Monitoring of Highly Pathogenic Swine Fever viruses (HPSFVs) in Kazakhstan - UNOG
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Central Reference Laboratory
The Republic of Kazakhstan
Monitoring of Highly Pathogenic
Swine Fever viruses (HPSFVs) in
Kazakhstan.
Primary Institute: Branch of “National Center for Biotechnology” in Almaty
Central Reference Laboratory
Project manager: профессор Мамадалиев С.М.
Project Collaborator: Dr. Guillermo R. Risatti
University of Connecticut, USA
Almaty, 2019
Project objectives: • Determine current trends of occurrence (prevalence) of HPSFVs in domestic pigs in Kazakhstan. • Describe virus associated with HPSFVs in different pig populations. • Genetically identify (partial sequencing) of selected HPSFVs detected in clinical samples obtained from swine.
North Kaz.
Pavlodar
Kostanay Akmola
54%
West Kazakhstan
East Kazakhstan 32%
Atyrau Aktobe
Karagandy
12%
Almaty
Mangystau Kyzylorda
Zhambyl
South KZ
Figure 1: Distribution of domestic swine population as percentage of total domestic
swine population (~500,000 heads in 2017) in Kazakhstan.
• The study will assess the prevalence of diseases in the Republic of Kazakhstan with significant consequences for pig health and public health, including swine influenza (SI), classical swine fever (CSF), African swine fever (ASF) and reproductive respiratory porcine syndrome (PRRS). • Despite the etiological difference of pathogens these swine diseases have similar clinical signs which often lead to errors in the diagnosis. As a result, when studying these diseases, it is necessary to have a large set of differential diagnosis methods and anamnestic data that allow a correct diagnosis.
Swine influenza (SI)
family Orthomyxoviridae,
genus Influenza А
• Swine influenza (SI) is caused by different serotypes of influenza A
viruses (IAV). The three most common viruses that affect pigs globally
are H1N1, H1N2 and H3N2
• Infections with these viruses result in rapid onset of disease that in
some cases is characterized by high morbidity and mortality events.
Immunity to influenza viruses is often short lived (6 months) and the
immunity profile in breeding herds varies considerably with time.
• SIV do not normally infect humans and it should be noticed that
transmission of SIV to humans is considered sporadic.
Figure 2: Main flyways for wild birds across Asia.
• Of important relevance to this proposal is that Southern Asia includes areas within the continent where wintering of wild birds takes place. Northward migration of wild birds through the three major Asian flyways converge during the summer time in Central and Northern Asia, including a fly pathways over Kazakhstan (Figure 2). Thus, in this way IAVs (influenza A viruses) are moved through the continent and eventually intercontinentally.
• Flu isolates belonging to the H3N6, H4N6, H1N1, H10N7, H3N8 and H13N8 subtypes were detected in Kazakhstan, Mongolia and Russia that are part of the CAF (Central Asia flyway) (Marchenko, et al., 2012). In Kazakhstan samples were collected at the Alakol Lake located in the East Kazakhstan Oblast close to the border with China. The array of flu viruses detected in the region were associated with birds of six orders, including species inhabiting aquatic and dry plain environments. • Overall the information accumulated so far indicates that the northern area of the CAF is the source of a variety of low-path and high-path flu viruses some of them with the potential to infect domestic and wild swine populations. Therefore, the location of wild bird flyways over Kazakhstan will be key for defining suitable areas for sampling swine in the country.
• In summary, due to its geographical location and certain swine
breeding practices (i.e. backyard, free- roaming), and the presence of
wild boars (Figure 3), Kazakhstan may be at the verge of HPSFVs spill-
over mainly from Russia and China— Kazakhstan’s largest neighbors.
Therefore, Northern and Eastern Kazakhstan are suitable grounds
for surveillance of HPSFs.
Figure 3: wild boar range; native (green) and
introduced (blue).
Classic swine fever virus (CSFV)
family Togaviridae,
genus Pestivirus
• Virulent CSFV infection in susceptible pigs causes severe and
widespread epidemic pig morbidity and mortality (Risatti and Borca,
2016).
• Less virulent CSFV strains or less susceptible pigs suffer decreased
growth and productivity.
• CSFV is endemic-to-epidemic in regions of South America, Central
America, and Mexico.
• In Western Europe the disease is endemic in wild boars with
occasional epidemics affecting domestic swine.
• CSFV is also widespread in Asia mainly in China (Luo et al., 2014) and
South East Asia (figure 4).Figure 4: Global distribution of CSF based on country reports to the OIE 2016 (World Animal Health Organization) (Source: “Current Policy Needs in the Effective Management in TADs in the Asia-Pacific Region -OIE Perspectives-” Trans-Boundary Animal Disease Workshop, July 2016, Manila, The Philippines).
African swine fever virus (ASFV)
family Iridoviridae
• ASFV induces a well characterized, economically important and acute,
clinically severe viral hemorrhagic fever of domestic and wild pigs
(Risatti et al., 2015).
• Thus, differential diagnostics is important since ASFV has not been
reported in most countries in Asia with the exception of the Asian
region of the Russian Federation (Figure 5).
• As on August 2018 China has reported outbreaks of ASF in different
regions of the country (Normile, 2018).Figure 5: Progression of ASF outbreaks in a period of 5 years [from: African swine fever (ASF): Five ears around Europe” 2013 Vet. Microbiol. 165(1-2): 45-60].
Porcine reproductive and respiratory syndrome virus (PRRSV)
genus Arterivirus,
family Arteriviridae
• PRRSV, was detected in early 2006 in the People’s Republic of China, following an
outbreak in which 2 million pigs were affected and 400,000 died (Tian et al.,
2007).
• The disease in domestic swine is characterized by high fever (41°C), and high
morbidity (50%-100%), and high mortality (20%-100%) rates for pigs of all ages.
• The virus originated from a type 2 PRRSV that was already circulating in China.
• New HP-PRRSVs continue to emerge and highly pathogenic forms seem to be
highly prevalent in China (Tian et al., 2007; Liu et al., 2017; Chen et al., 2018). The
virus has now been detected in China, Vietnam, the Laos People’s Democratic
Republic, Thailand, Cambodia, Russia and India.
• Prevalence of PRRSV in Kazakhstan has not been assessed. Clinically the disease
resembles CSF or ASF thus a confirmatory laboratory diagnostic test is required.Figure 6: Global distribution of PRRSV.
Selection of region for sample collection from pigs
during this project
• A study was conducted on the prevalence of the disease among
various groups of animals, which made it possible to determine the
current prevalence of highly pathogenic viral fever in pigs in two
priority regions of Kazakhstan.
• These regions are characterized as regions with marshy (rivers,
swamps, lakes) and forest landscapes, and which are located in close
proximity to the border regions of Russia and / or China (Fig. 7 and 8).Figure 7: Sampling area in North Kazakhstan Oblast in the. Petropavl area. This area is bordering with Russia could be considered a priority area The region encompasses lakes, and farming areas and major roads that go across the international border, where wild and domestic animals (swine and waterfowl) might be at close proximity and a risk of spill-over of diseases into domestic swine is possible
Figure 8: Sampling area in Eastern Kazakhstan. The selected area in East Kazakhstan Oblast including the Semey region close to the border with Russia and Lake Zaysan close to the border with China is considered at high-risk for swine disease. The Lake Alakol region in the Almaty Oblast is close to the international border with China will also be considered for surveillance program. These areas in Eastern Kazakhstan support a lot of movement particularly across the border with China.
Plan for sample collection from pigs in each region
Sample collection areas
North Kazakhstan and East Kazakhstan and Total number of samples
Pavlodar regions Almaty region for each season
Blood 400 400 800
Serum 400 400 800
Nasal swabs 400 400 800
If such an opportunity presents itself, samples from wild pigs can also be used. Samples will be
collected by four field teams.Research methods/Approaches • Sanger sequencing: specific areas/segments within the genomes of SIV/IAV (matrix, H and N genes), CSFV (NS5B, E2, 5’UTR), ASFV (p54, p72, B602L) and PRRSV (NS2, GP5) will be amplified. • Next-Generation Sequencing (NGS). Partial genome sequencing of detected viruses will be conducted by using a MinION (Oxford Nanopore technology) once the scope of positives to test becomes apparent. Genes of greatest interest will be specifically targeted for sequencing. The affordable technology is based on a device that can be used for DNA and RNA sequencing and is handled from a laptop computer. The system can produce 10- 20 gigabites of DNA sequence data. • Sample processing: RNA and DNA will be extracted from collected samples using commercial kits. Oxford Nanopore chemistry will be used for library preparation and sequencing including Direct cDNA Sequencing Kit, Direct RNA Sequencing Kit and Rapid Sequencing Kit. • Sequencing data processing: the MinION device is controlled by MinKNOW software. MinKNOW carries out several core tasks: data acquisition; real-time analysis and feedback; base-calling; data streaming; device control including selecting the run parameters; sample identification and tracking.
Results Figure 9. The areas where samples were collected at the first stage of the expedition are indicated by gray color.
The results of the ELISA analysis of samples from the North Kazakhstan
(NK) region
number of number of number of PRRS Influenza CSF ASF
№ Sample collection location
samples samples samples positive A positive positive positive
Blood Serum Swabs
NK, Magzhan Zhumabayev district,
1 Zolotaya Niva village 7 7 7 0 0 0 0
NK, Magzhan Zhumabayev district,
2 Vozvyshenka village 6 4 6 0 3 0 0
NK, Magzhan Zhumabayev district,
3 Sovetskoe village 14 17 10 0 14 0 0
4 NK, Esil district, Tarangul village 19 19 19 0 8 10 0
NK, Gabit Musrepov district,
5 Urozhainoe village 5 5 5 1 0 0 0
NK, Shalakyn district, Gorodninskoe
6 village, collective farm ”Ayna" 29 29 16 0 6 0 0
NK, Taiynshy district, Krasnaya polyana
7 village 31 31 31 0 3 0 0
8 NK, Taiynshy district, Chkalovo village 9 9 21 0 0 0 0
NK, Taiynshycity, collective farm
9 "AgroFirma" 0 45 0 0 0 11 0
Total number in North Kazakhstan 120 166 115 1 34 21 0
% of positive 1 20 13 0The results of the ELISA analysis of samples from Pavlodar region
number of number of number of PRRS Influenza CSF ASF
№ Sample collection location
samples samples samples positive A positive positive positive
Blood Serum Swabs
1 Pavlodar reg., Pavlodarskoe vil. 40 22 17 34 0
2 Pavlodar reg., Chernoyarka vil. 41 22 28 31 0
3 Pavlodar reg., Lvovka vil. 10 0 2 0 0
4 Pavlodar reg., Zhelezinka vil. 15 0 15 11 0
5 Pavlodar reg., Leninskiy vil. 20 1 20 17 0
Total number in Pavlodar region 0 126 0 45 82 93 0
% of positive 36 65 74 0The results of the ELISA analysis of samples from Karagandy region
Influenza
number of number of number of PRRS CSF ASF
№ Sample collection location A
samples samples samples positive positive positive
positive
Blood Serum Swabs
Karagandy region, Buhar Zhyrau
district, ”Doskei” village, collective
1 farm ”Medeu" 77 94 0 77 38 80 0
Total number in Karagandy region. 77 94 0 77 38 80 0
% of positive 82 40 85 0The results of the ELISA analysis of samples from East Kazakhstan (EK) region
number of number of number of PRRS Influenza CSF ASF
№ Sample collection location
samples samples samples positive A positive positive positive
Blood Serum Swabs
1 EK, Vostochnoe vil. 57 52 0 45 0
EK, Leshoz vil., collective farm
2 ”Rodnichok" 30 30 28 0 0
3 EK, Parygino vil. 5 1 0 0 0
4 EK, Donskoe vil. 10 10 4 0 0
5 EK, Teplyi kluych vil. 2 0 2 0 0
6 EK, Landmn vil. 10 10 10 4 1 0 0
7 EK, Urdjar city. 19 19 19 0 0 0 0
8 EK, Mirolyubovka vil. 5 5 7 0 0 0 0
9 EK, Vozdvizhenka vil. 4 4 4 0 0 0 0
10 EK, Preobrazhenka vil. 6 6 6 0 0 0 0
Total number in East Kazakhstan 0 148 0 97 35 45 0
% of positive 66 24 30 0The results of the ELISA analysis of samples from Almaty region
number of number of number of PRRS Influenza CSF ASF
№ Sample collection location
samples samples samples positive A positive positive positive
Blood Serum Swabs
1 Almaty reg., Tekeli city 5 5 6 0 0 0 0
2 Almaty reg., Aksu district, Kapal vil. 17 17 18 4 0 0 0
3 Almaty reg., Sarkand city 6 6 6 0 0 0 0
Almaty reg., Sarkand district,
4 Karaultobe vil. 6 6 6 0 1 0 0
5 Almaty reg., Sarkand district, Koilyk vil. 6 6 6 0 0 0 0
Almaty reg., Sarkand district, Sokolovka
6 vil. 7 7 8 0 0 0 0
7 Almaty reg., Alakol district, Akzhar vil. 2 2 3 0 0 0 0
8 Almaty reg., Alakol district, Konyr vil. 10 10 10 0 0 0 0
Almaty reg., Alakol district, Kabanbay
9 vil. 2 2 2 0 0 0 0
10 Almaty reg., Alakol district, Terekti vil 10 10 10 0 0 0 0
Total number in Almaty region 71 71 75 4 1 0 0
% of positive 6 1 0 0Conclusion on the KZ-35 project
• As a result of the study of collected samples for the detection of pathogens
of CSF, ASF, SI and PRRS the following were established :
1. No ASF pathogen was detected in all tested samples.
2. The causative agent of CSF was found in samples from the North
Kazakhstan region, in those farms where pigs are vaccinated against this
infection.
3. Antibodies against PRRS infection were also detected in samples
collected from the North Kazakhstan and Karaganda regions, where also
sows are routinely vaccinated.
4. Antibodies against swine influenza were detected within 20 to 65% of all
samples collected from the North Kazakhstan, Pavlodar, East Kazakhstan,
Karaganda regions.
5. Farms in these areas do not use any prophylactic drugs and vaccines
against swine inflienza.
6. The routine collection and testing/study of samples in these areas will
continue.KZ-35 project manager and staff Mamadaliyev S.M., professor, Zhigailov A.V., PhD Doctor of veterinary science Dmitrovsky A.M., professor, Nizkorodova A.S, Ph.D Doctor of medical science Skiba Y.A., Ph.D Maltseva E.R., PhD Ismagulova G.A., Ph.D Neupokoyeva A.S., MSc in Biotecnhology
Berdygulova Z.A., PhD Kuatbekova S., MSc in Veterinary
Ostapchuk E.O., PhD Naizabayeva D.A., MSc in Biotecnhology
Perfilyeva Y.V., Ph.D Bissenbay A.O., MSc in Biomedical
and Molecular sciences research
Kuznetsova T.V., MSc in VeterinaryInternational scientific projects on the study of biosafety problems and
infections in the Republic of Kazakhstan:
1. Project KZ-31 CBR: Rickettsia & Plague. Institute: M. AIKIMBAYEV KAZAKH SCIENTIFIC
CENTER OF QUARANTINE AND ZOONOTIC DISEASES (KSCQZD)
2. Project KZ-32 CBR: Bluetongue and brucellosis. Institute: Research Institute for Biological
Safety Problems (RIBSP)
3. Project KZ-33 CBR: MERS. Institute: Research Institute for Biological Safety Problems (RIBSP)
Project TAP-08: Saiga. Institute: Research Institute for Biological Safety Problems (RIBSP)
4. Project TAP-10: TBEV. Institute: Scientific and Practical Center for Sanitary and
Epidemiological Expertise and Monitoring.
5. Project TAP-11: Hantavirus. Institute: Ural anti-plague station.
6. Project TAP-12 Bioinformatics: Newcastle disease virus. Institute: Research Institute for
Biological Safety Problems (RIBSP)
7. ISTC Project 53: Strengthening the national legal framework and providing specialized
training in biosafety and biosecurity in the countries of Central Asia.
8. CDC Project: Study of rickettsia genotypes circulating in Pavlodar region of Kazakhstan.Conclusions • The work on ensuring biosafety of dangerous and highly dangerous infections in the Republic of Kazakhstan is carried out both through the Ministry of Health and the Ministry of Agriculture, as well as in the framework of state and international projects. • A network of reference laboratories for monitoring dangerous and ршпрдн dangerous infections in the Republic of Kazakhstan has been established and is functioning. • A legislative and legal base has been created for ensuring biosafety in the Republic of Kazakhstan, and this base is constantly being improved and many articles fully harmonize with international standards..
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