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Human respiratory syncytial virus (RSV) is a pneumovirus that causes severe infections in infants worldwide. Despite intensive research, safe and effective vaccines against RSV have remained elusive. The main reason is that RSV infection of children previously immunized with formalin-inactivated-RSV vaccines has been associated with exacerbated pathology, a phenomenon called RSV vaccine-enhanced respiratory disease. In parallel, despite the high RSV prevalence, only a minor proportion of children develop severe diseases. Interestingly, variation in the immune responses against RSV or following RSV vaccination could be linked with differences of exposure to microbes during childhood. Gammaherpesviruses (γHVs), such as the Epstein-Barr virus, are persistent viruses that deeply influence the immune system of their host and could therefore affect the development of pneumovirus-induced immunopathologies for the long term. Here, we showed that a previous ɣHV infection protects against both pneumovirus vaccine-enhanced disease and pneumovirus primary infection and that CD8 T cells are essential for this protection. These observations shed a new light on the understanding of pneumovirus-induced diseases and open new perspectives for the development of vaccine strategies.

During late summer 2016, in a northwest European region extending over Belgium, the Netherlands and the eastern border of the German state of North Rhine Westphalia, an outbreak of wild bird deaths occurred similar to those reported on the continent since 1996. Dead birds were necropsied and examined by complementary methods. Pathologic and immunohistological investigations strongly suggested an infection by Usutu virus. Subsequently, genomic segments of the said virus were detected, the virus was isolated and its complete genome was sequenced. The strain, designated Usutu-LIEGE, is a close phylogenetic relative of those isolated in Germany which form a distinct group within the USUV phylogeny, the so-called Europe_3 lineage. Should this outbreak recapitulate the characteristics of those in southwest Germany in 2011 and in/around Vienna (Austria) in 2001, it is expected that specific avian populations in the affected area will face a significant reduction in size for a few years.

In the present study, we compare a classical slow freezing (SLF) method and an aseptic vitrification (Vitrif) technique to cryopreserve a stable primordial germ cell (PGCs) line issued from the Ardennaise chicken breed. Viability immediately after warming was close to 80% and did not differ between the two cryopreservation methods. Proliferation tended to be slower for both cryopreservation methods compared with controls, but the difference was significant only for Vitrif. No difference was found between the two methods after flow cytometry analysis of stage-specific embryonic antigen-1 expression and reverse transcription–polymerase chain reaction on several factors related to PGC phenotype. After 1 week in culture, all cryopreserved cells reached controls' main morphologic and expanding (viability/proliferation) features. However, SLF generated more unwanted cells clusters than Vitrif. After injection of the PGCs into recipient embryos, vitrified PGCs reported a clear, yet not significant, tendency to colonize the gonad at a higher rate than slow frozen PGCs. SLF in cryovials remains simple, inexpensive, and less technically demanding than Vitrif. Nevertheless, the intrinsic advantages of our aseptic Vitrif method and the present study suggest that this should be considered as safer than classical SLF for cryopreserving chicken PGCs.

The pestivirus bovine viral diarrhea virus (BVDV) is known to bind to the CD46 molecule, which subsequently promotes entry of the virus. Mapping of the BVD-virion-binding site has shown that two peptides, 66EQIV69 and 82GQVLAL87, located on antiparallel beta sheets in the most distal complement control protein module (CCP1), provide the attachment platform. In the present study, we reveal new CD46-encoding transcripts that are predicted to encode CCP1-containing soluble forms. Further, we show that the serum of most adult cattle contains soluble CD46 (sCD46) and that a recombinant soluble isoform neutralizes BVDV infectivity in an in vitro assay. We have then established an ELISA for determination of plasma sCD46 in a large cohort of animals. Overall, serum sCD46 amounts to 8 ± 18 ng/mL (mean ± SD, n = 440), with a IC [95-105] ranging from 6,4 to 9,8 ng/mL and extreme values ​​between 0 and 178 ng/mL. We found that sCD46 is not detectable in fetal and neonatal sera and that its plasma concentration increases progressively up to adulthood. We also detected high- and low-sCD46 performers and show that this phenotype does not depend of environment. As modern rearing techniques make it possible to disseminate genetically-determined phenotypes very quickly in a population, a large-scale study examining whether high-sCD46 animals provide epidemiological protection against BVDV infection and transmission should be undertaken.

The pestivirus bovine viral diarrhea virus (BVDV) is known to bind to the CD46 molecule, which subsequently promotes entry of the virus. Mapping of the BVD-virion-binding site has shown that two peptides, 66EQIV69 and 82GQVLAL87, located on antiparallel beta sheets in the most distal complement control protein module (CCP1), provide the attachment platform. In the present study, we reveal the existence of ten distinct allelic versions of the CCP1 module, varying significantly in frequency among taurine and indicine races. A complex mRNA splicing pattern was also evidenced for bovine CD46, generating three different serine-threonine-proline segments and five different cytoplasmic domains. The four most frequent allelic variants and the six splice variants were then expressed in BVDV-nonpermissive porcine cells and the quantity of progeny virions generated by each cell preparation was measured 48 h post-infection. As expected, ectopic expression of the 10 bovine CD46 isoforms rendered the PK15 cells permissive to BVDV, as attested by the 100,000-fold greater recovery of virions from these cells than from non-transfected cells. This permissivity increase was significantly lower (-33%, P

As the inbred mouse strain SJL/J displays increased resistance to several pathogens and as its immune system shows multiple specificities, it is tempting to infer a causal link between these observations. The first question that comes to mind is whether adaptive immunity plays a role, and a way to answer this question is to see if the resistance phenotype persists when adaptive immunity is depressed. Although it has long been known that irradiation causes repression of leukopoiesis in mice, the technical data available in the literature are of no help in the case of strain SJL/J, because it displays exceptional radioresistance. Here we show that exposure of SJL/J to ∼9Gy, an intensity corresponding to the lethal dose 50 for the species Mus musculus, leads to serious but reversible alteration of leukopoiesis. This conclusion stems from an examination of the effects, 1-11 days post-exposure, of whole-body gamma-ray irradiation on leukocyte populations in the thymus and peripheral blood of young adult females. Immunodepression was most severe 4 days post-exposure. As in other strains, leukocyte populations displayed differential radiosensitivity, B (CD19(+)) cells being most sensitive, T (CD4(+)/CD8(+)) cells moderately sensitive, and natural killer (NK1.1(+)) cells most resistant. Surprisingly, however, the helper/inducer T lymphocytes proved more resistant than the cytotoxic/suppressor T lymphocytes, contrarily to what is observed in other strains. The procedure described will make it possible to refute or establish reliably the existence of causal links between SJL-specific phenotypic traits and immune aberrations and to elucidate further the respective roles of innate and acquired immunity in determining the resistance of this strain to an array of viral diseases.

In October 2012, a 3-year-old bullfinch (Pyrrhula pyrrhula) held in captivity for its entire lifespan and a wild adult great spotted woodpecker (Dendrocopos major), both with neurological signs, were found 4 km from each other and 5 days apart in the Meuse Valley, Belgium. Non-suppurative encephalitis and mild degeneration and necrosis were identified in the brain and cerebellum, and Usutu virus antigen and RNA were detected by immunohistochemistry and real-time reverse transcriptase PCR, respectively. The two cases reported here represent the most western distribution of clinical disease in birds due to Usutu virus in Europe.

Mannheimiosis is the major respiratory disease among some ruminants, whereas it is not pathogenic for other mammals, an observation that has been attributed to a specific interaction between Mannheimia haemolytica leukotoxin (Lkt) and bovine or ovine CD18 subunit of lymphocyte function-associated antigen-1 (LFA-1) and Mac-1. We therefore hypothesized that Lkt utilizes CD18 as its receptor on caprine leukocytes as well. We have transiently transfected the beta2-integrins-deficient K-562 cell line with cDNAs encoding caprine CD11a and caprine CD18 to determine the susceptibility of the transfectants to Lkt-induced cytolysis. Flow cytometric analysis of the transfectants revealed surface expression of caprine LFA-1 and lysis by Lkt in a concentration-dependent manner whereas the parent cells were not. Moreover, K562 cells expressing caprine CD18 and human or bovine CD11a were also sensitive to Lkt whereas K-562 cells expressing caprine CD11a and human CD18 were not. Taken together, these results indicate that CD18 on caprine leukocytes serves as a receptor for Lkt.

Clostridium perfringens has been implicated in a broad array of enteric infections including the fatal haemorrhagic enteritis/enterotoxaemia syndrome in cattle. The beta2 toxin (CPB2), encoded by cpb2 , is suspected to be implicated in this syndrome. However, among C. perfringens isolates from cattle suspected of clostridial disease, an atypical allele was recently found to predominate at the cpb2 locus and atypical corresponding CPB2 proteins were shown to be poorly expressed, thus arguing against a biologically significant role of the beta2 toxin in clostridial diseases in cattle. This study compared genotype and phenotype of the beta2 toxin between C. perfringens isolates from a group of healthy calves ( n = 14, 87 isolates) and from a group of enterotoxaemic calves ( n = 8, 41 isolates). PCR results revealed the exclusive presence of the typical “consensus” cpb2 in the enterotoxaemic group. Western blot analysis demonstrated that the typical variant of CPB2 was often expressed in isolates from enterotoxaemic calves (43.9%) and infrequently in isolates from healthy cattle (6.9%). These data suggest that the typical variant of the CPB2 toxin may play a role in the pathogenesis of cattle enterotoxaemia.

Lymphocyte function-associated antigen-1 (LFA-1, αLβ2, CD11a/CD18) plays a critical role in the complex and well-orchestrated molecular interactions responsible for cell adhesion events required for normal and pathologic functions of the immune system. This review focuses on the diseases from various etiologies (genetic, bacterial, viral, neoplastic, allergic, and autoimmune) that are associated to lymphocyte function-associated antigen-1 with a tremendous impact on human and animal health.

Lymphocyte function-associated antigen-1 (LFA-1, CD11a/CD18, αLβ2) actively contributes to the molecular interactions responsible for normal functions of the immune system but is also associated to several diseases from various etiology (genetic, bacterial, viral, neoplastic, allergic, and autoimmune). In this way, the interaction between lymphocyte function-associated antigen-1 and its major ligand intercellular adhesion molecule-1 (ICAM-1 or CD54) has been extensively studied, leading to the development of therapeutic antibodies, peptides, and small inhibitory molecules.

The β 2 -integrin lymphocyte function-associated antigen-1 (LFA-1, α L β 2 , CD11a/CD18) is made of the association of the CD11a and CD18 subunits that each possesses a large extracellular region and short transmembrane and cytoplasmic parts. A general comparison among species enlights the importance of especially conserved functional regions, as well as their role in folding and heterodimerization. This review also focuses on providing insights into structural aspects that lead to lymphocyte function-associated antigen-1 ability, central to its critical role in the molecular interactions responsible for leukocyte adhesion and migration in the immune system, to modulate dynamically its adhesiveness through avidity (affinity and valency)-based mechanisms.

Toll-like receptors (TLR) are an important component in the innate immune response to a wide variety of pathogens. Recently, a series of studies has addressed the hypothesis that TLR4 also participates in the host innate response against respiratory syncytial virus (RSV), the leading cause of lower respiratory tract infections in infants and young children. In most of the studies available, RSV, which is not a natural pathogen of mice, has been systematically used in mouse models of human bronchiolitis, with conflicting results. Pneumonia virus of mice (PVM), a member of the pneumovirus genus, shares many similarities with RSV. The serological and structural relationships that exist between them suggest that the immune response to these viruses may be similar in their respective natural hosts. To determine the role of TLR4 in host defense against PVM, TLR4-competent and TLR4-deficient mice were intranasally infected with PVM. Variation of body weight, pulmonary function values, histopathology, and pulmonary viral loads were analyzed. None of the investigated clinical, functional, histological and virological parameters was different between strains, which demonstrates that the sensitivity of the mouse to its natural pneumovirus infection is independent of the presence or absence of TLR4 sensing.

The leukocyte integrins play a critical role in a number of cellular adhesive interactions during the immune response. We describe here the isolation and characterization of the ovine β 2 (CD18) subunit, common to the leukocyte β 2 -integrin family. The deduced 770-amino-acid sequence reveals a transmembrane protein with 81%, 83% and 95% identity with its murine, human and bovine homologues, respectively. Comparisons of CD18 sequences emphasize the functional importance of the β 2 subunit I-like domain and included metal ion-dependent adhesion site (MIDAS)-like motif and confirm that of the cytoplasmic tail. The data provided here will offer the possibility to explore new avenues in studies based on the ovine model.

Some MX proteins are known to confer a specific resistance against a panel of single-stranded RNA viruses. Many diseases due to such viruses are known to affect cattle worldwide, raising the possibility that the identification of an antiviral isoform of a bovine MX protein would allow the implementation of genetic selection programs aimed at improving innate resistance of cattle. With this potential application in mind, the present study was designed to isolate the bovine Mx1 gene including its promoter region and to investigate its genomic organisation and promoter reactivity. The bovine Mx1 gene is made up of 15 exons. All exon-intron boundaries conformed to the consensus sequences. A PCR product that contained a approximately 1-kb, 5'-flanking region upstream from the putative transcription start site was sequenced. Unexpectedly, this DNA region did not contain TATA or CCAAT motifs. A computer scan of the region disclosed a series of putative binding sites for known cytokines and transcription factors. There was a GAAAN(1-2)GAAA(C/G) motif, typical of an interferon-sensitive responsive element, between -118 and -107 from the putative transcription start site. There were also a NF-kappaB, two interleukin-6 binding sites, two Sp1 sites and five GC-rich boxes. The region also contained 12 stretches of the GAAA type, as described in all IFN-inducible genes. Bovine Mx1 expression was assessed by Northern blotting and immunofluorescence in the Madin Darby bovine kidney cells (MDBK) cell line treated with several stimuli. In conclusion, the bovine Mx1 gene and promoter region share the major structural and functional characteristics displayed by their homologs described in the rainbow trout, chicken, mouse and man.

Bovine respiratory syncytial virus (BRSV) infection is an important cause of outbreaks of respiratory disease among calves. This virus commonly induces mild to severe respiratory signs but, in a substantial proportion of cases, is also reported to be associated with paroxystic respiratory distress syndrome (PRDS). The pathogenesis of this 'malignant' clinical form has not been fully elucidated. The present study aimed at determining whether mast cell (MC) degranulation plays a role in the physiopathologic cascade leading to the PRDS. Paired serum samples were taken in herds during outbreaks of severe respiratory diseases (acute sera) and 3 weeks after (convalescent sera). Based on seroconversion to BRSV and clinical picture, 67 pairs of sera were selected from calves with a BRSV-associated PRDS for circulating MC tryptase determination. A MC metachromatic score was measured in post-mortem lungs from animals died from a BRSV-associated PRDS (principals) and compared with reference scores obtained from healthy lungs (controls). Levels of tryptase were significantly higher in acute sera (26.6 +/- 12.4 microg/l) compared to convalescent sera (8.4 +/- 7.8 microg/l; P

The effect of platelet-activating factor (PAF) on bovine pulmonary mast cells (MCs) was investigated in calves aged 1–4 months. PAF was administered over a 5-min period at a rate of 50 ng/kg/min to six animals, which were killed approximately 10 min later. No macroscopical lesions were observed in the lung tissue of five control calves. On semi-thin toluidine-blue stained sections, MC cytoplasm showed profuse deep-blue granulation. In the lung tissues of PAF-treated calves, striking interstitial, alveolar and interlobular oedema and emphysema were observed. MC granules occurred at the periphery of the cytoplasm and MC nuclei were easily detectable. Moreover, the number of granules in the MC cytoplasm was remarkably low. A few granules with some metachromatic material were sometimes detected outside the cell limits. Ultrastructurally, the matrix pattern of the MC granules in lung tissues of control calves was either amorphous or granular. In lung tissues of the PAF-treated calves, the granular matrix pattern was pronounced, varying from fine- to coarse-grained, and the MCs were of reduced size, with many filiform processes and exocytotic vesicles. These findings indicate that pulmonary MC degranulation was induced by PAF in healthy calves.

The presence of chymase-like proteinase in bovine mast cells was investigated by an enzyme-histochemical technique (naphthol-AS-D-chloroacetate as substrate) in normal skin, primary bronchus, lung and duodenum. The counts and distribution of chymase-positive and toluidine blue-positive mast cells were compared by means of successive staining. Mast cells with chymase-like activity were detected in all areas, but their proportion was greater in connective than mucosal tissues, with the exception of the skin. These results contrast with those obtained in rodents, in which chymase-like proteinases are detected in all tissues and also in all mast cells. Bovine mast cells are closer to those of human beings, in which chymase-containing mast cells predominate in connective tissues, including skin. The results suggest that more than one chymase subset is present, at least in duodenum. The possible occurrence of dual-specific chymase mast cells, as in other ruminants, is discussed.

Mast cell (MC) phenotypes may vary with respect to tissue site, sensitivity to degranulating agents, dependency on T lymphocytes and, above all, the composition of their granules. Proteinases (either trypsin-like or chymotrypsin-like) are granule constituents which provide an important means of distinguishing subtypes of MCs in man and rodents. The purpose of this study was to compare the distribution of MC trypsin-like protease (tryptase) in a variety of bovine tissues with the aim of examining MC heterogeneity. Tryptase was found in MCs regardless of their location within tissues. With respect to tryptase content, bovine MC distribution resembled more that of human and canine tissues than that of mice and rats. Comparison of the results yielded by enzyme- and immuno-histochemical staining suggested that a tryptase-negative, dual-specific chymase-positive MC subset occurred, at least in duodenal lamina propria, around bronchioles and within alveolar septa. The study also suggested that monoclonal antibodies raised against human tryptase can be used for quantitation of bovine tryptase in biological fluids; this offers a promising tool for evaluating the role of MC activation in disease.

The cardiovascular response to 5-hydroxytryptamine (5-HT) challenge has been previously described in cattle. Abrupt bradycardia, followed by tachycardia, triphasic systemic blood pressure response, and pulmonary hypertension were the major changes elicited by 5-HT. The purpose of the present study was to determine whether the cardiovascular response to 5-HT in calves was attributable to 5-HT2receptors. A specific 5-HT2antagonist (metrenperone, 0.05mg/kg) was administered intramuscular to six unsedated Friesian calves 30min before the animals were given a 5-min intravenous 5-HT infusion. Mean systemic arterial (SAP), mean pulmonary arterial (PAP), pulmonary capillary wedge (PW) pressures were obtained by means of fluid-filled catheters, and cardiac output (CO) was measured by the thermodilution technique. Heart rate, stroke volume, systemic (SVR) and pulmonary (PVR) vascular resistances were calculated. Administration of 5-HT after metrenperone induced a short-lasting period of severe bradycardia followed by tachycardia and increased CO. The systemic blood pressure response was exclusively hypotensive and associated with a decrease in SVR. Conversely, PAP, PW, and PVR were not modified by 5-HT administration. The results establish that 5-HT induced systemic as well as pulmonary hypertension is mediated through the activation of type-2 serotonergic or a-adrenergic receptors, or both. In contrast, neither apnoea, bradycardia and hypotension, nor the positive chronotropic effect induced by 5-HT in cattle are mediated through such receptors.

Summary To explore the pathophysiology of respiratory failure in an experimental pneumonia, a Pasteurella haemolytica broth culture was injected intratracheally into 12 calves, which were then studied over a period of 10 h. Measurements were made of inspired minute ventilation (V E ), ventilatory pattern [inspiratory time (T I ), expiratory time (T E ), respiratory rate (RR) and tidal volume (V T )], transdiaphragmatic pressure (Pdi), occlusion pressure at the airway opening 100 milliseconds after onset of inspiration (Pawo 100ms ), arterial blood gas tensions and pH and recorded diaphragmatic electromyogram (EMG di ) and rectal temperature (Tr). On and after the third hour after inoculation, the animals varied in respect of clinical signs, Tr, RR, V E , Pawo 100ms /EMG di , and arterial gases and pH. In benign cases, diminished alertness, laboured respiration and fall of arterial oxygen pressure (PaO 2 ) worsened up to 7h after inoculation, but then progressively improved, V E being maintained at approximately 150% baseline throughout the study (10h). Neither arterial carbon dioxide pressure (PaCO 2 ) nor pH was altered. Moderate cases resembled benign cases in respect of laboured respiration, V E , PaO 2 and PaCO 2 ; however, pH was at first maintained at preinoculation levels, but declined thereafter. In severe cases, the animals were drowsy between hours 3 and 7, and became comatose between hours 8 and 10; in contrast to both benign and moderate cases: (1) RR was reduced by hour 5, (2) there was no trend towards recovery of PaO 2 and pH, (3) V E , Pdi, Pawo 100ms and Pawo 100ms /EMG di were severely decreased, and (4) PaCO 2 increased. These results suggest that pneumonia does not alter ventilatory neuromuscularpump function in calves, unless concomitant cardiovascular collapse occurs. It is not clear whether fatal ventilatory failure is caused mainly by deterioration in ventilatory muscle fibre processes or structures, altered central nervous system adjustment of ventilatory timing, or cardiovascular dysfunction. However, inspiratory pressures fall when excitation to the diaphragm is still growing, which suggests peripheral respiratory muscle fatigue.

Summary This study was designed to investigate whether 3-methylindole (3-Mi), a tryptamine analogue, could cause pulmonary injury in calves other than by cytotoxicity. Injection of 3-Mi resulted in a marked increase of respiratory rate, decrease of tidal volume and increase in minute ventilation. Pulmonary mechanics values were also profoundly affected, lung dynamic compliance being reduced to approximately one-third of its baseline value and total pulmonary resistance being increased two-fold. Arterial oxygen partial pressure was dramatically reduced. Successive challenges with 3-Mi after physiological saline pretreatment resulted in quantitatively identical alterations of pulmonary function values. Conversely, all these ventilatory, mechanical and gas exchange changes were abolished by pretreatment with serotonergic antagonists. It was concluded that intravenous administration of 3-Mi to healthy calves induced immediate and reversible bronchoconstriction which affected both central and peripheral airways. Because the effect was abolished by pretreatment with antiserotonin drugs, it is suggested that 3-Mi acts either directly by stimulating serotonergic receptors or indirectly through the release of serotonin from platelets. Current concepts of the physiopathological cascade underlying the toxicity of 3-Mi should, therefore, be re-evaluated in the light of this pharmacological mechanism.

Summary The purpose was to determine whether gastric overdistension leads to lifethreatening perturbations of pulmonary gas exchange in healthy calves. Six animals were studied with normal (0 kPa) and increased (1, 2, 3, 4 and 5 kPa) intragastric pressure (IGP). Changes in pleural pressures and peak expiratory flow paralleled those of IGP. Inspiratory pressure-time index remained stable throughout the insufflation process. Pulmonary function values were characterized by abrupt changes with increasing IGP. Tidal volume declined as IGP increased and, along with inspiratory flow, decreased abruptly with the highest pressure (5 kPa). Respiratory rate progressively increased up to an IGP of 4 kPa, then decreased by 30%, due to breath-holding at the end of inspiration. Minute volume increased with IGP up to 4 kPa, but dramatically declined at 5 kPa. Total pulmonary resistance remained stable throughout the insufflation process, whereas lung dynamic compliance fell abruptly to one-half of its baseline value at IGPs of 1 kPa and above. Arterial oxygen tension was maintained at an IGP of 1 kPa, slightly diminished at 2–3 kPa, and markedly decreased at 4–5 kPa. Hypercapnia and respiratory acidosis developed progressively with increasing IGP. Changes in arterial gases were probably due to a combination of (1) alveolar hypoventilation, caused by altered tidal to dead space volume ratio, inadequate central nervous system “drive”, altered effectiveness of inspiratory muscle action, or end-inspiratory breath-holding, and (2) ventilation to perfusion mismatch, caused by perfusion of collapsed lung units. In the range of IGPs used, standardized arterial pH did not decline below the control value, which suggests that perfusion of peripheral tissues remained sufficient, and that respiratory failure rather than cardiovascular failure may be the principal physiopathological effect of increased gastric pressure.

This study was designed to test the applicability and the reproducibility of the thermodilution method in the measurement of right ventricular ejection fraction (EF), end-diastolic volume (EDV) and end-systolic volume (ESV) in unsedated cattle. In the 16 healthy calves studied, it was possible, using a fast response thermodilution catheter, to obtain characteristic exponential steplike washout curves, allowing the calculation of right ventricular EF, EDV and ESV. Five to 10 successive thermodilution curves were recorded within a few minutes and two to five similar sets of measurements were performed throughout the same day to test the reproducibility of the technique. The same protocol was repeated the following day. Right ventricular EF, EDV and ESV were reproducible successively, throughout a given day and on following days. The mean intraset, intraday and interdays coefficients of variation ranged from a mean of 4.0 (SD 4.1) per cent to a mean of 18.2 (SD 7.9) per cent. Values of right ventricular volumes and EF agreed with those previously reported in various animal species. Thermodilution was, therefore, shown to be a useful method for measuring with a satisfactory reproducibility, right ventricular EF, EDV and ESV in the unsedated healthy calf.

Summary Blood samples were collected for lactate concentration determination (La cc ) at the arrival of the cross-country phase from 40 horses of various class levels competing in 6 different three-day events. Moreover, in 8 horses, heart rate (HR) was recorded every 5 seconds during the cross-country. Among them, 5 horses were submitted, at the beginning of the competition's season to a standardized exercise test (SET) on a track which allowed us to study the relationship between HR, blood lactate concentration and running speed on flat ground in these horses. The relationship between mean HR and mean speed, between La cc and mean speed and between La cc and HR during the cross-country were studied using regression analysis. Mean HR during the cross-country was linearly related to mean speed during this phase (r=0.87, p ≤ 0.01), but this relationship was shifted upwards in comparison with the corresponding curve obtained during the SET (r=0.97, p ≤ 0.001). In the same way, the relationship between La cc and mean speed during the cross-country was exponential (r=0.93, p ≤ 0.001), but blood lactate accumulation occurred at a lower speed than during the SET (r=0.94, p ≤ 0.001). The correlation between La cc and HR was stronger when La cc was related to HR during the 15 last minutes of the cross (r = 0.84) than to mean HR during the whole cross (r = 0.75) or to HR during the 2 last (r = 0.74) or the last (r = 0.80) minute of the course, which indicated that continuous recording of HR allowed a more accurate estimation of the severity of exercise performed during the cross-country than La cc . Heart rate ranged from 170 to 190 beats/min during the first part of the cross-country and reached or exceeded values of 190 to 200 beats/min at the end of the course. Therefore, the anaerobic threshold is reached during cross-country. Recruitment of anaerobic me tabolism at the end of the course was otherwise demonstrated by the high values of La cc . These aerobic-anaerobic meta bolic requirements should be taken into account to design the training program of an eventing horse.