V. Briones

Bovine Tuberculosis (Mycobacterium bovis) in Wildlife in Spain

ABSTRACT Mycobacterium bovis infection in wildlife and feral species is a potential source of infection for livestock and a threat to protected and endangered species. The aim of this study was to identify Spanish wild animal species infected with M. bovis through bacteriological culture and spacer oligonucleotide typing (spoligotyping) of isolates for epidemiological purposes. This study included samples from red deer (Cervus elaphus), fallow deer (Dama dama), wild boar (Sus scrofa), Iberian lynx (Lynx pardina), hare (Lepus europaeus), and cattle (Bos taurus). They were collected in several geographical areas that were selected for their unique ecological value and/or known relationships between wildlife and livestock. In the areas included in this survey, M. bovis strains with the same spoligotyping pattern were found infecting several wild species and livestock, which indicates an epidemiological link. A locally predominant spoligotype was found in these areas. Better understanding of the transmission and distribution of disease in these populations will permit more precise targeting of control measures.

A microbiological, histopathological and immunohistological study of the intragastric inoculation of Listeria monocytogenes in mice

The course of murine infection after intragastric inoculation of L. monocytogenes was investigated by immunocytochemical, histopathological and microbiological techniques. L. monocytogenes antigen was observed in epithelial cells of intestinal mucosa overlying Peyers patches, but not in mucosa devoid of them. This suggests that penetration of L. monocytogenes into the host organism may take place through epithelium overlying Peyers patches. The efficiency of bacterial penetration appeared to be low, as shown by the small amounts of L. monocytogenes antigen detected and the low counts of bacteria in organs. Gross or histopathological lesions in the intestinal tract were not observed. The presence of L. monocytogenes in spleen, liver and in maxillary and mesenteric lymph nodes, confirmed that the systemic course of infection by this route of inoculation is similar to that of the parenteral routes. The results emphasize the subclinical character of murine listeriosis by the oral route.

Choroiditis and meningitis in experimental murine infection withListeria monocytogenes

In a study of central nervous system involvement in experimental listeriosis 27 Swiss CD1 mice were inoculated subcutaneously withListeria monocytogenes. Systemic infection developed, as shown by the isolation ofListeria monocytogenes and histopathological lesions in the spleen and liver. In the central nervous system a mixed inflammatory infiltration in the ventricular system, especially in the choroid plexus, and leptomeningitis were the most relevant lesions. Inflammatory lesions were associated with the presence ofListeria monocytogenes, as demonstrated by a positive anti-Listeria monocytogenes immunoperoxidase reaction within phagocytic cells. It is suggested that choroiditis and meningitis developed as a consequence of hematogenous dissemination ofListeria monocytogenes within mononuclear phagocytes and penetration of these cells into the ventricular system through the choroid plexus.

Lymphatic drainage of Listeria inonocytogenes and Indian ink inoculated in the peritoneal cavity of the mouse

The lymphatic drainage of the peritoneal cavity has been investigated by intraperitoneal inoculation of an intracellular bacterium (Listeria monocytogenes) and an inert marker (Indian ink). The results reveal that both agents are transported, either after phagocytosis by intraperitoneal macro phages or in suspension in the lymph, towards the cranial sternal lymph nodes (Lymphonodi sternales craniales) of the ventral thoracic Iymphocentrum (Lymphocentrum thoracicum ventrale) and to the lymph nodes of the mediastinal lymphocentrum (Lymphocentrum mediastinale), prior to systemic dissemination. This mechanism of peritoneal lymph drainage has relevance on experimental studies involving the inoculation of pathogens, and on the investigation of metastatic diffusion of neoplasms from the peritoneum.

BENEFICIAL EFFECTS OF CLOACAL BACTERIA ON GROWTH AND FLEDGING SIZE IN NESTLING PIED FLYCATCHERS (FICEDULA HYPOLEUCA) IN SPAIN

Abstract Effects of bacteria on avian hosts in the wild have received little attention until recently. Whereas the pathogenic effects of bacteria are well known, positive effects of symbiotic bacteria are more rarely considered. Nestling growth has important repercussions for offspring fitness in avian populations and may be affected by microbial colonization of the gut. Enterococcus faecalis is a common opportunistic pathogen, whereas E. faecium has been used as a growth promoter because it interacts competitively with pathogenic bacteria, E. faecalis included. We followed the growth in tarsus length and mass of 18 Pied Flycatcher (Ficedula hypoleuca) broods. Chicks were weighed and measured on days 4, 8, 10, and 13 after hatching. On day 13, wing length was also measured and cloacal swabs were taken of two chicks in each brood for detection of enterococci. In all, the methods used allowed us to detect six species of bacteria among a possibly much richer community. Most chicks had E. faecalis, whereas E. faecium was less prevalent. There was a negative association between scores for E. faecalis and for the rest of the species pooled. The presence of E. faecalis showed no detectable association with nestling mass or size at any age, whereas the presence of the other species showed significantly positive associations with mass and size on day 13, but not before. Presence of E. faecium on its own was positively associated with nestling mass and size shortly before fledging. E. faecium may act as a growth promoter in the wild through its competitive interactions with facultative pathogenic bacteria. The presence of some microbes are critically important in avian growth and development.

A technique for the direct identification of haemolytic-pathogenic listeria on selective plating media

A technique based on the addition of a red cells top layer to a selective plating medium after listeria growth is proposed in order to detect directly the haemolytic activity of pathogenic listeria colonies. It was applied to different selective plating media (modified McBride agar, lithium chloride‐phenylethanol‐moxalactam, listeria selective medium–Oxford formulation, polymyxin‐acriflavine‐lithium chloride‐ceftazidime‐aesculin‐mannitol and LSAMM). The haemolytic activity of listeria colonies was more easily detected with the top layer than when red cells were incorporated in the selective plating medium. The LSAMM was the best medium for the recovery and identification of Listeria monocytogenes colonies by this technique (three Listeria monocytogenes colonies were distinguished among 2520 Listeria innocua colonies in raw milk).

Mycobacterium avium subspecies paratuberculosis in fallow deer and wild boar in Spain

Mycobacterium avium subspecies paratuberculosis is the aetiological agent of a chronic granulomatous enteritis in ruminants known as Johne’s disease or paratuberculosis. It is considered to be one of the most serious diseases affecting dairy cattle worldwide, being responsible for significant economic losses (Harris and Barletta 2001). Its clinical relevance is commonly recognised in captive or farmed wild ruminants, but some studies have suggested that wild ruminant populations could also be an important natural reservoir of M avium subspecies paratuberculosis. The pathogen has already been isolated from a number of wild ruminant species, such as red deer (Cervus elaphus), fallow deer (Dama dama) and tule elk (Cervus elaphus nannodes) (Cook and others 1997, Pavlik and others 2000). In Spain, there has been only one study of the presence of the pathogen in free-ranging fallow deer (Marco and others 2002). However, the host range of M avium subspecies paratuberculosis is not limited to ruminants, and it has also been isolated from a wide variety of wild non-ruminant species, such as fox (Vulpes vulpes), stoat (Mustela erminea), weasel (Mustela nivalis), crow (Corvus corone), rook (Corvus frugilegus), jackdaw (Corvus monedula), rat, wood mouse (Apodemus sylvaticus), rabbit (Oryctolagus cuniculus), hare (Lepus capensis) and badger (Meles meles) (Beard and others 2001). There have been very few studies on the presence of the pathogen in wild boar (Sus scrofa), and none in Spain, despite the fact this species is known to be a relevant reservoir of another important and related pathogen, Mycobacterium bovis (Aranaz and others 2004). This short communication describes a study to investigate the presence of M avium subspecies paratuberculosis in wild red deer, fallow deer and wild boar in the south and west of Spain. A total of 260 animals (101 red deer, 94 fallow deer and 65 wild boar) was examined for the presence of M avium subspecies paratuberculosis between 2001 and 2003. All animals had been hunted; no clinical signs of disease were observed and no gross lesions were detected at postmortem examination. Samples of intestine, ileocaecal valve and mesenteric lymph nodes were collected under aseptic conditions and kept refrigerated at 4°C until they were processed in the laboratory within eight hours. The tissue specimens from each animal were pooled and processed for M avium subspecies paratuberculosis isolation as described by Greig and others (1999). Selective media (Middlebrook 7H11 agar, Herrold’s egg yolk medium [HEYM], HEYM plus sodium pyruvate and Lowestein-Jensen medium) (Biomedics) supplemented with antimicrobials (Mycobacteria Selectatab; MAST Laboratories) and 2 mg/litre mycobactin J (Allied Monitor) were incubated aerobically at 37°C and checked monthly for up to six months for the presence of colonies compatible with M avium subspecies paratuberculosis. Identification of suspect colonies was confirmed by two PCR reactions, the first aimed at the M avium subspecies paratuberculosis-specific insertion sequence IS900 (Millar and others 1995), and the second targeting a novel DNA sequence present in M avium subspecies paratuberculosis isolates but absent in other strains from the M avium-Mycobacterium intracellulare complex (Collins and others 2002). M avium subspecies paratuberculosis was isolated from one wild boar and one fallow deer from the same area. No positive cultures were obtained from the red deer. Both isolates belonged to the cattle type, which is the most common in Europe regardless of the animal source (Whittington and others 2000). The low frequency of detection obtained in the fallow deer (1·1 per cent) was similar to that reported in the north of Spain by Marco and others (2002), as well as in other areas of Europe such as the Czech Republic (Pavlik and others 2000) or Scotland (Fawcett and others 1995). This suggests that, unlike farmed animals, free-ranging wild cervid populations are rarely infected by the pathogen (Jessup and Williams 1999). The present study is the first of wild boar carried out in Spain and the second one in Europe. Machackova and others (2003) investigated the prevalence of M avium subspecies paratuberculosis in wild boar in several central European countries (Bosnia and Herzegovina, Croatia, the Czech Republic, Hungary and Slovakia). The detection frequency of the pathogen reported by those authors was even lower than that found in the present study (1·5 per cent with a confidence interval of 95 per cent). From these data, it seems likely that a low prevalence of M avium subspecies paratuberculosis infections in wild boar should also be expected. Despite low frequencies of detection found in the three animal species under study, the epidemiological consequences should be considered with caution. It is well known that most infected dairy cows do not develop clinical signs despite shedding the pathogen in their faeces (Chiodini and others 1984). These infected animals represent a source of contamination of the environment, where the pathogen is able to persist for long periods of time (Larsen and others 1956), contributing to the infection of new hosts by the faecal-oral route (Chiodini and others 1984). A similar situation could also occur in wild deer and boar populations. Thus, the detection of deer and boar infected by M avium subspecies paratuberculosis is still epidemiologically relevant, as these animals might contribute to the persistence and spread of the pathogen, particularly where higher densities of animals occur (such as at drinking points and in dens and shelters). In particular, wild boar should be given greater consideration, as they have the potential to spread the pathogen in a broader range due to their active behaviour, high mobility and increasing populations in some areas.

Pathogenesis of lymphoid lesions in murine experimental listeriosis

Adult female Swiss albino mice were infected intraperitoneally or subcutaneously with Listeria monocytogenes Serovar 4b or 1/2a and killed at intervals. Thymus, spleen, Peyers patches and a variety of lymph nodes, including the jejunal (mesenteric), mediastinal, lumbar, mandibular and superficial inguinal, were examined by histopathology and by immunocytochemistry for detection of L. monocytogenes antigen. Similar results were obtained with both Serovars and by both routes of inoculation used. In the spleen, L. monocytogenes was detected, by immunoperoxidase staining, as soon as 4 h after inoculation, inside phagocytic cells located predominantly in the marginal zone of the white pulp. This was followed by inflammation, necrosis and depletion of lymphoid cells, which extended in extreme cases to the whole organ. Inflammatory lesions diminished progressively at 5 to 6 days after inoculation. In animals dying of the infection, a severe necrotizing splenitis was present. Depletion of lymphoid cells and inflammatory changes were widespread in the lymph nodes and to a lesser extent in the Peyers patches. An extensive necrotizing lymphadenitis was the prominent lesion in severely affected nodes. Inflammatory lesions and detection of L. monocytogenes antigen started around the venules of high endothelium. A thymus depletion, not associated with the multiplication of bacteria in the organ, was also a constant feature of the infection. This study suggests that L. monocytogenes (1) is transported to the spleen and to the lymph nodes by phagocytes, entering the organs by the marginal sinus in the spleen and by the venules of high endothelium in the lymph nodes; (2) multiplies in these cells as well as in neutrophilic granulocytes (the latter rapidly migrate to the affected zones); and (3) induce a splenitis and lymphadenitis, involving predominantly T cell-dependent areas, with a necrotizing component in severe cases. From our observations it is concluded that infection of the lymphoid system is a major feature in the pathogenesis of murine listeriosis.

Weissella confusa Infection in Primate (Cercopithecus mona)

We describe systemic infection by Weissella confusa in a mona monkey (Cercopithecus mona) on the basis of microbiologic, molecular genetic, and histologic data. The same strain of W. confusa, as determined by pulsed-field gel electrophoresis, was isolated in pure culture from the primate’s brain, liver, spleen, and intestine. Histologic lesions showed inflammatory infiltrates mainly composed of neutrophils, indicating an acute septicemic process.

Is nestling growth affected by nest reuse and skin bacteria in Pied Flycatchers Ficedula hypoleuca

Abstract. Bacteria may colonize avian nests with unknown repercussions on nestling growth and health, although bacteria on nest materials may easily colonize nestling skin and growing feathers. Cavity nesters may have to build their nests on top of used nest materials, given restrictions on cavity availability. Nest reuse may favour bacterial colonization of nest materials and nestling skin and thereby affect nestling feather growth. To test these possibilities, we conducted a study of Pied Flycatchers Ficedula hypoleuca breeding in nest-boxes in central Spain. We left a sample of nest-boxes without removing old nest materials in 2010 and compared bacterial loads of nest materials, control inert objects and nestling belly skin in reused nests with those in new nests in 2011. Nestlings raised in reused nests had higher bacterial loads on their belly skin than those in new nests, while no difference between nest types for nest materials and control inert objects were found. There was a marginally significant tendency for wing length before fledging to be lower in reused nests, but no trend for mass or tarsus length. The bacterial loads of nests showed a negative association with feather growth of nestlings as expressed through wing length but not with tarsus length or mass growth. These results indicate an association between nest reuse and bacterial growth on nestling skin not hitherto detected. They also suggest a possible impairment of flight capacity at fledging mediated by nest bacterial communities which are in direct contact with nestling skin and growing feathers.