Immunodiffusion Test Research Articles

Testing for Blastomycosis, Coccidioidomycosis, and Histoplasmosis at a Major Commercial Laboratory, United States, 2019-2024.

Blastomycosis, coccidioidomycosis, and histoplasmosis are environmentally acquired fungal diseases that clinically resemble bacterial and viral community-acquired pneumonia and require laboratory testing for diagnosis. Patients frequently present to primary care and experience diagnostic delays when a fungal etiology is not initially suspected. Current national-level public health surveillance for these diseases is limited and does not include laboratory data, so nationwide testing practices are unknown. We identified laboratory tests for blastomycosis, coccidioidomycosis, and histoplasmosis ordered during 1 March 2019-29 February 2024 and performed within a major national commercial laboratory system. We analyzed test results, patient and healthcare provider features, reasons for testing, and temporal trends. Results included 5693 Blastomyces complement fixation tests (of those, 12% were positive), 71 858 immunodiffusion tests (0.1% positive), and 1186 serum enzyme immunoassay (EIA) tests (11% positive); 154 989 Coccidioides EIA immunoglobulin M results (5% positive) and 154 968 immunoglobulin G results (8% positive); and 46 346 Histoplasma complement fixation tests (30% positive), 49 062 immunodiffusion tests (1% positive), 35 506 serum EIA tests (4% positive), and 82 489 urine EIA tests (2% positive). Most histoplasmosis (58%-74%) and blastomycosis (42%-68%) tests were ordered from hospitals, whereas coccidioidomycosis tests were most frequently ordered by primary care providers (40%). A yearly average of 2727 positive tests were ordered by healthcare providers in states without public health surveillance for these diseases. Blastomycosis, coccidioidomycosis, and histoplasmosis are likely underdetected in primary care settings or by public health surveillance. Increased testing by primary care providers and expanded surveillance are needed to reduce disease burden.

Deciphering immunodiffusion: In silico optimization for faster protein diagnostics

Immunodiffusion tests offer a simple yet powerful method for detecting protein antigens, but their long assay times hinder clinical utility. We unveil the complex interplay of parameters governing this process using finite element simulations. By meticulously validating our model against real-world data, we elucidate how initial concentrations and diffusivities of antigen and antibody shape the intensity, size, and formation time of the precipitin ring. Our key innovation lies in employing phase diagram analysis to map the combined effects of these parameters on assay performance. This framework enables rapid in silico parameter estimation, paving the way for the design of novel immunodiffusion assays with drastically reduced assay times. The presented approach holds immense potential for optimizing protein diagnostics for fast and reliable diagnostics.

Equine Infectious Anaemia: The Active Surveillance of an Entire Equid Population Reduces the Occurrence of the Infection

Equine infectious anaemia (EIA) is a life-long viral infection affecting equids, transmitted mechanically by biting flies and iatrogenic means. Despite its global distribution, active surveillance is limited, with passive clinical surveillance or control of specific equine sectors prevailing. In Italy, a national surveillance plan in horse, donkey, and mule populations has been established and includes mandatory passive and active surveillance through annual serological tests. During 2007–2010, the agar gel immunodiffusion (AGID) test served as both screening and confirmatory tests. Since 2011, a three-tier diagnostic pathway was introduced, utilizing the ELISA test for screening, AGID as the confirmatory test, and the immunoblot test for cases where ELISA was positive and AGID was negative. From a total equid population of 406,000 animals, 1,337,899 samples were analysed during 2007–2012, with 2,348 (0.18%) testing positive. EIA seroprevalence significantly decreased across all the species/hybrids during the study period. EIA occurrence was higher in mules (IRR = 48.90) and lower in donkeys (IRR = 0.56) compared to horses. The holding seroprevalence was 1.15%. Spatial analysis revealed clusters of infection in central Italy. These findings demonstrate that active systematic surveillance effectively reduces EIA prevalence in equid populations. Mules and working horses in wooded areas appeared to be at higher risk of infection and act as EIA reservoirs. Surveillance and control should be maintained and strengthened in these species/hybrids and in these areas to effectively control EIA. Passive surveillance alone is insufficient to eradicate the disease, and EIA remains a constant threat for the equine industry if active control is not implemented.

Outbreak Of Fowlpox In Commercial Brown Pullets Previously Vaccinated With Fowlpox Vaccine In Zaria, Kaduna State, Nigeria

Fowlpox is a contagious viral disease of poultry, ranked as the third most important cause of mortality in Nigerian indigenous chickens. The present study confirms an outbreak of fowlpox in a flock of 500 Isa brown, 11-weeks-old pullets in Zaria, Kaduna State, Nigeria. The pullets were managed under deep litter system and previously vaccinated with live attenuated vaccine (ABIC biological laboratories Ltd. Israel) at 8-weeks old. They had gross lesions suggestive of cutaneous fowlpox on skin of the head region with 58% morbidity but persistent low mortality rate of 13.8%. Fowlpox virus was isolated from the cutaneous lesions using chorioallantoic membrane (CAM) inoculation in 9 to 11-day-old embryonated chicken eggs based on the presence of opaque-white pock lesions, then confirmed by Agar Gel Immuno Diffusion test using homologous known fowlpox virus (antigen) and antisera. Specific antibodies to fowlpox virus were quantified from the sera of the chickens using an indirect ELISA (Abbkine, Inc, China). The case was managed using; Intramuscular injection of oxytetracycline (TLA), topical applications of oxytetracycline spray as well as oral administrations of Doxygen® and vitamins. Apparently healthy chickens were revaccinated against fowlpox and the pen was thoroughly cleaned, washed, and disinfected. This report had shown an occurrence of vaccine failure, alongside a promising management option and highlighted the significance of vaccination with strict biosecurity measures in the control of fowlpox in Nigeria.

A Review on Paragonimiasis and its Differential Diagnosis Technique

This review highlights the existence of Paragonimus (PRG) and how they are mistaken for Tuberculosis (TB) during diagnosis. PRG is a parasitic lung fluke (flatworm) that infect the lungs, human serves as the first hosts, crabs, crayfish, and snails as the second intermediate hosts. The north-eastern states of India are endemic to PRG and infection is acquired by consuming uncooked or partially cooked crustaceans. Due to their similar clinical presentation to TB, PRGs are usually misdiagnosed, leading to delayed treatment. Ziehl-Neelsen (ZN) stain, conventional wet film for staining the sputum, pleural fluid, and stool, and lung biopsy are different laboratory tests to determine PRG infection. In addition, efficient technique that can aid diagnosis are immunological assays, like Enzyme-linked immunosorbent assay (ELISA), dot-ELISA, complement fixation test (CFT), intradermal test (ID), Western blot, immunodiffusion and indirect haemagglutination test (IHA). In India, PRG infection is most likely to persist until medical professionals and governing bodies raise awareness to implement adequate management measures.

Epidemiology of coccidioidomycosis in Argentina, an update.

The National Reference Laboratory in Clinical Mycology of Argentina conducted a retrospective review of human coccidioidomycosis cases diagnosed by the National Mycology Laboratory Network of Argentina between 2010 and 2022 to determine the burden of the disease in the country. A total of 100 human coccidioidomycosis cases were documented, with a higher prevalence in male patients (male-to-female ratio of 1.9:1), with a median age of 41 years. Comparing the number of cases between two 10-year periods (2000-2009 and 2010-2019), the increase was 36.51% (from 63 to 86 cases). Among the 100 recorded cases, 79 tested positive using the double immunodiffusion test. Spherules were observed in 19 cases through histopathology or direct microscopic examination and the fungus was isolated in 39 cases. Thirty-six isolates were identified as Coccidioides posadasii through partial sequencing of the Ag2/PRA gene. Catamarca province had the highest number of cases, comprising 64% of the total, with an incidence rate above 1.0-2.5/100,000 inhabitants until 2018. However, there has been a recent downward trend in the region from 2018 to 2022. It is concerning that more than half of diagnosed cases were chronic pulmonary or disseminated forms, indicating a lack of early disease detection. To rectify this issue, it is imperative to conduct targeted training programs for healthcare personnel and enhance public awareness within the endemic area. This will contribute to a better understanding of the true burden of coccidioidomycosis and enable the implementation of appropriate sanitary control measures.

The Study of Bluetongue Virus (BTV) and Epizootic Hemorrhagic Disease Virus (EHDV) Circulation and Vectors at the Municipal Parks and Zoobotanical Foundation of Belo Horizonte, Minas Gerais, Brazil (FPMZB-BH).

Bluetongue Virus (BTV) and Epizootic Hemorrhagic Disease Virus (EHDV) are Orbiviruses primarily transmitted by their biological vector, Culicoides spp. Latreille, 1809 (Diptera: Ceratopogonidae). These viruses can infect a diverse range of vertebrate hosts, leading to disease outbreaks in domestic and wild ruminants worldwide. This study, conducted at the Belo Horizonte Municipal Parks and Zoobotany Foundation (FPMZB-BH), Minas Gerais, Brazil, focused on Orbivirus and its vectors. Collections of Culicoides spp. were carried out at the FPMZB-BH from 9 December 2021 to 18 November 2022. A higher prevalence of these insects was observed during the summer months, especially in February. Factors such as elevated temperatures, high humidity, fecal accumulation, and proximity to large animals, like camels and elephants, were associated with increased Culicoides capture. Among the identified Culicoides spp. species, Culicoides insignis Lutz, 1913, constituted 75%, and Culicoides pusillus Lutz, 1913, 6% of the collected midges, both described as competent vectors for Orbivirus transmission. Additionally, a previously unreported species in Minas Gerais, Culicoides debilipalpis Lutz, 1913, was identified, also suspected of being a transmitter of these Orbiviruses. The feeding preferences of some Culicoides species were analyzed, revealing that C. insignis feeds on deer, Red deer (Cervus elaphus) and European fallow deer (Dama dama). Different Culicoides spp. were also identified feeding on humans, raising concerns about the potential transmission of arboviruses at the site. In parallel, 72 serum samples from 14 susceptible species, including various Cervids, collected between 2012 and 2022 from the FPMZB-BH serum bank, underwent Agar Gel Immunodiffusion (AGID) testing for BTV and EHDV. The results showed 75% seropositivity for BTV and 19% for EHDV. Post-testing analysis revealed variations in antibody presence against BTV in a tapir and a fallow deer and against EHDV in a gemsbok across different years. These studies confirm the presence of BTV and EHDV vectors, along with potential virus circulation in the zoo. Consequently, implementing control measures is essential to prevent susceptible species from becoming infected and developing clinical diseases.

Standardization of Semi-Quantitative Dot Blotting Assay-Application in the Diagnosis, Follow-Up, and Relapse of Paracoccidioidomycosis.

This study standardized a semi-quantitative dot blotting assay (DB) and a quantitative real-time polymerase chain reaction (qPCR) to detect specific antibodies for Paracoccidioides brasiliensis and its DNA in PCM patients. We evaluated 42 confirmed PCM patients upon admission using a serological double agar gel immunodiffusion test (DID), DB, and molecular tests (qPCR in total blood). The control groups included 42 healthy individuals and 37 patients with other infectious diseases. The serological progress during treatment was evaluated in eight patients, and there was a relapse diagnosis in ten patients using the Pb B.339 strain antigen. The cut-off points for the serological tests were determined by a receiver operator characteristic curve. The DB and DID tests showed similar accuracy, but the DB identified lower antibody concentrations. Cross-reactions were absent in the DB assay. In the relapse diagnoses, DB exhibited much higher sensitivity (90%) than DID (30%). A DB assay is easier and faster than a DID test to be performed; DB and DID tests show the same accuracy, while blood qPCR is not recommended in the diagnosis at the time of admission; cross-reactions were not observed with other systemic diseases; DB and DID tests are useful for treatment monitoring PCM patients; and a DB assay is the choice for diagnosing relapse. These findings support the introduction of semi-quantitative DB assays in clinical laboratories.

Evaluation of specific chicken IgY antibody value developing diagnostic capture antibody ELISA kit against Foot and Mouth disease.

The most preferred method for the detection of foot-and-mouth disease (FMD) viral antigen and identification of viral serotype is the enzyme-linked immunosorbent assay (ELISA). Diagnostic tests with high sensitivity are necessary both to distinguish infected vaccinated animals and execute disease control programs for the identification of the carrier animals. The current strategies for the detection of FMD virus are mainly based on the capture antibody (sandwich) ELISA test. The usage of laying pullets as an animal bioreactor for the production of specific egg yolk antibodies (IgY) has increased in recent years due to its high yield, affinity, low price, and quick production turnover. The present study aimed to produce a concentrated and purified IgY polyclonal antibody to design a capture antibody ELISA kit against the FMD virus (FMDV) serotype A. At first, laying hens were immunized with inactivated FMDV serotype virus, and then, on days 14, 21, and 28 following vaccination, the eggs and sera were collected. Afterward, the IgY polyclonal antibodies were extracted and purified from the chicken egg yolk using a polyethylene glycol 6000-ethanol precipitation procedure. Extracts were filtered, purified by ion exchange chromatography, and dialyzed. The purified IgY concentration, estimated by Bradford assay, confirmed its presence by SDS-PAGE and Western blot and also its specific immune reaction by Ouchterlony double immunodiffusion and Dot blot tests. Moreover, for achieving the optimum concentration of antigen/antibody (sera) in sandwich ELISA, a checkerboardtitrationtest was set up based on indirect ELISA results. Eventually, 119 previously confirmed samples (including 80 positive and 39 negative) by both real-time polymerase chain reaction (quantitative PCR, qPCR) and a commercial ELISA kit were used for evaluation of the sensitivity and accuracy of our developed Capture antibody ELISA kit. In this manner, the sensitivity and specificity of our designed kit were 100% and 98%, respectively. Accordingly, the present developed capture ELISA kit based on IgY had high sensitivity and specificity for FMD virus detection and it could be used in the future for both commercial detecting and serotyping applications.

Development and evaluation of a test strip for the rapid detection of antibody against equine infectious anemia virus

Equine infectious anemia (EIA) is a contagious disease of horses caused by the equine infectious anemia virus (EIAV). The clinical signs at the acute phase include intermittent high fever, thrombocytopenia, hemorrhage, edema, and anemia. The clinical signs at chronic and relapsing subclinical levels include emaciation and progressive weakness. Surviving horses become lifelong carriers because of the integration of the viral genome into that of the host, and these horses can produce and transmit the virus to other animals. This increases the difficulty of imposing practical control measures to prevent epidemics of this disease. Serological tests measuring the antibodies in equine sera are considered to be a reliable tool for the long-term monitoring of EIA. However, the standard serological tests for EIV either have low sensitivity (e.g., agar gel immunodiffusion test, AGID) or are time consuming to perform (e.g., ELISA and western blotting). The development of a rapid and simple method for detecting the disease is therefore critical to control the spread of EIA. In this study, we designed and developed a colloidal gold immunochromatographic (GICG) test strip to detect antibodies against EIAV based on the double-antigen sandwich. Both the p26 and gp45 proteins were used as the capture antigens, which may help to improve the positive detection rate of the strip. We found that the sensitivity of the test strip was 8 to 16 times higher than those of two commercially available ELISA tests and 128 to 256 times higher than AGID, but 8 to 16 times lower than that of western blotting. The strip has good specificity and stability. When serum samples from experimental horses immunized with the attenuated EIAV vaccine (n = 31) were tested, the results of the test strip showed 100% coincidence with those from NECVB-cELISA and 70.97% with AGID. When testing clinical serum samples (n = 1014), the test strip surprisingly provided greater sensitivity and a higher number of “true positive” results than other techniques. Therefore, we believe that the GICG test strip has demonstrated great potential in the field trials as a simple and effective tool for the detection of antibodies against EIAV.Key points• A colloidal gold immunochromatographic (GICG) fast test strip was developed with good specificity, sensitivity, stability, and repeatability• The test strip can be used in point-of-care testing for the primary screening of EIAV antibodies• Both the p26 and gp45 proteins were used as the capture antigens, giving a high positive detection rate in the testing of experimentally infected animal and field samples

Актуальные проблемы в борьбе с болезнью Марека и пути их решения

Marek’s disease virus (MDV) causes a deadly lymphoproliferative disease in chickens, resulting in huge economic losses in the poultry industry. MDV causes an economically important disease in chickens, and there is no evidence of MDV infections in humans. Vaccination forms the basis of the current MD control program. MDV replicates and sheds from feather follicle epithelial cells and intermediate epidermal layers of the foot skin in cell-free form into the environment. Vaccination induces anti-MDV T cell responses, which reduce host symptoms and prolong survival, but it fails to inhibit virus replication and shedding. Failure to induce sterile immunity has led to the emergence of more virulent strain. The virus is currently being studied in more detail for the further development of new effective vaccines. Immunodiffusion test, PCR and real-time PCR are used to isolate the virus from pathologic material.

Prevalência do vírus da anemia infecciosa equina em cavalos e burros, determinada por comparação de ELISA e AGID, no México

ABSTRACT From February to December 2021, 960 serum samples from different climate regions in two Mexican states were analyzed for antibodies against EIAV using competitive enzyme-linked immunosorbent assay (cELISA) and agar gel immunodiffusion (AGID) tests. The overall seroprevalence of EIAV infection in equids determined by ELISA and AGID tests was 19.69% and 17.60%, respectively. The highest rate of seropositivity was observed in the state of Veracruz (23.67%, using AGID test), and the lowest rate was found in the state of Puebla (13.12%, using cELISA test). In total, 17.45% (85/487, using AGID test) and 15.40% (75/487, using cELISA test) of horses were positive for EIAV infection. Donkeys showed a prevalence of 19.87% (94/473) in cELISA and 21.99% (104/476) in AGID. The cELISA was compared with AGID as the gold standard test. The diagnostic sensitivity for horses and donkeys was 90.4% and 92.2%, and the specificity was 97.5% and 97.3%, respectively, with an almost perfect degree of agreement (kappa values of 0.871 and 0.888). Therefore, the overall seroprevalence rate of EIAV infection in equines in the sampled states of Puebla and Veracruz was 19.3%, with 91.4% sensitivity, 97.4% specificity, and an agreement of 96.25%, indicating almost perfect agreement (kappa = 0.880).

Characterization of infectious laryngotracheitis virus isolated from commercial layer chickens in Bangladesh during the year 2021-2022.

Infectious laryngotracheitis virus (ILTV) is responsible for causing infectious laryngotracheitis (ILT), which is a rapidly spreading and extremely transmissible disease in chickens. The current research aims to isolate and characterize ILTV from layer chickens in Bangladesh. A total of 345 samples (trachea, larynx, and lungs) were collected from ILT-suspected dead and sick layer chickens of 32 ILT-suspected farms in three different outbreak districts (Gazipur, Tangail, and Mymensingh) of Bangladesh during the outbreak year 2021-2022. Rapid detection kits examined the samples for avian influenza virus (AIV) and Newcastle disease virus (NDV). ILTV-specific primers were used to screen 72 NDV- and AIV-negative samples by polymerase chain reaction (PCR). Using chorioallantoic membrane (CAM), the study isolated the ILT virus from 9 to 10-day-old seronegative embryonated chicken eggs (ECEs) using selected PCR-positive samples. The virus was confirmed using nucleotide sequencing, agar gel immunodiffusion test (AGIDT), viral neutralization test (VNT), and pathogenicity evaluations using mortality index for chicken embryos (MICEs) and intra-tracheal pathogenicity index (ITPI). The results indicated that among the PCR-positive 10 samples, only two (Alim_ILT_1001 and Alim_ILT_1,000) were found positive using ECEs. There were two field isolates of ILTVs, as shown by the amplicon size of the ICP4 gene-based PCR. A phylogenetic study of the ICP4 gene revealed that the recent isolates have a close similarity with the ILTV isolates of Turkey, Bangladesh, and Australia. AGIDT revealed strong precipitation lines due to ILTV-specific antibodies reacting with field viruses, while VNT neutralized both isolates with conventional ILTV antibodies. The pathogenicity testing indicated that Alim_ILT_1001 had MICE and ITPI values of 0.77 and 0.63, whereas Alim_ILT_1,000 had 0.71 and 0.57. Both the ILTV isolates have similarities with the isolates of Turkey, Bangladesh, and Australia, and they are highly virulent for chickens.

Evaluation of candidate monovalent Bitis arietans antivenom using eggs from hyperimmunized layer chickens in Zaria Kaduna State, Nigeria

Aim: This research sought to experiment on a cheaper alternative to snake antivenom production using hyperimmunized chicken eggs. Methods: Twenty-two Swiss albino mice were used to determine the median lethal dose (LD50) and lethality neutralization assay. Twenty-layer chickens, aged 17-weeks-old were divided into two groups of 10 chickens each. Based on body weight, group A were hyperimmunized with 1/5th LD50 of crude Bitis arietans venom in adjuvant (Montanide™ ISA 201) at two-weeks interval over eight-weeks period; while group B were administered normal saline. Using polyethylene glycol 6000, immunoglobulin Y was extracted from 15 pooled egg yolks. Twelve Wistar rats were used for the haemorrhagic and anti-haemorrhagic assays. The haemolytic activity of the venom and anti-haemolytic effect of the candidate antivenom was determined in vitro using caprine erythrocytes. Results: The LD50 of the pooled Bitis arietans venom was 0.9 mg/kg. Agar gel immunodiffusion test confirmed the presence of precipitin antibodies in the candidate antivenom. EchiTab plus-ICP® neutralized lethal effect of all dilutions and the stock (10 mg/ml) venom of Bitis arietans while the candidate antivenom was limited to 3log2 dilution of the stock venom (1.25 mg/ml). In the haemorrhagic assay the venom exhibited haemorrhagic activity at 2.5, 5 and 10 mg/ml and the highest anti-haemorrhagic activity of the candidate antivenom at 10mg/ml. The candidate antivenom exhibited significantly higher (p < 0.05) anti-haemolytic activity against the venom of Bitis arietans at 10 mg/ml. Conclusion: The antibodies have inhibitory properties against the lethal, haemorrhagic and haemolytic properties of Bitis arietans venom.

Review on Virulence and Diagnostic Methods of Mareks Disease Virus in Chickens

Marek’s Disease is a highly contagious oncogenic and neuropathic disease of chickens caused by Marek’s Disease Virus which is a highly cell-associated Alpha herpes virus that replicates in chicken lymphocytes and establishes a latent infection within T cells. Evolution of Marek’s Disease Virus towards higher virulence have been a concern over the years from mild to virulent, very virulent and very virulent plus. Mareks Disease is diagnosed by clinical examination of infected chickens accompanied with demonstration of postmortem lesions. The technique for diagnosis of infection with Marek’s Disease Virus involves isolation and identification of Marek’s Disease Virus from infected tissues. Virus isolation is usually by virus propagation in cell culture and identification/ quantification by cytopathic changes. Further confirmatory diagnosis of Marek’s Disease Virus can be done by serological test such as Agar Gel Immuno Diffusion Test and Enzyme Linked Immuno Sorbent Assay and molecular based technique such as Polymerase Chain Reaction. High-throughput sequencing technology which is a second-generation sequencing, provide the possibility to rapidly obtain the full sequence of pathogen genome at once for studying virus evolution and development of novel vaccines. Mareks Disease can be controlled by live-attenuated vaccines. In Ethiopia, reports coming from various regions of the country indicated that Marek’s Disease possesses a great threat to the flourishing young poultry industry.

Epidemiological situation of Equine Infectious Anemia in the state of Paraná, Brazil

To assist decision making regarding the National Equine Health Program in the state of Paraná, a study was conducted to estimate the prevalence of infected farms and seropositive animals for Equine Infectious Anemia (EIA) and also identify possible risk factors for the disease. The state was divided into three regions, within which about 300 farms were randomly selected. On the selected farms, a minimum number of animals aged 6 months or older were examined to characterize them as infected or free of EIA. In the sampled farms, an epidemiological questionnaire was applied to investigate possible associations between the disease and its characteristics, including sanitary practices and livestock management. The test used was the Agar Gel Immunodiffusion Test. A total of 2818 equids from 889 farms were tested. In the state, the prevalence of infected farms was 1.55% [0.92; 3.00] and the prevalence of seropositive animals was 0.55% [0.27; 1.00]. Introduction of equids showed an association with EIA (OR=5.5 [1.9; 15.9]). Paraná equine owners should be alerted to the need to observe health precautions regarding EIA when introducing animals to their herd. In 2018, the sensitivity of the Surveillance System for EIA in Paraná was only 1.36%, probably insufficient to change the endemic balance of the disease, therefore, needs to be re-evaluated involving in the process all public and private agents interested in the subject.

Serotypes, virulence factors and multilocus sequence typing of Glaesserella parasuis from diseased pigs in Taiwan.

Glaesserella parasuis (G. parasuis) belongs to the normal microbiota of the upper respiratory tract in the swine, but virulent strains can cause systemic infections commonly known as Glässer's disease that leads to significant economic loss in the swine industry. Fifteen serotypes of G. parasuis have been classified by gel immunodiffusion test while the molecular serotyping based on variation within the capsule loci have further improved the serotype determination of unidentified field strains. Serovar has been commonly used as an indicator of virulence; however, virulence can be significantly differ in the field isolates with the same serotype. To date, investigations of G. parasuis isolated in Taiwan regarding antimicrobial resistance, serotypes, genotypes and virulence factors remain unclear. A total of 276 G.parasuis field isolates were collected from 263 diseased pigs at the Animal Disease Diagnostic Center of National Chiayi University in Taiwan from January 2013 to July 2021. Putative virulence factors and serotypes of the isolates were identified by polymerase chain reaction (PCR) and antimicrobial susceptibility testing was performed by microbroth dilution assay. Additionally, the epidemiology of G. parasuis was characterized by multilocus sequence typing (MLST). Serotype 4 (33.3%) and 5 (21.4%) were the most prevalent, followed by nontypable isolates (15.9%), serotype 13 (9.4%), 12 (6.5%), 14 (6.2%), 7 (3.3%), 1 (1.8%), 9 (1.1%), 11 (0.7%) and 6 (0.4%). Nine out of 10 putative virulence factors showed high positive rates, including group 1 vtaA (100%), fhuA (80.4%), hhdA (98.6%), hhdB (96.0%), sclB7 (99.6%), sclB11 (94.9%), nhaC (98.2%), HAPS_0254 (85.9%), and cirA (99.3%). According to the results of antimicrobial susceptibility testing, ceftiofur and florfenicol were highly susceptible (>90%). Notably, 68.8% isolates showed multidrug resistance. MLST revealed 16 new alleles and 67 new sequence types (STs). STs of these isolated G. parasuis strains were classified into three clonal complexes and 45 singletons by Based Upon Related Sequence Types (BURST) analysis. All the G. parasuis strains in PubMLST database, including strains from the diseased pigs in the study, were defined into two main clusters by Unweighted Pair Group Method with Arithmetic Mean (UPGMA). Most isolates in this study and virulent isolates from the database were mainly located in cluster 2, while cluster 1 included a high percentage of nasal isolates from asymptomatic carriers. In conclusion, this study provides current prevalence and antimicrobial susceptibility of G. parasuis in Taiwan, which can be used in clinical diagnosis and treatment of Glässer's disease.

Defining optimal thresholds for digital Brix refractometry to determine IgG concentration in ewe colostrum and lamb serum in Scottish lowland sheep flocks.

This research aimed to define thresholds for ewe colostrum and lamb serum Brix refractometer measurements in lowland Scottish sheep. This would facilitate the use of this convenient, sheep-side test, enabling quick and accurate identification of poor quality colostrum and prevention of failure of transfer of passive immunity (FTPI) in lambs. Secondary aims were to identify risk factors for poor colostrum quality and FTPI in lambs. Serum samples (n=233) were collected from lambs between 24 and 48hrs after birth, from four lowland Scottish meat sheep farms. Pre-suckle colostrum samples (n=112) were also collected from ewes on two of these farms. Farmers provided information on litter size, ewe body condition score, ewe breed and dystocia. Duplicate digital Brix refractometer measurements were compared with immunoglobulin G (IgG) radial immunodiffusion (RID) testing for all colostrum and serum samples. Receiver operating characteristic (ROC) curves were used to redefine thresholds for Brix testing in colostrum and serum. Linear regression models were constructed with colostrum and serum IgG concentration as the outcomes of interest. Colostrum and serum IgG concentrations were highly variable. The prevalence of inadequate colostrum quality (using <50g/L IgG on RID) was 4.5% (95% CI = 1.5 - 10.1) and the prevalence of FTPI (using <15g/L IgG in serum on RID) was 7.73% (95% CI = 4.64-11.93). A ewe's colostrum IgG concentration was significantly and positively associated with the serum IgG concentration of her lamb(s) (p=0.02). ROC analysis defined a Brix threshold for adequate colostrum quality of >22.10% (sensitivity 80% (95%CI=28.4-99.5), specificity 90% (95%CI=82.3-94.8)). ROC analysis defined a Brix threshold for serum of >8.65% for adequate passive transfer of immunity in Scottish lambs (sensitivity 94% (95%CI=72.7-99.8), specificity 82% (95%CI=76.6-87.2)). To optimise passive transfer of immunity in lambs, we suggest that ewe colostrum Brix measurements be defined as 'poor' (<22%); 'fair' (22-26%) and 'good' (>26%); and lamb serum as 'poor' (<8%); 'fair' (8-9%) and 'good' (>9%). It is recommended that these tests are used as for flock screening, using samples from multiple animals.

Study the Profile of Foot-and-mouth Disease Virus Protein by Electrophoresis and Identification of the Immunodominant Proteins.

Foot-and-mouth disease is an extremely infectious and occasionally fatal viral disease with a rapid onset and a short course that affects cloven-hoofed animals and results in considerable financial losses. Today, Foot-and-mouth disease is controlled by traditional inactivated vaccines. Due to the short duration of immunity, a study was conducted for proteins of the virus as well as obtaining immunodominant proteins to design more efficient vaccines against Foot-and-mouth disease virus. This research aims to study the profile of Foot-and-mouth disease virus protein by electrophoresis and identification of the immunodominant proteins. The purified Foot-and-mouth disease virus was purchased then the protein concentration of that solution was measured by Lowry method. SDS-PAGE was done to achieve the protein profiles of the virus and immunization of 5 guinea pigs was done, then blood samples were taken for obtaining serum. Finally, serology tests; double immunodiffusion, ELISA, and western blotting were used to evaluate antigen response to antibodies (antigenic immunization). The protein concentration was 3.5 mg/ml. In SDS-PAGE with 10% gel, the protein profile of the virus was observed. After immunization, by conducting double immunodiffusion tests, the sediment lines between the serum antibody and the antigen of the virus were formed. Also, The ELISA test showed that the antibodies were formed against the antigens. In the western blot test, two immunodominant proteins of the FMD virus were obtained. According to the results, the immunodominant proteins of the FMD virus were determined. These proteins can be used in immunological diagnostic methods and also novel vaccines.

Изучение генетической вариабельности выделенных изолятов вируса лейкоза крупного рогатого скота в Белгородской области

Abstract. The purpose of the research Was to study genetic diversity of bovine leukemia virus isolates obtained in the Belgorod Region using restriction fragment length polymorphism method (RFLP). Scientific novelty. Bovine leukosis is one of the most common chronic infectious diseases of cattle in many countries of the world, which causes significant economic losses in the livestock industry. The typing of bovine leukemia virus (BLV), the study of its genetic structure, the evaluation of the mutation vector and a more detailed disclosure of the biological properties of the pathogen represent fundamental and applied value. Methods. The object of the research was 3-4-year-old cows infected with leukemia virus (n = 10), identified by serological methods in disadvantaged dairy farms. The immunodiffusion (ID) test, hematologic studies, PCR, genotyping, statistical processing of obtained data were conducted. Results. Conducted hematological studies determined the stage of the leukemic process in each animal. The target env fragment of the BLV gene (444 bp) was amplificated by 2-stage nested PCR, and this region was genotyped for all studied leukemia virus isolates using the restriction fragment length polymorphism (RFLP) method. In the course of the work, specific regions of the BLV env (gp51) gene, 970 bp long, were also obtained. We have given a primary assessment of the genetic diversity of BLV with the establishment of a genetic group (Belgian genotype according to RFLP). In the course of the work, specific regions of the env gp51 BLV gene, 444 bp long, were obtained. These fragments will be used for further DNA sequencing followed by phylogenetic analysis and determination of amino acid changes in the structure of the surface glycoprotein (gp51) of the bovine leukemia virus. Monitoring studies of BLV genotypes and the study of antigenic changes in the pathogen will allow timely development of the latest means of controlling and restricting the spread of bovine leukosis and improvement of diagnostic serological and PCR test systems.