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Abstract
Objective—To evaluate the effectiveness of a commercial conventional blood culture system (BCS), a commercial resin-containing BCS, and a commercial lysis-centrifugation–based BCS for the recovery of Escherichia coli from equine blood samples inoculated with that organism.
Sample Population—Samples of blood obtained from a clinically normal horse that were inoculated with E coli.
Procedures—Blood samples were aseptically collected and inoculated with an E coli specimen (50 CFUs/mL) that had been previously isolated from a foal with sepsis. Subsequently, samples were spiked with gentamicin at a concentration of 30 μg/mL, and 10 mL of each mixture was inoculated into 1 bottle or tube of each BCS. Samples were processed and incubated according to the manufacturer's guidelines and inoculated onto 5% sheep blood agar plates. Plated samples were examined macroscopically at regular intervals for as long as 72 hours. Detection of E coli and time to detection were recorded for each medium.
Results—Detection frequency of E coli was significantly greater by use of the resin-containing BCS (14/23 bottles) than that achieved by use of the conventional BCS (7/23 bottles) or the lysis-centrifugation–based BCS (0/10 tubes). Mean detection time (6 hours after plating) did not differ between the BCS with conventional medium and the BCS with resincontaining medium.
Conclusions and Clinical Relevance—Results suggest that a BCS with resin-containing medium may provide clinical benefit in the successful recovery of E coli from the blood of foals with sepsis that have been previously administered gentamicin.
Abstract
Objective—To investigate the effects of short-chain fatty acids (SCFAs) and pH on neutrophil oxidative burst, phagocytosis, and morphology after exposure to acetate, propionate, butyrate, or succinate at pH 5.5 and 6.7.
Sample Population—Neutrophils isolated from bovine blood samples and Porphyromonas levii, Prevotella spp, and Bacteroides fragilis isolated from lesions of cattle with acute interdigital phlegmon (foot rot).
Procedures—Bacteria were cultured in strictly anaerobic conditions. Bacterial SCFA production was measured with high-performance liquid chromatography. Neutrophils were isolated, stimulated with phorbol 12-myristate 13-acetate (PMA) or opsonized zymosan (OZ), and incubated with dihydroethidium or dichlorofluorescein diacetate to measure production of O2−and H2O2, respectively. Phagocytosis was assessed after exposure to serum-opsonized bacteria. Cellular morphology was assessed with differential staining.
Results—All bacteria produced at least 3 of the 4 SCFAs. Production of both O2− and H2O2 was markedly curtailed in PMA-stimulated neutrophils exposed to SCFA at pH 5.5, compared with production at pH 6.7. Succinate caused a significant dose-dependent decrease in O2− production at pH 6.7 in OZ-stimulated neutrophils. Monoprotic SCFAs elicited a significant increase in H2O2 production in OZ-stimulated neutrophils at pH 6.7 but a significant decrease at pH 5.5. Monoprotic SCFAs significantly increased phagocytosis at pH 6.7 but decreased phagocytic activity at pH 5.5. Cellular necrosis was observed in cells exposed to SCFAs at pH 5.5.
Conclusions and Clinical Relevance—Establishment and persistence of anaerobic bacteria in cattle with foot rot infection may result in part from neutrophil dysfunction secondary to the effects of bacterially secreted SCFA in acidotic microenvironments.
Abstract
Objective—To evaluate activation of Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) pathway in bovine monocytes after incubation with Mycobacterium avium subsp paratuberculosis (Mptb) organisms.
Sample Population—Bovine monocytes obtained from 4 healthy adult Holstein dairy cows.
Procedures—Bovine monocytes were incubated with Mptb organisms with or without a specific inhibitor of the JNK/SAPK pathway (SP600125) for 2, 6, 24, or 72 hours. Expression of interleukin (IL)-1β, IL-10, IL-12, IL-18; transforming growth factor-β (TGF-β); and tumor necrosis factor-α (TNF-α) and the capacity of Mptb-infected monocytes to acidify phagosomes and kill Mptb organisms were evaluated. Phosphorylation status of JNK/SAPK was evaluated at 10, 30, and 60 minutes after Mptb incubation.
Results—Compared with uninfected control monocytes, Mptb-infected monocytes had increased expression of IL-10 at 2 and 6 hours after incubation and had increased expression of TNF-α, IL-1β, IL-18, and TGF-β at 2, 4, and 6 hours. Additionally, Mptb-infected monocytes had increased expression of IL-12 at 6 and 24 hours. Addition of SP600125 (specific chemical inhibitor of JNK/SAPK) resulted in a decrease in TNF-α expression at 2, 6, and 24 hours, compared with untreated Mptb-infected cells. Addition of SP600125 resulted in a decrease in TGF-β expression at 24 hours and an increase in IL-18 expression at 6 hours. Addition of SP600125 failed to alter phagosome acidification but did enhance the capacity of monocytes to kill Mptb organisms.
Conclusions and Clinical Relevance—Activation of JNK/SAPK may be an important mechanism used by Mptb to regulate cytokine expression in bovine monocytes for survival and to alter inflammatory and immune responses.
Abstract
Objective—To experimentally infect adult alpacas by ID inoculation of Corynebacterium pseudotuberculosis, follow the clinical and pathologic course of disease, and study the humoral response to infection.
Animals—13 adult alpacas.
Procedures—9 alpacas were inoculated with 1.1 X 106 CFUs of C pseudotuberculosis from llama (n = 4) or alpaca (5) origin, and 4 alpacas were sham inoculated as controls. Alpacas were clinically observed after inoculation and euthanatized on days 16, 58, 93, or 128 after inoculation; necropsy examination and histologic evaluation were performed. An indirect ELISA, which made use of the C pseudotuberculosis cell wall as the antigen, was used to measure antibody titers in serum samples.
Results—Alpacas had a persistent febrile response, a local severe inflammatory response, and leucocytosis (> 30 X 103 WBCs/μL). Internal abscesses that localized mainly in the renal lymph node were observed. Corynebacterium pseudotuberculosis was recovered from the inoculation site 1 week after inoculation and from internal abscesses at 58 days after inoculation. Initial lesions were typical pyogranulomas with central caseous necrosis, whereas later lesions consisted of connective tissue, mononuclear cells, abundant neutrophils, and liquefactive necrosis. Infected alpacas had detectable serum antibody titers starting on day 16 that persisted until day 93 after inoculation. Shaminoculated alpacas did not develop serum antibody titers, clinical signs of infection, or lesions.
Conclusions and Clinical Relevance—Alpacas inoculated with C pseudotuberculosis developed abscesses at the inoculation site and internally in the renal lymph nodes, without lung lesions. Corynebacterium pseudotuberculosis isolates from llama and alpaca origin were found to be pathogenically indistinct.
Abstract
Objective—To evaluate use of covered-rod (CR) silicone implants containing ivermectin for long-term prevention of infection with Dirofilaria immitisin dogs.
Animals—145 adult male and female dogs.
Procedures—Dogs received implants of different sizes, and ivermectin concentrations and serum ivermectin concentrations were monitored for 16, 57, and 56 weeks, respectively, in 3 preclinical dose selection studies. Ability of implants to prevent infection with D immitis was evaluated in 2 further studies; dogs were challenged with 50 infective third-stage larvae 52 weeks after implant administration and necropsied 145 days after challenge, and the total number of adult heartworms was counted. A field study was then undertaken in which client-owned dogs received an implant and plasma samples were collected at intervals until week 52 for ivermectin analysis and heartworm antigen determination.
Results—Use of the implants resulted in maintenance of an ivermectin concentration ≥ 0.2 ng/mL for 12 months. In challenge studies, no treated dogs had adult heartworms, in contrast to untreated dogs, which all had adult heartworms at necropsy. In the field study, dogs treated with an implant had negative results of heartworm antigen testing for 12 months.
Conclusions and Clinical Relevance—The CR silicone implant containing 7.3 mg of ivermectin was 100% effective in preventing experimental infection with D immitislarvae and resulted in negative results for heartworm antigen in a field trial. This product has the potential to alleviate poor owner compliance with monthly prevention regimens.
Abstract
Objective—To identify and partially characterize a coronaviruslike virus isolated from naturally infected pigeons.
Animals—50 specific pathogen-free (SPF) embryonated chicken eggs, 30 White Leghorn SPF chickens, and 12 clinically normal pigeons.
Procedures—Pancreatic tissue specimens from sick pigeons were inoculated into SPF embryonated chicken eggs for viral isolation and investigation of morphologic and hemagglutinating properties of the isolate, called PSH050513. Furthermore, virulence studies in SPF chickens and experimental pigeons were performed. The spike (S) glycoprotein gene of PSH050513 was further sequenced and analyzed.
Results—PSH050513 was isolated and identified from the experimentally infected pigeons by a routine method, which was in accordance with Koch's postulates. The complete S protein (1,167 amino acids) was compared with published S protein sequences of other avian and mammalian coronaviruses. A high degree of sequence identity (79.3% to 99.6%) was observed between the S protein sequence of PSH050513 and published sequences of avian infectious bronchitis virus (IBV); only limited identity (< 37.8%) was observed with turkey coronavirus and mammalian coronaviruses. Furthermore, when the virus was inoculated into SPF chickens, pancreatitis developed.
Conclusions and Clinical Relevance—PSH050513 has been tentatively identified as a novel member of group 3 coronaviruses that have close genetic relationships with IBV strains.
Abstract
Objective—To evaluate serovar and antimicrobial resistance patterns of Salmonella enterica isolated from preweaned calves and identify management risk factors associated with fecal shedding of S enterica.
Sample Population—Cohorts of 10 to 15 preweaned calves (1 to 84 days of age) on 26 dairies and 7 calf ranches and cross-sectional samples of preweaned calves on smaller farms.
Procedures—Calves were evaluated every 2 weeks during a 6-week period. Salmonella isolates obtained from rectal fecal swabs underwent antimicrobial susceptibility testing against 12 antimicrobials. Cluster analysis enabled description of antimicrobial susceptibility patterns. Calf, cohort, and farm risk factors associated with both the prevalence of S enterica and multiple-antimicrobial–resistant S enterica in preweaned calves were identified with repeated-measure logistic regression models.
Results—Salmonella enterica was detected on > 50% of farms and in 7.5% of 3,686 fecal samples. Many isolates (33%) were resistant to multiple antimicrobials. Shedding of Salmonella spp was negatively associated with increasing calf age, herds being closed to incoming cattle, and antimicrobial supplementation of milk replacer; prophylactic antimicrobial treatment in day-old calves increased shedding. No association between farm management and presence of multiple-antimicrobial–resistant S enterica or between calving management and presence of S enterica in calves ≤ 1 week old was detected.
Conclusions and Clinical Relevance—In preweaned calves, the most important factors associated with decreased likelihood of fecal shedding of S enterica were the use of antimicrobial-supplemented milk replacer and maintenance of a closed herd. Infection with multiple-antimicrobial–resistant S enterica was not associated with antimicrobial administration.
Abstract
Objective—To evaluate a technique for identifying horses latently infected with neuropathogenic strains of equine herpesvirus-1 (EHV-1).
Animals—36 adult mares, 24 of which were experimentally infected as weanlings with neuropathogenic or nonneuropathogenic EHV-1.
Procedures—Mandibular lymph node (MLN) tissue was obtained from each horse via biopsy during general anesthesia. Purified DNA from MLNs was tested for EHV-1 DNA by use of a magnetic bead, sequencecapture, nested PCR assay. For MLNs that contained EHV-1 DNA, the 256-bp DNA fragments amplified via sequence-capture nested PCR were sequenced to determine the nucleotide at the polymorphic site that determines pathotype (ie, neuropathotype [G2254] or non-neuropathotype [A2254]).
Results—Latent viral DNA was detected in 26 of the 36 (72%) mares tested. Neuropathogenic and nonneuropathogenic EHV-1 genotypes were detected in the latently infected horses. In each mare previously infected with known EHV-1 pathotypes, the open reading frame 30 genotype of latent EHV-1 was identical to that of the strain that had been inoculated 4 to 5 years earlier. Latent viral DNA was detected in 10 of the 12 mares that were inoculated as weanlings with neuropathogenic strains of EHV-1. The detection rate of the sequence-capture PCR method for EHV-1 latency was double that of conventional nested or realtime PCR assays performed on the same MLN DNA preparations.
Conclusions and Clinical Relevance—The magnetic bead, sequence-capture, nested PCR technique enabled low-threshold detection of DNA from latent neuropathogenic strains of EHV-1 in MLN specimens from live horses. The technique may be used to screen horses for latent neuropathogenic EHV-1 infection.
Abstract
Objective—To determine the effect of dietary supplemental lipoic acid (LA) on serum concentrations of metabolic hormones and acute-phase proteins of steers challenged with infectious bovine rhinotracheitis virus (IBRV).
Animals—32 steers.
Procedures—Steers were randomly assigned to 4 treatments: negative control (NC; no LA, no IBRV challenge), control (CON; no LA, IBRV challenge), 16 mg of LA/kg of body weight (BW)/d plus IBRV challenge (LA16), and 32 mg of LA/kg of BW/d plus IBRV challenge (LA32). Following a 21-day adaptation period, CON, LA16, and LA32 steers received IBRV (2 mL/nostril [day 0]); NC steers received saline (0.9% NaCl) solution. Blood samples, nasal swab specimens, BW, and rectal temperatures were obtained 0, 1, 3, 5, 7, 14, and 21 days after challenge. Serum was analyzed for concentrations of haptoglobin, amyloid-A, leptin, and anti-IBRV antibodies.
Results—Steers fed LA32 began gaining BW by day 7, whereas BW of CON and LA16 steers declined. Serum haptoglobin concentration of LA32 steers was lower than that of CON and LA16 steers on day 7. Serum neutralization titers for 30 of 32 steers were negative for anti-IBRV antibodies before challenge; however, all steers (including NCs) had antibodies on day 21.
Conclusions and Clinical Relevance—Results suggested that LA supplementation augmented certain aspects of the immune response; LA32 steers had a more rapid recovery from IBRV viral challenge than did others.
Abstract
Objective—To determine whether particular antimicrobial susceptibility profiles of bovine mastitis-causing Staphylococcus aureus isolates were associated with specific S aureus genotypes.
Sample Population—357 S aureus isolates recovered from milk samples submitted for diagnostic bacteriologic testing from 24 dairy herds.
Procedures—Antimicrobial susceptibility of S aureus isolates was assessed by determining minimum inhibitory concentrations (MICs) to 14 antimicrobial agents. After digestion of S aureus genomic DNA by SmaI, electrophoretic patterns were obtained via pulsed-field gel electrophoresis (PFGE) and used to classify isolates into types. Gels were analyzed, and data were used to prepare dendrograms.
Results—308 of 357 (86%) S aureus isolates were susceptible to all antimicrobials evaluated. Forty-nine S aureus isolates were resistant to 1 or more antimicrobials; of these isolates, 37 were resistant only to penicillin, 9 were resistant to penicillin and erythromycin, 2 were resistant to tetracycline, and 1 was resistant to erythromycin. Isolates were assigned to 7 PFGE types. An association was found between PFGE type and antimicrobial susceptibility profile. Organisms with resistance to at least one of the tested antimicrobial agents were identified in only 4 of the 7 types of S aureus.
Conclusions and Clinical Relevance—Antimicrobial resistance was uncommon among the mastitis-causing S aureus isolates identified in the milk samples. A limited number of genotypes were associated with mastitis in these herds. Antimicrobial resistance phenotypes were associated with particular S aureus PFGE types; this association may have implications for future treatment and control of S aureus–associated mastitis in cattle.
