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.

Recombinant LSDV Strains in Asia: Vaccine Spillover or Natural Emergence?

From 2017 to 2019, several vaccine-like recombinant strains of lumpy skin disease virus (LSDV) were discovered in Kazakhstan and neighbouring regions of Russia and China. Shortly before their emergence, the authorities in Kazakhstan launched a mass vaccination campaign with the Neethling-based Lumpivax vaccine. Since none of the other countries in the affected region had used a homologous LSDV vaccine, it was soon suspected that the Lumpivax vaccine was the cause of these unusual LSDV strains. In this study, we performed a genome-wide molecular analysis to investigate the composition of two Lumpivax vaccine batches and to establish a possible link between the vaccine and the recent outbreaks. Although labelled as a pure Neethling-based LSDV vaccine, the Lumpivax vaccine appears to be a complex mixture of multiple CaPVs. Using an iterative enrichment/assembly strategy, we obtained the complete genomes of a Neethling-like LSDV vaccine strain, a KSGP-like LSDV vaccine strain and a Sudan-like GTPV strain. The same analysis also revealed the presence of several recombinant LSDV strains that were (almost) identical to the recently described vaccine-like LSDV strains. Based on their InDel/SNP signatures, the vaccine-like recombinant strains can be divided into four groups. Each group has a distinct breakpoint pattern resulting from multiple recombination events, with the number of genetic exchanges ranging from 126 to 146. The enormous divergence of the recombinant strains suggests that they arose during seed production. The recent emergence of vaccine-like LSDV strains in large parts of Asia is, therefore, most likely the result of a spillover from animals vaccinated with the Lumpivax vaccine.

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.

Molecular Characterization of Bovine Leukemia Virus with the Evidence of a New Genotype Circulating in Cattle from Kazakhstan.

Bovine leukemia virus (BLV) is a retrovirus that causes enzootic bovine leukosis (EBL) and has worldwide distribution. Infections with BLV have been reported in cattle from Kazakhstan but the virus has not yet been thoroughly characterized. In this study, we detect and estimate the level of BLV proviral DNA by qPCR in DNA samples from 119 cattle naturally infected with BLV, from 18 farms located in four different geographical regions of Kazakhstan. Furthermore, we conducted the phylogenetic and molecular analysis of 41 BLV env-gp51 gene sequences from BLV infected cattle. Phylogenetic analysis showed the affiliation of sequences to two already known genotypes G4 and G7 and also to a new genotype, classified as genotype G12. In addition, a multivariate method was employed for analysis of the association between proviral load and different variables such as the geographical location of the herd, cattle breeds, age of animals, and the presence of particular BLV genotypes. In summary, the results of this study provide the first evidence on molecular characterization of BLV circulating in cattle from Kazakhstan.

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.

The Importance of Quality Control of LSDV Live Attenuated Vaccines for Its Safe Application in the Field.

Vaccination is an effective approach to prevent, control and eradicate diseases, including lumpy skin disease (LSD). One of the measures to address farmer hesitation to vaccinate is guaranteeing the quality of vaccine batches. The purpose of this study was to demonstrate the importance of a quality procedure via the evaluation of the LSD vaccine, Lumpivax (Kevevapi). The initial PCR screening revealed the presence of wild type LSD virus (LSDV) and goatpox virus (GTPV), in addition to vaccine LSDV. New phylogenetic PCRs were developed to characterize in detail the genomic content and a vaccination/challenge trial was conducted to evaluate the impact on efficacy and diagnostics. The characterization confirmed the presence of LSDV wild-, vaccine- and GTPV-like sequences in the vaccine vial and also in samples taken from the vaccinated animals. The analysis was also suggestive for the presence of GTPV-LSDV (vaccine/wild) recombinants. In addition, the LSDV status of some of the animal samples was greatly influenced by the differentiating real-PCR used and could result in misinterpretation. Although the vaccine was clinically protective, the viral genomic content of the vaccine (being it multiple Capripox viruses and/or recombinants) and the impact on the diagnostics casts serious doubts of its use in the field.

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.

Complete Coding Sequence of a Lumpy Skin Disease Virus Strain Isolated during the 2016 Outbreak in Kazakhstan.

Lumpy skin disease virus (LSDV) causes an economically important disease in cattle. Here, we report the complete coding sequence of the LSDV isolate Kubash/KAZ/16, detected in a clinical sample from an infected cow from the outbreak reported on 7 July 2016 in Kazakhstan (Atyrau Region).

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.

Genotyping of Brucella melitensis and Brucella abortus strains in Kazakhstan using MLVA-15.

Currently, although the prevalence of brucellosis in Kazakhstan remains high, there are limited data available on the genetic diversity of circulating Brucella strains. Here, MLVA was employed to genotype a panel of 102 Brucella isolates collected from eight Kazakh regions and neighboring countries (Russia, Kyrgyzstan) during the period 1935-2017. MLVA-11 analysis classified 64 B. abortus strains into genotypes 72, 82, 331, 71, 341 and 69, while one genotype was novel, having no correspondence within the MLVA international database. MLVA-11 analysis of 37 B. melitensis strains showed 100% identity with genotypes 116, 114 and 11. One B. suis strain was classified into genotype 33. Phylogeography based on MLVA-15 demonstrated that all B. abortus and B. melitensis strains belonged to "Abortus C" and "East Mediterranean" lineages, respectively. B. abortus strains from Kazakhstan and Russia resulted genetically related to Portuguese, Brazilian and US isolates, suggesting ancient spread of these lineages from Europe westwards to South America and eastwards to Turkey, Russia and Asia. Most of Kazakh B. melitensis isolates were related to strains circulating in China, likely due to long-term trading partnerships between the two countries. In fine-scale MLVA-15 analysis, 17 B. abortus and 12 B. melitensis genotypes were identified; among them 12 are novel. Interestingly, epidemiological information supporting molecular data were retrieved for two clusters within the B. abortus group, thus proving that MLVA is an appropriate tool for effective traceback analyses. Our findings suggest that molecular genotyping should be applied systematically to support control plans for eradication of brucellosis in Kazakhstan.

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.