Immunohistochemical characterization of tuberculous and non-tuberculous lesionsin naturally infected European badgers (Meles meles)

A panel of species cross-reactive antibodies was established for the immunohistochemical labelling of phagocytic and lymphoid cells in formalin-fixed normal badger tissues. These reagents were used to investigate the immunopathogenesis of both tuberculous and non-tuberculous granulomas in badgers. In normal badger tissues, antisera specific for the CD79a and CD79b epitopes strongly labelled follicular B lymphocytes and plasma cells in lymph nodes, bronchus-associated lymphoid tissue and Peyer's patches. Rabbit anti-dog IgG, IgM and IgA, and goat anti-human lambda light chain strongly labelled plasma cells, but goat anti-ferret IgA produced weak labelling. Interfollicular and occasional follicular lymphocytes and gut intraepithelial lymphocytes expressed the CD3 epitope. Mouse anti-human HLA-DR (MHC Class II) antigen strongly labelled macrophages, some follicular lymphocytes and some intestinal and respiratory epithelial cells. Mouse anti-human calprotectin (MAC387) labelled a limited number of macrophages. In infected badgers, all fusiform to angular macrophages (epithelioid cells) of all tuberculous granulomas strongly expressed HLA-DR antigen, but only a small, variable proportion of these were labelled by MAC387 antiserum. Lymphocytes in the peripheral rims of granulomas and those scattered sparsely amongst the epithelioid cells were labelled primarily with CD3 antiserum. Peripheral plasma cells were more common in larger than in smaller tubercles and usually expressed IgA or IgG. Small unencapsulated siliceous granulomas, which were present in both tuberculous and non-tuberculous badgers, consisted of aggregates of round to polyhedral epithelioid cells expressing the MHC Class II but not the MAC387 epitope. Granulomas caused by infection with presumed fungal adiaspores of Chrysosporium sp. consisted of aggregates of variably shaped macrophages that expressed MHC Class II antigen, but only a proportion expressed MAC387 antigen. The majority of lymphocytes within the peripheral rims of these granulomas were T cells, accompanied by sparse to moderate numbers of plasma cells that primarily expressed IgG or IgA. In conclusion, species cross-reactive antibodies can be used to identify the cellular components of tuberculous and non-tuberculous granulomas. Immunohistochemical examination failed to distinguish small tuberculous granulomas from adiaspiromycotic granulomas.     

A comparison of the antibody responses of badgers (Meles meles) and rabbits (Oryctolagus cuniculus) to some common antigens

The primary and secondary antibody responses of rabbits and badgers were compared after intravenous inoculation of inactivated influenza A virus, sheep erythrocytes (SRBCs), bovine serum albumin (BSA) or bacteriophage psi X174. BSA was also given as a primary injection by the intramuscular route in solution or in Freund's incomplete or complete adjuvant, followed by an intravenous secondary inoculation without adjuvant. Antibody responses were monitored by: haemagglutination inhibition and neutralization tests for influenza virus; direct and antiglobulin haemagglutination tests for SRBCs; indirect haemagglutination test and the Farr method for antigen-binding capacity (ABC) for BSA; neutralization of psi X174. Rabbits gave good responses to all antigens, but the response of badgers was generally poor. After intravenous administration, badgers gave a good response only to psi X174, but even then they produced less antibody than rabbits receiving 100 times less antigen; the immune elimination of phage was more rapid and antibody appeared about 48 h earlier in rabbits than in badgers. Intramuscular administration of BSA and the use of adjuvants improved the badgers' response, with greatest improvement in ABC. These results indicate that badgers display relatively poor immune responses to a variety of antigens.     

Molecular identification of Sarcocystis lutrae in the European otter (Lutra lutra) and the European badger (Meles meles) from the Czech Republic

Parasitology Research - Muscular sarcosporidial infections by Sarcocystis lutrae (Apicomplexa: Sarcocystidae) from the otter (Lutra lutra) and badger (Meles meles) (Carnivora: Mustelidae) were...     

Validation of the BrockTB stat-pak assay for detection of tuberculosis in Eurasian badgers (Meles meles) and influence of disease severity on diagnostic accuracy

A lateral-flow immunoassay (BrockTB Stat-Pak) for detecting tuberculosis in Eurasian badgers was 49% sensitive and 93% specific against culture for M. bovis (n = 1,464) at necropsy. However, the sensitivity was significantly higher (66 to 78%) in animals with more severe tuberculosis, indicating that the BrockTB Stat-Pak may be useful for the detection of badgers with the greatest risk of transmitting disease.     

Development and evaluation of a test for tuberculosis in live European badgers (Meles meles) based on measurement of gamma interferon mRNA by real-time PCR

A real-time PCR assay for the measurement of gamma interferon (IFN-gamma) mRNA in European badger (Meles meles) blood cultures was developed. The levels of IFN-gamma mRNA in blood cultures stimulated with either bovine or avian tuberculin or specific mycobacterial antigens were compared with those in a nonstimulated control blood culture as the basis for determining the tuberculosis (TB) status of live badgers. The assay was validated by testing 247 animals for which there were matching data from postmortem examination and culture of tissues. Relative changes in the levels of IFN-gamma mRNA in response to bovine tuberculin and specific antigens were found to be greater among badgers with tissues positive for TB on culture. The test was at its most accurate (87% of test results were correct) by using blood cultures containing bovine tuberculin as the antigen and when the response to avian tuberculin was taken into account by subtracting the avian tuberculin response from the bovine tuberculin response. At a specificity of 90.7%, the test was 70.6% sensitive. At the same specificity, the current serological enzyme-linked immunosorbent assay for TB in badgers was only 53% sensitive. This work demonstrates that measurement of IFN-gamma mRNA by real-time PCR is a valid method for the detection of TB in live badgers and may provide an alternative to the current serological methods of diagnosis, the Brock test. The testing procedure can be completed within 5 h of receipt of the blood culture samples. In addition, the use of a molecular biology-based test offers the potential to fully automate the testing procedure through the use of robotics.     

Red fox (Vulpes vulpes) cannibalistic behaviour and the prevalence of Trichinella britovi in NW Italian Alps

Food habits of the red fox (Vulpes vulpes) were studied in the Aosta Valley region (NW Italian Alps) and were related to the prevalence of Trichinella infection in the red fox itself and in two Mustelid species (the stone marten (Martes foina) and the badger (Meles meles)). The search of Trichinella by the automatic digestion of muscles samples led us to determine a prevalence of 3.5±1.2% in red foxes, 7.9±4.3% in stone martens and 1.9±1.8% in badgers, with no significant differences among the species. All larvae were identified as Trichinella britovi. The fox diet was assessed through the analysis of both faeces (n=180) and the stomach contents of road-killed animals (n=109). Our results confirmed the opportunistic feeding behaviour of the red fox, which is able to use various trophic resources, both of animal and vegetal origin: e.g. wild and cultivated fruits (F%=47.1; V%=67.3), rodents (F%=22.8; V%=64.8) and carrion (F%=15.6; V%=78.6) formed the bulk of the foxs diet. The frequency of occurrence of potential events of cannibalism was 1.0%, even if the complete absence of undigested remains, other than hairs, suggested the possibility of confusing cannibalism with coat-cleaning. We suggest that intra-specific necrophagy could not represent the unique way of transmission of the nematode in natural conditions.     

Performance of a Noninvasive Test for Detecting Mycobacterium bovis Shedding in European Badger (Meles meles) Populations

The incidence of Mycobacterium bovis, the causative agent of bovine tuberculosis, in cattle herds in the United Kingdom is increasing, resulting in substantial economic losses. The European badger (Meles meles) is implicated as a wildlife reservoir and is the subject of control measures aimed at reducing the incidence of infection in cattle populations. Understanding the epidemiology of M. bovis in badger populations is essential for directing control interventions and understanding disease spread; however, accurate diagnosis in live animals is challenging and currently uses invasive methods. Here we present a noninvasive diagnostic procedure and sampling regimen using field sampling of latrines and detection of M. bovis with quantitative PCR tests, the results of which strongly correlate with the results of immunoassays in the field at the social group level. This method allows M. bovis infections in badger populations to be monitored without trapping and provides additional information on the quantities of bacterial DNA shed. Therefore, our approach may provide valuable insights into the epidemiology of bovine tuberculosis in badger populations and inform disease control interventions.     

Mycobacterium bovis infection in the Eurasian badger (Meles meles): the disease, pathogenesis, epidemiology and control

Eurasian badgers (Meles meles) are an important wildlife reservoir of tuberculosis (Mycobacterium bovis) infection in Ireland and the United Kingdom. As part of national programmes to control tuberculosis in livestock, considerable effort has been devoted to studying the disease in badgers and this has lead to a rapid increase in our knowledge of tuberculosis in this host. Tuberculosis in badgers is a chronic infection and in a naturally-infected population the severity of disease can vary widely, from latent infection (infection without clinical signs and no visible lesions) to severe disease with generalized pathology. The high prevalence of pulmonary infection strongly supports the lungs as the principal site of primary infection and that inhalation of infectious aerosol particles is the principal mode of transmission. However, other routes, including transmission via infected bite wounds, are known to occur. The ante-mortem diagnosis of infection is difficult to achieve, as clinical examination and immunological and bacteriological examination of clinical samples are insensitive diagnostic procedures. Because infection in the majority of badgers is latent, the gross post-mortem diagnosis is also insensitive. A definitive diagnosis can only be made by the isolation of M. bovis. However, to gain a high level of sensitivity in the bacteriological examination, a large number of tissues from each badger must be cultured and sensitive culture methods employed. The transmission and maintenance of M. bovis in badger populations are complex processes where many factors influence within-population prevalence and rates of transmission. Badger social structures and the longevity of infected animals make them an ideal maintenance host for M. bovis infection. Badgers are directly implicated in the transmission of infection to cattle and the inability to eradicate the disease from cattle is, in part, a consequence of the interactions between the two species. A detailed understanding and knowledge of the epidemiology and pathogenesis of the disease are recognized as fundamental for devising new strategies to control infection with a view to limiting interspecies transmission. Vaccination, in spite of formidable challenges, is seen as the best long-term strategy option and studies with captive badgers have shown that vaccination with M. bovis bacillus Calmette-Guérin (BCG) induces protection when delivered by a variety of routes. Continued research is required to develop effective technologies to control the disease both in badgers and cattle. A combination of strategies, which employ the optimal use and targeting of resources, is likely to make a significant contribution towards eradication of the disease.     

First report of Trichinella pseudospiralis in Poland, in red foxes (Vulpes vulpes)

Nematode worms of the genus Trichinella are one of the most widespread zoonotic pathogens. Natural transmission between hosts can only occur through the ingestion of infected meat. To date, two Trichinella species are known to be etiological agents of disease among domestic animals and wildlife in Poland: T. spiralis and T. britovi. In the last decades, since the administration of an oral vaccination against rabies, the red fox population in Poland has increased exponentially. The study area covers the Nowy Targ region: a mountainous area (585–1138 m above the sea) in southern Poland. Of 24 red foxes examined in the study, four were infected with Trichinella isolates: three were identified as T. britovi and one as T. pseudospiralis. The muscle of red foxes infected with T. britovi harboured 2.75, 3.11, 4.4 LPG and with T. pseudospiralis 0.36 LPG. Trichinella larvae were identified at species level by genomic and mitochondrial multiplex PCR, the products of which were sequenced for comparison with other sequences available in GenBank. The sequences obtained from the Polish T. pseudospiralis isolate, deposited in GenBank under the accession numbers JQ809660.1 and JQ809661.1, matched sequences already published in GenBank. Sequence comparison showed a 100% match with the large subunit ribosomal RNA gene of T. pseudospiralis isolate ISS 013, and a 96–95% match with those of T. pseudospiralis isolates ISS 141 and ISS 470. This is the first report of the identification of T. pseudospiralis larvae from red fox in Poland.     

Evaluation of a Rapid Serological Test for the Determination of Mycobacterium bovis Infection in Badgers (Meles meles) Found Dead

Between October 2005 and May 2006, a total of 727 badgers found dead in Wales were reported, and 550 were delivered to the Regional Laboratories of the Veterinary Laboratories Agency (VLA). Of the 459 carcasses suitable for examination, 55 were deemed to be infected with Mycobacterium bovis on the basis of culture, spoligotyping, and variable-number tandem repeat typing. Acid-fast bacteria were observed histologically in a further six badgers, but these bacteria were not confirmed as M. bovis by culture. A rapid serological test (BrockTB Stat-Pak) performed on thoracic blood showed a sensitivity of 35% and a specificity of 99%. Presence of M. bovis infection was 45 times more likely to be confirmed postmortem by culture in BrockTB Stat-Pak-reactive animals than in seronegative ones. Using visible carcass lesions as a marker of bovine tuberculosis (bTB) infection had a similar sensitivity (38%) but was significantly less specific (84%) than serology. The overall accuracy of the antibody detection was 93% (346 correct results from 374 tests), whereas the accuracy of regarding visible lesions as a marker for bTB infection was 78% (354 correct from 453 carcasses examined). Culture remains the gold standard method for detecting M. bovis infection in badgers. However, where resources are limited and/or an instant result is preferred, the BrockTB Stat-Pak could be used in field surveillance efforts to identify animals which should be examined further by only submitting test-negative animals to more detailed postmortem examination and culture.Mycobacterium bovis infection is the cause of bovine tuberculosis (bTB) in a wide range of mammal species, including domestic livestock and captive and free-ranging wildlife. Bovine TB remains an important zoonotic disease with significant impacts on the economy in many countries (6, 22, 23). Eurasian badgers (Meles meles) are a wildlife maintenance host of bTB in Great Britain and Ireland (5, 15) and are implicated in the maintenance and onward transmission of M. bovis infection to cattle (10, 19).Surveillance of wildlife vectors of disease for prevalence estimates of infection may be valuable in disease control strategies and for the assessment of risk of transmission to livestock. Diagnosis of bTB in live badgers has been demonstrated using assays of both serological (4, 20) and cell-mediated (8, 9) immunity. While isolation of M. bovis from clinical samples is definitive, it is too insensitive for badgers, as infected animals yield positive samples infrequently and intermittently (3). A rapid serological test (BrockTB Stat-Pak; Chembio Diagnostic Systems, Inc.) has recently been developed for the diagnosis of bTB in multiple wildlife species (20). The test has modest sensitivity (46 to 55%) for antibody detection in live, infected badgers, but it has the advantages of being simple, rapid, inexpensive, and suitable for field application. Its utility as an animal-side test for badgers, however, is limited by the difficulties associated with obtaining a blood sample from a nonanesthetized animal.Where carcasses are recovered and submitted for mycobacterial culture, the sensitivity of diagnosis depends on the effort taken for careful examination and on the number of tissue samples submitted for culture testing and histopathology (7), as well as on the condition of the carcass. In many cases, the cost involved may prove prohibitive. Reliance on the presence of visible lesions as indicative of bTB is fraught with difficulties, as infected animals may present with no visible lesions or lesions may be the result of other infections while having the appearance of bTB (reviewed in reference 13). The purpose of this study was to determine whether the BrockTB Stat-Pak test could detect M. bovis antibody in blood collected from the carcasses of dead badgers as an alternative means of diagnosis and decision making. Animals were obtained as part of a separate government-funded study to determine the prevalence of bTB in badgers found dead in Wales (http://new.wales.gov.uk/depc/publications/environmentandcountryside/animalhealthandwelfare/diseasesurveillancecontrol/bovinetb/2567889/publicationindex/2326585/badgerfounddeadreport?lang=en). Our results reveal that the BrockTB Stat-Pak test used on thoracic blood samples was very specific (99%) but less sensitive (35%) than found previously for live badgers (2, 14). However, bTB was 45 times more likely to be confirmed in BrockTB Stat-Pak-positive animals than in BrockTB Stat-Pak-negative ones, whereas using visible carcass lesions as a marker of infection was less reliable.     

First report of a human case of trichinellosis due to Trichinella britovi after jackal (Canis aureus) meat consumption in Algeria

We report a single case of trichinellosis contracted in Algeria (Batna region), in a practising Moslim. Shortly after returning to France in November 2004, the patient developed the typical clinical and biological signs of the disease. Although the patient claimed having only eaten mutton, an unusual host for Trichinella, a meticulous investigation revealed that he also had eaten a grilled leg of jackal (Canis aureus). One of the four Trichinella larvae detected in a muscular biopsy enabled us to identify the parasite as Trichinella britovi by a multiplex PCR analysis. This is the first identification of the etiological agent of sylvatic trichinellosis occurring in North Africa and the first case of symptomatic trichinellosis due to jackal meat consumption in Africa.     

A new species and new host records of the quill mites (Acari: Syringophilidae) associated with sunbirds (Passeriformes: Nectariniidae)

Neoaulonastus cinnyris sp. nov. (Acari: Prostigmata: Syringophilidae) parasitising Cinnyris mediocris (Passeriformes: Nectariniidae) from Tanzania is described. Additionally, Picobia oritis Skoracki et al. 2009 was recorded on four new hosts belonging to the family Nectariniidae from Ethiopian region: Cinnyris oustaleti (Bocage) from Angola, Cinnyris venustus (Shaw) from West Somalia, Cinnyris talatala Smith from Botswana and Zambia and Cinnyris erythrocercus (Hartlaub) from Uganda. All known quill mite species from family Nectariniidae are summarized in table.     

Molecular identification and phylogenetic analysis of Trichinella isolates from different provinces in mainland China

Polymerase chain reaction (PCR) and sequencing are useful for species identification of Trichinella spp., especially when their morphological characteristics useful for identifying taxa are lacking. In the present study, nine Trichinella isolates from different provinces in mainland China were identified by the PCR-based method using the 5S ribosomal DNA intergene spacer region (5S ISR) and the mitochondrial large subunit ribosomal DNA genes as molecular markers. The results indicated that eight isolates originating from domestic pigs and one isolate originating from civet cat (Paguma larvata) showed identical DNA banding pattern to Trichinella spiralis. Sequence analysis of the 5S ISR gene further confirmed that the nine Trichinella isolates were T. spiralis and revealed the intraspecies genetic variation within T. spiralis.     

The naked-tailed armadillo Cabassous centralis (Miller 1899): a new host to Paracoccidioides brasiliensis. Molecular identification of the isolate.

The natural habitat of Paracoccidioides brasiliensis remains undefined but the repeated demonstration of infection by this fungus in the nine-banded armadillo Dasypus novemcinctus has opened interesting research avenues. We report here the isolation of this fungus from the spleen of a naked-tailed armadillo Cabassous centralis (Miller 1899) captured in a coffee farm localized in the Colombian endemic area for paracoccidioidomycosis. This particular isolate was identified by its dimorphism and also by comparison of the PbGP43 gene and ribosomal internal transcribed spacer regions (ITS) with recognized P brasiliensis strains. This finding extends the range of naturally acquired infections in mammals of the family Dasypodidae and confirms the existence of this human pathogen in areas where human paracoccidioidomycosis is known to occur.     

New host and distribution records of Pelecitus fulicaeatrae (Diesing, 1861) (Nematoda, Onchocercidae)

The nematode Pelecitus fulicaeatrae (Diesing, 1861) (Onchocercidae, Dirofilariinae) was found in the leg of a silvery grebe Podiceps occipitalis (Garnot, 1826) (Aves, Podicipediformes) at Puerto Madryn, Argentina. The specimens redescribed in this note are based on 7 females and 3 males. This is the first Argentinean record for this species and the second for South America, and represents the southern-most record of a species from this genus.