Flow cytometric characterization of Peyer's patch and cecal tonsil T lymphocytes in laying hens following challenge with Salmonella enterica serovar Enteritidis

Two trials were conducted to determine T cell changes in Peyer's patches (PP) and cecal tonsils (CT) of specific-pathogen-free Single-Comb White Leghorn hens challenged with Salmonella enterica serovar Enteritidis (SE). Each week, crop lavage samples were obtained from 4 or 3 hens in Trials 1 and 2, respectively. These birds were then sacrificed and their intestinal tracts excised. The crop sample and contents of one cecum from each hen were cultured for the presence of SE. Cells were purified from proximal and distal PP along with both CT and then aliquots of cells were incubated with antibodies to CD4, CD8, and the three T cell receptors (TCR). The T subsets were identified via flow cytometric analysis. Crop and cecal samples were 100% culture positive for SE at week 1 post challenge and a percentage of samples remained positive throughout the study. Some differences in TCR subsets between or within tissues were observed at various times relative to SE challenge but over-all the subsets remained similar during the study. The predominant TCR was TCR2 (vβ1) followed by TCR3 (vβ2). Low numbers of TCR1 (γδ) cells were observed. CD4/CD8 ratios increased in the PP and CT tissues by week 1 post challenge and the ratio elevation persisted throughout the experiment. These results indicate that T cell populations are comparable between PP and CT and enteric SE infection can affect the cellular dynamics of these lymphoid tissues.     

Temporal Relationships and Repeatability of Follicle Diameters and Hormone Concentrations within Individuals in Mares

Data were collected daily from 23 mares during two consecutive interovulatory intervals (IOIs). Several significant (p < 0.05) new observations on temporal relationships were made. The FSH increase that begins before ovulation temporarily plateaued on the day of discharge of follicular fluid into the peritoneal cavity in association with ovulation. During the declining portion of the pre-ovulatory oestradiol surge, an abrupt reduction in the rate of decrease occurred in synchrony with the peak of the LH surge and is consistent with a negative effect of LH on oestradiol. Repeatability within mares was based on the following positive and significant correlations between the two IOIs: (i) length of the interval between ovulations and between ovulation and the beginning of follicle deviation; (ii) diameter of the pre-ovulatory follicle on days -3 to -1; (iii) number of follicles in diameter classes of 2–5 mm (correlation for 22/23 days of the IOI), 5.1–10 mm (18/23 days), 10.1–15 mm (12/23 days) and 15.1–20 mm (12/23 days) and (iv) concentrations of FSH (18/23 days) and LH (22/23 days). The greatest repeatability for the follicle-diameter classes occurred in the 2–5 mm class, and thereafter the repeatability progressively decreased as the diameters for the classes increased. Results demonstrated measurable repeatability within mares for several end points between consecutive IOIs.     

Development of an indirect ELISA for detection of antibody to wobbly possum disease virus in archival sera of Australian brushtail possums (Trichosurus vulpecula) in New Zealand

AIMS: To develop an indirect ELISA based on recombinant nucleocapsid (rN) protein of wobbly possum disease (WPD) virus for investigation of the presence of WPD virus in Australian brushtail possums (Trichosurus vulpecula) in New Zealand.

METHODS: Pre- and post-infection sera (n=15 and 16, respectively) obtained from a previous experimental challenge study were used for ELISA development. Sera were characterised as positive or negative for antibody to WPD virus based on western-blot using WPD virus rN protein as antigen. An additional 215 archival serum samples, collected between 2000–2016 from five different regions of New Zealand, were also tested using the ELISA. Bayesian modelling of corrected optical density at 450?nm (OD₄₅₀) results from the ELISA was used to obtain estimates of receiver operating characteristic (ROC) curves to establish cut-off values for the ELISA, and to estimate the prevalence of antibody to WPD virus.

RESULTS: Western blot analysis showed 5/14 (36%) pre-infection sera and 11/11 (100%) post-infection sera from experimentally infected possums were positive for antibodies to WPD virus. Bayesian estimates of the ROC curves established cut-off values of OD₄₅₀≥0.41 for samples positive, and OD₄₅₀<0.28 for samples negative for antibody to WPD virus, for sera diluted 1:100 for the ELISA. Based on the model, the estimated proportion of samples with antibodies to WPD virus was 0.30 (95% probability interval=0.196–0.418). Of the 230 archival serum samples tested using the ELISA, 48 (20.9%) were positive for antibody to WPD virus, 155 (67.4%) were negative and 27 (11.7%) equivocal, using the established cut-off values. The proportion of samples positive for WPD virus antibody differed between geographical regions (p<0.001).

CONCLUSION: The results suggested that WPD virus or a related virus has circulated among possums in New Zealand with differences in the proportion of antibody-positive samples from different geographical regions. Antibodies to WPD virus did not seem to protect possums from disease following experimental infection, as one third of possums from the previous challenge study showed evidence of pre-existing antibody at the time of challenge. These results provide further support for existence of different pathotypes of WPD virus, but the exact determinants of protection against WPD and epidemiology of infection in various regions of New Zealand remain to be established.

CLINICAL RELEVANCE: Availability of the indirect ELISA for detection of WPD virus antibody will facilitate prospective epidemiological investigation of WPD virus circulation in wild possum populations in New Zealand.     

COMPARISON OF THE ACCURACY OF POSITIONING DEVICES FOR RADIATION THERAPY OF CANINE AND FELINE HEAD TUMORS

The purpose of this retrospective study was to evaluate the repositioning accuracy of different positioning devices in order to determine their applicability for potential use in conformal radiation therapy for animals. Forty-four animals with spontaneous tumors of the head were included. The animals were divided into 3 groups determined according to the positioning device used. Group 1 animals were positioned using a thermoplastic mask. Group 2 animals were positioned using a head holder. Group 3 animals were positioned using the head holder and an inflatable pillow. The time of presentation determined which position device was used. Port films of the 44 patients were reviewed retrospectively, and the repositioning precision was recorded by measurements in three orthogonal planes. Groups 2 and 3 had significantly better repositioning accuracy (P < or = 0.05) compared to Group 1. The position variation was not significantly different (P < or = 0.05) between Groups 2 and 3 in the lateral and longitudinal direction. Group 3 had a median reposition variation of 0.5 to 1.0 mm, with a standard deviation of 1.0 to 1.5 mm.     

Age-related differences in collagen crimp patterns in the superficial digital flexor tendon core region of untrained horses

Objective To measure collagen fibril crimp angles and lengths as well as collagen fibril mass-average diameters in central and peripheral regions of the superficial digital flexor tendon of wild horses, to ascertain any age-related changes in either region in the absence of imposed galloping exercise.
Design Measurements from a random cull of wild horses.
Sample population Superficial digital flexor tendon samples were taken from 23 wild horses ranging in age from two to ten years.
Procedure Horses were divided into 'young' (< 5 years, n = 10), 'middle-aged' (5 to < 10 years, n = 9) and 'old' groups (10+ years, n = 4) and the mean crimp angle, mean crimp length and collagen fibril mass-average diameter calculated for the central region, and for the peripheral region of each group. Differences between groups and regions were analysed using two-tailed t tests.
Results The crimp angle for the central region was found to decrease with age, so that in old horses it was smaller than that for the tendon periphery (P < 0.05). The crimp angle for the latter region did not alter significantly with age. Crimp period lengths and collagen fibril mass-average diameters did not show significant changes with age in either region.
Conclusion Reduction of the crimp angle in the core of the superficial digital flexor tendon occurs normally with age, as tendons of older animals would have undergone a higher number of loading cycles. It is possible that athletic training increases the frequency and/or the magnitude of high loading cycles experienced by the tendon, and may accelerate and worsen the normal load-related ageing process in the superficial digital flexor tendons of young performance horses, particularly in the central regions where lesions usually occur.     

Pathophysiology of humeral fractures in a sample of dairy heifers

AIMS: To investigate the pathophysiology of humeral fractures in first-lactation dairy heifers in the North Island of New Zealand.

METHODS: Ten 2-year-old dairy heifers with humeral fractures were subject to euthanasia and the fractured and non-fractured contralateral humeri were collected. Humeri were also collected from 10 unaffected 2-year-old dairy heifers sent for slaughter. Humeri from heifers with and without fractures were examined using computed tomography (CT), and four slices of the diaphysis and lower metaphysis (D1–4) were analysed using the Bone J plug-in for Image J. The humeri were sectioned sagittally and 5?mm bone slabs were processed for histopathology.

RESULTS: There were no differences in bone length between the humeri from heifers with or without fractures (p=0.31). Median cortical bone mineral density at D1 was increased in humeri from affected compared with unaffected heifers (810 vs. 783?mg/cm³; p=0.03), cortical area at D1 was reduced (816 vs. 1,037?mm²; p=0.04), the median stress strain index, a calculated theoretical measure of bone strength, at D1 was decreased (7,288 vs. 9,072?mm³; p<0.01), and the median ratio of overall bone volume (BV) to total volume (TV) was decreased (0.32 vs. 0.38; p<0.01). The median periosteal circumference at D1 was also reduced in humeri from affected compared with unaffected heifers (151 vs. 173?mm; p<0.01). Using a binary logistic regression model, BV/TV was the only variable associated with humeral fractures (p=0.03).In nine of 10 fractured humeri the fracture appeared to have started just distal to the head of the humerus and spiralled distally down the diaphysis to end just above the humeral condyles. Histopathological findings included a reduction in the number, and thickness, of trabeculae in the metaphysis; metaphyseal growth arrest lines, and osteoclastic resorption in fractured humeri. Concentrations of copper in serum from four of five animals with fractures were within, and one was below, normal reference ranges, while concentration of copper in the livers of three heifers with fractures were below adequate ranges.

CONCLUSIONS: The CT and histological findings were consistent with a diagnosis of osteoporosis. We propose that humeral fractures in dairy heifers are associated with osteoporosis, possibly as a result of insufficient deposition of bone during growth because of protein-calorie malnutrition. Increased osteoclastic resorption of bone associated with calcium mobilisation for lactation, and periods of copper deficiency may contribute to bone weakening.     

Crop immune response post-Salmonella enteritidis challenge in eight commercial egg-layer strains and specific-pathogen-free White Leghorn chickens

The crop immune response against Salmonella Enteritidis (SE) challenge in eight commercial egg-layer strains (five white-egg layer and three brown-egg layer) and specific-pathogen-free (SPF) White Leghorn (WL) hens was investigated. Pre- and post-SE challenge mucosal immune responses within the crops were evaluated. Commercial layers and SPF WL hens were orally challenged with 10(8) CFU/ml SE PT13a and SE nalR PT13, respectively. Crop lavage samples were collected at weekly intervals from day 0 (pre-challenge) to day 25-27 postinfection (PI), and bacteriological examination was performed to monitor progression of SE infection. Crop lavage samples were analyzed for SE-lipopolysaccharide (LPS)-specific IgA using enzyme-linked immunosorbent assay (ELISA). H&E-stained slides of crop sections from day 34 PI and uninfected controls were assessed for lymphoid tissue via light microscopy. Lymphoid areas were graded based on morphology, size, and cellularity using a score 0 to 5 scale. The 0 to 5 (low to high) numerical values represented progressive increases in size and cellular density of lymphoid tissue. Bacterial culture results showed the highest percentage of SE-positive crop lavage samples from all hen groups at day 5-6 PI and day 11-12 PI. A progressive decline in percentage of SE-positive crop lavage samples did occur as time PI lengthened; however, at day 25-27 PI SE persisted in crop lavage samples from SPF WL hens and three commercial white-egg layer strains. A marked increase in SE-LPS-specific IgA was measured in crop lavage samples between day 0 and day 11-12 PI for all hen groups. Crop SE-LPS-specific IgA response remained elevated above day 0 baseline for the duration of the experiment. Well-defined score 3 to 5 lymphoid tissue aggregates were observed in crop tissue sections harvested at day 34 PI. Comparison of crop sections determined a 1.2-4.0 times increase in ratio of lymphoid tissue in day 34 PI SE-challenged hens vs. uninfected control hens.     

Analysis of neurotransmitter metabolite concentrations in canine cerebrospinal fluid

The concentrations of dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindole-3-acetic acid (5-HIAA) in CSF obtained from the cisterna magna of 21 nonneurologically compromised dogs were determined by high-pressure liquid chromatography and electrochemical detection. A rapid method of sample preparation, which involved single filtration through a deproteinizing membrane, was used. Canine CSF obtained in this manner contained 5.78 +/- 0.78 ng of DOPAC/ml, 72.19 +/- 4.09 ng of HVA/ml, and 29.95 +/- 1.67 ng of 5-HIAA/ml. Linear regression analysis between HVA and 5-HIAA yielded a correlation coefficient of 0.4804. The neurotransmitter index, HVA/5-HIAA, was found to be more indicative of the dopaminergic metabolite HVA than the acid metabolite of serotonin, 5-HIAA (correlation coefficient with HVA = 0.5529 vs a correlation coefficient with 5-HIAA = -0.4462). A poor relationship (correlation coefficient = -0.1715) was found to exist between the 2 dopaminergic metabolites DOPAC and HVA in the CSF.