Pulmonary lesions and clinical disease response to Mannheimia haemolytica challenge 10 days following administration of tildipirosin or tulathromycin.

This clinical trial evaluated the impact of metaphylactic antimicrobial administration 10 d before experimental inoculation with Mannheimia haemolytica (MH) to mitigate pulmonary lesions. Thirty-three crossbreed heifers were procured as a single group and were randomly allocated to 1 of 3 blocks and to treatment, tildipirosin (ZUP; 4 mg/kg) or tulathromycin (DRX; 2.5 mg/kg) or saline (SAL; 1 mL/45.5 kg), within block on arrival at Kansas State University. All trial procedures were staggered by 7-d intervals for each block, resulting in all animals within a block receiving treatment, challenge, and necropsy on the same dates. Heifers within each block received an endoscopic MH challenge 10 d following treatment administration (d 0) and were housed in individual indoor stalls for 3 d postchallenge. Clinical illness scores (CIS), respiration quality scores, appetite scores, and injection site reactions were recorded on all animals from d 0 through d 13. Rectal temperatures were measured once daily on all animals from d 8 through d 13. Heifers were necropsied, and lung lesions were evaluated on d 13. Lung lesion data were evaluated using nonparametric methods (Kruskall-Wallis), and standard least squares models were used to evaluate the remaining variables. The pulmonary lesion scores (percentage of affected lung) ranged from 3.3% to 39.8% for all heifers with 92% (11/12) of ZUP-treated heifers having <10% lesions. Tildipirosin-treated heifers had lower (P < 0.05) lung lesion scores when compared with DRX- and SAL-treated heifers. Lung weight expressed as a percentage of BW was lower (P < 0.05) in ZUP heifers compared to DRX- and SAL-treated heifers. The probability of receiving abnormal CIS, appetite scores, and respiratory scores was lower (P < 0.05) in ZUP-treated heifers compared to DRX- and SAL-treated animals. This study showed that heifers treated with tildipirosin 10 d before MH challenge have less pulmonary damage and fewer clinical signs of illness compared to heifers treated with DRX or SAL.

Effect of Mannheimia haemolytica pneumonia on behavior and physiologic responses of calves during high ambient environmental temperatures.

The objective of this study was to determine the effect of pneumonia during conditions of high (maximum ≥ 32°C) ambient temperatures on physiological and behavioral responses of calves. Eighteen black beef heifers averaging 240 kg were blocked by BW and randomly assigned to 1 of 2 treatment groups: 1) pneumonia induced by bronchoselective endoscopic inoculation with Mannheimia haemolytica (MH; n = 10) and 2) noninoculated controls (CN; n = 8). Nasal passage and rectal temperatures were measured every 2 h for 24 h after challenge and then twice daily for 9 d. Accelerometers, pedometers, and positioning devices monitored cattle behavior within the pen for 9 d after challenge. Blood samples were collected on trial d 0, 0.5, 1, 2, 3, 7, and 9 and were analyzed to determine the concentration of substance P, cortisol, haptoglobin, and metalloproteinase. All calves in the MH group were euthanized and necropsied on trial d 9. All MH calves became clinically ill postchallenge. A treatment × time interaction (P < 0.05) was evident for nasal and rectal temperatures, behavior, weight, and blood analysis. Rectal temperatures in MH were higher (P < 0.01) than CN during the period from 6 to 24 h after challenge. Conversely, nasal passage temperatures were less in MH calves compared with CN at 12 to 22 h after challenge. Calves in MH spent less time at the grain bunk, less time at the hay feeder, and more time lying down during the early pneumonia period compared with CN calves. Also, MH calves had significantly greater concentrations of blood biomarkers of pain (substance P) on d 0.5 (P < 0.01); stress (cortisol) on d 0.5 and 1 (P < 0.01); haptoglobin on d 0.5, 1, 2, 3, 7 (P < 0.01); and metalloproteinase on d 1, 2, and 3 (P < 0.01) compared with CN calves. At necropsy, all MH calves had right cranioventral bronchopneumonia (median lung lesions = 6.8%). Mannheimia haemolytica pneumonia caused significantly more changes in behavior and increased biomarkers during high (maximum ≥32°C) ambient temperatures compared with control calves. The results of this study may guide research in the development of objective assessment tools for management of cattle affected with bovine respiratory disease during extreme summer conditions.

Effects of melengestrol acetate on the inflammatory response in heifers challenged with Mannheimia haemolytica.

Previous research from our laboratory has indicated that melengestrol acetate (MGA) added to the diet during the first 35 d after arrival in the feedlot improves growth rates and tends to reduce chronic respiratory disease in heifers naturally challenged with bovine respiratory disease. The current study was conducted to provide further insight into the possible immunomodulatory effects of MGA. Crossbred heifers (n = 48; 232 +/- 5.5 kg of BW) were used in a randomized complete block design to determine the effects of MGA on lung pathology and markers of inflammation after Mannheimia haemolytica challenge. On d 0, cattle were blocked by BW and randomly assigned, within block, to diets (54% concentrate) that provided 0 or 0.5 mg of MGA per heifer daily for the duration of the experiment. Inoculum containing from 1.3 x 10(9) to 1.7 x 10(9) cfu of M. haemolytica (20 mL) was instilled at the bifurcation of the trachea on d 14. Blood samples were collected, clinical observations were made, and rectal temperatures were recorded for each animal at 0, 12, 24, 48, 72, 96, 120, and 138 h after inoculation. Heifers fed MGA had greater circulating concentrations of eosinophils and postchallenge concentrations of segmented neutrophils and white blood cells (P < 0.01) than controls, as well as elevated plasma protein, serum haptoglobin, and fibrinogen after M. haemolytica challenge (P < 0.01). Heifers fed MGA had lower plasma glucose (P < 0.01), greater plasma urea N (P = 0.02), and elevated respiratory indices (P < 0.01) compared with controls. Necropsies performed on d 6 after inoculation suggested that M. haemolytica challenge was relatively mild, because lesions were confined to a small portion of the lungs. On a 0 to 100 scale, average lung lesion scores were 3 and 1 for MGA-fed and control groups, respectively (P < 0.06). Heifers fed MGA before mild M. haemolytica challenge were more susceptible to infection, as evidenced by a greater number of heifers fed MGA exhibiting pulmonary lesions 138 h after inoculation than controls (14 out of 23 vs. 6 out of 24 for MGA and controls, respectively; P < 0.02).

MHCII, Tlr4 and Nramp1 genes control host pulmonary resistance against the opportunistic bacterium Pasteurella pneumotropica.

MHCII, Tlr4, and Nramp1 genes are each independently important in pulmonary immunity. To determine the effect of these genes on host resistance, mice carrying various combinations of functional alleles for these three genes were experimentally challenged with the opportunistic bacterium, Pasteurella pneumotropica. MHCII-/-, Tlr4d/d, and Nramp1s/s mice were significantly more susceptible to experimental infections by P. pneumotropica after intranasal challenge compared to mice carrying functional alleles at only one of those genes. P. pneumotropica were cultured from the lungs of challenged mice, and the severity of the pneumonia strongly correlated with the number of isolated bacteria. Mice with the genotype MHCII-/- Tlr4n/n genotype were less susceptible to pneumonia than MHCII+/+, Tlr4d/d mice. It is interesting that the Nramp1 gene contribution to host resistance was apparent only in the absence of functional MHCII or Tlr4 genes. These data suggest that MHCII, Tlr4, and Nramp1 genes are important to pulmonary bacterial resistance.

FnrP interactions with the Pasteurella haemolytica leukotoxin promoter.

Pasteurella haemolytica FnrP is homologous to Fnr, the global transcriptional regulator of anaerobic respiration in Escherichia coli. To investigate the role of O(2) in the expression of P. haemolytica leukotoxin, we tested a lktC::lacZ fusion constructed in E. coli for a FnrP-mediated regulatory effect under aerobic and anaerobic growth conditions. Both E. coli Fnr and FnrP suppressed leukotoxin transcription under aerobic conditions. Under anaerobic conditions, Fnr suppressed transcription, while FnrP increased transcription. These results were confirmed using FnrP*, a mutant form of FnrP that activates anaerobically inducible genes under aerobic conditions. In mobility shift assays, partially purified FnrP bound to a potential regulatory site in a P. haemolytica lktC promoter fragment.

Cryptosporidiosis. A global challenge.

During the last 30 years, our concept of cryptosporidiosis has changed from that of a rare, largely asymptomatic disease, to an important cause of diarrhea in animals and humans worldwide. Significant disease first appeared in cattle. Subsequently, the zoonotic danger of the organism was recognized in HIV-infected persons and young children. Cryptosporidium are now ubiquitous and disease has been described in over 79 host species. Cryptosporidiosis has become a major cause of calfhood diarrhea worldwide. In humans it accounts for up to 20% of all cases of childhood diarrhea in developing countries and is a potentially fatal complication of AIDS. Waterborne contamination is a growing concern as a source of widespread outbreaks of disease. Factors that have contributed to the emergence of cryptosporidiosis in animals include biological features of the organism, the lack of an effective treatment or preventative, increased environmental contamination, and trends in livestock production. In humans the zoonotic nature of infection and an increased at-risk population have contributed to disease. Genetic characterization of Cryptosporidium, improved detection methods, and a better understanding of the factors that predispose to disease are important contributions to understanding the epidemiology of cryptosporidiosis.

Cloning and characterization of the gene encoding Pasteurella haemolytica FnrP, a regulator of the Escherichia coli silent hemolysin sheA.

A Pasteurella haemolytica A1 gene was identified from a recombinant library clone that expressed hemolysis in host Escherichia coli cells. The gene, designated fnrP, had sequence identity to E. coli fnr, a global transcriptional regulator of genes required for conversion to anaerobic growth. FnrP complemented anaerobic deficiencies of a fnr-null mutant strain of E. coli and increased expression of the Fnr-dependent, anaerobic terminal reductase gene, frdA. FnrP was purified, identified by immunoblotting, and shown to be nonhemolytic. When FnrP was expressed in E. coli DeltasheA, a null mutant of the cryptic hemolysin SheA, the transformants were nonhemolytic, indicating that FnrP activates this silent hemolysin.

Actin polymerization enhances Pasteurella haemolytica leukotoxicity.

Pasteurella haemolytica leukotoxin is cytotoxic to bovine leukocytes, causing increased cell membrane permeability, osmotic swelling, release of cytosolic proteins and cell lysis. These studies were designed to test if leukotoxin causes release of the cytoskeletal protein, actin, from bovine leukemia cells and if purified actin-influenced bacterial growth or leukotoxin production. Culture supernatants caused a 7-fold decrease in viability of bovine leukemia cells and increased cell permeability that was accompanied by release of beta-actin into the cell culture supernatant. Exposing P. haemolytica to purified actin solutions induced the conversion of monomeric G-actin to polymerized F-actin. This conversion was partially inhibited by bovine P. haemolytica immune, but not pre-immune, serum. Loss of streptomycin resistance following treatment of the organism with acridine orange ablated the polymerizing activity. Incubation of P. haemolytica in the presence of purified F-actin did not affect growth but resulted in culture supernatant that had 3.0-3.9-fold greater leukotoxicity compared to medium alone or medium containing G-actin, heat-denatured actin or albumin. The effect of actin on leukotoxicity was concentration-dependent and directly associated with increases in secreted leukotoxin. The interaction between P. haemolytica and actin is potentially detrimental to the host by inducing polymerization of actin into insoluble filaments and by enhancing leukotoxicity.

Actin enhances the haemolytic activity of Escherichia coli.

Actin is a major cytoskeletal protein of mammalian muscle and non-muscle cells. Exposure of cells to soluble factors that damage cell membranes results in the release of actin into the extracellular spaces. The alpha-haemolysin (HlyA) of Escherichia coli is the prototype RTX (repeat in toxin) toxin and is thought to be important in virulence because of its ability to lyse cells by formation of pores in the cell membrane. These studies were conducted to determine if actin influences growth and haemolytic activity of E. coli. Growth of E. coli in the presence of actin resulted in culture supernatant haemolytic activity that was 2.4-, 2.7- and 3.3-fold greater than that of E. coli grown in medium containing BSA, non-supplemented medium, or medium containing heat-denatured actin, respectively. The enhanced haemolytic activity occurred only when actin was present during the growth phase and there was no effect when actin was added to culture supernatants containing haemolysin. The increased haemolytic activity by actin was concentration-dependent, detectable in early-exponential-phase growth, and associated with increased concentrations of secreted HlyA by Western blotting. Actin induced a 2.9-fold increase in alkaline phosphatase activity in E. coli CC118 with a TnphoA insertion in the hlyB determinant of the recombinant haemolysin plasmid pWAM04. These results indicate that extracellular actin enhances haemolysin production by E. coli and may have implications in the pathogenesis of E. coli infections.

Comparison of serologic and protective responses induced by two Pasteurella vaccines.

Vaccine development for the prevention of pneumonic pasteurellosis remains a critical issue for the feedlot industry. Most currently available Pasteurella vaccines are formulated to stimulate immunity by either providing an adequate antigenic mass in the administered dose, or by relying on subsequent production of antigens by in vivo growth of live organisms. The ability of these different types of vaccines to stimulate rapid and high titres to key antigens is a key factor that will influence subsequent resistance to disease. The serologic and protective responses to a streptomycin-dependent, modified-live vaccine and a killed (bacterin-toxoid) vaccine against experimental pneumonic pasteurellosis were compared. Calves were vaccinated with a single injection of either a test vaccine or phosphate-buffered saline, challenged 14 d later by transthoracic injection with Pasteurella haemolytica, and euthanized 3 d post-challenge to evaluate the severity of pneumonia. On days 0, 7, and 14, serologic responses to various P. haemolytica antigens, including cell-associated and soluble antigens, were determined by enzyme-linked immunosorbent assays, and anti-leukotoxin antibody levels were determined by leukotoxin neutralization. The bacterin-toxoid elicited significantly greater serologic responses compared to controls for all antigens. The modified-live vaccine elicited a significantly greater response compared to controls for a whole-cell antigen preparation. Lesion scores were significantly smaller (greater protection) in calves that received the bacterin-toxoid, but not the modified-live vaccine, compared to controls.

In vivo effect of Pasteurella haemolytica infection on bovine neutrophil morphology.

OBJECTIVE: To determine whether characteristic changes in neutrophil morphology caused in vitro by Pasteurella haemolytica leukotoxin (LKT) can be observed in vivo by electron microscopic examination of infected tissue chamber fluids and pneumonic lungs. ANIMALS: 7 mixed-breed beef calves. PROCEDURE: Tissue chambers were implanted subcutaneously in 3 calves and were inoculated with P haemolytica or phosphate-buffered saline solution. Chamber fluid samples, obtained at 8 and 32 hours after inoculation, were examined, using electron microscopy. Experimental pneumonia was induced in an additional 4 calves by transthoracic inoculation with P haemolytica. These calves were euthanatized at 6, 12, 24, and 36 hours after inoculation and lung sections were examined, using transmission electron microscopy. RESULTS: On examination, using transmission electron microscopy, neutrophils in lung sections and tissue chamber fluids had cytoplasmic and nuclear changes indicative of irreversible cell injury, including cell swelling, loss of plasma membrane ruffling, mitochondrial swelling, autolytic vacuolation, disruption of plasma membrane, nuclear pyknosis, karyolysis, and karyorrhexis. On examination, using scanning electron microscopy, leukocytes obtained from tissue chambers did not have their typical convoluted surfaces, but appeared rounded and swollen or shrunken with pitted surfaces. CONCLUSIONS: Pasteurella haemolytica-induced changes in neutrophil morphology in vivo were similar to those previously induced by in vitro exposure of neutrophils to LKT. Changes were suggestive of injury initiated by damage to the plasma membrane, which is consistent with the mechanism of action of pore-forming cytolysins. CLINICAL RELEVANCE: Pasteurella haemolytica LKT appears to be an important virulence factor in vivo; a fact that should be addressed in the development of vaccines.

Bacterial pneumonia.

Bacteria play a critical role in the severe pneumonia and fatalities associated with the bovine respiratory disease complex. Although numerous bacteria have the potential to cause pneumonia, only a small number of these are responsible for the majority of cases of disease. Virulence and immunogenic characteristics of these organisms are important determinants of the host response to infection. These bacterial characteristics are reviewed and applied to a discussion of the epidemiology, pathogenesis, and prevention of bacterial pneumonia is also discussed.

Experimental cryptosporidiosis in adult and neonatal rabbits.

Neonatal and adult New Zealand White rabbits were infected experimentally with Cryptosporidium parvum. No histologic evidence of infection was found in adult rabbits. However, increased levels of anti-cryptosporidial serum IgG were present, and multiple antigens were detected by serum on immunoblots. In neonates, variably severe, transient infection was present from Days 3 to 21 postinoculation. Serum IgG was initially elevated, decreased until Day 10 postinoculation, then progressively increased for the remainder of the study. A prominent 15 kilodalton antigen was detected on immunoblots using serum obtained on Days 14 until 28 postinoculation. Neonate anti-cryptosporidial fecal IgA were slightly elevated on Days 14 and 21 postinoculation.

Differentiation of Cryptosporidium parvum, C. muris, and C. baileyi by PCR-RFLP analysis of the 18S rRNA gene.

We have developed a combined polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) assay for the detection and differentiation of Cryptosporidium parvum, Cryptosporidium muris, and Cryptosporidium baileyi. The assay utilizes PCR to amplify an approximately 1750 basepair product of the Cryptosporidium 18S rRNA gene. Following double digestion with restriction endonucleases Dral and Vsp1, this PCR product yields DNA fragments that can be resolved electrophoretically into RFLP profiles that are distinct for C. parvum, C. muris, and C. baileyi. Previous PCR-restriction analyses could not simultaneously differentiate all three species. Future application of this technique could include predicting the disease-causing potential of oocyst-contaminated environmental specimens and helping to determine the source of oocyst contamination.

Simplified method for recovery and PCR detection of Cryptosporidium DNA from bovine feces.

An assay to identify Cryptosporidium DNA in bovine feces has been developed emphasizing standardization of sample preparation and simplification of the DNA recovery process for PCR amplification and DNA hybridization detection. The Cryptosporidium DNA recovery-PCR detection procedure (CR-PCR) can recover DNA suitable for PCR amplification without using or generating hazardous chemicals or wastes. In comparisons with a commercial enzyme-linked immunoassay (Color Vue-Cryptosporidium; Seradyn, Indianapolis, Ind.), the CR-PCR could detect 10(3) to 10(4) times fewer purified oocysts diluted in solution (water or buffered saline) and 10(2) times fewer oocysts from diarrheic feces and showed earlier detectability from solid, nondiarrheic feces in an experimental infection. This assay may prove useful for detecting Cryptosporidium oocysts in feces and in clarifying the role of livestock in waterborne outbreaks of cryptosporidiosis.

A Cryptosporidium parvum genomic region encoding hemolytic activity.

Successful parasitization by Cryptosporidium parvum requires multiple disruptions in both host and protozoan cell membranes as cryptosporidial sporozoites invade intestinal epithelial cells and subsequently develop into asexual and sexual life stages. To identify cryptosporidial proteins which may play a role in these membrane alterations, hemolytic activity was used as a marker to screen a C. parvum genomic expression library. A stable hemolytic clone (H4) containing a 5.5-kb cryptosporidial genomic fragment was identified. The hemolytic activity encoded on H4 was mapped to a 1-kb region that contained a complete 690-bp open reading frame (hemA) ending in a common stop codon. A 21-kDa plasmid-encoded recombinant protein was expressed in maxicells containing H4. Subclones of H4 which contained only a portion of hemA did not induce hemolysis on blood agar or promote expression of the recombinant protein in maxicells. Reverse transcriptase-mediated PCR analysis of total RNA isolated from excysted sporozoites and the intestines of infected adult mice with severe combined immunodeficiency demonstrated that hemA is actively transcribed during the cryptosporidial life cycle.

Passive protection of calves with Pasteurella haemolytica antiserum.

Four colostrum-deprived calves each were immunized passively with antisera to whole Pasteurella haemolytica, leukotoxin-containing supernatants of P haemolytica, P haemolytica lipopolysaccharide, or newborn calf serum. Calves were challenge exposed intrabronchially with 5 x 10(9) P haemolytica, and 24 hours later, the resulting lesions were evaluated. The greatest protection against challenge exposure was provided by the antiserum to whole P haemolytica (lesion score = 6.3), whereas newborn calf serum provided the least protection (lesion score = 28.3). Calves that received antiserum to P haemolytica supernatants were moderately protected (lesion score = 16.3), and the antiserum to lipopolysaccharide provided minimal protection (lesion score = 21.8). Antibodies that were unique to whole P haemolytica antiserum and produced dense bands on immunoblots were detected to antigens at 66, 50, and 30 kd. Antibodies in the supernatant preparation that produced prominent bands reacted to antigens between 100 and 90 kd. Collectively, antibodies to these antigens may be responsible for enhancing resistance to experimentally induced pneumonic pasteurellosis. Antibodies to antigens in P haemolytica lipopolysaccharide provided little to no protection.