U.S. patent application number 11/494215 was filed with the patent office on 2010-05-13 for detection and measurement of blood-feeding activity.
Invention is credited to Nathan L. Smith.
Application Number | 20100120071 11/494215 |
Document ID | / |
Family ID | 37684005 |
Filed Date | 2010-05-13 |
United States Patent
Application |
20100120071 |
Kind Code |
A1 |
Smith; Nathan L. |
May 13, 2010 |
DETECTION AND MEASUREMENT OF BLOOD-FEEDING ACTIVITY
Abstract
This invention provides compositions and methods for detection
of hematophagous ectoparasitic activity in an enclosure or area.
The compositions comprise a reagent or reagents which are reactive
against antigens or markers as they appear in the excrement or
other ectoparasitic materials. Such markers or antigens may be
produced by the ectoparasite itself or may have been introduced
into the ectoparasite because of its blood feeding activity. The
method of the present invention comprises collecting from the
enclosure or area, a sample comprising environmental dust or
materials and subjecting the sample to tests for detecting the
presence of hematophagous ectoparasitic markers, host markers or
both.
Inventors: |
Smith; Nathan L.; (North
Andover, MA) |
Correspondence
Address: |
HODGSON RUSS LLP;THE GUARANTY BUILDING
140 PEARL STREET, SUITE 100
BUFFALO
NY
14202-4040
US
|
Family ID: |
37684005 |
Appl. No.: |
11/494215 |
Filed: |
July 27, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60702914 |
Jul 27, 2005 |
|
|
|
Current U.S.
Class: |
435/7.22 |
Current CPC
Class: |
G01N 21/76 20130101;
G01N 33/72 20130101; G01N 2333/43586 20130101; G01N 2333/43582
20130101; G01N 2333/76 20130101; G01N 2333/43591 20130101; G01N
33/5308 20130101; G01N 33/68 20130101 |
Class at
Publication: |
435/7.22 |
International
Class: |
G01N 33/569 20060101
G01N033/569 |
Claims
1. A method of determining a likelihood of blood feeding activity
by a hematophagous ectoparasite in an enclosure inhabited by
individuals of a host species, wherein the hematophagous
ectoparasite is a bedbug, the method comprising the steps of: a)
collecting a sample of environmental dust from the enclosure; b)
detecting for the presence or absence in the environmental dust
sample of a marker selected from the group of markers consisting of
host hemoglobin, hematophagous ectoparasite salivary protein
antigens, and combinations of host hemoglobin and hematophagous
ectoparasite salivary protein antigens, which host hemoglobin
marker or hematophagous salivary protein antigen marker is
detectable in excrement of the hematophagous ectoparasite, wherein
detecting the presence of the marker in the environmental dust
sample is indicative of a likelihood of blood feeding activity by
the hematophagous ectoparasites feeding on the inhabitants of the
host species in the enclosure and wherein the absence of the marker
in the environmental dust sample is indicative of a likelihood of a
lack of blood feeding activity by the hematophagous ectoparasites
feeding on the inhabitants of the host species in the
enclosure.
2.-7. (canceled)
8. The method of claim 1, wherein the environmental sample is
collected by vacuuming, swiping or mopping.
9. The method of claim 1, wherein the host species is selected from
the group consisting of human, dogs, cats, rodents and
chickens.
10. The method of claim 1, wherein the enclosure is inhabited by
humans.
11. The method of claim 1, wherein the enclosure is inhabited by
non-human animals or birds.
12.-20. (canceled)
Description
[0001] This application claims priority to U.S. Provisional
application No. 60/702,914, filed on Jul. 27, 2005, the disclosure
of which is incorporated herein by reference.
FIELD OF THE INVENTION
[0002] This invention relates to environmental sampling and testing
in structures, shelters, or dwellings for markers associated with
the presence of free-living organisms that feed on vertebrate
blood.
BACKGROUND OF THE INVENTION
[0003] Hematophagy is a behavior of feeding on blood taken from
vertebrate hosts. It is practiced by about 15,000 species and about
400 genera of arthropods (Mans, Louw et al. 2002). It has also been
reported that blood-feeding has evolved independently at least six
times. In addition to arthropods, members of other phyla including
annelids such as worms and leeches are hematophagous. While
independent evolution and wide-spread occurrence of taking
blood-meals have resulted in considerable biological diversity,
there are consistent characteristics pertinent to strategies for
hematophagy including: locating and accessing a suitable
blood-containing host, penetrating the integument and blood
circulatory system, counteracting the hemostatic system of the
host, taking a blood-meal and escaping detection so as to survive
to digest the meal.
[0004] To obtain blood, hematophagous ectoparasites cut or pierce
the epithelium and disrupt the integrity of the circulatory system
of the host. During this process of gaining access to blood and
feeding on blood, hematophages may inject substances into the host
such substances being brought to the symbiotic relationship by the
parasite. These injected substances may be either produced by the
parasite and assist in blood-feeding or present in the hematophage
as a residue of previous feeding.
[0005] Blood-feeding behavior results in numerous new infections in
humans per year and many more in animals. Among the most widely
known of these infections are cases of malaria resulting from
mosquito bites. While Yellow fever, Chagas disease, Dengue fever,
Ebola, and other diseases are transmitted by tropical insects,
human arthropod-borne infections also occur in non-tropical
regions. Infectious diseases such as Borrelia, Babesia, Rickettsia,
West Nile virus, and various types of encephalitis infections are
found in cold weather climates in the Northern Hemisphere.
Similarly, many non-human species are infected by hematophagous
parasites and may serve as alternate hosts or reservoirs for human
pathogens.
[0006] Among blood-feeing organisms, arthropods are possibly the
best known. Included in this group are arachnids (ticks and mites)
and insects, flies, bugs, and lice. Some hematophagous parasites
require blood-meals at every stage of life, such as is the case
with ticks, lice, and bedbugs, and others only as adults and in
some cases only females are parasitic. Some hematophages spend
their entire life on the hosts such as is the case with head lice.
Other species, such as bedbugs, get onto the source of blood only
to feed. The wide biological diversity of hematophagous organisms
presents a challenge to detecting and controlling these pests.
[0007] Sources of blood for these parasites are vertebrates
including, most importantly, terrestrial reptiles, birds, and
mammals because of their association with humans. Some species of
hematophages are opportunistic and will take meals from a variety
of host species whereas others may preferentially or obligately
feed on one host species. In many cases humans are more exposed to
hematophagous organisms upon leaving shelter. Well-known
blood-sucking insects, such as mosquitoes and biting flies, and
ticks, live primarily outdoors. While these parasites may enter
houses, primarily to feed, they usually do not infest human
shelters. Typically, arthropod life cycles are more compatible with
the environment outside of human dwellings and humans are
opportunistically used as sources of blood.
[0008] There are, however, animals that opportunistically share
houses with humans even in developed countries. These organisms are
commonly known as pests. Many arthropod pests enter a dwelling with
other pests; for example, fleas may come in on rodents. It is also
possible that pests enter dwellings on their own, with the humans
themselves, or companion animals such as dogs or cats carry pests
into the homes. Infestations by hematophagous ectoparasites can
cause annoyance, discomfort, health problems, and economic losses.
For example, pests in structures other than those for human
habitation, including but not limited to hen houses for avian egg
production, barns housing domestic animals, mammalian breeding
facilities, or other structures for continued occupation by birds
or mammals, may cause reduced productivity. Once inside, some
arthropods are able to establish breeding colonies in the home.
Often the homeowner is not aware of such infestations. For example,
house dust mite colonies exist in a high percentage of homes in the
United States without the human co-inhabitants being aware of their
presence. These organisms feed primarily on human skin flakes that
are sloughed constantly and are a component of house dust. House
dust can also serve as the main nutrient source for immature fleas,
obligate blood-feeders as egg producing adults.
[0009] Pesticide use to control many hematophagous species has
undergone significant changes with the virtual elimination of
"residual insecticides" (Spielman, Pollack et al. 2001).
Subsequently, current pest management practices have not eliminated
hematophagous ectoparasites from shelters: school children still
are infected by head lice (Pediculus capitas); bedbugs (Cimex
lectularius) infest henhouses and are being found with increasing
frequency in homes and lodging facilities in the United States;
fleas (Ctenocephalides sp.) and several genera of ticks enter
structures inhabited by humans, mice, dogs, cats, and other mammals
alone or in combination; and companion animals of humans are
frequently hosts for are variety arachnid and insect ectoparasites.
Because modern integrated pest management practices tend toward
more judicious use of pesticides, determining that an infestation
exists is a necessary first step in controlling pests and is useful
in evaluating the effectiveness of control measures taken.
Currently visual inspection either directly or using various
collection and magnification aids is the primary means to determine
the presence of pests. Frequent publication of articles and patents
describing new and better ways to detect the presence of pests
attests to the fact that current methods need improvement.
[0010] As mentioned above, blood-sucking parasites may inject
substances into the host during feeding. Compounds of parasite
origin have been found to have various physiological activities
including anticoagulation, platelet aggregation inhibition, pain
inhibition, and anti-inflammation. Such findings have lead to
published studies describing the structure and function of these
compounds of possible use as pharmaceuticals. Hirudin, produced by
the leech Hirudo medicinalis, has been cloned for mass production
by recombinant DNA technology and is available for treating humans
as "Refludan" (BERLEX Laboratories, Wayne, N.J.). This and other
therapeutic applications have lead to an expanding literature base
in pharmacognosy focused on proteins, especially of salivary
origin, elaborated by hematophagous invertebrates to assist in
blood-feeding (Valenzuela, Walker et al. 1995; Valenzuela, Charlab
et al. 1998; Valenzuela, Belkaid et al. 2001). These studies have
provided DNA sequences for many salivary proteins which served as
useful prior art for the present invention. Salivary biomolecules
are injected into the host circulatory system where the
physiological effects occur. The proteins of hematophage origin are
also carried by the blood into the parasite digestive system where
they become part of the blood-meal and are subject to degradation
by the digestive hydrolytic processes. As a result, the structure
and molecular characteristics of salivary proteins may be altered
during passage through the digestive system of parasites and be
different from those of the salivary proteins per se or produced
from recombinant genes.
[0011] Vertebrate blood is typically about 80% water. Many
hematophagous invertebrates, especially ectoparasitic species,
utilize diuresis mechanisms to eliminate much of the water from a
blood-meal very quickly usually as feces (Quinlan, Tublitz et al.
1997). This is necessary for many reasons including improved
mobility of the pest to avoid being detected and preyed upon or
swatted and more efficient digestion. The very liquid feces contain
compounds that have undergone little digestion during the short
time between feeding and excretion. These feces will contain
biomolecular markers of blood feeding activity derived from both
the host blood and those associated with the hematophagous organism
involved.
[0012] Various types of tests have proven to be reliable and
cost-effective for detecting biomolecules. Those skilled in the art
of detecting molecules of biological origin have developed various
methods to offer rapid, easy-to-use, and specific tests with
sufficient sensitivity to be useful for detecting markers for a
wide variety of organisms including but not limited to bacteria,
viruses, parasites, and arthropods in the environment. Many of
these assays take advantage of the chemical composition and of
specific binding properties of biochemicals including but not
limited to effectors, inhibitors, modulators, and hormones binding
with receptors; lipids, metals, proteins, hormones being
specifically bound to carrier proteins; antibodies binding to cell
surface binding domains; and the well-know interaction between
antigens and antibodies. Thus, while detection assays are know in
the art, such assays have not been successfully adapted to the
detection of hematophagous ectoparasites that live outside of the
body of the host by the methods and compositions of this
invention.
SUMMARY OF THE INVENTION
[0013] The present invention provides methods and compositions to
detect infestation of an enclosure or area by hematophagous
ectoparasites. This invention is based on the unexpected
observation that host specific or parasite specific markers can be
identified in environmental dust or other materials collected from
the enclosure or area. Therefore an identification of hematophagous
ectoparasitic blood feeding activity can be made in the absence of
visual detection of the ectoparasites.
[0014] Accordingly, this invention provides compositions comprising
a reagent or reagents to detect the presence of blood-feeding
behavior and/or to provide evidence as to the identity of the
hematophagous ectoparasite and/or the host. The reagents are those
that react with antigens or markers as they appear in the excrement
or other ectoparasitic materials (such as saliva). Such markers or
antigens may be produced by the ectoparasite itself (ectoparasitic
markers or antigens) or may have been introduced into the
ectoparasite because of its blood feeding activity (host markers or
antigens). In one embodiment, the composition comprises a regent
which can react with an ectoparasitic antigen. In another
embodiment, the composition further comprises a regent which is
capable of detecting a host antigen (including, but not limited to,
heme, hemoglobin, immunoglobulin or albumin).
[0015] The method of the present invention comprises collecting
from the enclosure or area, a sample comprising environmental dust
or materials and subjecting the sample to tests for detecting the
presence of hematophagous ectoparasitic markers, host markers or
both.
BRIEF DESCRIPTION OF THE FIGURES
[0016] FIG. 1 Photograph of bedbug culture on a cardboard substrate
showing dark colored spots of feces. Also shown are eggs shells as
light objects.
[0017] FIG. 2 Photograph of bedbug culture debris containing
exoskeleton molts (large objects) and small, dark colored fecal
matter.
[0018] FIG. 3 Photograph of samples cut from bedbug culture
substrate cardboard shown after being used in guaiac fetal occult
blood test (FOBT).
[0019] FIG. 4 FOBT (Biomerica) results with bedbug culture extract
("B") and office dust extract ("D"). Note positive reaction with B
and no color development with D.
[0020] FIG. 5 Photograph of FOBT (Immunostics) showing office dust
extract on left and bedbug culture extract on right. A positive
reaction is seen with only with the bedbug extract.
[0021] FIG. 6 Photograph of single-use cartridge (ImmoCARE) showing
the sample addition site (labeled "5") and the zones labeled "C"
where the control line develops in a valid test. If a line develops
in the area labeled "T", the test is positive and human hemoglobin
has been detected. No line is a negative result. In addition to the
cartridge, a vial of buffer is supplied to prepare a suspension and
extract of the sample.
[0022] FIG. 7 Photograph of office dust extract run on a cartridge
similar to one shown in FIG. 6. Negative result indicates no human
hemoglobin was detected.
[0023] FIG. 8 Photograph shows result of ImmoCARE FOBT with bedbug
culture debris extract. Strong line at "T" shows a positive result
indicating presence of human hemoglobin.
[0024] FIG. 9 Photograph showing bedbug culture debris placed on
plastic backed absorbent paper. Two (2) exoskeleton molts can be
seen on left. Feces and other debris are shown on right side of
paper.
[0025] FIG. 10 Bedbug culture debris preparation shown in FIG. 9
after treatment with luminol (BlueStar).
DETAILED DESCRIPTION OF THE INVENTION
[0026] The present invention provides methods and compositions to
detect infestation of an enclosure by hematophagous ectoparasites.
This invention is based on the observation that biological markers
specific for the host (immediate host or a prior host) or
biological markers specific to the hematophagous parasite can be
identified in the excrement of the parasites. Therefore, this
invention makes use of the presence, in environmental samples, of
identifiable biomolecules that serve as markers of blood-feeding
behavior of pests. The method comprises two steps: First, a sample
representative of the environment to be tested is collected by any
of many methods available including, but not limited to, vacuum
collection, swabbing, scraping, shaking, or otherwise causing
compounds, dust, and matter in the environment to be gathered,
collected, or obtained. Such material could be collected such that
it is suitable for transport. Second, the representative
environmental sample is subjected to testing to detect desired
biomolecule or biomolecules. From the results of the testing using
commercially available reagents or the reagents of the invention,
the presence or absence of hematophagous ectoparasites can be
ascertained. Further, by comparing the levels of the biomolecules
to a control level, the degree of infestation can also be
determined.
[0027] The invention has several advantages over presently
available methods of detecting ectoparasite infestations. Visual
inspection is the most commonly practiced method of locating pest
infestations. This is practiced by examining the environment for
evidence of the pests, such as the organisms, molted exoskeletons,
and/or fecal matter, by direct examination, combing, trapping, bait
and trapping, or vacuuming. Hematophages have developed many
different behaviors or traits to enable them to feed from the
vertebrate animal without being killed, removed, or otherwise
prevented obtaining blood. These behaviors or traits work against
the visual methods for establishing the presence of the
parasites.
[0028] Although hematophagous ectoparasites have different
behaviors of blood-feeding and avoiding detection, it was
unexpectedly observed that some common attributes could be used for
their detection. For example, it was observed that host proteins
(or partially degraded portions thereof), maintained enough
integrity to be detectable in the excrement of parasites. Thus,
because the pests to be detected by this invention feed on blood,
based on the observations of this invention, these pests will be
expected to excrete undigested and partially digested components of
blood in their feces. Because the presence of vertebrate blood in
the indoor environment is not normal, the presence of blood, in the
absence of other known causes, can be taken as presumptive evidence
of the activity of blood-feeding. In the present invention standard
reagent compositions or compositions developed as provided herein,
can be used detect any of a myriad of compounds derived from the
host blood. For example, in one embodiment heme, present in
vertebrate blood from all species, is detected to look for evidence
of blood-feeding in general. In a dwelling inhabited by humans and
any of a number of animal species such as dog, cats, birds,
ferrets, etc., individually or in combination, detection of heme in
the indoor environment indicates the possibility of blood-feeding
pest infestation. Conversely, the absence of heme in house dust or
other samples of the environment would provide strong evidence of
the lack of hematophagous ectoparasite infestation. In another
embodiment, reagent compositions could be used for detection of
blood components, for example blood proteins including but not
limited to hemoglobin, albumin, or immunoglobulins, with more
specificity than detecting heme which is present in hemoglobins of
all vertebrates.
[0029] A second common trait that enables and accompanies
blood-feeding is injection of salivary compounds of parasite origin
into the wound in the host. These compounds, mostly proteins as
discussed above, appear in the gut of the blood-sucking pest. As a
result of the diversity of hematophages, these compounds vary
highly in structure and composition even if not in function. The
invention teaches that specificity will be increased by taking
advantage of this biochemical diversity.
[0030] The method and selected reagents of this invention could be
used to detect hematophagous ectoparasites including bedbugs and
other bugs, lice, mites, fleas, mosquitos, flies, and ticks Those
species of particular interest are those that can infest dwellings
used by their hosts. In one embodiment, bedbugs (Cimex lectularius)
and Pediculus humanus (human head and body louse) are detected by
the method of this invention.
[0031] Broadly, it is an object of the instant invention to provide
compositions of prescribed reagents to detect the presence of
blood-feeding behavior and to also provide evidence as to the
identity of the ectoparasitic hematophages involved. The reagents
will be those that react with antigens or markers as they appear in
the excrement or other ectoparasitic materials (such as saliva).
Such markers or antigens may be originating from the ectoparasite
itself (ectoparasitic markers or antigens) or may have been
introduced into the ectoparasite because of its blood feeding
activity (host markers or antigens). Non-limiting examples of such
markers or antigens are those that are present in the excrement
and/or saliva of the ectoparasite--whether due to being produced by
the ectoparasite or derived from a host on whom the ectoparasite
has previously fed. In one embodiment, the composition comprises a
regent which can react with an ectoparasitic antigens. In another
embodiment, the composition further comprises a regent which is
capable of detecting a host antigen.
[0032] In the present invention, testing may be performed on an
environmental sample depending on the information that is to be
obtained. For example, testing may be directed to detecting all
blood feeding activity (such as detection of heme in the
environmental sample), detecting blood feeding on humans (such as
by using probes specific for human blood proteins, detecting for
the presence of all blood feeding pests (such as by detecting
salivary proteins of pest-origin), or the presence of specific
blood feeding pests (such as by detecting species specific salivary
proteins). Those skilled in the art will recognize that any
combination or routine modification of the above can be used
depending upon the information desired.
[0033] The method of the present invention can be useful in
detecting blood-feeding activity in such places as: multi-room
homes; hotels, motels, hostels, dormitories, boarding houses,
shelters, and any other facilities in which people live or
otherwise occupy; transportation related enclosures such as cruise
ships, railway sleeping cars, airplanes; and animal housing
enclosures such as henhouses, indoor rodent colonies, vivaria, and
non-carnivorous animal breeding establishments. In multi-room
establishments, samples, such as dust captured by vacuuming, can be
collected from each room and tested for the presence of, for
example, hemoglobin. Those rooms that test positive can be
subjected to treatment or additional testing.
[0034] In one embodiment, the invention can also be used to
discriminate between blood feeding organisms of different species
or blood feeding organisms feeding on more than one species.
Therefore, the method of the present invention could be used in
structures where more that one vertebrate species may be present
either intentionally or by happenstance. In these cases the
composition of the testing reagent can be selected to be specific
for detecting hemoglobin, or other blood substance, of the species
of interest. For example, a number or reagents are available (or
can be produced by standard techniques) which react specifically
with human and which do not react with hemoglobins from cats, dogs,
or rodents. Such reagents can be used to specifically test for
blood feeding parasite activity on humans. Alternatively, a reagent
specific for canine hemoglobin could be used to differentiate
between parasites feeding on dog blood from those feeding or
humans, cats, and rodents. Such compositions can be useful in homes
occupied by people and their vertebrate pets and in barns occupied
by a colony of domestic animals and also possibly by vertebrate
pests. In such a test, the presence of blood-feeding activity and
the host could be determined by using reagents similar to those
currently used to obtain species identity of blood stains in
forensic serology. For example, recently, a human hemoglobin
specific rapid field-use forensic test has been introduced by
BlueStar, ROC Import, Monte Carlo, Monaco. This test gives positive
results for human hemoglobin while being negative for blood from
non-primate animals. The same art has been utilized by several of
the immunological FOBT described herein to allow for detecting
human hemoglobin without giving false positive reactions with
non-human hemoglobin.
[0035] A second embodiment of the present invention is to test for
the presence of a marker of parasite origin in samples. This method
is preferred in situations in which detecting compounds derived
from host blood might not be sufficient to confirm the infestation.
This method would also be useful in determining the identity of the
pest. It is important in selecting the composition of the reagents
to practice this embodiment to identify the structure and binding
properties of the biomolecule or biomolecules to be detected as
they exist in the environment dust samples. These properties may or
may not differ from those of the native molecule as produced in the
digestive system, including salivary glands. Therefore, in spite of
knowing the gene sequence and protein structure, the properties of
the biochemicals of interest may be altered by passage through the
digestive system and release into the environment. This art has
been practiced to increase specificity of immunoassays including
the FOBT produced by Care Products, Waterbury, Conn. In this test,
antibodies selected for use are specific to hemoglobin released
into the distal gut and not to the more extensively degraded
hemoglobin emanating from mouth, stomach, and proximal gut
locations.
[0036] A third embodiment is detecting in the environment dust
samples comprising excrement of hematophagous ectoparasites, one or
more markers indicative of a previous feeding of the pest. For
example, a tick becomes infected with Borrelia, the Lyme Disease
organism, by feeding on a small rodent infected with the organism.
During a subsequent feeding on humans, the infected tick may inject
Borrelia microorganisms into the bloodstream of that host with
saliva. Some of the Borrelia organisms thus injected can then be
ingested by the ectoparasite that injected them. Therefore, the
excrement of the ecotoparasite may contain not only the markers for
the immediate past host (in this example a human), but also a
marker obtained by the ectoparasite from a prior host (in this
example Borrelia). In manner similar to that described for blood
components and salivary compounds, compounds of microbial origin
may be found in excreted materials. Microbes, molecules, or markers
derived therefrom are then available for detection in environmental
samples. Such tests could be very valuable for epidemiological
studies where catching organisms is not practical.
[0037] In the method of the present invention, collection of an
environmental sample is the first step in detecting presence of
hematophages. The terms "environmental dust sample" or
"environmental dust" as used herein refers to materials collected
from an enclosure that can be expected to contain the excrement,
which in turn contains markers of blood feeding by pests. Such
environmental dust sample or environmental dust may comprise
materials including flakes and smears from diuresis elimination of
water by pests described previously; shells, casts, casings, and
any other remnants of the blood-feeding pest bodies or eggs; and
fecal matter containing markers as described herein. Any method by
which an environmental sample or environmental dust can be
collected, can be used. One well-established method is the vacuum
trace evidence collection practiced in crime scene investigations.
These systems are commercially available (a vacuum evidence
collection kit is offered by, for example, 3M, Minneapolis, Minn.)
The utility of these systems has been well accepted in criminal
investigative work. Disposable filters and sealable plastic
containers, designed to maintain sample integrity and chain of
custody, might be well suited to collect samples in the instant
invention. In addition, other sample collection methods to be used
include collecting dust accumulated on home HVAC filters; filters
in room air conditioners in hotels and motels; dust accumulated on
or near ventilation fans in barns, stables, henhouses, etc.; dust
collected by electrostatic particle precipitators; and samples
collected by damp mopping non-porous floors such as tile floors in
schoolrooms. Specific sample collection methods are not necessary
to practice the invention but the validity and therefore the
utility of the results are dependent upon collecting and
maintaining the integrity of a representative Sample. For example,
in a room with a suspected blood-feeding pest infestation, it might
be deemed useful to vacuum the whole room to screen for markers; to
manually collect dark-colored flakes around the head of the bed; to
vacuum only around the head of the bed to look more specifically
for bedbugs due to their feeding habits; to lift materials with
water-dampened swabs; or possibly all or any combination of
collection methods. All samples thus obtained could be tested for
any or all markers depending upon judgment of the persons involved,
costs, desired thoroughness, etc. All or a portion of the collected
materials in the environmental dust sample is then used for
detection of blood feeding activity.
[0038] The second step in the invention is to test for marker or
markers indicative of blood feeding activity. The invention
provides for selecting reagents depending upon the specific
inquiry. For example, reagents can be used for detection of heme,
which is present in all vertebrate blood and is expected to be
present in the excrement of all blood-feeding parasites following
blood feeding activity. The invention also provides for species
specific screening for a marker or markers such as using monoclonal
or polyclonal antibody based immunoassays to differentiate human
hosts from animal hosts, for example. Examples of such reagents
include antibodies (including antigen binding fragments thereof) to
species specific blood proteins as described herein for human
hemoglobin and IgG. The invention also provides for parasite
specific screening, that is, testing for markers that differentiate
a parasite species from others without providing information on the
host species. The invention further provides for combinations of
reagents or test components designed to provide relevant
information for the conditions encountered. Further, the method
provides for detecting marker or markers released into the
environment by blood-feeding said marker or markers being present
in the pest as a result of previous feeding or feedings.
[0039] Detecting components of the blood of the host or those
derived from the parasitic organism may be accomplished by direct
chemical methods such as guaiac oxidation, cyanmethemoglobin tests
or more specific ligand-binding assay, such as immunoassay, nucleic
acid based detection methods or other suitable methods.
[0040] Examples 1, 2 and 3, provide details on the methods to
detect vertebrate hemoglobin in hematophagous insect feces using
chemical reactions involving the metalloporphyrin moiety present in
hemoglobin from all vertebrate species. One method is based on
reaction between heme of hemoglobin with guaiac after which a
developer solution containing hydrogen peroxide is added. If
hemoglobin is present, the guaiac is oxidized, turning the
detection reagent blue. In addition, several other methods that
might be used to detect hemoglobin in samples collected could be
used. One of particular interest, because of its well-established
sensitivity would be luminol spray used successfully by forensic
scientists to visualize bloodstains not visible to the human eye.
Samples can be collected by vacuum onto a barrier filter membrane
as described herein and as practiced in forensics. The membrane can
be removed and placed into a dark chamber in view of a sensitive
camera. Either film or digital photography could be employed. A
luminol solution (such as from BlueStar), can be sprayed onto the
membrane and sample material associated thereto and
chemiluminescence, if any, would be recorded by the camera suitably
setup and activated.
[0041] Liquid Samples, such as those obtained by mopping an
enclosure, or a water extract of a dust sample can also be used.
The use of the cyanomethemoglobin method (with Drabkin's reagent
described in Drabkin, D. L., Austin, J. H. Journal Biological
Chemistry 112, 51, (1935)) to detect and measure hemoglobin is well
established in diagnostic and veterinary medicine and can be used
in this embodiment by adding the sample to the reagent and test for
the development of the specified color.
[0042] In Examples 4 and 5, immunological determination of human
hemoglobin in hematophagous arthropod feces is disclosed. These
examples demonstrate that proteins, as exemplified by the globin
portion of hemoglobin, present in the blood-meal retain antigenic
characteristics that enable testing for the protein using
immunochemical reactions. In addition to the immunological fecal
occult blood test (iFOBT) other antigens including animal blood
proteins and salivary proteins of parasite origin, and antigens
associated with infectious disease causative agents such as
malaria, West Nile virus, trypanosomiasis, etc, can also be used.
To accomplish this, commercially available antibodies (such as
directed against canine hemoglobin or chicken hemoglobin) can be
used or antibodies specific for markers of interest may produced
using hematophages or parts or products of hematophages such as
feces. Commercially available antibodies, raised against intact
proteins isolated from host blood, can also be used as reagents of
the invention. Alternatively, or additionally, antibodies can also
be generated against proteins present in the feces. This will
address the issue of degradation of antigenic epitopes during the
digestive process. A similar approach can be used for salivary
proteins produced by the hematophagous arthropod. Once antibodies
are obtained either commercially or raised against the proteins or
peptides, the antibody can then be validated for use by
establishing reactivity with antigens in feces. Such antibodies can
be employed in any of various immunoassay techniques available to
those skilled in the art including but not limited to
immunochromatographic or lateral flow techniques, ELISA, double
diffusion, particle agglutination, immunoprecipitation, laser and
non-coherent light nephalometry, and immunoinhibitory assays. The
selection of the technique will be made based on the requirements
of the Sample, the information being sought, and the venue in which
the test will be performed.
[0043] Examples 6 and 7 describe results obtained with
environmental samples collected from a multiplicity of rooms in
hotel building operating commercially. In addition to testing for
hemoglobin using methods of preceding Examples, the versatility,
adaptability, and broad applicability of the present invention is
disclosed by successfully testing for IgG, a blood protein, in the
environmental samples (Examples 8). Examples 9 describes a
combination of immunological and chemical detection methods.
Example 10 describes results obtained from a sample collected from
non-human housing structure.
[0044] Examples 11-13 demonstrates the feasibility of generation of
antibodies to salivary antigens of hematophagous ectoparasites such
that the antibodies can be used for detection of the parasites in
parasitic excrements or environmental dust samples from
enclosures.
[0045] The following Examples describe the preliminary results
obtained in experiments conducted prior to filing this
application.
Example 1
Detecting Heme-Iron in Bedbug Culture Substrate
[0046] Cultures of bedbugs (Cimex lectularius) were maintained in
glass jars containing a pressed paper cardboard substrate (FIG. 1)
at room temperature and were allowed to feed at 7 to 14-day
intervals on human blood. The bedbugs were allowed access to human
skin through a 200-mesh nylon screen attached over the top of their
jars. The skin was allowed to be in contact with the mesh for at
least 10 minutes with each jar during which time the bedbugs fed to
repletion. Cardboard substrate was removed from each jar at least
10 days after feeding, put into plastic bags, sealed, and stored in
the freezer at temperatures below -10.degree. C. until
processed.
[0047] Fecal occult blood tests (FOBT) of two different peroxidase
activity visualization chemistries were used from three different
commercial suppliers (guaiac from Immunostics, Inc., Ocean, N.J.
and Care Products, Waterbury, Conn. and 3,3',5,5'-tetramethyl
benzidine (TMB) from Biomerica, Inc., Newport Beach, Calif.) were
used to qualitatively detect heme. Samples tested were bedbug
culture debris (an example of which is shown in FIG. 2) and pieces
of the cardboard cut from the substrate (such as shown in FIG.
3).
[0048] Extracts were made by putting bedbug culture debris into
vials of buffer supplied by Care Products for the immunologic FOBT
and shaken for about 5 minutes. Two drops of these extracts were
placed on EZ Detect pads (Biomerica, Inc.) employing the TMB
chemistry and processed according to manufacturers instructions.
FIG. 4 shows the results with clear development of color (positive
result) with the bedbug culture extract. FIG. 5 shows the results
of the same extracts run with Immunostics' guaiac FOBT. In
addition, pieces of the culture substrate, such as shown in FIG. 3,
were placed on the paper of both the Immunostics and Care Products
guaiac FOBT. Upon addition of the developing reagent, positive
results were observed. A molted exoskeleton gave a negative result
in guaiac FOBT.
[0049] It can be concluded from these results that the peroxidase
activity such as that of hemecan be detected in the feces of
bedbugs
Example 2
Detection of Blood in Bedbug Cultures Using Luminol
[0050] Debris collected from bedbug cultures as in Example I was
placed on absorbent plastic-backed paper (FIG. 9) and sprayed with
a commercially available luminol blood visualization solution
(BlueStar, ROC Import, Monte Carlo, Monaco). The resultant
chemiluminescence was seen to be associated with the debris and not
with the molted exoskeletons (FIG. 10). This visualization
technique can be used as in initial step to identify individual or
clusters of particles emitting light to be isolated and subjected
to further testing to confirm hematophage activity.
Example 3
Detection of Blood in Bedbug Cultures Using Urine Test Strips
[0051] Debris collected from bedbug cultures as in Example I was
dissolved in water and tested for the presence of blood using
commercially available urine dipsticks (such as from Roche or
Bayer). With each test the presence of detectable blood was
indicated by positive reaction on the appropriate pads.
Example 4
Detection of Human Hemoglobin in Bedbug Cultures with Monoclonal
Antibodies
[0052] The culture debris extracts from Example I were also tested
with ImmoCARE (immunological FOB from Care Products, Inc.
Waterbury, Conn.). The suspensions were added to cartridges (FIG.
6) provided with the kit and allowed to develop according to the
instructions. The results were read per the instructions and are
shown in FIGS. 7 and 8. The bedbug culture debris extract clearly
gave a positive result indicating that sufficient epitopes were
present of the hemoglobin in the feces to react with the monoclonal
antibodies in the iFOBT.
Example 5
Detection of Human Hemoglobin in Bedbug Cultures with Polyclonal
Antibodies
[0053] The culture debris extracts from Example I were tested for
reactivity with commercially available polyclonal raised against
human hemoglobin by direct and sandwich immunoassays. For direct
detection, 20 .mu.L of extract was applied to nitrocellulose
membranes and washed to remove unbound materials. The membranes
were blocked with 20% horse serum in buffered saline and washed 3
times. Spotted membranes were incubated with 2% horse serum-saline
alone or containing polyclonal anti-hemoglobin labeled with
horseradish peroxidase (HRP) or alkaline phosphatase (AP) and
washed to remove unbound enzyme-labeled antibody. Any enzyme
activity bound via antibody to antigen present in the debris
extract bound to the membrane was visualized with the appropriate
substrate. Clearly visible colored spots were observed when spotted
membranes were exposed to enzyme labeled anti-hemoglobin and the
appropriate substrate while no visible reaction was observed when
either substrate was exposed to spotted membranes not allowed to
react with antibody.
[0054] Sandwich immunoassays for hemoglobin in culture debris by
immobilizing polyclonal anti-hemoglobin to nitrocellulose
membranes, washing, and blocking as described above. The capture
antibody membranes were then allowed to incubate with culture
debris extract, washed, and exposed to enzyme labeled
anti-hemoglobin. After visualization with enzyme substrate, the
presence of hemoglobin in the culture debris was indicated by
developed color.
[0055] Bedbug culture debris was incubated with affinity purified
polyclonal anti-hemoglobin to occupy epitopes necessary for
positives reaction in iFOBT assays of Example 4 or immunoassays of
Example 5. In all cases, prior treatment of the antigen with
anti-hemoglobin prevented detection of hemoglobin by the
immunoassays described in the cited Examples.
Example 6
Detection of Hemoglobin in Environmental Samples
Sample Collection
[0056] Examples 1 through 5 demonstrated that evidence of blood
feeding by hematophagous organism can be obtained from the
organisms' fecal debris. To establish the utility of these methods
for detecting naturally occurring blood feeding by testing
environmental samples, samples were collected from offices, hotel
rooms, and homes. Samples were collected by methods described
herein including using standard vacuum cleaners to collect vacuum
bag dust, using handheld portable vacuum devices, manually removing
dust collected on filters of HVAC systems, air conditioners, and
fans, and collecting dust manually or by wiping. Dust samples thus
collected were used directly without further treatment or extracted
with any of a variety of aqueous solutions to prepare liquid
samples in the examples that follow.
Example 7
Samples from Office and Hotel Rooms
Testing for Environmental Hemoglobin as Evidence of Blood
Feeding
[0057] Vacuum dust collected by vacuuming and manually removed from
air conditioner filters from individual rooms were tested with
luminol by spraying the test solution onto dust samples as
collected. Some vacuum dust samples gave positive chemiluminescence
with various densities of light emitting particles per volume of
dust while other samples gave no visible chemiluninescence. Samples
of dust from filters of air conditioners, such dust being a capture
or aerosolized particles over time, were uniformly negative for
chemiluminescence when tested with luminol. These results indicate
that the luminol test has sufficient sensitivity to be useful in
the present invention.
[0058] The samples were also tested for hemoglobin using FOBT and
test strips giving results consistent with the luminol results.
These results also indicate that testing for the heme moiety is
useful for screening, that is to eliminate sites without detectable
blood-feeding activity and therefore possible infestation from
further investigation at a given time.
Example 8
Samples from Hotel Rooms
Testing for Human IgG as Evidence of Blood Feeding
[0059] Samples from bedbug cultures and environmental samples were
extracted with dilute (0.001 N) sodium hydroxide, neutralized by
addition of 0.2 M potassium phosphate in 0.9% NaCl, and tested at
various dilutions for the presence of human IgG, a blood serum
protein, using a commercially available enzyme linked immunosorbent
assay (ELISA) test kit (ZeptoMetrix Inc., Buffalo. NY). Positive
results for IgG in environmental samples as well as Cimex culture
debris indicated the potential utility of testing for blood
components other that hemoglobin for screening or detecting blood
feeding activity. These results demonstrate that that proteins
other than the globin moiety of hemoglobin remain sufficiently
structurally intact after the digestive process by Cimex to be
detectable by standard immunological reagents and means. The result
also provide support for the idea that the methods and compositions
of the present invention can be designed to be applicable to a
variety of testing requirements.
Example 9
Detecting Hemoglobin by Combined Immunological and Chemical
Methods
[0060] Culture debris and environmental samples from previous
examples were further tested with an immunoassay and luminol
combination method. Test membranes to which monoclonal antibodies
specific for human antibodies were removed from the iFOBT devices
previously described. In addition, approximately 1 microgram of
polyclonal anti-human-antibody obtained commercially from Bethyl
Labs, Montgomery, Tex., was immobilized on a nitrocellulose
membrane strip. Both sets of antibody test strips were washed in
PBS and coated with 10% normal horse serum in PBS for 30 min. Hotel
room and culture debris extracts, also in PBS, were added to the
strips for 2 hours at room temperature. Test strips not exposed to
the extracts were included in the experiment as negative controls.
After incubation with the samples or control, all strips were
washed 3 time with PBS. Each strip was sprayed with luminol
solution as previously described and examined for chemiluminescence
which was clearly observed with the antibody strips exposed to
samples. In the control strips no chemiluminescence was observed at
the location of the immobilized antibody. This experiment
demonstrates that analytical methods can be used alone or combined
to depending upon the testing needs.
Example 10
Samples Chicken Houses
Environmental Samples
[0061] Samples of dust from the floor, trays, fans, and fan filters
were collected from structures housing chickens. Some of the birds
were in individual cages and some were in groups of several dozens
free to move about contained in open rooms. Positive
chemiluminescence and dip stick tests were obtained with samples
but all tests using immunoassays specific for human hemoglobin were
negative. These results demonstrate the capability to design test
reagents and systems for detecting ectoparasitic blood-feeding on
animal hosts and the ability to differentiate such activity from
that occurring with human hosts.
Example 11
Antibodies Against Hematophage Antigens
[0062] Blood was drawn by a healthcare professional from a human
volunteer who had been bitten by Cimex repeatedly for more than 3
years and who had consented to provide blood and have it tested for
reactivity with samples obtained as described herein. Plasma was
prepared, separated and frozen. To determine the presence of
antibodies against antigens present in bedbug culture debris, a
debris extract was applied to nitrocellulose membranes as in
Example 5, washed, blocked with 2% normal horse serum in PBS, and
exposed to human plasma from above at various dilutions. The
membranes were then washed and allowed to incubate with alkaline
phosphatase labeled anti-IgG (human). The presence of antibodies to
antigens present in the bedbug culture debris was visualized by
exposing the membrane so treated with substrate for alkaline
phosphatase. The presence of color was observed at antiserum
concentrations higher than at a 1:500 dilution and was not observed
on membranes not exposed to the human antibody solutions. These
results indicate that antibodies produced in response to exposure
to hematophage saliva can be used for detecting the presence of
pest excrement. Such presence could indicate past or present blood
feeding activity.
Example 12
Collection of Cimex Saliva
[0063] Saliva was collected from bedbugs immobilized with adhesive
tape by positioning the mouthparts of the insect into a capillary
tube. A solution of pilocarpine in alcohol sufficient to induce
saliva excretion in the individual being used was applied to the
head of the insect. The saliva thereby secreted was collected in
the capillary, transferred to vials, sealed, and frozen until
used.
Example 13
Testing of Reactivity of Antibodies Against Hematophage Salivary
Antigens
[0064] Human antibodies preparation collected as described in
Example 11 was tested with Cimex saliva described in Example 12 by
immobilizing proteins on to nitrocellulose membranes, blocking and
washing as previously described. The membranes were incubated with
human antibodies at dilutions varying from 1:50 to 1:1000 in PBS
containing 2% normal horse serum and the reactivity was detected
with enzyme-labeled-anti-human IgG as previously described. Color
development indicated reaction between the saliva sample and human
antibodies. The reactivity was similar to that observed in Example
XI and was consistent with the presence of immunologically
detectable hematophage specific markers in saliva. This example
demonstrates that antibodies generated from individuals who have
been bitten by the hematophagous parasites (and therefore exposed
to the salivary proteins of the parasites) can be used for the
detection of parasites in an environmental sample. Thus polyclonal
or monoclonal antibodies from individuals (humans or animals) could
be obtained following exposure to the parasitic antigens such as
salivary proteins--whether by being bitten by the parasite or other
conventional immunization procedures. Because the volunteer donor
reported redness and itching as a result of bedbug bites--a
physiological reaction associated with an immunoreaction to
substances injected in saliva--these results support the conclusion
of the inventor that the invention will have utility in testing for
any blood-feeding ectoparasite to which the host mounts an immune
response.
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