Exogenous paraoxonase‐1 during oocyte maturation improves bovine embryo development in vitro

Paraoxonase‐1 (PON1) is an enzyme found in serum and follicular fluid that protects cell membrane and circulating lipids against oxidative damage. The aims of this study were to measure the direct effects of recombinant PON1 (rPON1) on bovine oocyte maturation at the molecular level (gene expression) and to measure the carry‐over effects of PON1 on pre‐implantation embryo development in vitro. COCs were submitted to IVM with the addition of 0.0, 0.02, 0.04 and 0.08 mg ml^?1 of rPON1, corresponding to an average PON1 arylesterase enzyme activity of 2.2 ± 0.4, 15.5 ± 1.5, 30.2 ± 3.0 and 57.9 ± 5.0 U ml^?1, respectively. The results indicated that addition of rPON1 during IVM improved embryo development in a dose‐dependent manner as D7 embryo development was 22.2%, 29.4%, 32.2% and 37.0% for the treatment groups, respectively (p = 0.02). In conclusion, addition of PON1 enzyme during IVM exerted dose‐related positive effects on embryo development rates to blastocysts.     

Evaluation of serological tests for the diagnosis of Neospora caninum infection in dogs: optimization of cut off titers and inhibition studies of cross-reactivity with Toxoplasma gondii

Diagnosis of Neospora caninum infection in dogs is based on serological assays such as the indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assays (ELISA). This study evaluated two serological tests (IFAT and ELISA) for the detection of IgG antibodies to N. caninum in 300 serum samples of dogs through the optimization of cut off titers by using the two-graph receiver-operating characteristic (TG-ROC) curve. In addition, the identification of major cross-reactive antigens with Toxoplasma gondii was investigated by inhibition ELISA and immunoblotting (IB) assays. IFAT and ELISA results showed 74% agreement, with a good negative concordance (P(neg)=0.83), but a poor positive concordance (P(pos)=0.42). The great majority (86%) of sera with positive concordant results (IFAT+/ELISA+) recognized at least two out of three N. caninum immunodominant antigens, particularly the 29-32 and 35-37 kDa bands. Optimization of cut off titers in IFAT and ELISA was performed considering the reactivity to at least two out of three N. caninum immunodominant antigens as infection markers, obtaining a titer of 50 for IFAT and 200 for ELISA. Seropositivity to N. caninum was significantly associated with T. gondii-seropositive samples, particularly in ELISA (55.4%). Inhibition ELISA curves for N. caninum showed a partial heterologous inhibition, indicating some degree of cross-reactivity between N. caninum and T. gondii antigens. Inhibition IB assays showed a moderate heterologous inhibition for N. caninum antigens above 45-50 kDa. These results indicate that ELISA should be used critically when crude tachyzoite antigen preparations are employed, due to possible cross-reactivity with other related parasites as T. gondii. Also, the cut off dilution of 1:50 in IFAT showed to be the most appropriated for N. caninum serology in dogs. Therefore, we suggest that N. caninum immunodominant antigens, specially the 17 and 29-32 kDa proteins, should be selected markers in serological assays for canine neosporosis.     

Evaluation of transformation growth factor beta1, interleukin-10, and interferon-gamma in male symptomatic and asymptomatic dogs naturally infected by Leishmania (Leishmania) chagasi

The aims of this study were to evaluate the immunomodulatory role of TGF-β₁, IL-10, and INF-γ in spleen and liver extracts and supernatant cultures of white spleen cells from male symptomatic and asymptomatic dogs, naturally infected by Leishmania (Leishmania) chagasi. Thirty dogs from Araçatuba, São Paulo, Brazil, an endemic leishmaniosis area, were selected by positive ELISA serological reaction for Leishmania sp. and divided into two groups: asymptomatic (n = 15) and symptomatic (n = 15) consisting of animals with at least three characteristic signs (fever, dermatitis, lymphoadenopathy, onychogryphosis, weight loss, cachexia, locomotion problems, conjunctivitis, epistaxis, hepatosplenomegaly, edema, and apathy). After euthanasia, spleen and liver fragments were collected for ex vivo quantification of TGF-β₁, IL-10, and INF-γ. Naturally active in vitro produced TGF-β₁ was also evaluated in spleen cell culture supernatant. Spleen and liver extract of asymptomatic dogs had higher mean TGF-β₁ levels than symptomatic dogs. High concentrations of IL-10 were found in spleen, and mainly in liver extract of both groups. Higher INF-γ concentrations were found in spleen extracts of symptomatic dogs, and in liver extracts of asymptomatic dogs. Extract of this cytokine was lower in spleen extract. Although INF-γ is being produced in canine infection, mean levels of TGF-β₁ and IL-10 from spleen and liver extracts were quantitatively much higher; suggesting that immune response in both asymptomatic and symptomatic dogs was predominantly type Th2.     

Comparison of serological assays for the diagnosis of canine visceral leishmaniasis in animals presenting different clinical manifestations

Three serological methods, indirect fluorescent immunoassay (IFI), enzyme-linked immunosorbent assay (ELISA) and direct agglutination test (DAT) that are commonly employed in the diagnosis of canine visceral leishmaniasis (CVL), have been assessed. A total of 234 domestic dogs, drawn from an area in the municipality of Belo Horizonte, Minas Gerais, Brazil, endemic for visceral leishmaniasis, were submitted to clinical and parasitological examinations and serological assay. Sera collected from confirmed non-infected dogs (n=20), and from dogs with other parasitic diseases including Trypanosoma cruzi (n=7), Leishmania braziliensis (n=5), Toxoplasma gondii (n=5) and Ehrlichia canis (n=3), were also included in the study. IFI presented a lower sensitivity (72%) than ELISA (95%), although the specificities of these assays were low (52 and 64%, respectively) and both exhibited cross-reactivity with sera from dogs infected with T. cruzi, L. braziliensis and E. canis. In contrast, DAT exhibited a high sensitivity (93%) and a high specificity (95%) and cross-reacted with only one serum sample derived from an E. canis-infected dog. The reproducibilities of the ELISA and DAT assays were excellent, whilst that of IFI was considered to be acceptable. The results produced by ELISA and DAT were in complete agreement, those between ELISA and IFI were at an acceptable level of agreement, whilst the concurrence between the IFI and DAT results were either acceptable or poor depending on the clinical conditions of the group of dogs examined. Since there is no readily accessible method for the diagnosis of CVL that offers 100% specificity and sensitivity, the choice of technique employed must depend on the aim of the investigation.     

Protective activity against oocyst shedding in cats vaccinated with crude rhoptry proteins of the Toxoplasma gondii by the intranasal route

This study evaluated a vaccine made from crude rhoptry proteins of Toxoplasma gondii with Quil-A, which was administered to cats by the intranasal route. Eleven short-hair domestic cats were divided into four groups: G1 (n=3) received three doses (200 microg/dose) of the rhoptry vaccine with Quil-A (20 microg); G2 (n=3) received PBS with Quil-A (20 microg); G3 (n=3) and G4 (n=2) received only PBS. Treatments were administered at days 0, 21, and 42 by the intranasal route. Challenge was done to G1, G2, and G3 animals with 600 cysts of the VEG strain on day 51 (challenge day); G4 animals were unchallenged. The anti-T. gondii IgG and IgA antibody levels from sera were measured by indirect enzyme-linked immunosorbent assay (ELISA). At challenge, two animals from G1 revealed antibody levels for both IgG and IgA; oocysts were not detected in feces of these two cats. There were no differences in hematological values between groups throughout the experiment (p>0.10). Preventable fractions were 67% in G1 and 0% in G2 and G3. Comparatively, G1 animals shed 89.3% and 90.8% less oocysts than G3 and G4, respectively. Two out of three cats were protected against T. gondii oocyst shedding when the rhoptry vaccine was administered by the intranasal route. This is the first study using crude rhoptry proteins as vaccine by the intranasal route in cats to evaluate protection against oocysts shedding.     

Agarose gel electrophoresis of cerebrospinal fluid proteins of dogs after sample concentration using a membrane microconcentrator technique

BACKGROUND: Cerebrospinal fluid (CSF) is produced in the cerebral ventricles through ultrafiltration of plasma and active transport mechanisms. Evaluation of proteins in CSF may provide important information about the production of immunoglobulins within the central nervous system as well as possible disturbances in the blood-brain barrier. OBJECTIVE: The objective of this study was to measure the concentration and fractions of protein in CSF samples using a membrane microconcentrator technique followed by electrophoresis, and to compare the protein fractions obtained with those in serum. METHODS: CSF samples from 3 healthy dogs and 3 dogs with canine distemper virus infection were concentrated using a membrane microconcentrator having a 0.5 to 30,000 d nominal molecular weight limit (Ultrafree, Millipore, Billerica, MA, USA). Protein concentration was determined before and after concentration. Agarose gel electrophoresis was done on concentrated CSF samples, serum, and serial dilutions of one of the CSF samples. RESULTS: Electrophoretic bands were clearly identified in densitometer tracings in CSF samples with protein concentrations as low as 1.3 g/dL. The higher CSF protein concentration in dogs with distemper was mainly the result of increased albumin concentration. CONCLUSION: The microconcentrating method used in this study enables characterization of the main protein fractions in CSF by routine electrophoresis and may be useful for interpreting the underlying cause of changes in CSF protein concentrations.     

Catheter-induced urethral trauma in cats with urethral obstruction

Fifteen cats were evaluated with urethral obstruction. Penile trauma by catheterization was the major indication for perineal urethrostomy. Ten cats had developed a urethral stricture and five had rupture of the urethra following medical management. All cats had abnormalities in penis and/or prepuce and/or scrotal sacs including hyperemia or swelling. Perineal urethrostomy was performed in all cases and they were evaluated for 6 months after surgery. Few complications were noted. Urinary tract infection was the most frequent complication observed. The clients considered their cats to have a good quality of life following surgery.     

Speciation of phosphorus in a tidal floodplain forest in the Amazon estuary

The processes of release and retention of phosphorus (P) in soils of a tidal floodplain were studied at Combu Island (Belém/Brazil). Three parcels of 220m² were chosen: low floodplain (LF), intermediate floodplain (MF) and high floodplain (HF). Two cores to 0.4 m depth were collected in each parcel, one week after the highest and lowest tides of the year. Organic phosphorus (P—OM) concentrations ranged from 0.02 ± 0.01 to 0.07 ± 0.02 mg/g, the highest concentrations were found in the HF parcel. On the other hand the HF parcel had the lowest concentrations of total inorganic phosphorus (TIP): 0.10 ± 0.03 to 0.11 ± 0.33 mg/g. The speciation of inorganic P suggested that P—Fe/Al is the main chemically bound P in soil (0.05 ± 0.01 to 0.20 — 0.04 mg/g). The tidal inundation of the floodplain appears to influence these chemical reactions involving phosphate retention and release.     

Measurements of the equation of state of deuterium at the fluid insulator-metal transition

A high-intensity laser was used to shock-compress liquid deuterium to pressures from 22 to 340 gigapascals. In this regime deuterium is predicted to transform from an insulating molecular fluid to an atomic metallic fluid. Shock densities and pressures, determined by radiography, revealed an increase in compressibility near 100 gigapascals indicative of such a transition. Velocity interferometry measurements, obtained by reflecting a laser probe directly off the shock front in flight, demonstrated that deuterium shocked above 55 gigapascals has an electrical conductivity characteristic of a liquid metal and independently confirmed the radiography.     

Combining Maltogenic Amylase with CMC or Wheat Gluten to Prevent Amylopectin Recrystallization and Delay Corn Tortilla Staling

Antistaling properties of a bacterial maltogenic amylase, sodium carboxymethylcellulose (CMC), and vital wheat gluten on quality of corn tortillas were evaluated during 14 days of storage. Amylopectin recrystallization was the driving force behind the staling of corn tortillas. Increasing levels of recrystallized amylopectin measured by differential scanning calorimetry (DSC) correlated significantly with increased tortilla stiffness (r = 0.43) and reduction in tortilla pliability (r = ‐0.42) during storage. Maltogenic amylase (275–1,650 activity units) made tortillas less stiff but did not preserve pliability and extensibility as effectively as CMC (0.25–0.5%). The combination of 825 MANU of maltogenic amylase (to interfere with intragranular amylopectin recrystallization) and 0.25% CMC (to create a more flexible intergranular matrix than retrograded amylose and amylopectin) produced less stiff, equally flexible, and less chewy tortillas than did 0.5% CMC. Vital wheat gluten was not as effective as CMC in preserving tortilla flexibility or as good as the maltogenic amylase in reducing stiffness. Further research is required to optimize the addition of maltogenic amylases in continuous processing lines that use fresh masa instead of nixtamalized corn flour (NCF) and to determine how these amylases interfere with amylopectin recrystallization.