Milk Containing <i>Mycobacterium bovisi> as a Source of Infection for White-Tailed Deer Fawns (<i>Odocoileus virginianusi>)

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.

Susceptibility of Raccoons (<i>Procyon lotori>) to Infection with <i>Mycobacterium bovisi>

Tuberculosis due to Mycobacterium bovis infection is endemic in white-tailed deer (Odocoileus virginianus) in the northeastern portion of the lower Michigan peninsula (USA). Various wild carnivores and omnivores, including raccoons (Procyon lotor), are infected with M. bovis within the endemic area. To investigate the pathogenesis of tuberculosis in raccoons and the likelihood of M. bovis transmission from infected raccoons to other susceptible hosts, we experimentally inoculated raccoons with single oral doses of M. bovis (ranging from 30 to 1.7 x 105 colony forming units [CFU]), five daily oral doses of M. bovis (ranging from 10 to 1 x 105 CFU), or a single intravenous (IV) dose of 1 x 105 CFU of M. bovis, from November 1998 through December 2000. Granulomatous lesions consistent with tuberculosis, or tissue colonization with M. bovis, were seen in one of five raccoons in the single low oral dose group, one of five raccoons in the multiple low oral dose group, two of five raccoons in the multiple medium oral dose group, five of five raccoons in the multiple high oral dose group, and five of five raccoons in the IV inoculated group. In orally inoculated raccoons, lesions were most common in the tracheobronchial and mesenteric lymph nodes and lung. Excretion of M. bovis in saliva or nasal secretions was noted in all IV inoculated raccoons and two of five multiple low oral dose raccoons. Mycobacterium bovis was not isolated from urine or feces from any experimentally inoculated raccoons. The need for multiple large oral doses to establish infection, and the low number of orally inoculated raccoons that excreted M. bovis in nasal secretions or saliva, suggest that widespread tuberculosis among raccoons is unlikely.

Lesion Development in White-tailed Deer (<i>Odocoileus virginianusi>) Experimentally Infected with <i>Mycobacterium bovisi>

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.

Tuberculin Skin Testing in White-Tailed Deer (<i>Odocoileus virginianusi>)

The comparative cervical skin test for antemortem diagnosis of tuberculosis was done 169 times on 116 different white-tailed deer of known Mycobacterium bovis infection status. The sensitivity and specificity were 97 and 81%, respectively. The magnitude of change in skin thickness at test sites was not significantly influenced by dosage of inoculum, dissemination of the disease process, or repeated skin testing. However, the magnitude of change in skin thickness was significantly greater in deer infected for less than 109 days than in deer infected for more than 109 days. As used in the present study, the comparative cervical skin test is a sensitive method of antemortem diagnosis of M. bovis infection in white-tailed deer.

Experimental Deer-To-Deer Transmission of <i>Mycobacterium bovisi>

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.

Host Specificity of <i>Calyptospora fundulii> (Apicomplexa: Calyptosporidae) in Atheriniform Fishes

Calyptospora funduli has a broad host specificity, infecting at least 7 natural and 10 additional experimental definitive hosts, all atheriniform fishes within 5 families, but most in the genus Fundulus. Barriers, apparently innate ones, prevent any development of C. funduli in perciform fishes but allow incomplete or abnormal development of the parasite in a few unnatural atheriniform hosts. In the freshwater species Fundulus olivaceus and Fundulus notti, these abnormalities consisted of asynchronous development, degeneration of the parasite in early stages of development, and the formation of numerous macrophage aggregates. Rivulus marmoratus has the ability to eliminate infections with a granulomatous inflammatory response. Additional barriers that limit natural infections of C. funduli in other hosts include feeding behavior, environmental conditions, and geographic isolation.

<i>Margolisianum bulbosumi> n. gen., n. sp. (Nematoda: Philometridae) from the Southern Flounder, <i>Paralichthys lethostigmai> (Pisces: Bothidae), in Mississippi Sound, USA

A new species of a philometrid nematode, Margolisianum bulbosum, is described from the subcutaneous tissue in the mouth (larvigerous females), head (males, ovigerous, and larvigerous females), and eye (preovigerous females) of the southern flounder, Paralichthys lethostigma, from Mississippi Sound. It is placed in a new genus diagnosed by the combination of 8 large, paired but separate cephalic papillae; no inner cephalic papillae; an esophagus with a separate, muscular anterior bulb; a prominent mononuclear esophageal gland; and variable, irregularly distributed cuticular bosses in the females, as well as a vestigial rectum, particularly in larvigerous females. Some female specimens exhibit rows of lateral grooves and longitudinal ridges near the posterior end. Males have two small slightly subequal spicules, a barbed gubernaculum, 4 pairs of small cephalic papillae, and a bipartite hypodermal extension within a membranous cuticle on the posterior end. Males, ovigerous females, and larvigerous females appear to be present year round in this sporadic infection in Mississippi.

<i>Myxobolus mississippiensisi> n. sp. (Myxosporea) from Gills of <i>Lepomis macrochirusi> in Mississippi

Myxobolus mississippiensis n. sp. is described from gill lamellae of the bluegill (Lepomis macrochirus) inhabiting the Pascagoula River System, Mississippi. Fresh spores measure 16.4-18.7 μm long, 3.9-6.2 μm wide, and 4.7-6.2 μm thick. Spore width to length ratio is 1:3.2. Polar capsules are 5.5-7.8 μm long and 1.5-2.3 μm wide, with 9-10 filament coils that when extruded measure 42.1 ± 4.2 μm. This parasite is unique among known species of Myxobolus,/i> in having spores that are lenticular in frontal view.

<i>Cucullanus palmerii> n. sp. (Nematoda: Cucullanidae) from the Batfish <i>Ogcocephalus nasutusi> in the Gulf of Mexico

Cucullanus palmeri n. sp. is described from the shortnose batfish, Ogcocephalus nasutus, in the Gulf of Mexico. This species is anatomically most similar to Cucullanus gendrei but differs from it in having a shorter esophagus and longer tail. Males differ in having a smaller gubernaculum and a different arrangement of a lateral caudal papilla. Cucullanus palmeri is readily distinguished from Cucullanus lophii, also from a lophiform fish host, in that the deirids and excretory pore are not posterior to the posterior end of the esophagus like in C. lophii.

Gonadal Maturation in the Cobia <i>Rachycentron canadumi> from the Northcentral Gulf of Mexico

Gonadal maturation of cobia, Rachycentron canadum, was evaluated by examining 508 specimens from its recreational fishery. Specimens were collected off southeast Louisiana to northwest Florida by hook-and-line during February through October 1987-1991. Fork lengths (FL) of these fish ranged from 580-1,530 mm, with corresponding weights of 2.0-43.5 kg. The female:male ratio was 1:0.37. Using a combination of oocyte size frequency and histological assessment of many of the fish, we determined that females were ripe from May through September, with atretic oocytes occurring in some fish from July through October. Degenerating hydrated oocytes in July and October and the presence of resting ovaries in July suggest two major spawning periods; however, monthly gonosomatic indices peaking in May, followed by a steady decline, do not support that finding. Ovaries were placed into undeveloped, early developing, mid-developing, or late developing categories based upon oocyte size-frequency distributions. Developing ovaries had two or three modes of oocytes larger than 30 μm. Batch fecundity was estimated to be 2.6×106 - 1.91×108 oocytes, depending on the size of fish/ovaries. The smallest female with oocytes exhibiting vitellogenesis was 834 mm FL. This fish was 2 years old based its otolith evaluation. The smallest male with an abundance of spermatozoa in its testes was 640 mm FL and 1 year old based on otolith evaluation; smaller males were not examined. Females larger than 840 mm FL had vitellogenic oocytes in March and April. A few fish still had vitellogenic oocytes in early October, but none did by late October. When Gilson’s fluid was used to assess ovarian tissue, the fresh weight of the tissue was reduced by 20% after being stored for 3 months. The diameter of oocytes shrunk about 25% in Gilson’s fluid which was 11% less than those fixed in formalin, embedded in paraffin, and sectioned. Tissue sections from specific individuals, each demonstrating a variety of different developmental stages, were similar regardless of whether they were obtained from the anterior, middle, or posterior portion of either ovary.

<i>Mycobacterium bovisi>–Infected White-Tailed Deer (<i>Odocoileus Virginianusi>): 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., ~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 Gross Pathology, Histopathology, and Mycobacterial Culture for the Diagnosis of Tuberculosis in Elk (<i>Cervus elaphusi>)

Using the isolation of Mycobacterium bovis as the reference standard, this study evaluated the sensitivity, specificity and kappa statistic of gross pathology (abattoir postmortem inspection), histopathology, and parallel or series combinations of the two for the diagnosis of tuberculosis in 430 elk and red deer. Two histopathology interpretations were evaluated: histopathology I, where the presence of lesions compatible with tuberculosis was considered positive, and histopathology II, where lesions compatible with tuberculosis or a select group of additional possible diagnoses were considered positive. In the 73 animals from which M. bovis was isolated, gross lesions of tuberculosis were most often in the lung (48), the retropharyngeal lymph nodes (36), the mesenteric lymph nodes (35), and the mediastinal lymph nodes (16). Other mycobacterial isolates included: 11 M. paratuberculosis, 11 M. avium, and 28 rapidly growing species or M. terrae complex. The sensitivity estimates of gross pathology and histopathology I were 93% (95% confidence limits [CL] 84,97%) and 88% [CL 77,94%], respectively, and the specificity of both was 89% [CL 85,92%]). The sensitivity and specificity of histopathology II were 89% (CL 79,95%) and 77% (CL 72,81%), respectively. The highest sensitivity estimates (93- 95% [CL 84,98%]) were obtained by interpreting gross pathology and histopathology in parallel (where an animal had to be positive on at least one of the two, to be classified as combination positive). The highest specificity estimates (94-95% [CL 91-97%]) were generated when the two tests were interpreted in series (an animal had to be positive on both tests to be classified as combination positive). The presence of gross or microscopic lesions showed moderate to good agreement with the isolation of M. bovis (Kappa = 65-69%). The results show that post-mortem inspection, histopathology and culture do not necessarily recognize the same infected animals and that the spectra of animals identified by the tests overlaps.

Vaccination with <i>Mycobacterium bovisi> BCG Strains Danish and Pasteur in White-tailed Deer (<i>Odocoileus virginianusi>) Experimentally Challenged with <i>Mycobacterium bovisi>

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 (<i>Odocoileus virginianusi>) with <i>Mycobacterium bovis i>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.

Evaluation of an In Vitro Blood-Based Assay to Detect Production of Interferon-γ By <i>Mycobacterium Bovisi>–Infected White-Tailed Deer (<i>Odocoileus Virginianusi>)

Tuberculosis due to Mycobacterium bovis in captive Cervidae was identified as an important disease in the United States in 1990 and prompted the addition of captive Cervidae to the USDA Uniform Methods and Rules for eradication of bovine tuberculosis. As well, M. bovis infection was identified in free-ranging white-tailed deer in northeast Michigan in 1995. Tuberculosis in both captive and free-ranging Cervidae represents a serious challenge to the eradication of M. bovis infection from the United States. Currently, the only approved antemortem tests for tuberculosis in Cervidae are the intradermal tuberculin skin test and the blood tuberculosis test (BTB). At present, the BTB is not available in North America. Tuberculin skin testing of Cervidae is time-consuming and involves repeated animal handling and risk of injury to animals and humans. This study evaluated the potential of a new blood-based assay for tuberculosis in Cervidae that would decrease animal handling, stress, and losses due to injury. In addition, a blood-based assay could provide a more rapid diagnosis. Twenty 6–9-month-old white-tailed deer, male and female, were experimentally inoculated by instillation of 300 colony-forming units of M. bovis in the tonsillar crypts. Seven, age-matched uninfected deer served as controls. Blood was collected on days 90, 126, 158, 180, 210, 238, 263, and 307 after inoculation and was analyzed for the production of interferon-γ (IFN-γ) in response to incubation with M. bovis purified protein derivative (PPDb), M. avium PPDa, pokeweed mitogen (PWM), or media alone. Production of IFN-g in response to PPDb was significantly greater (P < 0.05) at all time points in samples from M. bovis–infected deer as compared with uninfected control deer, whereas IFN-γ production to PWM did not differ significantly between infected and control deer. Measurement of IFN-γ production to PPDb may serve as a useful assay for the antemortem diagnosis of tuberculosis in Cervidae.