Commercial Indirect Enzyme-linked Immunosorbent Assay Research Articles

Caprine arthritis and encephalitis virus infection in goats of Bangladesh: Serological detection and its associated risk factors.

Caprine arthritis and encephalitis (CAE) is a multisystemic persistent viral disease of goat that causes significant economic losses to the farmers and livestock sector. However, no information in this country is available regarding CAE virus (CAEV) infection. Therefore, this study aimed to estimate the seroprevalence of CAEV infection among the goat population in the selected goat-prone districts in Bangladesh and to identify the associated risk factors of the disease. From July 2021 to June 2022, 446 goat serum samples were randomly collected from the study area. Goat owners were interviewed using a pretested questionnaire to determine the risk factors. A commercial indirect enzyme-linked immunosorbent assay kit was used to screen blood serum for CAEV antibodies. Logistic regression models were used to analyze risk factors and serological data to identify the potential risk factors. Out of 446 serum samples, 19 samples were seropositive against CAEV. The overall seroprevalence was 4.26% (95% confidence interval [CI]: 2.58-6.57). The multivariable logistic regression model identified sex (Female; odds ratio [OR]: 3.98; 95% CI: 1.13-13.95), animal age (12-48 months; OR: 4.93, 95% CI: 0.63-38.13), and biosecurity status (Poor biosecurity; OR: 1.66, 95% CI: 0.46-5.92) as potential risk factors for CAEV seropositivity. This study revealed the serological detection of CAEV in Bangladeshi goats where seroprevalence is found to be relatively low. To eradicate the disease, screening and culling of infected goats from the herd might be implemented.

HEALTHCARE WORKERS IN BULGARIA - ARE THEY PROTECTED FROM VACCINE-PREVENTABLE INFECTIONS?

Background: Healthcare workers (HCWs) are at increased risk of exposure to many viral infections, including vaccine preventable diseases (VPDs) such as measles, mumps and rubella (MMR) as compared to non-HCWs. Immunity of HCWs against these viruses is mandatory in a healthcare setting due to possible exposure from patients or colleagues. Aim: To provide an assessment of anti-measles, mumps and rubella IgG seropositivity among Bulgarian HCWs employed in hospitals and regional health inspectorates (RHI), as an indicator of protective immunity against MMR in this risk group.  Materials and Methods: In the current study, 181 HCWs from Infectious Units in regional hospitals in the country, and HCWs from the RHI, involved in the monitoring and surveillance of MMR cases in Bulgaria were screened. Serum specimens from all participants were tested by a commercial indirect enzyme-linked immunosorbent assay (Anti-Measles, Anti-Mumps, Anti-Rubella IgG EIA-Euroimmun®, Germany) for presence of IgG antibodies against measles, mumps and rubella, as an indicator of protective immunity.  Results: The study included 181 HCWs, 25 male and 156 female, aged 22 to 66 years. The average protective seroprevalence for measles, mumps and rubella was 82.9%, 76.2% and 92.3% percent, respectively. The highest share of negative results were obtained for mumps-specific IgG – 23.2% (42/181), followed by measles 16.6% (60/181) and rubella-specific IgG 7.7% (19/181). Regarding the age distribution, the highest number of HCWs non-immune to measles and mumps was found among the 31- 40-year olds, and against mumps – among the 41-50-year-olds. Conclusion: HCWs are at greater risk of contracting infections than the general population because of contact with sick patients or infectious material. Infected healthcare workers can spread nosocomial diseases to vulnerable patients with more severe illness, leading to complications and even death. Therefore, the vaccination status of HCWs must be strictly monitored.

Establishment of an indirect immunofluorescence assay for the detection of African swine fever virus antibodies

African swine fever (ASF) continues to cause enormous economic loss to the global pig industry. Since there is no safe and effective vaccine, accurate and timely diagnosis of ASF is essential to implement control measures. Indirect immunofluorescence assay (IFA) is a gold standard serological method recommended by the World Organization for Animal Health (WOAH). In this study, we used primary fetal kidney cells to establish a wild boar cell line (BK2258) that supported the efficient replication of ASF virus (ASFV) SD/DY-I/21 and showed visible cytopathic effect (CPE). Moreover, using BK2258, we established a sensitive and specific IFA for ASFV antibody detection. To standardize and evaluate the performance of this assay, we used serum samples from pigs infected with the low virulent genotype I SD/DY-I/21 and genotype II HLJ/HRB1/20, and immunized with the vaccine candidate HLJ/18-7GD, field samples, and negative serum samples. The IFA reacted with the ASFV-positive sera and displayed bright fluorescence foci. There was no non-specific green fluorescence due to cellular senescence or other cell damage-causing factors. Compared to a commercial indirect enzyme-linked immunosorbent assay (iELISA), ASFV antibodies were detected 1–4 days earlier using our IFA. The detection limits of the IFA and iELISA for the same ASFV-antibody positive serum samples were 1:25,600 and 1:6,400, respectively, indicating that the IFA is more sensitive than iELISA. The newly established IFA was highly specific and did not cross-react with sera positive for six other important porcine pathogens (i.e., Classical swine fever virus (CSFV), Porcine reproductive and respiratory syndrome virus (PRRSV), Porcme circovirus type 2 (PCV2), Pseudorabies virus (PRV), Foot-and-Mouth disease virus type O (FMDV/O), and Porcine epidemic diarrhea virus (PEDV)). This study thus provides a sensitive, specific, and reliable detection method that is suitable for the serological diagnosis of ASF.

Seroprevalence of Coxiella burnetii in Sheep and Goats and associated Risk ‎Factors

A cross-sectional seroprevalence study was conducted among flocks of sheep and goats in three agricultural zones of Borno State. Seven hundred sixty-eight small ruminants (384 sheep and goats each) of both sexes (282 males and 486 females) of different age groups from 90 flocks were randomly selected for blood collection and related epidemiological data. A commercial indirect enzyme-linked immunosorbent assay (iELISA; I.D. Vet) was used to test the sera samples for the presence of both phases I and II antibodies against Coxiella burnetii infections. The overall seroprevalence in sheep and goats was 10.9% (44/384) and 12.0% (46/384). There was no statistically significant association between the two species of the animals tested and the infection rates of coxiellosis (P = 0.734, OR= 0.924, 95% CI= 0.578–1.408). Bivariate analysis showed that female animals of all species tested were more seropositive for antibodies to Coxiella burnetii than males. There was a statistically significant association between the sex of both sheep and goats tested (P = 0.032, OR = 0.439, 95% CI = 0.204–0.9470) and (P = 0.022, OR = 0.434, 95% CI = 0.208–0.903). Age of sheep and goats were not statistically significant (P = 0.199, OR = 0.577, 95% CI = 0.247–1.346), (P = 0.304, OR = 0.670, 95% CI = 0.311–1.444. There was no statistically significant association between the breeds of sheep and goats tested (P= 0.861, OR= 0.787, 95% CI= 0.315–1.964), and (P= 0.742, OR= 0.736, 95% CI= 0.264–2.050). The study indicates that seroprevalence of coxiellosis was high in the studied small ruminant population, particularly among female sheep and goats, and can be considered a potential risk for both susceptible animals and humans in the study area

Validation of a Commercial Indirect ELISA Kit for the Detection of Bovine alphaherpesvirus1 (BoHV-1)-Specific Glycoprotein E Antibodies in Bulk Milk Samples of Dairy Cows.

Simple SummaryRecent studies have assessed the feasibility of using commercial indirect enzyme-linked immunosorbent assays (ELISA) on bulk milk (BM) samples. Today is a need to validate a diagnostic method for granting and maintaining a correct surveillance strategy for infectious bovine rhinotracheitis (IBR). However, the poor availability of reference materials for diagnostic tests on milk samples and, the low sensitivity of blocking glycoprotein E (gE) ELISAs using milk as a matrix, represent limitations for developing new assays to control infectious diseases in livestock. This study aimed to validate a commercial indirect ELISA kit to detect antibodies to gE of Bovine alphaherpesvirus 1 (BoHV-1) from BM samples, according to the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. The samples were collected from an IBR outbreak. The findings demonstrated high diagnostic performances. Furthermore, the validated kit is an easy-to-use and economical method to discriminate between BoHV-1 infected or immunised animals with gE-deleted markers vaccines using BM samples. Moreover, milk sampling represents a fast and non-invasive procedure for animals because it avoids negative effects, such as stress, and reduces the total cost of surveillance. In this study, we validated a commercial indirect enzyme-linked immunosorbent assay (ELISA) to detect antibodies to glycoprotein E (gE) of Bovine alphaherpesvirus 1 (BoHV-1) in bulk milk (BM) samples using the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. The assay performance characteristics were evaluated using a panel of positive (n = 36) and negative (n = 80) samples with known infectious bovine rhinotracheitis (IBR) status. The assay showed adequate repeatability (within-run and between-run), with a coefficient of variability (CV%) of replicates below 30%; only two 1:40 diluted samples had a CV% above 20%. Additionally, an agreement analysis of the qualitative results of replicates led to a Gwet’s agreement coefficient of 0.99 (95% confidence interval (CI): 0.96–1.00, p < 0.001). The estimated diagnostic sensitivity (DSe) and diagnostic specificity (DSp) were 100% (95% CI: 90.3–100%) and 97.5% (95% CI: 91.3–99.7%), respectively. Overall, a good level of agreement was observed between the assay results and the true IBR status of samples (weighted Cohen’s κ: 0.96, 95% CI: 0.78–1.00). The findings demonstrate that the indirect ELISA kit validated here is an easy-to-use and economical method to differentiate infected and gE-deleted marker vaccine-immunised animals using BM samples.

Comparison of Commercial Enzyme-linked Immunosorbent Assays for Diagnosis of Contagious Agalactia Caused By Mycoplasma Agalactiae.

IntroductionContagious agalactia (CA) is a disease affecting small ruminants with worldwide distribution and caused by several mycoplasmas, especially M. agalactiae. The main option for systematic diagnosis under monitoring control programmes is the enzyme-linked immunosorbent assay (ELISA) test.Material and MethodsThis study was designed to appraise the performance of two commercial indirect ELISA tests using M. agalactiae p48 protein and one using total protein, for antibody detection in small ruminants after natural infection with different M. agalactiae strains. We carried out the test evaluation using sera of confirmed M. agalactiae-positive goats with clinical signs. In addition, test agreement was assessed by kappa between the three commercial ELISA tests.ResultsAll three ELISA tests showed high validity scores (Youden’s J: 72.9–84%). The sensitivity values for the P48 protein-based tests were 76.9% and 84.6%, and was 79% for the total protein-based test. The specificity of all tests was 100%. In addition, between the total protein-based ELISA test and the other two ELISA tests based on the P48 protein, the agreement was substantial (kappa: 0.762–0.763) and the agreement between the latter two tests was almost perfect (kappa: 0.93).ConclusionThe validity parameters for all tests allowed their application for diagnostic purposes in lactating goats excreting M. agalactiae in milk and presenting clinical signs. The agreements show that any of these ELISA tests could be equally well used for diagnosis in programmes against CA.

Seroprevalence of bovine coronavirus and factors associated with the serological status in dairy cattle in the western region of Thailand.

Background and Aims:Bovine coronavirus (BCoV) is a pathogen affecting the productivities of dairy cattle worldwide. The present study aimed to determine the seroprevalence and factors associated with BCoV serological status using a commercial indirect enzyme-linked immunosorbent assay (ELISA).Materials and Methods:A cross-sectional study was conducted in the western region of Thailand. Blood samples were collected from 30 dairy herds. In total, 617 blood serum samples were tested using a commercial indirect ELISA for BCoV-specific immunoglobulin G antibodies. A questionnaire was used to collect data on the factors which have been identified as risk factors for BCoV antibody detection. The age and history of diarrhea of each animal were recorded. Fisher’s exact test was performed to univariately assess the association between BCoV serological status and possible risk factors. Variables with Fisher’s exact test p<0.10 were then evaluated using multivariate logistic regression to identify factors associated with BCoV serological status. The Bonferroni adjustment was used for multiple comparisons of significant variables in the final multivariate logistic regression model.Results:No herd was free from antibodies to BCoV. The individual seroprevalence of BCoV was 97.89% (604/617). The prevalence within herds was in the range of 45.45-100%. Cattle >3 years of age were more likely to be seropositive to BCoV compared to cattle <1 year of age (p=0.003), with the odds ratio being 81.96. Disinfecting diarrhea stools were a protective factor for being BCoV seropositive, with odds ratios of 0.08 and 0.06 compared to doing nothing (p=0.008) and to clean with water (p=0.002), respectively.Conclusion:BCoV seropositive dairy cattle were distributed throughout the western region of Thailand. The probability of being seropositive for BCoV increased with increasing animal age. Cleaning the contaminated stool with appropriate disinfectants should be recommended to farmers to minimize the spread of the virus.

Seroprevalence of Toxoplasma gondii and associated risk factors in domestic pigs raised from Cuba

A cross-sectional study was carried out to determine the seroprevalence of Toxoplasma gondii and associated risk factors in pigs in the largest pork-producing region in Cuba. Serum samples from 420 pigs, including 210 sows and 210 post-weaning pigs, were tested for antibodies against T. gondii using a commercial indirect enzyme-linked immunosorbent assay. Anti-T. gondii antibodies were detected in 56 animals (13.3%, 95% CI: 10.1–16.6). A generalized estimating equations model revealed that the risk factors associated with higher seropositivity in pigs were altitude (higher in farm’s location < 250 m above sea level (masl) versus ≥ 250 masl) and age (higher in sows compared to post-weaning pigs). The results indicated that this protozoan parasite is widely distributed on pig farms in the study area, which is a public health concern since the consumption of raw or undercooked pork meat products containing tissue cysts is considered one of the main routes of T. gondii transmission worldwide. Control measures should be implemented to reduce the risk of exposure to T. gondii in pigs in Cuba.

Seroprevalence of small ruminant lentivirus infection in goats in Thailand.

Small ruminant lentivirus (SRLV), which causes caprine arthritis encephalitis in goats and ovine progressive pneumonia (maedi-visna disease) in sheep, is classified in genus Lentiviruses belonging to Retroviridae family. It persists in infected goats and sheep, which mostly are sub- clinical. A serological survey was conducted to determine the prevalence of small ruminant lentivirus infection in Thai goat population. Serum samples were taken from 1,925 goats distributed throughout the country, then they were tested for the presence of SRLV antibodies using commercial indirect enzyme-linked immunosorbent assay (ELISA) test kits. Results revealed that a total of 68 goats were found seropositive, representing the apparent prevalence and true prevalence of 3.57% and 2.60%, respectively. The seroprevalence, revealed in this study, was lower than in the previous reports. The decreasing of seroprevalence might be caused by successful control strategies from Department of Livestock Development (DLD).

Prevalence of Toxoplasma gondii and Cryptosporidium in Feral and Farmed American Mink (Neovison vison) in Denmark

To investigate the prevalence of Cryptosporidium spp. infection and Toxoplasma gondii antibodies in farmed and feral mink in Denmark. We examined meat juice from 235 feral mink and 306 farmed mink for T. gondii antibodies, and faecal samples from 113 feral mink and 166 farmed mink for Cryptosporidium oocyst excretion. Meat juice was analysed using a commercial indirect enzyme-linked immunosorbent assay and oocyst excretion was identified by a modified Ziehl-Neelsen method. All farmed mink tested sero-negative, while 53.6% of feral mink were T. gondii sero-positive. The probability of being sero-positive for T. gondii was not associated with recent escapes from farms (p = 0.468), but was significantly higher for male feral mink (64.2%) than female feral mink (42.5%) (p = 0.0008). Only one feral mink and four farmed mink (2.4%) excreted Cryptosporidium oocysts. Farmed mink were all T. gondii sero-negative, whereas approximately half the feral mink were sero-positive. Cryptosporidium prevalence in farmed and feral mink were low. Overall, the public health risk of transmission of these two parasites via mink in Denmark is low.

Relationship between natural and infection-induced antibodies in systemic autoimmune diseases (SAD): SLE, SSc and RA.

Infection or vaccine-induced T cell-dependent immune response and the subsequent high-affinity neutralizing antibody production have been extensively studied, while the connection between natural autoantibodies (nAAbs) and disease-specific antibodies has not been thoroughly investigated. Our goal was to find the relationship between immunoglobulin (Ig)M and IgG isotype nAAbs and infection or vaccine-induced and disease-related autoantibody levels in systemic autoimmune diseases (SAD). A previously described indirect enzyme-linked immunosorbent assay (ELISA) test was used for detection of IgM/IgG nAAbs against citrate synthase (anti-CS) and F4 fragment (anti-F4) of DNA topoisomerase I in 374 SAD samples, with a special focus on systemic lupus erythematosus (SLE) (n=92), rheumatoid arthritis (n=73) and systemic sclerosis (n=157) disease groups. Anti-measles IgG and anti-dsDNA IgG/IgM autoantibodies were measured using commercial and in-house indirect ELISA tests. In all SAD groups the anti-measles IgG-seropositive cases showed significantly higher anti-CS IgG titers (P=0·011). In anti-dsDNA IgG-positive SLE patients, we detected significantly higher levels of anti-CS and anti-F4 IgG nAAbs (P=0·001 and<0·001, respectively). Additionally, we found increased levels of IgM isotypes of anti-CS and anti-F4 nAAbs in anti-dsDNA IgM-positive SLE patients (P=0·002 and 0·016, respectively). The association between IgG isotypes of pathogen- or autoimmune disease-related antibodies and the IgG nAAbs may underscore the immune response-inducible nature of the diseases investigated. The relationship between protective anti-dsDNA IgM and the IgM isotype of anti-F4 and anti-CS may provide immunoserological evidence for the beneficial roles of nAAbs in SLE patients.

Sero-Prevalence of Bovine Leukemia Virus Infection in Kosovo Cattle

Abstract A cross-sectional survey was conducted in Kosovo to determine the presence and prevalence of bovine leukemia virus antibodies in cattle. A total of 5,051 serum samples from 315 villages were collected during 2016. Samples were tested using commercial indirect enzyme-linked immunosorbent assay. At least one sero-positive animal was found in 55 (17.5%) of the villages sampled, of which 23 had more than one sero-positive detected. Overall individual sero-prevalence, corrected for geographic distribution of samples, was found to be 2.26% (95% c.i. 1.62% to 3.04%). There were no statistically significant associations between serological status and herd size or age of animal, although sero-prevalence in 5-year-old cattle was higher than in the other ages. There was a statistical significant heterogeneity in sero-prevalence between different geographic zones of Kosovo (chi-square value = 20.68 (4 d.f.); (p=0.0004). Pairwise comparisons showed that sero-prevalence in the south was significantly higher than in the east and in the north and sero-prevalence in the west was significantly higher than in the north. The 3.11% aggregated sero-prevalence for the two highest sero-prevalence zones, south and west, was significantly higher than the 1.57% aggregated sero-prevalence for the remaining zones, centre, east and north (Fisher exact p-value (2-tail) = 0.0004). The reason for higher prevalence in the south and west of Kosovo is uncertain. These results may serve to enrich the information of bovine leukemia virus distribution in the region, as well as a starting point for the future control and eradication strategy in Kosovo.

Detection of Coxiella burnetii antibodies in sheep and cattle on a veterinary campus in St. Kitts: Implications for one health in the Caribbean region

Coxiella burnetii is a ubiquitous zoonotic bacterium reported worldwide that causes Q-fever. Infections result in profound economic losses to livestock producers by causing abortions and low birth weights. Current information about the disease in the Caribbean region is scarce. With multiple small islands and territories, it is often considered that the bacterium is absent or circulates at a low prevalence. Our study aimed to determine whether sheep and cattle housed at a veterinary campus in St Kitts had previous exposure to C. burnetii. Blood samples were taken from cattle (n = 63; 72% of the herd) and sheep (n = 133; 71% of the flock). Antibodies to C. burnetii were detected by a commercial indirect enzyme-linked immunosorbent assay (IDvet® ELISA) test. The seroprevalence was estimated at 26.3% (95% CI: 19.1–34.7%) in sheep and 0% (95% CI: 0–5.7%) in cattle. Sheep importation to St. Kitts is very rare, thus, these results suggest that C. burnetii is present on the island. The seronegativity of all the cattle highlights the absence of the bacterium on the veterinary campus. The high seroprevalence in sheep, however, has potentially important implications for animal health and public health as well as for wildlife conservation. Further investigation about animal seroprevalence and human exposure are warranted in St. Kitts and in the Caribbean region.

Serological study on brucellosis in captive elephants (Elephas maximus) and stray dogs in North Thailand.

Background and Aim:Brucellosis is considered as an important zoonotic disease caused by various strains of Brucella in numerous host species. Although brucellosis has been reported in almost animal species, the relevance of brucellosis infection and diagnostic technique in Asian elephant (Elephas maximus) has been limited. The present serological investigation aimed to investigate the antibody response to Brucella abortus in captive Asian elephants in North Thailand. Moreover, further serological survey was also conducted to detect the antibody response to Brucella canis in stray dogs cohabiting the same area as the elephant herd.Materials and Methods:Serum samples were collected from 40 captive Asian elephants and submitted for serological analysis based on B. abortus antigen using Rose Bengal plate test (RBPT) in combination with ethylenediaminetetraacetic acid-tube agglutination test (EDTA-TAT) as a supplementary test and by commercial indirect enzyme-linked immunosorbent assay (iELISA). In addition, serum samples were also obtained from 16 stray dogs that live nearby the elephant-raising area and were tested using commercial Dot-ELISA based on B. canis antigen.Results:Serological analysis in captive Asian elephants showed 100% seronegative (40/40) from all serological tests response to B. abortus. For stray dogs, 12.5% (2/16) had a low positive reaction response to B. canis.Conclusion:The serological survey for brucellosis in Asian elephant was adapted and applied using RBPT, EDTA-TAT, and iELISA in the present study. For future evaluation, we recommended the use of a combination of serological tests with validation together with comparing by direct detection such as bacterial isolation to provide an appropriate brucellosis surveillance program in Asian elephants. In addition, the surveillance of stray dogs or multispecies habitation should be kept into considerations.

Seroprevalences of Rickettsia conorii, Ehrlichia canis and Coxiella burnetii in Dogs from Montenegro.

The incidence of vector-borne zoonoses has been increasing in Europe as a result of global climate change, and rickettsioses are a significant etiologic entity among these infections. The objective of this study was to investigate the seroprevalences of Rickettsia conorii, Ehrlichia canis and Coxiella burnetii in dogs in Montenegro. The seroepidemiological study covered 259 dogs, of which 155 were owned dogs suspected of infection with agents of rickettsial aetiology from the continental (Podgorica) or five coastal municipalities of Montenegro (Budva, Herceg Novi, Kotor, Ulcinj and Bar), and 104 dogs were from a public shelter in Podgorica. The presence of specific IgG antibodies against R. conorii was analysed using a commercial indirect enzyme-linked immunosorbent assay (ELISA) test, and the presence of antibodies against E. canis and C. burnetii was analysed using commercial indirect immunofluorescence tests (IFAT). Seroprevalences of 73.36, 19.3 and 1.16% were determined for R. conorii, E. canis and C. burnetii, respectively. R. conorii was significantly more prevalent (χ2 = 14.53; p < 0.001) in owned dogs (81.93%) than in dogs from the public shelter (60.6%), while E. canis was more prevalent (χ2 = 12.31; p < 0.001) in dogs from the public shelter (29.81%) than in owned dogs (12.26%). Coinfection with two pathogens was determined in 40 (15.44%) dogs, and the prevalence of R. conorii/E. canis (χ2 = 4.23; p < 0.05) was greater in dogs from the public shelter (20.19%) than in owned dogs (10.97%). The prevailing evidence from this study shows that dogs from Montenegro are exposed to pathogens of veterinary and public health importance. This calls for the One Health approach to sensitise the public on the risks of zoonoses from dogs and to formulate policies and strategies to mitigate their spread and safeguard public health.

Indirect Enzyme Linked Immunosorbent Assay Sebagai Metode untuk Melacak Bruselosis pada Sapi Perah (INDIRECT ENZYME IMMUNOSORBENT ASSAY (I-ELISA) AS METHOD FOR DETECT BRUCELLOSIS IN DAIRY COW)

Brucellosis has become a zoonotic disease that received attention in efforts to prevent and eradicate strategic infectious animal diseases in Indonesia. Brucellosis can be detected early by the rose bengal test (RBT), followed by complement fixation test (CFT) and by enzyme linked immunosorbent assay (ELISA). The aims of this research was to study the indirect enzyme linked immunosorbent assay test (I-ELISA) as an alternative test for detecting brucellosis in dairy cattle. The method was used by conducting tests of RBT, CFT, I-ELISA and commercial I-ELISA to test brucellosis. The test results were calculated sensitivity and specificity, as well as analyzed by calculating the kappa value. The method was used by conducting tests of RBT, CFT, I-ELISA and commercial I-ELISA to test brucellosis. The test results were calculated for sensitivity and specificity, as well as analyzed by calculating the Kappa statistical value. The results of the sensitivity and specificity calculation showed that the indirect enzyme linked immunosorbent assay (I-ELISA) test developed a higher sensitivity (100%) compared to RBT test (93.75%) and commercial I-ELISA (93.75%). The developed I-ELISA specificity (74.68%) was still lower than RBT (89.87%), but higher than commercial I-ELISA (70.52%). The calculation of the statistical value of kappa RBT with CFT showed the kappa value 0.7120 which meaned it had a good agreement, commercial I-ELISA with CFT showed kappa value 0.6165 which meaned it had good suitability, whereas I-ELISA developed with CFT showed kappa value 0.4984 which meaned having a moderate agreement.In conclusion, the indirect enzyme linked immunosorbent assay (I-ELISA) which had been developed had low specificity, but the sensitivity was the highest compared to the commercial I-ELISA test and RBT, so this test was appropriate to be used as a screening test, especially in dairy cows movement into brucellosis-free areas or regions.

First dengue virus seroprevalence study on Madeira Island after the 2012 outbreak indicates unreported dengue circulation

BackgroundIn 2012, the first dengue virus outbreak was reported on the Portuguese island of Madeira with 1080 confirmed cases. Dengue virus of serotype 1 (DENV-1), probably imported from Venezuela, caused this outbreak with autochthonous transmission by invasive Aedes aegypti mosquitoes.ResultsWe investigated the seroprevalence among the population on Madeira Island four years after the outbreak. Study participants (n = 358), representative of the island population regarding their age and gender, were enrolled in 2012 in a cross-sectional study. Dengue antibodies were detected with an in-house enzyme-linked immunosorbent assay (ELISA) using the dimer of domain III (ED3) of the DENV-1 envelope protein as well as commercial Panbio indirect and capture IgG ELISAs. Positive ELISA results were validated with a neutralization test. The overall seroprevalence was found to be 7.8% (28/358) with the in-house ELISA, whereas the commercial DENV indirect ELISA detected IgG antibodies in 8.9% of the individuals (32/358). The results of the foci reduction neutralization test confirmed DENV-1 imported from South America as the causative agent of the 2012 epidemic. Additionally, we found a higher seroprevalence in study participants with an age above 60 years old and probable secondary DENV infected individuals indicating unreported dengue circulation before or after 2012 on Madeira Island.ConclusionsThis study revealed that the number of infections might have been much higher than estimated from only confirmed cases in 2012/2013. These mainly DENV-1 immune individuals are not protected from a secondary DENV infection and the majority of the population of Madeira Island is still naïve for DENV. Surveillance of mosquitoes and arboviruses should be continued on Madeira Island as well as in other European areas where invasive vector mosquitoes are present.

ASSAYS FOR DETECTION AND IDENTIFICATION OF THE CAUSATIVE AGENT OF MANGE IN FREE-RANGING BLACK BEARS ( URSUS AMERICANUS).

Three mite species ( Demodex ursi, Ursicoptes americanus, and Sarcoptes scabiei) have been associated with mange in black bears ( Ursus americanus). Since the early 1990s, the number and geographic distribution of mange cases in black bears in Pennsylvania, US has increased; however, the causative mites have yet to be completely defined. We evaluated several diagnostic approaches for detection and identification of mites in 72 black bears with severe lesions consistent with mange. Sarcoptes scabiei was morphologically identified in skin scrapes from 66 of the bears; no mites were identified in the remaining six. Histopathologic lesions consistent with sarcoptic mange were observed in 39 of 40 bear skin samples examined, and intralesional mites were observed in samples from 38 of these bears. Samples were collected from a subset of the 72 bears for PCR testing targeting both the internal transcribed spacer (ITS)-2 region and cytochrome c oxidase I ( cox1) gene including 69 skin scrapes ( ITS-2 only), 56 skin biopsies ( ITS-2 and cox1), and 36 fecal samples ( ITS-2 and cox1). Skin scrapes were a more sensitive sample for PCR detection than either skin biopsies or fecal samples, and the ITS-2 primers proved more sensitive than cox1. Using a commercial indirect enzyme-linked immunosorbent assay, antibodies to S. scabiei were detected in 45/49 (92%) black bears with confirmed mange and 0/62 (0%) cubs with no gross lesions suggestive of mange and which were born to seronegative sows. Sarcoptes scabiei was the predominant mite associated with mange in black bears in Pennsylvania. Diagnostically, cytologic examination of skin scrapes was the most effective approach for diagnosing active mite infestations in black bears. The evaluated serologic assay accurately detected antibodies to S. scabiei in most bears with confirmed S. scabiei infestations. Additional research is needed to determine the usefulness of this approach for larger scale surveys and for asymptomatic bears.

Seroprevalence of Neospora caninum-specific antibodies in German breeding bitches

BackgroundNeospora caninum is an intracellular obligate apicomplexan parasite responsible for multisystemic lesions in dogs. Being definitive hosts and reservoirs, dogs excrete environmentally resistant oocysts. Breeding bitches represent a susceptible dog group and infected bitches may spread this parasite through transplacental transmission.ResultsA total of 218 serum samples of German breeding bitches were collected to determine the presence of N. caninum. Antibodies were detected in 16 (7.33%) bitches using a commercial indirect enzyme-linked immunosorbent assay (ELISA). Immunoblotting analysis confirmed all seropositive samples detected by ELISA, proving that the animals were infected with N. caninum. The owners were interviewed regarding breed, age, environment, type, vaccine status, feeding habits and the presence of reproductive disorders. Seropositive animals were between the ages of two to seven years; three of them were kept in kennels while the others were household dogs, one of which was additionally a hunting dog. Owners of four seropositive bitches reported one gestation, while multiple pregnancies had been recorded for the other twelve bitches. Fourteen bitches were regularly vaccinated and six were fed with fresh raw meat.ConclusionsAlthough the results confirmed a low incidence of N. caninum seropositive German breeding bitches, further epidemiological and surveillance studies are required to complement our findings regarding the current situation of neosporosis in this specific canine population of Germany.

Sensitivity and specificity of indirect ELISA for the detection of antibody titers against BVDV from beef cattle raised in Pará State

The aims of this study were to establish the prevalence of anti-bovine viral diarrhea virus (BVDV) antibodies (Ab) in beef cattle raised in Pará state, to compare the prevalence of seropositive animals to BVDV using a commercial indirect enzyme-linked immunosorbent assay kit (iELISA) and the virus neutralization (VN) test, and finally, to determine the sensitivity (Se) and specificity (Sp) of the iELISA for the detection of anti-BVDV Ab using VN as a gold standard. A total of 400 serum blood samples from Nelore cows aged at least 24 months from five farms in the Pará state from two mesoregions (Metropolitan Region of Belem and Northeast of Pará) were analyzed. All animals were vaccinated against brucellosis and foot-and-mouth disease. The examination of anti-BVDV Ab with VN was performed in the Laboratory of Bovine Viruses of the Biological Institute of Sao Paulo as described in the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. For VN, bovine kidney epithelial cells from the Madin Darby Bovine Kidney (MDBK) strain were used. The determinations of anti-BVDV Ab were performed with the iELISA test at the Laboratory of Immunology and Microbiology of the Federal Rural University of Amazonia according to the manufacturer's recommendations. The results were classified as follows: (a) correct positive diagnosis, (b) incorrect positive diagnosis, (c) correct negative diagnosis, and (d) incorrect negative diagnosis, according to the results obtained from VN. From the values obtained from VN and iELISA, Se [(a ÷ a + d) × 100], Sp [(c ÷ c + b) × 100], positive predictive value [(a ÷ a + B) × 100], and negative predictive value [(c ÷ c + d) × 100] were calculated for iELISA. The frequencies (%) of seropositive animals were determined and compared both between the different tests (iELISA and VN) and between the different farms (1, 2, 3, 4, and 5). The statistical analysis was performed with a significance level of 5%. The prevalence of seropositive animals was found to be different (P &lt; 0.0001) using VN (39.25% [157/400]) and iELISA (54.50% [218/400]). It was observed that the Se and Sp of the iELISA assay were 98.72% and 74.07%, respectively. Of the total, 25.93% (63/243) of the samples were considered false-positive and 1.27% false-negative (2/157). It was concluded that the BVDV infection is present in beef cattle herds of the state of Para. Based on the speed of execution, ease of handling, and high Se of the iELISA, it is suggested that this assay can be used as a screening test for the detection of anti-BVDV Ab with the aim of eliminating infected animals from large herds of beef cattle.