Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-10-31
2002-12-17
Fredman, Jeffrey (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S024330
Reexamination Certificate
active
06495325
ABSTRACT:
TECHNICAL FIELD
The present invention relates to methods and means for diagnosis of pathological or other detrimental conditions affecting the general health of a group of individuals, in particular vertebrates, and more typically mammals and/or birds, such as livestock. The invention further relates to methods and means for microbiological typing of substances comprising a variety of microbiological organisms such as, but not limited to, food, food additives, waste material, soils etc. The invention is exemplified by using feces as a source of microbiological populations. However, the methods and means of the invention are equally applicable to microbiological populations from other sources.
BACKGROUND
In livestock a number of pathological conditions and/or syndromes have been noticed that do not seem attributable to, e.g., an infection with a single micro-organism. These syndromes, however, do seem to have an effect on the equilibrium which normally exists within the different bacteria constituting the bacterial flora. A number of these syndromes are referred to as dysbacteriosis and bacterial overgrowth.
Although not wishing to be bound by theory, a possible explanation for the presence of these syndromes is that these diseases may occur due to:
1. feed intake/feed type.
The feed interferes with the digestion, resulting in changes in the bacteria. Some particular bacteria or groups of bacteria are abundant and overwhelming, some particular bacteria or groups of bacteria are inhibited and cannot survive.
2. Invasion of one (or more) bacterial species.
The bacteria need not be pathogenic themselves, to disturb the balance within the flora.
3. Invasion of one (or more) viral or fungal species,
4. Immune depression,
5. Environmental effects such as temperature and humidity.
Typically, these syndromes do have an influence on the general health and performances of an animal and often even on the whole of the group of animals. In poultry, there are even a number of syndromes which show clinical symptoms directly related to the condition of the intestinal bacterial flora. Therefore, it is of importance to be capable to check the general health of a population of livestock, and/or to have a means of at least determining the presence of such an ill-defined pathological condition in a group (herd, flock) of, e.g., livestock.
Examples of syndromes related to bacterial disequilibrium are, e.g., steathorroea in calves, ileitis in pigs, and also diarrhea at weaning, diarrhea in poultry.
DISCLOSURE OF THE INVENTION
The present invention provides novel means (e.g., kits) and methods for determining (relative) abundance as well as (estimates) of actual amounts of different bacteria present in intestinal bacterial flora, in order to determine, e.g., the presence or absence of equilibrium in said intestinal bacterial flora, especially when compared to a set of normal compositions of intestinal bacterial flora. Until the present invention analyses of bacterial flora occurred with methods used in classical bacteriology. In classical bacteriology, which is based on cultivation and identification of different bacterial strains. Various methods are used, including aerobic and anaerobic growth, pre-enrichment, etc., Furthermore, counting the number of colonies has generally been used to obtain information about the number of bacteria present in samples.
Using these methods, only an estimated 20% of the bacteria have been isolated and characterised until now. Nearly axiomatic in these classical bacterial techniques (as well as with techniques finding other micro-organisms, such as viruses, yeast or fungi) is the thinking that one disease has one causal agent, or at least that one or a distinct few agents can be considered instrumental in causing the distinct disease. However, these classical insights to not provide any practical manner of obtaining insight into the presence or absence of a “healthy” equilibrium in intestinal tracts.
Thus, the present invention provides a method for analysing the composition of microbiological (e.g. viral, fungal, yeast or bacterial) flora in an intestinal tract, comprising providing a sample of said flora, selectively amplifying nucleic acid in said sample, subjecting the amplificate to restriction digestion and analysing the resulting pattern of restriction fragments, and providing detection of interrelationships between flora constituents such as known and unknown bacteria, fungi, viruses and the like, for example under distinct disease conditions without the need to identify an organism. Said nucleic acid is selectively amplified, albeit usually from as many, or the greater part of, micro-organism species as possible present in said sample, and optionally supplemented with specific amplification of known organism species or parts thereof possibly present in said sample to provide optional identification, when so desired. Typical results include the detection of a cluster or clusters of amplified fragments (which may have been subjected to restriction), said fragments localised or detected in a pattern (governed for example by molecular mass) allowing the recognition of specific (cluster) patterns. These may be identical in various samples, may be different, or may be supplemented with additional clusters (typically nearly equally sized fragments). Typically, when the sample is representative of a “normal” population, a well balanced cluster pattern may be seen. In contrast, when dysbacteriosis or overgrowth has occurred, one cluster (commonly representing one micro-organism) may be over-represented, typically to the detriment of other clusters (representing others)
The sample may typically be derived from feces, or a biopsy, and in many cases can be taken post-mortem. Preferably, samples are taken under conditions which can be repeated, so that the differences in the flora are attributable to the conditions to be diagnosed and/or analyzed, and not to factors which are temporal, dietary or the like. Post-mortem samples and biopsies should be taken from the same area in the intestinal tract. When using this technique as provided by the invention, it is not per se the mere identification of a tentative causal micro-organism that matters. Rather as the recognition of distinct patters in the resulting analysis allow determinations of equilibrium with respect to a distinct or fitting condition (diseased or non-diseased) in said flora, and allow identification of similar pattern in animals from the same or different flock. Clearly, classical techniques have not paid attention to patterns in flora that, for example relate to multifactorial disease patterns, but have commonly tried to identify one or more distinct causal agents, which in general are sought after with predetermined and specific detection means, such as nucleic acid primers or probes, antibodies, and so on, for the detection of specific micro-organisms or specific components thereof. For example, U.S. Pat. No. 5,543,294; JP 05 317096; Ratcliff et al., Path. (1994) 26:477-479; Wood et al., Appl. Env. Microb. (1998) 64:3683-3689; and U.S. Pat. No. 5,571,674 all give specific and direct instructions on how to identify a specific bacterium, or a bacterium belonging to a specific genus or family, but do not pay attention to other micro-organisms that are or may be present in the test samples, clearly demonstrating that they are not interested in the whole content of the flora but only in specific micro-organisms therein. They will therefore never be able to detect interrelationships between flora constituents such as known and unknown bacteria, fungi, viruses and the like.
In the field of bacteriology, the invention, for example provides the means for quality control, identification and quantification of bacterial species of interest, as well as the detection of (presence or absence) of highly pathogenic and lethal bacterial species, or the detection of new, previously unknown, bacterial. The invention provides, for example, information on the total of bacteria in any given sample. Because of the nature of the t
van Haeringen Hendrick
van Haeringen Willem Anne
Chunduru Suryaprabha
Dr. van Haeringen Laboratorium B.V.
Fredman Jeffrey
TraskBritt
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