Seroprevalence of Ehrlichia canis and of canine granulocytic Ehrlichia infection in dogs in Switzerland

Serum samples from 996 dogs in Switzerland were examined for antibodies to Ehrlichia canis and to the agent causing canine granulocytic ehrlichiosis (CGE). Ehrlichiosis, borreliosis, and systemic illness not associated with ticks were suspected in 75, 122, and 157 of these dogs, respectively. The remainder of the serum samples were obtained from clinically healthy dogs which resided north (n = 235) or south (n = 407) of the Alps. The serum samples were tested by an indirect immunofluorescence technique for antibodies to the two agents incriminated, E. canis and Ehrlichia phagocytophila, a surrogate marker of the agent of CGE. Twenty-two of 996 (2.2%) serum samples had antibodies to E. canis and were distributed as follows: 20 of 75 (26.7%) samples from dogs suspected of having ehrlichiosis, 1 of 122 (0.8%) from dogs suspected of having borreliosis, and 1 of 407 (0.2%) from healthy dogs which resided south of the Alps. Of the 75 (7.5%) serum samples that had antibodies to E. phagocytophila, significantly more samples were from ill dogs than from healthy dogs. Among the sera from healthy dogs, antibodies to E. phagocytophila were significantly more prevalent in the north. Because seropositive dogs had a history of travel outside Switzerland and because Rhipicephalus sanguineus is found exclusively south of the Alps, it was presumed that, in contrast to the agent of CGE, E. canis is not indigenous to Switzerland.     

Ehrlichia canis infections of dogs in Germany. Epidemiology, diagnosis, therapy and prophylaxis

Between January and December 1997 infections with Ehrlichia canis were detected in 211 dogs in Germany. Of the 53 epidemiologically evaluable dogs, 19 animals born and raised in Germany had travelled with their owners abroad in endemic areas, 30 dogs originated from there and four dogs had never left Germany. As regards to the possible location of infection it has been registered that most dogs had been taken to countries of the Mediterranean Sea (Spain, France, Italy, Greece) or had been imported from there. On inquiry, ticks had been forwarded from four dogs only, which were determined as Rhipicephalus sanguineus. The subsequent serological investigation of these four dogs revealed a Rickettsia conorii infection in two of them.     

Persistence of Ehrlichia phagocytophila infection in lambs in relation to clinical parameters and antibody responses

Five lambs were infected experimentally with Ehrlichia phagocytophila and examined regularly during the next six months. The lambs all had recurrences of parasitaemia at various times but had a fever on only 21 per cent of these occasions. A reduced number of leucocytes was observed in all the lambs for at least eight weeks. All the lambs were still infected four months after inoculation with E phagocytophila. After six months, blood from four of the five lambs was infective when inoculated into susceptible lambs.     

Comparison of nested PCR with immunofluorescent-antibody assay for detection of Ehrlichia canis infection in dogs treated with doxycycline

A partial 16S rRNA gene was amplified in Ehrlichia canis-infected cells by nested PCR. The assay was specific and did not amplify the closely related Ehrlichia chaffeensis, Ehrlichia muris, Neorickettsia helminthoeca, and SF agent 16S rRNA genes. The assay was as sensitive as Southern hybridization, detecting as little as 0.2 pg of E. canis DNA. By this method, all blood samples from four dogs experimentally infected with E. canis were positive as early as day 4 postinoculation, which was before or at the time of seroconversion. One hundred five blood samples from dogs from Arizona and Texas (areas of E. canis endemicity) and 30 blood samples from dogs from Ohio (area of E. canis nonendemicity) were examined by nested PCR and immunofluorescent-antibody (IFA) test. Approximately 84% of dogs from Arizona and Texas had been treated with doxycycline before submission of blood specimens. Among Arizona and Texas specimens, 46 samples were PCR positive (44%) and 80 were IFA positive (76%). Forty-three of 80 IFA-positive samples (54%) were PCR positive, and 22 of 25 IFA-negative samples (88%) were negative in the nested PCR. None of the Ohio specimens were IFA positive, but 5 specimens were PCR positive (17%). Our results indicate that the nested PCR is highly sensitive and specific for detection of E. canis and may be more useful in assessing the clearance of the organisms after antibiotic therapy than IFA, especially in areas in which E. canis is endemic.     

Reisolation of Ehrlichia canis from blood and tissues of dogs after doxycycline treatment

We present evidence that supports the carrier status of dogs experimentally infected with Ehrlichia canis after treatment with doxycycline. Canine ehrlichiosis was induced in five dogs by intravenous inoculation with E. canis-infected DH82 cells. All animals developed mild clinical signs of transient fever, body weight loss, thrombocytopenia, and increased gamma globulin levels in plasma. An indirect fluorescent-antibody test (IFA) revealed that all dogs had seroconverted (titer, 5,120) by day 10 postinoculation (p.i.). E. canis was reisolated from blood samples collected at intervals throughout the 2-month period p.i. Doxycycline was administered orally once daily at 10 mg/kg of body weight per day for 1 week starting at 2 months p.i. Following treatment, gamma globulin levels in plasma were decreased. At necropsy on days 54 to 59 after the start of treatment, spleen, liver, kidney, and lymph nodes were collected for E. canis culture and histopathologic examination. Although the dogs did not show significant clinical signs during or after treatment with the antibiotic, E. canis was reisolated from the blood and tissue samples of three of five dogs. A 16-fold reduction in IFA titer was noted in two dogs which were negative for E. canis reisolation at day 49 after the start of treatment, whereas a zero- to fourfold reduction in IFA titer was seen in the remaining three dogs. Western immunoblot reactions to higher-molecular-size E. canis antigens in the sera of two dogs which were negative for E. canis on blood culture decreased, whereas they remained continuously high or only transiently decreased for the duration of the study for antigens in the sera of three dogs from which E. canis was reisolated. Histopathologically, prominent plasmacytosis in the kidney cortex was present in three dogs from which E. canis was reisolated, whereas the kidney cortices of two dogs had moderate to minor plasmacytosis. These findings pose questions regarding the efficacy, dosage and duration of doxycycline treatment in dogs with E. canis infection. In addition, it was shown that IFA and Western immunoblotting may aid in assessing the efficacy of antibiotic therapy when definitive reisolation procedures are not readily available.     

Application of the polymerase chain reaction for the detection of Ehrlichia canis in tissues of dogs

Doxycycline treatment resulted in a carrier status in 3 dogs and complete clearance of Ehrlichia canis in 2 dogs (Iqbal and Rikihisa, 1994). Using specimens obtained during that study applicability of polymerase chain reactions (PCRs) in detecting E. canis DNA in tissue specimens and correlation of PCR results with our previous cell culture isolation results were evaluated. PCRs using a pair of primers specific to E. canis 16SrRNA gene sequence were used to detect DNA of E. canis in tissues of 5 experimentally-infected dogs 2 months after doxycycline treatment. An approximately 600 bp product defined by the specific primers was amplified in blood, kidney, lymph nodes, liver, and/or spleen of 3 dogs from which E. canis was reisolated in cell culture. In contrast, E. canis DNA was not detected in tissue or blood specimens of the 2 dogs from which E. canis was not reisolated after doxycycline treatment or in 2 control uninfected dogs. The findings indicate PCR is effective in detecting E. canis in tissues.     

Identification of a granulocytic Ehrlichia strain isolated from a horse in Switzerland and comparison with other rickettsiae of the Ehrlichia phagocytophila genogroup

This case report describes a 12-year-old Arabian mare with granulocytic ehrlichiosis. Clinical signs included fever, apathy, anorexia, icterus, limb edema, and reluctance to move. Examination of buffy coat smears revealed Ehrlichia organisms in neutrophils and eosinophils. A band of 1,428 bp was amplified from DNA of leukocytes via nested PCR and was identified as part of the Ehrlichia 16S rRNA gene. It differed from the gene sequences of Ehrlichia phagocytophila and E. equi at two and three positions, respectively. Interestingly, the nucleotide sequence of the 16S rRNA was 100% identical to that of the agent of human granulocytic ehrlichiosis.     

Development of an experimental model of Microsporum canis infection in cats

An experimental infection model was developed for reliable induction of Microsporum canis skin infections in cats, using a defined number of macroconidia harvested from the fungus in culture. The strain of M. canis used produced highly fluorescent hairs under ultraviolet illumination. Kittens 8 to 9 weeks of age (n = 6) received 10(5) macroconidia applied topically to a closely-shaved area of skin. Sites were dressed with an occlusive bandage for 3 days, then grooming was restricted for an additional 4 weeks. Lesions were first observed 2 weeks after inoculation, enlarged over the following 6 to 8 weeks, then decreased in size and appeared healed at 12 to 14 weeks after inoculation. Cats often developed satellite lesions on the face, ears, or other body regions. The experimental infections strongly resembled moderately severe cases of naturally-occurring feline dermatophytosis in clinical patients. This experimental infection model will be useful for evaluation of topical and systemic treatments for feline M. canis infection.     

Granulocytic Ehrlichia infection in ixodid ticks and mammals in woodlands and uplands of the U.K

BACKGROUND: The number of patients waiting lung transplantation greatly exceeds the supply of donors. This study was conducted to determine the effect of high-dose steroid administration on oxygenation and donor lung recovery after brain death. METHODS: A retrospective analysis was conducted on 118 consecutive organ donors from January 1 through December 31, 1995. Eighty donors received high-dose steroids (methylprednisolone, mean 14.5+/-0.06 mg/kg) after organ procurement organization management began; a second group was composed of 38 patients who received no steroids. PaO2/FiO2 ratios were used to evaluate oxygenation. The number of single and double lungs transplanted served as the endpoint. RESULTS: No differences were noted in hemodynamics, most clinical or demographic variables and initial values of PaO2/FiO2 between groups. However, nonsteroid-treated donors showed an overall decrease in oxygenation (mean decrease in PaO2/FiO2 -34.2+/-14), whereas steroid-treated donors had a significant and progressive increase in oxygenation (mean increase in PaO2/FiO2: 16+/-14) before aortic cross-clamping (p = 0.01). Time before cross-clamping was longer in the steroid-treated patients (p = 0.003). The number of procured lungs was markedly greater in steroid-treated than nonsteroid-treated donors (25/80 patients vs 3/38; p < 0.01). CONCLUSIONS: High-dose methylprednisolone given during donor management results in improved oxygenation at organ recovery. This treatment resulted in a significant increase in the number of lungs transplanted and may have enabled donors to be treated longer.     

Multiple infection with Toxocara canis. Influence of antihistamines and corticosteroids on the histopathological response

The histopathology of the tissues of mice infected with ten doses of 100 embryonated eggs of Toxocara canis and the influence of antihistamines and corticosteroids on these findings is described. The main lesions were confined to the liver and lungs. In infected but untreated control mice hepatic lesions consisted of periportal infiltrates, widespread foci of liver cell necrosis and polymorphonuclear and mononuclear accumulation and a few fibrotic granulomata. Treatment with antihistamines did not change these findings significantly, but corticosteroids decreased the amount of cellular infiltration and the size of the lesions. Pulmonary lesions consisted mostly of confluent infiltrates of polymorphonuclears, eosinophils and mononuclear cells with some macrophage proliferation. Neither drug seemed to affect the lung involvement.     

Incidence of patent Toxocara canis infection in bitches during the oestrous cycle

The incidence of patent Toxocara canis infection as result of reactivation of somatic larvae with subsequent tracheal migration was investigated by faecal examination during 23 oestrous cycles of 15 bitches. Blood samples were collected for determination of total and differential leukocyte counts, prolactin concentration, and Toxocara titre. Five pregnant dogs were used as controls. In the cyclic dogs there were no alterations in white blood cell counts or prolactin concentration, in contrast with the pregnant dogs, in which both variables increased, starting 10 days after onset of the luteal phase. The difference was significant at day 40 and day 60 (both p < 0.005). No significant differences were observed in the number of eosinophils or in the Toxocara antibody titre. T. canis eggs were only found in the faeces of three 1-year-old, cyclic dogs at 1, 60, and 140 days, respectively, after the onset of the luteal phase. It is concluded that cyclic beagle bitches, in which prolactin levels increase in the second half of the luteal phase, are unlikely to be at higher risk for patent T. canis infection than in other phases.     

Piroplasms of domestic animals in the Macedonia region of Greece. 1. Serological cross-reactions

During a serological survey on haemoparasites in Macedonia, serum samples were collected from cattle, sheep and goats. All sera were tested by the indirect immunofluorescence test (IFAT); the cattle sera against Theileria orientalis, T. annulata, Babesia bigemina, B. bovis, B. divergens and B. major antigens; the sheep and goat sera against T. ovis, B. ovis, B. motasi and B. crassa antigens. Parallel tests of negative and positive control sera against all the antigens showed the existence of cross-reactions of different degrees between species of the same genus. In cattle, the most important cross-reactions were obtained against B. bigemina antigen, especially with the anti-B. bovis serum, in small ruminants against B. motasi with the anti-B. crassa serum. In the field sera, there was a high correlation between the antibody titres of B. bigemina and B. bovis, and also between the titres of these two Babesia spp. and B. divergens. A high correlation was also found between B. motasi and B. crassa, and lower ones between these two and B. ovis. The correlations of the sera titres were due to mixed infections or to cross-reactions. Therefore, the use of the IFAT is not always satisfactory for diagnosing infections in regions where animals are infected with different piroplasms.     

Cloning and characterization of multigenes encoding the immunodominant 30-kilodalton major outer membrane proteins of Ehrlichia canis and application of the recombinant protein for serodiagnosis

A 30-kDa major outer membrane protein of Ehrlichia canis, the agent of canine ehrlichiosis, is the major antigen recognized by both naturally and experimentally infected dog sera. The protein cross-reacts with a serum against a recombinant 28-kDa protein (rP28), one of the outer membrane proteins of a gene (omp-1) family of Ehrlichia chaffeensis. Two DNA fragments of E. canis were amplified by PCR with two primer pairs based on the sequences of E. chaffeensis omp-1 genes, cloned, and sequenced. Each fragment contained a partial 30-kDa protein gene of E. canis. Genomic Southern blot analysis with the partial gene probes revealed the presence of multiple copies of these genes in the E. canis genome. Three copies of the entire gene (p30, p30-1, and p30a) were cloned and sequenced from the E. canis genomic DNA. The open reading frames of the two copies (p30 and p30-1) were tandemly arranged with an intergenic space. The three copies were similar but not identical and contained a semivariable region and three hypervariable regions in the protein molecules. The following genes homologous to three E. canis 30-kDa protein genes and the E. chaffeensis omp-1 family were identified in the closely related rickettsiae: wsp from Wolbachia sp. , p44 from the agent of human granulocytic ehrlichiosis, msp-2 and msp-4 from Anaplasma marginale, and map-1 from Cowdria ruminantium. Phylogenetic analysis among the three E. canis 30-kDa proteins and the major surface proteins of the rickettsiae revealed that these proteins are divided into four clusters and the two E. canis 30-kDa proteins are closely related but that the third 30-kDa protein is not. The p30 gene was expressed as a fusion protein, and the antibody to the recombinant protein (rP30) was raised in a mouse. The antibody reacted with rP30 and a 30-kDa protein of purified E. canis. Twenty-nine indirect fluorescent antibody (IFA)-positive dog plasma specimens strongly recognized the rP30 of E. canis. To evaluate whether the rP30 is a suitable antigen for serodiagnosis of canine ehrlichiosis, the immunoreactions between rP30 and the whole purified E. canis antigen were compared in the dot immunoblot assay. Dot reactions of both antigens with IFA-positive dog plasma specimens were clearly distinguishable by the naked eye from those with IFA-negative plasma specimens. By densitometry with a total of 42 IFA-positive and -negative plasma specimens, both antigens produced results similar in sensitivity and specificity. These findings suggest that the rP30 antigen provides a simple, consistent, and rapid serodiagnosis for canine ehrlichiosis. Cloning of multigenes encoding the 30-kDa major outer membrane proteins of E. canis will greatly facilitate understanding pathogenesis and immunologic study of canine ehrlichosis and provide a useful tool for phylogenetic analysis.     

Relationship between epicardial ST-segment elevation and myocardial ischemic damage after experimental coronary artery occlusion in dogs

The relationship between early and late epicardial electrocardiographic changes as well as those in regional myocardial blood flow (MBF) and the severity of myocardial damage was determined in 12 anesthetized dogs with left anterior descending coronary artery ligation. Radioactive microspheres (15 mum) were used to measure regional MBF at 15 min (early) and 24 h (late) after coronary occlusion. Severity of myocardial damage was assessed by the extent of myocardial creatine phosphokinase depletion 24 h after coronary ligation. There was a close linear correlation between myocardial creatine phosphokinase activity and regional MBF both early (r=0.93, 2P less than 0.001) and late (r=0.88, 2P less than 0.001). An inverse but less precise relationship existed between acute epicardial ST-segment elevation and early (r=-0.41, 2P less than 0.001), or late (r=0.35, 2P less than 0.05) regional MBF. Similarly, a weak correlation was found between myocardial creatine phosphokinase (IU/mg protein) at 24 h and early epicardial ST (millivolt) elevation (r=-0.36, 2P less than 0.02). In the center zones of the infarct with MBF 1/10 of normal, about 35% of the areas with normal QRS width had no epicardial ST-segment elevation 15 min after coronary occlusion. About 44% of the areas which developed pathological Q-waves in the electrocardiogram at 24 h had no ST elevation 15 min after coronary ligation. Late evolution of abnormal Q-waves occurred almost invariably in areas in which the early MBF was reduced to less than 50% of normal and in areas which subsequently had myocardial creatine phosphokinase levels reduced to less than 60% of normal. After coronary occlusion, the severity of the ultimate myocardial damage, which was directly proportional to the degree of reduction in MBF, was therefore not reliably predicted by the early epicardial ST-segment elevation. The data obtained in these studies suggest the need for caution in the use of acute ST-segment elevation as a predictive index of the extent or severity of myocardial ischemic damage.     

Kinetics of subcutaneous versus intravenous epoetin-beta in dogs, rats and mice

The total body clearance of recombinant human erythropoietin (rhEPO) calculated per kilogram of body weight increased in the order man = dog < rat < mouse. The differences disappeared or were reversed when clearance was expressed per square meter of body surface. There was no similar species difference in terminal half life. Total body clearance increased with the dose in dogs and mice, but not in rats. The bioavailability from a subcutaneous depot was 80% in dogs, 76% in rats, and 70% in mice. The absorption from the subcutaneous depot is rapid in rats and mice, but slow in dogs. The pharmacodynamic activity of rhEPO injected by both routes was compared in polycythemic mice. The equipotent doses were 2.44 times higher with intravenous than with subcutaneous injection. Taking into account the bioavailability of 70% from a subcutaneous depot, one obtains a potency ratio of 3.5 for absorbed subcutaneous versus intravenous rhEPO.     

Toxocara canis infection in the paratenic host: a study on the chemosusceptibility of the somatic larvae in mice

A research on formulation and dosage strategies of anthelmintics have been conducted in mice experimentally infected with eggs of Toxocara canis. Multidose treatments commenced at days 2, 14, 81, 87 and 123 postinfection. Results indicated no detectable difference in the chemosusceptibility of the migrating early infection larvae and the resting hypobiotic chronic infection larvae. Application of medicated dry food (pellets) may be a simple way of improving efficacy of treatment compared to oral drenching. The larvicidal potential of fenbendazole (FBZ), albendazole (ABZ), flubendazole (FUBZ), oxibendazole (OBZ) and ivermectin was assessed. Reductions of 84.2 to 99.7 or 88.8 to 100% of group mean larval counts were recorded after 20-30-day courses of feeding pellets containing FBZ at 6 g kg-1 or ABZ at 1.6 g kg-1 food, respectively. Efficacies of 57.8 to 88.2 or 81.1 to 32.0% were achieved by 20-day courses of feeding pellets medicated with FUBZ and OBZ at 1.6 g kg-1 or 6.0 g kg-1 food, respectively. Ivermectin at various dosing regimens showed only moderate larvicidal potential. Efficacy rates were not closely correlated with the amount of drug taken by the animals. The blood-brain barrier is permeable for the anthelmintics tested, and the brain of mice does not provide a site promoting survival of larvae.     

A 16S rRNA-based PCR assay for detection and identification of granulocytic Ehrlichia species in dogs, horses, and cattle

A PCR-based assay was developed for detecting DNA of granulocytic ehrlichiae in blood samples from dogs, horses, and cattle, Primers were designed from 16S rRNA sequence information to specifically amplify DNA from a newly identified Swedish Ehrlichia species. The 16S rRNA nucleotide sequence of this Swedish species differs in only two and three positions from the sequences of Ehrlichia phagocytophila and Ehrlichia equi, respectively, which were also amplified by this PCR system. For evaluation, PCR results were compared with microscopic examination of stained blood smears for the detection of granulocytes containing ehrlichiae (morulae). Thirty-four of 36 microscopically positive samples were also positive by PCR, and 6 microscopically negative samples were negative by PCR as well. Six samples, in which morulae-like structures had been seen, were negative by PCR, also at a lower annealing temperature and when a reamplification of the first PCR products was performed. The identities of the PCR products from some canine and equine isolates were verified by direct DNA sequencing and were found to be identical with the Ehrlichia sequence found in these animal species that had been obtained earlier. The sequences of a segment of approximately 600 nucleotides from two bovine isolates were identical to that of E. phagocytophila, whereas the sequence of another bovine isolate differed in two positions from that of E. phagocytophila and in three positions from the sequences of the canine and equine isolates. Serum samples were analyzed by indirect fluorescent-antibody testing. Seventy-three percent of the animals which were positive by microscopy and PCR also had positive antibody titers. However, it was not possible to rely on a single serological result for diagnosis of present infection. It was, therefore, concluded that PCR was the most reliable method, useful in the clinical laboratory for specific and early diagnosis of granulocytic ehrlichiosis in animals.     

The relation between psychometric and experimental research in psychology.

A discussion of the distinction between experimental research (variables determined by experimental operations) and psychometric research (variables determined by psychometric operations). Indications of how these two traditionally distinct methods may be combined and the resultant advantages obtained are noted. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Influence of metal implants on infection. An experimental study in rabbits

We implanted cylinders of cobalt-chrome or titanium, with smooth or porous surfaces, into rabbit bones which had been inoculated with suspensions of Staphylococcus aureus in various doses. The bacterial concentration required to produce infection of porous-coated titanium implants was 2.5 times smaller than that necessary to infect implants with polished surfaces. Porous-coated cobalt-chromium implants required bacterial concentrations that were 40 times smaller than those needed to infect implants with polished surfaces, and 15 times smaller than those required to infect porous-coated titanium implants. The other advantages and disadvantages of the various implants, such as improved osseointegration, larger ion-release surfaces, surface wear and relative stiffness, must be weighed against the higher infection rates in the porous-coated implants, and particularly in the cobalt-chromium implants.