Overt Mycobacterium avium subsp. paratuberculosis Infection: An Infrequent Occurrence in Archived Tissue from False TB Reactor Cattle in Michigan, USA

The objective of this study was to retrospectively determine whether or not cattle from the state of Michigan which were classified as bovine tuberculosis reactors, based on currently approved field and laboratory testing methods, were overtly infected with Mycobacterium avium subsp. paratuberculosis (MAP). Included in this study were 384 adult cattle submitted to the Diagnostic Center for Population and Animal Health over a seven-year period. Cattle were tested utilizing standard methods to confirm that all cattle were lesion and culture negative for infection with Mycobacterium bovis at postmortem examination. Retrospective analysis of formalin-fixed, paraffin-embedded sections of ileum and ileocecal lymph node were evaluated by histopathology, acid-fast staining, and PCR assays to detect MAP. Overall, only 1.04 percent of cattle showed overt infection with MAP on visual examination of sections of ileum and/or ileo-cecal lymph node. This increased slightly to 2.1 percent of cattle likely infected with MAP after additional testing using a PCR assay. Based on these results, we found no evidence that overt infection with MAP plays a major role in the false tuberculosis reactor test results for cattle examined in this study

Differential Gene Expression Segregates Cattle Confirmed Positive for Bovine Tuberculosis from Antemortem Tuberculosis Test-False Positive Cattle Originating from Herds Free of Bovine Tuberculosis

Antemortem tests for bovine tuberculosis (bTB) currently used in the US measure cell-mediated immune responses against Mycobacterium bovis. Postmortem tests for bTB rely on observation of gross and histologic lesions of bTB, followed by bacterial isolation or molecular diagnostics. Cumulative data from the state of Michigan indicates that 98 to 99% of cattle that react positively in antemortem tests are not confirmed positive for bTB at postmortem examination. Understanding the fundamental differences in gene regulation between antemortem test-false positive cattle and cattle that have bTB may allow identification of molecular markers that can be exploited to better separate infected from noninfected cattle. An immunospecific cDNA microarray was used to identify altered gene expression (P ≤ 0.01) of 122 gene features between antemortem test-false positive cattle and bTB-infected cattle following a 4-hour stimulation of whole blood with tuberculin. Further analysis using quantitative real-time PCR assays validated altered expression of 8 genes that had differential power (adj  P ≤ 0.05) to segregate cattle confirmed positive for bovine tuberculosis from antemortem tuberculosis test-false positive cattle originating from herds free of bovine tuberculosis

Prolonged persistence of canine distemper virus RNA, and virus isolation in naturally infected shelter dogs.

Canine distemper virus remains an important source of morbidity and mortality in animal shelters. RT-PCR is commonly used to aid diagnosis and has been used to monitor dogs testing positive over time to gauge the end of infectious potential. Many dogs excrete viral RNA for prolonged periods which has complicated disease management. The goal of this retrospective study was to describe the duration and characteristics of viral RNA excretion in shelter dogs with naturally occurring CDV and investigate the relationship between that viral RNA excretion and infectious potential using virus isolation data. Records from 98 different humane organizations with suspect CDV were reviewed. A total of 5,920 dogs were tested with 1,393; 4,452; and 75 found to be positive, negative, or suspect on RT-PCR respectively. The median duration of a positive test was 34 days (n = 325), and 25% (82/325) of the dogs still excreting viral RNA after 62 days of monitoring. Virus isolation was performed in six dogs who were RT-PCR positive for > 60 days. Infectious virus was isolated only within the first two weeks of monitoring at or around the peak viral RNA excretion (as detected by the lowest cycle threshold) reported for each dog. Our findings suggest that peak viral RNA excretion and the days surrounding it might be used as a functional marker to gauge the end of infectious risk. Clarifying the earliest point in time when dogs testing positive for canine distemper by RT-PCR can be considered non-contagious will improve welfare and lifesaving potential of shelters by enabling recovered dogs to be cleared more quickly for live release outcomes

Mycoplasmosis in Ferrets

We report an outbreak of severe respiratory disease associated with a novel Mycoplasma species in ferrets. During 2009–2012, a respiratory disease characterized by nonproductive coughing affected ≈8,000 ferrets, 6–8 weeks of age, which had been imported from a breeding facility in Canada. Almost 95% became ill, but almost none died. Treatments temporarily decreased all clinical signs except cough. Postmortem examinations of euthanized ferrets revealed bronchointerstitial pneumonia with prominent hyperplasia of bronchiole-associated lymphoid tissue. Immunohistochemical analysis with polyclonal antibody against Mycoplasma bovis demonstrated intense staining along the bronchiolar brush border. Bronchoalveolar lavage samples from 12 affected ferrets yielded fast-growing, glucose-fermenting mycoplasmas. Nucleic acid sequence analysis of PCR-derived amplicons from portions of the 16S rDNA and RNA polymerase B genes failed to identify the mycoplasmas but showed that they were most similar to M. molare and M. lagogenitalium. These findings indicate a causal association between the novel Mycoplasma species and the newly recognized pulmonary disease

Experimental Induction of Pulmonary Fibrosis in Horses with the Gammaherpesvirus Equine Herpesvirus 5

<div><p>Gammaherpesviruses (γHV) are implicated in the pathogenesis of pulmonary fibrosis in humans and murine models of lung fibrosis, however there is little direct experimental evidence that such viruses induce lung fibrosis in the natural host. The equine γHV EHV 5 is associated with equine multinodular pulmonary fibrosis (EMPF), a progressive fibrosing lung disease in its natural host, the horse. Experimental reproduction of EMPF has not been attempted to date. We hypothesized that inoculation of EHV 5 isolated from cases of EMPF into the lungs of clinically normal horses would induce lung fibrosis similar to EMPF. Neutralizing antibody titers were measured in the horses before and after inoculation with EHV 5. PCR and virus isolation was used to detect EHV 5 in antemortem blood and BAL samples, and in tissues collected postmortem. Nodular pulmonary fibrosis and induction of myofibroblasts occurred in EHV 5 inoculated horses. Mean lung collagen in EHV 5 inoculated horses (80 µg/mg) was significantly increased compared to control horses (26 µg/mg) (<i>p</i> < 0.5), as was interstitial collagen (32.6% ± 1.2% vs 23% ± 1.4%) (mean ± SEM; p < 0.001). Virus was difficult to detect in infected horses throughout the experiment, although EHV 5 antigen was detected in the lung by immunohistochemistry. We conclude that the γHV EHV 5 can induce lung fibrosis in the horse, and hypothesize that induction of fibrosis occurs while the virus is latent within the lung. This is the first example of a γHV inducing lung fibrosis in the natural host.</p> </div

Experimental EHV 5 infection; gross pathology (horse E3, E4).

<p>Nodules of fibrosis (A, between arrows, and at asterisk; horse E4) were evident beneath the pleura (cut section, B; horse E4). The nodules of fibrosis extended into the underlying alveolar parenchyma (B,C; horse E3,E4 respectively).</p

Immunohistochemistry for α-smooth muscle actin for myofibroblasts (MFB) (horses C1, E4).

<p>In control horse lung smooth muscle actin is present around blood vessels and within occasional alveolar interstitial MFB (A). Numerous MFB are present within the nodules of alveolar fibrosis in the lungs of EHV 5 infected horses. Similar distribution of MFB is present within the foci of alveolar fibrosis in horses with naturally-acquired EMPF (C). Inset (A) negative control, mouse serum IgG substitution for primary antibody. Magnification: A-C - 20x.</p