Antigen Capture ELISA Research Articles

Fasciola hepatica Cathepsin L Zymogens: Immuno-Proteomic Evidence for Highly Immunogenic Zymogen-Specific Conformational Epitopes to Support Diagnostics Development.

Fasciola hepatica, the common liver fluke and causative agent of zoonotic fasciolosis, impacts on food security with global economic losses of over $3.2 BN per annum through deterioration of animal health, productivity losses, and livestock death and is also re-emerging as a foodborne human disease. Cathepsin proteases present a major vaccine and diagnostic target of the F. hepatica excretory/secretory (ES) proteome, but utilization in diagnostics of the highly antigenic zymogen stage of these proteins is surprisingly yet to be fully exploited. Following an immuno-proteomic investigation of recombinant and native procathepsins ((r)FhpCL1), including mass spectrometric analyses (DOI: 10.6019/PXD030293), and using counterpart polyclonal antibodies to a recombinant mutant procathepsin L (anti-rFhΔpCL1), we have confirmed recombinant and native cathepsin L zymogens contain conserved, highly antigenic epitopes that are conformationally dependent. Furthermore, using diagnostic platforms, including pilot serum and fecal antigen capture enzyme-linked immunosorbent assay (ELISA) tests, the diagnostic capacities of cathepsin L zymogens were assessed and validated, offering promising efficacy as markers of infection and for monitoring treatment efficacy.

Development of Highly Sensitive Sandwich ELISA for the Early-Phase Diagnosis of Chikungunya Virus Utilizing rE2-E1 Protein.

IntroductionChikungunya is caused by an alpha virus transmitted to humans by an infected mosquito. Infection is generally considered to be self-limiting and non-critical. Chikungunya infection may be diagnosed by severe joint pain with fever, but it is difficult to diagnose because the symptoms of chikungunya are common to many pathogens, including dengue fever. Diagnosis mainly depends on viral culture, reverse transcriptase polymerase chain reaction (RT-PCR), and IgM ELISA. Early and accurate diagnosis of the virus can be achieved by the application of PCR methods, but the high cost and the need for a thermal cycler restrict the use of such methods. On the other hand, antibody-based IgM ELISA is considered to be inexpensive, but antibodies against chikungunya virus (CHIKV) only develop after 4 days of infection, so it has limited application in the earlier diagnosis of viral infection and the management of patients. Because of these challenges, a simple antigen-based sensitive, specific, and rapid detection method is required for the early and accurate clinical diagnosis of chikungunya.MethodsThe amino acid sequence of CHIKV ectodomain E1 and E2 proteins was analyzed using bioinformatics tools to determine the antigenic residues, particularly the B-cell epitopes and their characteristics. Recombinant E2-E1 CHIKV antigen was used for the development of polyclonal antibodies in hamsters and IgG was purified. Serological tests of 96 CHIKV patients were conducted by antigen-capture ELISA using primary antibodies raised against rCHIKV E2-E1 in hamsters and human anti-CHIKV antibodies.ResultsWe observed high specificity and sensitivity, of 100% and 95.8%, respectively, and these values demonstrate the efficiency of the test as a clinical diagnostic tool. There was no cross-reactivity with samples taken from dengue patients.DiscussionOur simple and sensitive sandwich ELISA for the early-phase detection of CHIKV infection may be used to improve the diagnosis of chikungunya.

Development and evaluation of polyclonal antibodies based antigen capture ELISA for detection of porcine rotavirus

Rotaviruses are rising as zoonotic viruses worldwide, causing the lethal dehydrating diarrhea in children, piglets, and other livestock of economic importance. A simple, swift, cost-effective, highly specific, and sensitive antigen-capture enzyme–linked immunosorbent assay (AC-ELISA) was developed for detection of porcine rotavirus-A (PoRVA) by employing rabbit (capture antibody) and murine polyclonal antibodies (detector antibody) produced against VP6 of PoRVA (RVA/Pig-tc/CHN/TM-a/2009/G9P23). Reactivity of the both polyclonal antibodies was confirmed by using an indirect ELISA, western-blot analysis and indirect fluorescence assay against rVP6 protein and PoRVA. The detection limit of AC-ELISA was found 50 ng/ml of PoRVA protein. The relative sensitivity and specificity of this in-house AC-ELISA were evaluated for detection of PoRVA from 295 porcine diarrhea samples, and results were compared with that of RT-PCR and TaqMan RT-qPCR. The relative sensitivity and specificity of AC-ELISA compared with those of TaqMan RT-qPCR were found as 94.4 and 99.2%, respectively, with the strong agreement (κ − 0.58) between these two techniques. Furthermore, AC-ELISA could not detect any cross-reactivity with porcine epidemic diarrhea virus, transmissible gastro-enteritis virus, pseudo rabies virus and porcine circovirus-2. This in-house AC-ELISA efficiently detected PoRVA from clinical samples, which suggests that this technique can be used for large-scale surveillance and timely detection of rotavirus infection in the porcine farms.

Prevalence of Crimean-Congo Hemorrhagic Fever Virus among Livestock and Ticks in Zhambyl Region, Kazakhstan, 2017.

Crimean-Congo hemorrhagic fever (CCHF) is a highly fatal zoonotic disease endemic to Kazakhstan. Previous work estimated the seroprevalence of CCHF virus (CCHFV) among livestock owners in the Zhambyl region of southern Kazakhstan at 1.2%. To estimate CCHFV seroprevalence among cattle and sheep, we selected 15 villages with known history of CCHFV circulation (endemic) and 15 villages without known circulation (nonendemic) by cluster sampling with probability proportional to livestock population size. We collected whole blood samples from 521 sheep and 454 cattle from randomly selected households within each village and collected ticks found on the animals. We tested livestock blood for CCHFV-specific IgG antibodies by ELISA; ticks were screened for CCHFV RNA by real-time reverse transcription polymerase chain reaction and CCHFV antigen by antigen-capture ELISA. We administered questionnaires covering animal demographics and livestock herd characteristics to an adult in each selected household. Overall weighted seroprevalence was 5.7% (95% CI: 3.1, 10.3) among sheep and 22.5% (95% CI: 15.8, 31.2) among cattle. CCHFV-positive tick pools were found on two sheep (2.4%, 95% CI: 0.6, 9.5) and three cattle (3.8%, 95% CI: 1.2, 11.5); three CCHFV-positive tick pools were found in nonendemic villages. Endemic villages reported higher seroprevalence among sheep (15.5% versus 2.8%, P < 0.001) but not cattle (25.9% versus 20.1%, P = 0.42). Findings suggest that the current village classification scheme may not reflect the geographic distribution of CCHFV in Zhambyl and underscore that public health measures must address the risk of CCHF even in areas without a known history of circulation.

Development and Validation of Rapid In-House Diagnostic ELISA Kits for Detection of Human Orthopneumovirus in Clinical Samples.

Currently, the standard assay employed to diagnose human orthopneumovirus infection is real-time reverse transcriptase PCR assay (rRT-PCR), a costly and time-consuming procedure that requires the manipulation of infectious viruses. In addition to RT-PCR, serological tests can complement the molecular diagnostic methods and have proven to be important tools in sero-surveillance. In this study, we report the development, optimization, and validation of a novel and rapid in-house diagnostic ELISA kit to detect human orthopneumovirus in clinical samples. We developed three sensitive ELISA formats through the immunization of rats with novel recombinant pPOE-F or pPOE-TF vectors. The two vectors expressed either the full-length (pPOE-F) or the truncated form (pPOE-TF) of the fusion (F) protein. The developed ELISA kits were optimized for coating buffer, capture antibody, blocking buffer, sample antigen, detection antibodies, and peroxidase-conjugated antibody, and validated using 75 rRT-PCR-confirmed nasopharyngeal aspirate (NPA) human orthopneumovirus samples and 25 negative samples collected from hospitalized children during different epidemic seasons between 2014 and 2017. Our results indicate that rats immunized with pPOE-F or pPOE-TF showed significant induction of high levels of MPAs. Validation of the ELISA method was compared to the rRT-PCR and the sensitivity hierarchy of these developed ELISA assays was considered from highest to lowest: indirect competitive inhibition ELISA (93.3%) > indirect antigen-capture ELISA (90.6%) > direct antigen-capture ELISA (86.6%). The development of the rapid in-house diagnostic ELISA kits described in this study demonstrates that a specific, rapid and sensitive test for human orthopneumovirus antigens could be successfully applied to samples collected from hospitalized children during different epidemics and can help in the efficient diagnosis of respiratory syncytial viral infections.

Development of a dual antigen lateral flow immunoassay for detecting Yersinia pestis.

Yersinia pestis is the causative agent of plague, a zoonosis associated with small mammals. Plague is a severe disease, especially in the pneumonic and septicemic forms, where fatality rates approach 100% if left untreated. The bacterium is primarily transmitted via flea bite or through direct contact with an infected host. The 2017 plague outbreak in Madagascar resulted in more than 2,400 cases and was highlighted by an increased number of pneumonic infections. Standard diagnostics for plague include laboratory-based assays such as bacterial culture and serology, which are inadequate for administering immediate patient care for pneumonic and septicemic plague. The goal of this study was to develop a sensitive rapid plague prototype that can detect all virulent strains of Y. pestis. Monoclonal antibodies (mAbs) were produced against two Y. pestis antigens, low-calcium response V (LcrV) and capsular fraction-1 (F1), and prototype lateral flow immunoassays (LFI) and enzyme-linked immunosorbent assays (ELISA) were constructed. The LFIs developed for the detection of LcrV and F1 had limits of detection (LOD) of roughly 1-2 ng/mL in surrogate clinical samples (antigens spiked into normal human sera). The optimized antigen-capture ELISAs produced LODs of 74 pg/mL for LcrV and 61 pg/mL for F1 when these antigens were spiked into buffer. A dual antigen LFI prototype comprised of two test lines was evaluated for the detection of both antigens in Y. pestis lysates. The dual format was also evaluated for specificity using a small panel of clinical near-neighbors and other Tier 1 bacterial Select Agents. LcrV is expressed by all virulent Y. pestis strains, but homologs produced by other Yersinia species can confound assay specificity. F1 is specific to Y. pestis but is not expressed by all virulent strains. Utilizing highly reactive mAbs, a dual-antigen detection (multiplexed) LFI was developed to capitalize on the diagnostic strengths of each target.

Immunological characterization of chicken tumor necrosis factor-α (TNF-α) using new sets of monoclonal antibodies specific for poultry TNF-α

Tumor necrosis factor-α (TNF-α) is a type II transmembrane protein with either membrane-bound or soluble forms and is a prototypical member of the TNF superfamily. TNF-α is a pleiotropic cytokine associated with the regulation of systemic inflammation and host defense. Chicken TNF-α (chTNF-α) is a long-missed avian ortholog, and its immunological properties remain largely unknown compared to those of its mammalian counterparts. Here, we report the functional characterization and immunomodulatory properties of chTNF-α using a panel of newly developed anti-chTNF-α mouse monoclonal antibodies (mAbs). Using anti-chTNF-α mAbs, we determined the tissue expression of chTNF-α in lymphoid and non-lymphoid organs. A chTNF-α-specific antigen-capture sandwich ELISA was developed using compatible mAb partners by screening and validation of ten different mAbs. Employing 3G11 and 12G6 as capture and detection antibodies, respectively, the levels of native chTNF-α in the circulation of Clostridium perfringens, Eimeria, or dual C. perfringens/Eimeria-infected chickens were determined. Furthermore, intracellular expression of chTNF-α in primary immune cells or cell lines derived from chickens was validated by immunocytochemistry and flow cytometry assays using both 3G11 and 12G6 mAbs. Notably, both 3G11 and 12G6 neutralized chTNF-α-induced nitric oxide production in chicken HD11 cells in vitro. Collectively, our results enhance our understanding of the functional characteristics of chTNF-α, and these anti-chTNF-α mAbs will serve as valuable immune reagents to inform on inflammatory responses and disease pathogenesis in the fundamental and applied studies of avian species.

Establishment of an antigen-capture enzyme-linked immunosorbent assay for detecting avian reticuloendotheliosis virus

In this study, an antigen-capturing enzyme-linked immunosorbent assay (AC-ELISA) was established for the detection of avian reticuloendotheliosis virus (REV) using monoclonal and polyclonal antibodies against gp90. New Zealand white rabbits were immunized with recombinant REV-gp90 protein, and polyclonal antibodies were obtained after purification and used as the capture antibody. Mice monoclonal antibody 1A12D against REV-gp90 protein previously prepared in our laboratory was used as the detection antibody. The specificity of the AC-ELISA was confirmed with REV, avian leukosis virus subgroup J, Marek's disease virus serotype Ⅰ, avian hepatitis E virus and Fowl adenovirus serotype 4. The results showed that the AC-ELISA had specific binding reaction with REV, and did not react with other viruses. The detection limit of this assay was 195 TCID50 units of REV. Furthermore, commercial vaccine artificially contaminated with REV was detected by three methods: AC-ELISA, the TaqMan probe fluorescence real-time quantitative RT-PCR (RT-qPCR) and indirect immunofluorescence assay (IFA). The results showed that the positive coincidence rate of RT-qPCR and AC-ELISA was 90.63 %, and the positive coincidence rate of RT-qPCR and IFA was 96.88%, indicating that the AC-ELISA established in this study was effective and feasible. This method simplified the detection process for REV contamination in poultry attenuated vaccines, and provide necessary technical tools for high-throughput detection of REV.

Two Monoclonal Antibodies That Specifically Recognize Aspergillus Cell Wall Antigens and Can Detect Circulating Antigens in Infected Mice.

Invasive aspergillosis (IA) is a life-threatening disease mainly caused by Aspergillus fumigatus and Aspergillus flavus. Early diagnosis of this condition is crucial for patient treatment and survival. As current diagnostic techniques for IA lack sufficient accuracy, we have raised two monoclonal antibodies (1D2 and 4E4) against A. fumigatus cell wall fragments that may provide a platform for a new diagnostic approach. The immunoreactivity of these antibodies was tested by immunofluorescence and ELISA against various Aspergillus and Candida species in vitro and by immunohistochemistry in A. fumigatus infected mouse tissues. Both monoclonal antibodies (mAbs) showed intensive fluorescence with the hyphae wall of A. fumigatus and A. flavus, but there was no staining with other Aspergillus species or Candida species. Both mAbs also showed strong immunoreactivity to the cell wall of A. fumigatus hyphae in the infected liver, spleen and kidney of mice with IA. The antigens identified by 1D2 and 4E4 might be glycoproteins and the epitopes are most likely a protein or peptide rather than a carbohydrate. An antibody-based antigen capture ELISA detected the extracellular antigens released by A. fumigatus, A. flavus, A. niger and A. terreus, but not in Candida species. The antigen could be detected in the plasma of mice after 48 h of infection by double-sandwich ELISA. In conclusion, both 1D2 and 4E4 mAbs are potentially promising diagnostic tools to investigate invasive aspergillosis.

Retroviral Association with Ovarian Adenocarcinoma in Laying Hens

The laying hen has been used as a model for ovarian adenocarcinoma (OAC) in women. Previous work has shown an association between expression of endogenous retroviral proteins and elevated envelope mRNA and occurrence of OAC has been demonstrated in humans, but causality has not been demonstrated. The objective of this study was to determine whether there is a similar association between retrovirus presence and OAC in a commercial laying hen flock at the University of Illinois Poultry Research facility with a history of a high prevalence of OAC in its aged hens. Laying hens of three age strata were randomly selected for a cross-sectional study. Blood samples were collected, and serum was tested for antigens of endogenous or exogenous avian leukosis virus (ALV) by ELISA. Birds were humanely euthanized, and spleens, ovaries, and any tissues with gross lesions were sampled. Ovaries and tissues with gross lesions were examined histologically and spleens were used for RT-PCR to detect endogenous ALV via ALV-E env mRNA expression. Overall, hens with OAC were 5.2 times more likely to be ALV positive than hens without OAC (95% C.I. = 2.06-13.14). Holding age stratum constant, OAC positive hens were 3.6 times more likely to be positive for ALV via antigen-capture ELISA (95% C.I. 1.08- 11.96). Endogenous ALV-E in hens may be analogous to the human endogenous retroviruses, which have also been associated with OAC in women. Further studies to establish causation are warranted to better understand the potential for laying hens to serve as a laboratory model for viral-induced ovarian tumors in humans.

Antigen-capture ELISA and immunochromatographic test strip to detect the H9N2 subtype avian influenza virus rapidly based on monoclonal antibodies

BackgroundThe H9N2 subtype of avian influenza virus (AIV) has become the most widespread subtype of AIV among birds in Asia, which threatens the poultry industry and human health. Therefore, it is important to establish methods for the rapid diagnosis and continuous surveillance of H9N2 subtype AIV.MethodsIn this study, an antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) and a colloidal gold immunochromatographic test (ICT) strip using monoclonal antibodies (MAbs) 3G4 and 2G7 were established to detect H9N2 subtype AIV.ResultsThe AC-ELISA method and ICT strip can detect H9N2 subtype AIV quickly, and do not cross-react with other subtype AIVs or other viruses. The detection limit of AC-ELISA was a hemagglutinin (HA) titer of 4 for H9N2 subtype AIV per 100 μl sample, and the limit of detection of the HA protein of AIV H9N2 was 31.5 ng/ml. The ICT strip detection limit was an HA titer of 4 for H9N2 subtype AIV per 100 μl sample. Moreover, both detection methods exhibited good reproducibility and repeatability, with coefficients of variation < 5%. For detection in 200 actual poultry samples, the sensitivities and specificities of AC-ELISA were determined as 93.2% and 98.1%, respectively. The sensitivities and specificities of the ICT strips were determined as 90.9% and 97.4%, respectively.ConclusionsThe developed AC-ELISA and ICT strips displayed high specificity, sensitivity, and stability, making them suitable for rapid diagnosis and field investigation of H9N2 subtype AIV.

Zika Virus Non-Structural Protein 1 Antigen-Capture Immunoassay.

Infection with Zika virus (ZIKV), a member of the Flavivirus genus of the Flaviviridae family, typically results in mild self-limited illness, but severe neurological disease occurs in a limited subset of patients. In contrast, serious outcomes commonly occur in pregnancy that affect the developing fetus, including microcephaly and other major birth defects. The genetic similarity of ZIKV to other widespread flaviviruses, such as dengue virus (DENV), presents a challenge to the development of specific ZIKV diagnostic assays. Nonstructural protein 1 (NS1) is established for use in immunodiagnostic assays for flaviviruses. To address the cross-reactivity of ZIKV NS1 with proteins from other flaviviruses we used site-directed mutagenesis to modify putative epitopes. Goat polyclonal antibodies to variant ZIKV NS1 were affinity-purified to remove antibodies binding to the closely related NS1 protein of DENV. An antigen-capture ELISA configured with the affinity-purified polyclonal antibody showed a linear dynamic range between approximately 500 and 30 ng/mL, with a limit of detection of between 1.95 and 7.8 ng/mL. NS1 proteins from DENV, yellow fever virus, St. Louis encephalitis virus and West Nile virus showed significantly reduced reactivity in the ZIKV antigen-capture ELISA. Refinement of approaches similar to those employed here could lead to development of ZIKV-specific immunoassays suitable for use in areas where infections with related flaviviruses are common.

Development of Immunoassays for Detection of Francisella tularensis Lipopolysaccharide in Tularemia Patient Samples.

Francisella tularensis is the causative agent of tularemia, a zoonotic bacterial infection that is often fatal if not diagnosed and treated promptly. Natural infection in humans is relatively rare, yet persistence in animal reservoirs, arthropod vectors, and water sources combined with a low level of clinical recognition make tularemia a serious potential threat to public health in endemic areas. F. tularensis has also garnered attention as a potential bioterror threat, as widespread dissemination could have devastating consequences on a population. A low infectious dose combined with a wide range of symptoms and a short incubation period makes timely diagnosis of tularemia difficult. Current diagnostic techniques include bacterial culture of patient samples, PCR and serological assays; however, these techniques are time consuming and require technical expertise that may not be available at the point of care. In the event of an outbreak or exposure a more efficient diagnostic platform is needed. The lipopolysaccharide (LPS) component of the bacterial outer leaflet has been identified previously by our group as a potential diagnostic target. For this study, a library of ten monoclonal antibodies specific to F. tularensis LPS were produced and confirmed to be reactive with LPS from type A and type B strains. Antibody pairs were tested in an antigen-capture enzyme-linked immunosorbent assay (ELISA) and lateral flow immunoassay format to select the most sensitive pairings. The antigen-capture ELISA was then used to detect and quantify LPS in serum samples from tularemia patients for the first time to determine the viability of this molecule as a diagnostic target. In parallel, prototype lateral flow immunoassays were developed, and reactivity was assessed, demonstrating the potential utility of this assay as a rapid point-of-care test for diagnosis of tularemia.

Development of double antibody sandwich ELISA as potential diagnostic tool for rapid detection of Crimean-Congo hemorrhagic fever virus

Crimean-Congo hemorrhagic fever (CCHF) virus, a highly pathogenic viral agent is responsible for severe fatal hemorrhagic infections in many parts of the world. The early diagnosis of CCHF infection is important for successful clinical management and epidemiological control. The nucleoprotein (NP) of CCHFV being highly conserved and immunogenic is used as early diagnostic marker. In this study, we report a rapid and sensitive double antibody based antigen capture ELISA to detect Crimean-Congo hemorrhagic fever virus (CCHFV). Highly specific polyclonal and monoclonal antibody against NP has been generated and used as capture and detector antibody respectively. The assay was able to detect viral nucleoprotein in different matrices including human serum, ticks and culture supernatant. The detection limit of the developed sandwich ELISA assay was 25 ng of purified antigen. Comparison with a real time RT-PCR revealed its detection limit to be 1000 genome equivalents of CCHFV. Further the assay was comparatively evaluated with a commercial kit employing gamma irradiated CCHFV, revealing a sensitivity and specificity of 100%. This newly developed sandwich ELISA (sELISA) with high sensitivity and specificity could be used as an efficient method for the detection of CCHF virus in humans, ticks and culture supernatant. The assay will be useful as alternate tool for diagnosis of acute infection and is amenable for screening of large scale samples in resource limited settings.

Immunological studies on chicken interferon-kappa using an antigen-capture ELISA developed using new mouse monoclonal antibodies

Interferon (IFN)-κ is a type I IFN that plays a central role in anti-viral defense and host immune response. The functions of type I IFNs have not been clearly defined in chickens compared to those of their mammalian counterparts. In this study, we developed an antigen-capture ELISA using newly developed mouse monoclonal antibodies (mAbs) which are specific for chicken IFN-κ (chIFN-κ) and showed that this ELISA can measure native chIFN-κ production during the activation of macrophages by polyinosinic:polycytidylic acid (poly I:C). The recombinant chicken IFN-κ expressed in Escherichia coli was used to immunize mice. Five mAbs that specifically recognized chIFN-κ were selected and characterized based on their specificity and binding activity toward chIFN-κ via indirect ELISA and western blotting. To develop a capture ELISA for chicken IFN-κ, two sets of the best capture and detection mAbs combinations were identified via pairing assays. To validate the antigen-capture assay, the production of native IFN-κ was induced in chicken HD11 macrophages using poly I:C. Furthermore, qRT-PCR was used to confirm the transcript-level expression of IFN-κ in HD11 cells at 24 and 48 h. The neutralizing effects of anti-chIFN-κ mAbs were evaluated based on their ability to block the induction of IFN-stimulated genes (ISGs) in DF-1 fibroblast cells stimulated with recombinant chIFN-κ proteins. All five mAbs blocked the mRNA expression of ISGs in a dose-dependent manner. Our results validate the specificity and utility of these newly developed mAbs for the detection of native chicken IFN-κ. This novel antigen-capture ELISA will be a valuable tool for fundamental and applied research involving IFN-κ in the normal and diseased states.

Unveiling the immunological attributes of chicken tumor necrosis factor-α (TNF-α) using new sets of monoclonal antibodies specific for poultry TNF-α

Abstract Tumor necrosis factor-α (TNF-α) is a type II transmembrane protein with either membrane-bound or soluble forms and is a prototypical member of TNF superfamily. TNF-α is a pleiotropic cytokine associated with the regulation of systemic inflammation and host defense. Chicken TNF-α (chTNF-α) is a long missed avian ortholog and the immunological properties of chTNF-α remains largely unknown compared to the mammalian counterpart. Here, we report on the functional characterization and immunomodulatory roles of chTNF-α using a panel of new anti-chTNF-α mouse monoclonal antibodies (mAbs) that we developed. A chTNF-α-specific antigen-capture ELISA was developed using compatible mAb partners via the screening and validation of ten different mAbs (3G11, 4C4, 4F3, 6A52, 6E6, 10E8, 12G6, 12H7, 14H2 and 15G7). Employing 3G11 and 12G6 as capture and detection antibodies, respectively, the levels of native chTNF-α in the circulation of Clostridium perfringens-, Eimeria- or dual C. perfringens/Eimeria-infected chickens were determined. Furthermore, intracellular expressions of chTNF-α in primary cells or cell lines derived from chickens were validated in immunocytochemistry and flow cytometry assays using both 3G11 and 12G6 mAbs. Importantly, both 3G11 and 12G6 neutralized chTNF-α-induced nitric oxide production in chicken HD11 cells in vitro. Collectively, our results provide a better understanding of the functional characteristics of chTNF-α and these anti-chTNF-α mAbs will serve as valuable immunological tools for the fundamental and applied studies in avian species.

Avian Leukosis Virus in Hatchable Eggs of Commercial and Free-Range Chickens

Avian leukosis is caused by various avian retroviruses which induce a variety of benign and malignant neoplasm in chickens with subsequent economic losses to the poultry industry. A survey of avian leukosis in hatchable eggs of apparently healthy chickens was carried out in an attempt to determine the prevalence of avian leukosis virus (ALV) in exotic breeders and free-range indigenous chickens in Oyo state. A total of 188 (76 exotic breeders and 112 free-range chickens) albumen of hatchable eggs were obtained from 3 different commercial breeder farms and 12 free-range indigenous chicken farms. Eggs were tested by an antigen capture enzyme-linked immunosorbent assay (ELISA) technique. Eleven out of the 76 albumen obtained from eggs of exotic breeders tested positive to ALV antigen which represents 14.5% while 37 of the 112 albumen from free-range indigenous eggs were confirmed as positive representing 33%. Overall prevalence rate of 25% was recorded for all eggs sampled with the highest prevalence of ALV antigen detected in free-range indigenous chickens than the exotic chickens. The results emphasizes the control of the Avian Leukosis transmission by including both exotic and free-range indigenous birds as wells as the use of ALV free eggs in hatcheries

Molecular characterization of group A rotavirus strains detected in alpacas (Vicugna pacos) from Peru.

The alpaca is a very important social and economic resource for the production of fibre and meat for Andean communities. Peru is the main producer of alpacas. Group A rotavirus (RVA) has been sporadically detected in alpacas. In this study, a total of 1423 faecal samples from alpacas from different locations of the Puno department in Peru were collected and analysed by an antigen-capture ELISA in order to detect RVA. Four per cent of the samples were RVA-positive (57/1423). The genotype constellation of three selected alpaca RVA strains were G3/8 P[1/14]-I2-R2/5-C2/3-M2/3-A17-N2/3-T6-E3-H3. Two of the analysed strains presented a bovine-like genotype constellation, whereas the third strain presented six segments belonging to the AU-1-like genogroup (G3, M3, C3, N3, T3 and E3), suggesting reassorting events. Monitoring of the sanitary health of juvenile alpacas is essential to reduce the rates of neonatal mortality and for the development of preventive health strategies.

Isolation and identification of peste des petits ruminants virus from goats in Egyptian governorates.

Background and Aim:The peste des petits ruminants (PPR) is a highly contagious disease of small ruminants which negatively affects animal production and the socioeconomic status of farmers. Peste des petits ruminants virus (PPRV) encodes eight proteins, with the viral fusion protein (F) playing a role in virus virulence and stimulating an effective protective immune response. This study aimed to isolate and complete the identification of PPRV circulating in goats in different Egyptian governorates and perform molecular characterization of the PPRV F gene.Materials and Methods:Samples were collected from unvaccinated animals with clinical signs suggestive of PPR. A total of 256 sera were tested for the detection of PPRV antibodies using a competitive enzyme-linked immunosorbent assay (c-ELISA) kit, while 214 samples of blood buffy coat preparation, animal swabs (nasal, ocular, and saliva), and fecal and tissue samples were tested for the detection of the PPRV antigen using an antigen-capture ELISA kit. Molecular diagnosis, gene cloning, blast analysis, and phylogenetic analysis were performed for the molecular characterization of PPRV.Results:The seroprevalence results of PPRV antibodies in the tested sera showed a total of 67.9% positive samples. The rates of PPR antigen recorded by the antigen-capture ELISA in the swabs (nasal and ocular) and tissue samples were 44.3%, 46.8%, and 43.5%, respectively, with saliva swabs having the highest rate of PPRV positivity (76.4%) and fecal samples having the lowest (33.3%). Molecular characterization of the PPRV Vero cell culture revealed that the circulating PPRV strain belongs to the IV lineage. Blast analysis of the PPRV F gene showed 96.7% identity with the PPRV strain Egypt-2014 fusion protein (F) gene, KT006589.1, differing by 43 single-nucleotide polymorphisms.Conclusion:The results of this study indicate that the emerging PPRV belongs to the IV lineage among small ruminant animals. The findings also indicate the need for an innovative strategy to control and eliminate this disease based on a regularly administered and effective vaccine, a test to distinguish between infected and vaccinated animals, and the need for further study on the protein structure and PPRV F gene expression, which should help us to understand the molecular evolution of the virus and control and eliminate PPR disease.

Inducible expression of human papillomavirus-16 L1 capsomeres in the plastomes of Nicotiana tabacum: Transplastomic plants develop normal flowers and pollen.

Human papillomavirus type-16 (HPV-16) is the major HPV type involved in causing cervical cancer among women. The disease burden is high in developing and underdeveloped countries. Previously, the constitutive expression of HPV-16 L1 protein led to male sterility in transplastomic tobacco plants. Here, the HPV-16 L1 gene was expressed in chloroplasts of Nicotiana tabacum under the control of an ethanol-inducible promoter, trans-activated by nucleus-derived signal peptide. Plants containing nuclear component were transformed with transformation vector pEXP-T7-L1 by biolistic gun. The transformation and homoplasmic status of transformed plants was verified by polymerase chain reaction and Southern blotting, respectively. Protein was induced by spraying 5% ethanol for 7 consecutive days. The correct folding of L1 protein was confirmed by antigen-capture ELISA using a conformation-specific antibody. The L1 protein accumulated up to 3μg/g of fresh plant material. The L1 protein was further purified using affinity chromatography. All transplastomic plants developed normal flowers and produced viable seeds upon self-pollination. Pollens also showed completely normal structure under light microscope and scanning electron microscopy. These data confirm the use of the inducible expression as plant-safe approach for expressing transgenes in plants, especially those genes that cause detrimental effects on plant growth and morphology.