Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-07-22
2002-07-02
Smith, Lynette R. F. (Department: 1645)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C435S007200, C435S007220, C435S069200, C435S069400, C435S342000, C435S965000, C530S388600, C424S178100, C424S184100, C424S265100
Reexamination Certificate
active
06413721
ABSTRACT:
BACKGROUND
Infection with meningeal and tissue worms (Nematoda: Protostrongylidae) can result in serious neurological disease or death in certain ungulate hosts. The Protostrongylidae family of nematodes includes a variety of infectious species, for example,
Parelaphostrongylus tenuis
(meningeal worm),
Parelaphostrongylus andersoni, Parelaphostrongylus odocoilei, Elaphostrongylus rangiferi
and
Elaphostrongylus cervi
(tissue worm) (see, e.g., Platt (1984) for a discussion of various protostrongylidae). The nematode
Parelaphostrongylus tenuis
(
P. tenuis
) is widespread throughout wild white-tailed deer hosts (WTD;
Odocoileus virginianus
) in the eastern half of North America. (Anderson et al. (1992), Anderson and Prestwood (1981), Anderson and Streveli (1967)). Although symptoms may be mild in infected WTD,
P. tenuis
infection can result in fatal neurological disease in various cervids and camelids as well as in other wild and domesticated ruminants (Anderson and Prestwood, (1981)).
Development of a reliable and sensitive method for diagnosing infection with various meningeal worms has proven difficult. Currently, diagnosis is performed using the Baermann technique or a modified Baermann technique (see, e.g., Gajadhar et al. (1994)
Can. Vet. J.
35:433-437). In brief, this method involves detecting the presence of dorsal-spined, first-stage larvae (L1) in the feces of infected animals. However, there are several limitations to this technique. In particular, the method does not routinely allow detection of low level infection, although these animals may exhibit clinical signs and even die (Dew et al., 1992; Samuel et al., 1992). The reasons that infected animals may fail to shed detectable numbers of larvae has not been entirely elucidated. The parasite load may be low or larvae shed intermittently (Welch et al., 1991). Alternatively, animals infected with only one worm, or worms of the same gender, will not shed larvae.
The Baermann technique also fails to provide early detection of infection. Because the pre-patent period is long, between 82-137 days (Rickard et al., 1994), infection may not be diagnosed for many months and repeated testing is often required. Yet another drawback of the Baermann method is that is does not distinguish between various species of the protostrongylidae family. Many of the dorsal-spined larvae detected in feces are morphologically indistinguishable between species. (Pybus and Samuel, 1984; Lankester and Hauta, 1989; Lankester and Fong, 1989). Thus, available diagnostic techniques cannot routinely differentiate between infection with different nematodes.
Studies directed at identifying antibodies in the serum of infected animals have not resulted in reliable diagnostic methods. Although anti-
P. tenuis
antibodies have been detected in the serum of elk (wapiti;
Cervus elaphus canadensis,
Neumann et al., 1994; Bienek et al., 1998) and of goats (Dew et al., 1992), these antibodies are inconsistently detected in the serum of the definitive host, WTD (Dew et al., 1992; Duffy et al., 1993). Furthermore, even in those studies in which antibodies were detected, they were not measurable until at least 75 days post-exposure. (Duffy et al., supra). In addition, the serological cross-reactivity of anti-
P. tenuis
antibody against antigens of the other closely related nematodes has not been assessed.
Thus, there remains a need for early and accurate identification of parasitic nematode infections. In addition, there is a need for identification of antigens that specifically and uniquely identify parasites such as
P. tenuis
or
E. cervi.
SUMMARY OF THE INVENTION
Described herein are novel common and specific Protostrongylidea antigens, polynucleotides encoding these antigens and antibodies which recognize these antigens. Early and accurate diagnostic methods for parasitic infection are disclosed.
Thus, in one aspect, the invention includes isolated immunogenic Protostrongylidae antigens, for example a
P. tenuis
-specific 20 kDa antigen, a
P. tenuis
-specific 37 kDa antigen, an
E. cervi
-specific 37 kDa antigen, a 52 kDa antigen and a
P. tenuis
-specific 75 kDa antigen, a common 105 kDa antigen or a common 158 kDa antigen, as determined by SDS-PAGE gel electrophoresis. Thus, antigens specific for
P. tenuis
or
E. cervi
are also provided. Exemplary
P. tenuis
-specific antigens include a
P. tenuis
-specific 20 kDa antigen, a
P. tenuis
-specific 37 kDa antigen and a
P. tenuis
-specific 75 kDa antigen, as determined by SDS-PAGE gel electrophoresis. Exemplary
E. cervi
-specific antigens include an
E. cervi
-specific 37 kDa antigen and an
E. cervi
-specific 52 kDa antigen, as determined by SDS-PAGE gel electrophoresis.
In another aspect, antibodies that specifically recognize a common Protostrongylidae antigen are provided. Antibodies that specifically recognize
P. tenuis
-specific or
E. cervi
-specific antigens are also described.
In another aspect, the invention provides polynucleotides encoding common,
P. tenuis
-specific or
E. cervi
-specific antigens.
Methods of diagnosing Protostrongylidae infection in a vertebrate subject by detecting the presence of at least one common Protostrongylidae antigen in a biological sample, for example a serum sample, obtained from the subject are also provided. In some embodiments, the presence of one common antigen is detected, while in other embodiments, multiple common antigens are detected. Methods of specifically diagnosing
P. tenuis
or
E. cervi
infection using at least one
P. tenuis
-specific or
E. cervi
-specific antigens are also provided. The common or specific antigens can be detected, for example, using antibodies, using nucleic acid probes or using PCR. In one embodiment, the common or specific antigens are detected by (a) reacting the biological sample with one or more isolated common or one or more specific antigens under conditions which allow anti-Protostrongylidae,
P. tenuis
or
E. cervi
antibodies, when present in the sample, to specifically bind with said common antigens; (b) removing unbound antibodies; (c) providing one or more moieties capable of associating with the bound antibodies; and (d) detecting the presence or absence of the one or more moieties. The one or more moieties may comprise a detectably labeled immunoglobulin antibody.
In another aspect, methods of detecting, in a biological sample, antibodies to parasites comprising (a) reacting the biological sample with an antigen preparation selected from the group consisting of an ES-L3 antigen preparation and an sL3 antigen preparation, under conditions which allow parasitic antibodies to bind to an antigen in the antigen preparations and form an antigen:antibody complex; and (b) detecting the presence or absence of said complex are provided. The parasites may be a member of the Protostrongylidae family or may specifically be
P. tenuis
or
E. cervi.
Kits for use in the diagnostic methods described herein are also provided. The kits comprise, in a suitable packaging, one or more common or
P. tenuis
- or
E. cervi
-specific antigens immobilized on a solid support; and a reagent suitable for detecting, in a biological sample, the presence of antibodies to the one or more common or
P. tenuis
- or
E. cervi
-specific antigens.
In yet another aspect, the invention common antigens obtained by (a) providing a cDNA library which expresses protostrongylidae genes; (b) screening the expressed genes of the cDNA library with a source of anti-protostrongylidae antibodies to identify cDNA clones which express common antigens; and (c) transforming a host cell with the cDNA clones which express the common antigen. Also provided are
P. tenuis
-specific antigens obtained by (a) providing a cDNA library which expresses
P. tenuis
genes; (b) screening the expressed genes of the cDNA library with a source of anti-
P. tenuis
antibodies to identify cDNA clones which express
P. tenuis
-specific antigens; and (c) transforming a host cell with the cDNA clones which express the
P. tenuis
-specific antigen. Also provided are
E
Gajadhar Alvin A.
Ogunremi Oladele
Baskar Padma
Her Majesty the Queen in Right of Canada, as represented by the
Robins & Pasternak LLP
Smith Lynette R. F.
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