Fluorescent Antibody Assay Research Articles

Pestivirus bovis is implicated in repeated abortions and subfertility of vaccinated cattle in Egypt

The study of Pestivirus bovis, formerly known as Bovine Viral Diarrhea Virus (BVDV), is of significant importance due to the economic challenges it poses in cattle herds in several countries. This virus leads to decreased productivity, reproductive failures, and increased susceptibility to secondary infections. The current study aimed to investigate the role of BVDV in abortion and infertility among cattle in Egypt using advanced diagnostic techniques. We employed a one-step multiplex real-time reverse transcription polymerase chain reaction (rRT-PCR) to detect BVDV, and the results showed an overall prevalence of 11.2% among 178 tested samples. Notably, 11.2% tested positive for BVDV-1, with higher detection rates in adult cows (12.1%) compared to calves (9.85%). Importantly, 13.8% of samples from dams with subfertility and repeated abortions were positive for BVDV-1. The results of rRT-PCR guided virus isolation using Madin–Darby bovine kidney (MDBK) cells. Identifying cytopathic effects (CPEs) in 15% of the samples, consistent with cytopathic-BVDV (CP-BVDV). An indirect fluorescent antibody assay (IFA) confirmed the presence of CP-BVDV in these samples. For non-cytopathic (NCP)-strains, the immunoperoxidase (IP) test using bovine turbinate (BT) cells was more effective, detecting NCP-BVDV in samples without CPE in MDBK cells. The presence of mixed infection was indicated by an isolate from a diarrheic calf, showing positive results for both CP-BVDV and NCP-BVDV. Plaque assays further confirmed CP-BVDV and NCP-BVDV isolation from mixed infections, highlighting the selective amplification of both biotypes. These findings underscore the significant role of BVDV-1 in reproductive disorders in cattle and the importance of employing comprehensive diagnostic methods for effective control and management strategies. Our study provides valuable insights that can guide future studies and improve herd health and productivity.

Isolation of porcine circovirus 3 using primary porcine bone marrow-derived cells

Porcine circovirus 3 (PCV3) was first reported in the United States in 2016; this virus is considered to be involved in diverse pathologies, such as multisystem inflammation, porcine dermatitis and nephropathy syndrome, and reproductive disorders. However, successful isolation of PCV3 using cultured cells has been rare. In this study, we aimed to isolate PCV3 using primary porcine bone marrow-derived cells. Mononuclear cells were isolated from the femur bones of clinically healthy pigs. These primary cells were cultured for 6–10 days post-seeding and infected with PCV3-containing tissue homogenates. The cells were cultured for up to 37 days, and the culture medium was changed every 3–4 days. The growth curve of PCV3 in porcine bone marrow cells revealed a decline in growth during the first 10 days post-infection, followed by an increase leading to > 1010 genomic copies/mL of the cell culture supernatant; moreover, the virus was capable of passaging. The indirect fluorescent antibody assay for PCV3 infection revealed the presence of PCV3 capsid protein in the cytoplasm and nuclei of infected cells. Bone marrow cells were passaged for more than 20 generations (over 5 months), and PCV3 persistently infected the cells. PCV3-infected bone marrow cells expressed mesenchymal markers. These results reflect that primary porcine bone marrow-derived mesenchymal cells are permissive to PCV3 and continuously replicate a high copy number of the PCV3 genome. These findings regarding the high replication rate of PCV3 in bone marrow-derived mesenchymal cells could enhance our understanding of PCV3 pathogenicity.

Advances in Laboratory Diagnosis of Coronavirus Infections in Cattle.

Coronaviruses cause infections in humans and diverse species of animals and birds with a global distribution. Bovine coronavirus (BCoV) produces predominantly two forms of disease in cattle: a respiratory form and a gastrointestinal form. All age groups of cattle are affected by the respiratory form of coronavirus, whereas the gastroenteric form causes neonatal diarrhea or calf scours in young cattle and winter dysentery in adult cattle. The tremendous impacts of bovine respiratory disease and the associated losses are well-documented and underscore the importance of this pathogen. Beyond this, studies have demonstrated significant impacts on milk production associated with outbreaks of winter dysentery, with up to a 30% decrease in milk yield. In North America, BCoV was identified for the first time in 1972, and it continues to be a significant economic concern for the cattle industry. A number of conventional and molecular diagnostic assays are available for the detection of BCoV from clinical samples. Conventional assays for BCoV detection include virus isolation, which is challenging from clinical samples, electron microscopy, fluorescent antibody assays, and various immunoassays. Molecular tests are mainly based on nucleic acid detection and predominantly include conventional and real-time polymerase chain reaction (PCR) assays. Isothermal amplification assays and genome sequencing have gained increased interest in recent years for the detection, characterization, and identification of BCoV. It is believed that isothermal amplification assays, such as loop-mediated isothermal amplification and recombinase polymerase amplification, among others, could aid the development of barn-side point-of-care tests for BCoV. The present study reviewed the literature on coronavirus infections in cattle from the last three and a half decades and presents information mainly on the current and advancing diagnostics in addition to epidemiology, clinical presentations, and the impact of the disease on the cattle industry.

Assessment of the effect of long-term serum storage for retrospective serologic diagnosis of canine monocytic ehrlichiosis (Ehrlichia canis)

There is currently sparse information on the possible effect of long-term storage of serum specimens for the retrospective serodiagnosis of canine monocytic ehrlichiosis (CME). The aim of this study was to assess the agreement between the original serologic outcome and the results of a repeat indirect fluorescent antibody (IFA) assay for the detection of IgG antibodies against E. canis. A secondary aim was to compare the diagnostic performance of two commercially available point-of-care (POC) immunochromatographic (IC) assays. Archived serum samples originally tested as positive (n=66) or negative (n=19) for E. canis IgG antibodies and kept frozen at −20°C for a median of 22 years, were retrospectively examined by IFA and by two POC IC assays. Cohen’s Kappa coefficient (0.748, p < 0.0001), indicated a substantial agreement between the original and repeat serologic testing results. An almost identical high sensitivity and moderate specificity were established for the two POC IC assays. Canine serum specimens on long-term storage may still be of value for seroepidemiologic surveys investigating the exposure to E. canis.

MONOCLONAL ANTIBODIES AGAINST CTLA-4 AND PD-L1 RECEPTORS OF THE CATTLE IMMUNE SYSTEM

With the progression of bovine leukemia virus (BLV), the concentration of T-cells, as well as CTLA-4 and PD-1 receptors on their cytoplasmic membrane increases. Elevated concentrations of regulatory T-cells lead to increased production of transforming growth factor-β (TGF β), suppression of interferon-γ (IFN-γ) expression, tumor necrosis factor-α (TNF-α), and inhibition of natural killer (NK) cells. Effector and cytotoxic T-lymphocytes, as well as the production of cytokines IFN-γ and TNF-α, play a crucial role in immune response against viral infections. However, at late subclinical stages, T-lymphocyte activity decreases due to the activity of regulatory T-lymphocytes, contributing to infection growth and progression to clinical disease. Blockade of CTLA-4 and PD-1/PD-L1 receptors with specific antibodies restores the immune response against BLV. Monoclonal antibodies (mAbs) against recombinant bovine CTLA-4 and PD-L1 were obtained using hybridoma technology methods. The obtained mAbs were analyzed using enzyme-linked immunosorbent assay (ELISA) and western blotting methods. As a result of the study, hybridoma cell lines producing mAbs to recombinant bovine CTLA-4 and PD-L1 receptors were obtained. The hybridomas produced IgG1 class mAbs that specifically reacted with standard proteins and had a binding constant: 3b3 - 2.9×108 M-1, 4h10 - 1.9×108 M-1. The obtained mAbs effectively blocked the reaction of commercial bovine CTLA 4 and PD-L1 proteins with specific polyclonal antibodies in an indirect fluorescent antibody assay (IFA).

Bartonella spp. infection in people with Mild Cognitive Impairment: A pilot study.

Mild Cognitive Impairment (MCI) is a neurological disorder at the transition between normal cognitive decline and dementia. Despite the potential role of neuroinflammation in the pathogenesis of MCI, infectious triggers remain mostly unknown. Infection with Bartonella spp., a zoonotic bacterium, has recently been associated with diffuse neurological and psychiatric symptoms. Given the preferential endothelial localization of Bartonella spp. and the role of vascular changes in neurocognitive decline, we hypothesized that there is an association between Bartonella spp. infection and pathologically accelerated decline in cognitive function in aging. To test this hypothesis, we collected serological and molecular markers of past and present Bartonella spp. infection in a sample of older people with and without MCI. Samples were processed in a blinded way to exclude laboratory biases. Contrary to our hypothesis, people with MCI were not more likely than people without MCI to have an active Bartonella spp. infection as measured by droplet digital PCR (p = 0.735) and quantitative PCR (p = 1). In addition, there was no significant difference in positive serological results between cases and controls (p = 0.461). Overall, higher-than-expected active Bartonella spp. infection (37% by ddPCR) and seroreactivity (71% by indirect fluorescent antibody assay) were found in people without MCI. Conclusions require caution, as our study was limited by the small number of cases with MCI. Overall, our results identified a higher than previously recognized rate of exposure and infection with Bartonella spp. in this older study population but does not support a specific role for such infection in MCI.

Molecular Diagnostic Tools for Treponema pallidum

Syphilis, a common sexually transmitted disease, is caused by Treponema pallidum subsp. pallidum. Owing to the chameleonic behavior of syphilis, ocular involvement still presents a therapeutic problem. Direct detection of Treponema pallidum in the vitreous offers a potential diagnostic method because serodiagnosis has considerable limitations. The worldwide identification of T. pallidum substypes has occurred since the advent of molecular typing approaches. The purpose of this article is to provide more information on the development of a molecular approach for Treponema pallidum detection. A body of literature was gathered using automated database searches in Google Scholar, PubMed, and ScienceDirect. Although prior studies have focused on other genes, such as polA, 16S RNA, and the whole genome, there are still some that use the study of the arp and T. pallidum repeat (tpr) genes to subtype. Whole blood, vaginal ulcers, skin biopsies, and other samples can be used in molecular methods. Comparing quantitative reverse trascription-polymerase chain reaction (qRT-PCR) to traditional methods, such as reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), indirect fluorescent antibody (IFA) assay, and virus isolation, qRT-PCR has the advantage of being faster and more sensitive. Quick molecular methods, particularly polymerase chain reaction (PCR) results, will enable early detection of primary, secondary, and latent syphilis, which will lead to prompt treatment and prevention of disease progression as well as a reduction in the amount of time that the patient's sexual partners are exposed to the illness.

Bardoxolone methyl inhibits the infection of rabies virus via Nrf2 pathway activation in vitro

BackgroundRabies is a widespread, fatal, infectious disease. Several antivirals against rabies virus (RABV) infection have been reported, but no approved, RABV-specific antiviral drugs that inhibit RABV infection in the clinic after symptom onset are available. Therefore, more effective drugs to reduce rabies fatalities are urgently needed. Bardoxolone methyl (CDDO-Me), an FDA-approved compound that has long been known as an antioxidant inflammatory modulator and one of the most potent nuclear factor erythroid-derived 2-like 2 (Nrf2) activators, protects myelin, axons, and CNS neurons by Nrf2 activation. Therefore, we investigated the potency of its anti-RABV activity in vitro.MethodsThe mouse neuroblastoma cell line Neuro2a (N2a) and three RABV strains of different virulence were used; the cytotoxicity and anti-RABV activity of CDDO-Me in N2a cells were evaluated by CCK-8 assay and direct fluorescent antibody (DFA) assay. Pathway activation in N2a cells infected with the RABV strains SC16, CVS-11 or CTN upon CDDO-Me treatment was evaluated by western blotting (WB) and DFA assay.ResultsCDDO-Me significantly inhibited infection of the three RABV strains of differing virulence (SC16, CVS-11 and CTN) in N2a cells. We also examined whether CDDO-Me activates the Nrf2-associated pathway upon infection with RABV strains of differing virulence. Nrf2, phosphorylated sequestosome (SQSTM1), SQSTM1, hemoglobin oxygenase (HO-1) and NAD(P)H dehydrogenase quinone 1 (NQO1) expression in N2a cells increased to varying degrees with CDDO-Me treatment, accompanied by Kelch-like ECH-associated protein 1 (Keap1) dissociation, upon infection with SC16, CVS-11 or CTN. The activation of SQSTM1 phosphorylation was significantly associated with the degradation of Keap-1 in CDDO-Me-treated N2a cells upon RABV infection. Furthermore, N2a cells pretreated with the Nrf2-specific inhibitor ATRA showed a significant decrease in HO-1 and NQO1 expression and a decrease in the anti-RABV efficacy of CDDO-Me. These inhibitory effects were observed upon infection with three RABV strains of differing virulence.ConclusionCDDO-Me inhibited RABV infection via Nrf2 activation, promoting a cytoprotective defense response in N2a cells. Our study provides a therapeutic strategy for RABV inhibition and neuroprotection during viral infection.

B-110 Validation of Digital and Automated anti-dsDNA IFA Interpretations Using the dIFine Automated Fluorescent Microscope System

Abstract Background Detection of anti-double stranded DNA antibodies (anti-dsDNA) by the indirect fluorescent antibody (IFA) assay is a well-established tool for aiding in the diagnosis of systemic lupus erythematosus (SLE). ZEUS Scientific has manufactured an FDA-cleared anti-dsDNA IFA test system for decades, which relies upon manual interpretation of qualitative results using a standard fluorescent microscope. This same anti-dsDNA IFA test system was recently utilized to train software, in conjunction with a specialized fluorescent microscope (collectively termed dIFine), towards automating qualitative result interpretation. The objective of this study was to validate the reproducibility, sensitivity, and specificity of the ZEUS anti-dsDNA IFA test system, in conjunction with the dIFine automated microscope. Methods Multi-site reproducibility study–2 low-positive, 2 mid-positive, 2 high-positive, and 2 negative serum samples were assembled to create a reproducibility panel. The sample identities of the panel members were scrambled prior to testing. The panel was assayed via the ZEUS anti-dsDNA-IFA test system at a 1:10 screening dilution, in triplicate, twice per day, on five days, at three different laboratories (including ZEUS Scientific). At each laboratory, the processed slides were interpreted manually by two operators, then scanned by dIFine to generate digital images of the wells, as well as an automated qualitative result interpretation. The digital images of each slide well were also interpreted by two operators, resulting in 3 independent interpretations per sample, for each operator and instrument (Manual, Digital, Automated). The number of slide wells processed for interpretation at each laboratory was: 8 samples × 30 replicates each = 240 total. Clinical study–300 serum samples derived from donors previously diagnosed with SLE were assembled. A control cohort consisting of 360 samples from donors previously diagnosed with other rheumatic and non-rheumatic diseases was also assembled. The sample identities were scrambled for both cohorts prior to testing. The 660 samples were assayed at a 1:10 screening dilution using the ZEUS anti-dsDNA IFA test system at ZEUS Scientific. Manual, digital, and automated interpretations were carried out at ZEUS Scientific as described above. Results Reproducibility (Actual/Expected): [Site 1 - Operator A - Manual (240/240), Digital (240/240); Site 1 - Operator B - Manual (240/240), Digital (240/240); Site 1 - Automated - (231/240)]. [Site 2 - Operator A - Manual (240/240), Digital (240/240); [Site 2 - Operator B - Manual (240/240), Digital (240/240); Site 2 - Automated - (236/240)]. [Site 3 - Operator A - Manual (240/240), Digital (238/240); Site 3 - Operator B - Manual (240/240), Digital (238/240); Site 3 - Automated - (231/240)]. Sensitivity for SLE cohort (Positive/Total, %): [Operator A - Manual, (79/300, 26.3%), Digital (79/300, 26.3%)]; [Operator B - Manual, (79/300, 26.3%), Digital (79/300, 26.3%), Automated (81/300, 27.0%)]. Specificity for control cohort (Negative/Total): [Operator A - Manual, (358/360, 99.4%), Digital (358/360, 99.4%)]; [Operator B - Manual, (358/360, 99.4%), Digital (358/360, 99.4%), Automated (358/360, 99.4%)]. Conclusions These validation studies demonstrate that the ZEUS anti-dsDNA-IFA test system, in conjunction with the automated dIFine microscope, yields highly reproducible and accurate results derived from digital and automated interpretations.

Effect of a Commercially Available Electrolyte Solution on Acute, Non-Specific Diarrhea in Dogs

Purpose: To determine if a commercially available electrolyte solution is safe and lessens the duration and severity of diarrhea in shelter dogs in stressful situations. Methods: In Experiment 1, six healthy beagles were administered the protocol-approved dose of the electrolyte solution to evaluate clinical, biochemical, or fecal microbiome changes. In Experiment 2, 22 dogs with small or large bowel diarrhea were randomized into one of three groups: the electrolyte solution and a prescription veterinary diet, a placebo and a prescription veterinary diet, or the electrolyte solution and a standard diet. A fecal score was assigned by trained, masked observers through Day 5 using the Purina 7-point fecal scoring system. All dogs were screened for enteric parasites by fecal flotation and the use of a fluorescence antibody assay for Giardia spp. cysts and Cryptosporidium spp. oocysts and all dogs that were parasitized were administered fenbendazole for five days. Results: In Experiment 1, all dogs tolerated the electrolyte solution with no vomiting or diarrhea noted and there was no evidence of negative effects on the gastrointestinal microbiome. In Experiment 2, 16 of the 22 dogs enrolled in the study had a normal stool the day after the first dose of the electrolyte solution, prescription diet, or placebo. All six dogs with the first day of normal stool detected after Day 1 were parasitized. When the days to normalcy were compared, the parasitized dogs had a significantly slower resolution (P = 0.018) than dogs with no parasites regardless of the other treatment group. Conclusions and Relevance: The results of the study suggest that this electrolyte solution is safe for use in dogs and that adding the electrolyte solution to a standard diet is equivalent to using a therapeutic diet alone or the electrolyte solution combined with a therapeutic diet.

Prevalence of Anaplasma phagocytophilum and Borrelia burgdorferi infection in horses from Pennsylvania (2017-2019) using antibody and organism-based detection.

To determine the prevalence of Anaplasma phagocytophilum and Borrelia burgdorferi infections in Pennsylvania horses. 271 horses. A survey was conducted with PCR and serology to evaluate anaplasmosis and Lyme disease infections in horses from Pennsylvania that were suspected for tick-borne infection. A phagocytophilum was detected in 19/271 (7.0%) Pennsylvania horses tested by the duplex PCR. B burgdorferi was not detected in any horse blood tested by PCR. Overall, 120/271 (44.3%) horses tested positive for presence of A phagocytophilum antibodies by at least the IDEXX SNAP 4Dx Plus lateral flow immunosorbent (SNAP) or indirect fluorescent antibody (IFA) assay, with 69 (25.5%) testing positive by both SNAP and IFA; 43 (15.9%) tested positive by IFA only, and 8 (3.0%) tested positive by SNAP only. Similarly, 209/271 (77.1%) horses tested positive for the presence of B burgdorferi antibodies by at least 1 test, with 139 (51.3%) testing positive by both SNAP and IFA; 45 (16.6%) tested positive by SNAP only, and 25 (9.2%) tested positive by IFA. Both A phagocytophilum and B burgdorferi are important tick-borne infections. The study provides prevalence data for both A phagocytophilum and B burgdorferi and compares test performance. For serologic detection, IFA detected antibodies to A phagocytophilum in a higher proportion (41.3%) of horses compared to SNAP (28.4%), while SNAP detected antibodies to B burgdorferi in a higher proportion (67.9%) of horses compared to IFA (60.5%). Both diseases showed a high seroprevalence in all areas surveyed.

Development of an Indirect Fluorescent Antibody (IFA) Assay for the Detection of Leishmania RNA Virus 2 (LRV2) in Leishmania Parasites.

Detection of Leishmania RNA virus (LRV) in Old World Leishmania species and their possible role in the disease prognosis requires sensitive and specific methods, preferably independent of the viral genome. We aimed to develop an indirect immunofluorescence antibody (IFA) assay to detect LRV in the Old World Leishmania parasites. Clinical samples were collected from 86 cutaneous leishmaniasis (CL) patients in different endemic areas of CL in Iran, during 2017-2019. For antibody preparation, the viruses were obtained from sediment of an LRV-infected L. major culture-using freeze and thaw cycles followed by gradient cesium chloride centrifugation. The purified viruses were used to immunize a male 3-4 months rabbit. Various dilutions of the LRV-immunized rabbit's serum and a conjugated antibody were deployed to detect LRV in 48 isolates by IFA assay. LRV virus was detected in four of the 48 CL cases using IFA method. Amplification of a partial fragment of RNA-dependent RNA polymerase (RdRp) gene from the isolates confirmed the IFA results. In phylogeny, the generated RdRp sequences from four isolates were grouped with the other Old World LRVs, but separate from L. aethiopica LRVs, which appeared as a highly supported distinct clade. Further optimization of this approach to detect the LRV directly in lesion scrapings can make it a more reliable tool for field studies and disclosing the virus's possible role in disseminating and unusual clinical features.

Characterization of monoclonal antibodies that specifically differentiate field isolates from vaccine strains of classical swine fever virus.

Classical swine fever virus (CSFV) is a major animal pathogen threatening the global pork industry. To date, numerous anti-CSFV monoclonal antibodies (mAbs) and their recognizing epitopes have been reported. However, few mAbs were systematically characterized for the capacity to differentiate field CSFV isolates from CSF vaccine strains, and the molecular basis associated with antigenic differences between vaccines and field isolates is still largely unknown. In the present study, recombinant CSFV structural glycoproteins E2 of both virulent and vaccine strains and Erns of vaccine strain were expressed using eukaryotic cells and murine mAbs generated against E2 and Erns. After serial screening and cloning of the hybridomas, the viral spectra of mAbs were respectively determined by indirect fluorescent antibody assay (IFA) using 108 CSFVs, followed by Western blot analysis using expressed glycoproteins of all CSFV sub-genotypes including vaccine strains. The antigenic structures recognized by these mAbs were characterized by epitope mapping using truncated, chimeric, and site-directed mutated E2 and Erns proteins. We have identified two vaccine-specific, one field isolate-specific, and two universal CSFV-specific mAbs and five novel conformational epitopes with critical amino acid (aa) motifs that are associated with these five mAbs: 213EPD215, 271RXGP274, and 37LXLNDG42 on E2 and 38CKGVP42, W81, and D100/V107 on Erns. Particularly, E213 of E2 is field isolate-specific, while N40 of E2 and D100/V107 of Erns are vaccine strain-specific. Results from our study further indicate that N40D of E2 mutation in field strains was likely produced under positive selection associated with long-term mass vaccination, leading to CSFV evasion of host immune response. Taking together, this study provides new insights into the antigenic structure of CSFV E2 and Erns and the differentiating mAbs will contribute to the development of a diagnostic strategy to differentiate C-strain vaccination from natural infection (DIVA) of CSFV in terms of elimination of CSF in China.

Robust humoral immune response against rabies virus in rabbits and guinea pigs immunized with plasmid DNA vectors encoding rabies virus glycoproteins – An approach to the production of polyclonal antibody reagents

DNA-based immunization has been previously shown to be an efficient approach to induce robust immunity against infectious diseases in animals and humans. The advantages of DNA vaccines are simplicity of their construction and production, low cost, high stability, and ability to elicit a full spectrum of immune responses to target antigens. The goals of this study were (i) to assess the antibody immune response to rabies virus glycoproteins (rGPs) in rabbits and guinea pigs after intramuscular immunization with pTargeT and pVAC2-mcs mammalian expression vectors encoding either the wild-type (WT) or codon-optimized (cOPT) rGP genes; and (ii) to prepare in-house rabbit anti-rGP polyclonal antibody reagents suitable for in Single Radial Immunodiffusion (SRID) and Indirect Fluorescent Antibody (IFA) assays. The maximum antibody responses against rabies virus in rabbits and guinea pigs were observed after immunization series with 500 μg/dose of pVAC2-mcs vector encoding either the WT or cOPT rGP genes adjuvanted with Emulsigen-D. No significant difference in the anti-rabies virus neutralizing antibody titers was observed in rabbits immunized with the WT and cOPT rGPs. The in-house rabbit anti-rGP polyclonal antibody reagents reacted comparable to the current reference reagents in SRID and IFA assays. The results of the study demonstrated that the DNA immunization of animals with the WT or cOPT rGPs is a promising approach to either induction of high anti-rabies virus neutralizing antibody titers in vivo or for production of polyclonal antibody reagents against rabies.

A study on the infection status and transmission of avian leukosis virus subgroup J in Hy-line brown roosters.

Avian leukosis virus subgroup J (ALV-J) is the most prevalent subgroup in chickens and exhibits increased pathogenicity and stronger horizontal and vertical transmission ability among different breeds. Although vertical transmission of ALV-J from infected hens through artificial insemination has been inferred from the detection of the p27 antigen in swabs and serum, there has been no further research on the transmission pattern of ALVs in roosters. In the present study, the positive rate of ALV increased significantly in an indigenous flock after detecting the p27 antigen via enzyme-linked immunosorbent assay (ELISA) and virus isolation in DF-1 cells. Viral sequence comparisons and an indirect fluorescent antibody assay showed that these isolates belonged to the ALV-J subgroup but formed a new branch in a phylogenetic tree when compared to domestic and foreign referential strains. The gp85 gene of the ALV-J isolated from hens and albumen was 94.1-99.7% identical to that in roosters, revealing that these isolates were quite likely transmitted to the hens and their offspring through the semen of ALV-infected roosters by artificial inseminationfrom the Hy-line brown roosters. In addition, we defined four ALV-J infection states in plasma and semen of roosters (P+S+, P-S+, P+S-, and P-S-), which suggests that, in order to eradicate ALV in roosters, it is necessary to perform virus isolation using both semen and plasma. Additionally, ALV detection in semen by ELISA produced false-positive and false-negative results when compared to virus isolation in DF-1 cells. Collectively, our results suggested that an incomplete process of eradication of ALV from ALV-positive roosters led to the sporadic presence of ALV-J in laying hens.

Detection of porcine parainfluenza virus type-1 antibody in swine serum using whole-virus ELISA, indirect fluorescence antibody and virus neutralizing assays

BackgroundPorcine parainfluenza virus 1 (PPIV-1) is a respiratory virus in the family Paramyxoviridae and genus Respirovirus. It is closely related to bovine parainfluenza virus 3, human parainfluenza virus 1, and Sendai virus. Recent reports suggest PPIV-1 is widespread in swine herds in the United States and abroad. However, seroprevalence studies and the ability to evaluate cross neutralization between heterologous strains is not possible without validated antibody assays. This study describes the development of an indirect fluorescence antibody (IFA) assay, a whole virus enzyme-linked immunosorbent assay (wv-ELISA) and a serum virus neutralization (SVN) assay for the detection of PPIV-1 antibodies using 521 serum samples collected from three longitudinal studies and two different challenge strains in swine.ResultsThe area under the curve (AUC) of the wv-ELISA (95% CI, 0.93–0.98) was significantly higher (p = 0.03) compared to the IFA (95% CI, 0.90–0.96). However, no significant difference was observed between the IFA and wv-ELISA when compared to the SVN (95% CI, 0.92–0.97). All three assays demonstrated relatively uniform results at a 99% true negative rate, with only 11 disagreements observed between the IFA, wv-ELISA and SVN.ConclusionsAll three serology assays detected PPIV-1 antibody in swine serum of known status that was collected from experimental studies. The SVN detected seroconversion earlier compared to the IFA and the wv-ELISA. Both the wv-ELISA and the SVN had similar diagnostic performance, while the IFA was not as sensitive as the wv-ELISA. All three assays are considered valid for routine diagnostic use. These assays will be important for future studies to screen seronegative swine for research, determine PPIV-1 seroprevalence, and to evaluate vaccine efficacy against PPIV-1 under experimental and field conditions.

Experimental infection of cats with Cryptosporidium felis.

The aims of this study were to experimentally inoculate cats with Cryptosporidium felis oocysts and compare fecal detection by fluorescent antibody assay (FA) and quantitative PCR (qPCR), and document clinical signs associated with infection. Cryptosporidium felis oocysts were concentrated from the feces of a naturally infected cat and orally inoculated into six cats that tested negative for C felis by an FA and fecal flotation (FF). Cats were observed daily for the presence of clinical signs consistent with infection. Fecal samples from all cats on days 0 and 9, and one sample per cat (days 18-21), were evaluated by all assays. On day 31, two cats negative for C felis by FF and FA were administered methylprednisolone acetate and all assays were repeated on days 34, 36 and 38. Samples from all cats were tested by FF and FA on days 41, 43, 45 and 48. A total of 41 samples were tested, 25 of which were compared by FA and qPCR. Cryptosporidium felis was detected in 2/25 (8%) and in 19/25 (76%) samples by FA and by qPCR, respectively; the other 16 samples were tested by FF and FA. None of the cats was positive for C felis by FF or FA in samples collected on days 0, 9 or 18-21. One, five and six samples tested positive by qPCR on days 0, 9 and 18-21, respectively. The cats administered methylprednisolone acetate tested positive for C felis by FA on day 36 and by qPCR on days 31, 34, 36 and 38. None of the cats showed clinical signs of disease. Clinical signs were not recognized in any of the cats for the duration of the study. FA was insensitive compared with qPCR for detecting cats with subclinical C felis infection.

Epidemiology of Viruses Causing Pediatric Community Acquired Pneumonia in Shanghai During 2010-2020: What Happened Before and After the COVID-19 Outbreak?

IntroductionSince the global outbreak of COVID-19, there has been a significant reduction in pediatric outpatient and emergency visits for infectious diseases. The purpose of this study was to analyze the changes in respiratory viruses in children with community-acquired pneumonia (CAP) in Shanghai in the past 10 years, especially in the first year after COVID-19.MethodsWe conducted a retrospective, observational study; the results for eight common respiratory viruses (respiratory syncytial virus (RSV), influenza virus A and B, parainfluenza virus 1–3 (PIV), adenovirus (ADV) and human metapneumovirus) tested by direct fluorescent antibody assays in hospitalized CAP cases in Children’s Hospital of Fudan University during 2010–2020 were analyzed.ResultsOf the 5544 hospitalized CAP patients included in this study, 20.2% (1125/5544) were positive for the eight respiratory viruses. The top three pathogens were RSV, PIV3 and ADV, detected from 9.8% (543/5544), 5.3% (294/5544) and 2.0% (111/5544) of the samples, respectively. RSV had the highest positive rates among children < 2 years old. In 2020, the detection rate of all viruses showed a sharp decline from February to August compared with the previous 9 years. When the Shanghai community reopened in August 2020, the detection rate of eight viruses rebounded significantly in September.ConclusionsThese eight respiratory viruses, especially RSV and PIV, were important pathogens of CAP in Shanghai children in the past 10 years. The COVID-19 pandemic had a significant impact on the detection rates for eight respiratory viruses in children with CAP in Shanghai.

Development of a Multiplex Bead Assay To Detect Immunoglobulin G Antibodies to Babesia duncani in Human Serum.

Babesia duncani is the causative agent of babesiosis in the western United States. The indirect fluorescent antibody (IFA) assay is the diagnostic test of choice for detection of B. duncani-specific antibodies. However, this test requires parasitized red blood cells harvested from infected hamsters, and test results are often difficult to interpret. To simplify serological testing for B. duncani, a proteomics approach was employed to identify candidate immunodiagnostic antigens. Several proteins were identified by electrospray ionization mass spectrometric analysis, and four recombinant protein constructs were expressed and used in a multiplex bead assay (MBA) to detect B. duncani-specific antibodies. Two antigens, AAY83295.1 and AAY83296.1, performed well with high sensitivities and specificities. AAY83295.1 had a higher sensitivity (100%) but lower specificity (89%) than AAY83296.1, which had a sensitivity of 90% and a specificity of 96%. Combining these two antigens did not improve the performance of the assay. This MBA could be useful for diagnosis, serosurveillance, and blood donor screening for B. duncani infection.

Experimental infection of cats with Cystoisospora felis

Background Cystoisospora felis is a common parasite of cats and is diagnosed by fecal flotation, but false‐negative results can be common.Hypothesis/ObjectivesTo experimentally inoculate cats with C. felis oocysts, to compare fecal flotation and polymerase chain reaction (PCR) results, and to describe any clinical signs consistent with infection.AnimalsSix cats.Methods Cystoisospora felis oocysts were identified morphologically from feces of a naturally infected kitten with diarrhea, sporulated oocysts (5000) were inoculated to 6 cats that were negative for fecal parasites by fecal flotation and by a fluorescent antibody assay (FA) for Giardia spp. and Cryptosporidium spp. Cats were observed daily for the presence of clinical signs consistent with infection. Fecal samples were evaluated by fecal flotation and FA up to 3 times per week post inoculation (PI) to Day 27. Thirty‐six samples collected before inoculation and from Days 8, 10, 13, 15, and 20 PI were assayed using an internal transcribed spacer 1 (ITS1) PCR that amplifies DNA of C. felis.ResultsAll cats were negative for C. felis by both assays before inoculation. All cats shed C. felis oocysts by Day 10 PI, oocysts were not detected by fecal flotation after Day 15 PI. Cystoisospora felis DNA was amplified from 24/36 (66.6%) fecal samples from 6/6 (100%) of the cats. Oocysts were not detected by fecal flotation in 4 of the samples that were positive for C. felis DNA by PCR. Clinical signs were not recognized in any of the study cats.Conclusions and Clinical ImportanceFecal flotation is a convenient assay for detection of C. felis but could occasionally give false‐negative results when compared to this ITS1 PCR.