Inhibitory effect of naphthoquine phosphate on Babesia gibsoni in vitro and Babesia rodhaini in vivo

Background Drug resistance and toxic side effects are major challenges in the treatment of babesiosis. As such, new drugs are needed to combat the emergence of drug resistance in Babesia parasites and to develop alternative treatment strategies. A combination of naphthoquine (NQ) and artemisinin is an antimalarial therapy in pharmaceutical markets. The present study repurposed NQ as a drug for the treatment of babesiosis by evaluating the anti-Babesia activity of naphthoquine phosphate (NQP) alone. Methods An in vitro growth inhibition assay of NQP was tested on Babesia gibsoni cultures using a SYBR Green I-based fluorescence assay. In addition, the in vivo growth inhibitory effect of NQP was evaluated using BALB/c mice infected with Babesia rodhaini. The parasitemia level and hematocrit values were monitored to determine the therapeutic efficacy of NQP and the clinical improvements in NQP-treated mice. Results The half maximal inhibitory concentration of NQP against B. gibsoni in vitro was 3.3 ± 0.5 μM. Oral administration of NQP for 5 consecutive days at a dose of 40 mg/kg of body weight resulted in significant inhibition of B. rodhaini growth in mice as compared with that of the control group. All NQP-treated mice survived, whereas the mice in the control group died between days 6 and 9 post-infection. Conclusion This is the first study to evaluate the anti-Babesia activity of NQP in vitro and in vivo. Our findings suggest that NQP is a promising drug for treating Babesia infections, and drug repurposing may provide new treatment strategies for babesiosis. Graphical Abstract

Co-infection with Babesia canis and Babesia gibsoni in a dog

A four-year-old intact male Boxer, that had a history of travelling to Serbia, was referred for lethargy and anaemia. Shortly before the dog was referred, it was diagnosed twice with an infection with Babesia canis and was treated with imidocarb both times. A blood smear evaluation was indicative of the presence of intraerythrocytic piroplasms. After receiving inconclusive results regarding the type of piroplasm, the dog was diagnosed with simultaneous infections with B. canis and Babesia gibsoni via real-time polymerase chain reaction (rt-PCR) testing. The dog was treated with imidocarb, atovaquone and azithromycin, and in a follow-up examination, the PCR results were negative for B. canis and B. gibsoni. Several weeks later, the dog was presented again, and a PCR was positive for B. gibsoni. After atovaquone and azithromycin failed to eliminate the parasites, a therapy attempt using metronidazole, clindamycin and doxycycline was initiated. Six months after diagnosis, the treatment appeared successful in eliminating B. gibsoni. This case report describes the clinical findings of the co-infection and the initiated diagnostic and therapeutic approaches.

Inhibitory effect of naphthoquine phosphate against Babesia gibsoni in vitro and Babesia rodhaini in vivo

Background: Drug resistance and severe side effects are major challenges in the treatment of babesiosis as they lead to less choices for treatment. Development of new drugs to enrich the treatment strategies and delay the emergence of drug resistance in parasites is still needed. Naphthoquine (NQ) combined with artemisinin treats Plasmodium infection by rapid parasite clearance. The current study repurposed NQ as a babesiosis drug treatment by evaluating the effects of naphthoquine phosphate (NQP) as a single dose treatment for babesiosis. Methods: In vitro anti-Babesia activity of NQP was tested on Babesia gibsoni cultures. The inhibition of parasite growth was verified using a SYBR green I-based fluorescence assay. In vivo efficacy of NQP was evaluated using BALB/c mice infected with Babesia rodhaini. The parasitemia level and hematocrit values were monitored. Results: The half maximal inhibitory concentration of NQP against B. gibsoni in vitro was 3.3 ± 0.5 μM. Oral administration of NQP for 5 successive days at a dose of 40 mg/kg of body weight resulted in significant inhibition on parasite growth compared with the control group. All mice in NQP-treated group survived, whereas the mice in control group died between days 6 and 9 post infection. Conclusion: This is the first study to evaluate the anti-Babesia activity of NQP in vitro and in vivo. The results showed that NQP is a promising drug for babesiosis treatment and drug repurposing may provide new treatment strategies for babesiosis.

Clinical variations observed among the main hemoparasitosis caused by Rhipicephalus sanguineus in dogs

Hemoparasitosis are diseases of great importance in the practice of a small animal’s veterinarian, due to their high number of cases in the clinical routine of these species, and the wide occurrence of their vectors. Related to hemoparasitosis in dogs, the tick Rhipicephalus sanguineus has the greatest importance due to parasitizing dogs, perpetuating hemoparasitosis in this host group. The present work describes a literature review about the three main hemoparasitosis in the routine of a veterinarian in Brazil, ehrlichiosis, caused by Ehrlichia canis, babesiosis caused by the protozoa Babesia canis and Babesia gibsoni, and anaplasmosis caused by Anaplasma platys, comparing them regarding the differences between clinical signs and laboratory alterations, such as the different types of anemia caused by these conditions that culminate in clinical signs related to them. It also aims to describe the similarity in the diagnostic techniques used and infection treatments, which in all cases is used tetracycline, the most commonly used is doxycycline, and highlight the need for further studies about this topic.

Incidence of Canine Tick Vectors and Molecular Detection of Haemoparasites in Vectors and Hosts

Background: Ticks are of great importance in transmission of various canine tick borne diseases. Several characteristics of ticks make them outstanding vectors of pathogenic agents, the wide host range and slow feeding habit along with tendency to feed on several hosts during life cycle ensures ample opportunity to acquire and transmit pathogens. Methods: This study focuses on status of canine tick vectors and molecular detection of haemoparasites in these ticks and their host, in and around Kolkata. The blood and tick samples were collected from Dog Ward, Department of Teaching Veterinary and Clinical Complex; Faculty of Veterinary and Animal Sciences, WBUAFS at Belgachia; Veterinary Clinic of Kolkata Police Dog Squad at Alipore; Veterinary Clinic of Barrackpore Police dog Squad and samples from stray dogs were also collected from inside the University campus through a period of one year (August, 2016 to July, 2017). Result: The tick infestation was recorded at 41% with Rhipicephalus sanguineus, being the only tick. Nine primer sets were used for detection of Babesia spp, Ehrlichia canis, Ehrlichia chaffensis, Babesia gibsoni, Hepatozoon canis, Mycoplasma haemocanis, Anaplasma platys and Theileria annae from the respective tick samples and blood sample of hosts. Tick samples were found positive for Babesia spp, Ehrlichia spp. and H. canis where as the corresponding blood samples were found positive for Babesia spp, Ehrlichia spp., Mycoplasma spp. and H. canis. This study conclusively provides evidence of high rates of incidence of haemoparasitic infection or canine tick borne diseases infection and tick infestation, with at least four haemoparasites infecting the dog population and at least one tick species (Rhipicephalus sanguineus) infesting the dogs in and around Kolkata.

Stable transfection system for Babesia sp. Xinjiang

Background Stable transfection systems have been described in many protozoan parasites, including Plasmodium falciparum, Cryptosporidium parvum, Babesia bovis, Babesia ovata, and Babesia gibsoni. For Babesia sp. Xinjiang (Bxj), which is the causative pathogen of ovine babesiosis and mainly prevails across China, the platform of those techniques remains absent. Genetic manipulation techniques are powerful tools to enhance our knowledge on parasite biology, which may provide potential drug targets and diagnostic markers. Methods We evaluated the inhibition efficiency of blasticidin (BSD) and WR99210 to Bxj. Then, a plasmid was constructed bearing selectable marker BSD, green fluorescent protein (GFP) gene, and rhoptry-associated protein-1 3′ terminator region (rap 3′ TR). The plasmid was integrated into the elongation factor-1 alpha (ef-1α) site of Bxj genome by cross-over homologous recombination technique. Twenty μg of plasmid was transfected into Bxj merozoites. Subsequently, drug selection was performed 24 h after transfection to generate transfected parasites. Results Transfected parasite lines, Bxj-c1, Bxj-c2, and Bxj-c3, were successfully obtained after transfection, drug selection, and colonization. Exogenous genes were integrated into the Bxj genome, which were confirmed by PCR amplification and sequencing. In addition, results of western blot (WB) and indirect immunofluorescence assay (IFA) revealed that GFP-BSD had expressed for 11 months. Conclusions In our present study, stable transfection system for Bxj was successfully developed. We anticipate that this platform will greatly facilitate basic research of Bxj. Graphical abstract

HEMOPARASITOSE EM UMA CANINA- RELATO DE CASO

Introdução: A hemoparasitose é uma patologia de alta casuística na rotina clínica veterinária, desencadeada por bactérias, protozoários, helmintos, possuindo como principal transmissor o carrapato em períodos do ano de maior incidência de calor e umidade, tendo em vista o potencial zoonótico. A babesiose é causada pelos protozoários Babesia Canis e Babesia gibsoni transmitida pelo carrapato Rhipicephalus sanguineus, esses parasitas causam a hemólise das hemácias. Já a anaplasmose é oriunda de bactérias intracelulares Anaplasma platys ou Anaplasma phagocytophilum transmitidas pelo carrapato Rhipicephalus sanguineus, causando trombocitopenia. A rangeliose é ocasionada por um piroplasma Rangelia vitalli, transmitida por carrapatos Rhipicephalus sanguineus e Amblyomma aerolatum, se replicando nos eritrócitos. Objetivo: Relatar um caso clínico de uma canina. Relato de caso: canina castrada, sem raça definida, com 1 ano e dois meses, pesando 11,4 Kg, sendo atendida em uma clínica veterinária particular em Caxias do Sul/RS, apresentando apatia, hematoquezia, anorexia, prostração, epistaxe, sangramento em pontas de orelhas, mucosas hipocoradas com petéquias e febre. Durante o atendimento foi realizada coleta sanguínea para avaliação hematológica, esfregaço sanguíneo periférico da orelha, bioquímica sérica (creatinina, fosfatase alcalina, ureia, transaminase pirúvica) e urinálise, apresentando alterações, tais como anemia regenerativa, leucocitose por neutrofilia com desvio a esquerda, linfocitose, trombocitopenia, na urinálise houve presença de hemoglobina, leucócitos, proteinúria. No exame de esfregaço de sangue periférico apresentou-se negativo para hemoparasitas. O diagnóstico definitivo foi através da Reação em Cadeia da Polimerase (PCR), na qual evidenciou os agentes patogênicos de babesiose, rangeliose e anaplasmose. O tratamento instituído foi doxiciclina (5 mg/kg/q.12h durante 28 dias), azitromicina (10 mg/Kg/q.24h durante 10 dias), diaceturato de diminazeno (0,5ml/2Kg) repetindo em 14 dias a dose com prévia aplicação de atropina (0,044 mg/Kg), suplemento vitamínico mineral aminoácido (1 ml/q.24h). O animal se manteve bem clinicamente durante o tratamento, obtendo cura da enfermidade. Conclusão: Mesmo com o diagnóstico negativo de esfregaço sanguíneo periférico, com a suspeita clínica do animal foi solicitado PCR para confirmar a patologia, assim foi de suma importância a pesquisa de hemoparasitas pelos sinais clínicos do canino, juntamente do tratamento precoce e assertivo.