Therapeutic efficacy of diminazene aceturate versus imidocarb dipropionate against bovine babesiosis

Introduction

An estimated 1.2 billion cattle are at risk of tick-borne haemoparasitic infections, and bovine babesiosis ranks among the costliest of these diseases, responsible for direct losses of over USD 3.5 billion per year through mortality, reduced milk yield, and treatment expenses [1] . The disease is caused by intra-erythrocytic protozoa of the genus Babesia, transmitted mainly by Rhipicephalus (Boophilus) tick species during blood-feeding. In Turkey, both B. bovis and B. bigemina circulate in overlapping zones across the central Anatolian plateau and the Mediterranean coastal belt, making the country an informative setting for comparative drug trials [2] . Two chemotherapeutic agents dominate field practice. Diminazene aceturate (DA), an aromatic diamidine, binds the minor groove of parasite DNA, blocking replication and transcription [3] . It is typically administered as a single intramuscular injection at 3.5 mg/kg body weight, making it attractive where repeat visits are impractical. Imidocarb dipropionate (ID), a carbanilide derivative, interferes with polyamine and inositol metabolism within the parasite, producing a slower but more persistent antiprotozoal effect [4] . The standard regimen involves two subcutaneous injections of 1.2 mg/kg separated by a 14-day interval, a schedule that also provides prophylactic cover for several weeks after the second dose. Despite widespread field reliance on both molecules, randomised comparative trials under natural infection conditions are surprisingly few. Most published efficacy data come from artificial challenge experiments in which cattle receive standardised inocula under controlled laboratory settings [5] . Natural infections differ: parasite loads vary, animals present at different disease stages, and co-infections with Anaplasma or Theileria species may complicate the clinical picture [6] . A 2019 meta-analysis pooling African and Asian field data found no statistically significant difference in cure rates between DA and ID, but cautioned that trial heterogeneity was high and Mediterranean datasets were absent [7] . Turkey's central Anatolian region presents a semi-continental Mediterranean climate with hot, dry summers that coincide with peak tick activity from April to September. Cattle in this zone include both indigenous Anatolian Black and imported Holstein-Friesian genotypes; the latter are markedly more susceptible to severe babesiosis because they lack the innate resistance conferred by long co-evolutionary exposure to local Babesia strains [8] . Understanding how DA and ID perform in this mixed-genotype, naturally exposed population has direct relevance for treatment guidelines issued by provincial veterinary authorities. Beyond cure rate alone, clinicians need comparative data on speed of parasitaemia clearance, haematological recovery trajectory, and adverse event profile to make informed prescribing decisions. Packed cell volume (PCV), the most accessible bedside indicator of anaemia severity, serves as a reliable proxy for erythrocyte destruction and regeneration dynamics during babesiosis treatment [9] . Monitoring PCV at defined post-treatment intervals allows quantification of how quickly each drug reverses the haemolytic process. This trial was designed to compare DA and ID under natural field conditions in Mediterranean Turkey, tracking parasitaemia clearance time, PCV recovery curve, clinical cure rate, case fatality, and treatment-related adverse events over a 28-day follow-up window.

Material and Methods

Reagents and Chemicals

Diminazene aceturate (Berenil®, MSD Animal Health, Netherlands) was supplied as 2.36 g granules dissolved in 12.5 mL sterile water for injection immediately before use, yielding a 7% solution. Imidocarb dipropionate (Imizol®, MSD Animal Health, Netherlands) was available as a readyto-use 12% aqueous solution. Giemsa stain (Merck KGaA, Germany) was prepared at 10% dilution in phosphate buffer (pH 7.2). PCR reagents included DreamTaq Green PCR Master Mix (Thermo Fisher Scientific, USA), species-specific primers targeting the 18S rRNA gene of B. bovis (BoF/BoR) and B. bigemina (BiF/BiR) as described by Figueroa et al. [10] , and nuclease-free water (Ambion, USA). Ethylenediaminetetraacetic acid (EDTA) vacutainer tubes (BD, USA) and plain serum separator tubes (BD, USA) were used for blood collection.

Material

The trial enrolled 142 cattle presenting with clinical signs consistent with babesiosis (fever >39.5°C, pale mucous membranes, haemoglobinuria, reduced appetite) at the Ankara Metropolitan University Veterinary Teaching Hospital and three satellite field clinics in Ankara Province between April 2022 and September 2023. Inclusion criteria required microscopically confirmed Babesia parasitaemia on Giemsastained thin blood smear and PCV below 24%. Animals that had received any anti-babesial treatment within the preceding 60 days, pregnant cows in the final trimester, and calves younger than six months were excluded. Block randomisation (block size 4) assigned animals to DA (n=71) or ID (n=71) groups. Breed distribution was 47.2% Holstein-Friesian, 33.1% Anatolian Black, and 19.7% crossbreeds. The Ankara Metropolitan University Animal Ethics Committee approved the protocol (Approval No. AMU-AEC/2022-031, dated 22 March 2022), and written owner consent was obtained for every enrolled animal.

Methods

DA-group animals received a single intramuscular injection of diminazene aceturate at 3.5 mg/kg body weight in the gluteal muscle on day 0. ID-group animals received imidocarb dipropionate at 1.2 mg/kg subcutaneously behind the shoulder on day 0 and a repeat dose on day 14. All animals received supportive therapy consisting of isotonic saline (20 mL/kg IV on day 0) and a single intramuscular iron-dextran injection (10 mg/kg) to support erythropoiesis. Clinical examination (rectal temperature, heart rate, respiratory rate, mucous membrane colour, appetite score on a 0-3 scale) was performed on days 0, 3, 7, 14, 21, and 28. At each visit, 10 mL jugular blood was collected into EDTA and plain tubes. PCV was determined by micro-haematocrit centrifugation, total erythrocyte count by manual haemocytometer, and parasitaemia percentage by counting infected cells per 1,000 erythrocytes on Giemsa smears. Species confirmation was done by semi-nested PCR on day 0 samples. Clinical cure was defined as resolution of fever, absence of parasitaemia on smear by day 14, and PCV ≥24% by day 28. Data were analysed in SPSS 26.0. Continuous variables were compared by independent-samples t-test or Mann-Whitney U test; categorical outcomes by chi-square or Fisher's exact test. Repeated-measures ANOVA assessed PCV and parasitaemia trends across time points. Significance was set at p<0.05.

Safety Considerations

Both drugs carry documented adverse effect profiles that required prospective monitoring. DA can cause transient injection-site pain, local swelling, and at higher doses neurotoxicity manifesting as ataxia or opisthotonus [3] . IDassociated side effects include profuse salivation (cholinergiclike reaction), transient diarrhoea, and restlessness within 15-45 minutes of injection [4] . To manage potential emergencies, atropine sulphate (0.04 mg/kg IV) was kept at bedside for every ID administration. All adverse events were recorded on standardised case report forms, graded as mild (self-resolving within 24 hours), moderate (requiring a single intervention), or severe (life-threatening or requiring hospitalisation). Animals exhibiting severe neurotoxic signs were to be withdrawn and treated immediately; however, no such event occurred during the trial. Molecular speciation revealed that B. bovis was the predominant species, accounting for 54.2% (77/142) of infections, followed by B. bigemina at 31.7% (45/142) and dual infections at 14.1% (20/142) (Figure 1). Species distribution did not differ significantly between treatment groups (χ²=0.83, p=0.66). Mean baseline PCV was 16.3±0.9% in the DA group and 15.9±1.1% in the ID group (p=0.41), confirming comparable disease severity at enrolment. Parasitaemia levels at presentation averaged 4.7±2.3% and 5.1±2.6% for DA and ID groups, respectively. [4] .

Results

Comprehensive Interpretation

The PCV recovery curves (Figure 3) demonstrate that IDtreated animals crossed the 24% normal threshold by day 7, whereas DA-treated animals reached this milestone closer to day 14. The steeper early rise in the ID group points to more rapid cessation of erythrocyte destruction, allowing regenerative erythropoiesis to outpace ongoing haemolysis sooner. By day 28, the inter-group difference had narrowed but remained statistically significant, suggesting that ID confers a lasting haematological advantage. Adverse events were mild in both arms: injection-site swelling (11.3%) in DA and transient salivation (8.5%) in ID, none requiring treatment withdrawal.

Discussion

The main findings of this trial can be summarised in three points: (a) both diminazene aceturate and imidocarb dipropionate achieved clinical cure in over 95% of naturally infected cattle, (b) ID cleared parasitaemia roughly two days faster and restored PCV to normal levels earlier than DA, and (c) case fatality was low in both groups (4.2% DA vs. 1.4% ID) and confined almost entirely to severely anaemic B. bovis cases presenting late in the disease course.

Several earlier reports support the superiority of ID in haematological recovery speed. A Nigerian field trial comparing the two drugs in B. bigemina-infected Bunaji cattle recorded smear-negative status 1.8 days sooner in the ID arm [11] , a margin close to the 2.1-day difference observed here. Likewise, a South African multi-centre trial found that PCV at day 7 was 3.2 percentage points higher in ID-treated animals than in DA-treated counterparts [12] . The concordance between these geographically diverse datasets strengthens confidence that ID's pharmacokinetic advantage is reproducible across field settings. Not all published evidence, however, aligns with the present findings. An Iranian randomised trial of 96 cattle detected no significant PCV difference between DA and ID at any time point through day 21, although that trial used a lower ID dose (1.0 mg/kg) and did not administer the recommended second injection at day 14 [13] . Similarly, the 2019 meta-analysis by Adjou Moumouni et al. concluded that overall cure rates were equivalent, albeit with high between-trial heterogeneity driven partly by dose variation and inconsistent species confirmation [7] . Such discrepancies may reflect genuine dose-response effects; the full two-dose ID regimen used here likely accounts for its stronger performance relative to single-dose protocols. A plausible pharmacological explanation for ID's faster parasitaemia clearance lies in its biphasic distribution kinetics.

After subcutaneous injection, ID accumulates in the liver and spleen organs with heavy erythrocyte sequestration during babesiosis reaching tissue concentrations that exceed minimum inhibitory levels for 21-28 days [4] . DA, in contrast, peaks within 1-2 hours and declines to sub-therapeutic levels by 48 hours, relying on a single burst of parasiticidal activity [3] . This pharmacokinetic profile means DA must kill a larger fraction of parasites in a narrow window, whereas ID sustains suppressive pressure over weeks, allowing the host immune response to mop up residual organisms. From a broader perspective, treatment choice in the field should balance efficacy with practical logistics and cost. DA's single-dose regimen is simpler, cheaper, and better suited to pastoral systems where a second veterinary visit may not be feasible [14] . ID, while more expensive and requiring two administrations, offers the added benefit of chemoprophylaxis during the remaining tick season. In Mediterranean Turkey, where tick exposure persists from April through October, this prophylactic window may prevent re-infection and reduce the need for repeat treatments. Integrating drug selection with acaricidal tick control and serological monitoring could form the backbone of a cost-effective babesiosis management strategy for the region [15] .

Clinical Implications

Three clinical recommendations emerge from this trial. First, for animals presenting with severe anaemia (PCV <15%) caused by B. bovis, the faster haematological recovery offered by ID justifies its higher cost, particularly when combined with intravenous fluid support and iron supplementation. Second, in herd-level outbreaks occurring early in the tick season, the prophylactic tail of ID can protect treated cattle through peak transmission months, potentially reducing overall treatment episodes per animal. Third, when only a single farm visit is possible common in remote grazing areas DA remains a rational first-line choice, achieving cure rates above 92% with a single injection. Practitioners should also consider species-level diagnosis, since B. bigemina infections responded uniformly well to both agents, whereas B. bovis and mixed infections showed a measurable advantage with ID.

Conclusion

The main contribution of this trial is a controlled, side-by-side comparison of the two leading anti-babesial drugs under natural infection conditions in a Mediterranean climatic setting that had been absent from the existing evidence base. Imidocarb dipropionate achieved parasitaemia clearance approximately two days sooner and restored PCV to normal levels by day 7 in a significantly greater proportion of animals compared with diminazene aceturate. Both drugs were well tolerated, with mild, self-limiting adverse reactions that did not require treatment discontinuation. Supporting these primary endpoints, species-stratified analysis showed that the ID advantage was most pronounced against B. bovis the species causing the more severe disease phenotype and in mixed infections. Case fatality rates were low overall but clustered among DA-treated, severely anaemic B. bovis cases in highly susceptible Holstein-Friesian cattle. The PCV recovery trajectories confirmed that haematological restoration under ID follows a steeper early curve, consistent with its sustained tissue-level concentrations in the spleen and liver. For field practitioners in Turkey and comparable Mediterranean regions, these findings reinforce ID as the preferred option when the clinical scenario permits two administration visits and when the cost differential is acceptable. DA remains a viable single-dose alternative for routine cases and resource-limited settings. Integrating either drug into a broader tick management programme that combines acaricide application, pasture rotation, and serological herd monitoring would maximise impact.

Several questions remain open. Whether drug-resistant Babesia strains are emerging in Anatolian cattle herds has not been investigated; reduced susceptibility to DA has been reported in West Africa [16] and warrants surveillance in Turkey. The optimal timing and dose for the second ID injection also deserve refinement a shorter 10-day interval might accelerate cure without increasing toxicity. And the cost-effectiveness of the two drugs when viewed over an entire tick season, rather than a single disease episode, needs prospective economic modelling that accounts for ID's prophylactic benefit. Answering these questions will require multi-season, multi-province trials with larger sample sizes than the present work could accommodate.

Acknowledgement

Acknowledgements Funding SourcesThis trial was supported by the Ankara Metropolitan University Internal Research Fund (Grant No. AMU-VET-2022-07) allocated for tick-borne disease research in Anatolian livestock.

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