An update on the development of a bottlenose dolphin, Tursiops truncatus, immune reagent toolkit.

There are extensive immunological reagents available for laboratory rodents and humans. However, for veterinary species there is a need for expansion of immunological toolkits, with this especially evident for marine mammals, such as cetaceans. In addition to their use in a research setting, immune assays could be employed to monitor the health status of cetaceans and serve as an adjunct to available diagnostic tests. Such development of specific and sensitive immune assays will enhance the proper care and stewardship of wild and managed cetacean populations. Our goal is to provide immune reagents and immune assays for the research community, clinicians, and others involved in care of bottlenose dolphins. This review will provide an update on our development of a bottlenose dolphin immunological toolkit. The future availability and continued development of these reagents is critical for improving wild and managed bottlenose dolphin population health through enhanced assessment of their responses to alterations in the marine environment, including pathogens, and improve our ability to monitor their status following vaccination.

Monoclonal antibody development advances immunological research in horses.

Host immune analyses require specific reagents to identify cellular and soluble components of the immune system. These immune reagents are often species-specific. For horses, various immunological tools have been developed and tested by different initiatives during the past decades. This article summarizes the development of well characterized monoclonal antibodies (mAbs) for equine immune cells, immunoglobulin isotypes, cytokines, and chemokines.

Food antigen-specific IgE in dogs with suspected food hypersensitivity.

OBJECTIVE: Knowledge of cross-reactions in food-sensitive dogs will influence the choice of elimination diets and the long-term management of those patients. The objective of this study was to evaluate food allergen-specific IgE tests of suspected allergic dogs for concurrent positive reactions as possible evidence for cross reactions between allergens. MATERIAL AND METHODS: Results of serum IgE tests from 760 suspected allergic dogs submitted to 2 laboratories were evaluated statistically. After the tested allergens were grouped by their phylogenetic relationship, odds ratios as well as a sensitivity analysis of the odds ratios were performed to evaluate if concurrent positive IgE results to 2 allergens occurred more often than expected. RESULTS: Within related allergen pairs 27% (laboratory 1) and 72% (laboratory 2) of the pairs could be considered as associated. For the unrelated allergen pairs only 6.8% and 10.6% of the analyzed pairs were considered associated respectively. Strong correlations were shown in the group of ruminant allergens, especially beef and lamb, and grain allergens. High rates of concurrent reactions were also detected in the poultry group, especially between chicken and duck, as well as between pork and ruminant allergens, and soy and grain allergens. CONCLUSION: As our results showed not only correlations within related but also between non-related allergens, the possible relevance of carbohydrate moieties as well as panallergens for canine hypersensitivities warrants further study. Further investigations are necessary to distinguish co-sensitization from cross-reactions and determine the clinical relevance of food-specific IgE reactivity. CLINICAL RELEVANCE: Due to possible cross reactivity related allergens, especially beef and lamb as well as grain allergens, should not be used for an elimination diet to avoid false results.

[Prevalance of immunoglobulin E against cross-reactive carbohydrate determinants (CCD) and impact of a blocker in seasonal allergy tests]. / Prävalenz von Immunglobulin E gegen kreuzreagierende Kohlenhydrat-Seitenketten (CCD) und die Wirkung eines Blockers im saisonalen In-vitro-Allergietest.

OBJECTIVES: Cross-reactive carbohydrate determinants (CCD) cause false positive/clinically irrelevant results in seasonal in vitro allergy tests due to the binding of immunoglobulin IgE against CCD (anti-CCD IgE).There is no study regarding the presence of this phenomen in cats. The aim of this study was to investigate the prevalence of polysensitization in serum samples and evaluate the impact of a CCD inhibitor/blocker in multi-positive seasonal allergy test results in cats. MATERIALS AND METHODS: A total of 472 feline sera, submitted from July 2017 to June 2018 for seasonal in vitro allergy test via ELISA Fc-Ε receptor technology, were studied. Samples were grouped into polysensitized (group A) and non-polysensitized (group B). Polysensitized samples (A) were retested after adding a modified glycoprotein plants extract (blocker). To determine the impact of the blocking to each allergen, the results in 48 randomly selected samples in cats prior- and post-blocking were investigated. RESULTS: Polysensitization to seasonal allergens was diagnosed in 137 (29 %) samples. No discrepancy in presence of polysensitization was seen in different seasons. Blocking eliminated the binding of anti-CCD IgE and produced either negative test results (49 %) or a decrease of 1-4 reaction classes (41 %) which is indicative of the simultaneous presence of clinically relevant allergen specific IgE. Total negative reactions after blocking were less common in 6-grass mix (31 %), rye (23 %) and sheep sorrel (25 %) in comparison to willow und birch-hazel (67 %), mugwort-ragweed und nettle (65 %), as well as English plantain (54 %). CONCLUSION AND CLINICAL RELEVANCE: In order to improve the quality of seasonal in vitro allergy test, blocking should be employed in cases of polysensitized results resulting in an avoidance of the administration of non-offending allergens during allergen-specific immunotherapy (ASIT).

Development of a method to quantify endogenous IFNγ protein in amberjack species.

Interferon (IFN)γ is a pivotal cytokine that promotes and orchestrates innate cellular and adaptive cell-mediated immunity against intracellular pathogens. The capacity of T cells in mammals to produce IFNγ has been measured using specific antibodies in order to analyze cell-mediated immune responses against infection or immuno-stimulants. In fish, however, measurement of IFNγ protein levels has not been possible due to a lack of research tools. In the present study, therefore, we established antibodies that react with endogenous amberjack IFNγ. An enzyme-linked immunosorbent assay (ELISA) for IFNγ in amberjack species was developed using these antibodies. The ELISA could detect endogenous IFNγ at concentrations less than 100 pg/mL in PMA/ionomycin-stimulated leukocytes culture supernatant. IFNγ production was enhanced and lasted a long time following intracellular bacterial infection with Nocardia seriolae, which is thought to be targeted by cell-mediated immunity. These results demonstrate that quantification of IFNγ using the reported ELISA can be used to estimate the status of cell-mediated immunity in amberjack species.

Comparisons of Stereological and Other Approaches for Quantifying Macrophage Aggregates in Piscine Spleens.

Macrophage aggregates (MAs) are focal accumulations of pigmented macrophages in the spleen and other tissues of fish. A central role of MAs is the clearance and destruction of degenerating cells and recycling of some cellular components. Macrophage aggregates also respond to chemical contaminants and infectious agents and may play a role in the adaptive immune response. Tissue damage or physiological stress can result in increased MA accumulation. As a result, MAs may be sensitive biomarkers of environmental stress in fish. Abundance of MAs in tissues has been reported in a variety of ways-most commonly as density, mean size, and relative area-but the utility of these estimates has not been compared. In this study, four different types of splenic MA abundance estimates (abundance score, density, relative area, and total volume) were compared in two fish populations (Striped Bass Morone saxatilis and White Perch M. americana) with a wide range in ages. Stereological estimates of total volume indicated an increase in MA abundance with spleen volume, which generally corresponded to fish age, and with splenic infections (mycobacteria or trematode parasites). Abundance scores were generally limited in the ability to detect changes in MA abundance by these factors, whereas density estimates were greatly influenced by changes in spleen volume. In some instances, densities declined while the total volume of MAs and spleen volume increased. Experimentally induced acute stress resulted in a decrease in spleen volume and an increase in MA density, although the total volume of MAs remained unchanged. Relative area estimates accounted for the size and number of MAs but not for changes in organ volume. Total volume is an absolute measure of MA abundance irrespective of changes in organ volume or patterns of accumulation and may provide an improved means of quantifying MAs in the spleens of fish.

Biochemical and biological characterization of the Hypanus americanus mucus: A perspective on stingray immunity and toxins.

Stingrays skin secretions are largely studied due to the human envenoming medical relevance of the sting puncture that evolves to inflammatory events, including necrosis. Such toxic effects can be correlated to the biochemical composition of the sting mucus, according to the literature. Fish skin plays important biological roles, such as the control of the osmotic pressure gradient, protection against mechanical forces and microorganism infections. The mucus, on the other hand, is a rich and complex fluid, acting on swimming, nutrition and the innate immune system. The elasmobranch's epidermis is a tissue composed mainly by mucus secretory cells, and marine stingrays have already been described to present secretory glands spread throughout the body. Little is known about the biochemical composition of the stingray mucus, but recent studies have corroborated the importance of mucus in the envenomation process. Aiming to assess the mucus composition, a new non-invasive mucus collection method was developed that focused on peptides and proteins, and biological assays were performed to analyze the toxic and immune activities of the Hypanus americanus mucus. Pathophysiological characterization showed the presence of peptidases on the mucus, as well as the induction of edema and leukocyte recruitment in mice. The fractionated mucus improved phagocytosis on macrophages and showed antimicrobial activity against T. rubrumç. neoformans and C. albicans in vitro. The proteomic analyses showed the presence of immune-related proteins like actin, histones, hemoglobin, and ribosomal proteins. This protein pattern is similar to those reported for other fish mucus and stingray venoms. This is the first report depicting the Hypanus stingray mucus composition, highlighting its biochemical composition and importance for the stingray immune system and the possible role on the envenomation process.

Serological diagnosis of canine visceral leishmaniasis in Brazil: systematic review and meta-analysis.

OBJECTIVE: To evaluate the quality and accuracy of serological diagnosis of canine visceral leishmaniasis in the Americas. METHODS: A systematic review found original studies in the databases MEDLINE, EMBASE and LILACS up to November 2012 and in complementary sources up to February 2013. Studies were evaluated in accordance with QUADAS 2 and STARD parameters and recommended in accordance with GRADE parameters. Meta-analysis was carried out with Meta-DiSc software, using the random-effect model. RESULTS: Two hundred and eighty-four studies were identified, of which 25 met the inclusion criteria, comprising the final synthesis. All but one was conducted in Brazil, and only two were judged to be of good quality. 15 studies involving immuno-enzymatic tests with crude antigens (cELISA), 11 studies on indirect immunofluorescence tests (IFAT) and three on the immunochromatographic dual-path platform (DPP) test were meta-analysed. The combined results for sensitivity and specificity were cELISA: 0.89 (CI 95% 0.87-0.91) and 0.87 (CI 95% 0.86-0.88); IFAT: 0.88 (CI 95% 0.85-0.91) and 0.63 (CI 95% 0.61-0.65); and DPP: 0.83 (CI 95% 0.78-0.88) and 0.73 (CI 95% 0.70-0.75). CONCLUSION: Enzyme-linked immunosorbent assay with crude antigens and DPP tests have moderate accuracy for the diagnosis of canine visceral leishmaniasis, and the quality of the design, implementation and analysis of validation studies on diagnostic tests for this disease urgently require improvement. The recommendation for use of the evaluated tests is based on evidence that is scarce and restricted to Brazil.

Mycobacterium avium subspecies paratuberculosis: an insidious problem for the ruminant industry.

Mycobacterium avium subspecies paratuberculosis is considered as one of the most serious problems affecting the world's ruminant industry due to its significant impact on the global economy and the controversial issue that it may be pathogenic for humans. M. avium subspecies paratuberculosis is the causative agent of Johne's disease in animals and might be implicated in cases of human Crohn's disease. We provide an insight into M. avium subspecies paratuberculosis from some bacteriological, clinical, and molecular epidemiological perspectives.

[Detection of processed animal protein: European experience and perspectives]. / Détection des protéines animales transformées: expérience et perspectives européennes.

European Commission Regulation (EC) No. 152/2009 imposes optical microscopy as the reference method for official controls to detect traces of animal protein in animal feed. Since 1 July 2004, the one-solvent technique has been the only authorised variant of optical microscopy. Its detection limit is 0.1% of meat-and-bone meal. Other techniques--using molecular biology (polymerase chain reaction, immunology), microscopy or near-infrared imaging--have been developed in the past ten years to supplement the official method, which has certain limitations. This paper compares and discusses the different techniques, highlighting the strengths of each technique in order to propose a feasible control scheme to improve the sensitivity and specificity of the technique for the detection of processed animal protein in livestock feed.

Design and characterization of highly immunogenic heat-stable enterotoxin of enterotoxigenic Escherichia coli K99(+).

In this study, STa peptide of enterotoxigenic Escherichia coli K99(+) was purified and successfully covalently cross-linked to modified bovine serum albumin after thorough evaluation of three different hapten-carrier conjugation protocols. Dimethyformamide (DMF) based STa-conjugation protocol demonstrated higher biological activity (10×10(6) STa Total Mouse Units [MU]) and 100% conjugation efficiency. A range of conjugation ratio of 4-12 STa molecules per one molecule of BSA was achieved and confirmed by matrix-assisted laser desorption ionization-time of flight/mass spectroscopy (MALDI-TOF/MS). This conjugate was used for immunization of ten rabbits for STa antibody production. A high antibody binding titer (10(6)) against STa was obtained with a neutralization capacity of 3×10(4) STa MUs/ml serum. These levels of high STa binding and neutralizing antibodies titers propose the potential use of this conjugate for the development of immunotherapeutic reagents and/or STa-based vaccine against ETEC K99(+).

Identification of Brucella abortus genes in elk (Cervus elaphus) using in vivo-induced antigen technology (IVIAT) reveals novel markers of infection.

Elk in the Greater Yellowstone Area are a major reservoir for brucellosis, which represents an obstacle to eradication of the disease in domestic livestock. Furthermore, immune responses to Brucella abortus infection in the wild host are not well-understood. In this regard, in vivo-induced antigen technology (IVIAT) was employed to identify novel B. abortus antigens expressed during infection in elk. Sera collected from sero-positive Wyoming elk were pooled and absorbed against in vitro-grown cultures of B. abortus. Approximately 35,000 E. coli clones, expressing B. abortus DNA, were then screened by colony immunoblot, yielding ten genes with immuno-reactive products, to include seven proteins secreted beyond the inner membrane. Three products, an outer membrane protein (D15), malate dehydrogenase (Mdh), and an ion transporter (AfuA), were examined by Western blot against individual elk serum samples. Sero-reactivity was significantly more frequent for both Mdh and D15 in naturally infected animals, compared to vaccinated and uninfected elk, indicating that antibody to these two antigens is a predictor of natural infection. Cross-reactivity of all three proteins was next examined with serum samples from confirmed brucellosis-positive cattle. While variable patterns of reactivity were seen with the antigens, the sample group was equivalently reactive to AfuA and Mdh, compared to elk, suggesting that these antigens are commonly expressed during infection in both hosts. We conclude that the application of IVIAT to B. abortus may not only facilitate the identification of serologic markers for brucellosis in elk, but may provide further insight into biological processes of the pathogen in different hosts.

Evaluation of the Chagas Stat-Pak assay for detection of Trypanosoma cruzi antibodies in wildlife reservoirs.

An immunochromatographic assay (Chagas Stat-Pak) was evaluated for the detection of Trypanosoma cruzi antibodies in 4 species of wildlife reservoirs. Antibodies to T. cruzi were detected in raccoons (Procyon lotor) (naturally and experimentally infected) and degus (Octodon degu) (experimentally-infected) using the Chagas Stat-Pak. In naturally exposed wild raccoons, the Chagas Stat-Pak had a sensitivity and specificity of 66.7-80.0% and 96.3%, respectively. Compared with indirect immunofluorescent antibody assay results, seroconversion as determined by Chagas Stat-Pak was delayed for experimentally infected raccoons, but occurred sooner in experimentally infected degus. The Chagas Stat-Pak did not detect antibodies in naturally or experimentally infected Virginia opossums (Didelphis virginiana) or in experimentally infected short-tailed opossums (Monodelphis domestica). These data suggest that the Chagas Stat-Pak might be useful in field studies of raccoons and degus when samples would not be available for more-conventional serologic assays. Because this assay did not work on either species of marsupial, the applicability of the assay should be examined before it is used in other wild species.

Survey of dogs from Vietnam for antibodies to visceralizing Leishmania spp.

Cases of visceral leishmaniasis, one of the most neglected tropical diseases, are increasing globally. Dogs are considered an important reservoir host for visceral leishmaniasis in people. The first cases of human visceral leishmaniasis in Vietnam have recently been reported. Blood samples were collected from 41 dogs in rural Vietnam. Sera were examined for antibodies to visceralizing Leishmania spp. by canine immunochromatographic strip assays based on recombinant K39 antigen. Antibodies to Leishmania spp. were not detected in any of the dogs tested. Results from this study suggest that rural dogs are not likely to be involved in the emergence of human visceral leishmaniasis in Vietnam.

Construction and screening of a cDNA library from the triactinomyxon spores of Myxobolus cerebralis, the causative agent of salmonid whirling diseases.

The ZAP Express cDNA library was constructed using mRNA extracted from the triactinomyxon spores of Myxobolus cerebralis. First-strand cDNA was synthesized using Moloney Murine leukaemia virus reverse transcriptase. Following second-strand cDNA synthesis, the double-stranded cDNA was digested with Xho I restriction enzyme, cDNA fragments less than 400 bp were removed and the remaining cDNA was ligated with the lambda ZAP Express vector. The recombinants were packaged in vitro using Gigapack III gold packaging extract. The primary cDNA library titre contained 0.5 x 10(6) clones, with 97% recombinant and only 3% non-recombinant clones. The cDNA library was then screened using the anti-triactinomyxon antibodies. Positive clones were selected and re-screened twice more to give a final selection of 526 clones. One clone (46-5) was selected randomly and subjected to in vivo excision of the pBK-CMV phagemid from the ZAP express vector. The sequence of the entire clone was obtained using rapid amplification of the cDNA ends. A search of the clone sequence against GenBank revealed that it related to ribosomal protein L23 and it had a high percentage similarity to this protein from different species. A conserved domain for ribosomal protein L23 was also identified in the clone sequence.

Research and clinical aspects of immunology in Africa.

There are few immunologists in Africa. Researchers predominantly study the immunology of infectious diseases (HIV, malaria and tuberculosis), HLA genotypes and cytokine secretion patterns. Lack of research funding is the problem; continued, equitable international collaboration is a short-term answer. Sustainable development will come when African countries find ways of training and retaining scientists who will produce research and diagnostic tests. The Internet should be utilized to improve communication and as a conduit for online, virtual immunology courses.

Beta-hydroxybutyrate levels in peripheral blood and ketone bodies supplemented in culture media affect the in vitro chemotaxis of bovine leukocytes.

The role of ketone bodies on chemotactic capacities of leukocytes was characterized in two experiments. Experiment I was performed to investigate the association between serum beta-hydroxybutyrate concentrations (BHB) and in vitro chemotaxis of leukocytes. Cows were divided into low-BHB, medium-BHB, and high-BHB ones and classified according to their BHB. Leukocytes from high-BHB cows had a significantly lower chemotactic differential than leukocytes from low-BHB cows (p < 0.01). The effect of adding ketone bodies into in vitro chemotaxis cultures on leukocytes chemotaxis was studied in Experiment II. Either individual or a combination of commercial ketone bodies - sodium salts of BHB (BHBA), lithium salt of acetoacetate (ACAC), and acetone (Acetone) - were diluted in culture media and divided into eight concentrations corresponding to concentrations of bovine subclinical and clinical ketosis. For leukocytes from medium- and high-BHB cow, the chemotactic indexes of leukocytes were reduced by ACAC and Acetone. Chemotactic differentials of cultures with ACAC and acetone supplementation from both sources of leukocytes were significantly lower than that of the control culture (p < 0.05). For leukocytes from high-BHB cows, chemotactic indexes were suppressed in a ketone-body environment. In conclusion, leukocytes from naturally-occurring ketotic cows have lower chemotactic differentials than those from non-ketotic cows, and a chemotactic capacity indicated by a chemotactic differential is impaired when leukocytes migrate in an environment with ketone bodies in vitro.

[Serological studies in wild boars in the Veluwe area]. / Serologisch onderzoek bij wilde zwijnen op de Veluwe.

In the second part of 1994, blood samples from 115 wild swine are tested for gE-antibodies to Aujeszky Disease Virus. Three of the blood samples reacted positive. The tests for Classical Swine Fever and for Swine Vesicular Disease were negative in all samples. Perhaps the wild swine do not form a source of infection to the surrounding area with regard to these viral diseases but they may play a role as indicator for circulating viruses in the surrounding area.