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.

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.

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.

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.

Role of chicken melanoma differentiation-associated gene 5 in induction and activation of innate and adaptive immune responses to infectious bursal disease virus in cultured macrophages.

The objective of the present study was to determine if chicken melanoma-differentiation-associated gene 5 (MDA5) senses infectious bursal disease virus infection to induce innate immunity that bridges to adaptive immunity. During IBDV infection in HD11 cells, IBDV titers and RNA loads increased up to 3.4 × 10(7) plaque-forming units (PFU)/mL and 1114 ng/µL, respectively, at 24 hours postinfection (hpi). IBDV infection in HD11 cells induced significantly upregulated (p < 0.05) expression levels of chicken MDA5 (59-fold), interferon-ß (IFN-ß) (693-fold), dsRNA-dependent protein kinase (PKR) (4-fold), 2', 5'-oligoadenylate synthetase (OAS) (286-fold), myxovirus resistance gene (Mx) (22-fold), interleukin-1ß (IL-1ß) (5-fold), IL-6 (146-fold), IL-8 (4-fold), IL-10 (4-fold), inducible nitric oxide synthase (iNOS) (15-fold), and major histocompatibility complex class I (MHC class I) (4-fold). Nitric oxide production in the culture supernatants increased significantly (p < 0.05) up to 6.5 µM at 24 hpi. The expressed chMDA5 and IBDV-derived dsRNA were localized in the cytoplasm of HD11 cells during IBDV infection. ChMDA5-knockdown HD11 cells had significantly higher (p < 0.05) IBDV RNA loads at 24 hpi and significantly lower (p < 0.05) nitric oxide production and expression levels of chicken MDA5, IFN-ß, PKR, OAS, Mx, IL-1ß, IL-6, IL-8, IL-12(p40), IL-18, IL-10, iNOS, MHC class I and CD86 at 24 hpi. In addition, chMDA5 overexpression in HD11 cells resulted in significantly reduced (p < 0.05) IBDV titers and RNA loads and significantly increased (p < 0.05) nitric oxide production at 16 and 24 hpi. It also resulted in significantly higher (p < 0.05) expression levels of chicken MDA5, IFN-ß, PKR, OAS, Mx, IL-1ß, IL-6, IL-8, IL-12(p40), IL-10 and iNOS at 2 hpi. In conclusion, the results indicate that chMDA5 senses IBDV infection in chicken macrophages, and this is associated with IBDV-induced expression of IFN-ß and initiation of an innate immune response that in turn activates the adaptive immune response and limits IBDV replication.

Genotyping of turkey coronavirus field isolates from various geographic locations in the Unites States based on the spike gene.

Turkey flocks have experienced turkey coronaviral enteritis sporadically in the United States since the 1990s. Twenty-four field isolates of turkey coronavirus (TCoV) from multiple states in the United States were recovered from 1994 to 2010 to determine the genetic relationships among them. The entire spike (S) gene of each TCoV isolate was amplified and sequenced. Pairwise comparisons were performed using the Clustal W program, revealing 90.0% to 98.4% sequence identity in the full-length S protein, 77.6% to 96.6% in the amino terminus of the S1 subunit (containing one hypervariable region in S1a), and 92.1% to 99.3% in the S2 subunit at the deduced amino acid sequence level. The conserved motifs, including two cleavage recognition sequences of the S protein, two heptad repeats, the transmembrane domain, and the Golgi retention signal were identified in all TCoV isolates. Phylogenetic analysis based on the full-length S gene was used to distinguish North American TCoV isolates from French TCoV isolates. Among the North American TCoV isolates, three distinct genetic groups with 100% bootstrap support were observed. North Carolina isolates formed group I, Texas isolates formed group II, and Minnesota isolates formed Group III. The S genes of 24 TCoV isolates from the United States remained conserved because they contained predominantly synonymous substitutions. The findings of the present study suggest endemic circulation of distinct TCoV genotypes in different geographic locations.

Recombinant nucleocapsid protein-based enzyme-linked immunosorbent assay for detection of antibody to turkey coronavirus.

Nucleocapsid (N) protein gene of turkey coronavirus (TCoV) was expressed in a prokaryotic system and used to develop an enzyme-linked immunosorbent assay (ELISA) for detection of antibody to TCoV. Anti-TCoV hyperimmune turkey serum and normal turkey serum were used as positive or negative controls for optimization of the ELISA. Goat anti-turkey IgG (H+L) conjugated with horseradish peroxidase was used as detector antibody. Three hundred and twenty two turkey sera from the field were used to evaluate the performance of ELISA and determine the cut-off point of ELISA. The established ELISA was also examined with serum samples obtained from turkeys experimentally infected with TCoV. Those serum samples were collected at various time intervals from 1 to 63 days post-infection. The optimum conditions for differentiation between anti-TCoV hyperimmune serum and normal turkey serum were recombinant TCoV N protein concentration at 20 µg/ml, serum dilution at 1:800, and conjugate dilution at 1:10,000. Of the 322 sera from the field, 101 were positive for TCoV by immunofluorescent antibody assay (IFA). The sensitivity and specificity of the ELISA relative to IFA test were 86.0% and 96.8%, respectively, using the optimum cut-off point of 0.2 as determined by logistic regression method. Reactivity of anti-rotavirus, anti-reovirus, anti-adenovirus, or anti-enterovirus antibodies with the recombinant N protein coated on the ELISA plates was not detected. These results indicated that the established antibody-capture ELISA in conjunction with recombinant TCoV N protein as the coating protein can be utilized for detection of antibodies to TCoV in turkey flocks.

Contrasting clinical outcomes in two cohorts of cats naturally infected with feline immunodeficiency virus (FIV).

Despite over 25 years of feline immunodeficiency virus (FIV) research, relatively little is known about the longitudinal course of FIV infection following natural infection. In contrast to published reports of experimental infections using lethal strains of the virus, clinical signs of naturally acquired FIV infection can be mild or inapparent, rather than life-threatening. In this prospective, longitudinal controlled study, based in Chicago, IL (n=17) and Memphis, TN (n=27), we investigated two cohorts of privately owned, naturally infected cats kept under different housing conditions. Cats in the Chicago cohort (Group 1) were kept in households of ≤2 cats, while the Memphis cohort (Group 2) comprised part of a large multi-cat household of over 60 cats kept indoors only, with unrestricted access to one another. The majority of cats from Group 1 did not display clinical signs consistent with immunodeficiency during the 22-month observation period. In contrast, the outcome of infection in Group 2 was dramatically different; 17/27 (63%) of cats lost a median of 51.3% of their bodyweight (P<0.0005) and died during the study period, with lymphoma being the most common cause of mortality. Although the decrease in CD4+ T cell count between enrolment and terminal disease was significant (P=0.0017), the CD4:CD8 ratio at the time of enrolment did not reliably distinguish FIV-positive cats classified as 'healthy' and 'not healthy' at either cohort. FIV load at enrolment was significantly lower in Group 1 than in Group 2 (P<0.0001), but there were no significant differences at enrolment between healthy and not healthy cats at either group. In conclusion, the results of this study suggest that management and housing conditions impact on disease progression and survival times of FIV-positive cats.

Bursal transcriptome of chickens protected by DNA vaccination versus those challenged with infectious bursal disease virus.

Infectious bursal disease virus (IBDV) infection destroys the bursa of Fabricius, causing immunosuppression and rendering chickens susceptible to secondary bacterial or viral infections. IBDV large-segment-protein-expressing DNA has been shown to confer complete protection of chickens from infectious bursal disease (IBD). The purpose of the present study was to compare DNA-vaccinated chickens and unvaccinated chickens upon IBDV challenge by transcriptomic analysis of bursa regarding innate immunity, inflammation, immune cell regulation, apoptosis and glucose transport. One-day-old specific-pathogen-free chickens were vaccinated intramuscularly three times at weekly intervals with IBDV large-segment-protein-expressing DNA. Chickens were challenged orally with 8.2 × 10(2) times the egg infective dose (EID)50 of IBDV strain variant E (VE) one week after the last vaccination. Bursae collected at 0.5, 1, 3, 5, 7, and 10 days post-challenge (dpc) were subjected to real-time RT-PCR quantification of bursal transcripts related to innate immunity, inflammation, immune cell regulation, apoptosis and glucose transport. The expression levels of granzyme K and CD8 in DNA-vaccinated chickens were significantly (p < 0.05) higher than those in unvaccinated chickens upon IBDV challenge at 0.5 or 1 dpc. The expression levels of other genes involved in innate immunity, inflammation, immune cell regulation, apoptosis and glucose transport were not upregulated or downregulated in DNA-vaccinated chickens during IBDV challenge. Bursal transcripts related to innate immunity and inflammation, including TLR3, MDA5, IFN-α, IFN-ß, IRF-1, IRF-10, IL-1ß, IL-6, IL-8, iNOS, granzyme A, granzyme K and IL-10, were upregulated or significantly (p < 0.05) upregulated at 3 dpc and later in unvaccinated chickens challenged with IBDV. The expression levels of genes related to immune cell regulation, apoptosis and glucose transport, including CD4, CD8, IL-2, IFN-γ, IL-12(p40), IL-18, GM-CSF, GATA-3, p53, glucose transporter-2 and glucose transporter-3, were upregulated or significantly (p < 0.05) upregulated at 3 dpc and later in unvaccinated chickens challenged with IBDV. Taken together, the results indicate that the bursal transcriptome involved in innate immunity, inflammation, immune cell regulation, apoptosis and glucose transport, except for granzyme K and CD8, was not differentially expressed in DNA-vaccinated chickens protected from IBDV challenge.

Eliciting specific humoral immunity from a plasmid DNA encoding infectious bursal disease virus polyprotein gene fused with avian influenza virus hemagglutinin gene.

DNA vaccine coding for infectious bursal disease virus (IBDV) polyprotein gene and that for avian influenza virus (AIV) hemagglutinin (HA) gene have been shown to induce immunity and provide protection against the respective disease. The present study was carried out to determine whether an IBDV polyprotein gene-based DNA fused with AIV HA gene could trigger immune response to both IBDV and AIV. After transfection, VP2 and HA were detected in the cytoplasm and at cell membrane, respectively, by immunofluorescent antibody double staining method, suggesting the fusion strategy did not affect the location of protein expression. VP4 cleavage between VP2 and HA was confirmed by Western blot, indicating the fusion strategy did not affect VP4 function in transfected cells. After vaccination in chickens, the DNA construct VP24-HA/pcDNA induced ELISA and virus neutralizing antibodies against VP2 and hemagglutination inhibition antibody against the HA subtype. The results indicated that a single plasmid construct carrying IBDV VP243 gene-based DNA fused with AIV HA gene can elicit specific antibody responses to both IBDV and AIV by DNA vaccination.

Overexpression of type VI collagen in neoplastic lung tissues.

Type VI collagen (COL6), an extracellular matrix protein, is important in maintaining the integrity of lung tissue. An increase in COL6 mRNA and protein deposition was found in the lungs of patients with pulmonary fibrosis, a chronic inflammatory condition with a strong association with lung cancer. In the present study, we demonstrated overexpression of COL6 in the lungs of non-small cell lung cancers. We hypothesized that excessive COL6 in the lung interstitium may exert stimulatory effects on the adjacent cells. In vitro stimulation of monocytes with COL6 resulted in the production of IL-23, which may promote tumor development in an environment of IL-23-mediated lung inflammation, where tissue modeling occurs concurrently with excessive COL6 production. In addition, COL6 was capable of stimulating signaling pathways that activate focal adhesion kinase and extracellular signal­regulated kinase 1/2 in lung epithelial cells, which may also facilitate the development of lung neoplasms. Taken together, our data suggest the potential role of COL6 in promoting lung neoplasia in diseased lungs where COL6 is overexpressed.

Neglected leptospirosis in raccoons (Procyon lotor) in Indiana, USA.

BACKGROUND: Leptospirosis is a globally important zoonotic disease occurring clinically and subclinically in humans and animals. OBJECTIVES: To determine whether raccoons in Indiana carried leptospires in their kidneys. ANIMALS AND METHODS: Thirty-four raccoons were live-trapped from two forest patches in central Indiana. Following euthanasia, a portion of kidney (2 cm(2)) from each raccoon was homogenized and used for leptospiral culture. Leptospiral cultures were subjected to DNA extraction and various polymerase chain reaction (PCR) procedures reported previously. Serum sample from each raccoon was collected and antibody titers to leptospiral serovars were determined by microscopic agglutination test (MAT). RESULTS: All leptospiral cultures were positive for Leptospira by various PCR procedures. The PCR with the primers targeting the conservative region of LipL32 gene was the most sensitive PCR in the detection of pathogenic leptospires. The variable LipL32 PCR amplicons were sequenced and compared to the reference strains available in GenBank. Twelve kidney cultures had Leptospira interrogans, eight had Leptospira kirschneri and two had Leptospira borgpetersenii. They were predominantly Grippotyphosa serogroup. Antileptospire antibodies were detected in 16 out of 34 raccoons (47.1%) by MAT. There were titers ≥ 1:80 in 16 raccoons (47.1%) and titers ≥ 1:400 in 3 raccoons (8.8%). The highest leptospiral serovar-specific seroreactivity among 34 raccoons was L. interrogans Bratislava (38.2%) and L. interrogans Grippotyphosa (32.4%). CONCLUSIONS: Raccoons in Indiana carry leptospiral organisms in kidneys and the leptospires are predominantly L. interrogans species and of the Grippotyphosa serogroup. CLINICAL IMPORTANCE: The raccoons serve as reservoir hosts that exposure sources to wildlife, livestock, pets and humans.

An influenza A virus hemagglutinin (HA) epitope inserted in and expressed from several loci of the infectious bursal disease virus genome induces HA-specific antibodies.

The N-terminus of infectious bursal disease virus (IBDV) VP5 has been shown to be capable of tolerating the insertion of small epitopes. The objective of the present study was to determine if IBDV genomic sites, including the 5' end of vp5, could carry an influenza A virus hemagglutinin (HA) epitope. HA-expressing IBDVs were generated when the HA epitope was fused to the N-terminus of VP5 (HA5-IBDV) or VP4 (HA4-IBDV) or the C-terminus of VP1 (1HA-IBDV). Viral titers obtained after co-transfection with cDNA from the ha-containing segment and the complementary genomic segment were 1.3 × 10(4), 3.7 × 10(3) and 3.8 × 10(4) pfu/ml for HA5-IBDV, HA4-IBDV and 1HA-IBDV, respectively. The HA tag expression remained stable after 10 passages when the tag gene was inserted into the vp4 and vp1 genes. HA-IBDVs did not cause pathogenicity in specific-pathogen-free (SPF) chickens. However, only HA4-IBDV and 1HA-IBDV induced HA-specific antibodies, which were measured by ELISA with a maximum optical density (OD) value of 0.701 and 0.769, respectively, at 24 days after infection. Thus, IBDV can potentially be employed as a bivalent viral vector when the epitope is fused with VP4 or VP1.