Comparative Histopathology of Bovine Acute Interstitial Pneumonia and Bovine Respiratory Syncytial Virus-Associated Interstitial Pneumonia.

Acute interstitial pneumonia (AIP) is a significant disease of cattle and many aetiologies have been implicated on the basis of the characteristic pathological lesions. Bovine respiratory syncytial virus (BRSV) is one of the key aetiological factors in bovine respiratory disease complex and several studies have suggested, controversially, that BRSV may be an underlying cause of bovine AIP. BRSV infection is known to cause several distinctive histopathological changes, including epithelial syncytia formation and intracytoplasmic viral inclusions. However, distinguishing bovine AIP from BRSV-related pneumonia by clinical presentation, gross pathology or histopathology can sometimes be challenging. In order to identify the potential distinguishing features, we compared the histopathological findings of AIP that were, and were not, associated with BRSV infection in naturally occurring cases. We found that multinucleated giant cells were more frequently identified in cattle with AIP while bronchiolitis was more common in BRSV-infected cattle. However, this was not considered a sole indicator of either disease group. Statistically, we identified that a combination of several histopathological features, including alveolar septal necrosis, presence of multinucleated giant cells and bronchiolitis, can serve as an excellent indicator for distinguishing between idiopathic AIP and BRSV-related pneumonia, with a strong statistical significance (P = 0.0004). Based on the results of this retrospective study, we present a histopathological scoring system for predicting BRSV-associated AIP.

Sequential exposure to bovine viral diarrhea virus and bovine coronavirus results in increased respiratory disease lesions: clinical, immunologic, pathologic, and immunohistochemical findings.

Bovine coronaviruses (BoCVs) have been found in respiratory tissues in cattle and frequently associated with bovine respiratory disease (BRD); however, pathogenesis studies in calves are limited. To characterize the pathogenesis and pathogenicity of BoCV isolates, we used 5 different BoCV strains to inoculate colostrum-deprived calves, ~ 2-5 wk of age. Later, to determine if dual viral infection would potentiate pathogenicity of BoCV, calves were inoculated with BoCV alone, bovine viral diarrhea virus (BVDV) alone, or a series of dual-infection (BVDV-BoCV) schemes. A negative control group was included in all studies. Clinical signs and body temperature were monitored during the study and samples collected for lymphocyte counts, virus isolation, and serology. During autopsy, gross lesions were recorded and fixed tissues collected for histopathology and immunohistochemistry; fresh tissues were collected for virus isolation. Results suggest increased pathogenicity for isolate BoCV OK 1776. Increased body temperature was found in all virus-inoculated groups. Lung lesions were present in calves in all dual-infection groups; however, lesions were most pronounced in calves inoculated with BVDV followed by BoCV inoculation 6 d later. Lung lesions were consistent with mild-to-moderate interstitial pneumonia, and immunohistochemistry confirmed the presence of BoCV antigen. Our studies demonstrated that BVDV-BoCV dual infection may play an important role in BRD pathogenesis, and timing between infections seems critical to the severity of lesions.

Austwickiosis in Captive African Spurred Tortoises (Geochelone sulcata) Co-infected with Cryptosporidium ducismarci.

Dermatophilosis has been described in many animals since it was first reported in 1915. In 2010, the genus and species associated with chelonid dermatophilosis was reclassified as Austwickia chelonae. Here we discuss a series of four submissions consisting of eight juvenile African spurred tortoises (Geochelone sulcata) from a large breeding facility. Submissions began in December 2016 and continued to June 2017 and then again in November 2018. Clinical signs were originally noted in December of 2015 and consisted of facial swelling and mandibular necrosis that led to complete disarticulation of the mandibles from the skull. Affected animals were emaciated with minimal gastrointestinal contents and several had soft faeces. The facial and head lesions were caused by A. chelonae. Additionally, co-infection with Cryptosporidium ducismarci was discovered within the small intestine. This report gives information on the pathology and pathogenicity of A. chelonae and characterizes the first reported cases of cryptosporidiosis in African spurred tortoises.

Mannheimia haemolytica IgA-specific proteases.

Mannheimia haemolytica colonizes the nasopharynx of cattle and can cause severe fibrinous pleuropneumonia. IgA proteases are metalloendopeptidases released by bacteria that cleave IgA, enhancing colonization of mucosa. The objectives of these studies were to characterize M. haemolytica IgA1 and IgA2 proteases in vitro and in silico, to clone and sequence the genes for these proteases, and to demonstrate immunogenicity of components of the entire IgA protease molecule. Both IgA protease genes were cloned, expressed, and sequenced. Sequences were compared to other published sequences. Components were used to immunize mice to determine immunogenicity. Sera from healthy cattle and cattle that recovered from respiratory disease were examined for antibodies to IgA proteases. In order to assay the cleavage of bovine IgA with IgA1 protease, M. haemolytica culture supernatant was incubated with bovine IgA. Culture supernatant cleaved purified bovine IgA in the presence of ZnCl2. Both IgA proteases contain three domains, 1) IgA peptidase, 2) PL1_Passenger_AT and 3) autotransporter. IgA1 and IgA2 peptidases have molecular weights of 96.5 and 87 kDa, respectively. Convalescent bovine sera with naturally high anti-M. haemolytica antibody titers had high antibodies against all IgA1 & IgA2 protease components. Mouse immunizations indicated high antibodies to the IgA peptidases and autotransporters but not to PL1_Passenger_AT. These data indicate that M. haemolytica produces two IgA proteases that are immunogenic, can cleave bovine IgA, and are produced in vivo, as evidenced by antibodies in convalescent bovine sera. Further studies could focus on IgA protease importance in pathogenesis and immunity.

Genome Sequence of a Bovine Isolate of Pasteurella multocida Strain 232.

We present here the genome sequence of Pasteurella multocida 232, a bacterium that is associated with pneumonia in humans as well as in many animal species. The genome of Pasteurella multocida 232 has an N 50 value of 187.32 kb and a total size of 2.34 Mb.

Mannheimia haemolytica in bovine respiratory disease: immunogens, potential immunogens, and vaccines.

Mannheimia haemolytica is the major cause of severe pneumonia in bovine respiratory disease (BRD). Early M. haemolytica bacterins were either ineffective or even enhanced disease in vaccinated cattle, which led to studies of the bacterium's virulence factors and potential immunogens to determine ways to improve vaccines. Studies have focused on the capsule, lipopolysaccharide, various adhesins, extracellular enzymes, outer membrane proteins, and leukotoxin (LKT) resulting in a strong database for understanding immune responses to the bacterium and production of more efficacious vaccines. The importance of immunity to LKT and to surface antigens in stimulating immunity led to studies of individual native or recombinant antigens, bacterial extracts, live-attenuated or mutant organisms, culture supernatants, combined bacterin-toxoids, outer membrane vesicles, and bacterial ghosts. Efficacy of several of these potential vaccines can be shown following experimental M. haemolytica challenge; however, efficacy in field trials is harder to determine due to the complexity of factors and etiologic agents involved in naturally occurring BRD. Studies of potential vaccines have led current commercial vaccines, which are composed primarily of culture supernatant, bacterin-toxoid, or live mutant bacteria. Several of those can be augmented experimentally by addition of recombinant LKT or outer membrane proteins.

Canine amoebic meningoencephalitis due to Balamuthia mandrillaris.

A 1-year-old Siberian Husky dog with acute-onset of seizures, recumbency, paddling, and muscular fasciculations was autopsied. A locally extensive hemorrhagic and malacic focus was noted in the right cerebral frontal cortex, and severe necrotizing and hemorrhagic, neutrophilic meningoencephalitis was diagnosed microscopically. Amoebic trophozoites and cysts were identified within the affected cerebral parenchyma and confirmed by indirect immunofluorescence assay and real-time PCR as Balamuthia mandrillaris. B. mandrillaris is found in soil and water and the infection has been reported in both immunocompromised and immunocompetent humans and rarely in the dog.

Bovine viral diarrhea virus 1b fetal infection with extensive hemorrhage.

Bovine viral diarrhea virus (BVDV) 1b was isolated from tissues of a term bovine fetus with petechial hemorrhages noted throughout the body and placenta at autopsy. Fresh lung, kidney, thymus, and liver tissues were examined by direct fluorescent antibody testing and were positive for BVDV antigen and negative for bovine herpesvirus 1 antigen. An organ pool of fresh tissues was positive for noncytopathic (NCP) BVDV-1 by virus isolation. BVDV-1b was identified by sequencing of the 5'-UTR region of the genome. Fixed brain, placenta, thymus, lymph node, lung, kidney, skeletal muscle, liver, and bone marrow were positive for BVDV antigen by immunohistochemistry. Although BVDV hemorrhage and/or thrombocytopenia has been associated historically with NCP strains of BVDV-2, this case adds to more recent reports of BVDV-1 infections and hemorrhage in cattle. This BVDV-1b isolate should be investigated for its potential to cause hemorrhage in postnatal cattle.

Proteomic and bioinformatic analyses of putative Mannheimia haemolytica secretome by liquid chromatography and tandem mass spectrometry.

Mannheimia haemolytica is a major bacterial contributor to bovine respiratory disease complex that costs the livestock industry a billion dollars a year in USA. Commercial vaccines are only partially efficacious under field conditions. Earlier studies found that outer membrane protein preparations and culture supernatants can induce immune responses that enhance resistance to challenge by M. haemolytica strains. The objective of this study was to characterize secretome of two M. haemolytica stains grown under two different media. Bacteria-free concentrated supernatants from M. haemolytica culture was subjected to LC-MS/MS. The secretome of M. haemolytica from both strains yielded 923 proteins. Using bioinformatic tools, 283 were identified as secreted proteins. Further breakdown of 283 proteins showed that 114 (40.2%), 184 (65.0%), 138(48.7%), 151 (53.3%) and 172 (60.7%) were characterized as secreted proteins by SignalP 4.1, SecretomeP 2.0, LipoP, Phobius, and PRED-TAT, respectively. A total of 95 (33.56%) proteins were characterized as being secreted via non-classical pathway as opposed to the majority that were secreted in signal peptide dependent pathway. The demonstrated proteins include all previously immunologically characterized M. haemolytica proteins. The potential of using secretome analysis in the design and development of a multivalent vaccine is discussed.

Genome Sequence of a Spontaneous Nonhemolytic Mutant of Mannheimia haemolytica 16041065 GH.

We report here the draft genome sequence of a spontaneous nonhemolytic mutant of Mannheimia haemolytica 16041065 GH. This mutant arose during routine passage and was devoid of hemolytic activity on standard blood agars. This genome sequence had a total size of 2.7 Mb with an N50 of 117 kb.

Genome Sequence of Mannheimia haemolytica Serotype 1 Strain 16041065 BH.

Here, we report the genome sequence of Mannheimia haemolytica serotype 1 strain 16041065 BH, which was recently isolated from a Midwestern calf that died due to Mannheimia haemolytica-induced pneumonia. This genome comprised a total of 2.7 Mb, with an N50 of 122 kb, and maintained hemolytic activity when grown on blood heart infusion agar supplemented with 5% sheep's blood.

Disseminated phaeohyphomycosis in a dog.

Phaeohyphomycosis is a rare but emerging disease caused by dematiaceous fungi. Here we describe the case of an immunosuppressed dog with disseminated phaeohyphomycosis secondary to Bipolaris spicifera infection. Regionally extensive infiltration of the paw pads, skin, myocardium, liver, renal interstitium and diaphragm was identified on histopathology. Candida glabrata and Fusarium oxysporum were also cultured from multiple sites post-mortem. The dog was treated with fluconazole, itraconazole, terbinafine and liposomal amphotericin B, but was euthanized due to its poor prognosis after 12 days of therapy.

Clinical disease and lung lesions in calves experimentally inoculated with Histophilus somni five days after metaphylactic administration of tildipirosin or tulathromycin.

OBJECTIVE: To compare clinical disease and lung lesions in calves experimentally inoculated with Histophilus somni 5 days after metaphylactic administration of tildipirosin or tulathromycin. ANIMALS Twenty-four 3-month-old Holstein and Holstein-crossbreed steers. PROCEDURES: Calves were randomly allocated to 3 groups of 8 calves. On day 0, calves in group 1 received tildipirosin (4 mg/kg, SC), calves in group 2 received tulathromycin (2.5 mg/kg, SC), and calves in group 3 received isotonic saline (0.9% NaCl) solution (1 mL/45 kg, SC; control). On day 5, calves were inoculated with 10 mL of a solution containing H somni strain 7735 (1.6 × 10(9) CFUs/mL, intrabronchially; challenge). Calves were clinically evaluated on days 5 through 8 and euthanized on day 8. The lungs were grossly evaluated for evidence of pneumonia, and bronchial secretion samples underwent bacteriologic culture. RESULTS: The mean clinical score for each group was significantly increased 12 hours after challenge, compared with that immediately before challenge, and was significantly lower for tildipirosin-treated calves on days 6, 7, and 8, compared with those for tulathromycin-treated and control calves. The mean percentage of lung consolidation for tildipirosin-treated calves was significantly lower than those for tulathromycin-treated and control calves. Histophilus somni was isolated from the bronchial secretions of some tulathromycin-treated and control calves but was not isolated from tildipirosin-treated calves. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that metaphylactic administration of tildipirosin to calves 5 days prior to H somni challenge prevented subsequent culture of the pathogen from bronchial secretions and was more effective in minimizing clinical disease and lung lesions than was metaphylactic administration of tulathromycin.

Enteric disease in postweaned beef calves associated with Bovine coronavirus clade 2.

Bovine coronavirus (BoCV; Betacoronavirus 1) infections are associated with varied clinical presentations including neonatal diarrhea, winter dysentery in dairy cattle, and respiratory disease in various ages of cattle. The current report presents information on BoCV infections associated with enteric disease of postweaned beef cattle in Oklahoma. In 3 separate accessions from a single herd, 1 in 2012 and 2 in 2013, calves were observed with bloody diarrhea. One calf in 2012 died and was necropsied, and 2 calves from this herd died in 2013 and were necropsied. A third calf from another herd died and was necropsied. The gross and histologic diagnosis was acute, hemorrhagic colitis in all 4 cattle. Colonic tissues from all 4 animals were positive by fluorescent antibody testing and/or immunohistochemical staining for BoCV antigen. Bovine coronavirus was isolated in human rectal tumor cells from swabs of colon surfaces of all animals. The genomic information from a region of the S envelope region revealed BoCV clade 2. Detection of BoCV clade 2 in beef cattle in Oklahoma is consistent with recovery of BoCV clade 2 from the respiratory tract of postweaned beef calves that had respiratory disease signs or were healthy. Further investigations on the ecology of BoCV in cattle are important, as BoCV may be an emerging disease beyond the initial descriptions. Challenge studies are needed to determine pathogenicity of these strains, and to determine if current BoCV vaccines are efficacious against the BoCV clade 2 strains.

Interleukin-6 deficiency corrects nephritis, lymphocyte abnormalities, and secondary Sjögren's syndrome features in lupus-prone Sle1.Yaa mice.

OBJECTIVE: To assess disease features in Sle1.Yaa mice with genetic interleukin-6 (IL-6) deficiency. METHODS: Sera and tissues were collected from C57BL/6 (B6), Sle1.Yaa, and Sle1.Yaa.IL-6(-/-) mice and analyzed for various features of disease. Using serum samples, autoantibody specificities were determined by enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence, cytokine production was analyzed by Luminex and ELISA, and levels of blood urea nitrogen were determined by ELISA. Renal, lung, and salivary gland tissue sections were evaluated for pathologic changes. Lymphocyte phenotypes, including CD4+ T cell cytokine production, and those of follicular and extrafollicular T helper subsets, germinal center B cells, and plasma cells, were determined using flow cytometry. RESULTS: IL-6 deficiency not only ameliorated autoantibody production and renal disease in this model, but also effectively reduced inflammation of lungs and salivary glands. Furthermore, IL-6 deficiency abrogated differentiation of Th1 and extrafollicular T helper cells, germinal center B cells, and plasma cells in the spleen and eliminated renal T cells with IL-17, interferon-γ, and IL-21 production potential. CONCLUSION: Our findings highlight IL-6-mediated T cell aberrations in Yaa-driven autoimmunity and support the concept of therapeutic IL-6/IL-6 receptor blockade in systemic lupus erythematosus and Sjögren's syndrome by impairing the production of autoantibodies and lymphocytic infiltration of the kidneys, lungs, and salivary glands.

A retrospective study of equine actinobacillosis cases: 1999-2011.

Several Actinobacillus spp. are common commensal bacteria of the oral cavity, gastrointestinal tract, and reproductive tract of horses and can cause disease in both foals and adults. The current retrospective study was designed to review Actinobacillus spp. isolated from clinical samples or necropsies of 99 horses during 1999-2011. The cases consisted of 43 foals (<6 months of age), 4 young adults (6 months-2 years), 39 adults (>2 years of age), 2 aborted fetuses, and 11 with unspecified ages. Clinical history, signs, bacterial species isolated, and associated lesions were documented. Actinobacillus spp. were isolated 111 times. The most common isolates were Actinobacillus equuli subsp. equuli (38.7%) and hemolytic Actinobacillus spp. (24.3%). Other isolates were Actinobacillus lignieresii (5.4%), Actinobacillus pleuropneumoniae (1.8%), and unclassified Actinobacillus spp. (28.8%). Actinobacillus equuli subsp. equuli was most commonly isolated from clinical and necropsy cases of septicemia and respiratory disease in both foals and adults. Embolic nephritis, the classical septicemic lesion of equine neonatal actinobacillosis, was also present in several adult septicemic actinobacillosis cases. Predisposing factors such as failure of passive transfer of colostral antibodies as well as concurrent pathogenic bacterial or viral infections were present in numerous actinobacillosis cases. There were many cases, however, for which a predisposing factor or concurrent infection was not documented or apparent, suggesting that Actinobacillus spp. can be primary pathogens under the right circumstances and in the right location.

The OmpA family of proteins: roles in bacterial pathogenesis and immunity.

The OmpA family of outer membrane proteins is a group of genetically related, heat-modifiable, surface-exposed, porin proteins that are in high-copy number in the outer membrane of mainly Gram-negative bacteria. OmpA proteins are characterized by an N-terminal domain that forms an eight-stranded, anti-parallel ß barrel, which is embedded in the outer membrane. The C-terminal domain is globular and located in the periplasmic space. Escherichia coli OmpA is the best characterized of the proteins. Other well-characterized OmpA-equivalent proteins from pathogenic bacteria include Pseudomonas aeruginosa OprF, Haemophilus influenzae P5, Klebsiella pneumoniae OmpA, and Chlamydia trachomatis major outer membrane protein (MOMP). OmpA from the veterinary pathogens Mannheimia haemolytica, Haemophilus parasuis, Leptospira interrogans, and Pasteurella multocida have been studied to a lesser extent. Among many of the pathogenic bacteria, OmpA proteins have important pathogenic roles including bacterial adhesion, invasion, or intracellular survival as well as evasion of host defenses or stimulators of pro-inflammatory cytokine production. These pathogenic roles are most commonly associated with central nervous system, respiratory and urogenital diseases. Alternatively, OmpA family proteins can serve as targets of the immune system with immunogenicity related to surface-exposed loops of the molecule. In several cases, OmpA proteins are under evaluation as potential vaccine candidates.

Proteomic analysis and immunogenicity of Mannheimia haemolytica vesicles.

Mannheimia haemolytica, a major causative agent in bovine respiratory disease, inflicts extensive losses each year on cattle producers. Commercially available vaccines are only partially efficacious. Immunity to M. haemolytica requires antibodies to secreted toxins and outer membrane proteins (OMPs) of the bacterium. Gram-negative bacteria produce membrane blebs or vesicles, the membrane components of which are primarily derived from OMPs. Accordingly, vesicles have been used as immunogens with various degrees of success. This study characterized components of M. haemolytica vesicles and determined their immunogenicity in mice and cattle. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of vesicles from this bacterium identified 226 proteins, of which 58 (25.6%) were OMPs and periplasmic and one (0.44%) was extracellular. Vesicles were used to vaccinate dairy calves and BALB/c mice. Analyses of sera from calves and mice by enzyme-linked immunosorbent assay (ELISA) showed that circulating antibodies against M. haemolytica whole cells and leukotoxin were significantly higher on days 21 and 28 (P < 0.05) than on day 0. For control calves and mice, there were no significant differences in serum anti-whole-cell and leukotoxin antibody levels from days 0 and 21 or 28, respectively. Lesion scores of lungs from vaccinated calves (15.95%) were significantly (P < 0.05) lower than those from nonvaccinated calves (42.65%). Sera from mice on day 28 and calves on day 21 showed 100% serum bactericidal activity. Sera from vesicle-vaccinated mice neutralized leukotoxin.

A rapid microtiter plate serum bactericidal assay method for determining serum complement-mediated killing of Mannheimia haemolytica.

In this study, we describe a rapid microtiter serum bactericidal assay (RMSBA) that can be used to measure the functionality of immune sera. It quantifies bactericidal activity of immune sera in the presence of complement against a homologous bacterium, M. haemolytica in this case. There is high correlation between data from RMSBA and standard complement-mediated bacterial killing assay (r=0.756; p<0.0001). The RMSBA activity of sera can be generated in less than 5 h instead of overnight incubation. RMSBA costs substantially less in terms of time, labor, and resources and is highly reproducible.

Cytokine expression by pulmonary leukocytes from calves challenged with wild-type and leukotoxin-deficient Mannheimia haemolytica.

The objective of this study was to assess the role of leukotoxin (LKT) in modulating the pulmonary cytokine response of calves challenged with Mannheimia haemolytica. Thirty-six calves, seronegative to LKT and M. haemolytica whole cell antigen were divided into three groups (I, II and III). Calves in groups I and II were challenged by the intra-bronchial route with 25 mL of phosphate buffered saline (PBS) containing 0.44×10(9) cfu/mL of LKT deficient (lkt(-)) and 25 mL of PBS containing 0.31×10(9) cfu/mL of wild-type (wt) M. haemolytica serotype 1, respectively. Group III calves were challenged intra-bronchially with 25 mL of sterile PBS. Leukocytes were collected from broncho-alveolar lavage fluid (BALF) 4 days before and at 1, 3, and 6 days post-inoculation (p.i.). Expression of the following cytokines in the recovered leukocytes was measured using real-time PCR: interleukin (IL)-1ß, -8, -10, -12 (p40) and TNF-α. The amount of TNF-α produced was also quantified by ELISA. Although a statistically significant difference in the expression of these cytokines was not observed between groups challenged with the wt and lkt(-) strains, the wt infected group (II) did exhibit higher mean clinical scores. Overall, there was considerable variation in the composition of the BALF between the groups and by day 7 p.i., both lkt(-)- and wt-challenged calves had seroconverted to M. haemolytica.