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Biomedical subjects

D D Collie

Publications and source records attributed to D D Collie.

At least 19 recordsLinked to original sources
PubMed2001

Local lung responses following local lung challenge with recombinant lungworm antigen in systemically sensitized sheep.

BACKGROUND: Chronic mast cell-mediated inflammation may contribute significantly towards the extensive tissue remodelling that is a feature of lungworm infection in ruminants. Understanding the factors that control tissue remodelling is a necessary step toward effective management and treatment of conditions that feature such pathology. OBJECTIVE: We sought to define in a novel ovine model system, the cellular, immune and mast cell phenotypic events that occur following local lung challenge with a recombinant protein antigen, DvA-1, derived from the ruminant lungworm nematode, Dictyocaulus viviparus. METHODS: Two spatially disparate lung segments in systemically sensitized sheep were challenged on three occasions with DvA-1 (3xDVA) and two further segments were challenged with saline (3xSAL). Two months after the third challenge, one of the two segments previously repeatedly challenged with DvA-1 was challenged again with DvA-1 (3xDVA:DVA) whilst the other was challenged with saline (3xDVA:SAL). A similar protocol was followed with the saline challenged segments (3xSAL:SAL and 3xSAL:DVA). Bronchoalveolar lavage fluid (BALF) (n = 16) and tissue (n = 3) were collected after the last challenge. RESULTS: Cellular changes 24 h after the fourth challenge were characterized by an increase in the absolute numbers of neutrophils and eosinophils in BALF from 3xDVA:DVA and 3xSAL:DVA segments. Local antibody production was implied through increased levels of antibody in both 3xDVA:DVA and 3xDVA:SAL segments, with the latter being unaffected by inflammation. Levels of active transforming growth factor beta-1 (TGF-beta(1)) were significantly increased in 3xDVA:SAL segments and a trend towards an increase was apparent in 3xDVA:DVA segments. Total TGF-beta1 levels were significantly correlated with eosinophil counts in all except the 3xDVA:SAL segments. Such changes in the bronchoalveolar space were complemented by increased ratios of sheep mast cell proteinase-1 expressing cells and tryptase expressing cells, to toluidine blue positive cells in airways from 3xDVA:DVA segments. CONCLUSION: Mast cell phenotypic events occurring as a consequence of antigen challenge were limited to segments in which changes in BALF were characterized by neutrophil influx and increased local antibody production.

Animals↗
PubMed2001

Pulmonary and systemic effects of inhaled endotoxin in control and heaves horses.

To investigate whether inhaled endotoxin contributes to airway inflammation and dysfunction in stabled horses, control (n = 6) and asymptomatic heaves (previously termed chronic obstructive pulmonary disease)-susceptible (n = 7) horses were given inhalation challenges with 20, 200 and 2,000 microg of soluble Salmonella typhimurium Ra60 lipopolysaccharide (LPS). LPS inhalation induced a dose-dependent neutrophilic airway inflammatory response in both groups. Inhalation with 2,000 microg of LPS also induced detectable lung dysfunction in the heaves group. LPS inhalation did not alter clinical score, tracheal secretion volume or airway reactivity in either group. The no-response thresholds were lower for the heaves group (<20 microg for airway inflammation; 200 to 2,000 microg for lung dysfunction) than for the control group (20 to 200 microg for airway inflammation; >2,000 microg for lung dysfunction). To enable comparison of these threshold levels with airborne endotoxin concentrations in stables, horses also received a 5 h duration hay/straw challenge, during which the total and respirable airborne endotoxin concentrations were determined. Comparison of the effects of acute LPS inhalation and hay/straw challenges suggest that inhaled endotoxin is not the sole cause of heaves. However, it is likely that it contributes to airway inflammation, both in heaves horses in concert with other inhalants, and in normal horses when they are exposed to high levels in poor stable environments.

Air↗
PubMed2001

Ovalbumin aerosols induce airway hyperreactivity in naïve DO11.10 T cell receptor transgenic mice without pulmonary eosinophilia or OVA-specific antibody.

The pathobiology of allergic asthma is being studied using murine models, most of which use systemic priming followed by pulmonary challenges with the immunizing antigen. In general, mice develop eosinophilic pulmonary inflammation, increased antigen-specific immunoglobulins, and airway hyperreactivity (AHR), all of which are dependent on antigen-specific T cell activation. To establish a model of allergic asthma, which did not require systemic priming, we exposed DO11.10 T cell receptor transgenic mice, which have an expanded repertoire of ovalbumin (OVA), peptide-specific T cells, to limited aerosols of OVA protein. DO11.10 +/- mice developed AHR in the absence of increases in total serum IgE, OVA-specific IgG, or eosinophilia. The AHR was accompanied by pulmonary recruitment of antigen-specific T cells with decreased expression of CD62L and CD45RB and increased expression of CD69, a phenotype indicative of T cell activation. Our results support recent hypotheses that T cells mediate AHR directly.

Administration, Intranasal↗
PubMed2000

cDNA sequence of two sheep mast cell tryptases and the differential expression of tryptase and sheep mast cell proteinase-1 in lung, dermis and gastrointestinal tract.

BACKGROUND: Mast cell tryptases are a family of serine proteinases which are implicated in the proliferation of smooth muscle cells and fibroblasts, upregulation of interleukin-8 synthesis by endothelial cells, and recruitment of neutrophils and eosinophils. Trials in sheep showed that administration of a specific tryptase inhibitor reduced the late-phase response to inhaled allergen. OBJECTIVES: The aim of this study was to characterize the sequence and distribution of sheep tryptase(s), to validate the sheep model of allergic lung disease. METHODS: Reverse transcriptase PCR cloning was used to obtain cDNA sequences for two sheep tryptases. Lung and gut extracts were used as a source of tryptase for partial purification and characterization of the protein. The distribution of tryptase in skin, lung and gut was determined by immunohistochemistry, and compared with the distribution of sheep mast cell proteinase-1 (sMCP-1). RESULTS: Two highly similar cDNA sequences encoding sheep tryptase were found, indicating the presence of a 28 amino acid leader sequence, and a mature peptide of 245 amino acids. Partial purification of a putative sheep tryptase from lung and gut extracts was achieved using heparin-Sepharose affinity chromatography. Rabbit antihuman skin tryptase antiserum recognized the putative sheep tryptase on Western blot (approximate Mr 32-34 000) and paraformaldehyde-fixed tissue sections. Tryptase was detected in all lung, skin and gut mast cells by this antibody, and transcripts for tryptase were detected in all three tissues by RT PCR. Sheep mast cell proteinase-1, detected by a specific monoclonal antibody, was present in all intestinal and gastric mucosal mast cells, but was not found in mast cells of the muscularis, thus defining at least two mast cell phenotypes in the gut. Whereas all dermal and pulmonary mast cells were tryptase positive, only a low proportion in the lung, and almost none in the dermis, were positive for sMCP-1. CONCLUSION: In view of the structural and functional similarities of sheep and human tryptases, and their similarity in tissue distribution in normal sheep, the sheep lung appears to be a good model for in vivo studies relating to human tryptase.

Amino Acid Sequence↗
PubMed1999

Ovine bronchoalveolar lavage cellularity: reproducibility and the effect of multiple repeated lavage.

We sought to determine the normal within-sheep variability of bronchoalveolar cellular parameters and to infer the numbers of sheep required to detect a defined significant change within the same. We further sought to examine the effect of repeated bronchoalveolar lavage (BAL) on those parameters. Neither the total cell number nor the absolute numbers of any given cell type changed significantly following repeated BAL. Additionally, there was no significant change in percentage differential cell populations over time in randomly sampled lobes. However for lobes sampled repeatedly, a significant change in the percentage of lymphocytes was detected. Although the percentages of neutrophils in repeatedly and randomly sampled lobes were significantly correlated, the percentage found in the former tended to be greater and more variable than in the latter. As a consequence, larger group sizes are required to detect given changes in neutrophil percentages in repeatedly sampled lobes over time when compared with detecting equivalent changes in randomly selected lobes.

Animals↗
PubMed1999

Dissociation of airway hyperresponsiveness from immunoglobulin E and airway eosinophilia in a murine model of allergic asthma.

Nonspecific airway hyperresponsiveness (AHR) is a hallmark of human asthma. Both airway eosinophilia and high serum levels of total and antigen-specific immunoglobulin E (IgE) are associated with AHR. It is unclear, however, whether either eosinophilia or increased IgE levels contribute directly to, or predict, the development of AHR. Investigations conducted with various murine models of asthma and different mouse strains have resulted in conflicting evidence about the roles that IgE and airway eosinophilia play in the manifestation of AHR. We show that systemic priming with ovalbumin (OVA) in alum, followed by a single day of OVA aerosol challenge, is sufficient to induce AHR, as measured by increased pulmonary resistance in response to intravenously delivered methacholine in BALB/c, but not C57BL/6 or B6D2F1, mice. This was observed despite the fact that OVA-challenged BALB/c mice had less airway eosinophilia and smaller increases in total IgE than either C57BL/6 or B6D2F1 mice, and had less pulmonary inflammation and OVA-specific IgE than B6D2F1 mice. We conclude that airway eosinophilia, pulmonary inflammation, and high serum levels of total or OVA-specific IgE are all insufficient to induce AHR in C57BL/6 and B6D2F1 mice, whereas BALB/c mice demonstrate AHR in the absence of airway eosinophilia. These data confirm that the development of AHR is genetically determined, not only in naive mice, but also in actively immunized ones, and cannot be predicted by levels of airway eosinophilia, pulmonary inflammation, total IgE, or antigen-specific IgE.

Animals↗
PubMed1997

Evaluation of association of blood and bronchoalveolar eosinophil numbers and serum total immunoglobulin E concentration with the expression of nonspecific airway reactivity in dogs.

OBJECTIVE: To characterize the relation between bronchoalveolar and blood eosinophil numbers, serum total IgE concentration, and nonspecific airway reactivity in healthy dogs. ANIMALS: 26 healthy Beagles. PROCEDURE: Prior to measurement of nonspecific airway responsiveness, dogs were anesthetized and bronchoscopy was performed to recover bronchoalveolar lavage (BAL) fluid. Repeated measurements were made in 6 dogs. RESULTS: The percentage of blood eosinophils varied between 0 and 13 (mean +/- SD, 5.6 +/- 3.6) %, the percentage of eosinophils in BAL fluid ranged between 0 and 63.5 (8.8 +/- 12.9) %, and total serum IgE concentration was 0.1 to 107.5 (23.4 +/- 29.1) U/ml. A strong association was evident between numbers of blood eosinophils and total serum IgE concentration (R2 = 0.413, P < 0.001), and a trend toward an association between numbers of blood eosinophils and numbers of eosinophils in BAL fluid was apparent (R2 = 0.110, P < 0.053). Significant associations were not found between any other aspects of the blood and BAL fluid cell profiles and total serum IgE concentration or airway reactivity. Serum total IgE concentration was not associated with airway reactivity. Further, in dogs examined on repeated occasions, variation in BAL fluid eosinophil numbers was not associated with any change in serum total IgE concentration or airway reactivity. CONCLUSIONS: Neither numbers of bronchoalveolar or blood eosinophils nor serum total IgE concentration have a significant role in determining airway reactivity in health dogs.

Animals↗
PubMed1996

Early pulmonary cell response during experimental maedi-visna virus infection.

A model of experimental infection with EV1, a British isolate of maedi-visna virus (MVV), has been developed. Twelve male Texel sheep were allocated to three groups and inoculated by the respiratory route with different inocula. Six of the animals received 10(7.2) tissue culture infective dose (TCID50) of MVV EV1 strain. Two sheep were inoculated with the same dose of heat inactivated MVV EV1 strain. An additional group of four sheep was sham-inoculated with identically prepared virus-free culture media. Experimental infection was followed for 16 weeks. Prior to inoculation, routine haematology, bronchoalveolar lavage (BAL) and flow cytometric analysis of bronchoalveolar lavage fluid (BALF) lymphocytes were performed in all animals to provide baseline parameters. Flow cytometric analysis of BALF lymphocytes and differential BALF cell counts were performed. Precipitating antibodies to MVV developed in all MVV-inoculated animals during the first 4 weeks post-inoculation, while the rest remained seronegative to MVV. MVV-infected animals had significantly decreased (P < 0.05) percentages of macrophages and significantly increased (P < 0.05) percentages of lymphocytes in BALF 4 weeks post-inoculation. Phenotypic changes in BALF T lymphocytes from MVV-inoculated animals, compared with the other two groups, showed significantly decreased (P < 0.05) percentages of CD4+ and gamma delta + T lymphocytes, significantly increased (P < 0.05) percentages of CD8+ lymphocytes and significant inversion (P < 0.05) of the CD4+/CD8+ ratio at different sampling times, but between 2 and 12 weeks post-inoculation. These findings indicate that during experimental MVV-infection an early, short-term cellular reaction occurs in the lung, that is characterised by T lymphocyte phenotypic changes that are very similar, if not identical, to those observed in natural MVV infection.

Animals↗
PubMed1995

A study on lymphocyte activation in maedi-visna virus induced pneumonia.

The stage of activation of bronchoalveolar lavage fluid (BALF) lymphocytes and peripheral blood lymphocytes (PBL) from maedi-visna virus (MVV) infected (n = 7) and control (n = 7) sheep was investigated by assessing four parameters of lymphocyte activation; lymphocyte size and complexity, loss of CD5+ T cells, expression of cell surface interleukin-2 receptor (IL-2R) and expression of DR and DQ MHC Class II molecules. BALF lymphocytes from MVV-infected animals had a significant loss of CD5+ lymphocytes (P < 0.05) and upregulation of DR and DQ MHC Class II molecules compared with controls, consistent with BALF lymphocyte activation. No changes in cell size and complexity or expression of IL-2R were observed. No evidence of PBL activation was detected. These findings suggest an impaired BALF lymphocyte activation during MVV infection.

Animals↗
PubMed1995

Quantitation of transforming growth factor-beta in plasma and pulmonary epithelial lining fluid of sheep experimentally infected with maedi-visna virus.

A model of experimental infection with EV1, a lytic British isolate of maedi-visna virus (MVV), was developed. Ten Texel sheep were allocated to two groups and inoculated by the respiratory route with different inocula. Six of the animals received 10(7.2) TCID50 (tissue culture infective dose) of EV1 strain, while four sheep were sham-inoculated with identically prepared virus-free buffer solution. Experimental infection was followed for 8 weeks post-inoculation (PI), with development of precipitating antibodies to MVV developed in the MVV-inoculated animals during the first 4 weeks PI. Transforming growth factor-beta (TGF-beta) levels, in both bronchoalveolar lavage fluid supernatant and plasma samples, were measured. Concentrations of pulmonary epithelial lining fluid (PELF) TGF-beta were calculated. TGF-beta concentrations in PELF were approximately 165-fold higher than in plasma. No significant differences in the concentrations of plasma or PELF TGF-beta, either within or between groups, were observed.

Animals↗
PubMed1995

CD8+ lymphocytes in bronchoalveolar lavage and blood: in vivo indicators of lung pathology caused by maedi-visna virus.

A study to determine the putative relationship between lymphocyte phenotypic alterations in bronchoalveolar lavage fluid and stage of lung pathology in maedi-visna infected sheep has been carried out. Twenty-one ewes (16 Texel and five Scottish blackface) naturally infected by maedi-visna virus and three Oxford controls were used. Animals were killed, lungs were removed, bronchoalveolar lavage was performed and pathological studies were completed. Blood samples were also obtained from 16 animals. Lymphocytes in both bronchoalveolar lavage and peripheral blood were labelled with monoclonal antibodies against the main T lymphocyte subsets (CD4, CD8, CD5 and gamma delta TCR) in order to perform flow cytometric studies. Three aspects of pathology were studied: lymphoid interstitial pneumonia, lymphoid follicular hyperplasia and smooth muscle hyperplasia. Percentages of CD4+, CD5+, gamma delta + T cells and the value for the CD4+ / CD8+ ratio in bronchoalveolar lavage of maedi-visna infected animals were significantly decreased (P < 0.05) when compared to controls, while percentages of CD8+ lymphocytes were increased in bronchoalveolar lavage of infected sheep and they were very close to being significant (P = 0.07) when compared to controls. Lesions were evaluated and simple least-squares regression tests demonstrated that there were several significant correlations between various lymphocyte subsets and pathological parameters studied in this work. However, when a multiple regression test was applied to the data, it was observed that only the CD8+ T cell subset both in bronchoalveolar lavage and in blood was significantly correlated with severity of lung pathology. It is concluded that CD8+ lymphocytes are key cells in the development of the interstitial reaction and the lymphocytic alveolitis observed in maedi-visna infected ewes and that the CD8+ alveolitis is a parallel feature to the intensity of lung lesions. It is further suggested that the percentage of CD8+ lymphocytes in bronchoalveolar lavage and in blood may act as in vivo indicators of lung pathology in maedi-visna infected sheep.

Animals↗
PubMed1995

Distribution and quantitation of lung parenchymal contractile tissue in ovine lentivirus-induced lymphoid interstitial pneumonia. Do tissue forces limit lung distensibility?

BACKGROUND: Lymphoid interstitial pneumonia (LIP) is frequently identified in sheep infected with the ovine lentivirus, maedi-visna virus (MVV). Functional consequences of this condition include a reduction in lung distensibility that cannot be explained by the density of surface forces within the lung parenchyma. A potential source of tissue forces to account for this functional deficit is the substantial parenchymal smooth muscle hyperplasia that is a feature of the lung pathology. This investigation examines the relationship between lung distensibility and the quantity and distribution of smooth muscle hyperplasia in MVV-induced LIP. EXPERIMENTAL DESIGN: Immunohistochemical localization of alpha-smooth muscle actin (ASMA) was used to identify parenchymal contractile tissue. The distribution and morphometric quantitation of ASMA in lung parenchyma was determined in normal sheep lungs and in lungs from sheep seropositive for MVV. The relationship between the volume density of ASMA in lung parenchyma (Vv'ASMA') and static lung compliance (Cst) and lung distensibility (K) was examined. RESULTS: In normal lungs, ASMA was expressed by typical smooth muscle cells surrounding airways and blood vessels, by cells at the alveolar septal tips protruding into the alveolar ducts, and, rarely, by individual cells within septa. In MVV-seropositive sheep with minimal histopathology, increased ASMA expression occurred in association with early interstitial infiltrates and was located both at septal tips and within septa. With more severe pathology, ASMA-expressing cells became organized into bundles within obviously thickened septa and septal tips. In maedi, Vv'ASMA' is negatively correlated with K and Cst (rs = -0.614; p < 0.005; and rs = -0.504; p < 0.025, respectively). However, partial correlation coefficients indicate that Vv'ASMA' and lung parenchymal tissue density (Vvt) are strongly interdependent. CONCLUSIONS: ASMA expression in normal sheep lung parenchyma follows a similar pattern of distribution to that described for human lungs. The quantity of ASMA in lung parenchyma in LIP associated with MVV infection is negatively correlated with lung distensibility; however, whether this is a causal association remains undetermined.

Actins↗
PubMed1994

Exponential analysis of the pressure-volume characteristics of ovine lungs.

Static pressure-volume curves were generated from data obtained from 18 normal anaesthetized adult sheep. Lung volumes were determined by helium dilution. An exponential curve of the form V = Vmax - Ae-KP was fitted to the pressure-volume data from each sheep where P is the static recoil pressure, Vmax represents the volume asymptote, A is the difference between Vmax and the intercept on the volume axis and K defines the slope and hence the shape of the P-V curve. Quality of fit of the data was assessed visually, by means of a sign test and a runs test and by the coefficient of determination (r2). Exponential equations were found to adequately describe the shape of the pressure-volume curve in sheep. The exponent K was not correlated with effective alveolar volume (VAeff) (rs = 0.183; P > 0.05). Static lung compliance was determined over a volume range from the end-expiratory level (VEEL) to VEEL plus 400 ml. Measurements of static lung compliance were significantly correlated with measurements of effective alveolar volume (VAeff) (rs = 0.505; P < 0.025). In the ovine, the exponent K, an index of distensibility, is independent of lung volume and offers a means of assessing lung distensibility in this species.

Air Pressure↗
PubMed1994

Pathophysiologic correlations in lymphoid interstitial pneumonia.

Effective alveolar volume, diffusing capacity for carbon monoxide (DCOsb), volume-corrected diffusing capacity (D/VA), static lung compliance (Cst), and lung distensibility were measured in 16 sheep seropositive for maedi-visna virus (MVV) immediately before they were killed. Lungs were inflation-fixed, and the left lung was randomly sampled for morphometric analysis. The total lung weight, total fixed lung volume, volume densities of tissue (Vvt) and air (Vva), and the alveolar surface density were measured and correlated with the physiologic measurements. The density of surface forces could not account for the variation in the distensibility of the lungs, indicating that tissue-related forces may be important in determining lung distensibility in lymphoid interstitial pneumonia (LIP) associated with MVV infection. Possible sources of tissue-related forces are the contractile tissue associated with lung parenchyma, airways, or vasculature. When DCOsb was corrected for volume, a strong negative correlation with Vvt was noted, indicating that factors distinct from lung-volume reduction are important in limiting gas exchange in LIP associated with MVV infection. More sheep demonstrated abnormal D/VA values than any other physiologic measurement, with reduced values being apparent even in sheep considered clinically normal and with little or no morphometric evidence of lung disease. Measurements of diffusing capacity are thus considered the most sensitive functional index of disease progression.

Animals↗
PubMed1993

Lung compliance, lung volume and transfer factor for carbon monoxide in anaesthetised sheep: normal values and reproducibility of measurements.

Measurements of quasistatic compliance (Cqst), effective alveolar volume (VA,eff) and single-breath transfer factor for carbon monoxide (TL,CO, 'sb') were completed in 16 normal, anaesthetised, adult Texel ewes. Regression equations were computed for these variables as a function of bodyweight and the optimal equations selected. The 95 per cent prediction intervals for the equations were calculated such that normal lung function in similar sheep could be accurately predicted. The long term reproducibility of these measurements was assessed in nine sheep, measured at monthly intervals over a period of five months. Although measurements made in individual sheep were often highly variable, the variation between repeated measurements on the separate days for the group was insignificant.

Anesthesia, General↗
PubMed1993

Phenotypic analysis of cells in bronchoalveolar lavage fluid and peripheral blood of maedi visna-infected sheep.

A phenotypic analysis of bronchoalveolar lavage fluid (BALF) and peripheral blood (PB) cells in maedi visna virus (MVV)-infected sheep has been performed. The differential cell count in BALF from MVV-infected animals was characterized by a significant increase (P < 0.05) in lymphocytes and neutrophils. Lymphocyte phenotyping in BALF from MVV-infected sheep showed a significant decrease (P < 0.05) of CD4+ cells, a significant increase (P < 0.05) of CD8+ cells and a significant inversion (P < 0.001) of the CD4+/CD8+ ratio. CD5+ lymphocytes were also significantly decreased (P < 0.05). Gamma delta T cells and B cells did not differ significantly when compared with the controls. No correlation was observed between BALF and PB lymphocyte phenotypes. BALF macrophages from MVV-infected animals showed increased MHC class II expression and BALF lymphocytes from the same animals demonstrated up-regulation of LFA-1 and LFA-3 expression. These findings and their relationship with lentiviral pathogenesis are discussed.

Animals↗
PubMed1993

Effects on lung compliance, lung volume, and single-breath transfer factor for carbon monoxide in sheep with lentivirus-induced lymphoid interstitial pneumonia.

Static lung compliance, static lung volumes, and transfer factor for carbon monoxide were measured in 12 anesthetized adult Texel ewes seropositive for maedi-visna virus (MVV) and in 11 breed-, sex-, and age-matched seronegative controls. Median static lung compliance in MVV-infected sheep (1.24 L.kPa-1; range, 0.27 to 2.20 L.kPa-1) was not significantly different from that in controls (1.58 L.kPa-1; range, 0.82 to 2.08 L.kPa-1). Median body weight of MVV-infected sheep (56 kg; range, 40 to 75 kg) was significantly (P < 0.05) less than that of controls (65 kg; range, 53 to 87 kg). Median effective alveolar lung volume in MVV-infected sheep (3.36 L; range, 1.44 to 4.52 L) was significantly (P < 0.01) less than that in controls (4.12 L; range, 3.75 to 4.90 L). Median effective end expiratory lung volume in MVV-infected sheep (1.20 L; range, 0.56 to 1.99 L) was significantly (P < 0.001) less than that of controls (1.98 L; range: 1.76 to 2.78 L). Median lung volumes expressed per unit of body weight did not differ significantly between the groups. Median single-breath transfer factor for carbon monoxide in MVV-infected sheep (7.89 mmol.min-1.kPa-1; range, 3.45 to 12.74 mmol.min-1.kPa-1) was significantly (P < 0.001) less than that in controls (14.10 mmol.min-1.kPa-1; range, 10.02 to 18.30 mmol.min-1.kPa-1).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗