THE EFFECTS OF THE ALKALOID SCOPOLAMINE ON THE PERFORMANCE AND BEHAVIOR OF TWO CATERPILLAR SPECIES.

Plants have evolved many defenses against insect herbivores, including numerous chemicals that can reduce herbivore growth, performance, and fitness. One group of chemicals, the tropane alkaloids, is commonly found in the nightshade family (Solanaceae) and has been thought to reduce performance and fitness in insects. We examined the effects of the tropane alkaloid scopolamine, the alkaloid constituent of Datura wrightii, which is the most frequent host plant for the abundant and widespread insect herbivore Manduca sexta in the southwestern United States. We exposed caterpillars of two different species to scopolamine: M. sexta, which has a shared evolutionary history with Datura and other solanceous plants, and Galleria mellonella, which does not. We showed that the addition of ecologically-realistic levels of scopolamine to both the diet and the hemolymph of these two caterpillar species (M. sexta and G. mellonella) had no effect on the growth of either species. We also showed that M. sexta has no behavioral preference for or against scopolamine incorporated into an artificial diet. These results are contrary to other work showing marked differences in performance for other insect species when exposed to scopolamine, and provide evidence that scopolamine might not provide the broad-spectrum herbivore resistance typically attributed to it. It also helps to clarify the coevolutionary relationship between M. sexta and one of its main host plants, as well as the physiological mechanism of resistance against scopolamine.

Bighorn sheep × domestic sheep hybrids survive Mannheimia haemolytica challenge in the absence of vaccination.

Bighorn sheep (BHS, Ovis canadensis) are much more susceptible than domestic sheep (DS, Ovis aries) to pneumonia caused by leukotoxin (Lkt)-producing members of the Family Pasteurellaceae, particularly Mannheimia haemolytica and Bibersteinia trehalosi. Leukotoxin is widely accepted as the critical virulence factor of these bacteria since Lkt-negative mutants do not cause death of BHS. Typically, DS carry Lkt-positive M. haemolytica and/or B. trehalosi as commensal bacteria in their nasopharynx. In contrast, most BHS do not carry Lkt-positive M. haemolytica or B. trehalosi, or carry Lkt-negative strains in their nasopharynx. In previous studies, we demonstrated that unimmunized DS resist M. haemolytica challenge while BHS succumb to it. We hypothesized that Lkt-neutralizing antibodies, induced by Lkt-positive M. haemolytica and/or B. trehalosi innately carried by DS in their nasopharynx, render them less susceptible to infection by these bacteria. In this study we developed BHS×DS F1 hybrids by artificial insemination of domestic ewes with BHS semen. F1 hybrids were fertile, and produced F2 hybrids and back-crosses. The F1, F2, and back-crosses were raised together with domestic ewes. All these animals acquired Lkt-positive M. haemolytica and/or B. trehalosi, and developed high titers of Lkt-neutralizing antibodies in the absence of vaccination. Furthermore, all of these animals resisted challenge with lethal dose of M. haemolytica. These results suggest that lack of previous exposure to Lkt is at least partially responsible for fatal pneumonia in BHS when they acquire Lkt-positive M. haemolytica and/or B. trehalosi from DS when the two species commingle.

Spina bifida with associated malformations of the central nervous system in Dorper-cross sheep.

CASE HISTORY: In 2008, six lambs within a flock of Dorper-cross sheep were born with musculoskeletal and neurological disease. Clinical signs included hindlimb weakness, and urinary incontinence. CLINICAL FINDINGS: All lambs had focal, inverted areas of alopecic skin over the caudal sacrum, and short, often kinked tails. Four affected lambs were subject to euthanasia, and necropsied. On gross examination, the arches of sacral vertebrae were absent, and spinal nerves and meninges were adherent to the overlying subcutis. Other gross lesions included narrow, elongated skulls, herniation of the occipital lobes into the caudal fossas, hydrocephalus, and syringomyelia. One lamb had coning of the cerebellar vermis, but cerebellar herniation through the foramen magnum was not identified. DIAGNOSIS: Spina bifida, with associated malformations of the central nervous system. CLINICAL RELEVANCE: Examination of breeding records suggested either an autosomal recessive or partially penetrant autosomal dominant pattern of inheritance. Because of the associated tail lesions it is proposed that the pathogenesis of this syndrome involves a defect in development of the tail bud (secondary neurulation), that tethering of the spinal cord resulted in the clinical signs, and abnormal pressure of the cerebral spinal fluid resulted in the defects in the skull and brain.

Elastic fibers of musculoskeletal tissues in bovine Marfan syndrome: a morphometric study.

Bovine Marfan syndrome has all the major pathognomonic, clinical, and pathological features of the human syndrome. To further explore the alterations characterizing Marfan syndrome, musculoskeletal tissues from affected and healthy cattle were subjected to histochemical and histomorphometric analysis. Our findings demonstrated reduced elastic fiber content in the periosteum, capsule, interosseous ligament, and flexor tendons of the metatarsophalangeal joint of affected cattle. The elastic properties of a tissue are supplied by elastic fibers in the extracellular matrix; therefore, their reduced content in articular tissues suggests that these fibers could be functionally incompetent to withstand normal stress, causing a predisposition to joint laxity and dislocation. Moreover, perichondrial-periosteal membranes, which are made of collagen and elastic fibers, are known to affect the growth process of the long bones. The decrease in the number of elastic fibers in these membranes could result in reduced restraint of skeletal growth and explain some skeletal abnormalities of Marfan syndrome (i.e., dolichostenomelic habitus).

Mitoxantrone and cyclophosphamide combination chemotherapy for the treatment of various canine malignancies.

Thirteen dogs with histopathologically confirmed malignancies were treated with mitoxantrone and cyclophosphamide combination therapy. One to four doses were administered at 21-day intervals. Recombinant human granulocyte colony-stimulating factor was administered to ameliorate myelosuppression in dogs with neutrophil nadirs less than 1,000/microl. While the protocol appears to be safe for use in tumor-bearing dogs, an advantage over mitoxantrone single-agent protocols in terms of tumor response was not demonstrated in this initial pilot study.

Ocular pathology in bovine Marfan's syndrome with demonstration of altered fibrillin immunoreactivity in explanted ciliary body cells.

Cattle affected by bovine Marfan's syndrome demonstrate most clinical features of the human disease, which is caused by mutations in the fibrillin-1 gene. Immunohistochemical and metabolic labeling studies in affected cattle have demonstrated abnormalities in fibrillin metabolism. Clinically identified ocular features of the bovine disease, which are similar to human Marfan's syndrome, are ectopia lentis, microspherophakia, and myopia. The purpose of this study was to compare the ocular pathology of the human and bovine diseases and to evaluate fibrillin-1 immunoreactivity in the extracellular matrix of explanted ciliary body cells from affected cattle. Eyes from affected cattle and unrelated normal cattle were examined grossly, and portions of the anterior uvea and ciliary zonule were examined by light and scanning electron microscopy. Portions of the ciliary zonular fibers were examined by transmission electron microscopy. The results were compared between affected animals and normal controls. Explanted ciliary body cells from two affected cattle and one unaffected cow were grown on chambered microscope slides, and expression of fibrillin-1 in the extracellular matrix was compared. Eyes of affected cattle were characterized by megaloglobus, increased circumlental distance, asymmetrical ciliary processes, intact but fragile zonular fibers, and ectopia lentis. Affected animals had moderately hypoplastic ciliary bodies, compact filtration angles, and long thin irises with decreased fibrous stroma. As shown by scanning electron microscopy, the zonular fibers of affected animals were wavy and loosely arranged, with abnormal sites of insertion on the lens capsule. The ciliary processes of affected animals had flattened or smooth surfaces. Explanted ciliary body cells from affected animals demonstrated decreased fibrillin immunoreactivity when compared with a normal control. The ocular pathology observed in bovine Marfan's syndrome is, in most respects, similar to that described for the human disease and will be a useful model for studies of in vivo evaluation of abnormal microfibrillar aggregation within ocular structures.

Site-directed mutagenesis of intersubunit boundary residues in histidine decarboxylase, a pH-dependent allosteric enzyme.

Histidine decarboxylase (HDC) from Lactobacillus 30a forms a trimer around a central cavity or well. Three active sites are formed around the well at the interface of each of two adjacent molecules. HDC exhibits cooperative kinetics at pH 7.6 and can be described in terms of a two-state, T and R, model. At pH 4.8, protons stabilize HDC in the R form. Asp 198 and Asp 53, from a neighboring molecule, are the core of the pH-sensitive mechanism controlling the shift in quaternary state. Eight site-directed mutations have been made to analyze the region. Several mutants, including the conversion of Asp 53 to Asn, cause HDC to exhibit sigmoidal kinetics even at pH 4.8. Others lock the enzyme into the T state. Kinetic analysis suggests that kcat values for T and R states are similar. The Km for the T state, near 8 mM, exceeds that for the R state by 40-fold and shows HDC is primarily regulated by altering its affinity for substrate.

Sequence of the coding region of the bovine fibrillin cDNA and localization to bovine chromosome 10.

We report the cDNA sequence for the bovine gene for fibrillin corresponding to the human gene, fibrillin 1 (FBN1), and the localization of the gene to bovine chromosome 10 (syntenic group U5). The identity between the human and bovine sequences is 97.8% at the amino acid level and 92% at the nucleotide level. The bovine fibrillin sequence contains the same number and type of motifs as the human FBN1 sequence, including the same number of putative calcium binding sites. All of the motifs conform to the patterns demonstrated in the human sequence, and many of the differences in identity between the sequences are conservative.

Cardiovascular lesions in bovine Marfan syndrome.

Bovine Marfan syndrome is a genetic disease with many of the clinical and pathologic manifestations of human Marfan syndrome. Major manifestations include ectopia lentis and aortic dilatation, aneurysm, and rupture. Affected cattle have a defect in fibrillin metabolism similar to that in human patients. Ten cattle were followed and their disease progression and lesions documented. Ages ranged from a term fetus (No. 9) to a 4-year-old cow (No. 4); three animals were male (Nos. 1-3) and seven were female (Nos. 4-10). Of eight animals (80%) that died or were euthanatized (Nos. 1-3, 5-9), six (75%) had severe cardiovascular lesions identified at necropsy. Gross cardiovascular lesions of bovine Marfan syndrome included cardiac tamponade secondary to aortic rupture (animal Nos. 3, 6, 8), dissecting aneurysms of the aorta and pulmonary artery (animal No. 5), and intrauterine cardiac tamponade secondary to rupture of the pulmonary artery (animal No. 9). Microscopically, Verhoeff Van Gieson-stained sections of aorta contained severe fragmentation of the elastic laminae in the aortic media, but the cystic medial necrosis seen in human Marfan aortae was not identified, even in the chronic aortic dissection. Ultrastructurally, affected aortic tissue was characterized by thin, dark elastic fibers with abundant, tangled microfibrils on the periphery. Swirls of collagen fibers and bundles of hypertrophic smooth muscle cells replaced damaged elastic laminae. Gross and microscopic cardiovascular lesions in bovine Marfan syndrome are similar to those in human Marfan syndrome. Bovine Marfan syndrome is a valuable model for investigation of molecular pathogenesis and treatment of human Marfan syndrome.

Abnormal fibrillin metabolism in bovine Marfan syndrome.

Bovine Marfan syndrome is a disorder that closely resembles human Marfan syndrome in its clinical signs and pathological lesions. The similarities between the human and bovine diseases suggest that similar metabolic defects could be responsible. Although indirect immunofluorescent assays for fibrillin in skin biopsies did not distinguish affected cattle from control animals, cultures of skin fibroblasts of affected animals were distinguished from normal, unrelated control animals and normal half-siblings on the basis of fibrillin staining. After 72 to 96 hours in culture, stained with anti-fibrillin monoclonal antibody 201, hyperconfluent fibroblast cultures of affected cattle had less immunoreactive fibrillin than control cultures, and the staining pattern was granular rather than fibrillar. Under similar culture conditions, normal bovine aortic smooth muscle cells produced large amounts of immunoreactive fibrillin, but smooth muscle cells from a single affected cow showed markedly less fibrillin staining. In pulse-chase metabolic labeling experiments with [35S]cysteine, dermal fibroblasts from 6 affected calves, incorporated far less fibrillin into the extracellular matrix than control cells. These findings are similar to those reported in human Marfan syndrome, and they suggest that the bovine Marfan syndrome, like the human disorder, is caused by a mutation in fibrillin, leading to defective microfibrillar synthesis.

Normal elastin content of aorta in bovine Marfan syndrome.

Samples from the ascending aortae from two calves affected by bovine Marfan syndrome were subjected to biochemical analyses of the connective tissue and were compared to age-matched controls. Elastin was extracted from the aortic samples with 5 M guanidine-HCl, bacterial collagenase digestion, and dithiothreitol reduction. Amino acid analysis revealed that desmosine and isodesmosine levels were the same in Marfan calves as in control animals. Gravimetric measurements of elastin, amino acid composition, soluble protein, and uronic acid values also showed no significant difference between Marfan and control tissue. In contrast to elastin, collagen in aortae of Marfan calves was significantly higher than the mean of several controls. These findings, along with other observations of this animal model, support the conclusion that the microscopic and biochemical lesions of aortic elastin in bovine Marfan syndrome likely result from defective microfibrillar metabolism. Absence of cystic medial necrosis in bovine Marfan aortae may explain normal elastin content in the animal model.

An animal model of the Marfan syndrome.

A congenital syndrome of long, thin limbs, severe joint and tendon laxity, microspherophakia, ectopia lentis, heart murmurs and aortic dilatation was identified in 7 calves. All affected calves were sired by a single phenotypically normal bull suspected of germline mosaicism for a new mutation resulting in this disease. One of the calves subsequently died with ruptured aorta at age 16 months. Histopathologic and electron microscopic studies of the aortic media of affected calves demonstrated disorganized elastin and narrowed elastic lamina separated by widened spaces. This bovine disease provides a unique animal model of the human Marfan syndrome. A herd of cattle with this disease is being developed for further studies.

Suspected ciliary dysfunction in Chinese Shar Pei pups with pneumonia.

Chronic pneumonia was investigated in a litter of young Chinese Shar Pei in which 4 of 6 dogs were affected. Serum immunoglobulin concentrations (IgA, IgG, IgM) determined by radial immunodiffusion varied over time, but were not consistently lower in affected dogs, compared with control dogs. Two dogs that died had hydrocephalus and lymphoid depletion, in addition to severe broncho-pneumonia. Evaluation of ciliary ultrastructure in 2 affected dogs revealed random orientation of adjacent respiratory tract or oviductal cilia and a greater number of microtubular disarrangements, compared with control dogs. In vivo tracheal mucociliary clearance of 99mtechnetium macroaggregated albumin was absent in 1 dog examined. The ciliary abnormalities were suspected to have resulted in an inefficient mucociliary transport system predisposing to the development of pneumonia. Further evaluation of 1 Chinese Shar Pei revealed lymphocyte mitogenesis results that were not consistently less than those of a control dog, normal total hemolytic complement values, and normal blood neutrophil chemotaxis.

Generalized steatitis associated with selenium deficiency and normal vitamin E status in a foal.

A 7-month-old male Quarter Horse was examined because of severe cachexia, which had begun 2 months earlier. The foal was emaciated and weak despite an excellent appetite. Percutaneous biopsy revealed multiple areas of subcutaneous steatitis with mineralization. Radiography demonstrated calcification of the nuchal ligament. The foal died 9 days after admission. Necropsy revealed mineralized lesions throughout the fascial planes and adipose tissues of the thorax, abdomen, and skeletal muscles, with little to no involvement of muscular tissue. Serum magnesium, copper, and thyroxine concentrations were slightly lower than normal. The serum selenium concentration was unusually low. Renal selenium content was significantly lower (P less than 0.01) than that in similarly prepared sections of kidneys from 3 clinically normal ponies. Low dietary selenium intake was suspected as the cause of the problem.

Isolation and partial characterization of an eosinophil chemotactic factor from metacestodes of Taenia taeniaeformis (ECF-Tt).

Eosinophil chemotactic activity associated with protein extracts of Taenia taeniaeformis metacestodes was investigated. Chemotactic activity was associated with the nonbound protein after QAE cellulose chromatography of a 3 M KCl extract of homogenized larvae. When this material was precipitated with ammonium sulfate, activity was present in the 40 to 80% precipitate. Upon rechromatography on QAE cellulose equilibrated in a low ionic strength buffer, eosinophil chemotactic activity was retained by the gel and eluted after application of the NaCl gradient. Gel filtration of Sephacryl S-300 yielded an estimated m.w. of 91,000. Chromatofocusing revealed a broad peak of activity with a pI of 4.5 to 5.0. SDS-PAGE showed the active fraction migrated as a protein with a m.w. of 10,400. ECF-Tt had chemotactic and chemokinetic activity for equine eosinophils and murine eosinophils, but not for equine and murine neutrophils.

Stimulation of equine eosinophil migration by hydroxyacid metabolites of arachidonic acid.

Lipoxygenase products of arachidonic acid are important mediators of inflammation, affecting several aspects of cell function. Monohydroxyeicosatetraenoic acid (mono-HETE) and 5,12-dihydroxyeicosatetraenoic acid (LTB4) enhance migration of both neutrophils and eosinophils in several species. The relative ability of positional isomers of HETE and of LTB4 to affect migration of equine eosinophils was studied. The 5, 8, 9, 11, 12, and 15 isomers of HETE were prepared by autooxidation of arachidonic acid, separated by sequential normal phase and reverse phase high performance liquid chromatography, and their identities verified by gas chromatography-mass spectrometry. Equine eosinophils were isolated to 30-70% purity on discontinuous metrizamide gradients. All isomers of HETE stimulated directed migration (chemotaxis) at concentrations ranging from 10(-5) to 10(-8) M. The relative activities of isomers were 11 greater than 9 = 8 = 5 greater than 12 greater than 15. The dihydroxy acid LTB4 maximally stimulated chemotaxis of equine eosinophils at a concentration of 3 X 10(-7) M. The eosinophil migration that resulted was less than the maximal stimulation observed in response to isomers of HETE. The results of our study suggest that equine eosinophils are excellent indicator cells for assay of arachidonic acid metabolites with chemotactic activity. Equine eosinophils are more sensitive to chemotactic stimulation by HETEs than cells from other animal species but are far less sensitive to stimulation by LTB4.

Inflammation and mediators of lung injury.

This article covers the major pathways involved in acute inflammation in mammals with a particular emphasis on their relevance to the bovine species. It focuses on the potential and proven contributions of these systems to pulmonary defense mechanisms and lung pathology. The article also points out what is known and where gaps in our information exist as well as promising areas for research in the coming years.

Natural infection of captive coyote pups with a herpesvirus antigenically related to canine herpesvirus.

Herpesviruses were isolated from captive coyote pups with ocular discharge and hepatomegaly. The viruses were shown to be antigenically related to canine herpesvirus on the basis of specific virus neutralization with canine herpesvirus antiserum. The epizootiology of the outbreak suggested that the herpesvirus was acquired by indirect contact with guard dogs being cared for by the same animal technicians who cared for the coyotes.

Glutathione metabolism in canine hereditary stomatocytosis with mild erythrocyte glutathione deficiency.

Mild glutathione deficiency can accompany hereditary stomatocytosis in man and dogs. We studied several facets of glutathione metabolism in dogs to better define hereditary stomatocytosis. In vivo glutathione flux was measured with alpha-ketoglutarate-alpha-(2-14C) and glycine-(2-3H) in a double-labeled experiment. Glycine-labeled glutathione turnover was faster than that of alpha-ketoglutarate-labeled glutathione, and the glutathione flux was greater in HSt cells than in normal cells. The glutathione-reducing enzymes, glutathione reduction rate, glutathione precursors (except cysteine), oxidized glutathione percentage, and erythrocyte water were increased significantly in HSt cells. Glutathione synthetic enzymes, oxidized glutathione transport system, and glutathione-S-transferase did not differ significantly. These observations suggest that the mild glutathione deficiency in canine hereditary stomatocytosis is a reflection of increased catabolism.