S. Vázquez

Induction and Inhibition of Apoptosis by Pseudorabies Virus in the Trigeminal Ganglion during Acute Infection of Swine

ABSTRACT We examined the ability of pseudorabies virus (PRV) to induce and suppress apoptosis in the trigeminal ganglion during acute infection of its natural host. Eight pigs were intranasally inoculated with a virulent field strain of PRV, and at various early times after inoculation, the trigeminal ganglia were assessed histologically. PRV-infected cells were detected by use of immunohistochemistry and in situ hybridization, and apoptosis was identified by in situ terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling. Light and electron microscopy was also used for morphological studies. Apoptosis was readily detected among infiltrating immune cells that were located surrounding PRV-infected neurons. The majority of PRV-infected neurons did not show morphological or histochemical evidence of apoptosis, even including those neurons that were surrounded by numerous inflammatory cells and exhibited profound pathological changes. However, neuronal virus-induced apoptosis also occurred but at a sporadic low level. These findings suggest that PRV is able to block apoptosis of infected trigeminal ganglionic neurons during acute infection of swine. Furthermore, our results also suggest that apoptosis of infiltrating inflammatory cells may represent an important viral mechanism of immune evasion.

Light and electron microscopic studies on turbot Psetta maxima infected with Enteromyxum scophthalmi: Histopathology of turbot enteromyxosis

In the last decade, a new parasite that causes severe losses has been detected in farmed turbot Psetta maxima (L.), in north-western Spain. The parasite was classified as a myxosporean and named Enteromyxum scophthalmi. The aim of this study was to characterize the main histological changes that occur in E. scophthalmi-infected turbot. The parasite provoked catarrhal enteritis, and the intensity of the lesions was correlated with the progression of the infection and with the development of the parasite. Infected fish were classified into 3 groups, according to the lesional degree they showed (slight, moderate and severe infections). In fish with slight infections, early parasitic stages were observed populating the epithelial lining of the digestive tract, without eliciting an evident host response. As the disease progressed, catarrhal enteritis was observed, the digestive epithelium showed a typical scalloped shape and the number of both goblet and rodlet cells was increased. Fish with severe infections suffered desquamation of the epithelium, with the subsequent release of parasitic forms to the lumen. The dislodged enterocytes underwent anoikis, a mode of apoptosis triggered by the loss of anchorage, which might facilitate spreading of the parasite. Lymphohaematopoietic depletion was also observed, mainly in head kidney and spleen, which could contribute to the high virulence of this parasite.

Risk factors associated with Enteromyxum scophthalmi (Myxozoa) infection in cultured turbot, Scophthalmus maximus (L.).

An epidemiological cohort study of Enteromyxum scophthalmi in cultured turbot was performed on a farm in North Western Spain. Four different ongrowing stocks (A, B, C, D) were monitored monthly until market size. Fish from stocks C and D were divided into 2 subgroups, receiving filtered (CF and DF) or unfiltered (CUF and DUF) water. The lack of water filtration was positively associated with infection prevalence, as all fish kept in filtered water remained uninfected. Parasite abundance varied seasonally (P<0.05) in stock B and subgroup CUF. Infection was also associated (P<0.05) with host weight, and the highest prevalences and intensities were detected in 101-200 g and 201-300 g fish. Distribution pattern of E. scophthalmi in subgroups CUF and DUF had a variance higher than the mean, indicating overdispersion. The minimum period necessary for the first detection of the parasite and for the appearance of disease symptoms and mortality, varied depending on the stock and introduction date, although a long pre-patent period was always observed. Several factors, such as host density, parasite recruitment and parasite-induced fish mortality can contribute to the observed distribution pattern. Risk factors found to be associated with E. scophthalmi infection, including water quality and accumulation of infective stages in the culture tanks, should be considered when designing control strategies to prevent the introduction and spread of infective stages in the facilities.

L-Particle Production during Primary Replication of Pseudorabies Virus in the Nasal Mucosa of Swine

ABSTRACT Different tissue culture cell lines infected with a number of alphaherpesviruses produce, in addition to virions, light particles (L particles). L particles are composed of the envelope and tegument components of the virion but totally lack the proteins of the capsid and the virus genome; therefore, they are noninfectious. In this electron microscopy report, we show that L particles are produced during primary replication of the alphaherpesvirus pseudorabies virus (PRV) in the nasal mucosa of experimentally infected swine, its natural host. Although PRV infected different types of cells of the respiratory and olfactory mucosae, PRV L particles were found to be produced exclusively by epithelial cells and fibroblasts. We observed that formation of noninfectious particles occurred by budding of condensed tegument at the inner nuclear membrane and at membranes of cytoplasmic vesicles, resulting in intracisternal and intravesicular L particles, respectively. Both forms of capsidless particles were clearly distinguishable by the presence of prominent surface projections on the envelope and the higher electron density of the tegument, morphological features which were only observed in intravesicular L particles. Moreover, intravesicular but not intracisternal L particles were found to be released by exocytosis and were also identified extracellularly. Comparative analysis between PRV virion and L-particle morphogenesis indicates that both types of virus particles share a common intracellular pathway of assembly and egress but that they show different production patterns during the replication cycle of PRV.

Localized pleural mesothelioma causing cranial vena cava syndrome in a dog.

A 9-year-old female crossbred dog was presented to the Hospital Universitario Veterinario Rof Codina (Universidad de Santiago de Compostela, Lugo, Spain) for acute onset of severe, progressive swelling of the head, neck, and cranial trunk. Survey radiographs and ultrasonography revealed a large, heterogeneous mass in the cranial mediastinum, compressing or growing into a large blood vessel within the cranial mediastinum and displacing the heart dorsocaudally. At postmortem examination, the mass was diagnosed as a large, localized mesothelioma. Localized mesotheliomas are rare neoplasms in dogs but should be considered as a possible differential diagnosis for cranial vena cava syndrome. The anatomic distribution and clinical features of mesothelioma in the present report are similar to other cases in humans.

Host and environmental risk factors associated with Cryptosporidium scophthalmi (Apicomplexa) infection in cultured turbot, Psetta maxima (L.) (Pisces, Teleostei).

An epidemiological cohort study of Cryptosporidium scophthalmi in cultured turbot Psetta maxima L. of Northwestern Spain was conducted along a four-year period. Four different ongrowing cohorts were monitored monthly from introduction into the ongrowing tanks (10-50 g) until reaching market size (400-1400 g). The association of host and environmental factors with five categories of parasite abundance was assessed using a multivariable regression framework. Epidemiological factors assessed here were water temperature, weight, length, month of collection, season, age, origin, condition factor, water filtration, and status to the myxozoan Enteromyxum scophthalmi infection. E. scophthalmi was included into the analysis because it targets the same organ than C. scophthalmi and it was prevalent in the studied population. The multivariable analysis demonstrated the statistically significant association between several factors and parasite abundance. C. scophthalmi abundance was associated (P<0.05) with age, condition factor, season, and status to E. scophthalmi infection. Young animals, with poor condition factor, during spring or summer, and not infected with the myxozoan were most likely to be highly infected by C. scophthalmi. Inclusion of these four variables significantly (P<0.05) improved the model, compared to the model that did not include any of these epidemiological factors. Increasing levels of C. scophthalmi abundance were associated (P<0.01) with higher severity of C. scophthalmi-compatible lesions. The frequency of distribution of C. scophthalmi abundance was clearly right-skewed and fitted a negative binomial distribution, whereas the intensity of infection fitted a Poisson distribution. The quantification of the variance-to-mean ratio stratified by age demonstrated overdispersion for 8-16 months old fish, although this bivariate association is likely affected by several other factors, as suggested by the results of the multivariable analysis. The negative relation between C. scophthalmi abundance and status to E. scophthalmi infection suggests differences in the transmission, onset, and course of both infections. The coarse filtration used in some cohorts did not significantly affect the levels of infection. C. scophthalmi was probably introduced into the ongrowing tanks mainly with carrier fish, though the involvement of infective oocysts from the water supply cannot be disregarded. Infection prevalence and mean intensity decreased with fish age and a seasonal distribution was found. Results presented here will help to understand the epidemiology of C. scophthalmi in turbot, to estimate the expected levels of infection associated with presence or absence of epidemiological factors, and to quantify the impact that the disease may have on susceptible turbot populations. The multivariable model used here is more powerful than the visual inspection of graphics for exploring associations in cooperative processes and can be easily extended to the assessment of epidemiological associations in other population and parasitic diseases.

Skeletal anomalies in reared Senegalese sole Solea senegalensis juveniles: a radiographic approach

Reared Senegalese sole Solea senegalensis Kaup show a high incidence of vertebral anomalies; however, little is known about its skeletal anomaly profile in the later farming phases. The purpose of this study was to provide a detailed description and quantification of the most common skeletal anomalies in reared Senegalese sole in the juvenile stage by means of computed radiography. A total of 374 Senegalese sole were classified according to the external morphology of the fish as normal or altered and then radiographed in latero-lateral and in dorso-ventral projections. Radiographic evaluation of anomalies focused especially on vertebral body anomalies (VBA) and vertebral column deviations (VCD). The 2 orthogonal projections provided a more complete visualization of the skeleton. Approximately 75% of the individuals showed at least 1 anomaly, while VBA and/or VCD were detected in 48.9% of the specimens. Regarding external morphology, 88% of the fish were categorized as normal, although about 72% of these normal fish displayed abnormalities in radiographies. The most frequent anomalies consisted of deformations of the caudal complex plates (hypurals, parhypural and epural), preurals and caudal vertebrae. Scoliosis was the most prevalent among VCD, affecting the caudal area in almost 15% of the individuals. The anomaly profile at the juvenile stages showed some differences compared to what has been reported previously in earlier stages of development. In light of these results, further investigation into the progression of skeletal anomalies over time and the causative factors at later stages is required.

Intracranial epidural empyema due to Cryptococcus neoformans in a 5-year-old neutered male European short hair cat

A 5-year-old neutered male European short hair cat was examined at the Veterinary Teaching Hospital Rof Codina for a one-month history of progressive lethargy and purulent left nasal and ocular discharge that did not show improvement on treatment with antibiotics and glucocorticoids. Moreover, the owner reported that during the last two weeks the cat had shown loss of house training habits and some episodes of aggression. On physical examination, the cat showed unilateral nasal and ocular discharge with blepharospasm on the left side, hypothermia (37.8 C), a fluid containing non-painful subcutaneous mass over the frontal bones, more evident on the left side, and moderate dehydration (Figure 1). The neurological examination revealed depressed mental status, reduced postural reactions in all four limbs with normal spinal reflexes, reduced menace reaction in the left eye with normal pupillary light and palpebral reflexes and left facial hypalgesia. Neurological signs were consistent with a space-occupying lesion in the right supratentorial region or other multifocal or diffuse central nervous system (CNS) disease. The main differential diagnosis was an inflammatory disease, although a focal neoplasia was also considered possible. Results of haematology, biochemistry, urinalysis, feline leukemia virus (FeLV) and feline immunodeficienciy virus (FIV) testing, thoracic radiographs and abdominal ultrasound were unremarkable. Preand postcontrast cranial computed tomography (CT) showed extensive left frontal sinusitis with severe osteomyelitic destruction of the frontal bone and subcutaneous fluid collection with intracranial extension. The intracranial fluid accumulation was located in the right frontoparietal area and was seen as a well-demarcated lesion with medial rim enhancement and significant mass effect (Figure 2). These findings were interpreted as a frontal sinusitis with secondary intracranial extension and an associated epidural empyema. A right-sided rostrotentorial craniotomy was performed. A small defect in the temporal bone was created and during this approach, a viscous purulent material flowed out of the defect. A large quantity of this material was drained and after saline lavage purulent material was no longer observed in the subdural space. A sample of this material was taken, formalin-fixed and processed for histology by routine methods. Sections were stained by haematoxylin and eosin, periodic acid-Schiff and toluidine blue. Microscopic examination revealed a loosely organized tissue with necrotic debris mixed with a large number of inflammatory cells predominantly macrophages and neutrophils and marked accumulations of structures with morphology highly suggestive of Cryptococcus spp. (Belluco et al. 2008; Magalh~aes et al. 2012). The yeasts were ovoid-spherical in shape, 3 8 mm in diameter and rimmed by a thin cell wall, which was surrounded by a clear mucoid capsule negatively stained (halo) of variable thickness (10 30 mm) (Figure 3). Organisms were intracytoplasmatic within macrophages and extracellular within the necrotic tissue. Gram stain of the empyema showed low numbers of Gram-positive cocci. Bacterial growth was not observed on routine aerobic and anaerobic cultures. Fungal culture produced a heavy, pure growth of Cryptococcus spp. The isolated organism was confirmed to be Cryptococcus neoformans using the selective medium L-canavanine glycine bromothymol blue agar (Klein et al. 2009) and it was susceptible to itraconazole, fluconazole, amphotericin B and flucytosine. Treatment with cephadroxil (22 mg/kg body weight (BW) orally twice daily; Cefa Cure, MSD Animal Health, Salamanca, Spain) and fluconazole (50 mg orally twice daily; Fluconazol, Normon, Madrid, Spain) were instituted and neurological deficits resolved gradually over the next two weeks. On re-evaluation one month after the surgery, the owners reported a significant improvement in the cat’s status. No significant abnormali-

Skeletal Anomalies in Senegalese Sole (Solea senegalensis), an Anosteocytic Boned Flatfish Species:

Skeletal anomalies affect animal welfare and cause important economic problems in aquaculture. Despite the high frequency of skeletal problems in reared Solea senegalensis, there is lack of information regarding the histological features of normal and deformed vertebrae in this flatfish. The aim of this study was to describe the histopathological and radiographical appearance of vertebral body anomalies. Sixty-seven juvenile fish were radiographically examined 104 or 105 days after hatching. Through radiographic images, vertebral segments were selected and processed for histopathological examination from 7 normal and 7 affected fish. Alterations in bone shape and vertebral fusion were the most significant anomalies in the vertebral bodies. These alterations occurred most frequently between the last 3 abdominal vertebrae and the first 10 caudal centra. Radiographically, deformed vertebrae showed flattening of the endplates and narrowing of the intervertebral spaces. The radiographic findings concurred with the histological lesions where affected vertebrae exhibited irregular endplates and changes in trabecular bone. Radiolucent cartilaginous tissue was evident in the endplates of the deformed vertebra and, in some cases, the cartilaginous material extended from the growth zone into the intervertebral space. These changes were likely the primary alterations that led to vertebral fusion. Fused vertebrae were often reshaped and showed a reorganization of the trabeculae. The formation of metaplastic cartilage is frequent in a variety of anomalies affecting teleost species.