Diagnostic ELISA Research Articles

Lumpy Skin Disease: A Review on Etiopathogenesis, Transmission, Diagnosis and Treatment

Lumpy Skin Disease: A Review on Etiopathogenesis, Transmission, Diagnosis and Treatment

Antibody reactions of horses against various domains of the EHV-1 receptor-binding protein gD1.

Equid alphaherpesviruses 1 (EHV-1) and 4 (EHV-4) are closely related and both endemic in horses worldwide. Both viruses replicate in the upper respiratory tract, but EHV-1 may additionally lead to abortion and equine herpesvirus myeloencephalopathy (EHM). We focused on antibody responses in horses against the receptor-binding glycoprotein D of EHV-1 (gD1), which shares a 77% amino acid identity with its counterpart in EHV-4 (gD4). Both antigens give rise to cross-reacting antibodies, including neutralizing antibodies. However, immunity against EHV-4 is not considered protective against EHM. While a diagnostic ELISA to discriminate between EHV-1 and EHV-4 infections is available based on type-specific fragments of glycoprotein G (gG1 and gG4, respectively), the type-specific antibody reaction against gD1 has not yet been sufficiently addressed. Starting from the N-terminus of gD1, we developed luciferase immunoprecipitation system (LIPS) assays, using gD1-fragments of increasing size as antigens, i.e. gD1_83 (comprising the first 83 amino acids), gD1_160, gD1_180, and gD1_402 (the full-length molecule). These assays were then used to analyse panels of horse sera from Switzerland (n = 60) and Iceland (n = 50), the latter of which is considered EHV-1 free. We detected only one true negative horse serum from Iceland, whereas all other sera in both panels were seropositive for both gG4 (ELISA) and gD1 (LIPS against gD1_402). In contrast, seropositivity against gG1 was rather rare (35% Swiss sera; 14% Icelandic sera). Therefore, a high percentage of antibodies against gD1 could be attributed to cross-reaction and due to EHV-4 infections. In contrast, the gD1_83 fragment was able to identify sera with type-specific antibodies against gD1. Interestingly, those sera stemmed almost exclusively from vaccinated horses. Although it is uncertain that the N-terminal epitopes of gD1 addressed in this communication are linked to better protection, we suggest that in future vaccine developments, type-common antigens should be avoided, while a broad range of type-specific antigens should be favored.

SARS-CoV2 IgM/IgG 신속 진단 키트와 ELISA를 이용한 백신 접종 후 항체 형성 평가

SARS-CoV2 IgM/IgG 신속 진단 키트와 ELISA를 이용한 백신 접종 후 항체 형성 평가

TORCH serological marker on cholestasis with the occurrence of biliary atresia and its clinical manifestations

Background and objectives. Biliary atresia occurs due to multifactorial etiology, and it was recently suggested that perinatal infection with TORCH, especially Cytomegalovirus (CMV), triggers inflammation of the bile ducts. This study aims to evaluate the TORCH infection on cholestasis with the occurrence of biliary atresia and its clinical manifestations. Materials and methods. A prospective single-center study was performed on 113 cholestatic infants and classified into two groups based on their positivity of immunoglobulin M and G (IgM and IgG). They were tested for CMV, Rubella, and Toxoplasmosis by a Gold Standard Diagnostics ELISA kit in our laboratory. Clinical conditions, laboratory tests (Aspartate Aminotransferase (AST), Alanine Aminotransferase (ALT), γ-glutamyl transpeptidase (GGT), and liver histopathology between the two groups were analyzed with p <0.05 was considered significant. Results. Out of 113 infants of cholestasis, 94.7% (n = 107) were CMV IgG-positive, followed by Rubella IgG-positive (47.8%, n=54). Rubella and toxoplasma IgM antibodies were least commonly found in cholestasis (8% and 7.1%, respectively). CMV IgG-positive status was noted to be older at the onset of jaundice. Infants with Rubella IgG-positive had less incidence of extrahepatic cholestasis (37.7% vs 62.3%) and liver fibrosis (39% vs 61%) (p<0.05). There was no difference in sex, birth weight, gestational age, AST, ALT, GGT, coagulation hemostasis, and abdominal USG abnormality based on torch serological markers (p>0.05). Conclusion: CMV infection mostly occurs in infants with cholestasis, followed by rubella infection. TORCH infection screening may be necessary in all infants with cholestasis.

Comparative Evaluation of Serological, ELISA and Molecular Tests in Diagnosis of Brucella abortus in Human Serum

Background: Brucellosis is one of the most prevalent and common diseases between humans and animals.Prompt diagnosis and timely treatment of this disease can prevent many complications. In this regard, this study aims to comparatively evaluate ELISA, PCR and serological methods to identify Brucella abortus.
 Methods: In this study, the serum of 100 patients referred to Tonekabon private laboratory from July 2020 to January 2021 was examined by PCR, ELISA and Wright, Coombs Wright, and 2ME methods for the detection of Brucella abortus.
 Results: In this study, the mean age of the sample was 43.3 ± 18.2 of which 21% were infected with Brucella abortus according to the above serological methods. According to ELISA test, 22% of the samples were IgM, 6% of the samples were IgG and 16% were PCR positive. Kappa agreement coefficient in Wright and Coombs Wright test and 2me were significant (P <0.001). Serological diagnostic indices and ELISA sensitivity were 68.75% and 68.75%, respectively. The lowest prediction rate of Brucella abortus among diagnostic methods was related to Elisa (IgM). Based on Fisher's exact test, there was no significant relationship between the percentage of Brucella abortus positive PCR cases and age, sex, previous history of infection with Brucella, fever, body aches and dairy consumption.
 Conclusion: Based on the results of our study, the accuracy of all methods is comparative and the lowest accuracy is related to Elisa (IgM) which has a lower level of predictability than other methods. The highest level of prediction belonged to Wright and 2me tests.

Assessment of stn, sipB and sopB Virulence Genes in Various Salmonella Serovars.

Salmonella is a zoonotic bacterium that is considered to be one of the most common causes of foodborne infections worldwide. Bearing in mind the genes involved in its virulence, identifying these genes can enable experts to better understand bacterial pathogenicity, which could subsequently help develop more efficient means to control and prevent infections. This study aimed to analyze stn, sipB, and sopB genes in various Salmonella serovars. To carry out this study, 103 Salmonella serovars were extracted from livestock, poultry, and humans from existing samples at the Department of Microbiology of the Razi Serum and Vaccine Research Institute in Karaj, Iran. These samples were cultured in selection and differential media, and their serovars were identified using specific antibodies based on Kaufman-White Tables. Utilizing PCR and specific primers, stn, sopB, and sipB genes were detected among these serovars. In this investigation, the most common human serovars were Salmonella paratyphi A, Salmonella paratyphi B, and Salmonella enteritidis; the most common serovars among livestock consisted of Salmonella dublin and Salmonella typhimurium and the most common Salmonella serovars among poultry consisted of Salmonella infantis and Salmonella enteritidis. The results of PCR on stn, sipB, and sopB genes demonstrated segments with 617bp, 875 bp, and 220 bp on agar gel, respectively. Based on the obtained findings, stn, sipB, and sopB genes were detected in 96.11%, 99.02%, and 98.05% of Salmonella serovars, respectively. Considering the fact that the aforementioned genes play significant roles in bacterial virulence, they can be used to develop diagnostic ELISA kits and recombinant vaccines.

Examination of ELISA against PCR for assessing treatment efficacy against Cryptosporidium in a clinical trial context.

Cryptosporidium is a gastrointestinal pathogen that presents a serious opportunistic infection in immunocompromised individuals including those living with human immunodeficiency syndrome. The CRYPTOFAZ trial, previously published, was conducted in Malawi to evaluate the efficacy of clofazimine in response to an unmet need for drugs to treat cryptosporidiosis in HIV populations. A combination of rapid diagnostic tests, ELISA, qPCR, and conventional sequencing were employed to detect Cryptosporidium in 586 individuals during pre-screening and monitor oocyst shedding and identify enteric co-pathogens in 22 enrolled/randomized participants during the in-patient period and follow-up visits. Oocyst shedding as measured by qPCR was used to determine primary trial outcomes, however pathogen was detected even at trial days 41-55 in individuals randomized to either clofazimine or placebo arms of the study. Therefore, in this work we re-examine the trial outcomes and conclusions in light of data from the other diagnostics, particularly ELISA. ELISA data was normalized between experiments prior to comparison to qPCR. The amount of all identified enteric pathogens was examined to determine if co-pathogens other than Cryptosporidium were major causative agents to a participant's diarrhea. ELISA had higher sample-to-sample variability and proved to be equally or less sensitive than qPCR in detecting Cryptosporidium positive samples. Compared to qPCR, ELISA had equal or greater specificity in detecting Cryptosporidium negative samples. Sequencing identified several Cryptosporidium species including viatorum which has never been identified in Malawi and Southern Africa. In addition to Cryptosporidium, enterotoxigenic E. coli was also identified as a pathogen in diarrheagenic amounts in 4 out of 22 participants.

Chagas disease in the Brazilian Northeast: understanding health and living conditions in a population with a historical transmission risk in the rural area

Chagas disease (CD) is considered a neglected disease by the World Health Organization and requires priority from the health authorities of the American continent. In Brazil, few data are available to evaluate the real situation of endemic areas for CD, mainly in the Northeast region. Thus, the objective of the study was to evaluate the living conditions, clinical aspects and prevalence of infection in inhabitants of rural areas endemic to CD. Blood was collected and submitted to diagnostic ELISA and indirect immunofluorescence techniques. A questionnaire covering sociodemographic information and knowledge related to CD was applied, and general clinical evaluation and swallowing assessment were performed. Twenty-four (7.04%) seropositive individuals were identified, of whom 83.3% were women with most in the age range of 30-60 years old. The majority of the population has a low level of education. Regarding the risk variables for CD, an association was noted between the presence of kissing bugs (OR 9.8) and contact/manipulation (OR 20.2), transfusion (OR 10.6), and sugarcane juice intake (OR 5.1). Was possible to detect changes in swallowing. The present study revealed the existence of a population seropositive for CD without follow-up for their health condition. In addition, some of these patients present changes in swallowing that may be related to the chronic phase. Although no infected children were identified, the area may be considered at risk due to the environmental and socioeconomic conditions of its population and the presence of infected vectors previously found in the area.

PCR and peptide based PCMV detection in pig - development and application of a combined testing procedure differentiating newly from latent infected pigs.

Porcine cytomegalovirus (PCMV) is widely distributed in pigs and difficult to detect due to latency. PCMV infection of source pigs was associated with early graft failure after cardiac and renal xenotransplantation into nonhuman primates. Importantly, PCMV infection of the first genetically modified pig heart into a human may have contributed to the reduced survival of the patient. Sensitive and reliable assays for detection of latent PCMV infection are thus indispensable. Here, we report the development of five peptide-induced rabbit antisera specific for PCMV glycoprotein B (gB) and their validation for detection of PCMV in infected pig fallopian tube (PFT) cells by immunofluorescence and electron microscopy (EM). The anti-gB antibodies were also used for detection by Western blot analysis of PCMV purified from the supernatant of infected PFT cells. Sera of infected versus non-infected pigs have been compared. In parallel, PCMV viral load in blood samples of the animals was quantified by a novel highly sensitive nested-PCR and qPCR assay. A combination of four partly overlapping peptides from the gB C-terminus was used to establish a diagnostic ELISA for PCMV gB specific pig antibodies which is able to differentiate infected from non-infected animals and to quantify maternal antibodies in neonates. The combination of a highly sensitive nested PCR for direct virus detection with a sensitive peptide-based ELISA detecting anti-PCMV gB-antibodies, supplemented by Western blot analysis and/or immunohistochemistry for virus detection will reliably differentiate pigs with active infection, latently infected pigs, and non-infected pigs. It may significantly improve the virologic safety of xenotransplantation.

Assessment of stn, sipB and sopB virulence genes in various Salmonella serovars

Salmonella is a zoonotic bacterium that is considered to be one of the most common causes of foodborne infections worldwide. Bearing in mind the genes involved in its virulence, identifying these genes can enable experts to better understand bacterial pathogenicity, which could subsequently help develop more efficient means to control and prevent infections. This study aimed to analyze stn, sipB, and sopB genes in various Salmonella serovars. To carry out this study, 103 Salmonella serovars were extracted from livestock, poultry, and humans from existing samples at the Department of Microbiology of the Razi Serum and Vaccine Research Institute in Karaj, Iran. These samples were cultured in selection and differential media, and their serovars were identified using specific antibodies based on Kaufman-White Tables. Utilizing PCR and specific primers, stn, sopB, and sipB genes were detected among these serovars. In this investigation, the most common human serovars were Salmonella paratyphi A, Salmonella paratyphi B, and Salmonella enteritidis; the most common serovars among livestock consisted of Salmonella dublin and Salmonella typhimurium and the most common Salmonella serovars among poultry consisted of Salmonella infantis and Salmonella enteritidis. The results of PCR on stn, sipB, and sopB genes demonstrated segments with 617bp, 875 bp, and 220 bp on agar gel, respectively. Based on the obtained findings, stn, sipB, and sopB genes were detected in 96.11%, 99.02%, and 98.05% of Salmonella serovars, respectively. Considering the fact that the aforementioned genes play significant roles in bacterial virulence, they can be used to develop diagnostic ELISA kits and recombinant vaccines.

Potential diagnostic application of the baculovirus-expressed recombinant truncated nucleocapsid protein of peste des petits ruminants virus in ELISA

The study describes the expression of recombinant truncated nucleocapsid protein (NP) of peste des petits ruminants (PPR) virus in the baculovirus system (PPRV-rBNP) and its potential application as a diagnostic antigen in ELISA for diagnosis of PPR in sheep and goats. The PPRV N-terminal immunogenic region (1–266 aa) of the NP coding sequence was amplified and cloned into the pFastBac HT A vector. The PPRV-rBNP with a molecular weight of ∼30 kDa was expressed in an insect cell system using generated recombinant baculovirus through Bac-to-Bac® Baculovirus Expression System. The crude PPRV-rBNP or Ni-NTA affinity-purified NP was characterized by SDS-PAGE and immunoblot using standard PPRV-specific sera. The PPRV-rBNP reacted well with PPRV anti-N specific monoclonal and polyclonal antibodies and PPRV-specific antiserum, suggesting that the expressed PPRV-rBNP is in its native form. The crude PPRV-rBNP as a diagnostic antigen was evaluated either as a coating antigen or standard positive control antigen in the Avidin-Biotin ELISA using the known standard panel reagents. The results showed that the expressed PPRV-rBNP can be an alternative diagnostic antigen to E. coli expressed recombinant PPRV-NPN and the utility of PPRV-rBNP avoids the need to use live PPRV antigen in the diagnostic ELISA. Hence, this allows scope in the future for large-scale field application of the recombinant antigen-based assays for diagnosis/surveillance and monitoring of PPR at the eradication as well as post-eradication phases in endemic countries or PPR non-endemic countries.

Improving dengue diagnosis and case confirmation in children by combining rapid diagnostic tests, clinical, and laboratory variables

BackgroundDengue is the most widely distributed arboviral disease in tropical and subtropical countries. Most suspected cases are diagnosed according to the clinical criteria, and early diagnosis is difficult. Moreover, in underdeveloped countries, several factors continue to challenge the diagnosis and surveillance of dengue cases. This study aimed to design a diagnostic algorithm using rapid diagnostic tests (RDTs), ELISA tests, and clinical and hematological variables to confirm dengue cases in febrile patients in Colombia.MethodsAltogether, 505 samples were collected. Serum samples were evaluated by RDTs (IgM and IgG antibodies and NS1 antigen), capture IgM and IgG ELISAs, and endpoint hemi-nested RT-PCR assay (qualitative). We statistically analyzed the performance of individual tests to determine the most useful ones to confirm dengue cases accurately.ResultsIndividual results for IgM, IgG, and NS1 RDTs yielded lower sensitivity and specificity values than the reference standard. High sensitivity and specificity were obtained after combining IgM and NS1 ELISA results (96.3% and 96.4%) and NS1 RDT plus IgM ELISA results (90.3% and 96.2%), respectively. Adjusted odds ratios (aORs) were calculated for clinical variables and laboratory tests to differentiate dengue from other febrile illnesses (OFI). This approach showed that myalgia, abdominal tenderness, and platelet count were identified with higher sensitivity to confirm dengue cases. IgM RDT and NS1 RDT differentiated dengue cases from OFI. A positive IgM RDT or a positive NS1 RDT combined with specific signs or symptoms confirmed 81.6% of dengue cases. A combination of clinical findings and a positive NS1 RDT or positive ELISA IgM confirmed 90.6% of the cases.ConclusionOur findings showed that clinical diagnoses in pediatric population alone cannot confirm true dengue cases and needs to be complemented by laboratory diagnostic tests. We also demonstrate the usefulness of combining clinical criteria with RDTs, suggesting that their implementation with the IgM ELISA test improves dengue case confirmation.

High expression of LipL21 protein of Iranian Leptospira interrogans in E. coli, applicable for diagnostic ELISA

Leptospirosis is an emerging infectious zoonotic disease caused by pathogenic Leptospira. The disease is more prevalent among farmers in hot and humid areas of Iran. Lack of clear clinical signs have impeded the diagnosis of leptospirosis. In this study, we attempted to produce a recombinant LipL21 protein of Leptospira based on a dominant pattern of Iranian isolates and to evaluate it in ELISA test. One hundred and sixty-two complete sequences of LipL21 available in GenBank until January 1, 2019 were compared. One dominant LipL21 protein pattern was selected. The codon optimised sequence was cloned into the pET32a+ expression vector. Trx-LipL21 fusion protein was induced, purified and confirmed by 10% SDS-PAGE followed Coomassie blue staining and immune blotting. For evaluation of effectiveness of rLip21 in ELISA test, 200 µg rLip21 with Montanide ISA70 adjuvant was injected subcutaneously in rabbits three times. Results showed that rLipL21 protein was highly expressed in 2YT media in presence of 0.1 mM IPTG after 16 hours incubation at 37 oC. Recombinant protein was purified 36 mg per liter using affinity batch formation method by Ni-NTA resin. ELISA with micro plate coated with 250 ng rLipL21 protein demonstrated prominently diffe­rences between test and control groups (P<0.01). The rLipL21 protein produced large amounts of antibodies in the rabbit. The protein was also able to detect high levels of antibody in animals immu­nised with Leptospira vaccine. The rLipL21 might be a good candidate for diagnosis and evaluation of antibody levels against Leptospira.

Molecular Detection of Hepatitis B Virus Among HBsAg Non-Reactive Blood of Donors

Despite all blood donations being tested routinely for HBsAg as a clinical marker of transmissible HBV, cases of post-transfusion hepatitis B virus infection are still being reported because molecular studies using Polymerase Chain Reaction (PCR) are not routinely available for Transfusion Transmissible Infection (TTIs) testing. In this study, we sought to use the PCR technique to re-screen donated blood that had already been proven to be HBsAg non-reactive with rapid diagnostic testing and ELISA. One hundred and eighty-five samples were obtained from a proportion of the blood deposited at the blood bank of the Federal Medical Center, Birnin-Kebbi, Nigeria. Socio-demographic parameters such as age group, status, ethnicity, occupation, and group PCV were obtained from donors' records. Nested PCR was employed to detect HBV DNA. Furthermore, genotyping was performed to determine HBV genotypes in the positive samples using PCR with genotype-specific primers. Of the 185 donors, it was observed that five (2.7%) of the population were positive for HBV. HBV is more common among people aged 18–30, singles, Hausa, and self-employed. In addition, the five positive samples were of genotype E. This study suggests the need to complement antibody-based tests with DNA testing for effective HBV screening and consequent safe transfusion. Keywords: Hepatitis B Virus; HBsAg; PCR; DNA testing

Seroprevalence of Fasciola-infection using Enzyme-linked immunosorbent assay in large ruminants from Pakistan

Fasciolosis, caused by liver fluke species of the genus Fasciola, are well recognized because of its high veterinary impact. Stool examination for Fasciola eggs is not a sensitive method, and limited efforts to find a reliable and cheaper means of detection are available. The present study aimed to develop rapid diagnostic ELISA test against fasciolosis. The excretory/secretory (ES) and somatic (SA) products of Fasciola helminths were analysed using polyacrylamide gel electrophoresis (PAGE). Immunogenicity was evaluated by immunoblotting using hyperimmune sera raised in rabbits and seroprevalence was determined by indirect ELISA. The results of SA antigen of Fasciola species showed polypeptide bands ranged from 10kDa-100kDa, while ES antigen of Fasciola showed bands of 15kDa-55kDa. The immunoblotting results showed the most prominent bands against ES antibodies were 25, 35, 55-70, 100 and 250 kDa and SA antigens showed 10, 15-25, 35, 70, 100 and 250 kDa polypeptide bands. The sensitivity and specificity of developed indirect ELISA for SA antigens was 95.45% and 87.1%, while for ES antigens was 100% and 77.42% respectively. The overall seroprevalence recorded for fascioliasis based on SA antigen was 39.8% and 29.8% for ES antigen. The fasciolosis did not show significant association with host type, sex and age groups of examined animals, however significantly higher infection was found in months of September and October. The result provides sensitive in house immunodetection assay for diagnosis of fasciolosis alternative to commercial kits with high import cost.

Development of a diagnostic indirect ELISA test for detection of Brucella antibody using recombinant outer membrane protein 16 kDa (rOMP16).

Brucellosis is considered as one of the important global zoonotic diseases that causes medical as well as economic problems especially in tropical countries. The illness has no specific pathognomonic signs; therefore, the rapid and accurate diagnosis of the disease has a very important role in preventing the Brucella spillover and treatment. The purpose of this study was to design a new indirect ELISA test for detection of human brucellosis based on using recombinant Brucella abortus outer membrane protein 16 kDa (rOMP16) as an antigen. OMP16 gene of B. abortus was initially synthesized and cloned in pET-21d vector and then expressed in Escherichia coli cells. The expression was confirmed by the SDS-PAGE, western blotting and dot blotting. The purified protein was coated in ELISA plates and an indirect ELISA was performed on 70 human serum samples. The results were evaluated with a commercial IgG ELISA kit and Rose Bengal plate agglutination tests as reference tests. Diagnostic performance of designed OMP16 ELISA test in comparison with Rose Bengal plate test revealed 100% of sensitivity, 95.00% of specificity and good Fleiss kappa agreement, whereas, where it was compared to commercial ELISA kit, it revealed very good kappa agreement with 100% of sensitivity and 100% of specificity in cut-off value of 0.13. It was concluded that OMP 16 kDa could be acceptable alternative antigen for detecting Brucella IgG antibody with high accuracy.

Evaluation of a novel Echinococcus granulosus recombinant fusion B-EpC1 antigen for the diagnosis of human cystic echinococcosis using indirect ELISA in comparison with a commercial diagnostic ELISA kit

Cystic echinococcosis (CE) is a zoonotic parasitic disease caused by the metacestode of Echinococcus granulosus sensu lato (s.l.). A large proportion of the patients are asymptomatic at the early and late stages of the disease. CE diagnosis is mainly based on imaging techniques. Laboratory diagnosis including antibody-antigen (recombinant or fusion recombinant) can be used for the diagnosis and follow up of CE and alveolar echinococcosis (AE), but need optimization and standardization. This study aimed to evaluate the efficacy of a recombinant B-EpC1 (rB-EpC1) fusion antigen comprising B1, B2, B4, and EpC1 antigens of E. granulosus using indirect ELISA in comparison with a commercial ELISA kit for the serodiagnosis of CE. The recombinant protein was expressed in the expression host, E. coli BL21, and purified. This recombinant antigen was then evaluated by indirect ELISA and compared to the commercial CE diagnostic kit (Vircell, Spain). The study samples included 124 human sera consisting of 62 sera of patients with CE, and 62 sera of individuals without clinical evidences of CE and specific anti-CE antibodies in routine indirect ELISA. The diagnostic sensitivity and specificity of the indirect rB-EpC1-ELISA test for detection of specific anti-hydatid cyst antibodies in human CE were 95.2% and 96.8%, respectively. Also, the diagnostic sensitivity and specificity of the commercial ELISA test were 96.8% in this study. Initial evaluation of the recombinant fusion antigen (B-EpC1) was promising for the detection of CE by ELISA in clinical settings. Standardization and evaluation of recombinant fusion protein require further studies.

Corynebacterium pseudotuberculosis Infections in Alpacas (Vicugna pacos).

Simple SummaryAlpacas have a quite recent history in Europe. Only in the 1990’s were a considerable number of alpacas imported into the UK and then into continental Europe, where the number of animals increased significantly. This development has necessitated a more thorough knowledge of infectious diseases in alpaca husbandry. One of the most serious infectious diseases in alpacas is caseous lymphadenitis (CLA), which is caused by Corynebacterium (C.) pseudotuberculosis. As shown by the post-mortem examinations in this study, CLA is characterized by the formation of multiple, visible abscesses, particularly affecting the lungs and liver as the pathogen spreads throughout the body. While post-mortem examinations and isolation of the pathogen are pivotal for a proven diagnosis, serological examinations are the basis of epidemiological and monitoring studies. We examined 232 alpacas living in three independent herds for this current study. C. pseudotuberculosis was able to be isolated in several alpacas. All of the alpacas were tested serologically using a commercially available ELISA, an in-lab ELISA, and, in select animals, immunoblot. The immunoblot showed the highest sensitivity in the early phase of the infection in alpacas proven to be infected with C. pseudotuberculosis. Testing for antimicrobial susceptibility based on minimal inhibitory concentration using the broth microdilution method revealed uniform susceptibility to aminopenicillins, cephalosporines, macrolides, enrofloxacin, florfenicol, tetracycline, sulfonamid/trimethoprime, tiamulin, gentamicin, neomycin, spectinomycin, and vancomycin, but resistance to colistin, nitrofurantoin, and oxacillin. The present study shows that C. pseudotuberculosis poses an imminent health risk for alpaca husbandry. Laboratory diagnostics based on post-mortem, bacteriological and serological examinations are valuable tools for implementing specific measures as a basis for tackling this infectious disease in alpaca herds.Alpacas are the major camelid species in Europe held for hobbies, animal-aided therapy, and commercial reasons. As a result, health-related issues associated with alpacas are of growing significance. This especially holds true for one of the most serious infectious diseases, caseous lymphadenitis, which is caused by the bacterial pathogen Corynebacterium (C.) pseudotuberculosis. Our study focuses on post-mortem examinations, the laboratory diagnostic tool ELISA, and the immunoblot technique for the detection of specific antibodies against C. pseudotuberculosis and detection of the causative pathogen in alpaca herds. We examined a total of 232 alpacas living in three herds. Four of these alpacas were submitted for post-mortem examination, revealing abscesses, apostematous and fibrinous inflammation in inner organs, pleura, and peritoneum. Serological investigation using a commercial ELISA based on phospholipase D (PLD) as antigen and an in-lab ELISA based on whole cell antigens (WCA) revealed an overall seroprevalence of 56% and 61.2%, respectively. A total of 247 alpaca sera originating from 232 animals were tested comparatively using the in-lab and the commercial ELISA and showed a substantial degree of agreement, of 89.5% (Cohen’s kappa coefficient of 0.784), for both tests. Further comparative serological studies using the two ELISAs and the immunoblot technique were carried out on selected sera originating from 12 breeding stallions and six breeding mares for which epidemiological data and partial C. pseudotuberculosis isolates were available. The results showed the immunoblot to have a sensitivity that was superior to both ELISAs. In this context, it should be emphasized that evaluation of these investigations and the epidemiological data suggest an incubation period of one to two months. Antibiotic susceptibility testing of 13 C. pseudotuberculosis isolates based on the determination of minimal inhibitory concentrations using the broth microdilution method revealed uniform susceptibility to aminopenicillins, cephalosporines, macrolides, enrofloxacin, florfenicol, tetracycline, sulfonamid/trimethoprime, tiamulin, gentamicin, neomycin, spectinomycin, and vancomycin, but resistance to colistin, nitrofurantoin, and oxacillin.

Sublethal effects of acrylamide on thyroid hormones, complete blood count and micronucleus frequency of vertebrate model organism (Cyprinus carpio)

Abstract Objectives Acrylamide, a widely used chemical in industry, clinical laboratory and waste treatment plants, is considered a carcinogen in humans. The present study examined the hormonal, hematologic, and genotoxic responses in the invertebrate model common carp Cyprinus carpio after exposure to sublethal acrylamide. Methods Fish were exposed to acrylamide at 10 and 50 mg/L for 96 h, along with the respective control group. Serum levels of cortisol and thyroid hormones were measured using diagnostic ELISA direct immunoenzymatic kits. For micronucleus (MN) frequency assay, thin smears of the peripheral blood of fish were prepared. Results Serum levels of cortisol in both treatment groups considerably increased, which proposed that acrylamide caused a stress reaction of acrylamide exposed fish (p<0.05). Fish demonstrated significant decreases in triiodothyronine (T3), free thyroxine (FT4), and free triiodothyronine (FT3) concentrations in a dose-dependent manner after acrylamide exposure (p<0.05). However, serum thyroxine (T4) concentrations did not alter significantly in the treatment groups. Mean MN frequencies of fish erythrocytes increased significantly in acrylamide exposed groups suggesting that acrylamide is genotoxic in common carp (p<0.05). The hematocrit, hemoglobin, and erythrocyte numbers of carp increased significantly in exposure groups (p<0.05). Conclusions These results suggested that acrylamide can significantly affect the hemopoietic system. Furthermore, this study confirmed that the widespread use of acrylamide, even in sublethal concentrations, could affect the survival of non-target organisms, especially fish, in aquatic environments.

Relationship between skin snip and Ov16 ELISA: Two diagnostic tools for onchocerciasis in a focus in Cameroon after two decades of ivermectin-based preventive chemotherapy.

BackgroundOnchocerciasis elimination currently relies on repeated ivermectin-based preventive chemotherapy. Current World Health Organization’s guidelines strongly recommend, though with low evidence of certainty, the use of Ov16 serology testing in children younger than 10 years old to assess whether mass drugs administration can be safely stopped. Therefore, more evidences are needed to support the use of this marker as sero-evaluation tool. This study aimed at determining the relationship between microfilaridermia and anti-Ov16 IgG4, and their variation according to age, gender and ivermectin intake history.MethodologyA cross-sectional survey was conducted in an area where ivermectin-based MDA has been implemented since more than 20 years. A questionnaire was used to record ivermectin intake history for the last 5 years. All volunteers aged ≥2 years were tested for microfilaridermia. IgG4 antibodies against Ov16 antigen were determined using the Standard Diagnostic Ov16 IgG4 ELISA kits and the recombinant anti-Ov16 AbD19432 antibodies. Prevalences, microfilaridermia counts and IgG4 concentrations were compared with regards to age, gender and history of ivermectin intake.Principal findingsThe prevalence of skin microfilariae was 23.4% (95% CI: 23.4–30.8), whereas Ov16 seroprevalence was 53.2% (95% CI: 47.9–58.4). A moderate positive percentage agreement (50.4%) and a high negative percentage agreement (69.2%) was found between skin snip and Ov16 serology in the whole population, while in children aged <10 years, the agreements were higher (positive percentage agreement: 62.6%; negative percentage agreement: 83.5%). In addition, no associations were found between ivermectin intake, Mf counts and estimated IgG4 concentration of participants. Anti-Ov16 IgG4 were higher in individuals harboring microfilariae than their negative counterparts (p<0.0001), though a negative correlation was found between skin microfilarial counts and anti-Ov16 IgG4 levels (r = -0.2400; p = 0.03). No variation in microfilarial counts according to age and gender was observed. Though positively correlated with age (r = 0.4020; p<0.0001), IgG4 was significantly different between the different age classes (p<0.0001).Conclusion/SignificanceOur results revealed moderate positive and negative agreements between parasitological and immunological parameters of onchocerciasis infection after several rounds MDA. Anti-Ov16 IgG4 levels increased with age but decreased with microfilarial counts, suggesting a variation of anti-Ov16 IgG4 as a result of constant exposure and accumulation of infection. This brings evidence sustaining the use of Ov16 serology in children as evaluation tool. However, additional investigations are needed to further reshape the appropriate age range among children aged <10 years old.