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Biomedical subjects

K B Platt

Publications and source records attributed to K B Platt.

At least 19 recordsLinked to original sources

Parasympathetic preganglionic neurons in the spinal cord involved in uterine innervation are cholinergic and nitric oxide-containing.

BACKGROUND: The purpose of this study was to elucidate parasympathetic preganglionic neurons in the spinal cord that project axons in pathways to the uterus and to reveal their neurotransmitter phenotype. METHODS: "Uterine-related" neurons were identified by using a combination of retrograde axonal tracers: (1) Fluorogold injected into the ganglia of termination of preganglionic fibers, and (2) a transganglionic axonal tracer (pseudorabies virus) injected into the uterus. Immunohistochemistry was used to reveal virus-labeled neurons and their neurotransmitter marker. RESULTS: Double-labeled (Fluorogold+pseudorabies virus) "uterine" preganglionic neurons were identified in the sacral parasympathetic nucleus of the rat lumbosacral spinal cord. Subpopulations of neurons in the sacral parasympathetic nucleus were shown to be immunoreactive for choline acetyltransferase or nitric oxide synthase. Double-staining immunohistochemistry (for pseudorabies virus+neurotransmitter enzyme) revealed that some of the uterine-related preganglionic neurons were cholinergic and some nitric oxide synthase-containing. CONCLUSIONS: These results demonstrate a subpopulation of preganglionic parasympathetic neurons in the sacral parasympathetic nucleus that are involved in uterine innervation. In addition, both acetylcholine and nitric oxide could be used to modify activity in the postganglionic neurons, which directly innervate the uterus.

Acetylcholine

Preliminary survey of pseudorabies virus prevalence in swine herds of Thailand and comparison of the relative efficacy of gI indirect and blocking ELISAS.

A survey of 15 swine herds ranging in size from approximately 200 to 2,000 head in central Thailand was conducted to determine the prevalence of pseudorabies (PR) in the region and to determine the infection rate within herds. All swine in the survey had been routinely vaccinated with gI deletion mutant modified live virus PR vaccines. Sera from individual pigs were tested for PR virus specific antibodies with both the gI blocking ELISA and the gI indirect ELISA. Fourteen (93.3%) of the herds were found to be infected with wild type PR virus. The infection rate within these 14 herds was more than 20% in 10 (71.0%) herds and ranged from 2.1 to 15.6% in the remaining 4 herds. The per cent agreement between the gI indirect and blocking assays was 99.3% for all field sera tested. This high level of agreement between the epitope specific gI blocking ELISA and the multiple epitope specific indirect gI ELISA indicates that both tests can be used with equal efficacy and that it is unlikely that field strains that could go undetected by the gI blocking ELISA exist in central Thailand.

Animals

Characterization of the humoral immune response to porcine reproductive and respiratory syndrome (PRRS) virus infection.

The development of the humoral immune response against porcine reproductive and respiratory syndrome (PRRS) virus was monitored by an indirect fluorescent antibody (IFA) test, immunoperoxidase monolayer assay (IPMA), enzyme-linked immunosorbent assay (ELISA), and serum virus neutralization (SVN) test over a 105-day period in 8 pigs experimentally infected with ATCC strain VR-2402. Specific antibodies against PRRS virus were first detected by the IFA test, IPMA, ELISA, and the SVN test 9-11, 5-9, 9-13, and 9-28 days postinoculation (PI), respectively, and reached their maximum values by 4-5, 5-6, 4-6, and 10-11 weeks PI, respectively, thereafter. After reaching maximum value, all assays showed a decline in antibody levels. Assuming a constant rate of antibody decay, it was estimated by regression analysis that the ELISA, IFA, IPMA, and SVN antibody titers would approach the lower limits of detection by approximately days 137, 158, 324, and 356 PI, respectively. In this study, the immunoperoxidase monolayer assay appeared to offer slightly better performance relative to the IFA test, ELISA, and SVN test in terms of earlier detection and slower rate of decline in antibody titers. Western immunoblot analysis revealed that antibody specific for the 15-kD viral protein was present in all pigs by 7 days PI and persisted throughout the 105-day observation period. Initial detection of antibodies to the 19-, 23-, and 26-kD proteins varied among pigs, ranging from 9 to 35 days PI. Thereafter, the antibody responses to these 3 viral proteins of PRRS virus continued to be detected throughout the 105-day study period.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals

Expression of Aleutian mink disease parvovirus capsid proteins in a baculovirus expression system for potential diagnostic use.

A 2.3-kb cDNA clone encoding Aleutian mink disease parvovirus (ADV) structural proteins VP1 and VP2 was inserted into the polyhedron gene of Autographa californica nuclear polyhedrosis virus (AcNPV) and expressed by the recombinant virus, AcADV-1, in Spodoptera frugiperda-9 cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western immunoblot analysis (WIA) indicated that synthesis of both VP1 and VP2 was being directed by AcADV-1. Fluorescence microscopic examination of AcADV-1-infected S. frugiperda-9 cells indicated that the recombinant protein was present within the nucleus of the cells, and electron microscopic examination of these cells revealed the presence of small particles 23-25 nm in diameter. Structures resembling empty ADV capsids could be purified on CsCl density gradients, thus indicating that the ADV proteins were self-assembling. The antigenicity of recombinant VP1 and VP2 was evaluated by WIA. Sera collected from 16 mink prior to infection with ADV did not react with VP1 and VP2. Ten sera collected from mink with counter current immunoelectrophoresis (CIE) titers greater than 4 (log2) reacted with VP1 and VP2 in WIA. Two of 6 sera with CIE titers of 4 and 1 of 14 sera with CIE titers < 4 reacted with the recombinant proteins. These results suggest that baculovirus recombinant ADV capsid proteins may be useful as diagnostic antigens.

Aleutian Mink Disease

Peripheral and central pathways regulating the kidney: a study using pseudorabies virus.

We used the retrograde transneuronal transport of a neurotropic virus, pseudorabies virus (PRV), to identify the neurons in sympathetic ganglia, spinal cord and brain which regulate renal function and renal circulation. PRV was microinjected into the left kidney of 70, pentobarbital-anesthetized, male rats. After an incubation period of 1-4 days, rats were anesthetized and sacrificed. PRV-infected neurons were located immunocytochemically in pre- and paravertebral sympathetic ganglia, the intermediolateral cell column of the T10-T13 segments and several brainstem cell groups: the medullary raphe nuclei, rostral ventrolateral medulla, rostral ventromedial medulla, A5 cell group, and the paraventricular hypothalamic nucleus. In more heavily infected rats, additional labeling was found in the locus coeruleus, periaqueductal gray matter, lateral hypothalamic area, zona incerta, and anterior hypothalamic area. No infected propriospinal neurons were observed in the lateral spinal nucleus or gray matter of the caudal cervical, lumbosacral or thoracic spinal segments not containing infected putative sympathetic preganglionic neurons. The paucity of infected propriospinal neurons in the presence of infected brainstem neurons, even in lightly infected rats, is discussed in reference to the relative importance of descending vs spinal regulation of the sympathetic outflow to the kidney.

Animals

Central nervous system innervation of the penis as revealed by the transneuronal transport of pseudorabies virus.

Transneuronal tracing techniques were used in order to identify putative spinal interneurons and brainstem sites involved in the control of penile function. Pseudorabies virus was injected into the corpus cavernosus tissue of the penis in rats. After a four day survival period, rats were perfused with fixative and virus-labelled neurons were identified by immunohistochemistry. Postganglionic neurons were retrogradely labelled in the major pelvic ganglia. In the spinal cord, sympathetic and parasympathetic preganglionic neurons were labelled transneuronally. Presumptive interneurons were also labelled in the lower thoracic and lumbosacral spinal cord in locations consistent with what is currently known about such interneurons. In the brainstem, transneuronally labelled neurons were found in the medulla, pons and hypothalamus. Regions consistently labelled included the nucleus paragigantocellularis, parapyramidal reticular formation of the medulla, raphe pallidus, raphe magnus, A5 noradrenergic cell group, Barrington's nucleus and the paraventricular nucleus of the hypothalamus. This study confirmed previous studies from our lab and others concerning the preganglionic and postganglionic neurons innervating the penis. The number, morphology and location of these neurons were consistent with labelling seen following injection of conventional tracers into the penis. The brainstem nuclei labelled in this study were also consistent with what is currently known about the brainstem control of penile function. The labelling appeared to be highly specific, in that descending systems involved in other functions were not labelled. These results provide further evidence that the pseudorabies virus transneuronal tracing technique is a valuable method for identifying neural circuits mediating specific functions.

Afferent Pathways

Studies on the biology of Ixodes dammini in the upper midwest of the United States.

The seasonal activity pattern of Ixodes dammini was monitored in south-central Wisconsin in 1989 and 1990. Immature tick activity and abundance were assessed by small mammal trapping (732 Peromyscus leucopus examined) and supplemented with flagging. Tick survival and molting times were evaluated by maintaining ticks in environmental chambers at field sites. Results indicate the biology of I. dammini in the upper midwest is similar to that previously reported from the eastern United States. One notable difference was that seasonal larval activity appeared broader (late April through October) and peaked earlier (on 9 July 1989 mean was 7.7 larvae/mouse; on 9 June 1990 mean was 7.3 larvae/mouse). Nymphal activity on P. leucopus peaked in May of both years (mean = 2.5 in 1989; mean = 1.8 in 1990). Bloodfed females placed in the field in early May 1990 oviposited in early June, and larvae emerged by early August. All bloodfed stages successfully overwintered at field sites.

Animals

Transneuronal labeling of spinal interneurons and sympathetic preganglionic neurons after pseudorabies virus injections in the rat medial gastrocnemius muscle.

The distribution of retrogradely and transneuronally labeled neurons was studied in CNS of rats 4 days after injections of the Bartha strain of pseudorabies virus (PRV) into the medial gastrocnemius (MG) muscle. Tissue sections were processed for immunohistochemical detection of PRV. Retrogradely labeled cells were identified in the ipsilateral MG motor column in the caudal L4 and the L5 spinal segments. In order to evaluate the efficacy of PRV retrograde cell body labeling, the number of PRV retrogradely labeled neurons in the MG motor column was compared to the number labeled with two conventional retrograde cell body markers--Fluoro-Gold and cholera toxin-HRP. A ratio of 1:3 representing medium-sized (less than 30 microns) versus large neurons (greater than 30 microns) was found in the Fluoro-Gold dye experiments; a 1:2 ratio was seen in the PRV experiments. In contrast, when cholera toxin-HRP was used as a retrograde marker, mainly large neurons were labeled; the medium-to-large cell body ratio was 1:10 suggesting cholera toxin-HRP may have a greater affinity for the terminals of alpha-motoneurons as opposed to gamma-motoneurons. Transneuronally labeled cells were identified in the L1-L6 spinal gray matter, intermediolateral cell column (T11-L2), lateral spinal nucleus and medial part of lamina VII in C4 and C5 spinal segments, brainstem (caudal raphe nuclei, rostral ventrolateral medulla, A5 cell group, paralemniscal nucleus, locus coeruleus, subcoeruleus nucleus, red nucleus) and paraventricular hypothalamic nucleus. In the L5 spinal cord, transneuronally labeled neurons were seen in the ipsilateral spinal laminae I and II and bilaterally in spinal laminae IV-VIII, and X. Similar results were obtained in rats that had chronic unilateral L3-L6 dorsal rhizotomies indicating most of the labeling was due to retrograde transneuronal cell body labeling. In order to determine whether PRV was transported into the spinal cord by the dorsal root axons, the ipsilateral dorsal root ganglia (DRGs) were examined for PRV immunoreactivity; none was found. However, using the polymerase chain reaction, viral DNA was shown to be present in the ipsilateral DRGs indicating that some of spinal cord cell body labeling may have resulted from anterograde transneuronal labeling, as well.

Animals

Rates of Borrelia burgdorferi infection in Ixodes dammini (Acari: Ixodidae) in southwestern Wisconsin.

Ixodes dammini Spielman, Clifford, Piesman & Corwin collected from a study site in southwestern Wisconsin were examined for Borrelia burgdorferi infection. Adult spirochete infection rates varied seasonally (38.1%, spring 1990; 60.3%, fall 1990; 41.2%, spring 1991) and were similar to or higher than rates reported in various studies from the northeastern United States. Statistical comparison of seasonal infection rates indicated a significant difference between the fall 1990 adult population and the subsequent overwintered population of the same cohort (spring 1991). Additionally, a significant decrease in the intensity of spirochete infection was observed in the overwintered adult sample.

Animals

Detection of pseudorabies virus infection in subunit-vaccinated and nonvaccinated pigs using a nucleocapsid-based enzyme-linked immunosorbent assay.

The potential of a pseudorabies virus (PRV) nucleocapsid protein (NC)-based enzyme-linked immunosorbent assay (ELISA) as a screening assay for PRV infection in subunit-vaccinated and nonvaccinated pigs was studied. The NC-ELISA compared favorably to a commercial ELISA for detecting PRV infection in nonvaccinated pigs. Virus-specific antibody was first detected by the NC-ELISA between days 14 and 21 in 5 pigs challenged intranasally with 10(4) PFU of virus. Antibody continued to be detected in these pigs through day 42, when the experiment was terminated. The NC-ELISA also detected antibody in 23 of 24 pigs from PRV-infected herds. In contrast, the commercial ELISA detected antibody 1 week earlier than the NC-ELISA in experimentally infected pigs but failed to detect antibody in 3 naturally exposed pigs that were identified by the NC-ELISA. Infection in these animals was confirmed by radioimmunoprecipitation analysis. The potential usefulness of the NC-ELISA for detecting infection in vaccinated pigs was also evaluated. The nucleocapsid-specific antibody responses of 10 PRV envelope glycoprotein subunit-vaccinated pigs were monitored prior to and following nasal exposure to a low dose (10(2.3) PFU) of PRV. Sera were collected periodically for 113 days after infection. Nucleocapsid-specific antibody responses measured by the NC-ELISA remained below the positive threshold before challenge but increased dramatically following virus exposure. Maximum ELISA responses were obtained on day 32 postchallenge (p.c.). Mean ELISA responses decreased thereafter but remained well above the positive threshold on day 113 p.c. PRV nucleocapsid protein can be used effectively as antigen in the ELISA for detecting PRV infection in both nonvaccinated and subunit-vaccinated pigs.

Animals

Comparison of in vitro replication and cytopathology caused by strains of canine distemper virus of vaccine and field origin.

Three biological properties of canine distemper virus were examined to determine if any would consistently differentiate field from vaccine strains of the virus. The properties were the ability to (1) infect macrophages and epithelial cells, (2) produce distinct cytopathologic effect in alveolar and peritoneal macrophages and Vero cells, and (3) produce pocks on the chorioallantoic membrane of embryonated chicken eggs. Four vaccine strains and 5 field isolates were used in the study. The 5 field isolates were obtained directly from canine tissues. Of the 3 properties studied, only the comparison of the ability of the viruses to infect macrophages and epithelial cells was a consistent marker of virus origin. Virulent field isolates would only infect macrophage cultures, whereas the vaccine strains infected both types of cells. One avirulent field isolate from a case of old dog encephalitis reacted more like a vaccine strain by infecting both cell types.

Allantois

CNS projections to the pterygopalatine parasympathetic preganglionic neurons in the rat: a retrograde transneuronal viral cell body labeling study.

The retrograde transneuronal viral cell body labeling method was used to study the CNS nuclei that innervate the parasympathetic preganglionic neurons which project to the pterygopalatine ganglion. Small injections of a suspension of pseudorabies virus (PRV) were made in the pterygopalatine ganglion of rats and after 4 days their brains wer e processed for immunohistochemical detection of PRV. Some of the tissues were stained with a dual immunofluoresence method that permitted the visualization of PRV and neurotransmitter enzyme or serotonin immunoreactivity in the same cell. Retrograde cell body labeling was detected in the ipsilateral ventrolateral medulla oblongata in the region that has been termed the superior salivatory nucleus. This area was the same region that was retrogradely labeled after Fluoro-Gold dye injections in the pterygopalatine ganglion. Retrograde transneuronally infected cell bodies that provide putative afferent inputs to the pytergopalatine parasympathetic preganglionic neurons were mapped throughout the brain. In the medulla oblongata, transneuronally labeled neurons were seen in the nucleus tractus solitarii, dorsomedial part of the spinal trigeminal nucleus and gigantocellular reticular nucleus. In most experiments, some A1 catecholamine cells and serotonin neurons of the raphe magnus, raphe pallidus, raphe obscurus, and parapyramidal nuclei were labeled. In the pons, labeled cells were found in the parabrachial nucleus. A5 catecholamine cell group, and non-catecholamine part of the subcoeruleus region. In the midbrain, cell body labeling was located in the central gray matter and retrorubral field. In the diencephalon, labeling was found mainly in the hypothalamus. The areas included the lateral hypothalamic area, lateral preoptic area, dorsomedial and paraventricular hypothalamic nuclei, and ventral zona incerta. Contralateral second order cell body labeling was seen in the tuberomammillary nucleus of the hypothalamus. Some of these cells were histidine decarboxylase-immunoreactive. In the forebrain, the bed nucleus of the stria terminalis, substantia innominata, and an area of the cerebral cortex called the amygdalopiriform transition zone were labeled.

Animals

The effect of human recombinant interleukin-2 on the porcine immune response to a pseudorabies virus subunit vaccine.

The effect of human recombinant interleukin-2 (rIL-2) as an immune enhancing agent was evaluated in pigs vaccinated with a pseudorabies virus subunit vaccine (SV). Two groups of three pigs received two 25 micrograms doses of SV given 3 weeks apart. One group received 10(5) kg-1 day-1 of rIL-2 subcutaneously over two 5-day periods beginning on the day of the first and second vaccine inoculation. Six other pigs were immunized with two 5 micrograms doses of SV. Three of these pigs were treated as above with rIL-2. The effect of treatment was evaluated by comparing: the humoral response; the cell-mediated immune (CMI) response as measured by lymphocyte blastogenesis before and after virus challenge; and the weight response and virus excretion pattern after challenge with virulent pseudorabies virus (PRV). The humoral antibody response as detected by the serum virus neutralization (SN) assay and the enzyme linked immunosorbent assay (ELISA) was consistently higher in rIL-2 treated pigs than in non-treated pigs. These differences were significant (P less than 0.05) among high vaccine dose pigs prior to virus challenge when measured by the SN assay and during the anamnestic response period between days 3 and 10 after challenge when measured by both the SN assay and the ELISA. No differences were detected between treatment groups in the weight response, virus excretion pattern or the CMI response. These results suggest that human rIL-2 may have enhanced the immune response of pigs to the subunit vaccine.

Animals

CNS cell groups regulating the sympathetic outflow to adrenal gland as revealed by transneuronal cell body labeling with pseudorabies virus.

The CNS cell groups that innervate the sympathoadrenal preganglionic neurons of rats were identified by a transneuronal viral cell body labeling technique combined with neurotransmitter immunohistochemistry. Pseudorabies virus was injected into the adrenal gland. This resulted in retrograde viral infections of the ipsilateral sympathetic preganglionic neurons (T4-T13) and caused retrograde transneuronal cell body infections in 5 areas of the brain: the caudal raphe nuclei, ventromedial medulla, rostral ventrolateral medulla, A5 cell group, and paraventricular hypothalamic nucleus (PVH). In the spinal cord, the segmental distribution of virally infected neurons was the same as the retrograde cell body labeling observed following Fluoro-gold injections in the adrenal gland except there was almost a 300% increase in the number of cells labeled and a shift in cell group distribution. These results imply there are local interneurons that regulate the sympathoadrenal preganglionic neurons. In the medulla oblongata, serotonin (5-HT)-, substance P (SP)-, thyrotropin-releasing hormone-, Met-enkephalin-, and somatostatin-immunoreactive neurons of the raphe pallidus and raphe obscurus nuclei and the ventromedial medulla were infected. In the ventromedial and rostral ventrolateral medulla, immunoreactive phenylethanolamine-N-methyltransferase, SP, neuropeptide Y, somatostatin, and enkephalin neurons were infected. The A5 noradrenergic cells were labeled, as were some somatostatin-immunoreactive neurons in this area. In the were infected. The A5 noradrenergic cells were labeled, as were some somatostatin-immunoreactive neurons in this area. In the hypothalamus, tyrosine hydroxylase- and SP-immunoreactive neurons of the dorsal parvocellular PVH were infected. Only a few immunoreactive vasopressin, oxytocin, Met-enkephalin, neurotensin, and somatostatin PVH neurons were labeled.

Adrenal Glands

A general pattern of CNS innervation of the sympathetic outflow demonstrated by transneuronal pseudorabies viral infections.

Pseudorabies virus (PRV) injections of various sympathetic ganglia and the adrenal gland were made in rats. These produced immunohistochemically detectable retrograde viral infections of ipsilateral sympathetic preganglionic neurons (SPNs) and transneuronal infections of the specific sets of second order neurons in the spinal cord and brain that innervate the infected SPNs. Five cell groups in the brain appear to regulate the entire sympathetic outflow: the paraventricular hypothalamic nucleus (PVH), A5 noradrenergic cell group, caudal raphe region, rostral ventrolateral medulla, and ventromedial medulla. In addition, local interneurons in laminae VII and X of the spinal cord are also involved. Other CNS areas also became transneuronally labeled after infections of certain sympathetic ganglia, most notably the superior cervical and stellate ganglia. These areas include the central gray matter and lateral hypothalamic area. The zona incerta was uniquely labeled after stellate ganglion infections. The cell body labeling was specific. This specificity was demonstrated in the PVH where the neurons of the parvocellular PVH that form the descending sympathetic pathway were labeled in a topographic fashion. Finally, we demonstrate that the retrograde transneuronal viral cell body labeling method can be used simultaneously with either neuropeptide transmitter or transmitter synthetic enzyme immunohistochemistry.

Animals

Antibody response of pseudorabies virus subunit-vaccinated pigs to viral nucleocapsid proteins following low-dose virus challenge of immunity.

The antibody response to pseudorabies virus nucleocapsid proteins (NCP) was evaluated by the western immunoblot analysis before and after challenge of immunity by nasal inoculation of 10(2.3) plaque-forming units of virus in 10 pigs that had been vaccinated with pseudorabies virus envelope glycoproteins. Antibody to 5 NCP with molecular mass of 140, 63, 41, 34, and 23 kD was first detected in vaccinated and nonvaccinated pigs on day 14 after challenge of immunity. Antibody to 2 of the 5 NCP continued to be detected through day 113 in 9 of 10 vaccinated pigs. Beyond day 32, antibody to NCP was not detected in 1 vaccinated pig. The 23-, 34-, and 41-kD proteins were the most immunogenic. Antibody to each of these proteins was first detected on day 14 in 10, 10, and 8 pigs, respectively. Seven, 6, and 8 pigs, respectively, were antibody-positive for these proteins on day 113. The 140- and 63-kD proteins were the least immunogenic. Antibody to these proteins was detected in 8 and 9 pigs, respectively, on day 14, and in 4 and 5 pigs, respectively, on day 113. Chi-square analysis for dependency indicated that the antibody response to the 140- and 63-kD proteins was interdependent. These results suggested that combinations of NCP may be useful as nonvaccine diagnostic antigens.

Animals

Studies on the ability of a 98-kilodalton pseudorabies virus diagnostic antigen to detect latent infections induced by low-dose exposure to the virus.

The effect of low-dose challenge of immunity with pseudorabies virus (PRV) on subunit-vaccinated pigs was studied in 2 experiments. In the first experiment, we studied the effect of challenge dose on the antibody response to an early excreted 98-kilodalton PRV-glycoprotein that was used as a diagnostic antigen in the ELISA. In the second experiment, we studied the effect of low doses of virus on the establishment of latent infections in subunit-vaccinated pigs. The relationship of virus exposure dose and vaccine dose to the response of pigs to diagnostic antigen was studied in 18 pigs. Two groups of 3 pigs were vaccinated with a total of 200 micrograms of a lectin-derived PRV subunit vaccine over a 5-week period. Two groups of 3 pigs were similarly vaccinated with a total of 100 micrograms. Two groups of 3 pigs served as nonvaccinated controls. One group of pigs from each of the preceding categories was intranasally exposed to 10(6.0) and 10(2.7) plaque-forming units (PFU) of virus. Antibody to diagnostic antigen was detected by the ELISA and radioimmunoprecipitation 3 to 7 days earlier in pigs exposed to 10(6.0) PFU, demonstrating that the size of the virus challenge dose affects the antibody response to diagnostic antigen. The establishment of latent infections by low PRV doses and the ability to detect these infections was studied in 10 subunit-vaccinated pigs. Each pig was intranasally exposed to 10(2.3) PFU of virus (day 0).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals