Porcine circovirus 2 (PCV2) increases the expression of endothelial adhesion/junction molecules

Abstract Porcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus disease, a complex multisystem syndrome in domestic pigs. Despite the significant economic losses caused by porcine circovirus disease, the mechanisms of pathogenesis underlying the clinical findings remain largely unclear. As various reports have highlighted the potential key role of vascular lesions in the pathogenesis of porcine circovirus disease, the aim of this work was to investigate effects of PCV2 infection on vascular endothelial cells, focusing on cell viability and expression of adhesion/junction molecules. PCV2 infection reduced endothelial cell viability, while viral infection did not affected the viability of several other classical cell lines. Also, PCV2 infection in endothelial cells displayed a dual/biphasic effect: initially, infection increased ICAM-1 expression, which can favor leukocyte recruitment and emigration to tissues and possibly inducing characteristic porcine circovirus disease inflammatory lesions; then, secondarily, infection caused an increase in zonula occludens 1 tight junction protein (ZO-1) expression, which in turn can result in difficulties for cell traffic across the endothelium and a potential impairment the immune response in peripheral tissues. These virus-induced endothelial changes could directly impact the inflammatory process of porcine circovirus disease and associated vascular/immune system disturbances. Data suggest that, among the wide range of effects induced by PCV2 on the host, endothelial modulation can be a pivotal process which can help to explain PCV2 pathogenesis in some porcine circovirus disease presentations.

Porcine circovirus 2 (PCV2) increases the expression of endothelial adhesion/junction molecules.

Porcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus disease, a complex multisystem syndrome in domestic pigs. Despite the significant economic losses caused by porcine circovirus disease, the mechanisms of pathogenesis underlying the clinical findings remain largely unclear. As various reports have highlighted the potential key role of vascular lesions in the pathogenesis of porcine circovirus disease, the aim of this work was to investigate effects of PCV2 infection on vascular endothelial cells, focusing on cell viability and expression of adhesion/junction molecules. PCV2 infection reduced endothelial cell viability, while viral infection did not affected the viability of several other classical cell lines. Also, PCV2 infection in endothelial cells displayed a dual/biphasic effect: initially, infection increased ICAM-1 expression, which can favor leukocyte recruitment and emigration to tissues and possibly inducing characteristic porcine circovirus disease inflammatory lesions; then, secondarily, infection caused an increase in zonula occludens 1 tight junction protein (ZO-1) expression, which in turn can result in difficulties for cell traffic across the endothelium and a potential impairment the immune response in peripheral tissues. These virus-induced endothelial changes could directly impact the inflammatory process of porcine circovirus disease and associated vascular/immune system disturbances. Data suggest that, among the wide range of effects induced by PCV2 on the host, endothelial modulation can be a pivotal process which can help to explain PCV2 pathogenesis in some porcine circovirus disease presentations.

Targeted survey of Newcastle disease virus in backyard poultry flocks located in wintering site for migratory birds from Southern Brazil.

Newcastle disease virus (NDV) causes a fast-spreading, highly contagious infectious disease in several bird species. Commercial poultry farms in Brazil were considered free of virulent NDV. Data on NDV infection levels in backyard poultry flocks and the epidemiology of the disease are limited. The aim of this study was to perform a NDV survey in backyard poultry from households flocks located around one of the main wintering sites for migratory wild birds in Brazil, and to identify potential risk factors associated with NDV. Backyard poultry may be sentinels and a source of infection for commercial poultry, since they may have as much contact with these birds as with migratory wild birds. Data were collected from 48 randomly selected households using an epidemiological questionnaire. Serum samples from poultry were tested for NDV antibodies using an ELISA, and tracheal and cloacal swabs were collected for NDV molecular detection. The risk factors were assessed using a multivariate Poisson regression with robust variance. The ELISA showed that 33.8% of the serum samples were positive for anti-NDV antibodies and in 42 households (87.5%) at least one NDV-positive bird was found. Tracheal and cloacal swabs were negative for NDV by real time RT-PCR, possible because within this region there might flow a low pathogenicity NDV strain, which can induce seroconversion with innaparent clinical findings. The prevalence ratio (PR) increased when farmers used their own replacement poultry to restock their flock (PR=1.64; 95% CI: 1.11-2.42). Furthermore, the increasing distance of the household flock from the "Laguna do Peixe" estuary was associated with decreasing NDV seropositivity (PR=0.94; 95% CI: 0.90-0.99). This is the first study in Brazil evaluating the presence of NDV and the associated risk factors in households with backyard poultry flocks. The great number of farms with seropositive birds indicates that the virus circulates in backyard flocks, and this breeding system may be a source of NDV. These data can be used to establish appropriate biosecurity and husbandry measures for this type of breeding system to prevent NDV spread in Brazil.

Does Rhipicephalus microplus tick infestation increase the risk for myiasis caused by Cochliomyia hominivorax in cattle?

The larval phase of Cochliomyia hominivorax (screwworm) is an obligate parasite of vertebrate animals, particularly mammals, and widespread in South America, where it remains one of the most important parasitic diseases of domestic animals. The skin of cattle highly infested by ticks, with cutaneous lesions, exudation of tissue fluid and blood scent seems to produce the ideal environment for fly attraction. However, an association between these parasites was never investigated. The aim of this work was to verify if there is an association between Rhipicephalus microplus tick load and the occurrence of C. hominivorax myiasis in cattle, and to quantify the risk. Sixty bovine (Bos taurus taurus, Angus breed) under field conditions were observed for 24 weeks, during which weekly tick counts and examination for the presence of myiasis were performed. There was a significant association between a high tick burden (24-week mean above 50 ticks per animal) and myiasis occurrence (P=0.0102). The calculated relative risk (RR) for C. hominivorax myiasis occurrence in cattle with high tick burden was 3.85 (CI95%=1.23-12.13); indicating that cattle highly parasitized by R. microplus have about four times more risk of myiasis than those with a low parasite load. As far as we aware, this is the first statistically based evidence of the relationship between R. microplus parasitic load and occurrence of myiasis by C. hominivorax. This result could be useful for the design of integrated control strategies for these parasites and to provide more information for the understanding of cattle tick parasitism in cattle production.

Ornithodoros brasiliensis (mouro tick) salivary gland homogenates inhibit in vivo wound healing and in vitro endothelial cell proliferation.

Ornithodoros brasiliensis is a nidicolous tick only found in the southern Brazilian highlands region. O. brasiliensis parasitism is frequently associated with toxicosis syndrome, which can lead to severe reactions, ranging from local pruritus and pain to systemic disturbances both in humans and dogs. One of the most frequent findings associated with an O. brasiliensis bite is a slow healing lesion at the site of tick attachment, which can take several weeks to heal. This work tested the hypothesis that an O. brasiliensis salivary gland homogenate is able to modulate the skin wound-healing process in vivo, using a model of excisional skin lesion in rats, which are divided into two groups: (1) control group and (2) treated group, which topically received salivary gland homogenate equivalent to the protein amount of one whole salivary gland (≈5 µg protein). The hypothesis that O. brasiliensis salivary gland homogenates interfere with endothelial cell proliferation, a key role phenomenon in wound healing, was also tested. O. brasiliensis salivary gland homogenates significantly delay skin wound healing. The time to full healing of skin lesions in control rats was 15 days, contrasting with 24 days in rats topically treated with O. brasiliensis salivary gland homogenates. The calculated HT50 (healing time to recover 50% of the wound area) for control groups was 3.6 days (95% CI, 3.2-3.9) and for salivary gland treated rats was 7.7 days (95% CI, 7.0-8.4). Salivary gland homogenates have a strong cytotoxic activity on cultured endothelial cells (LC50, 13.6 mg/ml). Also, at sublethal concentrations (≤3 mg/ml), salivary gland homogenates have a remarkable anti-proliferative activity (IC50 0.7 mg/ml) on endothelial cells, equivalent to ≈0.03 salivary gland pairs, an activity which seems to be much greater than reported for any other tick species. This is the first report about the biological activities of O. brasiliensis salivary compounds and provides the first in vivo evidence to support the concept of wound-healing modulation by tick salivary secretions. Results shown here contribute to an understanding of O. brasiliensis tick toxicosis syndrome, and also increase our knowledge of tick salivary bioactive compounds.

Epidemiology of Ornithodoros brasiliensis (mouro tick) in the southern Brazilian highlands and the description of human and animal retrospective cases of tick parasitism.

Ornithodoros brasiliensis, also known as the "mouro" tick, is an argasid tick found exclusively in the southern Brazilian highlands. O. brasiliensis parasitism is frequently associated with severe symptoms directly induced by the tick bite, a condition compatible with the definition of tick toxicosis. The objectives of this work include (i) the determination of the distribution of O. brasiliensis in farms located in the tick-endemic region, (ii) the description of the characteristics of O. brasiliensis habitats, (iii) the analysis of risk factors associated with O. brasiliensis, and (iv) the retrospective description of cases of human and animal parasitism by O. brasiliensis. Of the 30 farms included in this study, O. brasiliensis was identified on 5 farms (frequency 16.7%), in which several ticks found in high density buried in soil were collected. Information regarding the tick habitats and the local population was recorded. The data indicated that O. brasiliensis feeds on humans, dogs, armadillos (Dasypus hybridus), and possibly skunks (Conepatus chinga). The analysis of risk factors indicated that the presence of house basements with an unpaved (natural soil) floor on farms and insufficient sanitary conditions significantly enhanced the probability of identifying O. brasiliensis. Additionally, we describe retrospectively cases of tick parasitism in 28 humans and 11 dogs including the most common symptoms associated with tick toxicosis. This is the first study concerning O. brasiliensis epidemiology, distribution, and habitat, and the report represents the most comprehensive characterization of Ornithodoros bite-associated toxicosis syndrome.

Detection and characterization of fibropapilloma associated herpesvirus of marine turtles in Rio Grande do Sul, Brazil / Detecção e caracterização do herpesvírus associado ao fibropapiloma de tartarugas marinhas no Rio Grande do Sul, Brasil

Fibropapillomatosis (FP) is a benign tumoral disease that affects sea turtles, hampering movement, sight and feeding, ultimately leading to death. In Brazil, the disease was described for the first time in 1986. Research suggests the involvement of a herpesvirus in association with environmental and genetic factors as causal agents of FP. The objective of the present study was to detect and characterize this herpesvirus in sea turtles living in the coast of state Rio Grande do Sul (RS), Brazil. From October 2008 to July 2010, 14 turtles were observed between the beaches of Torres and Tavares, of which 11 were green turtles (Chelonia mydas) and 3 were loggerhead turtles (Caretta caretta). All turtles were young and mean curved carapace length was 37.71±7.82cm, and varied from 31 to 55cm. Only one green turtle presented a 1cm, papillary, pigmented fibropapilloma. Skin and fibropapilloma samples were analyzed by conventional and real time PCR assays to detect and quantify herpesvirus. All skin samples were negative, though the fibropapilloma specimen was positive in both tests. Viral load was 9,917.04 copies of viral genome per milligram of tissue. The DNA fragment amplified from the fibropapilloma sample was sequenced and allocated in the Atlantic phylogeographic group. This study reports the first molecular characterization of herpesvirus associated with fibropapilloma in turtles from the coast of RS.

A fibropapilomatose (FP) é uma doença tumoral benigna que pode causar a morte das tartarugas marinhas por dificultar a sua locomoção, visão e alimentação. Pesquisas sugerem o envolvimento de um herpesvirus em associação com fatores ambientais e genéticos como agentes causais da FP. No Brasil, foi descrita pela primeira vez em 1986. O objetivo do presente trabalho foi detectar e caracterizar esse herpesvírus em tartarugas marinhas do litoral do Estado do Rio Grande do Sul (RS). De outubro de 2008 a julho de 2010, foram encontradas 14 tartarugas marinhas entre as praias de Torres e Tavares, das quais 11 eram tartarugas verdes (Chelonia mydas) e 3 eram tartarugas cabeçudas (Caretta caretta). Todas as tartarugas eram jovens e o comprimento curvilíneo de carapaça médio foi de 37,71±7,82cm, variando de 31 a 55cm. Apenas uma tartaruga verde apresentou um fibropapiloma de 1cm, pigmentado e de superfície papilar. Amostras de pele e do fibropapiloma foram submetidas a PCR convencional e PCR em tempo real para detecção e quantificação do herpesvírus. Todas as amostras de pele foram negativas e o fibropapiloma foi positivo em ambas as técnicas, apresentando uma carga viral de 9.917,04 cópias de genoma viral/mg de tecido. O fragmento de DNA amplificado na amostra de fibropapiloma foi sequenciado e revelou pertencer ao grupo filogeográfico do Atlântico. Essa é a primeira caracterização molecular do herpesvirus associado ao fibropapiloma em tartarugas do litoral do RS.

Porcine circovirus 2 (PCV2) induces a procoagulant state in naturally infected swine and in cultured endothelial cells.

Porcine circovirus 2 (PCV2) is the primary causative agent of porcine circovirus disease (PCVD). PCVD is an emerging disease that has been reported worldwide, associated with wasting, lymphoid depletion, enteritis, pneumonia, vasculitis, ischemic lesions, and necrotizing dermatitis. Although PCVD causes considerable economic losses, the pathogenesis of PCV2 has not been fully understood. The aim of the present work was to study the participation of hemostatic system and of vascular endothelium in PCV2 infection, as well as their possible role in PCVD pathogenesis. Our results showed that naturally PCV2-infected swine displayed a prothrombotic state in vivo, since a diminished coagulation time (recalcification time, activated partial thromboplastin time and prothrombin time), a higher platelet aggregation ability (despite a diminished platelet blood count), and an increased thrombin plasma activity (associated with a reduced fibrinogen level) were observed. The PCV2-infected animals showed vasculitis and positive staining for PCV2 antigen in capillary vessels. Furthermore, PCV2-infected endothelial cells displayed an activated phenotype, characterized by an increase in cell surface procoagulant activity. Moreover, the PCV2-infected endothelial cells pre-treated with exogenous thrombin displayed an increased viral load. This work reports, for the first time, the role of the hemostatic system and of endothelium in the pathogenesis and infectivity of PCV2. The study reinforces the importance of the phenomena which occur during PCV2 infection, and affords a better knowledge of the mechanisms behind the pathophysiology of PCVD.