Short communication: Seroprevalence of paratuberculosis in Italian water buffaloes (Bubalus bubalis) in the region of Campania.

Paratuberculosis is a chronic enteric disease affecting virtually all ruminants, but only anecdotal information is currently available about the occurrence of this disease in water buffaloes (Bubalus bubalis). We carried out a survey study aimed at determining the prevalence of paratuberculosis in 2 provinces in the region of Campania, Italy, where about half of all Italian buffaloes are reared. From May 2017 to December 2018, we collected 201,175 individual serum samples from 995 buffalo herds. The sera were collected from animals over 24 mo old and were tested using a commercial ELISA test. The herd-level apparent prevalence result was 54.7%, and the animal-level apparent prevalence was 1.8%. The herd-level true prevalence was estimated using a Bayesian approach, demonstrating a high herd-level prevalence of paratuberculosis in water buffaloes from the Campania area. These findings suggest that the urgent adoption of paratuberculosis herd-control programs for water buffaloes in this area would be beneficial.

Short communication: Persistent contamination by Listeria monocytogenes of bovine raw milk investigated by whole-genome sequencing.

Following the persistent detection of Listeria monocytogenes in raw bovine milk sold through a vending machine, the 120 lactating cows of the herd producing the milk were subjected to bacteriological investigation. A single cow with subclinical mastitis (1.2-1.3 × 105 somatic cells/mL) and persistent L. monocytogenes excretion was detected. The cow was subjected to antimicrobial therapy, but L. monocytogenes excretion remained high (>3.0 × 102 cfu/mL). Following culling of the infected cow, L. monocytogenes disappeared from the tank milk, and further isolates were recovered from the mammary parenchyma and lymph nodes of the infected cow. To investigate the clonal nature of the contamination, all isolates recovered in the study (n = 13) were analyzed by serogroup PCR, pulsed-field gel electrophoresis, and whole-genome sequencing. Our results demonstrated the clonal nature of the contamination. All isolates belonged to lineage II, serogroup IIa, sequence type 37, clonal complex 37 and harbored some virulence determinants. This case showed that, although relatively rare, prolonged milk contamination by L. monocytogenes can originate from subclinical and persistently infected cows, posing a health risk to consumers.

Survival of Mycobacterium avium subsp. paratuberculosis in the intermediate and final digestion products of biogas plants.

AIMS: To evaluate the survival of Mycobacterium avium subsp. paratuberculosis (MAP) during anaerobic digestion (AD), we studied two different biogas plants loaded with manure and slurry from paratuberculosis-infected dairy herds. METHODS AND RESULTS: Both plants were operating under mesophilic conditions, the first with a single digester and the second with a double digester. Mycobacterium avium subsp. paratuberculosis detection was performed by sampling each stage of the process, specifically the prefermenter, fermenter, liquid digestate and solid digestate stages, for 11 months. In both plants, MAP was isolated from the prefermenter stage. Only the final products, the solid and liquid digestates, of the one-stage plant showed viable MAP, while no viable MAP was detected in the digestates of the two-stage plant. CONCLUSIONS: Mycobacterium avium subsp. paratuberculosis showed a significant decrease during subsequent steps of the AD process, particularly in the two-stage plant. We suggest that the second digester maintained the digestate under anaerobic conditions for a longer period of time, thus reducing MAP survival and MAP load under the culture detection limit. SIGNIFICANCE AND IMPACT OF THE STUDY: Our data are unable to exclude the presence of MAP in the final products of the biogas plants, particularly those products from the single digester; therefore, the use of digestates as fertilizers is a real concern related to the possible environmental contamination with MAP.

Exploring MALDI-TOF MS approach for a rapid identification of Mycobacterium avium ssp. paratuberculosis field isolates.

AIMS: The aim of the study was to explore the suitability of matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) for a rapid and correct identification of Mycobacterium avium ssp. paratuberculosis (MAP) field isolates. METHODS AND RESULTS: MALDI-TOF MS approach is becoming one of the most popular tests for the identification of intact bacterial cells which has been shown to be fast and reliable. For this purpose, 36 MAP field isolates were analysed through MALDI-TOF MS and the spectra compared with two different databases: one provided by the vendor of the system employed (Biotyper ver. 3·0; Bruker Daltonics) and a homemade database containing spectra from both tuberculous and nontuberculous Mycobacteria. Moreover, principal component analysis procedure was employed to confirm the ability of MALDI-TOF MS to discriminate between very closely related subspecies. Our results suggest MAP can be differentiated from other Mycobacterium species, both when the species are very close (M. intracellulare) and when belonging to different subspecies (M. avium ssp. avium and M. avium ssp. silvaticum). CONCLUSIONS: The procedure applied is fast, easy to perform, and achieves an earlier accurate species identification of MAP and nontuberculous Mycobacteria in comparison to other procedures. SIGNIFICANCE AND IMPACT OF THE STUDY: The gold standard test for the diagnosis of paratuberculosis is still isolation of MAP by cultural methods, but additional assays, such as qPCR and subculturing for determination of mycobactin dependency are required to confirm its identification. We have provided here evidence pertaining to the usefulness of MALDI-TOF MS approach for a rapid identification of this mycobacterium among other members of M. avium complex.

Short communication: Investigation into Mycobacterium avium ssp. paratuberculosis in pasteurized milk in Italy.

This study investigated the presence of viable Mycobacterium avium ssp. paratuberculosis (MAP) in pasteurized milk produced by Italian industrial dairy plants to verify the prediction of a previously performed risk assessment. The study analyzed 160 one-liter bottles of pasteurized milk from 2 dairy plants located in 2 different regions. Traditional cultural protocols were applied to 500mL of pasteurized milk for each sample. The investigation focused also on the pasteurization parameters and data on the microbiological characteristics of raw milk (total bacterial count) and pasteurized milk (Enterobacteriaceae and Listeria monocytogenes). No sample was positive for MAP, the pasteurization parameters complied with European Union legislation, and the microbiological analysis of raw and pasteurized milk showed good microbiological quality. The results show that a 7-log (or >7) reduction could be a plausible value for commercial pasteurization. The combination of hygiene practices at farm level and commercial pasteurization yield very low or absent levels of MAP contamination in pasteurized milk, suggesting that pasteurized milk is not a significant source of human exposure to MAP in the dairies investigated.

Short communication: In vitro antimicrobial susceptibility of Mycoplasma bovis isolates identified in milk from dairy cattle in Belgium, Germany, and Italy.

The objective of this study was to assess the in vitro antimicrobial susceptibility of 73 isolates of Mycoplasma bovis isolated from milk of dairy cattle herds of Belgium, Germany, and Italy. Minimal inhibitory concentration (MIC) values were determined by the microbroth dilution method for the following antimicrobials: erythromycin, spiramycin, tilmicosin, tylosin, lincomycin, enrofloxacin, doxycycline, oxytetracycline, florfenicol, and tiamulin. Macrolides, florfenicol, oxytetracycline, and enrofloxacin, were chosen because they represent antimicrobials families commonly used in several countries for treatment of M. bovis, and their MIC values in cattle population are reported in several studies, allowing a comparison with previous data. Doxycycline and tiamulin were selected to assess the susceptibility of M. bovis to new antimicrobials, because they are not registered in the European Union for the treatment of dairy cattle. Among the agents of the different antimicrobial classes, the macrolides showed the highest concentration to inhibit 90% of isolates (MIC90), all above the highest concentration tested: >8µg/mL for erythromycin, >16µg/mL for spiramycin, and >32µg/mL for tilmicosin and tylosin. Also the MIC90 of lincomycin was above the highest concentration tested (>32µg/mL), but the distribution of the MIC values was almost perfectly bimodal: 41 isolates had a MIC ≤0.5µg/mL and 30 isolates >32µg/mL. Oxytetracycline had a 2-fold higher concentration to inhibit 50% of isolates (2 vs. 0.5µg/mL) and 1-fold higher MIC90 (4 vs. 2µg/mL) than doxycycline. Enrofloxacin and florfenicol had both a MIC90 of 2µg/mL, whereas tiamulin had a MIC90 of 0.5µg/mL. Significant differences on the MIC values were found among the 3 countries for several antimicrobials: compared with Germany, Belgium and Italy showed significantly higher MIC for lincomycin, spiramycin, and tylosin, and lower for oxytetracycline and florfenicol. The Belgian isolates showed the lowest MIC for enrofloxacin compared with Germany and Italy. The MIC results obtained in our study suggest the presence of a high level of resistance of M. bovis isolates originating from milk to macrolides in all countries involved in this study. On the contrary, a low level of resistance was found against the antimicrobials that are not used in cattle, such as tiamulin and doxycycline, highlighting a possible link between antimicrobial treatments and development of resistance in the studied M. bovis population.

A screening sampling plan to detect Mycobacterium avium subspecies paratuberculosis-positive dairy herds.

Mycobacterium avium ssp. paratuberculosis (MAP) is the etiological agent of paratuberculosis, a chronic contagious bacterial disease primarily affecting dairy cattle. Paratuberculosis represents a dual problem for the milk production chain: in addition to economic losses to affected herds, MAP may have zoonotic potential. Infected herds must be identified in order to implement programs designed to reduce the incidence of disease within and between herds and to prevent MAP from entering the food chain. The objective of this study was to evaluate the sensitivity and specificity of a screening sampling plan (SSP) to detect MAP-positive dairy herds by repetitive analysis of bulk tank milk (BTM) samples by ELISA and in-line milk filter (ILMF) samples by PCR. Samples from BTM and ILMF were collected twice from 569 dairy herds in southern Italy. Additionally, 12,016 individual milk samples were collected: 9,509 from 102 SSP-positive herds (SSP MAP-positive) and 2,507 from 21 randomly selected SSP-negative herds (SSP MAP-negative). There was a total of 126 SSP MAP-positive herds (i.e., 21.3% SSP MAP-positive herds; 95% confidence interval=18.0-24.9); the within-herd apparent prevalence (AP) ranged between 0.00 and 22.73% (mean 6.07%). A significant difference in within-herd AP was shown between SSP MAP-positive herds and SSP MAP-negative herds. A highly significant association was shown between the median AP herd status (>5%) and positivity to at least one ILMF or BTM sample. The SSP detected a minimum of 56.25% of low AP herds (AP ≤ 2.0%) up to a maximum of 100% of herds with a within-herd AP ≥ 8.0%. Overall, the SSP detected 85.57% of herds in which at least one individual milk sample was positive by ELISA. The proposed SSP was an inexpensive and useful tool to detect MAP-positive herds with a higher risk of infection diffusion and milk contamination. Although the SSP cannot be used for MAP-free certification of herds, it could be useful to prioritize appropriate control measures aimed at reducing the prevalence of infection in dairy herds and milk contamination.

A rapid real-time PCR/DNA resolution melting method to identify Prototheca species.

AIM: The study describes the development of a simple and rapid tool to identify yeast-like microalgae belonging to the genus Prototheca. METHODS AND RESULTS: The method, based on two-step Real Time PCR reaction followed by DNA Resolution Melting Analysis (qPCR/RMA), has been developed using reference strains belonging to both pathogenic (P. zopfii genotype 2, P. wickerhamii and P. blaschkeae) and non-pathogenic species (P. zopfii genotype 1, P. stagnora and P. ulmea). In order to validate the method, seventy recently isolated Prototheca strains were thus tested in parallel with both the first qPCR/RMA and the conventional genotype-specific PCR assay: they were classified as P. zopfii genotype 1, P. zopfii genotype 2 and P. blaschkeae, with a perfect accordance between the two above methodologies. Furthermore, we used the second qPCR/RMA to identify the other species (P. stagnora, P. ulmea and P. wickerhamii), which cannot be discriminated by conventional PCR assay. CONCLUSIONS: The assay two-step Real Time PCR is accurate, robust, cost-effective and faster than auxonographical, biochemical or conventional molecular biology methods. SIGNIFICANCE AND IMPACT OF THE STUDY: the rapid and high throughout two-step qPCR/RMA tool can be usefully used for the identification of clinical and environmental Prototheca species into the framework of the diagnosis of animal (e.g. bovine mastitis) or human protothecosis.

Transmission patterns of a Mycobacterium avium subsp. paratuberculosis clone within a single heard investigated by Whole Genome Sequencing.

Mycobacterium avium subsp. paratuberculosis (MAP) is characterized by a low genomic rate of mutation. Current subtyping tools, such as Mini-Micro-satellite analyses, do to have not sufficient discriminatory power to disclose MAP's evolution on small spatial and temporal scales. The aim of the study was to investigate the population structure of MAP inside a single dairy herd using whole genome sequencing (WGS) approaches. For this purpose, the genomes of 43 field isolates, recovered from the faeces of 36 cows of the same dairy herd from 2012 to 2016, were sequenced by WGS. The isolates' genomes showed a low number (43) of polymorphic sites (SNPs), confirming the clonal origin of the herd infection. However, despite the limited genomic diversity found in WGS, the phylogenetic analysis was discriminatory enough to detect the presence of different genomic clades and sub-clades inside the herd population. In addition, the phylodynamic reconstruction showed the existence of an ancestor clade from which the other clades and sub-clades originated. Moreover, by reconstructing the putative within-herd transmission networks using WGS data, we demonstrated that: (i) in a herd where MAP is endemic, multiple isolates recovered from a single animal and differing from each other by few (three/four) SNPs can originate from different transmission or passive shedding events and not from intra-host evolution; and (ii) variability of minisatellites coupled with a few microsatellites does not represent reliable tracers of within-herd infection chains. Our findings show that WGS, coupled with relevant epidemiological information, represents a valuable tool to work out fine epidemiological and micro-evolutionary relationships such as those at herd-level scale.

Molecular characterization of Prototheca strains isolated from Italian dairy herds.

One hundred sixty-one Prototheca spp. strains isolated from composite milk and barn-surrounding environmental samples (bedding, feces, drinking, or washing water, surface swabs) of 24 Italian dairy herds were characterized by genotype-specific PCR analysis. Overall, 97.2% of strains isolated from composite milk samples were characterized as Prototheca zopfii genotype 2, confirming its role as the main mastitis pathogen, whereas Prototheca blaschkeae was only sporadically isolated (2.8%). Regarding environmental sampling, 84.9% of isolates belonged to P. zopfii genotype 2, 13.2% to P. blaschkeae, and 1.9% to P. zopfii genotype 1. The data herein contradict previous hypotheses about the supposed exclusive role of P. zopfii genotype 2 as the causative agent of protothecal mastitis and, on the contrary, confirm the hypothesis that such pathology could be caused by P. blaschkeae in a few instances.

Evaluation of Mycobacterium avium subsp. paratuberculosis survival during the manufacturing process of Italian raw milk hard cheeses (Parmigiano Reggiano and Grana Padano).

Mycobacterium avium subsp. paratuberculosis (MAP), the agent of paratuberculosis in ruminants, is suspected to be involved in the aetiology of some human diseases. Notably, the consumption of milk and dairy products is considered to be the main route of human exposure to MAP because of its ability to survive during pasteurization and manufacturing processes. The aim of this study was to investigate, through a microbiological challenge test, the survival of MAP during the manufacturing and ripening period of two Italian hard cheeses, Parmigiano Reggiano and Grana Padano, made from raw bovine milk. The challenge test was performed in two different phases: the creaming phase and the manufacturing phase. The creaming phase, which is the first step of cheese production, was reproduced in the laboratory employing raw cow's milk spiked with a MAP reference strain at a final concentration of 5.58 log10 CFU/mL. After the creaming at 18 °C and 27 °C for 12 h, a decrease of 0.80 log10 and 0.77 log10 was observed in partially skimmed milk, respectively. In the second phase, two batches of raw cow's milk (1000 L each) were inoculated with MAP reference and wild strains, respectively. Then, the entire manufacturing process for Parmigiano Reggiano and Grana Padano, both of Protective Designation of Origin (PDO), was reproduced in an experimental cheese factory, starting from a concentration in milk of 5.19 ±â€¯0.01 and 5.28 ±â€¯0.08 log10 CFU/mL of MAP reference and wild strains, respectively. Heating the curd at 53 °C for 20 min did not affect MAP survival, however a significant decrease (p < 0.05) in MAP viability was observed during the moulding phase and after salting in brine, regarding the wild strains and the reference strain, respectively. In addition, a significant decrease was observed during the ripening period, at which time the MAP concentration dropped below the limit of detection from the second and the third month of ripening, for the wild and reference strains, respectively. Taking into account the poor data availability about MAP survival in hard cheeses, this study may improve the knowledge regarding the effect of the cheese manufacturing process on the MAP dynamics, supporting also the safety of traditional raw milk hard cheeses.

Mycobacterium porcinum strains isolated from bovine bulk milk: implications for Mycobacterium avium subsp. paratuberculosis detection by PCR and culture.

In this study, the isolation of 52 mycobactin-independent fast growing mycobacteria from 631 bulk milk samples (8.2%), is reported. These strains, isolated during a bulk milk survey for Mycobacterium avium subsp. paratuberculosis (Map), strongly affected Map detection both by PCR and by culture, as they gave a positive IS900 PCR signal and resulted to totally inhibit the growth of Map when spotted on HEYM slants already inoculated with 200 microl of 10-fold dilutions containing from 5 x 10 to 5 x 10(3)Map cells/ml. 16S rRNA gene sequencing, using the MicroSeq 500 16S rDNA Bacterial Sequencing Kit (Applied Biosystems), was performed on a subset of six strains, identifying Mycobacterium porcinum with 100% homology in all six cases. The 52 strains were characterized by PCR-restriction fragment length polymorphism (RFLP) analysis of the hsp65 gene, which confirmed the identification of M. porcinum for all the isolates. Using specific primers designed on the Map-IS900 sequence and on the M. porcinum sequence determined in this study, a 1385bp sequence from the M. porcinum genome was characterized. This IS900-like sequence showed 82% homology with Map IS900. From our findings the following results emerged: (a) any culture showing one or more M. porcinum colonies represents a potential "false negative" result and should therefore be considered as contaminated; (b) IS900-like elements could be more widespread than was previously thought; (c) IS900 PCR positive results should be interpreted cautiously, as confirmed by the evidence that the primer pair used in this study resulted not to be specific.

Short communication: isolation of Prototheca species strains from environmental sources in dairy herds.

Composite milk samples from 548 cows, and samples from feces, feed, bedding, water, liners (before and after milking), and the postdipping product were aseptically collected from 2 Italian dairy herds from February to November of 2006. Prototheca zopfii was isolated from 11.9% of milk samples, 15% of feces, and 33.3% of bedding samples. No viable cells of P. zopfii were observed in water before washing procedures, whereas 25 to 28.6% of samples from water used for washing both refrigeration tanks and milking equipment were contaminated with this yeast-like microalga. Analogously, the presence of P. zopfii was detected only on swabs collected from the liners after milking. Interestingly, in 1 of the 2 herds, water from the drinking trough was contaminated by viable cells of both P. zopfii and the related environmental species Prototheca stagnora. No viable cells were observed in cow feed. On the basis of the results presented herein, P. zopfii seemed to be widespread throughout the environments of dairy herds where outbreaks of bovine mastitis had occurred.

Survey on the presence of Mycobacterium avium subsp. paratuberculosis in ground beef from an industrial meat plant.

Paratuberculosis of ruminants is characterised by chronic enteritis but, at advanced stages of the disease, a systemic dissemination of Mycobacterium avium subsp. paratuberculosis (MAP) in tissues and organs can occur. MAP has been recovered from lymph nodes and muscles of clinical and sub-clinical cows. In most countries, dairy and beef cattle infected with paratuberculosis are routinely sent to slaughter and the consumption of their meat could be a possible route of human exposure to MAP. However, few studies on MAP in ground beef are currently available. During the period November 2013-March 2014 we carried out a survey on the ground beef produced in an industrial meat processing plant. One-hundred and forty samples of ground meat were analysed by IS900-qPCR and culture (VersaTrek System). The limit of detection (LOD) of qPCR was 630 MAP cells/g (107 CFU/g) while the LOD for culture was 170-230 MAP cells/g (62-115 CFU/g). No samples were positive by direct IS900 qPCR, while two samples were positive by liquid culture. Our data suggest that the presence of live MAP in raw minced meat is possible. In order to avoid exposure for humans through the consumption of contaminated meat, proper cooking of meat is recommended.

First outbreak of bovine mastitis caused by Prototheca blaschkeae.

The most important animal disease caused by yeast-like algae belonging to the genus Prototheca is bovine mastitis. Although the infection can be caused by both Prototheca zopfii genotype 2 and Prototheca blaschkeae, the bulk of prevalence of bovine protothecal mastitis has been so far attributed to the former, being P. blaschkeae only sporadically isolated. However, we report here the first outbreak of bovine mastitis caused by P. blaschkeae in an Italian dairy herd. One hundred and four individual milk samples, three bulk tank milk and 16 environmental samples within the herd were screened for the presence of Prototheca: five, one and four positive samples, were respectively observed. Molecular analysis revealed that, with the sole exception of one environmental isolate belonging to P. zopfii genotype 2, all Prototheca strains were identified as P. blaschkeae. Our results might suggest that even P. blaschkeae can induce mastitis outbreaks, while it is not clear if the higher incidence of P. zopfii genotype 2 as causative agent of protothecal mastitis could reflect an intrinsic higher pathogenicity or it could be simply the consequence of its, so far observed, higher diffusion in worldwide dairy herd ecosystems.

Prevalence of paratuberculosis infection in dairy cattle in Northern Italy.

Paratuberculosis is a chronic granulomatous infection caused by Mycobacterium avium subsp. paratuberculosis (MAP) that affects multiple ruminant species causing important economic losses. Therefore, control programmes at herd and regional levels have been established worldwide and prevalence estimates are needed for their implementation. Although different herd-level prevalence estimations for paratuberculosis have been reported in Europe, very few studies provided comparable and interpretable values, due to poor study designs and lack of knowledge about the accuracy of the diagnostic tests used. To overcome these problems we applied a latent class analysis to the results of two prevalence studies carried out in two neighbouring Northern Italian regions (Lombardy and Veneto) that account for over 50% of the Italian dairy cattle population. Serum samples from a randomly selected number of farms in the two regions were analyzed by different ELISA tests. The herd-level Apparent Prevalences (AP) were 48% (190/391) for Lombardy and 65% (272/419) for Veneto. Median within-herd APs were 2.6% and 4.0% for Lombardy and Veneto, respectively. Posterior estimates for the herd-level True Prevalences (TP) based on a Bayesian model were very similar between the two regions (70% for Lombardy and 71% for Veneto) and close to previous estimates of infected herds in Europe. The two 95% credibility intervals overlap each other, virtually showing only one distribution of the herd-level true prevalence for both regions. On the contrary, estimates of the within-herd TP distributions differed between the two regions (mean values: 6.7% for Lombardy and 14.3% for Veneto), possibly due to the different age distribution within the herds from the two regions.

Detrusor instability as an energy-saving device in prostatic obstruction.

PURPOSE: We assessed whether voiding dynamics differ in patients with infravesical obstruction from benign prostatic hyperplasia between those with detrusor instability and those with stable bladders. MATERIALS AND METHODS: A total of 50 such patients with (25) and without (25) detrusor instability was investigated urodynamically by cystometry and pressure-flow study. RESULTS: In the unstable group there was greater obstruction (as assessed by the linear passive urethral resistance relation pressure-flow nomogram) but the detrusor expended less energy (p = 0.001) for voiding the unit volume (54.4 +/- 22.8 mJ./ml versus 74.4 +/- 31.8 mJ./ml. in the stable series). Spared energy was converted into more powerful micturition contractions and more efficient voiding, with a mean maximum contraction power and mean fraction of bladder volume voided of 17.9 +/- 4.9 microW./mm.2 and 93 +/- 11%, respectively, in the unstable population versus 12.7 +/- 2.1 microW./mm.2 (p < 0.001) and 79 +/- 13% (p < 0.001) in the stable subjects. CONCLUSIONS: Detrusor instability possibly works within physiological limits as an energy-saving device, preventing voiding efficiency in patients with prostate disease from decreasing too much with increasing obstruction.

Peripheral plasma amino acid abnormalities in rehabilitation patients with severe brain injury.

OBJECTIVE: Acute severe brain injury causes an increased mobilization of amino acids from tissue. The plasma amino acid profile of patients undergoing rehabilitation after brain injury is unknown. This study was aimed at delineating the plasma amino acid profile of rehabilitation patients with brain injury. DESIGN: Peripheral plasma aminogram, lactate, pyruvate, glycerol, ketone body, and carnitine concentrations were determined in 11 patients with brain injury (34.6+/-15 years old, 60+/-16.8 days after injury) and in 8 controls. Resting energy expenditure and nitrogen balance were also determined. RESULTS: (1) All essential amino acids and about 50% of nonessential amino acids were significantly lower in brain injury patients than in controls (p < .05). (2) Plasma amino acids were lower irrespective of either energy and protein intake or nitrogen balance. (3) Total carnitine concentration and esterified/free carnitine ratio were higher in brain injury patients than in controls (p < .05). CONCLUSIONS: Rehabilitation patients with brain injury may have an important reduction of their plasma aminogram. Muscle tissue depletion and the persistence of a hypercatabolic state caused by subclinical infections, pressure sores, and immobility may contribute to this reduction.