Species-specific Antibodies Research Articles

An observational study identifying highly tuberculosis-exposed, HIV-1-positive but persistently TB, tuberculin and IGRA negative persons with M. tuberculosis specific antibodies in Cape Town, South Africa.

BackgroundMycobacterium tuberculosis (Mtb) infection is inferred from positive results of T-cell immune conversion assays measuring Mtb-specific interferon gamma production or tuberculin skin test (TST) reactivity. Certain exposed individuals do not display T-cell immune conversion in these assays and do not develop TB. Here we report a hitherto unknown form of this phenotype: HIV-1-positive persistently TB, tuberculin and IGRA negative (HITTIN).MethodsA community-based case-control design was used to systematically screen and identify adults living with HIV (HIV+), aged 35–60 years, who met stringent study criteria, and then longitudinally followed up for repeat IGRA and TST testing. Participants had no history of TB despite living in TB hyper-endemic environments in Cape Town, South Africa with a provincial incidence of 681/100,000. Mtb-specific antibodies were measured using ELISA and Luminex.FindingsWe identified 48/286 (17%) individuals who tested persistently negative for Mtb-specific T-cell immunoreactivity (three negative Quantiferon results and one TST = 0mm) over 206±154 days on average. Of these, 97·2% had documented CD4 counts<200 prior to antiretroviral therapy (ART). They had received ART for 7·0±3·0 years with a latest CD4 count of 505·8±191·4 cells/mm3. All HITTIN sent for further antibody testing (n=38) displayed Mtb-specific antibody titres.InterpretationImmune reconstituted HIV+ persons can be persistently non-immunoreactive to TST and interferon-γ T-cell responses to Mtb, yet develop species-specific antibody responses. Exposure is evidenced by Mtb-specific antibody titres. Our identification of HIV+ individuals displaying a persisting lack of response to TST and IGRA T-cell immune conversion paves the way for future studies to investigate this phenotype in the context of HIV-infection that so far have received only scant attention.

Targeting the cell wall: Preparation of monoclonal antibody for accurate identification of Alicyclobacillus acidoterrestris in apple juice

Alicyclobacillus acidoterrestris represents the predominant microorganism implicated in apple juice spoilage. Most of existing detection methods for this microorganism necessitate advanced equipment and skilled technicians, both of which are unsuitable for practical application. In this study, an effective approach for the identification of A. acidoterrestris was developed based on an indirect sandwich enzyme-linked immunosorbent assay (is-ELISA). This method utilizes the species-specific monoclonal antibody (mAb) targeting a cell wall protein of A. acidoterrestris. The is-ELISA was highly sensitive with a limit of detection (LOD) of 5.4 × 102 CFU/mL. Furthermore, the assay is capable of accurately differentiating between A. acidoterrestris and other species of genus Alicyclobacillus. The applicability of is-ELISA was also evaluated in artificially contaminated apple juice. The convenience, simplicity, sensitivity and specificity of this assay represents a robust method for the detection of A. acidoterrestris and the resultant prevention of the spoilage of fruit juices.

Sensitive and rapid lateral-flow assay for early detection of subclinical mammary infection in dairy cows

Detection of subclinical mastitis (SCM) in its initial stage can save great economic losses, improve milk quality and animal welfare. We have developed a semiquantitative lateral flow assay for the detection of SCM in dairy cows targeting myeloperoxidase (MPO) enzyme of milk neutrophils. A competitive immunoassay format was used, and colloidal gold nanoparticles (GNP) were prepared and used as a labelling agent. Monoclonal anti-MPO antibodies were used and assessed for its quality by enzyme-linked immunosorbent assay and dot blot. Conjugation method for GNP and anti-MPO antibodies was standardised, and the conjugate was placed over the conjugate pad. MPO coupled with a carrier protein (OVA) and the species-specific secondary antibodies were placed on test and control lines, respectively. The developed assay was verified with 75 milk samples collected from healthy, SCM and clinical mastitis cows. It displayed a high sensitivity as it could detect MPO as low as 1.5 ng/ml, an accuracy greater than 97% and showed no crossreactivity when crosschecked with other milk proteins. The developed assay can be used as an alternative for SCM diagnostic tests where lab structure are available for obtaining the lysate of milk SCC.

Serological evidence of Toxoplasma gondii infection in Melanosuchus niger (Spix, 1825) and Caimam crocodilus (Linnaeus, 1758).

Toxoplasma gondii is a protozoan with worldwide prevalence, known to affect a large variety of warm-blooded hosts. However, its ability to induce long-lasting infections in cold-blooded animals remains unclear. The most likely source of infection is through consumption of meat containing tissue cysts or by ingestion of food or water contaminated with oocysts. The current global climate change trend and the progressive degradation of natural habitats are prone to alter the distribution of ectotherm populations over a short period of time, which may favor contact between these animals and the protozoan. In association, alligator meat is considered a delicacy in many regions and its consumption has been previously related to a diversity of foodborne diseases. In that sense, we proposed in this study to search for specific antibodies against T. gondii in serum samples of two common species of alligators from the Brazilian fauna (Melanosuchus niger and Caimam crocodilus). We obtained the serum samples from 84 alligators from the Araguaia region, which were tested by agglutination assays that do not require species-specific secondary antibodies (Modified Agglutination Test – MAT; Indirect Hemagglutination Assay – IHA). From the 84 samples tested, eight (9.5%) were positive by MAT. From those, seven (87.5% of MAT+, 8.3% of the total) were also positive by IHA, reassuring a probable exposure of these animals to the parasite. Direct parasite detection in muscle fragments of one serologically reactive alligator did not yield positive results. Our results provide serological evidence that Brazilian alligators may be exposed to T. gondii and further studies should be performed to elucidate whether alligators are natural hosts of this ubiquitous protozoan parasite.

Screening of opportunistic Encephalitozoon intestinalis and Enterocytozoon bieneusi in immunocompromised patients in Slovakia

In recent years new infectious diseases, i.e. emerging or re-emerging diseases, have been coming to the forefront. Currently, microsporidia, considered to be a major cause of emerging and opportunistic infections particularly in immunocompromised individuals, are also included in this group. Therefore, the aim of our study was to map the prevalence of Encephalitozoon intestinalis and Enterocytozoon bieneusi infection in a group of patients and to compare it with the occurrence of specific antigens in immunocompetent people. Detection of spores of both pathogens in faecal samples was performed by an immunofluorescence test using species-specific monoclonal antibodies. Positivity to E. intestinalis in 91 examined immunosuppressed patients reached 33% (30/91), while only 4.3% (3/70) of the control group samples were found to be positive (relative risk 7.7, p < 0.001). In case of E. bieneusi 14.3% (13/91) of immunocompromised patients were positive, as were 5.7% (4/70) of people from the control group (relative risk 2.5, p = 0.095). In case of development of any opportunistic infection, the infection is detected and removed in most cases at an early stage. The incidence of clinically manifested microsporidiosis in patients with immunodeficiency is rare as they are under constant medical supervision. However, we must not forget about opportunistic infections, and in case of any non-specific symptoms it is necessary to exclude or confirm the diagnosis for immediate treatment.

Generation, Characterization and Pre-Clinical Drug Evaluation of Patient-Derived Xenograft Models of Pediatric Down Syndrome AML

Children with Down syndrome (DS) are at a 500-fold increased risk for developing acute myeloid leukemia (AML) before they reach five years of age. DS-AML blasts have hypersensitivity to chemotherapeutic drugs such as cytarabine and daunorubicin. However, therapy-induced toxicity results in greater morbidity and remains a major barrier in attaining higher survival rate. Thus, alternate therapy approaches to minimize toxicity and increase efficacy are needed. Patient-derived xenograft (PDX) models of DS-AML are very limited and have not been used for preclinical studies. We generated three distinct disseminated PDX models by successful engraftment and serial passage of primary DS-AML blasts in NSG-B2m mice. DS-AML is characterized by the pathognomonic mutation in the gene encoding the essential hematopoietic transcription factor GATA1, resulting in N-terminally truncated mutant GATA1s protein. Accordingly, we identified GATA1 mutations and GATA1s protein (Fig. 1) in the DS-AML PDX lines. In addition to GATA1 mutation, whole exome sequencing also identified mutations in one or more co-operating mutations reported previously in DS-AML (Table 1). These PDX lines were extensively characterized and authenticated to be highly concordant with the primary patient sample with respect to immunophenotype (Table 2) and transcriptome analysis (by targeted sequencing using Archer FusionPlex HemeV2 panel), signifying the validity of the PDX models. We evaluated the efficacy of cytotoxic chemotherapy and epigenetic therapy in mice transplanted with DS-AML PDX lines. Cytotoxic chemotherapy comprised daunorubicin 1.5 mg/Kg Qxd3 i.v. and cytarabine 50 mg/Kg Qxd5 i.p. Epigenetic therapy consisted of DNA hypomethylating agent azacitidine and HDAC inhibitor panobinostat, alone or in combination. NSG-B2m mice were injected with DS-AML PDX lines intravenously. Disease progression and engraftment of human cells was monitored by periodic determination of the percentage of human leukemic cells versus mouse cells in the peripheral blood by staining with species-specific fluorescent antibodies followed by flow cytometry. The mice were randomized into 5 treatment groups - 1) cytotoxic chemotherapy, 2) vehicle (5% dextrose), 3) azacitidine (2.5 mg/Kg), 4) panobinostat (2.5 mg/Kg), and 5) combination of azacitidine + panobinostat (2.5 mg/Kg each). The epigenetic drugs were dosed with four cycles lasting five days a week with two days rest. One-week rest period was included between cycles 2 and 3. Chemotherapy prolonged survival by 11 and 23 days respectively in mice injected with NTPL-386 and NTPL-662. Single agent azacitidine and panobinostat prolonged survival by 48 and 31 days respectively in NTPL-60 mice. Mice transplanted with NTPL-386 showed enhanced survival by 29 and 21 days when treated with azacitidine and panobinostat alone. NTPL-662 study is ongoing and so far panobinostat treated mice showed 10 day increase in cell survival, while both azacitidine and azacitidine + panobinostat groups are alive and healthy. Azacitidine was more efficient than panobinostat in inducing remission and increasing the median survival compared to vehicle treated mice in all 3 models (Fig. 2A-C). Mice treated with the combination of azacitidine and panobinostat showed the greatest benefit and survived 127 days (for NTPL-60) and 55 days (for NTPL-386). Although azacitidine and panobinostat synergized to reduce leukemic burden and prolong survival in both PDX lines, there was a remarkable difference between the responses. Epigenetic therapy appeared to be more effective than chemotherapy. In conclusion, using DS-AML PDX models generated and characterized in-house, we show the efficacy of epigenetic therapy in comparison to chemotherapy. Disclosures Druley: Washington University: Employment; ArcherDX Inc.: Employment, Equity Ownership.

Comparison Of Four Anti-Avian IgY Secondary Antibodies Used In Western Blot And Dot-Blot ELISA To Detect Avian Bornavirus Antibodies In Four Different Bird Species.

PurposeThis study evaluated the specificity of different avian secondary antibodies used in Western blot and dot-blot ELISA to detect avian bornavirus antibodies in bird plasma.MethodsPlasma samples were collected from: two Blue and gold macaws, one positive and one negative for avian bornavirus by RT-PCR; a Cockatiel and a Monk parakeet prior to and following experimental infection; and, two Mallards, one positive and one negative for avian bornavirus by RT-PCR Samples were analyzed by Western blot and dot-blot ELISA that incorporated recombinant avian bornavirus nucleoprotein as the target analyte. Four species-specific anti-IgY secondary antibodies were used in the assays: goat anti-macaw IgY, goat anti-bird IgY, goat anti-duck IgY, and rabbit anti-chicken IgY.ResultsIn the Western blot, anti-macaw IgY secondary antibody produced strong signals with Blue and gold macaw and Cockatiel positive plasma, but no signal with Mallard positive plasma. Anti-bird IgY secondary antibody produced strong signals with Blue and gold macaw, Cockatiel, and Mallard positive plasma. Anti-duck and anti-chicken IgY secondary antibody produced a strong and moderate signal, respectively, only with Mallard positive plasma. In the dot-blot ELISA, there was a distinct and significant difference (P<0.05) in the signal intensity between the different secondary antibodies within a bird species. Anti-macaw IgY secondary antibody produced significantly (P<0.05) stronger signals than the other secondary antibodies in Blue and gold macaw, Cockatiel, and Monk parakeet positive plasma, while anti-duck IgY secondary antibody produced significantly (P<0.05) stronger signals than the other secondary antibodies in Mallard positive plasma.ConclusionIn testing psittacines with immunoassays, and especially in assays that incorporate short incubation reaction times such as a dot-blot ELISA, species-specific anti-IgY secondary antibodies provided more accurate results.

Generation of rabbit polyclonal human and murine collagen VII monospecific antibodies: A useful tool for dystrophic epidermolysis bullosa therapy studies

High conservation of extracellular matrix proteins often makes the generation of potent species-specific antibodies challenging. For collagen VII there is a particular preclinical interest in the ability to discriminate between human and murine collagen VII. Deficiency of collagen VII causes dystrophic epidermolysis bullosa (DEB) – a genetic skin blistering disease, which in its most severe forms is highly debilitating. Advances in gene and cell therapy approaches have made curative therapies for genetic diseases a realistic possibility. DEB is one disorder for which substantial progress has been made toward curative therapies and improved management of the disease. However, to increase their efficacy further preclinical studies are needed. The early neonatal lethality of complete collagen VII deficient mice, have led researches to resort to using models maintaining residual collagen VII expression or grafting of DEB model skin on wild-type mice for preclinical therapy studies. These approaches are challenged by collagen VII expression by the murine host. Thus, the ability to selectively visualize human and murine collagen VII would be a substantial advantage. Here, we describe a novel resource toward this end. By immunization with homologous peptides we generated rabbit polyclonal antibodies that recognize either human or murine collagen VII. Testing on additional species, including rat, sheep, dog, and pig, combined sequence alignment and peptide competition binding assays enabled identification of the major antisera recognizing epitopes. The species-specificity was maintained after denaturation and the antibodies allowed us to simultaneously, specifically visualize human and murine collagen VII in situ.

An Antibody Specific for the Dog Leukocyte Antigen DR (DLA-DR) and Its Novel Methotrexate Conjugate Inhibit the Growth of Canine B Cell Lymphoma.

Canine B-cell lymphoma (CBL) is an incurable, spontaneous lymphoid malignancy constituting an accurate animal model for testing novel therapeutic strategies in human medicine. Resources of available species-specific therapeutic monoclonal antibodies (mAbs) targeting CBL are scarce. The aim of the present study was to evaluate the therapeutic potential of mAb B5, specific for the dog leukocyte antigen DR (DLA-DR) and its antibody-drug conjugate with methotrexate (B5-MTX). B5 induced caspase-dependent apoptosis of DLA-DR-expressing canine B cell lymphoma/CLBL1 and CLB70 leukemia lines, but not the GL-1 line not expressing DLA-DR. The cytotoxicity of B5-MTX to sensitive cells was further potentiated by a payload of MTX, but without any substantial off-target effects. The infusion of B5 and B5-MTX in a murine model of disseminated, advanced canine lymphoma, mediated >80% and >90% improvement in survival, respectively, and was well tolerated by the animals. Interestingly, the concentrations of soluble DLA-DR (sDLA-DR) antigens present in the blood serum of tumor-bearing mice were found proportional to the tumor burden. On this basis, sDLA-DR levels were evaluated as a potential biomarker using samples from canine lymphoma patients. In summary, the action of B5 and B5-MTX holds promise for further development as an alternative/complementary option for the diagnosis and treatment of canine lymphoma.

A multi-species indirect ELISA for detection group-specific antibodies against VP7 protein of bluetongue virus

Abstract In the present study, a multispecies indirect ELISA for the detection of serogroup-specific antibodies against the VP7 protein of bluetongue virus (BTV) has been developed. The assay was optimized using a peroxidase-conjugated protein G secondary antibody that can detect antibodies from multiple species. Serum samples (n = 515) from small and large domestic ruminants were used to optimize the assay. The percent positivity (PP) values were used for the estimation of cut-off values. On receiver operator characteristic (ROC) analysis, different cut-off values along with their diagnostic sensitivity (DSn) and diagnostic specificity (DSp) were obtained. Among these, >20.24% PP value was accepted as the cut-off at which DSn was 96.05% with 92.9–98.1 (95%) confidence interval and DSp was 98.47 with 96.1–99.6 (95%) confidence interval. The above results indicate that the multispecies i-ELISA assay is sensitive, specific and minimize the dependence of species-specific conjugated antibodies for serological investigation of bluetongue in small and large ruminants.

Detection of an Antigen on an Immunoblot.

Detection of the antigen on an immunoblot can be achieved by either enzyme-based detection systems or using detection reagents labeled with fluorochromes. Two major types of enzyme-based detection systems are used in immunoblotting based on either horseradish peroxidase (HRP)-or alkaline phosphatase (AP)-coupled antibodies, and a range of soluble substrates that yield insoluble colored products (chromogenic detection) or generate light (chemiluminescent detection). This protocol describes chromogenic detection with AP as well as both chromogenic and chemiluminescent methods of detection with HRP. Before the detection step, the nitrocellulose or polyvinylidene fluoride (PVDF) membrane is incubated with the antigen using enzyme-conjugated primary antibodies or secondary detection reagents (such as species-specific anti-immunoglobulin antibodies, streptavidin, or Protein A) and washed. An appropriate substrate solution is applied to the membrane, and the signal is detected in the course of the enzymatic reaction, resulting either in development of insoluble colored products (chromogenic substrates) or in generation of light (chemiluminescent substrates). This protocol also describes use of detection reagents labeled with fluorochromes. The nitrocellulose or PVDF membrane incubated with fluorochrome-labeled detection reagents is rinsed with PBS and processed for image capturing with appropriate imaging equipment.

Molecular and cellular mechanisms of osteoarthritis in experimental arterial hypertension and hyperlipidemia

Purpose: To study the role of vascular endothelial growth factor (VEGF-A), adiponekin (Adipo) and matrix metalloproteinase-9 (MMP-9) in remodeling of articular cartilage (AC) and subchondral bone (SCB) in experimental arterial hypertension (AH) and hyperlipidemia (HL). Methods: An experimental study was carried out on 18 adult males of purebred guinea pigs (28-30 weeks old, weight 750-900 g), which were divided into 3 groups of 6 individuals each: in the first group AH was modeled, in the second group - HL, the third group was intact animals (control). To assess the reproduction of a qualitative model in animals, the level of blood pressure, total cholesterol, and body weight were determined. On the 60th day all the animals were removed from the experiment, the tissues of the knee joints of the hind (hock) paws were taken. Immunohistochemical reaction was performed according to the standard protocol using primary species-specific antibodies to VEGF-A, Adipo, MMP-9. Results: In the control group of animals, the immunohistochemical reaction of AC to VEGF-A was determined as single low-intensity precipitates in the region of the pericellular matrix of the basal layers of chondrocytes. In the SCB, single precipitates of vascular endothelial growth factor were found in the central parts of the haversian systems. A high intensity response to VEGF was observed in the red bone marrow. In the group of animals with hypertension, the reaction of articular cartilage on VEGF-A was determined in all areas of cartilage. Its greatest intensity was in the perchondrium, on the periphery of the zones of destruction of the articular cartilage. Within the limits of the fuzzy osteochondral line, elements of ectopic vascular invasion were determined by the type of non-vascular expansion in AC with a positive response to VEGF. Maximum expression of VEGF-A was found in the region of contact of the subchondral bone with destruction of articular cartilage. In hyperlipidemia, AC was positive for VEGF-A in the area of chondrocyte lacunae of hypertrophied chondrocytes in all layers of the cartilage plate. In SCB, the vascular endothelial growth factor was found in the area of osteogenesis imperfecta foci, the red bone marrow. The histochemical reaction of AC and SCB to adiponectin in intact animals was practically not determined. There were isolated low-intensity precipitates. Tissue expression of Adipo in animals with AH in AC was determined in the form of multiple precipitates with a high density of IHC-reaction with a maximum response in the basal regions of the cartilage plate. In the SCB with experimental hypertension, adiponectin tissue precipitates were found in the area of haversian systems, osteochondral communication. In hyperlipidemia in AC, a positive reaction to Adipo was found in the area of chondrocyte lacunae predominantly in the upper and middle layers of the cartilage plate. in all layers of the cartilage plate. Under conditions of lipid metabolism impairment, the Adipo SCB was defined as diffuse deposits in the communicative regions with basal soreness, in the zones of the haversian systems and the bone matrix. The tissue response to MMP-9 SCB and AC in the control group was not very pronounced, showing up as single low-intensity cell-cell precipitates. Under the conditions of experimental arterial hypertension in the articular cartilage, MMP-9 was determined in the subcellular matrix of hypertrophied chondrocytes, mainly in the middle layer. In SCB, a positive response to matrix metalloproteinase-9 was found in the area of angiovasal integration into the basal cartilage, in the centers of imperfect osteogenesis. The highest expression density of MMP-9 was determined in the group of animals with HL. Deposits of MMP-9 were found in cartilage tissue, mainly in the upper and middle sections. In SCB at HL, the tissue reaction to the matrix metalloproteinase-9 was also detected in the foci of ectopic angiogenesis. Conclusions: The results of the study allowed to establish that under the influence of systemic AH and HL in the articular cartilage and subchondral bone cell stress develops, caused by endothelium-dependent dysmetabolism. In the present study, it was shown that extravasal expression of VEGF-A is observed in experimental AH, most pronounced in SCB. In addition, moderate expression of adiponectin and MMP-9 in the SCB is noted. In systemic AH, in experimental conditions, VEGF-A is overexpressed in articular cartilage. In addition to this, in addition to this, Adipo and MMP-9 are highly expressed in SCB. It is established that angiogenic factors contribute to the neovascularization of cartilage, non-adaptive osteoregeneration of SCB. At the same time, remodeling of the tissues of the joints is obviously provided by tissue metalloproteinases. The data obtained allow us to makethe assumption that SCB and AC are target organs of cardiovascular factors.

The MUB40 Peptide for Use in Detecting Neutrophil-Mediated Inflammation Events.

Here, we provide a protocol involving the use of MUB40, a synthesized peptide with the ability to bind glycosylated lactoferrin stored at high concentrations in specific and tertiary granules of neutrophils. This protocol details how MUB40conjugated directly to a fluorophore can be used to stain neutrophils in fixed/permeabilized tissues as well as how this can be used in live-cell imaging to assay for neutrophil activation and de-granulation. Neutrophil detection methods are limited to species-specific monoclonal antibodies, which are not always suitable for certain applications. MUB40 does not penetrate the cell membrane and is thus excluded from lactoferrin stored in non-activated/non-permeabilized neutrophils. MUB40 has the added benefit of recognizing lactoferrin from a broad host range, making it especially useful for comparing results in studies involving multiple research models, reducing the number of duplicate reagents, and simplifying protocols through single-step staining.

Assessing the performance of a Fasciola gigantica serum antibody ELISA to estimate prevalence in cattle in Cameroon

BackgroundCattle rearing in Cameroon is both economically and culturally important, however parasitic diseases detrimentally impact cattle productivity. In sub-Saharan Africa bovine fasciolosis is generally attributed to F. gigantica, although understanding of Fasciola species present and local epidemiology in individual countries is patchy. Partly limited by the lack of representative surveys and understanding of diagnostic test perfromance in local cattle populations. The aims of this paper were to determine the Fasciola species infecting cattle, develop a species specific serum antibody ELISA, assess the performance of the ELISA and use it to assess the prevalence of F. gigantica exposure in two important cattle-rearing areas of Cameroon.ResultsA random sample of Fasciola parasites were collected and were all identified as F. gigantica (100%, CI:94.0–100%, n = 60) using RAPD-PCR analysis. A F. gigantica antibody ELISA was developed and initially a diagnostic cut-off was determined using a sample of known positive and negative cattle. The initial cut-off was used as starting point to estimate an optimal cut-off to estimate the best combination of sensitivity and specificity. This was achieved through sampling a naturally infected population with known infection status (cattle slaughtered at Bamenda abattoir, North West Region (n = 1112) and Ngaoundere abattoir, Vina Division, Adamawa Region (n = 776) in Cameroon). These cattle were tested and results analysed using a Bayesian non-gold standard method. The optimal cut-off was 23.5, which gave a sensitivity of 65.3% and a specificity of 65.2%. The prevalence of exposure to F. gigantica was higher in cattle in Ngaoundere (56.4% CI: 50.2–60.0%) than Bamenda (0.6% CI: 0.0–1.4%).ConclusionFasciola gigantica was identified as the predominant Fasciola species in Cameroon. Although the sensitivity and specificity F. gigantica antibody ELISA requires improvement, the test has shown to be a potentially useful tool in epidemiological studies. Highlighting the need for better understanding of the impact of F. gigantica infections on cattle production in Cameroon to improve cattle production in the pastoral systems of Central-West Africa. This paper also highlights that non-gold standard latent class methods are useful for assessing diagnostic test performance in naturally-infected animal populations in resource limited settings.

Serological Detection of Ebola Virus Exposures in Native Non-human Primates of Southern Nigeria

Ebola viruses (family: Filoviridae) are the cause of Ebola virus disease (EVD), a highly fatal illness characterised by haemorrhagic fever syndrome in both humans and non-human primates (NHPs). West Africa was the epicentre of the 2013–2015 EVD epidemic which caused the death of over 11,000 people, including eight casualties in southern Nigeria. Antibodies to filoviruses have been detected among NHPs in some countries, but there is no documented evidence of exposures to filoviruses among NHPs in Nigeria. From August 2015 to February 2017, a total of 142 serum samples were obtained from individual captive and wild animals, belonging to 11 NHP species, in southern Nigeria, and screened for species-specific antibodies to filoviruses belonging to the species; Zaire ebolavirus [Ebola virus (EBOV)], Sudan ebolavirus [Sudan virus (SUDV)], and Marburg marburgvirus [Ravn virus (RAVV)]–using a modifed filovirus species-specific ELISA technique. Of the sera tested, 2.1% (3/142) were positive for antibodies to EBOV. The entire 142 sera were negative for SUDV or RAVV. These findings point to the existence of natural exposures of NHPs in southern Nigeria to EBOV. There is need to discourage, the uncontrolled hunting of NHPs in Nigeria for public health safety.

Mediators between oral dysbiosis and cardiovascular diseases.

Clinical periodontitis is associated with an increased risk for cardiovascular diseases (CVDs) through systemic inflammation as the etiopathogenic link. Whether the oral microbiota, especially its quality, quantity, serology, and virulence factors, plays a role in atherogenesis is not clarified. Patients with periodontitis are exposed to bacteria and their products, which have access to the circulation directly through inflamed oral tissues and indirectly (via saliva) through the gastrointestinal tract, resulting in systemic inflammatory and immunologic responses. Periodontitis is associated with persistent endotoxemia, which has been identified as a notable cardiometabolic risk factor. The serology of bacterial biomarkers for oral dysbiosis is associated with an increased risk for subclinical atherosclerosis, prevalent and future coronary artery disease, and incident and recurrent stroke. In addition to species-specific antibodies, the immunologic response includes persistent, cross-reactive, proatherogenic antibodies against host-derived antigens. Periodontitis may affect lipoprotein metabolism at all levels, and all lipoprotein classes are affected. Periodontitis or its bacterial signatures may be involved not only in increased storage of proatherogenic lipids but also in attenuation of the anti-atherogenic processes, thereby putatively increasing the net risk of atherosclerosis. In this review we summarize possible molecular mediators between the dysbiotic oral microbiota and atherosclerotic processes.

Multiple immunolabeling with antibodies from the same host species in combination with tyramide signal amplification

A general problem in immunocytochemistry is the development of a reliable multiple immunolabeling method with primary antibodies originating from the same host species. When primary antibodies are raised in the same host species, the secondary species-specific antibodies can cross-react with each of the primary antibodies. This obstacle can however be avoided with the use of striping buffers eluting the primary/secondary antibody complex. After elution of the previous primary/secondary antibody complex, the next primary antibody from the same host species can be applied. Recently, a group from VENTANA (Tucson, AZ, USA) presented a fully automated multiplex protocol for fluorescent immunohistochemistry on the platform of VENTANA’s BenchMark ULTRA slide stainer using the same species antibodies in combination with tyramide signal amplification. We adapted the automated protocol of VENTANA for the use in a routine histochemical laboratory and present here a standard procedure with a manual mode of operation for simultaneously detecting two or more antigens from the same host species.

Development of a polyclonal anti-dugong immunoglobulin G (IgG) antibody with evaluation of total plasma IgG in a living dugong (Dugong dugon) population

Species-specific antibodies (Ab) for the measurement of immunoglobulins (Ig) are valuable tools for determining the humoral immune status of threatened and endangered wildlife species such as dugongs. However, no studies have reported antibody reagents against dugong immunoglobulin. The object of this study was to develop an Ab with specificity for dugong IgG and apply this tool to survey total IgG levels in plasma samples from a live wild population of dugongs in southern Queensland, Australia. Dugong IgG was isolated from plasma by protein A/G column chromatography and a polyclonal antiserum was successfully raised against the dugong IgG through immunization of mice. The anti-dugong antiserum was reactive with dugong serum but not immunoglobulin from other species such as rats and humans. When tested against a panel of dugong plasma samples, relative IgG levels from dugongs (n = 116) showed biologically relevant relationships with pregnancy status and a principal component of Body Mass Index (BMI)/globulin/fecal glucocorticosteroid (chronic stress) levels combined, which together accounted for 9.2% of the variation in total Ig levels. Together these data suggest that dugongs show variation in total IgG and that this correlates with some physiological parameters of dugong health.

Flow cytometric immunophenotyping of canine adipose-derived mesenchymal stem cells (ADMSCs) and feline ADMSCs using anti-human antibodies

Various trials have been conducted to develop therapies for serious untreatable diseases. Among these, those using stem cells have shown great promise, and adipose-derived mesenchymal stem cells (ADMSCs) are easier to obtain than other types of stem cells. Prior to clinical trials, characterization of ADMSCs with monoclonal antibodies should be performed. However, it is difficult to use species-specific antibodies for veterinarians. This study was conducted to confirm the panel of human antibodies applicable for use in immunophenotypic characterization of canine adipose-derived stem cells and feline ADMSCs extracted from subcutaneous adipose tissue collected during ovariohysterectomy. For flow cytometric immunophenotyping, the third passages of canine ADMSC and feline ADMSC and human CD31, CD34, CD42, CD44, CD62 and CD133 antibodies were used. Of these, CD133 reacted with canine cells (3.74%) and feline cells (1.34%). CD133 is known as a marker related with more primitive stem cell phenotype than other CD series. Because this human CD133 was not a species-specific antibody, accurate percentages of immunoreactivity were not confirmed. Nevertheless, the results of this study confirmed human CD133 as a meaningful marker in canine and feline ADMSCs.

A novel synthetic peptide microarray assay detects Chlamydia species-specific antibodies in animal and human sera

Serological analysis of Chlamydia (C.) spp. infections is still mainly based on micro-immunofluorescence and ELISA. To overcome the limitations of conventional serology, we have designed a novel microarray carrying 52 synthetic peptides representing B-cell epitopes from immunodominant proteins of all 11 chlamydial species. The new assay has been validated using monospecific mouse hyperimmune sera. Subsequently, serum samples from cattle, sheep and humans with a known history of chlamydial infection were examined. For instance, the specific humoral response of sheep to treatment with a C. abortus vaccine has been visualized against a background of C. pecorum carriership. In samples from humans, dual infection with C. trachomatis and C. pneumoniae could be demonstrated. The experiments revealed that the peptide microarray assay was capable of simultaneously identifying specific antibodies to each Chlamydia spp. The actual assay represents an open platform test that can be complemented through future advances in Chlamydia proteome research. The concept of the highly parallel multi-antigen microarray proven in this study has the potential to enhance our understanding of antibody responses by defining not only a single quantitative response, but also the pattern of this response. The added value of using peptide antigens will consist in unprecedented serodiagnostic specificity.