Floyd D. Wilson

Effect of green tea and its polyphenols on mouse liver.

Increased consumption of green tea (GT) without enough scientific data has raised safety concerns. Epigallocatechin 3-gallate (EGCG) is the most prominent polyphenol of GT that has antioxidant activity. However, higher doses of EGCG have been shown to cause liver injury. This study was initiated to determine the effect of GT extracts in a mouse model. We also investigated the effects of EGCG in normal and health-compromised mice. Different doses of GT fractions and EGCG were administered for 5 days to mice. Also, a single dose of lipopolysaccharide (LPS) was combined with EGCG in order to investigate its effect in the presence of fever. Plasma ALT and ALP levels were determined along with liver histopathology. Combining a single high IG dose of EGCG with a single IP dose of LPS initiated liver injury. Furthermore, repeated administration of high IG doses of EGCG showed mild liver injury, but it was augmented under febrile conditions induced by LPS. This study confirms the safety of reasonable consumption of GT over a short term. However, it highlights a caution that high doses of EGCG can lead to mild liver injury, and this may be markedly enhanced under febrile conditions.

High incidence of glomerulonephritis associated with inclusion body hepatitis in broiler chickens: routine histopathology and histomorphometric studies.

Abstract During the routine histologic evaluation of an outbreak of inclusion body hepatitis (IBH) in Mississippi broilers, a high incidence of renal enlargement and glomerulonephropathy was observed in the birds presenting classic hepatic pathology. Characteristic intranuclear adenoviral inclusion bodies were demonstrated in the livers of these birds, and fowl adenovirus was identified by viral isolation and by PCR. The glomerular lesions were consistent with proliferative or membranoproliferative forms of glomerulonephritis. Histomorphometric evaluations were performed to generate a more quantitative analysis of altered glomerular size and cellularity, to detect statistically significant borderline changes, and to get a clearer insight into the incidence of the glomerular alterations. Marked increases in both the average glomerular size (area) and the total glomerular cellularity were observed for the affected glomeruli relative to normal controls. The average glomerular area values for normal glomeruli in the peripheral subcapsular cortical and central cortical kidney regions were 1791 µm2 and 5302 µm2, respectively. In contrast, glomerular measurements for kidneys exhibiting glomerulonephritis by routine histopathology, had average values for the two regions of 4429 µm2 and 11,063 µm2. The average glomerular cell counts for the two regions in controls were 44 and 107 cells/glomeruli, while averages for birds with glomerulonephritis were 85 and 193 cells/glomeruli. The proportion of IBH-associated glomeruli greater than two standard deviations above the mean glomerular size of the normal controls was 52% for the central region and 62% for the peripheral region.

Morphometric Documentation of a High Prevalence of Left Ventricular Dilated Cardiomyopathy in Both Clinically Normal and Cyanotic Mature Commercial Broiler Breeder Roosters with Comparisons to Market-Age Broilers

SUMMARY Previous studies documented the common occurrence of transitory cyanosis and echocardiographic aortic insufficiency in mature commercial broiler breeder roosters. During further investigations, we observed a high prevalence of hearts exhibiting extensive dilation of the left ventricle chamber compatible with dilated left ventricular cardiomyopathy present in both cyanotic and normal subpopulations. We conducted quantitative studies focused on documentation of cardiac ventricle parameters by using simple gross morphometric methods performed on formalin-fixed hearts obtained from both clinically normal roosters and those exhibiting variable transitory cyanosis, echocardiographic aortic insufficiency, or both. A high prevalence of often dramatic left ventricular dilation reflected in enlarged left ventricular chamber areas and elevated left ventricle-to-total ventricle area ratios was morphometrically documented. However, no statistically significant differences in the occurrence of ventricular abnormalities were observed between normal and cyanotic roosters. Age-associated changes were also demonstrated by comparative morphometric studies on hearts from normal market-age broilers (average age of 7 wk) and those of mature roosters (average age of 42 wk). Elevation in both left and right ventricular weight-to-total heart weight ratios dramatically increased with aging. In addition, values (average ± SD) for the left ventricle chamber area-to-total ventricle area ratios increased from 3.2 ± 2.0% in broilers up to 10.0 ± 8.8% in roosters. None of the normal broilers studied demonstrated left ventricular volume ratios above 10%, whereas 33% of the roosters had left ventricular volume ratios above 10%, including 13% with ratios of 20% or higher. However, the left ventricle wall area-to-body weight ratios were much closer for the two age groups (0.85 ± 0.18 cm2/kg in broilers and 0.79 ± 0.13 cm2/kg in roosters). Also, the standard right ventricle-to-total ventricle weight ratio (RV/TV) went from 0.18 ± 0.04 in broilers to 0.25 ± 0.12 in roosters, whereas the left ventricle-to-total ventricle weight ratios were similar for the two age groups (0.74 ± 0.12 and 0.75 ± 0.08 in broilers and roosters, respectively). Our results for RV/TV in normal broilers were similar to the reported values for normal market-age broilers. In contrast, 36% of the roosters had RV/TV above values reported for broilers considered reflective of right ventricular hypertrophy due to pulmonary hypertension, whereas 4% had values above the reported threshold for broilers dying with ascites (ratios greater than 0.0249 and 0.299, respectively). However, ascites was not observed for any of the roosters. Although essentially all cardiac morphometric parameters demonstrated statistically significant correlations with the age-class group comparisons, significance could not be documented for comparisons between cardiomorphometrics and the subjective occurrences of transitory cyanosis or echocardiographic aortic insufficiency.

A Natural Outbreak of Clinical Toxoplasmosis in a Backyard Flock of Guinea Fowl in Mississippi

SUMMARY. A case of a naturally occurring infection with Toxoplasma gondii in a backyard flock of guinea fowl in north Mississippi is reported. To our knowledge, this is the first worldwide report of a natural clinical infection in a flock of guinea fowl. This case was two of seven birds lost out of approximately 20 guinea fowl present in the flock. Birds reportedly exhibited lethargy prior to death. Necropsy examinations were performed on two of the dead birds. There were no gross lesions; however, intralesional protozoan cysts suggestive of T. gondii were observed microscopically. One of two guinea fowl demonstrated dramatic microscopic pathology consisting of variable multifocal necrosis, fibrin exudation, and inflammation of spleen, lung, and heart associated with protozoa cysts and tachyzoites compatible with toxoplasmosis. The bone marrow also exhibited multifocal necrosis and fibrin exudation, as well as marked erythroid and lesser granulocytic hyperplasia with intralesional protozoan cysts. The diagnosis of toxoplasmosis was confirmed with immunohistochemistry and PCR.

OCCURRENCE OF HEPATOCELLULAR CARCINOMA IN AN ADULT MALE NILE LECHWE (KOBUS MEGACEROS)

Abstract A 14-yr-old male Nile lechwe (Kobus megaceros) from the Jackson Zoo (Mississippi, USA) had chronic weight loss that was associated with elevated serum alkaline phosphatase levels. Approximately 4 yr after the first clinical signs of weight loss, the animal fractured its left rear femur and was subsequently euthanized; on the same day necropsy was performed. The liver was grossly enlarged and contained a smooth-surfaced nodular mass that occupied the majority of the right lobe of the liver. The mass had a liver-like appearance exhibiting a tan–red coloration but having a soft consistency. A single, approximately 0.6 cm, round, slightly elevated discrete red nodule was present in the left anterior lung. Microscopically, the hepatic mass and pulmonary nodule diffusely consisted of irregular cords of cells exhibiting hepatoid features, but containing no evidence of normal portal bile duct or portal triad development. To our knowledge, this is the first report in the scientific literature of a naturally occurring case of hepatocellular carcinoma in a Nile lechwe or in any antelope species.

Comparison of two methods for determination of intestinal villus to crypt ratios and documentation of early age-associated ratio changes in broiler chickens1,2,3

&NA; The determination of intestinal villus to crypt ratios (VCR) is a common method utilized to evaluate effects of various diet regimens on gut microanatomy and for the histologic quantification of intestinal responses to disease processes. Two methods for the determination of small intestinal VCR were compared in early age chickens. A standard method for VCR determination based on 10 villus and crypt length measurements in the jejunal region of the small intestine was employed for the calculation of villus to crypt length ratio (VCLR). That method was compared to a new approach based on a single histomorphometric determination of the crypt and total mucosal areas using image analysis software. Subtraction of the crypt area from the total area provided the villus area and allowed for the subsequent calculation of villus to crypt area ratio (VCAR). At 4 and 18 h posthatch, VCLR was higher than that of VCAR, but there was no significant difference between VCLR and VCAR at 0 h (hatch) and at 168 h (d 7) posthatch. Nevertheless, the pattern of age‐associated changes for VCLR and VCAR were comparable throughout the early posthatch period. Furthermore, the new method used in determining VCAR is subject to less human error, allows for an appreciable reduction in the number of measurements required, and facilitates a larger intestinal segment evaluation. Standard linear measurements require the selection of variable numbers of villi and crypts, whereas the area method only requires selection of a single region that incorporates numerous villi and crypts of variable sizes in providing a less subjective approach. This is particularly advantageous in studies on intestinal disease conditions resulting in marked multifocal variation in villus stature. This study further documented age‐associated changes occurring in the VCR of the small intestine during the early posthatch period. Across the 2 methods used for VCR determination, a major and highly significant reduction in the VCR was observed to occur between 18 h and 168 h posthatch.

Stem cell intervention ameliorates epigallocatechin-3-gallate/ lipopolysaccharide-induced hepatotoxicity in mice

Stem cells are identified as a novel cell therapy for regenerative medicine because of their ability to differentiate into many functional cell types. We have shown earlier a new model of hepatotoxicity in mice by administering (1500 mg/kg) epigallocatechin-3-gallate (EGCG) intragastric (IG) for 5 days after a single intraperitoneal dose (6 mg/kg) of lipopolysaccharide (LPS). In this study, we aimed to study the effect of intrahepatic (IH) injection of mouse embryonic stem cells (MESCs) on the hepatotoxicity induced by EGCG/LPS in mice. Mice were administered EGCG/LPS and rested for 3 days. MESCs were obtained from American Type Culture Collection and cultured in vitro for 4 days. Stem cells were injected IH. Seven days later, a single dose of LPS (6 mg/kg) followed by daily doses of IG administration of EGCG were re-administered for 5 days. At the end of the experiment, blood samples were collected for analysis of biochemical parameters associated with liver. Results showed that the group of mice that were administered MESCs prior to EGCG/LPS showed lower levels of alanine amino transferase, alkaline phosphatase, and bilirubin, higher albumin/globulin ratio, and less remarkable histopathological lesions. Also, that group of mice showed less expression of oxidative stress biomarkers (oxidized low-density lipoprotein Ox.LDL and chemokine CXCL16), less expression of nuclear protein receptors (retinoic acid receptor and retinoid X receptor), and less expression of inflammatory biomarkers (tumor necrosis factor α and transforming growth factor β1) compared with other groups of mice that were not given MESCs. In conclusion, MESCs can ameliorate EGCG/LPS-induced hepatotoxicity in mice.

Thymoquinone Protects Against EGCG/LPS Induced Hepatotoxicity in Mice

Green tea (GT) is a widely used beverage and folk medicine. Epigallocatechin-3-gallate (EGCG), the main polyphenol of GT has been demonstrated to impact a number of biological targets. Our previous work has shown that the combination of LPS and EGCG in male mice causes hepatotoxicity, perhaps related to a prooxidant effect. Thymoquinone (TQ) is a prominent constituent of “black seed”, Nigella sativa, and is known for a number of biological activities, including potent antioxidant and anti-inflammatory activities. It has been extensively used in the Middle East as a protectant against hepatic and renal injuries. In the current study we treated mice with oral doses of TQ before and concurrently with EGCG/LPS. Thymoquinone ameliorated the rise in the plasma concentrations of ALT, ALP, TB, Amylase and the decrease of A/G ratio to normal after exposure to EGCG/LPS. It also blunted the weight loss. No histopathological lesions or mortality was observed. It can be concluded from this study that TQ showed a hepatoprotective effect in a murine model of EGCG/LPS induced hepatotoxicity. This hepatoprotective effect may be related to the antioxidant properties of TQ, but this conclusion needs further evaluation.

Comparative effects of in ovo versus subcutaneous administration of the Marek's disease vaccine and pre-placement holding time on the intestinal villus to crypt ratios of Ross 708 broilers during early post-hatch development1,2,3

ABSTRACT Villus to crypt ratio (VCR) is used to quantify the microanatomical response of the intestine to various treatments. In early age chickens, comparative effects of the in ovo (i.o.) and s.c. methods of administration (moa) of the Mareks disease (MD) vaccine on 2 types of measurement of small intestinal VCR at 0 and 4 h post‐hatch (poh) were investigated. The effects of moa and 4 and 18 h pre‐placement holding times (pht) on the VCR measurements at 168 h (7 d) poh were also investigated. In the jejunum of the small intestine, a standard method for VCR determination, based on 10 villus and crypt length measurements, was utilized for the calculation of villus to crypt length ratio (VCLR). In that same region, a single histomorphometric determination of the crypt and total mucosa areas using image analysis software was also used. Subtraction of the crypt area from the total mucosa area provided the villus area, allowing for calculation of the villus to crypt area ratio (VCAR). Across 0, 4, and 18 h of poh bird age, the VCLR of birds that received an s.c. vaccination was higher in comparison to that of those that received an i.o. vaccination. The highest and lowest VCAR values were observed in the s.c. treatment at 0 h poh and in the i.o. treatment at 4 h poh, respectively. Furthermore, at 168 h poh, VCLR values in the 18 h pht and s.c. vaccination group were higher than those in the 4 h pht and s.c. vaccination or 18 h and i.o. vaccination groups. In conclusion, the effects of pht and MD vaccine moa on VCR were dependent on the use of either the VCLR or VCAR method of measurement. However, regardless of method, s.c. injection overall led to a higher VCR through 4 h poh in Ross 708 broilers, and the effects of moa on VCLR at 168 h were influenced by pht.

Testicular histomorphometrics including Sertoli cell quantitation for evaluating hatchability and fertility issues in commercial breeder-broiler roosters

&NA; Testicular histomorphometric methods were developed for the investigation of fertility and hatchability problems in commercial broiler‐breeder roosters. Sertoli cell (SC) evaluations were emphasized in view of their role in influencing embryonic testicular organogenesis and spermatogenesis in adults. H&E stained sections prepared from formalized tissues were examined. SC evaluations were mostly made from computer generated images using image analysis software, but were also compared to direct counting with ocular grid reticle. Other measurements were germinal epithelial and total seminiferous tubule widths, quantification of individual spermatozoa and sperm clusters, and determinations of stromal area. Age‐associated changes in SC occurred between 15 to 54 wk of age. A significant reduction in SC concentration occurred at 29 wk of age (P < 0.001) that corresponded with the histologic initiation of spermatogenesis. Expanded morphometric evaluations were performed on 59‐week‐old roosters comparing a flock having good body condition and footpad scores with normal testicular and body weights (optimal profile or OP group) to a second flock demonstrating relatively poor expression of these parameters and decreased fertility (sub‐optimal profile or SOP group). Significant reductions (P < 0.05) in SC concentration, spermatozoa, and tubular widths, but an increased percentage of stroma area occurred in SOP compared to OP flocks. SC results also correlated well with testes weight. A unique finding was a significant relationship between decreasing SC concentration and increasing severity scores for footpad lesions. Differences in all histomorphometric parameters occurred between flocks demonstrating good hatchability compared to flocks exhibiting either low or decreasing hatchability. Significant reductions in SC and other morphometric parameters were observed for the poor relative to good hatchability group. A strong positive association was observed for SC results using computer generated images and direct reticle ocular counting (R = 0.80). Replicate counts on control sections demonstrated generally good reproducibility of methods between experiments. The findings are supportive of the value of testicular histomorphometrics for evaluations of hatchability issues in commercial roosters.