The objective was to study the feasibility of granulocyte macrophage-colony stimulating factor (GM-CSF) delivery to the lung using an aerosol in humans. A Phase I dose escalation study provided GM-CSF at three dose levels as a twice-a-day (BID) x 7 days schedule. Pulmonary functions were monitored using a remote spirometry device. Blood counts were checked at the beginning and end of each week of GM-CSF nebulization. If no toxicity was encountered, patients rested for 7 days and then were treated at the next dose level. Six of seven patients were successfully dose escalated from 60 microg/dose BID x 7 days, to 120 microg/dose BID x 7 days, then 240 microg/dose BID x 7 days. No toxicity was seen. Comparison of day 0 and day 7 blood leukocyte counts showed no significant increases in either leukocyte numbers or percentage of neutrophils. Pulmonary functions test changes were minor. No significant change in forced vital capacity, FEV1, peak flow, or FEF 25-75 related to either time or dose level was observed. One patient's lung metastases progressed. The other five patients received an additional 2-6 months of intermittent aerosol GM-CSF at dose level 3 without side effects. One patient with Ewing's sarcoma has a complete response, and a patient with melanoma had a partial response; the other three had stabilization of pulmonary metastases for 2-6 months. Aerosol delivery of GM-CSF is feasible, safe, and possibly effective. Aerosol cytokine delivery may achieve effective immunological activation against cancer in the lung and is worthy of further study.
Abstract Background In a beef cattle facility an outbreak of abortions occurred over a 36-day period and included two aborted fetuses and 21 post-abortion clinical cases sampled. There are numerous etiologies, including clinical listeriosis. At the species level, Listeria monocytogenes is ubiquitous in cattle production environments, including soil, feed, and occasionally water sources, and is a common enteric resident of cattle and other mammals. There are four genetically distinct lineages of L. monocytogenes (I-IV), with most lineage III and IV isolates obtained from ruminants. Definitive diagnosis of L. monocytogenes as a causative agent in disease outbreaks relies upon case identification, appropriate sample collection, and laboratory confirmation. Furthermore, clearly establishing a relationship between a pathogen source and clinical disease is difficult.Results Of the two fetal and 21 clinical case submissions, 19 were positive for L. monocytogenes. Subsequent culture for L. monocytogenes from water and silage sources identified both as potential origins of infection. Using whole-genome sequencing and phylogenetic analyses, clinical, water and silage L. monocytogenes strains grouped into two of four lineages. All water and silage strains, plus 11 clinical strains placed in lineage III, with identical or nearly identical genomic sequences. The remaining eight clinical strains placed in lineage I, with seven having nearly identical sequences and one distinctly different.Conclusion Three genetically distinct strains within two lineages of L. monocytogenes caused the abortion outbreak. The etiology of abortion in 11 cases was directly linked to water and silage contamination from a lineage III L. monocytogenes strain. The source of infection for the remaining abortion cases with two different strains from lineage I is unknown. This is the first report of L. monocytogenes genomics being used as part of an outbreak investigation of cattle abortion.
Infectious bovine keratoconjunctivitis (IBK) is an economically significant disease caused by Moraxella bovis. Moraxella bovoculi, although not reported to cause IBK, has been isolated from the eyes of cattle diagnosed with IBK. Identification of M. bovis and M. bovoculi can be performed using biochemical or DNA-based approaches, both of which may be time consuming and inconsistent between laboratories. We conducted a comparative evaluation of M. bovoculi and M. bovis identification using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with a database provided by Bruker Daltonics (termed the BDAL database), the BDAL database supplemented with spectra generated in our study (termed the UNLVDC database), and with PCR–restriction-fragment length polymorphism (PCR-RFLP) typing. M. bovoculi ( n = 250) and M. bovis ( n = 18) isolates from cattle with or without IBK were used. MALDI-TOF MS using the UNLVDC database correctly identified 250 of 250 (100%) of M. bovoculi and 17 of 18 (94%) of M. bovis isolates. With the BDAL database, MALDI-TOF MS correctly identified 249 of 250 (99%) of M. bovoculi and 7 of 18 (39%) of M. bovis isolates. In comparison, the PCR-RFLP test correctly identified 210 of 250 (84%) of M. bovoculi and 12 of 18 (66%) of M. bovis isolates. Thus, MALDI-TOF MS with the UNLVDC database was the most effective identification methodology for M. bovis and M. bovoculi isolates from cattle.
PCR primers that target the bacterial 16S rRNA genes (or the tuf gene for the genus Enterococcus) were used to identify 10 putative bacterial pathogens in root canals with necrotic pulp. In addition, the associations of these microorganisms with symptoms and a history of diabetes mellitus were investigated. Microbial samples from the root canals of 24 teeth with necrotic pulp were included in the study. PCR with universal bacterial primers identified bacterial DNA in 22 specimens; the remaining 2 specimens were from intact teeth that had been traumatized 6 months prior to treatment. PCR with specific primers showed that preoperative symptoms were significantly associated with the presence of Streptococcus spp. (P < 0.001 by chi-square analysis). There was also a nonsignificant trend for symptoms to be associated with Fusobacterium nucleatum and Porphyromonas gingivalis (odds ratio, >2) and for diabetes mellitus to be associated with P. gingivalis and Porphyromonas endodontalis (odds ratio, >2). Cloning and sequencing of the universal PCR product in one specimen revealed the presence of an organism related to the genus Olsenella, which has not previously been described in endodontic infections.
Abstract Background In a beef cattle facility an outbreak of abortions occurred over a 36-day period and included samples from two aborted (non-viable) fetuses and 21 post-abortion clinical cases. There are numerous etiologies, including clinical listeriosis. At the species level, Listeria monocytogenes is ubiquitous in cattle production environments, including soil, feed, and occasionally water sources, and is a common enteric resident of cattle and other mammals. There are four genetically distinct lineages of L. monocytogenes (I-IV), with most lineage III and IV isolates obtained from ruminants. Definitive diagnosis of L. monocytogenes as a causative agent in disease outbreaks relies upon case identification, appropriate sample collection, and laboratory confirmation. Furthermore, clearly establishing a relationship between a pathogen source and clinical disease is difficult. Results Of the two fetal and 21 clinical case submissions, 19 were positive for L. monocytogenes . Subsequent culture for L. monocytogenes from water and silage sources identified both as potential origins of infection. Using whole-genome sequencing and phylogenetic analyses, clinical, water and silage L. monocytogenes strains grouped into two of four lineages. All water and silage strains, plus 11 clinical strains placed in lineage III, with identical or nearly identical genomic sequences. The remaining eight clinical strains placed in lineage I, with seven having nearly identical sequences and one distinctly different. Conclusion Three genetically distinct strains within two lineages of L. monocytogenes caused the abortion outbreak. The etiology of abortion in 11 cases was directly linked to water and silage contamination from a lineage III L. monocytogenes strain. The source of infection for the remaining abortion cases with two different strains from lineage I is unknown. This is the first report of L. monocytogenes genomics being used as part of an outbreak investigation of cattle abortion.
Abstract Small ruminant lentiviruses (SRLVs) are prevalent in North American sheep and a major cause of production losses for the U.S. sheep industry. Sheep susceptibility to SRLV infection is influenced by genetic variation within the ovine transmembrane 154 gene ( TMEM154 ). Animals with either of two distinct TMEM154 haplotypes that both encode glutamate at position 35 of the protein (E35) are at greater risk of SRLV infection than those homozygous with a lysine (K35) haplotype. Prior to this study, it was unknown if TMEM154 associations with infection are influenced by SRLV genetic subgroups. Accordingly, our goals were to characterize SRLVs naturally infecting sheep from a diverse U.S. Midwestern flock and test them for associations with TMEM154 E35K genotypes. Two regions of the SRLV genome were targeted for proviral amplification, cloning, sequence analysis, and association testing with TMEM154 E35K genotypes: gag and the transmembrane region of env . Independent analyses of gag and env sequences showed that they clustered in two subgroups (1 and 2), they were distinct from SRLV subtypes originating from Europe, and that subgroup 1 associated with hemizygous and homozygous TMEM154 K35 genotypes and subgroup 2 with hemi- and homozygous E35 genotypes ( gag p < 0.001, env p = 0.01). These results indicate that SRLVs in the U.S. have adapted to infect sheep with specific TMEM154 E35K genotypes. Consequently, both host and SRLV genotypes affect the relative risk of SRLV infection in sheep.
Bovine respiratory disease (BRD) is one of the most costly diseases to cattle production throughout the globe, and losses in the US are estimated at one billion dollars annually. Mannheimia haemolytica is the most commonly detected bacterial agent associated with BRD. Recently, whole genome sequencing was employed on 1,133 M. haemolytica isolates from North American cattle and a nucleotide polymorphism typing system was developed. Two major genotypes were discovered (1 and 2). Genotype 1 M. haemolytica were mostly isolated from the nasopharynx of cattle without BRD. In contrast, genotype 2 M. haemolytica predominantly associated with the lungs of cattle with BRD and integrative conjugative elements that contained antimicrobial resistance determinants. The objective of this study was to develop a rapid matrix-assisted laser desorption/ionization- time of flight mass spectrometry (MALDI-TOF MS) assay for the detection of M. haemolytica genotypes 1 and 2. MALDI-TOF MS is an emerging technology in diagnostic microbiology and is most frequently used for bacterial identification.
