Establishing a molecular toolbox of lineage-specific real-time RT-PCR assays for the characterization of foot-and-mouth disease viruses circulating in Asia.

Foot-and-mouth disease (FMD) is endemic in many Asian countries, with outbreaks occurring regularly due to viruses from serotypes O, A, and Asia1 that co-circulate in the region. The ability to rapidly characterize new virus occurrences provides critical information to understand the epidemiology and risks associated with field outbreaks, and helps in the selection of appropriate vaccines to control the disease. FMD lineage-specific characterization is usually determined through sequencing; however, this capacity is not always readily available. In this study, we provide a panel of real-time RT-PCR (rRT-PCR) assays to allow differentiation of the FMD virus (FMDV) lineages known to have been co-circulating in Asia during 2020. This panel included five new rRT-PCR assays designed to detect lineages O/ME-SA/PanAsia-PanAsia-2, O/ME-SA/Ind-2001, O/SEA/Mya-98, O/CATHAY, and A/ASIA/Sea-97, along with three published rRT-PCR assays for A/ASIA/Iran-05, A/ASIA/G-VII, and Asia1 serotypes. Samples of known FMD lineage (n = 85) were tested in parallel with all eight lineage-specific assays and an established 3D pan-FMD rRT-PCR assay, and comparative limit of detection (LOD) experiments were conducted for the five newly developed assays. All samples (85/85) were assigned to the correct serotype, and the correct lineage was assigned for 70 out of 85 samples where amplification only occurred with the homologous assay. For 13 out of 85 of the samples, there was amplification in two assays; however, the correct lineage could be designated based on the strongest Ct values for 12 out of 13 samples. An incorrect lineage was assigned for 3 out of 85 samples. The amplification efficiencies for the five new rRT-PCR assays ranged between 79.7 and 100.5%, with nucleic acid dilution experiments demonstrating broadly equivalent limits of detection when compared to the 3D pan-FMD rRT-PCR assay. These new tests, together with other published lineage-specific rRT-PCR assays, constitute a panel of assays (or molecular toolbox) that can be selected for use in FMD endemic countries (individually or a subset of the assays depending on region/lineages known to be circulating) for rapid characterization of the FMDV lineages circulating in Asia at a relatively low cost. This molecular toolbox will enhance the ability of national laboratories in endemic settings to accurately characterize circulating FMDV strains and facilitate prompt implementation of control strategies, and may be particularly useful in settings where it is difficult to access sequencing capability.

The progressive control of foot-and-mouth disease (FMD) in the Republic of Kazakhstan: Successes and challenges.

Foot-and-mouth disease (FMD) has historically caused far-reaching economic losses to many regions worldwide. FMD control has been problematic, and the disease is still prevalent in many West and Central Asia countries. Here, we review the progress made by Kazakhstan in achieving freedom from FMD and discuss some of the challenges associated with maintaining the FMD-free status, as evidenced by the occurrence of an outbreak in 2022. A combination of zoning, movement control, vaccination, and surveillance strategies led to eliminating the disease in the country. However, the circulation of the FMD virus in the region still imposes a risk for Kazakhstan, and coordinated strategies are ultimately needed to support disease elimination. The results presented here may help design effective pathways to progressively eliminate the disease in West and Central Asia while promoting the design and implementation of regional actions to support FMD control.

Allelic polymorphisms of the BoLA-DRB3 gene and resistance to brucellosis in Kazakh cattle.

The Authors investigated polymorphism of the bovine BoLA­DRB3 gene in connection with resistance or susceptibility to brucellosis of two Kazakh meat breeds, Auliekol and Kazakh Whiteheaded breeds, using PCR­RFLP. In Auliekol cattle (n = 158), 22 alleles were detected in the brucellosis group, and 24 alleles were shown in the healthy group. BoLA-DRB3 alleles *3, *4, *19, *21 were more common in healthy animals, while Brucella­positive cattle were more frequently carriers of alleles *7, *10, *18. In Kazakh Whiteheaded cattle (n = 146), 21 alleles were detected in infected and 23 alleles in healthy cattle. Alleles *3, *8, *21 significantly predominate in healthy cattle, while alleles *7, *11, *16 are typical for animals with brucellosis. This study identified BoLA-DRB3 alleles associated with genetic resistance (*3 and *21) and susceptibility (*7) to brucellosis; remarkably, resistance alleles are shared by two important meat breeds of Kazakhstan.

Toward the calibration of serological assays using sera collected from cattle and sheep following a single dose of foot-and-mouth disease vaccine.

Background and Aim: Serological assays are widely used to monitor the performance of foot-and-mouth disease (FMD) vaccines to estimate vaccination coverage and to ensure that vaccinated animals generate adequate immune responses. This study aimed to measure the FMD virus (FMDV)-specific responses in cattle and sheep after a single dose of a trivalent FMD vaccine containing serotypes A, O, and Asia-1, and to use these sera to calibrate virus neutralization tests (VNTs) and serotype-specific serological enzyme-linked immunoassays (ELISAs) that can measure post-vaccination responses. Materials and Methods: Sera were collected from cattle (n=10) and sheep (n=10) on 0, 21, and 56 days after immunization with an imported aqueous formulated FMD vaccine. These samples were tested by VNT using field FMDV isolates that are representative of the epidemiological risks in Central Asia (A/ASIA/Iran-05, A/ASIA/GVII, O/ME-SA/Ind-2001, O/SEA/Mya-98, O/ME-SA/PanAsia, and Asia-1 Shamir). Heterologous VNT antibody responses were compared to those measured using commercial FMDV-specific ELISAs for serotypes O, A, and Asia 1. Results: Administration of the FMD vaccine increased FMDV-specific antibody titers for both species in sera collected on day 21, but these elevated titers were short-lived and were decreased by day 56. Conclusion: These results highlight the short duration of immunity with a single dose of this aqueous vaccine and motivate further studies to assess immune responses in cattle and small ruminants after a two-dose course vaccination schedule. Further comparative data for VNT and serotype-specific ELISAs are needed to define cutoffs that can be used to monitor post-vaccination immune responses in low-containment laboratories where it is not possible to handle live FMDVs.

Mapping the risks of the spread of peste des petits ruminants in the Republic of Kazakhstan.

Peste des petits ruminants (PPR) is a viral transboundary disease seen in small ruminants, that causes significant damage to agriculture. This disease has not been previously registered in the Republic of Kazakhstan (RK). This paper presents an assessment of the susceptibility of the RK's territory to the spread of the disease in the event of its importation from infected countries. The negative binomial regression model that was trained on the PPR outbreaks in China, was used to rank municipal districts in the RK in terms of PPR spread risk. The outbreak count per administrative district was used as a risk indicator, while a number of socio-economic, landscape, and climatic factors were considered as explanatory variables. Summary road length, altitude, the density of small ruminants, the maximum green vegetation fraction, cattle density, and the Engel coefficient were the most significant factors. The model demonstrated a good performance in training data (R2  = 0.69), and was transferred to the RK, suggesting a significantly lower susceptibility of this country to the spread of PPR. Hot spot analysis identified three clusters of districts at the highest risk, located in the western, eastern, and southern parts of Kazakhstan. As part of the study, a countrywide survey was conducted to collect data on the distribution of livestock populations, which resulted in the compilation of a complete geo-database of small ruminant holdings in the RK. The research results may be used to formulate a national strategy for preventing the importation and spread of PPR in Kazakhstan through targeted monitoring in high-risk areas.

Risk for African Swine Fever Introduction Into Kazakhstan.

African swine fever (ASF) is a disease of swine that is endemic to some African countries and that has rapidly spread since 2007 through many regions of Asia and Europe, becoming endemic in some areas of those continents. Since there is neither vaccine nor treatment for ASF, prevention is an important action to avoid the economic losses that this disease can impose on a country. Although the Republic of Kazakhstan has remained free from the disease, some of its neighbors have become ASF-infected, raising concerns about the potential introduction of the disease into the country. Here, we have identified clusters of districts in Kazakhstan at highest risk for ASF introduction. Questionnaires were administered, and districts were visited to collect and document, for the first time, at the district level, the distribution of swine operations and population in Kazakhstan. A snowball sampling approach was used to identify ASF experts worldwide, and a conjoint analysis model was used to elicit their opinion in relation to the extent at which relevant epidemiological factors influence the risk for ASF introduction into disease-free regions. The resulting model was validated using data from the Russian Federation and Mongolia. Finally, the validated model was used to rank and categorize Kazakhstani districts in terms of the risk for serving as the point of entry for ASF into the country, and clusters of districts at highest risk of introduction were identified using the normal model of the spatial scan statistic. Results here will help to allocate resources for surveillance and prevention activities aimed at early detecting a hypothetical ASF introduction into Kazakhstan, ultimately helping to protect the sanitary status of the country.

Economic and health burden of brucellosis in Kazakhstan.

Brucellosis is a widespread zoonotic disease considered as an emerging and re-emerging disease with a resulting threat of public health and animal health. Official reports document an animal incidence in Kazakhstan of about 0.6% per year, and the country still registers high number of human cases annually . The main objective of this paper was to evaluate the distribution and economic impact of brucellosis in Kazakhstan. We analysed human disease incidence data obtained from the Government Sanitary & Epidemiological Service with the aim to estimate the burden of disease in terms of disability-adjusted life years (DALYs). We also estimated the economic impact in terms of monetary losses. Additionally, we mapped the geographical distribution of the disease throughout Kazakhstan. In total, 1,334 human cases of brucellosis were registered in 2015 in Kazakhstan that resulted in 713 DALYs. Around $21 million was spent on compensation for animals that had to be slaughtered due to brucellosis, and an additional $24 million was spent on testing animals. Animal brucellosis and human brucellosis occur throughout the whole country, some trends of which are reviewed in this paper. We estimated the burden of the disease and explored possible explanation for high human incidence rates. This paper is the first to estimate the human burden of disease and the economic costs in Kazakhstan. Both of these are substantial.

Development and evaluation of a novel real-time RT-PCR to detect foot-and-mouth disease viruses from the emerging A/ASIA/G-VII lineage.

A new lineage of foot-and-mouth disease virus (FMDV), called A/ASIA/G-VII, emerged from the Indian subcontinent in 2015 and continues to spread in Western Asia. Currently, the distribution of viruses belonging to this lineage is defined using sequencing approaches, but other cheaper and faster diagnostic methods are urgently needed. Thus, this study describes the development and validation of a novel A/ASIA/G-VII lineage-specific real-time RT-PCR (rRT-PCR). Diagnostic sensitivity and specificity were evaluated using representative field specimens and isolates from the A/ASIA/G-VII lineage, as well as samples comprising other FMDV lineages that co-circulate in Asia (n=54). This lineage-specific assay accurately detected all A/ASIA/G-VII samples tested (n=29), and no detection was observed for samples belonging to other FMDV lineages (n=25), namely A/ASIA/Sea-97, A/ASIA/Iran-05SIS-10, A/ASIA/Iran-05FAR-11, Asia1/ASIA/Sindh-08, O/CATHAY, O/ME-SA/PanAsia-2ANT-10, O/ME-SA/Ind-2001d, O/SEA/Mya-98. Additionally, the limit of detection was found to be at least equivalent to a pan-serotypic rRT-PCR assay. Therefore, these data indicate that this newly developed rRT-PCR assay can be applied to characterise field isolates in countries where the A/ASIA/G-VII lineage is endemic, as well as to monitor new incursions and outbreaks due to this lineage.

Zoning the territory of the Republic of Kazakhstan as to the risk of rabies among various categories of animals.

This paper presents the zoning of the territory of the Republic of Kazakhstan with respect to the risk of rabies outbreaks in domestic and wild animals considering environmental and climatic conditions. The national database of rabies outbreaks in Kazakhstan in the period 2003-2014 has been accessed in order to find which zones are consistently most exposed to the risk of rabies in animals. The database contains information on the cases in demes of farm livestock, domestic animals and wild animals. To identify the areas with the highest risk of outbreaks, we applied the maximum entropy modelling method. Designated outbreaks were used as input presence data, while the bioclim set of ecological and climatic variables, together with some geographic factors, were used as explanatory variables. The model demonstrated a high predictive ability. The area under the curve for farm livestock was 0.782, for domestic animals -0.859 and for wild animals - 0.809. Based on the model, the map of integral risk was designed by following four categories: negligible risk (disease-free or favourable zone), low risk (surveillance zone), medium risk (vaccination zone), and high risk (unfavourable zone). The map was produced to allow developing a set of preventive measures and is expected to contribute to a better distribution of supervisory efforts from the veterinary service of the country.