Expression of fibroblast growth factor 10 and its receptor, fibroblast growth factor receptor 2B, in the bovine corpus luteum

There is evidence that several fibroblast growth factors (FGFs) are involved in growth and development of the corpus luteum (CL), but many FGFs have not been investigated in this tissue, including FGF10. The objective of this study was to determine if FGF10 and its receptor (FGFR2B) are expressed in the CL. Bovine CL were collected from an abattoir and classed as corpus hemorrhagica (stage 1), developing (stage 11), developed (stage 111), and regressed (stage IV) CL. Expression of FGF10 and FGFR2B mRNA was measured by reverse transcription-polymerase chain reaction (RT-PCR). Both genes were expressed in bovine CL, and FGF10 expression did not differ between stages of CL development. FGF10 protein was localized to large and small luteal cells by immunohistochemistry. FGFR2B expression was approximately threefold higher in regressed compared to developing and developed CL (P < 0.05). To determine if FGF10 and FGFR2B expression is regulated during functional luteolysis, cattle were injected with PGF2 alpha and CL collected at 0, 0.5, 2, 4, 12, 24, 48, and 64 hr thereafter (n = 5 CL/time point), and mRNA abundance was measured by real-time RT-PCR. FGF10 mRNA expression did not change during functional luteolysis, whereas FGFR2B mRNA abundance decreased significantly at 2, 4, and 12 hr after PGF2a, and returned to pretreatment levels for the period 24-64 hr post-PGF2 alpha. These data suggest a potential role for FGFR2B signaling during structural luteolysis in bovine CL.

Ovulation rate and its relationship with follicle diameter and gene expression of the LH receptor (LHR) in Nelore cows

The objective was to determine the relationship among the diameter of ovarian follicles, ovulation rate, and gene expression of the LH receptor (LHR) in Nelore cattle. In Experiment 1, ovulation was synchronized in 53 Nelore cows. Three days after ovulation, ovaries were assessed with ultrasonography, all cows were given 6.25 mg LH im, and they were allocated into three groups, according to diameter of their largest ovarian follicle: G1 (7.0-8.0 mm); G2 (8.1-9.0 mm); and G3 (9.1-10.0 mm). For these three groups, ovulation rates were 9, 36, and 90%, respectively, (P < 0.03; each rate differed significantly from the other two). In Experiment 2, granulosa and theca cells were subjected to total RNA extraction, and gene expression of the LHR was determined by RT-PCR. Follicles were allocated in three groups based on their diameter (similar to the Experiment 1), which were denoted Groups A, B, and C. Expression of the LHR gene in granulosa cells was lower in Group A than Group C (P < 0.05). However, there were no significant differences among groups in expression of the LHR gene in theca cells. We concluded that ovulatory capacity in Nelore cattle was related to increased follicular diameter and expression of the LHR gene in granulosa cells. (C) 2012 Elsevier B.V. All rights reserved.

Replication of classical infectious bursal disease virus in the chicken embryo related cell line

Infectious bursal disease (IBD) is an acute, highly contagious viral disease. The diagnosis of IBD depends on time-consuming and costly procedures, like virus isolation on chick embryos and histopathological examination, A double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), immunoperoxidase and reverse transcription polymerase chain reaction (RT-PCR) were applied in this study to detect classical IBD virus (IBDV) after three blind passages of the Lukert strain on chicken embryo related (CER) cell monolayer after different periods of infection: 6, 12, 24 and 48 h, Cytophatic effects were most evident 12 h post-infection (p.i.) but were observed at 6 h p.i. The maximum discrimination between IBDV-infected and uninfected cell suspensions obtained by the use of DAS-ELISA for virus detection corresponded to 0.597+/-0.02 and 0.010+/-0.01 after 12h p.i., respectively. The RT-PCR was performed using the set of primers A3.1 and A3.2 to amplify the VP2 region of the IBDV genome, This molecular technique demonstrated that from 6 h p.i., it was possible to detect the viral RNA. The results show that the CER cell line can be used for classical IBDV propagation, confirmed by the DAS-ELISA, immunoperoxidase and RT-PCR assay.

Infectious bronchitis virus replication in the chicken embryo related cell line

The susceptibility of the chicken embryo related (CER) cell line to infectious bronchitis virus (IBV M41) was characterized after five consecutive passages in CER cells. Virus replication was monitored by cytopathic effect observation, electron microscopy, indirect immunofluorescence, and reverse transcription polymerase chain reaction (RT-PCR). At 96 h post-infection (p.i.), the cytopathic effect was graded 75% by cell fusion, rounding up of cells and monolayer detachment, and the electron microscopy image characterized by coronavirus morphology. Cytoplasmic fluorescence was readily observed by from 24 h p.i. onwards, and at all times the respective viral RNA from IBV-infected monolayers was demonstrated by RT-PCR. Extra-cellular virus was measured by virus titration performed on chicken kidney cells and embryonated chicken eggs, and respective titres ranged from 4.0 to 6.0 log(10) EID50/ml on embryonated chicken eggs, and from 2.0 to 6.0 log(10) TCID50/ml on both CER cells and chicken kidney cells studied from 24 to 120 h p.i. These results confirmed that the M41 strain replicated well in the CER cell line.

Detection of turkey coronavirus in commercial turkey poults in Brazil

Poult enteritis complex has been incriminated as a major cause of loss among turkey poults in other countries. We have observed this in Brazil, associated with diarrhoea, loss of weight gain and, commonly, high mortality In this study, we have used the reverse transcriptase polymerase chain reaction (RT-PCR) to detect turkey coronavirus (TCoV) in sick poults 30 to 120 days of age from a particular producer region in Brazil. The RT-PCR was applied to extracts of intestine tissue suspensions, and the respective intestinal contents, bursa of Fabricius, faecal droppings and cloacal swabs. Primers were used to amplify the conserved 3' untranslated region of the genome, and the nucleocapsid protein gene of TCoV Histo pathological and direct immunohistochemical examinations were performed to detect TCoV antigen in infected intestine and bursa slides. All the results from stained tissues revealed lesions as described previously for TCoV infection. The direct immunohistochemical positive signal was present in all intestine slides. However, all bursa of Fabricius tissues analysed were negative. RT-PCR findings were positive for TCoV in all faecal droppings samples, and in 27% of cloacal swabs. Finally, the best field material for TCoV diagnosis was faecal droppings and/or intestine suspensions.

Relative expression of insulin like growth factor I (IGFI) and follicle stimulating hormone receptor (FSHR) in follicles and ovarian tissue from Bos primigenius indicus (Nelore)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Susceptibility of mammalian cell line for isolation of IBDV from clinical samples

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Detection of turkey astrovirus in young poults affected with poult enteritis complex in Brazil

Reverse transcription polymerase chain reaction (RT-PCR) of turkey astrovirus (TAstV) capsid and polymerase genes was applied to the bursa of Fabricius (BF), thymus (TH), spleen (SP) and cloacal swabs (CS) of young poults with "Poult enteritis complex" (PEC). The histological lesions included atrophy, lymphoid depletion, cellular infiltration and necrosis of the BF, TH and SP, respectively. The RT-PCR reactions were positive for the polymerase gene of TAstV-2 in all 100 CSs, 7 out of 10 of BFs and 10 out of 20 THs and SPs, respectively. Five out of 10 THs and SPs samples, considered to be negative by RT-PCR, were positive when specific primers designed for the TAstV-2 capsid gene were applied. This is the first description of turkey astrovirus infection presenting PEC in Latin America.