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.     

Regional distribution and integrity of equine ovarian pre‐antral follicles

The goal of this study was to determine the distribution of pre‐antral follicles in the ovarian parenchyma of mares. For Experiment 1, each ovary was cut longitudinally at the greater curvature, performing two hemiovaries. After that, six fragments from each hemiovary were obtained, resulting in 12 fragments, which were divided into the innermost region of the parenchyma, the middle region and the outermost region. All the three obtained sections were cut transversally to obtain two fragments from each one. For Experiment 2, each ovary also submitted to a longitudinal cut on the greater curvature, forming two hemiovaries. Each hemiovary was sectioned into four symmetrical fragments, resulting in eight fragments per ovary. The fragments were related as being near to or far from the ovulatory fossa. The fragments of both experiments were immediately fixed in Carnoy for 12 hr and kept in 70% ethanol for 24 hr. Follicles were classified according to the stages of development and for morphological integrity according to oocyte morphology and granulosa cells. After the histological assessment, a total of 1,130 follicles were visualized from Experiment 1, being 1,054 (93.3%) primordial follicles and 76 (4.7%) follicles in development. The innermost region had the highest percentage of pre‐antral follicles compared to the other regions (p < .05). The middle and outermost regions showed higher percentages of intact primordial and developing follicles than the innermost region (p < .05). Considering Experiment 2, 938 follicles were found, being 894 (95.3%) primordial and 44 (4.7%) follicles in development. The region near the ovulatory fossa presented higher (58.7%; 551 of 938) follicular concentration compared to the region far from the ovulatory fossa (41.3%; 387 of 938; p < .05). As a conclusion, distribution of pre‐antral follicles in the equine ovary has a specific pattern through the parenchyma. Also, the follicular integrity differed in the studied ovarian areas.     

Follicular degeneration in the ovaries of goats experimentally infected with Trypanosoma vivax from the Brazilian semi-arid region

Infection by Trypanosoma vivax and other African trypanosomes plays an important role in reproductive disorders in male and female livestock. Outbreaks of T. vivax in the semi-arid region of northeastern Brazil are characterized by wasting disease in cattle, sheep and goats with hematological, cardiac and nervous compromises in addition to reproductive failures. Similar to reports from Africa, we previously observed a reduction in fertility rates and severe testicular degeneration and epididymitis in male sheep infected with T. vivax from this region. Although anestrus is frequently reported in goats and sheep infected with T. vivax, the effects of this infection on the female reproductive organs need clarification. In this study, we addressed this issue through a histopathological evaluation of ovarian follicular morphology and classification in goats experimentally infected with a T. vivax isolate from the Brazilian semi-arid region. The infected animals presented typical clinical signs of trypanosomosis by T. vivax, including anemia, hyperthermia, pallor of the mucous membranes, enlarged lymph nodes, and progressive loss of weight. All the infected goats remained anestrus throughout the experimental period and exhibited important disturbances in the ovaries, evidenced by reduced size and a smooth surface without follicles or corpora lutea, and abnormal follicular development. In addition, through PCR, we detected T. vivax DNA in the ovarian tissues of the infected goats. Our findings contributed to understand the female reproductive failure associated with trypanosomosis caused by T. vivax.     

FSH up-regulates angiogenic factors in luteal cells of buffaloes

Follicle-stimulating hormone has been widely used to induce superovulation in buffaloes and cows and usually triggers functional and morphologic alterations in the corpus luteum (CL). Several studies have shown that FSH is involved in regulating vascular development and that adequate angiogenesis is essential for normal luteal development. Angiogenesis is regulated by many growth factors, of which vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2) have an established central role. Therefore, we have used a combination of in vitro and in vivo studies to assess the effects of FSH on the expression of VEGF and FGF2 and their receptors in buffalo luteal cells. The in vivo model consisted of 12 buffalo cows, divided into control (n = 6) and superovulated (n = 6) groups, and CL samples were collected on day 6 after ovulation. In this model, we analyzed the gene and protein expression of FGF2 and its receptors and the protein expression of VEGFA systems with the use of real-time PCR, Western blot analysis, and immunohistochemistry. In the in vitro model, granulosa cells were collected from small follicles (diameter, 4–6 mm) of buffaloes and cultured for 4 d in serum-free medium with or without FSH (10 ng/mL). To induce in vitro luteinization, LH (250 ng/mL) and fetal bovine serum (10%) were added to the medium, and granulosa cells were maintained in culture for 4 d more. The progesterone concentration in the medium was measured at days 4, 5, and 8 after the beginning of cell culture. Cells were collected at day 8 and subjected to real-time PCR, Western blot analysis, and immunofluorescence for assessment of the expression of FGF2, VEGF, and their receptors. To address the percentage of steroidogenic and growth factor-expressing cells in the culture, flow cytometry was performed. We observed that in superovulated buffalo CL, the FGF2 system mRNA expression was decreased even as protein expression was increased and that the VEGF protein was increased (P < 0.05). In vitro experiments with granulosa cells showed an increase in the mRNA expression of VEGF and FGF2 and its receptors 1 and 2 and protein expression of VEGF, kinase insert domain receptor, FGF receptor 2, and FGF receptor 3 in cells treated with FSH (P < 0.05), in contrast to the in vivo experiments. Moreover, the progesterone production by FSH-treated cells was elevated compared with untreated cells (P < 0.05). Our findings indicate that VEGF, FGF2, and their receptors were differentially regulated by FSH in vitro and in vivo in buffalo luteal cells, which points toward a role of CL environment in modulating cellular answers to gonadotropins.     

Anatomicohistological Characteristics of Female Genital Tubular Organs of the South American Nutria (Myocastor coypus)

Morphohistological features of the tubular organs of the Myocastor coypus (coypu) female reproductive tract were studied. Specimens came from breeding farms with yard breeding systems. The analysis of the organs was made by histological methods and by macro and microscopic measurement techniques. The animals showed oviducts with macro and microscopically differentiable regions. Their mucosa showed primary branched folds in the ampullar sector. In the rest of the oviduct tract these folds were only of the primary type. The double uterus showed regional variations in the lumen, endometrial glands along the whole surface and a wide fibromuscular cervix with pseudoglands. The endocervical mucosa made clear a complex system of folds covered by a mucus-secreting epithelium. In the long vagina of the coypu a folded, rugose and irregular mucosa was observed.     

Brazilian foot and mouth disease status and meat exportation to the European Union

The aim of this study was to define the differences between the Brazilian states that export and do not export meat to the European Union (EU) and to identify the variables that are important to meet the export requirements. Infrastructure and computerization of the control of animal transit in Brazil that impact on regional health status were evaluated and linked to other variables such as status for foot and mouth disease (FMD) and qualification to export meat to EU. Variables related to transit control of bovines implemented by the state agencies of animal health and inspection in each Brazilian state were evaluated. Using a discriminant analysis, four variables were selected that explained the variation between Brazilian states that were “free” and “not free” of FMD while another four were selected to explain the variation between the zones “approved” and “not approved” to export meat to the EU, including number of official veterinarians, total transit of bovines and buffaloes, total number of animal transit certificates issued for bovine and buffaloes at the state or zone level, and total number of municipalities in the state or zone. It was possible to correctly discriminate between “free” and “not free” FMD states or zones. Variables related to animal transit are important in assessing the state for the classification of animal health situation and for EU approval for the exportation of meat.     

Performance and meat quality of broiler chickens that are fed diets supplemented with Agaricus brasiliensis mushrooms

This trial was performed to study the use of the mushroom Agaricus brasiliensis as an alternative additive to antimicrobial growth promoters in broiler chicken diets and to assess the quality of the broiler chicken breast meat of birds that are fed diets containing this fungus. Thus, 595 1-day-old chicks were reared in reused poultry litter without anticoccidial and antimicrobial additives. The results showed that a concentration of 1.6 g mushrooms/kg diet was ideal for these birds because it provided better bird performance. When the birds’ immune system organs were analyzed, it was found that the addition of both mushrooms influenced the immune system organs of these broiler chickens. Adding A. brasiliensis to broiler chicken diets did not compromise breast meat quality.