Purification of the receptor for the N-acetyl-D-glucosamine specific adhesin of Mannheimia haemolytica from bovine neutrophils.

The GlcNAc-specific adhesin from Mannheimia haemolytica (MhA) has been shown to participate in pathogenicity of mannheimiosis due to its capacity to adhere to tracheal epithelial cells and activate the oxidative burst of bovine neutrophils. In this work, we purified the MhA receptor from bovine neutrophils (MhAr) by affinity chromatography on MhA-Sepharose. The MhAr, which corresponded to approximately 2% of the protein from cell lysate, is a glycoprotein mainly composed of Glu, Ala, Ser, Gly, and Asp, without cysteine. The glycan portion, which corresponds to 20% by weight, is composed of GalNAc, GlcNAc, Man, Gal, and NeuAc. The receptor is a 165-kDa glycoprotein, as determined by molecular sieve chromatography under native conditions; SDS-PAGE analysis shows a heterodimer of 83 and 80 kDa subunits. This work suggests that the GlcNAc-containing receptor plays a relevant role by activating bovine neutrophils through non-opsonic mechanisms.

Fluorescence polarization assay for the detection of antibodies to Mycobacterium bovis in bovine sera.

The performance of a fluorescence polarization assay (FPA) that detects antibodies to Mycobacterium bovis in bovine sera is described. The FPA reported here is a direct binding primary screening assay using a small polypeptide derived from the M. bovis MPB70 protein. A secondary inhibition assay confirms suspect or presumed positive samples. Specificity studies involved five different veterinary laboratories testing 4461 presumed negative bovine samples. FPA specificity was 99.9%. The FPA was used to identify herd status as either M. bovis infected or non-infected. Herd surveillance studies (nine herds) were performed in Mexico and South Africa. The FPA had a specificity of 100% (two negative herds), and correctly identified six of seven infected herds. Finally, sera from 105 slaughter animals that had gross lesions in lymph nodes similar to those seen with bovine tuberculosis were tested by the FPA. Thin sections from the associated formalin-fixed paraffin-embedded samples of lymph nodes were stained using hematoxylin and eosin (H&E) for morphologic examination and using the Ziehl-Neelsen (ZN) method for detection of acid-fast bacilli. Of the 105 animals, 78 were classified as TB suspect based on lesion morphology, 21 were positive by ZN, 9 were positive by FPA and 13 were positive by PCR for the tuberculosis group of Mycobacterium. Among the 21 ZN positives, 11 (52.4%) were PCR positive. Among the 9 FPA positives, 8 (88.9%) were PCR positive. For the 13 PCR positives, 8 (61.5%) were FPA positive and 11 (84.6%) were ZN positives. These results show that use of the FPA for detection of M. bovis infection of cattle has value for bovine disease surveillance programs.

The N-acetyl-D-glucosamine specific adhesin from Mannheimia haemolytica activates bovine neutrophils oxidative burst.

In this work we identified specific bovine leukocytes that were bound by the Mannheimia haemolytica adhesin molecule (MhA) and the biological effect on the leukocytes. Histochemical staining and flow cytometry showed that MhA bind neutrophils (90%) and monocytes (5%). MhA induced an oxidative response in purified neutrophils; this effect was 1.5-fold higher than the effect observed with control cells activated with Zymosan. Cellular binding by MhA was inhibited with GlcNAc and its oligomers, as well as by glycoproteins containing tri- and tetra-antennary N-glycosydically linked glycans. MhA-induced oxidative burst was significantly inhibited by GlcNAc, iodoacetamide, superoxide dismutase, and piroxicam (p<0.05). Our findings suggest that among bovine leukocytes, neutrophils are the main target for MhA, inducing production of oxidative radicals by non-opsonic mechanism that seem to play an important role in tissue damage during mannheimiosis.

Pathologic findings in Western gray squirrels (Sciurus griseus) from a notoedric mange epidemic in the San Bernardino Mountains, California.

Notoedric mange, caused by the contagious, burrowing mite Notoedres centrifera, has been associated with several large-scale population declines of western gray squirrels (Sciurus griseus) and has been a significant obstacle to population recovery in Washington State where the species is listed as threatened. In 2009, residents and wildlife rehabilitators in the isolated San Bernardino Mountains of southern California reported a dramatic die-off of western gray squirrels, in what had been a previously dense and robust population. Individuals were observed suffering from abnormal neurologic behaviors (ataxia and obtundation) and severe skin disease. Full necropsy of five squirrels from the epidemic showed that all had moderate to severe infestation with mange mites and severe dermatitis characterized by hyperkeratosis, acanthosis, intralesional mites, intracorneal pustules and superficial bacteria. Mites from affected squirrels were evaluated by light and electron microscopy and identified as N. centrifera based on morphologic criteria. Additionally, the internal transcribed spacer-2 region of the mite was cloned, sequenced and accessioned in GenBank. The cause for the abnormal neurologic behavior was not confirmed on post-mortem examination. However, we hypothesize that mange can cause incoordination and obtundation as a result of malnutrition and dehydration, and intense pruritis may induce abnormal or erratic behavior that could be mistaken for neurologic signs. While we have characterized the severe impact this disease can have on individual animals, more work is needed to understand the impact on squirrel populations, particularly in view of the anecdotal reports of dramatic population declines that may take decades to recover.