Vertebrate Intestine Research Articles

Giardia\u2019s primitive GPL biosynthesis pathways with parasitic adaptation \u2018patches\u2019: implications for Giardia\u2019s evolutionary history and for finding targets against Giardiasis

Giardia is a worldwide spread protozoan parasite colonizing in small intestines of vertebrates, causing Giardiasis. The controversy about whether it is an extremely primitive eukaryote or just a highly evolved parasite has become a fetter to its uses as a model for both evolutionary and parasitological studies for years. Glycerophospholipid (GPL) synthesis is a conserved essential cellular process, and thus may retain some original features reflecting its evolutionary position, and this process should also have undergone parasitic adaptation to suit Giardia’s dietary lipid-rich environment. Thus, GPL synthesis pathways may be a perfect object to examine the controversy over Giardia. Here, we first clarified Giardia’s previously confusing GPL synthesis by re-identifying a reliable set of GPL synthesis genes/enzymes. Then using phylogenetic and comparative genomic analyses, we revealed that these pathways turn out to be evolutionarily primitive ones, but with many secondary parasitic adaptation ‘patches’ including gene loss, rapid evolution, product relocation, and horizontal gene transfer. Therefore, modern Giardia should be a mosaic of ‘primary primitivity’ and ‘secondary parasitic adaptability’, and to make a distinction between the two categories of features would restart the studies of eukaryotic evolution and parasitic adaptation using Giardia as a model system.

Proteolytic enzymes and their inhibitors in cestodes

The review summarizes the available published data on proteases and their inhibitors in cestodes. The intricate physiological relationships between a parasite and its host are confirmed by the ability of cestodes to produce their own proteolytic enzymes or to adsorb and utilize host proteases, on the one hand, and to secrete inhibitors of these enzymes, on the other. It is likely that the protease inhibitors primarily confront the host immune response in larval cestodes versus adult worms, living in the vertebrate intestine, where they mainly protect the parasites from hydrolysis by intestinal proteinases. In addition, the presence of the parasite in the intestine often entails changes in the activity of host proteolytic enzymes.

How thyroid hormone regulates transformation of larval epithelial cells into adult stem cells in the amphibian intestine

In the amphibian intestine during metamorphosis, a small number of larval epithelial cells dedifferentiate into adult stem cells that newly form the adult epithelium analogous to the mammalian counterpart, while most of them undergo apoptosis. Because this larval-to-adult intestinal remodeling can be experimentally induced by thyroid hormone (TH) both in vivo and in vitro, TH response genes identified in the Xenopus intestine provide us valuable clues to investigating how adult stem cells and their niche are formed during postembryonic development. Their expression and functional analyses by using the culture and recent transgenic (Tg) techniques have shed light on key signaling pathways essential for intestinal stem cell development. The present review focuses on such recent findings and discusses the evolutionally conserved roles of TH in development or maintenance of the stem cells which are common to the terrestrial vertebrate intestines.

Interactions between Giardia duodenalis Sm proteins and their association with spliceosomal snRNAs.

Giardia duodenalis is a parasite that colonises the intestines of humans and other vertebrates, causing diarrhoea and poor nutrient absorption. G. duodenalis is sometimes considered an early diverging eukaryote, and its genome exhibits simplified molecular machinery for many cellular processes, which makes it an interesting model to study. The spliceosome, one of the most complex molecular machines in the eukaryotic cell, is responsible for intron excision and exon splicing. Just over a decade ago, it was believed that the G. duodenalis genome did not contain introns or undergo splicing. Research now shows that this speculation was incorrect and that uncommon mechanisms, such as trans-splicing from different genes, occur. In silico studies of the parasite suggest the possibility of a simplified spliceosome and spliceosomal small nuclear RNA (snRNA) candidates; however, none of these components have been identified in vivo. Here, we developed a strategy to study the in vivo expression, interactions and localisation of these spliceosome components in G. duodenalis. Haemagglutinin (HA)-tagged SmB and SmD3 proteins, which form part of the spliceosome core, were overexpressed in the parasite. Immunoprecipitation with anti-HA revealed that the SmD3 protein is associated with the proteins SmB, SmD1, SmD2, SmE and SmF in vivo. In addition, the U1, U2 and U4 snRNA candidates reported previously were found in the protein complex, suggesting that these molecules are spliceosomal snRNAs of G. duodenalis and they contained a 2,2,7-trimethylguanosine modification at their 5' end. Our data indicate that the actively expressed spliceosome in G. duodenalis is similar to that of highly evolved protists and higher animals.

MT1-MMP and its potential role in the vertebrate intestinal morphogenesis

Membrane type 1-matrix metalloproteinase (MT1-MMP) is involved in numerous biological processes, including morphogenesis. However, the role of MT1-MMP in the development of the vertebrate intestine is poorly understood. This study aimed to evaluate the expression of MT1-MMP in the intestine of rats and chickens along the embryonic and postnatal periods using immunohistochemistry. Results revealed a remarkable spatiotemporal correlation between MT1-MMP expression and intestinal villi morphogenesis in both vertebrates. However, the villi morphogenesis process was found to be different in chickens to that of rats. Moreover, extensive MT1-MMP labeling was observed in the entire villus epithelium from birth until the complete maturation of the small intestinal mucosa in both vertebrates. From these results, we suggest that MT1-MMP contributes to intestinal development, particularly to villi morphogenesis, in both vertebrates. However, further studies are necessary to confirm the role of MT1-MMP in this cellular process. In addition, we performed validation of the primary antibody against human MT1-MMP for adult chickens.

Genetic characterization of commensal Escherichia coli isolated from laboratory rodents.

BackgroundEscherichia coli, a commensal in the intestines of vertebrates, is capable of colonizing many different hosts and the environment. Commensal E. coli strains are believed to be the precursor of pathogenic strains by means of acquisition of antimicrobial resistant and virulence genes. Laboratory rodents are inherently susceptible to numerous known infectious agents, which could transfer virulence determinants to commensal E. coli. Hence, in this study, the genetic structure of commensal E. coli found in laboratory rodents and their antimicrobial resistance profiles were investigated.ResultsE. coli strains belonging to phylogroup A were the predominant strain obtained from the animals used in the study. Four novel sequence types (ST746, ST747, ST748 and ST749) were discovered using the multi locus sequence typing, together with one common ST357 in the gastrointestinal tract, liver and, the trachea and lung. Serotyping demonstrated that these commensal E. coli strains were non-Shiga toxin-producers. Phenotypic and genotypic analyses of extended spectrum beta lactamases were also negative.ConclusionsThese findings implied that the E. coli strains recovered from the laboratory rodents were truly commensal in nature. Further study is required to investigate the possible influence of gender on the susceptibility of hosts to E. coli colonization in laboratory rodents.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-016-2745-9) contains supplementary material, which is available to authorized users.

Histochemical differences along the intestine of Corydoras paleatus (Siluriformes: Callichthyidae).

The Neotropical catfish Corydoras paleatus is a facultative air-breather and the caudal half of the intestine is involved in gas exchange. In South America, air-breathing fishes are found in tropical or sub-tropical freshwaters where the probability of hypoxia is high. The aim of this study was to characterize by traditional histochemical and lectinhistochemical methods the pattern of carbohydrate in the intestinal mucosa. Intestine samples were taken from 25 healthy adult specimens collected in Buenos Aires (Argentina). Samples were fixed by immersion in 10 % buffered formalin and routinely processed and embedded in paraffin wax. Subsequently, these sections were incubated in the biotinylated lectins battery. Labeled Streptavidin-Biotin (LSAB) system was used for detection, diaminobenzidine as chromogen and haematoxylin as a contrast. To locate and distinguish glycoconjugates (GCs) of the globet cells, we used the following histochemical methods: PAS; PAS*S; KOH/ PA*S; PA/Bh/KOH/PAS; KOH/PA*/Bh/PAS; Alcian Blue and Toluidine Blue at different pHs. Microscopically, the general structure of vertebrate intestine was observed and showed all the cell types characteristic of the intestinal epithelium. The cranial sector of catfish intestine is a site of digestion and absorption and its structure is similar to other fish groups. In contrast, enterocytes of the caudal portion are low cuboidal cells; and between these, globet cells and capillaries are observed, these latter may reach the mucosal lumen. Underlying the epithelium, observed a well-developed lamina propria-submucosa made of connective tissue; this layer was highly vascularized and did not exhibit glands. According to histochemistry, the diverse GCs elaborated and secreted in the intestine are associated with specific functions in relation to their physiological significance, with special reference to their role in lubrication, buffering effect and prevention of proteolytic damage to the epithelium together with other biological processes, such as osmoregulation and ion exchange. The lectinhistochemical analysis of the intestinal mucosa reveals the presence of terminal residues of glucose, mannose and galactose. In conclusion, this study has shown that GCs synthesized in the intestine of C. paleatus exhibit a high level of histochemical complexity and that the lectin binding pattern of the intestinal mucosa is characteristic of each species and the variations are related with the multiple functions performed by the mucus in the digestive tract. The information generated here may be a relevant biological tool for comparing and analyzing the possible glycosidic changes in the intestinal mucus under different conditions, such as changes in diet or different pathological stages.

Dietary lipid absorption and lipoprotein secretion by the intestine of the crocodilian Caiman yacare (Daudin, 1802)

Although it is stated that dietary lipids are absorbed proximally in the small intestine of vertebrates, there are variations of the primary site for lipid absorption even when closely related species are considered. Moreover, there are evidences suggesting that the small intestine distal segments are equally capable of absorbing lipids, although it is not known whether it is the case for crocodilians. The lipoprotein assembling process and secretion routes are also largely unknown for crocodilians and therefore, assumed to be similar to mammals. The aims of this study were to identify the crocodilian Caiman yacare intestinal segments where lipid absorption occurs, to characterize the intestinal lipoproteins secreted by enterocytes and to evaluate lymphatic system contribution to exportation of lipoproteins from the intestine. For this, soybean oil was injected into C. yacare stomach and intestinal lipid absorption process was characterized by light and electron microscopy 24, 48 and 72 h after oil injection. The same amount of lipid inclusions was present in the duodenum, in the proximal jejunum and in the distal jejunum. The colon also showed a few lipid inclusions. The bulk of lipoproteins secreted by the enterocytes was <200 nm in diameter and was observed inside the lymphatic central lacteals. Lipid inclusions were absent from the intestinal mucosa and from the lacteals of the control animals. Finally, the high amount of lipids ingested did not recruit innate immune cells to the mucosa in any intestinal segment, suggesting that soybean oil is not pro-inflammatory for intestinal mucosa of C. yacare in the short time analyzed.

Identification of Population Bottlenecks and Colonization Factors during Assembly of Bacterial Communities within the Zebrafish Intestine.

ABSTRACTThe zebrafish, Danio rerio, is a powerful model for studying bacterial colonization of the vertebrate intestine, but the genes required by commensal bacteria to colonize the zebrafish gut have not yet been interrogated on a genome-wide level. Here we apply a high-throughput transposon mutagenesis screen to Aeromonas veronii Hm21 and Vibrio sp. strain ZWU0020 during their colonization of the zebrafish intestine alone and in competition with each other, as well as in different colonization orders. We use these transposon-tagged libraries to track bacterial population sizes in different colonization regimes and to identify gene functions required during these processes. We show that intraspecific, but not interspecific, competition with a previously established bacterial population greatly reduces the ability of these two bacterial species to colonize. Further, using a simple binomial sampling model, we show that under conditions of interspecific competition, genes required for colonization cannot be identified because of the population bottleneck experienced by the second colonizer. When bacteria colonize the intestine alone or at the same time as the other species, we find shared suites of functional requirements for colonization by the two species, including a prominent role for chemotaxis and motility, regardless of the presence of another species.

Spatial and temporal features of the growth of a bacterial species colonizing the zebrafish gut.

ABSTRACTThe vertebrate intestine is home to microbial ecosystems that play key roles in host development and health. Little is known about the spatial and temporal dynamics of these microbial communities, limiting our understanding of fundamental properties, such as their mechanisms of growth, propagation, and persistence. To address this, we inoculated initially germ-free zebrafish larvae with fluorescently labeled strains of an Aeromonas species, representing an abundant genus in the zebrafish gut. Using light sheet fluorescence microscopy to obtain three-dimensional images spanning the gut, we quantified the entire bacterial load, as founding populations grew from tens to tens of thousands of cells over several hours. The data yield the first ever measurements of the growth kinetics of a microbial species inside a live vertebrate intestine and show dynamics that robustly fit a logistic growth model. Intriguingly, bacteria were nonuniformly distributed throughout the gut, and bacterial aggregates showed considerably higher growth rates than did discrete individuals. The form of aggregate growth indicates intrinsically higher division rates for clustered bacteria, rather than surface-mediated agglomeration onto clusters. Thus, the spatial organization of gut bacteria both relative to the host and to each other impacts overall growth kinetics, suggesting that spatial characterizations will be an important input to predictive models of host-associated microbial community assembly.

Exploring the physiology and pathology of aging in the intestine of Drosophila melanogaster

The gastrointestinal tract, due to its role as a digestive organ and as a barrier between the exterior and interior milieus, is critically impacted by dietary, environmental, and inflammatory conditions that influence health and lifespan. Work in flies is now uncovering the multifaceted molecular mechanisms that control homeostasis in this tissue, and establishing its central role in health and lifespan of metazoans. The Drosophila intestine has thus emerged as a productive, genetically accessible model to study various aspects of the pathophysiology of aging. Studies in flies have characterized the maintenance of regenerative homeostasis, the development of immune senescence, the loss of epithelial barrier function, the decline in metabolic homeostasis, as well as the maintenance of epithelial diversity in this tissue. Due to its fundamental similarity to vertebrate intestines, it can be anticipated that findings obtained in this system will have important implications for our understanding of age-related changes in the human intestine. Here, I review recent studies exploring age-related changes in the fly intestine, and their insight into the regulation of health and lifespan of the animal.

Competitive regulation of human intestinal β-carotene 15,15′-monooxygenase 1 (BCMO1) gene expression by hepatocyte nuclear factor (HNF)-1α and HNF-4α

AimAmong the pro-vitamin A carotenoids, β-carotene is an excellent source of vitamin A. β-Carotene 15,15′-monooxygenase 1 (BCMO1) is a critical enzyme involved in the conversion of β-carotene into vitamin A (retinal) in the small intestine of many vertebrates. In the present study, we investigated the regulation of human BCMO1 gene expression using human intestinal Caco-2 BBe cells. Main methodsWe performed electrophoretic mobility shift assays and chromatin immunoprecipitation assays to investigate the binding properties of hepatocyte nuclear factor (HNF)-1α and HNF-4α to the proximal promoter of the human BCMO1 gene. Caco-2 BBe cells were also transfected with HNF-1α and HNF-4α siRNAs, and BCMO1 gene expression levels and promoter activity were analyzed by real-time reverse transcription-polymerase chain reaction and luciferase reporter assays, respectively. Key findingsWe identified overlapping binding sites for HNF-1α and HNF-4α in the human BCMO1 gene proximal promoter. Endogenous nuclear HNF-1α and HNF-4α proteins competitively bound these sites in Caco-2 BBe cells. BCMO1 gene expression levels and promoter activity were significantly decreased in HNF-1α siRNA-transfected Caco-2 BBe cells. In contrast, HNF-4α siRNA-transfected cells exhibited a significant increase in BCMO1 gene expression and promoter activity. Mutation of these overlapping binding sites dramatically decreased BCMO1 promoter activity. SignificanceOur study indicates that the competitive actions of HNF-1α and HNF-4α on their overlapping binding sites in the human BCMO1 gene promoter oppositely regulate BCMO1 gene expression in the human small intestine.

Identification and characterization of neurotrophic factors in porcine small intestinal submucosa

“Small intestinal submucosa” (SIS) is a natural degradable biomaterial derived from the small intestine of vertebrates. Porcine SIS has been widely used in repairing various tissues and organs, especially in nervous tissues because intrinsic autonomic nerve plexuses are included in submucosal layer. However, little is known about active neurotrophic factors in this material. In this study, we proposed a method to prepare an extract from decellularized SIS and to identify and characterize the major neurotrophic factors in it. Decellularized SIS extract was obtained by sequential procedures: mechanical disassociation, degreasing, enzyme digestion, freeze-drying, freezer-milling, extracting, concentrating, and filtering. Contamination with cellular components in the SIS extract was evaluated by hematoxylin and eosin (H&E) staining and the content of genomic DNA. Major neurotrophic factors in SIS extract was assessed quantitatively by ELISA. The effects of SIS extracts on cells were evaluated by observation of neurite outgrowth from PC12 cells. Decellularized SIS extract was obtained successfully by the proposed method and no cellular component was found within it. Brain-derived neurotrophic factor (BDNF), erythropoietin (EPO), glial cellderived neurotrophic factor (GDNF), nerve growth factor (NGF), and platelet-derived growth factor (PDGF) were identified and characterized form SIS extract. The bioactivity of SIS extract was assessed in terms of neurite outgrowth from PC12 cells. In summary, we prepared a decellularized extract from porcine SIS and identified major neurotrophic factors present in it. SIS extract also had bioactivity with PC12 cells. From these results, we conclude that SIS may be useful to repair or improve nervous system function.

Phytoceramide in Vertebrate Tissues: One Step Chromatography Separation for Molecular Characterization of Ceramide Species

Ceramide is a precursor for complex sphingolipids in vertebrates, while plants contain phytoceramide. By using a novel chromatography purification method we show that phytoceramide comprises a significant proportion of animal sphingolipids. Total ceramide including phytoceramide from mouse tissue (brain, heart, liver) lipid extracts and cell culture (mouse primary astrocytes, human oligodendroglioma cells) was eluted as a single homogenous fraction, and then analyzed by thin layer chromatography, and further characterized by gas chromatography-mass spectrometry (GC-MS). We detected a unique band that migrated between non-hydroxy fatty acyl ceramide and hydroxy fatty acyl ceramide, and identified it as phytoceramide. Using RT-PCR, we confirmed that mouse tissues expressed desaturase 2, an enzyme that has been reported to generate phytoceramide from dihydroceramide. Previously, only trace amounts of phytoceramide were reported in vertebrate intestine, kidney, and skin. While its function is still elusive, this is the first report of phytoceramide characterization in glial cells and vertebrate brain, heart, and liver.

Expression Profiling of Intestinal Tissues Implicates Tissue-Specific Genes and Pathways Essential for Thyroid Hormone-Induced Adult Stem Cell Development

The study of the epithelium during development in the vertebrate intestine touches upon many contemporary aspects of biology: to name a few, the formation of the adult stem cells (ASCs) essential for the life-long self-renewal and the balance of stem cell activity for renewal vs cancer development. Although extensive analyses have been carried out on the property and functions of the adult intestinal stem cells in mammals, little is known about their formation during development due to the difficulty of manipulating late-stage, uterus-enclosed embryos. The gastrointestinal tract of the amphibian Xenopus laevis is an excellent model system for the study of mammalian ASC formation, cell proliferation, and differentiation. During T3-dependent amphibian metamorphosis, the digestive tract is extensively remodeled from the larval to the adult form for the adaptation of the amphibian from its aquatic herbivorous lifestyle to that of a terrestrial carnivorous frog. This involves de novo formation of ASCs that requires T3 signaling in both the larval epithelium and nonepithelial tissues. To understand the underlying molecular mechanisms, we have characterized the gene expression profiles in the epithelium and nonepithelial tissues by using cDNA microarrays. Our results revealed that T3 induces distinct tissue-specific gene regulation programs associated with the remodeling of the intestine, particularly the formation of the ASCs, and further suggested the existence of potentially many novel stem cell-associated genes, at least in the intestine during development.

Paracellular absorption is relatively low in the herbivorous Egyptian spiny-tailed lizard, Uromastyx aegyptia.

Absorption of small water-soluble nutrients in vertebrate intestines occurs both by specific, mediated transport and by non-specific, passive, paracellular transport. Although it is apparent that paracellular absorption represents a significant route for nutrient absorption in many birds and mammals, especially small, flying species, its importance in ectothermic vertebrates has not previously been explored. Therefore, we measured fractional absorption (ƒ) and absorption rate of three paracellular probes (arabinose, l-rhamnose, cellobiose) and of 3-O-methyl d-glucose (absorbed by both mediated and paracellular pathways) by the large herbivorous lizard, Uromastyx aegyptia, to explore the relative importance of paracellular and mediated transport in an ectothermic, terrestrial vertebrate. Fractional absorption of 3-O-methyl d-glucose was high (ƒ = 0.73±0.04) and similar to other vertebrates; ƒ of the paracellular probes was relatively low (arabinose ƒ = 0.31±0.03, l-rhamnose ƒ = 0.19±0.02, and cellobiose ƒ = 0.14±0.02), and decreased with molecular mass, a pattern consistent with other vertebrates. Paracellular absorption accounted for approximately 24% of total 3-O-methyl d-glucose uptake, indicating low reliance on this pathway for these herbivorous lizards, a pattern similar to that found in other terrestrial vertebrates, and different from small flying endotherms (both birds and bats).

Intestinal inflammation and stem cell homeostasis in aging Drosophila melanogaster

As a barrier epithelium, the intestinal epithelium has to coordinate physiological functions like digestion and nutrient resorption with the control of commensal bacteria and the prevention of pathogenic infections. It can therefore mount powerful innate immune and inflammatory responses, while, at the same time, maintaining tissue homeostasis through regenerative processes. How these different functions are coordinated remains unclear, and further insight is required to understand the age-related loss of homeostasis in this system, as well as the etiology of inflammatory and proliferative diseases of the gut. Recent work in Drosophila melanogaster has provided important new insight into the regulation of regenerative activity, innate immune homeostasis, commensal control, as well as age-related dysfunction in the intestine. Interestingly, many of the identified processes and mechanisms mirror similar homeostatic processes in the vertebrate intestine. This review summarized the current understanding of how innate immune responses, changes in commensal bacteria, and other challenges influence regenerative activity in the aging intestinal epithelium of flies and draws parallels to similar processes in mammals.

Investigating Bacterial-Animal Symbioses with Light Sheet Microscopy

Microbial colonization of the digestive tract is a crucial event in vertebrate development, required for maturation of host immunity and establishment of normal digestive physiology. Advances in genomic, proteomic, and metabolomic technologies are providing a more detailed picture of the constituents of the intestinal habitat, but these approaches lack the spatial and temporal resolution needed to characterize the assembly and dynamics of microbial communities in this complex environment. We report the use of light sheet microscopy to provide high-resolution imaging of bacterial colonization of the intestine of Danio rerio, the zebrafish. The method allows us to characterize bacterial population dynamics across the entire organ and the behaviors of individual bacterial and host cells throughout the colonization process. The large four-dimensional data sets generated by these imaging approaches require new strategies for image analysis. When integrated with other "omics" data sets, information about the spatial and temporal dynamics of microbial cells within the vertebrate intestine will provide new mechanistic insights into how microbial communities assemble and function within hosts.

Proteomic analysis of the ventral disc of Giardia lamblia

BackgroundGiardia lamblia is a multiflagellated protozoan that inhabits the small intestine of vertebrates, causing giardiasis. To colonize the small intestine, the trophozoites form of the parasite remains attached to intestinal epithelial cells by means of cytoskeletal elements that form a structure known as the ventral disc. Previous studies have shown that the ventral disc is made of tubulin and giardins.ResultsTo obtain further information on the composition of the ventral disc, we developed a new protocol and evaluated the purity of the isolation by transmission electron microscopy. Using 1D- and 2D-PAGE and mass spectrometry, we identified proteins with functions associated with the disc. In addition to finding tubulin and giardin, proteins known to be associated with the ventral disc, we also identified proteins annotated in the Giardia genome, but whose function was previously unknown.ConclusionsThe isolation of the ventral disc shown in this work, compared to previously published protocols, proved to be more efficient. Proteomic analysis showed the presence of several proteins whose further characterization may help in the elucidation of the mechanisms involved in the attachment of the protozoan to epithelial cells.

Diversity of Enterococcus cecorum from chickens

Enterococcus cecorum is a normal inhabitant of the intestine of birds and other vertebrates. It has recently emerged in Canada and other countries as an important cause of arthritis and osteomyelitis in chickens. The objectives of this study were to assess if this emergence was caused by a particular clone of E. cecorum and to assess the antimicrobial susceptibility of this organism.One hundred and thirteen E. cecorum isolates from infections in Canadian chickens (cases) and from the ceca of control chickens from Canada and Belgium were examined. Isolates were identified using biochemical tests and, for a number of them, identification was confirmed by partial 16S rRNA gene sequencing. Case and control isolates were typed by pulsed-field gel electrophoresis and tested for antimicrobial susceptibility using the broth microdilution method.Cecal isolates from control birds were genetically very diverse but the vast majority of those from cases belonged to a single major clonal lineage. Reduced susceptibility was widespread for tetracycline, bacitracin, and erythromycin. Isolates from cases were generally less susceptible to antimicrobial agents than isolates from control birds.