Francisella tularensis in Swedish predators and scavengers.

Tularaemia is a zoonotic disease, in Europe caused by Francisella tularensis subsp. holarctica. Many lagomorphs and a variety of small rodents are wildlife species prone to develop clinical disease, while predators and scavengers are relatively resistant and may serve as sentinels. Blood samples from 656 Swedish wild predators and scavengers were serologically investigated using slide agglutination and microagglutination. In the slide agglutination test, 34 seropositive animals were detected, and they were found among all species investigated: brown bear (Ursus arctos), Eurasian lynx (Lynx lynx), raccoon dog (Nyctereutes procyonoides), red fox (Vulpes vulpes), wild boar (Sus scrofa), wolf (Canis lupus) and wolverine (Gulo gulo). Due to haemolysis the microagglutination test was more difficult to read at low titres, and only 12 animals were classified as seropositive. F. tularensis subsp. holarctica was detected by a polymerase chain reaction in lymphatic tissues of the head in one brown bear, one red fox and one wolf. The significance of this finding regarding possible latency of infection is not clear. In conclusion, the results of this study indicate that all predator and scavenger species included in this study may serve as sentinels for tularaemia in Sweden. Their role as reservoirs is unclear.

Proposed attributes of national wildlife health programmes.

Wildlife health is important for conservation, healthy ecosystems, sustainable development and biosecurity. It presents unique challenges for national programme governance and delivery because wildlife health not only crosses jurisdictional responsibilities and authorities but also inherently spans multiple sectors of expertise. The World Organisation for Animal Health (OIE) encourages its Members to have wildlife disease monitoring and notification systems. Where national wildlife health surveillance programmes do exist, they vary in scope and size. Evidence-based guidance is lacking on the critical functions and roles needed to meet the OIE's recommendations and other expectations of a national programme. A literature review and consultation with national wildlife health programme leaders identified five key attributes of national programmes: 1) being knowledge and science based; 2) fostering cross-nation equivalence and harmonisation; 3) developing partnerships and national coordination; 4) providing leadership and administration of national efforts; and 5) capacity development. Proposed core purposes include: 1) establishment and communication of the national wildlife health status; 2) leading national planning; 3) centralising information and expertise; 4) developing national networks leading to harmonisation and collaborations; 5) developing wildlife health workforces; and 6) centralising administration and management of national programmes. A national wildlife health programme should aim to identify, effectively communicate and manage the risk to or from a country's wildlife populations. It should generate the appropriate knowledge required to improve the effectiveness of wildlife policies and systems, including identifying and assessing emerging priorities, thus facilitating early warning, preparedness and preventive actions.

La santé de la faune sauvage a un impact important sur la préservation des espèces et d'écosystèmes sains, sur le développement durable et sur la biosécurité. Les défis sont nombreux et complexes pour les programmes nationaux de gouvernance et de mise en oeuvre car les responsabilités et les compétences juridictionnelles sont croisées et les secteurs d'expertise sont multiples. L'Organisation mondiale de la santé animale (OIE) encourage ses Membres à mettre en place des systèmes de notification et de surveillance des maladies de la faune sauvage. Les programmes existants sont de tailles et de compétences variables et les orientations prises concernant les fonctions indispensables pour répondre aux recommandations de l'OIE et à ce qui est attendu d'un programme national ne sont pas toujours déterminées sur une base scientifique. Une revue de la littérature et des consultations auprès de responsables des programmes nationaux de santé de la faune sauvage ont permis d'identifier cinq attributs à proposer pour ces programmes nationaux. Ces programmes doivent :1) fonctionner sur la base de données scientifiques ; 2) favoriser l'équivalence et l'harmonisation transnationales ; 3) développer des partenariats et une coordination à l'échelle nationale ; 4) encadrer et administrer les efforts nationaux ; et 5) assurer le renforcement des capacités. Les missions essentielles sont : 1) déterminer et rendre publique la situation sanitaire de la faune sauvage dans le pays ; 2) encadrer le plan national ; 3) centraliser l'information et l'expertise ; 4) développer les réseaux nationaux d'harmonisation et de collaboration ; 5) former des personnels compétents dans le domaine de la faune sauvage ; et 6) centraliser l'administration et la gestion des programmes nationaux. Les objectifs d'un programme national de santé de la faune sauvage sont d'identifier, de rendre publics et de gérer les risques pour les populations d'animaux sauvages ainsi que les risques générés par ces mêmes populations. Ces programmes doivent promouvoir les connaissances nécessaires pour améliorer l'efficacité des politiques et des systèmes applicables à la faune sauvage, en particulier l'identification et l'évaluation des nouvelles priorités afin de faciliter la mise en oeuvre de systèmes d'alerte précoce, de préparation aux urgences et d'action préventive.

La salud de los animales silvestres, tan importante para la conservación del medio, el buen funcionamiento de los ecosistemas, el desarrollo sostenible y la seguridad biológica, presenta singulares dificultades desde el punto de vista de la gobernanza y aplicación de programas nacionales, dado que la fauna silvestre no solo toca a múltiples responsabilidades y atribuciones jurisdiccionales sino que, por su propia naturaleza, convoca a una plétora de especialidades técnicas. La Organización Mundial de Sanidad Animal (OIE) alienta a sus Miembros a que se doten de sistemas de vigilancia y notificación de enfermedades de la fauna silvestre. Allí donde existen, los programas nacionales en la materia son muy variopintos en cuanto a sus dimensiones y alcance. Faltan pautas científicamente sólidas sobre las funciones y atribuciones básicas que se requieren para cumplir las recomendaciones de la OIE y otras expectativas a las que pueda responder un programa nacional. Tras efectuar un estudio bibliográfico y consultar a los directivos de programas nacionales en la materia, los autores determinaron cinco atributos clave que debe reunir todo programa nacional: 1) estar basado en el saber y la ciencia; 2) favorecer la equivalencia y la armonización entre naciones; 3) crear alianzas y mecanismos de coordinación nacional; 4) encabezar y administrar las actividades a escala nacional; y 5) desarrollar los medios de acción del país. Los objetivos básicos propuestos son: 1) determinar y dar a conocer la situación sanitaria de la fauna silvestre del país; 2) dirigir las labores de planificación a escala nacional; 3) centralizar la información y las competencias especializadas; 4) instituir redes nacionales que propicien la armonización y las iniciativas de colaboración; 5) desarrollar los recursos humanos dedicados a la sanidad de la fauna silvestre; y 6) centralizar la gestión y administración de los programas nacionales. Todo programa nacional de sanidad de la fauna silvestre debe responder a la finalidad de detectar, comunicar eficazmente y gestionar los riesgos que amenacen a las poblaciones de animales silvestres del país o que provengan de ellas. A tal efecto debe generar el conocimiento adecuado y necesario para conferir más eficacia a las políticas y sistemas tocantes a la fauna silvestre, lo que supone, entre otras cosas, determinar y evaluar las nuevas prioridades, facilitando con ello la alerta anticipada y las labores de preparación y prevención.

Arrival of rabbit haemorrhagic disease virus 2 to northern Europe: Emergence and outbreaks in wild and domestic rabbits (Oryctolagus cuniculus) in Sweden.

Incursion of rabbit haemorrhagic disease virus (RHDV) into Sweden was documented in 1990 and it is now considered endemic in wild rabbit (Oryctolagus cuniculus) populations. Rabbit haemorrhagic disease virus 2 (RHDV2), a new, related lagovirus was first detected in France in 2010, and has spread rapidly throughout Europe and beyond. However, knowledge of RHDV2 in northern Europe is sporadic and incomplete, and in Sweden, routinely available diagnostic methods to detect rabbit haemorrhagic disease (RHD) do not distinguish between types of virus causing disease. Using RHDV2-specific RT-qPCR, sequencing of the VP60 gene and immunological virus typing of archived and prospective case material from the National Veterinary Institute's (SVA) wildlife disease surveillance programme and diagnostic pathology service, we describe the emergence of RHDV2 in Sweden in both wild and domestic rabbits. The earliest documented outbreak occurred on 22 May 2013, and from May 2013 to May 2016, 10 separate incidents of RHDV2 were documented from six different municipalities in the southern half of Sweden. Phylogenetic analysis of the VP60 gene shows clear clustering of Swedish isolates into three separate clusters within two different clades according to geographic location and time, suggesting viral evolution, multiple introduction events or both. Almost all cases of RHD examined by SVA from May 2013 to May 2016 were caused by RHDV2, suggesting that RHDV2 may be replacing RHDV as the predominant cause of RHD in Sweden.

Francisella tularensis in muscle from diseased hares - a risk factor for humans?

Tularemia caused by the bacterium Francisella tularensis is a zoonotic disease. Tularemia is a common disease in the hare, and as a game species can be an important source of infection for humans. In this study, hares diagnosed with tularemia were examined with the aim to investigate whether the muscle (meat) had any pathological changes and/or contained F. tularensis. Real-time PCR and/or immunohistochemistry (IHC) detected the bacteria in muscle samples from 40 out of 43 investigated hares. IHC showed that bacteria were few and most commonly located in the peri- and endomysium. Histopathology showed occasional perimysial necroses and mild inflammation in association to the bacteria. Attempts to culture from 14 muscle samples were successful in two cases, both stored in the freezer <1 year. The result of this study shows that since F. tularensis is present in the muscle of infected hares, there is a risk for human infection when consuming undercooked hare meat. The risk is enhanced by the fact that some hares do not have easily detected gross lesions. The study contributes to a better understanding of sources of infection and risk factors for humans to contract tularemia.

Tularaemia in European Brown Hares (Lepus europaeus) and Mountain Hares (Lepus timidus) Characterized by Histopathology and Immunohistochemistry: Organ Lesions and Suggestions of Routes of Infection and Shedding.

Tularaemia is an emerging zoonotic infectious disease caused by the bacterium Francisella tularensis. In Sweden, hares are considered to be key species in the epidemiology of tularaemia. The aim of this study was to characterize the pathology of natural tularaemia infection in European brown hares (EBHs) (Lepus europaeus) and mountain hares (MHs) (Lepus timidus) in Sweden, in order to better understand the presentation of disease and the routes of infection, body dissemination and shedding of F. tularensis. During 2000-2013, 49 EBHs and 37 MHs were diagnosed with tularaemia. Enlargement of the spleen was seen in 80% of EBHs and 62% of MHs. Necrosis was often obvious in the bone marrow, liver, lung and spleen, but 30% of the hares had no lesions or minimal gross lesions. On microscopical examination of tissues from 27 EBHs and three MHs, necrosis was seen in the majority of samples of liver, spleen, bone marrow, lymph node and adrenal glands and was common in the lungs and brain meninges. Immunohistochemistry for Francisella spp. detected bacteria in association with necrosis and inflammation. In several cases, Francisella spp. were also found inside blood vessels, in the renal pelvis, in lactating mammary glands, in bronchioles and in the skin, associated with tick bites. Using quantitative polymerase chain reaction, two genotypes of F. tularensis subsp. holarctica were found; canSNP group B.6, all belonging to subgroup B.7, and canSNP group B.12. There were no differences in pathology between the genotypes. Our results indicate that the urinary tract and mammary glands are important routes for the shedding of F. tularensis. Hunters may not be aware of the risks of contracting tularaemia while handling hares, since infected hares do not always show noticeable gross lesions.

Spillover Events of Infection of Brown Hares (Lepus europaeus) with Rabbit Haemorrhagic Disease Type 2 Virus (RHDV2) Caused Sporadic Cases of an European Brown Hare Syndrome-Like Disease in Italy and Spain.

Rabbit haemorrhagic disease virus (RHDV) is a lagovirus that can cause fatal hepatitis (rabbit haemorrhagic disease, RHD) with mortality of 80-90% in farmed and wild rabbits. Since 1986, RHDV has caused outbreaks in rabbits (Oryctolagus cuniculus) in Europe, but never in European brown hares (Lepus europaeus, EBH). In 2010, a new RHDV-related virus, called RHDV2, emerged in Europe, causing extended epidemics because it largely overcame the immunity to RHDV present in most rabbit populations. RHDV2 also was identified in Cape hare (Lepus capensis subsp. mediterraneus) and in Italian hare (Lepus corsicanus). Here, we describe two distinct incidents of RHDV2 infection in EBH that occurred in Italy (2012) and Spain (2014). The two RHDV2 strains caused macroscopic and microscopic lesions similar to European brown hare syndrome (EBHS) in hares, and they were genetically related to other RHDV2 strains in Europe. EBHs are common in Europe, often sharing habitat with rabbits. They likely have been exposed to high levels of RHDV2 during outbreaks in rabbits in recent years, yet only two incidents of RHDV2 in EBHs have been found in Italy and Spain, suggesting that EBHs are not a primary host. Instead, they may act as spillover hosts in situations when infection pressure is high and barriers between rabbits and hares are limited, resulting in occasional infections causing EBHS-like lesions. The serological survey of stocked hare sera taken from Italian and Spanish hare populations provided an understanding of naturally occurring RHDV2 infection in the field confirming its sporadic occurrence in EBH. Our findings increase the knowledge on distribution, host range and epidemiology of RHDV2.

Macroscopic and microscopic evaluation of Eurasian lynx (Lynx lynx) female tubular reproductive organs in relation to ovarian structures.

Although monitoring wild animals in the field is essential for estimations of population size and development, there are pitfalls associated with field monitoring. In addition, some detailed data about reproductive physiology can be difficult to obtain in wild live animals. Studying reproductive organs from the Eurasian lynx killed at hunting or found dead could be used as a valuable addition to other field data. We evaluated reproductive organs from 39 Eurasian lynx females (Lynx lynx) killed in Sweden during the hunting seasons in 2009, 2010, and 2011. According to notes on ovarian structures, the animals were categorized as being in one of four different reproductive stages: juvenile (n = 10), follicular stage (n = 8), luteal stage (n = 11), and anestrus (n = 10). Corpora lutea were classified as fresh CL from the present season or as luteal bodies from previous cycles. Microscopic evaluations were blindly coded while the outer measurements of the vagina and uterus were taken at the time of organ retrieval. The width of the endometrium, myometrium, outer width of the uterine horns, and the diameter of the vagina differed significantly with the reproductive stage (P < 0.001) and were largest in the follicular and luteal phases. The number of endometrial glands evaluated blindly coded on a subjective scale was significantly associated with the reproductive stage (P < 0.0001) and was significantly higher in the luteal phase than that in any other reproductive stages (P < 0.05). Cornification of the vaginal epithelium was only observed in females in the follicular stage or in females with signs of a recent ovulation. In conclusion, both macroscopic and histologic measurements are useful for a correct classification of the reproductive stage when evaluating reproductive organs in the Eurasian lynx killed during the hunting season. Routine evaluation of reproductive organs has a potential to be a useful additional tool to field studies of live lynx to monitor their reproduction.

The status of tularemia in Europe in a one-health context: a review.

The bacterium Francisella tularensis causes the vector-borne zoonotic disease tularemia, and may infect a wide range of hosts including invertebrates, mammals and birds. Transmission to humans occurs through contact with infected animals or contaminated environments, or through arthropod vectors. Tularemia has a broad geographical distribution, and there is evidence which suggests local emergence or re-emergence of this disease in Europe. This review was developed to provide an update on the geographical distribution of F. tularensis in humans, wildlife, domestic animals and vector species, to identify potential public health hazards, and to characterize the epidemiology of tularemia in Europe. Information was collated on cases in humans, domestic animals and wildlife, and on reports of detection of the bacterium in arthropod vectors, from 38 European countries for the period 1992-2012. Multiple international databases on human and animal health were consulted, as well as published reports in the literature. Tularemia is a disease of complex epidemiology that is challenging to understand and therefore to control. Many aspects of this disease remain poorly understood. Better understanding is needed of the epidemiological role of animal hosts, potential vectors, mechanisms of maintenance in the different ecosystems, and routes of transmission of the disease.

Forensic veterinary pathology, today's situation and perspectives.

To investigate the current status of forensic veterinary pathology, a survey was composed directed at pathology laboratories and institutes, mostly in Europe. The questions included number of and type of cases, resources available, level of special training of the investigating pathologists and the general view on the current status and future of the discipline. The surveys were sent to 134 laboratories and were returned by 72 respondents of which 93 per cent work on forensic pathology cases. The results indicate scarcity of training opportunities and special education, and insufficient veterinary-specific reference data and information on forensic analyses. More cooperation with human forensic pathology was desired by many respondents, as was more interaction across country borders.

Detection of herpesvirus DNA in Arctic foxes (Vulpes lagopus; syn. Alopex lagopus) with fatal encephalitis.

A captive breeding programme for the Fennoscandian Arctic fox (Vulpes lagopus; syn. Alopex lagopus) failed due to fatal encephalitis. The aim of this study was to identify the causative agent. Viral nucleic acid was detected by PCR and in situ hybridization in the brain of affected foxes. The results suggest that a herpesvirus might be the causative agent. Whether this infection also occurs in free-living Arctic foxes is unknown.

Establishing a European network for wildlife health surveillance.

Surveillance of wildlife health in Europe remains informal and reporting wildlife diseases is not yet coordinated among countries. At a meeting in Brussels on 15 October 2009, delegates from 25 countries provided an overview of the current status of wildlife health surveillance in Europe. This showed that every year in Europe over 18,000 wild animals are examined as part of general surveillance programmes and over 50,000 wild animals are examined in the course of targeted surveillance. The participants at the Brussels meeting agreed to set up a European network for wildlife health surveillance. The goals of this network, which was established in February 2010, are to improve procedures for the rapid exchange of information, harmonise procedures for investigation and diagnosis of wildlife diseases, share relevant expertise, and provide training opportunities for wildlife health surveillance.

Outbreaks of highly pathogenic avian influenza in Europe: the risks associated with wild birds.

The infection of wild birds by highly pathogenic strains of avian influenza (Al) virus was virtually unknown--apart from one instance of the disease appearing in common terns in South Africa in 1961--before the Asian strain of highly pathogenic AI virus (AIV), H5N1, began to expand across the world. Outbreaks of clinical disease in Eurasia have resulted in visible mortality among populations of free-ranging wild birds in a multitude of species. The circulation pattern of influenza viruses in natural ecosystems results from a selection pressure towards strains which are indirectly transmitted by droppings from water birds and contaminated fomites, and which exhibit low pathogenicity. Some of these viruses, of the subtypes H5 or H7, can mutate into highly pathogenic strains after being introduced into domestic poultry farms. The maintenance of highly pathogenic AIV (HPAIV) H5N1 in several parts of the world exposes wild birds to infected poultry, resulting in long-distance virus transmission. There is great concern that these wild birds may, in turn, propagate these HPAIV or introduce them into domestic birds. Rigorous disease control and biosecurity measures to protect poultry farms are the only solution presently available to mitigate such a risk.

A review of infection of wildlife hosts with Mycobacterium bovis and the diagnostic difficulties of the 'no visible lesion' presentation.

The pathology, frequency and diagnostic implications of 'no visible lesion' (NVL) tuberculosis (Tb), i.e. infection with Mycobacterium bovis in the absence of macroscopic lesions, are described in a wide taxonomic range of wildlife hosts. Information collected and evaluated on the definition and occurrence of NVL Tb, histopathological characteristics, post-mortem techniques to detect minimal lesions, and diagnostic difficulties revealed most Tb-infected individuals with NVL had minute tuberculous lesions, which were difficult to see by eye. Acid-fast organisms (AFO) were sometimes detected in the lesions. Ideally, mycobacterial culture of pools of lymph nodes and/or oropharyngeal tonsils is necessary for the accurate diagnosis of Tb in the absence of macroscopic lesions. At a very minimum, the diagnostic methods applied for studying the prevalence of Tb in the population should be clearly described, to allow comparison between studies.

An outbreak of type C botulism in herring gulls (Larus argentatus) in southeastern Sweden.

From 2000 to 2004, over 10,000 seabirds, primarily Herring Gulls (Larus argentatus), died from an undetermined cause in the Blekinge archipelago in southeastern Sweden. In June 2004, 24 affected Herring Gulls were examined clinically, killed humanely, and 23 were examined by necropsy. Seven and 10 unaffected Herring Gulls collected from a local landfill site and from Iceland, respectively, served as controls. All affected birds showed similar neurologic signs, ranging from mild incoordination and weakness to severe flaccid paralysis of legs and wings, but generally were alert and responsive. All affected gulls were in normal nutritional condition, but were dehydrated and had empty stomachs. No gross or microscopic lesions, and no bacterial or viral pathogens were identified. Type C botulinum toxin was detected in the sera of 11 of 16 (69%) affected gulls by mouse inoculation. Type C botulism was the proximate cause of disease in 2004. Sera from 31% of birds tested from outbreaks in 2000 to 2003 also had detectable type C botulinum toxin by mouse inoculation. No large-scale botulism outbreak has been documented previously in this area. The source of toxin, initiating conditions, and thus, the ultimate cause of this outbreak are not known. This epidemic might signal environmental change in the Baltic Sea.

Molecular characterization of novel circoviruses from finch and gull.

The purpose of this study was to molecularly characterize circoviruses that infect finches and gulls. Circovirus-specific DNAs were isolated using polymerase chain reaction methods from bursa of Fabricius tissues from a Gouldian finch (Chloebia gouldiae) and a herring gull (Larus argentatus) that were known to be circovirus-infected. Nucleotide sequence determination and analysis of cloned genomic DNAs showed that these circoviruses represented novel members of the genus Circovirus of the family Circoviridae, and have been tentatively named Finch circovirus (FiCV) and Gull Circovirus (GuCV). Both new circoviruses shared genome organizational features with previously characterized circoviruses, such that both contained two major, inversely-arranged open reading frames encoding the putative replication-associated and capsid proteins, and both contained a potential stem-loop and nonanucleotide motif. Phylogenetic analyses based on genome nucleotide sequences and involving the seven additional genus members indicated that FiCV and GuCV were more closely related to canary circovirus, beak and feather disease virus and pigeon circovirus, and that FiCV and canary circovirus were the most closely related avian circoviruses. Pairwise comparisons showed that the capsid proteins of FiCV and GuCV shared highest amino acid identity values with those of canary circovirus (62.0%) and pigeon circovirus (40.6%), respectively. The 5' intergenic region of GuCV was longer (207 nucleotides) and contained more direct and inverse repeated sequences than those of other circoviruses, while the 3' intergenic region of FiCV was notable in being longer (307 nucleotides) than its counterparts in other circoviruses and in containing two long repeats of 77 nucleotides.

Bovine spongiform encephalopathy: the effect of oral exposure dose on attack rate and incubation period in cattle.

The dose-response of cattle exposed to the bovine spongiform encephalopathy (BSE) agent is an important component of modelling exposure risks for animals and humans and thereby, the modulation of surveillance and control strategies for BSE. In two experiments calves were dosed orally with a range of amounts of a pool of brainstems from BSE-affected cattle. Infectivity in the pool was determined by end-point titration in mice. Recipient cattle were monitored for clinical disease and, from the incidence of pathologically confirmed cases and their incubation periods (IPs), the attack rate and IP distribution according to dose were estimated. The dose at which 50 % of cattle would be clinically affected was estimated at 0.20 g brain material used in the experiment, with 95 % confidence intervals of 0.04-1.00 g. The IP was highly variable across all dose groups and followed a log-normal distribution, with decreasing mean as dose increased. There was no evidence of a threshold dose at which the probability of infection became vanishingly small, with 1/15 (7 %) of animals affected at the lowest dose (1 mg).

Histopathogenesis of experimental Mycobacterium bovis infection in mice.

In-bred strains of mice are commonly used to model pathogenic infections due to their cost and utility. In order to understand better the nature of experimental tuberculosis in mice, we infected BALB/c mice with a virulent field isolate of Mycobacterium bovis. Mice were sacrificed at intervals in order to visualise the pathological lesions in major internal organs. Pathological lesions in tissues increased in number and severity over time and replicated many of the salient features observed in badgers and cattle infected with M. bovis. These similarities are discussed. Examination of pathological lesions at terminal stages of infection enabled us to suggest the lethal effects of M. bovis mediated through the host response. We conclude that the mouse is a relevant surrogate species in which to study the virulence of M. bovis, as well as the influence of vaccination on its pathogenicity.

Nasal boost with adjuvanted heat-killed BCG or arabinomannan-protein conjugate improves primary BCG-induced protection in C57BL/6 mice.

Today it is generally accepted that the Bacillus Calmette-Guerin (BCG) vaccine protects against childhood tuberculosis (TB) but this immunity wanes with age, resulting in insufficient protection against adult pulmonary TB. Hence, one possible strategy to improve the protective efficacy of the BCG vaccine would be to boost in adulthood. In this study, using the mouse model, we evaluated the ability of two new TB vaccine candidates, heat-killed BCG (H-kBCG) and arabinomannan-tetanus toxoid conjugate (AM-TT), given intransally in a novel Eurocine adjuvant, to boost a primary BCG-induced immune response and to improve protection. Young C57BL/6 mice were vaccinated with conventional BCG and, 6 months later, boosted intranasally with adjuvanted H-kBCG or AM-TT, or subcutaneously with BCG. Ten weeks after the booster, mice were challenged intravenously with M. tuberculosis (Mtb) strain H37Rv. In spleens, there was a significant reduction of cfu counts in mice boosted with either H-kBCG or AM-TT vaccines compared to the non-boosted BCG-vaccinated mice. None of the boosting regimens significantly reduced bacterial loads in lungs, compared to non-boosted BCG vaccination. However, the extent of granulomatous inflammation was significantly reduced in the lungs of mice that received two of the booster vaccines (AM-TT and conventional BCG), as compared with sham-vaccinated mice. All boosted groups, except for mice boosted with the AM-TT vaccine, responded with a proliferation of spleen T cells and gamma interferon production comparable to that induced by a single BCG vaccination.