Characterization, distribution, antimicrobial resistance and resistance risk factors in staphylococci isolated from cats from 2001 to 2014.

Relatively few studies have been published describing the patterns of staphylococcal isolation and antimicrobial resistance over time in cats. The objective of this retrospective study was to determine the frequency, location, characteristics and antimicrobial resistance profiles of staphylococci isolated by the Louisiana Animal Disease Diagnostic Laboratory between the years 2001 and 2014. All feline staphylococcal isolates were classified phenotypically. Isolates corresponding to known or possibly pathogenic species (Staphylococcus intermedius group (SIG) and Staphylococcus aureus (SA)) as well as Staphylococcus epidermidis (SE) and non-speciated coagulase-negative staphylococci (CNS) were further evaluated to determine antimicrobial resistance patterns. A total of 519 staphylococci were isolated. The largest percentage of isolates was CNS, representing 39.3% of the total, while SIG, SE, SA and non-speciated coagulase positive staphylococci (CPS) represented 18.1%, 10.2%, 8.3% and 7.3%, respectively. Methicillin resistance (MR) was identified in 57.1% of SA and 20.5% of SIG. Resistance to 3 or more antimicrobial classes (multidrug resistance; MDR) was demonstrated in 54.5% of SA and 23.9% of SIG. The prevalence of MDR increased over time in both SIG and SA, while the prevalence of MR increased over time in SIG. An increase in mean antimicrobial resistance score over time was seen in SIG. This study demonstrates a high and increasing prevalence of MDR in SIG and SA, as well as increasing prevalence of MR in SIG isolated from cats.

Collagen and Hydroxyapatite Scaffolds Activate Distinct Osteogenesis Signaling Pathways in Adult Adipose-Derived Multipotent Stromal Cells.

Osteogenic cell signaling pathway disruption varies among bone diseases. This investigation was designed to identify adipose-derived multipotent stromal cell (ASC) and bone graft scaffold combinations for local, targeted restoration of gene expression and extracellular matrix (ECM) deposition. Human ASC osteogenesis on bone graft materials was quantified following culture in stromal (S), osteogenic (O), or osteogenic for 48 h followed by stromal medium (OS) to test the two-part hypothesis: (1) identical ASC isolates on distinct bone graft scaffolds demonstrate unique viability, differentiation, ECM production, and gene expression in the same culture conditions; (2) identical ASC-bone graft scaffold combinations have different cell viability, differentiation, ECM production, and gene expression when cultured in S, O, or OS medium. Three commercially available bone graft scaffold materials, type I bovine collagen (C), hydroxyapatite + ß-tricalcium phosphate + type I bovine collagen (HT), and ß-tricalcium phosphate + type I bovine collagen (CT) were evaluated. Passage 3 ASCs were loaded onto scaffold blocks with a spinner flask bioreactor, and constructs were cultured up to 28 days. Cell viability, gene expression (alkaline phosphatase [ALPL], osteoprotegerin [TNFRSF11B], osteocalcin [BGLAP], cannabinoid receptors type I [CNR1] and II [CNR2], receptor activator of nuclear factor kappa ß ligand [TNFSF11]), as well as ECM DNA, collagen, sulfated glycosaminoglycan, and protein content were quantified. Matrix organization was evaluated with scanning electron microscopy. Effects of scaffold, medium, or culture duration on cell viability were minimal. Significantly higher initial ALPL expression decreased with time, while BGLAP expression increased in HT constructs in O medium, and the constructs had the most abundant ECM components and ultrastructural organization. There was a similar, although delayed, pattern of gene expression and greater ECM collagen with less organization in C constructs in O medium. Higher CNR1 expression in C versus higher TNFRSF11B/TNFSF11 expression in HT constructs throughout the study support stimulation of unique osteogenic signaling pathways by identical cell isolates. These results suggest that bone scaffold composition may be used to selectively target specific osteogenic cell signaling pathways in ASC constructs to stimulate ECM deposition based on therapeutic needs.

Determination of irritant threshold concentrations of multiple tree, grass, weed and mould allergens for intradermal testing of horses residing in the southern USA.

BACKGROUND: Appropriate allergen threshold concentrations (TCs) for intradermal testing (IDT) have not been established in horses for many pollen and mould allergens. OBJECTIVES: To determine the TCs in non-allergic horses and describe the frequency of late phase reactions for 26 allergens, including trees, grasses, weeds and moulds in horses residing in the southern Unites States. ANIMALS: Twenty four clinically normal horses in the southern United States. METHODS: Threshold concentrations for different allergens were determined using IDT subjective measurements at 30 minutes. Delayed reactions were evaluated at 4 and 24 h. RESULTS: Threshold concentrations (all PNU/mL) were established for eight tree allergens (black willow 1,000, box elder 1,000, live oak 1,000, pecan 2,000, white ash 4,000, red oak 4,000, red mulberry 2,000 and green ash 2,000); two grass allergens (Johnson grass 250 PNU/mL and Kentucky blue grass 500 PNU/mL); two weeds (carelessweed 1,000 PNU/mL, great ragweed 500 PNU/mL) and one mould (Curvularia 8,000 PNU/mL). The TC was not determined due to excessive reactivity at the lowest concentration tested (1,000 PNU/mL) for bahia and perennial rye grass. Eleven other allergens did not meet the criteria to establish a TC when evaluated at 30 min due to lack of positive reactions. Multiple allergens caused positive reactions in ≥10% of horses at 4 h. Reactions at 24 h were rare with the exception of one horse. CONCLUSIONS AND CLINICAL IMPORTANCE: This study identified intradermal TC for multiple pollen and mould allergens in horses. These values may prove useful for optimizing allergen concentrations for IDT of allergic horses.

Interaction of Francisella noatunensis subsp. orientalis with Oreochromis mossambicus bulbus arteriosus cell line.

Francisella noatunensis subsp. orientalis (Fno) (syn. F. asiatica) is an emergent warmwater fish pathogen and the causative agent of piscine francisellosis. Although Fno causes septicemia and can live extracellularly in infected tilapia (Oreochromis spp.), the early interaction of Fno with vasculature endothelium is unknown. In the present study, we examined the interaction of wild-type Fno (WT) and two Fno knockout [intracellular growth loci C (ΔiglC) and pathogenicity determinant protein A (ΔpdpA)] strains with the endothelial O. mossambicus bulbus arteriosus cell line (TmB) at 25 °C and 30 °C. Similar amounts of WT, ΔiglC, and ΔpdpA attached and were detected intracellularly after 5 h of incubation at both temperatures; however temperature affected attachment and uptake. While significantly greater amounts of Fno (WT, ΔiglC, and ΔpdpA) were detected intracellularly when TmB cells were incubated at 30 °C, bacteria attached to TmBs at greater levels at 25 °C. Only WT Fno was able to replicate intracellularly at 25 °C, which resulted in Fno mediated cytotoxicity and apoptosis at 24 and 72 h post-infection. WT Fno incubated at 30 °C as well as ΔiglC, and ΔpdpA incubated at 25 °C and 30 °C were all defective for survival, replication, and the ability to cause cytotoxicity in TmB. Taken together, these results demonstrate that temperature plays a vital role for Fno intracellular survival, persistence and cytotoxicity.

Joint stability after canine cranial cruciate ligament graft reconstruction varies among femoral fixation sites.

OBJECTIVE: To quantify stability in cranial cruciate ligament (CrCL) deficient canine stifles with hamstring grafts affixed at 3 femoral locations. STUDY DESIGN: Canine stifle motion study using a multi-cohort, repeated measures design. SAMPLE POPULATION: 27 canine cadaver stifles. METHODS: Hamstring grafts (HG) were affixed at the gracilis-semitendinosus insertion and on the lateral femur (1) proximal trochlear ridge (TR), (2) craniodistal to fabella (F), or (3) condyle center (CC). Total, cranial, and caudal tibial translation and total, medial, and lateral angular displacement, with and without translational load, were quantified with the CrCL intact, transected, and reconstructed. Angular displacement was quantified from points on the distal femur and proximal tibia. Graft strain was calculated from tissue displacement measured at joint angles of 30°, 60°, 90°, and 120°. RESULTS: Tibial translation was lowest in F constructs, which also achieved the least difference in tibial translation from intact stifles. Tibial translation was lower in intact stifles than in CrCL transected or reconstructed stifles. Less angular displacement of the proximal tibia was detected in the medial than in the lateral direction, and tibial displacement was lower in the cranial than the caudal direction. Angular displacement was lowest in the F treatment group. F constructs had the lowest graft strain at joint angles greater than 30°. CONCLUSIONS: Femoral fixation of a canine hamstring graft craniodistal to the lateral fabella conferred the best joint stability and lowest graft strain in vitro. No fixation method restored joint stability of the intact CrCL.

EX VIVO CORRELATION OF ULTRASONOGRAPHIC SMALL INTESTINAL WALL LAYERING WITH HISTOLOGY IN DOGS.

Canine ultrasonographic intestinal layers have been reported to correlate with histological layering. However, discrepancies have been reported in people, and additional layers visualized. The aim of this method comparison study was to describe ex vivo canine small intestinal layering and correlate it with histology. Small intestinal samples of 12 adult dogs euthanized for reasons unrelated to gastrointestinal disease were resected immediately following euthanasia, pinned on a Petri dish, and transverse ultrasonographic images acquired in a water bath, using a high-frequency linear transducer. Transverse histological sections were obtained at the same level. Measurements of the intestinal layers were performed on the ultrasonographic and histological images. No significant statistical differences were noted between the ultrasonographic and histological measurements and strong to very strong (r > 0.7) positive correlation was observed for all layers, except for the serosa, which had a low moderate positive correlation (r = 0.479). In addition to the five established layers, a dual mucosal echogenicity was consistently observed, with seven samples presenting an additional inner mucosal severe hyperechogenicity. Histologically, this dual echogenicity was attributed to the intestinal villi (mildly echogenic) and lamina propria (hypoechoic). The additional inner mucosal severe hyperechogenicity observed in seven samples was attributed to mild-to-moderate lacteal dilation histologically. In 4/12 ileal samples, an additional hyperechoic mucosal line was also observed parallel to the submucosa, corresponding histologically to prominent Peyer's patches. Finally, a hyperechoic line was observed within the muscularis of all samples, corresponding histologically to the interface between the muscularis longitudinal and circular layers.

Gait Changes Vary among Horses with Naturally Occurring Osteoarthritis Following Intra-articular Administration of Autologous Platelet-Rich Plasma.

Mechanisms to reduce lameness associated with osteoarthritis (OA) are vital to equine health and performance. This study was designed to quantify response to autologous, intra-articular platelet-rich plasma (PRP) in horses with OA. Kinetic gait analysis was performed on 12 horses with unilateral forelimb lameness and OA in the same limb before and after intra-articular anesthesia (IAA). Radiographs and kinetic data were obtained before and 6 and 16 weeks after PRP administration to same joint, 4 weeks after IAA. Statistical evaluations included filtration effect on platelet concentration, relationship between kinetic variable changes after IAA versus PRP in the affected limb, and associations between response to PRP and response to IAA, platelet concentration, and radiographic OA. A positive response to IAA or PRP was defined as ≥5% improvement in peak vertical force, vertical impulse, or breaking impulse of the affected limb. Out of 10 horses that responded to IAA, 3 responded to PRP at both time points and 4 responded at one. Of the two horses that did not respond to IAA, one responded to PRP at both time points. Filtration increased platelet concentration significantly. The relationship between kinetic variable alterations of the affected limb after IAA and PRP was not significant, and response to PRP was not associated with response to IAA, platelet concentration, or radiographic OA. Changes in kinetic variables following IAA in joints with naturally occurring OA provide a custom standard to assess intra-articular therapy. Kinetic gait changes after intra-articular PRP are variable in horses with moderate to severe forelimb OA.

Morphometric and Ultrastructure studies of Primordial Follicles and Expression of Estrogen-dependent Genes in the Ovaries of Domestic Cats.

The aim of this study was to compare and estimate the population of the primordial follicle morphometrically and ultrastructurally in the left and right side ovaries of 10 ovariohysterectomied healthy domestic shorthair cats. The ovaries were processed for light microscopy, electron microscopy, and estrogen-dependent gene expression for assessments. A total of 15,092 primordial follicles with and without a nucleus were examined and counted. A total of 6842 primordial follicles with a nucleus were examined and counted. The light-microscopy numerical data were collected from two histological sections per ovary for a total of 20 sections from the left ovary and 20 sections from the right ovary. The average surface area of the histological sections was 645.99 mm2. The number of tertiary follicles was found to be higher in the left ovaries than in the right ovaries. The primordial follicles are under the tunica albuginea at various levels. Some are crowded or scattered in one or two rows, although at times, there were areas without any primordial follicles. The primordial follicles varied in size, and were surrounded by 4-10 squamous granulosa cells. Some primordial follicles shared their ooplasm with one or two neighboring primordial follicles, forming a giant primordial follicle with two or three nuclei. The ultrastructure of the primordial follicles showed rounded nuclei with distinct nucleoli, rounded and elongated mitochondria, and a considerably thick basement membrane under the granulosa cells. The squamous granulosa cells showed well-developed microvilli intermingled with the microvilli of the oocyte oolemma. Elongated mitochondria, coated pits, multicytoplasmic vesicles, ribosomes, and Golgi apparatuses were obvious in the oocyte ooplasm. Large vesicles contain small multivesicles and some scattered lipid globules in the ooplasm. There were estrogen-dependent gene-expression differences between the right and left ovaries. Further gene research is in the plan, using a larger pool of cats, with a focus on age differences.

Biofilm formation of Francisella noatunensis subsp. orientalis.

Francisella noatunensis subsp. orientalis (Fno) is an emergent fish pathogen in both marine and fresh water environments. The bacterium is suspected to persist in the environment even without the presence of a suitable fish host. In the present study, the influence of different abiotic factors such as salinity and temperature were used to study the biofilm formation of different isolates of Fno including intracellular growth loci C (iglC) and pathogenicity determinant protein A (pdpA) knockout strains. Finally, we compared the susceptibility of planktonic and biofilm to three disinfectants used in the aquaculture and ornamental fish industry, namely Virkon(®), bleach and hydrogen peroxide. The data indicates that Fno is capable of producing biofilms within 24 h where both salinity as well as temperature plays a role in the growth and biofilm formation of Fno. Mutations in the iglC or pdpA, both known virulence factors, do not appear to affect the capacity of Fno to produce biofilms, and the minimum inhibitory concentration, and minimum biocidal concentration for the three disinfectants were lower than the minimum biofilm eradication concentration values. This information needs to be taken into account if trying to eradicate the pathogen from aquaculture facilities or aquariums.

The need for econometric research in laboratory animal operations.

The scarcity of research funding can affect animal facilities in various ways. These effects can be evaluated by examining the allocation of financial resources in animal facilities, which can be facilitated by the use of mathematical and statistical methods to analyze economic problems, a discipline known as econometrics. The authors applied econometrics to study whether increasing per diem charges had a negative effect on the number of days of animal care purchased by animal users. They surveyed animal numbers and per diem charges at 20 research institutions and found that demand for large animals decreased as per diem charges increased. The authors discuss some of the challenges involved in their study and encourage research institutions to carry out more robust econometric studies of this and other economic questions facing laboratory animal research.

Evaluation of a ferret-specific formula for determining body surface area to improve chemotherapeutic dosing.

OBJECTIVE: To use CT-derived measurements to create a ferret-specific formula for body surface area (BSA) to improve chemotherapeutic dosing. ANIMALS: 25 adult ferrets (19 live and 6 cadavers). PROCEDURES: Live subjects were weighed, and body measurements were obtained by each of 3 observers while ferrets were awake and anesthetized. Computed tomography was performed, and a 3-D surface model was constructed with open-source imaging software, from which BSA was estimated. The CT-derived values were compared with BSA calculated on the basis of the traditional tape method for 6 cadavers. To further validate CT analysis software, 11 geometric shapes were scanned and their CT-derived values compared with those calculated directly via geometric formulas. Agreement between methods of surface area estimation was assessed with linear regression. Ferret-specific formulas for BSA were determined with nonlinear regression models. RESULTS: Repeatability among the 3 observers was good for all measurements, but some measurements differed significantly between awake and anesthetized ferrets. Excellent agreement was found between measured versus CT-derived surface area of shapes, traditional tape- versus CT-derived BSA of ferret cadavers, and CT-derived BSA of cadavers with and without monitoring equipment. All surface area formulas performed relatively similarly. CONCLUSIONS AND CLINICAL RELEVANCE: CT-derived BSA measurements of ferrets obtained via open-source imaging software were reliable. On the basis of study results, the recommended formula for BSA in ferrets would be 9.94 × (body weight)(2/3); however, this represented a relatively minor difference from the feline-derived formula currently used by most practitioners and would result in little practical change in drug doses.

Identification of host proteins involved in rickettsial invasion of tick cells.

Tick-borne spotted fever group (SFG) Rickettsia species are obligate intracellular bacteria capable of infecting both vertebrate and invertebrate host cells, an essential process for subsequent bacterial survival in distinct hosts. The host cell signaling molecules involved in the uptake of Rickettsia into mammalian and Drosophila cells have been identified; however, invasion into tick cells is understudied. Considering the movement of SFG Rickettsia between vertebrate and invertebrate hosts, the hypothesis is that conserved mechanisms are utilized for host cell invasion. The current study employed biochemical inhibition assays to determine the tick proteins involved in Rickettsia montanensis infection of tick-derived cells from a natural host, Dermacentor variabilis. The results revealed several tick proteins important for rickettsial invasion, including actin filaments, actin-related protein 2/3 complex, phosphatidylinositol-3'-kinase, protein tyrosine kinases (PTKs), Src family PTK, focal adhesion kinase, Rho GTPase Rac1, and neural Wiskott-Aldrich syndrome protein. Delineating the molecular mechanisms of rickettsial infection is critical to a thorough understanding of rickettsial transmission in tick populations and the ecology of tick-borne rickettsial diseases.

Associations between early radiographic and computed tomographic measures and canine hip joint osteoarthritis at maturity.

OBJECTIVE: To evaluate associations of measures assessed by radiography, 2-D CT, and 3-D CT of the hip joints of immature dogs with osteoarthritis in the same joints at maturity. ANIMALS: 46 hound-type dogs from a colony predisposed to osteoarthritis. PROCEDURES: Images of hip joints (1/dog) were obtained at 16, 32, and 104 weeks of age. Radiographic measures included Norberg angle, distraction index, and osteoarthritis score. Two-dimensional CT measures included acetabular index, percentage of femoral head coverage, and center edge, horizontal toit externe, acetabular anteversion, and ventral, dorsal, and horizontal acetabular sector angles. Three-dimensional CT measures were femoral head and neck volume, femoral neck angle, and femoral head and neck radius. Differences among measures at 16 and 32 weeks in dogs with different osteoarthritis scores at later time points, relationships among variables at each time point, and relationships of single and combined measures with the presence of osteoarthritis at 104 weeks were evaluated. RESULTS: The 16- and 32-week distraction index, center edge angle, dorsal acetabular sector angle, horizontal acetabular sector angle, percentage of femoral head coverage, acetabular index, and Norberg angle and the 32-week femoral neck angle varied significantly with osteoarthritis severity at 104 weeks. Presence of osteoarthritis in mature dogs was most strongly associated with 16-week combined measures of distraction index and center edge angle and 32-week combined measures of dorsal acetabular sector angle and Norberg angle. CONCLUSIONS AND CLINICAL RELEVANCE: Changes in hip joint morphology associated with radiographic signs of osteoarthritis were detectable as early as 16 weeks of age and varied with osteoarthritis severity in adult dogs. The use of combined hip joint measures may improve early identification of dogs predisposed to hip joint osteoarthritis.

Effect of size and temperature at vaccination on immunization and protection conferred by a live attenuated Francisella noatunensis immersion vaccine in red hybrid tilapia.

Francisella noatunensis subsp. orientalis (Fno) is a pleomorphic, facultative intracellular, Gram-negative, emerging bacterial pathogen of marine and fresh water fish with worldwide distribution. In this study, the efficacy of an attenuated Fno intracellular growth locus C (iglC) mutant was evaluated for use as a live immersion vaccine, when administered to hybrid tilapia at two different stages of growth (5 g fry and 10 g fingerlings) and at two temperatures (25 °C and 30 °C). To determine vaccine efficacy, mortality, days to first death, and Fno genome equivalents (GE) in the spleens of survivors, as well as serum and mucus antibody levels, were evaluated after 30 d in fish challenged with a wild type virulent strain. Both size and temperature at vaccination played an important role in immunization and protection. Fry vaccinated at 25 °C were not protected when compared to non-vaccinated fry at 25 °C (p = 0.870). In contrast, 5 g fry vaccinated at 30 °C were significantly protected compared to non-vaccinated fry at 30 °C (p = 0.038). Although lower mortalities occurred, 10 g fingerlings vaccinated at 25 °C were not protected, compared to non-vaccinated fingerlings at 25 °C (p = 0.328), while, 10 g fingerlings vaccinated at 30 °C were significantly protected, compared to non-vaccinated fingerlings at 30 °C (p = 0.038). Additionally, overall mortality of 5 g fish was significantly higher than in 10 g fish. Mortality was also significantly higher in fish subjected to a 30 to 25 °C temperature change one week prior to challenge, than in fish maintained at the same temperature during vaccination and challenge. This information demonstrates that both temperature and size at vaccination are important factors when implementing immunization prophylaxis in cultured tilapia.

Phenotypic and genotypic heterogeneity among Streptococcus iniae isolates recovered from cultured and wild fish in North America, Central America and the Caribbean islands.

Streptococcus iniae, the etiological agent of streptococcosis in fish, is an important pathogen of cultured and wild fish worldwide. During the last decade outbreaks of streptococcosis have occurred in a wide range of cultured and wild fish in the Americas and Caribbean islands. To gain a better understanding of the epizootiology of S. iniae in the western hemisphere, over 30 S. iniae isolates recovered from different fish species and geographic locations were characterized phenotypically and genetically. Species identities were determined biochemically and confirmed by amplification and sequencing of the 16S rRNA gene. Repetitive-element palindromic PCR fingerprinting as well as biochemical and antimicrobial susceptibility profiles suggest that a single strain of S. iniae was responsible for two different disease outbreaks among reef fishes in the Caribbean, one in 1999 and another in 2008. Interestingly, a majority of the isolates recovered from cultured fish in the Americas were genetically distinct from the Caribbean isolates and exhibited a trend toward higher minimal inhibitory concentration with respect to several antibiotics as well as greater genetic variability. The biological significance of this genetic variability is unclear, but it could have implications for future vaccine development and treatment.

Effect of pectin, lecithin, and antacid feed supplements (Egusin®) on gastric ulcer scores, gastric fluid pH and blood gas values in horses.

BACKGROUND: The objectives of this study were to evaluate the effects of two commercial feed supplements, Egusin 250® [E-250] and Egusin SLH® [E-SLH], on gastric ulcer scores, gastric fluid pH, and blood gas values in stall-confined horses undergoing feed-deprivation. METHODS: Nine Thoroughbred horses were used in a three-period crossover study. For the three treatment groups, sweet feed was mixed with E-250, E-SLH, or nothing (control group) and fed twice daily. Horses were treated for 21 days, then an additional 7 days while on an alternating feed-deprivation model to induce or worsen ulcers (period one). In periods two and three, horses (n=6) were treated for an additional 7 days after feed-deprivation. Gastroscopies were performed on day -1 (n=9), day 21 (n=9), day 28 (n=9) and day 35 (n=6). Gastric juice pH was measured and gastric ulcer scores were assigned. Venous blood gas values were also measured. RESULTS: Gastric ulcers in control horses significantly decreased after 21 days, but there was no difference in ulcer scores when compared to the Egusin® treated horses. NG gastric ulcer scores significantly increased in E-250 and control horses on day 28 compared to day 21 as a result of intermittent feed-deprivation, but no treatment effect was observed. NG ulcer scores remained high in the control group but significantly decreased in the E-SLH- and E-250-treated horses by day 35. Gastric juice pH values were low and variable and no treatment effect was observed. Mean blood pCO2 values were significantly increased two hours after feeding in treated horses compared to controls, whereas mean blood TCO2 values increased in the 24 hour sample, but did not exceed 38 mmol/l. CONCLUSIONS: The feed-deprivation model increased NG gastric ulcer severity in the horses. However, by day 35, Egusin® treated horses had less severe NG gastric ulcers compared to untreated control horses. After 35 days, Egusin® products tested here ameliorate the severity of gastric ulcers in stall-confined horses after feed stress.

Characterization of IgE-mediated cutaneous immediate and late-phase reactions in nonallergic horses.

OBJECTIVE: To characterize the response of skin of nonallergic horses following ID injection of polyclonal rabbit anti-canine IgE (anti-IgE) and rabbit IgG. ANIMALS: 6 healthy horses. PROCEDURES: Skin in the cervical area was injected ID with anti-IgE and IgG. Wheal measurements and skin biopsy specimens were obtained before and 20 minutes and 6, 24, and 48 hours after injection. Tissue sections were evaluated for inflammatory cells at 4 dermal depths. Immunohistochemical analysis for CD3, CD4, and CD8 was performed, and cell counts were evaluated. RESULTS: Anti-IgE wheals were significantly larger than IgG wheals at 20 minutes and 6 and 24 hours after injection. There were significantly more degranulated mast cells after anti-IgE injection than after IgG injection. There were significantly more eosinophils at 6, 24, and 48 hours and neutrophils at 6 hours after anti-IgE injection, compared with cell numbers at those same times after IgG injection. There were significantly more eosinophils in the deeper dermis of anti-IgE samples, compared with results for IgG samples. No significant differences between treatments were detected for CD3(+), CD4(+), or CD8(+) cells. CONCLUSIONS AND CLINICAL RELEVANCE: Injection of anti-IgE antibodies was associated with the development of gross and microscopic inflammation characterized by mast cell degranulation and accumulation of inflammatory cells, particularly eosinophils and neutrophils. This pattern appeared to be similar to that of horses with naturally developing allergic skin disease, although lymphocytes were not increased; thus, ID injection of anti-IgE in horses may be of use for evaluating allergic skin diseases of horses.

Novel identification of Dermacentor variabilis Arp2/3 complex and its role in rickettsial infection of the arthropod vector.

Tick-borne spotted fever group (SFG) Rickettsia species must be able to infect both vertebrate and arthropod host cells. The host actin-related protein 2/3 (Arp2/3) complex is important in the invasion process and actin-based motility for several intracellular bacteria, including SFG Rickettsia in Drosophila and mammalian cells. To investigate the role of the tick Arp2/3 complex in tick-Rickettsia interactions, open reading frames of all subunits of the protein including Arp2, Arp3, ARPC1, ARPC2, ARPC3, ARPC4, and ARPC5 were identified from Dermacentor variabilis. Amino acid sequence analysis showed variation (ranging from 25-88%) in percent identity compared to the corresponding subunits of the complex from Drosophila melanogaster, Mus musculus, Homo sapiens, and Saccharomyces cerevisiae. Potential ATP binding sites were identified in D. variabilis (Dv) Arp2 and Arp3 subunits as well as five putative WD (Trp-Asp) motifs which were observed in DvARPC1. Transcriptional profiles of all subunits of the DvArp2/3 complex revealed greater mRNA expression in both Rickettsia-infected and -uninfected ovary compared to midgut and salivary glands. In response to R. montanensis infection of the tick ovary, the mRNA level of only DvARPC4 was significantly upregulated compared to uninfected tissues. Arp2/3 complex inhibition bioassays resulted in a decrease in the ability of R. montanensis to invade tick tissues with a significant difference in the tick ovary, indicating a role for the Arp2/3 complex in rickettsial invasion of tick cells. Characterization of tick-derived molecules associated with rickettsial infection is imperative in order to better comprehend the ecology of tick-borne rickettsial diseases.

Transthoracic lung ultrasound in normal dogs and dogs with cardiogenic pulmonary edema: a pilot study.

Pulmonary edema is the most common complication of left-sided heart failure in dogs and early detection is important for effective clinical management. In people, pulmonary edema is commonly diagnosed based on transthoracic ultrasonography and detection of B line artifacts (vertical, narrow-based, well-defined hyperechoic rays arising from the pleural surface). The purpose of this study was to determine whether B line artifacts could also be useful diagnostic predictors for cardiogenic pulmonary edema in dogs. Thirty-one normal dogs and nine dogs with cardiogenic pulmonary edema were prospectively recruited. For each dog, presence or absence of cardiogenic pulmonary edema was based on physical examination, heartworm testing, thoracic radiographs, and echocardiography. A single observer performed transthoracic ultrasonography in all dogs and recorded video clips and still images for each of four quadrants in each hemithorax. Distribution, sonographic characteristics, and number of B lines per thoracic quadrant were determined and compared between groups. B lines were detected in 31% of normal dogs (mean 0.9 ± 0.3 SD per dog) and 100% of dogs with cardiogenic pulmonary edema (mean 6.2 ± 3.8 SD per dog). Artifacts were more numerous and widely distributed in dogs with congestive heart failure (P < 0.0001). In severe cases, B lines increased in number and became confluent. The locations of B line artifacts appeared consistent with locations of edema on radiographs. Findings from the current study supported the use of thoracic ultrasonography and detection of B lines as techniques for diagnosing cardiogenic pulmonary edema in dogs.

Effects of pentoxifylline on immediate and late-phase cutaneous reactions in response to anti-immunoglobulin E antibodies in clinically normal dogs.

OBJECTIVE: To characterize the effects of pentoxifylline on the gross and microscopic variables associated with immediate and late-phase inflammation following injection of IgE-specific antibodies in the skin of clinically normal dogs. ANIMALS: 6 healthy adult mixed-breed dogs. PROCEDURES: Intradermal injections (0.1 mL each) of PBS solution, histamine phosphate, and cross-linking rabbit-origin anti-canine IgE antibodies (3 injections/dog) were administered at 0 hours on day 0; wheal sizes were evaluated at 20 minutes, 6 hours, and 24 hours. Biopsy specimens of injected and noninjected skin were collected 24 hours after injection. On day 2, treatment with pentoxifylline (20 mg/kg, PO, q 8 h) was initiated and continued until day 30. For each dog, injection, measurement, and biopsy procedures were repeated on days 30 to 31 and on days 37 to 38 (ie, after discontinuation of pentoxifylline administration). RESULTS: Pentoxifylline administration was associated with a significant decrease in wheal size at 6 and 24 hours (but not at 20 minutes) after injection of anti-canine IgE. Repeated injections performed 1 week after drug discontinuation revealed partial recovery of the 6-hour cutaneous reaction and complete recovery of the 24-hour cutaneous reaction. Pentoxifylline administration was also associated with inhibition of mast cell degranulation and significant decreases in the total numbers of cutaneous inflammatory cells and eosinophils, compared with pretreatment findings. CONCLUSIONS AND CLINICAL RELEVANCE: In clinically normal dogs, pentoxifylline effectively impaired late-phase reactions but not immediate reactions at sites of intradermal injection of IgE-specific antibodies by inhibiting mast cell degranulation and recruitment of cutaneous inflammatory cells, especially eosinophils.