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The Neospora caninum Calcium-dependent protein kinase 1 (NcCDPK1) inhibitor BKI-1294 had demonstrated excellent efficacy in a pregnant mouse model of neosporosis, and was also highly efficacious in a pregnant sheep model of toxoplasmosis. In this work, we present the efficacy of BKI-1294 treatment (dosed 5 times orally every 48 h) starting 48 h after intravenous infection of sheep with 105 Nc-Spain7 tachyzoites at mid-pregnancy. In the dams, BKI-1294 plasma concentrations were above the IC50 for N. caninum for 12–15 days. In treated sheep, when they were compared to untreated ones, we observed a minor increase in rectal temperature, higher IFNγ levels after blood stimulation in vitro, and a minor increase of IgG levels against N. caninum soluble antigens through day 28 post-infection. Additionally, the anti-NcSAG1 and anti-NcSAG4 IgGs were lower in treated dams on days 21 and 42 post-infection. However, BKI-1294 did not protect against abortion (87% foetal mortality in both infected groups, treated and untreated) and did not reduce transplacental transmission, parasite load or lesions in placentomes and foetal brain. The lack of foetal protection was likely caused by short systemic exposure in the dams and suboptimal foetal exposure to this parasitostatic drug, which was unable to reduce replication of the likely established N. caninum tachyzoites in the foetus at the moment of treatment. New BKIs with a very low plasma clearance and good ability to cross the blood-brain and placental barriers need to be developed., Graphical abstract Image 1, Highlights • BKI-1294 triggers a transiently decrease in anti-N. caninum IgG levels in sheep. • BKI-1294 does not protect against abortion and vertical transmission of N. caninum. • The efficacy of BKI-1294 is different in pregnant mice and sheep models of neosporosis.

In this work, an experimental model for chronic besnoitiosis in bovine was developed and characterized. Using a previously established calf model, two new variables (parasite stage and inoculation route) were combined and used. Twelve Holstein Friesian 3-month-old male calves were randomly divided into four groups of three animals each. Bradyzoites were obtained from a chronically infected bull and used for inoculation via three different inoculation routes. Three groups were inoculated with 106 bradyzoites by intravenous (G1), subcutaneous (G2) and intradermal (G3) routes, and a non-infected control group (G4) was inoculated with PBS. The trial lasted for 90 days and included daily clinical monitoring as well as weekly skin biopsies and blood sampling. Sera were obtained to analyse both cellular and humoral responses. Once the calves were euthanized, tissues from the skin, eyes, respiratory and reproductive tracts, among others, were collected to study presence of the parasite. Clinically, the infection was classified as mild to moderate for the acute stage since all infected calves showed lymphadenopathy from four days post-infection (pi) and fever from one week pi until 24 days pi. However, the most relevant results were achieved during the chronic stage that was classified as moderate to severe. In fact, pathognomonic conjunctival cysts were observed in all infected calves from 40 days pi onwards and were more abundant in G3. Moreover, one calf from this group developed skin lesions (49 days pi). The microscopic tissue cysts and Besnoitia DNA were detected primarily in skin, reproductive tract and respiratory tissue samples, and parasite load was higher in G3. In conclusion, the parasite stage (bradyzoite) and the inoculation route are key factors that influence the outcome of an infection. In particular, the intradermal route led to more severe clinical signs of the chronic phase in the inoculated calves.

Brainstem auditory evoked potentials (BAEP) are useful to diagnose neurologic and genetic disorders in cattle but normal parameters and its application to detect changes in hearing are not demonstrated. The aims of this study were to develop a protocol for obtaining BAEP in Holstein calves and to establish the normal parameters of peak latencies, interpeak latencies (IPL) and waveforms of BAEP in this species. Ten male Holstein calves, aged 2.5 months and weighting 90.7 ± 4.0 kg were used. All animals were previously sedated with 0.08 mg/kg xylazine hydrochloride administered intramuscularly. The BAEP analysis was recorded using the IHS SmartEP-ASSR program connected with electrode needles placed on the standardized locations of the animal's head. Monoaurally presented broadband click stimuli (0.1 ms) were used for BAEP stimulation, with contralateral masking of 30 dB SPL. Stimuli were presented at a rate of 35.5 pulses per second (PPS) and evoked responses were collected and averaged 450–1500 times for each sound intensity. Earphones were inserted into the ear canal to present the acoustic stimulus and to attenuate environmental noise. Peak latencies, IPL, waveforms and auditory thresholds were recorded and analyzed in the right ear and were like those described in previous papers in adult male bulls. Auditory thresholds obtained in all animals were 33 ± 1.5 dB SPL. Peak latencies were 1.4 ± 0.07 ms (Peak I), 2.4 ± 0.10 ms (Peak II), 3.3 ± 0.10 ms (Peak III), 4.4 ± 0.19 ms (Peak IV) and 5.6 ± 0.28 ms (Peak V). IPL were 1.9 ± 0.12 ms (IPL I-III), 4.1 ± 0.29 ms (IPL I-V), 2.2 ± 0.19 ms (IPL II-V) and 2.2 ± 0.19 ms (IPL III-V). Brainstem auditory evoked potentials are a minimally-invasive method to evaluate level of audition in Holstein calves. In addition, BAEP is a method that can be applied in young calves and it would work on-farm to select the animals with better conditions.

13 páginas, 2 tablas, 7 figuras., Neospora caninum is one of the main causes of abortion in cattle, and recent studies have highlighted its relevance as an abortifacient in small ruminants. Vaccines or drugs for the control of neosporosis are lacking. Bumped kinase inhibitors (BKIs), which are ATP-competitive inhibitors of calcium dependent protein kinase 1 (CDPK1), were shown to be highly efficacious against several apicomplexan parasites in vitro and in laboratory animal models. We here present the pharmacokinetics, safety and efficacy of BKI-1553 in pregnant ewes and foetuses using a pregnant sheep model of N. caninum infection. BKI-1553 showed exposure in pregnant ewes with trough concentrations of approximately 4 mu M, and of 1 mu M in foetuses. Subcutaneous BKI-1553 administration increased rectal temperatures shortly after treatment, and resulted in dermal nodules triggering a slight monocytosis after repeated doses at short intervals. BKI-1553 treatment decreased fever in infected pregnant ewes already after two applications, resulted in a 37-50% reduction in foetal mortality, and modulated immune responses; IFN. levels were increased early after infection and IgG levels were reduced subsequently. N. caninum was abundantly found in placental tissues; however, parasite detection in foetal brain tissue decreased from 94% in the infected/untreated group to 69-71% in the treated groups. In summary, BKI-1553 confers partial protection against abortion in a ruminant experimental model of N. caninum infection during pregnancy. In addition, reduced parasite detection, parasite load and lesions in foetal brains were observed., We gratefully acknowledge Jacobo Sampere from the SALUVETgroup (Complutense University of Madrid, Spain), Teresa Navarro andJosé María González (University of Zaragoza, Spain), Isabel Ayala,Lucas Troya, Clara Colmenero, Dolores González, Natalia Peral,Verónica Pérez, Ignacio Gómez and Isabel Santiago from the Clinical Veterinary Hospital (Complutense University of Madrid, Spain) for their excellent technical assistance, and also Rama Subba Rao Vidadala and Dustin Maly (Department of Chemistry, University of Washington) fortheir assistance in further purification of the study drug. The AnimalExperimentation Service (SEA) at the University of Zaragoza is ac-knowledged for providing their facilities to carry out the reproduction programme. Roberto Sánchez Sánchez is supported by a fellowship from the Spanish Ministry of Education, Culture and Sports (MECD), asa part of the Program of Training of University Teaching Staff (FPU,grant number FPU13/03438). Patricia Vázquez has a Juan de la Cierva-Formación post-doctoral contract (FJCI-2014-20982) from the Spanish Ministry of Economy and Competitiveness (MINECO). Andrew Hemphill is supported by the Swiss National Science Foundation grant(No. 310030 165782). This work was supported by the Public Health Service, National Institutes of Health, Bethesda, MD (grants R01 AI111341 and R01 HD 080670), the U.S. Department of Agriculture(grant 2014-67015-22106) and the Community of Madrid, Spain(PLATESA, S2013/ABI2906).

16 páginas, 3 tablas, 6 figuras., Previous studies on drug efficacy showed low protection against abortion and vertical transmission of Toxoplasma gondii in pregnant sheep. Bumped kinase inhibitors (BKIs), which are ATP-competitive inhibitors of calcium-dependent protein kinase 1 (CDPK1), were shown to be highly efficacious against several apicomplexan parasites in vitro and in laboratory animal models. Here, we present the safety and efficacy of BKI-1294 treatment (dosed orally at 100 mg/kg of body weight 5 times every 48 h) initiated 48 h after oral infection of sheep at midpregnancy with 1,000 TgShSp1 oocysts. BKI-1294 demonstrated systemic exposure in pregnant ewes, with maximum plasma concentrations of 2 to 3 M and trough concentrations of 0.4 M at 48 h after each dose. Oral administration of BKI-1294 in uninfected sheep at midpregnancy was deemed safe, since there were no changes in behavior, fecal consistency, rectal temperatures, hematological and biochemical parameters, or fetal mortality/morbidity. In ewes infected with a T. gondii oocyst dose lethal for fetuses, BKI-1294 treatment led to a minor rectal temperature increase after infection and a decrease in fetal/lamb mortality of 71%. None of the lambs born alive in the treated group exhibited congenital encephalitis lesions, and vertical transmission was prevented in 53% of them. BKI-1294 treatment during infection led to strong interferon gamma production after cell stimulation in vitro and a low humoral immune response to soluble tachyzoite antigens but high levels of anti-SAG1 antibodies. The results demonstrate a proof of concept for the therapeutic use of BKI-1294 to protect ovine fetuses from T. gondii infection during pregnancy., We gratefully acknowledge the SALUVET group (Complutense University of Madrid, Spain), Luis Miguel Ferrer, Jose Ventura, José Calasanz Jiménez, Francisco Saura, Teresa Navarro, and José María González from the University of Zaragoza (Spain), and Victor Herrero and Javier Blanco from Clinical Veterinary Hospital (Complutense University of Madrid, Spain) for their excellent technical assistance, Dale Kempf and Kennan Marsh, from AbbVie, Inc., for their suggestion of the Phosal vehicle used in the experiments reported here, Rama S. R. Vidadala and Dustin J. Maly, Department of Chemistry, University of Washington, for characterization and further purification of BKI-1294, and advice from Robert Choy and Eugenio L. de Hostos from the PATH Drug Development Program. The Animal Experimentation Service (SEA) at the University of Zaragoza is acknowledged for providing their facilities to carry out the reproduction program. R.S.-S. is supported by a fellowship from the Spanish Ministry of Education, Culture and Sports (MECD), as a part of the Program of Training of University Teaching Staff (FPU; grant number FPU13/03438). A.H. is supported by a Swiss National Science Foundation grant (no. 310030-165782). This work was supported by the Public Health Service, National Institutes of Health, Bethesda, MD (grants R01 AI 111341 and R01 HD 080670), the U.S. Department of Agriculture (grant 2014-67015-22106), and the Community of Madrid, Spain (PLATESA2, P2018/BAA-4370). I.F., A.H., K.K.O., W.C.V.V., and L.M.O.-M. conceived the study and participated in its design. R.S.-S. wrote the manuscript, with results interpretation and discussion inputs from I.F., J.R.-C., A.H., M.A.H., L.K.B., W.C.V.V., and L.M.O.-M. J.J.R. selected the animals and executed the reproductive program. R.S.-S., I.F., and M.R. carried out oocyst infection and drug administration. R.S.-S., I.F., M.R., M.P.D., M.G.-H., E.T., and J.B. participated in clinical examination and sampling of animals and performed necropsies and hematological, biochemical, and histopathological analyses. M.H., L.K.B., R.C., G.R.W., and K.K.O. determined the pharmacokinetics of the compound. R.S.-S. performed peripheral blood stimulation assay, serological assays, PCR analyses, statistical analysis, and interpretation of the results. All authors read and approved the final manuscript. W.C.V.V. is the president and co-owner of ParaTheraTech, Inc., a company that is developing BKIs for animal health. W.C.V.V. did not perform the experiments or interpret the results of the experiments, but he did edit the paper and helped plan the experiments. The other authors have no competing interests to declare.

OBJECTIVE To compare the effectiveness of an ultrasound-guided paravertebral nerve blockade technique (UGPNB) with distal and proximal paravertebral nerve blockade techniques without ultrasound guidance (DPNB and PPNB, respectively) in calves. ANIMALS 4 calf cadavers and 7 healthy calves. PROCEDURES A suitable acoustic window was identified to facilitate access to the T13, L1, and L2 spinal nerves in cadavers and live calves. In cadavers, nerves were injected with dye under ultrasound guidance. In calves, the UGPNB, DPNB, and PPNB were performed in random order at 10-day intervals by injection of an anesthetic solution containing 2% lidocaine hydrochloride. Nociceptive withdrawal responses were assessed to determine the effects of the blockades. RESULTS In cadavers, nerve staining success rates (ie, ≥ 2-cm-long dye path) achieved with ultrasound guidance were 88% (T13 [ventral branch]), 75% (T13 and L1 [dorsal branches] and L1 and L2 [ventral branches]), and 38% (L2 [dorsal branch]). The nerves were each identified as a hyperechoic band in a longitudinal plane. In calves, the UGPNB, DPNB, and PPNB reduced the withdrawal response to the noxious stimulus, mainly in the dorsal-cranial, dorsal-caudal, and ventral-cranial areas of the flank. Overall, the UGPNB resulted in a better nociceptive cumulative score, administering only one half of the local anaesthetic dose, compared with findings for the DPNB and PPNB. However, time to perform the UGPNB was longer. CONCLUSIONS AND CLINICAL RELEVANCE The UGPNB evaluated may be an improved alternative to the DPNB and PPNB for provision of anesthesia for flank surgery in calves. However, effectiveness of the UGPNB should be evaluated in a clinical setting and in adult cattle.

Superficial nerves can be visualized through ultrasonography in the cattle and facilitate local anesthetic disposition around nerve structures. Expected advantages include a higher successful rate of nerve block improving the degree and duration of the block. Among others, conduction nerves of clinical interest in cattle include the paravertebral nerves, nerves of the epidural space, the brachial plexus, and the sciatic and femoral nerves, and nerves of the head.

This study aimed to describe a suitable acoustic window to facilitate access to the sciatic and femoral nerves in calves and to study the effects of their blockade with local anaesthetics. The neuroanatomical and ultrasound (US) study was performed on the cadavers of 10 calves, and the effects of 2% lidocaine with epinephrine (0.2 mL/kg) were determined in five healthy calves. The sciatic nerve in the cadavers was easily visualised as a hyperechoic band distal to the femoral greater trochanter and caudal to the femoral shaft. The femoral nerve in the cadavers was not easily identified, and was visualised as a hyperechoic oval structure situated immediately medial to the psoas major muscle and lateral to the femoral artery. The sciatic nerve was stained by methylene blue, injected under US guidance, in 9/10 cases, and the femoral nerve was stained in 6/10 cases. Sciatic nerve blockade under US guidance produced adduction of the limb with metatarsophalangeal joint flexion, while the femoral nerve blockade produced reduced weight bearing. The sciatic nerve blockade produced a reduced response to the noxious stimulus, mainly in the phalanges, proximal and distal metatarsus, tarsus and tibia and, following the femoral nerve blockade, in the medial subarea of the femur. However, femoral nerve blockade produced a more variable degree of blockade. In conclusion, US -guided anaesthetic blockade of the sciatic nerve in calves may be considered for surgery in the distal pelvic limb, although further studies are necessary to determine its clinical application.

The immobilisation and anaesthesia of free-ranging cattle requires the administration of appropriate drugs in small volume via rifle or dart. The objective of this randomised controlled study was to test the capacity of a concentrated combination of tiletamine–zolazepam (TZ), ketamine (K) and detomidine (D) (TZKD) to immobilise/anaesthetise calves. Following administration of low, medium and high doses of TZKD to six healthy animals IM, the time-of-onset and duration of anaesthesia were recorded, in addition to standard cardio-respiratory parameters. Two noxious stimuli were applied to assess the analgesic effect of the combination. TZKD produced a dose-dependent anaesthetic action associated with respiratory depression and moderate hypoxaemia. Total recumbency lasted from 1 h (with low dose) to 2 h (with medium and high doses). The findings indicate that TZKD induces anaesthesia in calves, suitable not only for animal immobilisation, but also to carry out minor surgical procedures with or without additional local analgesia. Respiratory depression was the most severe side-effect and careful patient monitoring is recommended when using this drug combination.

Background Chronic kidney disease (CKD) is associated with poor renal and cardiovascular outcomes, and early identification largely depends on general practitioners' (GPs') awareness of it. To date, no study has evaluated CKD prevalence in patients with hypertension in primary care. Study Design Cross-sectional evaluation of the Italian GPs' database. Setting & Participants 39,525 patients with hypertension representative of the Italian hypertensive population followed up by GPs in 2005. Factor Estimated glomerular filtration rate (eGFR); eGFR 2 was defined as CKD. Outcomes GPs' awareness of CKD assessed using International Classification of Diseases, Ninth Revision, Clinical Modification diagnostic codes for CKD, and blood pressure (BP) control. Measurements Data concerning serum creatinine levels, BPs, and antihypertensive medications were obtained for each patient from the GPs' database; eGFR was calculated according to the 4-variable Modification of Diet in Renal Disease (MDRD) Study equation. Results CKD prevalence was 23%, but kidney disease was diagnosed by GPs in only 3.9% of patients. BP control was inadequate in patients with CKD and those with eGFR >60 mL/min/1.73 m 2 , with only 44% of patients reaching a BP target 2 whose GPs were aware of CKD were more likely to reach recommended BP target values (OR, 1.35; 95% CI, 1.15-1.59; P Limitations The prevalence of decreased eGFR may be overestimated because of the lack of creatinine calibration. Proteinuria data were not available. Conclusions Awareness of CKD by GPs is critical for achieving the recommended guideline BP targets. However, awareness of CKD by GPs is still far too low, highlighting the need to systematically adopt eGFR for more accurate identification of CKD in high-risk populations.

Chronic kidney disease (CKD) is associated with elevated cardiovascular morbidity and mortality. Cardiovascular disease (CVD) is highly prevalent in CKD and mortality due to CVD is 5- to 200-fold higher in dialysis patients than in the general population.

The objectives of this study were to determine: (1) the sedative effects of dexmedetomidine in combination with methadone, midazolam, or both, and (2) the propofol dose required to achieve endotracheal intubation in healthy dogs. Seven healthy Beagle dogs were included in a prospective experimental, crossover, randomised and masked design. All dogs received four treatments IM, with at least 1 week between sessions, as follows: dexmedetomidine 5 µg/kg (D) alone, or combined with methadone 0.3 mg/kg (DMe), midazolam 0.3 mg/kg (DMi), or both (DMeMi). The degree of sedation was evaluated using a numerical scale (maximum 15 points). The dose of propofol required for intubation was also calculated for each group. Recovery time and quality were determined. Statistical analysis was performed using parametric (ANOVA) and nonparametric tests (Friedman, Cochran Q), as appropriate. The degree of sedation obtained with DMe and DMeMi (13, [7–14]; 13, [6–14], respectively) was significantly higher than in the control group (2, [1–4]; P = 0.023, P = 0.006, respectively). The required dose of propofol was lower in all groups (DMi, 1.5 ± 0.5 mg/kg, P = 0.002; DMe, 1.2 ± 0.5 mg/kg, P 0.001; DMeMi, 0.9 ± 0.3 mg/kg) than in the control group (2.9 ± 0.9 mg/kg; P 0.001). Recovery quality was not different between groups ( P = 0.137). In healthy dogs, the addition of midazolam did not enhance the sedative effects of dexmedetomidine or a dexmedetomidine–methadone combination at the doses studied, and propofol requirements were reduced. The sedative effect of dexmedetomidine was enhanced with methadone, and the required dose of propofol was reduced.

Background. Chronic kidney disease (CKD) is associated with poor renal and cardiovascular (CV) outcome, and early identification largely depends on the general practitioners' (GPs) awareness of it. Only a few studies have evaluated the prevalence of CKD in type 2 diabetes in primary care, and no studies are available on hypertensive diabetics. Thus, the aim of this study was to assess the prevalence of CKD and its association with CV morbidity in such a population. Methods. On the basis of an Italian national project involving GPs and nephrologists, we retrieved demographic, laboratory and clinical data regarding 7582 hypertensive type 2 diabetics (3564 men; age 25-89 years) who were selected using the diagnostic code Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM) for diabetes and hypertension. Blood pressure (BP) values, serum creatinine, ECG-diagnosed left ventricular hypertrophy (LVH) and the occurrence of previous major CV events were obtained for each patient from the GPs' Health Search Database. Estimated glomerular filtration rate (GFR) was calculated according to the four-variable MDRD equation. CKD was defined as an estimated GFR < 60 mL/min/ 1.73 m 2 . Results. CKD prevalence was 26%, although renal disease was diagnosed by GPs in only 5.4% of cases. The prevalence of both LVH and major CV events was 8%. Adequate BP control was only achieved in 10.4% of patients. Patients whose GFR was

Arterial hypertension together with proteinuria is one of the most important factors associated with the progression of both diabetic and nondiabetic chronic kidney disease. In this review, the role of hypertension and proteinuria in renal disease progression, the BP target that should be achieved to slow the progression of renal damage, and the influence of baseline and current proteinuria on the renoprotective effects of antihypertensive therapy are discussed thoroughly. The interaction between the renoprotective effects of specific antihypertensive agents--mostly angiotensin-converting enzyme inhibitors and angiotensin receptor blockers--and the level of achieved BP also are evaluated. The body of evidence provided by several studies emphasizes the importance of both lowering BP and inhibiting the renin-angiotensin system as specific goals for renal and cardiovascular protection in chronic kidney disease.

There is a rising incidence and prevalence of ESRD as a result of diabetes, with poor outcome and growing costs. Recently, two large trials, the Irbesartan Diabetic Nephropathy Trial (IDNT) and Reduction of Endpoints in NIDDM with the Angiotensin II Antagonist Losartan (RENAAL), showed that angiotensin receptor blockers (ARB) are more effective than traditional antihypertensive therapies at reducing progression toward ESRD in hypertensive patients with type 2 diabetes and overt nephropathy, regardless of changes in BP. The results of these two trials were used to compare the costs of ARB with those of renal replacement therapy (dialysis and renal transplantation) in an effort to establish whether ARB are cost-saving because they delay ESRD. Two different pharmacoeconomic approaches were used. With regard to the RENAAL trial, the number of ESRD days on losartan therapy as compared with the number of ESRD days on standard antihypertensive therapy was calculated, and the difference between the two was combined with the costs of ESRD. In the IDNT trial, Markov models were applied to assess the economic impact of irbesartan and to extrapolate future clinical and cost outcomes. Several economic analyses were performed in the United States and in European countries. Applying pharmacoeconomic models showed that treatment with ARB was associated with a greater improvement in life expectancy and lower total costs compared with amlodipine and standard antihypertensive therapy. Therefore, treating patients with type 2 diabetes, nephropathy, and hypertension with ARB is life- and cost-saving compared with traditional antihypertensive therapy.