Increased incidence of DNA amplification in follicular than in uterine and blood samples indicates possible tropism of Neospora caninum to the ovarian follicle

This study evaluated the presence of Neospora caninum in ovarian follicle aspirates and uterine flushes obtained from N. caninum seropositive dairy cows. Ninety-two cows that aborted within the previous 90 days were sampled to determine the presence of antibodies against N. caninum. Thirteen seropositive cows were chosen for collection of blood leukocytes, uterine flushes (UF; n=12) and follicular aspirates (OPU; n=13). Samples were centrifuged and the cellular sediment from the follicular fluid, uterine flushes and blood leukocytes were used for DNA extraction and PCR. Follicular aspirates had the highest frequency of DNA amplification for N. caninum (p<0.05, 92.3%; 12/13). Whereas uterine (4/12) and blood leukocyte (5/13) samples had similar (p>0.05) rate of positive results. Nonetheless, there was no agreement between blood leukocytes and follicular samples taken from the same animal (Cohen Kappa=-0.16). Similarly, blood leukocytes and uterine results had moderate agreement between them (Cohen Kappa=0.47). This study indicates that N. caninum is present in the ovarian follicle and uterus of seropositive cows, suggesting a possible risk of neosporosis transmission between females during oocyte and embryo collection and transfer. Hence, precautions should be taken to minimize the risk of N. caninum transmission. Furthermore, the high incidence of positive results in follicle samples, that exceeded that of their paired blood leukocytes, suggests a possible tropism of N. caninum for the ovarian follicle.     

Hematologic abnormalities and flow cytometric immunophenotyping results in dogs with hematopoietic neoplasia: 210 cases (2002–2006)

Background: Growing interest in veterinary oncohematology has facilitated the recent development and advancement of new techniques, such as flow cytometry, for immunophenotyping hematopoietic neoplasia in animals. Objective: The aim of this retrospective study was to characterize hematologic abnormalities and flow cytometric immunophenotyping (FCI) results in cases of hematopoietic neoplasia in dogs. Methods: Signalment, CBC data, and FCI results were obtained for 210 dogs with blood samples submitted to our laboratory. Immunophenotyping was carried out using an Epics XL‐MCL flow cytometer and a panel of 10 antibodies (CD45, CD3, CD4, CD8, CD79, CD21, CD14, CD34, CD41/61, CD61). The prevalence and severity of hematologic abnormalities was determined for the different types of hematopoietic neoplasms. Results: Based on cell morphology and phenotype, cases were classified as: acute lymphoblastic leukemia (ALL, n=51), acute myeloid leukemia (AML, n=33), chronic lymphocytic leukemia (CLL, n=61), and leukemic high‐grade lymphoma (L‐HGL, n=65). Most cases of ALL (47/51) and L‐HGL (41/65) had a B‐cell phenotype, while most cases of CLL (54/61) had a T‐cell phenotype, with a high prevalence of the large granular lymphocyte subtype (49/61). Anemia was found in 85% of all cases and was significantly more severe in ALL and AML compared with CLL and L‐HGL. Neutropenia was seen in 64–78% of acute leukemias (AML and ALL) in contrast to no cases of CLL and 11% of L‐HGL. Thrombocytopenia was seen in 88–90% of acute leukemias in contrast to 15% of CLL and 40% of L‐HGL. Thrombocytopenia was more prevalent (71% vs 22%) and significantly more severe in T‐cell vs B‐cell L‐HGL. Conclusion: A standard CBC is useful in suggesting the type of hemoproliferative disorder and may also help to predict the phenotype, especially in cases of L‐HGL.     

Caprine herpesvirus-1-specific IgG subclasses in naturally and experimentally infected goats

Enzyme-linked immunosorbent assays (ELISAs) were developed to detect caprine herpervirus-1 (CpHV-1)-specific IgG1 and IgG2 in sera from 43 naturally infected goats. The analysis of the IgG subclasses showed a dual pattern of distribution in seropositive goats with a major group of animals (36 out of 43) exhibiting significantly higher levels of IgG2 over IgG1 and a minor group (7 out of 43) possessing equal levels of IgG1 and IgG2. Four goats were experimentally infected with a virulent CpHV-1 Ba.1 strain by the intranasal or the intravaginal route and the kinetics of appearance of CpHV-1-specific IgG, IgG1 and IgG2 in the serum were studied. Two weeks following infection, both IgG1 and IgG2 levels increased although convalescent sera (i.e., collected 5–8 weeks post-infection) showed a clear prevalence of the IgG2 subclass. To determine the contribution of the different IgG subclasses to herpesvirus immunity, serum neutralization (SN) assays were performed in both naturally and experimentally infected goats. The kinetics of SN showed that neutralization activity was mainly associated to the IgG1 subclass and this was also confirmed in naturally infected goats. The results are discussed from the standpoint that the profile of the IgG subclasses is instrumental to study immune responses to CpHV-1 and that vaccination strategies may benefit from this information.     

Clinical Use of Testicular Fine Needle Aspiration Cytology in Oligozoospermic and Azoospermic Dogs

Clinical investigation of canine testicular function is complicated by the difficulty in the evaluation of seminiferous tubules. Until recently, testicular biopsy was the only diagnostic option for dogs with persistent oligo/azoospermia. In human andrology, testicular fine needle aspiration (TFNA) is currently considered a useful method in the evaluation of azoospermia and severe oligozoospermia, and has long replaced classical biopsy to evaluate spermatogenesis. In order to verify its diagnostic efficacy for the clinical approach to canine oligo- or azoospermia, TFNA was performed in seven adult (two oligozoospermic and five azoospermic) dogs. After sedation, a fine (21–23 gauge) butterfly needle connected to a 50-ml syringe was inserted into each testicle; strong suction was applied and the aspirated fluid squirted on a glass slide, smeared out, air-dried and stained with a modified May–Grunwald–Giemsa. Under light microscopy, Sertoli cells (all those found in each investigated field) and spermatogenic cells (n = 100) were counted on each smear in order to differentiate spermatogonia, primary spermatocytes, secondary spermatocytes, early spermatids, late spermatids and spermatozoa, and calculate their relative percentages. Cytological analysis showed the following testicular pictures: normal spermatogenesis (compatible with obstruction of the seminal ducts), hypospermatogenesis, maturative disturbances and Sertoli cell-only syndrome. Two dogs with an obstructive lesion were treated with corticosteroids; one of them recovered and sired two litters of puppies.     

Campylobacter jejuni in the red squirrel (Sciurus vulgaris) population of Southern Italy

Rectal swab samples were collected from 60 red squirrels (Sciurus vulgaris) from July 2006 to April 2007 in Southern Italy. Samples were tested for Campylobacter jejuni and C. coli by culture methods and suspected colonies were then confirmed by polymerase chain reaction. C. jejuni was detected in 5/60 (8.3%) samples examined but infection status was not related to age or sex and C. coli was not isolated. This is believed to be the first report of C. jejuni infection in the red squirrel.     

Inhibitory properties and binding loop polymorphism in Bowman-Birk inhibitors from <Emphasis Type="Italic">Phaseolus</Emphasis> species

The primary structure of the double-headed Bowman-Birk inhibitor (BBI) family and the antitryptic activity were investigated in cultivated and wild Phaseolus species. Two BBI types were identified; the first one inhibits trypsin and chymotrypsin (tc-BBI), the second one elastase and trypsin (et-BBI). Only tc-BBI was found in P. lunatus and P. parvulus, while none of BBI types, identified in this study, was found in P. leptostachyus. The deduced amino acid sequences revealed some polymorphisms within both tc-BBI and et-BBI binding loops that could affect the inhibitory activity. The trypsin inhibitor content showed a high variation with the lowest value recorded in P. lepthostachyus and the highest one observed in P. oligospermus. Southern blot analysis confirmed the absence of both BBI types in P. leptostachyus and suggests that in P. coccineus, P. dumosus and P. costaricensis, the two genes were clustered in a narrow genomic region of 1.3 kbp.     

Induction of systemic resistance by a hypovirulent Rhizoctonia solani isolate in tomato

A polynucleate Rhizoctonia isolate (R3) was analysed for virulence, growth characteristics, enzyme production and presence of dsRNAs. Taxonomic position was assessed morphologically and by anastomosis group (AG) testing and ITS sequence analysis. Results indicated that R3 is a hypovirulent R. solani AG 4. Mechanisms underlying biocontrol towards virulent R. solani and Botrytis cinerea were investigated and plant-mediated resistance was followed using biochemical markers of defence (PR1, laminarinase, chitinase). Control apparently relies on spatial and nutrient competition in soil, and on systemic induced resistance. This is the first report on induction of systemic resistance and of defence markers by a hypovirulent strain of R. solani.     

Evidence for the Accelerated Degradation of Isoproturon in Soils

The herbicide isoproturon was degraded rapidly in a sandy loam soil under laboratory conditions (incubation temperature, 15°C; soil moisture potential, -33 kPa). Degradation was inhibited following treatment of the soil with the antibiotic chloramphenicol, but unaffected by treatment with cycloheximide, thus indicating an involvement of soil bacteria. Rapid degradation was not observed with other phenylurea herbicides, such as diuron, linuron, monuron or metoxuron incubated in the same soil under the same experimental conditions. Three successive applications of isoproturon to ten soils differing in their physicochemical properties and previous cropping history induced rapid degradation of the herbicide in most of them under laboratory conditions. There were, however, no apparent differences in ease of induction of rapid degradation between soils which had been treated with isoproturon for the last five years in the field and those with no pre-treatment history. A mixed bacterial culture able to degrade isoproturon in liquid culture was isolated from a soil in which the herbicide degraded rapidly.     

Brazilian biosafety law and the new breeding technologies

Globally, the area of land cultivated with genetically modified (GM) crops has increased a thousand-fold over the last two decades. Although this technology has become important for food production, the regulatory frameworks that underpin these outcomes are based on a list of requirements for a risk assessment that differ from country to country. In recent years, policymakers have had the opportunity to learn from the controversies over transgenics to create effective regulatory milestones for emerging technologies, allowing them to reach their potential for a more sustainable agriculture, ensuring food security. In Brazil, Law No. 11.105 of 24 March 2005 established a framework with four main organizations responsible for risk assessment and management. However, most of new breeding technologies did not exist at that time and were not considered in this law. In 2016, Normative Resolution No. 16 of the National Biosafety Technical Commission (CTNBio) was established to address this gap based on the evaluation of the products obtained through these techniques (termed Innovative Precision Improvement Techniques in the resolution), in a case-by-case consultation system. Briefly, if the product is designated to be a GM, the developer will have to go through the biosafety requirements and will be approved only after CTNBio risk assessment. If the product is designated not to be GM (for the purposes of the legislation), then it can be registered using the existing procedures. Currently, 152 GM products are commercially approved in Brazil. In 2018, CTNBio assessed the first consultation on commercial release of plants generated using the new breeding technologies and has subsequently approved six products. It is expected that many institutions would be able to participate in Brazilian and world markets, developing and introducing new biotechnological solutions and products through a more sustainable approach and without facing public disapproval, a common issue for GM crops.