A Potent Anti-Malarial Human Monoclonal Antibody Targets Circumsporozoite Protein Minor Repeats and Neutralizes Sporozoites in the Liver.

Discovering potent human monoclonal antibodies (mAbs) targeting the Plasmodium falciparum circumsporozoite protein (PfCSP) on sporozoites (SPZ) and elucidating their mechanisms of neutralization will facilitate translation for passive prophylaxis and aid next-generation vaccine development. Here, we isolated a neutralizing human mAb, L9 that preferentially bound NVDP minor repeats of PfCSP with high affinity while cross-reacting with NANP major repeats. L9 was more potent than six published neutralizing human PfCSP mAbs at mediating protection against mosquito bite challenge in mice. Isothermal titration calorimetry and multiphoton microscopy showed that L9 and the other most protective mAbs bound PfCSP with two binding events and mediated protection by killing SPZ in the liver and by preventing their egress from sinusoids and traversal of hepatocytes. This study defines the subdominant PfCSP minor repeats as neutralizing epitopes, identifies an in vitro biophysical correlate of SPZ neutralization, and demonstrates that the liver is an important site for antibodies to prevent malaria.

Change of voltage-gated sodium channel repertoire in skeletal muscle of a MuSK myasthenia gravis mouse model.

Muscle-specific kinase myasthenia gravis (MuSK MG) is caused by autoantibodies against MuSK in the neuromuscular junction (NMJ). MuSK MG patients have fluctuating, fatigable skeletal muscle weakness, in particular of bulbar muscles. Severity differs greatly between patients, in spite of comparable autoantibody levels. One explanation for inter-patient and inter-muscle variability in sensitivity might be variations in compensatory muscle responses. Previously, we developed a passive transfer mouse model for MuSK MG. In preliminary ex vivo experiments, we observed that muscle contraction of some mice, in particular those with milder myasthenia, had become partially insensitive to inhibition by µ-Conotoxin-GIIIB, a blocker of skeletal muscle NaV1.4 voltage-gated sodium channels. We hypothesised that changes in NaV channel expression profile, possibly co-expression of (µ-Conotoxin-GIIIB insensitive) NaV1.5 type channels, might lower the muscle fibre's firing threshold and facilitate neuromuscular synaptic transmission. To test this hypothesis, we here performed passive transfer in immuno-compromised mice, using 'high', 'intermediate' and 'low' dosing regimens of purified MuSK MG patient IgG4. We compared myasthenia levels, µ-Conotoxin-GIIIB resistance and muscle fibre action potential characteristics and firing thresholds. High- and intermediate-dosed mice showed clear, progressive myasthenia, not seen in low-dosed animals. However, diaphragm NMJ electrophysiology demonstrated almost equal myasthenic severities amongst all regimens. Nonetheless, low-dosed mouse diaphragms showed a much higher degree of µ-Conotoxin-GIIIB resistance. This was not explained by upregulation of Scn5a (the NaV1.5 gene), lowered muscle fibre firing thresholds or histologically detectable upregulated NaV1.5 channels. It remains to be established which factors are responsible for the observed µ-Conotoxin-GIIIB insensitivity and whether the NaV repertoire change is compensatory beneficial or a bystander effect.

Bovine Digital Dermatitis: Treponema spp. on trimming equipment and chutes - effect of washing and disinfection.

BACKGROUND: Digital dermatitis (DD) is a contagious bovine foot disease causing reduced animal welfare and negative economic consequences for the farmer. Treponema spp. are the most important causative agents. Studies indicate that trimming equipment can transfer DD-associated treponemes between cows. The aim of this observational study in 22 DD-positive Norwegian dairy herds was to investigate the risk of transferring Treponema spp. with trimming equipment and chutes after claw trimming, and after washing and disinfection. Swabs from the trimming equipment and chutes were collected from nine different locations, at five different time points. Bacterial DNA was extracted from 647 swabs and analysed by qPCR for Treponema spp. In addition, 172 swabs taken immediately after trimming, were analysed by a multiplex qPCR targeting T. phagedenis, T. pedis and T. medium/vincentii. Biopsy sampling from DD lesions was performed on cows in the same herds during trimming. Altogether 109 biopsies were analysed by FISH for confirmation of the DD diagnosis and identification of Treponema phylotypes (PTs). RESULTS: High numbers of Treponema spp. were detected from all nine locations on the trimming equipment and chutes immediately after trimming, and T. phagedenis was detected on two or more locations in all but two herds, 1 and 19. There was a decline in the amount of Treponema spp. after washing and disinfection. The belly belt, the cuff, and the footrest on the chute had the highest proportion of positive samples after disinfection. The belly belt had the highest copy numbers of all nine locations (median = 7.9, max = 545.1). No Treponema spp. was detected on the hoof knives after disinfection. Treponema phagedenis, T. pedis, and Treponema phylotype 3 (T. refringens) were detected by FISH analysis of the biopsies. Treponema phagedenis was detected in biopsies from all herds except 1 and 19. CONCLUSION: This study shows that DD-associated Treponema spp. were present on the trimming equipment and chutes after trimming cows in DD-positive herds. Washing and disinfection reduced the load of Treponema spp. However, large differences in Treponema spp. between different locations were documented. High copy numbers on the grinder and the chute after disinfection, indicates that sufficient cleaning and disinfection of these locations is difficult, and that passive transfer of DD-associated treponemes (viable or not) is possible.

The ability of a handheld near-infrared spectrometer to do a rapid quality assessment of bovine colostrum, including the immunoglobulin G concentration.

Portable infrared-based instruments have made important contributions in different research fields. Within the dairy supply chain, for example, most of portable devices are based on near-infrared spectroscopy (NIRS) and are nowadays an important support for farmers and operators of the dairy sector, allowing fast and real-time decision-making, particularly for feed and milk quality evaluation and animal health and welfare monitoring. The affordability, portability, and ease of use of these instruments have been pivotal factors for their implementation on farm. In fact, pocket-sized devices enable nonexpert users to perform quick, low-cost, and nondestructive analysis on various matrixes without complex preparation. Because bovine colostrum (BC) quality is mostly given by the IgG level, evaluating the ability of portable NIRS tools to measure antibody concentration is advisable. In this study we used the wireless device SCiO manufactured by Consumer Physics Inc. (Tel Aviv, Israel) to collect BC spectra and then attempt to predict IgG concentration and gross and fine composition in individual samples collected immediately after calving (<6 h) in primiparous and pluriparous Holstein cows on 9 Italian farms. Chemometric analyses revealed that SCiO has promising predictive performance for colostral IgG concentration, total Ig concentration, fat, and AA. The coefficient of determination of cross-validation (R2CV) was in fact ≥0.75). Excellent accuracy was observed for dry matter, protein, and S prediction in cross-validation and good prediction ability in external validation (R2CV ≥ 0.93; the coefficient of determination of external validation, R2V, was ≥0.82). Nonetheless, SCiO's ability to discriminate between good- and low-quality samples (IgG ≥ vs. < 50 g/L) was satisfactory. The affordable cost, the accurate predictions, and the user-friendly design, coupled with the increased interest in BC within the dairy sector, may boost the collection of extensive BC data for management and genetic purposes in the near future.

Mid-infrared spectroscopy for large-scale phenotyping of bovine colostrum gross composition and immunoglobulin concentration.

Immunoglobulin G is the fundamental antibody for acquisition of passive transfer of immunity in ruminant newborns. Colostrum, in fact, must be administered as soon as possible after birth to ensure a successful transfer of IgG from the dam to the calf. Assessment of colostrum Ig concentration and gross composition via gold standards is expensive, time consuming, and hardly implementable for large-scale investigations. Therefore, in the present study we evaluated the predictive ability of mid-infrared spectroscopy (MIRS) as an indirect determination method. A total of 714 colostrum samples collected within 6 h from parturition from Italian Holstein cows, 30% primiparous and 70% pluriparous, were scanned using a benchtop spectrometer after dilution in pure water. The prediction models were developed by correlating spectral information with the reference measurements: IgG concentration (93.54 ± 33.87 g/L), total Ig concentrations (102.82 ± 35.04 g/L), and content of protein (14.71 ± 3.51%), fat (4.61 ± 3.04%), and lactose (2.36 ± 0.51 mg/100 mg). We found a good to excellent performance in prediction of colostrum IgG concentration and traditional composition traits in cross-validation (R2CV ≥ 0.92) and a promising and good predictive ability in external validation with R2V equal to 0.84, 0.89, and 0.74 for IgG, protein, and fat, respectively. In the case of IgG and protein content, for example, the coefficient of determination in external validation was greater than 0.84. The other Ig fractions, A and M, presented insufficient prediction accuracy likely due to their extremely low concentration compared with IgG (4.56 and 5.06 g/L vs. 93.54 g/L). The discriminant ability of MIRS-predicted IgG and protein content was outstanding when trying to classify samples according to the quality level (i.e., low vs. high concentration of IgG). In particular, the cut-off that better discriminate low- from high-quality colostrum was 75.40 g/L in the case of the MIRS-predicted IgG and 13.32% for the MIRS-predicted protein content. Therefore, MIRS is proposed as a rapid and cheap tool for large-scale punctual IgG, protein, and lactose quantification and for the screening of low-quality samples. From a practical perspective, there is the possibility to install colostrum models in the MIRS benchtop machineries already present in laboratories in charge of official milk testing. Colostrum phenotypes collected on an individual basis will be useful to breeders for the definition of specific selection strategies and to farmers for management scopes. Finally, our findings may be relevant for other stakeholders, given the fact that colostrum is an emerging ingredient for the animal and human food and pharmaceutical industry.

Discovery of novel targets in a complex regional pain syndrome mouse model by transcriptomics: TNF and JAK-STAT pathways.

Complex Regional Pain Syndrome (CRPS) represents severe chronic pain, hypersensitivity, and inflammation induced by sensory-immune-vascular interactions after a small injury. Since the therapy is unsatisfactory, there is a great need to identify novel drug targets. Unbiased transcriptomic analysis of the dorsal root ganglia (DRG) was performed in a passive transfer-trauma mouse model, and the predicted pathways were confirmed by pharmacological interventions. In the unilateral L3-5 DRGs 125 genes were differentially expressed in response to plantar incision and injecting IgG of CRPS patients. These are related to inflammatory and immune responses, cytokines, chemokines and neuropeptides. Pathway analysis revealed the involvement of Tumor Necrosis Factor (TNF) and Janus kinase (JAK-STAT) signaling. The relevance of these pathways was proven by abolished CRPS IgG-induced hyperalgesia and reduced microglia and astrocyte markers in pain-associated central nervous system regions after treatment with the soluble TNF alpha receptor etanercept or JAK inhibitor tofacitinib. These results provide the first evidence for CRPS-related neuroinflammation and abnormal cytokine signaling at the level of the primary sensory neurons in a translational mouse model and suggest that etanercept and tofacitinib might have drug repositioning potentials for CRPS-related pain.

Heat treatment of colostrum at 60°C decreases colostrum immunoglobulins but increases serum immunoglobulins and serum total protein: A meta-analysis.

Calves are born hypogammaglobulinemic; thus, the newborn calf's immune defense relies on the ingestion and absorption of colostrum, which provides energy, immunoglobulins, immune cells, and cytokines to the newborn calf. A heat treatment applied to colostrum for 60 min at 60°C has been found to be effective at reducing the total bacterial count while preserving the colostrum IgG levels. The objective of this work was to perform a meta-analysis on the association between the characteristics of heat-treated colostrum and the concentration of colostrum IgG, serum IgG concentration, and serum total protein (STP). A meta-analysis was carried out based on existing peer-reviewed literature. Publications comparing colostrum IgG, serum IgG, and STP for heat-treated or raw frozen colostrum were included. The different heating temperatures applied to the colostrum were divided into 2 subgroups: high temperature (HT; >60°C) and low temperature (LT; ≤60°C). Twelve studies, including 21 trials, met the inclusion criteria for colostrum IgG concentration. The results indicated decreases in colostrum IgG by 20.6 g/L [95% confidence interval (CI) = 11.8-29.4] for HT and 5.38 g/L (95% CI = 2.9-7.8) for LT when colostrum was heat-treated compared with raw or frozen colostrum. Heterogeneity was high to moderate (I2 = 82% for HT and 65% for LT). The heat treatment of colostrum was also associated with a nonsignificant decrease in serum IgG by 3.40 g/L for HT (95% CI = 7.54-0.74) but a significant increase in serum IgG by 2.65 g/L for LT (95% CI = 1.51-3.79). The regression model indicated that heterogeneity was not explained by any moderators. The heat treatment of colostrum was also associated with a significant increase in STP by 0.21 g/dL for LT (95% CI = 0.07-0.35). In conclusion, the present work demonstrated that the heat treatment of colostrum ≤60°C decreased colostrum IgG by 5.38 g/L for LT and increased serum IgG by 2.65 g/L and STP by 0.21 g/dL. When compared with the range of values observed in the field for serum IgG, the present results are of high interest for the cattle industry. Because immune colostrum benefits also include cytokines and immune cells, further work is required to evaluate the effect of colostrum heat treatment on these 2 immune components of colostrum.

Associations between the metabolic status of the cow and colostrum quality as determined by Brix refractometry.

Supplying newborn calves with immunoglobins is critical for their health and a daily challenge in the dairy industry. Among various factors determining colostrum quality, the prepartum metabolic status of the cow might be of particular importance. The objective of this observational cross-sectional study was to evaluate relationships between cow-level variables and the colostrum quality as determined by Brix refractometry. A total of 873 cows of varying breed and parity from 124 German dairy herds were included in the study, and blood and urine samples were taken 3 to 1 wk before the expected calving date. Effectively, samples were collected on average 8.2 d (geometric mean) before calving, ranging from 2 to 45 d. The final variable set included body condition score, lameness score, breed, parity, vaccination of the cow, the activity of glutamate dehydrogenase and aspartate aminotransferase, the urine concentration of creatinine, net acid-base excretion, the serum concentration of cholesterol and calcium, and the difference in albumin and total protein concentration. Generalized linear mixed effects regression models with hierarchically structured random effects (cow within herd) using the maximum likelihood method were fitted to the data to identify associations between the Brix value as an outcome and cow-level variables as predictors. Cows entering second parity had lower Brix values compared with cows entering third or greater parity, and prepartum vaccination of cows led to higher Brix values compared with nonvaccinated cows. Cows with a moderate to high lameness score had lower Brix values than cows with low-grade lameness. An increase of glutamate dehydrogenase serum activity and serum calcium concentration were associated with lower Brix values, whereas an increase in the difference of total protein and albumin serum concentration led to higher Brix values. In conclusion, the metabolic health of the cow affects colostrum quality and may cause failure of passive immunoglobulin transfer as well as impaired calf health.

Colostrum source and passive immunity transfer in dairy bull calves.

Colostrum is essential for good neonate health; however, it is not known whether different calves absorb the nutrients from colostrum equally well. In this study, the absorption of protein, IgG, and γ-glutamyl transferase was compared in newborn dairy bull calves for 1 wk after feeding colostrum from different sources. Thirty-five Holstein-Friesian bull calves were randomly allocated into 3 groups and fed colostrum within 4 h after birth. Group A calves (n = 12) were bottle fed colostrum from their own dam for 3 d. Colostrum from these group A cows was also used as foster cow colostrum for the group B calves (n = 12), such that each group A and B calf pair received identical colostrum from each milking of the respective group A dam (10% of birth weight per day). The group C calves (n = 11) were fed 1 bottle (2 L) of pooled colostrum and transition milk (referred to as pooled colostrum), as was the standard practice on the dairy farm. The pooled colostrum was collected from the other dairy cows on the farm 0 to 4 d postpartum and stored at 4°C for less than 12 h. Blood was sampled from calves before the first feeding and at 1, 2, 3, and 7 d after birth. Levels of total solids, total protein, and IgG were higher in the dam colostrum than in the pooled colostrum. At birth, there were no differences between the calf groups for any measurements, and all calves had very low IgG levels. After receiving colostrum, the glucose, plasma γ-glutamyl transferase, serum total protein, and IgG concentrations increased significantly in all calves. There were no differences in any blood measurements at any time point between the pairs of group A and group B calves that received colostrum from the same cow except for the IgG concentration 2 d after birth. However, the group A calves had a higher total serum protein level and IgG concentration than the group C calves for all the time points after the first feeding. The group B calves had a higher IgG concentration than the group C calves on d 1, 2, and 7 after birth. Compared with groups A and B, there was no difference in the proportion of calves in group C that failed to have passive immunity transferred adequately based on the IgG threshold (<10 g/L). Thus, the calves receiving identical colostrum from the same cow had the same levels of IgG, and even the pooled colostrum provided sufficient transfer of IgG as the calves were fed within 4 h after birth.

Predicting colostrum and calf blood components based on refractometry.

Provision of good quality colostrum is essential for the passive immunity and nutrition of newborn calves. In order to better predict the quality of colostrum and the transfer of passive immunity, the relationships between colostrum components and between calf serum components were examined in this study. Samples of bulk tank milk, colostrum pooled from several cows 0-4 d postpartum, and colostrum collected from individual cows twice daily for 3 d post-partum were compared. With the exception of fat percentage, there were strong correlations between the levels of the components in the pooled colostrum and in the individual cow colostrum collected 0-1 d postpartum. The correlations between total solids as measured by Brix refractometry and total protein, immunoglobulin G (IgG), lactose % and protein % in colostrum within 1 d postpartum and pooled colostrum were 0.92, 0.90, -0.88 and 0.98, respectively. These high correlations enabled these colostrum components to be accurately predicted from Brix % and therefore, the volume of colostrum required to feed neonate calves can be optimised based on Brix refractometry to avoid failure of passive immunity transfer. To assess whether the components obtained from colostrum were correlated in calf blood, newborn calves were separated from their dams before suckling and blood sampled before feeding (day 0), and on days 1 and 7, after receiving colostrum or milk twice a day. The correlations between glucose, total protein, IgG, and gamma-glutamyl transferase (GGT) levels in the calf blood were lower than the correlations observed between the colostrum components. The highest correlation was between serum protein measured by refractometer and serum IgG within one week postpartum. GGT activity was not a good indicator of serum IgG levels. However, serum protein refractometer measurements predicted serum IgG level with high accuracy, providing an on-farm test to determine that calves have received sufficient passive immunity and colostrum components.

Passive Transfer of Blood Sera from ALS Patients with Identified Mutations Results in Elevated Motoneuronal Calcium Level and Loss of Motor Neurons in the Spinal Cord of Mice.

Introduction: Previously, we demonstrated the degeneration of axon terminals in mice after repeated injections of blood sera from amyotrophic lateral sclerosis (ALS) patients with identified mutations. However, whether a similar treatment affects the cell body of motor neurons (MNs) remained unresolved. Methods: Sera from healthy individuals or ALS patients with a mutation in different ALS-related genes were intraperitoneally injected into ten-week-old male Balb/c mice (n = 3/serum) for two days. Afterward, the perikaryal calcium level was measured using electron microscopy. Furthermore, the optical disector method was used to evaluate the number of lumbar MNs. Results: The cytoplasmic calcium level of the lumbar MNs of the ALS-serum-treated mice, compared to untreated and healthy-serum-treated controls, was significantly elevated. While injections of the healthy serum did not reduce the number of MNs compared to the untreated control group, ALS sera induced a remarkable loss of MNs. Discussion: Similarly to the distant motor axon terminals, the injection of blood sera of ALS patients has a rapid degenerative effect on MNs. Analogously, the magnitude of the evoked changes was specific to the type of mutation; furthermore, the degeneration was most pronounced in the group treated with sera from ALS patients with a mutation in the chromosome 9 open reading frame 72 gene.

Behaviour and neuropathology in mice injected with human contactin-associated protein 2 antibodies.

Serum antibodies that bind to the surface of neurons or glia are associated with a wide range of rare but treatable CNS diseases. In many, if not most instances, the serum levels are higher than CSF levels yet most of the reported attempts to reproduce the human disease in mice have used infusion of antibodies into the mouse cerebral ventricle(s) or intrathecal space. We used the intraperitoneal route and injected purified plasma IgG from either a CASPR2-antibody-positive patient (n = 10 mice) or healthy individual (n = 9 mice) daily for 8 days. Lipopolysaccharide was injected intraperitoneally on Day 3 to cause a temporary breach in the blood brain barrier. A wide range of baseline behaviours, including tests of locomotion, coordination, memory, anxiety and social interactions, were established before the injections and tested from Day 5 until Day 11. At termination, brain tissue was analysed for human IgG, CASPR2 and c-fos expression, lymphocyte infiltration, and neuronal, astrocytic and microglial markers. Mice exposed to CASPR2-IgG, compared with control-IgG injected mice, displayed reduced working memory during the continuous spontaneous alternation test with trends towards reduced short-term and long-term memories. In the open field tests, activities were not different from controls, but in the reciprocal social interaction test, CASPR2-IgG injected mice showed longer latency to start interacting, associated with more freezing behaviour and reduced non-social activities of rearing and grooming. At termination, neuropathology showed more IgG deposited in the brains of CASPR2-IgG injected mice, but a trend towards increased CASPR2 expression; these results were mirrored in short-term in vitro experiments where CASPR2-IgG binding to hippocampal neurons and to CASPR2-transfected HEK cells led to some internalization of the IgG, but with a trend towards higher surface CASPR2 expression. Despite these limited results, in the CASPR2-IgG injected mouse brains there was increased c-fos expression in the piriform-entorhinal cortex and hypothalamus, and a modest loss of Purkinje cells. There was also increased microglia density, morphological changes in both microglia and astrocytes and raised complement C3 expression on astrocytes, all consistent with glial activation. Patients with CASPR2 antibodies can present with a range of clinical features reflecting central, autonomic and peripheral dysfunction. Although the behavioural changes in mice were limited to social interactions and mild working-memory defects, the neuropathological features indicate potentially widespread effects of the antibodies on different brain regions.

Hypoxia induces an increase in intestinal permeability and pulmonary arterial pressures in neonatal Holstein calves despite feeding the flavonoid rutin.

The purposes of this study were to determine whether the naturally occurring flavonoid quercetin, as its glucorhamnoside rutin, reduces intestinal permeability and susceptibility to hypoxia-induced pulmonary hypertension in neonatal Holstein calves. A 2 × 2 between-subjects factorial design was conducted using Holstein steers (n = 16). Factors included oxygen level (simulated altitude of 4,572 m vs. 975 m) and quercetin supplementation as its glucorhamnoside rutin (4 g of quercetin per day vs. 0 g per day). Two days after arrival (d 0 of study) the calves were blocked by body mass into treatment groups, and treatments were initiated. Pulmonary arterial pressure, echocardiography, and serum concentrations of orally administered lactulose (0.45 g/kg) and mannitol (0.15 g/kg) were measured on d 12, 13, and 14, respectively. Calves were euthanized on d 15 and pulmonary tissues collected for semiquantitative scoring of histological lesions. Data were analyzed using linear regression, generalized estimating equations, and 2-sample proportion tests. Hypoxia, but not rutin, was found to be associated with intestinal permeability. The lactulose-mannitol ratio was 0.54 ± 0.13 (standard error) in hypoxic calves and 0.02 ± 0.13 in normoxic controls. Hypoxia increased mean pulmonary arterial pressure. Calves fed rutin under hypoxic conditions tended to have a lower mean pulmonary arterial pressure (59 ± 7 mmHg) than control calves (80 ± 7 mmHg) but similar pressures under normoxic conditions. Paradoxically, however, a greater proportion of calves fed rutin had histological evidence of pulmonary arteriolar medial hypertrophy and adventitial hyperplasia than did controls. In conclusion, the findings of this study indicate that hypoxia increased intestinal permeability in neonatal calves. The flavonoid quercetin, as its glucorhamnoside rutin, had no protective effect on intestinal permeability, and, although it tended to reduce the severity of hypoxia-induced pulmonary hypertension, a greater proportion of calves fed rutin had histological lesions consistent with pulmonary arteriolar remodeling.

Evaluation of different analytical methods to assess failure of passive transfer in neonatal calves.

The objective of this study was to evaluate different analytical methods of assessing failure of passive transfer (FPT) in neonatal calves. We hypothesized that 3 different media (i.e., centrifuged serum, centrifuged plasma, filtered plasma) and different analytical methods [i.e., ELISA, capillary electrophoresis (CE), Brix refractometer, and handheld optical refractometer] would be highly correlated with the gold standard radial immunodiffusion (RID) and would generate comparable results. Serum and plasma blood samples were collected from Holstein Friesian calves (n = 216) aged 1 to 7 d, from 2 commercial dairy herds in northeast Germany. The RID analysis showed that 59 of 216 calves (27%) had serum IgG concentrations of <10 mg/mL and 157 calves (73%) had serum concentrations of ≥10 mg/mL. The mean IgG concentration (± standard deviation) was 17.1 ± 9.8 mg/mL, and the range was 0.8 to 47.8 mg/mL. In serum, the correlation between RID and CE was r = 0.97, and between RID and ELISA was r = 0.90; CE and ELISA were also highly correlated (r = 0.89). Both refractometry methods were highly correlated with RID using centrifuged serum, centrifuged plasma, or filtered plasma (Brix refractometer: r = 0.84, 0.80, and 0.78, respectively; handheld optical refractometer: r = 0.83, 0.81, and 0.80, respectively). We determined test characteristics (optimum thresholds, sensitivity, specificity, positive predictive value, negative predictive value, and area under the curve) for CE, ELISA, and the handheld optical and digital refractometers using receiver operating characteristic curve analyses with RID as the reference value. Optimal thresholds for assessing FPT using plasma were higher than for serum, regardless of the method of plasma harvesting. The 4 different devices had comparable areas under the curve, irrespective of the medium used. All analytical methods can be used to assess FPT.

Validation of Brix refractometers and a hydrometer for measuring the quality of caprine colostrum.

On-farm assessment of caprine colostrum quality is important for goat farmers; the ability to quickly recognize whether colostrum is suitable to feed to kids helps achieve successful passive transfer of immunity. The study compared the use of optical and digital Brix refractometers and a hydrometer against the international gold standard radial immunodiffusion (RID), using both fresh and frozen samples. A locally available ELISA methodology was included for comparison. A total of 300 samples were collected from 2 farms (farm 1: n = 157, collected by research staff within 24 h of parturition; farm 2: n = 143, collected by the farmer within 12 h of parturition). Farm 1 provided doe age for a subset of samples (n = 86). Samples were tested fresh and then frozen for shipment and repeated testing. Specific gravity was measured using a hydrometer in a subset of samples (n = 22) from farm 2. Because no gold standard thresholds are currently available for caprine colostrum, RID-derived values of 30, 40, and 50 g/L IgG were used as potential "good quality" thresholds. Pearson (ρ) and Lin's concordance correlation coefficients (CCC) were calculated for comparison of methods. Optimum thresholds were established maximizing the Youden index and minimizing the "distance closest to the top left corner" of the receiver operator characteristic curves. Brix values were correlated with RID (optical Brix, fresh: ρ = 0.73; digital Brix, fresh: ρ = 0.71; digital Brix, frozen: ρ = 0.76) and with each other (range: ρ = 0.93 to 0.99; CCC = 0.91 to 0.99). Specific gravity measured by the hydrometer yielded a strong relationship with RID (ρ = 0.83) and with Brix values (range: ρ = 0.88 to 0.90). The ELISA method was not correlated with Brix methods (range: ρ = 0.02 to 0.09) or RID (ρ = 0.20). Depending on the colostrum IgG threshold, the hydrometer yielded high Youden indices (range: 0.78 to 0.93) and low distance closest to the top left corner criteria (0 to 0.05) at a threshold of 1.047 specific gravity. For all RID IgG thresholds, the best Brix threshold (regardless of type or whether the sample was fresh or frozen) was 18 or 19%, with the highest Youden indices (range: 0.47 to 0.61) and lowest distance to the top left corner criteria (range: 0.09 to 0.16); however, we recommend 19%, because this reduces the potential of feeding poor-quality colostrum. The ELISA method was the poorest predictor of colostrum concentration. Age was not found to affect colostrum quality; however, the sample size of this subset was small. Hydrometers are inexpensive and easy to use, whereas Brix methods use only a small amount of colostrum; we suggest that either method could be used on-farm.

Passive Transfer of Sera from ALS Patients with Identified Mutations Evokes an Increased Synaptic Vesicle Number and Elevation of Calcium Levels in Motor Axon Terminals, Similar to Sera from Sporadic Patients.

Previously, we demonstrated increased calcium levels and synaptic vesicle densities in the motor axon terminals (MATs) of sporadic amyotrophic lateral sclerosis (ALS) patients. Such alterations could be conferred to mice with an intraperitoneal injection of sera from these patients or with purified immunoglobulin G. Later, we confirmed the presence of similar alterations in the superoxide dismutase 1 G93A transgenic mouse strain model of familial ALS. These consistent observations suggested that calcium plays a central role in the pathomechanism of ALS. This may be further reinforced by completing a similar analytical study of the MATs of ALS patients with identified mutations. However, due to the low yield of muscle biopsy samples containing MATs, and the low incidence of ALS patients with the identified mutations, these examinations are not technically feasible. Alternatively, a passive transfer of sera from ALS patients with known mutations was used, and the MATs of the inoculated mice were tested for alterations in their calcium homeostasis and synaptic activity. Patients with 11 different ALS-related mutations participated in the study. Intraperitoneal injection of sera from these patients on two consecutive days resulted in elevated intracellular calcium levels and increased vesicle densities in the MATs of mice, which is comparable to the effect of the passive transfer from sporadic patients. Our results support the idea that the pathomechanism underlying the identical manifestation of the disease with or without identified mutations is based on a common final pathway, in which increasing calcium levels play a central role.

PARAMETERS FOR IDENTIFYING FAILURE OF PASSIVE TRANSFER IN SITATUNGA (TRAGELAPHUS SPEKII).

Failure of passive transfer of immunity (FPT) leads to increased calf morbidity and mortality and requires intensive, time-sensitive, and often expensive management for nondomestic ruminants. Without species-specific information with which to make informed decisions, neonatal data from domestic ruminants are often extrapolated to nondomestic zoo-housed species. To date, there have been no studies evaluating FPT in sitatunga (Tragelaphus spekii). The goal of the present study was to establish parameters to characterize adequate passive transfer in sitatunga calves and compare them to published reference intervals in other species. Medical records of 22 sitatunga calves (12 female, 10 male) were reviewed. Seventeen of these calves were defined as "healthy," having survived at least 60 days without colostrum administration or a plasma transfusion. Calf weight, serum glucose, serum gamma-glutamyl transferase (GGT), total protein (TP), globulin concentrations, and results of a zinc sulfate turbidity test (ZSTT) were noted where possible. Mean birth weight of healthy calves at 24 hr was 4.5 kg (range: 3.76.5 kg, n = 12). The mean blood glucose in healthy calves was 152 mg/dl (range: 80-182, n = 16), mean serum TP concentration was 5.9 g/dl (range: 4.9-7.5, n = 16), mean serum globulin concentration was 3.3 g/dl (range: 1.7-4.7, n = 17), and mean serum GGT concentration was 466 U/L (range: 91-1901, n = 16). A ZSTT was performed for 10 healthy calves, resulting in four negative ZSTT results despite having no clinical signs of FPT and the calves having been observed nursing before testing. Sitatunga appear to have lower values for normal FPT parameters than those developed for domestic cattle. This study illustrates the difficulty of cross-species comparisons, as even closely related species can vary greatly in biologic parameters.

Anti-CNTN1 IgG3 induces acute conduction block and motor deficits in a passive transfer rat model.

BACKGROUND: Autoantibodies against the paranodal protein contactin-1 have recently been described in patients with severe acute-onset autoimmune neuropathies and mainly belong to the IgG4 subclass that does not activate complement. IgG3 anti-contactin-1 autoantibodies are rare, but have been detected during the acute onset of disease in some cases. There is evidence that anti-contactin-1 prevents adhesive interaction, and chronic exposure to anti-contactin-1 IgG4 leads to structural changes at the nodes accompanied by neuropathic symptoms. However, the pathomechanism of acute onset of disease and the pathogenic role of IgG3 anti-contactin-1 is largely unknown. METHODS: In the present study, we aimed to model acute autoantibody exposure by intraneural injection of IgG of patients with anti-contacin-1 autoantibodies to Lewis rats. Patient IgG obtained during acute onset of disease (IgG3 predominant) and IgG from the chronic phase of disease (IgG4 predominant) were studied in comparison. RESULTS: Conduction blocks were measured in rats injected with the "acute" IgG more often than after injection of "chronic" IgG (83.3% versus 35%) and proved to be reversible within a week after injection. Impaired nerve conduction was accompanied by motor deficits in rats after injection of the "acute" IgG but only minor structural changes of the nodes. Paranodal complement deposition was detected after injection of the "acute IgG". We did not detect any inflammatory infiltrates, arguing against an inflammatory cascade as cause of damage to the nerve. We also did not observe dispersion of paranodal proteins or sodium channels to the juxtaparanodes as seen in patients after chronic exposure to anti-contactin-1. CONCLUSIONS: Our data suggest that anti-contactin-1 IgG3 induces an acute conduction block that is most probably mediated by autoantibody binding and subsequent complement deposition and may account for acute onset of disease in these patients. This supports the notion of anti-contactin-1-associated neuropathy as a paranodopathy with the nodes of Ranvier as the site of pathogenesis.

Reduced Cell-Associated DNA and Improved Viral Control in Macaques following Passive Transfer of a Single Anti-V2 Monoclonal Antibody and Repeated Simian/Human Immunodeficiency Virus Challenges.

A high level of V1V2-specific IgG antibodies (Abs) in vaccinees' sera was the only independent variable that correlated with a reduced risk of human immunodeficiency virus (HIV) acquisition in the RV144 clinical trial. In contrast, IgG avidity, antibody neutralization, and antibody-dependent cellular cytotoxicity each failed as independent correlates of infection. Extended analyses of RV144 samples demonstrated the antiviral activities of V1V2-specific vaccine-induced antibodies. V2-specific antibodies have also been associated with protection from simian immunodeficiency virus (SIV), and the V2i-specific subset of human monoclonal antibodies (MAbs), while poor neutralizers, mediates Fc-dependent antiviral functions in vitro The objective of this study was to determine the protective efficacy of a V2i-specific human MAb, 830A, against mucosal simian/human immunodeficiency virus (SHIV) challenge. V2i MAb binding sites overlap the integrin binding site in the V2 region and are similar to the epitopes bound by antibodies associated with reduced HIV infection rates in RV144. Because the IgG3 subclass was a correlate of reduced infection rates in RV144, we compared passive protection by both IgG1 and IgG3 subclasses of V2i MAb 830A. This experiment represents the first in vivo test of the hypothesis emanating from RV144 and SIV studies that V2i Abs can reduce the risk of infection. The results show that passive transfer with a single V2i MAb, IgG1 830A, reduced plasma and peripheral blood mononuclear cell (PBMC) virus levels and decreased viral DNA in lymphoid tissues compared to controls, but too few animals remained uninfected to achieve significance in reducing the risk of infection. Based on these findings, we conclude that V2i antibodies can impede virus seeding following mucosal challenge, resulting in improved virus control.IMPORTANCE Since the results of the HIV RV144 clinical trial were reported, there has been significant interest in understanding how protection was mediated. Antibodies directed to a subregion of the envelope protein called V1V2 were directly correlated with a reduced risk, and surprisingly low virus neutralization was observed. To determine whether these antibodies alone could mediate protection, we used a human monoclonal antibody directed to V2 with properties similar to those elicited in the vaccine trial for passive infusions in rhesus macaques and challenge with SHIV. The single V2 antibody at the dose given did not significantly reduce the number of infections, but there was a significant reduction in the seeding of virus to the lymph nodes and a decrease in plasma viremia in the HIV antibody-infused macaques compared with the control antibody-infused animals. This finding shows that V2 antibodies mediate antiviral activities in vivo that could contribute to a protective HIV vaccine.

Effect of butyrate on passive transfer of immunity in dairy calves.

The objectives of this study were to determine the effects of supplemental butyrate on (1) Ig production in dams and (2) Ig absorption in their calves. Twenty dry dams fed a close-up total mixed ration were assigned to either a control treatment (CTRL-D) or a butyrate treatment where the close-up total mixed ration was supplemented with butyrate at 1% of dry matter intake (wt/wt; BUT-D). At calving, calves were assigned to 1 of 2 treatments: a control group fed colostrum replacer only (CTRL-C) and a butyrate group fed colostrum replacer with supplemental butyrate at 2.5% (wt/vol; BUT-C). Serum IgG, glucose, and ß-hydroxybutyrate were measured weekly in both dams and calves. Additionally, calves were weighed weekly to determine average daily gain. In dams, serum IgG concentration was not different between CTRL-D and BUT-D (1,785 ± 117 vs. 1,736 ± 137 mg/dL, respectively), nor was there a change in Ig levels in the colostrum between control and butyrate groups. Serum total protein did not differ between CTRL-D and BUT-D dams. Dam dry matter intake did not differ between CTRL-D and BUT-D but did decrease 1 wk before parturition. Compared with CTRL-C calves, BUT-C calves had significantly decreased serum IgG concentration at 24 h (2,110 ± 124 vs. 1,400 ± 115 mg/dL), wk 1 (1,397 ± 121 vs. 866 ± 115 mg/dL), and wk 2 (1,310 ± 121 vs. 797 ± 115 mg/dL). Additionally, apparent efficiency of absorption was lower for the BUT-C group compared with the CTRL-C group (35.3 ± 2.1 vs. 25.9 ± 2.0). Differences in serum Ig concentrations between the CTRL-C and BUT-C groups did not affect average daily gain (0.59 ± 0.05 vs. 0.48 ± 0.05 kg/d, respectively), serum glucose concentrations, or serum ß-hydroxybutyrate concentrations. These data demonstrate that butyrate inclusion in colostrum negatively affects IgG absorption in newborn calves, whereas calf body weight gains were unaffected.