J. Hermoso de Mendoza

Comparative pathology of the natural infections by Mycobacterium bovis and by Mycobacterium caprae in wild boar (Sus scrofa).

The potential role of wild animals in the maintenance and spread of tuberculosis (TB) infection in domestic livestock is of particular importance in countries where eradication programs have substantially reduced the incidence of bovine tuberculosis but sporadic outbreaks still occur. Mycobacterium bovis is the agent mainly isolated in wildlife in Spain, but recently, infections by Mycobacterium caprae have increased substantially. In this study, we have analysed 43 mandibular lymph nodes samples containing TB-like lesions from 43 hunted wild boar from Madrid and Extremadura (central and south-western regions of Spain). After isolation, identification and typing of Mycobacterium tuberculosis complex isolates, we found that 23 mandibular lymph nodes involved M. caprae infections and 20 M. bovis. The lesions were compared for histopathology (different granuloma stage and number of multinucleated giant cells (MNGCs)), and acid-fast bacilli (AFBs) were quantified in the Ziehl-Neelsen-stained slides. Granulomas produced by M. caprae showed more stage IV granulomas, more MNGCs and higher AFBs counts than those induced by M. bovis. In conclusion, lesions caused by M. caprae would be more prone to the excretion of bacilli, and infected animals result as a high-risk source of infection for other animals.

Spleen size variations and reproduction in a Mediterranean population of wild boar (Sus scrofa)

We analysed spleen size variations of free-ranging wild boars from the west-central Iberian Peninsula during the hunting season (autumn and winter) in relation to the rut, the gestation effort and the attainment of sexual maturity by males and females. Females had larger spleens than males once they reached their sexual maturity. Individuals shot in winter had larger spleens than those shot in autumn, the start of the rutting period. In contrast to other reports, we found no influence of the reproductive status of adult females on their spleen sizes. Our findings may point to an influence of sex hormones, and possibly also stress hormones, and environmental factors on spleen development, mainly in adult and subadult males.

Management practices and their association with Mycobacterium tuberculosis complex prevalence in red deer populations in Southwestern Spain

Intensification of game management may increase the prevalence of tuberculosis (TB) in wildlife despite eradication programs implemented in cattle herds in the same areas. In this cross-sectional study, we investigated the association between wild game management practices and the presence of tuberculosis in red deer populations in Southwestern Spain. Five hundred and fifty-one animals were examined by necropsy to detect tuberculosis-like lesions in the main lymph nodes. Prevalence, as determined by TB-like lesions, was estimated to be 5.1% of animals, with 77% of TB-like lesions confirmed by PCR. Our results suggest that population density, in addition to factors which promote the local aggregation of animals, is factors associated with increased prevalence of TB in red deer populations. We suggest that management practices including supplementary feeding, fencing, water ponds and interaction with domestic livestock should be revised in order to prevent TB in wild deer both.

Enzymatic activities of Dermatophilus congolensis measured by API ZYM

API ZYM kit was used to test enzymatic activities on eighteen strains of Dermatophilus congolensis. All strains produced lipase and acid phosphatase, which act on lipids, and leucine arylamidase which act on proteins. Another 10 exoenzymes were present in at least one of the strains.

AETIOLOGY OF CAPRINE CONTAGIOUS AGALACTIA SYNDROME IN EXTREMADURA, SPAIN

ing fish were treated by adding sodium benzylpenicillin DAVIS, H. S. (1922) A new gill disease in trout. Transactions of the American (10,000 iu/litre) to the water for 24 hours. Two days later, the Fisheries Society 56, 156-159 antibiotic treatment was repeated. Following these meaDECOSTERE, A. (1997) Development of a gill perfusion model and consesurements, a marked drop in mortality was recorded. The quent study of the adherence of Flavobacterium columnare to the gill tissue. fish appeared to be much more active and skin lesions MSc thesis, Stirling, Scotland diminishapped. hmoeativeandskinlesons DECOSTERE, A., HAESEBROUCK, F. & DEVRIESE, L. A. (1997) Shieh medium supplemented with tobramycin for the selective isolation of F columnare was first described by Davis in 1922. Since Flavobacterium columnare (Flexibactercolumnaris) from diseased fish.Journal then, it has been stated that all freshwater fishes are susceptiof Clinical Microbiology 35, 322-324 ble to columnaris under environmental conditions favourable DECOSTERE, A., HAESEBROUCK, F., TURNBULL, J. F. & CHARLIER, G. to the bacterium and stressful to the fish (Dalsgaard 1993, (1998) Influence of environmental parameters on the adhesion of high and Wakabayashi 1993). In this case, the high level of nitrite most low virulence Flavobacterium columnare strains to isolated gill arches. Journal likely triggered the columnaris outbreak, as was found by ofFish Diseases 21, 1-11 Hanson and Grizzle (1985) in channel catfish (IctaluruspuncHANSON, L. A. & GRIZZLE, J. M. (1985) Nitrite-induced predisposition of adhesion ofF columnare to the ill tissue is indeed channel catfish to bacterial diseases. Progressive Fish Culturist 47, 98-101 tatus). Thne aaneslon or r columnare to tne glll lssueSHIEH, H. S. (1980) Studies on the nutrition of a fish pathogen, Flexibacter enhanced in the presence of nitrite (Decostere 1997, columnaris. Microbios letters 13, 129-133 Decostere and others 1998). TOMEY, W. A. (1985) Ziekten bij aquariumvissen. Best, Nederland, Zuid F columnare does not grow on blood agar. Special media Boekprodukties b.v. p 140 such as Cytophaga (Anacker and Ordal 1959) or Shieh WAKABAYASHI,H. (1993) Bacterial diseases of fish. EdsV. Inglis, R. J. Roberts, (1980) medium are needed for isolation. This explains why N. R. Bromage. Blackwell Scientific Publications, Oxford. p 23-33 columnaris disease frequently is misdiagnosed. Another reason is that columnaris disease resembles a parasitic (costiasis, white spot) and a fungal infection (saprolegniosis). In all cases, the presence of white-grey areas on the skin is evident. However, in columnaris disease, these areas are typically localised on the head and cranial dorsal part of the body and around the dorsal fin. The lesions are mostly circular as if o g of c p spreading from a single focus in all directions at the same Aetiology Of CaPrine rate. Differential diagnosis can easily be made by means of* microscopic examination of skin scrapings of the affected con iou ag-alact areas. In the case described, effective treatment consisted of two syndrome In parts: first, elimination of the predisposing factor, that is, decreasing the nitrite level; and, secondly, antibiotic treatExtremad ura, Spain ment. Sodium benzylpenicillin appeared to be effective, as was reported previously by Bassleer (1983) and Tomey (1985). This therapeutic agent is relatively cheap and does M. C. Gii, M. HERMOSO DE MENDOZA, not colour the water. Water change has to be complete after J. REY, J. M. ALONSO, J. B. POVEDA, 24 hours, since sodium benzylpenicillin might produce substances toxic for fish after this time (Bassleer 1983). Other J HERMOSO DE MENDOZA antimicrobial agents can be used to treat columnaris disease, both in the water and the feed. An excellent review has been CONTAGIOUS agalactia (CA) ofgoats and sheep is one of the provided by Amend (1970). most serious diseases affecting small ruminants. Endemic in In conclusion, the risk of a severe columnaris outbreak most Mediterranean countries (Lambert 1987), the disease is not a problem to be taken lightly, especially when dealing is widely distributed throughout most of Spain (Garrido and with live-bearing fish. Since F columnare can cause heavy others 1987, Real and others 1994). The disease in goats may mortalities over a short period of time, the condition must be be caused by several Mycoplasma species (DaMassa and othdiagnosed quickly and measures taken accordingly. ers 1992); this short communication describes the numbers Veterinary Record (1999) and types of mycoplasma isolated from goats affected by CA 144,24-25 in the Extremadura region of Spain. ACKNOWLEDGEMENTS This study was carried out between 1991 and 1995 in 43 M. C. Gil, PhD, goat flocks affected by CA outbreaks in Extremadura, southM. Hermoso de Mendoza, The authors appreciate the cooperation of the farmer conwest Spain. Five hundred and ninety-six adult and young PhD, cerned. The Fund for Scientific Research, Flanders, is goats, mainly of the Serrana breed, were sampled and a total J. Rey, PhD, acknowledged for providing a grant to A. D. of 1309 specimens were collected from both live and, after J. M. Alonso, PhD, routine necropsy, dead animals. Mycoplasmas were isolated J. Hermoso de Mendoza,

Preliminary characterisation of extracellular serine proteases of Dermatophilus congolensis isolates from cattle, sheep and horses.

Dermatophilus congolensis is a filamentous branching actinomycete that causes dermatophilosis, an exudative dermatitis in ruminants. The pathogenesis of this disease is poorly understood and virulence factors of D. congolensis have not been characterised. Culture filtrate (CF) of 14 D. congolensis isolates from cattle, 15 from sheep and four from horses were examined for proteolytic activity using azocasein as a non-specific substrate. The isolates were from a variety of geographical locations. All the isolates examined produced extracellular proteolytic activity. CF from 24 and 48 h cultures and from first and third passages contained proteases. Proteolytic activity was greatest in neutral to alkaline pH (pH 7-10). CF of bovine isolates contained more proteolytic activity than that of ovine isolates. Furthermore, in substrate SDS-PAGE gels containing azocasein the number of proteolytic bands and their molecular weights in CF of bovine, ovine and equine isolates were different, giving distinctive band patterns for isolates from each host species. Three out of four bovine isolates from Antigua gave a fourth band pattern. Bands of equivalent molecular weights to the proteases could not be identified in silver stained SDS-PAGE gels of CF. Serine protease inhibitors had a concentration-dependent inhibitory effect on proteolytic activity in CF and inhibited activity of all proteolytic bands in substrate gels. With the exception of EDTA which had a variable-enhancing effect on activity, inhibitors of other classes of protease had no effect on activity. We conclude that D. congolensis produces a number of extracellular alkaline serine proteases, our results suggest the presence of host-specific variation between isolates and to a lesser extent between isolates from the same host species.

Histological and immunohistochemical characterisation of Mycobacterium bovis induced granulomas in naturally infected fallow deer (Dama dama).

Mycobacterium bovis infections in fallow deer have been reported in different countries and play an important role in the epidemiology of bovine tuberculosis (bTB), together with other deer species. There is little knowledge of the pathogenesis of bTB in fallow deer. The aim of this study was to perform a histopathological characterisation of the granulomas induced by M. bovis in this species and the immunohistochemical distribution of different cell subsets (CD3+, CD79+, macrophages) and chemical mediators (iNOS, TNF-α, IFN-γ) in the different developmental stages of granulomas. Stage I/II granulomas showed a marked presence of macrophages (MAC387+) expressing high iNOS levels while stage III/IV granulomas showed a decrease in the number of these cells forming a rim surrounding the necrotic foci. This was correlated with the presence of IFN-γ expressing cell counts, much higher in stage I/II than in stage III/IV. The number of B cells increased alongside the developmental stage of the granuloma, and interestingly the expression of TNF-α was very low in all the stages. This characterisation of the lesions and the local immune response may be helpful as basic knowledge in the attempts to increase the vaccine efficacy as well as for disease severity evaluation and for the development of improved diagnostic tools. Immunohistochemical methods using several commercial antibodies in fallow deer tissues are described.

Rapid and easy method to extract and preserve DNA from Cryptococcus neoformans and other pathogenic yeasts

Summary. The mucopolysaccharide capsule of Cryptococcus neoformans and other pathogenic yeasts prevent the extraction of DNA from these important zoonotic agents. We report that the use of a lysis buffer containing a high concentration of urea is an easy, efficient and time‐saving technique to obtain high yields of good‐quality DNA for molecular diagnosis. The use of urea also prevents the degradation of DNA during storage of samples at room temperature for up to 6 months.

In vitro studies of Dermatophilus congolensis antimicrobial susceptibility by determining minimal inhibitory and bacteriocidal concentrations.

The Minimal Inhibitory Concentration (MIC) and Minimal Bacteriocidal Concentration (MBC) of 19 antimicrobials on 16 isolates of D. congolensis were determined. The potential field efficacy of the agents was evaluated by comparing the results with serum levels of drug unbound to proteins and the in vitro and in vivo findings of other authors. A modified standard microtechnique was used for serial dilution-antimicrobial sensitivity and found to be easy and reproducible. Erythromycin, spiramycin, penicillin G, ampicillin, chloramphenicol, the streptomycin, amoxicillin, the tetracyclines and novobiocin had high serum concentrations in comparison with their MBCs and were shown to have potential use for the treatment of dermatophilosis.

Influence of Porcine Circovirus Type 2 Infections on Bovine Tuberculosis in Wild Boar Populations

The wild boar is an important reservoir of bovine tuberculosis (bTB) in south-western Spain. Some risk factors such as wild boar density or age have been associated with the presence of high prevalences of bTB in wild boar. However, the influence of other risk factors such as co-infections with other pathogens has not yet been studied. This work aims to assess the influence of porcine circovirus type 2 (PCV-2) infection on bTB prevalence and bTB lesional patterns observed in wild boar. The presence of bTB-like lesions was evaluated in 551 hunted wild boar from 11 different game estates in south-western Spain, with a known history of bTB. Tuberculosis prevalences in each estate were calculated based on the percentage of animals found with bTB-like lesions. The percentage of animals with generalized bTB lesional patterns (bTB lesions in more than one organ) was also assessed. The prevalence of PCV-2 was studied in each estate using a specific PCR assay. The relationship between PCV-2 and bTB prevalences and between PCV-2 infections and the presence of generalized lesional patterns in wild boar were analysed. A statistical relationship between the prevalences of bTB and PCV-2 was found, with bTB prevalences being higher in estates where prevalences of PCV-2 were high. On the other hand, animals infected with PCV-2 were more likely to develop a generalized lesional pattern. Porcine circovirus type 2 prevalences seem to be associated with prevalences of bTB in wild boar. PCV-2 infection may aggravate the development and severity of bTB, favouring the presence of generalized lesional patterns and raising the risk of contagion in these estates. The implementation of sanitary measures that focus on the control of PCV-2 infection may be necessary as a preliminary measure in bTB control programmes for wild boar.