Mycobacterium ulcerans infections in two horses in south-eastern Australia

Two horses were diagnosed as having Mycobacterium ulcerans infections. The first was a 21-year-old Quarterhorse-cross mare living in Mallacoota (a coastal town near the border of New South Wales and Victoria, Australia) that presented with lichenification, hair-loss and oedema on a fetlock, which subsequently ulcerated, as well as a non-healing ulcer on the wither. The second horse was a 32 year-old Standardbred gelding from Nicholson, near Bairnsdale, Victoria, that had an ulcerated lesion on its caudal thigh. Histologically, there were characteristic changes seen with M. ulcerans infections in other species, including extensive necrosis without associated granulomatous inflammation. The organisms were seen in Ziehl-Neelsen-stained smears or sections of the lesions from both horses and were isolated in culture from the first horse. A definitive diagnosis was provided by real-time polymerase chain reaction targeting the M. ulcerans-specific insertion sequence, IS2404. Delayed identification of the infectious agent in the first case led to the use of suboptimal antimicrobial therapy, resulting in failure to control the infection and the horse was subsequently euthanased. The second horse was successfully treated following surgical debulking of the centre of the lesion and one session of aggressive cryosurgery. Mycobacterium ulcerans should be considered in the differential diagnosis of unexplained lichenification with oedematous and ulcerated skin lesions in horses living in regions where this organism is endemic.     

Differentiation of Australian isolates of Mycobacterium paratuberculosis using pulsed-field gel electrophoresis

Objectives To examine strain variation amongst Australian isolates of Mycobacterium paratuberculosis .
Design Pulsed field gel electrophoresis was optimised for differentiation of M paratuberculosis strains, and this typing technique was then applied to a collection of Australian isolates.
Procedure DNAs from 35 Australian isolates of M para-tuberculosis and a UK reference strain were digested with one or other of three restriction endonucleases. The banding patterns obtained after pulsed field gel electrophoresis of the DNA fragments were compared.
Results The Australian isolates were divided into two groups on the basis of their DNA banding pattern. Both were different from the UK reference strain. Seven isolates from cattle in Victoria and the Northern Territory had the same pattern as five isolates from alpacas in Victoria and Western Australia. Another 20 isolates from cattle in Victoria, Western Australia and the Northern Territory had the same pattern as isolates from two sheep and a goat in New South Wales.
Conclusion Pulsed field gel electrophoresis was a useful tool for strain typing of M paratuberculosis , and could be used to study the transmission of strains in Australia.     

Localised Mycobacterium ulcerans infection in a cat in Australia

A 10-year-old castrated male domestic cat domiciled in eastern Victoria (Australia) was presented for a subcutaneous mass on its nasal bridge in November 2006. Cytological examination of an aspirate demonstrated pyogranulomatous inflammation. At surgery, the lesion consisted of an encapsulated mass containing viscid fluid. Histological examination of the resected lesion revealed pyogranulomatous inflammation surrounding a central zone of necrosis. Sections stained with the Ziehl-Neelsen method revealed numerous acid-fast bacilli, intracellularly within macrophages and extracellularly. Molecular studies established the infection was caused by Mycobacterium ulcerans. As histology demonstrated that the infection extended to the margin of the excised tissues, the cat was treated subsequently with clarithromycin (62.5mg orally once daily for 7 days, then twice daily for 3 months). The surgical wound healed unremarkably. The infection has not recurred at the time of writing, 1 year following discontinuation of treatment. Although M ulcerans infections have been recorded in variety of mammals, this is the first known case in a cat.     

Real-time polymerase chain reaction testing for the detection of Mycobacterium genavense and Mycobacterium avium complex species in avian samples

Diagnosis of avian mycobacteriosis, caused by Mycobacterium genavense or species belonging to the Mycobacterium avium complex (MAC), is problematic. Polymerase chain reaction (PCR) offers rapid and sensitive detection of minute quantities of DNA, and conventional protocols have been used for evaluating avian specimens. The recent development of real-time PCR offers several advantages over conventional PCR. In attempts to improve diagnosing avian mycobacteriosis, a real-time TaqMan PCR assay was developed targeting the 65-kD heat shock protein gene of M. genavense and MAC spp. Nineteen reference isolates, 16 clinical isolates, and 32 avian tissue samples were used to evaluate the assay. When sufficient amplicons were produced, the species of mycobacteria was determined by standard sequencing of TaqMan PCR products and compared with results from commercial mycobacteriology laboratories and/or standard sequencing of conventional PCR products. The TaqMan PCR detected DNA from reference isolates of M. genavense, MAC spp., and Mycobacterium tuberculosis complex spp. Of the clinical isolates, the TaqMan PCR detected DNA from 10 of 12 Mycobacterium avium avium isolates and two of three Mycobacterium avium intracellulare isolates. For the tissue samples, the TaqMan PCR amplified DNA in six of nine samples that were identified by sequencing of conventional PCR products and/or by commercial mycobacteriology laboratories as being MAC spp. positive and three of four samples that were positive for M. genavense. There was some disagreement between speciation results from the TaqMan PCR and those from commercial mycobacteriology laboratories or conventional PCR or both. This disagreement was suspected to be because of relatively small numbers of base pairs in the TaqMan PCR products. The TaqMan PCR may provide a useful tool for evaluating clinical samples for DNA from mycobacteria species that most commonly infect birds; however, further refinement is needed in order to improve sensitivity and provide more accurate speciation.     

PCR for detection of Mycobacterium tuberculosis in experimentally infected dogs

The susceptibility of dogs to infection with Mycobacterium tuberculosis was studied through different ways of experimental infection. The examination shows, that in most cases the disease runs subclinically with pathological changes localized mainly in the lungs, lymph nodes, small intestines, liver, kidneys and spleen. Histological findings demonstrate granulomatous inflammation with caseosation and predominance of epitheloide macrophages and single lymphocytes. Tissue samples from internal organs of experimentally infected dogs as well as non-infected but contact animals were investigated by direct PCR. Specific PCR-products were obtained in 44 of 96 studied samples. Eighty-three (86.5%) of PCR results coincided with bacteriological finds, 82 (85.4%) with the pathological and 71 (74.0%) simultaneously with bacteriological and pathological results. The observed specific DNA products in tissue samples of infected and non-infected dogs demonstrate significant sensitivity of PCR method. It could be assumed that the transmission of M. tuberculosis infection is possible by close contact between ill and healthy dogs and that the naturally infected dogs or dogs suffering from tuberculosis may serve as a permanent source of infection to humans and other animals.     

Ulcerated and nonulcerated nontuberculous cutaneous mycobacterial granulomas in cats and dogs

Background –  Mycobacterial granulomas of the skin and subcutis can be caused by one of a number of pathogens. This review concentrates on noncultivable species that cause diseases characterized by focal granuloma(s), namely leproid granuloma (in dogs) and feline leprosy (in cats). Clinically indistinguishable lesions can be caused by tuberculous organisms (Mycobacterium bovis and Mycobacterium microti) and members of the Mycobacterium avium complex. Rapidly growing mycobacterial species that cause infection of the subcutaneous panniculus associated with draining tracts are not discussed. Disease caused by Mycobacterium ulcerans is an important emerging differential diagnosis for ulcerated cutaneous nodules in certain localized regions. Clinical lesions –  Lesions comprise one or multiple nodules in the skin/subcutis. These are generally firm and well circumscribed, and typically become denuded of hair. They may or may not ulcerate, depending on the virulence of the causal organisms and the immune response of the host. Diagnosis –  The most inexpensive, noninvasive means of diagnosis is by submission of methanol‐fixed, Romanowsky‐stained smears to a Mycobacterium Reference Laboratory after detecting negatively stained or acid‐fast bacilli on cytological smears. Scrapings of material from slides usually provide sufficient mycobacterial DNA to enable identification of the causal organism using sequence analysis of amplicons after PCR using specific mycobacterial primers. Therapy –  Therapy relies upon a combination of marginal resection of easily accessible lesions and treatment using two or three drugs effective against slowly growing mycobacteria, choosing amongst rifampicin, clarithromycin, clofazimine and pradofloxacin/moxifloxacin.     

Mycobacterium avium infection in a farmed deer herd

Tuberculous lesions were identified over a 2-year period in 36 clinically normal red deer from a single herd. The lesions were only present in the retropharyngeal lymph nodes and lymph nodes draining the intestinal tract, indicating infection by the oral route. Mycobacterium avium was isolated from 27 of 29 lesions examined by bacterial culture. Grossly and histologically, the lesions were indistinguishable from those caused by Mycobacterium bovis. DNA restriction endonuclease analysis revealed that all the 26 M. avium isolates available for examination had identical cleavage patterns. These patterns were identical to a New Zealand M. avium serotype 2 isolate from a pig and were very similar to a reference strain of M. avium serotype 2. The DNA examinations indicated that the deer were infected from a common source that was not identified.     

Putative transmission of Mycobacterium tuberculosis infection from a human to a dog

A 3.5-year-old Yorkshire Terrier was evaluated for anorexia and vomiting; infection with Mycobacterium tuberculosis was diagnosed by use of histology, bacteriologic culture, and polymerase chain reaction (PCR) assay on various tissues. The dog was living with a human with an established M. tuberculosis infection. Findings were unique in that diagnosis of M. tuberculosis infection was obtained via PCR techniques, and isolates from the owner and dog were matched via restriction fragment length polymorphism fingerprinting. Dogs infected with M. tuberculosis from humans are most commonly infected via the respiratory tract. Clinical signs in dogs are variable and depend on the integrity of the immune system and the degree of dissemination. Diagnosis can often be obtained through histopathology and bacteriologic culture; additional diagnostic techniques are also available. Treatment of a dog with confirmed M. tuberculosis infection is controversial, and at least 6 months of multidrug treatment is required.     

应用TaqMan荧光定量PCR检测牛分枝杆菌

为建立快速检测牛分枝杆菌(M.bovis)的TaqMan荧光定量PCR方法,本研究以GenBank登录的M.bovis特有229 bp基因为研究对象,设计并合成引物及探针。该方法具有较好的特异性,与标准质控菌株呈阳性反应,与其他微生物样品呈阴性反应;灵敏性最低检测值可达1 pg/mL;对20阳性临床样品进行荧光定量PCR检测,均为阳性;而对培养为阴性的20份临床样品进行检测,6份为阳性。该研究结果表明,建立的方法特异性强,敏感性高,稳定性好,能够用于M.bovis的鉴别检测,对牛分枝杆菌病的快速检测和早期诊断具有重要意义。     

The use of real-time PCR in the diagnosis and monitoring of Mycoplasma haemofelis copy number in a naturally infected cat

A 5-year-old male neutered cat was diagnosed with severe anaemia due to acute Mycoplasma haemofelis infection. Inflammatory respiratory disease was present concurrently. The cat was treated successfully using a fresh transfusion of whole blood and a 6 week course of doxycycline. The patient made a rapid recovery although the allergic airway disease subsequently required specific therapy consisting of inhaled fluticasone and salbutamol. Real-time quantitative PCR assays confirmed the presence of M. haemofelis DNA copies in the blood at presentation. Repeat PCR assays showed a reduction in copy number during treatment and negative PCR results were obtained both 91 and 425 days after presentation. The report describes, for the first time, the use of real-time PCR in the diagnosis and monitoring of natural M. haemofelis copy number, as well as the induction of long-term negative PCR status.     

Direct Detection of Mycobacterium bovis in Bovine Lymph Nodes by PCR

Thirty‐five lymph node samples were taken from animals with macroscopic lesions consistent with Mycobacterium bovis infection. The animals were identified by postmortem examination in an abattoir in the northwestern region of state of Paraná, Brazil. Twenty‐two of the animals had previously been found to be tuberculin skin test positive. Tissue samples were decontaminated by Petroff’s method and processed for acid‐fast bacilli staining, culture in Stonebrink and Lowenstein‐Jensen media and DNA extraction. Lymph node DNA samples were amplified by PCR in the absence and presence (inhibitor controls) of DNA extracted from M. bovis culture. Mycobacterium bovis was identified in 14 (42.4%) lymph node samples by both PCR and by culture. The frequency of PCR‐positive results (54.5%) was similar to that of culture‐positive results (51.5%, P > 0.05). The percentage of PCR‐positive lymph nodes increased from 39.4% (13/33) to 54.5% (18/33) when samples that were initially PCR‐negative were reanalysed using 2.5 μl DNA (two samples) and 1 : 2 diluted DNA (three samples). PCR sensitivity was affected by inhibitors and by the amount of DNA in the clinical samples. Our results indicate that direct detection of M. bovis in lymph nodes by PCR may be a fast and useful tool for bovine tuberculosis epidemic management in the region.     

Skin lesions caused by orthopoxvirus infection in a dog

A seven-year-old male dobermann was presented for examination of a non-pruritic ulcerated lesion occurring at the site of a suspected rat bite on the muzzle. Biopsy revealed focal ulcerative dermatitis, with cells in the epidermis, follicular infundibula and interposed sebaceous glands undergoing ballooning degeneration and containing large acidophilic intracytoplasmic structures resembling poxvirus inclusion bodies. The diagnosis of orthopoxvirus infection was confirmed by transmission electron microscopy and immunohistochemistry. The biopsy site healed uneventfully, without evidence of recurrence or development of further cutaneous or internal lesions, and a serum sample collected eight weeks after first presentation had a low titre of poxvirus antibodies. This report demonstrates that orthopoxvirus infection should be considered as a cause of ulcerative skin lesions in dogs, particularly if there has been recent contact with rodents or other small mammals.     

Outbreak of Mycobacterium avium subsp. avium infection in one flock of domestic pigeons

An outbreak of Mycobacterium avium subsp. avium infection was diagnosed in one breed of domestic pigeons (Columba livia f. domestica) in the Czech Republic. Nodular granulomatous lesions were found in 42 (9.7%) pigeons of the 435 examined; histopathologic examination of livers with gross lesions of mycobacteriosis from 15 randomly selected pigeons revealed granulomatous inflammation typical for avian mycobacteriosis in all samples. Direct Ziehl-Neelsen (ZN) microscopy and conventional culture were performed for a total of 117 liver samples (42 pigeons with nodular lesions, 55 randomly selected pigeons without nodular lesions, and 20 randomly selected squabs). Acid-fast bacilli were observed in 19 (16.2%), and conventional culture yielded growth of M. a. avium in 40 (34.2%) liver samples. A triplex quantitative real-time PCR assay based on the IS901 detection system was performed successfully in 115 liver samples and revealed M. a. avium in 63 (54.8%) of them. Mycobacterium a. avium was also detected in two squabs. Eight domestic rabbits (Oryctolagus cuniculus f. domestica) living in the breeding facility were also examined. Pyogranulomatous lesions were only found in one adult male rabbit. At necropsy, both direct ZN microscopy and culture gave negative results for mycobacteria in all examined rabbit tissues. Mycobacterium a. avium was diagnosed in a liver sample of one juvenile rabbit using triplex qPCR, suggesting that M. a. avium infection can occur as early as juvenile animals.     

Diagnosis of histoplasmosis by detection of the internal transcribed spacer region of fungal rRNA gene from a paraffin-embedded skin sample from a dog in Japan

The lesions of histoplasmosis in dogs in Japan differ from those in dogs in North America. Affected dogs in Japan have had multiple granulomatous or ulcerated foci in skin or gingiva and have not had pulmonary or gastrointestinal lesions. The present report introduces a polymerase chain reaction (PCR) diagnosis of canine histoplasmosis and the characteristic of disease in Japan. The surgically removed skin ulcerate samples from a 5-years-old female Shiba-inu native to Japan without traveling out of the country were evaluated. Tissue samples had many yeast-like organisms in the macrophages. DNA was extracted from paraffin-embedded tissue samples. A nested PCR technique was applied. The detected sequence of the internal transcribed spacer of ribosomal RNA gene had 99.7% in homology with Ajellomyces capsulatus (the teleomorph of Histoplasma capsulatum). Clinical manifestations, historical background of equine epizootic lymphangitis in Japan, and a human autochthonous case of histoplasmosis farciminosi indicated that this dog might have been infected with H. capsulatum var. farciminosum as a heteroecism.     

Detection of Leishmania infantum DNA by fret-based real-time PCR in urine from dogs with natural clinical leishmaniosis

The aim of this study was to detect Leishmania infantum DNA by real-time PCR in urine from different groups of dogs with clinical leishmaniosis. Urine from 10 clinically healthy dogs and 43 dogs with clinical leishmaniosis diagnosed by positive serology and/or bone marrow PCR were studied. The group of 43 dogs with clinical leishmaniosis was divided into three subgroups: 13 dogs with renal insufficiency and proteinuria (urine protein-creatinine ratio greater than one), 13 dogs with only proteinuria, and 17 dogs with neither renal insufficiency nor proteinuria. The detection of Leishmania DNA was performed by light cycler real-time PCR using hybridization probes in each urine sample. Leishmania positive PCR was found in 47% (20/43) of the urine from leishmaniotic dogs, while all urine from clinically healthy dogs were negative. The percentages of positive Leishmania PCR were 85% (11/13) in dogs with renal insufficiency and proteinuria, 23% (3/13) in dogs with proteinuria and 35% (6/17) in dogs with neither renal insufficiency nor proteinuria. Dogs with renal insufficiency and proteinuria presented a statistical significant greater percentage of positive Leishmania PCR in urine when compared with the other subgroups (P<0.02). This study demonstrates the presence of Leishmania DNA in urine of dogs with leishmaniosis. Those dogs with severe renal damage present a greater number of Leishmania parasites in urine.     

Detection of Mycobacterium avium subspecies paratuberculosis-specific DNA by PCR in intestinal biopsies of dogs

Background: Mycobacterium avium subspecies paratuberculosis (MAP) is the cause of paratuberculosis. MAP infections have not been reliably detected in dogs, but a reemerging debate about the link between MAP and Crohn's disease has renewed interest about the occurrence of MAP in pets.
Hypothesis: This study was undertaken to examine canine intestinal biopsies for the presence of MAP-specific DNA.
Animals: Forty-two dogs with chronic vomiting, diarrhea, or both; and 14 dogs with no gastrointestinal disease.
Methods: All dogs with signs of gastrointestinal disease had a standard work-up for chronic gastrointestinal disease. Endoscopically obtained intestinal biopsies were submitted for histopathologic and molecular investigations. Biopsies were screened for MAP-specific DNA by 3 polymerase chain reaction (PCR) methods (nested, seminested, and triplex real-time PCR). Samples from control dogs were obtained during necropsy.
Results: Histopathology of the biopsies was indicative of inflammatory bowel disease (IBD) in 17 and neoplasia in 6 dogs. Six dogs showing nonspecific changes responded to diet and were classified as having food-responsive enteropathy. In 13 dogs a final diagnosis was not established. MAP-specific DNA was detected and confirmed by sequencing in 8 dogs (19%). These dogs were diagnosed with food-responsive enteropathy (n = 3), IBD (n = 2), and open diagnosis (n = 3). MAP-specific DNA was not detected in dogs with no gastrointestinal disease.
Conclusions and clinical importance: MAP-specific DNA was detected in approximately one fifth of dogs with chronic gastrointestinal disease and might play a role as a pathogenic agent. Apart from animal welfare, the zoonotic aspect warrants further studies addressing the viability of MAP organism in canine intestinal biopsies by culture.     

Evaluation of tongue as a complementary sample for the diagnosis of parvoviral infection in dogs and cats.

Diagnosis of canine parvovirus type 2 and feline panleukopenia virus infection in dogs and cats may be hampered by the severity of enteric lesions, secondary bacterial overgrowth, and rapid onset of autolysis. In contrast to small intestine, tongue epithelium is less sensitive to postmortem changes. Sections of tongue and small intestine from 11 dogs and 11 cats with a clinical history and gross and microscopic lesions compatible with canine and feline parvoviral infection were examined for parvoviral infection using real-time polymerase chain reaction (PCR), immunohistochemistry (IHC), and direct fluorescent antibody testing (FA). Parvoviral DNA was detected by PCR in both small intestine and tongue of all but 1 dog. Nineteen of 22 animals (86%) with suspect or positive FA staining in the small intestine also had positive FA and IHC staining in the tongue. Three of 3 dogs (100%) whose carcasses had been frozen and thawed prior to necropsy had more consistently positive staining in tongue than in small intestine by FA and IHC. These data confirm tongue as an excellent complementary sample for parvoviral testing in dogs and cats, especially in cases in which postmortem autolysis has occurred.     

Molecular heterogeneity in clinical isolates of Malassezia pachydermatis from dogs

Molecular investigation of 16 strains, conventionally identified to be Malassezia pachydermatis, isolated from dogs in Japan was carried out by random amplification of polymorphic DNA (RAPD) and chitin synthase 2 (CHS2) gene sequence analyses. The RAPD band patterns of 13 clinical isolates were identical to that of standard strain of M. pachydermatis (CBS-1879). The other three clinical isolates were different from the standard strain of M. pachydermatis in RAPD patterns, and two of the three isolates were identical. About 620 bp genomic DNA fragments of the CHS2 gene were amplified from the same 16 clinical isolates of M. pachydermatis by polymerase chain reaction (PCR) and sequenced. The phylogenetic analysis of the nucleotide sequences of CHS2 gene fragments of the 16 clinical isolates revealed that the 13 strains were genetically very close to the standard strain of M. pachydermatis and the other two isolates were genetically close to the standard strain of M. furfur rather than M. pachydermatis. The remaining one isolate was phylogenetically distinct from all the seven Malassezia species reported so far.     

Use of the polymerase chain reaction assay for the detection of Mycobacterium avium subspecies paratuberculosis in blood and liver biopsies from experimentally infected sheep

OBJECTIVE: To evaluate a polymerase chain reaction-based assay for the detection of Mycobacterium avium subsp paratuberculosis in blood and liver biopsies from subclinically infected sheep. STUDY DESIGN: A direct PCR assay for the detection of M a paratuberculosis was applied to liver biopsy specimens and to samples of blood that were sequentially collected over 53 weeks from 14 sheep infected experimentally with the organism. RESULTS: Of 117 blood samples from the 14 experimentally infected sheep, two tested positive in the PCR assay. Both positive results were obtained in two subclinically infected sheep that had paratuberculosis later confirmed by histological examination at necropsy. However, the assay failed to detect the target DNA in samples of blood from five other sheep with histologically confirmed paratuberculosis. Similarly, the PCR assay on liver biopsy specimens collected 32 weeks after administration of M a paratuberculosis gave only two positive results, both of which were obtained in sheep with histological evidence of paratuberculosis. CONCLUSIONS: The PCR assay on blood and liver biopsies does not provide a useful method for the diagnosis of M a paratuberculosis infection in subclinically infected sheep.     

Disseminated tuberculosis in hyrax (Procavia capensis) caused by Mycobacterium africanum

Tuberculosis due to Mycobacterium africanum was diagnosed in an adult female hyrax (Procavia capensis). Pathologic examination revealed disseminated tuberculous lesions. The same pathologic changes were also found in a male hyrax that died a year later. Both animals were imported from the United Arab Emirates and were held in captivity at the Zagreb Zoo in Croatia. The source of infection remains unknown. The acid-fast bacteria isolated from the lungs of the female hyrax were identifyed by polymerase chain reaction as Mycobacterium tuberculosis complex and Geno Type MTBC test confirmed the strain to be M. africanum I.