A novel pooled milk test strategy for the herd level diagnosis of Dictyocaulus viviparus.

Current diagnostic methods for detecting the presence or absence of Dictyocaulus viviparus in dairy herds, are insensitive when based on detection of antibody levels in bulk tank milk (BTM). Here we present a novel technique to confirm the presence of the parasite based on a pooled-milk sample from 10 randomly selected first - lactation heifers (FLH). This study was run in two parts. First, a longitudinal study was performed to look at infection dynamics in milk samples across the grazing season using a prototype ELISA developed by Svanova (Boehringer-Ingelheim, Uppsala). We identified that mean ODR values in milk samples from FLH was significantly higher than that for older cows (0.13 versus 0.07 respectively, p < 0.001) suggesting that samples from the FLH cohort should be pooled to produce the test. Second, the pooled - milk test was evaluated on a cross-sectional survey of UK dairy herds (n = 25 grazing and n = 25 zero-grazing herds) to evaluate test performance under field conditions. The optical density ratio (ODR) cut-off value for our pooled-milk test using 10 FLH milk samples was optimal at a value of 0.16. Pooling 10 FLH samples created a sensitivity and specificity of 66.7% and 95.5% respectively. In comparison, whole-herd BTM samples had a maximum sensitivity of 37.5% and specificity of 63.6% at an ODR cut-off of 0.18. The area under the curve according to receiver-operative-characteristic (ROC) analysis was high for the 10-heifer test (0.87) but poor for the whole herd BTM testing (0.45). This study provides a more sensitive diagnostic test strategy for the screening of D.viviparus in dairy herds. Testing herds at the end of a grazing season would facilitate the planning of effective control measures, such as the use of the lungworm vaccination or strategic deworming, for the following grazing season. This may prove to be a useful test strategy for the diagnosis of a variety of parasitic diseases of livestock.

Development and validation of a pen side test for Rift Valley fever.

BACKGROUND: Rift Valley fever (RVF) is one of the main vector borne zoonotic diseases that affects a wide range of ruminants and human beings in Africa and the Arabian Peninsula. A rapid and specific test for RVF diagnosis at the site of a suspected outbreak is crucial for the implementation of control measures. METHODOLOGY/PRINCIPAL FINDINGS: A first-line lateral flow immunochromatographic strip test (LFT) was developed for the detection of the nucleoprotein (N) of the RVF virus (RVFV). Its diagnostic performance characteristics were evaluated using reference stocks isolates recovered from different hosts and in geographic regions mimicking clinical specimens and from known RVF negative serum samples. A high level of diagnostic accuracy (DSe (35/35), DSp (167/169)) was observed, including the absence of cross-reactivity with viruses belonging to different genera. CONCLUSION/SIGNIFICANCE: The fact no specialized reagents and laboratory equipment are needed, make this assay a valuable, first-line diagnostic tool in resource-poor diagnostic territories for on-site RVFV detection, however the staff require training.

A novel pooled milk test strategy for the herd level diagnosis of Dictyocaulus viviparus.

Current diagnostic methods for detecting the presence or absence of Dictyocaulus viviparus in dairy herds, are insensitive when based on detection of antibody levels in bulk tank milk (BTM). Here we present a novel technique to confirm the presence of the parasite based on a pooled-milk sample from 10 randomly selected first - lactation heifers (FLH). This study was run in two parts. First, a longitudinal study was performed to look at infection dynamics in milk samples across the grazing season using a prototype ELISA developed by Svanova (Boehringer-Ingelheim, Uppsala). We identified that mean ODR values in milk samples from FLH was significantly higher than that for older cows (0.13 versus 0.07 respectively, p < 0.001) suggesting that samples from the FLH cohort should be pooled to produce the test. Second, the pooled - milk test was evaluated on a cross-sectional survey of UK dairy herds (n = 25 grazing and n = 25 zero-grazing herds) to evaluate test performance under field conditions. The optical density ratio (ODR) cut-off value for our pooled-milk test using 10 FLH milk samples was optimal at a value of 0.16. Pooling 10 FLH samples created a sensitivity and specificity of 66.7% and 95.5% respectively. In comparison, whole-herd BTM samples had a maximum sensitivity of 37.5% and specificity of 63.6% at an ODR cut-off of 0.18. The area under the curve according to receiver-operative-characteristic (ROC) analysis was high for the 10-heifer test (0.87) but poor for the whole herd BTM testing (0.45). This study provides a more sensitive diagnostic test strategy for the screening of D.viviparus in dairy herds. Testing herds at the end of a grazing season would facilitate the planning of effective control measures, such as the use of the lungworm vaccination or strategic deworming, for the following grazing season. This may prove to be a useful test strategy for the diagnosis of a variety of parasitic diseases of livestock.

Bovine herpes virus type 4 alters TNF-α and IL-8 profiles and impairs the survival of bovine endometrial epithelial cells.

Bovine herpes virus type 4 (BoHV-4) can be transmitted by contaminated semen to cows at the time of breeding and may cause uterine disease. The aim of this study was to characterize the susceptibility of bovine endometrial epithelial cells (bEEC) to BoHV-4 by using an in vitro model. When bEEC were challenged with different multiplicity of infection (MOI; from 0.001 to 10) of BoHV-4 for 6days, a significant decrease in cell survival with increasing MOI was observed. The bEEC were subsequently challenged with BoHV-4 MOI 0.1 for 7days. During the first 4days, numbers increased in a similar way in controls and infected group (p<0.01 when compared to Day 0). After Day 4, numbers of live cells in infected samples decreased when compared to controls and were lower than control at Day 7 (p<0.01). From titration and qPCR, increasing number of viral particles was observed from Day 1, and reached a plateau at Day 5. Concentrations of IL-8 increased with time and were higher in supernatants from infected cells than in controls (p<0.0001). TNF-α concentrations presented similar profile as cell survival ones. In conclusion, the survival of bEEC was strongly impaired by BoHV-4 infection in a time and dose dependent manner and supernatant cytokine profiles were altered. This information supports BoHV-4 implication in clinical cases of uterine diseases and the existence of a risk of BoHV-4 transmission from infected males through animal breeding.

Molecular Diagnosis of Hemorrhagic Fever with Renal Syndrome Caused by Puumala Virus.

Rodent-borne hantaviruses cause two severe acute diseases: hemorrhagic fever with renal syndrome (HFRS) in Eurasia, and hantavirus pulmonary syndrome (HPS; also called hantavirus cardiopulmonary syndrome [HCPS]) in the Americas. Puumala virus (PUUV) is the most common causative agent of HFRS in Europe. Current routine diagnostic methods are based on serological analyses and can yield inconclusive results. Hantavirus-infected patients are viremic during the early phase of disease; therefore, detection of viral RNA genomes can be a valuable complement to existing serological methods. However, the high genomic sequence diversity of PUUV has hampered the development of molecular diagnostics, and currently no real-time reverse transcription-quantitative (RT)-PCR assay is available for routine diagnosis of HFRS. Here, we present a novel PUUV RT-PCR assay. The assay was validated for routine diagnosis of HFRS on samples collected in Sweden during the winter season from 2013 to 2014. The assay allowed detection of PUUV RNA in 98.7% of confirmed clinical HFRS samples collected within 8 days after symptomatic onset. In summary, this study shows that real-time RT-PCR can be a reliable alternative to serological tests during the early phase of HFRS.

Molecular investigations on the prevalence and viral load of enteric viruses in pigs from five European countries.

Enteric viral infections in pigs may cause diarrhea resulting in ill-thrift and substantial economic losses. This study reports the enteric infections with porcine astrovirus type 4 (PAstV4), porcine group A rotavirus (GARV), porcine group C rotavirus (GCRV), porcine circovirus type 2 (PCV2) and porcine kobuvirus (PKoV) in 419 pigs, comprising both healthy and diarrheic animals, from 49 farms in five European countries (Austria, Germany, Hungary, Spain and Sweden). Real-time RT-PCR assays were developed to test fecal samples and to compare the prevalence and viral load in relation to health status, farms of origin and age groups. The results showed that PAstV4 (70.4%) was the dominant virus species, followed by PKoV (56.7%), PCV2 (42.2%), GCRV (3%) and GARV (0.9%). Diarrheic pigs had a higher viral load of PAstV4 in the nursery and growing-finishing groups. Rotaviruses were mainly detected in diarrheic pigs, whereas PCV2 was more often detected in clinically healthy than in diarrheic pigs, suggesting that most PCV2 infections were subclinical. PAstV4, PCV2 and PKoV were considered ubiquitous in the European pig livestock and co-infections among them were frequent, independently of the disease status, in contrast to a low prevalence of classical rotavirus infections.

A semi-automated magnetic capture probe based DNA extraction and real-time PCR method applied in the Swedish surveillance of Echinococcus multilocularis in red fox (Vulpes vulpes) faecal samples.

BACKGROUND: Following the first finding of Echinococcus multilocularis in Sweden in 2011, 2985 red foxes (Vulpes vulpes) were analysed by the segmental sedimentation and counting technique. This is a labour intensive method and requires handling of the whole carcass of the fox, resulting in a costly analysis. In an effort to reduce the cost of labour and sample handling, an alternative method has been developed. The method is sensitive and partially automated for detection of E. multilocularis in faecal samples. The method has been used in the Swedish E. multilocularis monitoring program for 2012-2013 on more than 2000 faecal samples. METHODS: We describe a new semi-automated magnetic capture probe DNA extraction method and real time hydrolysis probe polymerase chain reaction assay (MC-PCR) for the detection of E. multilocularis DNA in faecal samples from red fox. The diagnostic sensitivity was determined by validating the new method against the sedimentation and counting technique in fox samples collected in Switzerland where E. multilocularis is highly endemic. RESULTS: Of 177 foxes analysed by the sedimentation and counting technique, E. multilocularis was detected in 93 animals. Eighty-two (88%, 95% C.I 79.8-93.9) of these were positive in the MC-PCR. In foxes with more than 100 worms, the MC-PCR was positive in 44 out of 46 (95.7%) cases. The two MC-PCR negative samples originated from foxes with only immature E. multilocularis worms. In foxes with 100 worms or less, (n = 47), 38 (80.9%) were positive in the MC-PCR. The diagnostic specificity of the MC-PCR was evaluated using fox scats collected within the Swedish screening. Of 2158 samples analysed, two were positive. This implies that the specificity is at least 99.9% (C.I. = 99.7-100). CONCLUSIONS: The MC-PCR proved to have a high sensitivity and a very high specificity. The test is partially automated but also possible to perform manually if desired. The test is well suited for nationwide E. multilocularis surveillance programs where sampling of fox scats is done to reduce the costs for sampling and where a test with a high sensitivity and a very high specificity is needed.

High-throughput screening of tick-borne pathogens in Europe.

Due to increased travel, climatic, and environmental changes, the incidence of tick-borne disease in both humans and animals is increasing throughout Europe. Therefore, extended surveillance tools are desirable. To accurately screen tick-borne pathogens (TBPs), a large scale epidemiological study was conducted on 7050 Ixodes ricinus nymphs collected from France, Denmark, and the Netherlands using a powerful new high-throughput approach. This advanced methodology permitted the simultaneous detection of 25 bacterial, and 12 parasitic species (including; Borrelia, Anaplasma, Ehrlichia, Rickettsia, Bartonella, Candidatus Neoehrlichia, Coxiella, Francisella, Babesia, and Theileria genus) across 94 samples. We successfully determined the prevalence of expected (Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, Rickettsia helvetica, Candidatus Neoehrlichia mikurensis, Babesia divergens, Babesia venatorum), unexpected (Borrelia miyamotoi), and rare (Bartonella henselae) pathogens in the three European countries. Moreover we detected Borrelia spielmanii, Borrelia miyamotoi, Babesia divergens, and Babesia venatorum for the first time in Danish ticks. This surveillance method represents a major improvement in epidemiological studies, able to facilitate comprehensive testing of TBPs, and which can also be customized to monitor emerging diseases.

Chemokine receptor expression by mast cells.

There is a growing interest in the role of chemokines and their receptors in the determination of mast cell tissue localization and how chemokines regulate mast cell function. At least nine chemokine receptors (CXCR1, CXCR2, CXCR3, CXCR4, CX3CR1, CCR1, CCR3, CCR4 and CCR5) have been described to be expressed by human mast cells of different origins. Seven chemokines (CXCL1, CXCL5, CXCL8, CXCL14, CX3CL1, CCL5 and CCL11) have been shown to act on some of these receptors and to induce mast cell migration. Mast cells have a unique expression pattern of CCR3, CXCR1 and CXCR2. These receptors are mainly expressed intracellularly on cytoplasmic membranes. Upon an allergic activation, CCR3 expression is increased on the cell surface and the cell becomes vulnerable for CCL11 treatment. Chemokines do not induce mast cell degranulation but CXCL14 causes secretion of de novo synthesized CXCL8. Because of the expression of CCR3, CCR5 and CXCR4 on mast cell progenitors, these cells are susceptible to HIV infection and mast cells might therefore be a persistent HIV reservoir in AIDS. In this review, we summarize the knowledge about chemokine receptor expression and function on mast cells.

Expression of CCL5/RANTES by Hodgkin and Reed-Sternberg cells and its possible role in the recruitment of mast cells into lymphomatous tissue.

HL is a malignant lymphoma characterized by a small number of malignant HRS cells among a major population of infiltrating reactive cells, e.g., lymphocytes and eosinophils. We previously reported that mast cells are present in HL-affected lymph nodes and therein are the predominant CD30L-expressing cells. The CD30L expressed on mast cells is functionally active and can provide stimulatory signals to HRS cells. Thus, mast cells constitute an important portion of the infiltrating reactive cells that contribute to tumor progression in HL. Control of the recruitment of this previously unrecognized cell and its interactions with tumor cells are essentially unknown. To elucidate if mast cells might be specifically attracted to the tumor area by chemokines produced by HRS cells, we investigated chemokine expression in HL cell lines and in vivo. By RNase protection assay, mRNA expression of several chemokines could be detected in the cell lines. Despite the heterogeneous expression profile exhibited by the cell lines, 4 of 5 expressed CCL5 (RANTES) mRNA. RT-PCR and immunohistochemistry confirmed expression of CCL5 in vivo. Furthermore, secreted CCL5 was detected in conditioned media from 3 of the cell lines. In a migration assay, we found that CCL5 present in conditioned medium could induce mast cell migration. Taken together, our results suggest that CCL5 produced by HRS cells is one mechanism by which mast cells can be attracted into the tumor tissue in HL.

Selective CCL5/RANTES-induced mast cell migration through interactions with chemokine receptors CCR1 and CCR4.

Mast cells (MCs) accumulate at sites of allergic mucosal inflammation where they act as central effector and regulatory cells. Because chemokines are of vital importance in directing inflammatory leukocytes to the sites of inflammations, we have investigated the expression and function of CC-chemokine receptor (CCR) on human MCs. Two previously unrecognized MC-chemokine receptors, CCR1 and CCR4, could be identified on cord blood-derived MCs (CBMCs). CCR1 and CCR4 expressed on CBMCs exhibited a unique response profile. Of seven CCR1 and CCR4 agonists tested, only CCL5/RANTES act as an agonist inducing chemotaxis. The migration could be partially blocked by specific antibodies against CCR1 or CCR4, while a complete inhibition was achieved when both CCR1 and CCR4 were blocked. These results demonstrate that both CCR1 and CCR4 are functional receptors on human mast cells with capacity to mediate migration towards CCL5.

Human mast cells express two leukotriene C(4) synthase isoenzymes and the CysLT(1) receptor.

Cysteinyl-leukotrienes (cys-LTs) are potent smooth muscle contracting agents, especially in the respiratory tract and microcirculation, and play a key role in inflammatory and allergic diseases. The final step in the biosynthesis of LTC(4), the parent compound of cys-LTs, is catalyzed by a specific GSH transferase termed LTC(4) synthase, which is typically expressed in certain bone marrow-derived cells such as eosinophils and mast cells. Here we report that the human mast cell line HMC-1 as well as human mast cells derived from cord blood (CBMC) express a second enzyme capable of synthesizing leukotriene C(4), i.e., microsomal GSH transferase type 2. Furthermore, these cells abundantly express CysLT(1) receptors that are mostly located at the surface of both types of mast cells, as judged by immunohistochemistry. In addition, stimulation of CBMC with LTC(4) and LTD(4) elicits an immediate and dose-dependent (10(-7)-10(-11) M) mobilization of intracellular Ca(2+), which can be blocked with specific CysLT(1) receptor antagonists. Taken together, our data suggest that human mast cells are equipped with two enzymes that can catalyze the committed step in the biosynthesis of cys-LTs. Moreover, the expression of the cognate receptor CysLT(1) suggests that these lipid mediators may be involved in autocrine signaling pathways regulating mast cell functions.