A MALDI-TOF MS database for fast identification of Vibrio spp. potentially pathogenic to marine mollusks.

In mollusk aquaculture, a large number of Vibrio species are considered major pathogens. Conventional methods based on DNA amplification and sequencing used to accurately identify Vibrio species are unsuitable for monitoring programs because they are time-consuming and expensive. The aim of this study was, therefore, to develop the MALDI-TOF MS method in order to establish a rapid identification technique for a large panel of Vibrio species. We created the EnviBase containing 120 main spectra projections (MSP) of the Vibrio species that are potentially responsible for mollusk diseases, comprising 25 species: V. aestuarianus, V. cortegadensis, V. tapetis and species belonging to the Coralliilyticus, Harveyi, Mediterranei, and Orientalis clades. Each MSP was constructed by the merger of raw spectra obtained from three different media and generated by three collaborating laboratories to increase the diversity of the conditions and thus obtain a good technique robustness. Perfect discrimination was obtained with all of the MSP created for the Vibrio species and even for very closely related species as V. europaeus and V. bivalvicida. The new EnviBase library was validated through a blind test on 100 Vibrio strains performed by our three collaborators who used the direct transfer and protein extraction methods. The majority of the Vibrio strains were successfully identified with the newly created EnviBase by the three laboratories for both protocol methods. This study documents the first development of a freely accessible database exclusively devoted to Vibrio found in marine environments, taking into account the high diversity of this genus. KEY POINTS: • Development of a MALDI-TOF MS database to quickly affiliate Vibrio species. • Increase of the reactivity when faced with Vibrio associated with mollusk diseases. • Validation of MALDI-TOF MS as routine diagnostic tool.

Identification and subtyping of clinically relevant human and ruminant mycoplasmas by use of matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) recently emerged as a technology for the identification of bacteria. In this study, we aimed to evaluate its applicability to human and ruminant mycoplasmal identification, which can be demanding and time-consuming when using phenotypic or molecular methods. In addition, MALDI-TOF MS was tested as a subtyping tool for certain species. A total of 29 main spectra (MSP) from 10 human and 13 ruminant mycoplasma (sub)species were included in a mycoplasma MSP database to complete the Bruker MALDI Biotyper database. After broth culture and protein extraction, MALDI-TOF MS was applied for the identification of 119 human and 143 ruminant clinical isolates that were previously identified by antigenic or molecular methods and for subcultures of 73 ruminant clinical specimens that potentially contained several mycoplasma species. MALDI-TOF MS resulted in accurate (sub)species-level identification with a score of ≥1.700 for 96% (251/262) of the isolates. The phylogenetically closest (sub)species were unequivocally distinguished. Although mixtures of the strains were reliably detected up to a certain cellular ratio, only the predominant species was identified from the cultures of polymicrobial clinical specimens. For typing purposes, MALDI-TOF MS proved to cluster Mycoplasma bovis and Mycoplasma agalactiae isolates by their year of isolation and genome profiles, respectively, and Mycoplasma pneumoniae isolates by their adhesin P1 type. In conclusion, MALDI-TOF MS is a rapid, reliable, and cost-effective method for the routine identification of high-density growing mycoplasmal species and shows promising prospects for its capacity for strain typing.

Diagnostic value of ultrasonography to assess stifle lesions in dogs after cranial cruciate ligament rupture: 13 cases.

The objective of this study was to evaluate the accuracy of ultrasonographic diagnosis of lesions in the canine stifle associated with cranial cruciate ligament rupture. Thirteen dogs that had a diagnosis of cranial cruciate ligament rupture were included in this prospective clinical study. Two ultrasonographers who were unaware of specific historical and clinical data performed the sonography with a high frequency (8-16 MHz) linear transducer. Surgical treatment of the affected stifle was performed within two days of ultrasonography by a surgeon who was unaware of the ultrasonographic findings. The lesions observed during ultrasonography and arthrotomy were compared at the completion of the study. Visualisation of the superficial tendons (quadriceps and long digital extensor) and ligaments (patellar ligament, collateral ligaments) of the stifle using ultrasonography was excellent. However, the detection of deep stifle ligaments (cranial cruciate ligament and caudal cruciate ligament) was extremely difficult to perform using ultrasonography. For cranial cruciate ligament rupture, the sensitivity for ultrasonographic diagnosis was 15.4%. For meniscal lesions, the sensitivity, specificity, positive and negative predictive values for ultrasonographic diagnosis were 82%, 93%, 90% and 88% respectively. High frequency ultrasonography is a non-invasive method for accurately and efficiently detecting superficial ligaments, tendons and meniscal lesions associated with cranial cruciate ligament rupture in the stifle of non-sedated dogs.

Analysis of the sheep MUC1 gene: structure of the repetitive region and polymorphism.

An investigation was undertaken with the aim of studying the repetitive region of the MUC1 gene and analyzing its polymorphisms in some Italian sheep breeds. Two primers previously used for the goat MUC1 gene analyses allowed for the amplification of 4 different alleles. The sequence analysis showed that the repetitive region of the sheep MUC1 gene is an array of 60-bp repeats, in accordance with the information reported in humans, cattle, and goats. The polypeptide sequence encoded by the consensus repeat was very similar to the corresponding sequences of goats and cattle. The average homology of all repeated units was 82%; when the repeats were compared with the derived consensus repeat, homology dropped to 78%. The repeats were not all perfectly conserved, but the sequence homology was nevertheless clearly sufficient to preserve the mechanism giving rise to the variable-number tandem-repeat polymorphism. In spite of their reduced sequence homology, the sheep repeats shared a high number of potential glycosylation sites. The conservation of the exact number and position of glycosylation sites did not seem to be very important for the purpose of functional integrity, but glycosylation appeared to be conserved as a bulk property. Analysis of the polymorphism in 6 Italian breeds showed that the sheep repetitive region seemed to be less variable and smaller in size than the repetitive region of the goat. The findings of this study suggest that ruminants can be a useful model to study the mechanisms by which the variation in the repeat number and the extracellular domain size can modulate the effectiveness of MUC1 as a cell-surface shield.

Carpal collateral ligament desmopathy in three horses.

The radiographic and ultrasonographic findings in three horses with severe damage to the collateral ligaments of the carpus, associated with degenerative joint disease, are described. The horses remained lame despite prolonged rest and medical management, primarily because of the progression of the joint disease. Ultrasonography was found to be an accurate method for diagnosing the injury to the ligaments and is recommended as part of a routine examination in suspected carpal injuries.

Casein haplotype structure in five Italian goat breeds.

The aim of this work was to investigate the genetic structure of the casein gene cluster in 5 Italian goat breeds and to evaluate the haplotype variability within and among populations. A total of 430 goats from Vallesana, Roccaverano, Jonica, Garganica, and Maltese breeds were genotyped at alphas1-casein (CSN1S1), alphas2-casein, (CSN1S2), beta-casein (CSN2), and kappa-casein (CSN3) loci using several genomic techniques and milk protein analysis. Casein haplotype frequencies were estimated for each breed. Principal component analysis was carried out to highlight the relationship among breeds. Allele and haplotype distributions indicated considerable differences among breeds. The haplotype CSN1S1*F- CSN1S2*F-CSN3*D occurred in all breeds with frequencies >0.100 and was the most common haplotype in the Southern breeds. A high frequency of CSN1S1*0-CSN1S2*C-CSN3*A haplotype was found in Vallesana population (0.162). Principal component analysis clearly separated the Northern and Southern breeds by the first component. The variability of the caprine casein loci and variety of resulting haplotypes should be exploited in the future using specific breeding programs aiming to preserve biodiversity and to select goat genetic lines for specific protein production.

Analysis of the MUC1 gene and its polymorphism in Capra hircus.

The objective of our study was to demonstrate the existence of a repetitive region in the goat MUC1 gene and to develop a polymerase chain reaction (PCR) protocol to analyze its polymorphism in different breeds. Using 2 primers derived from the bovine MUC1 sequence, a PCR fragment was obtained and cloned. The sequence analysis shows that the repetitive region of goat MUC1 is an array of 60 bp repeats in accordance with the data reported for other species. The polypeptide sequences encoded by the consensus repeats of goat and bovine were exactly alike. A PCR protocol to improve the detection of goat MUC1 polymorphism was developed, and a total of 178 animals from 6 Italian breeds were analyzed. Fifteen different alleles, ranging in size from 1500 to 3000 bp, were found. The high number of alleles observed shows that the goat MUC1 is a hypervariable gene. These results are the basis for further investigations on the possible role of MUC1 polymorphism in the genetic control of disease susceptibility and production traits in the goat species.

Comparison of radiography and scintigraphy in the diagnosis of dental disorders in the horse.

Scintigraphy, with 99mTechnetium methylenediphosphonate (99mTc-MDP) and 99mTc-labelled leucocytes, was compared to radiography in the diagnosis of dental disease in the horse in a prospective case-controlled study, comprising 30 horses with clinical signs of dental disease and 30 control horses. In each case, right and left lateral, ventral and dorsal soft tissue and bone phase scintigraphic images were obtained after i.v. injection of 1 GBq/100 kg bwt 99mTc-MDP, using a gamma camera. The same views were acquired in 10 horses with clinical signs of dental disease and 12 control horses after injection of 99mTc-labelled leucocytes. Standard radiographic projections of the paranasal sinuses and of the apices of the maxillary and mandibular teeth were obtained. The scintigraphs and radiographs were assessed subjectively by 2 board-certified surgeons and one board-certified radiologist, with extensive experience of equine radiology, from who the clinical history was withheld. Sensitivity, specificity and kappa, as a measure of agreement, were calculated for the different methods. Bone phase images were also scored subjectively on a scale from 0 to 3 on the basis of isotope uptake over the teeth. Regions of interest were defined over the teeth, and normal teeth compared to diseased counterparts. Total scintigraphic counts were related to the age of the animal and to the disease process. Differences in density ratios between left and right teeth were evaluated using the Mann-Whitney Test. Dental disease was confirmed in 22 horses at surgery or postmortem examination. Horses with dental disease showed a significant increase in scintigraphic activity over the affected tooth compared to the contralateral tooth, with a typical pattern for different diseases. The sensitivity of scintigraphy with 99mTc-MDP proved to be excellent (95.5%), whereas the specificity was moderate (86.4%). In contrast, radiography had excellent specificity (95.0%) and a low sensitivity (51.5%). The greatest sensitivity and specificity were achieved by evaluating radiographs and scintigrams together. The objective scintigraphic density ratios were found to be significantly different between diseased and control horses. The results of this study suggest that, if a density ratio of 1.5 or greater between a suspected diseased tooth and its contralateral number is regarded as abnormal, only 1% false positive diagnoses and 20% false negative diagnoses will occur. In contrast, scintigraphy with 99mTc-labelled leucocytes was not very successful, due to the lack of anatomical detail provided by this technique, which made identification of the diseased tooth impossible. Accurate radiographic interpretation of dental disease presents difficulties, both in terms of missed diagnoses and mistaken diagnoses. Scintigraphy complements radiographic examination of dental structures by providing information important for accurate diagnosis and is, therefore, conceived to be essential for selection of the appropriate treatment for dental disease.

Endoscopic examination of the tarsal sheath of the lateral digital flexor tendon in horses.

This study was designed to develop a reliable technique for endoscopic examination of the tarsal sheath of the lateral digital flexor tendon of horses. The anatomy of the tendon sheath and associated structures was studied in detail in cadavers before determining portals for the insertion of an arthroscope into the sheath. Approaches into the sheath through the proximal pouch and through the flexor retinaculum, at the level of the sustentaculum tali, were performed and compared in cadavers. The proximal pouch portal permitted visualisation only of the proximal half of the sheath, while the approach through the retinaculum allowed examination of the entire sheath. The normal endoscopic appearance of the tarsal sheath was studied. The endoscopic approach was subsequently used to examine and treat 5 horses with tarsal sheath tenosynovitis, including 2 cases of chronic, traumatic tenosynovitis and 3 of subacute septic tenosynovitis. Four of these horses had fragmentation of the sustentaculum tali. The technique allowed adequate examination of the sheath and debridement of adhesions and lesions within the lumen of the sheath. Fragments dorsal to the medioplantar edge of the sustentaculum tali could not be visualised endoscopically and had to be removed after widening of the wound. All 5 horses survived. Follow-up enquiries (8-31 months) revealed that the horses were all reported to be sound. Four were performing at their previous level of activity, 1 was used for hacking. The 2 cases presented with chronic tenosynovitis had residual sheath distension with no associated loss of function. A prospective study, including longer term follow-up investigation, is currently being performed.

Comparison of radiography, scintigraphy and ultrasonography in the diagnosis of a case of temporomandibular joint arthropathy in a horse.

Diagnosis of temporomandibular joint disease in the horse presents a challenge to the equine veterinarian. This case report illustrates a combined imaging approach using radiography, scintigraphy and ultrasonography in a horse with severe arthropathy of the left temporomandibular joint. Radiographs were inconclusive. Scintigraphy with Tc99m methylenediphosphonate localised, but failed to characterise the disease process; ultrasonography was the only imaging method which both localised and characterised the lesion. Ultrasonography proved to be a relatively cheap, technically easy to perform and non-invasive method for the assessment of the disease.

Septic osteitis of the distal phalanx: findings and surgical treatment in 18 cases.

Eighteen cases of septic osteitis of the distal phalanx were diagnosed clinically and confirmed at surgery over a 6 year period (1990-1996). Focal bone lysis was the most common radiographic finding. Sequestra were identified in 4 horses, one of which was associated with a fractured plantar process. All the horses were treated surgically with open debridement. The septic process recurred in 4 horses: one was subjected to euthanasia due to navicular bursa infection, secondary to a pathological fracture, the other 3 horses responded to further debridement. A pathological fracture occurred in another horse. All the survivors were not lame 3 months post operatively, although complete healing took 1-12 months. Long term follow up was available in 14 cases. One was retired, all the other horses were back in work at a similar level of performance. This series indicates that surgical treatment of septic pedal osteitis carries a fair to good prognosis despite a high rate of post operative complications and convalescence period of up to 12 months.

Gross and ultrasonographic anatomy of the carpal flexor tendon sheath in horses.

This study was undertaken to establish the gross anatomy and the ultrasonographic appearance of the carpal digital flexor tendon sheath (carpal sheath) and the palmar carpal region in normal horses. The isolated forelimbs from 15 horses were used to study the morphology of the sheath and associated structures, including a detailed study of the location of the main blood vessels and nerves in that region. These limbs and the forelimbs of five live, sound horses were also examined ultrasonographically. The examination yielded good soft tissue detail of the tendons and ligaments, synovial and perisynovial tissues and larger blood vessels. There was a good correlation between the ultrasonographic and gross anatomical appearance of the limbs. The sheath cavity was only identified after it had been distended with water, and the various synovial recesses at the level of the carpal canal were poorly imaged.

Endoscopic examination of the carpal flexor tendon sheath in horses.

This study was undertaken to design a safe technique to examine the carpal flexor tendon sheath (carpal sheath) of horses endoscopically, using an arthroscope. The limbs from 15 horses were used to study the normal anatomy of the carpal sheath and related structures, establish a safe approach and endoscopic technique, and determine the normal endoscopic appearance of the sheath. Major arteries, veins and nerves, present within and around the sheath, left few 'safe' areas to insert the endoscope. Several portals were assessed and a distal lateral approach was found to be safest and to allow adequate visualisation of most of the sheath. The surgical technique and normal endoscopic findings are described in detail and discussed.

Surgical treatment of a comminuted articular fracture of the accessory carpal bone in a thoroughbred horse.

The clinical, radiographic and ultrasonographic findings in a case of a comminuted articular fracture of the accessory carpal bone of a thoroughbred chaser are described, and its surgical treatment and aftercare are detailed. The horse made an uneventful recovery and successfully returned to racing.

Ultrasonographic examination of the femorotibial articulation in horses: imaging of the cranial and caudal aspects.

The aim of this study was to determine the normal ultrasonographic features of the cranial and caudal aspects of the femorotibial articulation and, in particular, to establish a method of examining the menisci, cruciate and meniscal ligaments ultrasonographically. Twenty hindlimbs isolated post mortem from 10 horses were used to study the normal ultrasonographic and gross anatomy of the femorotibial joint. Five stifles from 3 normal, live horses were also examined with B-mode, real-time ultrasound imaging. The results of the anatomical study are presented. The joint surfaces, menisci, cruciate and meniscal ligaments could be imaged adequately in all the specimens. No obvious variations in shape or echotexture, that might impair visualisation of potential lesions, were observed. The authors suggest that ultrasonography is a potential, noninvasive means of diagnosing soft tissue lesions in the femorotibial joint.