Previously we focused on the microbiology and the pathogenesis of Bacillus anthracis, but today we will focus on the epidemiology of the bacteria.

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Classification of Anthrax in humans can be classified by the spore entry into the host. Anthrax acquired via break in the skin is termed as cutaneous, eating contaminated meat is termed as gastrointestinal and inhaling spores causes inhalation anthrax. (Cendrowski, S. et al, 2004)

Cutaneous anthrax develops a day to 7 days after exposure, it is relatively less dangerous and prevalent. Gastrointestinal anthrax has an incubation period of 1-7 days and is rare in the United States. Inhalation anthrax is the deadliest types with an incubation period of 7-28 days. (CDC, 2014)

Fig 1-Transmission of anthrax.https://www.brainkart.com/article/Epidemiology—Bacillus-anthracis_18070/

The chance of transmitting Anthrax from one person acting as an intermediate fomites to another person is rare and might result in cutaneous form of anthrax. B. anthracis spores maintain their viability in calcium rich soil with at high pH hence highly infectious to their host. Alkaline soil at a temperature of 15.5 °C with high level of moisture and organic matter promotes the vegetative form of the bacteria, which subsequently increase the production of spores. These conditions can affect outbreak of infection. (Dragon & Rennie, 1995)

B. anthracis is found in the soil hence herbivores are vulnerable to anthrax. Anthrax is enzootic in the southern states of India and 1612 outbreaks were reported between 1991 to 1996.Other enzootic cases of anthrax were reported in China, Nepal, Australia, Namibia and South Africa. The zoonotic nature of Anthrax also makes farmers and people handling animal products susceptible to the infection.In 1957, a Plant that processes goat hair in Manchester in the US was hit with an anthrax outbreak which resulted in 9 cases and 4 fatalities. (Hinton, 1999)

B. anthracis can be used as a biological weapon by the deliberate release of anthrax spores. Anthrax spores are targeted for bioterrorism because, the spores are ubiquitous, tasteless, microscopic and are dispersed without detection. In 2001, mailed letter were contaminated with B. anthracis, 22 people were infected, lead to 5 fatalities. (CDC, 2014) The Infectious dose(ID50) of anthrax in humans ranges from 1000-10,000. B. anthracis have monomorphic strains have identical phenotype and genotype ,based on their location of their habitat.Hence the source of the anthrax used for an attack can be determined. (WHO, 2008)

Anthrax can be prevented with antibiotics, Ciprofloxacinand doxycycline or Anthrax Vaccine Adsorbed (AVA). Infections can also be treated antibiotics and antitoxins. (CDC, 2014)

It has been a remarkable journey, hope you enjoyed yourself as much I did. Thanks for indulging me.

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Reference

1. Centers for Disease Control and Prevention,(2014) National Center for Emerging and Zoonotic Infectious Diseases (NCEZID)
2. Dragon, D. C., & Rennie, R. P. (1995). The ecology of anthrax spores: tough but not invincible. The Canadian veterinary journal = La revue veterinaire canadienne, 36(5), 295–301.https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1686874/
3. Hinton MH (1999) Infections and intoxications associated with animal feed and forage which may present a hazard to human health. Vet J. 2000; 159:124–138. [Google Scholar]
4. World Health Organization (2008)Anthrax in humans and animals – 4th ed. All rights reserved. Publications of the World health organization. Who Press, World health organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland

Bacillus anthracis are gram-positive, non-mobile spores extracellular and inside their host, they exist as vegetative state.It has a single circular chromosome  with base pairs of 5,227,293 per DNA molecule with circular double-stranded DNA plasmids, pXO1 and pXO2. (Read,2003)

Video 1-https://www.youtube.com/watchv=7LRGT098lxc#action=share

The trophic level for B. anthracis is heterotrophic and since it is facultative anaerobe, it can be cultured in media with sources of amino acid, carbon, nitrogen and supplementary methionine and thiamine. A defined medium for B. anthracis culture will include glucose, adenine, guanine, thiamine, uracil, calcium and other amino acid sources. (Koehler, 2009)

The bacteria target the hemoglobin since it bound to iron and it is needed for metabolic processes such as growth and reproduction. B. anthracis utilizes hemophores, siderophores and transporter proteins for iron extraction.

Pathogenic mechanism such as inhibiting the normal function of the immune system ,Cytoloysis etc. of B.anthracis. courtesy of https://jamanetwork.com/journals/jama/article-abstract/194886

Inside a host cell, B. anthracis release lysosomal enzymes that lysed the host’s red blood cells, releasing its content, hence the bacterial hemophore takes up the iron released and transfer to their cytosol. Outside of a cell, at low iron levels, B. anthracis produce bacillibactin and petrobactin(siderophores) that have high affinity to iron. The minimal iron available readily binds to the siderophores and are transported via transporter proteins.(Eremenko, 2017; Koppisch et al, 2005; Cendrowski, S. et al, 2004)

B. anthracis thrives best at a temperature of 37°C and at temperatures above 43°C, bacterial growth is halted. At optimum temperature of 37°C, the Cell doubling times range from 30 minutes to 60 minutes in a complex media. (Charlton et al., 2007)

B. anthracis is the causative agent of anthrax, and the type of anthrax is determined by the spore entry into the host. The classification of Anthrax includes cutaneous, gastrointestinal and pulmonary and injection anthrax. (Cendrowski, S. et al,2004) (video 1) When conditions are not favorable, the infectious endospores of B. anthracis are produced .The dormant spores are ideal for bioterrorism since they can withstand any harsh conditions and are easily dispersed without trace.

The sporulation process of Bacillus anthracis.
http://slideplayer.com/slide/5677854/18/images/73/Bacterial+cell+structure+Endospores+(spores)+–+Sporulation.jpg

Binary fission occurs when bacteria g the copied DNA into two and it finally closes off, by forming a cross-wall. The two-daughter cell separate, and they are identical to the mother cell. B. anthracis undergo gene transfer by close contact via conjugation. The virulence plasmids pXO12 and pXO1 can be transferred during conjugation, the bacterial cell with the plasmids become the donor. (Green et al,1989)

We could go, on and on about this amazing bacteria, but time and cyberspace wouldn’t allow.

Bye for now.

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Reference:

1. Cendrowski, S., MacArthur, W. and Hanna P. (2004), Bacillus anthracis requires siderophore biosynthesis for growth in macrophages and mouse virulence. Molecular Microbiology, 51: 407-417. doi:10.1046/j.1365-2958.2003.03861.x

2.Charlton, S. , Herbert, M. , McGlashan, J. , King, A. , Jones, P. , West, K. , Roberts, A. , Silman, N. , Marks, T. , Hudson, M. and Hallis, B. (2007), A study of the physiology of Bacillus anthracis Sterne during manufacture of the UK acellular anthrax vaccine. Journal of Applied Microbiology, 103: 1453-1460. doi:10.1111/j.1365-2672.2007.03391.x

3. Eremenko, E.I. (2017)A Bacillus anthracis system for acquisition of heme-bound iron. Microbiol. Virol. (2017) 32: 1. https://doi.org/10.3103/S0891416817010037

4. Koehler T. M. (2009). Bacillus anthracis physiology and genetics. Molecular aspects of medicine, 30(6), 386–396. doi:10.1016/j.mam.2009.07.004

5.Koppisch, A.T., Browder, C.C., Moe, A.L. et al. Biometals (2005) 18: 577. https://doi.org/10.1007/s10534-005-1782-6

6.Read, T. et al (2003) The genome sequence of Bacillus anthracis Ames and comparison to closely related bacteria .NATURE | VOL 423 | © 2003 Nature Publishing Group 81 | www.nature.com/nature. https://deepblue.lib.umich.edu/bitstream/handle/2027.42/62580/nature01586.pdf?sequence=1&isAllowed=y

https://www.cdc.gov/anthrax/news-multimedia/animations-and-videos.html

The origin of Anthrax is believed to be Egypt and Mesopotamia. It was present during the times of Moses as the fifth Egyptian plagues that affected oven, human, camel etc. (CDC,2017)

The causative agent for Anthrax isBacillus anthracis. Its name was derived from the Greek word for coal, describing the black dry scab observed on affected organisms. B. anthracis is a nonmotile, rod-shaped gram- positive obligate bacteria is an aerobic that ranges from 1–1.5 × 3–10 μm in size. The bacteria exist as oval dormant spore naturally existing in the soil until, introduced via inhalation by their host. They are activated into their vegetative forms triggered by the availability of resources.(Fig 1)

Fig 1.Two forms of B. anthracis in the spore and vegetative form. https://www.cdc.gov/anthrax/basics/index.html

B. anthracis has virulence plasmids pXO1 that has a three toxins lethal factor, edema factor and protective antigen and pXO2 is involved in the production of polyglutamyl capsule. Hence pXO1 is responsible for necrosis ,edema and  bleeding  and PXO2 capsule production prevents phagocytes from engulfing the B. anthracis in its vegetative form in the host.(Leppla ,1982).The toxin produced is an A-B toxin ,where the protective antigen toxin is the medium that delivers the edema and lethal toxin from the outside to inside  of the host cell.(Lacy & Collier, 2002)

The dehydrated spore is surrounded by a membrane and a specialized cortex that limits the exchange of water and solute to and from the spore. This is the protective outer layer that withstands chemical, enzymatic and desiccation. (Setlow, 2006).

B. anthracis cultured at 37°C in sheep blood agar has an off-white appearance, non-hemolytic, comma shaped slightly cover with irregular edges. (Fig 2) It is observed purple rod unicellular or chain or rods when stained with gram stain and observed under the microscope. (Fig 1)

Photo of Bacillus anthracis colonies in blood agar. Courtesy of CDC/Larry Stauffer.

Fig 3. Mucoid colonies of Bacillus anthracis due to production of polyglutamyl capsule. http://textbookofbacteriology.net/Anthrax_2.html

This is all for now about the infamous Anthrax bacteria..

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REFERENCE

1.CDC ( 2017): Centers for Disease Control and Prevention, National Center for Emerging and Zoonotic Infectious Diseases (NCEZID)

2. Leppla SH.(1982)Anthrax toxin edema factor; a bacterial adenylate cyclase that increases cyclic AMP concentrations of eukaryotic cells. Proc Natl Acad Sci U S A1982;79:3162–6. [PMC free article][PubMed] [Google Scholar]

3.Lacy D.B. & Collier R.J. (2002) Structure and Function of Anthrax Toxin. In: Koehler T.M.  Anthrax. Current Topics in Microbiology and Immunology, vol 271. Springer, Berlin, Heidelberg. https://link.springer.com/chapter/10.1007%2F978-3-662-05767-4_4#citeas

4.Setlow P.(2006) Spores of Bacillus subtilis: their resistance to and killing by radiation, heat and chemicals. J Appl Microbiol101, 514–525.

Wiley Online LibraryCASPubMedWeb of Science®Google Scholar

Video 1-Showing representation of Wnt/β-Catenin signaling pathway ,including factors that affects its growth and development. Wnt proteins signal vital process in embryonic development. Courtesy of JJ medicine at https://www.youtube.com/watch?v=NGVP4J9jpgs.

Adipogenesis is the development process of adipocyte.It involves various signaling such as Wnt/β  and Notch signaling pathway. These involves communications between protein receptors, secondary messages and ligand such as growth factor β, interleukin 17, growth factor 1 (IGF1), activin.(Lowe C. E,2011)

Figure 1-Showing the overview of Adipogenesis and the various signaling pathways involved .Courtesy of http://jcs.biologists.org/content/124/16/2681.

Obesity can be studied by determining the role of  Wnt/β-catenin signaling pathway. Wnt/β-catenin signaling pathway is involved in maintaining balance in conditions in developed cells b controlling cell differentiation,  increase in number and its stability.If there was any mutation in any part of the proteins and secondary messengers involved in adipogenesis, it might lead to cancer, neurodegeneration and osteoporosis. The complexity of the cascading signally pathway has lead to more research to find link between obesity and Wnt/β-catenin signaling.

The Wnt/β-catenin signaling was discovered in 1982 in as the Wnt1 gene in mice which regulated the formation of wings , which led to further discovery of the Wnt/β-catenin signaling. Cysteine-rich Wnt proteins produce ligands( ie 40 kDa glycoproteins) that initiate   signaling involving receptors that control  cofactor β-catenin’s transcription activities. We studied about  β-catenin interacting with cadherins and  transmit signals to the cytoplasm. Wnts  regulates and stabilize  β-catenin .  Factors such as growth factor, protein kinase A influences the activity of β- catenin.(Chen et al., 2003)

Figure 2- Digram representing  the Wnt/β-catenin signaling in Adipogenesis.Courtesy of  Wnt proteins work with LRP, FZD, Wnt-FZD-LRP5/6 complex in regulating β-catenin by  preventing it’s phosphorylation. When  β-catenin enters the nucleus, and turns on transcription via binding to members of the lymphoid enhancer factor (TCF/LEF) family of transcription factors. ZNRF3/RNF43 is a negative feedback regulator of Wnt/β-catenin signaling. With a lack of R-spondin proteins (RSPOs), ZNRF3/RNF43 is recruited by DVL to Wnt-FZD-LRP5/6 complex to mediate their degradation (left panel).  RSPO’s presence together with  ZNRF3/RNF43 and LGR4/5/6, leads  to addition of ubiquinine, endocytosis of ZNRF3/RNF43

Wnts phosphorylates β- catenin by using the activities of seven-Transmembrane receptors, protein 5 (LRP5) , Frizzled (FZD) ,seven-Transmembrane receptors as Wnt-FZD-LRP5/6 complex. (Chen et al., 2003). Then β- catenin  binds to lymphoid enhancer factor (TCF/LEF by entering the nucleus thus becoming a transcription factor.(Figure 2)

Reference

Can you imagine, if the human skins could “see” a threat and respond accordingly, while our backs were turned (No seeing with eyes or touching involved)? Eye-independent, light-activated chromatophore expansion (LACE) and phototransduction genes in the skins of cephalopods make this possible. This mind blowing feature of Octopus bimaculoides and other cephalopods is what I would like to discuss on this final blog.

When the skin of O. bimaculoides is exposed to bright white light, the chromatophores present react by expanding, a behavior termed LACE.  LACE responses is evident that the skin of  O. bimaculoides can sense light, independent of eyes.

Ramirez and Oakley hypothesized that r-opsin, a protein responsible for detecting light found in the eyes of octopuses may also be present in their skin, thus activation of light-sensitive chromatophore in their skin was possible, without the input of the eyes (Ramirez et al., 2014).

11 adult Octopus bimaculoides and their hatchlings were obtained, killed and their funnels dissected. Ten dissected funnels were mounted in fresh seawater filled Petri dishes using insect pins. The mounts were set up at equal distance from a white light fiber optic source and the reaction of the chromatophores on the skins was observed and recorded using infrared CCD camera. The photon counts and absolute radiance were measured with spectrophotometer. Three trials for the set ups were performed to ensure accuracy.

There have been a lot of behavioral and physiology research in relations to light sensitivity of mollusks skins (Ramirez et al., 2011). This specific research paper is the best evidence till date for cephalopods’ light-sensitive skin and the role of LACE in controlling chromatophore for camouflage, alongside the main control of the central nervous system.

Further studies to determine on the specific functions of opsin-expressing cells in hatchling O. bimaculoides skin and the extent of importance of the role of opsin in their function/functions apart from photoreception. The evolutionary path to LACE in the skins of octopuses is worth  researching also.

REFERENCE

Ramirez, M. et al (2011). Understanding the dermal light sense in the context of integrative photoreceptor cell biology. Vis. Neurosci. 28, 265-279.

Ramirez M. D.& Oakley T. H. (2014) Eye-independent, light-activated chromatophore expansion (LACE) and expression of phototransduction genes in the skin of Octopus bimaculoides. Journal of Experimental Biology 2015, 218: 1513-1520; doi: 10.1242/jeb.110908    http://jeb.biologists.org/content/jexbio/218/10/1513.full.pdf

KEYWORDS

 Chromatophore is a pigment-containing cell in the deeper layers of the skin of animals. The distribution of the chromatophores and the pigments determine the color patterns of an organism.

Mechanoreceptors are specialized neurons that transmit mechanical deformation information into electrical signals.

Phototransduction is the conversion of light into a change in the electrical potential across the cell membrane. This process involves the sequential activation of a series of signaling proteins, leading to the eventual opening or closing of ion channels in the photoreceptor cell membrane.

Visual phototransduction is the photochemical reaction that take place when light (photon) is converted to an electric signal in the retina. 

In my previous blog while comparing two cladograms of Octopoda, Octopus bimaculoides and Hapalochlaena muculosa were shown as sister taxa in both relationships. A sister taxon relationship based on shared trait of ink sac and two sucker rows.There is an African proverb that embodies the importance of this blog,it states that “show me your friend and I’ll show you your character”.

Fig 1-Blue-ringed octopus, Hapalochlaena muculosa Marcello Di Francesco/courtesy of University of Miami 

 It is befitting to feature Hapalochlaena muculosa as organism of todays’ blog.Commonly known as the Blue-ringed octopus, it has numerous blue eyespots on its mantle as compared to the California two-spot octopus with only one blue eye spots below each eye.(Fig 1) (Lydia M. et al (2012)

Hapalochlaena sp. is also a small sized octopus but normally found along the southern coastal regions of Australia unlike O. bimaculoides found on the southern coast of California Santa Barbara stretching to San Quintin, Mexico. (Tranter, D.J.et al ,1973)

 Hapalochlaena sp. is venomous and displays its numerous vibrant blue rings on mantle when disturbed. This creates an effective conspicuous warning display. The venom contains primarily tetradotoxin, is used to kill or paralyze its prey i.e. crabs and crayfish. (Sheumack et al,1978) Hapalochlaena muculosa was the first species with venom contain primarily tetrodotoxin unlike others that occurs as a poison in the skin, liver, muscle, eggs or ovaries. Tetradotoxin is a very potent neurotoxin that can paralyze and even kill humans.

Fun fact

Copulation in  Hapalochlaena sp. is more vigorous than seen in other octopus and it lasts for about an hour. The male mounts and grasps the female securely and after a brief struggle the female subdues. Finally, the male deposits its sperm in the female’s oviduct using his hectocotylus.(Tranter, D.J.et al ,1973)

Joke of the day

I hope I’m not poisonous, says the first blue-ringed octopus

“Why?” asks the second blue-ringed octopus

“Because I just bit my lip.” said the first.

Reference

1.Lydia M. et al (2012) How does the blue-ringed octopus (Hapalochlaena lunulata) flash its blue rings? Journal of Experimental Biology 2012, 215: 3752-3757; doi: 10.1242/jeb.076869. Accessed on 4/10/2018 at http://jeb.biologists.org/content/215/21/3752.figures-only

2.Sheumack, D. D et al (1978). Maculotoxin: a neurotoxin from the venom glands of the octopus Hapalochlaena maculosa identified as tetrodotoxin. Science 199, 188-189. Accessed on 4/10/2018 at http://science.sciencemag.org/content/199/4325/188/tab-pdf

3.Tranter, D.J. & Augustine, O. (1973) Observations on the life history of the blue-ringed octopus Hapalochlaena maculosa. Marine Biology January 1973, Volume 18, issue 2 pp 115–128. Accessed on 4/10/2018 at https://doi.org/10.1007/BF00348686

FIG. 1. Maximum-parsimony tree obtained from analysis of the unweighted COI data Constraint of monophyly of the Octopodidae resulted in a significantly longer tree, whereas enforcement of monophyly of the Cirrata did not yield a significantly longer tree. Source-(Carlin D.A et al, 2001)

FIG. 2. Maximum-likelihood tree derived from a heuristic search of the COI data under the assumption of a general time-reversible model of substitution with site-specific rates (GTR ) estimated according to the gamma distribution. Branch lengths are drawn proportional to the probabilities of change occurring along each branch under the GTR model.

In today’s blog, let us explore phylogenetic relationships of the Octopoda comparing results from molecular data analyses with classification based on morphological features.

The Octopod phylogeny is complex and full of controversies. Research utilizing Molecular data did not support the Octopodidae as a monophyletic group (based on morphological features).(Carlin D.A et al, 2001)

 Notable disparities were seen at the order of branching.(Fig.1 and 2) At the base, Argonauta branched off first in the MP tree. Argonauta grouped based on smaller mature male compared to mature female, hectocotylus remain coiled inside a pocket until use, and hectocotylus break off during mating. (Nesis, 1987), Whereas in the ML tree, a clade consisting of Argonauta and three members of subfamily octopodinae (O. tetricus, O. bimaculoides, and Hapalochlaena sp.) initially branched off from the others.Based on the various positioning of  O. bimaculoides ,it’s evolutionary history is unconfirmed.

O. tetricus ;O. bimaculoides and Hapalochlaena were shown as sister taxa, in clade subfamily octopodinae .A sister taxon relationship based on shared trait of ink sac and two sucker rows. This was also supported by data from ML and MP trees.

In both tree,O. californicus was closely related to Benthoctopus sp. than O. bimaculoides, although O. californicus shared possession of ink sac and double  sucker rows with O. bimaculoides. Benthoctopus sp. on the other hand possess double sucker row with no  ink sac .(O’Shea S.1999)

MP tree was a  significantly longer tree compared to the ML tree especially since branch lengths were constructed proportionally to the number of parsimony steps between nodes.(Carlin D.A et al, 2001)

REFERENCE

Carlin D.A et al (2001) A molecular phylogeny of the Octopoda (Mollusca: Cephalopoda) evaluated in light of morphological evidence. Molecular phylogenetics and evolution, ISSN: 1055-7903, Vol: 21, Issue: 3, Page: 388-97 .

Nesis, K. N. (1987). “Cephalopods of the World: Squids, Cuttlefishes, Octopuses, and Allies,” Tropical Fish Hobbyist Publ. Inc., Neptune City, NJ. 

a-Genome of Octopus bimaculoides anatomy, highlighting the tissues sampled for transcriptome analysis: viscera (heart, kidney and hepatopancreas), yellow; gonads (ova or testes), peach; retina, orange; optic lobe (OL), maroon; supraesophageal brain (Supra), bright pink; subesophageal brain (Sub), light pink; posterior salivary gland (PSG), purple; axial nerve cord (ANC), red; suckers, grey; skin, mottled brown; stage 15 (St15) embryo, aquamarine. Skin sampled for transcriptome analysis included the eyespot, shown in light blue

The million-dollar question is why are octopuses are so intelligent?

 DNA obtained from Octopus bimaculoides was sequenced and it was discovered that octopus possessed unusually high number of protocadherins, genes that regulate neural development. (Specifically ,168 protocadherin genes, 10 X that found in an invertebrate, and 2X that found in mammals.)

The octopus have a large number of transposons, genes that can change their position in the genome. The genes present in O. bimaculoides were not arranged in the doubly conserved synteny nut rather in clusters along the genome. O. bimaculoides had only a single Hox complement unlike multiple copies. This was similar in Hox transcripts in the bobtail squid. Unique to octopus, the Hox genes were not in clusters but completely atomized. 

These genes are responsible for the unique behavioral and physiological traits such as possessing the largest nervous systems among invertebrates ,camera-like eyes, highly functional arms, and a remarkably sophisticated adaptive camouflaging .

Joke of the day

Why did the octopus beat the shark in a fight? – Because the octopus was well armed.

Reference

1. The octopus genome and the evolution of cephalopod neural and morphological novelties.
   Albertin CB, Simakov O, Mitros T, Wang ZY, Pungor JR, Edsinger-Gonzales E, Brenner S, Ragsdale CW, Rokhsar DS. 2015. Nature. 524:220-224.
2. Duboule, D. The rise and fall of Hox gene clusters. Development 134, 2549–2560 (2007). Retrived online at https://www.nature.com/articles/nature11696