Recombinant ELISA Research Articles

Avidin-Biotin recombinant nucleoprotein competitive ELISA for the detection of peste des petits ruminants virus antibodies in sheep and goats

The present study describes the development of a truncated recombinant peste des petits ruminants virus (PPRV) nucleoprotein (rPPRV-NPN) and its polyclonal antibodies-based immuno-diagnostic assay, Avidin-Biotin (AB) recombinant nucleoprotein competitive ELISA (ABrC-ELISA) for the detection of PPRV antibodies in the sheep and goats. The PPRV N-terminal immunogenic region (1–266 aa) of nucleoprotein (NPN) coding sequence was amplified and cloned into the pETite vector. The rPPRV-NPN with a molecular weight of ∼ 30 kDa was expressed in E. coli, purified, and characterized by SDS-PAGE and immunoblot using standard PPRV specific sera. The Ni-NTA affinity-purified rPPRV-NPN as coating antigen and its hyperimmune serum as competitive antibodies raised in guinea pigs were evaluated as diagnostic reagents in ABrC-ELISA using the known standard panel of sera. The threshold (cut-off) Percentage Inhibition (PI) value was determined as 45 (mean ± 3 SD) based on the reactivity of the known sheep and goats sera to PPRV antibodies [negative (n = 140) and positive (n = 98)] and the assay had a sensitivity of 97 % (95 % Confidence Interval (CI): 91.3–99.4 %) and specificity of 100 % (95 % CI: 97.4–100 %) with an excellent Area under curve (AUC) of 0.997 (95 % CI: 0.99–1.0). On evaluation of diagnostic performance of the assay using the sheep and goats sera (n = 391) from vaccinated, infected, and non-vaccinated animals, the ABrC-ELISA showed the relative diagnostic sensitivity of 95.88 % (95 % CI: 92.56–98.01 %) & 98.77 % (95 % CI: 96.43–99.74 %) and diagnostic specificity of 97.97 % (95 % CI: 94.19–99.58 %) & 90.54 % (95 % CI: 84.64–94.73 %) against indigenous PPR competitive ELISA kit & IDvet Screen® PPR Competition kit, respectively. The study showed that ABrC-ELISA is rapid, sensitive, and specific and can be a better alternative assay for the detection of the PPRV antibodies in the sera of small ruminants for serosurveillance / seromonitoring of PPR not only at the eradication and post-eradication phases in the disease-controlled endemic countries but also in the PPR non-endemic countries.

Evaluation of UMELISA CHAGAS™ with the incorporation of new monomeric and chimeric peptides representative of different regions of Trypanosoma cruzi

ResumenIntroducción. La mayoría de las personas con enfermedad de Chagas desarrolla anticuerpos específicos contra Trypanosoma cruzi. En la infección temprana se producen anticuerpos IgM contra T. cruzi que son reemplazados por IgG durante el curso de la enfermedad. Los primeros síntomas de la enfermedad suelen ser muy leves y atípicos, por lo que a menudo no se detecta en la fase aguda.Objetivos. Evaluar la sensibilidad y la especificidad clínica y analítica, la precisión y la eficacia del UMELISA CHAGAS® con la incorporación de nuevos péptidos sintéticos en la fase sólida representativos de la proteína SAPA (Shed Acute Phase Antigen) y del antígeno TSA (Trypomastigote Surface Antigen).Materiales y métodos. Se evaluó un panel de desempeño de título mixto anti-I cruzi y uno de seroconversión de Chagas, así como muestras de suero positivas y negativas provenientes de zonas endémicas de la enfermedad y muestras positivas de otras enfermedades que podían interferir con la prueba. Las pruebas Bioelisa CHAGAS, Chagatest ELISA recombinante v. 4.0, Chagatest HAI y SD BIOLINE CHAGAS Ab Rapid, se emplearon como referencia.Resultados. Los porcentajes de sensibilidad y especificidad clínica fueron de 97,73 % (IC95% 96,23-99,24) y 99,33 % (IC95% 98,88-99,78), respectivamente. Se obtuvo un 98,96 % de eficacia y una buena precisión.Conclusiones. Los resultados demuestran que la nueva fase sólida del UMELISA CHAGAS® puede utilizarse para el inmunodiagnóstico, la certificación de sangre y la vigilancia epidemiológica en países endémicos y no endémicos con población de alto riesgo.

Enhanced Protection by Recombinant Newcastle Disease Virus Expressing Infectious Bronchitis Virus Spike Ectodomain and Chicken Granulocyte-Macrophage Colony-Stimulating Factor.

We previously reported that recombinant Newcastle disease virus LaSota (rLS) expressing infectious bronchitis virus (IBV) Arkansas (Ark)-type trimeric spike (S) ectodomain (Se; rLS/ArkSe) provides suboptimal protection against IBV challenge. We have now developed rLS expressing chicken granulocyte-macrophage colony-stimulating factor (GMCSF) and IBV Ark Se in an attempt to enhance vaccine effectiveness. In the current study, we first compared protection conferred by vaccination with rLS/ArkSe and rLS/ArkSe.GMCSF. Vaccinated chickens were challenged with virulent Ark, and protection was determined by clinical signs, viral load, and tracheal histomorphometry. Results showed that coexpression of GMCSF and the Se from rLS significantly reduced tracheal viral load and tracheal lesions compared with chickens vaccinated with rLS/ArkSe. In a second experiment, we evaluated enhancement of cross-protection of a Massachusetts (Mass) attenuated vaccine by priming or boosting with rLS/ArkSe.GMCSF. Vaccinated chickens were challenged with Ark, and protection was evaluated. Results show that priming or boosting with the recombinant virus significantly increased cross-protection conferred by Mass against Ark virulent challenge. Greater reductions of viral loads in both trachea and lachrymal fluids were observed in chickens primed with rLS/ArkSe.GMCSF and boosted with Mass. Consistently, Ark Se antibody levels measured with recombinant Ark Se protein-coated ELISA plates 14 days after boost were significantly higher in these chickens. Unexpectedly, the inverse vaccination scheme, that is, priming with Mass and boosting with the recombinant vaccine, proved somewhat less effective. We concluded that a prime and boost strategy by using rLS/ArkSe.GMCSF and the worldwide ubiquitous Mass attenuated vaccine provides enhanced cross-protection. Thus, rLS/GMCSF coexpressing the Se of regionally relevant IBV serotypes could be used in combination with live Mass to protect against regionally circulating IBV variant strains.

Expression of Concern: Recombinant M2e Protein-Based ELISA: A Novel and Inexpensive Approach for Differentiating Avian Influenza Infected Chickens from Vaccinated Ones.

Expression of Concern: Recombinant M2e Protein-Based ELISA: A Novel and Inexpensive Approach for Differentiating Avian Influenza Infected Chickens from Vaccinated Ones.

Comparison of novel recombinant nucleoprotein ELISA with commercial ELISA for Newcastle disease

In the present study, a novel ELISA method used recombinant nucleocapsid protein (rNP) as the coating agent. Recombinant Newcastle disease virus (NDV) protein was cloned and expressed in Escherichia coli. Though the rNP-ELISA results were consistent with commercial ELISA results for the NDV-negative sera samples, qualitatively and quantitatively variable (often reduced) results were obtained with NDV-positive sera. Although the rNP-ELISA results for NDV detection were inconclusive, further improvement and standardization of the rNP-ELISA approach, such as using multiple recombinant proteins as the ELISA coating agent and performing comprehensive statistical analyses of combined recombinant protein ELISA, should help counter Newcastle disease outbreaks by improving NDV detection.

Autoantibodies Against the Purkinje Cell Protein RGS8 in Paraneoplastic Cerebellar Syndrome

ObjectiveTo describe the identification of regulator of G-protein signaling 8 (RGS8) as an autoantibody target in patients with cerebellar syndrome associated with lymphoma.MethodsSera of 4 patients with a very similar unclassified reactivity against cerebellar Purkinje cells were used in antigen identification experiments. Immunoprecipitations with cerebellar lysates followed by mass spectrometry identified the autoantigen, which was verified by recombinant immunofluorescence assay, immunoblot, and ELISA with the recombinant protein.ResultsThe sera and CSF of 4 patients stained the Purkinje cells and molecular layer of the cerebellum. RGS8 was identified as the target antigen in all 4 sera. In a neutralization experiment, recombinant human RGS8 was able to neutralize the autoantibodies' tissue reaction. Patient sera and CSF showed a specific reactivity against recombinant RGS8 in ELISA and immunoblot, whereas no such reactivity was detectable in the controls. Clinical data were available for 2 of the 4 patients, remarkably both presented with cerebellar syndrome accompanied by B-cell lymphoma of the stomach (patient 1, 53 years) or Hodgkin lymphoma (patient 2, 74 years).ConclusionOur results indicate that autoantibodies against the intracellular Purkinje cell protein RGS8 represent new markers for paraneoplastic cerebellar syndrome associated with lymphoma.Classification of EvidenceThis study provided Class IV evidence that autoantibodies against the intracellular Purkinje cell protein RGS8 are associated with paraneoplastic cerebellar syndrome in lymphoma.

Development of a novel recombinant ELISA for the detection of Crimean-Congo hemorrhagic fever virus IgG antibodies

Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne viral infection caused by Crimean-Congo hemorrhagic fever virus (CCHFV). Serological screening of CCHF is important and current ELISA use antigens prepared from virus which is expensive due to requirement of high bio-containment facilities. In this study, we aimed to develop a new recombinant ELISA. For this purpose, CCHFV genome were expressed as 13 proteins in E. coli and among them abundantly purified recombinant Nucleocapsid protein (rNP) and Mucin-like variable domain (rMLD) were used as antigen in ELISA (Rec-ELISA). Rec-ELISA using rNP, rMLD and a combination of both (rNP/rMLD) were probed with acute (n = 64; collected between days 1 and 7 after onset of symptoms), convalescent (n = 35; collected 8 days after onset of symptoms), consecutive sera (n = 25) of confirmed CCHF cases and control sera (n = 43). The sensitivity and specificity of Rec-ELISA using rNP/rMLD were 73% and 98% in acute cases and 97% and 98% in convalescent cases. The median interquartile absorbance value to discriminate the acute and convalescent phases of CCHF was significantly higher with ELISA using rNP/rMLD (P < 0.0001) compared to rNP (P > 0.05) and rMLD (P = 0.001). These results indicate that the Rec-ELISA using rNP/rMLD may be very useful to diagnose convalescent CCHF cases especially in field studies.

Avidin-Biotin recombinant antigen capture ELISA for the detection of peste des petits ruminants virus in the clinical specimens of sheep and goats

This study describes the development of Avidin-Biotin recombinant Antigen Capture ELISA (ABrAC ELISA) for the detection of the peste des petits ruminants virus (PPRV) antigens in the clinical specimens of sheep and goats. The assay uses the truncated recombinant PPRV N-terminal immunogenic region of nucleoprotein (rPPRV-NPN) as a reference positive antigen and its polyclonal antibodies as capture/detective antibodies and the rabbit PPRV polyclonal antibodies as coating antibodies. The cut-off value was determined as double times the mean reactivity of blank control based on the reactivity of the PPR confirmed negative and positive control panel samples. On assessing the specificity with the related differential diagnosis of the disease-causing viruses and bacteria, the assay showed specific detective reactivity to PPRV. Further, on evaluation using clinical specimens (n-274) of sheep and goats, the assay showed that the relative diagnostic sensitivity of 86.49 % (95 % confidence interval (CI): 71.23–95.46 %) and diagnostic specificity of 96.20 % (95 % CI: 92.91–98.25 %) against PPRV nucleoprotein-specific monoclonal antibody-based sandwich-ELISA (PPR s-ELISA) kit, with an accuracy of 94.89 % (95 % CI: 91.58–97.18 %) and Cohen's Kappa value of 0.791 + 0.055 SE (95 % CI: 0.68−0.90) with substantial agreements. The ABrAC-ELISA is an alternative method of an immunoassay for the rapid, sensitive, and specific detection of the PPRV antigens m the clinical specimens of sheep and goats for surveillance or diagnosis of PPR. This study also shows that the rPPRV-NPN and its specific polyclonal antibodies could be the sustainable source of safe diagnostic reagents without the need to handle the infectious virus during the eradication and post-eradication phases in endemic countries like India or PPR non-endemic countries.

Elicitation of Highly Pathogenic Avian Influenza H5N1 M2e and HA2-Specific Humoral and Cell-Mediated Immune Response in Chicken Following Immunization With Recombinant M2e-HA2 Fusion Protein.

The study was aimed to evaluate the elicitation of highly pathogenic avian influenza (HPAI) virus (AIV) M2e and HA2-specific immunity in chicken to develop broad protective influenza vaccine against HPAI H5N1. Based on the analysis of Indian AIV H5N1 sequences, the conserved regions of extracellular domain of M2 protein (M2e) and HA2 were identified. Synthetic gene construct coding for M2e and two immunodominant HA2 conserved regions was designed and synthesized after codon optimization. The fusion recombinant protein (~38 kDa) was expressed in a prokaryotic system and characterized by Western blotting with anti-His antibody and anti-AIV polyclonal chicken serum. The M2e–HA2 fusion protein was found to be highly reactive with known AIV-positive and -negative chicken sera by ELISA. Two groups of specific pathogen-free (SPF) chickens were immunized (i/m) with M2e synthetic peptide and M2e–HA2 recombinant protein along with one control group with booster on the 14th day and 28th day with the same dose and route. Pre-immunization sera and whole blood were collected on day 0 followed by 3, 7, 14, 21, and 28 days and 2 weeks after the second booster (42 day). Lymphocyte proliferation assay by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) method revealed that the stimulation index (SI) was increased gradually from days 0 to 14 in the immunized group (p < 0.05) than that in control chicken. Toll-like receptor (TLR) mRNA analysis by RT-qPCR showed maximum upregulation in the M2e–HA2-vaccinated group compared to M2e- and sham-vaccinated groups. M2e–HA2 recombinant protein-based indirect ELISA revealed that M2e–HA2 recombinant fusion protein has induced strong M2e and HA2-specific antibody responses from 7 days post-primary immunization, and then the titer gradually increased after booster dose. Similarly, M2e peptide ELISA revealed that M2e–HA2 recombinant fusion protein elicited M2e-specific antibody from day 14 onward. In contrast, no antibody response was detected in the chicken immunized with synthetic peptide M2e alone or control group. Findings of this study will be very useful in future development of broad protective H5N1 influenza vaccine targeting M2e and HA2.

Identification of immunodominant proteins of Leishmania infantum by immunoproteomics to evaluate a recombinant multi-epitope designed antigen for serodiagnosis of human visceral leishmaniasis

Visceral leishmaniasis (VL) is a protozoan disease caused by Leishmania infantum in the Mediterranean region including Iran. In 95% of cases, the disease can be fatal if not rapidly diagnosed and left untreated. We aimed to identify immunoreactive proteins of L. infantum (Iranian strain), and to design and evaluate a recombinant multi-epitope antigen for serodiagnosis of human VL. To detect the immunoreactive proteins of L. infantum promastigotes, 2DE immunoblotting technique was performed using different pooled sera of VL patients. The candidate immunoreactive proteins were identified using MALDI-TOF/TOF mass spectrophotometry. Among 125 immunoreactive spots detected in 2-DE gels, glucose-regulated protein 78 (GRP78), ubiquitin-conjugating enzyme E2, calreticulin, mitochondrial heat shock 70-related protein 1 (mtHSP70), heat shock protein 70-related protein, i/6 autoantigen-like protein, ATPase beta subunit, and proteasome alpha subunit 5 were identified. The potent epitopes from candidate immunodominant proteins including GRP78, mtHSP70 and ubiquitin-conjugating enzyme E2 were then selected to design a recombinant antigenic protein (GRP-UBI-HSP). The recombinant antigen was evaluated by ELISA and compared to direct agglutination test for detection of anti L. infantum human antibodies. We screened 34 sera of VL patients from endemic areas and 107 sera of individuals without L. infantum infection from non-endemic area of VL. The recombinant protein-based ELISA provided a sensitivity of 70.6% and a specificity of 84.1%. These results showed that GRP78, ubiquitin-conjugating enzyme E2, and mtHSP70 proteins are potential immunodominant targets of the host immune system in response to the parasite and they can be considered as potential candidate markers for diagnosis purposes.

Development of a ery-C recombinant protein-based ELISA approach for differentiating brucellosis infected cattle from vaccinated ones

Development of a ery-C recombinant protein-based ELISA approach for differentiating brucellosis infected cattle from vaccinated ones

Field Evaluation of Recombinant Antigen ELISA in Detecting Zoonotic Schistosome Infection Among Water Buffaloes in Endemic Municipalities in the Philippines.

In this study, we investigated the use of recombinant antigens thioredoxin peroxidase-1 (rSjTPx-1) and tandem repeat rSj1TR in evaluating the antibody positivity rates of Schistosoma japonicum infection among water buffaloes from four endemic areas in the Philippines, two municipalities with high endemicity (Calatrava, Negros Occidental and Catarman, Northern Samar) and two municipalities nearing elimination with no cases of human schistosomiasis (Talibon and Trinidad, Bohol). These recombinant antigen ELISA assays were compared with other diagnostic tests including SEA-ELISA, FECT, and fecal-based PCR. Results showed that rSj1TR-ELISA has the highest agreement with PCR in all study areas. Furthermore, significant positivity rates among water buffaloes were seen in Talibon and Trinidad, indicating that water buffaloes are maintaining the schistosome parasites in transmission areas even in the absence of human infection. Hence, serological assay using a more sensitive and specific rSj1TR-ELISA can be used for animal surveillance to prevent emergence and re-emergence of human schistosomiasis.

Testing for Lyme borreliosis: could serology tell more?

Purpose: Remnant antibodies might be prevalent in the general population, thus recognizing relevant seropositivity might be challenging. The sequential nature of the anti-Borrelia antibody response might be diagnostically utilized. We aimed to specify details which might potentially be useful in orientating the diagnostic schedule. Methods: We processed the sera of 1304 patients using a recombinant antigen-based ELISA between Aprils of 2017 and 2019. Seroreactivity (when coherent with the anamnestic data) was confirmed with a line immunoassay (LIA). ELISA testing (IgG and/or IgM) was reactive in 539 cases. 107 patients with persistent symptoms tested positive or borderline with IgG ELISA. Results: A significant difference was observed (Mann-Whitney U-test p=0.003) between the LIA scores of patients with characteristic (arthritis, acrodermatitis, neuropathy, other objective neurologic disorder; n=83; median LIA score: 16) and non-specific symptoms (entirely subjective complaints, other known disease, lone subfebrility, uveitis; n=24; median LIA score: 6). 101 of the 107 patients tested positive for IgG against any specific protein by LIA. Those with a LIA score reaching the group median of 15 (n=51) displayed strong anti-VlsE IgG positivity or a typical late IgG antibody (against p100, p18 or p39) more often than those below (88,2% vs. 30% and 100% vs. 38% respectively, Χ2 p<0.0001). Conclusions: Weak LIA positivity (especially anti-VlsE IgG) in combination with the lack of late antibodies in patients with persistent symptoms might suggest the presence of remnant antibodies and prompt scrupulous differential diagnosis.

Procalcitonin Detection in Veterinary Species: Investigation of Commercial ELISA Kits.

Simple SummaryAmong sepsis biomarkers, procalcitonin resulted to be a specific indicator of bacterial infection or severity of infection, and to be a good control of the success of a therapeutic procedure. The clinical studies on the relevance of procalcitonin as a sepsis predictor in veterinary patients are few, likely due to the total absence of validated assays. For this reason, this study aimed to investigate commercial ELISA kits for the detection of canine and equine procalcitonin. Validation was performed evaluating linearity, limits of detection (LOD), recovery, and intra-assay and inter-assay variability; furthermore, clinical samples were analyzed. The results of the present study demonstrate that the human PCT ELISA kit is suitable to detect equine procalcitonin with a LOD of 56 ng/mL, and the canine recombinant PCT ELISA kit can be used to measure canine procalcitonin in plasma samples, showing an intra-assay and inter-assay coefficient of variation less than 20% and a LOD of 11 pg/mL.In human medicine, procalcitonin (PCT), the precursor of calcitonin, is used for the rapid identification of the origin and severity of sepsis. In veterinary medicine, PCT has been studied in horses, cattle, and dogs, but the use of PCT in diagnostic and/or prognostic settings is not possible because of the lack of validated assays to obtain reference ranges. The aim of the present study was the investigation of commercially available ELISA kits for the detection of canine and equine PCT in plasma samples. Validation of the ELISA kits was performed by using species-specific recombinant proteins spiked both in plasma and buffer samples; linearity, limit of detection (LOD), recovery, and intra-assay and inter-assay variability were calculated. Moreover, clinical samples obtained from sick and healthy animals were also analyzed with the tested kits. Canine PCT was measured with a recombinant canine and a canine PCT ELISA kit. Equine PCT was measured with an equine and a human ELISA PCT kit. Our data demonstrate that the canine recombinant PCT ELISA kit can be used to measure canine PCT in plasma samples, showing an intra-assay and inter-assay coefficient of variation less than 20% and a LOD of 11 pg/mL, whereas the present results do not support the use of the canine PCT ELISA kit. The human PCT ELISA kit is suitable to detect equine PCT with a LOD of 56 ng/mL, whereas the equine PCT ELISA kit did not detect recombinant equine PCT.

Abstract 6292: Novel seroprofiling assay reveals previously undescribed autoantibody responses in immunotherapy treated cancer patients

Abstract In contrast to cell-mediated immunity, the potential role of antibody-mediated “humoral” immunity in the context of immunotherapy is greatly under-appreciated. This is in spite of clues that patient autoantibodies can dramatically affect cancer progression. For instance, patients who fortuitously produce autoantibodies that target their tumor (e.g., anti-HER2 and anti-MUC1) have enhanced survival. Autoantibodies also underlie numerous cancer-associated paraneoplastic syndromes and immune-related adverse events. Thus, the humoral immune system represents a rich source of natural products (antibodies) that can exert either anti-neoplastic activity or harmful toxicities. A major barrier to uncovering functional humoral responses in immunotherapy is the paucity of methods to comprehensively determine autoantibody targets, particularly for autoantibodies targeting extracellular proteins that could plausibly mediate disease-modifying effects. To address this, we developed a yeast-surface display-based method that enables rapid discovery of seroreactivities. In short, we created a library of ~3,500 extracellular proteins, with each protein displayed on genetically-barcoded yeast clones. We incubate a small sample (~50µL) of patient serum or plasma with the library, use magnetic selection to isolate antibody-coated yeast clones, and use next-generation sequencing of genetic barcodes to identify bound proteins. Hundreds of samples can be screened against thousands of proteins simultaneously. Compared to platforms such as peptide arrays and phage-display libraries, yeast-surface display enables sampling of conformational epitopes that benefit from enhanced folding in a eukaryotic secretory environment. Using this assay, we screened a longitudinal cohort of 63 NSCLC patients treated primarily with anti-PD-L1 and anti-PD-1 checkpoint inhibition along with a variety of other antibody immunotherapies (anti-OX40, anti-VEGFA, anti-TNF, etc.). We successfully detected the therapeutic antibodies administered to these patients, which served as internal positive controls. We also identified novel autoantibody reactivities against proteins that have not yet been described in the context of cancer and that could potentially have disease-modifying effects. These include autoantibodies targeting chemokines (e.g., CXCL1/2/3), type 1 interferons, growth factors (e.g., VEGFB), and adhesion receptors (e.g., MADCAM1). We validated many of these reactivities using recombinant protein-based enzyme-linked immunosorbent assays and luciferase immunoprecipitation systems. Ultimately, these data indicate that humoral immunity could have wide-ranging functional effects in the context of immunotherapy and may provide not only insights into potential therapeutic targets but also novel patient-derived immunotherapeutic antibody agents. Citation Format: Eric Y. Wang, Connor E. Rosen, Yile Dai, Miguel F. Sanmamed, Aaron M. Ring. Novel seroprofiling assay reveals previously undescribed autoantibody responses in immunotherapy treated cancer patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6292.

Prevalence of locally undetected acute infections of Flaviviruses in North-Eastern Nigeria

The burden of Arboviral infections is largely underestimated in Africa, particularly in North-Eastern Nigeria. A total of 200 serum samples were collected from patients exhibiting febrile illness who visited the State Specialist Hospital in Maiduguri for medical attention between March and April 2018. Sera were tested for Flavivirus RNA by a pan-flaviviral primer set using hemi-nested RT PCR. Twenty-six samples were positive for flaviviral RNA and sequence analysis indicated a high number of West Nile virus infections and one case of Zika virus. In-house recombinant NS1-based IgM ELISA indicated 47 % of WNV and 22 % of ZIKV infections. These data were also compared to commercially available assays for West Nile and Zika viruses. Finally, NS1 IgG ELISA was conducted for Dengue, Zika, West Nile and Usutu viruses. For serum samples detected by at least one flavivirus, 945% tested positive by NS1 IgG antibodies, while only 5.5 % of the patients were negative for all.To conclude, there is a high prevalence rate of arbovirus infections in the region, including Zika and Usutu viruses that were not previously detected. Interestingly, the analysis was conducted using in–house tools to allow the implementation of a sustainable surveillance protocol locally.

Exploring the relationship between susceptibility to canine leishmaniosis and anti-Phlebotomus perniciosus saliva antibodies in Ibizan hounds and dogs of other breeds in Mallorca, Spain

BackgroundCanine leishmaniosis caused by Leishmania infantum is a neglected zoonosis transmitted by sand flies like Phlebotomus perniciosus. Clinical signs and disease susceptibility vary according to various factors, including host immune response and breed. In particular, Ibizan hounds appear more resistant. This immunocompetence could be attributed to a more frequent exposure to uninfected sand flies, eliciting a stronger anti-sand fly saliva antibody response.MethodsThis study aimed to investigate the prevalence of anti-P. perniciosus saliva antibodies in Ibizan hounds and dogs of other breeds in the Leishmania-endemic area of Mallorca, Spain, and to correlate these antibody levels with clinical, immunological and parasitological parameters. Anti-sand fly saliva IgG was examined in 47 Ibizan hounds and 45 dogs of other breeds using three methods: P. perniciosus whole salivary gland homogenate (SGH) ELISA; recombinant protein rSP03B ELISA; and rSP03B rapid tests (RT). Additionally, diagnostic performance was evaluated between methods.ResultsResults indicate significantly higher anti-SGH antibodies (P = 0.0061) and a trend for more positive SGH ELISA and RT results in Ibizan hounds compared to other breeds. General linear model analysis also found breed to be a significant factor in SGH ELISA units and a marginally significant factor in RT result. Although infection rates were similar between groups, Ibizan hounds included significantly more IFN-γ producers (P = 0.0122) and papular dermatitis cases (P < 0.0001). Older age and L. infantum seropositivity were also considered significant factors in sand fly saliva antibody levels according to at least one test. Fair agreement was found between all three tests, with the highest value between SGH and rSP03B RT.ConclusionsTo our knowledge, this is the first study elaborating the relationship between anti-P. perniciosus saliva antibodies and extensive clinical data in dogs in an endemic area. Our results suggest that Ibizan hounds experience a higher frequency of exposure to sand flies and have a stronger cellular immune response to L. infantum infection than other breed dogs. Additional sampling is needed to confirm results, but anti-P. perniciosus saliva antibodies appear to negatively correlate with susceptibility to L. infantum infection and could possibly contribute to the resistance observed in Ibizan hounds.

Seroprevalence of small ruminant lentivirus (SRLV) infection in wild cervids in Poland

Small ruminant lentiviruses (SRLV) are widespread amongst domesticated sheep and goats worldwide. Infection of wild ruminants in close contact with affected domesticated small ruminants has been proposed as an actor in SRLV epidemiology, but studies are limited. The aim of this study was to estimate the apparent (AP) and estimated prevalence (EP) of exposure to SRLV infection in wild ruminants from Poland. Samples originating from 198 free-living cervids comprising 142 European red deer and 56 roe deer were serologically tested using a multi-epitope recombinant antigen ELISA representing subtypes A1, A13, B1, and B2 of SRLV and a commercial ELISA test. The estimated prevalence of SRLV infection was estimated using the Bayesian approach with models that adjusted for the misclassification of animals because of a small population and lack of sampling method, the imperfect performance of the ELISAs and because sera of different species were tested. The calculated estimated prevalence ranged from 5.3 % (95 % CI 0.3, 12.5) to 24.6 % (95 % CI 3.3, 38.5) for the ELISA with multi-epitope antigens while estimated prevalence using the commercial ELISA was 2.5 % (95 % CI 0.2, 6.6). These results may suggest the existence of a new SRLV reservoir in Poland and highlight the importance of surveilling and controlling SRLV infection in domestic and wild ruminants sharing pasture areas.

Surveillance of equine infectious anaemia (eia) in India: Moving towards freedom from eiav infection

Equine infectious anaemia virus (EIAV) causes persistent infection in equines known as EIA. All the members of the equidae family are susceptible to the virus and clinical cases of EIA have been reported in horses, ponies, mules, zebras and donkeys. Animals infected with a virulent strain of EIAV may develop an acute form of disease with high viremia. As there are no specific pathognomonic clinical signs in EIA, demonstration of EIAV-specific antibody in the serum confirms the EIAV infection. The study was conducted to determine prevalence of EIA in India using agar gel immunodiffusion (AGID) and in-house recombinant p26 ELISA. A total of 48,951 equids from 22 states and 2 union territories in India were surveyed between October, 2012 and March, 2020. The study demonstrated no EIAV case during the survey period. Considering the rare prevalence of EIA in India, a structured surveillance strategy supported by Department of Animal Husbandry, Dairying and Fisheries (DADF), Ministry of Agriculture & Farmers Welfare, Govt of India would be a welcome approach towards this goal.

Serological evidence of arboviruses and coccidia infecting horses in the Amazonian region of Brazil.

BackgroundArboviruses and protozoans can cause neurologic disorders in horses. In Brazilian Amazon, several horses presenting signs compatible with disorders caused by these infectious agents have been observed.ObjectiveTo contribute to the knowledge of this epidemiological picture, we sought to construct a serological diagnostic panel for neurotrophic infectious agents in local horses.Material and methodsA total of 213 blood samples from horses were collected from 29 farms in three municipalities. Samples were evaluated and considered positive when they met the following criteria: titers ≥ 1:80 with the indirect fluorescent antibody test (IFAT) for apicomplexan protozoans; positive recombinant enzyme-linked immunosorbent assay (ELISA) with subsequent titers ≥ 1:10 by the PRNt for viruses; and detection under direct microscopic examination for Trypanosoma evansi.ResultsNo horses were found to be infected by T. evansi, and only two were infected Toxoplasma gondii and/or Neospora spp. The highest protozoan infection rate was observed for Sarcocystis neurona (40.3%; n = 86/213). Among the positive ELISA samples tested by the plaque reduction neutralization test (PRNT90), 92% (n = 76/83) were positive for St Louis Encephalitis virus, 43% (n = 6/14) were positive for West Nile virus and 33% (n = 16/48) were positive for Mayaro virus. Eighteen percent (n = 39/213) of horses were co-infected by S. neurona and at least one arbovirus, particularly SLEV and/or MAYV.ConclusionSamples positive for SLEV associated with S. neurona, including samples from horses that had recovered from neurological signs were frequent, and must be considered when investigating the possible causes of neurological diseases in South Roraima horses.