Jim Cooley

Bartonella vinsonii subsp. berkhoffii and Bartonella henselae as potential causes of proliferative vascular diseases in animals

Bartonella species are highly fastidious, vector borne, zoonotic bacteria that cause persistent intraerythrocytic bacteremia and endotheliotropic infection in reservoir and incidental hosts. Based upon prior in vitro research, three Bartonella sp., B. bacilliformis, B. henselae, and B. quintana can induce proliferation of endothelial cells, and each species has been associated with in vivo formation of vasoproliferative tumors in human patients. In this study, we report the molecular detection of B. vinsonii subsp. berkhoffii, B. henselae, B. koehlerae, or DNA of two of these Bartonella species simultaneously in vasoproliferative hemangiopericytomas from a dog, a horse, and a red wolf and in systemic reactive angioendotheliomatosis lesions from cats and a steer. In addition, we provide documentation that B. vinsonii subsp. berkhoffii infections induce activation of hypoxia inducible factor-1 and production of vascular endothelial growth factor, thereby providing mechanistic evidence as to how these bacteria could contribute to the development of vasoproliferative lesions. Based upon these results, we suggest that a fourth species, B. vinsonii subsp. berkhoffii, should be added to the list of bartonellae that can induce vasoproliferative lesions and that infection with one or more Bartonella sp. may contribute to the pathogenesis of systemic reactive angioendotheliomatosis and hemangiopericytomas in animals.

Mutation tryptophan to leucine at position 222 of haemagglutinin could facilitate H3N2 influenza A virus infection in dogs

An avian-like H3N2 influenza A virus (IAV) has recently caused sporadic canine influenza outbreaks in China and Korea, but the molecular mechanisms involved in the interspecies transmission of H3N2 IAV from avian to canine species are not well understood. Sequence analysis showed that residue 222 in haemagglutinin (HA) is predominantly tryptophan (W) in the closely related avian H3N2 IAV, but was leucine (L) in canine H3N2 IAV. In this study, reassortant viruses rH3N2-222L (canine-like) and rH3N2-222W (avian-like) with HA mutation L222W were generated using reverse genetics to evaluate the significance of the L222W mutation on receptor binding and host tropism of H3N2 IAV. Compared with rH3N2-222W, rH3N2-222L grew more rapidly in MDCK cells and had significantly higher infectivity in primary canine tracheal epithelial cells. Tissue-binding assays demonstrated that rH3N2-222L had a preference for canine tracheal tissues rather avian tracheal tissues, whereas rH3N2-222W favoured slightly avian rather canine tracheal tissues. Glycan microarray analysis suggested both rH3N2-222L and rH3N2-222W bound preferentially to α2,3-linked sialic acids. However, the rH3N2-222W had more than twofold less binding affinity than rH3N2-222L to a set of glycans with Neu5Aca2-3Galb1-4(Fuca-)-like or Neu5Aca2-3Galb1-3(Fuca-)-like structures. These data suggest the W to L mutation at position 222 of the HA could facilitate infection of H3N2 IAV in dogs, possibly by increasing the binding affinities of the HA to specific receptors with Neu5Aca2-3Galb1-4(Fuca-) or Neu5Aca2-3Galb1-3(Fuca-)-like structures that are present in dogs.

Error-prone pcr-based mutagenesis strategy for rapidly generating high-yield influenza vaccine candidates.

Vaccination is the primary strategy for the prevention and control of influenza outbreaks. However, the manufacture of influenza vaccine requires a high-yield seed strain, and the conventional methods for generating such strains are time consuming. In this study, we developed a novel method to rapidly generate high-yield candidate vaccine strains by integrating error-prone PCR, site-directed mutagenesis strategies, and reverse genetics. We used this method to generate seed strains for the influenza A(H1N1)pdm09 virus and produced six high-yield candidate strains. We used a mouse model to assess the efficacy of two of the six candidate strains as a vaccine seed virus: both strains provided complete protection in mice against lethal challenge, thus validating our method. Results confirmed that the efficacy of these candidate vaccine seed strains was not affected by the yield-optimization procedure.

Zoonotic Risk, Pathogenesis, and Transmission of Avian-Origin H3N2 Canine Influenza Virus

ABSTRACT Two subtypes of influenza A virus (IAV), avian-origin canine influenza virus (CIV) H3N2 (CIV-H3N2) and equine-origin CIV H3N8 (CIV-H3N8), are enzootic in the canine population. Dogs have been demonstrated to seroconvert in response to diverse IAVs, and naturally occurring reassortants of CIV-H3N2 and the 2009 H1N1 pandemic virus (pdmH1N1) have been isolated. We conducted a thorough phenotypic evaluation of CIV-H3N2 in order to assess its threat to human health. Using ferret-generated antiserum, we determined that CIV-H3N2 is antigenically distinct from contemporary human H3N2 IAVs, suggesting that there may be minimal herd immunity in humans. We assessed the public health risk of CIV-H3N2 × pandemic H1N1 (pdmH1N1) reassortants by characterizing their in vitro genetic compatibility and in vivo pathogenicity and transmissibility. Using a luciferase minigenome assay, we quantified the polymerase activity of all possible 16 ribonucleoprotein (RNP) complexes (PB2, PB1, PA, NP) between CIV-H3N2 and pdmH1N1, identifying some combinations that were more active than either parental virus complex. Using reverse genetics and fixing the CIV-H3N2 hemagglutinin (HA), we found that 51 of the 127 possible reassortant viruses were viable and able to be rescued. Nineteen of these reassortant viruses had high-growth phenotypes in vitro, and 13 of these replicated in mouse lungs. A single reassortant with the NP and HA gene segments from CIV-H3N2 was selected for characterization in ferrets. The reassortant was efficiently transmitted by contact but not by the airborne route and was pathogenic in ferrets. Our results suggest that CIV-H3N2 reassortants may pose a moderate risk to public health and that the canine host should be monitored for emerging IAVs. IMPORTANCE IAV pandemics are caused by the introduction of novel viruses that are capable of efficient and sustained transmission into a human population with limited herd immunity. Dogs are a a potential mixing vessel for avian and mammalian IAVs and represent a human health concern due to their susceptibility to infection, large global population, and close physical contact with humans. Our results suggest that humans are likely to have limited preexisting immunity to CIV-H3N2 and that CIV-H3N2 × pdmH1N1 reassortants have moderate genetic compatibility and are transmissible by direct contact in ferrets. Our study contributes to the increasing evidence that surveillance of the canine population for IAVs is an important component of pandemic preparedness.

Horses With Pasture Asthma Have Airway Remodeling That Is Characteristic of Human Asthma

Severe equine asthma, formerly recurrent airway obstruction (RAO), is the horse counterpart of human asthma, affecting horses maintained indoors in continental climates. Equine pasture asthma, formerly summer pasture RAO, is clinically similar but affects grazing horses during hot, humid conditions in the southeastern United States and United Kingdom. To advance translational relevance of equine pasture asthma to human asthma, histologic features of airway remodeling in human asthma were scored in lung lobes from 15 pasture asthma-affected and 9 control horses of mixed breeds. All noncartilaginous airways were scored using a standardized grading rubric (0–3) in hematoxylin and eosin (HE) and Movat’s pentachrome-stained sections; 15 airways were chosen randomly from each lobe for analysis. Logistic regression identified disease, age, and lobe effects on probability of histologic outcomes. Airway smooth muscle (odds ratio [OR] = 2.5, P < .001), goblet cell hyperplasia/metaplasia (OR = 37.6, P < .0001), peribronchiolar elastic system fibers (OR = 4.2, P < .001), peribronchiolar fibrosis (OR = 3.8, P = .01), airway occlusion by mucus/inflammation (OR = 4.2, P = .04), and airway adventitial inflammation (OR = 3.0, P = .01) were significantly greater in diseased airways. A novel complex tissue disorganization, designated terminal bronchiolar remodeling, was overrepresented in diseased airways (OR = 3.7, P < .0001). Distribution of terminal bronchiolar remodeling corresponded to putative sites of air trapping in human asthma, at secondary pulmonary lobules. Age (>15 years) was an independent risk factor for increased peribronchiolar fibrosis, elastic system fibers, and terminal bronchiolar remodeling. Remodeling differed significantly between lung lobes, congruent with nonhomogeneous remodeling in human asthma. Equine pasture asthma recapitulates airway remodeling in human asthma in a manner not achieved in induced animal asthma models, endorsing its translational relevance for human asthma investigation.

Healing of canine skin incisions made with monopolar electrosurgery versus scalpel blade

Objective: To determine the influence of monopolar electrosurgery in cutting mode set at 10, 20, or 30 W on surgery time, hemostasis, and healing of cutaneous wounds compared to scalpel incisions. Study design: Randomized blinded control trial. Animals: Dogs (n = 15). Methods: Four skin incisions were created on either side of the dorsal midline with a scalpel, or monopolar electrosurgery at 10, 20, and 30 W. Surgical time and incisional bleeding were measured. Each incision was assessed daily for edema, erythema and discharge, and complications. Healing was evaluated via histology at 7 days. Results were analyzed for significance at P ≤ .05. Results: Surgical time and hemostasis were improved in all electrosurgery groups. Erythema was reduced in all electrosurgical incisions for days 1‐4, but was greater in wounds created via electrosurgery at 20 W than those made with a scalpel blade by day 7. No difference was noted in the degree of edema or presence of wound discharge. All histologic variables of tissue healing were lower in electrosurgical incisions than scalpel incisions (P < .001). Ten incisional complications occurred, all associated with electrosurgery. Conclusions: The use of monopolar electrosurgery at 10, 20, and 30 W in a cutting waveform improved hemostasis and surgical time when incising canine skin, but delayed healing and increased complications within the first 7 days compared to scalpel incisions. Clinical significance: Although monopolar electrosurgery improved hemostasis and surgery times, this modality is not recommended to create skin incisions.

Concurrent metastatic islet cell carcinoma, ACTH-producing pituitary carcinoma, adrenocortical necrosis and glucocorticoid-deficient hypoadrenocorticism in a dog

A 13-year-old female spayed boxer presented for severe hypoglycaemia. Electrolyte concentrations were within reference range, and adrenocorticotropic hormone (ACTH) stimulation test results were consistent with hypoadrenocorticism. Abdominal ultrasound revealed pancreatic and hepatic nodules. The left adrenal gland was of normal size, but the cranial pole of the right adrenal gland was enlarged. While the patient was severely hypoglycaemic, the insulin concentration was above reference range, consistent with insulinoma. The patient was hospitalised on dextrose supplementation and discharged on prednisone and directions to feed small, frequent meals. Surgery to remove the insulinoma was declined by the owner. When the hypoglycaemia became refractory to increasing doses of prednisone, diazoxide was added. Two months following initial presentation, the patient was euthanased due to refractory hypoglycaemia. Postmortem examination revealed a pancreatic islet cell carcinoma with metastatic lesions in the liver and pancreatic lymph nodes, a pituitary carcinoma that stained strongly positive with ACTH antibody, and adrenocortical necrosis.

A Y161F Hemagglutinin Substitution Increases Thermostability and Improves Yields of 2009 H1N1 Influenza A Virus in Cells

ABSTRACT Vaccination is the primary strategy for influenza prevention and control. However, egg-based vaccines, the predominant production platform, have several disadvantages, including the emergence of viral antigenic variants that can be induced during egg passage. These limitations have prompted the development of cell-based vaccines, which themselves are not without issue. Most importantly, vaccine seed viruses often do not grow efficiently in mammalian cell lines. Here we aimed to identify novel high-yield signatures for influenza viruses in continuous Madin-Darby canine kidney (MDCK) and Vero cells. Using influenza A(H1N1)pdm09 virus as the testing platform and an integrating error-prone PCR-based mutagenesis strategy, we identified a Y161F mutation in hemagglutinin (HA) that not only enhanced the infectivity of the resultant virus by more than 300-fold but also increased its thermostability without changing its original antigenic properties. The vaccine produced from the Y161F mutant fully protected mice against lethal challenge with wild-type A(H1N1)pdm09. Compared with A(H1N1)pdm09, the Y161F mutant had significantly higher avidity for avian-like and human-like receptor analogs. Of note, the introduction of the Y161F mutation into HA of seasonal H3N2 influenza A virus (IAV) and canine H3N8 IAV also increased yields and thermostability in MDCK cells and chicken embryotic eggs. Thus, residue F161 plays an important role in determining viral growth and thermostability, which could be harnessed to optimize IAV vaccine seed viruses. IMPORTANCE Although a promising complement to current egg-based influenza vaccines, cell-based vaccines have one large challenge: high-yield vaccine seeds for production. In this study, we identified a molecular signature, Y161F, in hemagglutinin (HA) that resulted in increased virus growth in Madin-Darby canine kidney and Vero cells, two cell lines commonly used for influenza vaccine manufacturing. This Y161F mutation not only increased HA thermostability but also enhanced its binding affinity for α2,6- and α2,3-linked Neu5Ac. These results suggest that a vaccine strain bearing the Y161F mutation in HA could potentially increase vaccine yields in mammalian cell culture systems.

Pathogenicity and transmission of a swine influenza A(H6N6) virus.

Subtype H6 influenza A viruses (IAVs) are commonly detected in wild birds and domestic poultry and can infect humans. In 2010, a H6N6 virus emerged in southern China, and since then, it has caused sporadic infections among swine. We show that this virus binds to α2,6-linked and α2,3-linked sialic acids. Mutations at residues 222 (alanine to valine) and 228 (glycine to serine) of the virus hemagglutinin (HA) affected its receptor-binding properties. Experiments showed that the virus has limited transmissibility between ferrets through direct contact or through inhalation of infectious aerosolized droplets. The internal genes of the influenza A(H1N1)pdm09 virus, which is prevalent in swine worldwide, increases the replication efficiency of H6N6 IAV in the lower respiratory tract of ferrets but not its transmissibility between ferrets. These findings suggest H6N6 swine IAV (SIV) currently poses a moderate risk to public health, but its evolution and spread should be closely monitored.

Identification of histamine receptors in the canine gastrointestinal tract

The important role of histamine in chronic gastrointestinal diseases has been increasingly recognized over the last two decades in human medicine. Histamine is released following mast cell activation and exerts its action through binding to four different histamine receptors (H1, H2, H3, and H4). Histamine receptors are dispersed throughout the body, and each different receptor mediates a unique response. Documentation of the presence and type of histamine receptors in the differing sections of the canine gastrointestinal tract will provide additional research opportunities to further explore the role of histamine and its receptors in chronic canine enteropathies, as well as potential therapeutic options. Full thickness gastric, duodenal, jejunal, ileal, and colonic biopsies were obtained from 6 clinically normal adult dogs immediately after humane euthanasia. Commercially available histamine receptor antibodies predicted to react with canine tissues were applied to paraffin-embedded tissue sections using standard immunohistochemistry techniques to identify different histamine receptors. Staining intensity was graded from negative to strong, and the specificity of each antibody was evaluated with western blot. The presence and distribution of histamine receptors varied by anatomic site and histologic level within sections of the canine gastrointestinal tract. All 4 histamine receptors were readily identified, although the distribution of H4 receptors was decreased in comparison to the other histamine receptors. The distribution of the various histamine receptors was similar to that seen in the normal human gastrointestinal tract. H1 receptors were located in the stomach, lymphoid tissue of the ileum and colon, and the smooth muscle and ganglia of all sections. H2 receptors were located in all sections of the gastrointestinal tract, with greatest staining intensity in the gastric mucosa. H3 receptors were located in the stomach and colonic mucosa, smooth muscle and ganglia of all sections, and ileal and colonic lymphoid tissue. H4 receptors were located in the ganglia and smooth muscle of the gastrointestinal tract, as well as the gastric and colonic mucosal and ileal lymphoid tissue. Western blot demonstrated both specific and non-specific staining with the H1 and H3 receptor antibody, but good specificity with the H4 receptor antibody. The H2 receptor antibody was not compatible with western blot techniques, despite excellent immunohistochemical specificity and consistency. Further studies to compare the density and distribution of the various histamine receptors in dogs with gastrointestinal disease are warranted.