BackgroundThis study provides biochemical and molecular genetic characteristics of P. multocida isolated from dead saigas in 1988, 2010–2015 on the territory of the Republic of Kazakhstan.ResultsBacteriological samples taken from carcasses of saiga antelope during mortality events recorded in West Kazakhstan in both 2010 and 2011 and in Kostanay in 2012 and 2015 confirmed the presence of P. multocida, according to morphological and biochemical characterisation. Only in the event of 2015 was the agent proven to be the causative agent of the disease observed, haemorrhagic septicaemia. In the other mortality events it is not certain if the organism was a primary aetiology or an incidental finding as confirmatory pathological investigation was not undertaken. The implemented phylogenetic analysis of ribosomal RNA 16S gene allowed us to identify Pasteurella strains isolated in 2010–2015 as P. multocida subspecies multocida. Capsular typing by PCR showed that the studied strains isolated from dead saiga in 2010, 2011, 2012 and 2015 belonged to serotype B. MLST analysis showed that these strains of P. multocida are of the capsule type B and form one clonal grouping with isolates ST64, ST44, ST45, ST46, ST44, ST47 which isolated from cases of hemorrhagic septicemia of animals in Hungary, Burma, Sri Lanka, Pakistan and Spain. Sixteen virulence genes of the five strains of P. multocida, isolated from saigas were studied using multiplex PCR. ptfA, ompA, ompH, oma87, plpB, fimA, hsf-2, pfhA, exbB, tonB, hgbA, fur, nanB, nanH and pmHAS genes were detected in all strains. The toxA gene was not identified in the studied strains. The phylogenies of these isolates is compared across saiga populations and years and the 2015 isolate was compared to that of an isolate from a disease outbreak in 1988 and the findings suggest that these isolated bacteria are stable commensals, opportunistically pathogenic, being phylogenetically uniform with very little genetic variation notable over the last 4 decades.ConclusionIsolation, phenotypic and genetic characterization of the P. multocida isolates inform understanding of the epidemiology of infection in saigas and predict virulent potential of these opportunistic bacteria.

Peste des petits ruminant (PPR) is endemic in many Asian countries with expansion of the range in recent years including across China during 2013-2014 (OIE, 2014). Till the end of 2014, no cases of PPR virus (PPRV) were officially reported to the Office Internationale des Epizooties (OIE) from Kazakhstan. This study describes for the first time clinicopathological, epidemiological and genetic characterization of PPRV in 3 farm level outbreaks reported for the first time in Zhambyl region (oblast), southern Kazakhstan. Phylogenetic analysis based on partial N gene sequence data confirms the lineage IV PPRV circulation, similar to the virus that recently circulated in China. The isolated viruses are 99.5-99.7% identical to the PPRV isolated in 2014 from Heilongjiang Province in China and therefore providing evidence of transboundary spread of PPRV. There is a risk of further maintenance of virus in young stock despite vaccination of adult sheep and goats, along livestock trade and pastoral routes, threatening both small livestock and endangered susceptible wildlife populations throughout Kazakhstan.

bIn this study, we assessed in humans the immunogenicity and safety of one dose (7.5 or 15 g of hemagglutinin [HA]) of a whole-virion inactivated prepandemic influenza vaccine adjuvanted with aluminum hydroxide. The vaccine strain was made by reverse genetics from the highly pathogenic avian A/Chicken/Astana/6/05 (H5N1) clade 2.2 strain isolated from a dead bird in Kazakhstan. The humoral immune response was evaluated after a single vaccination by hemagglutination inhibition (HI) and microneutralization (MN) assays. The vaccine was safe and immunogenic, inducing seroconversion in 55% of the evaluated patients, with a geometric mean titer (GMT) of 17.1 and a geometric mean increase (GMI) of 3.42 after a dose of 7.5 g in the HI test against the vaccine strain. The rate of seroconversion increased up to 70% when the dose of 15 g was used. The percentages of individuals achieving anti-HA titers of >1:40 were 52.5% and 57.5% for the 7.5-and 15-g dose groups, respectively. Similar results were obtained when antibodies were analyzed in an MN test. Substantial cross-neutralization titers (seroconversion in 35% and 52.5% of subjects in the two dose groups, respectively) were detected against heterologous clade 1 strain NIBRG14 (H5N1). Thus, one dose of this whole-virion prepandemic vaccine adjuvanted with aluminum has the potential to be effective against H5N1 viruses of different clades.

We previously created a live vaccine against equine influenza based the new reassortant cold-adapted (Ca) strain A/HK/Otar/6:2/2010. The live vaccine contains surface proteins (HA, NA) from the wild-type virus A/equine/Otar/764/2007 (Н3N8; American Lineage Florida Clade 2), and internal proteins (PB2, PB1, PA, NP, M, NS) from the attenuated Ca donor virus A/Hong Kong/1/68/162/35CA (H3N2). To determine the safety and duration of the protective immune responses, 90 yearlings were intranasally vaccinated in single mode, double mode at an interval of 42 days (10(7.0) EID50/animal for both vaccinations), or with PBS (control group). Ten animals from each group were challenged with the homologous wild-type virus A/equine/Otar/764/07 (Н3N8) at 1, 2, 3, 4, 5, 6, 9 and 12 months after vaccination. Similarly, 10 animals from each group were challenged with the heterologous wild-type virus A/equine/Sydney/2888-8/07 (Н3N8; American Lineage Florida Clade 1) 12 months after vaccination. The vaccine was completely safe, and single intranasal vaccination of yearlings was capable of inducing statistically significant (from P=0.03 to P<0.0001) clinical and virological protection against the homologous virus; however, only double mode vaccination generated significant (from P=0.02 to P<0.0001) protection against the heterologous virus at 12 months (observation period). Interestingly, this vaccine enables the differentiation of infected and vaccinated animals. On this basis of this study, we recommend double intranasal administration of this vaccine at an interval of 42 days in veterinary practice.

Single intranasal administration of a modified-live EIV vaccine caused no adverse effects and vaccinated yearlings and pregnant mares did not form detectable levels of antibodies by days 7, 14 and 28 PV, as indicated by the HI reaction and ELISA. Secretory antibodies could be detected on day 7 and reached maximal levels on day 14 PV. In vitro studies showed that the yearlings and pregnant mares both formed a cell-mediated immune response by day 14 PV. The vaccine protected yearlings against challenge with wild-type virus. We conclude that single intranasal administration of the modified-live EIV vaccine was safe in the yearlings and pregnant mares that we treated, and was immunogenic and protective in the yearlings.

The development of influenza virus vectors with long insertions of foreign sequences remains difficult due to the small size and instable nature of the virus. Here, we used the influenza virus inherent property of self-optimization to generate a vector stably expressing long transgenes from the NS1 protein ORF. This was achieved by continuous selection of bright fluorescent plaques of a GFP-expressing vector during multiple passages in mouse B16f1 cells. The newly generated vector acquired stability in IFN-competent cell lines and in vivo in murine lungs. Although improved vector fitness was associated with the appearance of four coding mutations in the polymerase (PB2), haemagglutinin and non-structural (NS) segments, the stability of the transgene expression was dependent primarily on the single mutation Q20R in the nuclear export protein (NEP). Importantly, a longer insert, such as a cassette of 1299 nt encoding two Mycobacterium tuberculosis Esat6 and Ag85A proteins, could substitute for the GFP transgene. Thus, the inherent property of the influenza virus to adapt can also be used to adjust a vector backbone to give stable expression of long transgenes.

The efficacy of a novel BEI-inactivated porcine reproductive and respiratory syndrome virus (PRRSV) candidate vaccine in pigs, developed at RIBSP Republic of Kazakhstan and delivered with an adjuvant Montanide™ Gel 01 ST (D/KV/ADJ) was compared with a commercial killed PRRSV vaccine (NVDC-JXA1, C/KV/ADJ) used widely in swine herds of the Republic of Kazakhstan. Clinical parameters (body temperature and respiratory disease scores), virological and immunological profiles [ELISA and virus neutralizing (VN) antibody titers], macroscopic lung lesions and viral load in the lungs (quantitative real-time PCR and cell culture assay) were assessed in vaccinated and both genotype 1 and 2 PRRSV challenged pigs. Our results showed that the commercial vaccine failed to protect pigs adequately against the clinical disease, viremia and lung lesions caused by the challenged field isolates, Kazakh strains of PRRSV type 1 and type 2 genotypes. In contrast, clinical protection, absence of viremia and lung lesions in D/KV/ADJ vaccinated pigs was associated with generation of VN antibodies in both homologous vaccine strain LKZ/2010 (PRRSV type 2) and a heterogeneous type 1 PRRSV strain (CM/08) challenged pigs. Thus, our data indicated the induction of cross-protective VN antibodies by D/KV/ADJ vaccine, and importantly demonstrated that an inactivated PRRSV vaccine could also induce cross-protective response across the viral genotype.

The aim of this work was to evaluate the immunogenicity and neutralizing activity of sheep pox virus (SPPV; genus Capripoxvirus, family Poxviridae) structural proteins as candidate subunit vaccines to control sheep pox disease. SPPV structural proteins were identified by sequence homology with proteins of vaccinia virus (VACV) strain Copenhagen. Four SPPV proteins (SPPV-ORF 060, SPPV-ORF 095, SPPV-ORF 117, and SPPV-ORF 122), orthologs of immunodominant L1, A4, A27, and A33 VACV proteins, respectively, were produced in Escherichia coli. Western blot analysis revealed the antigenic and immunogenic properties of SPPV-060, SPPV-095, SPPV-117 and SPPV-122 proteins when injected with adjuvant into experimental rabbits. Virus-neutralizing activity against SPPV in lamb kidney cell culture was detected for polyclonal antisera raised to SPPV-060, SPPV-117, and SPPV-122 proteins. To our knowledge, this is the first report demonstrating the virus-neutralizing activities of antisera raised to SPPV-060, SPPV-117, and SPPV-122 proteins.