Histological and genotypical characterization of feline cutaneous mycobacteriosis: a retrospective study of formalin-fixed paraffin-embedded tissues

Twenty-nine cases presumptively diagnosed as feline cutaneous mycobacteriosis were evaluated microscopically with haematoxylin and eosin and modified Fite's stained sections using archived formalin-fixed paraffin-embedded tissue specimens. Lesions were characterized histologically as feline leprosy (7 cases lepromatous and 16 cases tuberculoid) or atypical mycobacteriosis (3 cases); three cases did not fit these criteria and were classified as 'miscellaneous'. Actinomycetales-specific polymerase chain reaction (PCR) of variable regions 1, 2 and 3 of the 16S ribosomal RNA (rRNA) gene and subsequent sequence analysis of the amplicons were performed to identify the species of mycobacteria associated with each case. Together, this study identified 10 different Actinomycetales organisms with greater than 98% nucleotide sequence identity to named species, nine were of the genus Mycobacterium and eight were associated with feline leprosy (both lepromatous and tuberculoid). Based on this study, we conclude that feline cutaneous mycobacteriosis should be considered as a syndrome with varied clinical and histological presentations associated with a variety of different Mycobacterium species, organisms other than Mycobacterium sp. may be associated with feline cutaneous mycobacteriosis lesions, and molecular diagnostic techniques can be an important tool for identifying agents associated with lesions of feline cutaneous mycobacteriosis.     

Feline leprosy: two different clinical syndromes

Feline leprosy refers to a condition in which cats develop granulomas of the subcutis and skin in association with intracellular acid-fast bacilli that do not grow on routine laboratory media. In this study, the definition was extended to include cases not cultured, but in which the polymerase chain reaction (PCR) identified amplicons characteristic of mycobacteria. Tissue specimens from 13 such cases from eastern Australia were obtained between 1988 and 2000. This cohort of cats could be divided into two groups on the basis of the patients' age, histology of lesions, clinical course and the sequence of 16S rRNA PCR amplicons.One group consisted of four young cats (less than 4 years) which initially developed localised nodular disease affecting the limbs. Lesions progressed rapidly and sometimes ulcerated. Sparse to moderate numbers of acid-fast bacilli were identified using cytology and/or histology, typically in areas of caseous necrosis and surrounded by pyogranulomatous inflammation. Organisms did not stain with haematoxylin and ranged from 2 to 6 microm (usually 2 to 4 microm). Mycobacterium lepraemurium was diagnosed in two cases based on the sequence of a 446 bp fragment encompassing the V2 and V3 hypervariable regions of the 16S rRNA gene a different sequence was obtained from one additional case, while no PCR product could be obtained from the remaining case. The clinical course was considered aggressive, with a tendency towards local spread, recurrence following surgery and development of widespread lesions over several weeks. The cats resided in suburban or rural environments. A second group consisted of nine old cats (greater than 9 years) with generalised skin involvement, multibacillary histology and a slowly progressive clinical course. Seven cats initially had localised disease which subsequently became widespread, while two cats allegedly had generalised disease from the outset. Disease progression was protracted (compared to the first group of cats), typically taking months to years, and skin nodules did not ulcerate. Microscopically, lesions consisted of sheets of epithelioid cells containing large to enormous numbers of acid-fast bacilli 2 to 8 microm (mostly 4 to 6 microm) which stained also with haematoxylin. A single unique sequence spanning a 557 bp fragment of the 16S rRNA gene was identified in six of seven cases in which it was attempted. Formalin-fixed paraffin-embedded material was utilised by one laboratory, while fresh tissue was used in another. The same unique sequence was identified despite the use of different primers and PCR methodologies in the two laboratories. A very slow, pure growth of a mycobacteria species was observed on Lowenstein-Jensen medium (supplemented with iron) and semi-solid agar in one of three cases in which culture was attempted at a reference laboratory. Affected cats were domicile in rural or semi-rural environments. These infections could generally be cured using two or three of rifampicin (10-15 mg/kg once a day), clofazimine (25 to 50 mg once a day or 50 mg every other day) and clarithromycin (62.5 mg per cat every 12 h).These findings suggest that feline leprosy comprises two different clinical syndromes, one tending to occur in young cats and caused typically by M lepraemurium and another in old cats caused by a single novel mycobacterial species.     

A case of feline leprosy caused by Mycobacterium lepraemurium originating from the island of Kythira (Greece): diagnosis and treatment

A 2-year-old, 4 kg, healthy, domestic shorthair female cat presented with ulcerated subcutaneous nodules on the commissures of its mouth. The cat was negative for feline leukaemia virus and feline immunodeficiency virus. Skin mycobacteriosis was diagnosed after detection of numerous acid-fast bacilli in Ziehl Neelsen-stained smears from the ulcers. Feline leprosy was suspected following preliminary polymerase chain reaction results: positive for Mycobacterium genus but negative for Mycobacterium tuberculosis and Mycobacterium avium complexes. Mycobacterium lepraemurium was later identified following DNA sequence analysis of the 5' end of the 16S rRNA gene and the 16S-23S internal transcribed spacer region. Microscopic lesions consisted of pyogranulomas containing mainly large foamy macrophages with 10-100 intra-cellular acid-fast bacilli per field. The cat was cured after surgery and a 14-week course of clofazimine (30 mg daily) and clarithromycin (50 mg twice daily).     

Two genetic clusters in swine hemoplasmas revealed by analyses of the 16S rRNA and RNase P RNA genes

Only two hemoplasma species, Eperythrozoon parvum and Mycoplasma suis, have been recognized in pigs. Here we demonstrate the genetic variations among six hemoplasma strains detected from pigs, by analyzing the 16S rRNA and RNase P RNA (rnpB) genes, and propose a novel hemoplasma taxon that has not been described previously. Phylogenetic trees based on the nucleotide sequence of the 16S rRNA gene indicated that these six hemoplasmas were divided into two clusters representing M. suis and a novel taxon. We further examined the primary and secondary structures of the nucleotide sequences of the rnpB gene of the novel taxon, and found it distinct from that of M. suis. In conclusion, we unveiled a genetic cluster distinct from M. suis, suggesting a new swine hemoplasma species or E. parvum. Our findings also suggest that this novel cluster should be included in the genus Mycoplasma.     

The feline oral microbiome: A provisional 16S rRNA gene based taxonomy with full-length reference sequences

The human oral microbiome is known to play a significant role in human health and disease. While less well studied, the feline oral microbiome is thought to play a similarly important role. To determine roles oral bacteria play in health and disease, one first has to be able to accurately identify bacterial species present. 16S rRNA gene sequence information is widely used for molecular identification of bacteria and is also useful for establishing the taxonomy of novel species.The objective of this research was to obtain full 16S rRNA gene reference sequences for feline oral bacteria, place the sequences in species-level phylotypes, and create a curated 16S rRNA based taxonomy for common feline oral bacteria.Clone libraries were produced using “universal” and phylum-selective PCR primers and DNA from pooled subgingival plaque from healthy and periodontally diseased cats. Bacteria in subgingival samples were also cultivated to obtain isolates. Full-length 16S rDNA sequences were determined for clones and isolates that represent 171 feline oral taxa. A provisional curated taxonomy was developed based on the position of each taxon in 16S rRNA phylogenetic trees.The feline oral microbiome curated taxonomy and 16S rRNA gene reference set will allow investigators to refer to precisely defined bacterial taxa. A provisional name such as “Propionibacterium sp. feline oral taxon FOT-327” is an anchor to which clone, strain or GenBank names or accession numbers can point. Future next-generation-sequencing studies of feline oral bacteria will be able to map reads to taxonomically curated full-length 16S rRNA gene sequences.     

Experimental transmission of a feline mycobacterial skin disease (feline leprosy)

Non-culturable acid-fast bacteria from two spontaneous cases of so-called feline leprosy were transmitted to rats and cats and further passaged in rats or cats. Two to six months after infection, cats developed cutaneous lesions that were indistinguishable from spontaneous cases, including the occurrence of nasal granulomata in one cat. When injected into rats, the mycobacteria caused a generalized mycobacteriosis and the granulomatous reaction was composed chiefly of macrophages without polymorphonuclear granulocytes. Infection of cats with Mycobacterium lepraemurium did not produce any lesions. The feline disease may be a suitable model for the study of human leprosy (Hansen's Disease).     

Molecular detection of feline hemoplasmas in feral cats in Korea

The purpose of this study was to determine if Mycoplasma haemofelis, 'Candidatus Mycoplasma haemominutum' exist in Korea. Three hundreds and thirty one feral cats were evaluated by using PCR assay targeting 16S rRNA gene sequence. Fourteen cats (4.2%) were positive for M. haemofelis, 34 cats (10.3%) were positive for 'Candidatus M. haemominutum' and 18 cats (5.4%) were positive for both species. Partial 16S rRNA gene sequences were closely (>98%) related to those from other countries. This is the first molecular detection of feline hemoplasmas in Korea.     

Determination of intraspecies variations of the V2 region of the 16S rRNA gene of Streptococcus equi subsp. zooepidemicus

The 16S rRNA gene of 39 S. equi subsp. zooepidemicus strains and two S. equi subsp. equi strains was amplified by polymerase chain reaction and subsequently digested with the restriction enzyme Hinc II. A restriction profile with two fragments with sizes of 1250 bp and 200 bp could be observed for both S. equi subsp. equi strains and for 30 of the 39 S. equi subsp. zooepidemicus strains indicating a sequence variation within the V2 region of the 16S rRNA gene of the remaining nine S. equi subsp. zooepidemicus isolates. A segment of the 16S rRNA gene including the hypervariable V2 region of 11 S. equi subsp. zooepidemicus and two S. equi subsp. equi could be amplified by PCR and sequenced. The sequence of the V2 region of eight S. equi subsp. zooepidemicus strains appeared to be identical or almost identical to the sequence of the two S. equi subsp. equi strains. The sequence of the remaining three S equi subsp. zooepidemicus strains differed significantly from the sequence of S. equi subsp. equi. These differences allowed a division of S. equi subsp. zooepidemicus strains into two 16S rRNA types and might possibly have consequences for the taxonomic position of these phenotypically indistinguishable strains of one subspecies. A molecular typing could additionally be performed by amplification of the gene encoding the 16S-23S rRNA spacer region. A single amplicon of the spacer gene of 1100 bp could be observed for one S. equi subsp. zooepidemicus, an amplicon of 950 bp for two S. equi subsp. equi strains and 10 S. equi subsp. zooepidemicus strains, a amplicon of 780 bp for 27 S. equi subsp. zooepidemicus strains and a single amplicon of 600 bp for one S. equi subsp. zooepidemicus strain. The variations of the V2 region of the 16S rRNA gene and the size variations of the 16S-23S rRNA spacer gene were not related to each other. Both variations could be used for molecular typing of this species, possibly useful in epidemiological aspects.     

Intraspecific variation in the 16S rRNA gene sequences of Mycoplasma agalactiae and Mycoplasma bovis strains

Intraspecific variation in the 16S rRNA genes of 17 Mycoplasma agalactiae and eight Mycoplasma bovis isolates was investigated to determine the degree of sequence variation in these two species and to determine whether the polymorphisms in the 16S rRNA genes could be used for the construction of an evolutionary tree and as epidemiological markers. A high degree of variation was found within isolates (between operons) and between isolates of both species. In contrast to M. capripneumoniae no distinct evolutionary pattern could be seen, probably because there are functional systems for gene conversion in M. agalactiae and M. bovis. However, the non-European isolates of M. agalactiae shared three characteristic nucleotides and European isolates from the same or neighbouring countries were very similar. Differences within isolates included both polymorphic positions and sequence length differences between operons. The amount of variation within isolates of the respective species ranged from zero to seven polymorphisms for M. agalactiae and from zero to four polymorphisms for M. bovis. The high degree of variation suggests the potential for misdiagnosis of species in diagnostic PCR assays based on the 16S rRNA gene sequences. All isolates of both species had a thymidine in position 912 (E. coli numbering) that causes streptomycin resistance in several bacterial species and which is characteristic for the members of the hominis group. As expected, when five M. agalactiae and three M. bovis isolates were tested for streptomycin susceptibility, they all demonstrated streptomycin resistance. M. agalactiae and M. bovis were found to have high intraspecific variation in their 16S rRNA gene and the polymorphisms patterns indicate that gene conversion takes place.     

Mycobacterial disease in cats in Great Britain: I. Culture results, geographical distribution and clinical presentation of 339 cases

This study investigated 339 cases of feline mycobacterial disease from cats with cutaneous lesions or masses found at exploratory laparotomy. Tissue samples were submitted to the Veterinary Laboratories Agency for mycobacterial culture over a 4-year period to December 2008. The study assessed which species of culturable mycobacteria were involved, where the cats lived, and their clinical presentation (physical findings, serum biochemistry, radiography, feline leukaemia virus and feline immunodeficiency virus status). Mycobacterium microti was cultured from 19%, Mycobacterium bovis 15%, Mycobacterium avium 7%, non-M avium non-tuberculous mycobacteria 6%, with no growth in 53% of samples. M microti, M bovis and M avium were found in almost mutually exclusive clusters within Great Britain (GB) (ie, M bovis in South-West England/Wales/Welsh Border, M avium in eastern England and M microti south of London and in South-West Scotland). While differences were seen in the clinical presentation and distribution of lesions caused by the different infections, these were not sufficiently different to be diagnostic. Cats commonly presented with single or multiple cutaneous lesions (74%), which were sometimes ulcerated or discharging, located most frequently on the head (54%). Lymph nodes were usually involved (47%); typically the submandibular nodes. Systemic or pulmonary signs were rarely seen (10-16%). When a cat is suspected of having mycobacteriosis, accurate identification of the species involved helps to determine appropriate action. Our findings show that knowing the cat's geographic location can be helpful, while the nature of the clinical presentation is less useful. Most cases of feline mycobacterial disease in GB are cutaneous.     

Widespread infection with hemotropic mycoplasmas in bats in Spain,including a hemoplasma closely related to “Candidatus Mycoplasma hemohominis”

Molecular analyses of blood samples revealed infection with hemoplasmas in 97% of 31 cave bats captured in three caves in North-Eastern Spain. The characterization of 1250 bp of the 16S rRNA gene in 29 of the positive bats identified two different groups of sequences. Twenty-two Schreibers’ bats (Miniopterus schreibersii) and one long-eared bat (Myotis capaccinii) shared one group, composed of seven closely related sequences. These sequences showed an identity of about 97% with “Candidatus Mycoplasma hemohominis” and the phylogenetic branch including bat and human sequences showed a 100% bootstrap value, supporting a close phylogenetic relationship between these hemoplasmas. The second group, representing a potentially novel species, was composed of a single sequence shared by six Schreibers’ bats that had 91% identity with the recently reported hemoplasma from little brown bats in North America. Large bat aggregations in roosting caves probably benefits intra and inter-species transmission explaining the high observed prevalence.     

Mycobacterial dermatitis.

Mycobacterial skin disease in cats associated with atypical mycobacteria is an uncommon disease in small animal practice, and the disease is rarely encountered in dogs. A mycobacterial etiology should be considered in cases of chronic nodular dermatitis, draining tracts, and panniculitis. Cats and dogs affected with atypical mycobacterial infections are usually otherwise healthy, and systemic illness is unusual. In most cases, a diagnosis is made based on histopathological findings and growth of a causative organism. Group IV atypical mycobacteria can usually be grown following submission of affected tissue. Treatment should be based on antibiotic sensitivity test results. Treatment is prolonged and is unsuccessful in many cases. In cases of feline leprosy and canine leproid granuloma syndrome, organisms are difficult if not impossible to grow, and clinical and histopathological findings should be used to make a diagnosis. Spontaneous resolution of disease has been reported in atypical mycobacteriosis, feline leprosy, and canine leproid granuloma syndrome.     

Molecular analyses of mycobacteria other than the M. tuberculosis complex isolated from Northern Ireland cattle

Mycobacteria other than the Mycobacterium tuberculosis complex (MOTT), isolated from Northern Ireland cattle, were identified by PCR amplification of the 16S rRNA gene, and subsequent reverse cross blot hybridisation and sequence analyses. Elucidation of the MOTT species was to facilitate specificity testing of new and existing diagnostic test reagents for bovine tuberculosis. The presence of the genes for potential diagnostic antigens: MPB70, MPB64, ESAT-6 and CFP-10 in the isolated MOTT species was investigated. Molecular analyses of cultured isolates from bovine lymph node specimens of 48 cattle identified a wide variety of mycobacterial species including Mycobacterium nonchromogenicum, Mycobacterium malmoense, Mycobacterium bohemicum, Mycobacterium paratuberculosis, Mycobacterium avium, Mycobacterium kansasii, Mycobacterium holsaticum, Mycobacterium palustre, Mycobacterium sp. IWGMT 90210, Mycobacterium sp. LIV-2129, a potentially novel mycobacterial species (EMBL/GenBank/DDBJ Accession Number AJ617495) and Rhodococcus equi. Apart from M. kansasii, the results of traditional (standard phenotypic and biochemical) and molecular identification methods did not correlate well, with traditional methods identifying fewer species. Most of the species identified were either recognised pathogenic or potential pathogenic species. The genes for ESAT-6, CFP-10 and, unusually, MPB64 were detected in M. kansasii only. The MPB70 gene was not detected in any of the species. This study supported restricted species distribution of these genes as well as identifying a different range of MOTT species that could be included in specificity testing of new diagnostic reagents for bovine tuberculosis.     

红腹锦鸡大肠杆菌病的细菌学诊断

无菌采集病死红腹锦鸡肝脏、脾脏和心血,接种多种培养基分离细菌。以生化试验和小鼠攻毒试验,鉴定分离细菌及其致病性。采用PCR方法扩增分离细菌16S rRNA,测定核苷酸序列,与GenBank中大肠杆菌相应基因序列进行分析比较,绘制系统进化树。结果表明,分离细菌为大肠杆菌,可以致小鼠死亡。其16S rRNA基因序列与GenBank登录的大肠杆菌相应基因同源性超过99%。由此证明,该红腹锦鸡死于大肠杆菌病。     

Polymerase chain reaction identification of Mycobacterium avium in formalin-fixed, paraffin-embedded animal tissues.

A PCR procedure previously developed for identification of Mycobacterium bovis in formalin-fixed tissues was used to identify mycobacteria of the M. avium complex. Tissues were examined from 100 culture-positive cases of M. avium complex infection, including 86 in which the subspecies was not identified and 14 that had been identified as M. avium subsp. paratuberculosis. Each sample was tested with 5 primer sets, 16S ribosomal RNA (rRNA), IS900, IS901, IS1245, and a heat shock protein (hspX), that detect 1 or both M. avium subspecies. The success rate of PCR detection varied with the primers used and the animal species tested. Among the 86 cases with no M. avium subspecies designation, primers for the 16S rRNA gene were clearly the most efficient because they produced amplicons from all samples that reacted with any other primer set. The overall detection rate in this group of samples was 71%: highest in avian tissues (89%) followed by swine (72%) and ruminants (57%) None of the avian or swine tissues reacted with primers for IS900 or hspX, which identify M. a. paratuberculosis. In contrast, 7 of the 12 ruminant samples that were 16S rRNA positive reacted with 1 or both of these primers. All of the 14 cases shown by culture to be M. a. paratuberculosis infections were positive with IS900 primers, whereas only 11 were positive for 16S rRNA. These results indicate that 16S rRNA primers are the most useful for PCR identification of M. avium in formalin-fixed tissues of nonruminant species. However, IS900 primers should also be used when ruminant tissues are examined because these primers provide the greatest sensitivity for detection of M. a. paratuberculosis infections.     

Mycobacterium szulgai infection in a captive population of African clawed frogs (Xenopus tropicalis).

A colony of captive Xenopus tropicalis became infected with Mycobacterium szulgai. Clinical signs, when observed, were lethargy, weight loss, and emaciation. Visceral granulomas were common findings at laparoscopy and necropsy. The diagnosis of mycobacteriosis was based on histologic appearance and Ziehl-Neelsen staining of tissues. The identification of M. szulgai organisms was based on comparison of the 16S rRNA gene sequence with several GenBank databases. There have been no reports of this mycobacterial species as the causative agent of naturally occurring disease in amphibians.     

The GroES antigens of Mycobacterium avium and Mycobacterium paratuberculosis.

The GroES antigen provokes a strong immune response in human beings with tuberculosis or leprosy. We cloned and sequenced the Mycobacterium avium and Mycobacterium paratuberculosis GroES genes. M. avium and M. paratuberculosis have identical GroES sequences which differ from other mycobacterial species. This supports the current formal designation of M. paratuberculosis as M. avium subsp. paratuberculosis. Immunodominant epitopes from Mycobacterium tuberculosis GroES are conserved in M. avium, but some Mycobacterium leprae epitopes are distinct. GroES is unlikely to be specific as a serologic or skin test reagent, but may be an appropriate component of a broad mycobacterial vaccine.     

Development of a polymerase chain reaction for the detection of Mycoplasma felis in domestic cats

Mycoplasma felis is associated with conjunctivitis and respiratory disease in domestic cats. Currently no rapid diagnostic test is available for the detection of M. felis in clinical samples that does not rely on prior cultivation of the organism. The objective of this study was to determine whether a polymerase chain reaction (PCR) based upon the 16S/23S rRNA intergenic spacer sequence is suitable for the identification of M. felis directly in feline clinical samples. The high conservation between the 16S/23S rRNA intergenic spacers (IGS) of differing isolates of M. felis was established by sequence analysis and a PCR was developed to this region by comparison to IGS of other mycoplasmas. The PCR was found to be highly specific for M. felis and further PCR analysis on clinical samples showed the PCR to be highly sensitive and more rapid than the other methods of identification currently available.