Detection and phylogenetic analysis of porcine enteric calicivirus, genetically related to the Cowden strain of sapovirus genogroup III, in Brazilian swine herds

Sapovirus of the Caliciviridae family is an important agent of acute gastroenteritis in children and piglets. The Sapovirus genus is divided into seven genogroups (G), and strains from the GIII, GVI and GVII are associated with infections in swine. Despite the high prevalence in some countries, there are no studies related to the presence of porcine enteric sapovirus infections in piglets in Brazil. In the present study, 18 fecal specimens from piglets up to 28 days were examined to determine the presence of sapovirus genome by RT-PCR assay, using primers designed to amplify a 331 bp segment of the RNA polymerase gene. In 44.4% (8/18) of fecal samples, an amplified DNA fragment was obtained. One of these fragments was sequenced and submitted to molecular and phylogenetic analysis. This analysis revealed high similarity, with nucleotides (87%) and amino acids (97.8%), to the Cowden strain, the GIII prototype of porcine enteric calicivirus. This is the first description of sapovirus in Brazilian swine herds.

Genital immunization of heifers with a glycoprotein Edeleted, recombinant bovine herpesvirus 1 strain confers protection upon challenge with a virulent isolate

Venereal infection of seronegative heifers and cows with bovine herpesvirus type 1.2 (BoHV-1.2) frequently results in vulvovaginitis and transient infertility. Parenteral immunization with inactivated or modified live BoHV-1 vaccines often fails in conferring protection upon genital challenge. We herein report an evaluation of the immune response and protection conferred by genital vaccination of heifers with a glycoprotein E-deleted recombinant virus (SV265gE-). A group of six seronegative heifers was vaccinated with SV265gE- (0,2mL containing 10(6.9)TCID50) in the vulva submucosa (group IV); four heifers were vaccinated intramuscularly (group IM, 1mL containing 10(7.6)TCID50) and four heifers remained as non-vaccinated controls. Heifers vaccinated IV developed mild, transient local edema and hyperemia and shed low amounts of virus for a few days after vaccination, yet a sentinel heifer maintained in close contact did not seroconvert. Attempts to reactivate the vaccine virus in two IV vaccinated heifers by intravenous administration of dexamethasone (0.5mg/kg) at day 70 pv failed since no virus shedding, recrudescence of genital signs or seroconversion were observed. At day 70 pv, all vaccinated and control heifers were challenged by genital inoculation of a highly virulent BoHV-1.2 isolate (SV56/90, 10(7.1)TCID50/animal). After challenge, virus shedding was detected in genital secretions of control animals for 8.2 days (8-9); in the IM group for 6.2 days (4-8 days) and during 5.2 days (5-6 days) in the IV group. Control non-vaccinated heifers developed moderate (2/4) or severe (2/4) vulvovaginitis lasting 9 to 13 days (x: 10.7 days). The disease was characterized by vulvar edema, vulvo-vestibular congestion, vesicles progressing to coalescence and erosions, fibrino-necrotic plaques and fibrinopurulent exudate. IM vaccinated heifers developed mild (1/3) or moderate (3/4) genital lesions, lasting 10 to 12 days (x: 10.7 days); and IV vaccinated heifers developed mild and transient vulvovaginitis (3/4) or mild to moderate genital lesions (1/4). In the IV group, the clinical signs lasted 4 to 8 days (x: 5.5 days). Clinical examination of the animals after challenge revealed that vaccination by both routes conferred some degree of protection, yet IV vaccination was clearly more effective in reducing the severity and duration of clinical disease. Furthermore, IV vaccination reduced the period of virus shedding in comparison with both groups. Taken together, these results demonstrate that SV265gE- is sufficiently attenuated upon IV vaccination in a low-titer dosis, is not readily reactivated after corticosteroid treatment and lastly, and more importantly, confers local protection upon challenge with a high titer of a virulent heterologous BoHV-1 isolate. Therefore, the use of this recombinant for genital immunization may be considered for prevention of BoHV-1-associated genital disease in the field.

Immunogenicity of an inactivated bovine herpesvirus type 5 strain defective in thymidine kinase and glycoprotein E

The immunogenicity of an inactivated, experimental vaccine based on a bovine herpesvirus type 5 strain defective in thymidine kinase and glycoprotein E (BoHV-5 gE/TKΔ) was evaluated in cattle and the results were compared with a vaccine containing the parental BoHV-5 strain (SV507/99). To formulate the vaccines, each virus (wildtype SV507/99 and BoHV-5 gE/TK∆) was multiplied in cell culture and inactivated with binary ethyleneimine (BEI). Each vaccine dose contained approximately of 10(7.5) TCID50 of inactivated virus mixed with an oil-based adjuvant (46:54). Forty calves, 6 to 9-months-old, were allocated into two groups of 20 animals each and vaccinated twice (days 0 and 22pv) by the subcutaneous route with either vaccine. Serum samples collected at day 0 and at different intervals after vaccination were tested for virus neutralizing (VN) antibodies against the parental virus and against heterologous BoHV-5 and BoHV-1 isolates. The VN assays demonstrated seroconversion to the respective homologous viruses in all vaccinated animals after the second vaccine dose (mean titers of 17.5 for the wildtype vaccine; 24.1 for the recombinant virus). All animals remained reagents up to day 116 pv, yet showing a gradual reduction in VN titers. Animals from both vaccine groups reacted in similar VN titers to different BoHV-1 and BoHV-5 isolates, yet the magnitude of serological response of both groups was higher against BoHV-5 field isolates. Calves vaccinated with the recombinant virus did not develop antibodies to gE as verified by negative results in a gE-specific ELISA, what would allow serological differentiation from naturally infected animals. Taken together, these results indicate that inactivated antigens of BoHV-5 gE/TK recombinant virus induced an adequate serological response against BoHV-5 and BoHV-1 and thus can be used as an alternative, differential vaccine candidate.

Vaccination protocol and bacterial strain affect the serological response of beef calves against blackleg

The serological response of beef calves was evaluated with different vaccination regimens against blackleg, using an official strain (MT) and a field-collected strain of Clostridium chauvoei as antigens. Sixty calves were randomly allocated to four different groups and were submitted to distinct vaccination protocols with a commercial polyvalent vaccine. Group G1 was first vaccinated at four months of age and a booster shot was given after weaning, at eight months. Group G2 was given the first dose at eight months and a booster shot 30 days later. Group G3 was vaccinated only once at eight months and the control group was not vaccinated. These alternative vaccination regimens were proposed in an effort to adequately protect cattle under open-field farming conditions. Serological evaluations were made by Elisa at 4, 8, 9 and 10 months of age. Both groups receiving booster shots had a significantly increased serological response 30 days later. However, the serum IgG levels against C. chauvoei were significantly higher in the calves that were first vaccinated at four months. At 10 months, the two booster shot groups (G1 and G2) had similar serological responses, while the calves that were treated with a single dose of vaccine at weaning (G3) had a response that was similar to that of the control group. The serological response of the calves was significantly inferior at several of the evaluation times when the field strain of the bacteria was used as a challenge antigen instead of the official MT strain. The serological response of calves that are vaccinated twice was found to be satisfactory, independent of the first injection being made at four or eight months of age. It was also concluded that it would be useful to include local bacterial strains in commercial vaccine production.

Pain symptoms and sleep problems among school-aged children. Long-term prevalence changes, and pain symptoms as predictors of later mental health

Children’s pain symptoms and sleep problems are among the most common health complaints. They distract children from activities, decrease the quality of life, contribute to a significant economic burden, and have shown continuity into adulthood. The main aims of this thesis were to investigate long-term changes in the prevalence of pain symptoms and sleep problems among Finnish school-aged children, and the later mental health of those who in childhood experience pain. Prevalence, co-occurrence, and associated psychosocial factors of pain symptoms and sleep problems were also assessed. In study I, prevalence changes in eight-year-old children’s pain symptoms and sleep problems were investigated in three cross-sectional population-based samples (years 1989: n=1038, 1999: n=1035, and 2005: n=1030). In study II, cross-sectional associations between pain symptoms, sleep problems, and psychosocial factors were assessed among 13-18-year-old adolescents (n=2476). In studies III and IV, associations between pain symptoms at age eight (n=6017), and register-based data on antidepressant use and severe suicidality by age 24, were examined in a nationwide birth cohort. Pain symptoms and sleep problems were common and often co-occurred. A considerable number of children’s pain symptoms remained unrecognized by the parents. The prevalence of pain symptoms, sleep problems, and multiple concurrent symptoms approximately doubled from 1989 to 2005. Psychiatric difficulties or demographic factors did not explain the increase. Psychosocial factors that were associated with pain, sleep problems, and a higher number of symptoms, were female sex, psychological difficulties, emotional symptoms, smoking, victimization, and feeling not cared about by teachers. In longitudinal analyses, the child’s own report of headache, and to a smaller degree the parental report of the child’s abdominal pain predicted later antidepressant use. Parental report of the child’s abdominal pain predicted severe suicidality among males. If one of the symptoms is present, health care professionals should inquire about other symptoms as well. Questions should be directed to the children, not only to their parents. Inquiring about psychiatric difficulties, substance use, victimization, and relations with teachers should be included as a part of the assessment. Further studies are needed to clarify the reasons that underlie the increased prevalence rates, and the factors that may increase or decrease the risk for later mental health problems among pain-suffering children.

Prediction of 3D structure and ligand-binding properties : ABC transporters and flavodiiron proteins from Synechocystis sp. strain PCC 6803

Cyanobacteria are unicellular, non-nitrogen-fixing prokaryotes, which perform photosynthesis similarly as higher plants. The cyanobacterium Synechocystis sp. strain PCC 6803 is used as a model organism in photosynthesis research. My research described herein aims at understanding the function of the photosynthetic machinery and how it responds to changes in the environment. Detailed knowledge of the regulation of photosynthesis in cyanobacteria can be utilized for biotechnological purposes, for example in the harnessing of solar energy for biofuel production. In photosynthesis, iron participates in electron transfer. Here, we focused on iron transport in Synechocystis sp. strain PCC 6803 and particularly on the environmental regulation of the genes encoding the FutA2BC ferric iron transporter, which belongs to the ABC transporter family. A homology model built for the ATP-binding subunit FutC indicates that it has a functional ATPbinding site as well as conserved interactions with the channel-forming subunit FutB in the transporter complex. Polyamines are important for the cell proliferation, differentiation and apoptosis in prokaryotic and eukaryotic cells. In plants, polyamines have special roles in stress response and in plant survival. The polyamine metabolism in cyanobacteria in response to environmental stress is of interest in research on stress tolerance of higher plants. In this thesis, the potd gene encoding an polyamine transporter subunit from Synechocystis sp. strain PCC 6803 was characterized for the first time. A homology model built for PotD protein indicated that it has capability of binding polyamines, with the preference for spermidine. Furthermore, in order to investigate the structural features of the substrate specificity, polyamines were docked into the binding site. Spermidine was positioned very similarly in Synechocystis PotD as in the template structure and had most favorable interactions of the docked polyamines. Based on the homology model, experimental work was conducted, which confirmed the binding preference. Flavodiiron proteins (Flv) are enzymes, which protect the cell against toxicity of oxygen and/or nitric oxide by reduction. In this thesis, we present a novel type of photoprotection mechanism in cyanobacteria by the heterodimer of Flv2/Flv4. The constructed homology model of Flv2/Flv4 suggests a functional heterodimer capable of rapid electron transfer. The unknown protein sll0218, encoded by the flv2-flv4 operon, is assumed to facilitate the interaction of the Flv2/Flv4 heterodimer and energy transfer between the phycobilisome and PSII. Flv2/Flv4 provides an alternative electron transfer pathway and functions as an electron sink in PSII electron transfer.

Strain and strain rate by two-dimensional speckle tracking echocardiography in a maned wolf

The measurement of cardiovascular features of wild animals is important, as is the measurement in pets, for the assessment of myocardial function and the early detection of cardiac abnormalities, which could progress to heart failure. Speckle tracking echocardiography (2D STE) is a new tool that has been used in veterinary medicine, which demonstrates several advantages, such as angle independence and the possibility to provide the early diagnosis of myocardial alterations. The aim of this study was to evaluate the left myocardial function in a maned wolf by 2D STE. Thus, the longitudinal, circumferential and radial strain and strain rate were obtained, as well as, the radial and longitudinal velocity and displacement values, from the right parasternal long axis four-chamber view, the left parasternal apical four chamber view and the parasternal short axis at the level of the papillary muscles. The results of the longitudinal variables were -13.52±7.88, -1.60±1.05, 4.34±2.52 and 3.86±3.04 for strain (%), strain rate (1/s), displacement (mm) and velocity (cm/s), respectively. In addition, the radial and circumferential Strain and Strain rate were 24.39±14.23, 1.86±0.95 and -13.69±6.53, -1.01±0.48, respectively. Thus, the present study provides the first data regarding the use of this tool in maned wolves, allowing a more complete quantification of myocardial function in this species.

Evaluation of resistance in a selected field strain of Haemonchus contortus to ivermectin and moxidectin using the Larval Migration on Agar Test

Haemonchus contortus is one of the most common and economically significant causes of disease in small ruminants worldwide, and the control programs of parasitic nematodes - including H. contortus - rely mostly on the use of anthelmintic drugs. The consequence of the use of this, as the sole sanitary strategy to avoid parasite infections, was the reduction of the efficacy of all chemotherapeutic products with a heavy selection for resistance. The widespread of anthelmintic resistance and the difficulty of its early diagnosis has been a major concern for the sustainable parasite management on farms. The objective of this research was to determine and compare the ivermectin (IVM) and moxidectin (MOX) effect in a selected field strain of H. contortus with a known resistance status, using the in vitro larval migration on agar test (LMAT). Third stage larvae of the selected isolate were obtained from faecal cultures of experimentally infected sheep and incubated in eleven increasing diluted concentrations of IVM and MOX (6, 12, 24, 48, 96, 192, 384, 768, 1536, 3072 and 6144µg/mL). The dose-response sigmoidal curves were obtained using the R² value of >0.90 and the lethal concentration (LC50) dose for the tested anthelmintic drugs using a four-parameter logistic model. The LC50 value for MOX was significantly lower than IVM (1.253µg/mL and 91.06µg/mL), identifying the H. contortus isolate as considerably less susceptible to IVM compared to MOX. Furthermore, the LMAT showed a high consistency (p<0.0001) and provided to be a useful diagnostic tool for monitoring the resistance status of IVM and MOX in H. contortus field isolate, as well as it may be used for official routine drug monitoring programs under the Ministry of Agriculture (MAPA) guidance.

Development of an indirect ELISA to detect Corynebacterium pseudotuberculosis specific antibodies in sheep employing T1 strain culture supernatant as antigen

Corynebacterium pseudotuberculosis is the etiologic agent of caseous lymphadenitis (CLA), a chronic disease that affects goats and sheep, characterized by granuloma formation in subcutaneous and internal lymph nodes. CLA causes significant economic losses to commercial goat herds. In this study, we aimed to test secreted antigens secreted from T1 strain bacteria grown in brain heart infusion (BHI) broth in an indirect ELISA system to determine the presence of specific immunoglobulins against C. pseudotuberculosis. We analyzed the BHI antigen electrophoretic profile and the recognition pattern by infected sheep sera samples. The ELISA results were compared with multiplex PCR assay and IFN-gamma production. The ELISA was able to discriminate between negative and positive animals, with a sensitivity of 89% and a specificity of 99%, using microbiological isolation as gold standard. When this assay was compared with multiplex PCR and specific IFN-gamma quantification, six discrepant results were found among thirty-two samples. We concluded that the ELISA using antigens secreted from C. pseudotuberculosis T1 strain growth in BHI broth culture can be used for the serodiagnosis of CLA in sheep.

Evaluation of a PCR multiplex for detection and differentiation of Mycoplasma synoviae, M. gallisepticum, and M. gallisepticum strain F-vaccine

Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) are the mycoplasma infections of most concern for commercial poultry industry. MG infection is commonly designated as chronic respiratory disease (CRD) of chickens and infections sinusitis of turkeys. MS causes sub clinical upper respiratory infection and tenosynovitis or bursitis in chickens and turkeys. The multiplex PCR was standardized to detect simultaneously the MS, MG field strains and MG F-vaccine strain specific. The generic PCR for detection of any species of Mollicutes Class was performed and compared to the multiplex PCR and to PCR using species-specific primers. A total of 129 avian tracheal swabs were collected from broiler-breeders, layer hens and broilers in seven different farms and were examined by multiplex PCR methods. The system (multiplex PCR) demonstrated to be very rapid, sensitive, and specific. Therefore, the results showed a high prevalence of MS in the flocks examined (27.9%), and indicate that the MS is a recurrent pathogen in Brazilian commercial poultry flocks.

Exploring the Impact of Mental Health Education on Adolescents’ Perceptions About Mental Health and Mental Illness. Improving community health

The purpose of this study was to explore perceptions of mental health and mental illness as well as the perceptions towards people with mental illness among adolescents, and further to examine the impact that a mental health educational intervention has on these perceptions. The review of the literature revealed a small number of publications on mental health educational interventions among adolescents which aimed at increasing knowledge and affecting attitudes towards mental illness with positive results. Fifty nine pupils (13-16 years old) from two randomly selected secondary schools around Athens, Greece, participated in this study. These schools were randomly selected as the experimental group (n=28) which participated in the mental health educational intervention, and the comparison group (n=31), which did not receive any intervention. Data were collected using individual interviews with open-ended questions, drawings and a questionnaire (Opinions about Mental Illness - O.M.I. scale). The participants described mental health and mental illness before and after the intervention, using the same expressions for both terms. Among the experimental group, changes were seen within the same expressions after the intervention, although some descriptions did not change. However, after the intervention, participants in the experimental group did not confuse mental health with mental illness and they also included specific diagnostic examples or stated that mental illness can happen to anyone and it can be managed. Moreover, they expressed positive attitudes towards mentally ill people, which they had not done before the intervention. The analysis of the drawings before the intervention showed that mentally ill persons were drawn similarly in both groups. After the intervention, the drawings of the participants in the experimental group changed, including fewer negative elements, while the drawings of the comparison group did not change. Regarding the results on the O.M.I. scale, it was found that the score on the Social Discrimination factor significantly decreased from pre-test to post-test in both study groups. The experimental group had higher levels on Social Discrimination at pre-test compared to the comparison group, but this difference was not significant at post-test. No significant changes were found for the Social Restriction factor for either study group. Scores of the Social Care and Social Integration factors increased significantly only in the intervention group. Overall, the results of this study indicate that the mental health educational intervention had a positive impact on the perceptions about mental health and mental illness among adolescents, and (mental) health professionals can use these results for implementing similar interventions and further research.

Immunization against the colonization factor antigen I of enterotoxigenic Escherichia coli by administration of a bivalent Salmonella typhimurium aroA strain

An expression plasmid (pCFA-1) carrying the cfaB gene that codes for the enterotoxigenic Escherichia coli (ETEC) fimbrial adhesin colonization factor antigen I (CFA/I) subunit was constructed and used to transform a derivative of the attenuated Salmonella typhimurium aroA vaccine strain SL3261 carrying an F'lacIq. Treatment of the transformed strain with isopropyl-ß-D-thiogalactopyranoside (IPTG) resulted in elevated in vitro expression of the CFA/I subunit. Although flagellar function and lipopolysaccharide (LPS) synthesis were similar in both the parental and the recombinant strains, spleen colonization was reduced in the recombinant strain. All BALB/c mice parenterally inoculated with the recombinant strain developed significant anti-CFA/I and anti-LPS serum antibody titers (P<0.05). Moreover, 2 of 5 mice orally inoculated with the engineered Salmonella strain developed anti-CFA/I intestinal IgA (P>0.05) while 4/5 of the same mice developed anti-LPS IgA (P<0.05). The results indicate that the vaccine strain elicited an antibody response against the bacterial host both after oral and intravenous immunization while the response against the CFA/I antigen was significant only after inoculation by the intravenous route