Detecção molecular de Ehrlichia canis em cães e em órgãos de seus respectivos carrapatos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

JAVMA37-43 Anthrax in Swine

Anthrax is not commonly considered so important a menace to swine as it is to other forms of live stock, in which the disease is apt to declare itself with terrifying severity. In comparison with the marked mortality frequently observed in cattle, the tangible losses caused by anthrax in swine seem negligible. As recent as 25 years ago, there still were observers who sincerely doubted the occurrence of the disease in hogs and this opinion found some support in the many reported failures to induce the infection in that animal species by artificial methods.

Naturally Occurring Tuberculosis in White-Tailed Deer

Objective—To determine the distribution of lesions and extent of tissues infected with Mycobacterium bovis in a captive population of white-tailed deer. Design—Cross-sectional study. Animals—116 captive white-tailed deer. Procedure—Deer were euthanatized, and postmortem examinations were performed. Tissues with gross lesions suggestive of tuberculosis were collected for microscopic analysis and bacteriologic culture. Tissues from the head, thorax, and abdomen of deer with no gross lesions were pooled for bacteriologic culture. Tonsillar, nasal, oral, and rectal swab specimens, fecal samples, and samples of hay and pelleted feed, soil around feeding sites, and water from 2 natural ponds were collected for bacteriologic culture. Results—Mycobacterium bovis was isolated from 14 of 116 (12%) deer; however, only 9 of 14 had lesions consistent with tuberculosis. Most commonly affected tissues included the medial retropharyngeal lymph node and lung. Five of 14 tuberculous deer had no gross lesions; however,M bovis was isolated from pooled tissue specimens from the heads of each of these deer. Bacteriologic culture of tonsillar swab specimens from 2 of the infected deer yielded M bovis. Mean (± SEM) age of tuberculous deer was 2.5 ± 0.3 years (range, 0.5 to 6 years). Mycobacterium bovis was not isolated from feed, soil, water, or fecal samples. Conclusions and Clinical Relevance—Examination of hunter-killed white-tailed deer for tuberculosis commonly includes only the lymph nodes of the head. Results of such examinations may underestimate disease prevalence by as much as 57%. Such discrepancy should be considered when estimating disease prevalence.

Evaluation of Blood Assays for Detection of Mycobacterium bovis in White-Tailed Deer (Odocoileus virginianus) in Michigan

Surveillance and control activities related to bovine tuberculosis (TB) in free-ranging, Michigan white-tailed deer (Odocoileus virginianus) have been underway for over a decade, with significant progress. However, foci of higher TB prevalence on private lands and limited agency ability to eliminate them using broad control strategies have led to development and trial of new control strategies, such as live trapping, testing, and culling or release. Such strategies require a prompt, accurate live animal test, which has thus far been lacking. We report here the ability of seven candidate blood assays to determine the TB infection status of Michigan deer. Our aims were twofold: to characterize the accuracy of the tests using field-collected samples and to evaluate the feasibility of the tests for use in a test-and-cull strategy. Samples were collected from 760 deer obtained via five different surveys conducted between 2004 and 2007. Blood samples were subjected to one or more of the candidate blood assays and evaluated against the results of mycobacterial culture of the cranial lymph nodes. Sensitivities of the tests ranged from 46% to 68%, whereas specificities and negative predictive values were all .92%. Positive predictive values were highly variable. An exploratory analysis of associations among several host and sampling-related factors and the agreement between blood assay and culture results suggested these assays were minimally affected. This study demonstrated the capabilities and limitations of several available blood tests for Mycobacterium bovis on specimens obtained through a variety of field surveillance methods. Although these blood assays cannot replace mass culling, information on their performance may prove useful as wildlife disease managers develop innovative methods of detecting infected animals where mass culling is publicly unacceptable and cannot be used as a control strategy.

Shared Feed as a Means of Deer-to-Deer Transmission of Mycobacterium bovis

To determine the ability of experimentally inoculated white-tailed deer (Odocoileus virginianus) to transmit Mycobacterium bovis to naive deer through the sharing of feed, four deer were intratonsillarly inoculated with 4x105 colony-forming units of M. bovis. On a daily basis, feed not consumed by inoculated deer after approximately 8 hr was offered to four naıve deer maintained in a separate pen, where direct contact, aerosol transmission, or transmission through personnel were prevented. After 150 days, naıve deer were euthanized and examined. All naıve deer had lesions consistent with tuberculosis and M. bovis was isolated from various tissues. The most commonly affected tissues were lung, tracheobronchial lymph nodes, and mediastinal lymph nodes. This study demonstrates the potential for indirect transmission of M. bovis through the sharing of feed. Intentional or unintentional feeding of deer by wildlife or agricultural interests in regions where M. bovis infection is endemic should be avoided because both direct and indirect transmission through sharing of feed are enhanced.

Aerosol Exposure of White-Tailed Deer (Odocoileus virginianus) to Mycobacterium Bovis

Tuberculosis due to Mycobacterium bovis affects both captive and free-ranging Cervidae in the United States. Various animal models have been developed to study tuberculosis of both humans and animals. Generally, tuberculosis is transmitted by aerosol and oral routes. Models of aerosol exposure of large animals to M. bovis are uncommon. In order to develop a reliable method of aerosol exposure of white-tailed deer (Odocoileus virginianus) to M. bovis, 12 healthy white-tailed deer, aged 8–10 mo, were infected by aerosol exposure to 2x105 to 1x106 colony forming units (CFU) (high dose, n=4) of M. bovis or 6x102 to 1.6 x 103 CFU (low dose, n=8) of M. bovis. Tuberculous lesions were more widely disseminated in deer receiving the high dose, while lesions in deer receiving the low dose were more focused on the lungs and associated lymph nodes (tracheobronchial and mediastinal). Aerosol delivery of M. bovis to white-tailed deer results in a reliable manner of experimental infection that may be useful for studies of disease pathogenesis, immune response, mycobacterial shedding, and vaccine efficacy.

Milk Containing Mycobacterium bovis as a Source of Infection for White-Tailed Deer Fawns (Odocoileus virginianus)

Setting:White-tailed deer represent the first wildlife reservoir of Mycobacterium bovis in the United States. The behavior of does with nursing fawns provides several potential mechanisms for disease transmission. Little information exists concerning transmission between doe and fawn, specifically transmammary transmission. Objective: Determine if fawns can become infected by ingestion of milk replacer containing M. bovis, thus simulating transmission from doe to fawn through contaminated milk. Design: Seventeen, 21-day-old white-tailed deer fawns were inoculated orally with 2x108 CFU (high dose, n=5), 2.5 x 105 to 2.5 x 106 CFU (medium dose, n=5), and 1x104 CFU (low dose, n=5) of M. bovis in milk replacer. Dosages were divided equally and fed daily over a 5-day period. Positive control fawns (n=2) received 1x105 CFU of M. bovis instilled in the tonsillar crypts. Fawns were euthanized and examined 35-115 days after inoculation and various tissues collected for bacteriologic and microscopic analysis. Results: All fawns in the tonsillar, high oral and medium oral dose groups developed generalized tuberculosis involving numerous organs and tissues by 35-84 days after inoculation. Three of five fawns in the low-dose oral group had tuberculous lesions in the mandibular lymph node, and one of five had lesions in the medial retropharyngeal lymph node when examined 115 days after inoculation. Conclusion: White-tailed deer fawns can become infected through oral exposure to M. bovis. Therefore, the potential exists for fawns to acquire M. bovis while nursing tuberculous does.

Lesion Development in White-tailed Deer (Odocoileus virginianus) Experimentally Infected with Mycobacterium bovis

The recent discovery of tuberculosis in free-living white-tailed deer in northeastern Michigan underscores the need for increased understanding of the pathogenesis of tuberculosis in wildlife species. To investigate lesion development in white-tailed deer, 32 deer were experimentally infected by intratonsilar instillation of 300 colony-forming units of Mycobacterium bovis. Three deer each were euthanatized and examined at days 15, 28, 42, and 56 after inoculation, and five deer each were euthanatized and examined at days 89, 180, 262, and 328 after inoculation. Microscopic lesions first were seen in the medial retropharyngeal lymph node and lung 28 and 42 days after inoculation, respectively. Lung lesions were present in 12 (38%) of 32 deer, involving 23 lung lobes. Left caudal and right middle and caudal lobes were involved in 17 (74%) of the 23 affected lung lobes. Lesions in the medial retropharyngeal lymph node first appeared as granulomas composed of aggregates of macrophages and Langhans-type giant cells. Some early granulomas contained centrally located neutrophils. As granulomas developed, neutrophils were replaced with a central zone of caseous necrosis that first showed signs of mineralization 42 days after inoculation. Granulomas increased in size as the zone of caseous necrosis expanded. Peripheral fibrosis, first seen at 56 days after inoculation, progressed to only a thin fibrous capsule by 328 days after inoculation. By the termination of the study, the central necrotic core of the granuloma contained abundant liquefied necrotic material and grossly resembled an abscess. Although tuberculous lesions in white-tailed deer follow a developmental pattern similar to that in cattle, fibrosis is less pronounced and the advanced lesions may liquefy, a change seldom reported in cattle. An understanding of lesion development will aid in the identification of the spectrum of disease that may be seen in this important wildlife reservoir of tuberculosis.

Experimental Deer-To-Deer Transmission of Mycobacterium bovis

Objective—To determine whether Mycobacterium bovis can be transmitted from experimentally infected deer to uninfected in-contact deer. Animals—Twenty-three 6-month-old white-tailed deer. Procedure—On day 0, M bovis (2 X 108 colony-forming units) was administered by intratonsillar instillation to 8 deer; 3 control deer received saline (0.9% NaCl) solution. Eight in-contact deer were comingled with inoculated deer from day 21. On day 120, inoculated deer were euthanatized and necropsied. On day 180, 4 in-contact deer were euthanatized, and 4 new incontact deer were introduced. On day 360, all in-contact deer were euthanatized. Rectal, oral, and nasal swab specimens and samples of hay, pelleted feed, water, and feces were collected for bacteriologic culture. Tissue specimens were also collected at necropsy for bacteriologic culture and histologic analysis. Results—On day 90, inoculated and in-contact deer developed delayed-type hypersensitivity (DTH) reactions to purified protein derivative of M bovis. Similarly, new in-contact deer developed DTH reactions by 100 days of contact with original in-contact deer. Tuberculous lesions in in-contact deer were most commonly detected in lungs and tracheobronchial and medial retropharyngeal lymph nodes. Mycobacterium bovis was isolated from nasal secretions and saliva from inoculated and in-contact deer, urine and feces from in-contact deer, and hay and pelleted feed. Conclusions and Clinical Relevance—Mycobacterium bovis is efficiently transmitted from experimentally infected deer to uninfected in-contact deer through nasal secretions, saliva, or contaminated feed. Wildlife management practices that result in unnatural gatherings of deer may enhance both direct and indirect transmission of M bovis.

Monitoring of Transmission of Tuberculosis between Wild Boars and Cattle: Genotypical Analysis of Strains by Molecular Epidemiology Techniques

An epidemiological survey for the monitoring of bovine tuberculosis transmission was carried out in western Liguria, a region in northern Italy. Fifteen Mycobacterium bovis strains were isolated from 63 wild boar samples (62 from mandibular lymph nodes and 1 from a liver specimen). Sixteen mediastinal lymph nodes of 16 head of cattle were collected, and 15 Mycobacterium bovis strains were subsequently cultured. All M. bovisstrains isolated from cattle and wild boars were genotyped by spoligotyping and by restriction fragment length polymorphism (RFLP) analysis with the IS6110 and IS1081 probes. All M. bovis strains showed the typical spoligotype characterized by the absence of the 39 to 43 spacers in comparison with the number in M. tuberculosis. A total of nine different clusters were identified by spoligotyping. The largest cluster included 9 strains isolated from wild boars and 11 strains isolated from cattle, thus confirming the possibility of transmission between the two animal species. Fingerprinting by RFLP analysis with the IS6110 probe showed an identical single-band pattern for 29 of 30 strains analyzed, and only 1 strain presented a five-band pattern. The use of IS1081 as a second probe was useful for differentiation of M. bovis from M. bovis BCG but not for differentiation among M. bovis strains, which presented the same undifferentiated genomic profile. In relation to the epidemiological investigation, we hypothesized that the feeding in pastures contaminated by cattle discharges could represent the most probable route of transmission of M. bovis between the two animal species. In conclusion, our results confirmed the higher discriminatory power of spoligotyping in relation to that of RFLP analysis for the differentiation of M. bovis genomic profiles. Our data showed the presence of a common M. bovis genotype in both cattle and wild boars, confirming the possible interspecies transmission of M. bovis.

Bovine Tuberculosis in Free-Ranging White-Tailed Deer From Michigan

A 4.5 yr-old male white-tailed deer (Odocoileus virginianus) killed by a hunter during the 1994 firearm hunting season in northeastern Michigan (USA) had lesions suggestive of tuberculosis and was positive on culture for Mycobacterium bovis the causative agent for bovine tuberculosis. Subsequently, a survey of 354 hunter-harvested white-tailed deer for tuberculosis was conducted in this area from 15 November 1995 through 5 January 1996. Heads and/or lungs from deer were examined grossly and microscopically for lesions suggestive of bovine tuberculosis. Gross lesions suggestive of tuberculosis were seen in 15 deer. Tissues from 16 deer had acid-fast bacilli on histological examination and in 12 cases mycobacterial isolates from lymph nodes and/or lungs were identified as M. bovis. In addition, lymph nodes from 12 deer (11 females and 1 male) without gross or microscopic lesions were pooled into 1 sample from which M. bovis was cultured. Although more male (9) than female (3) deer had bovine tuberculosis infections, this difference was not statistically significant. Mycobacterium bovis culture positive deer ranged in age from 1.5 to 5.5 yr with a mean of 2.7 yr (median 2.5 yr) for males and 3.2 yr (median 3.5 yr) for females. This appears to be the first epidemic occurrence of M. bovis in free-ranging cervids in North America. A combination of environmental (high deer density and poor quality habit) and management-related factors (extensive supplemental feeding) may be responsible for this epizootic.

Bovine Tuberculosis in a Free-ranging Mule Deer (Odocoileus hemionus) from Montana

A survey of 41 mule deer (Odocolleus hemionus) and three white-tailed deer (O. virginianus) for bovine tuberculosis was conducted on a Montana (USA) cattle ranch from 2 November 1993 through January 1994. Gross and microscopic lesions typical of tuberculosis were present in tonsil and lymph nodes of the head, thorax, and abdomen of one adult female mule deer. Additionally, a single microgranuloma considered morphologically suggestive of tuberculosis was present in one lymph node of the head of a second mule deer. Mycobacterial isolates from lymph nodes of the head and thorax of the first deer were identified as Mycobacterium bovis.

Vaccination with Mycobacterium bovis BCG Strains Danish and Pasteur in White-tailed Deer (Odocoileus virginianus) Experimentally Challenged with Mycobacterium bovis

Wildlife reservoirs of Mycobacterium bovis represent serious obstacles to the eradication of tuberculosis in domestic livestock and the cause for many faltering bovine tuberculosis eradication programs. One approach in dealing with wildlife reservoirs of disease is to interrupt inter-species and intraspecies transmission through vaccination of deer or cattle. To evaluate the efficacy of BCG vaccination in white-tailed deer, 35 deer were assigned to one of three groups; one s.c. dose of 107 CFU of M. bovis BCG Pasteur (n = 12); 1 s.c. dose of 107 CFU of M. bovis BCG Danish (n = 11); or unvaccinated deer (n = 12). After vaccination, deer were inoculated intratonsilarly with virulent M. bovis. Lesion severity scores of the medial retropharyngeal lymph node, as well as all lymph nodes combined, were reduced in vaccinated deer compared to unvaccinated deer. BCG Danish vaccinated deer had no late stage granulomas characterized by coalescent caseonecrotic granulomas containing numerous acid-fast bacilli compared to BCG Pasteur vaccinated or unvaccinated deer where such lesions were present. Both BCG strains were isolated as late as 250 days after vaccination from deer that were vaccinated but not challenged. In white-tailed deer, BCG provides protection against challenge with virulent M. bovis. Issues related to vaccine persistence, safety and shedding remain to be further investigated.

Vaccination of White-Tailed Deer (Odocoileus virginianus) with Mycobacterium bovis Bacillus Calmette Guerin

Wildlife reservoirs of Mycobacterium bovis represent serious obstacles to the eradication of tuberculosis in domestic livestock. In Michigan, USA tuberculous white-tailed deer transmit M. bovis to cattle. One approach in dealing with this wildlife reservoir is to vaccinate deer in order to interrupt the cycle of deer to deer and deer to cattle transmission. Thirty-one white-tailed deer were assigned to one of three groups; 2 SC doses of 107 CFU of M. bovis BCG (n = 11); 1 SC dose of 107 CFU of M. bovis BCG (n = 10); or unvaccinated deer (n = 10). After vaccination, deer were inoculated intratonsilarly with 300 CFU of virulent M. bovis. Gross lesion severity scores of the medial retropharyngeal lymph node were significantly reduced in deer receiving 2 doses of BCG compared to unvaccinated deer. Vaccinated deer had fewer lymph node granulomas than unvaccinated deer, and most notably, fewer late stage granulomas characterized by coalescent caseonecrotic granulomas containing numerous acid-fast bacilli. BCG was isolated from 7/21 vaccinated deer as long as 249 days after vaccination. In one case BCG was transmitted from a vaccinated deer to an unvaccinated deer. In white-tailed deer BCG provides measurable protection against challenge with virulent M. bovis. However, persistence of vaccine within tissues as well as shedding of BCG from vaccinates remain areas for further investigation.

Detecção molecular de Ehrlichia canis em cães e em órgãos de seus respectivos carrapatos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)