Efficacy of an oral combination of afoxolaner and milbemycin oxime for the prevention of transmission of Babesia canis by Dermacentor reticulatus ticks to dogs

Efficacy of an oral combination of afoxolaner and milbemycin oxime for the prevention of transmission of Babesia canis by Dermacentor reticulatus ticks to dogs | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Efficacy of an oral combination of afoxolaner and milbemycin oxime for the prevention of transmission of Babesia canis by Dermacentor reticulatus ticks to dogs Eric Tielemans, Carin Rautenbach, Alta Viljoen, Frederic Beugnet This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5796842/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 15 Apr, 2025 Read the published version in Parasites & Vectors → Version 1 posted 6 You are reading this latest preprint version Abstract Background: Canine babesiosis is a tick-borne disease of significant veterinary importance in dogs. It is caused by Babesia canis in Europe, where it is transmitted by Dermacentor reticulatus ticks. Methods : A blinded, randomized, Good Clinical Practice (GCP) and negative control experimental study was conducted to verify the efficacy of NexGard Spectra ® in reducing the transmission of B. canis by D. reticulatus to dogs. NexGard Spectra ® (IVP) is an oral product for dogs combining afoxolaner an acaricide/insecticide compound from the isoxazoline class and milbemycin oxime a nematicide compound from the macrocyclic lactone class. Three groups of eight dogs were used, one group orally treated on Day 0 with the IVP at the minimum recommended dose and two untreated control groups. On Day 1, dogs from the treated group and from the control group 1 were infested with 50 D. reticulatus adult ticks of 50/50 sex ratio infected with B. canis at a 23% infection rate. On Day 28, dogs from the treated group and from the control group 2 were infested similarly to those on Day 1. Ticks were removed 6 days after each infestation. Results: Seven to nine days after each infestation, all untreated control dogs displayed clinical signs of canine babesiosis, i.e. lethargy, and/or dark urine, and/or >39.5°C rectal temperature. Blood was collected for microscopical blood smear examination, and for PCR analysis. The blood smears from all untreated control dogs were positive for Babesia and all the PCR analyses were positive for B. canis. The control dogs were rescue treated. All control dogs were confirmed positive for B. canis by IFA on Day 21 (control group 1) and on Day 42 (control group 2). None of the IVP-treated dogs expressed any clinical sign of canine babesiosis following each of the two infestations of Days 1 and 28 and until Day 56. Blood was collected for IFA and PCR analyses from the treated dogs on Days 21, 28, 42 and 56, and all results were negative. Conclusion: In this study, the antiparasitic treatment prevented the transmission of B. canis to dogs following induced infestations. Afoxolaner Dog Babesia canis Dermacentor reticulatus Prevention of transmission Background Blood parasites of the genus Babesia are mainly transmitted by ixodid ticks (Family Ixodidae) to a broad range of mammalian species, including domestic and wild animals, as well as humans [1-5]. Babesia spp. have a significant medical and economic importance and are considered the second most common blood parasite of mammals after trypanosomes [6]. Babesia species occur worldwide, where most species appear to be geographically constrained by the distribution of their reservoir hosts and vectors [7]. The pathological effect of Babesia spp. in the vertebrate host is caused by the invasion and destruction of erythrocytes in which sporozoites undergo asexual reproduction by binary fission, resulting in a replicative cycle with multiplied infective merozoites. Consequently, Babesia infections commonly cause systemic inflammatory reactions and hemolysis-related symptoms such as febrile syndrome, anemia, hemoglobinuria, jaundice, lethargy, and organ failure in the longer term. The severity of babesiosis can vary in intensity, depending on the host’s inherent conditions (genetic factors, age, concurrent infection with other pathogens, immunological status) and the infecting Babesia species [8-10]. Canine babesiosis is a disease of major veterinary importance, affecting dogs worldwide. Several identified canine Babesia species are variable in term of geographical distribution, vector, severity, clinical signs, and treatment sensitivity [8,9,11]. Large form species, e.g. B. canis, B. vogeli ( and B. rossi as imported cases) and small form species, e.g. B. vulpes, B. gibsoni, are reported in Europe [12]. Babesia canis is described across most of Europe, especially in wet and temperate to cold central regions, where Dermacentor reticulatus , its recognized vector, thrives [13]. Canine babesiosis caused by B. canis is usually characterized by an acute onset. It causes moderate to severe clinical symptoms, often associated with hemolytic anemia and thrombocytopenia, sometimes with poor prognosis [9,10]. NexGard Spectra ® , is an endectoparasiticide oral product for dogs, combining afoxolaner and milbemycin oxime. Afoxolaner is an insecticide and acaricide of the isoxazoline class. Isoxazolines antagonize the GABA-gated chloride ion channels of the hematophagous arthropods that fed on the treated host. Milbemycin oxime, the other active ingredient of the product, is a nematicide of the macrocyclic lactone class, with negligible efficacy on arthropods (unpublished data). The effectiveness of afoxolaner for the prevention of B. canis experimentally transmitted by Dermacentor reticulatus was already demonstrated in an experimental study with oral afoxolaner administered in a mono-product (NexGard ® ), at label dose, and in comparison to a single untreated control group [14]. This manuscript describes a new study, designed according to the most recent relevant guideline for studies evaluating the efficacy of parasiticides in reducing the risk of vector-borne pathogen transmission [15], namely requiring treatment at the minimum recommended dose, infective challenges repeated at the beginning and at the end of efficacy claim duration, and with a distinct control group for these two challenges. Methods Ethics Animals were managed with due regard for their wellbeing, and the Institutional Animal Care and Use Committee reviewed and approved the study design. Study design This study, conducted in 2024, was designed in accordance with Good Clinical Practices as described in International Cooperation on Harmonization of Technical Requirements for Registration of Veterinary Medicinal Products (VICH) guideline GL9, and was designed in accordance with the “World Association for the Advancement of Veterinary Parasitology (W.A.A.V.P.) guidelines for studies evaluating the efficacy of parasiticides in reducing the risk of vector-borne pathogen transmission in dogs and cats [15]. This study was conducted under a blinded and randomized design. The efficacy assessment was based on a comparison of Babesia infection level in a treated group and untreated control groups following infestations with D. reticulatus infected with B. canis , one day and twenty-eight days after treatment. The study outline and most animal characteristics per study group are illustrated in Table 1. Animals and husbandry Twenty-four healthy purpose-bred adult dogs (fourteen Beagles, four beagle-crosses and six mongrels), 11 males and 13 females, aged 19 to 88 months, and weighing 12.1 to 23.8 kg were included in the study. The containment of the dogs complied with the South African National Standard SANS 10386: latest version “The care and use of animals for scientific purposes”. The animals were kept individually in cages and had visual and auditory contact with conspecifics. Dogs were socialized weekly. The cages had concrete floors to facilitate cleaning and each cage was fitted with a sleeping bench. At least one toy/chew was made available to each dog and was replaced weekly. The dog cages were part of an environmentally controlled indoor animal unit; temperatures ranged between 16°C to 25°C. A photoperiod of 12 hours light - 12 hours darkness was maintained. The floor size of each dog cage was 3.0 m x 1.9 m. No substances with an insecticidal or acaricidal activity were used on the animals or in their environment. Standard commercially available diets (VetsBrands Premium adult maintenance dog food or Hill’s Adult Large Breed Dog Food) that met the nutritional requirements of the animals were provided at the recommended rates. Any dog diagnosed positive for Babesia was immediately rescue treated and provided Hill’s a/d for 3 days. Potable water, as supplied by the local municipality, was provided in stainless steel bowls and replenished at least twice daily. Dermacentor reticulatus and B. canis isolates The D. reticulatus ticks used in this study were originally obtained from BLE Laboratories (Ireland) in 2007. They had been genetically enriched with D. reticulatus, obtained from the Utrecht Centre for Tick-borne Diseases (UCTD), Netherlands, in 2009, 2012, 2014, and 2017. The B. canis parasites used in this study originated from the Netherlands. A laboratory-bred dog was inoculated with blood derived from a clinical case of canine babesiosis, and blood from this dog served as infectious material for the ticks used in this study. A batch of D. reticulatus nymphs were fed on the infected dog. The adult ticks obtained from the engorged nymphs were infected at a 34% rate, as determined by Polymerase Chain Reaction (PCR) on a sample of 50 ticks. Pre-treatment activities Thirty dogs were acclimated to study conditions since Day -7. A clinical examination was performed on all dogs on Day -7. To evaluate their suitability for tick infestation, all dogs were infested on Day -7, following the method described in the ‘tick infestation and removals’ paragraph, with the exceptions that the D. reticulatus were uninfected and were removed and counted two days after infestation, on Day -5. On Day -5, blood was collected from all dogs for Immuno Fluorescent Assay (IFA) and PCR analyses to ensure they were not positive for B. canis before inclusion. The 24 dogs with the highest live attached tick count and that were suitable for the study, as determined by the Investigator, were selected for inclusion. Eight blocks with three dogs each were defined based on live attached tick numbers, allowing a randomization to 3 groups of 8 dogs each, one treated group and two control groups. The numbers of live attached ticks in the included dogs ranged from 13 to 35, the average numbers of ticks were 22.9, 23.0 and 22.9, in the control groups 1, 2 and the IVP-treated group, respectively. Treatment On Day 0, dogs from the treated group were orally administered a combination of whole tablets defined according to their bodyweight and to target the minimum label dose of the product, i.e. 2.5 mg/kg afoxolaner and 0.5 mg/kg milbemycin oxime, as described in Table 2. The mean administered afoxolaner dose was 2.75 mg/kg. Tick infestations and removals The treated group was infested on Days 1 and 28. Two control groups were used to verify the adequacy of each B. canis transmission and were thus infested on Day 1 (control group 1) or Day 28 (control group 2). Each time, dogs were infested with 50 (± 2) adult ticks of 50/50 sex ratio. To obtain 50 ticks with a consistent B. canis infection rate for each individual infestation, 34 ticks from the infected batch (17 males and 17 females) with a 34% infection rate as determined by PCR, were combined with 16 ticks (8 males and 8 females) from the same origin, but that had not been exposed to B. canis infected blood. Therefore, the B. canis infection rate for each batch of 50 ticks used for infestations was 23%. To facilitate tick infestation, dogs were sedated and placed in an infestation crate for one to two hours. The ticks were released on the back or flank of the animal and were allowed to disperse into the hair coat. Six days after each infestation, on Days 7 and 34, the remaining ticks were removed and counted from each infested group (the treated group and control group 1 on Day 7, and the treated group and control group 2 on Day 34). Health and Babesia infection evaluations General health observations were performed once daily from Day -7 to Day 6 and twice daily from Day 7 to Day 56. Clinical examinations were performed weekly from Day 7 to Day 56. Rectal temperature was measured once daily from Day 1 to Day 56. Whenever the rectal temperature exceeded 39.5°C and/or babesiosis was suspected based on clinical signs (e.g. lethargy, dark urine, diarrhea, anorexia), blood was collected for blood smear processing and microscopic search of Babesia in red blood cells, and for B. canis PCR analysis, recognized methods for babesia diagnosis [16]. Irrespective of clinical signs, blood collection was scheduled for B. canis IFA and PCR analyses on Days 21 and 28 (treated group and control group 1), and on Days 42 and 56 (all dogs). For B. canis PCR analysis, total genomic DNA was isolated from whole blood samples using a commercial genomic DNA isolation kit (GeneJET Genomic DNA Purification Kit, Thermo Fisher Scientific, USA). PCR entailed using primers specific to the 18S Ribosomal DNA region of B. canis , and as referenced [17]. The PCR procedure followed a recognized method [18]. For IFA analysis, serum was collected from blood tubes and was assayed for B. canis antibodies using a commercially available IFA test kit (MegaFLUO ® BABESIA canis, Megacor Diagnostik, Austria). Statistical analysis The primary efficacy criterion was the number of dogs that became infected with B. canis (when tested positive for B. canis by both PCR analysis and IFA serology). The infection rate of dogs in groups that became infected during the study, were summarized descriptively. The percentage blocking efficacy for the IVP-treated group was calculated as follows in relation to the Day 1 and Day 28 infestations: Blocking efficacy (%) = 100 x (Tc - Tt)/Tc Tc = Total number of untreated control dogs that became infected (control group 1 for Day 1, control group 2 for Day 28) Tt = Total number of dogs that became infected in the IVP group (group 3) SAS Version 9.4 was used for all the statistical analyses. Endpoints Whenever a dog was diagnosed positive for Babesia infection on a blood smear, blood for PCR analysis was collected, and the dog was rescue treated. All study activities for rescue treated dogs were stopped except the scheduled blood collections for IFA and PCR analyses. A rescue-treated dog was removed from the study once a positive IFA result was obtained. Results The individual results of tick counts and Babesia infection diagnosis are described in Table 3. The tick infestations on Days 1 and 28 were demonstrated adequate by their corresponding control groups 1 and 2, as 6 days later, on Days 7 and 34 respectively, a significant number of live attached ticks were found. The mean number of live attached ticks was 36 (72%) in the untreated control group 1 and 30.4 (60.8%) in the untreated control group 2. No live attached tick was found on any IVP treated dog on Days 7 and 34, demonstrating a reduction of 100% of live attached ticks, 6 days after infestation on Days 1 and 28. A small number of dead attached ticks, ranging from 0 to 5, were found in this group. A clear diagnosis of canine babesiosis was made for all 16 untreated control dogs, 7 to 9 days following their respective D. reticulatus infestation. All dogs expressed abnormal clinical signs, i.e. lethargy, and/or dark urine, and/or rectal temperature exceeding 39.5°C. On the same day, the diagnosis of canine babesiosis was confirmed for each dog by a positive blood smear microscopical examination and B. canis PCR analysis. These 16 dogs were rescue treated on the day of diagnosis with injections of imidocarb dipropionate (7.2 mg/kg), diminazene aceturate (3.5 mg/kg) and several days of supportive medications, i.e. corticosteroid (prednisolone acetate 0.1 mg/kg), antiemetic (marbopitant 1 mg/kg), and hepatoprotectors. All dogs were confirmed positive by IFA analysis for B. canis on Day 21 (control group 1) or Day 42 (control group 2). The PCR analyses of these dogs on Days 21 and 42 had turned negative, demonstrating the efficacy of rescue treatments. None of the IVP-treated dogs expressed any abnormal clinical signs of babesiosis, and their PCR and IFA bioanalyses performed on Days 21, 28, 42 and 56 remained negative. Discussion The data obtained in this study with the untreated animals demonstrated a reliable and consistent B. canis infection model and showed with the treated animals that afoxolaner completely blocked the B. canis transmission by D. reticulatus ticks. Dermacentor reticulatus is a vector of numerous other protozoal, bacterial, and viral diseases [19], out of scope in this study, as the investigated model was limited to pathogen-free ticks experimentally infected only with B. canis. Canine babesiosis is a highly significant and challenging disease. Several therapeutic or preventive veterinary strategies can be implemented alone or in combination. The protozoan can be directly targeted through pharmaceutical products or vaccinations. As described in this manuscript, the ixodid vector can be targeted through acaricidal products. The use of repellents can also reduce tick infestations. Pharmaceutical treatment of dogs affected with canine babesiosis is challenging. A limited number of drugs used alone or in combination are available on- or off-label, with variable species and strain-related efficacy [12,20,21]. The mechanism of action of these drugs is mostly unclear. Injectable imidocarb dipropionate is registered for treating B. canis, nevertheless with cholinergic adverse effects and pain at injection. Intensive care is also necessary for severe manifestations of the disease, and relapses are possible. In this study, diminazene aceturate, a compound of poorly understood mechanism of action [22] and registered in South Africa for the treatment of B. canis in dogs, was used as treatment in combination with imidocarb dipropionate and with corticosteroid, hepato-protector and antiemetic supportive medications. Some vaccines containing soluble parasite antigens derived from in vitro cultures are registered against B. canis but with variable strain-related efficacy [23]. Babesia vaccines do not affect the infection but alter the disease progression and reduce the severity of the clinical signs. Research on recombinant antigens is currently underway and may provide new vaccines in the future [24,25]. Repellent and acaricide products, such as permethrin and flumethrin, have been described as effective for the reduction of B. canis transmission [26-28] and, more generally, for the prevention of several vector-borne diseases, including Babesia spp. [29]. The approach of blocking a pathogen transmission by using an acaricide targeting the vector has become an important veterinary strategy since the appearance of isoxazolines in 2014 [14,30-34]. Contrarily to repellent products, isoxazolines do not prevent an arthropod vector from biting or attaching to its host and start a fluid exchange process. Nevertheless, Babesia spp . usually require several days after host attachment for the ookinetes to undergo several sporogony cycles, leading to the formation of infective sporozoites in the salivary gland of their ixodid vector [2,13]. As examples, it was demonstrated in humans that B.microti was transmitted by Ixodes scapularis 36 to 48h after host attachment [35], or in cattle that B. bigemina was transmitted during the last 16 to 24h before vector ( Rhipicephalus microplus ) detachment [36]. Interestingly, in the present study, some dead attached D. reticulatus were found on the treated dogs but probably did not survive long enough to transmit their infective B. canis sporozoites . Afoxolaner, alone or combined with milbemycin oxime was demonstrated effective against D. reticulatus within 48 hours of infestations and for 5 weeks after a treatment at minimum dose [37]. Afoxolaner was also effective to prevent B. canis transmission by D. reticulatus , when administered at label dose in a mono-product (NexGard ® ) and with one control group verifying validity of weekly challenges [14]. The study described in this manuscript provides an upgraded design. It verified the efficacy of a single afoxolaner treatment administered at the minimum dose of its registered products and with a distinct control group verifying adequacy of infection after each challenge at the beginning (Day 1) and end (Day 28) of the efficacy claimed period. The 100% level of B. canis infection observed in both untreated control groups after challenges at Day 1 or at Day 28 supported the robustness of the model. The total absence of infection after the two infective tick challenges in the treated group supported the preventive efficacy of afoxolaner administered once and at minimum dose of its registered products. Conclusion In this study, the killing effect of the investigated oral formulation combining afoxolaner and milbemycin oxime was fast and complete enough to interrupt the vector-host fluid exchange process, thus blocking tick capability to transmit the infectious Babesia sporozoites. Declarations Animal Ethics and Consent to Participate declarations: Not applicable. Funding Not applicable References Akel T, Mobarakai N. Hematologic manifestations of babesiosis. Ann Clin Microbiol Antimicrob. 2017 Feb 15;16(1):6. doi: 10.1186/s12941-017-0179-z. PMID: 28202022; PMCID: PMC5310009. Hunfeld KP, Hildebrandt A, Gray JS. Babesiosis: recent insights into an ancient disease. Int J Parasitol. 2008 Sep;38(11):1219-37. doi: 10.1016/j.ijpara.2008.03.001. Epub 2008 Mar 20. PMID: 18440005. Malgwi SA, Ogunsakin RE, Oladepo AD, Adeleke MA, Okpeku M. A Forty-Year Analysis of the Literature on Babesia Infection (1982-2022): A Systematic Bibliometric Approach. Int J Environ Res Public Health. 2023 Jun 16;20(12):6156. doi: 10.3390/ijerph20126156. PMID: 37372744; PMCID: PMC10298011. Uilenberg G. Babesia --a historical overview. Vet Parasitol. 2006 May 31;138(1-2):3-10. doi: 10.1016/j.vetpar.2006.01.035. Epub 2006 Feb 28. PMID: 16513280. Vannier EG, Diuk-Wasser MA, Ben Mamoun C, Krause PJ. Babesiosis. Infect Dis Clin North Am. 2015 Jun;29(2):357-70. doi: 10.1016/j.idc.2015.02.008. PMID: 25999229; PMCID: PMC4458703. Schnittger L, Rodriguez AE, Florin-Christensen M, Morrison DA. Babesia: a world emerging. Infect Genet Evol. 2012 Dec;12(8):1788-809. doi: 10.1016/j.meegid.2012.07.004. Epub 2012 Jul 31. PMID: 22871652. Dear JD, Birkenheuer A. Babesia in North America: An Update. Vet Clin North Am Small Anim Pract. 2022 Nov;52(6):1193-1209. doi: 10.1016/j.cvsm.2022.07.016. PMID: 36336417. Leisewitz AL, Mrljak V, Dear JD, Birkenheuer A. The Diverse Pathogenicity of Various Babesia Parasite Species That Infect Dogs. Pathogens. 2023 Dec 11;12(12):1437. doi: 10.3390/pathogens12121437. PMID: 38133320; PMCID: PMC10746086. Solano-Gallego L, Sainz Á, Roura X, Estrada-Peña A, Miró G. A review of canine babesiosis: the European perspective. Parasit Vectors. 2016 Jun 11;9(1):336. doi: 10.1186/s13071-016-1596-0. PMID: 27289223; PMCID: PMC4902949. Eichenberger RM, Riond B, Willi B, Hofmann-Lehmann R, Deplazes P. Prognostic Markers in Acute Babesia canis Infections. J Vet Intern Med. 2016 Jan-Feb;30(1):174-82. doi: 10.1111/jvim.13822. Epub 2016 Jan 4. PMID: 26727465; PMCID: PMC4913656. Malgwi SA, Ogunsakin RE, Oladepo AD, Adeleke MA, Okpeku M. A Forty-Year Analysis of the Literature on Babesia Infection (1982-2022): A Systematic Bibliometric Approach. Int J Environ Res Public Health. 2023 Jun 16;20(12):6156. doi: 10.3390/ijerph20126156. PMID: 37372744; PMCID: PMC10298011. Baneth G. Antiprotozoal treatment of canine babesiosis. Vet Parasitol. 2018 Apr 30;254:58-63. doi: 10.1016/j.vetpar.2018.03.001. Epub 2018 Mar 7. PMID: 29657012. Beugnet F, Moreau Y. Babesiosis. Rev Sci Tech. 2015 Aug;34(2):627-39. doi: 10.20506/rst.34.2.2385. PMID: 26601462. Beugnet F, Halos L, Larsen D, Labuschagné M, Erasmus H, Fourie J. The ability of an oral formulation of afoxolaner to block the transmission of Babesia canis by Dermacentor reticulatus ticks to dogs. Parasit Vectors. 2014 Jun 23;7:283. doi: 10.1186/1756-3305-7-283. PMID: 24957215; PMCID: PMC4078974. Otranto D, Dantas-Torres F, Fourie JJ, Lorusso V, Varloud M, Gradoni L, Drake J, Geurden T, Kaminsky R, Heckeroth AR, Schunack B, Pollmeier M, Beugnet F, Holdsworth P. World Association for the Advancement of Veterinary Parasitology (W.A.A.V.P.) guidelines for studies evaluating the efficacy of parasiticides in reducing the risk of vector-borne pathogen transmission in dogs and cats. Vet Parasitol. 2021 Feb;290:109369. doi: 10.1016/j.vetpar.2021.109369. Epub 2021 Jan 24. PMID: 33548595. Lempereur L, Beck R, Fonseca I, Marques C, Duarte A, Santos M, Zúquete S, Gomes J, Walder G, Domingos A, Antunes S, Baneth G, Silaghi C, Holman P, Zintl A. Guidelines for the Detection of Babesia and Theileria Parasites. Vector Borne Zoonotic Dis. 2017 Jan;17(1):51-65. doi: 10.1089/vbz.2016.1955. PMID: 28055573. Troskie M, de Villiers L, Leisewitz A, Oosthuizen MC, Quan M. Development and validation of a multiplex, real-time PCR assay for Babesia rossi and Babesia vogeli . Ticks Tick Borne Dis. 2019 Feb;10(2):421-432. doi: 10.1016/j.ttbdis.2018.12.004. Epub 2018 Dec 14. PMID: 30591405. Heylen D, Day M, Schunack B, Fourie J, Labuschange M, Johnson S, Githigia SM, Akande FA, Nzalawahe JS, Tayebwa DS, Aschenborn O, Marcondes M, Madder M. A community approach of pathogens and their arthropod vectors (ticks and fleas) in dogs of African Sub-Sahara. Parasit Vectors. 2021 Nov 16;14(1):576. doi: 10.1186/s13071-021-05014-8. PMID: 34784947; PMCID: PMC8594167. Földvári G, Široký P, Szekeres S, Majoros G, Sprong H. Dermacentor reticulatus : a vector on the rise. Parasit Vectors. 2016 Jun 1;9(1):314. doi: 10.1186/s13071-016-1599-x. PMID: 27251148; PMCID: PMC4888597. Ji S, Galon EM, Rizk MA, Yi Y, Zafar I, Li H, Ma Z, Iguchi A, Asada M, Liu M, Xuan X. Efficacy of the Antimalarial MMV390048 against Babesia Infection Reveals Phosphatidylinositol 4-Kinase as a Druggable Target for Babesiosis. Antimicrob Agents Chemother. 2022 Sep 20;66(9):e0057422. doi: 10.1128/aac.00574-22. Epub 2022 Aug 4. PMID: 35924942; PMCID: PMC9487540. Weingart C, Krücken J, Kohn B. Repeated imidocarb treatment failure suggesting emerging resistance of Babesia canis in a new endemic area in north-eastern Germany. Ticks Tick Borne Dis. 2024 May;15(3):102315. doi: 10.1016/j.ttbdis.2024.102315. Epub 2024 Jan 31. PMID: 38301344. Kuriakose S, Uzonna JE. Diminazene aceturate (Berenil), a new use for an old compound? Int Immunopharmacol. 2014 Aug;21(2):342-5. doi: 10.1016/j.intimp.2014.05.027. Epub 2014 Jun 2. PMID: 24893117. Schetters TH, Kleuskens J, Scholtes N, Bos HJ. Strain variation limits protective activity of vaccines based on soluble Babesia canis antigens. Parasite Immunol. 1995 Apr;17(4):215-8. doi: 10.1111/j.1365-3024.1995.tb00891.x. PMID: 7542765. Moubri K, Kleuskens J, Van de Crommert J, Scholtes N, Van Kasteren T, Delbecq S, Carcy B, Précigout E, Gorenflot A, Schetters T. Discovery of a recombinant Babesia canis supernatant antigen that protects dogs against virulent challenge infection. Vet Parasitol. 2018 Jan 15;249:21-29. doi: 10.1016/j.vetpar.2017.11.002. Epub 2017 Nov 6. PMID: 29279083. Nathaly Wieser S, Schnittger L, Florin-Christensen M, Delbecq S, Schetters T. Vaccination against babesiosis using recombinant GPI-anchored proteins. Int J Parasitol. 2019 Feb;49(2):175-181. doi: 10.1016/j.ijpara.2018.12.002. Epub 2019 Jan 24. PMID: 30684517 Fourie JJ, de Vos C, Crafford D, Pollmeier M, Schunack B. A study on the long-term efficacy of Seresto® collars in preventing Babesia canis (Piana & Galli-Valerio, 1895) transmission to dogs by infected Dermacentor reticulatus (Fabricius, 1794) ticks. Parasit Vectors. 2019 Mar 22;12(1):139. doi: 10.1186/s13071-019-3393-z. PMID: 30902104; PMCID: PMC6431040. Jongejan F, de Vos C, Fourie JJ, Beugnet F. A novel combination of fipronil and permethrin (Frontline Tri-Act ® /Frontect ® ) reduces risk of transmission of Babesia canis by Dermacentor reticulatus and of Ehrlichia canis by Rhipicephalus sanguineus ticks to dogs. Parasit Vectors. 2015 Nov 19;8:602. doi: 10.1186/s13071-015-1207-5. PMID: 26586365; PMCID: PMC4653891. Navarro C, Reymond N, Fourie J, Hellmann K, Bonneau S. Prevention of Babesia canis in dogs: efficacy of a fixed combination of permethrin and fipronil (Effitix ® ) using an experimental transmission blocking model with infected Dermacentor reticulatus ticks. Parasit Vectors. 2015 Jan 17;8:32. doi: 10.1186/s13071-015-0645-4. PMID: 25595325; PMCID: PMC4302435. Otranto D, de Caprariis D, Lia RP, Tarallo V, Lorusso V, Testini G, Dantas-Torres F, Latrofa S, Diniz PP, Mencke N, Maggi RG, Breitschwerdt E, Capelli G, Stanneck D. Prevention of endemic canine vector-borne diseases using imidacloprid 10% and permethrin 50% in young dogs: a longitudinal field study. Vet Parasitol. 2010 Sep 20;172(3-4):323-32. doi: 10.1016/j.vetpar.2010.05.017. Epub 2010 May 25. PMID: 20591573. Baker CF, McCall JW, McCall SD, Drag MD, Mitchell EB, Chester ST, Larsen D. Ability of an oral formulation of afoxolaner to protect dogs from Borrelia burgdorferi infection transmitted by wild Ixodes scapularis ticks. Comp Immunol Microbiol Infect Dis. 2016 Dec;49:65-69. doi: 10.1016/j.cimid.2016.09.004. Epub 2016 Sep 29. PMID: 27865266. Cavalleri D, Murphy M, Seewald W, Drake J, Nanchen S. Two randomized, controlled studies to assess the efficacy and safety of lotilaner (Credelio™) in preventing Dermacentor reticulatus transmission of Babesia canis to dogs. Parasit Vectors. 2017 Nov 1;10(1):520. doi: 10.1186/s13071-017-2473-1. PMID: 29089056; PMCID: PMC5664908. Geurden T, Six R, Becskei C, Maeder S, Lloyd A, Mahabir S, Fourie J, Liebenberg J. Evaluation of the efficacy of sarolaner (Simparica ® ) in the prevention of babesiosis in dogs. Parasit Vectors. 2017 Sep 6;10(1):415. doi: 10.1186/s13071-017-2358-3. PMID: 28877743; PMCID: PMC5588728. Otranto D, Mendoza-Roldan JA, Beugnet F, Baneth G, Dantas-Torres F. New paradigms in the prevention of canine vector-borne diseases. Trends Parasitol. 2024 Jun;40(6):500-510. doi: 10.1016/j.pt.2024.04.009. Epub 2024 May 13. PMID: 38744542. Taenzler J, Liebenberg J, Roepke RK, Heckeroth AR. Prevention of transmission of Babesia canis by Dermacentor reticulatus ticks to dogs treated orally with fluralaner chewable tablets (Bravecto™). Parasit Vectors. 2015 Jun 4;8:305. doi: 10.1186/s13071-015-0923-1. PMID: 26040319; PMCID: PMC4465614. Piesman J, Spielman A. Human babesiosis on Nantucket Island: prevalence of Babesia microti in ticks. Am J Trop Med Hyg. 1980 Sep;29(5):742-6. doi: 10.4269/ajtmh.1980.29.742. PMID: 7435782. Hodgson JL. Biology and transmission of Babesia bigemina in Boophilus microplus . Ann N Y Acad Sci. 1992 Jun 16;653:42-51. doi: 10.1111/j.1749-6632.1992.tb19628.x. PMID: 1626891. Rehbein S, Fourie JJ, de Vos C, Anderson A, Larsen DL, Jeannin P. Efficacy of oral afoxolaner plus milbemycin oxime chewables against induced infestations with Dermacentor reticulatus in dogs. Parasitol Res. 2016 May;115(5):1845-51. doi: 10.1007/s00436-016-4924-6. Epub 2016 Jan 27. PMID: 26815036. Tables Table 1: Animal characteristics and study outline Groups Animal characteristics at inclusion (on Day -2) IVP treatment D. reticulatus Daily rectal temperatures 1 PCR and IFA analyses 2 n sex age (months) Bodyweight (kg) Infestation(s) Removal(s) Control 1 8 4 m, 4 f 19 - 88 12.1 - 20.0 NA Day 1 Day 7 Days 1 to 56 Days 21 and 28 Control 2 8 3 m, 5 f 31 - 88 12.9 - 23.4 NA Day 28 Day 34 Days 1 to 56 Days 42 and 56 IVP 8 4 m, 4 f 28 - 82 14.2 - 23.8 Day 0 Days 1 and 28 Days 7 and 34 Days 1 to 56 Days 21, 28, 42 and 26 Animals were examined for clinical signs once a day until Day 6 and twice daily from Day 7 to Day 56. 1 whenever the rectal temperature exceeded 39.5°C, and/or babesiosis was suspected on the basis of clinical signs, blood was collected for blood smear microscopical examination and PCR analysis. A dog with a positive blood smear was rescue treated. 2 A dog was removed from the study once a PCR and an IFA analysis was positive. Table 2: individual treatment on Day 0 Dog Tablet combination* Dose (mg) Dosage (mg/kg) # Bodyweight (kg) Afoxolaner MO Afoxolaner MO 1 15.95 9/2 + 38/8 46.875 9.375 2.94 0.59 2 21.55 19/4 + 38/8 56.250 11.250 2.61 0.52 3 18.75 19/4 + 38/8 56.250 11.250 3.00 0.60 4 18.45 9/2 + 38/8 46.875 9.375 2.54 0.51 5 19.65 19/4 + 38/8 56.250 11.250 2.86 0.57 6 14.15 38/8 37.500 7.500 2.65 0.53 7 23.80 9/2 + 19/4 + 38/8 65.625 13.125 2.76 0.55 8 14.35 38/8 37.500 7.500 2.61 0.52 Mean 2.75 0.55 *NexGard Spectra ® strengths: 9/2 = 9.375 mg afoxolaner & 1.875 mg milbemycin oxime (for dogs 2-3.5 kg) 19/4: 18.75 mg afoxolaner & 3.75 mg milbemycin oxime (for dogs >3.5-7.5 kg) 38/8: 37.5 mg afoxolaner & 7.5 mg milbemycin oxime (for dogs >7.5-15 kg) Table 3: Individual results of tick counts and Babesia diagnosis Group 1 # Live (dead) attached tick count 2 Original diagnosis of canine babesiosis Scheduled bioanalyses of Babesia canis Day of diagnosis, clinical signs of babesiosis and/or rectal temperature of >39.5°C Blood smear PCR IFA PCR Day 7 Day 34 Day 21 3 Day 28 Day 42 3 Day 56 Day 21 Day 28 Day 42 Day 56 Control 1 1 25 - Day 9: Lethargic positive positive positive - - - negative - - - Control 1 2 37 - Day 9: 39.8°C positive positive positive - - - negative - - - Control 1 3 13 - Day 9: 40.6°C positive positive positive - - - negative - - - Control 1 4 44 - Day 8: 40.2°C, Lethargic positive positive positive - - - negative - - - Control 1 5 41 - Day 8: 40.0°C, Lethargic positive positive positive - - - negative - - - Control 1 6 44 - Day 8: 39.8°C, Lethargic positive positive positive - - - negative - - - Control 1 7 42 (2) - Day 9: 40.1°C positive positive positive - - - negative - - - Control 1 8 42 - Day 9: 39.9°C, dark urine positive positive positive - - - negative - - - Control 2 1 - 27 Day 36: Lethargic positive positive - - positive - - - negative - Control 2 2 - 29 Day 37: Dark urine positive positive - - positive - - - negative - Control 2 3 - 25 Day 35: 39.7°C positive positive - - positive - - - negative - Control 2 4 - 40 Day 36: 40.2°C positive positive - - positive - - - negative - Control 2 5 - 34 Day 35: Lethargic positive positive - - positive - - - negative - Control 2 6 - 23 Day 36: Lethargic, dark urine positive positive - - positive - - - negative - Control 2 7 - 26 Day 35: 39.6°C positive positive - - positive - - - negative - Control 2 8 - 39 Day 36: 40.0°C positive positive - - positive - - - negative - IVP 1 0 0 No abnormal sign or rectal temperature - - negative negative negative negative negative negative negative negative IVP 2 0 (2) 0 (5) No abnormal sign or rectal temperature - - negative negative negative negative negative negative negative negative IVP 3 0 (3) 0 (3) No abnormal sign or rectal temperature - - negative negative negative negative negative negative negative negative IVP 4 0 (3) 0 (4) No abnormal sign or rectal temperature - - negative negative negative negative negative negative negative negative IVP 5 0 (1) 0 (2) No abnormal sign or rectal temperature - - negative negative negative negative negative negative negative negative IVP 6 0 0 (2) No abnormal sign or rectal temperature - - negative negative negative negative negative negative negative negative IVP 7 0 (5) 0 (1) No abnormal sign or rectal temperature - - negative negative negative negative negative negative negative negative IVP 8 0 (4) 0 (1) No abnormal sign or rectal temperature - - negative negative negative negative negative negative negative negative 1 Control 1 dogs were infested on Day 1 with 50 (± 2) D. reticulatus with a B. canis infection rate of 23% Control 2 dogs were infested on Day 1 with 50 (± 2) D. reticulatus with a B. canis infection rate of 23% Treated group: dogs were treated on Day 0 with NexGard Spectra® at a mean afoxolaner dose of 2.75 mg/kg and were infested on Days 1 and 28 with 50 (± 2) D. reticulatus with a B. canis infection rate of 23%. 2 this column describes the counts of live (and dead) attached ticks. Otherwise, no live free ticks were found, and 3 dead free ticks were found on Day 7 and Day 34, each time in the treated group. 3 all dogs from Control group 1 and Control group 2 were removed from the study on days 21 and 42, respectively, and no further evaluation was performed as they had been confirmed positive for B. canis by IFA analysis. Additional Declarations Competing interest reported. E.T. and F.B. are employees of Boehringer Ingelheim Animal Health, manufacturer of the investigated product (NexGard Spectra®) and sponsor of this study Cite Share Download PDF Status: Published Journal Publication published 15 Apr, 2025 Read the published version in Parasites & Vectors → Version 1 posted Editorial decision: Accepted 28 Mar, 2025 Reviews received at journal 28 Mar, 2025 Reviewers agreed at journal 28 Mar, 2025 Reviewers invited by journal 28 Mar, 2025 Submission checks completed at journal 28 Mar, 2025 First submitted to journal 28 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5796842","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":435487328,"identity":"db93e0d2-e33b-4d9c-9a46-e06bf29c435a","order_by":0,"name":"Eric Tielemans","email":"data:image/png;base64,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","orcid":"","institution":"Boehringer Ingelheim Animal Health","correspondingAuthor":true,"prefix":"","firstName":"Eric","middleName":"","lastName":"Tielemans","suffix":""},{"id":435487329,"identity":"3116b42e-8e8e-4a74-9086-e83cbb24bc62","order_by":1,"name":"Carin Rautenbach","email":"","orcid":"","institution":"Clinvet","correspondingAuthor":false,"prefix":"","firstName":"Carin","middleName":"","lastName":"Rautenbach","suffix":""},{"id":435487330,"identity":"87c96ddd-149c-49aa-a156-46c3ae1be2e5","order_by":2,"name":"Alta Viljoen","email":"","orcid":"","institution":"Clinvet","correspondingAuthor":false,"prefix":"","firstName":"Alta","middleName":"","lastName":"Viljoen","suffix":""},{"id":435487331,"identity":"03ad1fc6-9d35-4594-b1e0-9e4d2348e0a8","order_by":3,"name":"Frederic Beugnet","email":"","orcid":"","institution":"Boehringer Ingelheim Animal Health","correspondingAuthor":false,"prefix":"","firstName":"Frederic","middleName":"","lastName":"Beugnet","suffix":""}],"badges":[],"createdAt":"2025-01-09 13:23:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5796842/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5796842/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13071-025-06787-y","type":"published","date":"2025-04-15T15:57:11+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":81050935,"identity":"022fee72-5908-4427-bc7e-9277b019ae6d","added_by":"auto","created_at":"2025-04-21 16:07:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1005678,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5796842/v1/ff3a4784-37d0-498c-9603-f07c75214c15.pdf"}],"financialInterests":"Competing interest reported. E.T. and F.B. are employees of Boehringer Ingelheim Animal Health, manufacturer of the investigated product (NexGard Spectra®) and sponsor of this study","formattedTitle":"Efficacy of an oral combination of afoxolaner and milbemycin oxime for the prevention of transmission of Babesia canis by Dermacentor reticulatus ticks to dogs ","fulltext":[{"header":"Background","content":"\u003cp\u003eBlood parasites of the genus \u003cem\u003eBabesia\u003c/em\u003e are mainly transmitted by ixodid ticks (Family Ixodidae) to a broad range of mammalian species, including domestic and wild animals, as well as humans [1-5]. \u003cem\u003eBabesia\u003c/em\u003e spp. have a significant medical and economic importance and are considered the second most common blood parasite of mammals after trypanosomes [6]. \u003cem\u003eBabesia\u0026nbsp;\u003c/em\u003especies occur worldwide, where most species appear to be geographically constrained by the distribution of their reservoir hosts and vectors [7]. The pathological effect of \u003cem\u003eBabesia\u003c/em\u003e spp. in the vertebrate host is caused by the invasion and destruction of erythrocytes in which sporozoites undergo asexual reproduction by binary fission, resulting in a replicative cycle with multiplied infective merozoites. Consequently, \u003cem\u003eBabesia\u003c/em\u003e infections commonly cause systemic inflammatory reactions and hemolysis-related symptoms such as febrile syndrome, anemia, hemoglobinuria, jaundice, lethargy, and organ failure in the longer term. The severity of babesiosis can vary in intensity, depending on the host\u0026rsquo;s inherent conditions (genetic factors, age, concurrent infection with other pathogens, immunological status) and the infecting \u003cem\u003eBabesia\u003c/em\u003e species [8-10].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCanine babesiosis is a disease of major veterinary importance, affecting dogs worldwide. Several identified canine \u003cem\u003eBabesia\u003c/em\u003e species are variable in term of geographical distribution, vector, severity, clinical signs, and treatment sensitivity [8,9,11]. Large form species, e.g. \u003cem\u003eB. canis, B. vogeli (\u003c/em\u003eand \u003cem\u003eB. rossi\u003c/em\u003e as imported cases) and small form species, e.g. \u003cem\u003eB. vulpes, B. gibsoni,\u003c/em\u003e are reported in Europe [12].\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eBabesia canis\u003c/em\u003e is described across most of Europe, especially in wet and temperate to cold central regions, where\u003cem\u003e\u0026nbsp;Dermacentor reticulatus\u003c/em\u003e, its recognized vector, thrives [13]. Canine babesiosis caused by \u003cem\u003eB. canis\u003c/em\u003e is usually characterized by an acute onset. It causes moderate to severe clinical symptoms, often associated with hemolytic anemia and thrombocytopenia, sometimes with poor prognosis [9,10].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNexGard Spectra\u003csup\u003e\u0026reg;\u003c/sup\u003e, is an endectoparasiticide oral product for dogs, combining afoxolaner and milbemycin oxime. Afoxolaner is an insecticide and acaricide of the isoxazoline class. Isoxazolines antagonize the GABA-gated chloride ion channels of the hematophagous arthropods that fed on the treated host. Milbemycin oxime, the other active ingredient of the product, is a nematicide of the macrocyclic lactone class, with negligible efficacy on arthropods (unpublished data). The effectiveness of afoxolaner for the prevention of \u003cem\u003eB. canis\u0026nbsp;\u003c/em\u003eexperimentally\u003cem\u003e\u0026nbsp;\u003c/em\u003etransmitted by \u003cem\u003eDermacentor reticulatus\u003c/em\u003e was already demonstrated in an experimental study with oral afoxolaner administered in a mono-product (NexGard\u003csup\u003e\u0026reg;\u003c/sup\u003e), at label dose, and in comparison to a single untreated control group [14]. This manuscript describes a new study, designed according to the most recent relevant guideline for studies evaluating the efficacy of parasiticides in reducing the risk of vector-borne pathogen transmission [15], namely requiring treatment at the minimum recommended dose, infective challenges repeated at the beginning and at the end of efficacy claim duration, and with a distinct control group for these two challenges.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eEthics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnimals were managed with due regard for their wellbeing, and the Institutional Animal Care and Use Committee reviewed and approved the study design.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStudy design\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study, conducted in 2024, was designed in accordance with Good Clinical Practices as described in International Cooperation on Harmonization of Technical Requirements for Registration of Veterinary Medicinal Products (VICH) guideline GL9, and was designed in accordance with the \u0026ldquo;World Association for the Advancement of Veterinary Parasitology (W.A.A.V.P.) guidelines for studies evaluating the efficacy of parasiticides in reducing the risk of vector-borne pathogen transmission in dogs and cats [15].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis study was conducted under a blinded and randomized design.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe efficacy assessment was based on a comparison of \u003cem\u003eBabesia\u003c/em\u003e infection level in a treated group and untreated control groups following infestations with \u003cem\u003eD. reticulatus\u0026nbsp;\u003c/em\u003einfected with\u003cem\u003e\u0026nbsp;B. canis\u003c/em\u003e ,\u003cem\u003e\u0026nbsp;\u003c/em\u003eone day and twenty-eight days after treatment.\u003c/p\u003e\n\u003cp\u003eThe study outline and most animal characteristics per study group are illustrated in Table 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimals and husbandry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTwenty-four healthy purpose-bred adult dogs (fourteen Beagles, four beagle-crosses and six mongrels), 11 males and 13 females, aged 19 to 88 months, and weighing 12.1 to 23.8 kg were included in the study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe containment of the dogs complied with the South African National Standard SANS 10386: latest version \u0026ldquo;The care and use of animals for scientific purposes\u0026rdquo;. The animals were kept individually in cages and had visual and auditory contact with conspecifics. Dogs were socialized weekly. The cages had concrete floors to facilitate cleaning and each cage was fitted with a sleeping bench. At least one toy/chew was made available to each dog and was replaced weekly. The dog cages were part of an environmentally controlled indoor animal unit; temperatures ranged between 16\u0026deg;C to 25\u0026deg;C. A photoperiod of 12 hours light - 12 hours darkness was maintained. The floor size of each dog cage was 3.0 m x 1.9 m. No substances with an insecticidal or acaricidal activity were used on the animals or in their environment.\u0026nbsp;Standard commercially available diets (VetsBrands Premium adult maintenance dog food or Hill\u0026rsquo;s Adult Large Breed Dog Food) that met the nutritional requirements of the animals were provided at the recommended rates. Any dog diagnosed positive for \u003cem\u003eBabesia\u003c/em\u003e was immediately rescue treated and provided Hill\u0026rsquo;s a/d for 3 days. Potable water, as supplied by the local municipality, was provided in stainless steel bowls and replenished at least twice daily.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDermacentor reticulatus\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eand\u003cem\u003e\u0026nbsp;B. canis\u003c/em\u003e isolates\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eD. reticulatus\u003c/em\u003e ticks used in this study were originally obtained from BLE Laboratories (Ireland) in 2007. They had been genetically enriched with \u003cem\u003eD. reticulatus,\u003c/em\u003e obtained from the Utrecht Centre for Tick-borne Diseases (UCTD), Netherlands, in 2009, 2012, 2014, and 2017.\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eB. canis\u003c/em\u003e parasites used in this study originated from the Netherlands. A laboratory-bred dog was inoculated with blood derived from a clinical case of canine babesiosis, and blood from this dog served as infectious material for the ticks used in this study. A batch of \u003cem\u003eD. reticulatus\u003c/em\u003e nymphs were fed on the infected dog. The adult ticks obtained from the engorged nymphs were infected at a 34% rate, as determined by Polymerase Chain Reaction (PCR) on a sample of 50 ticks.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePre-treatment activities\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThirty dogs were acclimated to study conditions since Day -7. A clinical examination was performed on all dogs on Day -7. To evaluate their suitability for tick infestation, all dogs were infested on Day -7, following the method described in the \u0026lsquo;tick infestation and removals\u0026rsquo; paragraph, with the exceptions that the \u003cem\u003eD. reticulatus\u0026nbsp;\u003c/em\u003ewere uninfected and were removed and counted two days after infestation, on Day -5. On Day -5, blood was collected from all dogs for Immuno Fluorescent Assay (IFA) and PCR analyses\u0026nbsp;to ensure they were not positive for \u003cem\u003eB. canis\u003c/em\u003e before inclusion. The 24 dogs with the highest live attached tick count and that were suitable for the study, as determined by the Investigator, were selected for inclusion. Eight blocks with three dogs each were defined based on live attached tick numbers, allowing a randomization to 3 groups of 8 dogs each, one treated group and two control groups. The numbers of live attached ticks in the included dogs ranged from 13 to 35, the average numbers of ticks were 22.9, 23.0 and 22.9, in the control groups 1, 2 and the IVP-treated group, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTreatment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOn Day 0, dogs from the treated group were orally administered a combination of whole tablets defined according to their bodyweight and to target the minimum label dose of the product, i.e. 2.5 mg/kg afoxolaner and 0.5 mg/kg milbemycin oxime, as described in Table 2. The mean administered afoxolaner dose was 2.75 mg/kg.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTick infestations and removals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe treated group was infested on Days 1 and 28. Two control groups were used to verify the adequacy of each \u003cem\u003eB. canis\u003c/em\u003e transmission and were thus infested on Day 1 (control group 1) or Day 28 (control group 2). Each time, dogs were infested with 50 (\u0026plusmn; 2) adult ticks of 50/50 sex ratio. To obtain 50 ticks with a consistent \u003cem\u003eB. canis\u003c/em\u003e infection rate for each individual infestation, 34 ticks from the infected batch (17 males and 17 females) with a 34% infection rate as determined by PCR, were combined with 16 ticks (8 males and 8 females) from the same origin, but that had not been exposed to \u003cem\u003eB. canis\u003c/em\u003e infected blood. Therefore, the \u003cem\u003eB. canis\u003c/em\u003e infection rate for each batch of 50 ticks used for infestations was 23%. To facilitate tick infestation, dogs were sedated and placed in an infestation crate for one to two hours. The ticks were released on the back or flank of the animal and were allowed to disperse into the hair coat.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSix days after each infestation, on Days 7 and 34, the remaining ticks were removed and counted from each infested group (the treated group and control group 1 on Day 7, and the treated group and control group 2 on Day 34).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHealth and\u003cem\u003e\u0026nbsp;Babesia\u0026nbsp;\u003c/em\u003einfection evaluations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGeneral health observations were performed once daily from Day -7 to Day 6 and twice daily from Day 7 to Day 56. Clinical examinations were performed weekly from Day 7 to Day 56. Rectal temperature was measured once daily from Day 1 to Day 56.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWhenever the rectal temperature exceeded 39.5\u0026deg;C and/or babesiosis was suspected based on clinical signs (e.g. lethargy, dark urine, diarrhea, anorexia), blood was collected for blood smear processing and microscopic search of \u003cem\u003eBabesia\u003c/em\u003e in red blood cells, and for \u003cem\u003eB. canis\u003c/em\u003e PCR analysis, recognized methods for babesia diagnosis [16]. Irrespective of clinical signs, blood collection was scheduled for \u003cem\u003eB. canis\u003c/em\u003e IFA and PCR analyses on Days 21 and 28 (treated group and control group 1), and on Days 42 and 56 (all dogs).\u003c/p\u003e\n\u003cp\u003eFor \u003cem\u003eB. canis\u003c/em\u003e PCR analysis, total genomic DNA was isolated from whole blood samples using a commercial genomic DNA isolation kit (GeneJET Genomic DNA Purification Kit, Thermo Fisher Scientific, USA). PCR entailed using primers specific to the 18S Ribosomal DNA region of \u003cem\u003eB.\u0026nbsp;canis\u003c/em\u003e, and as referenced [17]. The PCR procedure followed a recognized method [18]. For IFA analysis, serum was collected from blood tubes and was assayed for \u003cem\u003eB. canis\u003c/em\u003e antibodies using a commercially available IFA test kit (MegaFLUO\u003csup\u003e\u0026reg;\u003c/sup\u003e BABESIA canis, Megacor Diagnostik, Austria).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe primary efficacy criterion was the number of dogs that became infected with \u003cem\u003eB. canis\u003c/em\u003e (when tested positive for \u003cem\u003eB. canis\u003c/em\u003e by both PCR analysis and IFA serology). The infection rate of dogs in groups that became infected during the study, were summarized descriptively. The percentage blocking efficacy for the IVP-treated group was calculated as follows in relation to the Day 1 and Day 28 infestations:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBlocking efficacy (%) = 100 x (Tc - Tt)/Tc\u003c/p\u003e\n\u003cp\u003eTc = Total number of untreated control dogs that became infected (control group 1 for Day 1, control group 2 for Day 28)\u003c/p\u003e\n\u003cp\u003eTt = Total number of dogs that became infected in the IVP group (group 3)\u003c/p\u003e\n\u003cp\u003eSAS Version 9.4 was used for all the statistical analyses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEndpoints\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhenever a dog was diagnosed positive for \u003cem\u003eBabesia\u003c/em\u003e infection on a blood smear, blood for PCR analysis was collected, and the dog was rescue treated. All study activities for rescue treated dogs were stopped except the scheduled blood collections for IFA and PCR analyses. A rescue-treated dog was removed from the study once a positive IFA result was obtained.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThe individual results of tick counts and \u003cem\u003eBabesia\u003c/em\u003e infection diagnosis are described in Table 3.\u003c/p\u003e\n\u003cp\u003eThe tick infestations on Days 1 and 28 were demonstrated adequate by their corresponding control groups 1 and 2, as 6 days later, on Days 7 and 34 respectively, a significant number of live attached ticks were found. The mean number of live attached ticks was 36 (72%) in the untreated control group 1 and 30.4 (60.8%) in the untreated control group 2. No live attached tick was found on any IVP treated dog on Days 7 and 34, demonstrating a reduction of 100% of live attached ticks, 6 days after infestation on Days 1 and 28. A small number of dead attached ticks, ranging from 0 to 5, were found in this group.\u003c/p\u003e\n\u003cp\u003eA clear diagnosis of canine babesiosis was made for all 16 untreated control dogs, 7 to 9 days following their respective \u003cem\u003eD. reticulatus\u003c/em\u003e infestation. All dogs expressed abnormal clinical signs, i.e. lethargy, and/or dark urine, and/or rectal temperature exceeding 39.5\u0026deg;C. On the same day, the diagnosis of canine babesiosis was confirmed for each dog by a positive blood smear microscopical examination and \u003cem\u003eB. canis\u003c/em\u003e PCR analysis. These 16 dogs were rescue treated on the day of diagnosis with injections of\u0026nbsp;imidocarb dipropionate (7.2 mg/kg), diminazene aceturate (3.5 mg/kg) and several days of supportive medications, i.e. corticosteroid (prednisolone acetate 0.1 mg/kg), antiemetic (marbopitant 1 mg/kg), and hepatoprotectors. All dogs were confirmed positive by IFA analysis for \u003cem\u003eB. canis\u003c/em\u003e on Day 21 (control group 1) or Day 42 (control group 2). The PCR analyses of these dogs on Days 21 and 42 had turned negative, demonstrating the efficacy of rescue treatments. None of the IVP-treated dogs expressed any abnormal clinical signs of babesiosis, and their PCR and IFA bioanalyses performed on Days 21, 28, 42 and 56 remained negative.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe data obtained in this study with the untreated animals demonstrated a reliable and consistent \u003cem\u003eB. canis\u003c/em\u003e infection model and showed with the treated animals that afoxolaner completely blocked the \u003cem\u003eB. canis\u003c/em\u003e transmission by \u003cem\u003eD. reticulatus\u0026nbsp;\u003c/em\u003eticks.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eDermacentor reticulatus\u003c/em\u003e is a vector of numerous other protozoal, bacterial, and viral diseases [19], out of scope in this study, as the investigated model was limited to pathogen-free ticks experimentally infected only with \u003cem\u003eB. canis.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eCanine babesiosis is a highly significant and challenging disease. Several therapeutic or preventive veterinary strategies can be implemented alone or in combination. The protozoan can be directly targeted through pharmaceutical products or vaccinations. As described in this manuscript, the ixodid vector can be targeted through acaricidal products. The use of repellents can also reduce tick infestations.\u003c/p\u003e\n\u003cp\u003ePharmaceutical treatment of dogs affected with canine babesiosis is challenging. A limited number of drugs used alone or in combination are available on- or off-label, with variable species and strain-related efficacy [12,20,21]. The mechanism of action of these drugs is mostly unclear. Injectable imidocarb dipropionate is registered for treating \u003cem\u003eB. canis,\u0026nbsp;\u003c/em\u003enevertheless with cholinergic adverse effects and pain at injection. Intensive care is also necessary for severe manifestations of the disease, and relapses are possible. In this study, diminazene aceturate, a compound of poorly understood mechanism of action [22] and registered in South Africa for the treatment of \u003cem\u003eB. canis\u003c/em\u003e in dogs, was used as treatment in combination with imidocarb dipropionate and with corticosteroid, hepato-protector and antiemetic supportive medications.\u003c/p\u003e\n\u003cp\u003eSome vaccines containing soluble parasite antigens derived from \u003cem\u003ein vitro\u003c/em\u003e cultures are registered against \u003cem\u003eB. canis\u0026nbsp;\u003c/em\u003ebut with variable strain-related efficacy [23]. \u003cem\u003eBabesia\u003c/em\u003e vaccines do not affect the infection but alter the disease progression and reduce the severity of the clinical signs. Research on recombinant antigens is currently underway and may provide new vaccines in the future [24,25].\u003c/p\u003e\n\u003cp\u003eRepellent and acaricide products, such as permethrin and flumethrin, have been described as effective for the reduction of \u003cem\u003eB. canis\u003c/em\u003e transmission [26-28] and, more generally, for the prevention of several vector-borne diseases, including \u003cem\u003eBabesia\u003c/em\u003e spp. [29].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe approach of blocking a pathogen transmission by using an acaricide targeting the vector has become an important veterinary strategy since the appearance of isoxazolines in 2014 [14,30-34]. Contrarily to repellent products, isoxazolines do not prevent an arthropod vector from biting or attaching to its host and start a fluid exchange process. Nevertheless, \u003cem\u003eBabesia\u0026nbsp;\u003c/em\u003espp\u003cem\u003e.\u0026nbsp;\u003c/em\u003eusually require several days after host attachment for the ookinetes to undergo several sporogony cycles, leading to the formation of infective sporozoites in the salivary gland of their ixodid vector [2,13]. As examples, it was demonstrated in humans that \u003cem\u003eB.microti\u003c/em\u003e was transmitted by \u003cem\u003eIxodes scapularis\u003c/em\u003e 36 to 48h after host attachment [35], or in cattle that\u003cem\u003e\u0026nbsp;B. bigemina\u003c/em\u003e was transmitted during the last 16 to 24h before vector (\u003cem\u003eRhipicephalus microplus\u003c/em\u003e) detachment [36]. Interestingly, in the present study, some dead attached \u003cem\u003eD. reticulatus\u003c/em\u003e were found on the treated dogs but probably did not survive long enough to transmit their infective \u003cem\u003eB. canis\u0026nbsp;\u003c/em\u003esporozoites\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAfoxolaner, alone or combined with milbemycin oxime was demonstrated effective against \u003cem\u003eD. reticulatus\u003c/em\u003e within 48 hours of infestations and for 5 weeks after a treatment at minimum dose [37]. Afoxolaner was also effective to prevent \u003cem\u003eB. canis\u003c/em\u003e transmission by \u003cem\u003eD. reticulatus\u003c/em\u003e, when administered at label dose in a mono-product (NexGard\u003csup\u003e\u0026reg;\u003c/sup\u003e) and with one control group verifying validity of weekly challenges [14]. The study described in this manuscript provides an upgraded design. It verified the efficacy of a single afoxolaner treatment administered at the minimum dose of its registered products and with a distinct control group verifying adequacy of infection after each challenge at the beginning (Day 1) and end (Day 28) of the efficacy claimed period. The 100% level of \u003cem\u003eB. canis\u003c/em\u003e infection observed in both untreated control groups after challenges at Day 1 or at Day 28 supported the robustness of the model. The total absence of infection after the two infective tick challenges in the treated group supported the preventive efficacy of afoxolaner administered once and at minimum dose of its registered products.\u0026nbsp;\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, the killing effect of the investigated oral formulation combining afoxolaner and milbemycin oxime was fast and complete enough to interrupt the vector-host fluid exchange process, thus blocking tick capability to transmit the infectious \u003cem\u003eBabesia\u003c/em\u003e sporozoites.\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAnimal Ethics and Consent to Participate declarations:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAkel T, Mobarakai N. Hematologic manifestations of babesiosis. Ann Clin Microbiol Antimicrob. 2017 Feb 15;16(1):6. doi: 10.1186/s12941-017-0179-z. PMID: 28202022; PMCID: PMC5310009.\u003c/li\u003e\n\u003cli\u003eHunfeld KP, Hildebrandt A, Gray JS. Babesiosis: recent insights into an ancient disease. Int J Parasitol. 2008 Sep;38(11):1219-37. doi: 10.1016/j.ijpara.2008.03.001. Epub 2008 Mar 20. PMID: 18440005.\u003c/li\u003e\n\u003cli\u003eMalgwi SA, Ogunsakin RE, Oladepo AD, Adeleke MA, Okpeku M. A Forty-Year Analysis of the Literature on \u003cem\u003eBabesia\u003c/em\u003e Infection (1982-2022): A Systematic Bibliometric Approach. Int J Environ Res Public Health. 2023 Jun 16;20(12):6156. doi: 10.3390/ijerph20126156. PMID: 37372744; PMCID: PMC10298011.\u003c/li\u003e\n\u003cli\u003eUilenberg G. \u003cem\u003eBabesia\u003c/em\u003e--a historical overview. Vet Parasitol. 2006 May 31;138(1-2):3-10. doi: 10.1016/j.vetpar.2006.01.035. Epub 2006 Feb 28. PMID: 16513280.\u003c/li\u003e\n\u003cli\u003eVannier EG, Diuk-Wasser MA, Ben Mamoun C, Krause PJ. Babesiosis. Infect Dis Clin North Am. 2015 Jun;29(2):357-70. doi: 10.1016/j.idc.2015.02.008. PMID: 25999229; PMCID: PMC4458703.\u003c/li\u003e\n\u003cli\u003eSchnittger L, Rodriguez AE, Florin-Christensen M, Morrison DA. Babesia: a world emerging. Infect Genet Evol. 2012 Dec;12(8):1788-809. doi: 10.1016/j.meegid.2012.07.004. Epub 2012 Jul 31. PMID: 22871652.\u003c/li\u003e\n\u003cli\u003eDear JD, Birkenheuer A. \u003cem\u003eBabesia\u003c/em\u003e in North America: An Update. Vet Clin North Am Small Anim Pract. 2022 Nov;52(6):1193-1209. doi: 10.1016/j.cvsm.2022.07.016. PMID: 36336417.\u003c/li\u003e\n\u003cli\u003eLeisewitz AL, Mrljak V, Dear JD, Birkenheuer A. The Diverse Pathogenicity of Various \u003cem\u003eBabesia\u003c/em\u003e Parasite Species That Infect Dogs. Pathogens. 2023 Dec 11;12(12):1437. doi: 10.3390/pathogens12121437. PMID: 38133320; PMCID: PMC10746086.\u003c/li\u003e\n\u003cli\u003eSolano-Gallego L, Sainz \u0026Aacute;, Roura X, Estrada-Pe\u0026ntilde;a A, Mir\u0026oacute; G. A review of canine babesiosis: the European perspective. Parasit Vectors. 2016 Jun 11;9(1):336. doi: 10.1186/s13071-016-1596-0. PMID: 27289223; PMCID: PMC4902949.\u003c/li\u003e\n\u003cli\u003eEichenberger RM, Riond B, Willi B, Hofmann-Lehmann R, Deplazes P. Prognostic Markers in Acute \u003cem\u003eBabesia canis\u003c/em\u003e Infections. J Vet Intern Med. 2016 Jan-Feb;30(1):174-82. doi: 10.1111/jvim.13822. Epub 2016 Jan 4. PMID: 26727465; PMCID: PMC4913656. \u003c/li\u003e\n\u003cli\u003eMalgwi SA, Ogunsakin RE, Oladepo AD, Adeleke MA, Okpeku M. A Forty-Year Analysis of the Literature on \u003cem\u003eBabesia\u003c/em\u003e Infection (1982-2022): A Systematic Bibliometric Approach. Int J Environ Res Public Health. 2023 Jun 16;20(12):6156. doi: 10.3390/ijerph20126156. PMID: 37372744; PMCID: PMC10298011.\u003c/li\u003e\n\u003cli\u003eBaneth G. Antiprotozoal treatment of canine babesiosis. Vet Parasitol. 2018 Apr 30;254:58-63. doi: 10.1016/j.vetpar.2018.03.001. Epub 2018 Mar 7. PMID: 29657012.\u003c/li\u003e\n\u003cli\u003eBeugnet F, Moreau Y. Babesiosis. Rev Sci Tech. 2015 Aug;34(2):627-39. doi: 10.20506/rst.34.2.2385. PMID: 26601462.\u003c/li\u003e\n\u003cli\u003eBeugnet F, Halos L, Larsen D, Labuschagn\u0026eacute; M, Erasmus H, Fourie J. The ability of an oral formulation of afoxolaner to block the transmission of \u003cem\u003eBabesia canis\u003c/em\u003e by \u003cem\u003eDermacentor reticulatus\u003c/em\u003e ticks to dogs. Parasit Vectors. 2014 Jun 23;7:283. doi: 10.1186/1756-3305-7-283. PMID: 24957215; PMCID: PMC4078974.\u003c/li\u003e\n\u003cli\u003eOtranto D, Dantas-Torres F, Fourie JJ, Lorusso V, Varloud M, Gradoni L, Drake J, Geurden T, Kaminsky R, Heckeroth AR, Schunack B, Pollmeier M, Beugnet F, Holdsworth P. World Association for the Advancement of Veterinary Parasitology (W.A.A.V.P.) guidelines for studies evaluating the efficacy of parasiticides in reducing the risk of vector-borne pathogen transmission in dogs and cats. Vet Parasitol. 2021 Feb;290:109369. doi: 10.1016/j.vetpar.2021.109369. Epub 2021 Jan 24. PMID: 33548595. \u003c/li\u003e\n\u003cli\u003eLempereur L, Beck R, Fonseca I, Marques C, Duarte A, Santos M, Z\u0026uacute;quete S, Gomes J, Walder G, Domingos A, Antunes S, Baneth G, Silaghi C, Holman P, Zintl A. Guidelines for the Detection of \u003cem\u003eBabesia\u003c/em\u003e and \u003cem\u003eTheileria\u003c/em\u003e Parasites. Vector Borne Zoonotic Dis. 2017 Jan;17(1):51-65. doi: 10.1089/vbz.2016.1955. PMID: 28055573.\u003c/li\u003e\n\u003cli\u003eTroskie M, de Villiers L, Leisewitz A, Oosthuizen MC, Quan M. Development and validation of a multiplex, real-time PCR assay for \u003cem\u003eBabesia rossi\u003c/em\u003e and \u003cem\u003eBabesia vogeli\u003c/em\u003e. Ticks Tick Borne Dis. 2019 Feb;10(2):421-432. doi: 10.1016/j.ttbdis.2018.12.004. Epub 2018 Dec 14. PMID: 30591405.\u003c/li\u003e\n\u003cli\u003eHeylen D, Day M, Schunack B, Fourie J, Labuschange M, Johnson S, Githigia SM, Akande FA, Nzalawahe JS, Tayebwa DS, Aschenborn O, Marcondes M, Madder M. A community approach of pathogens and their arthropod vectors (ticks and fleas) in dogs of African Sub-Sahara. Parasit Vectors. 2021 Nov 16;14(1):576. doi: 10.1186/s13071-021-05014-8. PMID: 34784947; PMCID: PMC8594167.\u003c/li\u003e\n\u003cli\u003eF\u0026ouml;ldv\u0026aacute;ri G, \u0026Scaron;irok\u0026yacute; P, Szekeres S, Majoros G, Sprong H. \u003cem\u003eDermacentor reticulatus\u003c/em\u003e: a vector on the rise. Parasit Vectors. 2016 Jun 1;9(1):314. doi: 10.1186/s13071-016-1599-x. PMID: 27251148; PMCID: PMC4888597.\u003c/li\u003e\n\u003cli\u003eJi S, Galon EM, Rizk MA, Yi Y, Zafar I, Li H, Ma Z, Iguchi A, Asada M, Liu M, Xuan X. Efficacy of the Antimalarial MMV390048 against \u003cem\u003eBabesia\u003c/em\u003e Infection Reveals Phosphatidylinositol 4-Kinase as a Druggable Target for Babesiosis. Antimicrob Agents Chemother. 2022 Sep 20;66(9):e0057422. doi: 10.1128/aac.00574-22. Epub 2022 Aug 4. PMID: 35924942; PMCID: PMC9487540.\u003c/li\u003e\n\u003cli\u003eWeingart C, Kr\u0026uuml;cken J, Kohn B. Repeated imidocarb treatment failure suggesting emerging resistance of \u003cem\u003eBabesia canis\u003c/em\u003e in a new endemic area in north-eastern Germany. Ticks Tick Borne Dis. 2024 May;15(3):102315. doi: 10.1016/j.ttbdis.2024.102315. Epub 2024 Jan 31. PMID: 38301344.\u003c/li\u003e\n\u003cli\u003eKuriakose S, Uzonna JE. Diminazene aceturate (Berenil), a new use for an old compound? Int Immunopharmacol. 2014 Aug;21(2):342-5. doi: 10.1016/j.intimp.2014.05.027. Epub 2014 Jun 2. PMID: 24893117.\u003c/li\u003e\n\u003cli\u003eSchetters TH, Kleuskens J, Scholtes N, Bos HJ. Strain variation limits protective activity of vaccines based on soluble \u003cem\u003eBabesia canis\u003c/em\u003e antigens. Parasite Immunol. 1995 Apr;17(4):215-8. doi: 10.1111/j.1365-3024.1995.tb00891.x. PMID: 7542765.\u003c/li\u003e\n\u003cli\u003eMoubri K, Kleuskens J, Van de Crommert J, Scholtes N, Van Kasteren T, Delbecq S, Carcy B, Pr\u0026eacute;cigout E, Gorenflot A, Schetters T. Discovery of a recombinant \u003cem\u003eBabesia canis\u003c/em\u003e supernatant antigen that protects dogs against virulent challenge infection. Vet Parasitol. 2018 Jan 15;249:21-29. doi: 10.1016/j.vetpar.2017.11.002. Epub 2017 Nov 6. PMID: 29279083.\u003c/li\u003e\n\u003cli\u003eNathaly Wieser S, Schnittger L, Florin-Christensen M, Delbecq S, Schetters T. Vaccination against babesiosis using recombinant GPI-anchored proteins. Int J Parasitol. 2019 Feb;49(2):175-181. doi: 10.1016/j.ijpara.2018.12.002. Epub 2019 Jan 24. PMID: 30684517\u003c/li\u003e\n\u003cli\u003eFourie JJ, de Vos C, Crafford D, Pollmeier M, Schunack B. A study on the long-term efficacy of Seresto\u0026reg; collars in preventing \u003cem\u003eBabesia canis\u003c/em\u003e (Piana \u0026amp; Galli-Valerio, 1895) transmission to dogs by infected \u003cem\u003eDermacentor reticulatus\u003c/em\u003e (Fabricius, 1794) ticks. Parasit Vectors. 2019 Mar 22;12(1):139. doi: 10.1186/s13071-019-3393-z. PMID: 30902104; PMCID: PMC6431040.\u003c/li\u003e\n\u003cli\u003eJongejan F, de Vos C, Fourie JJ, Beugnet F. A novel combination of fipronil and permethrin (Frontline Tri-Act\u003csup\u003e\u0026reg;\u003c/sup\u003e/Frontect\u003csup\u003e\u0026reg;\u003c/sup\u003e) reduces risk of transmission of \u003cem\u003eBabesia canis\u003c/em\u003e by \u003cem\u003eDermacentor reticulatus\u003c/em\u003e and of \u003cem\u003eEhrlichia canis\u003c/em\u003e by \u003cem\u003eRhipicephalus sanguineus\u003c/em\u003e ticks to dogs. Parasit Vectors. 2015 Nov 19;8:602. doi: 10.1186/s13071-015-1207-5. PMID: 26586365; PMCID: PMC4653891.\u003c/li\u003e\n\u003cli\u003eNavarro C, Reymond N, Fourie J, Hellmann K, Bonneau S. Prevention of \u003cem\u003eBabesia canis\u003c/em\u003e in dogs: efficacy of a fixed combination of permethrin and fipronil (Effitix\u003csup\u003e\u0026reg;\u003c/sup\u003e) using an experimental transmission blocking model with infected \u003cem\u003eDermacentor reticulatus\u003c/em\u003e ticks. Parasit Vectors. 2015 Jan 17;8:32. doi: 10.1186/s13071-015-0645-4. PMID: 25595325; PMCID: PMC4302435.\u003c/li\u003e\n\u003cli\u003eOtranto D, de Caprariis D, Lia RP, Tarallo V, Lorusso V, Testini G, Dantas-Torres F, Latrofa S, Diniz PP, Mencke N, Maggi RG, Breitschwerdt E, Capelli G, Stanneck D. Prevention of endemic canine vector-borne diseases using imidacloprid 10% and permethrin 50% in young dogs: a longitudinal field study. Vet Parasitol. 2010 Sep 20;172(3-4):323-32. doi: 10.1016/j.vetpar.2010.05.017. Epub 2010 May 25. PMID: 20591573.\u003c/li\u003e\n\u003cli\u003eBaker CF, McCall JW, McCall SD, Drag MD, Mitchell EB, Chester ST, Larsen D. Ability of an oral formulation of afoxolaner to protect dogs from \u003cem\u003eBorrelia burgdorferi\u003c/em\u003e infection transmitted by wild \u003cem\u003eIxodes scapularis\u003c/em\u003e ticks. Comp Immunol Microbiol Infect Dis. 2016 Dec;49:65-69. doi: 10.1016/j.cimid.2016.09.004. Epub 2016 Sep 29. PMID: 27865266.\u003c/li\u003e\n\u003cli\u003eCavalleri D, Murphy M, Seewald W, Drake J, Nanchen S. Two randomized, controlled studies to assess the efficacy and safety of lotilaner (Credelio\u0026trade;) in preventing \u003cem\u003eDermacentor reticulatus\u003c/em\u003e transmission of \u003cem\u003eBabesia canis\u003c/em\u003e to dogs. Parasit Vectors. 2017 Nov 1;10(1):520. doi: 10.1186/s13071-017-2473-1. PMID: 29089056; PMCID: PMC5664908. \u003c/li\u003e\n\u003cli\u003eGeurden T, Six R, Becskei C, Maeder S, Lloyd A, Mahabir S, Fourie J, Liebenberg J. Evaluation of the efficacy of sarolaner (Simparica\u003csup\u003e\u0026reg;\u003c/sup\u003e) in the prevention of babesiosis in dogs. Parasit Vectors. 2017 Sep 6;10(1):415. doi: 10.1186/s13071-017-2358-3. PMID: 28877743; PMCID: PMC5588728. \u003c/li\u003e\n\u003cli\u003eOtranto D, Mendoza-Roldan JA, Beugnet F, Baneth G, Dantas-Torres F. New paradigms in the prevention of canine vector-borne diseases. Trends Parasitol. 2024 Jun;40(6):500-510. doi: 10.1016/j.pt.2024.04.009. Epub 2024 May 13. PMID: 38744542.\u003c/li\u003e\n\u003cli\u003eTaenzler J, Liebenberg J, Roepke RK, Heckeroth AR. Prevention of transmission of \u003cem\u003eBabesia canis\u003c/em\u003e by \u003cem\u003eDermacentor reticulatus\u003c/em\u003e ticks to dogs treated orally with fluralaner chewable tablets (Bravecto\u0026trade;). Parasit Vectors. 2015 Jun 4;8:305. doi: 10.1186/s13071-015-0923-1. PMID: 26040319; PMCID: PMC4465614. \u003c/li\u003e\n\u003cli\u003ePiesman J, Spielman A. Human babesiosis on Nantucket Island: prevalence of \u003cem\u003eBabesia microti\u003c/em\u003e in ticks. Am J Trop Med Hyg. 1980 Sep;29(5):742-6. doi: 10.4269/ajtmh.1980.29.742. PMID: 7435782.\u003c/li\u003e\n\u003cli\u003eHodgson JL. Biology and transmission of \u003cem\u003eBabesia bigemina \u003c/em\u003ein\u003cem\u003e Boophilus microplus\u003c/em\u003e. Ann N Y Acad Sci. 1992 Jun 16;653:42-51. doi: 10.1111/j.1749-6632.1992.tb19628.x. PMID: 1626891.\u003c/li\u003e\n\u003cli\u003eRehbein S, Fourie JJ, de Vos C, Anderson A, Larsen DL, Jeannin P. Efficacy of oral afoxolaner plus milbemycin oxime chewables against induced infestations with \u003cem\u003eDermacentor reticulatus\u003c/em\u003e in dogs. Parasitol Res. 2016 May;115(5):1845-51. doi: 10.1007/s00436-016-4924-6. Epub 2016 Jan 27. PMID: 26815036.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1: Animal characteristics and study outline\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"924\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGroups\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"bottom\" style=\"width: 268px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAnimal characteristics at inclusion\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(on Day -2)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"bottom\" style=\"width: 79px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIVP treatment\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" style=\"width: 213px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eD. reticulatus\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"bottom\" style=\"width: 116px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDaily rectal temperatures\u003csup\u003e1\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"bottom\" style=\"width: 150px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePCR and IFA analyses\u003csup\u003e2\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 2px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 81px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 14px;\"\u003e\n \u003cp\u003e\u003cstrong\u003en\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 55px;\"\u003e\n \u003cp\u003e\u003cstrong\u003esex\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 96px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eage (months)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 117px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBodyweight (kg)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 107px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eInfestation(s)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 107px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRemoval(s)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 2px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 81px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eControl 1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 14px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 55px;\"\u003e\n \u003cp\u003e4 m, 4 f\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 96px;\"\u003e\n \u003cp\u003e19 - 88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 117px;\"\u003e\n \u003cp\u003e12.1 - 20.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 79px;\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 107px;\"\u003e\n \u003cp\u003eDay 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 107px;\"\u003e\n \u003cp\u003eDay 7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 116px;\"\u003e\n \u003cp\u003eDays 1 to 56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 150px;\"\u003e\n \u003cp\u003eDays 21 and 28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 2px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 81px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eControl 2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 14px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 55px;\"\u003e\n \u003cp\u003e3 m, 5 f\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 96px;\"\u003e\n \u003cp\u003e31 - 88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 117px;\"\u003e\n \u003cp\u003e12.9 - 23.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 79px;\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 107px;\"\u003e\n \u003cp\u003eDay 28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 107px;\"\u003e\n \u003cp\u003eDay 34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 116px;\"\u003e\n \u003cp\u003eDays 1 to 56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 150px;\"\u003e\n \u003cp\u003eDays 42 and 56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 2px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 81px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIVP\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 14px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 55px;\"\u003e\n \u003cp\u003e4 m, 4 f\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 96px;\"\u003e\n \u003cp\u003e28 - 82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 117px;\"\u003e\n \u003cp\u003e14.2 - 23.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 79px;\"\u003e\n \u003cp\u003eDay 0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 107px;\"\u003e\n \u003cp\u003eDays 1 and 28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 107px;\"\u003e\n \u003cp\u003eDays 7 and 34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 116px;\"\u003e\n \u003cp\u003eDays 1 to 56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 150px;\"\u003e\n \u003cp\u003eDays 21, 28, 42 and 26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 2px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"10\" valign=\"bottom\" style=\"width: 922px;\"\u003e\n \u003cp\u003eAnimals were examined for clinical signs once a day until Day 6 and twice daily from Day 7 to Day 56.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 2px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"10\" rowspan=\"2\" valign=\"bottom\" style=\"width: 922px;\"\u003e\n \u003cp\u003e\u003csup\u003e1\u003c/sup\u003e whenever the rectal temperature exceeded 39.5\u0026deg;C, and/or babesiosis was suspected on the basis of clinical signs, blood was collected for blood smear microscopical examination and PCR analysis. A dog with a positive blood smear was rescue treated.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 2px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"30\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 2px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"10\" valign=\"bottom\" style=\"width: 922px;\"\u003e\n \u003cp\u003e\u003csup\u003e2\u003c/sup\u003e A dog was removed from the study once a PCR and an IFA analysis was positive.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 2px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd height=\"20\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;Table 2: individual treatment on Day 0\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"602\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" style=\"width: 124px;\"\u003e\n \u003cp\u003eDog\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"bottom\" style=\"width: 157px;\"\u003e\n \u003cp\u003eTablet combination*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" style=\"width: 183px;\"\u003e\n \u003cp\u003eDose (mg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" style=\"width: 139px;\"\u003e\n \u003cp\u003eDosage (mg/kg)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17px;\"\u003e\n \u003cp\u003e#\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 106px;\"\u003e\n \u003cp\u003eBodyweight (kg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 107px;\"\u003e\n \u003cp\u003eAfoxolaner\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003eMO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 95px;\"\u003e\n \u003cp\u003eAfoxolaner\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 44px;\"\u003e\n \u003cp\u003eMO\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e15.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 157px;\"\u003e\n \u003cp\u003e9/2 + 38/8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 107px;\"\u003e\n \u003cp\u003e46.875\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003e9.375\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e2.94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 44px;\"\u003e\n \u003cp\u003e0.59\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e21.55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 157px;\"\u003e\n \u003cp\u003e19/4 + 38/8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 107px;\"\u003e\n \u003cp\u003e56.250\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003e11.250\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e2.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 44px;\"\u003e\n \u003cp\u003e0.52\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e18.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 157px;\"\u003e\n \u003cp\u003e19/4 + 38/8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 107px;\"\u003e\n \u003cp\u003e56.250\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003e11.250\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e3.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 44px;\"\u003e\n \u003cp\u003e0.60\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e18.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 157px;\"\u003e\n \u003cp\u003e9/2 + 38/8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 107px;\"\u003e\n \u003cp\u003e46.875\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003e9.375\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e2.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 44px;\"\u003e\n \u003cp\u003e0.51\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e19.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 157px;\"\u003e\n \u003cp\u003e19/4 + 38/8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 107px;\"\u003e\n \u003cp\u003e56.250\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003e11.250\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e2.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 44px;\"\u003e\n \u003cp\u003e0.57\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e14.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 157px;\"\u003e\n \u003cp\u003e38/8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 107px;\"\u003e\n \u003cp\u003e37.500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003e7.500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e2.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 44px;\"\u003e\n \u003cp\u003e0.53\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e23.80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 157px;\"\u003e\n \u003cp\u003e9/2 + 19/4 + 38/8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 107px;\"\u003e\n \u003cp\u003e65.625\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003e13.125\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e2.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 44px;\"\u003e\n \u003cp\u003e0.55\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e14.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 157px;\"\u003e\n \u003cp\u003e38/8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 107px;\"\u003e\n \u003cp\u003e37.500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003e7.500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e2.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 44px;\"\u003e\n \u003cp\u003e0.52\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 106px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 157px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 107px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eMean\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.75\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 44px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e0.55\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;*NexGard Spectra\u003csup\u003e\u0026reg;\u0026nbsp;\u003c/sup\u003estrengths:\u003c/p\u003e\n\u003cp\u003e9/2 = 9.375 mg afoxolaner \u0026amp; 1.875 mg milbemycin oxime (for dogs 2-3.5 kg)\u003c/p\u003e\n\u003cp\u003e19/4: 18.75 mg afoxolaner \u0026amp; 3.75 mg milbemycin oxime (for dogs \u0026gt;3.5-7.5 kg)\u003c/p\u003e\n\u003cp\u003e38/8: 37.5 mg afoxolaner \u0026amp; 7.5 mg milbemycin oxime (for dogs \u0026gt;7.5-15 kg)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3: Individual results of tick counts and \u003cem\u003eBabesia\u003c/em\u003e diagnosis\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"1160\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"bottom\" style=\"width: 75px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGroup\u003csup\u003e1\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"bottom\" style=\"width: 19px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e#\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" rowspan=\"2\" valign=\"bottom\" style=\"width: 109px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLive (dead) attached tick count\u003csup\u003e2\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" valign=\"bottom\" style=\"width: 435px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eOriginal diagnosis of canine babesiosis\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"8\" valign=\"bottom\" style=\"width: 521px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eScheduled bioanalyses of \u003cem\u003eBabesia canis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" rowspan=\"2\" valign=\"bottom\" style=\"width: 278px;\"\u003e\n \u003cp\u003eDay of diagnosis, clinical signs of babesiosis and/or rectal temperature of \u0026gt;39.5\u0026deg;C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"bottom\" style=\"width: 84px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBlood smear\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"bottom\" style=\"width: 73px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePCR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" valign=\"bottom\" style=\"width: 261px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIFA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" valign=\"bottom\" style=\"width: 261px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePCR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 55px;\"\u003e\n \u003cp\u003eDay 7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 55px;\"\u003e\n \u003cp\u003eDay 34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003eDay 21\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003eDay 28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003eDay 42\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003eDay 56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003eDay 21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003eDay 28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003eDay 42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003eDay 56\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 75px;\"\u003e\n \u003cp\u003eControl 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 19px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 55px;\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 55px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003eDay 9:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 202px;\"\u003e\n \u003cp\u003eLethargic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 84px;\"\u003e\n \u003cp\u003epositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 73px;\"\u003e\n \u003cp\u003epositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003epositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 75px;\"\u003e\n \u003cp\u003eControl 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 19px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 55px;\"\u003e\n \u003cp\u003e37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 55px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003eDay 9:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 202px;\"\u003e\n \u003cp\u003e39.8\u0026deg;C\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 84px;\"\u003e\n \u003cp\u003epositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 73px;\"\u003e\n \u003cp\u003epositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003epositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 75px;\"\u003e\n \u003cp\u003eControl 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 19px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 55px;\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 55px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003eDay 9:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 202px;\"\u003e\n \u003cp\u003e40.6\u0026deg;C\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 84px;\"\u003e\n \u003cp\u003epositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 73px;\"\u003e\n \u003cp\u003epositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003epositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 75px;\"\u003e\n \u003cp\u003eControl 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 19px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 55px;\"\u003e\n \u003cp\u003e44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 55px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003eDay 8:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 202px;\"\u003e\n \u003cp\u003e40.2\u0026deg;C, Lethargic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 84px;\"\u003e\n \u003cp\u003epositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 73px;\"\u003e\n \u003cp\u003epositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003epositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 75px;\"\u003e\n \u003cp\u003eControl 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 19px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 55px;\"\u003e\n \u003cp\u003e41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 55px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003eDay 8:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 202px;\"\u003e\n \u003cp\u003e40.0\u0026deg;C, Lethargic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 84px;\"\u003e\n \u003cp\u003epositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 73px;\"\u003e\n \u003cp\u003epositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003epositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 75px;\"\u003e\n \u003cp\u003eControl 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 19px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 55px;\"\u003e\n \u003cp\u003e44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 55px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003eDay 8:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 202px;\"\u003e\n \u003cp\u003e39.8\u0026deg;C, Lethargic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 84px;\"\u003e\n \u003cp\u003epositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 73px;\"\u003e\n \u003cp\u003epositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003epositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 75px;\"\u003e\n \u003cp\u003eControl 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 19px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 55px;\"\u003e\n \u003cp\u003e42 (2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 55px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003eDay 9:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 202px;\"\u003e\n \u003cp\u003e40.1\u0026deg;C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 84px;\"\u003e\n \u003cp\u003epositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 73px;\"\u003e\n \u003cp\u003epositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003epositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 75px;\"\u003e\n \u003cp\u003eControl 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 19px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 55px;\"\u003e\n \u003cp\u003e42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 55px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003eDay 9:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 202px;\"\u003e\n \u003cp\u003e39.9\u0026deg;C, dark urine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 84px;\"\u003e\n \u003cp\u003epositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 73px;\"\u003e\n \u003cp\u003epositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003epositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 75px;\"\u003e\n \u003cp\u003eControl 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 19px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 55px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003eDay 36:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 202px;\"\u003e\n \u003cp\u003eLethargic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 84px;\"\u003e\n \u003cp\u003epositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 73px;\"\u003e\n \u003cp\u003epositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003epositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 75px;\"\u003e\n \u003cp\u003eControl 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 19px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 55px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003eDay 37:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 202px;\"\u003e\n \u003cp\u003eDark urine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 84px;\"\u003e\n \u003cp\u003epositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 73px;\"\u003e\n \u003cp\u003epositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003epositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 75px;\"\u003e\n \u003cp\u003eControl 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 19px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 55px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003eDay 35:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 202px;\"\u003e\n \u003cp\u003e39.7\u0026deg;C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 84px;\"\u003e\n \u003cp\u003epositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 73px;\"\u003e\n \u003cp\u003epositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003epositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 75px;\"\u003e\n \u003cp\u003eControl 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 19px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 55px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003eDay 36:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 202px;\"\u003e\n \u003cp\u003e40.2\u0026deg;C\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 84px;\"\u003e\n \u003cp\u003epositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 73px;\"\u003e\n \u003cp\u003epositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003epositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 75px;\"\u003e\n \u003cp\u003eControl 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 19px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 55px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003eDay 35:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 202px;\"\u003e\n \u003cp\u003eLethargic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 84px;\"\u003e\n \u003cp\u003epositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 73px;\"\u003e\n \u003cp\u003epositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003epositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 75px;\"\u003e\n \u003cp\u003eControl 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 19px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 55px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003eDay 36:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 202px;\"\u003e\n \u003cp\u003eLethargic, dark urine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 84px;\"\u003e\n \u003cp\u003epositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 73px;\"\u003e\n \u003cp\u003epositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003epositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 75px;\"\u003e\n \u003cp\u003eControl 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 19px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 55px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003eDay 35:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 202px;\"\u003e\n \u003cp\u003e39.6\u0026deg;C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 84px;\"\u003e\n \u003cp\u003epositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 73px;\"\u003e\n \u003cp\u003epositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003epositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 75px;\"\u003e\n \u003cp\u003eControl 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 19px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 55px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003eDay 36:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 202px;\"\u003e\n \u003cp\u003e40.0\u0026deg;C\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 84px;\"\u003e\n \u003cp\u003epositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 73px;\"\u003e\n \u003cp\u003epositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003epositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 75px;\"\u003e\n \u003cp\u003eIVP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 19px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 55px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" style=\"width: 278px;\"\u003e\n \u003cp\u003eNo abnormal sign or rectal temperature\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 84px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 73px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 75px;\"\u003e\n \u003cp\u003eIVP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 19px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 55px;\"\u003e\n \u003cp\u003e0 (2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e0 (5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" style=\"width: 278px;\"\u003e\n \u003cp\u003eNo abnormal sign or rectal temperature\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 84px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 73px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 75px;\"\u003e\n \u003cp\u003eIVP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 19px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 55px;\"\u003e\n \u003cp\u003e0 (3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e0 (3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" style=\"width: 278px;\"\u003e\n \u003cp\u003eNo abnormal sign or rectal temperature\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 84px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 73px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 75px;\"\u003e\n \u003cp\u003eIVP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 19px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 55px;\"\u003e\n \u003cp\u003e0 (3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e0 (4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" style=\"width: 278px;\"\u003e\n \u003cp\u003eNo abnormal sign or rectal temperature\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 84px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 73px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 75px;\"\u003e\n \u003cp\u003eIVP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 19px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 55px;\"\u003e\n \u003cp\u003e0 (1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e0 (2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" style=\"width: 278px;\"\u003e\n \u003cp\u003eNo abnormal sign or rectal temperature\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 84px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 73px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 75px;\"\u003e\n \u003cp\u003eIVP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 19px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 55px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e0 (2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" style=\"width: 278px;\"\u003e\n \u003cp\u003eNo abnormal sign or rectal temperature\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 84px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 73px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 75px;\"\u003e\n \u003cp\u003eIVP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 19px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 55px;\"\u003e\n \u003cp\u003e0 (5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e0 (1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" style=\"width: 278px;\"\u003e\n \u003cp\u003eNo abnormal sign or rectal temperature\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 84px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 73px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 75px;\"\u003e\n \u003cp\u003eIVP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 19px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 55px;\"\u003e\n \u003cp\u003e0 (4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e0 (1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" style=\"width: 278px;\"\u003e\n \u003cp\u003eNo abnormal sign or rectal temperature\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 84px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 73px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eControl 1 dogs were infested on Day 1 with 50 (\u0026plusmn; 2) \u003cem\u003eD. reticulatus\u003c/em\u003e with a \u003cem\u003eB. canis\u003c/em\u003e infection rate of 23%\u003c/p\u003e\n\u003cp\u003eControl 2 dogs were infested on Day 1 with 50 (\u0026plusmn; 2) \u003cem\u003eD. reticulatus\u003c/em\u003e with a \u003cem\u003eB. canis\u003c/em\u003e infection rate of 23%\u003c/p\u003e\n\u003cp\u003eTreated group: dogs were treated on Day 0 with NexGard Spectra\u0026reg; at a mean afoxolaner dose of 2.75 mg/kg and were infested on Days 1 and 28 with 50 (\u0026plusmn; 2) \u003cem\u003eD. reticulatus\u003c/em\u003e with a \u003cem\u003eB. canis\u003c/em\u003e infection rate of 23%.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e2\u003c/sup\u003ethis column describes the counts of live (and dead) attached ticks. Otherwise, no live free ticks were found, and 3 dead free ticks were found on Day 7 and Day 34, each time in the treated group.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e3\u003c/sup\u003eall dogs from Control group 1 and Control group 2 were removed from the study on days 21 and 42, respectively, and no further evaluation was performed as they had been confirmed positive for \u003cem\u003eB. canis\u003c/em\u003e by IFA analysis.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Afoxolaner, Dog, Babesia canis, Dermacentor reticulatus, Prevention of transmission","lastPublishedDoi":"10.21203/rs.3.rs-5796842/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5796842/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCanine babesiosis is a tick-borne disease of significant veterinary importance in dogs. It is caused by \u003cem\u003eBabesia canis\u003c/em\u003e in Europe, where it is transmitted by \u003cem\u003eDermacentor reticulatus\u003c/em\u003e ticks.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003eA blinded, randomized, Good Clinical Practice (GCP) and negative control experimental study was conducted to verify the efficacy of NexGard Spectra\u003csup\u003e®\u003c/sup\u003e in reducing the transmission of\u003cem\u003e B. canis \u003c/em\u003eby \u003cem\u003eD. reticulatus\u003c/em\u003e to dogs. NexGard Spectra\u003csup\u003e®\u003c/sup\u003e (IVP) is an oral product for dogs combining afoxolaner an acaricide/insecticide compound from the isoxazoline class and milbemycin oxime a nematicide compound from the macrocyclic lactone class. Three groups of eight dogs were used, one group orally treated on Day 0 with the IVP at the minimum recommended dose and two untreated control groups. On Day 1, dogs from the treated group and from the control group 1 were infested with 50 \u003cem\u003eD. reticulatus\u003c/em\u003e adult ticks of 50/50 sex ratio infected with \u003cem\u003eB. canis\u003c/em\u003e at a 23% infection rate. On Day 28, dogs from the treated group and from the control group 2 were infested similarly to those on Day 1. Ticks were removed 6 days after each infestation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSeven to nine days after each infestation, all untreated control dogs displayed clinical signs of canine babesiosis, i.e. lethargy, and/or dark urine, and/or \u0026gt;39.5°C rectal temperature. Blood was collected for microscopical blood smear examination, and for PCR analysis. The blood smears from all untreated control dogs were positive for \u003cem\u003eBabesia\u003c/em\u003e and all the PCR analyses were positive for \u003cem\u003eB. canis.\u003c/em\u003e The control dogs were rescue treated. All control dogs were confirmed positive for \u003cem\u003eB. canis\u003c/em\u003e by IFA on Day 21 (control group 1) and on Day 42 (control group 2). None of the IVP-treated dogs expressed any clinical sign of canine babesiosis following each of the two infestations of Days 1 and 28 and until Day 56. Blood was collected for IFA and PCR analyses from the treated dogs on Days 21, 28, 42 and 56, and all results were negative.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, the antiparasitic treatment prevented the transmission of \u003cem\u003eB. canis\u003c/em\u003e to dogs following induced infestations.\u003c/p\u003e","manuscriptTitle":"Efficacy of an oral combination of afoxolaner and milbemycin oxime for the prevention of transmission of Babesia canis by Dermacentor reticulatus ticks to dogs","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-31 09:04:00","doi":"10.21203/rs.3.rs-5796842/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Accepted","date":"2025-03-28T17:26:06+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-28T14:43:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"325277857646385814794504189630082137233","date":"2025-03-28T14:36:28+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-28T14:18:53+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-28T14:15:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"Parasites \u0026 Vectors","date":"2025-03-28T13:59:09+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"55d444d4-278e-4ec5-ab37-8db04e8710a3","owner":[],"postedDate":"March 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-04-21T16:02:18+00:00","versionOfRecord":{"articleIdentity":"rs-5796842","link":"https://doi.org/10.1186/s13071-025-06787-y","journal":{"identity":"parasites-and-vectors","isVorOnly":false,"title":"Parasites \u0026 Vectors"},"publishedOn":"2025-04-15 15:57:11","publishedOnDateReadable":"April 15th, 2025"},"versionCreatedAt":"2025-03-31 09:04:00","video":"","vorDoi":"10.1186/s13071-025-06787-y","vorDoiUrl":"https://doi.org/10.1186/s13071-025-06787-y","workflowStages":[]},"version":"v1","identity":"rs-5796842","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5796842","identity":"rs-5796842","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}