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During the United States Department of Agriculture (USDA) National Animal Health Monitoring System's (NAHMS) 2007-2008 beef study, 567 producers from 24 US States were offered the opportunity to collect fecal samples from weaned beef calves and have them evaluated for the presence of parasite eggs (Phase 1). Participating producers were provided with instructions and materials for sample collection. Up to 20 fresh fecal samples were collected from each of the 99 participating operations. Fresh fecal samples were submitted to one of 3 randomly assigned laboratories for evaluation. Upon arrival at the laboratories, all samples were processed for the enumeration of strongyle, Nematodirus, and Trichuris eggs using the modified Wisconsin technique. The presence or absence of coccidian oocysts and tapeworm eggs was also noted. In submissions where the strongyle eggs per gram exceeded 30, aliquots from 2 to 6 animals were pooled for DNA extraction. Extracted DNA was subjected to genus level polymerase chain reaction (PCR) identification for the presence of Ostertagia, Cooperia, Haemonchus, Oesophagostomum, and Trichostrongylus. In this study, 85.6% of the samples had strongyle type, Nematodirus, and Trichuris eggs. Among the samples evaluated, 91% had Cooperia, 79% Ostertagia, 53% Haemonchus, 38% Oesophagostomum, 18% Nematodirus, 7% Trichuris, and 3% Trichostrongylus. The prevalence of coccidia and tapeworm eggs was 59.9% and 13.7%, respectively.

Four species of Cryptosporidium are routinely found in cattle: Cryptosporidium parvum, Cryptosporidium bovis, Cryptosporidium ryanae, and Cryptosporidium andersoni. It is important to determine the species of Cryptosporidium in infected cattle because C. parvum is the only serious pathogen for humans as well as cattle. Identification of Cryptosporidium species and genotypes currently relies on molecular methods such as polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) or gene sequencing. Incorporation of these techniques in a routine veterinary diagnostic laboratory is cost prohibitive. As such, their applications are limited primarily to research and a few public health laboratories. To overcome this problem, a multiplex PCR assay was developed for simultaneously detecting the 4 species of Cryptosporidium that commonly infect cattle. This assay specifically identifies Cryptosporidium oocysts present in cattle feces, improves the detection of mixed infections, reduces the time and cost relative to current sequencing methods, and further demonstrates the shortcomings of sequencing as the definitive method for identification when analyzing samples containing mixed infections.

CD14 is a high-affinity receptor protein for the complex of bacterial LPS (LPS) and LPS binding protein in animals. Binding of the soluble form of CD14 (sCD14) to LPS, found in the outer membrane of Escherichia coli and other Gram-negative bacteria, enhances host innate immune responses, reduces the severity of mastitis, and facilitates clearance and neutralization of LPS, thus protecting against an excessive immune response to LPS and development of endotoxic shock. A truncated form of sCD14, carrying a histidine residue affinity tag for purification, was incorporated into Potato virus X for transient expression in Nicotiana benthamiana plants. Western blots probed with CD14-specific antibodies demonstrated that crude plant extracts and affinity-purified samples contained immunoreactive sCD14. Biological activity of plant-derived recombinant bovine sCD14 (PrbosCD14) was demonstrated in vitro by LPS-induced apoptosis and interleukin (IL) -8 production in bovine endothelial cells, and in vivo by enhancement of LPS-induced neutrophil recruitment. Finally, in PrbosCD14-infused glands subsequently infected with E. coli, lower numbers of viable bacteria were recovered and there was an absence of clinical symptoms, demonstrating prophylactic efficacy of PrbosCD14. This is the first report of a functionally active animal receptor protein made in plants and the prophylactic use of a plant-derived protein to reduce the severity of bacterial infections in animals.

A multiplex PCR assay for differentiating strongyle eggs from cattle has recently been described; however, the egg disruption and DNA extraction procedures, though effective, are inadequate for large studies or clinical application. The purpose of this research was to evaluate methods for disrupting trichostrongyle eggs, then assess commercial kits for extracting egg DNA using Ostertagia ostertagi as a model species. Egg disruption procedures tested included probe sonication, bath sonication, bead beating, boiling, microwaving, proteinase K/SDS digestion, freezing, and various combinations of the above with the incorporation of sodium dodecyl sulfate. These procedures were evaluated in conjunction with four commercial DNA extraction kits: DNA Stool mini kit and DNeasy Plant kit (Qiagen), Fast DNA kit (QBiogene), and the MAP extraction kit (Tetracore). Results showed that egg disruption was best accomplished with the bead beater and ceramic beads, resulting in 100% disruption within 1min. When DNA extraction was preceded by the isolation of eggs from feces, all procedures except the Fast DNA kit produced PCR-ready DNA from at least two eggs. The DNeasy Plant kit allowed consistent detection of DNA released from one egg. Due to the morphological similarities among trichostrongyle eggs in ruminants, strongyle eggs in equids, and hookworm eggs, the methods described herein may have broad application to other nematodes.

Objective: To characterize cytokine messenger RNA (mRNA) expression in intranasally vaccinated calves after bovine respiratory syncytial virus (BRSV) challenge., Animals: Twelve 8- to 12-week-old calves., Procedures: Calves received modified-live BRSV vaccine (vaccinated) or spent tissue culture medium (mock-vaccinated) intranasally, followed by challenge 30 days later with BRSV, or mock challenge with spent tissue culture medium (mock-challenge controls). Interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) mRNA was measured in lungs, bronchoalveolar lavage (BAL) fluid cells, pharyngeal tonsils, and tracheobronchial lymph nodes, and tumor necrosis factor-alpha (TNF-alpha) mRNA was measured in lungs and BAL fluid cells by reverse transcriptase-competitive polymerase chain reaction assay., Results: Resistance to clinical signs of disease was conferred in vaccinated calves. Expression of TNF-alpha mRNA in lungs and BAL fluid cells was higher in mock-vaccinated calves than control or vaccinated calves. In the lung, IL-4 mRNA expression was higher in vaccinated calves than control or mock-vaccinated calves. In pharyngeal tonsils, expression of mRNA for IL-4 and IFN-gamma was higher in mock-vaccinated calves than control calves. In tracheobronchial lymph nodes, IFN-gamma mRNA expression was higher in mock-vaccinated calves than vaccinated calves., Conclusions and Clinical Relevance: Although vaccinated calves had decreased clinical signs of disease after BRSV challenge, compared with mock-vaccinated calves, this difference was not related to a T helper type 1 bias, as determined by increased expression of interferon-gamma mRNA relative to interleukin-4 mRNA in lungs, BAL fluid cells, or tracheobronchial lymph nodes of vaccinated calves. Pulmonary inflammation was decreased in vaccinated calves as determined by decreased expression of TNF-alpha mRNA.

Background: Cooperia oncophora and Ostertagia ostertagi are among the most important gastrointestinal nematodes of cattle worldwide. The economic losses caused by these parasites are on the order of hundreds of millions of dollars per year. Conventional treatment of these parasites is through anthelmintic drugs; however, as resistance to anthelmintics increases, overall effectiveness has begun decreasing. New methods of control and alternative drug targets are necessary. In-depth analysis of transcriptomic data can help provide these targets. Results: The assembly of 8.7 million and 11 million sequences from C. oncophora and O. ostertagi, respectively, resulted in 29,900 and 34,792 transcripts. Among these, 69% and 73% of the predicted peptides encoded by C. oncophora and O. ostertagi had homologues in other nematodes. Approximately 21% and 24% were constitutively expressed in both species, respectively; however, the numbers of transcripts that were stage specific were much smaller (~1% of the transcripts expressed in a stage). Approximately 21% of the transcripts in C. oncophora and 22% in O. ostertagi were up-regulated in a particular stage. Functional molecular signatures were detected for 46% and 35% of the transcripts in C. oncophora and O. ostertagi, respectively. More in-depth examinations of the most prevalent domains led to knowledge of gene expression changes between the free-living (egg, L1, L2 and L3 sheathed) and parasitic (L3 exsheathed, L4, and adult) stages. Domains previously implicated in growth and development such as chromo domains and the MADF domain tended to dominate in the free-living stages. In contrast, domains potentially involved in feeding such as the zinc finger and CAP domains dominated in the parasitic stages. Pathway analyses showed significant associations between life-cycle stages and peptides involved in energy metabolism in O. ostertagi whereas metabolism of cofactors and vitamins were specifically up-regulated in the parasitic stages of C. oncophora. Substantial differences were observed also between Gene Ontology terms associated with free-living and parasitic stages Conclusions: This study characterized transcriptomes from multiple life stages from both C. oncophora and O. ostertagi. These data represent an important resource for studying these parasites. The results of this study show distinct differences in the genes involved in the free-living and parasitic life cycle stages. The data produced will enable better annotation of the upcoming genome sequences and will allow future comparative analyses of the biology, evolution and adaptation to parasitism in nematodes. [ABSTRACT FROM AUTHOR]

Abstract: Gram-negative bacteria are responsible for approximately one-third of the clinical cases of bovine mastitis and can elicit a life-threatening, systemic inflammatory response. Lipopolysaccharide (LPS) is a membrane component of Gram-negative bacteria and is largely responsible for evoking the inflammatory response. Antibiotic and anti-inflammatory therapy for treating Gram-negative infections remains suboptimal. Bactericidal/permeability-increasing protein (BPI) is a neutrophil-derived protein with antimicrobial and LPS-neutralizing properties. Select peptide derivatives of BPI are reported to retain these properties. The objective of this study was to evaluate the antimicrobial activity of a human BPI-derived synthetic peptide against clinical bovine mastitis isolates of Gram-negative bacteria. A hybrid peptide was synthesized corresponding to two regions of human BPI (amino acids 90–99 and 148–161), the former of which has bactericidal activity and the latter of which has LPS-neutralizing activity. The minimum inhibitory (MIC) and bactericidal (MBC) concentrations of this peptide against various genera of bacteria were determined using a broth microdilution assay. The MIC''s were determined to be: 16–64μg/ml against Escherichia coli; 32–128μg/ml against Klebsiella pneumoniae and Enterobacter spp.; and 64–256μg/ml against Pseudomonas aeruginosa. The MBC''s were equivalent to or 1-fold greater than corresponding MIC''s. The peptide had no growth inhibitory effect on Serratia marcescens. The antimicrobial activity of the peptide was retained in the presence of serum, but severely impaired in milk. Further functional evaluation of the peptide demonstrated its ability to completely neutralize LPS. Together, these data support additional investigations into the therapeutic application of BPI to the treatment of Gram-negative infections in cattle. [Copyright &y& Elsevier]