Failure of imidocarb dipropionate to clear experimentally induced Ehrlichia canis infection in dogs

The recommended treatment for canine ehrlichiosis is tetracycline or its analog doxycycline, although recent reports have documented ineffective clearing of Erchlichia canis after doxycycline administration. Imidocarb dipropionate is used as an alternative treatment to tetracycline or is used in conjunction with doxycycline. The effectiveness of imidocarb dipropionate in clearing Ehrlichia species from the blood and tissues of dogs with E. canis infection has not been thoroughly evaluated. Fifteen dogs were experimentally infected with E. canis. Ten dogs were treated with imidocarb dipropionate (6.6 mg/kg, IM, 2 injections given 2 weeks apart). Five infected control dogs were not treated. Blood samples from all 15 dogs were E. canis DNA positive by PCR assay by 3 weeks after inoculation (PI), and E. canis antibodies were detected by IFA assay by 1 week PI. Blood platelet counts in all dogs were below the reference interval by 4 weeks PI. E. canis DNA was detected in bone marrow and splenic aspirates by PCR assay 4 weeks PI but not before infection. Bone marrow aspirates were E. canis DNA positive by PCR assay in 14/15 dogs, and splenic aspirates were E. canis DNA positive by PCR assay in 13/15 dogs. Blood samples from all treated and control dogs remained positive for E. canis DNA by PCR assay, and platelet counts remained below preinoculation values 13 weeks PI (6 weeks after 2nd treatment). As administered in this study, imidocarb dipropionate did not clear experimental E. canis infection in dogs.     

Doxycycline clearance of experimentally induced chronic Ehrlichia canis infection in dogs

BACKGROUND: Ineffective clearance of Ehrlichia canis after doxycycline administration has been reported despite the fact that the recommended treatment for canine ehrlichiosis is doxycycline. The effectiveness of doxycycline in clearing E canis infection from the blood and tissues of dogs requires additional evaluation. HYPOTHESIS: Doxycycline (5 mg/kg PO q12h), administered for 4 weeks, will eliminate E canis infection from the blood and tissues of experimentally infected dogs. ANIMALS: Fifteen Walker hound-mixed breed dogs were inoculated subcutaneously with E canis-infected canine histiocytic cells 4 months before doxycycline treatment. METHODS: Four dogs were treated with doxycycline (5 mg/kg PO q12h for 3 weeks), 5 dogs were treated with doxycycline at the same dosage for 4 weeks, and 5 control dogs were not treated. Dexamethasone (0.4 mg/kg i.v.) was given after treatment to precipitate recrudescence of any remaining E canis organisms. Platelet counts, anti-E canis immunofluorescent antibodies, and polymerase chain reaction (PCR) detection of E canis deoxyribonucleic acid (DNA) in blood and tissues were evaluated. RESULTS: E canis DNA was not detected in the blood and tissues of doxycycline-treated dogs after treatment. Platelet counts were within reference intervals, and E canis antibodies decreased. Spontaneous clearance of E canis infection occurred in 2 of 5 control dogs. Three control dogs had E canis DNA detected in blood and tissues, platelet counts remained low or within the reference interval, and E canis antibodies remained high. CONCLUSIONS AND CLINICAL IMPORTANCE: As administered in this study, doxycycline cleared E canis from the blood and tissues of experimentally infected dogs.     

Humoral immunity and reinfection resistance in dogs experimentally inoculated with Babesia canis and either treated or untreated with imidocarb dipropionate

It is proposed that the chronic asymptomatic carrier state produced by Babesia canis infection could make dogs more resistant against subsequent infections. This suggests that treatment with imidocarb dipropionate, which removes the organism, can make dogs more susceptible to reinfection in a short period of time. Ten male and female dogs of approximately 4-5 months of age were inoculated with B. canis. Half of them received treatment with imidocarb dipropionate (7 mg/kg) on days 15 and 27 post-infection and the other half were untreated. All the animals were examined using clinical and laboratory methods (CBC, platelet counts and serological study by indirect immunofluorescence test) for a 6-month period. Antibodies were first detected on day 7 post-injection and remained at high levels (1:2560) over the period in the non-treated group. This result was significantly different (P<0.001) from the treated group in which antibodies titers declined after day 34 post-infection. Six months later, after a homologous challenge infection only the dogs of treated group showed parasitaemia, thrombocytopenia and splenomegaly, which was significantly different (P<0.05) from the non-treated group. The sterilizing treatment with imidocarb dipropionate was effective in clearing the infection, but inhibited the maintenance of protective antibodies, making the animals more susceptible to reinfection.     

硝唑尼特治疗犬贾第虫病的研究

选择试验犬8只,将其随机分为4个小组,每组2只.连续6 d对8只试验犬分剐进行粪检,确定无贾第虫感染后,用体外纯培养的犬贾第虫滋养体接种试验犬,然后每天采集试验犬新鲜粪便40 g,用硫酸锌漂浮法进行粪检,当检测犬贾第虫感染呈阳性时,分别用1、2、4 mg/kg体质量剂量的硝唑尼特时1、2、3组试验犬进行灌服治疗,第4组试验犬不用药作为对照.用药后,每天以同样的方法检测贾第虫包囊,并计数.结果表明,以2、4 mg/kg体质量给药的试验犬1 d后粪检结果转为阴性,以1 mg/kg体质量给药的试验犬4 d后粪检结果转为阴性.结果表明以2、4 mg/kg体质量剂量的硝唑尼特对犬贾第虫痛有很好的疗效.     

Experimental and clinical trials of long acting oxytetracycline in the treatment of canine ehrlichiosis

Summary

Four splenectomized dogs were experimentally infected with Ehrlichia canis and treated at the point of illness with long acting (LA) Oxytetracycline at different dosages. Terramycin LA, when given at a dose of 20mg/kg body weight (deep intramuscularly) twice, at a four‐day interval, was found to have effectively controlled the disease and has replaced the usual 7–14 successive days’ treatment regimen when other groups of tetracycline drugs arc used.

Predej^® 2X* at the rate of 2 mg given intramuscularly concurrently eliminated the local inflammatory reaction caused by the injection of Terramycin LA.

Twenty‐four out of the 26 naturally infected dogs which were treated in a similar way were completely cured; one died before it could receive the full treatment, and another received a second medication five weeks after the first treatment, showing recurrent epistaxis; blood samples taken from this dog were negative for E. canis.     

Clinicopathological changes and effect of imidocarb therapy in dogs experimentally infected with Babesia canis

In this study one spleen-intact dog (A) and two splenectomised dogs (BSE, CSE) were infected with Babesia canis. All animals developed an acute disease characterised by fever, haemoglobinuria and anaemia, the latter being more severe in the splenectomised dogs. Fever and parasitised red blood cells were detected for three days after imidocarb treatment in the splenectomised animals. Haematological abnormalities included regenerative anaemia, thrombocytopenia and leukopenia (due to neutropenia and lymphopenia) in the acute phase, soon followed by leukocytosis, neutrophilia and left shift a few days later. Acute hepatopathy was detected in all dogs with elevated ALT activity, which was more seriously altered in the splenectomised dogs. Diffuse changes in liver structure and hepatomegaly were seen by ultrasonography. Liver biopsy and histology revealed acute, non-purulent hepatitis in the splenectomised dogs. Both splenectomised dogs were successfully cured after collection of 400 ml highly parasitised blood, proving that large-amount antigen production is possible with rescuing the experimental animals. Whole blood transfusion, imidocarb and supportive care with infusions, antipyretics, glucocorticoids and diuretics were applied. The spleen-intact dog clinically recovered after receiving supportive treatment, with no imidocarb therapy. Microbial infections developed in both splenectomised animals (BSE: haemobartonellosis, CSE: osteomyelitis caused by Escherichia coli), probably as a consequence of immunosuppression after splenectomy and glucocorticoid therapy.     

Patent Toxocara canis infections in previously exposed and in helminth-free dogs after infection with low numbers of embryonated eggs

The outcome of Toxocara canis infections in the canine host depends on the migratory pathway of parasite larvae (somatic or tracheal) which is considered to be related to the host's age and its immune status. However, field studies attest high prevalences of patent T. canis infections in adult animals. The controlled induction of patent infections with low doses of embryonated eggs was investigated in 18 beagles in a 7-month study until their 16th life month. The animals were assigned to three groups, each consisting of three vertically infected dogs (with a short patent infection as pups before anthelmintic treatment) and three helminth-free dogs. At study days 10 and 40, the animals of groups 1 and 3 were given each 100 embryonated T. canis eggs. In each case, group 1 was treated 10 days post-infection with Milbemax, while dogs of group 3 remained untreated. Control group 2 was not experimentally infected but treated as group 1. Two weeks after first egg administration, a sharp increase of specific antibody reactions in ELISA and increased eosinophilic counts indicated larval invasion in all infected dogs. 42-56 days following first infection, patent infections were detected coproscopically in all animals of group 3, but in none of the uninfected dogs (group 2) or the infected and treated dogs (group 1). Following a 3-month observation period, all animals of the three groups were treated with piperazine citrate to eliminate intestinal infections and all were administered 100 embryonated eggs. Subsequently, patent infections developed in animals of all groups: in one of the infected and treated animals of group 1, in five of the so far not infected control group 2 and in four of the dogs with previous patent infections (group 3). Susceptibility to patent infections was not significantly altered in T. canis-free dogs compared to dogs with previous patent infection (vertically acquired or experimentally induced). However, dogs of group 1 treated with Milbemax after repeated egg administration developed a significantly increased resistance to patent infections as compared to control dogs (group 2). Observed prepatency periods were between 40 and 56 days and did not differ in the three groups. Even in urban areas, facing high infection pressure with Toxocara eggs maintained by a high dog and fox population, dogs of all ages are at risk to develop patent T. canis infections.     

Transmission of Brucella canis by contact exposure

Transmission studies demonstrated that canine brucellosis can spread from infected to susceptible males maintained in close contact after 4 to 6 months of cohabitation. Spread by males occurred after epididymitis was observed in the infected dogs. Transmission via contaminated urine was suspected, but not proved. The bladder urine of infected males, probably contaminated with seminal fluid, contained higher numbers of B. canis organisms than did that of female dogs. Highest concentrations of bacteria in urine were found between postinfection weeks 8 and 12. Infected females transmitted the infection to contact females after 5 months. Immature females or males infected with B. canis did not transmit brucellosis until after an estrus or a mating was observed--about post-contact months 10-12.     

Effect of fenbendazole on Toxocara canis larvae in tissues of infected dogs.

The effect of fenbendazole therapy was studied in six dogs fed 10,000 embryonated Toxocara canis eggs. At 47 days after they were fed T canis, four dogs were given fenbendazole in two divided doses totaling 50 mg/kg of body weight each day for 14 days. Two infected dogs were not given fenbendazole. All dogs were necropsied at the end of treatment and the foci were counted in the lungs; their skeletal muscles were digested in 1% trypsin for the recovery of larvae. The T canis larvae were not recovered from the skeletal muscle of the four infected dogs treated with fenbendazole; 15 and 42 larvae/100 g of skeletal muscle were recovered from the two nonmedicated infeected dogs. The number of grossly visible foci on surfaces of lungs in treated dogs was markedly less than in the nonmedicated infected dogs. The results indicate that fenbendazole might be effective in preventing prenatal infection in dogs.     

Evaluation of neutrophil oxidative metabolism in canine monocytic ehrlichiosis

BACKGROUND: Canine monocytic ehrlichiosis (CME) is a tick-borne disease caused by Ehrlichia canis, a rickettsia that infects the monocytes of dogs. This infection can result in a chronic and life-threatening disease. Thrombocytopenia, mild anemia, and leukopenia are the most common hematologic findings in CME. OBJECTIVE: To investigate the role of peripheral blood neutrophils in CME, an evaluation was conducted of their functional state during the acute phase of the disease in dogs experimentally infected by E canis. METHODS: Seven dogs were inoculated with E canis, and 3 remained as uninfected controls. All dogs had physical exams and hematologic tests (CBC and nitroblue tetrazolium [NBT] reduction) during a 6-week period. RESULTS: There was no difference (P > .05) in spontaneous NBT reduction results between the 2 groups of dogs throughout the 6-week period of observation. Nevertheless, when stimulated, the neutrophils showed higher activity in the infected group (P = .01) on weeks 4 and 5 after infection. CONCLUSION: Infection by E canis has no influence on neutrophil oxidative metabolism even though during the remission period of the acute phase of the disease, the neutrophils seem to be more reactive under stimulation.     

Effects of milbemycin oxime on adult hookworms in dogs with naturally acquired infections

Previous work indicated that adult Ancylostoma caninum can be removed from experimentally infected dogs, using a formulation of milbemycin oxime at dosage of 0.5 mg/kg of body weight. To determine the efficacy of this treatment in dogs naturally infected with adult hookworms, 24 mixed-breed dogs with patent hookworm infections were purchased from an out-of-state vendor, and 6 male and 6 female dogs were assigned to either a control group or a group that would be treated. Dogs were treated 10 days after their arrival and were euthanatized 1 week after treatment. Beginning 3 days before treatment, fecal samples were collected daily from all dogs, and the number of Ancylostoma eggs per gram of dry weight of feces was determined from each sample. By 1 week after treatment, the mean number of eggs being passed by the treated dogs had dropped from 12,700 to 10 eggs/g of dried feces; there was no apparent change in fecal egg counts for dogs of the control group. At necropsy, the mean number of adult A caninum in dogs of the treated and control groups was 1.3 and 56, respectively; in these naturally infected dogs, efficacy of treatment was calculated to be 97.8%. The mean number of adult Trichuris vulpis recovered in dogs of the control and treated groups at necropsy was 24 and 0, respectively, which yielded treatment efficacy of 100%. Although Uncinaria stenocephala and Toxocara canis appeared also to be removed by use of this dosage, too few dogs were in the study to calculate meaningful efficacies.(ABSTRACT TRUNCATED AT 250 WORDS)     

A preliminary study to evaluate the immune responses induced by immunization of dogs with inactivated Ehrlichia canis organisms

Ehrlichia canis is an intracellular pathogen that causes canine monocytic ehrlichiosis. Although the role of antibody responses cannot be discounted, control of this intracellular pathogen is expected to be by cell mediated immune responses. The immune responses in dogs immunized with inactivated E. canis organisms in combination with Quil A were evaluated. Immunization provoked strong humoral and cellular immune responses, which were demonstrable by Western blotting and lymphocyte proliferation assays. By Western blotting antibodies to several immunodominant E. canis proteins were detected in serum from immunized dogs and antibody titres increased after each immunization. The complement of immunogenic proteins recognized by the antisera were similar to those recognized in serum from infected dogs. Upon challenge with live E. canis, rapid anamnestic humoral responses were detected in the serum of immunized dogs and primary antibody responses were detected in the serum from control dogs. Following immunization, a lymphocyte proliferative response (cellular immunity) was detected in peripheral blood mononuclear cells (PBMNs) of immunized dogs upon stimulation with E. canis antigens. These responses were absent from non-immunized control dogs until after infection with live E. canis, when antigen specific-lymphocyte proliferation responses were also detected in the PBMNs of the control dogs. It can be thus concluded that immunization against canine monocytic ehrlichiosis may be feasible. However, the immunization regimen needs to be optimized and a detailed investigation needs to be done to determine if this regimen can prevent development of acute and chronic disease.     

Experimental and clinical trials of long acting oxytetracycline in the treatment of canine ehrlichiosis

Four splenectomized dogs were experimentally infected with Ehrlichia canis and treated at the point of illness with long acting (LA) Oxytetracycline at different dosages. Terramycin LA, when given at a dose of 20 mg/kg body weight (deep intramuscularly) twice, at a four-day interval, was found to have effectively controlled the disease and has replaced the usual 7-14 successive days' treatment regimen when other groups of tetracycline drugs are used. Predef 2X at the rate of 2 mg given intramuscularly concurrently eliminated the local inflammatory reaction caused by the injection of Terramycin LA. Twenty-four out of the 26 naturally infected dogs which were treated in a similar way were completely cured; one died before it could receive the full treatment, and another received a second medication five weeks after the first treatment, showing recurrent epistaxis; blood samples taken from this dog were negative for E. canis.     

Evidence of an acute phase response in dogs naturally infected with Babesia canis

The erythrocyte sedimentation rate (ESR), white blood cell count (WBC), haematocrit (HCT) and platelet number (PLT) were quantified and compared with the acute phase proteins (APPs) in dogs naturally infected with Babesia canis and healthy dogs. Both groups were treated with imidocarb dipropionate on the day of admission and both groups were monitored for all parameters on the admission day and on the first, second, third, fourth and seventh days in order to determine the presence of an acute phase reaction, to assess the diagnostic value of these markers in uncomplicated canine babesiosis and to evaluate the use of APPs in treatment monitoring. It was demonstrated that an acute phase response occurs in dogs naturally infected with Babesia canis, with significant increases in the concentration of major acute phase proteins. The serum concentration of C-reactive protein (CRP), serum amyloid A (SAA) and the erythrocyte sedimentation rate (ESR) decreased daily after treatment and approached reference range values by the eighth day. PLT and haematocrit (HCT) increased daily after treatment and approached reference range values by the fourth day. WBC and haptoglobin increased after treatment and then decreased from the third and fourth days, respectively, to the eighth day. The diagnostic sensitivity of CRP, SAA and PLT was significantly higher compared to haptoglobin, ESR, HCT and the WBC count. CRP and SAA were of clinical use in monitoring the response to antibabesial treatment.     

Transplanted infections with Toxocara canis as a model for the effectiveness testing of anthelmintics

Patent infections of adult dogs with Toxocara canis induced by transplantation of immature, intestinal stages were examined for their suitability for testing of anthelmintics. Each of 5 dogs were infected four times by transplantation of 80 immature, intestinal stages of Toxocara canis. The dogs were treated with various anthelmintics of well established efficacy (pyrantel, nitroscanate, mebendazole, piperazine) 20 dpi. All anthelmintics tested showed the same efficacy as had been assessed earlier by treatment of dogs infected prenatally with Toxocara canis.     

Efficacy of a modified polymerase chain reaction assay for detection of Ehrlichia canis infection.

Detection of Ehrlichia canis in acutely infected and convalescent dogs is important for effective treatment and control. However, accurate detection has been difficult to achieve, in part because dogs that have been treated therapeutically often remain seropositive for extended periods. A new method, polymerase chain reaction (PCR) assay using biotinylated E. canis-specific primers (PCR-BP), was developed for detection of E. canis. Four dogs experimentally infected with E. canis by intravenous inoculation of whole blood from carrier dogs and 2 naturally infected convalescent carriers were used to compare the specificity and sensitivity of the new method with that of microscopy/blood smear evaluation, serologic test, and conventional PCR assay using E. canis-specific primers. In experimentally infected animals, infection was detected as early as 7 days post-exposure using PCR-BP. Although the 2 naturally infected dogs were positive by serologic test and PCR-BP, both were negative by conventional PCR. Results suggest that the new method is a sensitive assay for detection of E. canis infection. In addition, results were obtained more rapidly than with other PCR-based assays.     

Detection of platelet-bound antibodies in beagle dogs after artificial infection with Ehrlichia canis

Six dogs were infected with Ehrlichia canis by intravenous injection of heavily infected DH82 cells. All dogs developed typical signs of canine monocytic ehrlichiosis. Using flow cytometric technology, platelet-bound IgG (PBIgG) were detected in 5 of the 6 dogs after experimental infection with E. canis over a period of 3-10 days post infection (PI). The first detection of PBIgG was made as early as day 3 PI in 2 out of 6 dogs, and on day 5 PI in 1 dog. On day 7 PI, PBIgG was detected in 2 dogs, and on day 10 PI in 3 out of 6 dogs. This is the first report documenting the presence of PBIgG following E. canis infection in dogs. This finding further supports the theory that the thrombocytopenia seen in canine monocytic ehrlichiosis has an immunological component and that exposure to an infectious agent, in this case the rickettsia E. canis, can trigger autoimmune mechanisms. Due to the heterogeneous appearance of PBIgG among the infected dogs it was concluded that other non-immunological mechanisms are probably also involved in the pathogenesis of the thrombocytopenia seen in canine monocytic ehrlichiosis.     

Vaccination of dogs against heterologous Babesia canis infection using antigens from culture supernatants

Soluble parasite antigens (SPA) from European Babesia canis can be used to protect dogs against a homologous but not heterologous challenge infection. In this study it is shown that when dogs are vaccinated with a mixture of SPA from both, a European B. canis isolate and a South African Babesia rossi isolate, protective immunity against heterologous B. canis infection is induced. Three groups of five beagle dogs each were vaccinated twice with graded doses of SPA derived from in vitro cultures of B. canis and B. rossi, with a 3-week interval. Saponin was used as adjuvant. Three weeks after booster vaccination immunological responsiveness against heterologous B. canis antigen was measured by seroconversion against infected erythrocytes and lymphocyte transformation using SPA. Upon vaccination dogs produced antibodies against infected erythrocytes and lymphoblastogenic responses against SPA in a dose-dependent manner. Dogs were then challenged with heterologous B. canis parasites. Dogs appeared to be protected against challenge infection, which was reflected in less severe decrease of packed cell volume (PCV) and reduced clinical signs. The level of protection to clinical signs (but not excessive PCV drop) was related to the level of SPA in plasma and spleen size, and not related to peripheral parasitaemia. The results suggest that vaccination with this bivalent vaccine primes T-helper cells that recognise common epitopes on SPA from an antigenically distinct B. canis isolate. These cells provide the essential Th signal to mount an effective and timely antibody response against SPA and parasites or parasitised erythrocytes, which prevents the further development of clinical babesiosis.     

Concurrent bartonellosis and babesiosis in a dog with persistent thrombocytopenia

A 12-year-old castrated male West Highland White Terrier was referred because of recurrent episodes of collapsing. The dog was mildly anemic and severely thrombocytopenic and had high serum alanine aminotransferase activity. Infection with Bartonella vinsonii (berkhoffii) was initially diagnosed on the basis of serologic testing. Despite treatment with a series of antimicrobials and prolonged use of immunosuppressive drugs, thrombocytopenia persisted. After 5 months of treatment, Babesia canis organisms were seen during examination of a direct blood smear. The dog was treated with imidocarb dipropionate for babesiosis, after which thrombocytopenia resolved, and administration of immunosuppressive drugs was discontinued. Retrospective review of blood smears failed to identify organisms; however, polymerase chain reaction (PCR) analysis of multiple stored blood samples obtained during the 5-month period of persistent thrombocytopenia identified DNA of B. canis vogeli. Babesiosis may cause persistent, unexplained thrombocytopenia in dogs that are not anemic. A PCR assay can facilitate a diagnosis of babesiosis when organisms are not evident or when serologic testing fails to detect Babesia-specific antibodies.     

A blind test of the serological response of dogs to infection with Taenia ovis

Fifty six dogs of mixed age and sex were acquired from farms in the Otago/Southland region, and maintained at the Hydatid Research Unit, Taieri, where 43 were each fed two Taenia ovis cysts. All were bled fortnightly for six or 12 weeks. Coded sera were sent to Wallaceville Animal Research Centre for testing using ELISA, with antigen from T. ovis scoleces. Dog treatments were identified after all tests were complete. A discriminant level was derived from the mean absorbance value plus three standard deviations of 56 sera taken at time zero and 78 sera from serially bled uninfected dogs. None of these 134 sera registered as a false positive using this discriminant level. The data showed no significant deviation from normality, and the expected frequency of the occurrence of false positives is therefore less than 0.14%. Four weeks after infection 63% of dogs proved to be infected were serologically positive, rising to 78% after 6 weeks. When worms were removed by anthelmintic treatment, ELISA absorbance levels decreased. Four weeks after removal 70% of previously infected dogs remained positive, decreasing to 30% after 6 weeks.

Six weeks after infection the sensitivity of the test was 78%, and the specificity 63%. However, if dogs with positive ELISA absorbance levels, but which did not purge worms, were regarded as having had worms, the respective figures would be 82% and 100%. The latter figures are similar to our previously published laboratory results. The test is of comparable efficiency to arecoline purgation for surveillance, and has the additional advantage of detecting infection in the majority of those dogs that have been infected for three weeks or more but fail to pass worms on purgation, and a substantial proportion of those infected dogs that were treated by their owners prior to presenting them for purgation in order to avoid detection of infection.