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Background Procalcitonin (PCT) is an important biomarker for sepsis in human medicine, but there is little information regarding PCT as a biomarker for sepsis in dogs. There are no controlled studies evaluating serial concentrations of PCT in dogs. Hypothesis/objective That PCT would be rapidly detectable in serum after injection of LPS and would remain increased for at least 24 hours. Objective was to evaluate serial serum PCT concentrations in dogs after a single IV injection of LPS compared to placebo. Animals Six healthy mixed breed dogs. Methods A nonrandomized, placebo-controlled, crossover study was performed. Dogs were initially injected with placebo (0.9% NaCl; 1 mL, IV) and then experimental endotoxemia was induced by injecting lipopolysaccharide (LPS; 2 μg/kg, IV, once) after a 5-day washout period. Serial blood samples were collected for measurement of serum PCT after each injection. Difference in median PCT concentration between serial time points was assessed using a mixed effects model. Results After LPS administration, blood pressure decreased and body temperature increased along with the development of lethargy, vomiting, and diarrhea. Procalcitonin was significantly increased compared to baseline by 2 hours after injection of LPS (median = 67.9 versus 172.8, range = 46.0-74.1 versus 99.5-295.9, P = .0002) and remained significantly increased for 12 hours (median = 205.9, range = 119.9-297.4) with return to baseline by 48 hours. Procalcitonin was significantly higher than placebo 2, 4, 6, 8, 10, 12, and 24 hours after injection. There were no significant differences in PCT between time 0 and any of the subsequent time points in the saline group. Conclusions and clinical importance Procalcitonin expression is likely to be a clinically useful biomarker for sepsis in dogs and might have an additional role in prognostication and therapeutic decision-making.

Diagnosis, treatment, and prevention of vector-borne disease (VBD) in pets is one cornerstone of companion animal practices. Veterinarians are facing new challenges associated with the emergence, reemergence, and rising incidence of VBD, including heartworm disease, Lyme disease, anaplasmosis, and ehrlichiosis. Increases in the observed prevalence of these diseases have been attributed to a multitude of factors, including diagnostic tests with improved sensitivity, expanded annual testing practices, climatologic and ecological changes enhancing vector survival and expansion, emergence or recognition of novel pathogens, and increased movement of pets as travel companions. Veterinarians have the additional responsibility of providing information about zoonotic pathogen transmission from pets, especially to vulnerable human populations: the immunocompromised, children, and the elderly. Hindering efforts to protect pets and people is the dynamic and ever-changing nature of VBD prevalence and distribution. To address this deficit in understanding, the Companion Animal Parasite Council (CAPC) began efforts to annually forecast VBD prevalence in 2011. These forecasts provide veterinarians and pet owners with expected disease prevalence in advance of potential changes. This review summarizes the fidelity of VBD forecasts and illustrates the practical use of CAPC pathogen prevalence maps and forecast data in the practice of veterinary medicine and client education.

The vertebrate immune response comprises multiple molecular and cellular components that interface to provide defense against pathogens. Because of the dynamic complexity of the immune system and its interdependent innate and adaptive functionality, an understanding of the whole-organism response to pathogen exposure remains unresolved. Zebrafish larvae provide a unique model for overcoming this obstacle, because larvae are protected against pathogens while lacking a functional adaptive immune system during the first few weeks of life. Zebrafish larvae were exposed to immune agonists for various lengths of time, and a microarray transcriptome analysis was executed. This strategy identified known immune response genes, as well as genes with unknown immune function, including the E3 ubiquitin ligase tripartite motif-9 (Trim9). Although trim9 expression was originally described as “brain specific,” its expression has been reported in stimulated human Mϕs. In this study, we found elevated levels of trim9 transcripts in vivo in zebrafish Mϕs after immune stimulation. Trim9 has been implicated in axonal migration, and we therefore investigated the impact of Trim9 disruption on Mϕ motility and found that Mϕ chemotaxis and cellular architecture are subsequently impaired in vivo. These results demonstrate that Trim9 mediates cellular movement and migration in Mϕs as well as neurons.

Lyme disease (LD) is the most common vector-borne disease in the United States. Early confirmatory diagnosis remains a challenge, while the disease can be debilitating if left untreated. Further, the decision to test is complicated by under-reporting, low positive predictive values of testing in non-endemic areas and travel, which together exacerbate the difficulty in identification of newly endemic areas or areas of emerging concern. Spatio-temporal analyses at the national scale are critical to establishing a baseline human LD risk assessment tool that would allow for the detection of changes in these areas. A well-established surrogate for human LD incidence is canine LD seroprevalence, making it a strong candidate covariate for use in such analyses. In this paper, Bayesian statistical methods were used to fit a spatio-temporal spline regression model to estimate the relationship between human LD incidence and canine seroprevalence, treating the latter as an explanatory covariate. A strong non-linear monotonically increasing association was found. That is, this analysis suggests that mean incidence in humans increases with canine seroprevalence until the seroprevalence in dogs reaches approximately 30%. This finding reinforces the use of canines as sentinels for human LD risk, especially with respect to identifying geographic areas of concern for potential human exposure.

Background Dogs in the United States are hosts to a diverse range of vector-borne pathogens, several of which are important zoonoses. This paper describes factors deemed to be significantly related to the prevalence of antibodies to Ehrlichia spp. in domestic dogs, including climatic conditions, geographical factors, and societal factors. These factors are used in concert with a spatio-temporal model to construct an annual seroprevalence forecast. The proposed method of forecasting and an assessment of its fidelity are described. Methods Approximately twelve million serological test results for canine exposure to Ehrlichia spp. were used in the development of a Bayesian approach to forecast canine infection. Data used were collected on the county level across the contiguous United States from routine veterinary diagnostic tests between 2011–2015. Maps depicting the spatial baseline Ehrlichia spp. prevalence were constructed using Kriging and head-banging smoothing methods. Data were statistically analyzed to identify factors related to antibody prevalence via a Bayesian spatio-temporal conditional autoregressive (CAR) model. Finally, a forecast of future Ehrlichia seroprevalence was constructed based on the proposed model using county-level data on five predictive factors identified at a workshop hosted by the Companion Animal Parasite Council and published in 2014: annual temperature, percentage forest coverage, percentage surface water coverage, population density and median household income. Data were statistically analyzed to identify factors related to disease prevalence via a Bayesian spatio-temporal model. The fitted model and factor extrapolations were then used to forecast the regional seroprevalence for 2016. Results The correlation between the observed and model-estimated county-by-county Ehrlichia seroprevalence for the five-year period 2011–2015 is 0.842, demonstrating reasonable model accuracy. The weighted correlation (acknowledging unequal sample sizes) between 2015 observed and forecasted county-by-county Ehrlichia seroprevalence is 0.970, demonstrating that Ehrlichia seroprevalence can be forecasted accurately. Conclusions The forecast presented herein can be an a priori alert to veterinarians regarding areas expected to see expansion of Ehrlichia beyond the accepted endemic range, or in some regions a dynamic change from historical average prevalence. Moreover, this forecast could potentially serve as a surveillance tool for human health and prove useful for forecasting other vector-borne diseases.

This paper forecasts the 2016 canine Anaplasma spp. seroprevalence in the United States from eight climate, geographic and societal factors. The forecast’s construction and an assessment of its performance are described. The forecast is based on a spatial-temporal conditional autoregressive model fitted to over 11 million Anaplasma spp. seroprevalence test results for dogs conducted in the 48 contiguous United States during 2011–2015. The forecast uses county-level data on eight predictive factors, including annual temperature, precipitation, relative humidity, county elevation, forestation coverage, surface water coverage, population density and median household income. Non-static factors are extrapolated into the forthcoming year with various statistical methods. The fitted model and factor extrapolations are used to estimate next year’s regional prevalence. The correlation between the observed and model-estimated county-by-county Anaplasma spp. seroprevalence for the five-year period 2011–2015 is 0.902, demonstrating reasonable model accuracy. The weighted correlation (accounting for different sample sizes) between 2015 observed and forecasted county-by-county Anaplasma spp. seroprevalence is 0.987, exhibiting that the proposed approach can be used to accurately forecast Anaplasma spp. seroprevalence. The forecast presented herein can a priori alert veterinarians to areas expected to see Anaplasma spp. seroprevalence beyond the accepted endemic range. The proposed methods may prove useful for forecasting other diseases.

Bleeding heterogeneity amongst patients with immune thrombocytopenia (ITP) is poorly understood. Platelets play a role in maintaining endothelial integrity, and variable thrombocytopenia-induced endothelial changes may influence bleeding severity. Platelet-derived endothelial stabilizers and markers of endothelial integrity in ITP are largely underexplored. We hypothesized that, in a canine ITP model, thrombocytopenia would lead to alterations in the endothelial ultrastructure and that the Von Willebrand factor (vWF) would serve as a marker of endothelial injury associated with thrombocytopenia. Thrombocytopenia was induced in healthy dogs with an antiplatelet antibody infusion; control dogs received an isotype control antibody. Cutaneous biopsies were obtained prior to thrombocytopenia induction, at platelet nadir, 24 hours after nadir, and on platelet recovery. Cutaneous capillaries were assessed by electron microscopy for vessel thickness, the number of pinocytotic vesicles, the number of large vacuoles, and the number of gaps between cells. Pinocytotic vesicles are thought to represent an endothelial membrane reserve that can be used for repair of damaged endothelial cells. Plasma samples were assessed for vWF. ITP dogs had significantly decreased pinocytotic vesicle numbers compared to control dogs (P = 0.0357) and the increase in plasma vWF from baseline to 24 hours correlated directly with the endothelial large vacuole score (R = 0.99103; P 0.0001). This direct correlation between plasma vWF and the number of large vacuoles, representing the vesiculo-vacuolar organelle (VVO), a permeability structure, suggests that circulating vWF could serve as a biomarker for endothelial alterations and potentially a predictor of thrombocytopenic bleeding. Overall, our results indicate that endothelial damage occurs in the canine ITP model and variability in the degree of endothelial damage may account for differences in the bleeding phenotype among patients with ITP.

Background Serum N-terminal pro-C-natriuretic peptide (NT-proCNP) concentration at hospital admission has sufficient sensitivity and specificity to differentiate naturally occurring sepsis from nonseptic systemic inflammatory response syndrome (SIRS). However, little is known about serum NT-proCNP concentrations in dogs during the course of sepsis. Objective To determine serum NT-proCNP and cytokine kinetics in dogs with endotoxemia, a model of canine sepsis. Samples Eighty canine serum samples. Methods Eight healthy adult Beagles were randomized to receive Escherichia coli lipopolysaccharide (LPS, 5 μg/kg) or placebo (0.9% NaCl) as a single IV dose in a randomized crossover study. Serum collected at 0, 1, 2, 4, and 24 hours was stored at −80°C for batch analysis. Serum NT-proCNP was measured by ELISA and 13 cytokines and chemokines by multiplex magnetic bead-based assay. Results Serum NT-proCNP concentrations did not differ significantly between LPS- and placebo-treated dogs at any time. When comparing serum cytokine concentrations, LPS-treated dogs had higher interleukin-6 (IL-6), IL-10, TNF-α and KC-like at 1, 2, and 4 hours; higher CCL2 at 1, 2, 4, and 24 hours; and higher IL-8 and CXCL10 at 4 hours compared to placebo-treated dogs. There were no differences in serum GM-CSF, IFN-γ, IL-2, IL-7, IL-15 or IL-18 between LPS- and placebo-treated dogs. Conclusions and Clinical Importance Serum NT-proCNP concentration does not change significantly in response to LPS administration in healthy dogs. Certain serum cytokine and chemokine concentrations are significantly increased within 1–4 hours after LPS administration and warrant further investigation as tools for the detection and management of sepsis in dogs.

Canine immune thrombocytopenia (ITP) is analogous to human ITP, with similar platelet counts and heterogeneity in bleeding phenotype among affected individuals. With a goal of ultimately investigating this bleeding heterogeneity, a canine model of antibody-mediated ITP was developed. Infusion of healthy dogs with 2F9, a murine IgG2a monoclonal antibody to the canine platelet glycoprotein GPIIb (a common target of autoantibodies in ITP) resulted in profound, dose-dependent thrombocytopenia. Model dogs developed variable bleeding phenotypes, e.g. petechiae and haematuria, despite similar degrees of thrombocytopenia. 2F9 infusion was not associated with systemic inflammation, consumptive coagulopathy, or impairment of platelet function. Unexpectedly however, evaluation of cytokine profiles led to the identification of platelets as a potential source of serum interleukin-8 (IL8) in dogs. This finding was confirmed in humans with ITP, suggesting that platelet IL8 may be a previously unrecognized modulator of platelet-neutrophil crosstalk. The utility of this model will allow future study of bleeding phenotypic heterogeneity including the role of neutrophils and endothelial cells in ITP.

This paper models the prevalence of antibodies to Borrelia burgdorferi in domestic dogs in the United States using climate, geographic, and societal factors. We then use this model to forecast the prevalence of antibodies to B. burgdorferi in dogs for 2016. The data available for this study consists of 11,937,925 B. burgdorferi serologic test results collected at the county level within the 48 contiguous United States from 2011-2015. Using the serologic data, a baseline B. burgdorferi antibody prevalence map was constructed through the use of spatial smoothing techniques after temporal aggregation; i.e., head-banging and Kriging. In addition, several covariates purported to be associated with B. burgdorferi prevalence were collected on the same spatio-temporal granularity, and include forestation, elevation, water coverage, temperature, relative humidity, precipitation, population density, and median household income. A Bayesian spatio-temporal conditional autoregressive (CAR) model was used to analyze these data, for the purposes of identifying significant risk factors and for constructing disease forecasts. The fidelity of the forecasting technique was assessed using historical data, and a Lyme disease forecast for dogs in 2016 was constructed. The correlation between the county level model and baseline B. burgdorferi antibody prevalence estimates from 2011 to 2015 is 0.894, illustrating that the Bayesian spatio-temporal CAR model provides a good fit to these data. The fidelity of the forecasting technique was assessed in the usual fashion; i.e., the 2011-2014 data was used to forecast the 2015 county level prevalence, with comparisons between observed and predicted being made. The weighted (to acknowledge sample size) correlation between 2015 county level observed prevalence and 2015 forecasted prevalence is 0.978. A forecast for the prevalence of B. burgdorferi antibodies in domestic dogs in 2016 is also provided. The forecast presented from this model can be used to alert veterinarians in areas likely to see above average B. burgdorferi antibody prevalence in dogs in the upcoming year. In addition, because dogs and humans can be exposed to ticks in similar habitats, these data may ultimately prove useful in predicting areas where human Lyme disease risk may emerge.

Background This paper forecasts next year’s canine heartworm prevalence in the United States from 16 climate, geographic and societal factors. The forecast’s construction and an assessment of its performance are described. Methods The forecast is based on a spatial-temporal conditional autoregressive model fitted to over 31 million antigen heartworm tests conducted in the 48 contiguous United States during 2011–2015. The forecast uses county-level data on 16 predictive factors, including temperature, precipitation, median household income, local forest and surface water coverage, and presence/absence of eight mosquito species. Non-static factors are extrapolated into the forthcoming year with various statistical methods. The fitted model and factor extrapolations are used to estimate next year’s regional prevalence. Results The correlation between the observed and model-estimated county-by-county heartworm prevalence for the 5-year period 2011–2015 is 0.727, demonstrating reasonable model accuracy. The correlation between 2015 observed and forecasted county-by-county heartworm prevalence is 0.940, demonstrating significant skill and showing that heartworm prevalence can be forecasted reasonably accurately. Conclusions The forecast presented herein can a priori alert veterinarians to areas expected to see higher than normal heartworm activity. The proposed methods may prove useful for forecasting other diseases.

Oral vaccines that elicit a mucosal immune response may be effective against human immunodeficiency virus type 1 (HIV-1) because its transmission occurs mainly at the mucosa. The aim of this study was to construct recombinant Lactobacillus for oral delivery of oral vaccines against HIV-1 and to evaluate their immunogenicity. A recombinant Lactobacillus acidophilus strain expressing the HIV-1 Gag on the bacterial cell surface was established by fusion with the signal peptide and anchor motif of a mucus binding protein (Mub) from L. acidophilus with or without coexpression of Salmonella enterica serovar Typhimurium flagellin (FliC) fused to a different Mub signal peptide and anchor. Using HEK293 cells engineered to express Toll-like receptor 5 (TLR5), the biological activity of FliC on the bacterial cell surfaces was determined. The surface-exposed flagellin retained its TLR5-stimulating activity, suggesting that the recombinant strain with Gag and FliC dual display might provide a different immunopotency than the strain expressing only Gag. The immunological properties of the recombinant strains were assessed by coculture with human myeloid dendritic cells (DCs). The heterologous antigens on the cell surface affected maturation and cytokine responses of DCs. Acquired immune responses were also investigated by intragastric immunization of mice. The enzyme-linked immunosorbent spot assay showed induction of gamma interferon-producing cells at local mucosa after immunization of mice with the Gag-producing strain. Meanwhile, the immunization with L. acidophilus displaying both Gag and FliC resulted in an increase of Gag-specific IgA-secreting cells. These results suggested that the Gag-displaying L. acidophilus elicited specific immune responses and the coexistence of FliC conferred an adjuvant effect on local IgA production.

The host inflammatory response to HIV invasion is a necessary component of the innate antiviral activity that vaccines and early interventions seek to exploit/enhance. However, the response is dependent on CD4+ T-helper cell 1 (Th1) recruitment and activation. It is this very recruitment of HIV-susceptible target cells that is associated with the initial viral proliferation. Hence, global enhancement of the inflammatory response by T-cells and dendritic cells will likely feed viral propagation. Mucosal entry sites contain inherent pathways, in the form of natural regulatory T-cells (nTreg), that globally dampen the inflammatory response. We created a model of this inflammatory response to virus as well as inherent nTreg-mediated regulation of Th1 recruitment and activation. With simulations using this model we sought to address the net effect of nTreg activation and its specific functions as well as identify mechanisms of the natural inflammatory response that are best targeted to inhibit viral spread without compromising initial antiviral activity. Simulation results provide multiple insights that are relevant to developing intervention strategies that seek to exploit natural immune processes: (i) induction of the regulatory response through nTreg activation expedites viral proliferation due to viral production by nTreg itself and not to reduced Natural Killer (NK) cell activity; (ii) at the same time, induction of the inflammation response through either DC activation or Th1 activation expedites viral proliferation; (iii) within the inflammatory pathway, the NK response is an effective controller of viral proliferation while DC-mediated stimulation of T-cells is a significant driver of viral proliferation; and (iv) nTreg-mediated DC deactivation plays a significant role in slowing viral proliferation by inhibiting T-cell stimulation, making this function an aide to the antiviral immune response.

Display of heterologous antigens on the cell surface is considered a useful technique for vaccine delivery by recombinant lactobacilli. In this study, two recombinant Lactobacillus acidophilus derivatives displaying Salmonella flagellin (FliC) were constructed using different anchor motifs. In one instance, the FliC protein was fused to the C-terminal region of a cell envelope proteinase (PrtP) and was bound to the cell wall by electrostatic bonds. In the other case, the same antigen was conjugated to the anchor region of mucus binding protein (Mub) and was covalently associated with the cell wall by an LPXTG motif. These two recombinant L. acidophilus cell surface displays resulted in dissimilar maturation and cytokine production by human myeloid dendritic cells. The surface-associated antigen was highly sensitive to simulated gastric and small intestinal juices. By supplementation with bicarbonate buffer and soybean trypsin inhibitor, the cell surface antigen was protected from proteolytic enzymes during gastric challenge in vitro . The protective reagents also increased the viability of the L. acidophilus cells upon challenge with simulated digestive juices. These results demonstrate the importance of protecting cells and their surface-associated antigens during oral immunization.

Lactobacillus species are commensal bacteria that have long been recognized as probiotic microbes and are generally regarded as safe (GRAS) for human consumption. We have investigated the use of L. gasseri as a vaccine vector for oral immunization against mucosal pathogens. Recent research has shown that the immune response to different lactobacilli can vary widely depending on the species or subspecies of Lactobacillus being studied. While some lactobacilli seem to induce oral tolerance, others induce an adaptive immune response. This study characterized the systemic and mucosal immune response to wild-type and genetically modified L. gasseri. L. gasseri primarily activates TLR2/6, with additional activation through the TLR2 homodimer. To expand the Toll-like receptor (TLR) activation profile of L. gasseri and the immunogenicity of the vector, a plasmid containing fliC , the gene encoding bacterial flagellin, was introduced which resulted in the strong activation of TLR5. The treatment of human myeloid dendritic cells with recombinant lactobacilli expressing flagellin triggered phenotypic maturation and the release of proinflammatory cytokines. In contrast, bacterial treatment also resulted in a statistically significant increase in IL-10 production. In vivo studies established that treatment with L. gasseri led to a diversification of B-cell populations in the lamina propria of the murine colon. Furthermore, treatment with genetically modified L. gasseri led to a significant decrease in the percentage of FoxP3 + colonic lymphocytes. Taken together, these data clarify the interaction of L. gasseri with the host immune system and support further investigation of the in vivo immunogenicity of L. gasseri expressing both flagellin and candidate vaccine antigens.

Impaired dendritic cell (DC) function is thought to be central to human immunodeficiency virus-associated immunodeficiency. In this study, we examined the effect of chronic feline immunodeficiency virus (FIV) infection on DC cytokine production in response to microbial and T-cell stimulation. Cytokine production after either Toll-like receptor (TLR) or CD40 ligation in bone marrow-derived DCs (BM-DCs) was measured in naïve and chronically FIV-infected cats. The BM-DCs were stimulated with ligands to TLR-2, -3, -4, -7 and -9 or cocultured with 3T3 cells expressing feline CD40 ligand. Ligation of TLR-4 and TLR-9 in BM-DCs from infected cats resulted in a significant decrease in the ratio of interleukin-12 (IL-12) to IL-10. Conversely, TLR-7 ligation produced a significant increase in the IL-12 : IL-10 ratio in BM-DCs from infected cats. No difference was noted for TLR-3 ligation. RNA expression levels of TLR-2, -3, -4, -7 and -9 were not significantly altered by FIV infection. CD40 ligation significantly elevated both IL-10 and IL-12 messenger RNA production but did not alter the IL-12 : IL-10 ratio. Chronic FIV infection alters the ratio of immunoregulatory cytokines produced by BM-DCs in response to certain pathogen-derived signals, which is probably relevant to the increased risk of opportunistic infections seen in lentiviral infection.

Cytauxzoonosis is an emerging infectious disease of domestic cats (Felis catus) caused by the apicomplexan protozoan parasite Cytauxzoon felis. The growing epidemic, with its high morbidity and mortality points to the need for a protective vaccine against cytauxzoonosis. Unfortunately, the causative agent has yet to be cultured continuously in vitro, rendering traditional vaccine development approaches beyond reach. Here we report the use of comparative genomics to computationally and experimentally interpret the C. felis genome to identify a novel candidate vaccine antigen for cytauxzoonosis. As a starting point we sequenced, assembled, and annotated the C. felis genome and the proteins it encodes. Whole genome alignment revealed considerable conserved synteny with other apicomplexans. In particular, alignments with the bovine parasite Theileria parva revealed that a C. felis gene, cf76, is syntenic to p67 (the leading vaccine candidate for bovine theileriosis), despite a lack of significant sequence similarity. Recombinant subdomains of cf76 were challenged with survivor-cat antiserum and found to be highly seroreactive. Comparison of eleven geographically diverse samples from the south-central and southeastern USA demonstrated 91-100% amino acid sequence identity across cf76, including a high level of conservation in an immunogenic 226 amino acid (24 kDa) carboxyl terminal domain. Using in situ hybridization, transcription of cf76 was documented in the schizogenous stage of parasite replication, the life stage that is believed to be the most important for development of a protective immune response. Collectively, these data point to identification of the first potential vaccine candidate antigen for cytauxzoonosis. Further, our bioinformatic approach emphasizes the use of comparative genomics as an accelerated path to developing vaccines against experimentally intractable pathogens.

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Myristoylated alanine-rich C kinase substrate (MARCKS) is a ubiquitously expressed protein kinase C substrate that has emerged as a potential therapeutic target for the amelioration of mucin secretion and inflammation in patients with chronic obstructive pulmonary disease. MARCKS also plays a key role in regulating the adhesion, migration, and degranulation of neutrophils. Moreover, given its biological role in epithelial and immune cells, we hypothesized that MARCKS may play an integral role in cytokine secretion by neutrophils. Because the amino terminus of MARCKS is highly conserved across vertebrate species, we successfully applied the well-characterized human MARCKS inhibitory peptide, myristoylated N-terminal sequence (MANS), to attenuate the function of MARCKS in isolated canine neutrophils. Pretreatment of canine neutrophils with MANS peptide significantly reduced both mRNA and protein expression in a broad range of LPS-induced cytokines, including IL-8, a chemokine (C-X-C motif) ligand–1 orthologue, and TNF-α, in comparison with untreated cells or those treated with a control peptide. This reduction in cytokine expression was observed even when neutrophils were treated with MANS 2 hours after LPS exposure. The observed reduction in cytokine secretion was not attributable to protein retention or cell death, but was associated with reduced cytokine transcript synthesis. These observations identify MARCKS protein as a promising therapeutic target in the treatment of inflammatory diseases or syndromes attributed to neutrophil influx and inflammatory cytokine production, such as sepsis, acute lung injury, and acute respiratory distress syndrome.

HIV infection is associated with intestinal mucosal dysfunction and probiotics offer the therapeutic potential to enhance the mucosal barrier in HIV+ patients. To evaluate the response of immunocompromised hosts to probiotics, we orally administered Lactobacillus acidophilus to cats with chronic feline immunodeficiency virus (FIV) infection. FIV infection significantly affected transcellular, but not paracellular, transport of small molecules across the intestinal epithelium. Additionally, probiotic treatment of FIV+ cats resulted in changes in cytokine release and mucosal leukocyte percentages that were not paralleled in FIV- cats. These results suggest a novel role for FIV in upregulating transcellular transport across the gastrointestinal epithelial barrier and demonstrate the potential therapeutic use of probiotic bacteria to restore intestinal homeostasis.

The novel immune-type receptors (NITRs), which have been described in numerous bony fish species, are encoded by multigene families of inhibitory and activating receptors and are predicted to be functional orthologs to the mammalian natural killer cell receptors (NKRs). Within the zebrafish NITR family, nitr9 is the only gene predicted to encode an activating receptor. However, alternative RNA splicing generates three distinct nitr9 transcripts, each of which encodes a different isoform. Although nitr9 transcripts have been detected in zebrafish lymphocytes, the specific hematopoietic lineage(s) that expresses Nitr9 remains to be determined. In an effort to better understand the role of NITRs in zebrafish immunity, anti-Nitr9 monoclonal antibodies were generated and evaluated for the ability to recognize the three Nitr9 isoforms. The application of these antibodies to flow cytometry should prove to be useful for identifying the specific lymphocyte lineages that express Nitr9 and may permit the isolation of Nitr9-expressing cells that can be directly assessed for cytotoxic (e.g. NK) function., Journal Article, info:eu-repo/semantics/published

Background Hypocalcemia is a documented electrolyte disturbance in people and animals with sepsis, but its mechanism is poorly understood. Objective To investigate mechanisms of hypocalcemia in dogs with experimentally induced endotoxemia. Animals Six healthy mixed breed dogs were included in this nonrandomized, placebo-controlled, crossover study. Methods Dogs initially were injected with placebo (0.9% NaCl; 1 mL, IV) and then lipopolysaccharide (LPS; 2 μg/kg, IV) after a 5-day washout period. Blood and urine samples were collected for measurement of serum total calcium (tCa), ionized calcium (iCa), total magnesium (tMg), ionized magnesium (iMg), parathyroid hormone (PTH), 25-hydroxyvitamin D (vitamin D), venous blood gases, and fractional excretion (FE) of calcium. Results After LPS administration, body temperature increased and blood pressure decreased. Both iCa and tCa decreased (P

The dog is both a valued veterinary species and a widely used translational model for sepsis research. However, relatively little work has been performed evaluating potential biomarkers present during canine infection. Triggering receptor expressed on myeloid cells-1 (TREM-1) has shown promise as a biomarker for infection and pneumonia in humans. Here we describe, for the first time, the expression and function of the canine orthologue of TREM-1. Expression of TREM-1 on canine neutrophils is significantly up-regulated by stimulation with microbial agonists of TLR2/6, TLR1/2, and TLR4/MD2. Kinetics of TREM-1 protein up-regulation are rapid, with significant increases observed within 2 hr of neutrophil activation. Functionally, canine TREM-1 synergistically enhances LPS-induced production of IL-8, TNF-α and a canine orthologue of CXCL1. Collectively, these data suggest that TREM-1 expression in dogs, as it is in humans, is an amplifier of pro-inflammatory responses to microbial products. These results have direct application to veterinary diagnostics as well as the potential to enhance the utility of canine disease models in the assessment of potential therapeutics in the treatment of human sepsis.

We hypothesized that unrecognized differences in epithelial expression of inducible nitric oxide synthase (iNOS), resulting from engineered immunodeficiency, could explain the contradictory findings of prior studies regarding the importance of nitric oxide (NO) in murine models of Cryptosporidium parvum infection. Severe combined immunodeficient mice (SCID) failed to constitutively or inducibly express epithelial iNOS or increase NO synthesis in response to C. parvum infection. In contrast, mice lacking IFN-gamma alone induced both epithelial iNOS expression and NO synthesis in response to infection. Accordingly, lymphocytes mediate epithelial expression of iNOS and NO synthesis independent of IFN-gamma in response to C. parvum infection. These findings in large part explain the contradictory conclusions of prior studies regarding the role of iNOS in C. parvum infection.

Toll-like receptors (TLRs) are germline-encoded pattern recognition receptors (PRRs) that activate the innate immune system. While it is clear that TLRs are important in the immune response against pathogens, they may also be exploited by some pathogens. Our objective is to determine whether feline immunodeficiency virus (FIV) infection affects TLR expression or function thereby resulting in innate immune dysfunction. To this end, we cloned partial sequences for feline TLRs 1--3, 5--8, and developed real-time PCR assays to quantify feline TLRs 1--9. TLR expression was quantified in normal cat lymphoid tissues, purified lymphocyte subsets, and FIV-infected cell lines. Different expression patterns of TLRs were found in spleen, mesenteric lymph node, retropharyngeal lymph node, thymus, intestinal intraepithelial lymphocytes, and lamina propria lymphocytes. B lymphocytes, CD4+ T cells, and CD8+ T cells all expressed TLRs 2--5, 7--9; however, the relative levels of expression varied among lymphocyte phenotypes. Infection of cell lines with FIV resulted in altered TLR expression levels that differed depending on cell type. These results demonstrate that tissue distribution of TLRs is associated with the immunological role of a particular tissue, that lymphocytes may also express these 'innate immune' receptors, and that FIV infection can alter TLR expression.

Listeria monocytogenes is an attractive biologic vaccine vector against HIV because it induces a strong cell mediated immune response, can be delivered by mucosal routes, can be readily manipulated to express viral antigens, and is easy and inexpensive to produce. Proof of concept studies have been performed using HIV Gag expressing recombinant L. monocytogenes in the mouse. Here we report the development and validation of recombinant L. monocytogenes to be evaluated in the FIV/cat model of HIV. Using a simplified approach to introduce individual and polyprotein FIV gag genes, we show that recombinant L. monocytogenes containing the entire gag expresses the full-length Gag polyprotein in a soluble secreted form. A DNA vaccine plasmid (pND14-Lc-env) that replicates in Gram positive bacteria and contains the FIV SU (gp100) and the ectodomain of TM (gp40) in a eukaryotic expression cassette was transfected into LM-gag to create LM-gag/pND14-Lc-env. After infection of target cells with LM-gag/pND14-Lc-env in vitro, both FIV Gag and Env proteins were detected in soluble cell lysates. Whether previous exposure to L. monocytogenes affects the immunogenicity of LM-gag/pND14-Lc-env was determined in cats infected with wild-type L. monocytogenes orally and/or subcutaneously. After a single oral dose of LM-gag/pND14-Lc-env, cats with existing anti-L. monocytogenes immune responses developed anti-FIV Gag IgA titers in vaginal secretions, saliva, and feces. Similarly, FIV Gag and Env specific IFN-γ ELISPOT responses were measurable in spleen and lymph node but at a statistically higher frequency in cats exposed to a single subcutaneous dose of wild-type L. monocytogenes versus cats exposed both subcutaneously and orally. The FIV/cat model will provide a useful challenge system to determine whether recombinant L. monocytogenes can protect against a lentivirus in its natural host after challenge by the routes common to HIV transmission.

Regrettably, an error appeared in the ‘Injectable vaccine administration’ box on page 798: the pictures in Figures 8 and 9 were swapped with each other. ( The error appears in the printed copies of the journal, and in online versions downloaded before mid-October 2013.) The amended box is reproduced here in full. DOI of original article: 10.1177/1098612X13500429

Abstract 3326 Immune thrombocytopenia (ITP) is a relatively prevalent disease in dogs with significant morbidity and mortality. Canine ITP is clinically analogous to human ITP, with heterogeneity in bleeding manifestations in individuals with similar platelet counts. With a view to ultimately investigate this bleeding heterogeneity, we set out to develop a canine model of ITP. There are currently no existing large animal models of ITP. An induced canine ITP model would be representative of ITP without the confounding co-morbidities seen in clinical cases. Since spontaneous ITP occurs in both dogs and humans, the dog is an ideal translational model. We hypothesized that 2F9, a murine IgG2a monoclonal antibody to the canine platelet glycoprotein GPIIb (a common target of autoantibodies in ITP), would induce predictable dose-dependent thrombocytopenia (TCP) in healthy dogs. 2F9 had not been previously administered in vivo. We produced highly purified 2F9 and αYFA antibodies from the 2F9 hybridoma (gift of David Wilcox, Blood Research Institute, Wisconsin) and an isotype control murine anti-yellow fever antibody (αYFA) hybridoma. A dose titration (2 dogs) and a dose repeatability study (3 dogs) were performed in healthy adult research dogs by repeated intravenous infusion (≤ 6 doses) of 2F9 antibody until a target nadir of 5–30 × 103 platelets/μl was reached. Platelet counts were performed hourly until the platelet count reached the desired nadir range (t=0 hrs), after which complete blood counts were performed at 2, 4, 6, 8, 12, 24 hours, then q 24 hours for 10 days. The following were evaluated throughout the study: physical examination, buccal mucosal bleeding time (BMBT, baseline and t=0 only), serum cytokines and chemokines (INFγ, Interleukin (IL) 2, 6, 7, 8, 10, 15, 18, KC, IP-10, MCP-1, GM-CSF, TNFα; Milliplex CCYTOMAG-90K), fibrinogen, and D-dimers. Specificity of the 2F9 effect was confirmed by IV infusion of the isotype control (αYFA) to 3 dogs at the highest cumulative effective dose of 2F9 (167 μg/kg); all parameters were measured as above (t=0 hrs was one hour after αYFA dosing). Within 2 hours of a median cumulative 2F9 administration of 63 μg/kg (range 50.0–166.6 μg/kg), all dogs developed profound TCP (range 11–28 × 103/μl). Compared to the control group, platelet nadir was significantly lower (median (range): 6 (4–11) × 103/μl vs. 200 (179–209) × 103/μl; p= 0.036) and change in platelet count from baseline to nadir was significantly greater in the 2F9-treated group (median (range): 238 (179–325) × 103/μl vs. 4 (0–10) × 103/μl; p=0.036) (Fig 1); p-values were calculated using the exact Wilcoxon rank-sum test. Platelet nadir was in our target range and platelet count remained < 40 × 103/μl in all 2F9-treated dogs for 24 hours. Dosing was predictable: in each dog, after an initial dose of 50 μg/kg 2F9, the second dose needed to reach the target nadir could be accurately calculated from the initial platelet decrease. 2F9-treated dogs developed a range of clinical bleeding from none to petechiae, ecchymoses, melena, and hematuria. At t=0 hrs, BMBT increased 3–8 fold in treated dogs, compared to < 2 fold in control dogs. Dogs had no changes in vital signs or demeanor and did not require any transfusion support. The model does not appear pro-thrombotic as fibrinogen and D-dimers were similar over time in 2F9-treated vs. control dogs. 2F9 infusion also generated negligible systemic inflammation, as assessed by white blood cell count and serum cytokine measurement. Unexpectedly, however, serum IL8 tracked faithfully with platelet count, demonstrating that platelets are a major source of serum IL8 in dogs (Fig 2). Although α granules are known to contain IL8, platelets have not been previously described as a significant serum IL8 source. Since IL8 is an important neutrophil chemokine, our finding may illuminate a novel mechanism of platelet-neutrophil cross-talk. In summary, we have developed a novel large animal ITP model that is highly representative of the spontaneous disease. Like naturally-occurring ITP, dogs demonstrate bleeding heterogeneity despite similar platelet counts (data not shown). We expect our model to lead to further insights into bleeding mechanisms in ITP. Ultimately, understanding what factors predispose certain patients to bleed will allow us to exploit these factors therapeutically as novel ITP treatments. Disclosures: No relevant conflicts of interest to declare.

Abstract 2193 In both canine and human patients with Immune Thrombocytopenia (ITP), bleeding risk is challenging to predict, and potentially leads to over-treatment of patients at low risk. Conversely, recent studies have highlighted the risk of thrombosis in ITP during platelet recovery. Given these clinical observations, we hypothesized that in ITP, changes in platelet response to agonists may occur in addition to changes in platelet numbers. In response to dual agonist activation (thrombin and convulxin), a subpopulation of platelets in both humans and dogs develops enhanced procoagulant activity. This subpopulation is termed coated platelets, and differences in individuals' potential to form coated platelets have been correlated with both hemorrhagic and thrombotic outcomes. In this exploratory study, we serially evaluated ex vivo platelet responsiveness to both thrombin and dual agonists (termed coated platelet potential) in a novel canine model of ITP. Dogs (n=4) were infused with a murine monoclonal anti-GPIIb antibody (2F9) in order to model ITP and generate predictable severe thrombocytopenia. Control dogs (n=3) were infused with a control antibody. Platelet count, thrombin responsiveness, and coated platelet potential were measured at baseline, time zero, 6 hours, 24 hours, and every 24hrs thereafter until the platelet count was ≥ baseline for at least two consecutive measures (recovery). Time zero was defined as the time when platelet count first fell to ≤ 30,000/μl following 2F9 infusion, or 1 hour following control antibody infusion. For platelet thrombin responsiveness, a monoclonal antibody to P-selectin was used to determine platelet P-selectin surface expression by flow cytometry after stimulation with graded doses of thrombin. The ED50 Thrombin was defined as the concentration of thrombin required for half-maximal P-selectin expression. Coated platelet potential was defined as the percent of platelets activated to the highly procoagulant state after dual stimulation with thrombin and convulxin, as determined by binding of biotinylated fibrinogen by platelets by flow cytometry. All dogs in the treated group developed severe thrombocytopenia (median=6×103, range=4–11×103 platelets/uL); no dogs in the control group developed thrombocytopenia. All treated dogs had platelet recovery by 240 hours (median=132 hours, range 120–240hours). Of interest, at 6 hours, ED50 Thrombin in the treated group increased nearly twofold (fig 1A) (ratio of median ED50 Thrombin treated/baseline=1.6, range 1.3–2.3), which correlated with a decline in coated platelet potential by nearly half of baseline (fig 1B) (median 52.4% of baseline, range 19.6–61.5%); minimal change from baseline was observed in controls. In both groups, ED50 Thrombin was lower at recovery than baseline (fig 1A) (treated median ED50 Thrombin=71.5% of baseline; control median ED50 Thrombin=67% of baseline). A trend of rising coated platelet potential was also noted as platelets recovered in the treated group. In conclusion, in this exploratory study of a canine model of ITP, we observed dynamic changes in platelet responsiveness. During severe thrombocytopenia, we observed a rise in ED50, indicating a decline in response to thrombin, which correlated with a fall in coated platelet potential. We speculate that this early fall in platelet thrombin response and coated platelet potential could contribute to hemorrhage risk in ITP. As a complement to this finding, in the treated group, there was a rise in coated platelet potential as platelets rebounded and coated platelet potential was slightly greater than baseline at recovery. This is consistent with others' observation that younger platelets are more likely to have coated platelet potential. We also observed a decline in ED50 Thrombin at recovery, not only in the treated dogs, but also control dogs. Thus, at recovery, the decline in ED50 Thrombin was independent of treatment group. However, this may be an artifact of our small sample size. Our observed increase in coated platelet potential during platelet recovery could potentially contribute to the thrombotic tendency of some ITP patients. Future studies are planned to explore the relationship of hemorrhagic and thrombotic risk with platelet thrombin responsiveness and coated platelet potential in this model of ITP and clinical studies of canine and human ITP. Disclosures: No relevant conflicts of interest to declare.

The triggering receptor expressed on myeloid cells-1 (TREM-1) is a recently discovered cell surface molecule expressed on neutrophils, mature monocytes, and macrophages [1]. Activation of TREM-1 synergistically enhances proinflammatory cytokine production induced by toll-like receptor (TLR) stimulation [2]. A soluble form of TREM-1 has shown promise as a sensitive and specific biomarker for sepsis in humans [3-5]. Expression and function of TREM-1 in the dog has yet to be characterized. We hypothesize that the expression of canine TREM-1 will be upregulated after stimulation with TLR agonists. We assessed TREM-1 expression on canine neutrophils after exposure to TLR agonists in vitro and in vivo after i.v. LPS administration. In vitro, expression of TREM-1 on neutrophils is significantly upregulated by stimulation with microbe-derived agonists against TLR2/6 (Pam2CSK4), TLR1/2 (Pam3CSK4), and TLR4/MD2 (ultrapure LPS and wildtype LPS) (paired t test, P < 0.05). The TLR5 agonist flagellin did not significantly upregulate TREM-1 expression at any time point. See Figure ​Figure1.1. In contrast, i.v. administration of LPS to dogs resulted in a significant decrease in both TREM-1 expression and the percentage of TREM-1-positive neutrophils from 6 hours through 12 hours post LPS administration. See Figure ​Figure2.2. The disparity between in vitro and in vivo effects of LPS suggest other factors, such as systemic and local cytokine production and neutrophil turnover, may influence expression and shedding of TREM-1 on canine neutrophils. We suggest that naturally occurring sepsis in the dog represents the ideal model for defining diagnostic biomarkers and discovering efficacious therapeutics for use in human sepsis. Figure 1 Kinetics of TREM-1 expression after TLR agonist stimulation in vitro. Figure 2 Kinetics of TREM-1 expression post LPS administration in vivo.

Background Transmission of HIV-1 across mucosal surfaces is the most prevalent mode of viral infection. Therefore, a successful vaccine must induce broad anti-viral immunity at the mucosal surface. In the present study, we investigated the immunogenicity of a novel Lactobacillus gasseri mucosal vaccine vector for use in oral delivery of HIV antigens. L. gasseri was genetically engineered to express Salmonella spp. flagellin (L. gasseri-FliC) – the agonist for Toll-like receptor 5 (TLR5) and a potent activator of innate immune cells.

Plasmacytoid Dendritic Cells (PDC) produce copious amounts of IFN-a in response to TLR-7 or -9 stimulation and serve as a link between the innate and adaptive immune systems. In HIV infection, there is a gradual depletion of PDC as well as a functional deficiency in remaining circulating PDC. The mechanism of PDC depletion is not yet understood. We have demonstrated using imaging flow cytometry (ImageStream®) that PDC preferentially take up membrane and cytoplasm from HSV and Influenza infected cells in an endocytic process called "nibbling" that results in IFN-α production by the PDC. Using acutely HIVIIIB infected CD4 T cells or chronically HIVIIIB infected H9 T cells, PDC preferentially form conjugates with HIVIIIB infected vs uninfected T cells, but, rather than being nibbled, the majority of these encounters lead to PDC/infected T cell fusions. Acutely infected CD4 T cells but not chronically-infected H9 cells stimulated PDC to produce IFN-α, albeit it lower levels than other TLR7/9 inducers. Fusions were blocked with the addition of the specific fusion inhibitors (T-20 or AMD-3100), but there was no increase in conjugate formation or uptake of material. We hypothesize that this successful subversion of PDC nibbling and IFN-α production by HIV-infected cells contributes to loss of PDC and the deficient IFN-α production seen in HIV-infected patients. Supported by AI26806.