Improved detection of Mycobacterium tuberculosis and M. bovis in African wildlife samples using cationic peptide decontamination and mycobacterial culture supplementation

In South Africa, mycobacterial culture is regarded as the gold standard for the detection of Mycobacterium tuberculosis complex (MTBC) infection in wildlife even though it is regarded as “imperfect.” We compared a novel decontamination and mycobacterial culture technique (TiKa) to the conventional mycobacterium growth indicator tube (MGIT) system using known amounts of bacilli and clinical samples from MTBC-infected African buffaloes (Syncerus caffer), white rhinoceros (Ceratotherium simum), and African elephants (Loxodonta africana). Use of the TiKa-KiC decontamination agent on samples spiked with 10,000 to 10 colony forming units (cfu) of M. bovis (SB0121) and M. tuberculosis (H37Rv) had no effect on isolate recovery in culture. In contrast, decontamination with MGIT MycoPrep resulted in no growth of M. bovis samples at concentrations < 1,000 cfu and M. tuberculosis samples < 100 cfu. Subsequently, we used the TiKa system with stored clinical samples (various lymphatic tissues) collected from wildlife and paucibacillary bronchoalveolar lavage fluid, trunk washes, and endotracheal tube washes from 3 species with known MTBC infections. Overall, MTBC recovery by culture was improved significantly (p < 0.01) by using TiKa compared to conventional MGIT, with 54 of 57 positive specimens versus 25 of 57 positive specimens, respectively. The TiKa mycobacterial growth system appears to significantly enhance the recovery of MTBC members from tissue and paucibacillary respiratory samples collected from African buffaloes, African elephants, and white rhinoceros. Moreover, the TiKa system may improve success of MTBC culture from various sample types previously deemed unculturable from other species.     

Assessment of molecular detection of Fusarium circinatum in insects and passive spore traps in Pinus radiata plantations

Fusarium circinatum is the causal agent of pitch canker, a destructive disease that threatens natural and planted pine forests around the world. Although pitch canker has caused problems in Spain and Portugal, concerning Europe as a whole, the fungus is not established across the pine distribution area. Its dispersion by wind and/or insect vectors could nevertheless play a role in the colonization of currently uninfected stands. It is therefore crucial to develop monitoring tools for its detection. To this end, we assessed the molecular detection of the pathogen in environmental samples of bark beetles and passive spore traps, collected in two infected Pinus radiata plantations in Basque country, Spain. The spread pattern of F. circinatum was assessed by an experimental design that included insect and spore traps installed at the centre, at the edge and outside the plots. Our results showed that F. circinatum was detected in both types of samples, at almost all collection dates. In both type of samples, positive detections were mainly found at the centre of the plots, a lower proportion at the edge, and very few outside. This suggests that long‐distance dispersion of Fusarium circinatum does not rely on wind spore dispersal neither on insect flight. Our study also shows that molecular methods are a powerful tool to monitor the pathogen in environmental samples.     

Using knowledge of behaviour and optic physiology to improve fish passage through culverts

Culverts reduce connectivity for aquatic animals by being both a hydraulic and physical barrier. However, altered light intensity may also be a behavioural barrier to fish movement, especially for diurnal species that have adapted to moving when it is light. We propose that knowledge of optical physiology and fish behaviour, two important mechanisms underpinning movement, can inform efforts to improve fish movement through culverts. We firstly review the sensory systems of fish with reference to visual sense and explore how this affects fish movement. We then highlight theoretical knowledge that can help us understand fish behaviour and the potential mismatch between the conditions under which fish have evolved and altered conditions within culverts. We describe potential knowledge gaps and directions for future research to improve our understanding of how culverts may affect fish movement. Finally, we explore the potential costs and ecological benefits of different mitigation options to identify those with the most promise for managing the light environment in culverts to facilitate movement. For researchers and managers exploring this subject, we suggest an approach that: (a) identifies light requirements for movement by different species, (b) tests movement under different light conditions and (c) considers an integrative assessment method for testing fish behaviour around culverts. Understanding how optical physiology, fish behaviour and culvert design influence fish movement can improve connectivity for a range of species.     

High‐throughput Cryopreservation of Sperm from Sex‐reversed Southern Flounder,Paralichthys lethostigma

The Southern flounder, Paralichthys lethostigma, is a valuable aquaculture fish with established markets in the USA. All‐female production in this species is an important technology for aquaculture because the females usually have body sizes twice those of males at the same age, and sex‐reversed males (genotypic XX neomales) are used for all‐female production by crossing with genetically normal females. However, sperm volume from the neomales is usually small (<0.5 mL) and limits their application for all‐female fish production. Cryopreservation of sperm from these sex‐reversed neomales will provide access on demand with increased efficiency to extend the application of neomales. The goal of this study was to develop a protocol for cryopreservation of sperm from the Southern flounder by using an automated high‐throughput processing system. The objectives were to: (1) determine the effect of osmolality on activation of sperm motility; (2) evaluate the effect of extender solutions on sperm motility capacity; (3) evaluate the acute toxicity of cryoprotectants (dimethyl sulfoxide [DMSO], propylene glycol, and polyethylene glycol) on sperm motility, and (4) estimate the effect of cooling rate on sperm cryopreservation and post‐thaw fertilization. Sperm motility was activated when osmolality was 400 mOsmol/kg or higher. Of the three extender buffers tested, HEPES4‐(2‐hydroxyethyl)‐1‐piperazineethanesulfonic acid (HEPES) at 300 mOsmol/kg resulted in better protection for sperm motility than did Hanks' balanced salt solution and Mounib solution at 300 mOsmol/kg during 7 d of refrigerated storage. After 30 min equilibration with the cryoprotectant of 15% DMSO, sperm motility was 24 ± 21% (fresh sperm motility without any cryoprotectants was 42%). After cooling at a rate of 20 C/min, post‐thaw sperm motility was 8 ± 5% and fertilization was 63 ± 40% evaluated at the 32–64 cell stage (5 × 10⁵ sperm per egg). Overall, a protocol was developed for sperm cryopreservation in the Southern flounder with high‐throughput processing, which provides a tool to preserve the valuable genetic resources from neomale flounders, and enables germplasm repository development for the Southern flounder.     

In vitro characterization andin vivo detection ofRigidoporus lignosus,the causal agent of white root disease inHevea brasiliensis,by ELISA techniques

The aim of these studies is to develop a method for early detection ofRigidopoms lignosus (Basidiomycete, Polyporaceae), the causative agent of white root disease of rubber tree. Two polyclonal sera were produced against soluble mycelial proteins of twoR. lignosus isolates, one from Africa (FCI2), the other from Asia (FID2). The specificity of the antisera was tested using isoelectric focusing (IEF)/Western-blot and DAS-ELISA. The two sera recognized all 20R. lignosus isolates from various geographical origins. The banding patterns obtained by Western-blot enabled a distinction to be made between isolates from Africa and those from Asia. In DAS-ELISA and Western-blot analyses, strong cross reactions were observed withR. ulmarius. Only slight reactions were observed in Western-blot analysis toR. lineatus andP. noxius, both causative agents of root rot inHevea. These cross reactions were not observed under our DAS-ELISA analysis conditions. Finally, no cross reactions were obtained with 9 otherPolyporaceae orHevea root pathogen species. The sensitivity threshold of the DAS-ELISA method was 5 ng ml^–1 ofR. lignosus protein. An initial approach to using the DAS-ELISA test for the detection ofR. lignosus in infected plants was carried out on artificially inoculated root samples. The DAS-ELISA protocol enabled detection ofR. lignosus in the root systems of diseased plants. Moreover, no cross reaction was observed with healthy plant extracts.     

Day-blindness in three dogs: clinical and electroretinographic findings

A 6-month-old Rhodesian ridgeback-cross, a 6-year-old Chihuahua and a 12-month-old Australian cattle dog were presented to the authors with a history of colliding with obstacles in daylight. Ophthalmic examination was normal and all three dogs successfully negotiated obstacle courses in dim light. In daylight the dogs became suddenly blind and repeatedly collided with obstacles. Elecroretinography (ERG) revealed no retinal activity to high frequency (30 Hz), bright intensity blue light retinal stimulation by any dog, confirming cone dysfunction. Achromatopsia has previously been recorded in Alaskan malamutes and miniature poodles. This clinical case series illustrates the characteristic behavioral presentation and the electroretinographic findings of severe day-blindness and demonstrates that this condition may exist in other breeds of dogs.     

Osteomyelitis secondary to trauma involving the proximal end of the radius in horses: five cases (1987-2001)

OBJECTIVE: To determine clinical, radiographic, and scintigraphic abnormalities in and treatment and outcome of horses with trauma-induced osteomyelitis of the proximal aspect of the radius. DESIGN: Retrospective study. ANIMALS: 5 horses. PROCEDURE: Data collected from the medical records included signalment; history; horse use; degree of lameness; radiographic, ultrasonographic, and scintigraphic findings; treatment; and outcome. RESULTS: Duration of lameness prior to referral ranged from 14 to 60 days. Mean severity of lameness was grade 3 of 5, and all horses had a single limb affected. All horses had signs of pain during elbow joint manipulation and digital palpation over the lateral aspect of the proximal end of the radius. Radiographic lesions consisted of periosteal proliferation, osteolysis, and subchondral bone lysis. Scintigraphy in 3 horses revealed intense pharmaceutical uptake diffusely involving the proximal end of the radius. Two horses had sepsis of the elbow joint. All horses were treated with antimicrobials long-term; 1 horse was also treated by local perfusion of the radial medullary cavity through an indwelling cannulated screw. At follow-up, all horses had returned to their previous function. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that osteomyelitis of the proximal end of the radius can result from a traumatic injury to the antebrachium. Because lesions may be an extension of septic arthritis, a thorough examination of the wound area and elbow joint is recommended. Prolonged systemic antimicrobial treatment can result in a successful outcome.     

Medical management of biological warfare and bioterrorism: place of the immunoprevention and the immunotherapy

Biological weapons are considered as mass destruction and terror weapons. Terrorism including bioterrorism is the major threat in the future conflicts for our nations. The aim of bioterrorism is more related to the potential disorganisation of the society than to the lethal effects of the agents used. The dramatic consequences cannot be discarded, especially if contagious agents such viral are used. The preparation of specific defence measures is a major challenge for our countries.

The knowledge acquired from the struggle against natural infectious diseases and recent events are essential to improve behaviours to face the biological weapon threats. The defence attitude is based on the anticipation of the threat, the management of the victims, and the restoration of the operational capabilities. This global defence attitude implies six important functions: (i) alert, (ii) detection and diagnosis, (iii) availability of pharmaceutical countermeasures such as vaccine, sera and anti-infectious medicine and products, (iv) medical management of victims, (v) training and information, (vi) research and development.

Passive and active immunoprevention and immuntherapy belong to the approaches discussed in the context of bioterrorism countermeasures. Further researches might be focused on these topics.     

Isolation and identification of Mycobacterium kansasii from pleural fluid of a dog with persistent pleural effusion

A 3-year-old spayed female Whippet was examined for cough and respiratory distress. Lung lobe torsion with pleural effusion was diagnosed, and lung lobectomy was performed. Pleural effusion recurred during the following 27 months; conventional bacteriologic cultures of pleural effusion did not result in bacterial growth. A second lung lobectomy, pleuroperitoneal shunt placement. and pericardectomy were subsequently performed. Mycobacterium kansasii was eventually isolated from pleural fluid and identified by polymerase chain reaction amplification and DNA sequencing. The dog was euthanatized before therapeutic response could be evaluated. To our knowledge, this is the first report of M. kansasii infection in a dog. Additionally, this is the first report of mycobacterial isolation from pleural fluid, and one of few reports of antemortem mycobacterial isolation from a body fluid, as opposed to identification in specimens during histologic examination. Routine bacteriologic culture methods are insufficient to isolate mycobacterial agents, and special methods are indicated in dogs with persistent pleural effusion.