Indirect Fluorescent Antibody Assay Research Articles

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).

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.

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.

A STUDY ON THE DIAGNOSTIC METHODS OF SCRUB TYPHUS COMPARITIVELY

Scrub typhus is one of the causes of acute undifferentiated fever.During the study period from October 2012 to August 2014, a total of 633 cases of suspected Scrub typhus presented to our hospital. Out of these, 95 serum samples were positive for scrub typhus either by Weil Felix or by ELISA (IgM antibody to Orientia tsutsugamushi). Predominant clinical feature was fever with an average of 10 days followed by headache, vomiting, cough, myalgia, breathlessness, loose stools, reduced urine output, abdominalpain and nausea. Eschar was observed in 6 out of the 95 patients (6.3%) in our study. The percentages of patients with Scrub typhus having eschars were variable with different studies citing different results. In our study we observed an almost equal incidence of Scrub typhus in both genders. Other studies showed variable reports with some showing male predominance and others showing female predominance. Maximal incidence of Scrub typhus according to our study wasin the age group 41 – 50 yrs with decreasing incidence as one goes further from this peak to either side. Increased numbers of Scrub typhus cases were observed from September to February, which coincided with the cooler months of the year. Scrub Typhus diagnosis is made more complex by the presence of dual infections. Dual infections should be suspected when the patients present with atypical clinical features of either diseaseor when patient responds poorly to treatment. The platelet levels were low in Scrub typhus, but it was lower when the patient was infected with both Scrub typhus and Dengue infection. Main stay of Scrub typhus diagnosis remains serology. The gold standard for the diagnosis is Indirect Fluorescence Antibodyassay. Both Weil Felix and ELISA tests were done and compared withIFA in 100 samples. The sensitivity of Weil Felix OX K at a break point of 1:160 was found to be 40% and specificity 94%, Positive predictive value was 86.9% and negative predictive value 61.0%. The ELISA tests showed sensitivity to be 96% and specificity 88%, Positive predictive value was 88.8% and negative predictive value 95.6 %. All currently available serological methods have theirlimitations. There is an urgent need for newer diagnostic methods. The treatment given is Doxycycline or azithromycin. Most of our cases responded to these drugs except one case where combination therapy was attempted. Scrub typhus responds well to treatment and if not treated intime the patient can go in for complications emphasizing the need for early diagnosis and treatment. Keywords:- Doxycycline, azithromycin, Scrub Typhus, ELISA.

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.

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.

Respiratory syncytial virus seropositivity at birth is associated with adverse neonatal respiratory outcomes.

More than 60 years since the discovery of the respiratory syncytial virus (RSV), the effects of prenatal exposure to this virus remain largely unknown. In this investigation, we sought to find evidence of RSV seroconversion in cord blood and explore its clinical implications for the newborn. Offspring from 22 pregnant women with a history of viral respiratory infection during the third trimester of pregnancy (respiratory viral illness [RVI] group) and 40 controls were enrolled in this study between 1 September 2016 and 31 March 2019. Cord blood sera were tested for anti-RSV antibodies by indirect fluorescent antibody assay. RSV seropositivity was defined as the presence of anti-RSV immunoglobulin M (IgM) or immunoglobulin A (IgA), in addition to IgG in cord blood serum at ≥1:20 dilution. Anti-RSV IgG was present in all cord blood serum samples from infants born to RVI mothers (95% confidence interval [CI] = 82%-100%), with 16 samples also having elevated titers for either anti-RSV IgA or IgM (73%; 95% CI = 52%-87%). No controls had evidence of anti-RSV antibodies. Eight (50%) seropositive newborns developed at least one respiratory tract finding, including respiratory distress syndrome (N = 8), respiratory failure (N = 3), and pneumonia (N = 1). RSV seropositive newborns also required more days on oxygen, had leukocytosis and elevated C-reactive protein (P = .025, P = .047, and P < .001, respectively). This study provides evidence of acute seropositivity against RSV in cord blood of newborns delivered from mothers with a history of upper respiratory tract illness in the third trimester. Cord blood seropositivity for anti-RSV IgA or IgM was associated with adverse clinical and laboratory outcomes in newborns.

663. Efficacy and Safety of Lefamulin (LEF) vs. Moxifloxacin (MOX) for Legionella pneumophila (LP) in Patients with Community-Acquired Bacterial Pneumonia (CABP): Pooled Results From the Lefamulin Evaluation Against Pneumonia (LEAP) 1 and LEAP 2 Phase 3 Clinical Trials

Background LP is associated with severe CABP, rapid onset, and high morbidity/mortality. Poor outcomes in CABP have been linked to receiving inappropriate empiric therapy or delayed treatment (tx). LEF, a novel IV/oral pleuromutilin, demonstrated efficacy/safety in noninferiority studies (LEAP 1/2) vs. MOX in adults with CABP. We report efficacy/safety of LEF in patients with LP based on a pooled analyses of LEAP 1/2 data.MethodsIn LEAP 1, PORT III–V patients received LEF 150 mg IV q12h for 5–7 days or MOX 400 mg IV q24h for 7 days, with optional IV-to-oral switch (600 mg LEF q12h or 400 mg MOX q24h). In LEAP 2, PORT II–IV patients received oral LEF for 5 days or oral MOX for 7 days. Both studies assessed early clinical response (ECR) at 96 ± 24 hours after first dose in the intent-to-treat (ITT; all randomized patients) population and investigator assessment of clinical response (IACR) at test-of-cure (TOC; 5–10 days after last dose) in the modified ITT (received ≥1 dose) and clinically evaluable (met predefined evaluability criteria) populations. LP was identified from baseline (BL) samples by culture, serology (IgG, Zeus L. pneumophila group 1–6 indirect fluorescent antibody assay), urine antigen testing (BinaxNOW), and real-time PCR (positive for ssrA). Efficacy analyses herein were done in the microbiological ITT (microITT, treated patients with BL CABP-causing pathogen), microITT-2 (no PCR), and microbiologically evaluable populations; safety analyses included all randomized/treated patients.ResultsOf 65 pooled microITT patients, median age was 60 y, 66% were male, 51% had a normal renal function, and 54%/25% were PORT III/IV. LP was identified in 9.3% (34/364) of LEF patients (7 [20.6%]/19 [55.9%]/8 [23.5%] PORT II/III/IV) and in 9.0% (31/345) of MOX patients (7 [22.6%]/16 [51.6%]/8 [25.8%] PORT II/III/IV), primarily by urine antigen or serology (table). Patients with LP in both tx groups achieved high and similar responses across all endpoints (Figures 1 and 2). In both tx groups, TEAE rates were low and comparable (~32%) and most were mild to moderate; 5 patients (3 LEF; 2 MOX) had treatment-emergent SAEs, all unrelated to tx. No patients died due to TEAEs; no LEF patients and 2 MOX patients discontinued tx due to TEAEs.ConclusionLEF appears to be as safe and effective as MOX in treating patients with LP, including when given as short-course (5 days) oral therapy. Disclosures All authors: No reported disclosures.

Borrelia burgdorferi Antibody Test Results in Dogs Administered 4 Different Vaccines

Vaccines against Borrelia burgdorferi are administered frequently to dogs in areas endemic for the infection. These vaccines produce an antibody response to spirochetal proteins that cross-react in many antibody tests, including immunofluorescence assay, Western blot, and whole cell ELISA used to document exposure to B. burgdorferi. The purpose of this study was to determine whether the in-clinic C6 ELISA assay (SNAP® 4Dx® Plus; IDEXX Laboratories, Inc., Westbrook, Maine) and the quantitative format C6 ELISA (Lyme Quant C6® Antibody Test, IDEXX Laboratories, Inc., Westbrook, ME) react to sera from dogs that have been vaccinated with 1 of 4 different commercially available B. burgdorferi vaccines. Four groups of 3 dogs each were each administered one of the 4 vaccines and sera evaluated over time by indirect fluorescent antibody assay, western blot immunoassay, the in-clinic C6 ELISA assay, and the quantitative format C6 ELISA. While all dogs developed B. burgdorferi antibodies detectable by indirect fluorescent antibody assay and western blot immunoassay after vaccination, none of the samples were positive in either of the C6 peptide-based assays. Based on these results, positive anti-C6 antibody results in client-owned dogs are likely to reflect exposure to B. burgdorferi rather than vaccination.

Tick-borne pathogens in carthorses from Foz do Iguaçu City, Paraná State, southern Brazil: A tri-border area of Brazil, Paraguay and Argentina

Tick-borne diseases (TBD) constitute an important group of illness affecting animals and humans worldwide. In Brazil, carthorses are frequently exposed to ticks and tick-borne pathogens, leading to impairment of horse performance and imposing restrictions by the international veterinary authorities for the importation of horses. Accordingly, this study has aimed to i) determine the prevalence of the TBD agents Theileria equi, Babesia caballi, Ehrlichia spp., and hemotropic mycoplasmas in carthorses, ii) identify the tick species parasitizing the animals, and iii) determine factors associated with exposure/infection in Foz do Iguaçu City, Parana state, southern Brazil. A total of 103 carthorses were screened for anti-T. equi and anti-Ehrlichia spp. antibodies by indirect fluorescent antibody assays (IFA). Samples were also tested by PCR assays targeting the 18S rRNA gene of T. equi and B. caballi, and 16S rRNA gene of hemoplasmas. Additionally, PCR assays targeting the 16S rRNA, disulfide bond formation protein (dsb) and tandem repeat proteins 36 (trp36) genes of Ehrlichia spp. were also performed. Antibodies to T. equi and Ehrlichia spp. were detected in 43/103 (41.75%; 95% CI: 32.10–51.88%) and 5/103 (4.85%; 95% CI: 1.59–10.97%) horses by IFA, respectively. DNA of T. equi and B. caballi were found in 25/103 (24.27%; 95% CI: 16.36–33.71%) and 10/103 (9.71%; 95% CI: 4.75–17.13%) carthorses, respectively, and all tested negative for Ehrlichia spp. and hemoplasmas. All sequences showed ≥99% identity with multiple T. equi and B. caballi 18S rRNA gene sequences deposited in GenBank. Overall, 191 Dermacentor nitens ticks were collected from 25/103 (24.27%) animals. Carthorses older than 5 years were more likely to be positive for T. equi (p < 0.05). In conclusion, equine piroplasmosis agents are highly prevalent in carthorses from Foz do Iguaçu City. The low prevalence of Ehrlichia spp. found may be due to the absence of Amblyomma ticks infesting animals, which should be further investigated.

Growth characteristics of the novel goose parvovirus SD15 strain in vitro

BackgroundShort beak and dwarfism syndrome (SBDS) was caused by novel goose parvovirus (NGPV)--a variant of goose parvovirus (GPV). Ducks infected with NGPV shows clinical signs including growth retardation and protrusion of the tongue from an atrophied beak. SBDS outbreak was first reported at the northern coastal provinces of China during 2015 and it was again reported in Sichuan, an inland province of China in 2016. The disease caused a huge economic loss in Chinese duck feeding industry.ResultsThe SD15 strain of NGPV was isolated from liver and intestinal tract tissue samples of infected ducks. Real-time quantitative PCR (qPCR) was used to estimate viral load in embryonated eggs and cells infected with adapted virus. The data showed that duck embryo fibroblasts (DEFs) were permissive to NGPV, while goose embryo fibroblasts (GEFs) cells were not, and the copy numbers of SD15 in the allantoic fluid of infected eggs remained at 105.0–106.5 copies/ml. The adaption procession of the virus was determined via qPCR, and viral proliferation was detected through indirect fluorescent antibody assay (IFA) in DEFs. It was further determined that viral copy numbers peaked at 96 h post-inoculation (hpi), which is the best time to harvest the virus in DEFs. Cytotoxic effects and cell death were observed at 72 hpi in SD15 infected DEFs, yet SD15 did not induce apoptosis.ConclusionsThe growth characteristics of SD15 strain of NGPV determined would be beneficial for further molecular characterization of these viruses and develop potential vaccines if required.

High seroprevalence of Babesia antibodies among Borrelia burgdorferi-infected humans in Sweden

In northern Europe, tick-borne diseases such as Lyme borreliosis (LB) and tick-borne encephalitis (TBE) are well known. The actual incidence of Babesia infections, however, has remained elusive. In this study, the prevalence of antibodies against two Babesia spp. was investigated in a cohort of patients that were seropositive for Borrelia (B.) burgdorferi sensu lato (s.l.). Data were compared to a control group of healthy individuals. Sera were collected from 283 individuals residing in the southernmost region of Sweden, Skåne County. Almost one third of the sera were from patients with a confirmed seropositive reaction against B. burgdorferi s.l. All sera samples were assessed for IgG antibodies against Babesia (Ba.) microti and Ba. divergens by indirect fluorescent antibody (IFA) assays. Seropositive IgG titers for at least one of the Babesia spp. was significantly more common (p < 0.05) in individuals seropositive for Borrelia (16.3%) compared to the healthy control group (2.5%). Our findings suggest that Babesia infections may indeed be quite common among individuals who have been exposed to tick bites. Furthermore, the results indicate that human babesiosis should be considered in patients that show relevant symptoms; particularly for splenectomized and other immunocompromised individuals. Finally, the data challenges current blood transfusion procedures and highlights the current lack of awareness of the parasite in northern Europe.

THE DYNAMICS OF ORTHOHANTAVIRUS HANTAAN STRAINS REPLICATION ON THE MODEL OF MOUSE PERITONEAL MACROPHAGES

Orthohantaviruses (Orthohantavirus genus, Hantaviridae family) are the causative agents of a widespread natural focal infection in the  Russian Federation, hemorrhagic fever with renal syndrome (HFRS).  An important role in the persistence of orthohantavirus in reservoir  hosts among other immunological responses, as well as in the  spread of the virus in the infected organism, is played by infected  macrophages, which, along with the vascular endothelium, are the main targets for orthohantaviruses.The aim of our study was to investigate the characteristics of replication of orthohantavirus Hantaan strains isolated from  Apodemus mice and detect the influence of different values of  multiplicity of infection (MOI) on replication dynamics of orthohantaviruses on cell culture.Materials and methods. We used 4 strains of Hantaan virus, isolated from A. agrarius (n = 2) and A. peninsulae (n = 2), captured in the different areas of Primorsky Krai of Russia. The  modeling of infection was performed on the primary cell culture of  mouse peritoneal macrophages with different MOI (from 10 to 0.1).  The assessing of infection was conducted via indirect fluorescent  antibody assay, and results were expressed as rate of antigen- positive cells per all cells in the field of vision. Results. Common dynamics of orthohantavirus infection on this in vitro model was characterized by periodically increased rates of  infected cells after 2, 4, 6 и 8 hours post infection (p.i.). Replication  of A. agrarius-borne strains was more intensive compare with  viruses, isolated from A. peninsulae, in the time point 4 hour p.i. on  the background the same MOI the statistically significant difference  of rate of antigen-positive cell 24.9 ± 2.38 % vs 15.2 ± 1.87 % (t =  3.20; p = 0.001414) was observed. Additionally, the decrease of MOI was followed by determined decrease of replication effectivity.Conclusion. The results of our study showed the significant phenotyping heterogeneity of orthohantavirus Hantaan strains,  isolated from Apodemus mice, resulting in different rates of  replication in the culture of mouse peritoneal macrophages.

Visceral leishmaniasis: clinical and therapeutic aspects

Introduction : visceral leishmaniasis (VL) is endemic in areas bordering the Mediterranean Sea, including Tunisia. It represents a major public health problem not only in developing countries but also in developed ones. It is caused by Leishmania infantum and it is transmitted by the bite of hematophagous sand fly belonging to phlebotomus spp; dog constitutes the main reservoir of the infection. Methods : systemic review of the national and international medical literature concerning this disease. Results : clinical presentation of VL ranges from asymptomatic or subclinical infection to severe and complicated symptomatic disease. Classical manifestations of visceral leishmaniasis include chronicfever, pallor and splenomegaly. Pancytopenia is present very often. Rarely, it may be complicated by hemophagocytic lymphohistiocytosis (HLH) that has varied presentation and course. Diagnosis is established by serological tests (indirect fluorescent-antibody assay, immunoassay test, indirect hemagglutination assay) and by demonstration of Leishmania parasites by microscopy or polymerase chain reaction (PCR) in the bone marrow aspirates. Visceral Leishmaniasis is fatal if left untreated. Although pentavalent antimonial drugs have been used for many decades as standard treatment for VL, newer therapeutic options are nowadays considered the first-line treatment in many countries. They include conventional or liposomal amphotericin B, paromomycin and miltefosine. Liposomal amphotericin B seems to be the most tolerated drug. Unfortunately, it is not available in Tunisia. Recently, an international scientific seminar has been held in Pasteur institute of Tunis from the 2nd-4th April 2015, about visceral leishmaniasis in Maghreb. Three recommendations have been considered by the experts attending this international scientific seminar. Firstly, the necessity to decrease the delay between onset of symptoms and getting a proper diagnosis This aim can be reached by: The training of medical and nursing staff and education of high risk population, the use of rapid diagnostic tests in area with a high incidence of VL, the use of real time PCR on blood samples for diagnosis of VL to avoid marrow aspiration. Secondly, the usefulness of liposomal amphotericin as first-line therapeutic option in management of visceral leishmaniasis. The experts participating in the international scientific seminar agreed on the need to replace the meglumine antimoniate by liposomal amphotericin as first-line therapeutic option in Maghreb countries. This new drug will shorten considerably the duration of therapeutic cures and improve the effectiveness of the treatment and its safety. For immunocompromised patients (HIV(+), Infection and Solid Organ Transplant Recipients), it was strongly recommended to implement a post treatment long term follow-up because of the risk of relapse. And thirdly, the urgent need of fighting against the zoonosis by the control of reservoir host (dog) and the vector (sandflies). The actions include: The eradication of stray and semi-stray dogs, the early detection of canine leishmaniasis, the prevention of canine leishmaniasis (insecticide-impregnated dog collars), the setting up of clean environment (garbage control, whitewashing the walls, nets). Conclusion : despite many previous studies realized on Leishmaniases in Maghreb, they are still considered as neglected diseases because of absence of efficient control strategies. Poor vector control strategies, limited diagnostic services, drugs, treatments and lack of community awareness are the most important challenges in VL control and elimination especially in endemic areas such as Tunisia. Absence of highly sensitive and specific tests, lack of trained man power, and community awareness are the major challenges in VL control. Therefore, proper case diagnosis, community mobilization and launching of