Detection of Leishmania (L.) infantum in stray dogs by molecular techniques with sensitive species-specific primers

Background: Canine visceral leishmaniasis (CVL) is a worldwide parasitic zoonosis caused by Leishmania (Leishmania) infantum around the world. Canids are the definitive hosts and sand flies the intermediate hosts.

Objective: To test the hypothesis that a new species-specific primers (Lch14:Lch15, targeting a multiple alignment for L. infantum kDNA minicircle) is an efficient diagnostic tool for L. infantum.

Methods: The presence of L. infantum DNA was assessed in blood samples of 69 stray dogs using the conventional PCR (cPCR) and quantitative PCR (qPCR). Additional 50 lymph nodes and 50 bone marrow samples (positive and negative samples for parasitological tests) from dogs from endemic and nonendemic areas for CVL were also used.

Results: L. infantum strains, and all positive lymph node and bone marrow samples for parasitological test gave positive results for cPCR and qPCR, presenting analytical sensitivity of ～10⁰ parasite mL^?1. For the blood samples, 40/69 (58%; CI 95%; 46%–69%) resulted positive for L. infantum in both tests. All positive samples were confirmed by sequencing.

Conclusion: This study showed the importance of the specific detection of L. infantum based on species-specific primers by molecular techniques, highlighting the application as a confirmation method in epidemiological studies and to adopt the best control measures.     

Detection of <Emphasis Type="Italic">Brucella</Emphasis> sp. infection through serological,microbiological, and molecular methods applied to buffaloes in Maranhão State,Brazil

The aim of the current study is to diagnose Brucella spp. infection using methods such as serology, bacterial isolation, and molecular analysis in buffaloes bred in Maranhão State. In order to do so, 390 samples of buffalo serum were subjected to serological tests, to Rose Bengal Plate Test (RBPT) and to 2-mercaptoethanol (2-ME) combined with slow agglutination test (SAT). Vaginal swabs were collected from seropositive animals and subjected to bacterial isolation and to generic PCR. According to the serological test, 16 animals had a positive reaction to the confirmatory test (2-ME/SAT). As for bacterial isolation, three samples resulted in the isolation of Brucella spp.-characteristic colonies, which were confirmed through PCR. These results confirmed Brucella spp. infection in the buffalo herd from Maranhão State.     

Performance and carcass characteristics of lambs fed diets with increasing levels of <Emphasis Type="Italic">Mimosa tenuiflora</Emphasis> (Willd.) hay replacing Buffel grass hay

This study evaluated the performance and carcass characteristics of lambs fed diets with increasing levels of Mimosa tenuiflora (Willd.) hay replacing Buffel grass (Cenchrus ciliaris). Twenty-eight Santa Inês male lambs with an average body weight (BW) of 20.3 ± 1.49 kg(mean ± SD) were allocated in individual stalls and distributed in a completely random design with four treatments (0, 20, 40, and 60 g/100 g total DM M. tenuiflora hay replacing Buffel grass hay in diet) with seven replications. M. tenuiflora hay at the level of 20% dry matter (DM) total replacing Buffel grass hay increased final weight (P = 0.006), total weight gain (P < 0.001), average daily weight gain (ADWG; P < 0.001), DM intake (P < 0.001), and feed efficiency (P < 0.001). Intake of crude protein, NDF_ap, ADF_ap, ash, ether extract, total and non-fibrous carbohydrates, and total digestible nutrients presented a positive quadratic effect with M. tenuiflora hay replacing Buffel grass hay and 40 g/100 g total DM level presented greater intake. There were positive quadratic effects by M. tenuiflora hay inclusion at 20 g/100 g total DM level on slaughtering weight (P = 0.005), hot carcass weight (P = 0.002), cold carcass weight (P = 0.002), empty body weight (P = 0.001), hot carcass yield (P = 0.002), cold carcass yield (P = 0.003), and increase linear on biological yield (P = 0.003). There was no influence on cooling weight loss (P = 0.284). M. tenuiflora hay may be included in lamb diets at amounts up to 20 g/100 g total DM substitution of Buffel grass hay because increase in the nutrients intake, growth performance, and carcass characteristics.     

Cardiopulmonary effects of reverse Trendelenburg position at 5° and 10° in sevoflurane-anesthetized steers

Objective

To assess the cardiopulmonary effects caused by reverse Trendelenburg position (RTP) at 5° and 10° in sevoflurane-anesthetized yearling steers.

Serum biochemical analytes in captive Amazonian manatees (Trichechus inunguis)

Background: Establishment of reference values for serum biochemical analytes is important for monitoring health and physiological status of captive animals. Objective: The purpose of this study was to measure and report ranges for serum biochemical analytes in Amazonian manatees (Trichechus inunguis). Methods: Blood samples were collected from 24 healthy captive Amazonian manatees that comprised a mixture of adults, subadults, and calves and males and females; serum analytes were measured and analyzed using a dry reagent bench‐top chemical analyzer. Comparisons were made between sexes and with previously published values of closely related species. Results: Medians and ranges (minimum–maximum) of values for the analytes were: lactate dehydrogenase (LDH), 151 (111–278) U/L (n=20); creatine kinase, 144 (76–315) U/L (n=11); alanine aminotransferase, 10 (2–28) U/L (n=18); aspartate aminotransferase, 14 (5–28) U/L (n=21); γ‐glutamyltransferase, 47 (36–73) U/L (n=21); amylase, 1428 (1010–1874) U/L (n=21); alkaline phosphatase, 73 (36–141) U/L (n=19); total protein, 6.8 (6.2–8.0) g/dL (n=24); albumin, 3.3 (2.6–4.1) g/dL (n=21); cholesterol, 188 (101–399) mg/dL (n=21); triglycerides, 126 (60–236) mg/dL (n=21); glucose, 47 (22–69) mg/dL (n=21); urea, 43 (21–69) mg/dL (n=21); uric acid, 1.1 (0.5–1.8) mg/dL (n=22); creatinine, 2.2 (1.5–3.3) mg/dL (n=22); total bilirubin, 0.2 (0.2–2.0) mg/dL (n=21); calcium, 12.7 (10.2–18.6) mg/dL (n=24); iron, 282 (207–457) μg/dL (n=13); and magnesium, 6.9 (4.3–8.9) mg/dL (n=20). With the exception of LDH, no differences were observed between sexes. Conclusions: The ranges obtained in this study provide important preliminary estimates for concentrations and activities of serum analytes in Amazonian manatees until a larger reference interval study can be conducted.     

Response of Thawed Epidi dymal Red Deer Spermatozoa to Increasing Concentrations of Hydrogen Peroxide,and Importance of Individual Male Variability

Oxidative stress represents a challenge during sperm manipulation. We have tested the effect of increasing hydrogen peroxide (H₂O₂) levels on red deer spermatozoa after cryopreservation, and the role of male‐to‐male variation in that response. In a first experiment, eight thawed samples were submitted to 0, 25, 50, 100 and 200 μm H₂O₂ for 2 h at 37°C. Intracellular reactive oxygen species (H₂DCFDA‐CM) increased with H₂O₂ concentration, but we only detected a decrease in sperm function (motility by CASA and chromatin damage by sperm chromatin structure assay) with 200 μm . Lipoperoxidation assessed by the thiobarbituric acid reactive substance (TBARS) method increased slightly with 50 μm H₂O₂ and above. In a second experiment, samples from seven males were submitted to 0 and 200 μm H₂O₂ for 2 h, triplicating the experiment within each male. Males differed at thawing and regarding their response to incubation and H₂O₂ presence. We found that the kinematic parameters reflected male‐to‐male variability, whereas the response of the different males was similar for lipid peroxidation and viability. A multiparametric analysis showed that males grouped differently if samples were assessed after thawing, after incubation without H₂O₂ or after incubation with H₂O₂. Red deer spermatozoa are relatively resilient to H₂O₂ after thawing, but it seems to be a great male‐to‐male variability regarding the response to oxidative stress. The acknowledgement of this individual variability might improve the development of optimized sperm work protocols.     

Montenegro's skin reactions and antibodies against different Leishmania species in dogs from a visceral leishmaniosis endemic area

In this study, humoral (circulating anti-Leishmania antibodies) and cellular (Montenegro's skin test) immune responses of dogs from an endemic area of visceral leishmaniosis were tested using Leishmania chagasi, Leishmania amazonensis and Leishmania braziliensis antigens. The antibody response was tested in three animal groups, selected according to their anti-L. chagasi antibody activity, as measured by ELISA in the serum: 19 negative (O.D. below 0.30), seven with undefined (O.D. between 0.40 and 0.70) and 12 positive (O.D. above 1.0) ELISA result. In the group of animals with positive ELISA, the antibody activity against L. chagasi antigens (mean O.D.=1.31) was significantly higher (ANOVA, P<0.01) than against L. amazonensis (mean O.D.=0.88) or L. braziliensis (mean O.D.=0.87) antigens. The Montenegro's skin test results obtained with L. chagasi and L. braziliensis antigens showed a fair agreement (kappa=0.309). The same was observed when antigens from L. braziliensis and L. amazonensis were compared (kappa=0.374), whereas a moderate agreement between the results from tests performed with L. chagasi and L. amazonensis antigens was observed (kappa=0.530). The induration areas obtained with L. braziliensis antigen were smaller than those obtained with the other antigens. The data presented herein indicate that the use of antigens from different Leishmania species may interfere with the results of the immunological tests performed in dogs in an endemic area of visceral leishmaniosis.     

Isolation and characterization of an adventitious avian leukosis virus isolated from commercial Marek's disease vaccines

Commercial Marek's disease (MD) vaccines produced by two manufacturers were tested for possible contamination with avian leukosis virus (ALV). Samples of MD vaccines manufactured by two companies (A and B) were received from a breeder company; samples were also received directly from vaccine company B. Using virus isolation tests, samples initially tested positive for subgroup E (endogenous) ALV. However, upon repassage, the vaccines also tested positive for exogenous ALV. The isolated exogenous ALV proved to be a subgroup A virus, as determined by flow cytometry using polyclonal chicken antibodies specific for various subgroups of ALV, and by DNA sequencing of the envelope glygoprotein (gp85). The exogenous ALV isolated from MD vaccines was inoculated in chickens from ADOL lines 15I(5) x 7(1) and 0 to determine its pathogenicity and compare it with that of Rous-associated-virus-1 (RAV-1), the prototype strain of ALV-A. Each chicken from each line was inoculated with approximately 10,000 infectious units of RAV-1 or the ALV-A isolated from vaccines termed B-39 virus at 7th day of embryonation. At hatch, and at 4, 8, and 16 wk of age, chickens were tested for viremia and cloacal shedding; chickens were also observed for ALV-induced tumors within 16 wk of age. Viremia and cloacal shedding results suggest that chickens from both lines were susceptible to infection with either virus. Within 16 wk of age, the proportion of ALV tumors induced by strain B-39 in line 0 and line 15I5 x 7(1) chickens was 0% and 12%, respectively, compared with 62% and 67% in chickens inoculated with RAV-1. The data indicate that commercial MD vaccines produced by two manufacturers were contaminated with endogenous subgroup E and an exogenous subgroup A ALV. Further, data from biological characterization suggest that the ALV-A isolated from commercial MD vaccines is of low oncogenicity, compared with that of RAV-1. GenBank accession numbers: The gp85 gene sequences of ALV isolated from commercial Marek's disease vaccines have been deposited in GenBank and assigned the following accession numbers: A46 subgroup A, DQ412726 ; B53 subgroup A, DQ412727; A46 subgroup E, DQ412728; B53 subgroup E, DQ412729.     

B‐Mode and Doppler Sonography of the Mammary Glands in Dairy Goats for Mastitis Diagnosis

This study aimed to evaluate the sonographic characteristics of the udder and teats and to determine the Doppler indexes of mammary artery in healthy and undergoing subclinical and clinical mastitis goats. Thirty animals among Saanen and Alpine Brown goats were arranged in three groups, healthy goats (HG), goats with subclinical mastitis (SMG) and goats with clinical mastitis (CMG). Using the B‐mode, the sonographic characteristics (echotexture and echogenicity) and biometry (diameter and area of the udder cistern, diameter and area of the teat cistern and thickness of the teat wall) were evaluated. Using Doppler ultrasonography, the vascular indexes of the mammary artery were obtained. It was observed hyperechogenicity with solid component in the gland cistern when comparing animals with clinical mastitis and healthy mammary tissue. Regarding the echotexture of the breast tissue, there was heterogeneity in the mammary parenchyma on the three groups, for the milk, it was observed homogeneity for animals on HG and SMG and heterogeneity for animals on CMG. Grey‐scale quantitative assessment revealed increase in echogenicity (mean value) for all the structures when comparing the three groups. Biometry did not reveal statistical difference between groups, for none of the evaluated structures. Doppler examination of the mammary artery showed the decrease of end diastolic velocity and raise of pulsatility index between groups. The association of B‐mode and Doppler ultrasonography is useful for the evaluation of the udder of dairy goats with mastitis. It is a sensitive and specific method for the study of this disease. Doppler mode was unable to establish reliable criteria for diagnosis of subclinical mastitis. Moreover, the quantification of echogenicity is a useful technique for the evaluation of the milk in animals with mastitis; therefore, it is suggested that it can be used as complementary technique for the diagnosis of mastitis in goats.