Understanding the survival of Zika virus in a vector interconnected sexual contact network.

The recent outbreaks of the insect-vectored Zika virus have demonstrated its potential to be sexually transmitted, which complicates modeling and our understanding of disease dynamics. Autochthonous outbreaks in the US mainland may be a consequence of both modes of transmission, which affect the outbreak size, duration, and virus persistence. We propose a novel individual-based interconnected network model that incorporates both insect-vectored and sexual transmission of this pathogen. This model interconnects a homogeneous mosquito vector population with a heterogeneous human host contact network. The model incorporates the seasonal variation of mosquito abundance and characterizes host dynamics based on age group and gender in order to produce realistic projections. We use a sexual contact network which is generated on the basis of real world sexual behavior data. Our findings suggest that for a high relative transmissibility of asymptomatic hosts, Zika virus shows a high probability of sustaining in the human population for up to 3 months without the presence of mosquito vectors. Zika outbreaks are strongly affected by the large proportion of asymptomatic individuals and their relative transmissibility. The outbreak size is also affected by the time of the year when the pathogen is introduced. Although sexual transmission has a relatively low contribution in determining the epidemic size, it plays a role in sustaining the epidemic and creating potential endemic scenarios.

Evaluation of Fluorescence Microsphere Immunoassay for Detection of Antibodies to Rift Valley Fever Virus Nucleocapsid Protein and Glycoproteins.

Rift Valley fever virus (RVFV) is a mosquito-borne, zoonotic virus that infects ruminants, including cattle, sheep, goats, camels, and buffalo. Multiplexing diagnostic assays that can simultaneously detect antibodies against multiple RVFV antigens offer a high-throughput test for disease surveillance and vaccine evaluations. We describe the improvement and evaluation of a previously developed fluorescence microsphere immunoassay (FMIA) for the detection of IgG and IgM antibodies against the RVFV glycoprotein (Gn) and the immunogenic nucleocapsid protein (Np). Well-characterized vaccinated and experimentally infected ruminant sera were used for the evaluation of the assay. Recombinant viral proteins were produced and then coupled to polystyrene magnetic beads for analysis using the Luminex MAGPIX system with xMAP technology. The FMIA was performed in parallel with virus neutralization tests. Our results revealed the highest median fluorescence intensity (MFI) values for the detection of IgG antibodies against RVFV Np, indicating that this antigen would be a good candidate for a screening assay. The Np and Gn targets could differentiate infected animals from animals vaccinated with a candidate subunit vaccine formulation based on the RVFV Gn and Gc proteins. The results presented in this report demonstrate that FMIA provides a rapid and robust serological diagnostic tool for the detection of antibodies against RVFV. The targets developed in this assay provide the basis for the development of a companion diagnostic test for an RVFV Gn/Gc subunit vaccine that is capable of differentiating infected from vaccinated animals (DIVA), as well as a multiplex serodiagnostic assay that can simultaneously screen for several ruminant diseases.

Spatial multi-criteria decision analysis for modelling suitable habitats of Ornithodoros soft ticks in the Western Palearctic region.

Ticks are economically and medically important ectoparasites due to the injuries inflicted through their bite, and their ability to transmit pathogens to humans, livestock, and wildlife. Whereas hard ticks have been intensively studied, little is known about soft ticks, even though they can also transmit pathogens, including African Swine Fever Virus (ASFV) affecting domestic and wild suids or Borrelia bacteria causing tick-borne relapsing fever (TBRF) in humans. We thus developed a regional model to identify suitable spatial areas for a community of nine Ornithodoros tick species (O. erraticus, O. sonrai, O. alactagalis, O. nereensis, O. tholozani, O. papillipes, O. tartakovskyi, O. asperus, O. verrucosus), which may be of medical and veterinary importance in the Western Palearctic region. Multi-Criteria Decision Analysis was used due to the relative scarcity of high-quality occurrence data. After an in-depth literature review on the ecological requirements of the selected tick community, five climate-related factors appeared critical for feeding activity and tick development: (i) a spring temperature exceeding 10°C to induce the end of winter soft tick quiescent period, (ii) a three-months summer temperature above 20°C to allow tick physiological activities, (iii) annual precipitation ranging from 60mm to 750mm and, in very arid areas, (iv) dry seasons interrupted by small rain showers to maintain minimum moisture inside their habitat along the year or (v) residual water provided by perennial rivers near habitats. We deliberately chose not to include biological factors such as host availability or vegetation patterns. A sensitivity analysis was done by performing multiple runs of the model altering the environmental variables, their suitability function, and their attributed weights. To validate the models, we used 355 occurrence data points, complemented by random points within sampled ecoregions. All models indicated suitable areas in the Mediterranean Basin and semi-desert areas in South-West and Central Asia. Most variability between models was observed along northern and southern edges of highly suitable areas. The predictions featured a relatively good accuracy with an average Area Under Curve (AUC) of 0.779. These first models provide a useful tool for estimating the global distribution of Ornithodoros ticks and targeting their surveillance in the Western Palearctic region.

Functional Validation of Apoptosis Genes IAP1 and DRONC in Midgut Tissue of the Biting Midge Culicoides sonorensis (Diptera: Ceratopogonidae) by RNAi.

Culicoides biting midges transmit multiple ruminant viruses, including bluetongue virus and epizootic hemorrhagic disease virus, causing significant economic burden worldwide. To further enhance current control techniques, understanding vector-virus interactions within the midge is critical. We developed previously a double-stranded RNA (dsRNA) delivery method to induce RNA interference (RNAi) for targeted gene knockdown in adult Culicoides sonorensis Wirth & Jones. Here, we confirm the C. sonorensis inhibitor of apoptosis 1 (CsIAP1) as an anti-apoptotic functional ortholog of IAP1 in Drosophila, identify the ortholog of the Drosophila initiator caspase DRONC (CsDRONC), and demonstrate that injection of dsRNA into the hemocoel can be used for targeted knockdown in the midgut in C. sonorensis. We observed CsIAP1 transcript reduction in whole midges, with highest transcript reduction in midgut tissues. IAP1knockdown (kd) resulted in pro-apoptotic caspase activation in midgut tissues. In IAP1kd midges, midgut tissue integrity and size were severely compromised. This phenotype, as well as reduced longevity, was partially reverted by co-RNAi suppression of CsDRONC and CsIAP1. Therefore, RNAi can be directed to the midgut of C. sonorensis, the initial site of virus infection, using dsRNA injection into the hemocoel. In addition, we provide evidence that the core apoptosis pathway is conserved in C. sonorensis and can be experimentally activated in the midgut to reduce longevity in C. sonorensis. This study thus paves the way for future reverse genetic analyses of midgut-virus interactions in C. sonorensis, including the putative antiviral properties of RNAi and apoptosis pathways.

Lesser-known bunyavirus infections.

This paper reviews less well-known or less widely distributed viruses of the Bunyaviridae family that are nonetheless of significant veterinary and public health concern. These include: Cache Valley fever, Main Drain, Ingwavuma, Bhanja and Heartland viruses. A description of the agents, clinical signs of infection, epidemiology, and insect transmission is provided for each, and the authors discuss current diagnostic strategies plus the lack of control measures.

West Nile virus.

This review covers the basic biology of the West Nile virus and the host-vector-pathogen interactions that result in significant disease in wild birds, horses and humans. The review describes the basic properties of the virus, cellular infection and the pathogenesis of the disease, and the ecology of virus maintenance, amplification and transmission. Disease epidemiology and risk estimation strategies that are currently in use are also examined, and host immune responses and vaccination practices described. The principles of vector control, exposure control and long-term risks caused by climatic and habitat factors are also included.

Diagnostic approaches for Rift Valley fever.

Disease outbreaks caused by arthropod-borne animal viruses (arboviruses) resulting in significant livestock and economic losses world-wide appear to be increasing. Rift Valley fever (RVF) virus is an important arbovirus that causes lethal disease in cattle, camels, sheep and goats in Sub-Saharan Africa. There is concern that this virus could spread because of global warming, increased animal trade or through bioterrorism. This paper discusses the current and developing approaches to diagnosis of RVF. Diagnostic assays are available for RVF, but availability can be limited and there is a need for global harmonization. Continued improvement of standard serological and viral genome amplification approaches, including new embedded/syndromic testing, biosensor, emerging virus detection and characterization technologies is needed.

Inflammatory markers in bronchoalveolar lavage fluid of standardbred racehorses with inflammatory airway disease: response to interferon-alpha.

Protein and eicosanoid concentrations and procoagulant activity were determined in bronchoalveolar lavage fluid (BALF) from 32 Standardbred racehorses with inflammatory airway disease (IAD) and 6 control horses. Total protein, albumin and immunoglobulin G (IgG) concentrations were high (P < 0.05) in the BALF from horses with IAD, a finding consistent with exudation of plasma protein into the airway. Immunoglobulin A (IgA) concentrations also were increased (P < 0.05) which may signify local immunoglobulin production. Difference was not detected in prostaglandin E2 and 6-ketoprostaglandin F1 alpha concentrations in BALF of IAD-affected and control horses. Procoagulant activity was identified in the majority (66%) of BALF samples from IAD-affected horses and was not detected in control horses. Natural human interferon-alpha (nHulFN alpha) (placebo, 50, 150, or 450 units) was administered orally for 5 days to IAD-affected horses in a double-blind, randomised block design. Total protein, IgG, and IgA concentrations in BALF were reduced (P < 0.05) 8 days after administration of 50 u and 150 u nHuIFN alpha, and 15 days after administration of 50 u nHuIFN alpha. Procoagulant activity and albumin concentrations in BALF were lower 8 days after administration of 50 u nHuIFN alpha. Oral administration of low-dose nHuIFN alpha appeared to ameliorate these parameters of lower respiratory tract inflammation in Standardbred racehorses with IAD.

Antibacterial activity of a synthetic peptide (PR-26) derived from PR-39, a proline-arginine-rich neutrophil antimicrobial peptide.

PR-39 is a proline-arginine-rich (PR) neutrophil antibacterial peptide originally identified and purified from the porcine small intestine. We report on the synthesis of a functional antibacterial domain of PR-39, the first 26 amino acid residues of the NH2 terminus. PR-26 was as potent as or more potent than PR-39 against enteric gram-negative bacteria. This truncated form of PR-39 potentiated neutrophil phagocytosis of Salmonella choleraesuis and decreased the level of S. typhimurium invasion into intestinal epithelial cells. Scanning electron microscopy confirmed that these peptides did not lyse cells by pore-forming mechanisms; however, they potentiated the antibacterial capabilities of a pore-forming peptide, magainin A. In addition, PR-26 was not toxic to epithelial cells at concentrations several times greater than its bactericidal concentration. These data suggest that PR-39 and its functional domain, PR-26, may potentiate the host's defense capabilities against gram-negative infections.

Development and characterization of a flow cytometric assay for detection of platelet-bound immunoglobulin G in dogs.

OBJECTIVE: To develop a flow cytometric assay for detection of platelet-bound IgG in dogs. SAMPLE POPULATION: Negative-control platelets were obtained from 5 clinically normal Greyhounds. Positive-control platelets were platelets from 1 clinically normal dog, sensitized with dog anti-canine platelet alloantibodies. PROCEDURE: Washed platelets were incubated with mouse anti-canine IgG conjugated to fluorescein isothiocyanate and analyzed by flow cytometry. Optimal dilution of antibody reagent and dose-response were determined, as were effects on platelet-bound IgG detection of storage time and temperature of K3EDTA-anticoagulated blood samples, variable platelet numbers, and variable filling of K3EDTA evacuated tubes. RESULTS: A 1:128 dilution of antibody reagent was optimal. There was a linear increase in platelet-bound IgG when normal canine platelets were incubated with increasing concentrations of positive-control serum. Variable numbers of positive-control platelets tested and variable filling of K3EDTA evacuated tubes had no significant effect on platelet-bound IgG concentration. Platelet-bound IgG concentration increased with storage time at room temperature (P = 0.0003), but not when blood was kept cool. Sufficient platelets for assay were able to be isolated from 3 ml of blood from 5 dogs with < 10,000 platelets/microliters. CONCLUSIONS: This assay for platelet-bound IgG in dogs is simple, repeatable, and practical. The assay is not affected by platelet count or variable filling of evacuated tubes, and requires only 3 ml of K3EDTA-anticoagulated blood. Blood samples for testing require packaging on ice and overnight delivery but, after arrival at the laboratory, can be refrigerated and analyzed within 72 hours of collection. CLINICAL RELEVANCE: Assays for platelet-bound IgG may help in assessing causes and treatment of thrombocytopenia.

Vaccination of racing greyhounds: effects on humoral and cellular immunity.

Greyhound kennel owners frequently employ multiple vaccination schedules in an attempt to reduce financial losses incurred as a result of infectious diseases. In order to determine the effects of multiple vaccination schedules on the immune system of racing greyhounds, three litters of greyhound pups raised in laboratory conditions were divided into two groups and subjected to either a maximum or a minimum vaccination schedule. Blood samples were collected biweekly for 6 months beginning at 2 weeks of age and analyzed to establish 'baseline' values for the lymphatic system of greyhounds. Lymphocyte transformation, total and differential leukocyte counts, and flow cytometry were used to evaluate cellular immunity. Humoral immunity was evaluated using serum neutralization and hemagglutination inhibition tests. Proliferation of peripheral blood lymphocytes in response to the mitogen concanavalin A (Con A) was higher for the maximum vaccination groups. The frequency distribution of circulating CD4 and IgG labeled lymphocytes was higher in the minimum vaccination groups. A significant treatment by time interaction in CD4, IgG, and IgM labeled cells was observed, This interaction, however, was not significant at any point in time for CD4 and IgG labeled cells. The percentage of lymphocytes expressing surface IgM was significantly higher in the minimum vaccination groups at 10 and 14 weeks of age. No significant differences were detected in humoral immunity between the maximum and minimum groups of each litter. Results of this study indicate that maximum vaccination schedules do not appear to be more effective or more immunosuppressive than minimum vaccination schedules.

Dietary modulation of Kupffer cell and splenocyte function during a Salmonella typhimurium challenge in mice.

Oils from cold-water fish are rich in (n-3) polyunsaturated fatty acids, in particular eicosapentaenoic acid (20:5) and docosahexaenoic acid (22:6). Although those fatty acids are beneficial in the prevention of cardiac disease and have anti-inflammatory properties, they can also decrease survival rates of mice during challenges with food-borne pathogens. This study was designed to determine dietary fat effects on Kupffer cells and splenocytes during a Salmonella typhimurium challenge. Mice were fed a low corn oil diet (3%, control), high corn oil diet (20%, HCO), or a menhaden fish oil diet (17% + 3% corn oil, FO) for 28 days and then orally given 3.1 x 10(8) colony-forming units of S. typhimurium. Kupffer cells and splenocytes were separated immediately prior to and on days 6, 10, and 14 postchallenge. Fish oil decreased Kupffer cell phagocytosis and oxidative burst early in the infection and adhesion molecule (CD18) expression at the end of the infection. In splenocytes, fish oil affected Ia expression prior to and late in the infection and depressed CD18 expression late in the infection. These data suggest that the diet affected Kupffer cells most early in the infection but affected splenocytes primarily later in the infection. Therefore, because the greatest death rate during an S. typhimurium infection occurs early, the reduced function of the Kupffer cells is probably a major factor.

Lipopolysaccharide modulation of a CD14-like molecule on porcine alveolar macrophages.

Cluster of differentiation antigen 14 (CD14) functions as a receptor for lipopolysaccharide (LPS) LPS-binding protein (LBP) complexes. Because LPS has varying effects on CD14 expression in vitro, we evaluated CD14 expression in response to LPS with a fully differentiated macrophage phenotype, the alveolar macrophage. By using flow microfluorometric analysis and a radioimmunoassay with an anti-human CD14 monoclonal antibody (My4) that cross-reacts with porcine CD14, we found that macrophages stimulated with LPS for 24 h exhibited a two- to fivefold increase in CD14-like antigen compared with unstimulated cells. At low concentrations of LPS, up-regulation of the CD14-like antigen was dependent on serum; at higher concentrations of LPS, serum was not required. In the absence of serum a 10-fold higher dose of LPS (10 ng/ml) was required to increase CD14-like expression. In addition, LPS-induced CD14-like up-regulation correlated with secretion of tumor necrosis factor-alpha, regardless of serum concentration. Blockade with My4 antibody significantly inhibited LPS-induced tumor necrosis factor-alpha secretion at 1 ng/ml of LPS. However, inhibition decreased as we increased the LPS concentration, suggesting the existence of CD14-independent pathways of macrophage activation in response to LPS. Alternatively, My4 may have a lower affinity for the porcine CD14 antigen than LPS, which may have only partially blocked the LPS-LBP binding site at high concentrations of LPS. Therefore, these data suggest that LPS activation of porcine alveolar macrophages for 24 h increased CD14-like receptor expression. The degree of CD14-like up-regulation was related to LPS concentration, however, activation did not require the presence of serum at high concentrations of LPS.

Influence of recombinant bovine interleukin-1 beta and interleukin-2 in pigs vaccinated and challenged with Streptococcus suis.

An experiment was conducted to determine the adjuvanticity of recombinant bovine IL-1 beta (rBoIL-1 beta) and recombinant bovine IL-2 (rBoIL-2) administered in conjunction with a single Streptococcus suis vaccination in pigs. Sixty 4-week-old pigs were allotted to eight groups: nonvaccinated controls; vaccinated controls; rBoIL-beta at 0.1, 1, and 10 micrograms kg-1; rBoIL-2 at 2.5, 25, and 250 micrograms kg-1. All pigs (except nonvaccinated controls) were vaccinated on Day 0 with a commercial Streptococcus suis vaccine (serotypes 1 and 2). At vaccination, pigs were injected intramuscularly with their respective cytokine treatments. Pigs received additional cytokine injections on 2 consecutive days. On Day 21, all pigs were challenged intravenously with 3.2 x 10(9) colony forming units of a log phase culture of S. suis (serotype 2). The highest dose of rBoIL-1 beta exceeded the maximum tolerable dose for the cytokine; however, this dose of rBoIL-1 beta protected pigs from the S. suis challenge. Pigs administered rBoIL-1 beta at 10 micrograms kg-1 had higher antibody responses to S. suis, less severe clinical signs of the disease after challenge, better growth performance during the infection, and less severe gross pathological lesions caused by the bacteria. No pigs in this treatment group died from the bacterial challenge. These data suggest that rBoIL-1 beta (10 micrograms kg-1), administered intramuscularly for 3 consecutive days at vaccination, is more effective than a single S. suis vaccination alone in protecting pigs against a S. suis challenge.

Workshop studies on monoclonal antibodies reactive against porcine myeloid cells.

Investigators from eight laboratories analyzed the reactivity of 22 monoclonal antibodies (mAb) against porcine myeloid cells. Based on binding data, clustering analysis and inhibition studies, workshop mAb 74-22-15 (003) and 6F3 (007) were assigned a swine workshop cluster number 3 (SWC3). These mAb recognized macrophages and neutrophils; neutrophils; a monocyte/macrophage-specific mAb was not identified by this workshop.

Competitive binding analysis of monoclonal antibodies reactive with porcine alveolar macrophages using anti-CD14 and anti-CD18.

Four monoclonal antibodies (mAb) from the myeloid subset panel of the First International Swine CD Workshop (74-22-15, DH59B, PM16-6, and MUC21A) were analyzed using competitive inhibition studies with anti-human CD14 (My4) and anti-human/anti-porcine CD18 (MHM23) on porcine alveolar macrophages. Results suggested that none of the mAb tested recognized CD14 or CD18 on porcine alveolar macrophages. Additionally, the cross-reactivity of My4 with porcine alveolar macrophages was established.

Influence of interleukin-1 on neutrophil function and resistance to Streptococcus suis in neonatal pigs.

Nonspecific immunity is usually lower in neonates than adults. Consequently, enhancing the neonate's nonspecific immune capability may be beneficial for the health and growth performance of young animals. We conducted two experiments in which neonatal pigs were injected with recombinant bovine interleukin-1 beta (rBoIL-1 beta) at 9 to 11 days of age. Three consecutive daily injections of rBoIL-1 beta increased neutrophil and monocyte numbers, which remained elevated until the animals were challenged with Streptococcus suis at 19 days of age. Neutrophil bactericidal activity was greater in interleukin-1-treated pigs than in saline-injected controls. At lower ratios of effector to target cells, neutrophil-mediated, antibody-dependent cellular cytotoxicity was increased in neonates treated with IL-1. However, natural killer cell activity and neutrophil production of superoxide anion were not affected by treatment with IL-1. Expression of CD18 was increased transiently on neutrophils from IL-1-treated pigs at 15 days of age. Severity of the streptococcal infection was less in pigs that were treated with IL-1 at 9 to 11 days of age. These data suggest that IL-1 treatment in neonates may augment nonspecific immune function and disease resistance.

Primary immunodeficiencies of food animals.

Although there are few, well-characterized PIDs of food animals, these diseases are important because they tend to be severe and with no cure. Most animals with PID do not receive the intensive and aggressive care required for survival: Veterinarians may be consulted only when the animals are in the terminal stages of illness; it is generally not economically practical for livestock producers or practitioners to pay for the exhaustive laboratory tests required to detect and characterize these anomalies. Another reason for the small numbers of characterized clinical cases of PID is that they are rare. It is possible, however, that intensive artificial insemination and embryo transfer could select for heterozygous carriers of these autosomal traits. As seen with bovine leukocyte adhesion deficiency, as the frequency of an allele increases in the population, the numbers of affected animals increase. Furthermore, other immunodeficient syndromes are likely to exist. Veterinarians therefore should be aware of these disorders and should seek laboratory assistance to arrive at a correct diagnosis. Because of the inheritable nature of PID, livestock producers need assistance from veterinarians to identify carriers and establish sound breeding and control programs. One positive outcome from studies of PID is that research scientists and veterinarians learn much about immune systems from these afflicted animals. In fact, these animals may become models for gene therapy or marrow reconstruction procedures.

Immunotoxicity in the bovine animal: a review.

The immune system is a complex biological system involving cellular defenses as well as endogenous and exogenous factors. The potential for immune system exposure to immunotoxins in the environment is well documented. However, caution should be exercised when extrapolating meaningful conclusions from experimental data to assess risk factors to the bovine. Mycotoxins alter immune-mediated activities in cattle and are major immunotoxic risks. Another risk to the bovine is lead. Poor disposal of pollutants in the environment enhances this risk factor. One of the most controllable risk factors is the administration of immunotoxic drugs and biologics. Most of these compounds have minimal immunotoxic activity at recommended dosages. The extra-label use of drugs resulting in super-therapeutic concentrations has increased the probability that certain drugs may act immunotoxicologically.

Effects of antiorthostatic suspension and corticosterone on macrophage and spleen cell function.

The purpose of this study was to determine whether antiorthostatic suspension of C3HeB/FeJ mice for a period of 11 days affected macrophage and spleen cell function. We found that antiorthostatic suspension did not alter macrophage secretion of prostaglandin E2, tumor necrosis factor alpha, and interleukin-1. Antiorthostatic suspension also did not affect macrophage-mediated contact-dependent cytotoxicity, TNF-mediated cytotoxicity, expression of class II histocompatibility molecules, or concanavalin A and Bandeiraea simplicifolia lectin binding sites. The proliferative response of splenic T cells in response to mitogens and staphylococcal exotoxins was significantly enhanced in antiorthostatically suspended mice. We detected significantly higher concentrations of corticosterone in the plasma of antiorthostatically suspended mice. Therefore, there did not appear to be any direct immunosuppressive effects of corticosterone on the parameters tested.