Differential effects of P2Y1 deletion on glial activation and survival of photoreceptors and amacrine cells in the ischemic mouse retina.

Gliosis of retinal Müller glial cells may have both beneficial and detrimental effects on neurons. To investigate the role of purinergic signaling in ischemia-induced reactive gliosis, transient retinal ischemia was evoked by elevation of the intraocular pressure in wild-type (Wt) mice and in mice deficient in the glia-specific nucleotide receptor P2Y1 (P2Y1 receptor-deficient (P2Y1R-KO)). While control retinae of P2Y1R-KO mice displayed reduced cell numbers in the ganglion cell and inner nuclear layers, ischemia induced apoptotic death of cells in all retinal layers in both, Wt and P2Y1R-KO mice, but the damage especially on photoreceptors was more pronounced in retinae of P2Y1R-KO mice. In contrast, gene expression profiling and histological data suggest an increased survival of amacrine cells in the postischemic retina of P2Y1R-KO mice. Interestingly, measuring the ischemia-induced downregulation of inwardly rectifying potassium channel (Kir)-mediated K(+) currents as an indicator, reactive Müller cell gliosis was found to be weaker in P2Y1R-KO (current amplitude decreased by 18%) than in Wt mice (decrease by 68%). The inner retina harbors those neurons generating action potentials, which strongly rely on an intact ion homeostasis. This may explain why especially these cells appear to benefit from the preserved Kir4.1 expression in Müller cells, which should allow them to keep up their function in the context of spatial buffering of potassium. Especially under ischemic conditions, maintenance of this Müller cell function may dampen cytotoxic neuronal hyperexcitation and subsequent neuronal cell loss. In sum, we found that purinergic signaling modulates the gliotic activation pattern of Müller glia and lack of P2Y1 has janus-faced effects. In the end, the differential effects of a disrupted P2Y1 signaling onto neuronal survival in the ischemic retina call the putative therapeutical use of P2Y1-antagonists into question.

Osteopontin inhibits osmotic swelling of retinal glial (Müller) cells by inducing release of VEGF.

Osmotic swelling of retinal neurons and glial cells is an important pathogenic factor of retinal edema formation. Here, we show that the neuroprotective factor osteopontin (OPN), which is released from retinal glial (Müller) cells after stimulation of the cells with glial cell line-derived neurotrophic factor (Del Río et al., 2011, Glia 59:821-832), inhibits the swelling of rat Müller cells induced by hypoosmotic exposure of retinal slices in the presence of barium ions and H2O2, respectively, and in slices of postischemic retinas. OPN did not inhibit the hypoosmotic swelling of bipolar cells in slices of control and postischemic retinas. The inhibitory effect of OPN on Müller cell swelling was dose-dependent, with a half-maximal effect at ∼0.6 ng/ml. The effect of OPN was abrogated in the presence of pharmacological blockers of vascular endothelial growth factor (VEGF) receptor-2, metabotropic glutamate receptors, and purinergic receptors (P2Y1, adenosine A1 receptors), as well as of a neutralizing anti-VEGF antibody. The data suggest that OPN induces the release of VEGF, glutamate, ATP, and adenosine from Müller cells. The effect of OPN was also prevented by blockers of voltage-gated sodium channels (tetrodotoxin), T-type voltage-gated calcium channels (kurtoxin), potassium channels (clofilium), and chloride channels 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). The swelling-inhibitory effect of OPN was dependent on intracellular calcium signaling, activation of phospholipase C and protein kinase C, and vesicular exocytosis of glutamate. In retinal slices, Müller glial cells display immunoreactivity of OPN. The data suggest that Müller cell-derived OPN has (in addition to the effects on photoreceptors and retinal neurons) autocrine effects. The neuroprotective effects of OPN may be in part mediated by the prevention of cytotoxic Müller cell swelling and the release of VEGF and adenosine from Müller cells.

Region-specific expression of vesicular glutamate and GABA transporters under various ischaemic conditions in mouse forebrain and retina.

There is accumulating evidence that glutamate and GABA release are key mechanisms of ischaemic events in the CNS. However, data on the expression of involved transporters for these mediators are inconsistent, potentially impeding further neuroprotective approaches. Here, we applied immunofluorescence labelling to characterise the expression pattern of vesicular glutamate (VGLUT) and GABA transporters (VGAT) after acute focal cerebral ischaemia and in two models of retinal ischaemia. Mice were subjected to filament-based focal cerebral ischaemia predominantly involving the middle cerebral artery territory, also leading to retinal ischaemia due to central retinal artery occlusion (CRAO). Alternatively, retinal ischaemia was induced by a transient increase of the intraocular pressure (HIOP). One day after ischaemia onset, diminished immunolabelling of neuronal nuclei and microtubule-associated protein 2-positive structures were found in the ipsilateral neocortex, subcortex and the retina, indicating neuronal degeneration. VGLUT1 expression did not change significantly in ischaemic tissues whereas VGLUT2 was down-regulated in specific areas of the brain. VGLUT3 expression was only slightly down-regulated in the ischaemia-affected neocortex, and was found to form clusters on fibrils of unknown origin in the ischaemic lateral hypothalamus. In contrast, retinae subjected to CRAO or HIOP displayed a rapid loss of VGLUT3-immunoreactivity. The expression of VGAT appears resistant to ischaemia as there was no significant alteration in all the regions analysed. In summary, these data indicate a region- and subtype-specific change of VGLUT expression in the ischaemia-affected CNS, whose consideration might help to generate specific neuroprotective strategies.

Peritoneal inflammatory response to surgical correction of left displaced abomasum using different techniques.

The objective of this study was to compare the inflammatory response within the abdominal cavity between three surgical methods. The study comprised 45 cows with left displacement of the abomasum, which were allocated into three groups (n = 15). Right flank laparotomy and omentopexy (group R), left flank laparotomy and omentopexy (group L), and laparoscopic abomasopexy (group J) have been applied. Laparoscopic abomasopexy was the only technique that requires perforation of the abomasal wall. Blood and peritoneal fluid (PF) samples were obtained before, and on days 1, 2 and 3 after surgery. Macroscopic and microscopic evaluation of PF were performed. Cytological and biochemical parameters were analysed in blood and PF. No bacteria were present in PF after surgery. The number of PF leukocytes increased in all groups on day 1 after surgery with the highest value after laparoscopy (median, 1st quartile, 3rd quartile, R: 13.1, 6.4, 16.0; L: 13.6, 9.9, 17.4; J: 33.7, 21.1, 46.9 G/l). Laparotomy resulted in an increase of blood and PF CK on day 1 after surgery, whereas, laparoscopy caused an increased PF CK only. All groups had elevated PF D-dimer concentrations before surgery, with further increase in groups R and L on day 1 after surgery.

Peritoneal fluid analysis in dairy cows with left displaced abomasum and abomasal volvulus.

Peritoneal fluid (PF) was evaluated in 40 cows with left displaced abomasum (LDA) and 15 cows with abomasal volvulus (AV). PF was obtained by abdominocentesis at the right ventral abdomen at admission. Simultaneously, a blood sample was taken from the jugular vein. Biochemical and cytological variables in blood and PF specific for ischaemia, inflammation and cell damage were compared. Total protein, albumin, glucose and cholesterol were normal in PF of cows with LDA and AV. Although L-lactate increased in both groups, cows with AV had significantly higher values (LDA: 1.47/0.69/2.68 mmol/l; AV: 6.45/4.55/12.89 mmol/l (median/1. quartile/3. quartile)). D-dimer (LDA: 0.50/0.22/0.88 mg/l; AV: 1.11/0.40/1.85 mg/l) and LDH (LDA: 663/437/943 U/l; AV: 1099/750/1439 U/l) were only increased in PF of cows with AV. The number of leucocytes was normal; however, significantly more peritoneal neutrophils appeared necrotic or apoptotic after AV. PF of cows with abomasal displacement showed distinctive features of ischaemia and inflammation. Characteristics of haemostatic dysfunction and cell damage were mainly evident in PF of cows with AV. The results suggest that anti-inflammatory therapy is indicated in each cow with abomasal displacement. Additionally, medical treatment should be directed to prevent complications of ischaemia and reperfusion in cows with AV.

Mucosal injury and inflammatory cells in response to brief ischaemia and reperfusion in the equine large colon.

REASON FOR PERFORMING STUDY: Intestinal ischaemia and reperfusion (I/R) can activate inflammatory cells in the equine colon, although effects on different types of inflammatory cells have received little attention. OBJECTIVES: To assess early mucosal injury, the reaction of mucosal neutrophils, eosinophils, mast cells and macrophages, and cyclooxygenase (COX)-1 and -2 expression in response to I/R in the equine large colon. METHODS: Large colon ischaemia was induced for 1 h (1hI) followed by 4 h of reperfusion in 6 horses, and mucosal biopsies were sampled before and after ischaemia, and after 1, 2 and 4 h of reperfusion. Semithin sections (500 nm) of epon-embedded biopsies were stained with toluidine blue for histomorphometric evaluation. The number and distribution of mucosal macrophages (CD163), neutrophils (calprotectin), eosinophils (LUNA) and mast cells (toluidine blue) were determined, and mucosal COX-1 and -2 expression was identified. RESULTS: Ischaemia caused epithelial cell and nuclear swelling (mean ± s.e. nuclear width; control: 2.7 ± 0.2 µm vs. 1hI: 4.2 ± 0.2 µm; P<0.01), subepithelial oedema (control: 0.2 ± 0.1 µm vs. 1hI: 3.2 ± 0.2 µm; P<0.01) and increased epithelial apoptosis (control: 14.3 ± 4.1 apoptotic cells/mm mucosa vs. 1hI: 60.4 ± 14.0 apoptotic cells/mm mucosa; P<0.01). COX-2 expression (P<0.01) was evident after ischaemia. Reperfusion caused paracellular fluid accumulation (control: 0.9 ± 0.1 µm vs. 1hI: 0.6 ± 0.6 µm vs. 1hI + 4hR: 1.6 ± 0.2 µm; P<0.05). Epithelial repair started at 1 h of reperfusion (P<0.001), followed by migration of neutrophils into the mucosa after 2 h (control: 72.3 ± 18.4 cells/mm(2) mucosa vs. 1hI + 2hR: 1149.9 ± 220.6 cells/mm(2) mucosa; P<0.01). Mucosal eosinophils, mast cells and macrophages did not increase in numbers but were activated. CONCLUSIONS: Epithelial injury and COX-2 expression caused by short-term hypoxia were followed by intense inflammation associated with epithelial repair during reperfusion. POTENTIAL RELEVANCE: Equine colonic mucosa subjected to a brief period of ischaemia can repair during reperfusion, despite increased mucosal inflammation.

Ultrastructural changes in the equine colonic mucosa after ischaemia and reperfusion.

REASON FOR PERFORMING STUDY: Ultrastructural changes in the epithelium can provide information on early changes in barrier properties, repair and inflammation in equine colon after ischaemia and reperfusion (I/R). OBJECTIVES: To describe the morphology and ultrastructure of the epithelium in equine large colonic mucosa after I/R, and the response of inflammatory cells to injury. METHODS: Ischaemia was induced for 1 h followed by 4 h of reperfusion in a 40 cm segment of the pelvic flexure in 6 horses. Mucosal biopsies before and after ischaemia, and after 1, 2 and 4 h of reperfusion were fixed in glutaraldehyde/paraformaldehyde and osmium tetroxide, and embedded in epon. Morphological and ultrastructural changes were evaluated in toluidine blue-stained semithin sections by light microscopy and in thin sections stained with uranyl acetate/lead citrate by transmission electron microscopy. RESULTS: Ischaemia caused swelling of epithelial cells and their organelles, opening of tight junctions, detachment from the basement membrane, early apoptosis and single cell necrosis. Autophagy was a prominent feature in epithelial cells after ischaemia. Reperfusion was characterised by apoptosis, epithelial regeneration and restoration of apical cell junctions. Phagocytic-like vacuoles containing cellular debris and bacteria were evident in epithelial cells after reperfusion. Paracellular and subepithelial clefts formed, accompanied by infiltration of neutrophils, lymphocytes and eosinophils into the epithelium. Subepithelial macrophages and luminal neutrophils had increased phagocytic activity. CONCLUSIONS: Ischaemia caused ultrastructural damage to the colonic epithelium, but epithelial cells recovered during reperfusion. POTENTIAL RELEVANCE: Transmission electron microscopy can demonstrate subtle ultrastructural damage to epithelial cells and evidence of recovery after I/R in equine colon.

Effects of flunixin meglumine on the recovery of ischaemic equine colonic mucosa in vitro.

REASONS FOR PERFORMING STUDY: The effects of prostaglandins and nonsteroidal anti-inflammatory drugs (NSAIDs) on repair of equine intestinal mucosa are important since most horses with gastrointestinal diseases are routinely treated with NSAIDs, such as flunixin meglumine (FM), and these drugs can be toxic to equine gastrointestinal mucosa. HYPOTHESIS: Flunixin meglumine would not affect recovery of equine colonic mucosa in vitro, 18 h after a reversible ischaemic injury. METHODS: In 14 anaesthetised horses, a segment of pelvic flexure was subjected to 2 h of ischaemia and the horses were allowed to recover for 18 h. Seven horses received normal saline and 7 received FM, 1.1 mg/kg bwt i.v., at the end of ischaemia and 12 h later. Colonic mucosa was harvested during a second anaesthesia, 18 h after recovery from ischaemia and then horses were subjected to euthanasia. Transepithelial electrical resistance (TER) and transepithelial flux of tritiated mannitol were used to measure mucosal permeability during 4 h of incubation in Ussing chambers, with the following in vitro treatments: 1) no addition, 2) FM 14 µmol/l as powder, 3) FM 14 µmol/l in injectable form and 4) diluent for injectable FM. Histomorphological changes were assessed by light microscopy. RESULTS: There were no significant differences in any of the measurements between saline and FM treated horses. The mucosal height of the ischaemic FM tissues incubated in diluent was significantly decreased compared to the nonischaemic tissues. CONCLUSIONS: Flunixin meglumine did not adversely affect barrier integrity in ischaemic equine colonic mucosa.

Cardiovascular effects of N-butylscopolammonium bromide and xylazine in horses.

REASONS FOR PERFORMING STUDY: N-butylscopolammonium bromide (NBB) and xylazine are commonly used medications for the treatment of spasmodic colic and other forms of abdominal pain in horses. Both NBB and xylazine exert significant effects on the cardiovascular system and other vital systems of horses. OBJECTIVE: To evaluate the effects of i.v. administration of NBB, xylazine, and the combination of NBB and xylazine on heart rate, other commonly measured physiological parameters, cardiac rhythm and blood pressure. METHODS: Six mature horses of mixed breed were used. In a random cross-over design, each horse was given 0.3 mg/kg bwt of NBB i.v., 0.25 mg/kg bwt xylazine i.v., and a combination of 0.3 mg/kg bwt NBB and 0.25 mg/kg bwt xylazine. Heart rate, physiological parameters, cardiac rhythm and indirect blood pressure were recorded at timed intervals before and 60 min following administration. RESULTS: Heart rate and blood pressure were significantly elevated immediately following administration of NBB or NBB with xylazine. Administration of NBB with xylazine resulted in significantly greater initial and peak blood pressure values than with NBB alone. Administration of xylazine resulted in a decrease in heart rate, with an initial increase in blood pressure followed by a decrease in blood pressure. Sinus tachycardia was seen with NBB, and NBB and xylazine administration. First and second degree atrioventricular block was identified with xylazine administration. Ventricular tachycardia was identified in one horse following NBB and xylazine administration. CONCLUSIONS: Results of this study suggest that the effects of administration of NBB alone or in combination with xylazine to horses with colic, especially to those with systemic cardiovascular compromise, should be considered carefully to assess condition and predict prognosis accurately, and to avoid potential adverse effects.

Diagnostic accuracy of d-dimer and other peritoneal fluid analysis measurements in dairy cows with peritonitis.

BACKGROUND: Peritoneal fluid analysis in cattle traditionally includes the classic parameters despite the fact that they have only moderate diagnostic accuracy and often fail to identify the pathogenesis or etiological factors. Therefore additional parameters recently have been established to improve diagnostic precision. In a recent study, reference ranges for several of these parameters have been proposed in dairy cows. HYPOTHESIS/OBJECTIVES: The aim of this observational study was to assess the diagnostic value of D-Dimer and other measurements of peritoneal fluid analysis in dairy cows with peritonitis. ANIMALS: The study included 110 Holstein-Friesian cows grouped into cows with peritonitis (n = 47) and cows without peritonitis (n = 63). METHODS: Peritoneal fluid was obtained by abdominocentesis. Total protein, albumin, glucose, cholesterol, fibrinogen, l-lactate, D-Dimer, lactate dehydrogenase (LDH), alkaline phosphatase, creatine phosphokinase, white blood cell, and red blood cell were determined in peritoneal fluid and venous blood. Serum-ascites albumin gradient (SAAG) and ratios of peritoneal fluid-venous blood were calculated. Sensitivity (SN) and specificity (SP) were calculated and receiver operating characteristic curve analysis performed. RESULTS: Peritoneal fluid D-Dimer was most accurate in diagnosing peritonitis in cows (SN and SP>95.0%). Total protein concentration, LDH and LDH ratio, and SAAG had sensitivities between 49.0 and 67.1%, and specificities between 88.4 and 95.5%. A low-peritoneal fluid glucose concentration was found to be highly indicative of septic peritonitis. CONCLUSIONS AND CLINICAL IMPORTANCE: Measurement of the recently introduced parameters may increase the diagnostic value of peritoneal fluid analysis and provide additional specific information. Therefore these measurements should be included in the routine procedure.

Biochemical constituents of peritoneal fluid in cows.

Samples of peritoneal fluid and jugular venous blood were taken simultaneously from 95 clinically healthy Holstein-Friesian cows. The concentrations of total protein, albumin, glucose, cholesterol, fibrinogen, L-lactate and D-dimer, the activities of lactate dehydrogenase (LDH), alkaline phosphatase and creatine kinase, and the white blood cell count were determined in the samples. Light's criteria, the serum-ascites albumin gradient (SAAG) and the ratios of the concentration of each parameter in peritoneal fluid to its concentration in blood were calculated. The mean concentrations of total protein, albumin and D-dimer, the activity of LDH and the SAAG were different from the reference values for monogastric animals and human beings.

Detection of calprotectin and its correlation to the accumulation of neutrophils within equine large colon during ischaemia and reperfusion.

REASON FOR PERFORMING STUDY: The cytosolic protein complex, calprotectin, is abundant in neutrophils and could be used to improve the ability to localise and assess neutrophil infiltration in the equine intestine during ischaemia and reperfusion (I/R), but further study is required. OBJECTIVES: To assess the number of calprotectin-containing cells by immunohistochemistry in correlation with direct counting and scoring of neutrophils in the equine colon during I/R. METHODS: One and 2 h ischaemia of the left dorsal colon were induced, followed by 30 min reperfusion under general anaesthesia or by 18 h reperfusion after anaesthetic recovery. Biopsies were processed for light microscopy and stained with H/E for detection of neutrophils. To identify calprotectin-containing cells, immunohistochemistry was performed on formalin-fixed tissues with the murine MAC 387 antibody and a biotin-free peroxidase staining procedure. The number of neutrophils within submucosal venules and the colonic mucosa were calculated and compared with the number of calprotectin-positive cells. RESULTS: The number of calprotectin-positive cells within submucosal venules and within the colonic mucosa correlated significantly with the accumulation of neutrophils within the corresponding tissue segments. Within the submucosal venules, both calprotectin-positive cells and H/E-stained neutrophils increased with duration of ischaemia and peaked after 30 min of reperfusion. After 18 h reperfusion the number of these cells declined within the vessels. After 2 h ischaemia, neutrophils started to migrate into the mucosa towards the epithelium, with a significant increase over time during reperfusion, and peak infiltration after 18 h reperfusion. CONCLUSIONS: Neutrophil infiltration into the colon after I/R is a time-dependent process, involving migration through the submucosa towards the epithelium.

Effect of a novel solution for organ preservation on equine large colon in an isolated pulsatile perfusion system.

REASONS FOR PERFORMING STUDY: Several therapeutic agents have been tested in models of ischaemia and reperfusion injury (IRI) in equine jejunum, with mixed results. This study was based on the use of an organ perfusion solution (OPS) designed to protect human allografts from IRI. HYPOTHESIS: A modified OPS can preserve the integrity of equine large colon during 12 h of isolated pulsatile perfusion, in the absence of oxygen and blood. METHODS: Segments of large colon were removed from anaesthetised horses, the contents removed and the mucosa rinsed with 0.9% saline. Experimental segments were perfused for 12 h with one litre modified OPS (n = 7) delivered by pulsatile flow through an extracorporeal circuit. Control segments (n = 4) were perfused on the same circuit with one litre of autologous blood. Vascular resistance, flow and pressure were measured serially, and aliquots of OPS and blood drawn hourly for routine biochemical analyses. Mucosal biopsies of the experimental and control segments were taken at 0, 6 and 12 h and in vivo mucosal tissue at 0 h for baseline comparison. All biopsies underwent histomorphometric analysis and immunohistochemical assessment of calprotectin activity. RESULTS: All colon segments were machine perfused without technical complications. Vascular and biochemical indices remained constant over 12 h in the OPS group, and were constant over 6 h in the control group, but deteriorated later. Mucosal integrity, expression of cyclooxygenases-1 and -2, and expression of mucosal calprotectin were unchanged in the OPS group compared with the baseline tissues, and mucosal integrity was superior to the control tissues. CONCLUSIONS: A modified OPS designed to target specific pathways of damage from IRI can preserve colonic mucosal integrity for 12 h in the absence of blood and oxygen.

Morphometric analysis of the retina from horses infected with the Borna disease virus.

Borna disease (BD) is a fatal disorder of horses, often characterized by blindness. Although degeneration of retinal neurons has been demonstrated in a rat model, there are controversial data concerning whether a similar degeneration occurs in the retina of infected horses. To investigate whether BD may cause degeneration of photoreceptors and possibly of other neuronal cells at least at later stages of the disease, we performed a detailed quantitative morphologic study of retinal tissue from Borna-diseased horses. BD was diagnosed by detection of pathognomonic Joest-Degen inclusion bodies in the postmortem brains. Paraffin sections of paraformaldehyde-fixed retinae were used for histologic and immunohistochemical stainings. Numbers of neurons and Müller glial cells were counted, and neuron-to-Müller cell ratios were calculated. Among tissues from 9 horses with BD, we found retinae with strongly altered histologic appearance as well as retinae with only minor changes. The neuron-to-Müller cell ratio for the whole retina was significantly smaller in diseased animals (8.5 +/- 0.4; P < .01) as compared with controls (17.6 +/- 0.8). It can be concluded that BD in horses causes alterations of the retinal histology of a variable degree. The study provides new data about the pathogenesis of BD concerning the retina and demonstrates that a loss of photoreceptors may explain the observed blindness in infected horses.

Osmotic stimulation of the supraoptic nucleus: central and peripheral vasopressin release and blood pressure.

Experiments were performed to determine the effect of direct osmotic stimulation of the supraoptic nucleus (SON) on central and peripheral vasopressin (AVP) release and arterial pressure. A microdialysis method was used to deliver hyperosmotic NaCl, mannitol or urea bilaterally into the SON and to sample SON extracellular fluid and blood. Simultaneous brain and blood microdialysis showed that hyperosmotic NaCl increased central and peripheral AVP release and increased mean arterial pressure (MAP). The pressor response was not blocked by intravenous injection of a V1-receptor antagonist, D(CH2)5Tyr(Me)AVP, suggesting that circulating AVP was not involved in that response. Hyperosmotic mannitol or urea caused an increase in central peptide release, but failed to affect MAP or peripheral AVP release. The results suggest that central AVP release within the SON may be due to osmoreceptor stimulation while the peripheral effects on AVP release and MAP are specific for sodium. The results also demonstrate the utility of brain and blood microdialysis for the delivery of stimuli into specific brain regions with simultaneous monitoring of central and peripheral peptide release.

Comparison of humoral immune responses to trivalent influenza split vaccine in young, middle-aged and elderly people.

A total of 296 volunteers in five different groups were immunized with one dose of the commercial 1991-1992 trivalent split influenza vaccine formulation A/Singapore/6/86 (H1N1), A/Beijing/353/89 (H3N2) and B/Yamagata/16/88. The groups differed in age (young adults, middle-aged and elderly) and history of previous vaccination. Antibodies were determined in pre- and postvaccination sera by haemagglutination inhibition assay and the results were evaluated as geometric mean titre, mean fold antibody increase, protection and response rates. No significant age-related differences among the protection rates were found. The proportion of vaccinees with antibodies > or = 40 ranged between 70 and 95%. Compared with the H3N2 and B components the antibody response to the H1N1-component was low. Residents of a nursing home fully vaccinated the previous year developed 7.6-8.4-fold antibody increases and showed 96-100% protection rates.