Isolation of Mycobacterium avium and other nontuberculous mycobacteria in chickens and captive birds in peninsular Malaysia.

BACKGROUND: Mycobacterium avium complex (MAC) causes a chronic infectious in the birds known as avian mycobacteriosis. Almost all species of the birds are susceptible to MAC which consists of two closely related species of mycobacteria, that is, M. avium and M. intracellulare. This study aimed to determine the occurrence of Mycobacterium avium subsp. avium (MAA) in chickens and captive birds in selected states of Peninsular Malaysia. RESULTS: A 300 fecal samples were collected from village chickens (n = 100), layer chickens (n = 100) and captive birds (n = 100). Fecal samples were split into two aliquots for microbiological and molecular detection of MAA. Microbiology detection consisted of microscopy (Ziehl-Neelsen staining) and culture of samples decontaminated with 1% Cetylperidinium chloride and vancomycin, nalidixic acid and amphotericin B (VNA) antibiotic cocktail [vancomycin (VAN) 100 µg/ml, nalidixic acid (NAL) 100 µg/ml and amphotericin B (AMB) 50 µg/ml] onto Löwenstein-Jensen (L-J). Molecular detection (PCR-IS901) was performed to detect MAA DNA from the feces and PCR-16S rRNA and IS901 for identification of genus Mycobacterium and Mycobacterium avium sub species avium isolated onto L-J. All samples (296) were AFB negative smear. M. avium was isolated in 0.3% (1/296) samples by culture and detected in 2.5% (6/242) samples by PCR (IS901). Other mycobacteria were found in 1.7% (5/296) chickens. Of five isolates, two were identified as Mycobacterium terrae and M. engbaekii and remaining isolates were not sequenced. Birds positive for M. avium included White Pelican (n = 1) Black Hornbill (n = 1), Macaw (n = 2), Cockatoo (n = 2) and village chicken (n = 1). CONCLUSION: It is concluded that chickens and birds were infected with M. avium in selected areas of Peninsular Malaysia. Although, PCR is rapid, reliable and cost effective method for detection of M. avium in a subclinical stage, the culture of the avian feces should still be used as a reference test for the diagnosis of avian tuberculosis.

Case Study and Attempt of Treatment of Mycobacteriosis Caused by Mycobacterium avium in a Parental Flock of Meat-Breed Pigeons.

Mycobacteriosis caused by Mycobacterium avium subsp. avium was observed in a parental loft of 70 meat-breed pigeons. It was decided to undertake treatment as the birds represented a substantial value to the owner. A multiagent therapy using azithromycin, marbofloxacin, and ethambutol was administered. After 4 mo of therapy, the desired results were not obtained. At the end of treatment, the birds were in poor general condition, with white blood cells above 20 g/L, and after clutching, 2-yr-old and older birds were euthanatized. Overall, postmortem lesions were found in 17 out of 49 necropsied individuals. Slide agglutination tests with a M. avium subsp. avium lysate were conducted in all examined pigeons. In 28 pigeons, blood count was conducted once a month during therapy, while in 24 pigeons, a tuberculin sensitivity test was conducted before the planned euthanatization. The tuberculin sensitivity test did not prove useful in the diagnosis of ill individuals. Slide agglutination yielded positive results in only four birds, all of which also had postmortem lesions. Blood count in a large number of cases allowed distinguishing between ill and healthy individuals, which was used for subsequent selection. The comparison of cultured strains with the (CCG)4-based PCR method showed the variation of M. avium isolates up to a maximum of 30%. The described case proves that the treatment of mycobacteriosis in pigeon flocks is not effective, mainly due to the high resistance to M. avium subsp. avium. In addition, therapy may contribute to an even greater increase in mycobacterial resistance to antibiotics, which may pose a potential risk to public health.

Avian Gastric Yeast (Macrorhabdus ornithogaster) and Mycobacterium genavense Infections in a Zoo Budgerigar (Melopsittacus undulatus) Flock.

Over a 4-mo period, a Michigan zoo had 32 budgerigars, Melopsittacus undulatus, from their flock die. Whole animals or formalin-fixed tissues were submitted to Michigan State University Veterinary Diagnostic Laboratory for diagnosis. Avian gastric yeast infection, Macrorhabdus ornithogaster, was diagnosed in seven birds. There was atrophy of breast musculature and no subcutaneous or coelomic fat stores in six necropsied birds. Only two birds had proventricular dilatation grossly. Histologic examination of the proventriculus of all seven birds revealed abundant 3 × 50-µm septate, parallel-walled, nonbranching yeast organisms morphologically consistent with Macrorhabdus ornithogaster. Mycobacteriosis was diagnosed in three budgerigars, two of which were necropsied. Both necropsied birds had hepatomegaly and one also had splenomegaly. No acid-fast bacilli were found in the livers of either bird but were found in splenic macrophages of the bird with splenomegaly and in the intestine of the other bird. Mycobacterium species were cultured from the enlarged spleen and identified by DNA sequence as Mycobacterium genavense. Pulmonary granulomas with acid-fast bacilli were found in the bird submitted as fixed tissues. None of the budgerigars had a dual infection. The remainder of the budgerigars died from hepatitis, nephrosis, oviductal prolapse, exclusion from food and water by flock mates, or tumors.

Amyloid A amyloidosis secondary to avian tuberculosis in naturally infected domestic pekin ducks (Anas platyrhynchos domestica).

To investigate the correlation between avian tuberculosis and duck amyloidosis, the liver, lung, spleen, kidney, duodenum and pectoralis muscle of ducks naturally infected with Mycobacterium avium subsp. avium were used to detect amyloidosis by Congo red staining and potassium permanganate-Congo red staining. The expression level of IL-1ß, IL-6, IL-10, TNF-α and SAA2 were detected by quantitative real-time RT-PCR (qRT-PCR). The results showed that the liver, lung, spleen, kidney, duodenum and pectoralis muscle of the infected ducks exhibited amyloid proteins under ordinary light microscopy and the polarization light under polarized light microscopy. However, no amyloid deposition in potassium permanganate-Congo red staining sections indicated that the amyloidosis was AA amyloidosis. In addition, the expression level of IL-1ß, IL-6, IL-10, TNF-α and SAA2 increased from 4 to 43. This study showed that avian tuberculosis could induce secondary amyloidosis in naturally infected ducks.

Avian mycobacteriosis in captive brolgas (Antigone rubicunda).

CASE SERIES: Avian mycobacteriosis is a significant disease of a wide range of bird species worldwide. The most common causative agent, Mycobacterium avium, is reported to also infect a range of mammals, including humans. Of 11 brolgas (Antigone rubicunda) submitted to the University of Melbourne for postmortem examination over a 10-year period, 7 were diagnosed with mycobacteriosis. All were from a wildlife park and kept in permanent enclosures as part of a breeding program. Most of the brolgas with mycobacteriosis were in poor body condition and had widely disseminated granulomas throughout the body, especially within the liver, spleen and gastrointestinal tract. Respiratory tract involvement was common, with all disseminated cases having pulmonary or air sac granulomas. Rare to moderate numbers of acid-fast organisms were detected in granulomas by histological examination. Where examined by appropriate bacteriological examinations, M. avium complex was isolated from affected tissues. CONCLUSION: This case series is the first known report of mycobacteriosis in brolgas and highlights the pathological changes seen. The complications in maintaining an avian mycobacteriosis-free breeding program and in eradication of the disease from an enclosed wildlife environment are discussed.

Prevalence of bovine and avian tuberculosis in camel herds and associated public health risk factors in Isiolo County, Kenya.

A cross-sectional study was conducted among 308 lactating camels selected from 15 herds from three different camel milk clusters in Isiolo County, Kenya, to determine prevalence of bovine and avian tuberculosis using Single Comparative Intradermal Tuberculin Skin test. Seventy-five (75) questionnaires were administered to pastoralists/herders, and focus group discussions were conducted among 3-5 pastoralists/herders selected from each camel herd to collect information on camel husbandry and health management practices and knowledge on tuberculosis in livestock and wildlife. An overall prevalence of bovine and avian reactors was 3.57 and 18.18%, respectively, with bovine and avian reactors for different clusters being 2.38, 3.82, and 4.48% and 25, 17.2, and 11.94%, respectively. There was significant difference (p < 0.05) in prevalence of bovine and avian reactors between different clusters. There was a negative correction (r = -0.1399) between herd size and bovine reactors, while there was a positive correlation (r = 0.0445) between herd size and avian reactors. The respondents indicated that camel herds are exposed to several risk factors like close contact with other herds or livestock or wildlife during grazing and at watering points. Pastoralists have poor knowledge on mode of infection and transmission of bovine or avian tuberculosis. The high prevalence of bovine and avian reactors and pastoralists' poor knowledge on mode of transmission signify potential risk to public health.

Molecular Detection of Mycobacterium avium avium and Mycobacterium genavense in Feces of Free-living Scarlet Macaws ( Ara macao) in Costa Rica.

We conducted a study of the two main populations of free-living Scarlet Macaws ( Ara macao) in Costa Rica to detect the causal agents of avian tuberculosis using noninvasive techniques. We analyzed 83 fecal samples collected between February and May 2016 from the central and southern Pacific areas in the country. Using PCR, we first amplified the 16S region of the ribosomal RNA, common to all Mycobacterium species. Then, products from the insertion sequence IS901 and from a 155-base pair DNA fragment evidenced the presence of the avian pathogenic Mycobacterium avium subsp. avium strain and a Mycobacterium genavense strain, respectively. Seven of 38 (18%) samples collected in the central Pacific area were positive for Mycobacterium spp. and 3 of 38 (8%) were positive for M. genavense, with one sample amplifying regions for both. Two of the 45 (4%) samples collected in the south Pacific area of Costa Rica were positive to M. a. avium. Our detection of avian tuberculosis pathogens in free-living Scarlet Macaws suggests that free-living macaws could excrete in their feces M. genavense, bird-pathogenic M. a. avium, and possibly other Mycobacteria (not detected in our study).

Isolation, Identification, and Characterization of a New Highly Pathogenic Field Isolate of Mycobacterium avium spp. avium.

Avian tuberculosis is a chronic, contagious zoonotic disease affecting birds, mammals, and humans. The disease is most often caused by Mycobacterium avium spp. avium (MAA). Strain resources are important for research on avian tuberculosis and vaccine development. However, there has been little reported about the newly identified MAA strain in recent years in China. In this study, a new strain was isolated from a fowl with symptoms of avian tuberculosis by bacterial culture. The isolated strain was identified to be MAA by culture, staining, and biochemical and genetic analysis, except for different colony morphology. The isolated strain was Ziehl-Zeelsen staining positive, resistant to p-nitrobenzoic acid, and negative for niacin production, Tween-80 hydrolysis, heat stable catalase and nitrate production. The strain had the DnaJ gene, IS1245, and IS901, as well. Serum agglutination indicated that the MAA strain was of serotype 1. The MAA strain showed strong virulence via mortality in rabbits and chickens. The prepared tuberculin of the MAA strain had similar potency compared to the MAA reference strain and standard tuberculin via a tuberculin skin test. Our studies suggested that this MAA strain tends to be a novel subtype, which might enrich the strain resource of avian tuberculosis.

Outbreak of Avian Tuberculosis in Commercial Domestic Pekin Ducks ( Anas platyrhynchos domestica).

Avian tuberculosis is a contagious disease affecting various domestic and wild bird species, and is caused by Mycobacterium avium . It is reported extremely rarely in commercial poultry flocks and has not been reported in commercial domestic ducks to date, with domestic ducks reported to be moderately resistant to M. avium infection. Here, we report the outbreak of avian tuberculosis in commercial Pekin duck ( Anas platyrhynchos domestica) flocks. Postmortem and histopathologic findings included nodules presenting in the visceral organs of ducks, and granulomas with central caseous necrosis surrounded by infiltrating lymphocytes. The M. avium pathogen was isolated and further identified by Ziehl-Neelsen staining and PCR based on insert sequence IS901 and the 16S rRNA gene. We highlight that avian tuberculosis not only has economic significance for the duck industry, but also presents a potential zoonotic hazard to humans.

Respiratory tract infection caused by Mycobacterium bovis in a black swan (Cygnus atratus).

A 3-year-old male black swan (Cygnus atratus), belonging to a private collection, died suddenly and was subjected to post mortem examination. At necropsy, caseous exudate was observed in the lungs and air sacs; granulomatous lesions characterized by epithelioid macrophages and abundant mycobacteria were observed microscopically. Avian tuberculosis associated with Mycobacterium bovis was confirmed by bacteriologic isolation, biochemical tests and molecular methods. The organism was identified as spoligotype SB0140, which is frequently found in cattle and people in North America. In this case, interspecies transmission could have been the source of infection because the swan cohabited with cattle.

Genotyping of Mycobacterium avium subsp. avium isolates from naturally infected lofts of domestic pigeons in Ahvaz by IS901 RFLP.

BACKGROUND AND OBJECTIVES: Avian tuberculosis is one of the most important infections affecting most species of birds. Mycobacterium avium can not only infect all species of birds, but also infect some domesticated mammals. The most crucial aspect of control and eradication scheme is identification of infection sources and transmission routs. Molecular techniques such as restriction fragment length polymorphism and pulse field gel electrophoresis have been shown to be much more discriminatory and suitable for use in the epidemiological study. MATERIALS AND METHODS: Eighty suspected pigeons to avian tuberculosis based on their clinical signs, were subjected to the study. Forty Mycobacterium avium subsp. avium isolates out of a total of 51 identified isolates were subjected to the test. RESULTS: IS901-RFLP using Pvu II was successfully conducted and produced 7 patterns. The majority of isolates (60%) were RFLP type PI.1. This type was the most similar type to standard strain. However, all the patterns obtained in this study were different from the standard strain. CONCLUSION: The result of this study indicate that these isolates probably are limited to Khuzestan region. We recommend DNA fingerprinting differentiation of non tuberculous Mycobacteria particularly Mycobacterium avium complex isolated from infected birds and human to possibly find source of infections.

Genetic IS901 RFLP diversity among Mycobacterium avium subsp. avium isolates from four pheasant flocks.

IS901 RFLP analysis of 36 Mycobacterium avium subsp. avium (MAA) isolates from 15 pheasants (Phasianus colchicus) and two goshawks (Accipiter gentilis) from four pheasant farms was performed. Using this method, six different IS901 RFLP types (E, F, G, M, Q, and V) were identified. The distribution of IS901 RFLP profiles was tightly linked to individual flocks. Matching IS901 RFLP profiles observed in the present study indicate MAA transmission between pheasants and goshawks in the same locality. In two flocks, different pheasants within a flock as well as in various organs of five individual pheasants were found to have two distinct IS901 RFLP profiles.

Genetic IS901 RFLP diversity among Mycobacterium avium subsp. avium isolates from four pheasant flocks

IS901 RFLP analysis of 36 Mycobacterium avium subsp. avium (MAA) isolates from 15 pheasants (Phasianus colchicus) and two goshawks (Accipiter gentilis) from four pheasant farms was performed. Using this method, six different IS901 RFLP types (E, F, G, M, Q, and V) were identified. The distribution of IS901 RFLP profiles was tightly linked to individual flocks. Matching IS901 RFLP profiles observed in the present study indicate MAA transmission between pheasants and goshawks in the same locality. In two flocks, different pheasants within a flock as well as in various organs of five individual pheasants were found to have two distinct IS901 RFLP profiles.

Estudio de la tuberculosis aviar en un zoológico de la sabana de Bogotá, mediante la técnica de la tuberculinización y la vigilancia epidemiológica activa / Study of avian tuberculosis in a zoo at the Bogota savannah through tuberculin testing and active epidemiologic surveillance

El flujo de aves silvestres constituye una debilidad en la vigilancia epidemiológica, pues se desconoce su potencial como fuente de diseminación de enfermedades. Se hizo un seguimiento epidemiológico de la población aviar existente en un encierro mixto silvestre de un zoológico ubicado en la Sabana de Bogotá, donde tres aves murieron con diagnóstico presuntivo de tuberculosis. A fin de comprobar la presencia de Mycobacterium avium y proyectar las medidas para evitar los factores de riesgo, se utilizó un grupo control de exposición previa de cinco aves domésticas raza Hy line Brown, un grupo centinela de diez aves domésticas del mismo lote del grupo control, un grupo control externo, constituido por 102 aves de los encierros vecinos al área problema. Se realizaron estudios retrospectivos y prospectivos, mediante análisis histopatológicos, microbiológicos, epidemiológicos y moleculares. El 100% de las aves del encierro original que murieron se les confirmó genotípicamente tuberculosis, causada por Mycobacterium avium. Del grupo centinela, 35,5% resultaron afectadas por Mycobacterium avium, 28,6% por Mycobacterium gordonae, 14,3% por Mycobacterium chelonae y un 21,4% por una micobacteria de difícil clasificación. Los animales de fuera del encierro no mostraron ningún indicio de infección. Se concluye que el encierro problema se encuentra afectado por tuberculosis aviar y se constituye en un foco de alto riesgo, tanto para aves como para los humanos. También, las aves domésticas utilizadas como centinelas son muy buenos detectores de agentes infecciosos, en particular, de las microbacterias presentes en el medio ambiente y la prueba de tuberculinización es un buen indicador de infección con este tipo de microorganismos en aves domésticas.

The flow of wild birds is a weakness in epidemiologic surveillance because of its unknown potential as a source of disease dissemination. The investigation focused on an epidemiological tracking of the mixed wild bird population in a zoo in the Bogota Savannah, where three birds died with a presumptive diagnosis of tuberculosis. In order to verify the presence of Mycobacterium avium and to plan the required measures to avoid risk factors, a control group of five poultry birds of the Hy Line Brown variety that had already been exposed was used, as well as a sentinel group of the poultry birds from the same batch as the control group, and an external control group of 102 birds from cages near the area of the problem. Retrospective and prospective studies were carried out through histopathological, microbiological, epidemiological and molecular analysis. One hundred percent (100%) of the birds from the original cages that died were genotypically diagnosed with tuberculosis caused by Mycobacterium avium. Thirty-five percent (35.5%) of the sentinel group was affected by Mycobacterium avium, 28.6% by Mycobacterium gordonae, 14.3% by Mycobacterium chelonae and 21.4% by a mycobacterium that is very difficult to classify. The other animals outside the cage showed no evidence of infection. It is concluded that the problematic enclosure is affected by avian tuberculosis, which is of high risk both for birds and for humans. The poultry used as sentinels are excellent infective agent detectors, particularly of mycobacteria present in the environment, and the tuberculin test is a good indicator of infection with this type of microorganisms in poultry.