First isolation of Mycobacterium microti (Llama-type) from a dog

We report the first isolation of Mycobacterium microti from a dog with lesions of acute peritonitis. The isolate was demonstrated to be M. microti of Llama-Type by spoligotyping. Epidemiological implications of the isolation of this possibly zoonotic agent from a dog are discussed.     

Comparative study of IFNγ and antibody tests for feline tuberculosis

This study describes the comparison of the cell-based interferon-gamma (IFNγ) test with serological rapid antibody tests (STAT-PAK and DPP VetTB) for the ante mortem testing of tuberculosis in domestic cats. The antibody specificities of rapid antibody test-positive cats were further discerned using multi-antigen print immunoassay. A total of 62 cats with culture-confirmed Mycobacterium bovis, Mycobacterium microti, Mycobacterium avium and Mycobacterium malmoense, as well as negative controls and dangerous-contact cats were tested. Tests were also applied longitudinally to one further cat undergoing TB chemotherapy for suspected M. bovis infection. Our data from this small study show excellent test specificity (100% for all tests) and encouraging levels of test sensitivity for M. bovis and TB Complex infections (IFNγ 70-100% depending upon test interpretation criteria; rapid tests both 90% for M. bovis infection and up to 46.2% for M. microti infection). The differential diagnosis of very pathogenic TB Complex (M. bovis, Mycobacterium tuberculosis), as opposed to less-pathogenic TB Complex (M. microti) was possible where positive responses to the protein cocktail ESAT6/CFP10 were observed (80% of M. bovis-infected cats in this study showed positive IFNγ responses to ESAT6/CFP10, while 20% had antibody responses to ESAT6/CFP10 using MAPIA). Finally, preliminary data from a longitudinal study of one M. bovis-exposed cat with a positive IFNγ test pre-treatment suggest that a decrease in bacterial burden may be reflected in the IFNγ response, and thus the IFNγ test may provide a monitor for TB chemotherapy.     

Mycobacterium microti infection in the cat: a case report, literature review and recent clinical experience

OVERVIEW: Mycobacterium microti infection is infrequently described in cats in the veterinary literature. It can be one of a large number of possible differential diagnoses in a feline patient with dermal nodules and non-healing draining ulcers, and can occasionally spread to involve the lungs and/or other areas of the body. CASE SUMMARY: This report describes the clinical signs, eventual diagnosis and variable response to treatment in a cat in Switzerland with recurrent cutaneous M microti infection. Only after several diagnostic and therapeutic attempts, over more than 2 years, was the species of Mycobacterium finally identified and targeted therapy given. PRACTICAL RELEVANCE: For any cat in which there is even a low suspicion of mycobacterial infection, the authors recommend that an aggressive diagnostic approach is taken. Tissue specimens should be collected and frozen early on, and, as soon as acid-fast bacilli are detected, samples should be sent to a mycobacterial reference laboratory for definitive identification. LITERATURE REVIEW: A review of the literature relating to the aetiopathogenesis, diagnosis and management of M microti infection in cats and dogs is included. This is supplemented with clinical and therapeutic experience gained from this case and other, unpublished cases managed over the past 15 years by one of the authors (DGM).     

Antibody responses in New World camelids with tuberculosis caused by Mycobacterium microti

Antibody responses in New World camelids (NWC) infected with Mycobacterium microti were studied by two serological methods, multiantigen print immunoassay (MAPIA) and lateral-flow-based rapid test (RT). Serum samples were collected during 2004-2006 from 87 animals including 1 alpaca and 7 llamas with confirmed or suspected M. microti infection, 33 potentially exposed but clinically healthy animals from known infected herds, and 46 control NWC from herds where infection had not been previously diagnosed. The serological assays correctly identified infection status in 97% (MAPIA) or 87% (RT) cases. In three llamas with confirmed M. microti infection and one llama with gross pathology suggestive of disease, for which multiple serum samples collected over time were available, the antibody-based tests showed positive results 1-2 years prior to the onset of clinical signs or being found dead. In MAPIA, MPB83 protein was identified to be an immunodominant serological target antigen recognized in NWC infected with M. microti. With the limited number of animals tested in this study, the serological assays demonstrated the potential for convenient, rapid, and accurate diagnosis of M. microti infection in live llamas and alpacas.     

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.     

Identification of Mycobacterium bovis isolates using a monoclonal antibody

A rapid immunoperoxidase slide assay for the identification of Mycobacterium bovis culture isolates is described. The monoclonal antibody used in this assay is specific for the M. tuberculosis complex of organisms. All M. bovis isolates tested, including 151 separate field isolates of M. bovis were positive as were 11 out of 12 M. tuberculosis strains and 4 out of 6 Bacillus Calmette Guerin (BCG) strains. One strain each of M. africanum and M. microti was negative. This assay provides a considerable improvement in both time and expense over the conventional methods of biochemical typing of M. bovis.     

Tuberculosis survey of free-ranging marsh deer (Blastocerus dichotomus) in Brazil.

Esophageal-pharyngeal fluids from 53 free-ranging marsh deer (Blastocerus dichotomus) captured for a research program in the state of Mato Grosso do Sul, Brazil, were assayed for tuberculosis. Total DNA was extracted. amplified by polymerase chain reaction using specific primers for Mycobacterium tuberculosis complex (M. tuberculosis, M. bovis, M. microti, and M. africanum), and observed by agarose gel electrophoresis stained with ethidium bromide. All samples were negative. This, along with necropsy and histopathology data, suggests that these animals are not shedding and probably do not have active disease.     

Suppression of Babesia microti infection in mice concurrently infected with Fasciola hepatica

Blood cell parasitaemia of Babesia microti and the associated haematological changes were examined in mice harbouring patent Fasciola hepatica infections and in fluke-free control mice. B. microti parasitaemia was markedly suppressed in mice harbouring primary 7-week F. hepatica infections, as reflected in a reduction in the percentage of erythrocytes parasitised and in the incidence of multiple B. microti infections in the red cells. This suppression was accompanied by an annulment of B. microti induced reductions in haemoglobin and haematocrit levels in F. hepatica infected mice. In naive recipient mice, inoculated with blood from mice concurrently infected with F. hepatica and B. microti, the course of B. microti infection was characterised by a prolonged pre-parasitaemia period, a reduced peak parasitaemia and a delayed fall in haematocrit levels as compared to those inoculated with blood from mice infected with B. microti only. This feature may presumably be dose-related. The present study does not reveal the actual mechanism(s) involved in the suppression of the blood protozoan by F. hepatica. However, since B. microti has a preference for mature erythrocytes, the suppression may be a result of the altered erythrocyte kinetic state induced by the removal of erythrocytes by the blood-sucking fluke resulting in high levels of reticulocytes.     

Changes of splenic lymphocyte subpopulation in mice inoculated with Babesia microti and Babesia rodhaini.

Changes of splenic lymphocyte subpopulation after Babesia microti and Babesia rodhaini inoculation in mice were examined by flow cytometric analysis. The B. microti inoculated mice showed a longer period of time from inoculation to the onset of increase or decrease parasitaemia (%), packed cell volume, total spleen cell numbers and surface immunoglobulin positive splenic cell numbers than respective periods in B. rodhaini inoculated mice. The Thy-1 positive cell numbers in B. microti inoculated mice and B. rodhaini inoculated mice pre-immunized with homologous parasites were significantly higher than that of B. rodhaini inoculated mice. The ratio of L3T4 positive cell/Lyt-2 positive cell after inoculation with B. microti was quite similar to that in B. rodhaini mice pre-immunized. However, the ratio in B. rodhaini inoculated mice revealed a lack of an increasing phase. These results suggested that the T-cell dependent early immune response, especially suppressor activity, was closely related to the difference in the course of infection between the non-lethal B. microti and the lethal B. rodhaini infection in mice.     

Suppression of Babesia microti infection in mice concurrently infected with Schistosoma mansoni

Blood cell panasitaemias of Babesai microti and associated haematological changes were studied in mice concurrently infected with Schistosoma mansoni and in controls with no schistosome infections. In comparison to the controls B. microti parasit-aemias were suppressed markedly in mice harbouring 6 and 8 weeks old patent S. mansoni infections at the time of infection with B. microti. The suppression was accompanied by an alleviation/annulment of the B. microti induced reductions in haemoglobin and haematocrit levels and in its erythropoetic stimulation. The suppressive effect of patent S. mansoni infections on B. microti is suggested to be at least partly attributable to non-specific immunological factors although the altered erythrocyte kinetic state induced by the schistosoma infection in combination with the preference of B. microti for mature (old) erythrocytes might also play a role in the suppression. In mice harbouring 2 weeks old prepatent S. mansoni infections B. microti was not suppressed.     

Mycobacterial disease in a population of 339 cats in Great Britain: II. Histopathology of 225 cases, and treatment and outcome of 184 cases

This study investigated 339 cases of feline mycobacterial infection, with histopathology findings from 225 cases, and treatment and outcome information from 184 cases. Tissue samples from cats with cutaneous lesions or suspicious masses at exploratory laparotomy were submitted to the Veterinary Laboratories Agency for mycobacterial culture over a 4-year period to December 2008. The study reviewed the files for information about histopathology, treatment and outcome, and blindly reviewed histopathological changes (including staining for acid-fast bacteria [AFB]) in a sub-set of 45 cases. When a cat is suspected of having a mycobacterial infection, accurate identification of the species involved helps to determine possible treatment options and prognosis. The study confirmed that histopathology and the presence of AFB are useful tools in the recognition of mycobacterial infection. Unfortunately, they did little to help determine the species of mycobacteria involved. The study identified a group of cats that were negative for AFB at the primary laboratory, but from which mycobacteria could be cultured; commonly Mycobacterium bovis or Mycobacterium microti. The study also identified a group of cats which where culture negative, despite typical signs of mycobacterial infection and positive AFB staining. Many cases responded favourably to treatment (56% of the cases where information was available), and many cats gained complete remission (42%). However, relapses were common (64%) and often followed by pulmonary and/or systemic spread that may have resulted from treatment with short courses of single drugs. This study shows that the diagnosis and treatment of feline mycobacteriosis is complex and challenging.     

Use of DNA amplification for the rapid identification of Mycobacterium bovis

DNA amplification using the polymerase chain reaction technique was evaluated for rapid identification of Mycobacterium bovis. Two oligonucleotide primers of 20 bases in length were constructed to target a region of the gene encoding the M. bovis secretory protein, MPB70. The amplification reaction produced a single product 372 bp in size which was readily detected by agarose gel electrophoresis. All 84 strains of M. bovis tested produced a positive signal in the amplification reaction. In addition all isolates fro the M. tuberculosis complex tested, with the exception of M. microti, gave a single band at 372 bp. No amplified product was detected when 24 other species of mycobacteria and species from four other genera were tested. The sensitivity of the test was such that a single viable cell could be detected in the reaction. This technique provides a simple and extremely sensitive method of identifying isolates of M. bovis and other pathogenic M. tuberculosis complex organisms.     

Epizootiologic survey for Babesia microti among small wild mammals in northeastern Eurasia and a geographic diversity in the beta-tubulin gene sequences

We previously reported that small wild rodents in Japan harbor two types of novel Babesia microti-like parasites (designated as Hobetsu and Kobe types), but not the type commonly found in the northeastern United States (U.S. type) where human babesiosis is endemic. To determine whether these new types of parasites are distributed in places surrounding Japan, an epizootiologic survey was undertaken in three geographically distant areas in northeastern Eurasia; South Korea, Vladivostok in Russia, and Xinjiang in China. Blood samples were collected from a total of 387 animals comprising 24 species. DNAs extracted from the samples were tested by nested PCR targeting babesial nuclear small-subunit rRNA gene (rDNA), which revealed that small rodents harboring B. microti exist in all three survey areas. Sequence analysis showed that all PCR-positive samples had rDNA sequences virtually identical to that of U.S.-type B. microti. However, when beta-tubulin gene sequences were compared, evident geographic variations were seen. By use of primers specific for each of the beta-tubulin genes of Kobe-, Hobetsu-, and U.S.-type parasites, a type-specific PCR was developed. Parasite with Hobetsu- or Kobe-type sequence was not detected from any of the three survey areas. These findings suggest that U.S.-type B. microti is widely distributed among small wild mammals in temperate zones of not only North America, but also Eurasia, whereas that Hobetsu- and Kobe-type parasites may be uniquely distributed in Japan.     

Glucose uptake activity in murine red blood cells infected with Babesia microti and Babesia rodhaini

The glucose uptake activity in Babesia rodhaini and B. microti - infected red blood cell (IRBC) was investigated in mice using 2-deoxy-D-glucose (2DOG) and L-glucose (L-Glc), a non-metabolizable analogue of D-glucose and non-incorporative glucose to non-infected RBC (NRBC), respectively. The uptake activities of both DOG and L-Glc were higher in IRBCs than those in NRBC. The concentration dependent uptake of 2DOG and L-Glc in both IRBC revealed a linear curve, indicating non-transporter mediated uptake. In addition, B. microti IRBC showed higher 2DOG uptake than B. rodhaini IRBC, whereas no difference was observed in L-Glc uptake. These results indicated that some new glucose uptake system, at least two systems, developed in both IRBC. The new systems were sodium independent, non-competitive to L-Glc, and sensitive to temperature. One of two systems had no kinetical difference between B. rodhaini and B. microti IRBC, however another one might have higher uptake activity in B. microti IRBC compared to that in B. rodhaini IRBC.     

Adaptation of IFN-gamma ELISA and ELISPOT tests for feline tuberculosis

There are currently no reliable immunodiagnostic tests for feline tuberculosis. Infection of domestic cats in the UK is thought to occur via their contact with the relevant reservoir of infection, e.g. cattle and badgers for Mycobacterium bovis, and rodents for M. microti. In the African National Parks, where M. bovis infection of Bovidae is an increasing problem, transmission to big cats is occurring via their ingestion of infected carcasses. We have adapted feline ELISA and ELISPOT assays to potentially provide the first cell-based diagnostic test for the detection of tuberculosis in cats. We tested peripheral blood mononuclear cell antigen-specific IFN-gamma responses of 18 cats suspected of mycobacterial infection for which biopsy material was co-submitted to the Veterinary Laboratories Agency for mycobacterial culture and identification. Seventeen cats were tested by ELISA while seven cats were tested by ELISPOT (six cats were tested by both ELISA and ELISPOT). Six healthy control cats provided baseline data for these tests. Responses to bovine and avian tuberculins (PPDB and PPDA) and a protein cocktail of ESAT6 and CFP10 were measured, together with positive mitogen (PMA and calcium ionophore) and negative (medium) controls. Overall, both ELISPOT and ELISA tests were found to be suitable for generating rapid results (2 and 4 days, respectively), which provided good predictive information for M. bovis and M. microti infections, but were unable to reliably discern M. avium infection.     

Babesia microti-like rodent parasites isolated from Ixodes persulcatus (Acari: Ixodidae) in Heilongjiang Province, China

A Babesia microti-like rodent parasite was isolated from the tick, Ixodes persulcatus, collected from the northern forest area of Heilongjiang province, China. The collected I. persulcatus were allowed to feed on specific pathogen-free SCID mice and red blood cells from the mice were used to isolate Babesia spp. with the microareophilous stationary-phase culture technique. Paired and tetrad forms of merozoites were observed by light microscope in red blood cells of SCID mice. In vitro growth of the parasites was also achieved in mice erythrocytes, which indicated the presence of Babesia spp. in I. persulactus. To further identify the Babesia species, polymerase chain reaction screening and subsequent sequencing of nuclear small subunit ribosomal RNA (nss-rRNA) was employed. The results indicate that the observed parasites might be an isolate strain responsible for human babesiosis -B. microti - which has 99.3% identity with that of B. microti isolate RcM5201 (AB112050) from Mishan in Heilongjiang and Kobe isolates from Japan. In addition, the infection rate of B. microti in I. persulcatus ticks in the region was 3.6-4.0% in adult females and no infection in males. Though the infection rate is low, the high attack frequency of tick species on local residents indicates the risk of human babesiosis in the region and the necessity of precautionary measures.     

Horse anti-bovine lymphocyte serum. Its effect in calves

Summary Anti-bovine lymphocyte serum (ABLS) had been prepared in horses with calf thymocytes as antigen and its effects in calves following parenteral administration were studied. The optimal dose was found to be one ml/kg body weight. The A BLS suppressed both the T and B cell functions. The former was indicated by the disturbed response to sheep erythrocytes injections, by the decreased number of spontaneous E rosette forming lymphocytes, the prolonged survival of skin allografts and the significant inhibition of the delayed hypersensibility skin reaction (tuberculination) following administration of Mycobacterium microti. The latter was based on the disturbed response to a subcutaneous dose of tetanus toxoid. The reaction of lymphocytes of ABLS treated calves to phytohemagglutinin and poke weed mitogen was also inhibited. The disturbed reactions of the T and B cells might be among others based on the strong reduction of lymphocytes in the blood circulation by ABLS (up to 10-20%). Suggestions with regard to further applications and studies with ABLS were given.     

Influence of zinc deficiency to the mice infected with Babesia microti

Zinc (Zn) is an essential trace element for DNA synthesis and for cell growth and differentiation. The deficiency induces a wide range of disorders including immunodeficiency. In this study, the influence of Zn deficiency to the mice infected with Babesia microti was examined, and was compared with the influence in the rats infected with B. rodhaini previously reported. Experiments of B. microti infection were conducted using Zn-deficient (ZD; allowed to eat ad libitum on the ZD diet), Zn-adequate (ZA; allowed to eat ad libitum on the ZA diet), and diet-restricted (DR; supplied 2 g/day on the ZA diet) mice. It was suggested that the Zn deficiency exacerbated the infection dynamics of the mice with B. microti by the growth retardation, the reduction of immunity and the decrease in PCV. The results in the mice supported the consequences in the rats previously reported.     

Radiographic findings in cats with mycobacterial infections

This study describes radiographic changes associated with mycobacterial infection in 33 domestic cats confirmed by culture or interferon-gamma testing. Infection was seen most frequently in adult (average age 5.7 years; range 1.5-12 years), non-pedigree (87%; 27/31), neutered male cats (69%; 22/32). The most common infections were Mycobacterium microti (60%; 18/30) and Mycobacterium bovis (37%; 11/30); Mycobacterium avium and Mycobacterium malmoense were infrequently cultured (3% of each; 1/30). Radiographs were available for the thorax (24 cats), abdomen (eight), appendicular skeleton (11) and head (three). Radiographic changes affected the thorax most commonly, consisting of bronchial (46%; 11/24), alveolar (38%; 9/24), nodular unstructured interstitial (38%; 9/24) or unstructured interstitial (25%; 6/24) lung patterns, which were often mixed. Perihilar or sternal lymphadenopathy were common (42%; 10/24), particularly perihilar lymphadenopathy (25%; 6/24). Skeletal changes were found in the distal antebrachium (three), pes (two), maxilla, scapula, spine, manus, femur, and tarsus (one each). Changes were typically osteolytic (73%; 8/11), often permeative osteolytic (64%; 7/11). Osteoproliferative changes were seen in three cats and soft tissue swelling in five cats, which were adjacent to the bony abnormality in four cats. Other changes included submandibular soft tissue swelling, marked aortic, aortic root and brachiocephalic trunk calcification, and soft tissue swelling with calcification in the distal antebranchium which was not involving bone. Abdominal changes were uncommon (seen in 2/8 cats) and consisted of hepatomegaly and hepatosplenomegaly. In summary, radiographic changes were varied, no lesion was pathognomic for mycobacterial infection, and pathology was seen most commonly in the thorax.