Agar Gel Immunodiffusion Assay Research Articles

Seroprevalence of Bovine Leukemia Virus Infection in Cattle in Muş Province, Türkiye

Bovine leukemia virus (BLV) is known as the causative agent of enzootic bovine leukosis (EBL), which is a worldwide distributed disease and has also been detected in marketed beef and dairy products. BLV causes significant economic losses due to the loss of milk and yield or the slaughter of animals without adequate development. It has been reported in epidemiological studies that this infection is common in Türkiye, especially in the western provinces. There is no data on the possible presence or prevalence of BLV infection and its seroepidemiology in Muş province. The aim of this study is to determine the possible presence and prevalence of the infection, its role in yield losses, and to obtain epidemiological data on cattle farming in the Muş district. For this purpose, 300 blood serum samples were collected from cattle aged six months and older in the province of Muş and its different districts. The blood serum samples taken were tested for the presence of BLV-specific antibodies by agar gel immunodiffusion (AGID) and competitive enzyme-linked immunosorbent assay (C-ELISA) methods. As a result of the study, all of the controlled districts were evaluated as negative in the AGID and C-ELISA tests for the presence of BLV-specific antibodies. In conclusion, for the first time, it was demonstrated that cattle farming in the Muş province were BLV-free during the sampling period. Although BLV seropositivity was not detected in the tested animals, it is emphasized that the control of infection and eradication program should not be ignored.

Serological Examinations of Significant Viral Infections in Domestic Donkeys at the Special Nature Reserve "Zasavica", Serbia.

The paper presents the findings of specific antibodies in the blood sera of donkeys against the following viruses: equine infectious anemia virus (EIAV), African horse sickness virus (AHSV), equine herpesvirus type 1 (EHV-1), equine influenza virus subtype H3N8 (EIV) and equine arteritis virus (EAV). The analyses were conducted during the year 2022. From a total of 199 donkeys bred in "Zasavica", blood was sampled from 53 animals (2 male donkeys and 51 female donkeys), aged 3 to 10 years. Specific antibodies against EIAV were not detected in any of the tested animals using the agar-gel immunodiffusion (AGID) assay. No specific antibodies against AHSV, tested by enzyme-linked immunosorbent assay (ELISA), or antibodies against EAV, tested by the virus neutralization test (VNT) and ELISA were detected in any of these animals. A positive serological result for EHV-1 was determined by the VNT in all animals, with antibody titer values ranging from 1:2 to 1:128, while a very low antibody titer value for EIV (subtype H3N8) of 1:16 was determined in 18 donkeys using the hemagglutination inhibition test (HI test).

Modified, innovative method for detection of adulteration of meat pro­ducts with meat of other species

The aim of this study was to detect illegal adulteration of beef meat products with meat from other species. Samples (n=120) of industrial and handmade beef products were randomly collected from retail outlets in Assiut city, Egypt: raw beef burger, oriental beef sausage, beef kofta, and beef luncheon (30 samples each). Samples were analysed using agar gel immunodiffusion (AGID) and modified AGID (MAGID) assays. Results showed that 17.5% of examined products were adulterated with chicken meat. MAGID detected that 14.1% of samples were adulterated with donkey meat, whereas all AGID results were negative. Human tissue was detected in 8.3% (AGID) and 10% (MAGID) of examined samples. Histological examination was then used to detect foreign tissue, and all categories of products were found to be adulterated, and some of them - contaminated with human blood cells. Polymerase chain reaction analysis confirmed that MAGID was more accurate and sensitive than AGID, especially for false negative AGID results. Consumers are advised not to consume too much of the studied meat products to avoid exposure to adulterated or contaminated products that might constitute a health hazard.

Two Novel Avian Influenza Virus Subtypes Isolated from Domestic Ducks in North of Iran.

Avian Influenza Viruses (AIV) are the causative agents of Avian Influenza (AI), which is a contagious and zoonotic disease in birds. Among birds, wild waterfowls and ducks are the primary and natural reservoirs of low pathogenic avian influenza viruses (LPAI). This study aimed to identify and differentiate between two AIV subtypes (i.e., hemagglutinin and neuraminidase from domestic ducks by hemagglutinin inhibition (HI) and neuraminidase inhibition (NI) assays. To this end, 962 cloacal swabs were collected from domestic ducks being sold at different Iranian Live Bird Markets in Gilan, Mazandaran, and Golestan provinces, located at the southern coast of the Caspian Sea. The samples were inoculated in 10-day-old embryonated specific pathogen-free chicken eggs, and subsequently, harvested allantoic fluids were subjected to agar gel immunodiffusion, HI, and NI assays. In total, five positive samples, including two H4N2 and three H3N2 AIV subtypes were identified. Isolation of H4N2 and H3N2 viruses has never been reported from Iranian domestic ducks previously. This finding further suggests the diversity of LPAI viruses in Iranian ducks and also shows that the HI and NI assays are highly efficient in determining AIV subtypes.

Immunogenic protein profiling of pathogenic Escherichia coli strains isolated from infants with diarrhea in Quetta Balochistan

Escherichia coli (E. coli) is a widespread bacterial species that comprise a broad variety of strains and can be highly pathogenic often. Diarrhea caused by E. coli pathotypes is a leading public health problem in underdeveloped countries such as Pakistan. Five different diarrheagenic E. coli pathotypes (Enterohaemorrhagic, Enteropathogenic, Enteroaggregative, Enteroinvasive and Enterotoxigenic E. coli) were isolated from 200 samples of diarrheal children in Quetta, Pakistan. Total-cell protein profiles of all the strains isolated were resolved via SDS-Polyacrylamide Gel Electrophoresis (PAGE). More than 15 protein bands were found ranging from 15 kDa to 153 kDa in size. A dendrogram plot explained the relatedness of the pathotypes amongst them. The protein bands of 70 and 15 kDa, analyzed through GelAnalyzer 2010a© were found common among all the isolated pathotypes. Purified bands of these two proteins (70 and 15 kDa) from the gel were injected in experimental mice groups for immunization purposes. During immunogenicity tests, the mice that were immunized with 70 and 15 kDa proteins stayed alive for seven days despite subsequently being challenged with the E. coli pathotypes, while higher mortality 80% were observed in the non-immunized control group. Upon the postmortem it was revealed that all the vital organs were covered by the E. coli strains injected for challenge studies. The immunized mice and control mice sera subjected to the Agar Gel Immunodiffusion assay (AGID) test for antibody/antigen reaction were found positive for antibodies presence. The exploration of immunogenic proteins among all the prevalent pathotypes can be a breakthrough in the development of a combined, effective, and safe vaccine. Further investigation of these immunogenic proteins would be of great importance for future immunization endeavors.

Re-current Epizootics of Highly Pathogenic Avian Influenza in Nigeria: Status of Vaccination as Alternate control

Epizootic of Highly Pathogenic Avian Influenza subtype H5N1 in Nigeria was successfully contained during the first wave that lasted from 2006 to 2008 without the use of vaccine. Re-current and more severe outbreak was witnessed in 2015 and there are suspicions that some farmers may have resorted to vaccination to prevent infections in their flocks. We investigate evidence of vaccination in farms and the status of vaccination as alternate control for HPAI in Nigeria. The study was carried out in a cross section of 24 commercial poultry farms in four States in South West and North Central Nigeria. Five hundred and one sera collected randomly were screened by agar gel immunodiffusion (AGID) assay for antibody to group specific influenza A nucleoprotein. One hundred and eight sera obtained from five H5N1 infected poultry farms were also concurrently screened. Reactive sera were further analysed by Hemagglutinin Inhibition (HI) test against H5 antigen using 1% suspension of pooled washed chicken red blood cells. Only 8 out of 501 sera (1.6%) had evidence of influenza A antibody. All of the 8 samples were from one farm with 20 samples collected representing 40% seroconversion at farm level. Three out of those sera were positive for H5 at HI titer of 3log2. All other sera including those obtained from HPAI infected farms were negative for influenza antibody. This study confirms limited antibody response to avian influenza subtype H5 most likely due to vaccination in one commercial flock. Vaccination against avian influenza by farmers desperate to protect their investments may lead to unregulated and suboptimal application of vaccines requiring farmers’ and stakeholders’ engagement to forestall negative impact.
 Keywords: Avian influenza; Control measures; Recurrent outbreaks; Vaccination status.

Western Blot in Immunodiagnosis of Small Ruminant Lentivirus

Background: Small ruminant lentiviruses (SRLV) belong to genus Lentivirus, family Retroviridae. These viruses cause caprine arthritis encephalitis (CAE) and maedi visna (MV), infectious diseases that cause economic, production, and reproductive losses. There are no effective treatments or vaccines for these diseases. Thus, early detection via serology has great importance for control of SRLV. Therefore, the objective of this review is to demonstrate the potential of the western blot (WB) test as an immunodiagnostic test for SRLV. Review: In general, immunodiagnosis of SRLV is performed via agar gel immunodiffusion (AGID) and indirect enzyme-linked immunosorbent assay (ELISA), which can detect antibodies in several different biological samples but is used preferably with serum and blood plasma. However, WB has demonstrated efficacy in the early diagnosis of immunoglobulins against SRLV, presenting higher sensitivity and specificity than the serological tests usually used, because this technique can detect antibodies at a dilution as much as 256 times greater than that of AGID and 32 times greater than that of ELISA. SRLV infection and consequent immunological activation result in the induction of cellular and humoral responses. Additionally, around the third week, production of antibodies directed mainly toward viral capsid proteins (p25 and p28) occurs. After the fifth week, production of immunoglobulins directed toward other viral proteins occurs. Because of the persistence of SRLV infection, serology is considered to be the most practical means to diagnosis. Each serological test has a percentage specificity and distinct sensitivity, as well as advantages and disadvantages in its applicability. It should be noted that there is no gold standard test for diagnosis of SRLV infection. Moreover, SRLV are characterized by escape mechanisms such as genetic diversity, mutagenic potential, viral intermittence, and the process of compartmentalization, which make immunodiagnosis more difficult. In addition, positive animals tend to present unstable levels of antibodies over weeks, months, and even years. In this context, WB, with early antibody detection, has been proven to be a refined and more accurate technique than other immunodiagnostic tests for SRLV. WB allows the simultaneous resolution of several immunogenic antigens present in a sample, and this feature provides it with greater reliability, differentiates it from other immunological methods, and accredits it as a test of wide applicability. Epidemiological and immunological dynamics studies often use WB in the immunodynamic diagnosis of SRLV. Serum, blood plasma, and seminal plasma are typical biological materials used in the serological diagnosis of SRLV with WB, expanding its potential as an immunodiagnosis method.Conclusion: WB is the most accurate serological technique for SRLV. It is more capable of accurate diagnosis because the genetic diversity that characterizes such lentiviruses and their various immune system escape mechanisms routinely hinder traditional diagnosis. Additionally, this test has been used widely in studies of SRLV for various purposes, but mainly in studies of epidemiological and immunological dynamics, using serum, blood plasma, or seminal plasma. However, independently of the biological sample tested, WB maintains high sensitivity and precision in immunodiagnosis, making it a refined and valid technique for SRLV control programs.

Detection of Myxoma Virus in the Classical Form of Myxomatosis Using an AGID Assay: Statistical Assessment of the Assay's Diagnostic Performance.

IntroductionThe aim of the study was to estimate the diagnostic sensitivity (DSe) and specificity (DSp) of an agar gel immunodiffusion (AGID) assay for detection of myxoma virus (MYXV) in the classical form of myxomatosis and to compare its diagnostic performance to that of molecular methods (IAC-PCR, OIE PCR, and OIE real-time PCR).Material and MethodsA panel of MYXV-positive samples of tissue homogenates with low (1 PCR unit – PCRU) and high (3,125 PCRU) virus levels and outbreak samples were used for method comparison studies. The validation parameters of the AGID assay were assessed using statistical methods.ResultsThe AGID attained DSe of 0.65 (CI95%: 0.53–0.76), DSp of 1.00 (CI95%: 0.40–1.00), and accuracy of 0.67 (CI95%: 0.55–0.76). The assay confirmed its diagnostic usefulness primarily for testing samples containing ≥3,125 PCRU of MYXV DNA. However, in the assaying of samples containing <3,125 PCRU of the virus there was a higher probability of getting false negative results, and only molecular methods showed a 100% sensitivity for samples with low (1 PCRU) virus concentration. The overall concordance of the results between AGID and IAC-PCR was fair (ĸ = 0.40). Full concordance of the results was observed for OIE PCR and OIE real-time PCR when control reference material was analysed.ConclusionsFindings from this study suggest that AGID can be used with some limitations as a screening tool for detection of MYXV infections.

Serological survey of bluetongue virus in sheep from Minas Gerais

ABSTRACT: Bluetongue is an infectious, non-contagious disease that affects domestic and wild ruminants, caused by a virus from the Orbivirus genus, Reoviridae family, transmitted by arthropod vectors of the Culicoides genus. This paper aims to be the first serological survey of bluetongue in sheep from the Meso-regions of Campo das Vertentes and South and Southeast of Minas Gerais. Samples were collected from sheep from different properties. The serum samples were submitted to Agar Gel Immunodiffusion (AGID) and competitive Enzyme-Linked Immunosorbent Assay (cELISA). 303 serum samples were submitted to AGID and cELISA. In these samples, 164 (54.13%) were positive in the AGID technique, and 171 (56.44%) positive in the cELISA technique, with an almost perfect agreement between the techniques (kappa index = 0.887). In all visited properties, positive animals have been found in the herd. Animals acquired from properties of the studied mesoregions were more likely to be positive in IDGA and cELISA tests than animals acquired from properties in other regions of Brazil (p&lt;0.001). These results suggest that bluetongue virus (BTV) is widespread in the mesoregions of Campo das Vertentes and South and Southeast of Minas Gerais.

Avian Influenza Virus Isolation, Propagation, and Titration in Embryonated Chicken Eggs.

Avian influenza virus and some mammalian influenza A viruses can be isolated, propagated, and titrated in embryonated chicken eggs (ECEs). Most sample types can be accommodated in ECE culture with appropriate processing. Isolation may also be accomplished in cell culture, and if a mammalian lineage influenza A is expected to be in an avian sample, for example swine influenza in turkey specimens, mammalian cell may be preferable. Culture in ECEs is highly sensitive but is not specific for influenza A, which may be an advantage because a sample may be screened for several agents at once. Once an agent is isolated in culture, the presence of influenza viruses can be confirmed by hemagglutination inhibition assay, antigen immunoassay, agar gel immunodiffusion assay, or RT-PCR. Finally, ECEs may be used to propagate and titrate an avian influenza virus.

Seroprevalence of ovine brucellosis in the microregion of Teresina, Piauí, Brazil

ABSTRACT: Among the diseases that affect the reproductive system of domestic animals, brucellosis in the sheep species is important because it generates significant economic losses to sheep rearing. Thus, it is a threat to the growth and productivity of sheep herds. In the face of this problem, the objective of the present research was to identify the prevalence of ovine brucellosis in herds in municipalities of the Teresina, Piauí, Brazil microregion by using the agar gel immunodiffusion assay (AGID) and indirect enzyme-linked immunosorbent assay (ELISA) serological tests. Fourteen municipalities were included in the research. Blood samples were collected from 521 pubescent animals by puncturing the jugular vein. After collection, the samples were submitted to the serological techniques, AGID and indirect ELISA, to detect anti B. ovis antibody. Of the 521 samples submitted to the AGIDtest, 23 (4.41%) were sera reagent and 498 (95.58%) negative. The indirect ELISA tests, 24 (4.61%) suspect samples and 497 (95.39%) negative samples were obtained, and there were no reagent animals in this test, only suspect. The seroprevalence of ovine brucellosis in the Teresina, Piauí microregion was 4.41%. Thus, it is possible to identify sheep with reagent serology to infection by B. ovis, that is present in municipalities in the state of Piauí, Brazil. Furthermore, AGIDwas shown to be more sensitive in detecting animals that had had contact with the etiological agent of the disease.

Genetic and phylogenetic analysis of Chinese sacbrood virus isolates from Apis mellifera.

BackgroundSacbrood virus (SBV) is one of the most pathogenic honeybee viruses that exhibits host specificity and regional variations. The SBV strains that infect the Chinese honeybee Apis cerana are called Chinese SBVs (CSBVs).MethodsIn this study, a CSBV strain named AmCSBV-SDLY-2016 (GenBank accession No. MG733283) infecting A. mellifera was identified by electron microscopy, its protein composition was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and agar gel immunodiffusion assay, and its nucleotide sequence was identified using a series of reverse-transcription polymerase chain reaction fragments of AmCSBV-SDLY-2016 generated using SBV/CSBV-specific primers. To investigate phylogenetic relationships of the CSBV isolates, a phylogenetic tree of the complete open reading frames (ORF) of the CSBV sequences was constructed using MEGA 6.0; then, the similarity and recombination events among the isolated CSBV strains were analyzed using SimPlot and RDP4 software, respectively.ResultsSequencing results revealed the complete 8,794-nucleotide long complete genomic RNA of the strain, with a single large ORF (189–8,717) encoding 2,843 amino acids. Comparison of the deduced amino acid sequence with the SBV/CSBV reference sequences deposited in the GenBank database identified helicase, protease, and RNA-dependent RNA polymerase domains; the structural genes were located at the 5′ end, whereas the non-structural genes were found at the 3′ end. Multiple sequence alignment showed that AmCSBV-SDLY-2016 had a 17-amino acid (aa) and a single aa deletion at positions 711–729 and 2,128, respectively, as compared with CSBV-GD-2002, and a 16-aa deletion (positions 711–713 and 715–728) as compared with AmSBV-UK-2000. However, AmCSBV-SDLY-2016 was similar to the CSBV-JLCBS-2014 strain, which infects A. cerana. AmCSBV-SDLY-2016 ORF shared 92.4–97.1% identity with the genomes of other CSBV strains (94.5–97.7% identity for deduced amino acids). AmCSBV-SDLY-2016 was least similar (89.5–90.4% identity) to other SBVs but showed maximum similarity with the previously reported CSBV-FZ-2014 strain. The phylogenetic tree constructed from AmCSBV-SDLY-2016 and 43 previously reported SBV/CSBV sequences indicated that SBV/CSBV strains clustered according to the host species and country of origin; AmCSBV-SDLY-2016 clustered with other previously reported Chinese and Asian strains (AC genotype SBV, as these strains originated from A. cerana) but was separate from the SBV genomes originating from Europe (AM genotype SBV, originating from A. mellifera). A SimPlot graph of SBV genomes confirmed the high variability, especially between the AC genotype SBV and AM genotype SBV. This genomic diversity may reflect the adaptation of SBV to specific hosts, ability of CSBV to cross the species barrier, and the spatial distances that separate CSBVs from other SBVs.

Performance assessment of imported ELISA in the serodiagnosis of the enzootic bovine leukosis in herds of Pernambuco state, Brazil

ABSTRACT: Enzootic bovine leukosis (EBL) is an infectious disease of cosmopolitan distribution and chronic character caused by a virus of the Retroviridae family, bovine leukemia virus (BLV). The epidemiological situation of EBL in Brazil has motivated studies to improve its diagnosis, based on the recommended serological techniques: agar gel immunodiffusion (AGID) and enzyme-linked immunosorbent assay (ELISA). This study was designed to evaluate the use of imported ELISA for the detection of BLV in dairy herds raised in Pernambuco, Brazil, comparing it to AGID. Blood serum samples from 327 dairy cattle from the state of Pernambuco were tested to AGID and the imported commercial ELISA CHEKIT-Leucose-serum, produced by the IDEXX® laboratory for the diagnosis of EBL. Discarding 25 inconclusive samples from one or both tests, 302 samples were analyzed, being 24.1% positive (73/302) in the AGID and 45% (136/302) in the ELISA, which compared to the AGID, a technique considered standard, presented sensitivity of 98.6%, specificity of 72% and Kappa coefficient of 0.55. The lack of agreement in the diagnostic methods was probably due to the high sensitivity of the ELISA, which makes it possible to detect antibodies even in situations with low serum levels. Although AGID has been shown to be an efficient test so far, in more advanced stages of an EBL control and eradication program, with low prevalence rates, ELISA will present better performance, due to its higher sensitivity, avoiding the permanence of animals that spread the disease in the herds.

Spatial distribution and risk factors for equine infectious anaemia in the state of Mato Grosso, Brazil

The prevalence of equine infectious anaemia (EIA) in the three biomes of the state of Mato Grosso (Amazon, Cerrado and Pantanal) was estimated. Serum samples were collected from 3,858 equines in 1,067 herds between Septemberand December 2014. The agar gel immunodiffusion (AGID) assay was used to detect EIA virus antibodies, and if a herd contained a seropositive animal itwas classified as a focus. The prevalence rates were 17.2% (95% confidence interval [CI]: 14.9–19.8%) for herds and 6.6% (95% CI: 5.8–7.5%) for animals. The Pantanal region showed the highest prevalence rates: 36.1% (95% CI: 30.8–41.7%) for herds and 17.0% (95% CI: 14.7–19.6%) for animals. The spatial distribution of relative risk was calculated according to the kernel density, which revealed three major clusters with the highest prevalence rates occurring in the north-western(Amazon biome), north-eastern (Cerrado biome) and southern (Pantanal biome)regions. A high spatial correlation was found among ranches, with high intra-herd prevalence rates located in the Amazon and Cerrado biomes, but the highest spatial correlation with EIA foci was found in the Pantanal biome. Variables related to ranch management, reflecting human influence, were associated with positive equines. Based on the results, it can be concluded that EIA is present in all biomes of the state, and that the risk factors are associated with human interference in the transmission process. Given this situation, the EIA control programme should be re-evaluated and more prophylactic measures should be adopted to control the disease.

Spatial distribution and risk factors for equine infectious anaemia in the state of Mato Grosso, Brazil.

The prevalence of equine infectious anaemia (EIA) in the three biomes of the state of Mato Grosso (Amazon, Cerrado and Pantanal) was estimated. Serum samples were collected from 3,858 equines in 1,067 herds between Septemberand December 2014. The agar gel immunodiffusion (AGID) assay was used to detect EIA virus antibodies, and if a herd contained a seropositive animal itwas classified as a focus. The prevalence rates were 17.2% (95% confidence interval [CI]: 14.9-19.8%) for herds and 6.6% (95% CI: 5.8-7.5%) for animals. The Pantanal region showed the highest prevalence rates: 36.1% (95% CI: 30.8-41.7%) for herds and 17.0% (95% CI: 14.7-19.6%) for animals. The spatial distribution of relative risk was calculated according to the kernel density, which revealed three major clusters with the highest prevalence rates occurring in the north-western(Amazon biome), north-eastern (Cerrado biome) and southern (Pantanal biome)regions. A high spatial correlation was found among ranches, with high intra-herd prevalence rates located in the Amazon and Cerrado biomes, but the highest spatial correlation with EIA foci was found in the Pantanal biome. Variables related to ranch management, reflecting human influence, were associated with positive equines. Based on the results, it can be concluded that EIA is present in all biomes of the state, and that the risk factors are associated with human interference in the transmission process. Given this situation, the EIA control programme should be re-evaluated and more prophylactic measures should be adopted to control the disease.

Johne’s disease: a successful eradication programme in a dairy goat herd

This retrospective analysis and report describes the successful eradication and posteradication surveillance programme for Johne’s disease (Mycobacterium avium subspecies paratuberculosis (MAP)) in a closed herd of dairy goats. In 1994,...

Chinese sacbrood virus infection in Apis mellifera, Shandong, China, 2016

The Chinese sacbrood virus (CSBV) was first isolated from Apis cerana in 1972. However, the biological characteristics of the CSBV that naturally infects Apis mellifera, causing larval death, have not been reported yet. In the present study, natural CSBV infection was evaluated using clinical symptoms of A. mellifera larvae, RT-PCR, electron microscopy, agar gel immunodiffusion assays, and virus analysis in inoculated A. cerana larvae. The isolated CSBV strain was named AmCSBV-SDLY-2016. Subsequently, AmCSBV-SDLY-2016 was analyzed by constructing a phylogenetic tree using VP1. Data from the phylogenetic tree suggested that AmCSBV-SDLY-2016 is evolutionarily close to JLCBS-2014. It was also observed that CSBV crossed the species barrier, causing the death of A. mellifera larvae.

Enzyme-Linked Immunosorbent Assays Using the Recombinant gp51 and p24 of Bovine Leukemia Virus for Immunodetection of the Disease

Enzyme-linked immunosorbent assay (ELISA) is often used to test bovine leukemia virus (BLV) infection. However, commercially available kits test in South America detect only antibodies against the gp51 protein. With the aim to improve the sensitivity of the test, we developed here a two-step indirect dual ELISA test that included both proteins p24 and gp51, expressed and produced in E. coli and baculovirus expression system respectively. Two hundred ten BLV sera, stated as double positive or double negative by the combination of commercial agar gel immunodiffusion (AGID) assay and a gp51-ELISA test, were tested with our in house dual rp24/rgp51 ELISA. Firstly, we checked the purified, optimized and standardized proteins as antigen by the checkerboard technique, and set up our in house ELISA test. The concordance correlation coefficient (CCC) and coefficient of variation (CV) intraplate repeatability levels were within the values established by the international standards. The statistical analysis demonstrated the value of sera correctly ranked highest (93.48%), and for 0.3 cutoff, the sensitivity was 95.65% and the specificity 91.30%. In conclusion, the rp24/rgp51 ELISA developed and standardized here demonstrated to have good analytical characteristics to be considered for screening of BLV.

Leptospirosis and brucellosis seroepidemiology in sheep and dogs from non-mechanized rural properties in the northwestern region in the state of Paraná

Sheep breeding has been important in agribusiness, transforming the Brazilian productive scenario. However, it is still deficient due to the damages caused by infectious diseases. Leptospirosis is a severe disease with global distribution, caused by bacteria from the Leptospira genre affecting both humans and animals. The general infection is unapparent, or its clinical signs, when present, are similar to other infections. Brucellosis is an infectious disease caused by bacteria from the Brucella genre responsible for reproductive disorders in animals, especially ruminants. The purpose of this paper was to seroepidemiological study of Leptospira spp. and Brucella ovis in sheep and dogs from nonmechanized rural properties from the northwestern region in the state of Paraná, Brazil. In order to detect anti-Leptospira antibodies, microscopic agglutination (MAT) was performed. For anti-Brucella antibodies, the agar gel immunodiffusion assay (AGID) was performed. From the total 542 samples from sheep sera analyzed, 11.25% were considered reagent to Leptospira spp. and 18.26% to Brucella ovis. From the 36 dog samples, 25% were reagent to MAT and AGID. From the 32 properties analyzed, 75% were considered positive for leptospirosis and 56.25% for brucellosis. Antibodies against the most probable serovars were Hardjo (34.42%) and Butembo (44.44%) in sheep and dogs, respectively, and the variable exchange of animals among properties was associated to leptospiric infection (p=0.028) in sheep. Leptospirosis and brucellosis are present in the sheep herd and dogs in the rural properties studied, and such result is a warning of the zoonotic importance and the need to establish sanitary programs directed to these animal species.

Evaluation of an enzyme immunoassay for antibodies to a recombinant Blastomyces adhesin-1 repeat antigen as an aid in the diagnosis of blastomycosis in dogs.

To evaluate the sensitivity and specificity of an enzyme immunoassay (EIA) for antibodies to a recombinant Blastomyces adhesin-1 repeat antigen (rBAD-1) to aid in the diagnosis of blastomycosis in dogs and compare the findings with results from other tests used for this purpose. Prospective analytic study. Serum and urine from 70 dogs with and without blastomycosis. Serum and urine samples were collected from dogs with blastomycosis (n = 21), histoplasmosis (8), or nonfungal pulmonary disease (21) and from healthy control dogs living in a blastomycosis-endemic area (20). Serum was tested for antibodies against Blastomyces dermatitidis with the rBAD-1 antibody EIA and an A-antigen antibody agar gel immunodiffusion (AGID) assay. Serum and urine were tested for B dermatitidis antigen with a quantitative EIA. Sensitivity of the quantitative antigen EIA was 100% in serum and urine samples from dogs with blastomycosis, with specificity of 95% in urine samples from dogs with nonfungal pulmonary disease and 100% in urine samples from healthy dogs. Sensitivity of the rBAD-1 antibody EIA (95%) was significantly greater than that of the A-antigen antibody AGID assay (65%). Specificity of the antibody EIA was 88% in dogs with histoplasmosis, 95% in healthy dogs, and 100% in dogs with nonfungal pulmonary disease. The rBAD-1 antibody EIA had greater sensitivity than the A-antigen antibody AGID assay in dogs with blastomycosis. This antibody EIA may assist in distinguishing histoplasmosis from blastomycosis. Further evaluation in a larger prospective study is needed to verify these results.