Repeated imidocarb treatment failure suggesting emerging resistance of Babesia canis in a new endemic area in north-eastern Germany.

Canine babesiosis has been increasingly diagnosed in various regions of Germany such as north-eastern Germany in recent years. A dog with several relapses of Babesia canis infection after treatment with imidocarb is described. A 9-year-old male Magyar Viszla with B. canis infection was referred after two treatments with imidocarb (dosage 2.1 mg/kg SC) because of lethargy, fever and pancytopenia (additional treatments with prednisolone and doxycycline). Merozoites were detected in the blood smear and imidocarb treatment was repeated. Clinical signs, pancytopenia and a positive B. canis PCR occurred after the 3rd (6 mg/kg SC), 4th (7.7 mg/kg SC) and 5th (7.5 mg/kg SC and doxycycline for 4 weeks in addition) imidocarb injection and thorough tick prevention with isoxazoline and permethrin products. 12 days after the 5th injection, the PCR was negative for the first time. The dog was again presented with fever 35 days after the 5th injection. The B. canis PCR was positive and laboratory examination revealed pancytopenia. Treatment with atovaquone/azithromycin for 18 days was performed and no further relapse occurred for 32 weeks. In the case of suspected imidocarb resistance in B. canis infection, treatment with atovaquone/azithromycin can be an alternative.

Diminazene aceturate and imidocarb dipropionate-based combination therapy for babesiosis - A new paradigm.

In the present study, the effect of a combination therapy consisting of diminazene aceturate (DA) and imidocarb dipropionate (ID) on the in vitro growth of several parasitic piroplasmids, and on Babesia microti in BALB/c mice was evaluated using a fluorescence-based SYBR Green I test. We evaluated the structural similarities between the regularly used antibabesial medications, DA and ID, and the recently found antibabesial drugs, pyronaridine tetraphosphate, atovaquone, and clofazimine, using atom pair fingerprints (APfp). The Chou-Talalay approach was used to determine the interactions between the two drugs. A Celltac MEK-6450 computerized hematology analyzer was used to detect hemolytic anemia every 96 hours in mice infected with B. microti and in those treated with either mono- or combination therapy. According to the APfp results, DA and ID have the most structural similarities (MSS). DA and ID had synergistic and additive interactions against the in vitro growth of Babesia bigemina and Babesia bovis, respectively. Low dosages of DA (6.25 mg kg-1) and ID (8.5 mg kg-1) in conjunction with each other inhibited B. microti growth by 16.5 %, 32 %, and 4.5 % more than 25 mg kg-1 DA, 6.25 mg kg-1 DA, and 8.5 mg kg-1 ID monotherapies, respectively. In the blood, kidney, heart, and lung tissues of mice treated with DA/ID, the B. microti small subunit rRNA gene was not detected. The obtained findings suggest that DA/ID could be a promising combination therapy for treating bovine babesiosis. Also, such combination may overcome the potential problems of Babesia resistance and host toxicity induced by utilizing full doses of DA and ID.

Autochthonous Babesia canis infections in 49 dogs in Germany.

BACKGROUND: Vector-borne diseases are of increasing importance in Germany. Since 2015, autochthonous cases have been increasingly documented in Berlin/Brandenburg. OBJECTIVES: Describe autochthonous Babesia canis infection in the Berlin/Brandenburg region. ANIMALS: Forty-nine dogs with autochthonous B. canis infection. METHODS: Evaluation of history, clinical signs, laboratory abnormalities, treatment, and outcome. RESULTS: Dogs were presented between March and August (9) and September and January (40) in the years 2015-2021. Historical and clinical findings were lethargy (100%), pale mucous membranes (63%), fever (50%), and pigmenturia (52%). Common clinicopathological findings were thrombocytopenia (100%), anemia (85%), intravascular hemolysis (52%), pancytopenia (41%), and systemic inflammatory response syndrome (SIRS; 37%). Babesia detection was based on blood smear evaluation (n = 40) and PCR targeting the 18S rRNA gene of piroplasms (n = 49). Sequencing indicated 99.47% to 100% identity to B. canis sequences from GenBank. All dogs were treated with imidocarb (2.4-6.3 mg/kg; median, 5 mg/kg); 8 dogs received 1, 35 received 2, and 1 dog each received 3, 4, or 5 injections, respectively. Continued PCR-positive results were detected in 7 dogs after the 1st, in 5 after the 2nd, in 2 after the 3rd, and in 1 28 days after the 4th injection. Four dogs were euthanized and 3 dogs died. CONCLUSIONS AND CLINICAL IMPORTANCE: Autochthonous B. canis infections in Berlin/Brandenburg were associated with severe clinicopathological changes, SIRS, and multiorgan involvement. Testing by PCR during and after treatment is advisable to monitor treatment success. Screening of blood donors in high-risk areas and year-round tick protection is strongly recommended.

Efficacy of long-acting oxytetracycline and imidocarb dipropionate for the chemosterilization of Anaplasma marginale in experimentally infected carrier cattle in Argentina.

The expansion of anaplasmosis to non-endemic areas in Argentina has created the need for specific treatments to eliminate Anaplasma marginale from carriers. The most recent studies have failed to chemosterilize A. marginale infections. In this work, we compare the efficacy of long-acting oxytetracycline (OTC) and imidocarb dipropionate (IMD) to chemosterilize the A. marginale infection. For this purpose, twenty steers were randomly clustered into two groups of ten animals each 78 days after A. marginale experimental infection (day 0). Cattle from group 1 (G1) were treated with three doses of 20 mg kg-1 of OTC (Terramycin® LA, 200 mg/ml) 7 days apart by intramuscular injection. Cattle from G2 were treated with two doses of 5 mg kg-1 of IMD (Imizol®, 120 mg/ml) 14 days apart by intramuscular injection. The efficacy of sterilizing treatments was evaluated by detection of DNA by nested PCR, anti-MSP5 antibodies by ELISA and by inoculation of splenectomized calves with blood from the steers 104 days post-treatment (dpt). The results showed 50% efficacy of the OTC treatment to chemosterilize persistent A. marginale infections in cattle and the failure of the IMD treatment under the evaluated conditions. The persistence of specific antibody levels in the sterilized animals (56 dpt) was shorter than the period of DNA detection. The ELISA was the test of choice to confirm the sterilizing outcome after 60 dpt. In spite of its limitations, the sterilization of A. marginale carrier status using OTC, could be useful for high-value bovines in non-endemic areas.

A tick cell line as a powerful tool to screen the antimicrobial susceptibility of the tick-borne pathogen Anaplasma marginale.

Anaplasma marginale is the causative agent of the severe bovine anaplasmosis. The tick Rhipicephalus microplus is one of the main vectors of A. marginale in tropical and subtropical regions of the world. After the tick bite, the bacterium invades and proliferates within the bovine erythrocytes leading to anemia, impairment of milk production and weight loss. In addition, infection can cause abortion and high mortality in areas of enzootic instability. Immunization with live and inactivated vaccines are employed to control acute bovine anaplasmosis. However, they do not prevent persistent infection. Consequently, infected animals, even if immunized, are still reservoirs of the bacterium and contribute to its dissemination. Antimicrobials are largely employed for the prophylaxis of bovine anaplasmosis. However, they are often used in sublethal doses which may select pre-existing resistant bacteria and induce genetic or phenotypic variations. Therefore, we propose a new standardized in vitro assay to evaluate the susceptibility of A. marginale strains to different antimicrobials. This tool will help health professionals to choose the more adequate treatment for each herd which will prevent the selection and spread of resistant strains. For that, we initially evaluated the antimicrobial susceptibility of two field isolates of A. marginale (Jaboticabal and Palmeira) infecting bovines. The least susceptible strain (Jaboticabal) was used for the standardization of an antimicrobial assay using a culture of Ixodes scapularis-derived tick cell line, ISE6. Results showed that enrofloxacin (ENRO) at 0.25, 1 or 4 µg/mL and oxytetracycline (OTC) at 4 or 16 µg/mL are the most efficient treatments, followed by OTC at 1 µg/mL and imidocarb dipropionate (IMD) at 1 or 4 µg/mL. In addition, this proposed tool has technical advantages compared to the previously established bovine erythrocyte culture. Thereby, it may be used to guide cattle farmers to the correct use of antimicrobials. The choice of the most suitable antimicrobial is essential to eliminate persistent infections, prevent the spread of resistant strains and help controlling of bovine anaplasmosis.

Ehrlichia canis in dogs experimentally infected, treated, and then immune suppressed during the acute or subclinical phases.

BACKGROUND: Concerns for recrudescence of Ehrlichia canis infection arise when immunosuppressive drugs are used to treat immune-mediated diseases in dogs previously infected with E. canis. OBJECTIVES: Determine whether administration of prednisolone and cyclosporine would reactivate E. canis infection in dogs previously treated with doxycycline during the acute or subclinical phases. ANIMALS: Seven beagles previously experimentally infected with E. canis and administered doxycycline for 4 weeks were included. Three of the 7 dogs were incidentally concurrently infected with Anaplasma platys and Babesia vogeli and were administered 2 doses of imidocarb 2 weeks apart before enrollment in the current study. METHODS: Experimental study. Each dog was administered prednisolone and cyclosporine for 6 weeks. Clinical signs, complete blood cell count (CBC), polymerase chain reaction (PCR) assays for E. canis, A. platys, and B. vogeli DNA in blood, E. canis indirect fluorescent antibodies (IFA) titers, and flow cytometry for antiplatelet antibodies were monitored. RESULTS: All dogs completed the immunosuppressive protocol. No evidence for recrudescence of E. canis, A. platys, or B. vogeli were detected based on clinical signs or results of CBC, PCR, IFA, and flow cytometry for antiplatelet antibodies. E. canis IFA titers were negative in 5/7 dogs at the end of immunosuppressive protocol and were negative 6 months after the protocol in 5/5 dogs available for testing. CONCLUSIONS AND CLINICAL IMPORTANCE: Dogs administered with a 4-week course of doxycycline with or without imidocarb failed to show evidence of activation of E. canis infection after administration of a commonly used immune suppressive protocol.

[Theileria equi-infection in 2 German horses returning from a trail ride in southern France]. / Theileria equi-Infektion bei 2 Pferden nach einem Wanderritt in Südfrankreich.

Equine piroplasmosis, caused by the protozoan pathogens Theileria equi or Babesia caballi, is endemic in many (sub-)tropical regions worldwide, including Southern Europe. In Germany, it mainly plays a role as an imported parasitosis. Nevetherless, tick species with known vector potential for equine piroplasms occur in Germany. Thus, a long-term establishment of autochthonous cycles after introduction via infected equids or ticks cannot be excluded. In the present case, 2 horses developed clinical T. equi infection after returning from a trail riding trip to southern France. During treatment, a feeding tick was discovered on one of the horses, which was identified as a Rhipicephalus bursa specimen. This tick species cannot establish in Germany under the current climatic conditions; nevertheless, this discovery illustrates the potential of introducing infected ticks to Germany. The 2 horses were treated with imidocarb (3 mg/kg i. m.; 3 treatments in 3-day intervals) and no adverse drug effects were recorded. To prevent establishment of autochthonous infection cycles in Germany as well as a chronic manifestation of piroplasmosis, treatment should aim at eliminating the parasites despite possible adverse effects of imidocarb.

Fatal acute babesiosis associated with Babesia venatorum infection (Babesia sp. EU1) in a captive reindeer calf in Switzerland.

Babesia venatorum was isolated from a captive reindeer calf in Switzerland. The clinical signs consistent with acute babesiosis included hemolytic anemia and hemoglobinuria. The diagnosis was made based on visualization of intraerythrocytic parasites in the stained blood smears and confirmed by PCR analysis of the 18S rRNA gene, with subsequent species identification within Babesia confirmed by sequencing. The reindeer calf was initially treated with supportive care and an antiprotozoal drug (imidocarb dipropionate) but died a few days after hospitalization. Babesia venatorum is also known as Babesia sp. EU1 and can infect different mammalian species, including humans. The current case report aims to increase awareness among veterinarians and reindeer owners about the presence and the associated risk of this zoonotic pathogen. Considering the high morbidity and possible mortality associated with acute babesiosis, captive reindeer should receive tick prevention measures and be tested for subclinical infections in endemic area.

Clinical Occurrence of Canine Babesiosis in the Coastal Area of the Black Sea (Dobrogea) in Southeastern Romania and Associated Epidemiological Implications.

Canine babesiosis (CB) is a significant tick-borne disease caused by intra-erytrocytic protozoa of the genus Babesia (Apicomplexa: Piroplasmida) and is currently emerging or re-emerging in many European countries. Despite the increasing incidence of clinical CB, little is known of the epidemiology of this disease in Romania. Therefore, the present study was conducted to provide epidemiological data on CB in the coastal area of the Black Sea (Dobrogea), southeastern Romania. For this, 306 owned dogs presented during 2016-2018 at a veterinary clinic in the area and clinically suspected for CB were tested for the presence of intra-erytrocytic piroplasms using Giemsa-stained thin blood smears. Overall, 27.8% (95% CI: 22.83-33.16) of dogs were positive for large piroplasms. The Babesia spp.-positive dogs were 51 males and 34 females aged from 3 mo to 13 yr (mean 4.2 yr; SD = 3.6). A higher rate of infection was registered in Bichon Maltese (n = 13) and mixed-breed (n = 28) dogs. Most cases (76/85; 89.4%) were diagnosed from March to June, with only a few cases registered in November and December (1 case each). Infected dogs displayed different clinical presentations, varying from mild (38.8%) to moderate (29.4%) and severe disease (29.4%). Based on clinical- pathological changes, 38 (44.7%), 34 (40.0%), and 13 (15.3%) dogs were diagnosed with uncomplicated babesiosis, complicated babesiosis with a single organ dysfunction, and complicated babesiosis with multiple-organ-dysfunction syndrome (MODS), respectively. The recovery rate (81.6%, 70.1%, and 38.4%, respectively) was significantly different in these groups. The age of dogs appeared to be a risk factor for both severe disease (mean age of 5.8 yr) and MODS (mean age 6.8 yr). These findings document the common occurrence of CB in Dobrogea, southeastern Romania, and suggest that appropriate control measures need to be taken.

Hepatozoon canis infection causing a strong monocytosis with intra-monocytic gamonts and leading to erroneous leukocyte determinations.

In this case report, a Swedish flat-coated retriever was diagnosed with an extensive Hepatozoon canis infection. The dog had a prominent monocytosis (14.0 × 109 /L) with H canis gamonts detected in most monocytes, but none were found in the neutrophils. On the hematology system ADVIA 2120 peroxidase (PEROX) cytogram, most leukocytes were seen as a distinct cell population above the lymphocytes, which indicated that most of the cells were larger than lymphocytes and had weak myeloperoxidase staining. This distinct cell cluster appeared to be of a single cell type but was incorrectly divided by the ADVIA 2120 into lymphocytes, monocytes, and large unstained cells (LUC). The total leukocyte counts on the ADVIA 2120 WBC basophil (BASO) channel were much higher than that on the WBC PEROX count. The WBC BASO cytogram appeared abnormal with two parallel cell populations, so the BASO WBC count was considered erroneous. Polymerase chain reaction and DNA sequencing verified H canis infection. The dog was treated with subcutaneous imidocarb dipropionate (6 mg/kg) injections every other week. Post-treatment hematology analyses indicated that the percentage of parasitized leukocytes decreased from 40% to 5% about 4 weeks after the start of treatment and were not found in any monocytes 6 weeks after the beginning of the treatment. In conclusion, H canis infection in this dog was associated with a strong monocytosis, and gamonts were present in many monocytes, which caused aberrant automated leukocyte counts to occur.

First autochthonous case of clinical Hepatozoon felis infection in a domestic cat in Central Europe.

Three different Hepatozoon (Apicomplexa, Hepatozoidae) species have been described infecting domestic cats in Europe (i.e. H. felis, H. canis and H. silvestris), however, reports on clinical hepatozoonosis are uncommon and treatment protocols are not clearly defined. A six-year-old male European short-hair cat from Austria presented poor general condition, lethargy, anorexia, icterus, a painful abdomen, fever, ruffled hair and a tick infestation, and it had never left Austria. Laboratory tests revealed leukopenia, thrombocytopenia and increased serum levels of symmetric dimethylarginine (SDMA) and bilirubin. In May Grünwald-Giemsa-stained blood smears, structures resembling Hepatozoon gamonts were observed inside neutrophil granulocytes. A PCR targeting a fragment of the 18S rRNA gene of Hepatozoon spp. and DNA sequencing allowed the diagnosis of H. felis-DNA in blood samples. The cat was treated with imidocarb dipropionate (6 mg/kg body weight, repeated after 14 days) and doxycycline monohydrate (5 mg/kg body weight twice a day, p.o., for four weeks) and recovered completely. A broad haematological and biochemical laboratory control after six months showed all evaluated parameters under normal ranges. Coinfection with other feline pathogens (i.e. feline leukaemia virus, feline immunodeficiency virus, feline Coronavirus, Leishmania and Dirofilaria immitis) could not be detected. This study reveals the presence of H. felis in Austria and provides more evidence on the geographical distribution and pathogenicity of this parasite for domestic cats. To the authors' knowledge, this is the first autochthonous case of feline hepatozoonosis in Central Europe.

Presentation of hemolytic and hemorrhagic rangeliosis in Cerdocyon thous.

Rangeliosis, caused by protozoan Rangelia vitalii, is transmitted by the tick Amblyomma aureolatum. The disease is characterized by hemolytic and hemorrhagic disorder and has been described in dogs and other wild canids. The aim of this study was to compare clinicopathological findings and laboratory results of a Rangelia infection in a crab-eating fox (Cerdocyon thous) with those of canine rangeliosis. The zoo of Universidade de Caxias do Sul, received a crab-eating fox with marked jaundice in mucous membranes, dark-colored stools and neurological signs. The animal underwent an ear tip smear examination and blood collection for complete blood counts, serum biochemistry and PCR. Free-living and intraerythrocytic pyriform structures consistent with R. vitalii were found in the blood smear of the ear tip. The erythrogram revealed normocytic normochromic anemia, moderate macrocytosis, polychromasia and metarubricytosis. The leukogram revealed leukocytosis with neutrophilia and monocytosis, as well as severe thrombocytopenia. Serum biochemistry showed hypoproteinemia, hypoalbuminemia and elevated levels of urea and creatinine. The treatment was performed with imidocarb hydrochloride and dexamethasone, however 24 h after initiation of treatment the animal died. Macroscopic examination revealed jaundice, subcutaneous edema, enlarged superficial lymph nodes, splenomegaly, and hemorrhage of internal organs. Histological sections of the cerebellum, lung, pancreas, intestine and heart were consistent with R. vitalii infection of the vascular endothelium. Pathological and hematological findings were similar to those found in infected dogs, with clinical presentation characterized by hemolytic anemia and hemorrhage. The description of this case showed that C. thous does not only serve as reservoir of R. vitalii but may also develop disease.

Hepatozoon canis in three imported dogs: a new tickborne disease reaching the United Kingdom.

An increasing number of non-endemic vectorborne pathogens have been described in dogs imported to the UK in the past two decades. Recently, an outbreak of canine babesiosis in south-east England has raised veterinary awareness with regard to the impact of such diseases on the UK canine population. Canine hepatozoonosis, caused by Hepatozoon canis and transmitted by the ingestion of Rhipicephalus sanguineus ticks, is widespread in the Mediterranean basin. Herein we describe the first three molecularly confirmed clinical cases of canine hepatozoonosis in dogs imported into the UK. Veterinarians in the UK should be aware of H canis as a potential infection in imported dogs, especially in the face of the expanding distribution of R sanguineus ticks in Europe.

Parasitological and molecular diagnostic of a clinical Babesia caballi outbreak in Southern Romania.

Equine piroplasmosis (EP) is a tick-borne disease of equids caused by Babesia caballi and/or Theileria equi, which is endemic in many tropical and temperate areas of the world. However, clinical outbreaks of EP in Romania during the last decades have not been reported Therefore, the aim of this paper is (i) to describe a clinical B. caballi outbreak in horses on several farms in Southern Romania using a diagnostic and therapeutic approach and (ii) the molecular diagnostic of EP in an endemic area of Romania. In the first case, a 10-month-old stallion male was presented with lethargy, anorexia, fever (40.9 °C), pale mucosal/mucous/membranes and a marked anemia. In the subsequent weeks, three horses from other farms located in the same area, displayed similar clinical signs. B. caballi was diagnosed in all the horses based on Giemsa-stained blood smears and the diagnosis was further confirmed by polymerase chain reaction (PCR), using a single-round and multiplex PCR and sequencing. All four horses were treated with imidocarb dipropionate, at a dose rate of 2.2 mg/kg body weight (two injections at 48 h apart), and all horses clinically recovered within 24-48 h, post-treatment. This report presents the first molecularly characterized B. caballi outbreak in Romania in clinically affected horses, confirmed by DNA sequencing.

Verification of post-chemotherapeutic clearance of Theileria equi through concordance of nested PCR and immunoblot.

Certain countries including the United States remain non-endemic for particular infectious diseases such as equine piroplasmosis through import restrictions and surveillance. Endemic regions often employ premunition as the primary method to control disease, however in non-endemic countries, chemosterilization combined with methods to confirm parasite elimination are required to maintain disease-free status. The ability of imidocarb diproprionate (ID) to clear persistent Theileria equi infection from infected horses has been shown through the inability of treated horses to transmit via blood transfer. However, the common lengthy persistence of anti-T. equi antibody causes regulatory tests such as cELISA or IFA to remain positive for extended periods. Persistence of positive testing creates challenges for regulatory veterinary medicine and international trade. Concordance between nested polymerase chain reaction (nPCR) targeting the ema1 gene and immunoblotting (IB) measuring declination in anti-EMA1 and anti-EMA2 antibody were used to verify clearance of T. equi from 179 ID-treated horses. These data support the use of IB to demonstrate declining anti-EMA1 and EMA2 titers in T. equi-infected horses subsequent to successful ID treatment. Such data provide concordant support to a negative nPCR and allow for a more timely determination of effective ID clearance of T. equi. The post ID treatment results indicate that while nPCR was consistently negative by 14 days and cELISA generally remained positive after 1 year, immunoblot was on average negative after 4 months and 100% in agreement with nPCR.

Microscopic and molecular analysis of Babesia canis in archived and diagnostic specimens reveal the impact of anti-parasitic treatment and postmortem changes on pathogen detection.

BACKGROUND: Classification of Babesia parasites has traditionally relied on morphological differentiation based on piroplasm size and shape. Molecular typing has subsequently revealed a more complex taxonomy for these piroplasms than previously thought. To evaluate the factors that influence the morphology of Babesia species upon microscopic examination and hence, their taxonomic classification, we performed detailed characterizations of piroplasms from archival and prospective collections of cytological samples of dogs with piroplasmosis before and after death. Merozoite morphology and time of parasite disappearance following imidocarb dipropionate was also investigated. METHODS: The study was divided into a (i) review of archived cytological slides from confirmed cases of canine piroplasmosis, and (ii) a prospective study of smears and tissue imprints from 15 recently necropsied dogs. The latter group could be further sub-divided into a non-treated group and an imidocarb dipropionate-treated group. Exact times of treatment before death were reviewed. Additional blood smears prepared from the live dogs and taken before therapy were also evaluated in the latter group. Parasite burden per each slide was determined in both studies. The shape and size of merozoites were described from blood smears taken while the dogs were alive and from different organs during necropsy. The results of all measurements were statistically analyzed. RESULTS: The morphology and size of merozoites from live dogs corresponded to that of previously described 'large' Babesia. The morphology and size of merozoites were significantly different (P < 0.001) in postmortem samples, however, and more consistent in shape and size with piroplasm cells previously referred to as 'small' Babesia. PCR and sequencing confirmed B. canis as the causative agent of disease in all investigated dogs, including in postmortem negative tissue imprints from dogs treated at least 24 h before death. CONCLUSIONS: Changes in the morphology of 'large' B. canis to 'small'-like Babesia observed by light microscopy appear to represent a common postmortem change. Classification of Babesia parasites into 'large' and 'small' Babesia using only microscopy of postmortem slides should be treated with caution. PCR-based methodologies for detection and molecular typing of Babesia spp. may prove valuable for investigating suspected cases of babesiosis following necropsy.

First clinical case report of Cytauxzoon sp. infection in a domestic cat in France.

BACKGROUND: Feline cytauxzoonosis is an emerging infection caused by tick-transmitted apicomplexan parasites of the genus Cytauxzoon. The association of clinical disease with Cytauxzoon infection appears to be limited to C. felis infections in the Americas. Sporadic infections of wild and domestic felids with Cytauxzoon sp. were recently described in European countries but clinical reports of the infection are rare and incomplete. This case report brings new interesting information on cytauxzoonosis expression in Europe. CASE PRESENTATION: A 9-years-old castrated European shorthair cat living in rural area of north-eastern France (Saint Sauveur, Bourgogne-Franche-Comté region), without any travel history was presented for consultation due to hyperthermia, anorexia, depression and prolonged fever that didn't respond to antibiotic therapy. The cat had outdoor access with a history of vagrancy and was adequately vaccinated (core vaccines and FeLV vaccine). During biological investigations, intraerythrocytic inclusions were observed on blood smear and were further investigated by PCR analysis and sequencing. Molecular analyses confirmed Cytauxzoon sp. infection. The cat was treated with a subcutaneous injection of imidocarb dipropionate (3.5 mg/kg). One week after treatment, the cat improved clinically, although parasitic inclusions within erythrocytes persisted, and only a mild lymphocytosis was found. Two weeks after treatment, the cat appeared in excellent health, appetite was normal and parasitemia was negative. However, one month after treatment the cat relapsed with hyperthermia, anorexia, and depression. Blood smears and PCR were once again positive. Subsequently, the cat received an additional dose of imidocarb dipropionate (3.5 mg/kg SC) and recovered rapidly without other clinical signs. Two weeks after the second imidocarb injection, the cat was hit by a car and died. CONCLUSION: This case provides the first clinical description of infection by Cytauxzoon sp. in a domestic cat in France. These findings support the fact that cytauxzoonosis should be considered in the differential diagnosis of acute febrile illness which does not respond to antibiotic in cats with outdoor access especially in areas where populations of wild felids are present.

Efficacy, safety and tolerance of imidocarb dipropionate versus atovaquone or buparvaquone plus azithromycin used to treat sick dogs naturally infected with the Babesia microti-like piroplasm.

BACKGROUND: Piroplasmosis caused by the Babesia microti-like piroplasm (Bml) is increasingly being detected in dogs in Europe. Sick dogs show acute disease with severe anaemia associated with thrombocytopenia with a poor response to current available drugs. This study assesses the safety and tolerance of three treatments and compares their efficacy over a full year of follow up in dogs naturally infected with Bml. METHODS: Fifty-nine dogs naturally infected with Bml were randomly assigned to a treatment group: imidocarb dipropionate (5 mg/kg SC, 2 doses 14 d apart) (IMI); atovaquone (13.3 mg/kg PO q 8 h, 10 d)/azithromycin (10 mg/kg PO q 24 h, 10 d) (ATO); or buparvaquone (5 mg/kg IM, 2 d apart)/azithromycin (same dosage) (BUP). Before and after treatment (days 15, 45, 90 and 360), all dogs underwent a physical exam, blood tests and parasite detection (blood cytology and PCR). Clinical efficacy was assessed by grading 24 clinical and 8 clinicopathological signs from low to high severity. RESULTS: Before treatment, most dogs had severe regenerative anaemia (88.13%) and thrombocytopenia (71.4%). On treatment Day 45, clinical signs were mostly reduced in all dogs, and by Day 90, practically all dogs under the ATO or BUP regimen were clinically healthy (76.4 and 88%, respectively). Highest percentage reductions in laboratory abnormalities (82.04%) were detected in animals treated with ATO. Over the year, clinical relapse of Bml was observed in 8 dogs (8/17) treated with IMI. However, on Day 360, these animals had recovered clinically, though clinicopathological abnormalities were still present in some of them. Parasitaemia was PCR-confirmed on Days 90 and 360 in 47.05 and 50% of dogs treated with ATO, 68 and 60.08% with BUP, and 94.1 and 73.3% with IMI, respectively. Even after 360 days, 13.3% of the dogs treated with IMI returned a positive blood cytology result. CONCLUSIONS: IMI showed the worse clinical and parasitological, efficacy such that its use to treat Bml infection in dogs is not recommended. The treatments ATO and BUP showed better efficacy, though they were still incapable to completely eliminate PCR-proven infection at the recommended dose. All three treatments showed good tolerance and safety with scarce adverse events observed.

Changes in selected subpopulations of lymphocytes in dogs infected with Babesia canis treated with imidocarb.

OBJECTIVE: The purpose of this study was to track changes in selected subpopulations of lymphocytes in the blood of dogs infected with Babesia (B.) canis and treated with imidocarb. MATERIAL AND METHODS: The study included 16 dogs divided into two groups. The first group (n = 6) consisted of healthy control animals. Dogs of the se- cond group (n = 10) were infected with B. canis and after establishment of the diagnosis each animal received a single dose of imido- carb (5 mg/kg). Flow cytometry was used to enumerate several immune cell phenotypes. RESULTS: It was concluded that the invasion of B. canis contributes to the decreased percentage of CD3+, CD4+, CD8+, CD21+ lymphocytes in the blood of infected animals. The decreased level of tested subpopulations of lymphocytes in group 2 persisted for the entire 12-day period of the test. After the administration of imidocarb, each tested lymphocyte fraction in the blood of the dogs with babesiosis increased, but did not reach physiological values. CONCLUSION: The presented results indicate that the resolution of clinical signs associated with babesiosis may be related to the stimulation and intensity of cellular immunity, dependent on the CD4+ T cells profile. After administration of imidocarb, the parasitemia is cleared which allows the recovery of the lymphocyte populations.

Theileria equi isolates vary in susceptibility to imidocarb dipropionate but demonstrate uniform in vitro susceptibility to a bumped kinase inhibitor.

BACKGROUND: The apicomplexan hemoparasite Theileria equi is a causative agent of equine piroplasmosis, eradicated from the United States in 1988. However, recent outbreaks have sparked renewed interest in treatment options for infected horses. Imidocarb dipropionate is the current drug of choice, however variation in clinical response to therapy has been observed. METHODS: We quantified the in vitro susceptibility of two T. equi isolates and a lab generated variant to both imidocarb dipropionate and a bumped kinase inhibitor compound 1294. We also evaluated the capacity of in vitro imidocarb dipropionate exposure to decrease susceptibility to that drug. The efficacy of imidocarb dipropionate for clearing infection in four T. equi infected ponies was also assessed. RESULTS: We observed an almost four-fold difference in imidocarb dipropionate susceptibility between two distinct isolates of T. equi. Four ponies infected with the less susceptible USDA Florida strain failed to clear the parasite despite two rounds of treatment. Importantly, a further 15-fold decrease in susceptibility was produced in this strain by continuous in vitro imidocarb dipropionate exposure. Despite a demonstrated difference in imidocarb dipropionate susceptibility, there was no difference in the susceptibility of two T. equi isolates to bumped kinase inhibitor 1294. CONCLUSIONS: The observed variation in imidocarb dipropionate susceptibility, further reduction in susceptibility caused by drug exposure in vitro, and failure to clear T. equi infection in vivo, raises concern for the emergence of drug resistance in clinical cases undergoing treatment. Bumped kinase inhibitors may be effective as alternative drugs for the treatment of resistant T. equi parasites.