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This study demonstrates the detection and dynamics of macrocyclic trichothecene mycotoxin (MTM) tissue loading using a commercially available assay in a goat model. The detection of MTMs has been difficult and complex due to the uncertainty of what tissues to examine and when to sample. Twelve goats (two groups of each) were instilled with Stachybotrys chartarum conidial suspension via the trachea. The first group was challenged repeatedly with fungal conidia containing 1 mg/kg of MTM per instillation whereas the second group was exposed once, to spores with a calculated concentration of 5 microg/kg of mycotoxin. These toxin estimates were generated by the QuantiTox(TM) Kit assay; a conidium of S. chartarum possessed 8.5 pg of MTM. After repeated exposure of 3 days, MTM was detected in one of six animals. This animal and two others from the same group had mycotoxin detected in their serum 24 hours after challenge at a comparable level (1.69 ng/mL) to the six animals challenged with a single dose (2.02 ng/mL) at the same time post-instillation. Results showed that MTMs are detectable in experimental animals soon after challenge and contribute to the understanding of the role of these mycotoxins in the disease process following mold exposure.

Objective: To investigate the effects of sterile fine dust aerosol inhalation on antibody responses and lung tissue changes induced by Mucor ramosissimus or Trichoderma viride spores following intratracheal inoculation in goats., Animals: 36 weanling Boer-Spanish goats., Procedures: 6 goats were allocated to each of 2 M ramosissimus-inoculated groups, 2 T viride-inoculated groups, and 2 control (tent or pen) groups. One of each pair of sporetreated groups and the tent control group were exposed 7 times to sterilized fine feedyard dust (mean+/-SD particle diameter, <7.72+/-0.69 microm) for 4 hours in a specially constructed tent. Goats in the 4 fungal treatment groups were inoculated intratracheally 5 times with a fungal spore preparation (30 mL), whereas tent control goats were intratracheally inoculated with physiologic saline (0.9% NaCl) solution (30 mL). Pen control goats were not inoculated or exposed to dust. Goats received an IV challenge with equine RBCs to assess antibody responses to foreign antigens. Postmortem examinations were performed at study completion (day 68) to evaluate lung tissue lesions., Results: 5 of 7 deaths occurred between days 18 and 45 and were attributed to fine dust exposures prior to fungal treatments. Fine dust inhalation induced similar lung lesions and precipitating antibodies among spore-treated goats. Following spore inoculations, dust-exposed goats had significantly more spores per gram of consolidated lung tissue than did their nonexposed counterparts., Conclusions and Clinical Relevance: Fine dust inhalation appeared to decrease the ability of goats to successfully clear fungal spores from the lungs following intratracheal inoculation.

Objective: To determine effects of repeated aerosol exposures to fly ash dust on respiratory tracts of tent-confined goats., Animals: 12 weanling Boer-Spanish crossbred goats., Procedure: Goats were randomly assigned to 2 groups: fly ash treatment group (principal goats, n = 6) or control group (control goats, 6). Aerosolized fly ash dust was provided during a 4-hour period for each of 6 applications given over 3 months and one 2-hour application prior to necropsy. Fly ash particle diameters ranged from 0.1 to 130 microm and averaged 17.8 microm, with 1.5% of fly ash particles in the 0.1- to 5-microm-diameter range. A mean +/- SD of 748 +/- 152 g/treatment was delivered inside a tent containing principal goats; control goats were placed inside a similar tent for 4-hour treatments without dust. Following treatment, rectal temperatures were taken at 0, 4, 6, 8, 24, and 72 hours; Hcts were recorded at 0, 24, and 72 hours., Results: Rectal temperatures were significantly increased at 4, 6, and 8 hours and decreased at 72 hours, compared with 0 hours. Mean +/- SEM Hct values were significantly increased for principal goats (3747 +/- 0.39%), compared with control goats (36.17 +/- 0.42%). A significant increase in the mean area of gross atelectatic lung lesions (1,410 mm2) was found in principal goats (n = 6), compared with control goats (440 mm2; 5)., Conclusions and Clinical Relevance: An increase in atelectatic lung lesions was observed in principal goats, compared with control goats; however, overall, fly ash dust effects were nontoxic.

Objective: To compare the virulence of spores of 7 fungi by tracheal inoculation of goats following exposure of goats to an aerosol of sterilized feedyard dust. Animals-54 weanling Boer-Spanish goats., Procedure: A prospective randomized controlled study was conducted. There were 7 fungal treatment groups, a tent control group, and a pen control group (n = 6 goats/group). Goats in the 7 treatment and tent control groups were exposed to autoclaved aerosolized feedyard dust for 4 hours in a specially constructed tent. Goats in the 7 treatment groups were then inoculated intratracheally with 30 mL of a fungal spore preparation, whereas tent control goats were intratracheally inoculated with 30 mL of physiologic saline (0.9% NaCI) solution. These treatments were repeated each week for 6 weeks., Results: Severity of pathologic changes differed significantly among the 7 fungal treatment groups as determined on the basis of gross atelectatic and consolidated lung lesions and histologic lesions of the lungs. Descending order for severity of lesions was Mucor ramosissimus, Trichoderma viride, Chaetomium globosum, Stachybotrys chartarum, Aspergillus fumigatus, Penicillium chrysogenum, and Monotospora lanuginosa. Trichoderma viride spores were the most invasive and were isolated from the bronchial lymph nodes and thoracic fluid of all 6 goats administered this organism. Spores were observed-histologically in lung tissues harvested 72 hours after inoculation from all treatment groups., Conclusions and Clinical Relevance: 4 of 7 fungal spore types induced significantly larger lung lesions, compared with those induced by the other 3 spore types or those evident in control goats.

Objective: To determine the effect of transportation stress on serum concentrations of oxidative stress biomarkers of calves., Animals: 105 crossbred beef steer calves (mean [+/-SD] body weight, 207 +/- 21.2 kg)., Procedure: Calves were assembled at 1 location in Tennessee, and pretransit (day -3) blood samples were collected. Calves were allotted randomly by body weight into 2 groups. Calves were transported 1,930 miles to a feedlot in Texas, and 1 group received tilmicosin phosphate (33 microg/kg, s.c.) upon arrival. Calves were weighed and blood samples collected on the day of arrival (day 1) and on days 15, 22, and 28. Calves were scored daily for signs of bovine respiratory disease (BRD). Serum total antioxidant capacity (TACA) and serum malondialdehyde (MDA) concentrations were determined., Results: Transportation stress significantly decreased mean serum TACA concentrations (from 147 +/- 31.2 U/mL to 133 +/- 20.1 U/mL) and significantly increased serum MDA concentrations (from 10.9 +/- 18.3 microg/mL to 30.2 +/- 50.5 microg/mL). Calves that died had a 43% increase in serum MDA concentration on day 1, compared with calves that lived (42.2 +/- 67.0 microg/mL vs 29.4 +/- 49.4 microg/mL, respectively). Calves that had > or =3 episodes of BRD had 2-fold higher serum MDA concentrations on day 1 than healthy calves. Tilmicosin-treated calves had a 20.8% significantly greater average daily gain and significantly greater serum TACA concentration than nontreated calves on day 28., Conclusions and Clinical Relevance: Transportation stress increases serum concentrations of oxidative stress biomarkers that are related to episodes of BRD and mortality in calves.

Dust is an environmental stressor and can become extensive in agricultural production systems. Thirty-six female, Spanish goats (average BW 21.1 kg, SEM = 1.31; age = 4 mo) were randomly assigned to simulated dust events or no dust, with or without tilmicosin phosphate treatment in a 2 x 2 factorial arrangement of treatments to determine effects on performance, rectal temperature, and leukocyte changes. All goats were fed a standard growing diet (13.6% CP) consisting of 37% roughage and 63% concentrate (DM basis). Feed intake was measured daily, and BW (unshrunk) measured individually every 7 d. The tilmicosin-treated group received tilmicosin phosphate (10 mg/kg BW s.c.) before starting the study. Goats exposed to dust were enclosed as a group inside a canvass tent for 4 h each day and ground feed yard manure dust (mean particle size 100 microm) was aerosolized inside the tent to simulate a dust event. There was one single dust event (Phase I) followed by rectal temperature measurement, and heparinized blood collection for complete cell counts at 0 (pretrial), 4, 12, 20, 44, 68, and 210 h after dust exposure. This was followed by 21 d of chronic dust events (Phase II). The sampling procedures for Phase II were exactly the same as in Phase I, except that samples were obtained daily at 0 (before dust application), 4, 8, and 12 h after each dust event. Dust treatment had no effect (P > 0.05) on feed intake or ADG, but the gain:feed (G:F) ratio was lower (P < 0.05) in the control goats than the dust exposed group. Tilmicosin phosphate-treated goats had a higher (P < 0.05) G:F ratio than untreated goats. Dust exposure increased (P < 0.002), but tilmicosin treatment decreased (P < 0.05) rectal temperature at 4 and 8 h. Dust exposure increased (P < 0.02) blood lymphocyte counts compared with controls. These results suggest that simulated dust events altered rectal temperature and leukocyte counts of goats.

Objectives: To determine the bacterial, fungal, and endotoxin concentrations in aerosolized ambient air during the winter and summer in feedyards located in the Southern High Plains, identify aerosolized microbial pathogens, and determine the size of microbial and dust components., Sample Population: Aerosol samples were obtained from 7 feedyards., Procedure: Aerosol samples were collected upwind, on-site, and downwind from each feedyard at a point 1 m above the ground by use of biological 2- and 6-stage cascade impactors., Results: Significantly more microbes were cultured from on-site and downwind samples than upwind samples. There were significantly more microbes during the summer than during the winter. However, mean endotoxin concentration was significantly higher during the winter (8.37 ng/m3) than the summer (2.63 ng/m3). Among 7 feedyards, mean +/- SE number of mesophilic bacteria (1,441 +/- 195 colony-forming units [CFUs]/m3) was significantly higher than mean number of anaerobic bacteria (751 +/- 133 CFUs/m3) or thermophilic bacteria (54 +/- 10 CFUs/m3) in feedyard air. Feedyard aerosol samples contained more mesophilic fungi (78 +/- 7 CFUs/m3) than thermophilic fungi (2 +/- 0.2 CFUs/m3). Eighteen genera of bacteria were identified by use of an automated identification system., Conclusions and Clinical Relevance: It appeared that gram-negative enteric pathogens offered little risk to remote calves or humans via ambient aerosols and that gram-positive pathogens of the Bacillus, Corynebacterium, and Staphylococcus spp can be spread by aerosols in and around feedyards. It was common to detect concentrations of endotoxin in the ambient air of 7 feedyards.

Objective: To detect bovine adenovirus serotype 7 (BAV-7) infections in calves by use of viral isolation and serologic testing., Animals: 205 postweaning calves., Procedure: 121 calves were assembled by an order buyer through auction markets in eastern Tennessee and transported to New Mexico where they were commingled with 84 healthy ranch-reared calves. Tests included viral isolation in cell culture from peripheral blood leukocytes (PBL) and detection of serum BAV-7 antibodies by use of microtitration viral neutralization., Results: BAV-7 was isolated from PBL of 8 calves and seroconversion to BAV-7 was detected for 38 of 199 (19.1%) calves. Concurrent bovine viral diarrhea virus infections were detected in most calves from which BAV-7 was isolated., Conclusions and Clinical Relevance: Results of our study indicate that BAV-7 infections can be found in postweaning commingled calves and may develop more commonly in calves with concurrent infections with viruses such as bovine viral diarrhea virus (BVDV).

The prevalence of bovine viral diarrhea virus (BVDV) infections was determined in 2 groups of stocker calves with acute respiratory disease. Both studies used calves assembled after purchase from auction markets by an order buyer and transported to feedyards, where they were held for approximately 30 d. In 1 study, the calves were mixed with fresh ranch calves from a single ranch. During the studies, at day 0 and at weekly intervals, blood was collected for viral antibody testing and virus isolation from peripheral blood leukocytes (PBLs), and nasal swabs were taken for virus isolation. Samples from sick calves were also collected. Serum was tested for antibodies to bovine herpesvirus-1 (BHV-1), BVDV1a, 1b, and 2, parainfluenza 3 virus (PI3V), and bovine respiratory syncytial virus (BRSV). The lungs from the calves that died during the studies were examined histopathologically, and viral and bacterial isolation was performed on lung homogenates. BVDV was isolated from calves in both studies; the predominant biotype was noncytopathic (NCP). Differential polymerase chain reaction (PCR) and nucleic acid sequencing showed the predominant subtype to be BVDV1b in both studies. In 1999, NCP BVDV1b was detected in numerous samples over time from 1 persistently infected calf; the calf did not seroconvert to BVDV1a or BVDV2. In both studies, BVDV was isolated from the serum, PBLs, and nasal swabs of the calves, and in the 1999 study, it was isolated from lung tissue at necropsy. BVDV was demonstrated serologically and by virus isolation to be a contributing factor in respiratory disease. It was isolated more frequently from sick calves than healthy calves, by both pen and total number of calves. BVDV1a and BVDV2 seroconversions were related to sickness in selected pens and total number of calves. In the 1999 study, BVDV-infected calves were treated longer than noninfected calves (5.643 vs 4.639 d; P = 0.0902). There was a limited number of BVDV1a isolates and, with BVDV1b used in the virus neutralization test for antibodies in seroconverting calves' serum, BVDV1b titers were higher than BVDV1a titers. This study indicates that BVDV1 strains are involved in acute respiratory disease of calves with pneumonic Mannheimia haemolytica and Pasteurella multocida disease. The BVDV2 antibodies may be due to cross-reactions, as typing of the BVDV strains revealed BVDV1b or la but not BVDV2. The BVDV1b subtype has considerable implications, as, with 1 exception, all vaccines licensed in the United States contain BVDV1a, a strain with different antigenic properties. BVDV1b potentially could infect BVDV1a-vaccinated calves.

Objective: To determine effects of vaccination prior to transit and prophylactic administration of florfenicol at time of arrival at a feedyard on health of cattle and colonization of the nasopharynx by Mannheimia haemolytica (MH)., Animals: 121 steers from Tennessee and 84 steers from New Mexico., Procedure: Half of the steers were vaccinated before transport to a feedyard. Steers from Tennessee were vaccinated with MH bacterin-toxoid, and steers from New Mexico were vaccinated intranasally with modified-live leukotoxin-deficient MH. Half of the vaccinates and nonvaccinates were randomly selected to receive florfenicol on arrival at the feedyard. Steers were observed daily for respiratory tract disease (RTD)., Results: Administration of florfenicol at time of arrival reduced the incidence of RTD, delayed the interval before onset of RTD, and reduced the incidence of MH colonization of the nasopharynx for at least 4 days, but vaccination did not have any effect. Vaccination elicited an increase in serum antibody titers to MH. Administration of florfenicol at time of arrival reduced the development of serum antibody titers in intranasally vaccinated steers and both groups of nonvaccinated steers, but intranasal vaccination did not affect colonization by wild-type MH., Conclusions and Clinical Relevance: Administration of florfenicol at time of arrival decreased the incidence of MH organisms in the nasopharynx and delayed the onset of RTD. Prophylactic use of suitable antibiotics is likely to reduce the incidence of acute RTD in calves for several days after arrival at feedyards, which is the period when they are most susceptible to infectious organisms.

Objective: To determine the clinical, clinicopathologic, and histologic effects of aerosolized feedyard dust that contains natural endotoxins on adult sheep., Animals: Eighteen 3-year-old Saint Croix sheep., Procedure: A prospective randomized controlled study was conducted. There were 2 treatment groups (dust-endotoxin group, n = 9; control group, 9). Aerosolized feedyard dust was provided continuously during a 4-hour period for each application (once in week 1, 3 times in week 2, and 7 times in week 3) to sheep in a semiairtight tent. All sheep were euthanatized and necropsied 8 hours after the treatment group received the last dust treatment. Variables measured before and after each dust treatment were rectal temperature, total WBC count, and concentrations of fibrinogen and haptoglobin., Results: Mean amount of dust administered during each treatment was 451 g/4 h. Filter collection indicated 51 mg of dust/m3 and 7,423 ng of endotoxin. Mean rectal temperature at 8 hours (40.4 C) and mean WBC counts 12 and 24 hours after dust treatment were significantly higher for the treated group than the means of the respective variables for the control group. Similar responses were observed with repeated dust-endotoxin treatments; however, with each subsequent treatment, there was a diminished response. Sheep in the treatment group had generalized alveolar septal thickening and hypercellularity., Conclusions and Clinical Relevance: Feedyard dust induced a temporary febrile response and leukocytosis in sheep in the treatment group. Exposure to dust that contains endotoxins may be a stressor preceding acute infectious respiratory tract disease of marketed sheep.

Objective: To determine the impact of feedyards on endotoxin concentration, fecal coliform count, and other water quality measurements during winter and summer in feedyard playas (shallow lakes)., Sample Population: Water samples obtained from 7 feedyard playas and 3 nonfeedyard control playas., Procedure: Surface water samples were collected from each playa and at various depths from 3 feedyard playas. Endotoxin concentrations, 22 water quality variables, and fecal coliform counts were determined in samples collected in summer and winter from various combinations of playas., Results: Cattle numbers per feedyard ranged from 40,000 to 175,000 head/y. Mean endotoxin concentrations were significantly lower in control playas than in feedyard playas in winter and summer. Endotoxin concentration appeared to be homogenous at various water depths. Values for 20 of 22 water quality variables were higher in the feedyard playas than in control playas in winter and summer. In winter only, mean total fecal coliform concentration in feedyard playas was significantly greater than in control playas., Conclusions and Clinical Relevance: Results indicated that feedyards have the potential to impact water quality in playas, and cattle should not be allowed access to them. Feedyard playa water should not be used under high pressure to settle dust in pens with cattle or to cool cattle, because aerosols containing pathogens and high concentrations of endotoxin are a health hazard for humans and cattle?

Objective: To determine whether increased conglutinin titers are evident in stressed calves that do not develop respiratory tract disease in feedlots, compared with respiratory tract disease, and to determine the increase in immunoconglutinin titers., Animals: 101 mixed-breed beef calves., Procedure: Calves were processed at 4 farms of origin and allowed to remain with their dams for another 100 days. Calves from each farm were brought to a centrally located order-buyer barn. In a feedlot, 101 calves were assigned to pens and observed daily for clinical signs of acute respiratory tract disease. When sick calves were detected, they were treated with antibiotics and isolated in a pen for 4 days. Conglutinin and immunoconglutinin titers were determined for all calves., Results: During the 28-day study, 73 calves developed respiratory tract disease, whereas 28 calves remained healthy. Mean conglutinin titers differed significantly among calves from the 4 farms. Significant differences were not detected in conglutinin titers among calves on the basis of sex, morbidity, or vaccination status against Mannheimia haemolytica at each farm, the order-buyer barn, or the feedlot on days 8, 15, and 28 after arrival. Immunoconglutinin titers in calves differed significantly among farms and morbidity status., Conclusions and Clinical Relevance: Mean conglutinin titers in calves do not appear to be associated with the incidence of acute respiratory tract disease; however, increased immunoconglutinin titers appear to be associated with recovery of stressed calves from respiratory tract disease during the first 15 days after arrival in a feedlot.

Respiratory tract infections with viruses and Pasteurella spp. were determined sequentially among 26 cattle that died during two severe epizootics of shipping fever pneumonia. Nasal swab and serum samples were collected prior to onset of the epizootics, during disease progression, and after death, when necropsies were performed and lung samples were collected. Eighteen normal control cattle also were sampled at the beginning of the epizootics as well as at weekly intervals for 4 weeks. Respiratory bovine coronaviruses (RBCV) were isolated from nasal secretions of 21 and 25 cattle before and after transport. Two and 17 cattle nasally shed Pasteurella spp. before and after transport, respectively. RBCV were isolated at titers of 1 x 10(3) to 1.2 x 10(7) PFU per g of lung tissue from 18 cattle that died within 7 days of the epizootics, but not from the lungs of the remaining cattle that died on days 9 to 36. Twenty-five of the 26 lung samples were positive for Pasteurella spp., and their CFU ranged between 4.0 x 10(5) and 2.3 x 10(9) per g. Acute and subacute exudative, necrotizing lobar pneumonia characterized the lung lesions of these cattle with a majority of pneumonic lung lobes exhibiting fibronecrotic and exudative changes typical of pneumonic pasteurellosis, but other lung lobules had histological changes consisting of bronchiolitis and alveolitis typical of virus-induced changes. These cattle were immunologically naive to both infectious agents at the onset of the epizootics, but those that died after day 7 had rising antibody titers against RBCV and Pasteurella haemolytica. In contrast, the 18 clinically normal and RBCV isolation-negative cattle had high hemagglutinin inhibition antibody titers to RBCV from the beginning, while their antibody responses to P. haemolytica antigens were delayed. Evans' criteria for causation were applied to our findings because of the multifactorial nature of shipping fever pneumonia. This analysis identified RBCV as the primary inciting cause in these two epizootics. These viruses were previously not recognized as a causative agent in this complex respiratory tract disease of cattle.

The prevalence of bovine viral diarrhea virus (BVDV) infections was determined in a group of stocker calves suffering from acute respiratory disease. The calves were assembled after purchase from Tennessee auctions and transported to western Texas. Of the 120 calves, 105 (87.5%) were treated for respiratory disease. Sixteen calves died during the study (13.3%). The calves received a modified live virus BHV-1 vaccine on day 0 of the study. During the study, approximately 5 wk in duration, sera from the cattle, collected at weekly intervals, were tested for BVDV by cell culture. Sera were also tested for neutralizing antibodies to BVDV types 1 and 2, bovine herpesvirus-1 (BHV-1), parainfluenza-3 virus (PI-3V), and bovine respiratory syncytial virus (BRSV). The lungs from the 16 calves that died during the study were collected and examined by histopathology, and lung homogenates were inoculated onto cell cultures for virus isolation. There were no calves persistently infected with BVDV detected in the study, as no animals were viremic on day 0, nor were any animals viremic at the 2 subsequent serum collections. There were, however, 4 animals with BVDV type 1 noncytopathic (NCP) strains in the sera from subsequent collections. Viruses were isolated from 9 lungs: 7 with PI-3V, 1 with NCP BVDV type 1, and 1 with both BVHV-1 and BVDV. The predominant bacterial species isolated from these lungs was Pasteurella haemolytica serotype 1. There was serologic evidence of infection with BVDV types 1 and 2, PI-3V, and BRSV, as noted by seroconversion (> or = 4-fold rise in antibody titer) in day 0 to day 34 samples collected from the 104 survivors: 40/104 (38.5%) to BVDV type 1; 29/104 (27.9%) to BVDV type 2; 71/104 (68.3%) to PI-3V; and 81/104 (77.9%) to BRSV. In several cases, the BVDV type 2 antibody titers may have been due to crossreacting BVDV type 1 antibodies; however, in 7 calves the BVDV type 2 antibodies were higher, indicating BVDV type 2 infection. At the outset of the study, the 120 calves were at risk (susceptible to viral infections) on day 0 because they were seronegative to the viruses: 98/120 (81.7%), < 1:4 to BVDV type 1; 104/120 (86.7%) < 1:4 to BVDV type 2; 86/120 (71.7%) < 1:4 to PI-3V; 87/120 (72.5%) < 1:4 to BRSV; and 111/120 (92.5%) < 1:10 to BHV-1. The results of this study indicate that BVDV types 1 and 2 are involved in acute respiratory disease of calves with pneumonic pasteurellosis. The BVDV may be detected by virus isolation from sera and/or lung tissues and by serology. The BVDV infections occurred in conjunction with infections by other viruses associated with respiratory disease, namely, PI-3V and BRSV. These other viruses may occur singly or in combination with each other. Also, the study indicates that purchased calves may be highly susceptible, after weaning, to infections by BHV-1, BVDV types 1 and 2, PI-3V, and BRSV early in the marketing channel.

Objective: To identify cytocidal viruses and Pasteurella spp that could be isolated from cattle involved in 2 natural outbreaks of shipping fever., Animals: 105 and 120 castrated male 4- to 8-month-old feedlot cattle involved in 1997 and 1998 outbreaks, respectively., Procedures: Nasal swab specimens and blood samples were collected, and cattle were vaccinated on arrival at an order-buyer barn from 4 local auction houses. Four days later, they were transported to a feedlot, and additional nasal swab specimens and blood samples were collected. Nasal swab specimens were submitted for virus isolation and bacterial culture; blood samples were submitted for measurement of respiratory bovine coronavirus (RBCV) hemagglutinin inhibition titers., Results: 93 of 105 cattle and 106 of 120 cattle developed signs of respiratory tract disease during 1997 and 1998, respectively, and RBCV was isolated from 81 and 89 sick cattle, respectively, while at the order-buyer's barn or the day after arrival at the feedlot. During the 1997 outbreak, bovine herpesvirus 1 was isolated from 2 cattle at the order-buyer's barn and from 5 cattle 7 and 14 days after arrival at the feedlot, and parainfluenza virus 3 was isolated from 4 cattle 14 days after arrival at the feedlot. During the 1998 outbreak, bovine herpesvirus 1 was isolated from 2 cattle at the order-buyer's barn and on arrival at the feedlot and from 5 cattle 7 and 14 days after arrival at the feedlot, and parainfluenza virus 3 was isolated from 1 animal the day of, and from 18 cattle 7 and 14 days after, arrival at the feedlot. Pasteurella spp was cultured from 4 and 6 cattle at the order-buyer's barn and from 92 and 72 cattle on arrival at the feedlot during the 1997 and 1998 outbreaks, respectively., Conclusions and Clinical Relevance: Results suggest that RBCV may play a causative role in outbreaks of shipping fever in cattle. More than 80% of the sick cattle shed RBCV at the beginning of 2 outbreaks when the Pasteurella spp infection rate was low.

Thirteen clinical isolates of Pasteurella multocida from a variety of different animals and humans were examined for their ability to produce lipase. Lipase substrates used included Tween 20, Tween 40, Tween 80, and Tween 85. Lipase activity was detected in the filtrates of organisms grown to the exponential phase in Roswell Park Memorial Institute-1640 defined media (RPMI-1640), but activity increased in the filtrates when the cultures were allowed to proceed to the stationary phase. All strains examined (except for serotype 2) showed lipase activity against at least one of the Tweens. Tween 40 was the best substrate to demonstrate lipase activity. Pasteurella multocida serotype 8 produced the most active lipase against Tween 40 (3,561.7 units of activity/microgram of protein). This activity continued to increase after P. multocida entered a stationary growth phase. P. multocida lipase activity was optimal at pH 8.0. Lipase activity of P. multocida serotype 8 was eluted from a Sepharose 2B column at several points, indicating that several lipases may be produced in vitro by this organism. These data demonstrate that clinical isolates of P. multocida produce lipase; therefore, this enzyme should be considered a potential virulence factors for this organism.

Objective: To determine the effect of tilmicosin treatment on number of Pasteurella haemolytica (PH) organisms in nasal secretion specimens of calves with respiratory tract disease., Animals: 206 British mixed-breed beef calves, 2 to 5 months old., Procedure: In 2 separate studies of outbreaks, calves (study 1, n = 101; study 2, n = 105) that developed respiratory tract disease after transport to a feedlot were treated with tilmicosin. Nasal secretion specimens were examined for PH organisms to determine the status of colonization., Results: In both studies, PH serotypes A1 and A6 were isolated. In study 1, tilmicosin treatment eliminated or markedly reduced the number of PH organisms in calves on days 1, 4, and 5 after treatment. In study 2, tilmicosin treatment eliminated PH organisms in calves on days 1, 2, 5, and 6 after treatment., Conclusions and Clinical Relevance: Overall, tilmicosin treatment increased the number of culture-positive calves that became culture-negative and decreased the number of culture-negative calves that became culture-positive for up to 6 days after treatment. Tilmicosin treatment decreased the number of PH organisms in nasal secretion specimens, which indicated that fewer PH organisms were available to infect the lungs or to infect other calves. By reducing colonization, prophylactic use of tilmicosin before transport or at the time of arrival at a feedlot is likely to reduce the incidence of acute respiratory tract disease in calves for the initial several days after arrival, which is the period when they are most susceptible to infectious organisms.

Antibody responses against respiratory bovine coronavirus (RBCV) infections were monitored in cattle from the onset of a naturally occurring severe shipping fever (SF) epizootic to complete recovery of affected cattle or fatal outcomes. The infection with RBCV was detected in nasal secretions of 86 cattle, and 81 of them developed acute respiratory tract disease, including fatal pneumonia. Cattle nasally shedding RBCV at the beginning of the epizootic experienced characteristic primary immune responses with specific antibodies for hemagglutinin-esterase (HE) and spike (S) glycoproteins. Virus shedding in nasal secretions of the majority of the cattle ceased between days 7 and 14 with the appearance of HE- and S-specific antibodies. Nasal samples and lung tissues from 9 of the 10 fatal cases had high titers of RBCV, but these cattle had only IgM responses to RBCV infections. Cattle remaining negative in RBCV isolation tests entered this epizootic with antibodies against HE and S. Protection against respiratory tract disease was apparently associated with high level of opsonic and virus-neutralizing IgG2. The HE and S glycoproteins were recognized earliest by the bovine immune system while the N protein induced antibody responses during the later stage of initial infection and the early stage of reinfection. The membrane (M) glycoprotein was the least immunogenic of the major viral structural proteins.

Pasteurella haemolytica (Ph) is the most important cause of the bovine acute fibrinohemorrhagic pneumonia that occurs in market stressed calves after shipment to feedyards. Recent characterization of neuraminidase production by these organisms has shown that all 16 serotypes produce an immunologically similar form of the enzyme. Anti-neuraminidase antibody against PhA1 and PhA6 was determined in 101 2- to 5-month-old calves, on their farms of origin, at the order buyer barn (OBB), and through 28 days in the feedyard. Half of the calves were vaccinated with a killed Ph serotype-A1 (PhA1) product. Nasal secretion and tonsil wash specimens were cultured for Ph and Pasteurella multocida (Pm). Serum antibody against PhA1 and PhA6 was measured by indirect hemagglutination (IHA), and anti-neuraminidase antibody was determined by the neutralization assay. At the feedyard, 73 calves had respiratory tract disease. IHA values ranged between 1:2 and 1:1024 for PhA1 and between 1:2 and 1:512 for Ph serotype A6 (PhA6). Forty-two, 24, and 28% of the calves were infected with PhA1, PhA6, and Pm, respectively. Ninety-six percent of the calves experienced an increase in anti-PhA1 neuraminidase antibody when sera drawn on feedyard day 28 were compared with sera drawn on the farm. These data demonstrate that the enzyme neuraminidase is produced in vivo in market stressed cattle after a natural Ph infection.

Objective: To determine the rate and mode of infectious spread of Pasteurella haemolytica among calves maintained under typical conditions during collection, transport, and the first month of feeding., Animals: 101 two- to five-month-old Angus-crossbred calves., Procedure: Samples obtained from cattle prior to and after they were transported to a feedlot were used for isolation and characterization of P haemolytica. Samples were also obtained from additional calves, some of which were sick, and these calves were then commingled with the transported calves for 3 days. A strain of P haemolytica that contains a rare deletion of the 4.2-kilobase streptomycin- and sulfonamide-resistance plasmid was inoculated into both palatine tonsils of 12 calves. Nasal secretions were aspirated from the ventral nasal meatus. Tonsillar wash specimens were procured. Pasteurella haemolytica organisms were quantitatively cultured and identified on the basis of colony morphology and response to specific antisera. Plasmids were isolated by an alkaline lysis procedure and identified by agarose gel electrophoresis., Results: A single plasmid profile was observed from P haemolytica isolated from samples obtained prior to shipment. Commingled calves were shedding P haemolytica containing each known plasmid profile. After shipment, samples contained P haemolytica isolates with each known plasmid profile. The plasmid profile of the unique P haemolytica isolate was recovered from all 12 inoculated calves and 10 other calves. Some calves simultaneously shed P haemolytica isolates with differing plasmid profiles., Conclusions and Clinical Relevance: Pasteurella haemolytica serotype 1 was horizontally transmitted among calves within days of commingling, which continued after calves were transported to a feedlot.

Cross-protection studies employing three serotypes of Pasteurella haemolytica (Ph) were performed in goats, with challenge exposure by transthoracic injection. Indirect hemagglutination (IHA) serum titers showed that the herd had been naturally infected with Ph biovar A, serovar 2 (PhA2) prior to the study. Sixty-four weanling male Spanish goats were randomly allotted to 16 groups. Fifteen goats were given two transthoracic injections into the lungs 21 days apart with live Pasteurella haemolytica biovar A, serovar 1 (PhA1) in agar beads. Fifteen goats were given two transthoracic injections into the lungs 21 days apart with live PhA2 in agar beads. Sixteen goats were given two transthoracic injections into the lungs 21 days apart with live P. haemolytica biovar A, serovar 6 (PhA6) in agar beads. Eighteen control (CON) goats were given two transthoracic injections into the lungs 21 days apart with agar beads alone. Fourteen days after the second injection, goats were challenge-exposed to either live PhA1, PhA2, or PhA6 by transthoracic injection into the lung, and 4 days later, all goats were euthanatized and necropsied. Serum antibody to P. haemolytica antigens was measured throughout the experiment. Mean volumes of consolidated lung tissue for the CON goats challenged with PhA1, PhA2, and PhA6 were 28.29 cm3, 8.36 cm3, and 16.29 cm3, respectively. Mean volumes of consolidated lung tissue for the PhA1-immunized goats challenged with PhA1, PhA2, and PhA6 were 4.38 cm3, 0.25 cm3, and 1.90 cm3, respectively. Mean volumes of consolidated lung tissue for the PhA2-immunized goats challenged with PhA1, PhA2, and PhA6 were 9.68 cm3, 0.05 cm3, and 3.39 cm3, respectively. Mean volumes of consolidated lung tissue for the PhA6-immunized goats challenged with PhA1, PhA2, and PhA6 were 14.05 cm3, 1.27 cm3, and 4.53 cm3, respectively. These data demonstrate protection in immunized goats challenged with the homologous serotype of P. haemolytica. PhA1-immunized animals were protected against serotype 2 challenge as well as against serotype 6 challenge. PhA2-immunized animals were not protected against serotype 1 challenge, but were protected against transthoracic PhA6 challenge. PhA6-immunized animals were not protected against serotype 1 challenge, but were protected against transthoracic PhA2 challenge. There appears to be some cross-protection among the P. haemolytica serotypes, and this fact should be taken into consideration when developing vaccines against this organism.

Objective: To determine the effectiveness of Pasteurella multocida biovar A, serovar 3 (Pm A:3) killed by exposure to UV light and incorporated with a polyacrylate bead carrier as a vaccine., Animals: 18 weanling male Spanish goats., Procedure: Prospective, randomized controlled study with 3 treatment groups: positive-control (PC), negative-control (NC), and principal Pm A:3 bacterin (PA) groups. Six PC goats each received live Pm A:3 and polyacrylate beads twice, 22 days apart, by transthoracic injection into the left lung. Six NC goats each received only PA beads twice, 22 days apart, by transthoracic injection. Six principal goats each received Pm A:3 vaccine SC twice, 22 days apart. Fourteen days after the second vaccination, all goats were challenge exposed with live Pm A:3 by transthoracic injection into the right lung, and 4 days later they were euthanatized and necropsied., Results: Mean volume of consolidated lung tissue at the challenge site was 1.75 cm3 for the PC group, 15.18 cm3 for the NC group, and 3.9 cm3 for the PA vaccine group. The NC group had a significantly (P < or = 0.002) larger mean volume of consolidated lung tissue than did the PC and PA groups after challenge exposure., Conclusions: The PA bacterin and the PC groups developed protective immunity against live Pm A:3 challenge exposure. An SC administered, UV light-killed, Pm A:3 bacterin induced protective immunity similar to that induced by virulent live Pm A:3 injected into the target organ, the lung.

Six goats were injected transthoracically with live Pasteurella multocida A:3 to examine if an extracellular enzyme, neuraminidase, was produced in vivo during infection with this organism. The principal group of goats (n = 6) each received 1 ml of live 7.5 x 10(4) cfu of P. multocida mixed with polyacrylate beads transthoracically in the left lung on day 0 and 1 ml of live P. multocida (2.2 x 10(8) cfu) mixed with polyacrylate beads transthoracically in the left lung on day 22. Six goats were used as negative controls and received 0.3 g of polyacrylate beads subcutaneously in the right flank on days 0 and 22. Serum was obtained from all animals on days 0, 7, 14, 22, 29, and 36. Preimmune sera from all animals showed no detectable antibody to P. multocida A:3 neuraminidase in an enzyme neutralization assay. None of the sera from the negative control animals demonstrated a significant antibody titer against the P. multocida A:3 neuraminidase. On day 36, serum samples from the six infected animals possessed complete enzyme-neutralizing activity. Anti-neuraminidase antibody could be detected as early as day 14 in the infected animals. These data show that neuraminidase is produced in vivo during an active P. multocida A:3 lobar infection.

Objectives: To follow incidence of Pasteurella haemolytica (PH) in the upper respiratory tract of healthy calves at the farm and through the marketing process, and to determine the effect of vaccination on PH colonization of the upper respiratory tract and on the incidence of respiratory tract disease (RTD)., Animals: 2- to 5-month-old calves (n = 104) from 4 farms., Procedure: Calves were vaccinated with a killed PH serotype-1 product. Nasal secretion and tonsil wash specimens were cultured for PH, and serum antibody was measured by indirect hemagglutination. Calves with RTD were treated with tilmicosin phosphate., Results: At the feedyard, 73 calves had RTD. The incidence of RTD was significantly related to the farm of origin, and was inversely related to the PH serum titer at the farm, but was not influenced by vaccination. Isolations of PH serotype 1, however, were reduced by vaccination. The major serotypes of PH encountered were 1 and 6., Conclusion: Vaccination can reduce the frequency of colonization of the upper respiratory tract by PH.

Objective: To determine the effectiveness of Pasteurella haemolytica biovar A, serovar 1 (Ph A1) killed by UV light and incorporated with an oil adjuvant or carriers., Animals: 40 weaning male Spanish goats., Procedure: Goats were randomly allotted to 1 of 6 treatment groups: 4 Ph A1 bacterins (agar beads, polyacrylate beads [PA], phosphate-buffered saline solution, Freund's incomplete adjuvant), live Ph A1 with polyacrylate beads (LiPhPA), and polyacrylate beads (UnVac). Each of 4 Ph A1 vaccines was administered SC twice, 21 days apart, to 1 of 4 groups; another group received only PA beads SC, and the last group received live Ph A1 with PA beads by transthoracic injection into the left lung. 14 days after the second vaccination, all goats were challenge exposed with live Ph A1 by transthoracic injection into the right lung, and 4 days later, all goats were euthanatized and necropsied., Results: Mean volume of consolidated right lung tissue was 1.02 cm3 for the LiPhPA group, 168.1 cm3 for the UnVac group, 2.3 cm3 for the Freund's incomplete adjuvant bacterin group, 5.53 cm3 for the PA bacterin group, 9.01 cm3 for the agar beads bacterin group, and 7.51 cm3 for the phosphate-buffered saline solution bacterin group. Mean volume of consolidated lung tissue was significantly different between the UnVac group and the other 5 groups., Conclusion: The LiPhPA group and 4 bacterin groups developed protective immunity against live Ph A1 challenge exposure., Clinical Relevance: An s.c. administered, UV light-killed Ph A1 bacterin induced protective immunity equal to that induced by virulent live Ph A1 injected into the target organ, the lung.

Neuraminidases produced by 16 strains of Pasteurella multocida (serotypes 1 to 16) were characterized by molecular weight, substrate specificity, and antigenic identity. After growth in a chemically defined medium, stage I (lyophilized) culture supernatants were assayed for activity with N-acetylneuramin lactose, human alpha-1-acid glycoprotein, fetuin, colominic acid, and bovine submaxillary mucin. Neuraminidase produced by P. multocida A:3 was purified by a combination of salt fractionation, ion-exchange chromatography on DEAE-Sephacel, and gel filtration on Sephadex G-200. Purified P. multocida A:3 neuraminidase was employed to immunize rabbits, and the resulting antiserum reduced the activity of the P. multocida A:3 enzyme by 40.3%. This antiserum also reduced the activities of the neuraminidases produced by other serotypes by between 30.8 and 59.6%. Molecular weight estimates of the neuraminidases produced by the various serotypes were obtained by gel filtration chromatography on Sephadex G-200. Each of the 16 serotypes examined produced a neuraminidase with a molecular weight of approximately 500,000. In addition, all 16 high-molecular-weight neuraminidases showed similar substrate specificities. On the basis of these data, it appears that the high-molecular-weight neuraminidases produced by different P. multocida serotypes are quite similar.

A total of 721 field isolates of various Pasteurella species (haemolytica, multocida, and testudinis) from various regions of the United States were examined for extracellular neuraminidase production. All strains were grown and tested in the same way. Included were 372 P. haemolytica serotype 1 isolates, 181 P. haemolytica serotype 2 isolates, 63 P. haemolytica serotype 6 isolates, 101 Pasteurella multocida isolates, and 4 Pasteurella testudinis isolates. All Pasteurella species examined produced the enzyme. The data revealed the following: (1) Several transfers of P. haemolytica strains on blood agar medium did not cause a decrease in enzyme activity. (2) P. haemolytica serotypes 2 and 6 produce more neuraminidase than P. haemolytica serotype 1, P. multocida, and P. testudinis isolates. (3) There was no apparent change in neuraminidase production by P. haemolytica serotypes 1 and 2 obtained from the same animal taken on different days in the feedyard. (4) There was no significant change in neuraminidase production by P. haemolytica serotype 2 isolates taken from the same animal at the auction market and later at the feedyard.

The properties of an extracellular neuraminidase produced by a Pasteurella multocida A:3 strain that was isolated in a case of bovine pneumonia were examined during growth in a defined medium. This enzyme (isolated from concentrated culture supernatants of P. multocida A:3) was active against N-acetylneuramin lactose, human alpha-1-acid glycoprotein, fetuin, colominic acid, and bovine submaxillary mucin. Enzyme elaboration was correlated with the growth of the organism in a defined medium, with maximum quantities produced in the stationary phase. The enzyme was purified by a combination of ammonium sulfate fractionation, ion exchange on DEAE-Sephacel, and gel filtration on Sephadex G-200. The purified neuraminidase possessed a specific activity of 9.36 mumol of sialic acid released per min per mg of protein against fetuin. The enzyme possessed a pH optimum of 6.0 and a Km of 0.03 mg/ml. The P. multocida A:3 neuraminidase had a molecular weight of approximately 500,000 as estimated by gel filtration. The enzyme was stable at 4 and 37 degrees C for 3 h. Approximately 75% of the neuraminidase activity was lost within 30 min at 50 degrees C. Greater than 90% of the enzyme activity was destroyed within 10 min at temperatures of > or = 65 degrees C. The P. multocida neuraminidase does not appear to be serologically related to the Pasteurella haemolytica A1 neuraminidase since antiserum prepared against the purified P. haemolytica enzyme did not neutralize the P. multocida enzyme.

Nine goats were injected transthoracically with Pasteurella haemolytica A1 to determine if an extracellular bacterial enzyme, neuraminidase, was produced in vivo during infection with this organism. The principal group of goats (n = 9) each received 1 ml of 7.25 x 10(5) live P. haemolytica A1 cells in polyacrylate beads transthoracically in the left lung on days 0 and 21. Six goats were used as negative controls and received 0.3 g of polyacrylate beads subcutaneously in the right flank on days 0 and 21. Serum was obtained from all animals on days -4, 3, 7, 14, 21, 24, and 32. Preimmune serum from all animals showed no detectable antibody to P. haemolytica A1 neuraminidase in an enzyme neutralization assay. None of the sera from the negative control animals possessed a significant antibody concentration in response to the P. haemolytica A1 neuraminidase. On day 32, serum samples from the nine infected animals possessed enzyme neutralizing activity that ranged from 62% to 100%. Anti-neuraminidase antibody could be detected as early as day 14 by the enzyme neutralization assay. These data demonstrate that the enzyme neuraminidase is produced in vivo during an active P. haemolytica A1 lobar infection.