Distribution of lesions in cattle infected with Mycobacterium bovis

Recent extension of the use of recombinant growth hormone (rhGH) to non-growth hormone-deficient patients necessitates close attention to possible complications in these patients, including effects on bone. Recent studies on the use of rhGH in children with chronic renal failure (CRF) provide some early data. No significant differences in radiographic osteodystrophy scores, serum calcium, phosphorus, or parathyroid hormone (PTH) levels were found between treated and untreated groups. Alkaline phosphatase increased transiently. The effect of renal osteodystrophy on growth response has not yet been reported. Animal models demonstrate that GH stimulates chondrocyte proliferation. Experimental data further suggest that GH can weaken the epiphyseal plate. Slipped capital femoral epiphysis has been reported in GH-deficient patients, before, during, and after GH therapy. In CRF patients treated with GH, slipped capital femoral epiphysis has also been reported. As renal osteodystrophy and hypocalcemia are risk factors for this condition, the relationship to GH therapy is unclear in these patients. Avascular necrosis, known to be associated with slipped capital femoral epiphysis and CRF, has also been reported in patients receiving GH, although the relationship to the therapy is unknown. Children with CRF treated with rhGH should be serially monitored for renal osteodystrophy, slipped capital femoral epiphysis, and avascular necrosis with serial radiographs and serum calcium, phosphorus, alkaline phosphatase, and PTH levels.     

Effect of dietary restriction on cell-mediated immune responses in cattle infected with Mycobacterium bovis

Nine M. bovis-infected cattle on a diet deficient in both protein and energy for 133 days lost approximately 17% of their original body weight. However, dietary restriction did not result in any significant reduction in skin sensitivity to PPD, in vitro production of IFN-gamma or lymphocyte blastogenesis. The number of circulating BoCD4+ cells and B cells were similar in both the malnourished and the control cattle. However, significantly lower numbers (P < 0.01) of circulating BoCD2+ cells, BoCD8+ cells, WC1+ gamma delta T cells and ACT2+ cells were found in the malnourished cattle. With the exception of inorganic phosphate, the changes in plasma biochemical parameters were unremarkable.     

Evaluation of an ELISA for Mycobacterium bovis infection in badgers (Meles meles)

The performance of an indirect ELISA for diagnosing Mycobacterium bovis infection in live badgers was evaluated by examining blood samples collected from 1982 badgers captured during statutory badger removal operations in south west England. The Validity of the test and the factors affecting the prevalence of infection are described. The sensitivity of the ELISA was 40.7 percent, its specificity was 94.3 percent, the predictive value of a positive test was 67.5% percent and the predictive value of a negative test was 84.6 percent. Its sensitivity was significantly higher in males and animals with gross lesions typical of tuberculosis. The sensitivity and positive predictive values were enhanced when the results were grouped by control operation. Variables of significance for prevalence were the county, the time of year, the age and sex of animal, and the time after the start of a control operation. The possible use of the ELISA as a screening test is discussed.     

Tuberculous lymphadenitis caused by Mycobacterium bovis

According to the literature, bovine tuberculosis in man has been an illness of minor importance in Norway, unlike in Sweden and Denmark. This situation cannot be explained, since in former days infected cattle were a problem in the southeastern part of the country in particular. No case of bovine tuberculosis in humans has been reported in Norway since 1940. Recently we have observed a 29-year-old female immigrant from India with cervical lymph node tuberculosis caused by M. bovis. A second case is a 77-year-old Norwegian male. In the 1920s, at the age of ten, he was infected in Norway by drinking raw milk from tuberculous cattle. He developed tuberculosis of the mesenterial lymph nodes. Neither of these patients had tuberculosis in any other part of their body. Previous Norwegian reports, covering eight cases, are summarized.     

Immunoblot analysis of humoral immune responses to Mycobacterium bovis in experimentally infected cattle: early recognition of a 26-kilodalton antigen

Development of a serodiagnostic test for bovine tuberculosis necessitates an understanding of the humoral immune responses of animals following infection with Mycobacterium bovis. The antibody responses in groups of calves challenged intranasally with different doses of M. bovis (approximately 10(2), 10(4), and 10(6) CFU) or placed in contact with the infected animals were analyzed by immunoelectrophoretic blotting in which a whole-cell sonicate of M. bovis was utilized as an antigen. Antibody responses were evident early in infections in which calves were exposed to high doses of M. bovis, while in groups exposed to lower doses, the time until antibody was detected increased as the challenge dose decreased. In cattle exposed to M. bovis, immunoblot analysis showed antibody responses to three main antigens of 26, 22, and 16 kDa. It was further demonstrated that antibody responses to the 26-kDa antigen appeared earliest in the course of infection. Preliminary investigations in this study have identified a 26-kDa antigen for potential use in improved serodiagnosis by enzyme-linked immunosorbent assays.     

Antigenic characterization of Mycobacterium bovis BCG soluble antigens

The proteins in Mycobacterium bovis BCG sonicated antigens were separated by gel filtration chromatography. A total of 6 regions were observed. All the regions were analyzed separately for their antigenic reactivity using enzyme linked-immunosorbent assay (ELISA), delayed type hypersensitivity (DTH) skin testing and an in vitro lymphocyte transformation test (LTT). Region I of high molecular weight was found to have more reactivity in comparison to regions of lower molecular weight. When region I was subjected to anion exchange chromatography, it resolved into 5 further regions. Of these regions only region III was found to have highest reactivity when tested in ELISA, DTH and LTT. This was interpreted to indicate that region III of anion exchange chromatography has immunodominant epitopes. On sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, this region was found to be a homogeneous polypeptide of 71 kDA molecular weight. All regions were found to crossreact with various mycobacteria used in this study.     

Detection of Mycobacterium bovis in milk by polymerase chain reaction

A simple method was developed for DNA extraction of Mycobacterium bovis in milk and further detection of the bacterium by polymerase chain reaction (PCR). Milk previously seeded with M. bovis was used as the starting material. The procedure involved overnight digestion of a milk sample with proteinase K at 56 degrees C and phenol extraction, followed by ethanol precipitation and PCR. The amplification pattern obtained was analyzed with primers BW8-BW9 which amplify a 248 bp in strains of M. bovis. By using the BW8-BW9 primers, 10(3) CFU were detected on silver-stained PAGE gels. The procedure was validated by PCR analysis of milk in tuberculin-positive animals. It is anticipated that this method can be used for routine diagnosis of M. bovis in milk samples.     

Monitoring Babesia bovis infections in cattle by using PCR-based tests

The sensitivity and specificity of PCR tests based on the small-subunit rRNA gene sequence of Babesia bovis were compared in a blind study of experimentally infected cattle with the corresponding parameters of the complement fixation (CF) test currently used in the United States to screen for bovine babesiosis. Cattle were experimentally infected with a single inoculum of a cloned laboratory strain of B. bovis. Blood samples were collected and tested over a period covering from the day of infection to 10 months postinfection. The level of parasitemia (percent infected erythrocytes) present in each sample was estimated from test results and was plotted as a function of time postinfection. These data are the first describing the course of infection by methods capable of detecting parasitemias in the range of 10(-7)%, which frequently occur in the carrier state. Parasitemias in the samples tested strongly influenced the sensitivity and negative predictive value of the PCR-based tests which varied with time postinfection. The average sensitivities of the three PCR-based tests for B. bovis ranged from 58 to 70% for a single determination, while the sensitivity of the CF test was only 6%. Both PCR-based and CF tests for B. bovis had high specificity values ranging from 96 to 100%.     

Human Mycobacterium bovis infection in the south-west of Ireland 1983-1992: a comparison with M. tuberculosis

Epidemiological and bacteriological aspects of human Mycobacterium bovis disease were investigated in south-west Ireland (counties Cork & Kerry, population 536,000) over the years 1983-92 inclusive and compared to M. tuberculosis. Results showed a small, stable incidence of culture positive M. bovis human disease, mean annual incidence 0.56 per 100,000 population compared to a higher but declining incidence of culture positive M. tuberculosis (15.3 per 100,000 in 1983, 9.0 per 100,000 in 1992). Male patients were the majority, 63.4 per cent of M. bovis; 62.4% of M. tuberculosis (p = 0.03). Fifty three per cent of M. bovis cases (n = 30) were pulmonary, compared to 85% of M. tuberculosis (n = 626; p = 0.0001). M. bovis patients were older (p = 0.02), mean age 58.4 years (SD 18.9) compared to 48.5 (SD 22.2). The mycobacterial smear positive rate was similar in both groups taken as a whole. No rural-urban difference in incidence was found in either disease, suggesting in the case of M. bovis initial infection in childhood via contaminated milk in the pre-pasteurisation era.     

Aetiology and Pathogenesis of abomasal displacement in dairy cattle

A clinical chemical survey is given of the complex of factors which are involved during development of clinically manifest hypotony or atony of the abomasum prior to abomasal displacemnts. The importance of a change in the acid-base balance of the animals is especially stressed as a predisposing factor. The further pathogenesis of abomasal displacement is supposed to follow different ways, according to the feeding and to prevailing periods of indigestion.     

Specific differentiation between Mycobacterium bovis BCG and virulent strains of the Mycobacterium tuberculosis complex

A PCR procedure based on the intergenic region (IR) separating two genes encoding a recently identified mycobacterial two-component system, named SenX3-RegX3, was developed and was shown to be suitable for identifying Mycobacterium bovis BCG. The senX3-regX3 IR contains a novel type of repetitive sequence, called mycobacterial interspersed repetitive units (MIRUs). All tested BCG strains exclusively contained 77-bp MIRUs within the senX3-regX3 IR, whereas all non-BCG M. tuberculosis complex strains contained a 53-bp MIRU, in addition to the 77-bp MIRUs. All 148 strains analyzed so far could be divided into eight different groups according to the copy numbers of the 77-bp MIRU and to the presence or absence of the 53-bp MIRU. BCG strains contained either one, two, or three 77-bp MIRUs. The other strains contained one to five 77-bp MIRUs invariably followed by a 53-bp MIRU. The consistent absence of the 53-bp MIRU in BCG strains and its presence in virulent strains allowed us to develop an enzyme-linked immunosorbent assay using specific capture oligonucleotide probes to distinguish between BCG and other M. tuberculosis complex strains.     

An mpb-64 flanking sequence specific for Mycobacterium bovis

A clone carrying a plasmid with the mpb-64 gene and 3' flanking sequences (plasmid pMBA122) was detected during the screening of a Mycobacterium bovis genomic library with sera from infected cattle. When the pMBA122 insert was used as a probe in Southern blots against PvuII-digested mycobacterial DNA, it distinguished the different M. tuberculosis complex species. This probe hybridized with a 7-kb band in M. tuberculosis, a 5-kb band in M. bovis and a 3-kb band in M. tuberculosis complex strains from wild seals. Smal genomic digestions enabled us to locate this polymorphic region downstream of the mpb-64 gene. In order to clone this particular region, we designed a pair of PCR primers. Unexpectedly, these primers amplified only M. bovis DNA; no amplification was seen in M. tuberculosis DNA. When the annealing temperature was lowered from 70 to 55 degrees C, an amplification product of the same size was obtained with M. tuberculosis. This product was cloned and sequenced, and showed partial homology to the M. bovis amplified fragment. Therefore, this region comprises M. bovis sequences with a lower homology with M. tuberculosis than other compared sequences. This suggests that a more precise differentiation method at the species level for the M. tuberculosis complex could be achieved using PCR directed to this region.     

Pathogenesis, diagnosis, and management of trichomoniasis in cattle

Trichomoniasis is a disease of the pregnancy, but apparently not of either the cow or the bull, except in the case of postcoital pyometra. Its self-limiting nature in the cow and chronic nature in the bull mean that a positive diagnosis for the herd can more easily be obtained from bulls than from cows. Incubation of preputial scrapings or washings (or pyometritic fluid, if available) in a selective growth medium such as the InPouch system is the diagnostic method of choice. The diagnosis is based on identification of the morphology and characteristic rolling motility of the trichomonad. "High tech" molecular approaches may eventually offer greater diagnostic sensitivity than can culture methods, but currently they are no more accurate. In addition, serologic screening of the female herd (but interestingly, not the bulls) may become possible and may allow the practitioner to at least determine whether exposure has occurred in an unvaccinated herd. Control in an infected herd involves no pharmacologic treatment but rather culling of infected bulls, retention of younger, culture-negative bulls, and segregation of the female herd by reproductive status.     

Mycobacterium bovis (BCG) infection of the lymph nodes of normal, immune, and cortisone-treated guinea pigs

Strain-2 inbred guinea pigs were infected intradermally with 10(5)-10(7) viable BCG (Pasteur) organisms by means of multiple scarifications of shaven midflank skin. The spread of the BCG to the draining lymph nodes and on to the spleen was followed quantitatively for 28 days. The population of bacilli at the inoculation site increased as much as tenfold the first 14 days. The number of viable BCG organisms recovered from the primary draining superficial dorsal axillary and inguinal lymph nodes varied from 0.1 to 1.0% of the inoculum, with a further tenfold to 100-fold drop in counts for the secondary subclavian and lumbar lymph nodes. The bacterial counts for the various nodes increased substantially the first 14 days. By 28 days, as many as 1,000 viable bacilli were recovered from the spleen. Increasing the inoculum size or the number of inoculation sites increased the primary node counts and promoted a more extensive and rapid spread by the BCG population to the secondary lymph nodes and spleen. Prior vaccination of the host with living BCG decreased the spread of the BCG inoculum from the scarification site to the various draining lymph nodes. Multiple injections of cortisone tended to reverse this effect.     

Pathogenesis of wild-type and leaderless foot-and-mouth disease virus in cattle

Four calves were experimentally infected via aerosol with foot-and-mouth disease virus. Two were infected with a wild-type virus derived from a full-length infectious clone (A12-IC), and two were infected with a clone-derived virus lacking the leader gene (A12-LLV2), with euthanasia and tissue collection at 24 and 72 h postexposure (hpe). Clinical disease was apparent only in the animal given A12-IC and euthanized at 72 hpe. In situ hybridization revealed that the animal infected with A12-IC and euthanized at 24 hpe had abundant viral nucleic acid in the lung, present in clusters of positive cells in the respiratory bronchiolar epithelium and associated subepithelial regions. At 72 hpe in the A12-IC-infected calf, viral nucleic acid in the lung was present in interstitial areas, and in addition, viral nucleic acid was detectable in epithelial tissues around histologically apparent vesicles. In animals infected with A12-LLV2, viral nucleic acid was detectable in the lung at both 24 and 72 hpe, but staining revealed a more localized distribution with less nucleic acid than was found in animals given A12-IC. Therefore, it appears that after aerosol exposure to A12-IC, early replication is in the region of the lung, with subsequent dissemination to distal sites. In comparison, the A12-LLV2 virus is much less widely disseminated in the lung at 24 hpe, with no lesions or virus detectable in secondary sites at 72 hpe. The greatly reduced pathogenicity of A12-LLV2 may make it an excellent candidate for a modified live viral vaccine.     

Protection by CD4 or CD8 T cells against pulmonary Mycobacterium bovis bacillus Calmette-Guérin infection

Mice deficient in CD8 T cells demonstrated levels of Th1 cytokines and granulomatous responses in the lungs very similar to those demonstrated by normal control mice and were fully capable of controlling pulmonary mycobacterial infection by Mycobacterium bovis BCG as assessed at day 37 postinfection. In comparison, mice deficient in CD4 T cells had similar levels of interleukin-12 (IL-12) and tumor necrosis factor alpha but lower levels of gamma interferon in the lungs and were still able to mount tissue granulomatous responses and control pulmonary mycobacterial infection. In contrast, IL-12(-/-) mice with impaired CD4 and CD8 T-cell responses had a markedly weakened control of infection, whereas SCID mice deficient in all T cells succumbed to such pulmonary mycobacterial infections.     

Osteoarticular Mycobacterium xenopi infection

BACKGROUND: Mycobacterium xenopi is a potential pathogen for man and can cause bone and joint infections, particularly spondylodiscitis. Most cases of infection occur in fragilized patients and are found more and more often in AIDS patients. CASE REPORT: A 41-year-old HIV+ woman developed cervical spondylodiscitis due to Mycobacterium xenopi infection. The strain was isolated from a discovertebral biopsy and was resistant to several antibiotics. Outcome was unfavorable. DISCUSSION: Most of the cases reported to date have involved spondylodiscitis of the thoracic or lumbar spine. To our knowledge, this is the first report of cervical spondylodiscitis dut to Mycobacterium xenopi in an HIV+ patient. Antibiotic combinations using fluoroquinolones and new macrolides are usually prescribed. Such protocols may provide cure of these opportunistic infections in immunodeficient patients.     

Pathogenesis of Babesia caballi infection in experimental horses

OBJECTIVE: The spread of nosocomial multiresistant microorganisms is affected by compliance with infection control measures and antibiotic use. We hypothesized that "colonization pressure" (ie, the proportion of other patients colonized) also is an important variable. We studied the effect of colonization pressure, compliance with infection control measures, antibiotic use, and other previously identified risk factors on acquisition of colonization with vancomycin-resistant enterococci (VRE). METHODS: Rectal colonization was studied daily for 19 weeks in 181 consecutive patients who were admitted to a single medical intensive care unit. A statistical model was created using a Cox proportional hazards regression model including length of stay in the medical intensive care unit until acquisition of VRE, colonization pressure, personnel compliance with infection control measures (hand washing and glove use), APACHE (Acute Physiology and Chronic Health Evaluation) 11 scores, and the proportion of days that a patient received vancomycin or third-generation cephalosporins, sucralfate, and enteral feeding. RESULTS: With survival until colonization with VRE as the end point, colonization pressure was the most important variable affecting acquisition of VRE (hazard ratio [HR], 1.032; 95% confidence interval [C1], 1.012-1.052; P=.002). In addition, enteral feeding was associated with acquisition of VRE (HR, 1.009; 95% CI, 1.000-1.017; P=.05), and there was a trend toward association of third-generation cephalosporin use with acquisition (HR, 1.007; 95% CI, 0.999-1.015; P=.11). The effects of enteral feeding and third-generation cephalosporin use were more important when colonization pressure was less than 50%. Once colonization pressure was 50% or higher, these other variables hardly affected acquisition of VRE. CONCLUSIONS: Acquisition of VRE was affected by colonization pressure, the use of antibiotics, and the use of enteral feeding. However, once colonization pressure was high, it became the major variable affecting acquisition of VRE.