Immunoassay for detection of Dipylidium caninum coproantigen in dogs and cats.

Dipylidium caninum infections in dogs and cats are underestimated because of a lack of proglottid observations and poor recovery of parasite elements by centrifugal flotation. We developed an immunoassay that employs a pair of monoclonal antibodies to capture D. caninum-specific coproantigen in fecal extracts from dogs and cats. Real-time PCR for D. caninum DNA in perianal swabs and observation of proglottids were used as reference methods. In 6 experimentally infected dogs, parasite DNA, coproantigen, and proglottid segments were first detected at 22, 23, and 26 d post-infection, respectively. Praziquantel treatment of 3 experimentally infected dogs resulted in the elimination of both coproantigen and proglottid shedding within 1-5 d post-treatment; however, parasite DNA persisted for 14 d. Immunohistochemistry on immature and mature tapeworm segments using an antibody against the coproantigen supports the premise that the antigen is produced in mature segments. We assessed the performance of our coproantigen test in natural infections in 78 dogs from a flea-endemic area. Of the 12 antigen-positive samples, 11 were confirmed with a positive PCR test and/or proglottid observation. Finally, we evaluated a convenience sample set of 730 canine and 163 feline fecal samples obtained from a commercial diagnostic laboratory; D. caninum antigen was detected in 4.1% of the canine and 12.9% of the feline samples, whereas parasite elements were observed in only 0.028% of samples. Our coproantigen immunoassay provides a sensitive method for the detection of D. caninum infection in dogs and cats.

Diagnosis of feline whipworm infection using a coproantigen ELISA and the prevalence in feral cats in southern Florida.

Trichuris felis, the whipworm of cats, is a relatively rare parasite, although more common in tropical and sub-tropical regions such as the Caribbean and South America. In southern Florida, T. felis is known to occur, but estimating prevalence can be challenging using fecal egg counts due to low intensity and single sex infections. A microplate enzyme-linked immunosorbent assays (ELISAs) for parasite-specific antigen in feces could increase the ability to detect these infections. In this study, the IDEXX Fecal Dx™ antigen ELISA for whipworm (designed for detection of Trichuris vulpis) was evaluated for detection of T. felis using 35 feral cats from St. Kitts, euthanized for non-study related reasons. Twenty-nine of the cats were positive for T. felis with worm counts ranging from 1 to 66 per cat (mean 9.6) and egg counts ranging from 0 to >500 (mean 109.8). The ELISA detected 26 of 29 positive cats while flotation (centrifugation with Sheather's sugar solution) detected 24 of the 29 positive cats. To estimate prevalence in southern Florida, feces from 65 feral cats from the greater Miami area were tested using the ELISA and fecal flotation (centrifugation with zinc sulfate). Twenty-five cats (38%) were identified as positive with the ELISA compared to 17 using fecal flotation. This prevalence was surprising and further investigated by reviewing results of feline samples from Florida submitted to IDEXX Reference Laboratories between 2010 and 2017 and analyzed using fecal flotation. While prevalence was below 1%, there was an apparent trend in increasing prevalence. The results of this study confirm that the IDEXX Fecal Dx™ antigen test for whipworm ELISA, while developed for T. vulpis, can detect T. felis infections. It also suggests that prevalence might be higher than previously known in Florida and might be increasing. Further studies are required to determine the distribution of this higher prevalence and if the distribution and prevalence of T. felis are changing.

Enzyme-linked immunosorbent assays for coproantigen detection of Ancylostoma caninum and Toxocara canis in dogs and Toxocara cati in cats.

We report the development and field validation of 2 ELISAs for the detection of Ancylostoma caninum or Toxocara canis coproantigens in the feces of dogs with experimental and natural infections, and evidence of cross-reactivity with respective feline counterparts. A. caninum-specific coproantigens were detected in feces of experimentally infected dogs starting at 9 d post-infection (dpi), whereas eggs were not seen until 23 dpi. T. canis-specific coproantigens were detected in 3 of 5 experimentally infected dogs by 31 dpi, and 4 of the 5 animals by 38 dpi. T. canis eggs were seen in feces of 4 of the 5 animals by 38 dpi. One dog had delayed coproantigen detection and low egg output. Additionally, 817 canine and 183 feline fecal samples from naturally infected animals tested by flotation were subjected to coproantigen ELISA testing. Of these 1,000 canine and feline samples, 13 and 23 samples, respectively, were positive for "hookworm" or "roundworm" eggs; 19 and 26 samples were ELISA positive, respectively. The T. canis ELISA detected T. cati coproantigen in cat fecal samples. Discrepant ELISA and flotation results were obtained for 16 hookworm- and 13 roundworm-positive samples. Re-examination of the egg-positive, ELISA-negative samples indicated several instances of possible misidentification or coprophagy, whereas detection of antigen in samples without egg observations is likely a reflection of true infection status with egg shedding below detection levels. There is good indication, based on accumulated field data, that these antigen tests also detect other hookworm and ascarid species.

Secretion of a novel developmentally regulated chitinase (family 19 glycosyl hydrolase) into the perivitelline fluid of the parasitic nematode, Ascaris suum.

The early development of the parasitic nematode, Ascaris suum, occurs within a chitinous eggshell and an abundant chitinase (As-p50) has been identified in the perivitelline fluid (PVF) surrounding the infective larva prior to hatching. A cDNA encoding As-p50 was cloned, sequenced and the protein expressed in Escherichia coli. As-p50 is a member of glycosyl hydrolase family 19, previously identified only in plants, making the characterization of As-p50 the first family 19 glycosyl hydrolase from any animal species. As expected, the chitinase activity of recombinant As-p50 or isolated PVF was insensitive to allosamidin. As-p50 expression was developmentally regulated. As-p50 mRNA appeared between days 5 and 8 of development prior to the formation of the first-stage larva (L1). The As-p50 protein and chitinase activity appeared later between days 8 and 15 and remained at constant levels until hatching. GFP-promoter constructs of C08B6.4, the most closely related Caenorhabditis elegans As-p50 homologue, were expressed in hypodermal cells of 3-fold stage larvae and L1s with a timing similar to that of As-p50 and the fusion protein was secreted into the space between the hypodermis and the cuticle. Taken together, these results suggest that As-p50 is involved in the formation of the L1 cuticle and/or the initial molt; however, As-p50 may be multifunctional and also responsible for the digestion of the eggshell during hatching.

Enzyme-linked immunosorbent assay for coproantigen detection of Trichuris vulpis in dogs.

Infections with Trichuris vulpis, the canine whipworm, may be challenging to diagnose even though characteristic bipolar eggs are shed by mature worms and may be recovered from feces. Decreased detection sensitivities because of using flotation solutions with specific gravities <1.3 and a lengthy prepatent period can lessen the diagnostician's ability to detect infection. Coproantigen detection in feces is becoming an accepted form of diagnosing parasitic infections and can circumvent some of the factors that affect egg recovery. The development of an enzyme-linked immunosorbent assay (ELISA) for the detection of whipworm-specific coproantigens in the feces of dogs with experimental and natural T. vulpis infections is reported herein. Whipworm-specific coproantigens were evidenced in feces from experimentally infected dogs using the newly developed ELISA starting as early as day 23 postinfection, while eggs were not detected in feces until day 69. In addition, 1,156 field fecal samples were tested using fecal flotation methods and the newly developed whipworm ELISA. Of these, 27 samples were found by flotation to be whipworm egg positive, while 35 had detectable antigen on the ELISA. Discrepant results were obtained in 12 samples; 2 egg-positive samples tested ELISA negative, and 10 ELISA-positive samples did not contain detectable egg levels. Using the fecal ELISA for the detection of whipworms in dogs should allow for earlier detection of infection, aid the identification of cases in the face of low egg shedding, and increase detection sensitivity as most commercial laboratories are using flotation solutions not optimal for T. vulpis egg detection.

Two methods for improved purification of full-length mammalian proteins that have poor expression and/or solubility using standard Escherichia coli procedures.

Many mammalian proteins are multifunctional proteins with biological activities whose characterization often requires in vitro studies. However, these studies depend on generation of sufficient quantities of recombinant protein and many mammalian proteins cannot be easily expressed and purified as full-length products. One example is the Wilm's tumor gene product, WT1, which has proven difficult to express as a full-length purified recombinant protein using standard approaches. To facilitate expression of full-length WT1 we have developed approaches that optimized its expression and purification in Escherichia coli and mammalian cells. First, using a bicistronic vector system, we successfully expressed and purified WT1 containing a C-terminal tandem affinity tag in 293T cells. Second, using a specific strain of E. coli transformed with a modified GST vector, we successfully expressed and purified N-terminal GST tagged and C-terminal 2x FLAG tagged full-length human WT1. The benefits of these approaches include: (1) two-step affinity purification to allow high quality of protein purification, (2) large soluble tags that can be used for a first affinity purification step, but then conveniently removed with the highly site-specific TEV protease, and (3) the use of non-denaturing purification and elution conditions that are predicted to preserve native protein conformation and function.

Saccharomyces cerevisiae Rab-GDI displacement factor ortholog Yip3p forms distinct complexes with the Ypt1 Rab GTPase and the reticulon Rtn1p.

Rab GTPases are crucial regulators of organelle biogenesis, maintenance, and transport. Multiple Rabs are expressed in all cells, and each is localized to a distinct set of organelles, but little is known regarding the mechanisms by which Rabs are targeted to their resident organelles. Integral membrane proteins have been postulated to serve as receptors that recruit Rabs from the cytosol in a complex with the Rab chaperone, GDI, to facilitate the dissociation of Rab and GDI, hence facilitating loading of Rabs on membranes. We show here that the yeast (Saccharomyces cerevisiae) Golgi Rab GTPase Ypt1p can be copurified with the integral membrane protein Yip3p from detergent cell extracts. In addition, a member of the highly conserved reticulon protein family, Rtn1p, is also associated with Yip3p in vivo. However, Ypt1p did not copurify with Rtn1p, indicating that Yip3p is a component of at least two different protein complexes. Yip3p and Rtn1p are only partially colocalized in cells, with Yip3p localized predominantly to the Golgi and secondarily to the endoplasmic reticulum, whereas Rtn1p is localized predominantly to the endoplasmic reticulum and secondarily to the Golgi. Surprisingly, the intracellular localization of Rabs was not perturbed in yip3Delta or rtn1Delta mutants, suggesting that these proteins do not play a role in targeting Rabs to intracellular membranes. These data indicate that Yip3p may have multiple functions and that its interaction with Rabs is not critical for their recruitment to organelle membranes.

Targeting of the FYVE domain to endosomal membranes is regulated by a histidine switch.

Specific recognition of phosphatidylinositol 3-phosphate [PtdIns3P] by the FYVE domain targets cytosolic proteins to endosomal membranes during key signaling and trafficking events within eukaryotic cells. Here, we show that this membrane targeting is regulated by the acidic cellular environment. Lowering the cytosolic pH enhances PtdIns3P affinity of the FYVE domain, reinforcing the anchoring of early endosome antigen 1 (EEA1) to endosomal membranes. Reversibly, increasing the pH disrupts phosphoinositide binding and leads to cytoplasmic redistribution of EEA1. pH dependency is due to a pair of conserved His residues, the successive protonation of which is required for PtdIns3P head group recognition as revealed by NMR. Substitution of the His residues abolishes PtdIns3P binding by the FYVE domain in vitro and in vivo. Another PtdIns3P-binding module, the PX domain of Vam7 and p40phox is shown to be pH-independent. This provides the fundamental functional distinction between the two phosphoinositide-recognizing domains. The presented mode of FYVE regulation establishes the unique function of FYVE proteins as low pH sensors of PtdIns3P and reveals the critical role of the histidine switch in targeting of these proteins to endosomal membranes.

SER-7b, a constitutively active Galphas coupled 5-HT7-like receptor expressed in the Caenorhabditis elegans M4 pharyngeal motorneuron.

Serotonin plays a key role in the regulation of pharyngeal pumping in nematodes. We have isolated a Caenorhabditis elegans cDNA (C09B7.1b, ser-7b) with greatest identity to the 5-HT7 receptor family. Membranes from COS-7 cells expressing SER-7b exhibit saturable [3H]-LSD binding (Kd = 45 nm) that is inhibited by serotonin (5-HT) and tryptamine, but not by other physiological biogenic amines. Expression of SER-7b in COS-7 cells results in dramatic increase in basal cAMP levels over untransfected cells that is dependent on expression level. 5-HT further elevates cAMP levels in a dose-dependent manner (pEC50 = 7.5 +/- 0.5). Mammalian 5-HT7 receptor inverse agonists reduce constitutive activity, with methiothepin the most potent (pIC50 = 7.8 +/- 0.1). Ser-7::GFP transcriptional fusions reveal that SER-7b appears to be expressed solely in the M4 pharyngeal motorneuron after hatching. This is the first report of a Galphas coupled biogenic amine receptor in nematodes and the localization of SER-7b in the M4 pharyngeal motorneuron suggests that SER-7b may play a role in the regulation of pharyngeal pumping.