Design,optimization, and application of a conventional PCR assay with an internal control for detection of ‘Candidatus Mycoplasma turicensis’ 16S rDNA in domestic cats from Brazil

Background: ‘Candidatus Mycoplasma turicensis’ (CMtc) is a hemotrophic bacterial species that can, alone or in combination, induce anemia in cats. The diagnostic test of choice for hemoplasma infections is PCR. Conventional PCR assays have been developed for the detection of Mycoplasma haemofelis (Mhf) and ‘Candidatus M. haemominutum’ (CMhm) but not for CMtc. Although real‐time PCR assays have been reported for all of the feline hemoplasmas, the expense of necessary instrumentation precludes its use in Brazil and many other countries. Objectives: The goals of this study were to develop and optimize a conventional PCR assay to diagnose CMtc using an internal control to detect false‐negative results, and to evaluate the occurrence of CMtc infection in domestic cats from Brazil. Methods: Species‐specific primers were designed and a PCR assay was developed for the detection of CMtc 16S rDNA in cat blood. Sensitivity was determined by serial 10‐fold dilutions of plasmid and DNA extracted from blood from an experimentally infected cat. EDTA blood samples from 373 cats were collected. DNA was extracted using a silica‐based protocol and tested using the PCR assay. Results: Primer concentration, annealing temperature, and MgCl₂ concentration were optimized in the presence and absence of the internal control. Two samples negative for the internal control were excluded. Of the remaining 371 samples (117 healthy and 254 unhealthy cats), 17 (4.6%) were positive for CMtc. Conclusion: These results demonstrate the utility of an optimized PCR assay to detect CMtc in feline blood samples. We also report for the first time the prevalence of CMtc infection in domestic cats in Brazil.     

In vitro effect of isoprinosine on rabies virus

The in vitro effect of isoprinosine, an antiviral compound, was evaluated on ERA and VA319 rabies viral strains. Isoprinosine diminished the number of immunofluorescent cells, viral titers, and structures involved in rabies virus replication. Mechanism of antiviral action was probably mediated by modification of the polyribosome conformation and function.     

Detection of Ehrlichia canis by polymerase chain reaction in dogs from Portugal

Antibodies against Ehrlichia canis, the cause of canine monocytic ehrlichiosis, have been reported previously in clinically ill and stray dogs from Portugal. In this study, the 16S rRNA gene of E. canis was detected by the polymerase chain reaction (PCR) in 12/55 (22%) of dogs with suspected tick-borne disease in the Algarve region in Portugal.     

Protected fatty acid supplementation during estrus synchronization treatment on reproductive parameters of dairy goats

This study evaluated the effect of the protected fatty acid inclusion during estrus synchronization on reproductive parameters. Goats (n = 32) received progestagen sponges for 6 days and 200 IU equine chorionic gonadotropin and 30 µg d‐cloprostenol were given on Day 5. No difference was found among control (C), 1% protected fatty acid inclusion (C + 1%) or 4% protected fatty acid inclusion (C + 4%) groups, respectively, in estrus (100.0, 100.0 or 90.9%), estrus duration (31.6 ± 12.3; 43.2 ± 12.9 or 40.8 ± 14.1 h), animals ovulating (100.0, 90.0 or 100.0%) or ovulation rate (1.3 ± 0.5; 1.1 ± 0.3 or 1.2 ± 0.4). The interval from sponge removal to ovulation and from estrus to ovulation, respectively, were shorter for C + 4% (45.2 ± 8.0 h; 18.3 ± 11.0 h) compared with C (56.3 ± 12.6 h; 30.6 ± 10.5 h) or C + 1% (57.7 ± 8.7 h; 30.3 ± 11.1 h). The average ovulatory follicle diameter was smaller for C + 4% (6.2 ± 0.7 mm) than C (7.5 ± 0.8 mm), but similar to C + 1% (7.0 ± 1.5 mm). Insulin, insulin‐like growth factor 1, glucose and progesterone concentrations were similar among groups. The inclusion of protected fatty acid during synchronization treatment promoted no benefits on ovulation rate, but 4% anticipated the ovulation time.     

Comprehensive diversity assessment of walnut-associated xanthomonads reveal the occurrence of distinct Xanthomonas arboricola lineages and of a new species (Xanthomonas euroxanthea) within the same tree

Xanthomonas arboricola pv. juglandis (Xaj) is the aetiological agent of walnut diseases causing economic losses on walnut production worldwide. This phytopathogen is spread around the world where walnuts are produced and has a considerable genetic diversity. Using a comprehensive sampling methodology, focusing on factors that could influence the diversity of walnut-colonizing Xaj in Portugal, this work provides new insights on xanthomonad populations on walnut. Genetic diversity was assessed by multilocus sequence analysis (MLSA) and dot blot hybridization patterns on 131 Xanthomonas isolates obtained from 64 walnut trees considering epidemiological metadata such as year of isolation, distinct bioclimatic regions, production regimes, and host-related features. The results showed that the majority of isolates were split into 17 lineages of Xaj, while the other isolates clustered in four MLSA groups that did not include Xaj strains. These four groups were represented by three lineages of X. arboricola, and 11 lineages of Xanthomonas spp., including strains assigned to the recently proposed new species Xanthomonas euroxanthea. Furthermore, distinct Xaj, X. arboricola, and Xanthomonas spp. were isolated from the same walnut tree, suggesting possible genetic admixture within the same host. Phylogenetic analysis through geoBurst revealed the high diversity of these Xanthomonas spp. populations. Assessment of type III effector genes gave the indication that some Xanthomonas spp. strains were nonpathogenic on walnut, with the exception for X. euroxanthea CPBF 424. Altogether, these findings add to the thorough characterization of walnut-associated xanthomonads in Portugal, providing a comprehensive snapshot of the current diversity that could contribute to risk assessment analysis and improve phytosanitary control.     

Effects of dietary energy level and supplemental protein source on performance of growing steers and nutrient digestibility and nitrogen balance in lambs

Four experiments were conducted to evaluate three crude protein (CP) sources (urea, U; soybean meal, SBM; corn gluten meal, CGM) in diets based on corn silage (high energy) or grass hay (low energy). In Exp. 1 and 2, growing steers were fed all combinations of energy and protein source at 10.5 or 12% CP. Steers fed high energy diets or 12% CP had improved (P less than .05) daily gains and feed:gain over 84 d. Protein source had no effect (P greater than .05) on performance except that steers fed U consumed more (P less than .05) feed than those fed CGM. Steers were fed experimental diets to a common weight and switched to an 85% concentrate diet for finishing. During finishing, steers fed low energy diets in the growing period consumed more (P less than .05) feed and had increased (P less than .05) feed:gain compared with those fed high energy diets. Growing lambs were fed the same diets as steers. At 10.5% CP, lambs fed high energy diets had higher (P less than .05) digestibilities of dry matter (DM), organic matter (OM), nitrogen (N) and fiber components, and retained more (P less than .05) N. For lambs on 12% CP, high energy diets had higher (P less than .05) DM and OM digestibilities and lower (P less than .05) N digestibilities. At 12% CP, energy level had no effect (P greater than .05) on N retained. Protein source had no effect (P greater than .05) on N retention. There appeared to be no advantage in supplementing with ruminally undegradable proteins, i.e. CGM, in these experiments.     

Pregnancy rates in repeat-breeder heifers following multiple artificial inseminations during spontaneous oestrus

Hormonal asynchronies during oestrus, related to the presence of suprabasal plasma-progesterone (P4) concentrations and a delayed ovulation, interfere with the fertility of repeat-breeder heifers (RBH). Since tubal dysfunction can occur in connection with hormonal asynchronies and constrained availability of fertile spermatozoa at the time of ovulation, the present study tested the hypothesis that frequent sperm deposition from onset of oestrus to ovulation may improve pregnancy rates in RBH. Five RBH and five virgin heifers (VH; controls) were repeatedly artificially inseminated (AI) at 6 h intervals from onset of oestrus to spontaneous ovulation. Hormone analyses revealed suprabasal P4 concentrations and a delay in the occurrence of the luteinising hormone (LH) surge, but a normal cortisol profile in RBH. Compared with controls, RBH presented longer interval from onset of oestrus to ovulation, and therefore, received more AIs. Pregnancy rates in RBH reached control levels (60%; NS), indicating that the hypothesis might be correct. Pregnancy rates in VH were below the expected range, presumably attributed to a deleterious influence of the frequent handling. The study suggests that pregnancy rates can be improved in RBH by frequent AI in relation to spontaneous ovulation. However, this practice of repeated manipulations, while seeming not to show adverse effects, lacks practicality for routine use.     

Hemangiomatosis associated with osteolysis of the mandible in a dog resembling Gorham-Stout disease in humans

A 6-month-old female German Shepherd Dog died as a result of profuse oral bleeding. At postmortem examination, the oral cavity showed visible roots of the right mandibular fourth premolar and first molar teeth and, in addition, they were very mobile and compressible. Radiographs showed a generalized radiolucency in the body of the right mandible, with evidence of resorption of the affected alveolar bone. Histologically, the lesion of the right mandible was characterized by the lysis of bony structures and a non-malignant proliferation of blood-filled vascular spaces lined by a single layer of well-differentiated endothelial cells. The clinical, radiographic, and histologic presentation of this dog is consistent with that associated with Gorham-Stout disease, a rare bone disorder in humans.