Serologic responses to peptides of Anaplasma phagocytophilum and Borrelia burgdorferi in dogs infested with wild-caught Ixodes scapularis.

Anaplasma phagocytophilum and Borrelia burgdorferi are both transmitted by Ixodes spp. and are associated with clinical illness in some infected dogs. This study evaluated canine antibody responses to the A. phagocytophilum p44 peptides APH-1 and APH-4 as well as the B. burgdorferi C6 peptide before and after doxycycline treatment. A total of eight dogs were infested with wild-caught I. scapularis for 1 week. Blood was collected prior to tick attachment and from Days 3-77 to 218-302 with doxycycline treatment beginning on Day 218. Blood was assayed for A. phagocytophilum DNA by PCR assay. Sera was assessed for antibodies by immunofluorescent antibody (IFA) test and ELISA. Anaplasma phagocytophilum DNA was amplified from blood of all dogs by Day 7. Antibodies to APH-4 were detected in serum as early as 14days after tick exposure and six dogs had APH-4 antibodies detected 3-7 days before antibodies against APH-1. All dogs were seropositive for A. phagocytophilum from Days 218 to 302. Antibodies to B. burgdorferi were detected in 6/8 dogs beginning 21days after I. scapularis infestation. Among the five dogs that remained seropositive at Day 218, C6 antibody levels declined on average 81% within 84days of initiating treatment. The results suggest that the APH-4 peptide may be more useful than APH-1 for detecting antibodies earlier in the course of an A. phagocytophilum infection. After doxycycline administration, C6 antibody levels but not APH-1 or APH-4 antibody levels decreased, suggesting a treatment effect on C6 antibody production.

Persistent Ehrlichia ewingii infection in dogs after natural tick infestation.

BACKGROUND: Ehrlichia ewingii, which causes disease in dogs and people, is the most common Ehrlichia spp. infecting dogs in the United States, but little is known about how long E. ewingii infection persists in dogs. HYPOTHESIS/OBJECTIVES: To evaluate the persistence of natural infection with E. ewingii in dogs. ANIMALS: Four Class A Beagles; no previous exposure to ticks or tick-borne infectious agents. METHODS: Dogs were exposed to ticks by weekly walks through tick habitat in north central Oklahoma; dogs positive for infection with Ehrlichia spp. by sequence-confirmed PCR and peptide-specific serology were evaluated for 733 days (D). Whole blood was collected once weekly for PCR, and serum was collected once monthly for detection of antibodies to Ehrlichia canis (peptide p16), Ehrlichia chaffeensis (indirect fluorescence antibody [IFA] and variable-length PCR target [VLPT]), and E. ewingii (peptide p28). RESULTS: All dogs (4/4) became infected with Ehrlichia spp. as evidenced by seroconversion on IFA to E. chaffeensis (4/4); PCR detection of E. ewingii (4/4) and E. chaffeensis (2/4) DNA using both nested and real-time assays; and presence of specific antibodies to E. ewingii (4/4) and E. chaffeensis (2/4). Infection with E. chaffeensis was not detected after D55. Intermittent E. ewingii rickettsemia persisted in 3 of 4 dogs for as long as 733 days. CONCLUSIONS AND CLINICAL IMPORTANCE: Our data demonstrate that dogs infected with E. ewingii from tick feeding are capable of maintaining infection with this pathogen long-term, and may serve as a reservoir host for the maintenance of E. ewingii in nature.

Experimental Dirofilaria immitis infection in dogs: effects of doxycycline and Advantage Multi® administration on immature adult parasites.

To better understand the efficacy of doxycycline and 10% imidacloprid+2.5% moxidectin (Advantage Multi(®); Bayer Animal Health, Shawnee Mission, Kansas) on immature adult Dirofilaria immitis parasites and the results of antigen tests, 12 healthy, randomly selected dogs were experimentally infected with D. immitis and monitored for 407 days. Two dogs in each of three subgroups of four dogs were each infected with six (total of 6 dogs) or 12 (total of 6 dogs) D. immitis infective third-stage larvae (L3) obtained from infected mosquitoes. Doxycycline (10mg/kg per os twice daily×30 days) and 10% imidacloprid+2.5% moxidectin (1ml/kg by topical application every 30 days) treatment was initiated at 105 (Group A) and 149 (Group B) days post infection (PI) in two groups. One subgroup of two dogs given 6 L3 and one subgroup of two dogs given 12 L3 remained as untreated controls (GroupC). Serum obtained regularly throughout the study was evaluated by ELISA (PetChek(®) Heartworm-PF Antigen Test, IDEXX Laboratories, Inc.) for D. immitis adult circulating antigens. Six of the eight dogs in the treated groups had detectable antigenemia starting between 148 and 240 days post infection, but antigen was not detected in any treated dog at the end of the study. In the control subgroups, the dogs that received 6 L3 had no detectable antigen while the two dogs that received 12 L3 had detectable antigen beginning on Day 180 that persisted until the end of the study. None of the infected dogs had evidence of circulating microfilariae. At necropsy, no heartworms were recovered from the treated dogs, but all dogs in the untreated group had viable adult heartworms. These results indicate that early immature adult worms (3.5 and 5 months of age) of D. immitis were susceptible to a combined treatment regimen of doxycycline and 10% imidacloprid+2.5% moxidectin.

Human transforming growth factor-beta activates a receptor serine/threonine kinase from the intravascular parasite Schistosoma mansoni.

The biology of the helminth parasite Schistosoma mansoni is closely integrated with that of its mammalian host. SmRK1, a divergent type I transforming growth factor-beta (TGF-beta) receptor of unknown ligand specificity, was previously identified as a candidate for a receptor that allows schistosomes to respond to host-derived growth factors. The TGF-beta family includes activin, bone morphogenetic proteins (BMPs), and TGF-beta, all of which can play crucial roles in metazoan development. The downstream signaling protein of receptors that respond to TGF-beta and activin is Smad2, whereas the receptors that respond to BMPs signal via Smad1. When a constitutively active mutant of SmRK1 was overexpressed with either schistosome Smad1 (SmSmad1) or SmSmad2, a receptor-dependent modulation of SmSmad phosphorylation and luciferase reporter activity occurred only with SmSmad2. To evaluate potential ligand activators of SmRK1, a chimeric receptor containing the extracellular domain of SmRK1 joined to the intracellular domain of the human type I TGF-beta receptor was used. The chimeric receptor bound radiolabeled TGF-beta and could activate a luciferase reporter gene in response to both TGF-beta 1 and TGF-beta 3 but not BMP7. Confirmatory results were obtained using full-length SmRK1. These experiments implicate TGF-beta as a ligand for SmRK1 and as a potential host-derived regulator of parasite growth and development.

Conserved role for 14-3-3epsilon downstream of type I TGFbeta receptors.

Schistosoma mansoni receptor kinase-1 (SmRK1) is a divergent type I transforming growth factor beta (TGFbeta) receptor on the surface of adult parasites. Using the intracellular domain of SmRK1 as bait in a yeast two-hybrid screen we identified an interaction with S. mansoni 14-3-3epsilon. The interaction which is phosphorylation-dependent is not specific to schistosomes since 14-3-3epsilon also binds to TbetaRI, the human type I TGFbeta receptor. 14-3-3epsilon enhances TGFbeta-mediated signaling by TbetaRI and is the first TbetaRI-interacting non-Smad protein identified that positively regulates this receptor. The interaction of 14-3-3epsilon with schistosome and human TbetaRI suggests a conserved, but previously unappreciated, role for this protein in TGFbeta signaling pathways.

Functional conservation of Schistosoma mansoni Smads in TGF-beta signaling.

To begin to understand the molecular basis of communication between the parasite Schistosoma mansoni and its mammalian host, we are studying the signaling pathway downstream of S. mansoni receptor kinase-1 (SmRK1), a divergent type I transforming growth factor-beta (TGF-beta) receptor found on the tegumental surface of the parasite. In this study, we have used a homology based PCR approach to clone two S. mansoni Smad (SmSmad) genes; Smads play a pivotal role in the most well understood signaling pathways initiated by the TGF-beta family of ligands in other organisms. Comparison of the amino acid sequences with those of other Smads reveals that the conserved MH1 and MH2 domains of SmSmads show a high degree of identity to homologues in Drosophila. Transcripts for both SmSmads are detected in the same developmental stages as SmRK1, and both are capable of interacting with the intracellular domain of the receptor in vitro. Functional characterization using the human type I TGF-beta receptor further confirms the highly conserved nature of these proteins, as both SmSmads show TGF-beta dependent enhancement of luciferase activity and nuclear translocation in mammalian cells. These data are the first to show a TGF-beta-like receptor/Smad signaling pathway in parasitic helminths and by analogy with other systems, is likely important in regulating schistosome development.

Retrospective analysis of the clinical utility of ambulatory electrocardiographic (Holter) recordings in syncopal dogs: 44 cases (1991-1995).

The objective of this study was to evaluate retrospectively the clinical utility of ambulatory (Holter) electrocardiographic monitoring in syncopal dogs and to compare the Holter recording with the clinic electrocardiogram (ECG) in these animals. Fifty Holter reports and 44 medical records from 44 dogs were evaluated. A syncopcal episode occurred during monitoring in 24% of the recordings. No obvious relationship was found between the frequency of syncope occurring before Holter recording and the likelihood of a dog having an episode during recording. Holter recordings were helpful in establishing a diagnosis 42% of the time, but no relationship was detected between the frequency of episodes occurring before Holter recording and the likelihood of a diagnostically useful Holter. An arrhythmia was ruled out as the cause of syncope in 12% of the recordings and was implicated as the cause of syncope in 30% of recordings. Of these, 20% were ventricular tachyarrhythmias and 10% were bradyarrhythmias including pacemaker failure. Ambulatory electrocardiographic recordings led to a therapeutic change in 38% of cases. A comparison of the Holter recordings and clinic ECGs documented the expected increased sensitivity for Holter detection of arrhythmias. The average clinic ECG heart rate consistently exceeded the average Holter heart rate with a mean difference between the average heart rates recorded by the two techniques of 31 bpm (range -8-87 bpm).

Melanocortin mediated inhibition of feeding behavior in rats.

Melanocortinergic neurons are believed to play a role in the control of food intake. Melanocortin receptor agonists and antagonists modulate feeding in several mouse models of chemically and genetically induced hyperphagia. To date, little information is available describing the role of this neurological system in the control of the natural feeding cycle in genetically intact rats. To evaluate the involvement of melanocortins in spontaneous nocturnal feeding, the synthetic melanocortin receptor agonist, MTII and the antagonist, SHU9119 were administered ICV (third ventricle) alone and in combination. Dose-dependent inhibition or stimulation of food intake was observed with MTII or SHU9119, respectively. Co-injections containing equal concentrations of MTII and SHU9119 resulted in food intake that was indistinguishable from controls. Food intake patterns observed in studies in which various dose combinations of MTII and SHU9119 were co-injected are consistent with the concept that both affect feeding by acting on similar melanocortin receptors. The hypothesis that effects of melanocortins on feeding may be mediated via an NPY related pathway was tested by co-injecting MTII and NPY in a 2-h satiated food intake paradigm. MTII inhibited food intake induced by 5.0 microg hNPY in a dose dependent manner with the highest dose tested abolishing the NPY feeding response. The studies suggest that melanocortins act via specific receptors to control food intake in rats, possibly via an NPY related pathway. If similar neurochemical processes operate in humans, selectively modulating specific melanocortin receptor signaling may be an approach to the treatment of human obesity.

Heterogeneity of layer II neurons in human entorhinal cortex.

Abnormalities in the layer II neurons of human entorhinal cortex have been implicated in the pathophysiology of Alzheimer's disease and schizophrenia. The reported abnormalities are not homogeneously distributed throughout the entorhinal cortex, suggesting that layer II of entorhinal cortex may contain different subpopulations of neurons, each with a different susceptibility to pathological mechanisms. In order to investigate the possible heterogeneity of neurons in layer II of human entorhinal cortex, we first identified distinct subdivisions of human entorhinal cortex by adapting the cytoarchitectonic criteria for subdivisions of monkey entorhinal cortex described by Amaral et al. (J Comp Neurol 264:326, 1987). The morphology and regional distribution of distinct subpopulations of human layer II neurons were determined through the use of immunohistochemical techniques. Multipolar, stellate, and modified pyramidal neurons in the characteristic cell clusters or islands of layer II were immunoreactive for nonphosphorylated neurofilament proteins. The intensity of immunoreactivity for the nonphosphorylated neurofilament proteins gradually increased along the rostrocaudal axis of entorhinal cortex and was primarily due to a similar gradient in the density of labeled neurons per island. The calcium-binding protein calbindin D-28K was found in both pyramidal and nonpyramidal neurons in layers II and superficial III. The distribution of calbindin-immunoreactive neurons also depended upon the region of entorhinal cortex. In rostral entorhinal cortex, labeled neurons were scattered throughout the superficial layers, whereas in caudal entorhinal cortex, distinctive patches of small calbindin-immunoreactive neurons were found among the layer II islands. Another calcium-binding protein, parvalbumin, was present in nonpyramidal neurons in layers II and III that were distinct from those containing calbindin. The regional distribution of parvalbumin-positive neurons was very similar to that of the neurofilament immunoreactive neurons; in rostral entorhinal cortex very few parvalbumin-labeled neurons were present but their frequency gradually increased in the caudal direction. In addition, punctate parvalbumin immunoreactivity was frequently encountered in the location of the nonphosphorylated neurofilament protein-positive layer II islands. These findings demonstrate that layer II of human entorhinal cortex contains distinct subpopulations of neurons, that the relative density of each subpopulation differs across cytoarchitectonic regions, and that the patterns of distribution of these subpopulations are in some cases similar and in other cases complementary. This heterogeneity in the organization of layer II of human entorhinal cortex has important implications for the study of some neuropsychiatric disorders.