Management of encephalomyocarditis virus infection in Italian pig farms: a case report.

BACKGROUND: Encephalomyocarditis virus (EMCV) has been isolated from many animals, frequently as the cause of fatal myocarditis, but pigs are the most susceptible domestic specie. The virus was isolated in swine farms since 1958 in Panama and Europe from cases of sudden death in young pigs, and the main origin of outbreaks has been assumed to be local rodent populations. There is no treatment for the disease. CASE PRESENTATION: The clinical case describes an outbreak of encephalomyocarditis virus in a farrowing (farm A) and a weaning (farm B) site, with mortality that reached 24.2% in suckling piglets and 7.7% in weaners. The farms were located in an endemic Italian area, and the outbreak was characterised by high mortality with sudden death and clinical signs due to heart failure (trembling, dispnea and fever). The rodents control program was the key action in managing the outbreak. However, in the weaning site, the lack of rodent program in some unexplored areas of the barn (false ceiling) was responsible of a longer time of resolution of the outbreak. An unusual support treatment approach from human medicine suggestion was also applied using acetylsalicylic acid for its antiphlogistic and antithrombotic effects. CONCLUSIONS: To control the rodent population in a pig farm is often difficult and requires a deep knowledge also of the rodents habits. Considering the lack of treatment for the disease and the absence of available vaccines in several Countries, acetylsalicylic acid might be of interest for further studies as an important support for pigs' recovery.

Mitogenomic analysis of extant condor species provides insight into the molecular evolution of vultures.

The evolution of large vultures linked to mountainous habitats was accompanied by extreme physiological and behavioral specializations for energetically efficient flights. However, little is known on the genetic traits associated with the evolution of these obligate soaring scavengers. Mitochondrial DNA plays a vital role in regulating oxidative stress and energy production, and hence may be an important target of selection for flight performance. Herein, we characterized the first mitogenomes of the Andean and California condors, the world's heaviest flying birds and the only living representatives of the Vultur and Gymnogyps genus. We reconstructed the phylogenetic relationships and evaluated possible footprints of convergent evolution associated to the life-history traits and distributional range of vultures. Our phylogenomic analyses supported the independent evolution of vultures, with the origin of Cathartidae in the early Paleogene (~ 61 Mya), and estimated the radiation of extant condors during the late Miocene (~ 11 Mya). Selection analyses indicated that vultures exhibit signals of relaxation of purifying selection relative to other accipitrimorph raptors, possibly indicating the degeneration of flapping flight ability. Overall, our results suggest that the extreme specialization of vultures for efficient soaring flight has compensated the evolution of large body sizes mitigating the selection pressure on mtDNA.

Prevalence of fibrinous pericarditis in heavy pigs (170 kg) and its association with other pluck lesions at slaughter inspection.

This study determined the prevalence of fibrinous pericarditis and its correlation with other pluck lesions in 658 batches of pigs from 236 intensive farms located in Northern Italy over a 12-month period. All pigs were slaughtered at 170 kg, and a total 57,943 plucks (approximately 90 pigs/batch) were individually assessed for the presence of fibrinous pericarditis, pneumonia, pleuritis, and liver milk spots. There was no seasonal variation in the prevalence of plucks with fibrinous pericarditis and annual mean prevalence was 5.6% (range, 0-26.3% at batch level; median, 4.71%). Farm of origin, evaluated as a random effect, accounted for 17.7% batch variation. Batches with a high prevalence of fibrinous pericarditis (≥7.7%) had higher prevalences of pleural, pulmonary, and liver lesions than those with low-middle prevalence of pericarditis; high prevalence of pericarditis was predictive of pluck lesions (P < 0.001). There was a highly significant association between fibrinous pericarditis and severe pleuritis, and 55% of plucks with the highest score for pleuritis also had ongoing fibrinous pericarditis, with a positive correlation at batch level (r2 = 0.52; P < 0.001). The co-existence of pericarditis and pleuritis (73.5% of all pericarditis cases) suggests that pleuritis plays a role in the pathogenesis of pericarditis. Based on the prevalence fibrinous pericarditis, and the role of pleuritis as a potential comorbidity, abattoir data on pluck lesions with accompanying farm history, could aid the interpretation and management of on-farm health problems, and inform diagnostic protocols.

A novel blood-sampling technique in lactating sows: The mammary vein route.

In the pig industry, labour efficiency and animal welfare have become two of the most important factors for achieving technical goals and farming competitiveness. Blood sampling is one of the most common sample-collecting techniques, but routine on-field blood collection can be very demanding for farm operators and the welfare of the animals, in particular for lactating sows. The aim of this study was to describe and investigate the mammary vein as a novel means of blood access in lactating sows that does not require coercive restraint. The study involved a total of 68 sows: 34 animals were sampled from the jugular vein (Group J) and the other 34 sows from the mammary vein (Group M). Labour time and indicators of the sow welfare (vocalizations during collection and serum cortisol concentration in the 30min after the procedure) were collected from the two groups. The total amount of labour required, calculated as the time employed to perform blood collection multiplied by the number of operators involved in performing the technique (one for Group M and two for Group J, one for restraint and one for sampling), was significantly lower in Group M than Group J (Group M, 39.83±29.45s; Group J, 82.73±55.34s; P<0.001). Mean blood volume collected at T0 was 4.81±2.00mL and 4.84±1.73mL in Group J and Group M, respectively (P>0.05). The percentage of sows that vocalized in Group M was less than in Group J (2.94% vs. 94.12%; P<0.001). Serum cortisol concentrations were not statistically different between the two groups (P>0.05). The present study suggests greater efficiency in terms of saving labour time and reducing sow vocalization during blood collection with the use of mammary vein access compared with jugular vein access.

Effect of prior insemination of dead sperm and gestation housing management on gilt fertility.

Danbred gilts at about 120 kg were group housed for estrous detection. At detection of estrus, gilts either remained in pens (P) or were re-housed into individual gestation stalls (S) and were inseminated (DS), or not (SC), with a dose of frozen/thawed dead semen. Groups were P-DS (n = 81), P-SC (n = 70), S-DS (n = 98) and S-SC (n = 90). All gilts were inseminated with semen containing viable sperm at the second detected estrus and 24 h later. Pregnant gilts that were stall housed were moved to pens 35 d after insemination. There were no effects of insemination or housing management on farrowing rates or litter sizes.

A new hybrid approach for MHC genotyping: high-throughput NGS and long read MinION nanopore sequencing, with application to the non-model vertebrate Alpine chamois (Rupicapra rupicapra).

The major histocompatibility complex (MHC) acts as an interface between the immune system and infectious diseases. Accurate characterization and genotyping of the extremely variable MHC loci are challenging especially without a reference sequence. We designed a combination of long-range PCR, Illumina short-reads, and Oxford Nanopore MinION long-reads approaches to capture the genetic variation of the MHC II DRB locus in an Italian population of the Alpine chamois (Rupicapra rupicapra). We utilized long-range PCR to generate a 9 Kb fragment of the DRB locus. Amplicons from six different individuals were fragmented, tagged, and simultaneously sequenced with Illumina MiSeq. One of these amplicons was sequenced with the MinION device, which produced long reads covering the entire amplified fragment. A pipeline that combines short and long reads resolved several short tandem repeats and homopolymers and produced a de novo reference, which was then used to map and genotype the short reads from all individuals. The assembled DRB locus showed a high level of polymorphism and the presence of a recombination breakpoint. Our results suggest that an amplicon-based NGS approach coupled with single-molecule MinION nanopore sequencing can efficiently achieve both the assembly and the genotyping of complex genomic regions in multiple individuals in the absence of a reference sequence.

Prevalence and risk factors for gastric ulceration in pigs slaughtered at 170 kg.

Oesophago-gastric ulcers (OGU) are a production and welfare problem in pigs. Stomach condition was scored for 22 551 pigs in 228 batches over a 7-month period at an abattoir in Italy processing heavy pigs for ham production. Mild or severe ulceration was observed in 20.7% of pigs, of which 13% had scar tissue. Variation between batches was high (0% to 36% prevalence of severe ulcers) and showed a significant effect of farm of origin (P<0.001). Overnight lairage increased the prevalence of mild ulcers (P<0.001), but not severe or scarred ulcers. Scarred ulcers increased in the hottest summer months. Prevalence of ulcers showed only few and weak correlations at batch level with pathologies of the pleura, lungs and liver, but a strong correlation with on-farm mortality of the batch. Analysis of farm risk factors for OGU was assessed by questionnaire with a response rate of 17% of farms. Risk factors retained in a multivariable model included a protective effect of anthelmintic treatment (risk ratio (RR)=5.1, P=0.03), increased risk in farms using Mycoplasma vaccination (RR=5.6, P=0.04) and a tendency for association with type of flooring (P=0.06). Univariable analyses also highlighted possible influences of other stress-inducing factors including lack of enrichment objects and mixing of pigs during fattening, suggesting that the role of on-farm stressors merits further investigation. It is concluded that abattoir screening of OGU in future programmes for the assessment of well-being on farm should encompass only severe lesions and scarring, and results be returned to farmers to facilitate improvement of production and welfare.

Pig surgery: cryptorchidectomy using an inguinal approach.

The objective of the study was to determine whether a less-invasive surgical technique used in cryptorchid horses, dogs and cats can be used successfully to remove retained testicles in pigs on farm. In total, 284 monolateral cryptorchid pigs underwent surgery on-farm condition, during which an incision was made over the inguinal ring and the undescended testicle was located for removal via identification of the vaginal process and the embryonic gubernaculum. A traction of these structures allowed the testis to pass through the deep and the superficial rings up to its exteriorisation outside the abdominal wall through the inguinal canal. The undescended testicle was located in the abdomen in 258 cases (90.8 per cent) and in the inguinal region in the remaining 26 cases (9.1 per cent). In none of the pigs was the abdominal cavity breached or the inguinal rings enlarged. However, in 23 pigs (8.1 per cent) the gubernaculum testis was thin and it frayed and ruptured when traction was applied, requiring a recovering by inserting a finger and Kelly curved forceps into the abdomen through the inguinal ring. In two pigs (0.7 per cent), the undescended testicle was not found. All surgical procedures were completed within 6-12 minutes. Four pigs died within two days after surgery (1.4 per cent). Major intraoperative or long-term complications did not occur. Results suggested that this surgical method is highly effective and could be used as a primary surgical approach in cryptorchid pigs as it is in cryptorchid dogs, cats and horses.

An impedance method for spatial sensing of 3D cell constructs--towards applications in tissue engineering.

We present the characterisation and validation of multiplexed 4-terminal (4T) impedance measurements as a method for sensing the spatial location of cell aggregates within large three-dimensional (3D) gelatin scaffolds. The measurements were performed using an array of four rectangular chambers, each having eight platinum needle electrodes for parallel analysis. The electrode positions for current injection and voltage measurements were optimised by means of finite element simulations to maximise the sensitivity field distribution and spatial resolution. Eight different 4T combinations were experimentally tested in terms of the spatial sensitivity. The simulated sensitivity fields were validated using objects (phantoms) with different conductivity and size placed in different positions inside the chamber. This provided the detection limit (volume sensitivity) of 16.5%, i.e. the smallest detectable volume with respect to the size of the measurement chamber. Furthermore, the possibility for quick single frequency analysis was demonstrated by finding a common frequency of 250 kHz for all the presented electrode combinations. As final proof of concept, a high density of human hepatoblastoma (HepG2) cells were encapsulated in gelatin to form artificial 3D cell constructs and detected when placed in different positions inside large gelatin scaffolds. Taken together, these results open new perspectives for impedance-based sensing technologies for non-invasive monitoring in tissue engineering applications providing spatial information of constructs within biologically relevant 3D environments.

Polar bear encephalitis: establishment of a comprehensive next-generation pathogen analysis pipeline for captive and free-living wildlife.

This report describes three possibly related incidences of encephalitis, two of them lethal, in captive polar bears (Ursus maritimus). Standard diagnostic methods failed to identify pathogens in any of these cases. A comprehensive, three-stage diagnostic 'pipeline' employing both standard serological methods and new DNA microarray and next generation sequencing-based diagnostics was developed, in part as a consequence of this initial failure. This pipeline approach illustrates the strengths, weaknesses and limitations of these tools in determining pathogen caused deaths in non-model organisms such as wildlife species and why the use of a limited number of diagnostic tools may fail to uncover important wildlife pathogens.

Apoptosis and aging in mitochondrial morphology mutants of S. cerevisiae.

Cell viability during chronological aging and after apoptotic stimuli in some yeast mutants with altered mitochondrial morphology was followed; a function for the corresponding genes in the apoptotic process was assessed. MDM30 and DNM1, the genes encoding an F-box protein and the dynamin-related GTPase, respectively, are involved in triggering aging and apoptosis. In contrast, YME1, encoding a subunit of the mitochondrial inner membrane i-AAA proteinase complex, has a protective role in these processes. FIS1, the mitochondrial fission gene, might play a protective role after an apoptotic insult while it seems to promote cell death in aging cells.

Análise do perfil do paciente portador de doenca osteomuscular relacionada ao trabalho (DORT) e usuário do serviço de saúde do trabalhador do SUS em Belo Horizonte / Analysis of the patient`s profile who bears DORT and is watched by the worker`s health reference center of general health service from Belo Horizonte

O presente trabalho pretende descrever o perfil do paciente portador de DORT atendido pelo Sistema Unico de Saude(SUS) em Belo Horizonte. Conhece-lo e de fundamental importancia, pois essas patologias sao responsaveis por cerca de (85 por cento) dos afastamentos do trabalho alem, de causarem grandes impactos psicologicos e socioeconomicos. Realizou-se o levantamento de 145 prontuarios de pacientes do Centro de Referencia de Saude ddo Trabalhador (CERSAT), no periodo de 1998 a 2001. Dados como sexo, faixa etaria, escolaridade, profissao, situacao de trabalho e diagnosticos foram analisados. Os resultados apontam que as DORT acometem mais as mulheres entre 21 e 50 anos de idade com baixo grau de escolaridade. Dados importantes foram observados no item ``diagnosticos``, pois estes tem-se mostrado inconclusivos, impedindo intervencao fisioterapica adequada. Constatou-se, contudo, que as tendinites continuam sendo as mais frequentes (49 por cento) e que as tenossinovites, as epicondilites e a fibromialgia tem aumentado nos ultimos anos, sendo que, na maioria dos casos, os pacientes chegam ao tratamento em fases avancadas. Essa situacao e um problema de saude publicae tem trazido prejuizos aos trabalhadores, tornando evidente a importancia de conhecer as necessidades dos pacientes e de oferecer-lhes tratamento mais adequado

Molecular evolution of the period gene in sandflies.

The molecular evolution of the clock gene period was studied in Phlebotomine sandflies (Diptera: Psychodidae). Comparison of the synonymous and nonsynonymous substitution rates between sandflies and Drosophila revealed a significantly higher evolutionary rate in the latter in three of the four regions analyzed. The differences in rate were higher in the sequences flanking the Thr-Gly repetitive domain, a region that has expanded in Drosophila but remained stable and short in sandflies, a result consistent with the coevolutionary scenario proposed for this region of the gene. An initial phylogenetic analysis including eight neotropical sandfly species and one from the Old World was also carried out. The results showed that only the subgenus Nyssomyia is well supported by distance (neighbor-joining) and maximum parsimony analysis. The grouping of the other species from the subgenus Lutzomyia and Migonei group shows very low bootstrap values and is not entirely consistent with classical morphological systematics of the genus Lutzomyia.

Isolation and study of KlLSM4, a Kluyveromyces lactis gene homologous to the essential gene LSM4 of Saccharomyces cerevisiae.

We have isolated the KlLSM4 gene as a multicopy suppressor of a Kluyveromyces lactis mutant which shows a rag(-) phenotype (resistance to antimycin A on glucose). This gene is homologous to the ScLSM4 of Saccharomyces cerevisiae, which codes for an essential 187 amino acid protein containing Sm-like domains. These motifs are present in the evolutionarily conserved family of the Sm-like proteins, which are involved in a large number of cellular processes, including pre-mRNA splicing and mRNA decapping. We demonstrated that the first 72 amino acids of KlLsm4p, which contain the Sm-like domains, can restore cell viability in both K. lactis and S. cerevisiae cells lacking the wild-type protein. However, the absence of the carboxy-terminal region resulted in a remarkable loss of cell viability in the stationary phase. The KlLSM4 sequence has been deposited in the EMBL Data library under Accession No. AJ311719.

Tracking ability in subjects symptomatic of cumulative trauma disorder: does it relate to disability?

Symptoms of upper extremity cumulative trauma disorders (CTDs) often include weakness, discomfort, pain, numbness and stiffness, which are generally assessed clinically by using static tests or isolated movements. Little is known about the dynamic, functional ability of the upper extremity in CTD, yet, more than impairment, performance variables may relate to disability. The objectives of this study were to determine whether a manual tracking task was sensitive to the presence of symptoms associated with CTD and whether tracking performance related to disability. Forty-five volunteers who had frequently experienced one or more symptoms consistent with upper extremity CTD for at least 1 year and 22 control subjects performed the manual tracking task. Using a hand-held stylus over a digitizing tablet, subjects tracked a target that moved pseudo-randomly and was displayed on a computer screen. The root mean square error of the linear difference between target and stylus positions provided a measure of overall performance accuracy. Quadrant specific performance was also calculated to determine whether the location of the target (hence hand and wrist position) influenced performance. Additionally, the symptomatic group completed the Disability of the Arm, Shoulder and Hand (DASH) questionnaire reflecting physical disability level. Performance accuracy was poorer in symptomatic subjects than controls (p<0.001) and was influenced by target location (p<0.0001). The overall performance was associated with physical disability (r = 0.54). The findings suggest that tracking performance is sensitive to the presence of CTD symptoms and related to disability level. Further validation is required to determine whether the performance measure is sensitive to disease progression or intervention-induced changes.

Regulation of primary carbon metabolism in Kluyveromyces lactis.

In the recent past, through advances in development of genetic tools, the budding yeast Kluyveromyces lactis has become a model system for studies on molecular physiology of so-called "Nonconventional Yeasts." The regulation of primary carbon metabolism in K. lactis differs markedly from Saccharomyces cerevisiae and reflects the dominance of respiration over fermentation typical for the majority of yeasts. The absence of aerobic ethanol formation in this class of yeasts represents a major advantage for the "cell factory" concept and large-scale production of heterologous proteins in K. lactis cells is being applied successfully. First insight into the molecular basis for the different regulatory strategies is beginning to emerge from comparative studies on S. cerevisiae and K. lactis. The absence of glucose repression of respiration, a high capacity of respiratory enzymes and a tight regulation of glucose uptake in K. lactis are key factors determining physiological differences to S. cerevisiae. A striking discrepancy exists between the conservation of regulatory factors and the lack of evidence for their functional significance in K. lactis. On the other hand, structurally conserved factors were identified in K. lactis in a new regulatory context. It seems that different physiological responses result from modified interactions of similar molecular modules.

Molecular analysis of UAS(E), a cis element containing stress response elements responsible for ethanol induction of the KlADH4 gene of Kluyveromyces lactis.

KlADH4 is a gene of Kluyveromyces lactis encoding a mitochondrial alcohol dehydrogenase activity, which is specifically induced by ethanol and insensitive to glucose repression. In this work, we report the molecular analysis of UAS(E), an element of the KlADH4 promoter which is essential for the induction of KlADH4 in the presence of ethanol. UAS(E) contains five stress response elements (STREs), which have been found in many genes of Saccharomyces cerevisiae involved in the response of cells to conditions of stress. Whereas KlADH4 is not responsive to stress conditions, the STREs present in UAS(E) seem to play a key role in the induction of the gene by ethanol, a situation that has not been observed in the related yeast S. cerevisiae. Gel retardation experiments showed that STREs in the KlADH4 promoter can bind factor(s) under non-inducing conditions. Moreover, we observed that the RAP1 binding site present in UAS(E) binds KlRap1p.

How to bring orphan genes into functional families.

In the framework of the B1 Consortium of the EUROFAN-1 project, we set up a series of simple phenotypic tests that can be performed on a large number of strains at a time. This methodological approach was intended to help assign functions of putative genes coding for unknown proteins to several specific aspects of cell biology. The tests were chosen to study phenotypes which should be affected by numerous genes. In this report, we examined the sensitivity/resistance or the adaptation of the cell to physical or chemical stresses (thermotolerance, osmotolerance and ethanol sensitivity), the effects of the alteration of the level of protein phosphorylation (sensitivity or resistance to compounds affecting the activity of protein kinases or phosphatases) and the effects of compounds interfering with synthesis of nucleic acids or proteins. Deletions in 66 genes of unknown function have been tested in 21 different conditions. In many deletant strains, phenotypes were observed and, for the most promising candidates, tetrad analysis was performed in order to verify co-segregation of the deletion marker with the phenotype.