Vaccination Of Animals Research Articles

Rabbit Spleen and Medicinal Herbs: Animal Infectious Diseases, Grassroots Communes, and the State in Maoist China

summary: This article explores how Mao-era China responded to major epizootic and zoonotic diseases. It foregrounds a series of patterns in fighting contagious animal diseases—lockdowns, quarantines, disinfection, mass animal vaccination, mass education, and prioritizing the treatment of infected animals over mass culling—which were together called the Comprehensive Prevention and Treatment (CPT). Shedding light on this understudied topic in the fields of the history of medicine and of the People’s Republic of China (PRC), the author argues that it was not the central or provincial governments but rather local communes that led the effort to protect livestock from animal infectious diseases. This article critically demonstrates how the story of the CPT highlights the resilience of communal actors as well as the possibilities and limitations of the Maoist ideal of self-reliance.

Assessing the impact of preventative measures to limit the spread of Toxoplasma gondii in wild carnivores of Madagascar.

Novel multihost pathogens can threaten endangered wildlife species, as well as humans and domestic animals. The zoonotic protozoan parasite Toxoplasma gondii is transmitted by members of Felidae and can infect a large number of animal species, including humans. This parasite can have significant health consequences for infected intermediate hosts and could further endanger wild carnivore populations of Madagascar. Building on an empirical characterization of the prevalence of the pathogen in local mammals, we used mathematical models of pathogen transmission in a multihost community to compare preventative measures that aim to limit the spread of this parasite in wild carnivores. Specifically, we examined the effect of hypothetical cat vaccination and population control campaigns on reducing the risk of infection by T. gondii in wild Eupleridae. Our model predicted that the prevalence of exposure to T. gondii in cats would be around 72% and that seroprevalence would reach 2% and 43% in rodents and wild carnivores, respectively. Reducing the rodent population in the landscape by half may only decrease the prevalence of T. gondii in carnivores by 10%. Similarly, cat vaccination and reducing the population of definitive hosts had limited impact on the prevalence of T. gondii in wild carnivorans of Madagascar. A significant reduction in prevalence would require extremely high vaccination, low turnover, or both in the cat population. Other potential control methods of T. gondii in endangered Eupleridae include targeted vaccination of wild animals but would require further investigation. Eliminating the threat entirely will be difficult because of the ubiquity of cats and the persistence of the parasite in the environment.

Comparative structural and immunological analysis of outer membrane proteins and dermonecrotic toxin in Bordetella bronchiseptica canine isolate

Bordetella bronchiseptica is a pathogen causing respiratory infections in mammals. With the improving understanding of companion animals’ welfare, addressing the side effects of bordetella vaccine gains importance in dogs. Studies on diverse subunit vaccines are actively pursued in humans to safely and effectively control bordetellosis. Therefore, our objective was to develop a canine bordetella vaccine inspired by human vaccine development. We evaluated the immunogenicity of the two bacterial components: the outer membrane proteins (OMPs) and the dermonecrotic toxin (DNT) from a canine isolate of B. bronchiseptica. In-silico analysis identified eight domains of DNT, and Domain 3 was selected as the most promising antigen candidate. Additionally, the OMPs were extracted and examined using SDS-PAGE and Western blot analysis. The distinct immunological characteristic of OMPs and DNT-3 were examined individually and in combination. Gene expression and cytokine production were also evaluated in DH82 cells after stimulation with those antigens. Treatment with OMPs resulted in higher level of Th1 related cytokines, while DNT-3 induced a predominant response associated with Th17 and Th2 in the cytokine production. Synergistic effects were observed exclusively on IL-23, indicating increase of a potential risk of side effects when OMPs and DNT act together. These findings provide valuable insights into the reactogenicity of conventional Bordetella vaccines. Further, the presented preclinical data in this study offer an alternative method of the development for an optimal next-generation Bordetella vaccine for companion animals and humans, replacing the acellular vaccines containing both toxin and protein components.

Organization of veterinary services in the territories of the Leningrad region

The problem of organizing veterinary activities in the territories of horticultural, horticultural and other non-profit associations of citizens needs attention. Many land owners keep agricultural, pet companions. They often do not comply with the norms of legislation in the field of veterinary medicine, there is a concealment of facts and violation of the rules of maintenance, animal diseases, we see ignorance of the owners about the signs of manifestation, lack of awareness of the threats of a particular disease. At the same time, there is no centralized accounting of animals kept on the territory of the non-commercial gardening, as there is no work to perform veterinary knowledge. It is necessary to plan veterinary and educational work: organize meetings with representatives of the non-commercial gardening administration, animal owners, and present information material in the form of posters and leaflets on the ground. It is necessary to place sections on the electronic resources of the animal disease control station, non-commercial gardening containing in an accessible form the information on the maintenance, feeding of agricultural and domestic unproductive animals, carrying out antiepizootic measures, it is advisable to provide regulatory and legal information, inform in an accessible form about the need for veternary and sanitary examination of products obtained on the territory of the non-commercial gardening. The issue of registration and vaccination of animals can also be solved by organizing field events of representatives of the district animal disease control station in the non-commercial gardening. It seems necessary to change the approach to the list of acceptable ways of using land plots belonging to the category of agricultural land with an established type of permitted use for horticulture, so that among them there is the maintenance of farm animals.

Cell-engineered virus-mimetic nanovesicles for vaccination against enveloped viruses.

Enveloped viruses pose a significant threat to human health, as evidenced by the recent COVID-19 pandemic. Although current vaccine strategies have proven effective in preventing viral infections, the development of innovative vaccine technologies is crucial to fortify our defences against future pandemics. In this study, we introduce a novel platform called cell-engineered virus-mimetic nanovesicles (VNVs) and demonstrate their potential as a vaccine for targeting enveloped viruses. VNVs are generated by extruding plasma membrane-derived blebs through nanoscale membrane filters. These VNVs closely resemble enveloped viruses and extracellular vesicles (EVs) in size and morphology, being densely packed with plasma membrane contents and devoid of materials from other membranous organelles. Due to these properties, VNVs express viral membrane antigens more extensively and homogeneously than EVs expressing the same antigen. In this study, we produced severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) VNVs expressing the SARS-CoV-2 Spike glycoprotein (S) on their surfaces and assessed their preclinical efficacy as a COVID-19 vaccine in experimental animals. The administration of VNVs successfully stimulated the production of S-specific antibodies both systemically and locally, and immune cells isolated from vaccinated mice displayed cytokine responses to S stimulation.

A case study on anthrax in an eight-month old infant at Kyrgyz Republic.

Anthrax remains a threat, especially in countries like Kyrgyzstan with developed livestock farming. Despite preventive efforts, sporadic outbreaks endure on an annual basis, transmitted from infected animals to humans. Here, we report a severe anthrax case in an 8-month-old child known to be caused when a sick calf was slaughtered in the neighborhood without proper protocols, resulting in intra-family infection. This underscores the importance of swift diagnosis, treatment, preventive measures, and awareness of zoonotic infections, animal vaccination and adherence to sanitary and veterinary protocols.

WSV2023 - The second meeting of the world society for virology: One health - One world - One virology

The Second International Conference of the World Society for Virology (WSV), hosted by Riga Stradiņš University, was held in Riga, Latvia, on June 15–17th, 2023. It prominently highlighted the recent advancements in different disciplines of virology. The conference had fourteen keynote speakers covering diverse topics, including emerging virus pseudotypes, Zika virus vaccine development, herpesvirus capsid mobility, parvovirus invasion strategies, influenza in animals and birds, West Nile virus and Marburg virus ecology, as well as the latest update in animal vaccines. Discussions further explored SARS-CoV-2 RNA replicons as vaccine candidates, SARS-CoV-2 in humans and animals, and the significance of plant viruses in the ‘One Health' paradigm. The presence of the presidents from three virology societies, namely the American, Indian, and Korean Societies for Virology, highlighted the event's significance. Additionally, past president of the American Society for Virology (ASV), formally declared the partnership between ASV and WSV during the conference.

Knowledge, attitudes, and practices regarding brucellosis in a rural population: A cross-sectional study

Brucellosis is recognized as a significant global zoonotic infection that disproportionately affects low-income settings. A community cross-sectional survey was carried out in a rural setting of northwest Iran between February and May 2023. Rural individuals were interviewed according to a questionnaire with questions about demographic characteristics, knowledge, attitudes, and practices relating to human brucellosis. We employed descriptive statistics to study knowledge, attitudes, and practice patterns. In addition, we applied a multivariable logistic regression model to assess potential predictors of knowledge, attitudes, and practice related to brucellosis using odds ratios and 95% confidence intervals. Among 700 participants, 91.1% were aware of brucellosis, and 85.7% identified cows, sheep, and goats as primary transmission sources. 75.6% and 78.7% recognized transmission risks via unpasteurized and raw milk products. 60.5% understood the benefits of boiling milk and using gloves. Attitudinally, 64.3% perceived higher risk for livestock owners and supported practices like milk pasteurization (70.5%), protective gear (84.4%), handwashing (92.2%), and animal vaccination (71.0%). 90% expressed a need for more information. In practice, 44.8% exclusively consumed boiled and pasteurized milk, 37.8% consumed fresh cheese, and 92.2% solely consumed thoroughly cooked animal meat. Logistic regression revealed that higher education and no prior history of the disease correlated with good knowledge and attitudes, while older participants displayed inappropriate practices compared to their younger counterparts. This study highlights the reasonably high awareness of brucellosis, particularly regarding transmission sources and preventive measures. However, the gaps in knowledge, attitudes, and practices still exist, with education level emerging as a significant factor. To improve prevention practices, tailored interventions addressing age-related disparities are necessary.

The first commercially approved efficacious cryptosporidium vaccine protecting New-Born calves from severe diarrhea

Neonatal calf diarrhea (NCD) is an important cause of morbidity and mortality in calves. Four major pathogens, including Enterotoxigenic Escherichia coli (ETEC), bovine rotavirus (BRV), bovine coronavirus (BCV), and Cryptosporidium spp., are associated with NCD, with Cryptosporidium parvum (C. parvum) recognized as a major contributor. Despite various treatment options available, there is no effective vaccine for bovine cryptosporidiosis yet. In this study, recombinantly expressed C. parvum gp40 was evaluated as a candidate vaccine in pregnant animals. The vaccine was administered to pregnant cows during the last trimester of their pregnancy resulting in highly statistically significantly increased anti-gp40 antibody titers. The resulting anti-gp40 antibodies were transmitted to the calves via colostrum. Both suckling (n = 29; test:14, control:15) and non-suckling (n = 16; 8 per group) calves were challenged with live C. parvum oocysts. The calves that received the colostrum from gp40 vaccinated cows showed a highly statistically significant improvement in health (p < 0.0001), reduced incidence (p < 0.0001), and duration of severe diarrhea (p < 0.001) and increased weight gain in suckling calves (p < 0.0001). This study demonstrates that immune bovine colostrum, induced by immunization of late-gestation cows with C. parvum gp40, provided substantial protection to calves against cryptosporidiosis. These findings suggest that the gp40 vaccine has potential for preventing outbreaks of neonatal diarrhea associated with cryptosporidiosis in calves, thereby improving animal welfare and performance.

Rabies Clinical Perspective: Case Description and Care Strategies

The information in the article is useful for a wide range of medical professionals, including infectious disease physicians, epidemiologists, traumatologists, family doctors, as well as medical university students. Rabies is a highly contagious and incurable viral disease that affects the central nervous system that leads to severe encephalitis. This disease is common in 150 countries and territories of the world on all continents except Antarctica. Although the virus can be present in a variety of mammals, the main reservoirs in the wild nature are two specific genera. The most global threat is rabid dogs. The article analyzes the current situation regarding rabies in Ukraine, with a special emphasis on the Vinnytsia region. By collecting information on the prevalence of rabies cases and analyzing medical records of hospitalized patients, we aim to raise awareness of the severity of the disease and highlight prevention measures. The clinical case presented in this study is a tool to draw attention to the problem of the spread of rabies. By telling about this case, we hope to inform the society about the frightening consequences of the disease and promote preventive measures. Solving this issue requires a multifaceted approach. For the effective combat of the rabies spread, it is necessary to review the current restrictions and bans on hunting in the entire territory of Ukraine, in particular in the Vinnytsia region. In addition, more attention should be paid to informing the population through mass media and strengthening the preventive work of medical workers. It is advisable to strengthen control over the vaccination of domestic animals, as this can significantly contribute to the prevention of transmission of the virus. At the same time, it is extremely important to ensure that sufficient quantities of rabies vaccine and immunoglobulin are available for effective treatment and prevention of further cases.

Safety and immunogenicity of an adjuvanted recombinant spike protein-based severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine, SpikeVet™, in selected Carnivora, Primates and Artiodactyla in Australian zoos.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can infect a broad range of animal species and has been associated with severe disease in some taxa. Few studies have evaluated optimal strategies to mitigate the risk to susceptible zoo animals. This study evaluated the safety and immunogenicity of a protein-based veterinary SARS-CoV-2 vaccine (SpikeVet™) in zoo animals. Two to three doses of SpikeVet™ were administered intramuscularly or subcutaneously 3-4 weeks apart to 354 zoo animals representing 38 species. SpikeVet™ was very well tolerated across all species. Minor adverse effects were observed in 1.69% of animals vaccinated, or 1.04% of vaccine doses administered. Preliminary immunogenicity analyses in representative carnivores (meerkats, lions) and an artiodactylid (domestic goat) showed SpikeVet™-immunized animals developed serum antibodies able to neutralize a range of SARS-CoV-2 variants, including the vaccine-homologous Wuhan and Mu variants, as well as vaccine-heterologous Omicron BA.2 and XBB.1 strains. Prior to vaccination, all eight lions were seropositive for Wuhan strain by surrogate viral neutralization testing, suggesting past infection with SARS-CoV-2 or cross-reactive antibodies generated by another closely related coronavirus. These results from a range of zoo species support the ongoing development of SpikeVet™ as a safe and effective veterinary SARS-CoV-2 vaccine.

Serological detection of Newcastle disease virus in backyard poultry production system in Woliso District South West Shewa, Central Ethiopia.

Newcastle disease (ND)is one of the most important respiratory viral diseases. The disease is endemic in many parts of Ethiopia. However, there is no clear record about the introduction of the virus to the country (Ethiopia). Hence, detail about the ND is very important in its (ND) control and prevention. Despite these facts, there is no available research work done on ND in the current research area that would help either as references for researchers or that could help in the control and prevention of the disease. Therefore, the objective of this study was to detect the ND virus (NDV), using serological methods in from December 2018 to November 2019. A cross-sectional type of study was conducted to detect the NDV. The convenience sampling method was used for sample data collection. Before data collection, chicken with previous history of vaccination against the NDV was excluded from the sampling animals. Then, a total of 348 blood samples of 2 mLwere collected from the brachial vein in 3 mLdisposable syringes. The serum was collected in labeled 2 mL cryovial tubes. Indirect enzyme-linked immunosorbent assay(ELISA) tests were performed to detect antibodies against NDV and to determine its antibody titer. The test was performed using (ID.vetinnovative version 2) procedure. In the indirect ELISA test, 37.64% (131/348) were positive and antibody titer mean value of (1761.9088) was scored. The standard deviation of 2592.42160 and a percentage coefficient of variation of147% was scored. From the finding, we conclude that indirect ELISA test detected the presence of the NDV in the study area and the heterogeneousity of antibody titer in the study area. Therefore, further molecular characterization and epidemiological investigation should be carried and vaccination of animals is critical in the study area.

The effectiveness of vaccination in acute postpartum endometritis of cows

The article presents the results of studies of the vaccine against infectious mastitis and acute postpartum endometritis of cows inactivated. The specific efficacy of the vaccine was determined by the number of cases of cows with endometritis. The disease was recorded in 14.2% of the experimental and 52.5% of the animals in the control group within a month after calving.An experiment was conducted comparing the effectiveness of two immunization regimens, the first of which provides for primary vaccination of pregnant animals, the second — the beginning of immunization of heifers from 6–8 months of age, followed by a single injection every 6 months at a dose of 3 ml. During the experiment, endometritis was detected in 10% of cows in the 1st and 2nd experimental groups, which is 3.7 times lower than in animals in the control group (36.6%). Thus, it was established that both animal immunization schemes are effective in combating the incidence of calving cows with endometritis.When studying the intrauterine secretions of cows with endometritis, a significant effect of immunization on the species composition of pathogens was established. Thus, in cows of the control group, E.coli with endometritis was isolated 3.4 times more often than in animals of the experimental group. A similar pattern was observed for other microorganisms: the number of S. aureus isolates detected was 2.2 times less in the experimental group, S. agalactiae — 15.0 times, S. dysgalactiae — 2.0 times, S. uberis — 6.0 times, S. pyogenes — 3.0 times, K. pneumoniae — 3.4 times less in comparison with the control group.

Review of Knowledge and Practice of Nurses Regarding Biomedical Waste in Healthcare Settings

Biomedical Waste may also refer to trash that is created during the development and testing of biological. Following the laws passed in 1998 regarding managing and handling bodily waste, biological waste refers to any waste produced during the diagnosis, treatment or vaccination of human patients, animals, or research activities. The study's primary goal is to review nurses' knowledge and practice regarding managing biological waste in hospital settings. We compiled the systematic review based on: A secondary source is any book, previously published paper, or journal with a significant impact. After reviewing 30 publications, 16 of them were evaluated. It is necessary to emphasize the significance of training regarding managing biomedical Waste; The widespread availability of inaccurate and incomplete information regarding biological waste management affects the proper disposal of medicinal waste.

Immune response and protective efficacy of Streptococcus agalactiae vaccine coated with chitosan oligosaccharide for different immunization strategy in nile tilapia (Oreochromis niloticus)

In the past decade, the outbreak of Streptococcus agalactiae has caused significant economic losses in tilapia farming. Vaccine immunization methods and strategies have gradually evolved from single-mode to multi-mode overall prevention and control strategies. In this study, an inactivated vaccine of S. agalactiae with a chitosan oligosaccharide (COS) adjuvant was constructed using different administration methods: intraperitoneal injection (Ip), immersion combined with intraperitoneal injection (Im + Ip), immersion combined with oral administration (Im + Or), and oral administration (Or). Safety analysis revealed no adverse effects on tilapia, and the vaccine significantly promoted fish growth and development when administered through Im + Or or Or immunization. Following vaccination, innate immunity parameters including SOD, ACP and CAT activities were all significantly enhanced. Additionally, specific serum IgM antibodies reached their highest level at the 6th week post vaccination. Skin and intestinal mucus IgT antibodies reached peaked at the 6th and 7th week post vaccination, respectively. The relative peak expression values for IL-8, IL-12, MHC-I, MHC-II, IgM, IgT, CD4, CD8, TNFα, IFNγ from Im + Ip group were significantly higher than those in Ip group, Im + Or group and Or group in most cases (p < 0.05). Importantly, the relative protection survival of Im + Ip group was the highest (78.6%), followed by the Ip group (71.4%), the Or group (64.3%) and the Im + Or group (57.1%). In summary, this study encourages further research on multi-channel immunization strategies of other kinds of vaccines in other aquatic economic animals to improve their disease resistance.

Production of Recombinant Viral Antigens Using the Baculovirus-Insect Cell Expression System.

Insect cell expression has been successfully used for the production of viral antigens as part of commercial vaccine development. As expression host, insect cells offer advantage over bacterial system by presenting the ability of performing post-translational modifications (PTMs) such as glycosylation and phosphorylation thus preserving the native functionality of the proteins especially for viral antigens. Insect cells have limitation in exactly mimicking some proteins which require complex glycosylation pattern. The recent advancement in insect cell engineering strategies could overcome this limitation to some extent. Moreover, cost efficiency, timelines, safety, and process adoptability make insect cells a preferred platform for production of subunit antigens for human and animal vaccines. In this chapter, we describe the method for producing the SARS-CoV2 spike ectodomain subunit antigen for human vaccine development and the virus like particle (VLP), based on capsid protein of porcine circovirus virus 2 (PCV2d) antigen for animal vaccine development using two different insect cell lines, SF9 & Hi5, respectively. This methodology demonstrates the flexibility and broad applicability of insect cell as expression host.

Fasciola hepatica infection modifies IgG1 specific immune response to foot-and-mouth disease virus induced by vaccination

Fasciola hepatica, a worldwide distributed helminth, has a robust immunoregulatory effect in the host, increasing the susceptibility to secondary infections. Foot and mouth disease (FMD) is a highly contagious acute vesicular viral disease effectively controlled by vaccination in endemic regions. Despite the evidence of immunoregulatory effects, the impact of fasciolosis on the immune response induced by FMD vaccination in cattle has never been assessed. Our objective was to evaluate whether the infection by F. hepatica in cattle influences the long-term immunity elicited by the currently used commercial FMD-inactivated vaccines. Aberdeen Angus steers negative for F. hepatica were vaccinated twice against FMD virus (FMDV) during the first 6 months of age using a commercial oil vaccine formulated with A24/Cruzeiro and O1/Campos strains. When maternal antibodies against F. hepatica were weaned (18––20 months of age) animals were divided into groups of 12 and infected or mock-infected with 500 metacercariae/animal. Individual serum samples were collected at 0-, 28-, 59-, 87- and 157-days post-infection (dpi). Indirect ELISAs were used to detect A24/Cruzeiro specific bovine IgG and IgG subtypes. The total IgG antibody levels and avidity against FMDV did not show significant differences between all the groups. The commercial vaccine induced higher IgG2 than IgG1 titers in vaccinated animals. Anti-FMDV IgG1 levels significantly decreased in the infected group at 28 dpi. In addition, the avidity of IgG1 FMDV-specific antibodies at day 28 in the infected group was reduced compared to the control. These results show that F. hepatica infection modified anamnestic responses against FMDV, reducing serum IgG1 titers and avidity. To our knowledge, this is the first report of immune-regulation of F. hepatica altering the immune response of FMD vaccines, one of the most globally used animal vaccines.

FIRST OUTBREAK OF LUMPY SKIN DISEASE IN CATTLE AT DISTRICT KHAIRPUR MIRS-SINDH

The research study was conducted to examine the Lumpy skin disease in Cattle, Bos taurus L. cloven-hooved herbivores. Capripox virus infection is characterized by enlarged lymph nodes, fever, and circumscribed nodules particularly on hairless areas. The age-wise data of male and female (3 to 6) months weaning stage, (15 to 25) months bull/heifer (25 to above) months adults were kept under observation from April to September 2022 in all talukas of district Khairpur, Sindh-Pakistan. The maximum overall mean positive cases of LSD throughout the district level were reported from Kot Diji (73.63), followed by Kingri (66.53), Gambat (51.47), Thari Mirwah (45.65), Faiz Ganj (41.84), Sobho Dero (37.83), Naro (31.97) and Khairpur (29.17), talukas of district Khairpur. The age-wise specific data revealed that, the highest infection of lumpy skin diseases was recorded at the weaning stage in females (85.35) as compared to males (65.78), female heifer (47.67), bull (38.47), adults female (71.24) and male (57.00). The maximum number of cases was reported in females (67.57) and males (53.46), respectively. Both male and female cattle throughout the district of Khairpur were found with vigorous infestation. Generally, the clinical severity of LSD depends on immunological status, susceptibility, age of the host, and route of virus inoculation. Effective control measures against this economically divesting viral disease can be achieved through mass vaccination and separation of infected animals. Furthermore; it is recalling earlier understanding of the epidemiology, economic significance, and immediate control mechanisms of the disease is strongly needed to design suitable control measures.

Пространственно-временной анализ природно-очаговых заболеваний (сибирская язва) на территории Северо-Казахстанской области

The article presents the results of study of actual focal disease for the North Kazakhstan region – anthrax. The historically established livestock specialization and excessive concentration of livestock in a limited area (albeit short-term), the presence of favorable natural and landscape conditions for the preservation of the causative agent of anthrax infection (primarily the soil and climatic factor) led to the formation of numerous foci of this disease within the region. The questions of spatio-temporal manifestation and spread of anthrax infection on the territory of the region are considered. The retrospective analysis of the epizootic and epidemic processes of anthrax for 1938– 2022 was performed using archival, stock, and statistical materials. The study was carried out using GIS technologies, then a database, a series of electronic maps and a regional thematic geoinformation system “Anthrax in the North Kazakhstan region” were created. It has been determined that the greatest potential danger (epizootic and epidemic intensity) of anthrax manifestations is typical for the northern and central parts of the region. Within the boundaries of natural landscape units, the maximum risk of anthrax infection was recorded in the forest and steppe area and the valley of the river Ishim. Practical recommendations are proposed for improving measures aimed at preventing anthrax: annual full vaccination of farm animals, sanitary and epidemiological control over slaughtering points and the state of anthrax burials, establishing and maintaining a buffer zone when grazing livestock near stationary-unfavorable foci.

TaqMan real-time RT‒PCR assay with an internal amplification control for rapid detection of Bluetongue virus

Bluetongue (BT) is an arbovirus disease caused by Bluetongue virus (BTV), which has spread all over the world. In this study, a qRT-PCR assay with an internal amplification control was established for preventing false-negative results in Bluetongue virus (BTV) detection. The primers and probes for BTV NS3 were based on the qRT-PCR method for the detection of BTV in the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (2022) of the World Organization for Animal Health (WOAH). The GAPDH gene was selected as the internal amplification control, and the primers and probes for the GAPDH gene were designed based on the analysis of conserved sequences in the genome sequences of cattle, sheep and goats. Optimization of the reaction conditions, specificity, sensitivity and repeatability tests were conducted, and a qRT-PCR method was established for the simultaneous detection of BTV and the reference gene GAPDH. The results showed that the optimal primers and probe concentrations of BTV-NS3 were 0.30 μmol/L and 0.25 μmol/L, and were 0.15 μmol/L and 0.20 μmol/L for the reference gene GAPDH. The established method has no cross-reaction with other viruses, which showed good specificity. The minimum detection of BTV- and GAPDH-positive plasmid standard copies was 6.685 copies/μL and 209 copies/μL, respectively. The intra-assay and interassay CVs of the Ct values measured by BTV were less than 1.2%, and the intra-assay and interassay CVs of the Ct values measured by GAPDH were less than 0.7%, which suggested a high degree of repeatability and stability for this assay. The established BTV qRT-PCR method was used to detect BTV in 88 sheep serum samples stored in the laboratory, revealing 4 BTV-positive samples with a positive rate of 4.55%, which was consistent with the result obtained by nested PCR for detecting BTV recommended by WOAH. The qRT-PCR method established in this study for simultaneously detecting BTV and the internal reference gene GAPDH has high sensitivity, strong specificity and high repeatability. The addition of an internal reference gene can effectively control the detection accuracy and enable false-negative results to be avoided. It can provide a reference for the standardization of qRT-PCR methods for Bluetongue disease.