Pathology in Practice.

In collaboration with the American College of Veterinary Pathologists.

Pathology in Practice.

In collaboration with the American College of Veterinary Pathologists.

Prenatal Serotonin Fluctuation Affects Serotoninergic Development and Related Neural Circuits in Chicken Embryos.

The placenta is the primary source of serotonin (5-HT) for fetal development, programming fetal neural wiring in humans and other mammals. The fluctuation in maternal 5-HT affects fetal neurogenesis with life-long consequences, however, its mechanisms have not been well known. The chicken embryo, independent of maternal neurohormonal influence, may offer an ideal model for studying the mechanisms of prenatal 5-HT exposure altering postnatal physiological homeostasis and behavioral exhibition. To investigate the fine-tuning of 5-HT to the early embryonic neurodevelopment, 10 µg and 20 µg 5-HT were secretively injected to chicken embryos before incubation. 5-HT exposure mainly affected the neural development in the pons and midbrain, altered the serotoninergic and dopaminergic (DAergic) neuronal morphology, nucleus distribution, and their metabolisms and related gene expressions. The comprehensive effect of 5-HT exposure was not dosage-dependent but the working pathways differed, 10 µg 5-HT exposure reduced 5-HT turnover rate, increased 5-HT 1a receptor expression, and facilitated the ventral tegmental area neuronal development; while 20 µg 5-HT exposure increased the serotoninergic and DAergic neurotransmission and enhanced serotoninergic regulation to the hypothalamus. These findings indicate that the 5-HT exposure effect can be achieved via different paths by modifying the embryonic serotonergic (5-HTergic) and DAergic systems and altering fetal 5-HTergic influence on the thalamocortical circuit and hypothalamic-pituitary-adrenal axis. These results may offer a novel sight for understanding the function of 5-HT during neurodevelopment and raise the possibility for using selective 5-HT reuptake inhibitors to regulate emotional and mental wellness during early pregnancy and possible risks of complications for babies.

The development of the serotonergic and dopaminergic systems during chicken mid-late embryogenesis.

Serotonin (5-HT) acts as a morphogen influencing embryonic brain development, and as a neurotransmitter regulating multiple biological functions with lifelong effects on animal physical, physiological and mental health, especially during the rapid growth phase prior to birth when embryos face many challenges to reach structural and functional completion. In this study, the development of the serotoninergic (5-HTergic) system and its modulatory effect on the dopaminergic (DAergic) system and related neural circuits were investigated during the mid-late embryogenesis, embryonic day (E)12-E20, in the chicken's brain. During 5-HTergic neuronal maturation, a growth-related anatomical and functional remodeling was highlighted: the 5-HT neurons continuously grew during E12-E20 except for a remarkable regression during E14-E16. Correspondingly, there was a time-dependent change in the 5-HT synthetic capacity. Specifically, 5-HT concentrations in the raphe nuclei increased from E12 to E14, reaching a first plateau during E14-E16, then continuously increased up to E19, and reaching a second plateau between E19-E20. The second plateau of the 5-HT concentration was in correspondence with the establishment of the 5-HTergic autoregulatory loop during E19-E20 and the development of the DAergic system. The DA concentrations remained unchanged from E12 to E16, then started to increase at E16, reaching a maximum at E19, and diminished before hatching. The unique developing time sequence between the 5-HTergic and DAergic systems suggests that the 5-HTergic system may play a critical role in forming the 5-HT - DA neural circuit during chicken embryogenesis. These results provide new insights for understanding the functional organization of the 5-HTergic system during embryonic development and raise the possibility that prenatally modulating the 5-HTergic system may lead to long-lasting brain structural and functional alterations.

A free VP3 C-terminus is essential for the replication of infectious bursal disease virus.

Green fluorescent protein (GFP) has been successfully incorporated into the viral-like particles of infectious bursal disease virus (IBDV) with a linker at the C-terminus of VP3 in a baculovirus system. However, when the same locus in segment A was used to express GFP by a reverse genetic (RG) system, no viable GFP-expressing IBDV was recovered. To elucidate the underlying mechanism, cDNA construct of segment A with only the linker sequence (9 amino acids) was applied to generate RG IBDV virus (rIBDV). Similarly, no rIBDV was recovered. Moreover, when the incubation after transfection was extended, wildtype rIBDV without the linker was recovered suggesting a free C-terminus of VP3 might be necessary for IBDV replication. On the other hand, rIBDV could be recovered when additional sequence (up to 40 nucleotides) were inserted at the 3' noncoding region (NCR) adjacent to the stop codon of VP3, suggesting that the burden of the linker sequence was not in the stretched genome size but the disruption of the VP3 function. Finally, when the stop codon of VP3 was deleted in segment A to extend the translation into the 3' NCR without introducing additional genomic sequence, no rIBDV was recovered. Our data suggest that a free VP3 C-terminus is essential for IBDV replication.

Infectious bursal disease virus as a replication-incompetent viral vector expressing green fluorescent protein.

Infectious bursal disease virus (IBDV) has been established as a replication-competent viral vector capable of carrying an epitope at multiple loci in the genome. To enhance the safety and increase the insertion capacity of IBDV as a vector, a replication-incompetent IBDV vector was developed in the present study. The feasibility of replacing one of the viral gene loci, including pvp2, vp3, vp1, or the polyprotein vp243, with the sequence of green fluorescent protein (GFP) was explored. A method combining TCID50 and immunoperoxidase monolayer assay (IPMA) determined the most feasible locus for gene replacement to be pvp2. The genomic segment containing gfp at the pvp2 locus was able to be encapsidated into IBDV particles. Furthermore, the expression of GFP in GFP-IBDV infected cells was confirmed by Western blotting and GFP-IBDV particles showed similar morphology and size to that of wildtype IBDV by electron microscopy. By providing the deleted protein in trans in a packaging cell line (pVP2-DF1), replication-incompetent GFP-IBDV particles were successfully plaque-quantified. The gfp sequence from the plaque-forming GFP-IBDV in pVP2-DF1 was confirmed by RT-PCR and sequencing. To our knowledge, GFP-IBDV developed in the present study is the first replication-incompetent IBDV vector which expresses a foreign protein in infected cells without the capability to produce viral progeny. Additionally, such replication-incompetent IBDV vectors could serve as bivalent vaccine vectors for conferring protection against infections with IBDV and other economically important, or zoonotic, avian pathogens.

Necrotizing and haemorrhagic hepatitis and enteritis in commercial layer pullets.

This case report describes an episode of recurring severe necrotizing and haemorrhagic hepatitis and enteritis experienced in a flock of commercial layer pullets at 12 weeks of age and again at 18 weeks of age in Indiana. Pullets had been vaccinated at 10 weeks old using a trivalent Salmonella Enteritidis (SE)/Newcastle disease/infectious bronchitis oil-emulsion-inactivated vaccine. The pullets were found dead at 12 weeks with firm but friable, enlarged, haemorrhagic livers, enlarged spleens, and necrohaemorrhagic intestines. Histopathologic findings were consistent with a necrotizing and haemorrhagic enteritis and hepatitis. Livers had multiple intra-sinusoidal thrombi, intestines contained Gram-positive bacterial colonies, and spleens had marked lymphoid depletion. The pullets seemed to improve after antibiotic treatment. Pullets were vaccinated with an inactivated SE vaccine at 14 weeks of age. A second spike of mortality occurred at 18 weeks of age. Although clostridial enteritis and hepatitis were highly suspected in the two cases based on macroscopic and microscopic findings, no significant bacterial or viral agents were isolated from the livers and intestines. In summary, lesions in the liver and intestines are speculated to be due to repetitive vaccination, leading to an anamnestic response by the immune system, and resulting in an immune-mediated response. However, much of the pathogenesis is still unclear, and other causes such as unidentified infectious aetiology, transmissible amyloidosis, and hypersensitivity may need further investigation.

An embryonic atrazine exposure results in reproductive dysfunction in adult zebrafish and morphological alterations in their offspring.

The herbicide atrazine, a suspected endocrine disrupting chemical (EDC), frequently contaminates potable water supplies. Studies suggest alterations in the neuroendocrine system along the hypothalamus-pituitary-gonadal axis; however, most studies address either developmental, pubertal, or adulthood exposures, with few investigations regarding a developmental origins hypothesis. In this study, zebrafish were exposed to 0, 0.3, 3, or 30 parts per billion (ppb) atrazine through embryogenesis and then allowed to mature with no additional chemical exposure. Reproductive function, histopathology, hormone levels, offspring morphology, and the ovarian transcriptome were assessed. Embryonic atrazine exposure resulted in a significant increase in progesterone levels in the 3 and 30 ppb groups. A significant decrease in spawning and a significant increase in follicular atresia in the 30 ppb group were observed. In offspring, a decrease in the head length to body ratio in the 30 ppb group, along with a significant increase in head width to body ratio in the 0.3 and 3 ppb groups occurred. Transcriptomic alterations involved genes associated with endocrine system development and function, tissue development, and behavior. This study provides evidence to support atrazine as an EDC causing reproductive dysfunction and molecular alterations in adults exposed only during embryogenesis and morphological alterations in their offspring.

IBDV particles packaged with only segment A dsRNA.

Multi-segmented dsRNA viruses have been suggested to utilize cis-acting elements in the plus-strand RNA to accomplish genomic RNA assortment during viral packaging. It is not clear if bi-segmented dsRNA birnavirus uses the same strategy. By applying a reverse genetic technique, we generated IBDV particles packaged with only segment A by co-transfection DF-1 cells of cDNA from segment A and VP1 (without 5' and 3' noncoding region of segment B) supporting random assortment mechanism and indicating the packaging elements of segment B include sequences in the 5' and 3' NCR. However, gfp-containing IBDV could not be generated in the presence of gfp cDNA constructs flanked by 5' and 3' NCR from segment A or segment B. The data suggest additional packaging signals are required for IBDV genomic packaging. The presence of VP1 protein in the IBDV-A particles also suggests the formation of ribonucleoprotein (RNP) complexes might be involved in the assembly of viral particles.