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Incidences of major feline viral diseases provide basic information for preventing viral disease in cats. Despite the growing interest in feline viral diseases, sero-surveillances have been lacking. In this study, we analyzed the diagnoses of feline viral diseases and conducted a sero surveillance of feline panleukopenia virus (FPV), feline calicivirus (FCV), feline herpesvirus-1 (FHV-1), and feline infectious peritonitis virus (FIPV) in Korean cats. Of the 204 confirmed cases since 2015, the numbers of diagnoses for FPV, FIPV, FCV, feline influenza virus, and FHV-1 were 156, 32, 12, 3, and 1 case, respectively. In total, 200 sera, collected between 2019 and 2021, were screened for the presence of antibodies against FPV, 2 FCVs, FHV-1, and FIPV using a hemagglutination inhibition test and a virus-neutralizing assay (VNA). The overall seropositive rates in cats tested for FPV, the 2 FCVs, FHV-1, and FIPV were 92.5%. 42.0%, 37.0%, 52.0%, and 14.0%, respectively. A low correlation (r = 0.466) was detected between the VNA titers of 2 FCV strains. The highest incidence and seropositive rate of FPV reveal that FPV is circulating in Korean cats. The low r-value between 2 FCVs suggests that a new feline vaccine containing the 2 kinds of FCVs is required.
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[Objective] To prepare broad-spectrum monoclonal antibody against N protein of avian infectious bronchitis virus (IBV), so as to lay a foundation for identifying conservative domain epitope of N protein and establish a universal IBV detection method. [Method] N protein of GX-YL5, a representative strain of IBV dominant serotype in Guangxi, was expressed in prokaryote. BALB/c mice were immunized with the purified protein. After the serum titer of the immunized mice reached 104 or more, the splenocytes were fused with SP2/0 myeloma cells. After screening by indirect ELISA, monoclonal antibody was prepared by ascites-induced method. Western blotting, IFA and indirect ELISA were used to identify the titer, subtype, reaction specificity and cross-reaction spectrum. And the prepared monoclonal antibody was used for immunohistochemical detection. And the prepared monoclonal antibody was used to detect the IBV in the trachea and kidney tissues of SPF chickens artificially infected with 4 representative IBV variants (GX-N130048, GX-N160421, GX-QZ171023 and GX-QZ170728). [Result] The prepared monoclonal antibody N2D5 had a titer greater than 217 and its subtype was IgG2b. The Western blotting and IFA results showed that the monoclonal antibody N2D5 only reacted with IBV, and were negative with Newcastle disease virus (NDV), infectious laryngotracheitis virus (ILTV), avian metapneumovirus (aMPV), infectious bursal disease virus (IBDV), avian leukosis virus (ALV) and Marek's disease virus (MDV). Monoclonal antibody N2D5 reacted with many genotypes in China and all 7 serotypes of IBV currently prevalent in Guangxi, including commonly used standard strains, vaccine strains and field strains. Immunohistochemistry showed that the virus signals could be detected in the trachea and kidney tissues of SPF chickens at different time after artificial infection of 3 representative IBV strains from chicken and 1 isolated strain from duck, which further proved its broad spectrum. [Conclusion] The monoclonal antibody N2D5 of IBV prepared based on hybridoma technology belongs to the IgG2b subtype. It has the characteristics of high specificity, wide response spectrum and strong binding ability with IBV. It can be used as a specific diagnostic antibody for clinical diagnosis of IBV and the study of virus distribution.
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In order to develop monoclonal antibody against Feline infectious peritonitis virus (FIPV) S1 protein, the truncated S1 protein (rS1) was expressed through Escherichia coli and subsequently purified. Then BALB/c mice were immunized with purified rSl. Three hybridoma cell strains, named 2D7,3D8 and 5G1, stably secreting antibodies against rSl were obtained by cell fusion and indirect ELISA screening. The identification of antibody subtype showed that antibody subtypes of 2D7,5G1 and 3d8 strains were IgG2a,IgG2a and IgGl,respectively. And the light chain of those three hybridoma cell strains was Kappa. Result of karyotype identification of hybridoma cells showed that the chromosome numbers of those three hybridoma cells were about 102,101 and 103, which was belonged to the karyotype of hybridoma. The titer of ascites antibody for indirect ELISA was 1 : 204 800, and monoclonal antibodies were purified. Moreover, all of 2D7,3D8 and 5G1 could react with rS1 by Western-blot and FIPV in cells by IFA. These data suggest that three monoclonal antibodies against rSl with good activities were ideal materials in the study of early diagnosis of FIPV and the biological function of FIPV in the future.
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This paper describes the clinical signs and use of differential laboratory diagnostic techniques (computed tomography, cytology, histopathology, antigen/antibody detection and polymerase chain reaction) for infectious (viral, bacterial, fungal and parasitic) and non-infectious (inflammatory/immune mediated, neoplastic, cardiac, malformation, foreign body, smoke inhalation, aspiration of caustic material, non-cardiogenic, pulmonary oedema, traumativ, pneumothorax, pulmonary contusions and idiopathic) causes of respiratory diseases in cats and dogs in Ontario, Canada.
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Feline coronavirus (FCoV) belongs among pathogens with common occurrence in the cats population in the whole world. FCoV is ubiquitous in environments with a higher concentration of cats, e.g. in shelters, multicat households and kennels. FCoV primarily attacks the digestive feline tract, replicates in its cells and is excreted in the feces to surroundings of permanently or transiently infected cats. The aim of the study was the detection of FCoV in the feces of newly admitted cats to the shelter by the qPCR method and by means of commercial rapid immunochromatographic (antigen) tests from three different producers. For each of the antigen tests, sensitivity and specifity were determined by comparison with the qPCR analysis result. Out of 70 examined fecal samples, viral RNA was by the qPCR analysis identified in 44 samples (62.9%). Neither the age nor the gender of cats played a significant role in the viral excretion. Found sensitivity of the antigen tests was at a low (< 35%;tests A and C) to a satisfactory level (> 50%, test B). The number of viral particles in the samples determined by the qPCR method did not correlate significantly with the result of the antigen tests. The results of this study suggest that the use of rapid antigen tests for routine screening of FCoV shedding in feline shelters is limited due to the high rate of false-negative results.
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The aim of this study is to establish an indirect ELISA technique for detecting the SIgA antibody against porcine epidemic diarrhea virus (PEDV) to evaluate its mucosal immunity. Firstly, the S1D gene (534-789 aa) of PEDV was cloned into the pET-28a(+) vector, and induced in Escherichia coli BL21 (DE3) by IPTG, the product of which was in the form of inclusion bodies. According to Western-blot, the target protein S1D with antigenic activity was 32 ku in molecular weight and could be well detected. Then, the S1D protein was denatured by 8 mol/L urea, purified and gradient as the coating antigen to establish an indirect ELISA for detecting the PEDV specific SIgA antibody in nasal or oral mucus by optimizing conditions. And the optimal antigen coating concentration of ELISA was 2 micro g mL, the working concentrations of nasal mucus was 1:1 and the optimal blocking solution was 50 g/L skimmed milk, while the working concentrations and optimal blocking solution were 1:2 and 30 g/L BSA in oral mucus, the working concentrations of the enzyme-labeled antibody was 1:2 000 in nasal and oral mucus. Finally, 84 samples of oral and nasal mucus from immunized pigs were detected by S1D of ELISA, and the coincidence rate could reach 95.2% compared with purified PEDV of ELISA. In conclusion, the indirect ELISA established in this study provided a quick, simple, sensitive, and specific method to detect PEDV specific SigA for evaluating the level of PEDV mucosal immunity.
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The recombinant expression plasmid pIRES-S1 was constructed according to the gene sequence of PEDV S1 in NCBI (GenBank:JQ517274). The plasmid pIRES-S1 was transfected into ST cells by electrotransfer. After G418 pressurization screening, western-blot detection and suspension domestication, a stable transduction cell pool expressing S1 protein was obtained. The results of Western-blot showed that S1 protein have good reactivity. An indirect ELISA was established by using S1 protein as coating antigen, and the ELISA was used to detect PEDV clinical serum and PEDV negative serum of imported breeder pigs. Take the serum neutralization test as the standard, the results showed that the sensitivity of the ELISA was 96.3% and the specificity was 97.7%.It was significantly consistent with the serum neutralization test (kappa value=0.882, P < 0.05). The ELISA was used to detect the tracking serum of PEDV back-feeding pigs. The results showed that it could accurately evaluate the growth and decline of PEDV Ig G antibody level in infected pigs. Our results suggested that the ELISA based on S1 protein established in this study has high sensitivity and specificity. It could be used to detect PEDV antibody in clinical serum samples and provide an effective basis for immune evaluation of PEDV in pigs.
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Objective: To prepare monoclonal antibodies against porcine epidemic diarrhea virus (PEDV) N protein, and develop an indirect immuno-fluorescence assay method used for detecting PEDV. Method: The expressed recombinantly PEDV N protein was used as an immunogen and 8-week-old female BALB/c mice were immunized. Then their spleen cells with high antibody titer were isolated and fused with SP2/0 cells. The hybridoma cell lines secreting monoclonal antibodies against PEDV N protein were screened. In Vero cells infected with PEDV, monoclonal antibody of anti-PEDV N protein was used as the primary antibody and FITC-goat-anti-mouse IgG was used as the secondary antibody to develop indirect immuno-fluorescence assay method used for detecting PEDV. Result: The prepared hybridoma cell lines could stably secrete anti-PEDV N protein antibodies, ELISA antibody titer in cell supernatant was above 1:3 200, and in mouse ascites above 1:1 000 000. While monoclonal antibodies were applied in established indirect immuno-fluorescence assay, the optimal conditions were that cells were fixed with 80% () acetone at -20 degrees C for 30 min;The primary antibody was diluted 1 000 times by PBS buffer solution and incubated at 4 degrees C overnight;The secondary antibody was diluted 100 times by PBS buffer solution and incubated at 37 degrees C for 1 h. Transmissible gastroenteritis virus (TGEV), classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine reproductive virus (PRV), porcine enteric a corone virus (PEAV), porcine rotavirus (PoRV) and PEDV were detected by established indirect immuno-fluorescence assay method, only PEDV showed positive, all the else viruses showed negative.
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MERS-CoV was isolated from nasal swabs for 10 days from an adult female camel which displayed clear nasal discharge from both nostrils. When MERS-CoV ELISA antibodies appeared in the camel's blood, the virus was no longer present in its nasal cavities.
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We have evaluated the diagnostic performance of immunochromatographic point-of-care tests (POCT) for the detection of rotavirus, coronavirus, Escherichia (E.) coli F5, Cryptosporidium (C.) parvum, Clostridium (Cl.) perfringens and Giardia (G.) intestinalis in fresh and thawed faecal samples from calves aged up to six months with diarrhoea. We performed POCTs to detect rotavirus, coronavirus, E. coli F5, C. parvum, Cl. perfringens and G. intestinalis on fresh samples in a field study and re-evaluated the performance for C. parvum, Cl. perfringens and G. intestinalis using thawed samples. We calculated the performance based on the results of the reference methods, which were RT-qPCR for the detection of rota- and coronavirus and bacteriological culturing and PCR to detect E. coli F5 and Cl. perfringens a and ss2 toxins. C. parvum was detected by phase-contrast microscopy and G. intestinalis by immunofluorescence microscopy. We collected 177 faecal samples from diarrhoeic calves. We found good performance for the POCT targeting rotavirus (sensitivity (SE)=92.9%;specificity (SP)=95.6%) and C. parvum (SE=63.3%;SP=96.2%). For E. coli F5, the number of true positive samples (n=1) was too low to evaluate the performance. The POCT to detect coronavirus gave a poor performance (SE=3.3%;SP=96.6%) and the POCT to detect Cl. perfringens a moderate performance (SE=52.8%;SP=78.2%). G. intestinalis POCT showed a higher sensitivity to immunofluorescence microscopy in thawed than in fresh faecal samples (SE=43.9% versus SE=29.2%). There are substantial differences in diagnostic performance between the commercially available immunochromatographic POCTs. Still, POCT can make a valuable contribution to the diagnosis and prevention of calf diarrhoea.
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The purpose of this study is to establish a blocking ELISA antibodies detection method for porcine epidemic diarrhea virus (PEDV). The purified N protein was used as the coating antigen, and the ELISA reaction conditions were optimized by the chess rboard titration. A blocking ELISA method for detecting PEDV antibodies was established, and its specificity, sensitivity and repeatability tests were carried out. One hundred and forty clinical serum samples were tested, and the results were compared with commercially IDvet PEDV indirect ELISA antibodies detection kit. The results showed that the best antigen coating concentration was 625 ng.mL-1, and the best dilution ratio of serum was 1:1;The best dilution of the HRP-conjugated antibody working solution was 1:5 000;There was no cross-reaction with healthy pig serum and the positive sera of common pig disease pathogens, such as classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), and transmissible gastroenteritis virus (TGEV). The sensitivity of PEDV positive serum was 1:16, which was equivalent to that of IDvet ELISA kit (titer 1:32). The coefficient of variation of within-run and between-run repeatability test is less than 10%, so it showed that the blocking ELISA established in this study had good repeatability and stability;the kappa value of detected 140 clinical porcine serum using this method was 0.87 when compared with IDvet ELISA. The above results indicated that the established blocking ELISA method for detecting PEDV antibodies in this study could be applied to the prevention and control of PEDV, epidemiological investigation and the monitoring of antibody levels after vaccine immunization.
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[Objective] To identify the B-cell epitope peptide of porcine epidemic diarrhea virus (PEDV) S2 gene by combinative use of bioinformatics software and monoclonal antibody technology. [Method] The B-cell epitope of PEDV S2 gene was screened using CLC Sequence viewer 6.8 software and IEDB online database, and the obtained epitope peptide was synthesized artificially. Female BALB/c mice were immunized with the conjugate of epitope peptide and keyhole hemocyanin (KLH) as antigen. Mice with higher antibody titers were identified by ELISA assay and then received an additional immunization. The spleen of the mice was taken 3 days post immunization to prepare the splenocyte suspension for cell fusion. The cells were grown on HAT selective medium to screen for effective hybridoma cells. The positive clones screened by ELISA assay were then used for expanding culture. Positive hybridoma cells were intraperitoneally injected to mice and ascites were collected. ELISA assay was used to determine the antibody titers in mice ascites and in the supernatants of monoclonal cell strains. The cells with the highest antibody titers was used as cell strain for subsequent use. [Result] The selected B-cell epitope peptide sequence was MQYVYTPTYYML Following immunization with the peptide antigen, the serum antibody titer before cell fusion reached 1:2 000. The ELISA assay of ascites from BALB/c mice and the supernatants from monoclonal cell strain cultures demostrated that the antibody liter reached 1:4 000. [Conclusion] The B-ell epitope of PEDV S2 gene was identified, which may be helpful for the vector construction of a epitope based peptide vaccine against PEDV.
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In December 2019, the coronavirus disease COVID-19 began in China. Since then, millions of infections and deaths from this cause have been reported worldwide, particularly among health workers who have suffered the harsh onslaught of the pandemic in the context of healthcare systems collapsed by demand. In this sense, the objective of this work was to determine the prevalence, sociodemographic, epidemiological, and clinical characteristics of COVID-19 present in health workers of the "Instituto Aut..nomo Hospital Universitario de Los Andes" in M..rida-Venezuela. An observational, retrospective, single-center, documentary study was carried out, where 297 clinical-epidemiological records corresponding to 285 health workers were analyzed, in a period between March 16 and November 30, 2020. The records were separated into two groups, front-line workers and support workers. The overall positivity of the RT-PCRs performed was 31.6%. The frequency of positive confirmatory results was highest among support workers at 33.9%. Nursing staff had the highest positivity (39.5%). A seroprevalence of 34.3% was found in immunological tests. The prevalence of SARS-CoV-2 infection among health care workers was higher among support workers compared to front-line workers. Therefore, general and work-specific prevention strategies should be strengthened to limit the spread of SARS-CoV-2 among staff to ensure that they perform safely and effectively.
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Aim: The test, based on the detection of SARS-CoV-2 antigen, is often seen as an alternative to the PCR method in connection with the need for screening of larger populations. In order to assess the suitability of such an approach, we evaluated the sensitivity of two antigenic assays on a group of individuals, including both patients with symptoms of covid-19 and asymptomatic and healthy individuals.
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A 1:1 matched case-control study of calves under 1 month of age was carried out by weekly visits to 7 dairy farms in Mashhad, Iran. Fecal samples were collected from 112 calves with diarrhea and 112 controls matched assessed by scoring. Rotavirus and Coronavirus were isolated by antigen capture ELISA test. We found Rotavirus antigen in 29.5% in diarrheic and 17% in normal calves. We detected Coronavirus antigen in 2.7% and 1.8%, respectively. In diarrheic calves Rotavirus was most prevalent at 3rd week age whereas Coronavirus was found in very few cases by 2nd week. Excretion of Rotavirus in feces of scouring calves was greater (P < 0.03) than controls with odds ratio of 1.9 (1.05 - 3.76). We found no relationship between shedding Coronavirus in feces and diarrhea (P value =0.66) with odds ratio equals to 1.4 (0.24 -9.05). We find that Rotavirus is associated with newborn calf diarrhea in industrial dairy farms in Iran.
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Nationwide shortages of tests that detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and diagnose coronavirus disease 2019 (COVID- 19) have led the US Food and Drug Administration (FDA) to significantly relax regulations regarding COVID-19 diagnostic testing. To date the FDA has given emergency use authorization (EUA) to 48 COVID-19 in vitro diagnostic tests and 21 high complexity molecular-based laboratory developed tests, as well as implemented policies that give broad authority to clinical laboratories and commercial manufacturers in the development, distribution, and use of COVID-19 diagnostic tests. Currently, there are 2 types of diagnostic tests available for the detection of SARS-CoV-2: (1) molecular and (2) serological tests. Molecular detection of nucleic acid (RNA or DNA) sequences relating to the suspected pathogen is indicative of an active infection with the suspected pathogen. Serological tests detect antibodies against the suspected pathogen, which are produced by an individual's immune system. A positive serological test result indicates recent exposure to the suspected pathogen but cannot be used to determine if the individual is actively infected with the pathogen or immune to reinfection. In this article, the SARS-CoV-2 diagnostic tests currently approved by the FDA under EUA are reviewed, and other diagnostic tests that researchers are developing to detect SARS-CoV-2 infection are discussed.
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Generally, rapid detection of viral infection is necessary for preventing the virus from spreading among people in a society as a pandemic. Although there are many effective standard techniques used for virus identification, they are laborious, required skilled person to handle and time-consuming. Particularly, the detection of viral infection involved in the isolation and nucleic acid detection by collecting specimens (sample) from the appropriate sites. For instance, oral or nasal swab, nasopharyngeal or tracheal extract, lung tissue, blood, sputum and feces are collected in order to investigate the pandemic, COVID-19 for the effective and rapid diagnosis and eventually for the treatment. In this mini-review, it is summarized that the advanced testing methods which include RNA, immunologic and radiological based tests that could be used to detect COVID-19 and their cost, reliability and functionality are discussed in this review. This mini-review might help the researcher and health care sector to plan the diagnostic procedures as per the severity of the new infection, COVID-19.