experience. Clinical Infectious Diseases (1988) 10:S697–S702. https://doi.org/10.1093/clinids/10.supplement_4.s697 PMID: 3206083

50. Sihvonen L, Kulonen K, Neuvonen E, Pekkanen K. Rabies antibodies in vaccinated dogs. Acta Vet Scand (1995) 36:87–91. https://doi.org/10.1186/BF03547705 PMID: 7572461

51. Bahloul C, Taieb D, Diouani M, Ahmed S, Chtourou Y, Bchir B, Kharmachi H, Dellagi K. Field trials of a very potent rabies DNA vaccine which induced long lasting virus neutralizing antibodies and protection in dogs in experimental conditions. Vaccine (2006) 24:1063–1072. https://doi.org/10.1016/j.vaccine.2005.09.016 PMID: 16213633

52. Tepsumethanon W, Polsuwan C, Lumlertdaecha B, Khawplod P, Hemachudha T, Chutivongse S, Wilde H, Chiewbamrungkiat M, Phanuphak P. Immune response to rabies vaccine in Thai dogs: A preliminary report. Vaccine (1991) 9:627–630. https://doi.org/10.1016/0264-410x(91)90186-a PMID: 1950096

53. Watanabe I, Yamada K, Aso A, Suda O, Matsumoto T, Yahiro T, Ahmed K, Nishizono A. Relationship between Virus-Neutralizing Antibody Levels and the Number of Rabies Vaccinations: a Prospective Study of Dogs in Japan. Jpn J Infect Dis (2013) 66:17–21. https://doi.org/10.7883/yoken.66.17 PMID: 23429079

54. Pimburage RMS, Gunatilake M, Wimalaratne O, Balasuriya A, Perera KADN. Sero-prevalence of virus neutralizing antibodies for rabies in different groups of dogs following vaccination. BMC Vet Res (2017) 13:133. https://doi.org/10.1186/s12917-017-1038-z PMID: 28521804

55. Wera E, Warembourg C, Bulu PM, Siko MM, Dürr S. Loss of binding antibodies against rabies in a vaccinated dog population in Flores Island, Indonesia. PLOS Neglected Tropical Diseases (2021) 15: e0009688. https://doi.org/10.1371/journal.pntd.0009688 PMID: 34492033