I. de Blas

WIN EPISCOPE 2.0: improved epidemiological software for veterinary medicine

Recent changes in veterinary medicine have required quantitative epidemiological techniques for designing field surveys, identifying risk factors for multifactorial diseases, and assessing diagnostic tests. Several relevant techniques are brought together in the package of veterinary epidemiological computer software, WIN EPISCOPE 2.0, described in this paper. It is based on Microsoft Windows and includes modules for the design and analysis of field surveys, control campaigns and observational studies, and a simple mathematical model. It provides comprehensive ‘Help’ screens and should therefore be useful not only in field investigations but also for teaching veterinary epidemiology.

Host-microbiota interactions within the fish intestinal ecosystem.

Teleost fish are in direct contact with the aquatic environment, and are therefore in continual contact with a complex and dynamic microbiota, some of which may have implications for health. Mucosal surfaces represent the main sites in which environmental antigens and intestinal microbiota interact with the host. Thus, the gut-associated lymphoid tissues (GALT) must develop mechanisms to discriminate between pathogenic and commensal microorganisms. Colonization of intestinal mucosal surfaces with a normal microbiota has a positive effect on immune regulatory functions of the gut, and disturbance in these immune regulatory functions by an imbalanced microbiota may contribute to the development of diseases. Significant attention has therefore been recently focused on the role of probiotics in the induction or restoration of a disturbed microbiota to its normal beneficial composition. Given this, this article explores the fascinating relationship between the fish immune system and the bacteria that are present in its intestinal microbiota, focusing on the bacterial effect on the development of certain immune responses.

Anti-oxidant supplementation improves boar sperm characteristics and fertility after cryopreservation: comparison between cysteine and rosemary (Rosmarinus officinalis).

Anti-oxidants partially ameliorated the detrimental effects of reactive oxidative substances produced during cryopreservation. The objective of the study was to determine the effect of anti-oxidant addition to the freezing extender on boar semen qualities and fertility capacity. Ejaculates were collected from a previously selected boar and semen samples were processed using the straw freezing procedure. In experiment 1, semen samples were cryopreserved in lactose-egg yolk solution supplemented with various concentrations of cysteine (0, 5 and 10mM) to determinate a cysteine concentration capable of producing a protective effect during cryopreservation. Semen quality (total motility, progressive motility, viability, acrosome integrity and hypoosmotic swelling test) was evaluated after freezing and thawing and then every hour for 3h. In experiment 2, ejaculates were cryopreserved with lactose-egg yolk extender with or without the following anti-oxidants: cysteine, rosemary (Rosmarinus officinalis) and cysteine plus rosemary. Semen quality was evaluated. In the experiment 3, fertility capacity of semen frozen in anti-oxidant supplementation extenders was examined in vitro. A total of 2232 oocytes were in vitro matured and inseminated with frozen-thawed sperm. In summary: (i) the effective concentration of cysteine in freezing extender was 10mM; (ii) the addition of exogenous rosemary or cysteine to the freezing extender positively affected post-thawed viability and acrosome integrity. Only rosemary supplementation improved total motility at 3h and progressive motility at any time; (iii) the inclusion of rosemary into the extender was effective in penetration and cleavage rate and also in the efficiency of the fertilization system.

Enteric calicivirus and rotavirus infections in domestic pigs

We report the prevalence of rotavirus and calicivirus infections, along with their respective association with diarrhoea in the porcine population of the region of northern Spain. A total of 221 samples were collected at random from different farms in the region and from the main slaughterhouse facility in the city of Zaragoza. Faecal samples were scored as diarrhoeic or normal and grouped into five groups to match general farm management and age criteria: group I (suckling 0-4 weeks), group II (weaning >4-8 weeks), group III (transition >8-16 weeks), group IV (fattening >16-24 weeks) and group V (adults >24 weeks). Group A rotavirus detection and caliciviruses were investigated by reverse transcription-polymerase chain reaction (RT-PCR). Conventional RT-PCR was performed using primers designed to detect rotavirus group A, caliciviruses and/or human noroviruses. A real-time RT-PCR was carried out using TaqMan probes for genogroups GI and GII of noroviruses. Rotaviruses and caliciviruses were detected with an overall prevalence of 16.7% and 12.2%, respectively. Rotavirus detection in faecal samples was associated with both age and faecal consistency, being more frequent in piglets aged <8 weeks with odds ratios (ORs) equal to 4.3 and 4.9, respectively. Calicivirus shedding in faecal samples was homogenously distributed in all ages, showing no significant association with age or faecal consistency (OR 0.87 and 0.99, respectively). A selection of rotavirus-positive stools were genotyped by multiplex nested PCR. G10, P[6], G12 P[8], G9 [p8] and G4 P[23] genotype combinations were found. Three isolates showed a G3 genotype, but their VP4 gene could not be amplified. It should be noted that the G9 genotype was the major G genotype circulating during that period in Spain. None of the porcine samples was positive for norovirus by real-time RT-PCR, despite the ability of this technique to detect at least 18 human norovirus genotypes. Our data indicate that human noroviruses are unlikely to be circulating in the porcine population; however, sapoviruses have been found. Contrary to rotavirus infection, Calicivirus infection is asymptomatic. Specific primers to detect porcine noroviruses are needed.

Comparing sugar type supplementation for cryopreservation of boar semen in egg yolk based extender

Cryopreservation of boar semen is still considered suboptimal due to lower fertility when compared to fresh semen. The aim of this study was to evaluate the effects of the addition of different sugars (lactose, trehalose and glucose) on boar spermatozoa cryopreserved in an egg yolk based extender. Ejaculates were collected from a boar previously selected and semen samples were processed using the straw freezing procedure. In experiment 1, subsamples of semen were frozen in three different extenders: recommended lactose egg yolk extender (LEY); trehalose egg yolk extender (TEY) and glucose egg yolk extender (GEY). Sperm quality was assessed for motility, viability, acrosome integrity and hypoosmotic swelling test response upon collection, after freezing and thawing and then every hour for 3h. Results showed that total motility at 1 and 3h, progressive motility at 3h, positive hypoosmotic response at 2 and 3h and acrosome integrity at all times were significantly improved when trehalose was added to the extender. In experiment 2, sugar influence was also demonstrated in vitro fertilization. A total of 1691 oocytes were in vitro matured and inseminated with frozen-thawed sperm at 2000:1 sperm:oocyte ratio and coincubated for 6h. Presumptive zygotes were cultured in NCSU-23 medium to assess fertilization parameters and embryo development. Both penetration and monospermy rates were significantly higher for trehalose frozen semen. A significant increase was observed in efficiency and blastocyst formation rates from TEY to the other groups. Our results demonstrated that trehalose extender enhances spermatozoa viability and its in vitro fertilization parameters in boar ejaculates with good sperm freezability. Further studies are necessary to assess the impact of sugars on the entire population.

Mucosal immunization against ovine lentivirus using PEI-DNA complexes and modified vaccinia Ankara encoding the gag and/or env genes

Sheep were immunized against Visna/Maedi virus (VMV) gag and/or env genes via the nasopharynx-associated lymphoid tissue (NALT) and lung using polyethylenimine (PEI)-DNA complexes and modified vaccinia Ankara, and challenged with live virus via the lung. env immunization enhanced humoral responses prior to but not after VMV challenge. Systemic T cell proliferative and cytotoxic responses were generally low, with the responses following single gag gene immunization being significantly depressed after challenge. A transient reduction in provirus load in the blood early after challenge was observed following env immunization, whilst the gag gene either alone or in combination with env resulted in significantly elevated provirus loads in lung. However, despite this, a significant reduction in lesion score was observed in animals immunized with the single gag gene at post-mortem. Inclusion of IFN-gamma in the immunization mixture in general had no significant effects. The results thus showed that protective effects against VMV-induced lesions can be induced following respiratory immunization with the single gag gene, though this was accompanied by an increased pulmonary provirus load.

Systemic DNA immunization against ovine lentivirus using particle-mediated epidermal delivery and modified vaccinia Ankara encoding the gag and/or env genes

To determine whether systemic immunization with plasmid DNA and virus vector against visna/maedi virus (VMV) would induce protective immune responses, sheep were immunized with VMV gag and/or env sequences using particle-mediated epidermal bombardment and injection of recombinant modified vaccinia Ankara. The results showed that immunization induced both humoral and cell-mediated responses prior to and after virus challenge. The vaccination protocol did not prevent infection, but immunization with the gag gene or a combination of gag and env genes resulted in significantly reduced provirus loads in blood and mediastinal lymph node, respectively. Provirus loads in lung and draining lymph node were unaffected, but p25 expression was undetectable in lungs of animals immunized with a combination of gag and env genes. Analysis of target tissues for lesions at post-mortem showed that immunization with the env gene caused a significant increase in lesion score, while the gag gene or a combination of gag and env genes had no effect. Inclusion of the ovine interferon-gamma gene in the initial priming mixture had minimal effect on immune responses, provirus load, or lesion development, although it resulted in a decreased p25 expression in the lung. The results thus show that systemic immunization with gag or a combination of gag and env genes reduces provirus load in blood and lymphoid tissue, respectively whereas env immunization has no effect on provirus load but increased lesion development.

Percutaneous treatment of intrabdominal abscess: urokinase versus saline serum in 100 cases using two surgical scoring systems in a randomized trial

The purpose of this study was to assess whether regular instillation of urokinase during abscess drainage leads to an improved outcome compared to saline irrigation alone. One hundred patients referred for image-guided abdominal abscess drainage were randomized between thrice daily urokinase instillation or saline irrigation alone. At the end of the study, patient medical records were reviewed to determine drainage, study group, Altona (PIA II) and Mannheim (MPI) scoring, duration of drainage, procedure-related complications, hospital stay duration, and clinical outcome. The technical success rate of the percutaneous abscess drainage was 100%. The success or failure of abscess remission did not differ significantly between groups (success rate of 91.5% in the urokinase group vs. 88.8% in the saline group; failure rate was of 8.5 vs. 21.2%, respectively); however, days of drainage, main hospital stay, and overall costs were significantly reduced in patients treated with urokinase compared to the control group (P < 0.05). No adverse effects from urokinase were observed. Surgical scores were a useful homogeneity factor, and MPI showed a good correlation with prognosis, while PIA results did not have a significant correlation. For drainage of complex abscesses (loculations, hemorrhage, viscous material), fibrinolytics safely accelerate drainage and recovery, reducing the length of the hospital stay and, therefore, the total cost.

Clinical characterisation of natural scrapie in a native Spanish breed of sheep

SCRAPIE is the prototype of a group of diseases referred to as transmissible spongiform encephalopathies; it affects sheep and goats and is caused by the accumulation of an abnormal protease-resistant isoform (PrPSc) of cellular prion protein (PrPc) in tissues of the central nervous system (Prusiner 1982). The clinical signs of scrapie may differ between countries: in the UK pruritus is the most common sign; in the USA, wasting, debility, tremor and incoordination are distinctive signs of scrapie, with pruritus, if observed at all, as a subtle sign (Detwiler 1992). In Spain, the disease has been reported as having a slow and progressive course of three to six months’ duration from the onset of clinical signs (Garcia de Jalon and others 1987). This short communication describes a study to characterise the clinical features observed in sheep naturally affected with scrapie in Aragon, Spain, and to compare the results with previously described findings. A group of 24 adult sheep of the Rasa Aragonesa breed with clinical signs compatible with scrapie were selected from five flocks located in the Aragon region of Spain in which laboratory confirmation of the disease had been made. Another 26 adult sheep without clinical signs, or showing clinical signs not compatible with scrapie, were selected from the same flocks to make up a control group. All the scrapie-affected sheep were between two and five years old and they were homozygous for A136R154Q17 (ARQ/ARQ), the genotype associated with a high risk of suffering scrapie (Dawson and others 1998). The control animals also had genotypes conferring a high or intermediate risk for suffering the disease (ARQ/ARQ, ARQ/ARH, ARR/ARQ, ARQ/AHQ and ARQ/VRQ) (Monleon and others 2004). A complete clinical examination of all the animals was carried out, including their clinical history, a general examination and special examination of all systems, with particular attention to the nervous system (Lisle 1996). The animals’ body condition score (BCS) was evaluated on a scale of 1 to 5: 1 Emaciated, 2 Thin, 3 Average, 4 Fat, 5 Obese (Russel 1991). Electrocardiography was carried out using a bipolar lead base-apex electrocardiogram (Cardiofax ECG 6511; Nihon Kohden) (Reef and McGuirk 1996). The animals were euthanased by an overdose of barbiturates, according to ethical recommendations. Routine histopathological, immunohistochemical and Western blotting analysis of the brain tissues were used to make a definitive diagnosis of scrapie by previously described methods (Schaller and others 1999, Monleon and others 2003). Statistical analysis was performed by the Pearson chi-squared test or Fisher’s exact test, using SSPS software; the level of significance was established as P<0·05. If possible, the odds ratio (OR) was calculated as an indicator of the relationship grade between the clinical signs and the presence of the disease (Daniel 1997). The course of the disease lasted on average three months (range one to six months) from the onset of clinical signs to euthanasia when the animal was in the final stages of the disease. The most important clinical features observed could be classified as alteration of the animal’s mental status, alteration of superficial sensitivity or alteration of deep sensation and motor functions. Other clinical features observed in the scrapie-affected animals were alteration of cranial nerve function, cardiac rhythm and BCS. The first clinical sign observed in 70·8 per cent of the affected animals (P=0·251) (Fig 1) was alteration of mental status, a feature which has also been observed by other researchers (Stamp 1980, Parry 1983, Detwiler 1992, Capucchio and others 2001). Alterations were mostly characterised by excitatory signs such as restlessness, anxiety and a tendency to avoid restraint. However, hyperexcitability to external stimuli, such as a hand clap and/or approach, were observed in 50 per cent of affected animals (P<0·0001) (Fig 1), and teeth grinding was also observed in 41·7 per cent of them (OR=3·93, P=0·039) (Fig 1), this finding being in accordance with that of Healy and others (2003). Alteration of superficial sensitivity was characterised by hypoaesthesia or total anaesthesia at the distal extremes of the hindlimbs and sometimes the forelimbs; this was observed in 54·2 per cent of the scrapie-positive animals (OR=29·54, P=0·0001) (Fig 1). Pruritus was also observed in 70·8 per cent of them (P<0·0001) (Fig 1), mainly on the rump and tail areas, but sometimes also on the head, thorax and both the internal and external sides of the hindlimbs (Detwiler 1992, Schreuder 1998, Ulvund 1999, Capucchio and others 2001). However, pruritus was mostly a subtle sign, which sometimes could only be observed indirectly, either by wool loss (OR=13·36, P=0·0001) (Fig 1) in all the abovementioned areas or by inducing the nibbling reflex, which was only observed in 45·8 per cent of the affected animals (P=0·0001) (Fig 1). Alteration of deep sensation and motor functions were characterised by hyporeflexia, which was observed in 41·7 per cent of the scrapie-affected sheep (OR=5·48, P=0·017) (Fig 1), mainly in the hindlimbs (patellar reflex) but sometimes in the forelimbs (carporadial extensor reflex). Head tremors were observed in 41·7 per cent of the positive animals (OR=17·86, P=0·001) (Fig 1) and were more evident when the animal was stressed. Ataxia and gait abnormalities were observed in 54·2 per cent of the positive animals (OR=4·96, P=0·011) (Fig 1) and were characterised by a high-stepping gait (trotting) of the forelimbs, jumping of the hindlimbs (bunny hop) and hypermetria, which progressed to ataxia of the hindlimbs, generalised ataxia, falls, difficulty in rising and total recumbency, as has been described by other authors (Stamp 1980, Detwiler 1992). Short Communications

Freeze-dried dog sperm: Dynamics of DNA integrity

Freeze-drying (FD) has been proposed as an alternative method to preserve spermatozoa. During the FD procedure, sperm DNA might become damaged by both freezing and drying stresses caused by the endonucleases, the oxidative stress and the storage conditions. We examined the DNA integrity of dog sperm freeze-dried with two kinds of chelating agents in FD buffers and storage at two different temperatures. Ejaculated sperm from four dogs were suspended in basic medium (10 mM Tris-HCl buffer+50 mM NaCl) supplemented with 50 mM EGTA or with 50 mM EDTA and then freeze-dried. Sperm samples were stored at 4°C as room temperature, and the analysis of DNA damage was performed after a month and 5 months of storage using a Sperm Chromatin Dispersion test. We found four different sperm populations according to the size of the halos around the sperm head: (1) absent halo, (2) <6 μm, (3) 6-10 μm, (4) >10 μm. All of them coexisted in each freeze-dried dog semen samples and differed significantly among different treatments. The highest percentage of spermatozoa with halo >10 μm was obtained when the semen samples were freeze-dried in EDTA medium and stored at room temperature for five months. Results suggested that both, the kind of chelating agent as well as storage temperature and period, influenced DNA integrity of freeze-dried dog sperm.