Search Results (72)

Equine piroplasmosis (EP) is an endemic parasitic disease in southern European countries, such as Spain. Andalusia, the most southwestern region of Europe, is the community with the highest number of registered horses and farms in Spain and one of the main international exporters of Andalusian (Spanish Purebred) horses worldwide. Considering the current expansion of this disease and the possible effect of climate change on its prevalence, studying the EP prevalence in this region is compelling. Molecular (PCR) and serological methods (cELISA and IFAT) were used to study the true and apparent prevalences during a period of three consecutive years, evaluating the effects of age, sex, season, year of testing, and province. Results showed different EP prevalences between western and eastern provinces, as well as among seasons. Moreover, a positive association was observed between age and T. equi seropositivity, without any effect of sex. Our findings demonstrate that Andalusia is an EP endemic region, but prevalences were lower compared to central and northern Spanish regions. Moreover, EP prevalence has not increased in Andalusia in recent years despite climate changes. Full article

Interleukin-12 (IL-12), a crucial biomarker for immune and inflammatory responses, plays a pivotal role in diagnosing and managing diverse pathological conditions. Although colorimetric enzyme-linked immunosorbent assays (CELISAs) have been extensively employed to detect IL-12 in biological samples, their sensitivity is inherently limited by the catalytic efficiency of enzyme labels, presenting substantial challenges in achieving ultrasensitive detection and enabling pre-symptomatic diagnosis of diseases. In this study, we address this limitation by developing a novel peroxidase nanozyme, featuring ultrathin Pt skins consisting of only ~4 atomic layers, coated on Au nanoparticles (denoted as Au@Pt_4LNPs). These Au@Pt_4LNPs exhibit remarkable catalytic performance, achieving a ~1063-fold enhancement in peroxidase-like activity compared to horseradish peroxidase (HRP), while minimizing Pt consumption, thereby improving Pt utilization efficiency and reducing costs. This advancement facilitates the construction of an ultrasensitive CELISA capable of detecting IL-12 at femtomolar concentrations. Using Au@Pt_4LNPs as the signal labels, the developed CELISA demonstrates a quantitative detection range from 0.1 to 100 pg mL⁻¹, with a limit of detection (LOD) as low as 0.084 pg mL⁻¹ (1.1 fM), offering ~10 times greater sensitivity than the HRP-based CELISA. This study highlights the potential of Au@Pt_4LNP nanozymes as advanced signal labels, opening new avenues for next-generation ultrasensitive bioassays. Full article

Outbreaks of the West Nile Virus (WNV) have increased significantly in recent years in the Mediterranean region, including Tunisia. To understand the risks for animal and human health and to mitigate the impact of future outbreaks, comprehensive viral surveillance in vertebrate hosts and vectors is needed. We conducted the first serosurvey for the WNV in ruminants in southern Tunisia using the ELISA test and confirmed it with the micro-virus neutralization test (VNT). Antibodies were detected by the ELISA test in camels (38/112), sheep (9/155), and goats (7/58), and six samples were doubtful (five camels and one sheep). The ELISA positive and doubtful sera (n = 60) were further analyzed to confirm the presence of specific anti-WNV and anti-Usutu virus (USUV) antibodies using the micro-virus neutralization test (VNT). Out of the 60 sera, 33 were confirmed for specific WNV antibodies, with an overall seroprevalence of 10.15% [95% CI: 7.09–13.96]. The high seroprevalence observed in camels (22.3%) suggests their potential use as sentinel animals for WNV surveillance in southern Tunisia. The viral genome, and consequently active circulation, could not be detected by real-time RT-qPCR in blood samples. Ongoing surveillance of the WNV in animals, including camels, sheep, and goats, may be used for the early detection of viral circulation and for a rapid response to mitigate potential outbreaks in horses and humans. Full article

Rose Bengal antigen and smooth lipopolysaccharide (s-LPS) were produced from a field strain of Brucella ceti (“homologous” antigens) and from the reference strain B. abortus S99 (“heterologous” antigens); they are currently used for the diagnosis of brucellosis in cattle, water buffaloes, sheep, goats, and pigs, as recommended in the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals of the World Organization for Animal Health (WOAH). “Homologous” and “heterologous” antigens were used in a rapid serum agglutination test (Rose Bengal test, RBT) and a competitive ELISA assay (c-ELISA) to test a panel of sera, blood, and other body fluids (cerebrospinal fluid, pericardial fluid, tracheal fluid, and aqueous humor) collected from 71 individuals belonging to five cetacean species (Stenella coeruleoalba; Tursiops truncatus; Grampus griseus; Globicephala melas; and Ziphius cavirostris), which were found stranded on the Italian coastline. Six animals were positive for Brucella spp. for bacterial isolation and/or PCR, and 55 animals were negative; for the remaining 10 animals, no PCR/isolation data were available. A total of 90 samples were tested. Results obtained from the two tests were compared in order to identify the most suitable antigen for the serological diagnosis of Brucella infection in cetaceans. The RBT performed with the “homologous” antigen showed the best results in comparison with the “heterologous” antigen: diagnostic sensitivity, specificity, and accuracy were 80.0%, 44.1%, and 46.9% for the “homologous” antigen and 80.0%, 17.0%, and 21.9% for the “heterologous” antigen. For the c-ELISA, “homologous” and “heterologous” s-LPS showed similar results (diagnostic sensitivity 66.7%, diagnostic specificity 97.3%, and diagnostic accuracy 95.0%). Therefore, the RBT using the “homologous” antigen and c-ELISA with “homologous” or “heterologous” s-LPS could be used in parallel for the detection of antibodies against Brucella spp. in cetaceans. Full article

Bovine brucellosis (bB) is a zoonosis mainly caused by the Brucella abortus species in cattle. Bovine brucellosis can present with either a range of clinical symptoms, including spontaneous abortions in the last trimester of pregnancy, retained fetal membranes, and decreased milk production, or it can be asymptomatic. In Ecuador, vaccination against bB with S19 and/or RB51 is not mandatory and is the responsibility of the farmer. As serology is a convenient method for detecting antibodies against Brucella, evaluating the diagnostic performance and discriminative ability of such tests in various epidemiological settings is required. To estimate and compare the diagnostic sensitivity (Se) and specificity (Sp) of two screening tests, a new competitive (cELISA) and an indirect ELISA based on a new synthetic antigen (iELISA), a randomized, stratified, cross-sectional, serological survey was performed on the cattle population (3299 bovine sera from 223 farms) in continental Ecuador. A Bayesian approach was used to evaluate the two tests by estimating their respective diagnostic Se and Sp, as well as the true prevalence of bB in different sub-populations (non-vaccinated, vaccinated with S19 or RB51). The Se of both tests was similar across Bayesian models, with values around 94%. In contrast, the Sp of the iELISA, ranging between 97 and 98%, was significantly higher than that of the cELISA, which was approximately 94–95%. The true prevalence of bB was 1.63% (95% CrI: 0.56–2.54) in non-vaccinated cattle, decreased to 0.97% (95% CrI: 0.005–2.54) in S19-vaccinated cattle and was 2.75% (95% CrI: 0.50–5.32) in RB51-vaccinated cattle. The results of this study suggest that, with similar Se and higher Sp, the iELISA based on an innovative synthetic antigen (which is more standardizable) should be recommended as a possible screening test for bB in Ecuador. Also, the proposed approach suggests insights into the quality of the vaccination campaign and highlights the need for refining the Ecuadorian national brucellosis control program. Full article

Anaplasmosis is an infectious disease transmitted by ticks and caused by obligate intracellular pathogen of belonging to genus Anaplasma Infections of one-humped camels (Camelus dromedarius) and llamas (Lama glama) have been reported previously. The aim of this study was to investigate the seroprevalence and risk factors of anti-Anaplasma spp. antibodies in Camelus dromedarius of the Punjab, Pakistan. A cross-sectional study was conducted during 2017–2018 to study the seroprevalence of anaplasmosis in Camelus dromedarius of 13 districts in Punjab province of Pakistan and to assess the associated risk factors including age, breed, gender, body condition score, tick infestation, location, season and management type. Serum samples from 728 camels (433 females and 295 males) were examined for anti-Anaplasma antibodies using a commercially available competitive enzyme-linked immunosorbent assay (cELISA) test kit. A univariable analysis was conducted and extended to multivariate logistic regression to find potential risk factors associated with the disease. Overall, the seroprevalence of anti-Anaplasma antibodies was 8.5% (8.5%, CI 6.6–10.8) with 62 positives in 728 camels. The highest seroprevalence was recorded for camels of the Central Punjab districts (16.1%, CI 11.5–21.7) followed by those of the Northwestern (5.4%, 2.8–9.3) and Southern Punjab (5.2%, 2.9–8.4) districts (p < 0.001). Multivariable analysis showed that location (Central Punjab: OR 2.78, p = 0.004), season (summer: OR 7.94, p = 0.009), body condition score (BCS 2: OR 14.81, p = 0.029) and tick infestation (OR 38.59, p < 0.001) are potential risk factors in the corresponding camel populations. The results showed that the camel population in Pakistan is seropositive for Anaplasma spp. The geographical zone, season, body condition and tick infestation were identified as significantly associated risk factors for seroprevalence of anaplasmosis in dromedary camels. To the best of our knowledge, the results of this current study provide the first evidence of exposure of camels to anaplasmosis in Pakistan. Molecular investigations in the future are highly recommended to determine the dynamics of the disease in camels. Full article

A newly formatted enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to bluetongue virus (BTV) was developed and validated for bovine and ovine sera and plasma. Validation of the new sandwich ELISA (sELISA) was achieved with 949 negative bovine and ovine sera from BTV endemic and non-endemic areas of Australia and 752 BTV positive (field and experimental) sera verified by VNT and/or PCR. The test diagnostic sensitivity (DSe) and diagnostic specificity (DSp) were 99.70% and 99.20%, respectively, for bovine sera, and 97.80% and 99.50%, respectively, for ovine sera. Comparable diagnostic performances were noted for the sELISA compared to four competition ELISAs. While the sensitivity of the sELISA remained unaffected by BTV-15 positive sera, the cELISAs were not as sensitive. BTV-15 is endemic to Australia, and early warning depends on sensitive diagnoses of all serotypes: endemic or incurring. The sELISA failed to discriminate against epizootic hemorrhagic disease virus (EHDV) antibodies, the most serologically related orbivirus to BTV. The ACDP cELISA and the IDEXX kit showed cross-reactivity with some EHDV serotypes, with the least cross-reactive being the VMRD and the IDVet kits. Cross-reactivities, however, were also detected in sera raised experimentally from 10 isolates of the 21 known non-BTV orbiviruses. In this case, the sELISA was the least affected, followed equally by the VMRD and IDVet kits, and the IDEXX kit and the ACDP cELISA were the least discriminatory. In addition to exclusivity assessment of the ELISAs, an inclusivity assessment was made for all ELISAs using well characterized reference sera positive for antibodies to all serotypes BTV-1 to BTV-24. Full article

Aflatoxins (AFs), secondary metabolites produced by fungi, pose significant health risks, especially to children and elderly individuals. In developing countries such as Nepal, the tropical climate promotes fungal growth, leading to elevated levels of AF in animal feed and milk. In this study, we aimed to investigate the occurrence of aflatoxin M1 (AFM1) in dairy milk from the Kathmandu District and to assess husbandry practices contributing to contamination. We collected 84 milk samples, including raw milk from farms, retailers’ milk, and packet milk, and analyzed them using the competitive enzyme-linked immunosorbent assay (c-ELISA) technique. We also interviewed farmers to gather information on feeding and storage practices. All the collected milk samples were contaminated with AFM1, with 97.6% of the samples exceeding the European Union (EU) maximum permissible limit of 50 ppt (0.05 μg/kg). The majority (98.5%) of the farms included paddy straw, and all farms (100%) included concentrate in their feed regimens. Only half (52%) of the farms had proper storage facilities. Straw was mostly stored in sacks outdoors or left open in a shed, while concentrates were stored in a closed room or shed. This study reveals very high levels of AFM1 contamination in the milk samples, presenting a serious public health issue, and recommends comprehensive surveillance and further investigations across the country, especially given the limited research and literature available. Full article

Galectins are a class of lectins that are extensively expressed in all organisms. Galectins are involved in a range of functions, including early development, tissue regeneration, cancer and inflammation. It has been shown that galectin-8 is expressed in the villous and extravillous trophoblast (EVT) cells of the human placenta; however, its physiological role in pregnancy establishment has not been elucidated. Taking these factors into account, we investigated the functional role of galectin-8 in HTR-8/SVneo cells—a human EVT cell line—and human primary cytotrophoblast cells isolated from a first-trimester placenta. We analyzed the effects of recombinant human galectin-8 (rh galectin-8) on the adhesion, migration and invasion of HTR-8/SVneo cells. We used qPCR, cell-based ELISA (cELISA) and gelatin zymography to study the effects of galectin-8 on mediators of these processes, such as integrin subunits alpha-1 and beta-1 and matrix metalloproteinases (MMPs)-2 and -9, on the mRNA and protein levels. Further, we studied the effects of galectin-8 on primary cytotrophoblast cells’ invasion. Galectin-8 stimulated the adhesion, migration and invasion of HTR-8/SVneo cells, as well as the invasion of primary cytotrophoblasts. In addition, the MMP-2 and -9 levels were increased, while the expression of integrins alpha-1 and beta-1 was not affected. Galectin-8 has the ability to positively affect EVTs’ invasion, so it can be considered a significant factor in the trophoblast cell invasion process. Full article

Neurobrucellosis in cetaceans, caused by Brucella ceti, is a relevant cause of death in striped dolphins (Stenella coeruleoalba) from the Mediterranean Sea. Serological tests are not used as a routinary technique for the diagnosis of this infection. We briefly describe the pathological findings of nine free-ranging stranded cetaceans diagnosed with Brucella disease or infection in our veterinary necropsy service from 2012 to 2022. The findings included focal diskospondylitis and non-suppurative meningitis, choroiditis and radiculitis. Additionally, an exploratory serological study was conducted in sixty-six frozen sera collected in the period 2012–2022 from fifty-seven striped dolphins, five Risso’s dolphins (Grampus griseus), two common bottlenose dolphins (Tursiops truncatus), one common dolphin (Delphinus delphis) and one pilot whale (Globicephala melas) to compare antibody levels in Brucella-infected (n = 8) and non-infected (n = 58) animals, classified by the cause of death, sex, age class and cetacean morbillivirus (CeMV) infection status. The authors hypothesized that active infection in cases of neurobrucellosis would elicit a stronger, detectable humoral response compared to subclinical infections. We performed a commercial competition ELISA (cELISA) using serial serum dilutions for each sample, considering a percentage of inhibition (PI) of ≥40% as positive. A titer of 1:160 was arbitrarily determined as the seropositivity threshold. Seropositive species included striped dolphins and Risso’s dolphins. Seroprevalence was higher in animals with neurobrucellosis (87.5%) compared to the overall seroprevalence (31.8%) and to other causes of death, indicating, likely, a high sensitivity but low specificity for neurobrucellosis. Animals with chronic CeMV seemed to have higher seroprevalences, as well as juveniles, which also had a higher disease prevalence. These results indicate, as in other studies, that antibodies are not decisive against clinical brucellosis, although they may indicate a carrier state, and that CeMV may influence Brucella epidemiology. More research is required to elucidate the epidemiology and pathogenesis and to resolve the complicated host–pathogen interaction in Brucella species. Full article

West Nile virus (WNV) is a re-emerging flavivirus, primarily circulating among avian hosts and mosquito vectors, causing periodic outbreaks in humans and horses, often leading to neuroinvasive disease and mortality. Spain has reported several outbreaks, most notably in 2020 with seventy-seven human cases and eight fatalities. WNV has been serologically detected in horses in the Community of Madrid, but to our knowledge, it has never been reported from wild birds in this region. To estimate the seroprevalence of WNV in wild birds and horses in the Community of Madrid, 159 wild birds at a wildlife rescue center and 25 privately owned equines were sampled. Serum from thirteen birds (8.2%) and one equine (4.0%) tested positive with a WNV competitive enzyme-linked immunosorbent assay (cELISA) designed for WNV antibody detection but sensitive to cross-reacting antibodies to other flaviviruses. Virus-neutralization test (VNT) confirmed WNV antibodies in four bird samples (2.5%), and antibodies to undetermined flavivirus in four additional samples. One equine sample (4.0%) tested positive for WNV by VNT, although this horse previously resided in a WN-endemic area. ELISA-positive birds included both migratory and resident species, juveniles and adults. Two seropositive juvenile birds suggest local flavivirus transmission within the Community of Madrid, while WNV seropositive adult birds may have been infected outside Madrid. The potential circulation of flaviviruses, including WNV, in birds in the Madrid Community raises concerns, although further surveillance of mosquitoes, wild birds, and horses in Madrid is necessary to establish the extent of transmission and the principal species involved. Full article

The discovery and biotechnological application of new antimicrobial peptides are impeded by a lack of sensitive methods for peptide quantification. Paenibacillin is an emerging antimicrobial lantibiotic that was discovered in Paenibacillus polymyxa OSY-DF ATCC PTA-7852, isolated from the fermented vegetable Kimchee. This lantibiotic has potency against many foodborne pathogenic and spoilage bacteria. To advance the research and application of paenibacillin, a rapid, specific, and sensitive detection and quantification immunoassay was developed. After anti-paenibacillin polyclonal antibodies (pAbs) were generated and purified, a competitive enzyme-linked immunosorbent assay (cELISA) was developed and optimized for paenibacillin quantification. The dynamic range of the cELISA was determined by using a three-parameter nonlinear regression model, achieving a correlation (R²) value of 0.95. The cELISA displayed high sensitivity, with the ability to detect paenibacillin at levels as low as 15.6 ng/mL, which is significantly lower than the limit of detection of the conventional antimicrobial assay (20 µg/mL paenibacillin). The cELISA successfully differentiated paenibacillin concentrations in cell-free crude supernatants of P. polymyxa wild type and its mutant strain when grown at 30 °C and 37 °C; higher paenibacillin levels were found in the mutant (0.248–0.276 µg/mL) than in the wild type (0.122–0.212 µg/mL) culture. These findings were validated by the transcriptional analysis of 11 paenibacillin biosynthetic genes, which were significantly upregulated (≥2-fold increase) in the mutant compared with the wild strain. Additionally, the cELISA exhibited high sensitivity by recovery of paenibacillin titers spiked at 2.5 and 10 µg/mL in de Man, Rogosa, and Sharpe (MRS) broth and diluted skim milk. These results suggest that the anti-paenibacillin pAbs and the developed cELISA could be valuable in quantifying paenibacillin in complex matrices and in aiding the discovery of paenibacillin-producing natural microbiota. Full article

West Nile virus (WNV) is a globally significant mosquito-borne Flavivirus that causes West Nile disease (WND). In Libya, evidence of WNV circulation has been reported in humans but never in animals. The aim of this study was to determine the seroprevalence of WNV infection in horses and dogs in Libya. In total, 574 and 63 serum samples were collected from apparently healthy, unvaccinated horses and dogs, respectively, between 2016 and 2019. A commercially available competitive enzyme-linked immunosorbent assay (c-ELISA) kit was initially used to test the collected samples for the presence of WNV Ig-G antibodies. Positive and doubtful sera were also tested using a more specific virus neutralisation assay to confirm whether the ELISA-positive results were due to WNV or other Flavivirus antibodies. The seroprevalence of WNV IgG antibodies according to ELISA was 13.2% out of 574 of total horses’ samples and 30.2% out of 63 of total dogs’ samples. The virus neutralisation test (VNT) confirmed that 10.8% (62/574) and 27% (17/63) were positive for WNV-neutralising titres ranging from 1:10 to 1:640. Univariable analysis using chi-square tests was conducted to measure the statistical significance of the association between the hypothesized risk factors including city, sex, breed, and age group and were then analyzed using the subsequent multivariable logistic regression model for horse samples. Age group was found to be the only significant risk factor in this study. The results of the present study provide new evidence about WNV circulation in Libya. Full article

Bovine brucellosis is a worldwide zoonotic contagious disease. According to World Animal Health Information System reports Ecuador has presented an increasing number of bovine brucellosis outbreaks in the continental territory over the past years (756 in 2018 versus 964 in 2021), generating economic losses for producers and causing a risk to public health. A cross-sectional study was conducted to investigate the seroprevalence of bovine brucellosis and associated risk or protective factors between May and June 2018. This stratified random study was implemented in 290 cattle herds located in the 23 provinces of continental Ecuador, which represents a total of 3737 cows aged 24 months or older. A competitive ELISA was used to detect Brucella antibodies. Simultaneously, an epidemiological survey was implemented to assess the brucellosis risk or protective factors. The apparent prevalence of bovine brucellosis at the herd level was 21.3% (95% CI: 16.8–26.6) and 6.2% (95% CI: 5.5–7) at the animal level. Univariate and multivariate logistic regression analyses were performed to determine the relationship between the potential factors associated with the presence of bovine brucellosis. The risk factors identified after multivariate analysis were a surface in ha per herd > 70 ha (OR = 2.73; 95% CI: 1.18–6.32) and the number of parturitions per animal (two or more with OR ≥ 1.8 and p-value ≤ 0.047). On the contrary, the protective factors were the region (farms located in the eastern region) and the absence of reported clinical signs. In addition, in herds where extensive production predominates, farmers have a low level of knowledge, and the farm biosecurity level is low. These results can guide the authorities in managing the risk factors identified, understanding the current epidemiological situation in Ecuador, improving the bovine brucellosis control program and food safety, as well as increase the one-health approach. Full article

Serological testing is an important method for the diagnosis of pseudorabies virus (PRV) infection. We aimed to investigate the envelope glycoprotein I (gI) of PRV, a strong immunogen, and its potential as an efficient and low-cost diagnostic reagent. In this study, the DNA of the PRV SC strain was used as the template, and the recombinant fragment of gI (633 bp) was amplified via PCR using synthetic primers, and was then ligated into the pET-30a expression vector. The constructs were transferred into Escherichia coli (E. coli) for prokaryotic expression, and the antigenicity of the expression products was identified by Western blot analysis with pig positive serum against PRV. The recombinant protein was purified by a Ni column, and BALB/c mice were immunized with purified gI protein to obtain anti-gI-positive serum. After PK-15 cells had been infected by PRV for 48 h, the immunogenicity of purified gI protein was identified with a fluorescence immunoassay using anti-gI mouse serum. The recombinant plasmid (pET-30a-gI) was expressed, and the native gI protein was obtained after denaturation by urea and renaturation by dialysis. A small-scale ELISA test containing 1.0 µg/mL of purified gI protein was designed to evaluate pig serum (80 samples), and the results of the ELISA test were compared to those of competitive ELISA (cELISA) tests using IDEXX Kits, which resulted in 97.5% consistency. The results suggested that the truncated gI protein may be a potential diagnostic reagent. Full article