Ixodid ticks are vectors of pathogens that cause dangerous infectious and parasitic diseases in animals, leading to reduced productivity and, in some cases, mass mortality. In Kazakhstan, information on tick fauna and their epizootological role in the transmission of hemoparasites is limited. This study aimed to determine the species composition of ixodid ticks parasitizing cattle in the Almaty, Zhambyl, and Turkistan oblasts, and to assess their potential role in the transmission of Theileria and Anaplasma spp. A total of 3121 ixodid ticks were collected from cattle, belonging to the genera Hyalomma (86.9%; 2711/3121), Rhipicephalus (8.5%; 266/3121), Dermacentor (3.2%; 101/3121), and Haemaphysalis (1.4%; 43/3121). Morphological identification revealed that ticks of the genus Hyalomma, including Hyalomma scupense (31.7%), Hyalomma asiaticum (27.9%), and Hyalomma anatolicum (19.6%), were the predominant species. Rhipicephalus annulatus was recorded for the first time in the Almaty and Zhambyl oblasts. Partial sequencing of the cox1 gene confirmed the species identification obtained by morphological examination. A total of 113 representative ticks were subjected to DNA extraction for the identification of Theileria and Anaplasma species using conventional PCR with primers targeting the 28SrRNA and GroEL genes, respectively. No Anaplasma spp. were detected in the analyzed tick samples. Theileria annulata DNA was identified in seven nymphs of Hy. scupense (6.1%) collected in the Zhambyl oblast, and for the first time in Kazakhstan, in one female Rhipicephalus annulatus (0.9%) collected in the Almaty oblast. The overall infection prevalence of Theileria annulata was 7.0% (8/113).

Background and Aim: Bovine leukemia virus (BLV) is a globally distributed retrovirus that causes enzootic bovine leu-kosis, a chronic infection associated with significant economic losses in cattle. Conventional serological diagnostic tools such as agar gel immunodiffusion and enzyme-linked immunosorbent assay detect anti-BLV antibodies but cannot identify proviral DNA, especially in early infections or in calves with maternal antibodies. This study aimed to develop a sensitive and specific duplex quantitative polymerase chain reaction (qPCR) assay targeting the env gene of BLV with β-actin as an internal control and apply it for molecular surveillance and genotyping of BLV in cattle from six regions of Kazakhstan. Materials and Methods: A total of 1,680 bovine DNA samples from cattle aged over 3 years were collected from six administrative regions of Kazakhstan. A duplex qPCR assay was developed using primers targeting a conserved region of the BLV env gene and bovine β-actin. Sensitivity was assessed using plasmid and genomic DNA dilutions, and specificity was tested against existing WOAH-recommended and commercial polymerase chain reaction (PCR) protocols. Positive samples with cycle threshold <28 were subjected to nested PCR and Sanger sequencing for genotyping. Phylogenetic analysis was conducted using the maximum likelihood method. Results: The developed qPCR assay demonstrated a sensitivity of 20 plasmid copies for the env gene and 6 genomic equivalents for β-actin per reaction, with high specificity comparable to international standards. The overall BLV infection rate was 38.9%, ranging from 13% in Pavlodar to 60.5% in East Kazakhstan. Among 149 sequenced positive samples, four genotypes (G1, G4, G7, and G8) were identified. Genotype G4 was predominant, comprising 79.2% of sequences and present in all six regions. Conclusion: The duplex qPCR assay is a robust, sensitive, and cost-effective diagnostic tool for detecting BLV provirus, including in animals with maternal antibodies or early-stage infections. The regional genotypic distribution underscores the need for tailored control strategies. This molecular surveillance provides essential baseline data for national BLV eradication programs and contributes to global BLV epidemiological mapping. Keywords: bovine leukemia virus epidemiology, bovine leukemia virus, cattle, env gene, genotyping, Kazakhstan, molecular diagnostics, proviral DNA, quantitative polymerase chain reaction, β-actin.

In the pediatric population, coronavirus infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is more commonly observed in asymptomatic and moderately symptomatic cases. However, when COVID-19 coexists with other infections, it can result in a more severe disease progression. Various subtypes of the SARS-CoV-2 virus are associated with different clinical presentations, with certain subtypes exhibiting a predominance of specific symptoms or manifestations. Clinical manifestations of COVID-19 in children, both during the acute phase of the disease and in the post-COVID-19 period, are not fully understood and different compared to adults. This article presents four clinical cases in which PCR-confirmed COVID-19 was observed in combination with infections caused by viruses such as Ebstein-Barr, varicella, and rotavirus. The severity of the disease in these cases was attributed to symptoms related to intoxication, as well as skin and intestinal syndromes. To understand important genomic signatures of SARS-CoV-2 virus circulating in the area, the genome-wide sequencing was performed for the SARS-CoV-2 samples isolated from nasopharyngeal swabs collected from four patients. Based on the genotyping results, all four samples were found belonging to the SARS-CoV-2 virus Omicron lineage. Three months after the initial testing, follow-up evaluations of the children indicated a higher likelihood of complete recovery. However, some underlying disease manifestations, including anemia and atopic dermatitis, persisted despite the resolution of COVID-19 symptoms.

Для эффективной борьбы с тейлериозом крупного рогатого скота необходимы мероприятия по предотвращению распространения инфекции на благополучные регионы. В этой ситуации важным моментом является проведение систематических и достаточно масштабных серологических исследований восприимчивого поголовья, для чего необходимо наличие у ветеринарных специалистов высокочувствительных и специфичных диагностикумов. В рамках данных исследований была создана экспрессионная векторная система и отработан протокол очистки и рефолдинга рекомбинантного поверхностного протеина Theileria annulata (surface protein of T.annulata, TaSP). SDS-PAGE анализ очищенных проб показал достаточно высокий выход чистых препаратов рекомбинантного белка TaSP T. annulata с молекулярной массой 32 кДа. Полученный рекомбинантный протеин был использован в качестве антигена при оптимизации протокола иммуноферментного анализа (ИФА) для определения специфических антител в пробах плазмы крови в которых методом полимеразной цепной реакции (ПЦР), было определено наличие ДНК T.annulata. Результаты тестирования выборки условно положительных и отрицательных проб, определили сероактивность и достаточно высокий уровень специфичности полученного рекомбинантного антигена. Предварительные результаты показали перспективность использованного протеина для дополнительных исследований диагностической ценности и определения возможности его использования в качестве компонента иммунологических тест-систем.

Introduction.Phenylketonuria (PKU) is an autosomal recessive disorder of phenylalanine metabolism caused by pathogenic variants of the PAH gene. Allelic heterogeneity of the gene and the need for differential diagnosis of forms of hyperphenylalaninemia require molecular confirmation. Sanger sequencing remains valuable as an accessible and reliable method for variant verification and family analysis.The objectiveof the study was to develop and validate a unified protocol for PCR and Sanger sequencing of all coding exons of PAH with extended coverage of exon -intron boundaries.Materials and methods. A panel of 31 primers was developed to amplify 13 PAH exons, including overlapping amplicons for exon 13. Standardized PCR conditions (60 °C) were used. Validation was performed on five DNA samples, including a family trio. Sequencing was performed on an Applied Biosystems 3730xl.Results. The developed primer panel ensured reproducible and highly specific amplification of all target regions of the expected size. Sequencingdemonstrated high-quality chromatograms and complete coverage of the analyzed fragments, including overlapping regions. In the family trio, two pathogenic missense variants of the PAH gene in a state of compound heterozygosity were identified in the proband: c.728G>A (p.Arg243Gln) in exon 7 and c.1238G>C (p.Arg413Pro) in exon 12. Family analysis showed that the c.728G>A variant was paternally inherited, and c.1238G>C was maternally inherited. Additionally, a synonymous substitution c.735G>A was identified in the paternal allele. The established genotype fully corresponded to the clinical diagnosis of classical PKU and confirmed the diagnostic suitability of the developed approach.Conclusions. The proposed protocol is a reproducible, accessible and clinically informative method for molecular diagnostics of PKU and can be used both as a standalone tool and for validation of results obtained by other approaches.Keywords: phenylketonuria, hyperphenylalaninemia, exome sequencing, DNA mutation analysis.

Enteroviral infection is a common cause of aseptic meningitis, herpangina, and hand, foot, and mouth disease in children. Limited data are available on the enteroviral subtypes associated with hospitalization for these conditions in Kazakhstan. We collected cerebrospinal fluid (CSF) and nasopharyngeal swabs (NSW) from children (N=152, median age=8 years) hospitalized with symptoms of aseptic meningitis (AM, N=139) or herpangina (HA, N=13) disease. We then genotyped enteroviral subtypes associated with AM (n=50) and HA (n=9) using next-generation sequencing (NGS) on the viral protein 1 (VP1), followed up by whole-genome sequencing of the isolated viral species. All identified EVs were species B EV, consisting of five echovirus (E6, E9, E11, E21 and E25) and three coxsackievirus (CVA9, CVB3 and CVB5) serotypes within the cohort. The most abundant EVs were CVA9 (38.5%), CVB5 (21.5%) and E6 (13.8%). Most HA samples (6/9) were genotyped with coxsackievirus CVA9, while AM was associated with a variety of both echovirus and coxsackievirus serotypes. The results suggest that coxsackievirus CVA9 may be the dominant serotype circulating in the HA population, while AM is more diverse in terms of circulating echovirus and coxsackievirus serotypes. Further studies are needed to determine the clinical implications of these findings and to investigate potential differences in disease severity or outcomes associated with different EV serotypes.