Increased susceptibility to Mycobacterium avium complex infection in miniature Schnauzer dogs caused by a codon deletion in CARD9.

Mammals are generally resistant to Mycobacterium avium complex (MAC) infections. We report here on a primary immunodeficiency disorder causing increased susceptibility to MAC infections in a canine breed. Adult Miniature Schnauzers developing progressive systemic MAC infections were related to a common founder, and pedigree analysis was consistent with an autosomal recessive trait. A genome-wide association study and homozygosity mapping using 8 infected, 9 non-infected relatives, and 160 control Miniature Schnauzers detected an associated region on chromosome 9. Whole genome sequencing of 2 MAC-infected dogs identified a codon deletion in the CARD9 gene (c.493_495del; p.Lys165del). Genotyping of Miniature Schnauzers revealed the presence of this mutant CARD9 allele worldwide, and all tested MAC-infected dogs were homozygous mutants. Peripheral blood mononuclear cells from a dog homozygous for the CARD9 variant exhibited a dysfunctional CARD9 protein with impaired TNF-α production upon stimulation with the fungal polysaccharide ß-glucan that activates the CARD9-coupled C-type lectin receptor, Dectin-1. While CARD9-deficient knockout mice are susceptible to experimental challenges by fungi and mycobacteria, Miniature Schnauzer dogs with systemic MAC susceptibility represent the first spontaneous animal model of CARD9 deficiency, which will help to further elucidate host defense mechanisms against mycobacteria and fungi and assess potential therapies for animals and humans.

The Mycobacterium avium complex - an underestimated threat to humans and animals.

INTRODUCTION AND OBJECTIVE: The Mycobacterium avium complex (MAC) is a group of acid-resistant bacteria within the Mycobacteriaceae. Their cell walls have a specific structure impervious to many disinfectants. Mycobacteria are widespread in the environment and can also be found in food. This aim of the article is to review the current state of knowledge about the sources of infection, symptoms and treatment of MAC diseases in humans and animals, and summarizes the available methods for identifying the bacteria. It pays a special attention to the zoonotic potential of MAC bacteria and possible routes of transmission between humans and animals, including possible food-borne routes. BRIEF DESCRIPTION OF THE STATE OF KNOWLEDGE.: MAC bacterial infections occur both in immunocompetent people and those with functional predispositions and compromised immunity, particularly during HIV infection or immunosuppressive treatment. The incidence of MAC infections in humans is growing, with the most common form of infection being pulmonary disease (MTC-PD); however, there are conflicting reports on the role of Mycobacterium avium paratuberculosis (MAP) in the development of Crohn's disease. MAC bacteria can also attack livestock, household pets, and wild animals. Unfortunately, treatment is lengthy and often fails due to microbiological relapse; there is also increasing evidence of MAC bacteria are developing multi-drug resistance. CONCLUSIONS: Although new antibiotics are being created to inhibit the growth and division of Mycobacterium avium, there is clearly a need for further research into the virulence factors associated with MAC bacteria. Further studies should also examine the role of MAP in the etiopathogenesis of Crohn's disease.

Oesophageal Obstruction in a Donkey Due to Mediastinal Lymphadenitis Caused by Mycobacterium avium Complex.

Mycobacterial infections are rare in horses, donkeys and mules. Although there are a few reports in horses, mycobacterial disease is poorly documented in the donkey. Mycobacterial infection of equine species typically affects the alimentary tract, causing granulomatous enterocolitis resulting in diarrhoea and chronic weight loss, while lymph nodes and liver may also be affected. We now document recurrent oesophageal obstruction, secondary to cranial mediastinal lymphadenitis caused by Mycobacterium avium complex (MAC). To the best of our knowledge, this is the first report of MAC infection in a donkey in the UK.

Intestinal Mycobacterium avium Infection in Pet Dwarf Rabbits (Oryctolagus cuniculus).

Mycobacteriosis has been rarely described in pet rabbits (Oryctolagus cuniculus). Here we present two cases of intestinal mycobacteriosis from north-eastern Germany. The first adult rabbit was euthanized due to severe cardiovascular failure, hypothermia and chronic weight loss. Necropsy revealed cachexia and a focal, fibrinonecrotic lesion in the caecum. Histologically, severe granulomatous inflammation, with numerous multinucleated giant cells and abundant acid-fast bacilli, was detected under the fibrinonecrotic material in the abdominal wall adjacent to the caecal lesion, caecal lymph nodes, spleen, liver and lungs. Microbiological culture detected Mycobacterium avium subspecies hominissuis, Escherichia coli, Clostridium disporicum and Bacteroides ovatus. A retrospective assessment of 2,013 other pet rabbit necropsies, performed between 1995 and 2019, revealed one additional case of intestinal mycobacteriosis. This animal had been euthanized due to persistent hindlimb lameness and necropsy revealed comminuted fractures of the pelvic bones and multiple large liquefied abscess-like lesions in the caecal and colonic walls. Histology revealed granulomatous inflammation with acid-fast bacilli. Polymerase chain reaction on formalin-fixed, paraffin-embedded tissue identified the presence of M. avium spp. In contrast to European wild rabbits (Oryctolagus cuniculus) from Scotland, these findings indicate that intestinal mycobacteriosis is rare in pet rabbits from north-eastern Germany. Zoonotic potential should be considered due to the close contact between pets and their owners and the chronic course of the disease with an initial lack of clinical signs.

DIAGNOSIS AND MANAGEMENT OF MYCOBACTERIOSIS IN A COLONY OF LITTLE PENGUINS (EUDYPTULA MINOR).

A group of zoo-housed little penguins (Eudyptula minor) was diagnosed with mycobacteriosis. While undergoing multidetector computed tomography (MDCT) imaging for an unrelated research project, pulmonary lesions were detected in multiple individuals. In general, birds appeared healthy and free of outward signs of disease. After the loss of three individuals, polyclonal mycobacterial disease due to Mycobacterium avium-intracellulare complex was confirmed. Surviving birds were treated with rifampin (45 mg/kg), ethambutol (30 mg/kg), clarithromycin (10 mg/kg), and enrofloxacin (30 mg/kg) compounded into a single capsule administered once a day in food. After 3 mo of therapy, MDCT imaging documented a decrease in nodule size and number in all remaining birds, with further improvement documented after 13 mo of treatment. MDCT imaging was invaluable for diagnosing disease, documenting disease progression over time, and assessing response to therapy. Early initiation of therapy before the development of outward signs of disease led to resolution of mycobacterial pulmonary lesions in multiple penguins. Mycobacterial disease in this group of little penguins, as well as previously published reports, suggests that the species is at increased risk for developing mycobacteriosis.

Granulomatous Rhinitis in a Horse due to Mycobacterium intracellulare Infection.

Mycobacterial infections in horses are uncommon, but are caused most frequently by Mycobacterium bovis of the Mycobacterium tuberculosis complex or Mycobacterium avium of the M. avium complex. Disease caused by Mycobacterium intracellulare, the second most common species within the M. avium complex, has not been reported in horses to date. Mycobacteriosis in horses most often presents as enteric, pulmonary or, rarely, systemic disease. Here we report a case of M. intracellulare infection in a horse presenting as a granulomatous nasal mass.

Mycobacterium avium complex infection in pigs: A review.

Mycobacterial infections in pigs are caused particularly by the Mycobacterium avium complex (MAC) and these infections lead to great economic losses mainly within the countries with high pork meat production. The importance of the MAC infections in humans is rising because of its higher prevalence and also higher mortality rates particularly in advanced countries. In addition, treatment of the MAC infections in humans tends to be complicated because of its increasing resistance to antimicrobial agents. Several studies across Europe have documented the MAC occurrence in the slaughtered pigs - not only in their lymph nodes and tonsils, which are the most frequent, but also in the diaphragmas, other organs and not least in meat. This is why we need both more specific and more sensitive methods for the MAC infection detection. Different PCR assays were established as well as advanced intravital testing by the gamma interferon release test. On the other hand, tuberculin skin test is still one of the cheapest methods of mycobacterial infections detection.

Resolution of a Localized Granuloma Caused by Mycobacterium avium-intracellulare Complex on the Cere of a Bruce's Green Pigeon (Treron waalia).

A 3-year-old female Bruce's green pigeon (Treron waalia) was presented with granulomatous inflammation of the cere and underlying tissues with osteomyelitis and bone proliferation of the dorsal premaxilla. Biopsy and culture revealed the presence of Mycobacterium avium-intracellulare complex, and multi-antimicrobial treatment was initiated with clarithromycin, ethambutol, rifabutin, and enrofloxacin. The cere lesion improved and no evidence of systemic granulomas was observed over 4 months of treatment, although leukocytosis and monocytosis persisted. Five months after discontinuation of antibiotic therapy, the white blood cell count had normalized, but distal beak irregularities and partial recurrence of the mass were present. The bird died 15 months after discontinuation of antibiotic therapy and necropsy revealed no evidence of active mycobacteriosis of the beak or cere. This report documents an unusual clinical presentation of mycobacteriosis, in addition to its successful resolution.

MYCOBACTERIUM INTRACELLULARE INFECTION CAUSING A RETROPERITONEAL MASS IN A BINTURONG (ARCTICTIS BINTURONG).

A 19-yr-old castrated male binturong ( Arctictis binturong ) with a history of recurrent pyogranulomatous panniculitis, lymphangitis, and dermatitis was presented for evaluation of hyporexia and tenesmus. A large caudal abdominal mass was palpated on physical examination. On ultrasound, the mass encircled and obstructed the left ureter, resulting in hydroureter and hydronephrosis. The animal was euthanized, and necropsy revealed a large retroperitoneal pyogranuloma with acid-fast organisms identified in both the mass and the perineal skin. The acid-fast organisms within the retroperitoneal mass were identified as Mycobacterium intracellulare by PCR. This case represents an unusual presentation of M. intracellulare in a novel species.

Genetic diversity of Mycobacterium avium complex strains isolated in Argentina by MIRU-VNTR.

Mycobacterium avium sp. avium (MAA), M. avium sp. hominissuis (MAH), and M. avium sp. paratuberculosis (MAP) are the main members of the M. avium complex (MAC) causing diseases in several hosts. The aim of this study was to describe the genetic diversity of MAC isolated from different hosts. Twenty-six MAH and 61 MAP isolates were recovered from humans and cattle, respectively. GenoType CM® and IS1311-PCR were used to identify Mycobacterium species. The IS901-PCR was used to differentiate between MAH and MAA, while IS900-PCR was used to identify MAP. Genotyping was performed using a mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) scheme (loci: 292, X3, 25, 47, 3, 7, 10, 32) and patterns (INMV) were assigned according to the MAC-INMV database (http://mac-inmv.tours.inra.fr/). Twenty-two (22/26, 84·6%) MAH isolates were genotyped and 16 were grouped into the following, INMV 92, INMV 121, INMV 97, INMV 103, INMV 50, and INMV 40. The loci X3 and 25 showed the largest diversity (D: 0·5844), and the global discriminatory index (Hunter and Gaston discriminatory index, HGDI) was 0·9300. MAP (100%) isolates were grouped into INMV 1, INMV 2, INMV 11, INMV 8, and INMV 5. The HGDI was 0·6984 and loci 292 and 7 had the largest D (0·6980 and 0·5050). MAH presented a higher D when compared with MAP. The MIRU-VNTR was a useful tool to describe the genetic diversity of both MAH and MAP as well as to identify six new MAH patterns that were conveniently reported to the MAC-INMV database. It was also demonstrated that, in the geographical region studied, human MAC cases were produced by MAH as there was no MAA found among the human clinical samples.

Systemic infection of Mycobacterium avium subspecies hominissuis and fungus in a pet dog.

A 3-year-old neutered female poodle with a long history of dermatophytic skin disease was presented with lethargy, anorexia and progressive weight loss. Abdominal ultrasonography revealed markedly enlarged mesenteric lymph nodes and multiple hypoechoic foci in the spleen. Cytology of the mesenteric lymph nodes and spleen showed granulomatous inflammation with fungal organisms and negatively stained intracytoplasmic bacterial rods consistent with Mycobacteria spp. Based on culture, multiplex polymerase chain reaction and sequence analysis, the bacterium was identified as Mycobacterium avium subspecies hominissuis. Despite treatment with antibiotics, the dog's condition deteriorated, and it died approximately 3 weeks after first presentation.

Real time detection of farm-level swine mycobacteriosis outbreak using time series modeling of the number of condemned intestines in abattoirs.

Mycobacteriosis in swine is a common zoonosis found in abattoirs during meat inspections, and the veterinary authority is expected to inform the producer for corrective actions when an outbreak is detected. The expected value of the number of condemned carcasses due to mycobacteriosis therefore would be a useful threshold to detect an outbreak, and the present study aims to develop such an expected value through time series modeling. The model was developed using eight years of inspection data (2003 to 2010) obtained at 2 abattoirs of the Higashi-Mokoto Meat Inspection Center, Japan. The resulting model was validated by comparing the predicted time-dependent values for the subsequent 2 years with the actual data for 2 years between 2011 and 2012. For the modeling, at first, periodicities were checked using Fast Fourier Transformation, and the ensemble average profiles for weekly periodicities were calculated. An Auto-Regressive Integrated Moving Average (ARIMA) model was fitted to the residual of the ensemble average on the basis of minimum Akaike's information criterion (AIC). The sum of the ARIMA model and the weekly ensemble average was regarded as the time-dependent expected value. During 2011 and 2012, the number of whole or partial condemned carcasses exceeded the 95% confidence interval of the predicted values 20 times. All of these events were associated with the slaughtering of pigs from three producers with the highest rate of condemnation due to mycobacteriosis.

Tuberculosis in swine co-infected with Mycobacterium avium subsp. hominissuis and Mycobacterium bovis in a cluster from Argentina.

SUMMARY In Argentina little is known about the epidemiology of tuberculosis (TB) infection in swine. We characterized the epidemiological dynamics of Mycobacterium avium complex (MAC) infection in a swine population of Argentina using molecular tools and spatial analysis techniques. Isolates (n = 196) obtained from TB-like lesions (n = 200) were characterized by polymerase chain reaction. The isolates were positive to either M. bovis (IS6110) (n = 160) or M. avium (IS1245) (n = 16) while the remaining 20 (10.2%) isolates were positive to both M. bovis and M. avium. The detection of both bacteria together suggests co-infection at the animal level. In addition, MAC-positive isolates (n = 36) were classified as M. avium subsp. avium (MAA) (n = 30) and M. avium subsp. hominissuis (MAH) (n = 6), which resulted in five genotypes when they were typed using mycobacterial interspersed repetitive unit, variable number of tandem repeats (MIRU-VNTR). One significant (P = 0.017) spatial clustering of genotypes was detected, in which the proportion of MAH isolates was larger than expected under the null hypothesis of even distribution of genotypes. These results show that in Argentina the proportion of TB cases in pigs caused by M. avium is larger than that reported in earlier studies. The proportion of M. bovis-MAC co-infections was also higher than in previous reports. These results provide valuable information on the epidemiology of MAC infection in swine in Argentina.

Relatedness of Mycobacterium avium subspecies hominissuis clinical isolates of human and porcine origins assessed by MLVA.

Mycobacterium avium subsp. hominissuis (MAH) is an important opportunistic pathogen, infecting humans and animals, notably pigs. Several methods have been used to characterize MAH strains. RFLP and PFGE typing techniques have been used as standard methods but are technically demanding. In contrast, the analysis of VNTR loci is a simpler, affordable and highly reliable PCR-based technique, allowing a numerical and reproductive digitalization of typing data. In this study, the analysis of Mycobacterium avium tandem repeats (MATRs) loci was adapted to evaluate the genetic diversity of epidemiological unrelated MAH clinical strains of human (n=28) and porcine (n=69) origins, collected from diverse geographical regions across mainland Portugal. These MAH isolates were found to be genetically diverse and genotypes are randomly distributed across the country. Some of the human strains shared identical VNTR profiles with porcine isolates. Our study shows that the VNTR genotyping using selected MATR loci is a useful analysis technique for assessing the genetic diversity of MAH isolates from Portugal. This typing method could be successfully applied in other countries toward the implementation of a worldwide open-access database of MATR-VNTR profiles of MAH isolates, allowing a better assessment of the global epidemiology traits of this important pathogenic species.

Polymerase chain reaction-restriction fragment length polymorphism of the rpoB gene for identification of Mycobacterium avium subsp. paratuberculosis and differentiation of Mycobacterium avium subspecies.

Mycobacterial speciation by polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis (PRA) of the rpoB gene was evaluated for identification of Mycobacterium avium subsp. paratuberculosis (MAP) and other Mycobacterium avium complex (MAC) members to the species or subspecies level by comparison with conventional methods including hsp65 sequencing, high-performance liquid chromatography, and PCR for accepted species- or subspecies-specific genomic targets. A total of 185 type and clinical mycobacterial strains from humans, animals, and environments were tested. A 360-bp PCR product was subsequently digested with MspI, HaeIII, and SmaI restriction enzymes. The PRA using SmaI restriction showed a unique digestion pattern for MAP distinguishing it from other MAC members and other Mycobacterium spp. Moreover, HaeIII and MspI restriction of the rpoB gene enabled MAC-species and -subspecies discrimination. The rpoB-PRA using SmaI or MspI and HaeIII restriction of the rpoB gene is a simple, convenient, and reliable confirmatory assay for simultaneous identification of MAP and other MAC members.

Infection of Eurasian badgers (Meles meles) with Mycobacterium avium complex (MAC) bacteria.

There are few reports of infection with Mycobacterium avium complex (MAC) bacteria in badgers. In this study archive data relating to the isolation of MAC organisms from badgers in the UK is presented, and information derived from recent cases of such infection in Spain is used to illustrate the associated pathology and to characterise strain types. Tissue samples were cultured for mycobacteria and, in the case of Spanish badgers, were examined both histopathologically and using immunohistochemistry, and DNA typing of M. avium isolates was also carried out. A total of 5 (7.35%) and 281 (0.51%) isolates of M. avium spp. were recovered from badgers from the studies in Spain and the UK, respectively. DNA typing of the isolates from Spain identified the sub-species M. avium hominissuis and M. avium avium. These findings provide new information on the prevalence of MAC organisms in badgers in the UK and Spain. The extent to which infected badgers may be involved in the epidemiology of M. avium in other wild or domestic hosts remains unknown.

Efficient differentiation of Mycobacterium avium complex species and subspecies by use of five-target multiplex PCR.

Infections caused by the Mycobacterium avium complex (MAC) are on the rise in both human and veterinary medicine. A means of effectively discriminating among closely related yet pathogenetically diverse members of the MAC would enable better diagnosis and treatment as well as further our understanding of the epidemiology of these pathogens. In this study, a five-target multiplex PCR designed to discriminate MAC organisms isolated from liquid culture media was developed. This MAC multiplex was designed to amplify a 16S rRNA gene target common to all Mycobacterium species, a chromosomal target called DT1 that is unique to M. avium subsp. avium serotypes 2 and 3, to M. avium subsp. silvaticum, and to M. intracellulare, and three insertion sequences, IS900, IS901, and IS1311. The pattern of amplification results allowed determination of whether isolates were mycobacteria, whether they were members of the MAC, and whether they belonged to one of three major MAC subspecies, M. avium subsp. paratuberculosis, M. avium subsp. avium, and M. avium subsp. hominissuis. Analytical sensitivity was 10 fg of M. avium subsp. paratuberculosis genomic DNA, 5 to 10 fg of M. avium subsp. avium genomic DNA, and 2 to 5 fg of DNA from other mycobacterial species. Identification accuracy of the MAC multiplex was evaluated by testing 53 bacterial reference strains consisting of 28 different mycobacterial species and 12 nonmycobacterial species. Identification accuracy in a clinical setting was evaluated for 223 clinical MAC isolates independently identified by other methods. Isolate identification agreement between the MAC multiplex and these comparison assays was 100%. The novel MAC multiplex is a rapid, reliable, and simple assay for discrimination of MAC species and subspecies in liquid culture media.

Pulmonary mycobacteriosis caused by Mycobacterium intracellulare in a Tasmanian devil (Sarcophilus harrisii).

The health of captive Tasmanian devils (Sarcophilus harrisii) is currently of increased interest because wild populations are being decimated by the spread of devil facial tumour disease. This report describes the pathology of an aged captive Tasmanian devil that had a pulmonary mycobacterial infection caused by Mycobacterium intracellulare in addition to multiple neoplastic processes.