METHODOLOGY FOR AUTOMATICALLY DELIMITING PERMANENT PRESERVATION AREAS ALONG WATER COURSES - THE USE OF GIS IN THE HYDROLOGICAL BASIN OF THE SERGIPE RIVER, BRAZIL

ABSTRACT Permanent Preservation Areas (PPAs) along watercourses have been the focus of numerous studies, not only because of the fragility and ecological relevance of riverine vegetation, but also because of the inefficiency demonstrated in conforming to the legislation protecting it. One of the major difficulties encountered in terms of guaranteeing the effective conservation of these riverside areas is the absence of methodologies that can be used to define them rapidly and accurately without manually determining the widths of the rivers or assigning only uniform linear values for the entire watercourse. The present work sought to develop a spatial analysis methodology capable of automatically defining permanent preservation areas along watercourses using geographic information system (GIS) software. The present study was undertaken in the Sergipe River basin, "considering the river itself and its principal affluents. We used the database of the Digital Atlas of Hydrological Resources (SEMARH/SE), and the delimitations of the PPAs were performed using ArcGIS 10.1 and the XToolPro 9.0 extension. A total of 5,003.82 hectares of Permanent Preservation Areas were delimited along the margins of the rivers analyzed, with a margin of error of <1% in delimiting the widths of the rivers within the entire area considered. The methodology described here can be used to define PPAs efficiently, relatively rapidly, and with very small margins of error, thus representing a technological advance in terms of using GIS for land management.

Analysis of land use and occupancy in permanent preservation areas according to the hydrography of Ribeirão Água Fria - Bofete, SP - Brazil

The use conflicts are determined by the inadequate occupations of the soil, as it is the case of soil occupation inside of permanent preservation areas. This study aimed to determine the classes of the soil use and if there are conflicts inside of permanent preservation areas along the drainage network of the Água Fria Stream watershed, located in Bofete city - São Paulo, Brazil. It locates geographically between the coordinates: 48°09'30" to 48°18'30" longitude WGr., 22°58'30" to 23°04'30" latitude S, with an area of 15242.84 ha. The map of soil use was elaborated through the interpretation directly in the computer screen of satellite digital image. In the orbital data, the study area is inserted in the quadrant A, of image TM/Landsat - 5, orbit 220, point 76, passage 9/8th/2007. The Geographical Information System used was CartaLinx. The conflict areas of the watershed were obtained from the crossing between the maps of soil use and of PPAs. The results allowed the conclusion that more than half of the area (51.09%) is occupied by pastures, reflex of sandy soils and low fertility. It was also verified that although almost half of the watershed is covered with some type of vegetation (48.78% of natural forest /reforestation), it has approximately a third of permanent preservation areas used inappropriately by pastures (88.15%), reforestation (10.42%) and exposed soil (1.43%), totaling 343.07ha of conflicting areas, in a total of 993.26 ha of PPAs.

Vocalization of broilers can be used to identify their sex and genetic strain

In order to reach higher broiler performance, farmers target losses reduction. One way to make this possible is by rearing sexed broilers as male and female present diverse performance due to their physiological differences. Birds from different genetic strain also have a distinct performance at the same age. Considering that sexed flocks may present higher performance this study aimed to identify one-day-old chicks’ sex throughout their vocalization. This research also investigated the possibility of identifying the genetic strain by their vocalization attributes. A total of 120 chicks, half of them were from Cobb® genetic strain and the other half from Ross® genetic strain. From each group, a total of 30 were males and 30 females, which were previously separated by sex using their secondary physiological characteristics at the hatchery. Vocalizations audio recording was done inside a semi-anechoic chamber using a unidirectional microphone connected to an audio input of a digital recorder. Vocalizations were recorded for two minutes. Acoustic characteristics of the sounds were analyzed being calculated the fundamental frequency Pitch, the sound intensity, the first formant, and second formant. Results indicated that the vocalizations of both sexes could be identified by the second formant, and the genetic strain was detected by both the second formant and the Pitch.

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.

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.

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

The role of natural killer cells in the early period of infection in murine cutaneous leishmaniasis

In order to study the role of natural killer (NK) cells during the early period of Leishmania infection, BALB/c mice were selectively and permanently depleted of NK cells by injection with 90Sr and subsequently infected with Leishmania (Leishmania) amazonensis (HSJD-1 strain). 90Sr is known to selectively deplete NK cells, leaving an intact T- and B-cell compartment and preserving the ability to produce both interferon alpha and IL-2. This method of depletion has advantages when compared with depletion using anti-NK cell monoclonal antibodies because the effect is permanent and neither activates complement nor provokes massive cell death. In the present study, after one month of treatment with 90Sr, the depletion of NK cells was shown by a more than ten-fold reduction in the cytotoxic activity of these cells: 2 x 106 spleen cells from NK-depleted animals were required to reach the same specific lysis of target cells effected by 0.15 x 106 spleen cells from normal control animals. The histopathology of the skin lesion at 7 days after Leishmania infection showed more parasites in the NK cell-depleted group. This observation further strengthens a direct role of NK cells during the early period of Leishmania infection.

Properties of a constitutive alkaline phosphatase from strain 74A of the mold Neurospora crassa

A constitutive alkaline phosphatase was purified to apparent homogeneity as determined by polyacrylamide gel electrophoresis from mycelia of the wild strain 74A of the mold Neurospora crassa, after growth on acetate and in the presence of saturating amounts of inorganic phosphate (Pi) for 72 h at 30ºC. The molecular mass was 58 kDa and 56 kDa as determined by exclusion chromatography and SDS-PAGE, respectively. This monomeric enzyme shows an apparent optimum pH ranging from 9.5 to 10.5 and Michaelis kinetics for the hydrolysis of p-nitrophenyl phosphate (the Km and Hill coefficient values were 0.35 mM and 1.01, respectively), alpha-naphthyl phosphate (the Km and Hill coefficient values were 0.44 mM and 0.97, respectively), ß-glycerol phosphate (the Km and Hill coefficient values were 2.46 mM and 1.01, respectively) and L-histidinol phosphate (the Km and Hill coefficient values were 0.47 mM and 0.94, respectively) at pH 8.9. The purified enzyme is activated by Mg2+, Zn2+ and Tris-HCl buffer, and is inhibited by Be2+, histidine and EDTA. Also, 0.3 M Tris-HCl buffer protected the purified enzyme against heat inactivation at 70ºC(half-life of 19.0 min, k = 0.036 min-1) as compared to 0.3 M CHES (half-life of 2.3 min, k = 0.392 min-1) in the same experiment.

A single strain of Mycobacterium bovis bacillus Calmette-Guérin (BCG) grown in two different media evokes distinct humoral immune responses in mice

Two attenuated bacillus Calmette-Guérin (BCG) preparations derived from the same Moreau strain, Copenhagen but grown in Sauton medium containing starch and bacto-peptone (onco BCG, O-BCG), or asparagine (intradermal BCG, ID-BCG), exhibited indistinguishable DNA sequences and bacterial morphology. The number of viable bacilli recovered from spleen, liver and lungs was approximately the same in mice inoculated with the vaccines and was similarly reduced (over 90%) in mice previously immunized with either BCG vaccine. The humoral immune response evoked by the vaccines was, however, distinct. Spleen cell proliferation accompanying the growth of bacilli in tissue was significantly higher in mice inoculated with O-BCG. These cells proliferated in vitro upon challenge with the corresponding BCG extract. Previous cell treatment with mAb anti-CD4 T cells abolished this effect. Anti-BCG antibodies, as assayed either in serum by ELISA or by determining the number of antibody-producing spleen cells by the spot-ELISA method, were significantly higher in mice inoculated with ID-BCG. Anti-BCG antibodies were detected in all immunoglobulin classes, but they were more prevalent in IgG with the following distribution among its isotypes: IgG1>(IgG2a = IgG2b)>IgG3. When some well-characterized Mycobacterium tuberculosis antigens were used as substitutes for BCG extracts in ELISA, although antibodies against the 65-kDa and 96-kDa proteins were detected significantly, antibodies against the 71-kDa, 38-kDa proteins and lipoarabinomannan were only barely detected or even absent. These results indicate that BCG bacilli cultured in Sauton-asparagine medium permitted the multiplication of bacilli, tending to induce a stronger humoral immune response as compared with bacilli grown in Sauton-starch/bacto-peptone-enriched medium.