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.     

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.     

Epidemiologic investigation of Mycobacterium bovis in a population of cats

OBJECTIVE: To determine whether cats exposed at a residence were infected with Mycobacterium bovis, whether the tuberculin skin test can identify cats infected with M bovis, and whether an ELISA could identify tuberculosis-infected cats. ANIMALS: 20 domestic cats exposed to a cat with laboratory-confirmed disseminated M bovis infection. PROCEDURE: Cats were administered a tuberculin skin test and monitored for 72 hours. Blood and fecal samples were collected. Cats were then euthanatized, and postmortem examinations were performed. Tissues were examined grossly and histologically for signs of mycobacteriosis. Pooled tissue samples and fecal samples were submitted for mycobacterial culture. Blood samples were examined for evidence of tuberculosis by use of a comparative ELISA. RESULTS: 4 cats had positive responses for the ELISA, and 2 cats had suspicious responses. All tuberculin skin tests yielded negative results. No gross or histologic lesions of tuberculosis were detected in any tissues, and mycobacteria were not isolated from tissues or feces obtained from the 20 cats. CONCLUSIONS AND CLINICAL RELEVANCE: All cats that had positive or suspicious responses for the ELISA were offspring of the cat with tuberculosis. Evidence of tuberculosis was not seen in other cats at the residence, the owner, or the attending veterinarian. The most likely source of tuberculosis for the infected cat was through the consumption of M bovis-infected wildlife carcasses or offal. Because M bovis is endemic in wildlife in northeastern Michigan, there is a risk of exposure to tuberculosis in companion animals, their owners, and attending veterinarians.     

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.     

Mycobacterium bovis infection in a captive herd of Sika deer.

Infection with Mycobacterium bovis was diagnosed in a small privately owned herd of Sika deer. After postmortem examination of a deer with progressive pulmonary disease, diagnosis of infection with M bovis was confirmed by bacteriologic culture. The 2 remaining deer in this herd were euthanatized, necropsied, and confirmed to be infected with M bovis. Three cats in contact with the deer were also euthanatized and necropsied. One of these cats had lesions suggestive of mycobacterial infection in the colon and mesenteric lymph nodes. Infection of this cat with M bovis was not confirmed by bacterial culture. Mycobacteriosis, infrequently encountered in clinical veterinary practice, may be confused with disease caused by other infective agents or neoplasia. The zoonotic potential of these bacteria and a recent increase in human tuberculosis warrants continued surveillance of companion and food animal populations for mycobacterial infection.     

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).     

Antigenic characterization of mycobacteria from South American wild seals.

Tuberculosis-producing mycobacteria have been previously described in marine mammals (Cousins et al., 1990, 1993; Romano et al., 1995; Bernardelli et al., 1996). The strains belonged to the M. tuberculosis complex (M. tuberculosis, M. bovis, M. microti and M. africanum), but showed genetic and biochemical differences. The antigenic composition of mycobacteria isolated from wild seals was analyzed by Western blots, using antibodies against some selected antigens. The antigenic content was compared with that of M. bovis, M. tuberculosis and M. microti isolates. The lack of Hsp65 protein in supernatants suggested a low degree of cell lysis in the three-week cultures used. SOD, P27 lipoprotein, MPB64 and antigen 85 were observed in all the strains studied. The wild seal strains, as well as M. tuberculosis, did not produce MPB70 and MPB83. Only very weak bands of P36 antigen were observed in culture supernatants from wild seal mycobacteria. Summarizing, the antigenic composition of mycobacterial strains from wild seals is different from M. bovis strains.     

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.     

An outbreak of tuberculosis by Mycobacterium bovis in coatis (Nasua nasua)

Mycobacterium tuberculosis complex, which includes Mycobacterium bovis, infrequently causes severe or lethal disease in captive wildlife populations. A dead coati from a wildlife triage center showing pulmonary lesions compatible with tuberculosis had raised suspicion of a potential disease caused by mycobacteria species and was further investigated. Four native coatis (Nasua nasua) with suspected mycobacterial infection were sedated, and bronchoalveolar lavages and tuberculin skin tests (TSTs) were performed. All animals tested positive upon TST. Mycobacterial culturing, Ziehl-Neelsen staining, and genetic testing were performed on postmortem samples and the etiologic agent was identified as M. bovis. Molecular genetic identification using a polymerase chain reaction panel was crucial to achieving a definitive diagnosis.     

Further evaluation of an indirect enzyme-linked immunosorbent assay for the diagnosis of bovine tuberculosis

The sensitivity and specificity of an ELISA for the detection of bovine IgG anti-Mycobacterium bovis antibodies were 73.6% and 94.1%, respectively, as determined in 53 bacteriologically confirmed tuberculous cattle and 101 healthy cattle from a tuberculosis-free area. In addition, the results of ELISA and tuberculin tests in 149 cattle were compared with those of subsequent necropsy studies. Both tests failed to detect 2 animals with tuberculous lesions and positive culture; 3/12 cattle with M. bovis isolation and no lesions, and 2/7 with atypical mycobacterial infection reacted to tuberculin, but none had antibodies; in 128 cattle with neither lesions nor mycobacterial isolation, 6 were tuberculin reactors and 7 others had antibodies. Negative results were obtained by ELISA in 21/22 paratuberculous cattle. Antibodies were not detected in 88.9% to 96.4% of 697 cattle from two tuberculin negative herds of an endemic area. In a herd with proved M. bovis infection, distribution of seropositive animals in tuberculin and non-tuberculin reactors was similar. Antibody responses to cutaneous tuberculin stimuli were observed in 4 experimentally infected cattle, but only in 2/10 healthy controls after repeated PPD stimuli. Nine controls which had either received a single tuberculin dose or none showed no increase in antibody levels. The low sensitivity of this ELISA limits its usefulness as a diagnostic tool for bovine tuberculosis eradication campaigns. However, it could be helpful in epidemiological surveillance if its efficiency to identify infected herds is demonstrated.     

Identification of mycobacterial infections in wild boars in Northern Sardinia, Italy

During a six-month period a region of Northern Sardinia was monitored to check the presence of mycobacterial infections in wild boars. Forty-eight serum and 229 biopsy samples were collected from different animals and examined by both traditional diagnostic techniques (culture, bacterioscopic and molecular tests) and enzyme-linked immunosorbent assay (ELISA). The latter was used to determine the antibody response against both methylated and nonmethylated Heparin-Binding Haemagglutinin (HBHA) protein. Nine mycobacterial strains were isolated: three M. avium ssp. paratuberculosis (Map), three M. avium, one M. interjectum and two M. scrofulaceum strains. By PCR, only one animal was positive for M. bovis, whereas 10 animals were positive for Map. Out of the 48 sera tested, 19 showed a good humoral response to methylated HBHA and 17 to nonmethylated HBHA. Our data provide new information on the prevalence of mycobacterial infection among wild boars in Northern Sardinia and suggest that a more effective program should be developed to monitor mycobacterial infections in the wild animal population.     

Value of existing serological tests for identifying badgers that shed Mycobacterium bovis

In the UK there has been a sharp rise in the incidence of bovine tuberculosis since the early 1990s and the badger has been identified as an important wildlife reservoir for this infection. Infected badgers can excrete Mycobacterium bovis, putting other badgers and cattle at risk of becoming infected. Vaccination has been proposed as an approach to reducing the excretion of M. bovis by tuberculous badgers. In order to evaluate the efficacy of a badger vaccine it will be necessary to accurately determine the number of badgers excreting M. bovis without removing them for post-mortem evaluation. The existing live tests for tuberculosis in the badger (culture, indirect ELISA, Western blot) have not been assessed for their ability to detect badgers excreting M. bovis. Over the past 18 years, badgers from 31 social groups have been trapped and sampled in a study area of the Cotswold escarpment. We have examined the serological responses of 128 badgers trapped between 1985 and 1998 from social groups where M. bovis infection was endemic. These responses were compared with culture from faeces, urine, tracheal aspirates and bite wound swabs taken from these animals while alive. ELISA was found to be more sensitive than Western blot in detecting badgers excreting M. bovis. The majority of culture-positive badgers excreted M. bovis intermittently over the period of study. As a result, there was only a 27.5% chance of sampling a badger for culture when it was excreting M. bovis. In contrast, a positive ELISA result correctly predicted 68.2% of badgers with a history of excreting M. bovis. In the absence of alternative live tests for the badger, the Brock Test indirect ELISA appears to be more valuable than culture for measuring the effect of vaccination on reducing the number of badgers at risk of transmitting tuberculosis.     

Comparative genomics and proteomics to study tissue-specific response and function in natural Mycobacterium bovis infections

Bovine tuberculosis (bTB) is an established zoonotic disease which affects cattle and wildlife worldwide and new strategies are required to control and eradicate the disease. The European wild boar (Sus scrofa) is a major reservoir of bTB in Spain. The objective of this paper was to review tissue-specific response and function of mandibular lymph nodes (MLN) and oropharyngeal tonsils (OT) in European wild boar naturally infected with Mycobacterium bovis. Genomics and proteomics data were used to compare differential gene expression and global protein patterns in OT and MLN of M. bovis-infected and uninfected European wild boar and the results were analyzed considering previous reports of experimental infections in laboratory and domestic animals. The results showed tissue-specific differences in OT and MLN in response to M. bovis infection. Tissue-specific differences in gene expression and protein profiles suggested different functions for OT and MLN during mycobacterial infection and provided information to characterize the pathobiology of M. bovis infection in European wild boar with important implications for the control of bTB in Spain. The characterization of molecular events in tissues that play different roles during mycobacterial infection in naturally infected individuals may be relevant to understand the pathobiology of M. bovis infection and to design effective strategies for the control of bTB in wildlife reservoirs.     

Tuberculosis in domesticated red deer: Comparison of purified protein derivative and the specific protein MPB70 for in vitro diagnosis

The use of a Mycobacterium bovis-specific protein, mycobacterial protein bovis 70 (MPB70), was compared with complex, M bovis-derived purified protein derivative (bovine PPD), for its ability to improve the diagnostic precision of in vitro assays for tuberculosis in farmed deer. A combination of lymphocyte transformation and enzyme-linked immunosorbent assay (ELISA) was used to differentiate between specific M bovis reactivity and crossreactivity due to sensitisation with saprophytic mycobacteria such as Mycobacterium avium. In the lymphocyte transformation assay the response of mononuclear cells, from red deer, to MPB70 was found to be more specific, but less sensitive, as an indicator of infection by M bovis when compared with the complex antigen bovine PPD. When used in conjunction with bovine PPD alone, MPB70 was found to increase the specificity of the ELISA in diagnosing animals with disease.     

Modification of the QuantiFERON-TB Gold (In-Tube) assay for the diagnosis of Mycobacterium bovis infection in African buffaloes (Syncerus caffer)

African buffaloes (Syncerus caffer) are the most significant wildlife maintenance hosts of Mycobacterium bovis, the causative organism of bovine tuberculosis (BTB). Current diagnostic tests for the detection of M. bovis infection in free-ranging buffaloes have numerous limitations and we wished to evaluate a modification to a human TB assay, the QuantiFERON-TB Gold (In-Tube) assay (QFT), as a practical diagnostic test for BTB in buffaloes. One hundred and seventy-five buffaloes were tested using the single intradermal comparative tuberculin test (SICTT) and a modified QFT (mQFT). An appropriate cut-off point for the mQFT was derived from SICTT results using receiver operator characteristic curve analysis. Twenty-six SICTT-positive buffaloes were killed and subjected to necropsy, and selected tissues were processed for mycobacterial culture and speciation. An optimal cut-off point for the mQFT was calculated as 66pg/ml. The assay correctly detected 39/40 SICTT-positive buffaloes and 129/134 TST-negative buffaloes and M. bovis was cultured from 21/26 slaughtered SICTT/mQFT-positive animals. The mQFT shows promise as a practical test for M. bovis infection in buffaloes and shows a sensitivity and specificity at least similar to that of the TST.     

Mycobacterium bovis-infected white-tailed deer (Odocoileus virginianus): detection of immunoglobulin specific to crude mycobacterial antigens by ELISA.

White-tailed deer (Odocoileus virginianus) have recently emerged as a source of Mycobacterium bovis infection for cattle within North America. The objective of this study was to evaluate the antibody response of M. bovis-infected deer to crude mycobacterial antigens. Deer were experimentally inoculated with M. bovis strain 1315 either by intratonsilar instillation or by exposure to M. bovis-infected (i.e., in contact) deer. To determine the time course of the response, including the effects of antigen administration for comparative cervical skin testing, serum was collected periodically and evaluated by enzyme-linked immunosorbent assay (ELISA) for immunoglobulin (i.e., IgG heavy and light chains) reactivity to mycobacterial antigens. The reactivity to M. bovis purified protein derivative (PPDb) exceeded (P < 0.05) the reactivity to M. avium PPD (PPDa) only after in vivo administration of PPDa and PPDb for comparative cervical testing of the infected deer. The mean immunoglobulin response, as measured by ELISA, of intratonsilar-inoculated deer to a proteinase K-digested whole-cell sonicate (WCS-PK) of M. bovis strain 1315 exceeded (P < 0.05) the mean of the prechallenge responses to this antigen at approximately 1 month after inoculation and throughout the remainder of the study (i.e., approximately 11 months). This response also exceeded (P < 0.05) that of the uninfected deer. Although this is encouraging, further studies are necessary to validate the use of the proteinase K-digested M. bovis antigens in the antibody-based assays of tuberculosis.     

Comparison of postmortem techniques for the detection of Mycobacterium bovis in white-tailed deer (Odocoileus virginianus).

A retrospective study of various diagnostic postmortem techniques used in a 4-year surveillance program for detection of Mycobacterium bovis infection in wild white-tailed deer (Odocoileus virginianus) was conducted. The tests evaluated were routine histopathology, acid-fast staining, detection of acid-fast bacilli in culture, and an M. tuberculosis group-specific genetic probe applied to pure cultures. Each of these techniques were compared with a reference or "gold standard" of mycobacterial culture and identification. Histopathology, the most rapid form of testing for M. bovis infection in white-tailed deer samples, had a sensitivity of 98% and a specificity of 87%, resulting in a positive predictive value of 94%. The detection of acid-fast bacilli by staining was less sensitive than histopathology (90%), but its higher specificity (97%) resulted in a positive predictive value of 99%. The detection of acid-fast bacilli on culture was both highly specific (93%) and sensitive (100%). The group-specific genetic probe had the highest sensitivity and specificity and produced results in complete agreement with those of mycobacterial culture, suggesting that this technique could be used as the new "gold standard" for this particular wildlife tuberculosis surveillance program.     

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.     

Serologic prevalence of selected infectious diseases in cats with uveitis.

Serologic evidence of infection by Toxoplasma gondii, feline leukemia virus, feline coronaviruses, or feline immunodeficiency virus (FIV) is commonly found in cats with uveitis. Serum samples from 124 cats with uveitis were assayed by use of ELISA for the detection of T gondii-specific immunoglobulin M (IgM), IgG, and circulating antigens (Ag), as well as an ELISA for feline leukemia virus Ag, an ELISA for antibodies to FIV, and an indirect fluorescent antibody assay for antibodies to feline coronaviruses. Serologic evidence of infection by 1 or more of the infectious agents was detected in 83.1% of the samples. Serologic evidence of T gondii infection, defined as the detection of T gondii-specific IgM, IgG, or Ag in serum, was found in 74.2% of the samples. The seroprevalence of T gondii infection was significantly greater in cats with uveitis than in healthy cats from a similar geographic area. Serum samples from cats with serologic evidence of both T gondii and FIV infections were more likely to contain T gondii-specific IgM without IgG than samples from cats with serologic evidence of T gondii infection alone. Cats with serologic evidence of FIV and T gondii coinfection had a higher T gondii-specific IgM titer geometric mean and a lower T gondii-specific IgG titer geometric mean than did cats with serologic evidence of T gondii infection alone. Serologic evaluation for T gondii infection should include assays that detect IgM, IgG, and Ag, particularly in cats coinfected with FIV.     

Evaluation of three serological assays for the diagnosis of Mycobacterium bovis infection in brushtail possums

Three serological tests for the diagnosis of Mycobacterium bovis infection were evaluated on 29 possums (Trichosurus vulpecula) with tuberculosis and on 100 possums from a tuberculosis-free area. An indirect ELISA using M. bovis culture filtrate as the antigen had a sensitivity of 45% and a specificity of 96%, while an indirect ELISA using a M. bovis specific antigen (MPB70) had a sensitivity of 21% and a specificity of 98%. A blocking ELISA which utilised a monoclonal antibody against MPB70 had a sensitivity of 28% and a specificity of 99%. Combination of the test results of the three ELISAs resulted in an increase in sensitivity to 51% and a decrease in specificity to 93%. A previous study has shown that possums experimentally infected with M. bovis produced cellular responses to M. bovis antigens relatively early in the infection, but these responses decreased in the terminal stages of the disease. In contrast, analysis of serological responses in the current study from sequentially collected sera of possums experimentally and naturally infected with M. bovis showed that antibody was first detected late in the disease.