Haemophilus somnus

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Christina Tam BIMM 120

Contents

Classification

Haemophilus somnus (H. somnus; histophilus somni) belongs to bacteria domain, proteobactereia Phylum, Class of gamma proteobacteria, Order of pasteurellales, family of pasteurellaceace, genus Histophilus, species of somnus


Description and significance

Haemophilus somnus is a small, rod-shaped, gram-negative pleomorphic coccobacillus, (derived from the word cocci meaning “spherical shaped” and bacilli meaning “elongated”), with gram stain morphology similar to other species in this genus (as well as other genera of family Pasteurellaceae) [1] . It is an encapsulated, non acid-fast, and non-motile organism with no observable flagella or pili for motility. First discovered in 1960, H. somnus was described as a “haemophillus-like organism” that caused infectious meningo-encephalitis (IME) of feedlot cattle in California and was also believed to be the infectious agent that caused the reported bovine central nervous system disease in Colorado of 1956. Although the organism does not produce a capsule, which is associated with the pathogenic properties of many bacteria, under certain conditions it does form an exopolysaccharide (slime layer). H. somnus can be either opportunistic pathogens or commensal of bovine mucosal surfaces [4]. The bacteria commonly lives in the upper respiratory tract, prepuce and vagina of cattle [5] and can also be found circulating in the bloodstream. H. somnus typically colonize in the respiratory tract, reproductive tract, and circulatory system of many herd animals such as cattle, sheep, and American bison. Cattle infections caused by this bacteria have been reported in many parts of the world including North and South America, Europe, Russia, Japan, Australia, New Zealand, South Africa, and Zimbabwe. Having the bacteria’s genome sequenced is useful because it can be compared to virulent strains to aid in the development of protective strategies and vaccines for cattle against diseases caused by H. somnus [4] .


Genome Structure

The genome of H. somnus consists of a circular chromosome (seq: RS: NC_008309) containing 2,007,700 base pairs and plasmids (an autonomously replicating piece of DNA separate from the chromosome) including plasmid pHS129, which is approximately 5178 bp in length. The organisms genome is made up of roughly 34% GC content and 66% AT and contains genes coding for approximately 1798 different proteins and 65 RNA genes. One of the plasmids isolated from nasal discharge of cattle with respiratory disease was found to be 1065 base pairs long and a 39.2 percent G-C content [11]. It is unable to code for any protein longer than 43 residues and it is currently unclear whether the plasmid codes for any H. somnus factors.

Cell structure and metabolism

Lipooligosaccharides (LOS) are the major outer membrane component of H. somnus. It is composed of 3 main compartments: endotoxin lipid A, an inner core, and an outer core. Both lipid A and the inner core are generally conserved across species as opposed to the outer core, which is species specific. The outer core is made up of up to typically 10 non-repeating hexose resides, which are highly variable in their number of residues and linkage. The LOS is similar to enteric lipo polysaccharides (LPS) except that it does not consist of the O antigen, which confers hydrophilic properties of bacteria and allows for their survival in the gastrointestinal tract. LOS, on the other hand, is less hydrophilic, making it better suited to the mucosal environment[1]. In addition, LOS can also have attached phosphocholine (Pcho) and phosphoethanolamine along with phosphate groups. Pcho is also found across many other species and infections agents such as H. influenzae and other respiratory pathogens.

H. somnus also contains two bacterial transferrin receptors (105 and 73 kDa in size), which are structures that contributes to its virulence (typically in cattle). These function as iron repressible outer membrane proteins which typically bind bovine transferins and are likely to function as iron binding proteins in the host, allowing H. somnus to survive in iron limiting environments[8].

Four immunoglobulin binding proteins have been observed in H. somnus[2]. Three out of the four proteins, which are of molecular weights 350, 270, and 120 kDa are able to strongly bind the bovine IgG (immnuoglobulin G) subclasses, IgA and IgM. The receptor of molecular weight 41 kDa is only able to weakly bind the two subclasses. All four receptors are antigenic related and the 41 kDa receptor seems to be a subunit of the 350, 270, and 120 kDa proteins[2].

Like many bacteria, H. somnus is able to utilize many pathways for energy metabolism including oxidative phosphorylation, carbon fixation , reductive carboxylate cycle (CO2 fixation); methane, nitrogen, and sulfur metabolism. It is also able to metabolize lipids including glycerophospholipids, androgens, and estrogens and amino acids such as glutamate, alanine, glycine, and serine. For a full list of metabolic pathways, refer to http://www.genome.jp/kegg-bin/show_organism?menu_type=pathway_maps&org=hso .

In laboratory environments, H. somnus growth requirements include enriched medias such as brain, heart infusion agar with a supplement of 5 to 10 percent bovine or sheep blood containing under 5 to 20 percent carbon dioxide; additional nutritional requirements for many strains of H. somnus include thiamine pyrophoshate. It is grown at 37 degress celcius and does not require hemin or NAD for growth. Colonies grown on blood agar grow to be around 1-2 mm in size in about 2 to 3 days of growth. They are moist, round, and convex with a butyraceous consistency and a slight gray-yellow color.

Ecology

H. somnus is a parasitic organism that typically lives as a commensal organism in its host. It is commonly found in upper respiratory tract, prepuce and vagina of cattle [5] and can also be found circulating in the bloodstream. Like many haemophilus species, such as Haemophilus influenzae, H. somnus is a pathogen and is the cause of many fatal bovine diseases. They take over many of the host’s mechanisms in order to survive. For instance, H. somnus obtains its iron in iron low environments by attaching to bovine transferrin.

Pathology

Haemophilus somnus causes a wide variety of diseases ranging from pneumonia, abortion, arthritis, myocarditis, and septimia in cattle, sheep, and American bison. It is able to spread throughout cattle herds undetected until symptoms begin to occur. There are 3 primary forms that H. somnus is able to assume: reproductive tract, urinary tract, respiratory tract form.

1.Reproductive and urinary tract form[3]

In this form, H. somnus attacks the reproductive tract of pregnant cows, which may lead to the death of a fetus and subsequent abortion. The attack may also lead infection of the uterus and has been found in the urinary tract and prepuce of bulls which contracted the organism during mating. The excretion of uterine or vaginal discharge and urine of the infected animal also acts to transmit the disease to other unaffected cattle in close proximity, which become exposed to the organism by sniffing or by physically being contacted with the discharge. This can then lead to the respiratory form.

2. Respiratory form[3]

The respiratory form of this infection is able to attack both the upper and lower portions of the respiratory tract. Typically in calves, the upper respiratory tract is infected, leading to what is known as calf diptheria. The surface tissues of the larynx, also known as the voice box, become infected due to the interruption in blood supply, causing the tissue to die and become degraded, making it extremely difficult to breath. If the disease is allowed to spread, the windpipe may also become infected as well as the lungs. If the infection occurs in the lungs, pneumonia may be the result, which can cause rapid death. However, although this is typically the primary cause of pneumonia, it is often outgrown by other organisms such as Pasteurella multocida and Pasteurella haemolytica.

3. The septic form[3]

This form is developed from circulation of infected blood. Clinical signs depend on the location in the bovine body that the organism colonizes while the severity depends on the amount of tissue death as a result of blood clots. If the blockage occurs in the brain or spinal cord, the disease can become nervous disorders.

Application to Biotechnology

At Iowa State University (ISU), a protein from H. somnus that had been cloned into an E. coli vector was used to immunize mice. The recombinant protein, when injected into a mouse, resulted in the generation of antibodies against H. somnus that is now used as a vaccine in cattle to prevent infections by the bacteria.

Haemophilus somnus 129PT, which lacks the surface - binding protein for immunoglobulins, is also being used as a vaccine strain of H. somnus. The vaccine can be used to prevent spontaneous abortions and other systematic diseases that have lead to the widespread death of cattle, and has severely impacted the economic vitality of the agricultural sector worldwide.

Current Research

1. J.F. Challacombe (et. Al) has sequenced the genome of H. somnus 129Pt, a nonpathogenic commensal preputial isolate, and compared it to the genomes of H. somnus, H. influenzae Rd, and H. ducreyi 35000HP. They found genes in H. somnus 129Pt that involved amino acid metabolism, cell surface adhesion, biosynthesis of cofactors, energy metabolism, and electron transport and also determined that there were differences in the numbers and composition of the genes that are involved in metabolism and host colonization among the species. This information was proposed to be potentially useful in determining host specificities, niche preferences, and development of vaccines and protective strategies against these organisms.

2. It was determined that the proteins, transferrin and lactoferrin, found in bovine plasma increased the virulence of H. somnus. The organism was pre-incubated in fetal calf serum for 5 minutes which lead to the increase in virulence. In another set of experiments, H. somnus was pre-incubated in either purified bovine serum or plasma proteins before innoculation in mice; it was found that transferrin led to increased virulence as did lactoferrin. Increased amounts of whole cells and culture supernatant bound to transferrin was detected when the organism was grown in media with restricted iron levels; this property was not observed with lactoferrin although binding was observed. It was thus concluded that the increase of virulence in H. somnus was due to the binding of transferrin receptors to the organisms iron-regulated outer-membrane proteins (IROMPs) by providing the organism with iron and that lactoferrin increased the virulence by some undetermined mechanism. (Geertsema RS et al.) [9]

3. A series of experiments conducted by Kelly E. Beheling, et al. determined that H. somnus adheres to bovine brain endothelial cells (BBECs) in vitro but is unable to invade the cells. The activation of BBEC with tumor necrosis factor alpha (TNF-alpha) significantly increased the number of adherent H. somnus while the addition of exogenious glycosaminoglycans, heparinase digestion of the cell’s glycocalyx, and sodium chlorate inhibition of endothelial sulfated glycan synthesis significantly reduced H. somnus adherence to BBEC. The experiment suggests that heparin-binding proteins on the pathogen could serve as the initial adhesions to sulfated proteoglycans on the BBECs, leading to the ability of H. somnus to infect the bovine CNS. [10]



References

[1] Howard, Michael D.[et al]; “Antigenic Characterization of Haemophilus somnus Lipooligosaccharide"; http://scholar.lib.vt.edu/theses/available/etd-101398-081516/unrestricted/etd.pdf; Virginia Polytechnic Institute, Veterinary Medical Science 1998

[2] Yarnall M., Widders P.R. ,Corebeil L.B. “Isolation and Characterization of Fc Receptors from Haemophilus somnus” Scandinavian Journal of Immunology 28 (2), 129–137. doi:10.1111/j.1365-3083.1988.tb02424.x Volume 28 Issue 2 Page 129 - August 1988

[3] Richey, E. J. "Haemophilus somnus Disease in Cattle" http://edis.ifas.ufl.edu/VM066 ; University of Florida, IFAS Extension; Veterinary Medicine-Large Animal Clinical Sciences Department, Florida Cooperative Extension Service, Institute of Food and Agricultural Sciences, University of Florida. Original publication date December 1, 1990. Revised June 8, 2002.E.J.

[4]Challacombe JF (et al.),“Complete genome sequence of Haemophilus somnus (Histophilus somni) strain 129Pt and comparison to Haemophilus ducreyi 35000HP and Haemophilus influenzae Rd.” http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17172329 Department of Energy Joint Genome Institute, Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM 87545, USA., Epub 2006 Dec 15

[5]Alton C.S. Ward (et al.) “Haemophilus somnus (Histophilus somni) in bighorn sheep” http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=16548330 University of Idaho, College of Agriculture, Caine Veterinary Teaching Center, 1020 East Homedale Road, Caldwell, Idaho, 83607-8098, USA (Ward, Weiser, Anderson); Received March 3, 2005; Accepted August 11, 2005

[6] http://www.genome.jp/kegg-bin/show_organism?org=hso ; Haemophilus somnus; KEGG

[7] Genamics http://genamics.com/cgi-bin/genamics/genomes/genomesearch.cgi?field=ID&query=763; est. January 1999

[8] Andrew Ekins, Andrew (et al.), “Haemophilus somnus Possesses Two Systems for Acquisition of Transferrin-Bound Iron” Microbiology Unit, Department of Natural Resource Sciences, Macdonald Campus, McGill University, Sainte Anne de Bellevue, Quebec, Canada H9X 3V9, Received 31 October 2003/ Accepted 2 April 2004

[9] Geertsema RS, Kimball RA, Corbeil LB. “Bovine plasma proteins increase virulence of Haemophilus somnus in mice.” Department of Pathology, School of Medicine, University of California, San Diego, 200 West Arbor Drive, San Diego, CA 92103-8416, USA. Epub 2006 Nov 27.

[10] Behling-Kelly E, Vonderheid H, Kim KS, Corbeil LB, Czuprynski CJ.”Roles of cellular activation and sulfated glycans in Haemophilus somnus adherence to bovine brain microvascular endothelial cells.” http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=16926425; Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin, 2015 Linden Drive, Madison, WI 53706, USA. 2006 Sep

[11] Izadpanah R. (et al.), “DNA sequence of a small, unidentified plasmid isolated from a Haemophilus somnus strain: short communication” Department of Microbiology and Infectious Diseases, Faculty of Veterinary Science, Szent István University, H-1581 Budapest, P.O. Box 22, Hungary, 2006 Nov. 6






Edited by Christina Tam student of Rachel Larsen and Kit Pogliano

Edited KMG

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