Koch's Postulates

157,158 Fulfilling Koch's postulates, application of the superantigen staphylococcal enterotoxin B (SEB) to the peel tin induce peel changes of erythema and induration accompanied by the infiltration of T cells that are selectively expanded in response to SEB.

From: Pediatric Allergy: Principles and Practice (2nd Edition) , 2010

The Evolution of Koch's Postulates

Jonathan Cohen , in Infectious Diseases (Fourth Edition), 2017

Conclusions – and a Note of Circumspection

Koch's postulates were invaluable at the time they were developed and remain largely valid for a relatively small-scale number of defined circumstances in which bacteria can be precisely tied to the cause of a particular clinical syndrome. But in a globe in which viruses crusade cancer and noncultivable leaner can be demonstrated by molecular probes, Koch's postulates are no longer fit for purpose. What is more, used uncritically they have the potential to mislead. 16 Their principal purpose now is to provide a framework to ensure that scientific rigor is applied when proposing an organism as the cause of a illness – exactly as Koch intended when he kickoff conceived them.

References available online at expertconsult.com .

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New technological developments in identification and monitoring of new and emerging infections

Gert Uves van Zyl , in Reference Module in Biomedical Sciences, 2021

Identifying agents and proving an etiological role in disease

Koch's postulates, which require the presence of an infectious amanuensis in cases with disease, and its absence in those without, and the isolation of the agent in pure cultures, were initially very important to place infectious etiologies. All the same, it had limitations. Many organisms cannot be readily grown in cultures. Moreover, the association between infectious agent and disease is non always a simple unmarried agent deterministic clan merely could be more complex—infections could be present without causing disease or may not be the only (or necessary cause) of a disease. In addition to infection, it may require other factors such every bit host risk factors to cause affliction. In the latter case the infectious agent is non a sufficient cause ( Byrd and Segre, 2016; Falkow, 2004). Illness complications may also be the upshot of chronic infections with a long fourth dimension lag between infection and disease (O'Connor et al., 2006) which makes it more hard to directly associate the infectious amanuensis and affliction.

An adaptation of Koch's postulates was therefore proposed based on molecular criteria that relied on identification of pathogen sequences (Falkow, 1988). Yet, complex etiologies crave epidemiological testify that shows an increased risk in individuals who have particular infections compared to those without. In the example of organisms that consequence in pathologies such equally neoplasms, or other delayed complications of infections a combination of molecular and epidemiological evidence is oft required. Cervical cancer is almost always the consequence of infection with oncogenic types of human papillomaviruses (Nicolás et al., 2019) and hepatitis C virus and hepatitis B virus infections are strongly associated with liver cancer. However, other infections such every bit homo herpes virus eight (HHV8) and Epstein bar virus (EBV) require cofactors before oncogenesis is likely (Mesri et al., 2014). Proving the association of chronic Helicobacter pylori stomach infection with duodenal ulcers and stomach cancer has required epidemiological and intervention studies, in addition to identifying the infection in people with disease (Choi et al., 2018; Preda et al., 2009). Recent developments in human genetics, the analysis of mRNA expression (transcriptomics), the report of protein structure, function and interaction (proteinomics), and metabolic pathways (metabolomics) have resulted in increasingly rich data, which help to explain the interaction of host genetics, infections, and the environment. In future, novel approaches to analyze such rich data could allow for a better agreement of cellular level pathways of diseases with complex etiologies. This could also provide novel drug targets to treat these diseases (Karczewski and Snyder, 2018).

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Changing how nosotros think about infectious diseases

Jules J. Berman , in Taxonomic Guide to Infectious Diseases (Second Edition), 2019

Department 8.1 Abandoning Koch's postulates

It isn't that they can't encounter the solution. It'due south that they can't run into the problem.

Yard.K. Chesterton

Robert Koch's postulates, published in 1890, are a set of criteria that establish whether a particular organism is the cause of a item disease. Today, Koch's postulates are taught in high school and higher classrooms as a sit-in of the rigor and legitimacy of clinical microbiology. To review, the iv postulates of Koch are as follows:

–1.

The microorganism must be found in the diseased animate being, and non found in healthy animals.

–2.

The microorganism must be extracted and isolated from the diseased animal and later grown in culture.

–3.

The microorganism must cause disease when introduced to a healthy experimental animal.

–4.

The microorganism must be extracted from the diseased experimental creature and demonstrated to exist the same microorganism that was originally isolated from the first diseased animal.

Let'southward go over these four postulates in one case more, this time explaining how they ignore or contradict what we at present know about infectious diseases.

–ane.

The microorganism must exist found in the diseased animal, and not found in healthy animals.

As previously discussed, lots of pathogenic organisms are establish in good for you animals, producing disease in only a tiny fraction of the individuals who are infected. For case, Bartonella species can live in blood without causing disease, producing an asymptomatic bacteremia in the broad array of animals that they may infect. Hence, nosotros can no longer assume that claret samples from healthy animals are sterile. The mechanism of Bartonella transmission from animal to animal is not fully understood, but arthropod vectors (ticks, fleas, and lice) are suspected, every bit well as scratches and bites from infected animals (e.thousand., cats and rats) [1] [Glossary Vector].

There are now most eight species of Bartonella that are known or suspected to exist human pathogens. Until but a few decades ago, only ii such species were known. Today, the species of Bartonella, which are ubiquitous among mammals, are known or suspected to crusade a variety of phenotypically dissimilar diseases [1]:

–

Bartonella bacilliformis → Carrion disease

–

Bartonella quintana → Bacillary angiomatosis, trench fever, endocarditis

–

Bartonella henselae → Bacillary angiomatosis, cat-scratch disease, peliosis hepatis B. henselae

–

Bartonella clarridgeiae → True cat-scratch disease [2]

–

Bartonella elizabethae → Endocarditis

–

Bartonella vinsonii var berkhoffii → endocarditis

–

Bartonella vinsonii var arupensis → fever and a valvulopathy

–

Bartonella grahamii → uveitis

The precise diagnosis of Bartonella species in homo claret and lesions has provided u.s. with the names of infectious organism associated with a number of diseases, but this new noesis has non shed much light on why Bartonella can broadcast in the blood without causing any reaction, for indefinite periods of time, or why whatsoever given Bartonella species may be associated with any of several diverse clinical manifestations. Furthermore, Koch'due south tertiary postulate fails miserably for genus Bartonella; injecting any of these Bartonella species into experimental animals, volition more than probable produce no symptoms.

–2.

The microorganism must exist extracted and isolated from the diseased animal (and grown in culture).

Many pathogens do not grow in nutrient medium civilization. This applies generally to common Mollicute bacteria, including Erysipelothrix, Mycoplasma, and Ureoplasma. This would also apply to viruses, none of which grow in cell-complimentary media. Paradoxically, some of the organisms known to produce bacteremias in man blood abound very poorly in blood cultures, and this would include the aforementioned Bartonella species and the HACEK organisms [1, 3]. The HACEK organisms are a group of proteobacteria, found in otherwise salubrious individuals, that are known to cause some cases of endocarditis, especially in children, and which do not abound well in culture. The term HACEK is created from the initials of the organisms of the grouping: Haemophilus, particularly Haemophilus parainfluenzae; Aggregatibacter, including Aggregatibacter actinomycetemcomitans and Aggregatibacter aphrophilus; Cardiobacterium hominis; Eikenella corrodens; and Kingella, peculiarly Kingella kingae.

–iii.

The microorganism must cause disease when introduced to a healthy experimental animal.

Once more, some of the worst microorganisms will not produce disease in healthy animals. To misfile matters farther, nosotros now have examples of nonliving agents that will produce transmissible affliction in healthy animals (prions).

This third postulate of Koch presumes that each occurrence of an infectious disease has a particular organism that is "the cause" of the disease. We must return here to our often-repeated theme that diseases practice not have "a cause," and infectious diseases are no exception to the dominion that pathogenesis is a multistep procedure. Nosotros take already seen that myocardial infarction results from a multitude of weather condition that occur through time. In some cases, the last event is infectious, wherein a focal bacterial endocarditis precipitates a thrombus that blocks a narrowed coronary avenue. Information technology would be folly to believe that the sequence of events that atomic number 82 to a myocardial infarction can be precipitated simply by injecting an organism into an animal. Later in this chapter, we will run into two examples of rare infections for which several conditions must prevail before a disease emerges [4, 5].

–4.

The microorganism must exist extracted from the infected experimental animal and demonstrated to be the same microorganism that was originally isolated from the original diseased animal.

Many infections, considered the underlying cause of a disease, are absent from the lesions that ultimately develop. For example, Group A streptococcus infection is considered to be the underlying cause of rheumatic fever. The infection is long gone prior to the appearance of the valvular and endocardial lesions of rheumatic fever. Equally some other example, several species of human papillomavirus are considered to be the underlying crusade of nearly all cases of squamous carcinoma of the uterine cervix. Morphologic cytopathic effects are visible in the primeval precancers that precede the evolution of invasive carcinoma. The cancers, which may occur years following the early on papillomavirus infections, may lack recoverable virus.

Let's look at an example of an communicable diseases that violates every ane of Koch's postulates. Whipple disease, previously a disease of unknown etiology, is characterized by organ infiltration with foamy macrophages (i.eastward., specialized reticuloendothelial cells that "eat" bacteria and debris). The organ nearly often compromised in Whipple affliction is the modest intestine, where infiltration of infected macrophages in the lamina propria (i.east., a strip of loose connective tissue subjacent to the epithelial lining of the small intestine) causes malabsorption. Whipple disease is rare. It occurs almost often in farmers and gardeners who piece of work with soil.

Whipple illness was first described in 1907 [6], only its cause was unknown until 1992, when researchers isolated and amplified, from Whipple disease tissues, a 16s ribosomal RNA sequence that could only have a bacterial origin [7]. Based on molecular features of the ribosomal RNA molecule, the researchers assigned information technology to Grade Cellulomonadacea, and named the species Tropheryma whipplei, later on the homo who outset described the disease, George Hoyt Whipple.

Specially noteworthy, in the example of Whipple disease, is that Koch'south postulates never came close to being satisfied. For the experimentalist, the virtually of import of Koch's postulates require the extraction of the organism from a lesion (i.e., from diseased, infected tissue), the isolation and civilisation of the organism in the laboratory, and the consistent reproduction of the lesion in an creature injected with the organism. In the case of Whipple affliction, none of these criteria were satisfied. The consequent identification in Whipple affliction tissue of a particular molecule, feature of a particular species of bacteria, was deemed sufficient to establish the infectious origin of the disease.

In the general scheme of events, bacteria in the human torso are eaten by macrophages, wherein they are degraded. In the case of T. whipplei, but a small population of susceptible individuals lack the power to destroy T. whipplei organisms. In susceptible individuals, the organisms multiply inside macrophages. When organisms are released from dying macrophages, additional macrophages arrive to feed, but this only upshot in the local accumulation of macrophages bloated by bacteria. Whipple disease is a good example of a affliction acquired past an organism simply dependent on a genetic predisposition, expressed as a defect in innate immunity; specifically, a reduction of macrophages expressing CD11b (also known as macrophage-ane antigen) [8]. Whipple affliction cannot exist consistently reproduced in humans or any other animals, because it can only infect and grow in a small portion of the human population.

Every bit nosotros learn more and more about the complexity of illness causation, formerly useful paradigms, such every bit Koch's postulates, seem burdensome and useless. When we meet rare diseases of infectious cause, we might wait to find that the pathogenesis of disease (i.eastward., the biological steps that atomic number 82 to a clinical phenotype) may crave several independent causal events to occur in sequence. In the example of Whipple disease, the infected individual must be exposed to a soil organism, limiting the disease to farmers and gardeners. The organism, residing in the soil, must be ingested, perhaps by the inhalation of dust. The organism must evade degradation by gut macrophages, limiting affliction to individuals with a specific blazon of defect in cell-mediated amnesty, and the individual must have disease that is sufficiently active to produce clinical symptoms. Information technology is unlikely that we could reproduce a complex sequence of steps, leading to a disease, by simply inoculating an organism into an experimental animal [Glossary Underlying cause, Proximate cause, Root cause].

Side-stepping Koch'southward postulates has get de rigueur in the practice of modern medicine. For instance, the United States has experienced a recent increase in cases of acute flaccid myelitis, a rare disease of children [9]. Diagnosis is based on a metagenomic analysis (i.e., civilization-independent sequence searches conducted on an assemblage of microbial gene sequences in a biologic sample) of DNA obtained from nasopharyngeal swabs. The organism that is present in most of the examined cases is enterovirus-D68, and this virus is the presumed causal organism of astute flaccid myelitis, until proven otherwise.

Genotyping species of organisms has get quite easy, merely in that location are many millions of microorganism species, and information technology may never be feasible to consummate a database of genome sequences of all living species. Though the number of private species is too large to sequence, we tin do a fair job at sequencing near of the unlike genera of living species. We now have a fairly accurate way of identifying the genus of any organism establish within a tissue sample, by sequencing its ribosomal RNA and comparing the sequence against references sequences in public databases [10–13]. In that location are limitations to this technique, simply when nosotros combine our assay of ribosomal RNA with our accumulated cognition of clinical features of the infection, we can often arrive at candidate pathogen [xi, 14, 15].

Mod medicine has changed the vocabulary of infection. Familiar terms such as primary pathogen, opportunistic infection, and immunocompetent patient need to be reexamined in low-cal of what we have come to know. Even a fundamental concept, such as "the organism causing the disease" should probably be abased in low-cal of the multistep pathogenesis of all diseases. Because a microorganism may contribute to the pathogenesis of a affliction at a single moment of time, long before the disease becomes clinically manifest, nosotros tin expect to see cases in which screening tests for a putative causal organism will exist negative in affected patients [xvi]. Koch, in his own fourth dimension, understood the practical limitations of his postulates. Maybe it's time to reconsider Koch's postulates in light of the analytic methods at present bachelor that assign a taxonomic grade to an infective organism, without isolating or characterizing the agent [17].

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INTRODUCTION

GEORGE N. AGRIOS , in Plant Pathology (5th Edition), 2005

Serious Constitute Diseases of Unknown Etiology

Although pathogens equally large and complex every bit fungi and nematodes or as tiny and simple as viroids and prions take been discovered, at that place are many severe diseases of plants, particularly of copse, for which we still practise not know their existent cause, despite years of searching and research. Some of them, such as peach short life in the southeastern The states, waldsterben, or forest decline in central Europe and various wood tree declines in the northeastern and northwestern Usa, may be caused by more than one pathogen or past combinations of pathogens and adverse environment. Others, such as citrus blight in Florida and Due south America, spear rot in oil palm in Suriname and Brazil, and mango malformation in India and other mango-growing countries, seem to accept a biotic agent as the primary cause, only the activity of the agent seems to be strongly affected by environmental factors such equally soil or temperature. Despite more than 100 years of research on some establish diseases, the causes of these diseases remain unknown.

BOX five

Koch's postulates

Robert Koch (1843–1910) (Fig. 1-16C) was a medical doctor and a bacteriologist. He was the starting time to testify, in 1876, that anthrax, a disease of sheep and other animals, including humans, was acquired past a bacterium that he chosen Bacillus anthracis. He subsequently discovered, in 1882, that tuberculosis and, in 1883, that cholera are each acquired past a different bacterium, which led to the full general conclusion that each disease is acquired by a specific microbe. These experiments confirmed for the first fourth dimension the germ theory of disease proposed earlier by Louis Pasteur.

Before Koch's experiments, and while Koch himself was carrying out the piece of work on the diseases mentioned earlier, there was confusion and doubt well-nigh the occurrence and the crusade of each disease. Much of the time when bacteria or fungi were isolated from diseased or dead human, animal, or plant tissues, the isolated bacteria or fungi were later shown to be saprophytes, i.e., they coexisted with the microorganism that caused the illness but could non by themselves cause the illness for which they were beingness considered. Based on his experiences, in 1887, Koch set out the four steps or criteria that must be satisfied before a microorganism isolated from a diseased human, animal, or plant tin can be considered as the cause of the illness. These 4 steps, rules, or criteria are known equally "Koch'due south postulates."

1.

The suspected causal agent (bacterium or other microorganism) must be present in every diseased organism (e.g., a found) examined.

ii.

The suspected causal agent (bacterium, etc.) must exist isolated from the diseased host organism (found) and grown in pure civilization.

3.

When a pure culture of the suspected causal agent is inoculated into a healthy susceptible host (plant), the host must reproduce the specific disease.

four.

The same causal agent must exist recovered once again from the experimentally inoculated and infected host, i.due east., the recovered agent must accept the same characteristics every bit the organism in step 2.

Koch'south rules are possible to implement, although non always easy to carry out, with such pathogens as fungi, bacteria, parasitic higher plants, nematodes, most viruses and viroids, and the spiroplasmas. These organisms can be isolated and cultured, or tin can be purified, and they can so be introduced into the plant to see if they crusade the disease. With the other pathogens, however, such as some viruses, phytoplasmas, captious phloem-inhabiting leaner, protozoa, and even some establish pathogenic fungi that are obligate parasites of plants (such as the powdery mildew, downy mildew, and rust fungi), culture or purification of the pathogen is non notwithstanding possible and the pathogen ofttimes cannot be reintroduced into the institute to reproduce the disease. Thus, with these pathogens, Koch's rules cannot exist carried out, and their acceptance equally the bodily pathogens of the diseases with which they are associated is more than or less tentative. In well-nigh cases, notwithstanding, the circumstantial evidence is overwhelming, and information technology is assumed that farther improvement of techniques of isolation, culture, and inoculation of pathogens will anytime evidence that today'due south assumptions are justified. Nonetheless, in the absence of the proof demanded by Koch's rules and as a outcome of bereft information, all plant diseases caused past phytoplasmas (e.yard., aster yellows) and fastidious vascular bacteria (due east.g., Pierce's disease of grape) were for years idea to be caused by viruses.

Despite the difficulties of carrying out Koch'due south postulates with some causal agents, they accept been and continue to be applied, sometimes with certain modifications, in all cases of disease. They have had and continue to have a tremendous result in deciding and in convincing others that a particular microorganism is the cause of a specific disease. By attempting to carry out Koch'south postulates in all newly discovered diseases, a groovy bargain of piece of work with potential saprophytes has been avoided, while, at the aforementioned time, dubiousness and criticism are reduced to a minimum while conviction in and utilize of the identification increase greatly and quickly.

BOX 6

Viruses, Viroids, and Prions

Although they have been with united states of america forever, we know relatively little well-nigh how these pathogen operate. There are many mutual characteristics among viruses and viroids. The human relationship of prions to others is only in their small size but they are contrasted to the other two in that they do non depend on whatsoever kind of nucleic acid (RNA or DNA). Viruses cause numerous severe diseases in all types of organisms, have been studied the longest, and nosotros know the most about them. Viroids cause more forty diseases in plants, some of them lethal. Prions seem to impact merely humans and animals in which they cause degenerative diseases of the brain, such equally the recently much publicized "mad cow disease."

Viruses are submicroscopic spherical, rod-shaped, or filamentous entities (organisms) (Figs. 1-22A–one-22C) that consist of only ane blazon of nucleic acid (DNA or RNA). The nucleic acid is surrounded by a coat consisting of i or more kinds of poly peptide molecules. Viruses infect and multiply inside the cells of humans, animals, plants, or other organisms and usually cause illness.

Figure 1-22. (A–C) Relative shapes and sizes of plant viruses: spherical, rod shaped, and flexuous. (D) T. O. Diener. (Eastward) Potatoes infected with potato spindle tuber viroid. (F) Circular and linear particles of the coconut cadang-cadang viroid. (G) Stanley Prusiner. (H) Schematic presentation of a normal protein and of a plain-featured inactive i, i.due east., a prion. (I) Plaques in the brain of an animal affected past a prion.

 [Photographs courtesy of (East) H. D. Thurston, Cornell University, (F) J. Westward. Randles, University of Adelaide, Australia, and (H and I) S. Prusiner, University of California.]

Viroids were discovered by Diener (Fig. 1-22D) and colleagues in 1971 while they were studying the potato spindle tuber affliction (Fig. i-22E). Viroids are the smallest infectious agents that multiply autonomously in constitute cells; they consist only of small, circular RNA molecules (Fig. i-22F) that are likewise minor to code for even ane small-scale protein and therefore lack a poly peptide coat. Viroids infect plant cells and are replicated in their nucleus, using the substances and enzymes of plant cells. Viroids infect only plants and in many of them they ordinarily cause illness. Viroids have non withal been detected in any other kind of organism besides plants.

Prions were proposed for the first time in 1972 by Prusiner (Fig. 1-22G) who, for that and subsequent work, received the Nobel Prize in Physiology or Medicine in 1997. Prions are at start normal small protein molecules produced in nerve and other cells of the encephalon. Prions get pathogenic, i.e., they cannot carry out their normal functions and, instead, accept adverse effects on the brain and cause disease. This occurs when prions are forced past weather in the brain to change shape (Fig. 1-22H). The change in shape signals the onset of infection. Prions are not associated with any nucleic acrid. Abnormal prions appear to increase in number and to cause the appearance of amyloid fibrils and plaques, besides as the appearance of small cavities (Fig. 1-22I) in the brain of diseased animals and humans. Prions have not been observed in plants or other organisms.

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The Zebrafish: Disease Models and Chemical Screens

Kathryn Milligan-Myhre , ... Carol H. Kim , in Methods in Cell Biology, 2011

B Host–Microbiota Interactions

As noted previously, Koch's postulates are useful for establishing causation between a putative pathogen or virulence determinant and a disease, but the aforementioned logic can exist applied to investigations into mutualisms between microbes and animals. There are several beautiful examples of creature mutualisms in which the presence of a single microbe greatly shapes the evolution or physiology of an brute host, such as the gut endosymbionts of insects and the bioluminescent symbionts of squid ( Fraune and Bosch, 2010). Vertebrates, nonetheless, are typically associated with complex microbial communities (microbiota) that are hard to characterize and oft recalcitrant to civilization in the lab. With a loosening of the requirement for the growth of the microorganism in pure culture, Koch's postulates can be applied to understanding the effects of these circuitous microbial communities on their hosts. The collective consequence of the microbial community can exist evaluated by the comparison of developmental, physiological, and immune markers between conventionally colonized and "sanitary" animals (which lack the microbial customs). Alternatively, the effects of individual or subsets of culturable microbes tin can be evaluated in monoassociated animals, in which a single microbe is introduced into an otherwise germ-free animal, or animals with simple, divers microbial communities. Finally the microbiota's collective effects tin can be approximated past transplantation of microbial communities harvested from one donor host into a germ-complimentary recipient host. All together, these experiments can provide powerful evidence for the roles of microbial associations in normal animal development and physiology.

The mouse has been the traditional animal used in the field of gnotobiology (Greek for "known life"), in which the microbial associates of animals are entirely defined. Recently, the zebrafish has emerged as a powerful new gnotobiotic model. The ex utero evolution of the zebrafish allows for like shooting fish in a barrel surface sterilization of the embryo'south chorion, facilitating the derivation of thousands of sanitary animals at a time. Although we have not however established methods to rear sanitary zebrafish to adulthood, as is possible for germ-free mice, contempo husbandry advances have resulted in maintenance of germ-free fish through a month of age, potentially to the onset of adaptive immunity. Studies of germ-free mice and zebrafish have revealed a number of common differences from their conventionally reared counterparts, indicating a conserved vertebrate program of responses to their microbiota. These responses are listed in Table II.

Table 2. Germ-complimentary traits shared between zebrafish and rodents

Germ-gratuitous trait Zebrafish Rodents
Reduced cell proliferation (as measured by incorporation of nucleotide analogues) Cheesman et al. (2011), Rawls et al. (2004, 2006) Barbarous et al. (1981)
Reduced numbers of goblet cells Bates et al. (2006) Kandori et al. (1996)
Contradistinct expression of genes involved in metabolism (e.g., fasting-induced adipose factor) Kanther et al. (2011), Rawls et al. (2004, 2006) Hooper et al. (2001)
Reduced expression of genes involved in innate immunity (east.1000., serum amyloid A1) Kanther et al. (2011), Rawls et al. (2004, 2006) Hooper et al. (2001)
Reduced numbers of abdominal associated immune cells Bates et al. (2007)
(fewer intestinal neutrophils)		 Bouskra et al. (2008), Cebra et al. (1998)
(fewer lamina propria cells and lymphoid follicles)
Differences in glycan expression Bates et al. (2006) Bry et al. (1996)
Contradistinct gut motility Bates et al. (2006)
(increased in germ-free)		 Husebye et al. (1994) (decreased in germ-free)

This chapter provides the tools for the zebrafish researcher to fulfill Koch's postulates to establish a functional connection between the presence of a microorganism and an issue on the host. We will begin past addressing the showtime postulate of characterizing the microbial associates of the zebrafish. We provide protocols for culture-dependent and -independent enumeration of associated bacteria from the intestine, merely these can be extended to other anatomical sites and classes of microorganisms. We and so provide protocols for the derivation and rearing of germ-free zebrafish, and methods for the generation of zebrafish with defined microbial associates. Finally we provide a series of protocols for infecting zebrafish at different ages and via unlike routes with unlike classes of infectious agents. These protocols are designed to provide researchers with the starting signal for a multifariousness of experiments. We leave the final analysis of the experiments – the particular methodologies of 16S rRNA cistron sequence assay and the endpoint analyses of gnotobiotic and infection experiments, including microbiological measurements, pathological assessments, and molecular measures of host responses – to the experimenter.

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Respiratory Tract Viral Infections

EURICO ARRUDA , ... FREDERICK G. HAYDEN , in Tropical Infectious Diseases (Second Edition), 2006

AGENT

The HCoV that fulfills Koch'south postulates equally the causative amanuensis of SARS 127 shares structural features and genome organisation of the family unit Coronaviridae (Fig. 59-ane). The prompt recognition of the peculiar morphology of a coronavirus in the electron microscopic studies of Vero E6 cells inoculated with oropharyngeal material from a patient was the initial finding that resulted in the identification of SARS-CoV. 128 The viral genome is 29,727 nucleotides in length, with more than 11 open reading frames coding for 23 putative proteins, some of which accept unknown functions. SARS-CoV is phylogenetically different and equidistant from all previously known coronaviruses, but isolates from different origins are relatively homogeneous genetically. Genome analysis reveals that SARS-CoV is neither a host-range mutant nor a recombinant of previously known coronaviruses but rather an independently emerged virus. SARS-CoV seems to have evolved from an animal SARS-like virus, acquiring greater fitness in humans during the course of the outbreaks, probably through the appearance of nucleotide deletions in open up reading frame 8. 129 It is also noteworthy that genetic signatures present in the genomes allow for differentiation of isolates obtained from different clusters. 129 The replicative cycle of SARS-CoV is thought to follow the aforementioned main steps as other coronaviruses.

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The potential role of infectious agents in diseases of unknown etiology

Steven M. Opal , in Infectious Diseases (Third Edition), 2010

Civilisation-INDEPENDENT TECHNIQUES TO Detect NOVEL PATHOGENS

In the genomic age in which we at present live, Koch's postulates need to be modified or even discarded equally an obsolete arrangement to define the essential elements of what identifies a microbial pathogen. The evolving definition of what constitutes a human pathogen has moved from a formal requirement to fulfill all of Koch'due south postulates to a genomic expression of Descartes' assertion, 'I clone, therefore I am.' This is best evidenced by the remarkable discovery of hepatitis C every bit the most common cause of non-A, non-B post-transfusion hepatitis by Choo and colleagues in 1989. 35 After decades of concerted, yet futile, efforts to isolate the implicated virus using standard virologic techniques, Choo et al. decided to effort to clone the genome of the unknown agent straight from the serum of a chimpanzee experimentally infected and known by serial serum dilution and beast challenge to incorporate high titers of infectious non-A, non-B hepatitis.

Afterward treating the serum with reverse transcriptase, they generated a large number of sequences of amplified nucleic acids to create a comprehensive cDNA library. It was not known at the time if the presumed hepatitis-causing amanuensis was an RNA or DNA virus or perhaps a noncultivable bacterium. The resulting bombardment of lambda phage cDNA clones were then tested against the serum of a patient with chronic severe non-A, non-B hepatitis. It was hoped that this human serum would provide a source of antibodies that would recognize at least some of the expressed viral peptides derived from the cloned sequences. After screening nearly 1 1000000 clones, the reactive peptide sequence was identified. They and then showed that this was an RNAse- merely not DNAse-sensitive genome and, by overlapping hybridization experiments, they confirmed that this was a positive-stranded RNA virus of approximately 10 000 base pairs. This newly discovered virus shared unexpected homologies with a number of arthropod-borne viral pathogens. The viral agent was confirmed to be a previously unknown fellow member of the flavivirus family. The mystery of post-transfusion, not-A, non-B hepatitis was finally solved by cloning, unencumbered by the need to fulfill Koch'due south postulates. 3, 35

A similar cloning strategy has now been adapted from work washed with genome searches for comparative microbial ecology, ecology biology and molecular phylogeny studies. 2, three Microbial Dna sequences are isolated and amplified by polymerase chain reaction (PCR) using broad range bacterial primers for essential target sequences such every bit ribosomal RNA genes. These amplified, novel bacterial sequences are then cloned in a Deoxyribonucleic acid library where comparative sequence assay is performed with highly conserved and well-characterized sequences from known classes of micro-organisms. The genes for 16S RNA, other essential ribosomal RNA sequences, or highly conserved enzymes are scanned for sequence homologies throughout the Bacteria and Archaea domains.

Sequence homologies and genomic groupings can at present be exploited to place shared genetic infinite and evolutionary distance between essentially any unknown pathogen and all identifiable, genome-characterized micro-organisms. These culture-independent methodologies are now employed to search for novel, hard to civilization or noncultivable microbial pathogens that might cause man disease. Such unbiased genome searches have been instrumental to the work of Relman, Fredricks and others 3 discovering the etiology of longstanding diseases of known microbial cause but with noncultivable organisms such as the Whipple's disease bacillus (Tropheryma whipplei) 36, 37 or newly recognized, emerging pathogens such every bit the causative agents of bacillary angiomatosis (Bartonella henselae) and human monocytic ehrlichiosis (Ehrlichia chaffeensis). 3 The resulting genomic show about the predicted metabolic requirements of T. whipplei was then exploited to develop a cell-free medium to successfully civilization the Whipple's disease bacillus for the first time. 38 The basic strategy employed in these genome searches is depicted in Figure 64.1. Electric current sequencing machinery now permits the simultaneous sequencing of over 200 000 short unique nucleotides from small samples of DNA without the need for preparatory cloning. iii This facilitates rapid genome screens with known sequences of viruses, rickettsia and numerous other difficult to civilization microbial genomes for potential pathogens.

Some other genomic technique that tin exist used to discover and define novel microbial pathogens is DNA subtraction techniques. This methodology was successfully employed to link a previously unknown herpes-like virus (HHV-8), now known every bit Kaposi'due south sarcoma-associated herpes virus (KSHV) as the cause of HIV-related Kaposi'southward sarcoma. 2, 3 The DNA sequences derived from diseased tissues and a similar set of Dna sequences from normal tissues are subjected to subtractive hybridization and PCR amplification to enrich the DNA complement of DNA sequences found in the diseased sample just. Unique sequences are and then analyzed to determine if any sequence homologies exist with known pathogens. As the known genomic universe of micro-organisms continues to expand, it is probable that this and related culture-independent methods will reveal new pathogens associated with human illness.

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Tumor necrosis factor

Haichao Wang , ... Kevin J. Tracey , in The Cytokine Handbook (Fourth Edition), 2003

Septic shock

TNF plays a pivotal role in septic shock, because it fulfils Koch'southward postulates, a logical chain of evidence that can exist modified hither for considering cytokine biological science. First, TNF is released systemically during over-whelming sepsis, and in some cases leads to lethality ( Girardin et al., 1988; Waage et al., 1989). Persistent increases in TNF are associated with the development of multiple organ failure and mortality (Girardin et al., 1988; Calandra et al., 1990; van der Poll and Lowry, 1995). 2nd, administration of exogenous TNF causes shock and tissue injury that is physiologically, metabolically, hematologically and pathologically indistinguishable from septic shock syndrome (Tracey et al., 1986a, 1987a). 3rd, neutralization of TNF with antibodies prevents septic shock syndrome during lethal bacteremia (Tracey et al., 1987b). In support of this view, TNFR1-scarce knockout mice are resistant to endotoxemic shock (Pfeffer et al., 1993). TNF induces other mediators of sepsis such every bit IL-one, and infusion of anti- TNF antibody attenuates the activeness of these mediators (Tracey et al., 1987b; Fong et al., 1989).

TNF and soluble receptors of TNF (sTNFRs) are liberated during sepsis (Girardin et al., 1992). As mentioned above, sTNFRs are inhibitors of TNF biologic activities merely may also serve every bit a reservoir of TNF, and increased levels of sTNFRs are plant in many TNF-associated diseases. In fact, increased levels of sTNFRs correlate with TNF levels and outcome in severe meningococcemia (Girardin et al., 1992). In full general, sTNFR concentrations are associated with TNF levels when the TNF level is relatively low, only non when it is high. The imbalance between TNF and sTNFRs is probably pathophysiologically important, as reflected by the observation that their ratio on admission may be of predictive value for clinical outcome (Girardin et al., 1992).

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Rheumatoid Arthritis, Human being

Antonio La Cava , Salvatore Albani , in Encyclopedia of Immunology (2d Edition), 1998

Infectious agents

To engagement, no disarming evidence has been provided for a unmarried infectious agent fulfilling Koch's postulates every bit an etiologic agent for rheumatoid arthritis. Nevertheless, some interesting observations have been made that correlate some pathogens with rheumatoid arthritis. This is the example, for instance, with EBV. Patients with rheumatoid arthritis have increased titers of unusual antibodies to EBV, such as antirheumatoid arthritis nuclear antigens (anti-RANA). EBV antigens have been identified in the synovium and as discussed later on, EBV may besides participate in mechanisms of abnormal reactivity to the 'shared epitope'.

Some other family of viruses that some authors claim to play a role in etiopathogenesis of rheumatoid arthritis is represented past retroviruses, which might actuate 'autogenes' that may trigger autoimmune affliction. Bacterial antigens are believed to exist triggers of various arthritides, such as Reiter'south affliction. In these diseases, chronic arthritis is associated with aberrant allowed responses to bacteria such every bit Shiggella, Salmonella or Yersinia. These responses can be detected at the synovial sites of inflammation, without demonstration of the intact microorganism in the synovial infinite. Bacterial antigens with autoimmune potential may be transported into the joint, and initiate, and/or sustain, abnormal immune responses. Among the possible candidates, special attention has been given to heat stupor proteins. Bacterial oestrus stupor proteins, including hsp65 and Escherichia coli dnaJ, are targets of specific humoral and cellular responses in patients with rheumatoid arthritis.

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Research methods, statistics and prove-based practice

Andrew M. McIntosh , ... Stephen Thousand. Lawrie , in Companion to Psychiatric Studies (8th Edition), 2010

Criteria for causality

Establishing causation in multifactorial disorders is clearly complex and hard. Erstwhile models, such every bit Koch'south postulates and causes beingness 'necessary and sufficient', are inadequate. Bradford Hill indirectly proposed some criteria for deciding whether an exposure caused an event ( Box 9.eight). Of these, maybe the about important is that of establishing a temporal sequence, in demonstrating that the cause precedes the outcome, and that of an increase in the risk as the degree of exposure increases (a dose–response human relationship). The strength of the association, i.e. size of the odds ratio or relative take chances, is another important guide. Some authorities recommend that newly identified exposures or risk factors should only be taken seriously if the odds ratio is at least three, at least until replication. Replication with different study designs and unlike methods is crucial. Finally, any observed association should have biological plausibility, based on what is known about pathophysiology.

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