Efficacy of a multivalent modified-live virus vaccine containing a Mannheimia haemolytica toxoid in calves challenge exposed with Bibersteinia trehalosi Terry L. Bowersock, DVM, PhD; Brian E. Sobecki, BS; Sarah J. Terrill, MS; Nathalie C. Martinon, DVM; Todd R. Meinert, PhD; Randy D. Leyh, DVM, PhD
Objective—To determine the efficacy of a multivalent modified-live virus (MLV) vaccine containing a Mannheimia haemolytica toxoid to reduce pneumonia and mortality rate when administered to calves challenge exposed with virulent Bibersteinia trehalosi. Animals—74 Holstein calves. Procedures—Calves were assigned to 2 treatment groups. Calves in the control group (n = 36) were vaccinated by SC administration of 2 mL of a commercial 5-way MLV vaccine, and calves in the other group (38) were vaccinated by SC administration of a 2-mL dose of a 5-way MLV vaccine containing M haemolytica toxoid (day 0). On day 21, calves were transtracheally administered B trehalosi. Serum was obtained for analysis of antibody titers against M haemolytica leukotoxin. Nasopharyngeal swab specimens were collected from calves 1 day before vaccination (day –1) and challenge exposure (day 20) and cultured to detect bacterial respiratory pathogens. Clinical scores, rectal temperature, and death attributable to the challenge-exposure organism were recorded for 6 days after challenge exposure. Remaining calves were euthanized at the end of the study. Necropsy was performed on all calves, and lung lesion scores were recorded. Results—Calves vaccinated with the MLV vaccine containing M haemolytica toxoid had significantly lower lung lesion scores, mortality rate, and clinical scores for respiratory disease, compared with results for control calves. Conclusions and Clinical Relevance—Administration of a multivalent MLV vaccine containing M haemolytica toxoid protected calves against challenge exposure with virulent B trehalosi by reducing the mortality rate, lung lesion scores, and clinical scores for respiratory disease. (Am J Vet Res 2014;75:770–776)
B
ibersteinia trehalosi (previously known as Pasteurella trehalosi and Pasteurella haemolytica type T) is a gram-negative bacterial respiratory tract pathogen that causes pneumonia (as well as septicemia) in domestic sheep, goats, and bighorn sheep.1–3 The organism is the most common pathogen found in tonsils of clinically normal American Bison.4,5 It has been identified as a major cause of pneumonia and septicemia in cattle.6–8 Bibersteinia trehalosi can cause peracute bacterial pneumonia in adult dairy cattle, feedlot cattle, and young calves.9 Clinical signs range from pneumonia to sudden death. Lungs often have exudative fibrinous pneumonia, bronchiolitis, and alveolitis.9,10 Received February 25, 2014. Accepted April 9, 2014. From Global Biological Research (Bowersock, Leyh) and Global Development and Operations (Sobecki, Terrill, Martinon, Meinert), Zoetis Inc, Building 300 Dock, 333 Portage St, Kalamazoo, MI 49007. Supported by Zoetis Inc. Presented in abstract form at the Bovine Respiratory Disease Symposium 2014: New Approaches to Bovine Respiratory Disease Prevention, Management and Diagnosis, Denver, July 2014. Address correspondence to Dr. Bowersock (terry.l.bowersock@zoetis. com). 770
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AHDRCC BRD CI lkt MLV OD PBST PFGE
ABBREVIATIONS
Animal Health Developmental Research Culture Collection of Zoetis Inc Bovine respiratory disease Confidence interval Leukotoxin Modified-live virus Optical density PBS solution and 0.05% Tween 20 Pulsed-field gel electrophoresis
Bibersteinia trehalosi has been identified in healthy as well as pneumonic or septicemic cattle in the United Kingdom since 2003.10 Increasingly, diagnostic laboratories are isolating B trehalosi from clinically affected cattle.6–8 A report10 from the United Kingdom indicated that B trehalosi was the only pathogen in 18 of 65 (28%) cattle with pneumonia. From 2004 to 2009, B trehalosi was identified in 22 to 45 dairy cattle with pneumonia/y,11–16 and from 2008 to 2011, 149 cases of respiratory disease attributable to B trehalosi were identified at a diagnostic laboratory in California.a In the past, field isolates of B trehalosi were likely to be classified within the Mannheimia haemolytica comAJVR, Vol 75, No. 8, August 2014
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plex, which may have obscured the role of B trehalosi in disease in cattle.2 Some of the increase in apparent prevalence is the result of previous misdiagnosis of B trehalosi, which is phenotypically similar to M haemolytica. The use of PFGE has been beneficial in the assessment of the source of respiratory Pasteurellaceae in epidemiological investigations of wild and domestic ruminants and has confirmed the role of B trehalosi as a primary pathogen.17,18 The name B trehalosi was proposed by scientists following molecular evaluation of this group of organisms and the determination that the organism differs sufficiently from other Pasteurella spp and thus deserves a group to itself on the basis of DNA-DNA relatedness19 and on the basis of amplified fragment length polymorphism and 16s rRNA sequencing.2 Although genetically different to the extent that it is now a separate genus and species, B trehalosi shares some pathogenic characteristics with M haemolytica. The gene lktA is key to expression of lkt, which is a major virulence factor of M haemolytica; lktA has been found in B trehalosi.20 There is evidence that rabbit antisera to all serotypes of M haemolytica 1 through 12 (including what is currently referred to as B trehalosi) cytotoxin (lkt) cross neutralizes toxin from M haemolytica A1.21 Fibrinogen-binding proteins have been found in supernatants of both M haemolytica and B trehalosi, and evaluation of western blots has revealed that the fibrinogen molecules are of a similar size.22 A protease has been identified in B trehalosi that is similar to a protease in M haemolytica. For both organisms, prechallenge sera neutralizes protease activity, which results in lower lung lesion scores and suggests an immune response to protease is important in protection against both B trehalosi and M haemolytica.20 In the study2 that was intrinsic to the name change of this organism to B trehalosi, 9 of 43 isolates evaluated were isolated from cattle with pneumonia. Antimicrobial treatment cannot reliably control the peracute-acute nature of infections attributable to B trehalosi9; therefore, a vaccine is needed to aid in control of this bacterial pathogen in domestic as well as wild ruminants. The purpose of the study reported here was to evaluate the efficacy of a commercial vaccine containing multivalent MLV plus an M haemolytica toxoid for protection of calves against pneumonia after challenge exposure with B trehalosi. Materials and Methods Animals—Young (1 to 2 days old) top grade Holstein bull calves (n = 74) were purchased from sale barns and transported to a commercial farm. Requirements were that calves weigh > 45.5 kg and be vigorous and healthy. At the time of purchase, all calves had negative results for persistent infection with bovine viral disease virus, as determined by use of an antigencapture ELISA performed by personnel at a commercial laboratory.b Calves were raised on the commercial farm until they were 9 to 10 weeks old, when they were moved to a Zoetis Research farm in Richland, Mich. Before transport to the research farm, calves were judged to be healthy and had negative results for M haemolytica, Pasteurella multocida, and B trehalosi on nasopharynAJVR, Vol 75, No. 8, August 2014
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geal swab specimens. In addition, serum antibody titers against M haemolytica lkt and P multocida outer membrane proteins were tested with ELISAs by use of cutoff values that had been used to indicate cattle would be susceptible to challenge exposure with Pasteurellaceae in preliminary studies conducted by our research group. At the research farm, calves were housed in groups in rooms (14.94 X 8.23 X 3.05 m). Calves were housed such that they could not directly or indirectly contact calves in other rooms for the duration of the study. Animal identification and assignment per room were provided to the investigators. Calves then were randomly allocated to treatments within each room in accordance with a generalized randomized block design with the blocking factor based on room. The random treatment allocation plan was generated with a computer programc that used a random number generator function. There were 26 calves in one room, 24 calves in a second room and 24 calves in a third room; the 2 latter rooms had 12 calves of each treatment group, whereas the other room had 12 calves of one treatment group and 14 calves of the other treatment group. Animals remained in their assigned room for the duration of the study. The study was approved by the Animal Use and Care Committee of Zoetis Inc. Study design—Before assignment to study groups, all calves were clinically assessed (Appendix). Each calf had a clinical score of 0 and rectal temperature < 39.4°C and was free of B trehalosi and M haemolytica. Calves (n = 36) in one group were injected SC with a single 2-mL dose of a commercially available 5-way MLV vaccine containing bovine herpesvirus 1, bovine viral diarrhea virus types 1 and 2, parainfluenza virus type 3, and bovine respiratory syncytial virus,d and calves in the other group (38) were injected SC with a single 2-mL dose of a 5-way MLV vaccine containing the same 5 viral agents and M haemolytica toxoid.e Day of vaccination was designated as day 0. Twenty-one days after vaccination, calves were challenge-exposed with B trehalosi strain AHDRCC 40499. The challenge organism was acquired at a diagnostic laboratoryf from a calf with pneumonia. The challenge organism was identified by use of biochemical methods including a commercial microbial identification systemg and 16sRNA; subsequently, identity of a subset was confirmed by use of matrix-assisted laser desorption ionization–time-of-flight (ie, MALDI-TOF) and to possess lkt genes by use of PFGE. Challenge exposure—Calves were challenge exposed by transtracheal injection with 120 mL of a logarithmic-phase broth culture of B trehalosi AHDRCC 40499. Culture of B trehalosi was initiated by inoculating 50 mL of warmed HP mediumh into a 250-mL baffled flask that contained a loop (approx 100 µL) of frozen stock. The flask was incubated at 37°C with shaking (150 revolutions/min) for 15 to 18 hours. An aliquot (2 mL) of the culture was added to each 100 mL of HP medium in an appropriately sized baffled flask, which was incubated at 37°C with shaking (150 revolutions/min) until the OD (measured at 650 nm) approxi771
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mated that for a solution containing 1.0 X 109 CFUs/ mL. The flask was immediately placed on ice, and the contents were used as a stock solution that was diluted in PBS solution to achieve a final challenge-exposure dose of 6.4 X 1010 CFUs in 120 mL. Diluted challengeexposure material was maintained on ice until administered. Aliquots of the challenge-exposure material as well as the stock solution were retained in the laboratory and used to perform plate counts to determine the actual dose used for challenge exposure. Challenge exposure was conducted in the rooms used to house the calves. Briefly, calves were restrained in a standing position. Hair over the tracheal region of each calf was clipped, and the area was disinfected with alcohol wipes. Then, 4 mL of lidocaine solutioni was infused SC into the tissues over the trachea (diameter of infiltrated area, 1 to 2 cm). A 16-gauge trocharj was directed caudally through the skin and into the trachea at the location of the local anesthesia. A cannula was inserted through the trochar and advanced to the bifurcation of the trachea; it was then retracted 2 to 4 cm to ensure the tip was located in the distal aspect of the trachea and not in a major bronchus. A total of 120 mL of diluted challenge-exposure material was then infused through the cannula, which was followed by flushing with 60 mL of cold HP medium. The cannula was then removed. Clinical monitoring—Calves were observed, clinical scores of disease were assigned, and rectal temperatures were recorded daily after challenge exposure by individuals who were unaware of the treatment group of each calf. Calves that died or were euthanized because of clinical disease after challenge exposure were submitted for necropsy. All remaining calves were euthanized by captive bolt 6 days after challenge exposure (ie, day 27) and submitted for necropsy. Sample collection—Blood samples were collected from each calf on days –1, 20, and day of death or euthanasia (day 27 for most calves in the study). Samples were allowed to clot at room temperature (22.2°C). Serum was harvested, divided into 2 aliquots, and submitted for testing or stored frozen. Serum titers against M haemolytica lkt—Serum samples were assayed for antibodies against M haemolytica lkt by use of a sandwich ELISA developed at Zoetis Inc. Each well of a 96-well plate was coated with 100 µL of capture antibody by use of 0.43 mg of murine monoclonal antibodyk against M haemolytica lkt/ mL in 0.01M borate buffer. The plate was sealed and incubated for 16 to 32 hours at 4°C. The contents of each well were decanted, and 200 µL of 1% casein in PBST was added to each well. The plate was sealed and incubated for 1 to 4 hours at 37°C. The buffer was then decanted, and 100 µL of M haemolytica lkt supernatantk diluted 1:500 in 1% casein in PBST was added to each well. The plate was sealed and incubated for a mean ± SD of 60 ± 10 minutes at 37°C. The plate was decanted, and 100 µL of calf serum, control samples, and blanks was added to each well. The plate was then sealed and incubated for another 60 ± 10 minutes at 37°C. Serum samples from each calf and positive and 772
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negative control sera were prepared as serial 1:2 dilutions in 1% casein in PBST. Briefly, samples from each calf were plated in duplicate at dilutions of 1:100 to 1:3,200, the positive internal assay control sample was plated in duplicate at dilutions of 1:1,600 to 1:51,200, and the negative internal assay control sample was plated in duplicate at dilutions of 1:100 to 1:3,200. Each positive and negative internal control sample was also included (dilution of 1:800) in 5 wells of each plate and as a full dilution series (1:1,600 to 1:51,200) to duplicate wells in a separate plate. At the end of the incubation period, each well of every plate was washed 3 times with PBST (300 µL/well). The PBST was decanted, and 100 µL of goat anti-bovine IgGl diluted 1:5,000 in 1% casein was added to each well. The plate was sealed and incubated for 60 ± 10 minutes at 37°C. The plate then was washed 3 times with PBST (300 µL/well). The PBST was decanted, and 100 µL of substrate was added to each well. Substrate was prepared with equal parts of 2 components of a peroxidase substrate.m The plate was protected from light and incubated at 27 ± 2°C for approximately 10 minutes. The OD of plates was determined at 405 and 490 nm by use of a spectrophotometer; the OD for 490 nm was subtracted from the OD for 405 nm. The assay was considered valid and the plate value determined when the positive control sample was between 0.8 and 1.2 net absorbance units, the negative control sample was < 0.3 net absorbance units, and the blank was < 0.12 net absorbance units. The cutoff for the positive control sample was determined as the mean absorbance for the positive control sample divided by 5. A sample titer was calculated as the last titer point at which the mean absorption value for a sample minus the positive cutoff value was greater than zero. Nasopharyngeal swab specimensn were collected on days –1 (before vaccination) and 20 (before challenge exposure). Samples were submitted for standard bacterial culture to detect B trehalosi, P multocida, M haemolytica, Histophilus somni, and Trueperella pyogenes. Swab specimens and tissues were collected from the lungs of calves on the day of necropsy. Lung swab specimens were cultured to detect B trehalosi, P multocida, M haemolytica, H somni, and T pyogenes. Determination of lung lesion scores—Lung lesion scores (total percentage of consolidated lung in each lobe) were determined by an individual who was unaware of the treatment group of each calf. Percentage consolidation in each lung lobe was determined manually and was weighted by use of the following ratios of individual lung lobe to total lung mass: cranial part of the left cranial lobe, 5%; caudal part of the left cranial lobe, 6%; left caudal lobe, 32%; cranial part of the right cranial lobe, 6%; caudal part of the right cranial lobe, 5%; right medial lobe, 7%; right caudal lobe, 35%; and accessory lobe, 4%. Weighted lung lobe values were summed for all lobes to yield the percentage consolidation of the lungs for each calf. Data analysis—Arc-sin square root transformation was applied to the percentage consolidation of lung values, which was followed by analysis with a general linear mixed modelc that had treatment as a fixed efAJVR, Vol 75, No. 8, August 2014
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fect and block (room) as a random effect. When there was a significant (P ≤ 0.05) overall treatment effect, linear combinations of parameter estimates were used in a priori contrasts to make comparisons between treatments. Back-transformed least squares means, SEMs, and 95% CIs were calculated from least squares parameter estimates obtained from the analyses. For mortality data, a calf was described as having survived (euthanized at the end of the study on day 27) or as having died or been euthanized (died or euthanized before day 27) on the basis of data collected on the final disposition form and summarized by treatment. When death or euthanasia of a calf was related to the challenge exposure (ie, before day 27), then death was coded as a binary variable (0 = survived; 1 = died or was euthanized) and analyzed with a generalized mixed model proceduren by use of a binomial error and logit link. The model included treatment as a fixed effect and block (room) as a random effect. Least squares means and back-transformed least squares means and 95% CIs were reported. When there was a significant (P ≤ 0.05) treatment effect, comparison of mortality rates was performed. Death or euthanasia related to challenge exposure was summarized by use of the prevented fraction with 95% confidence limits. For clinical observation data, the number of calves with a clinical score ≥ 2 (acute or severe BRD) after challenge exposure was summarized. Results Clinical signs of respiratory tract disease, mortality rate, and lung lesion scores—Calves developed typical clinical signs of respiratory tract disease after challenge exposure. Acute BRD (clinical score ≥ 2 on
at least 1 day after challenge exposure) was observed during the 6 days after challenge exposure in significantly (P = 0.002) more control calves (26/36 [72.2%]) than in calves vaccinated with the 5-way MLV vaccine containing M haemolytica toxoid (13/38 [34.2%]; Table 1). Control calves had a significantly (P = 0.049) higher mortality rate (12/36 [33.3%]), compared with the mortality rate for the calves vaccinated with the 5-way MLV vaccine containing M haemolytica toxoid (5/38 [13.2%]). Mean percentage consolidated lung for the control calves (43%) was significantly (P = 0.012) higher than the percentage for the calves vaccinated with the 5-way MLV vaccine containing M haemolytica toxoid (30%). There were no significant differences in mean rectal temperature between groups during the study. Serum testing and bacterial culture of nasopharyngeal swab specimens and lung tissues—All of the calves had serum anti-lkt antibody titers ≤ 1:3,200 at the start of the study (Table 2). This titer had been found to be the break point at which animals were susceptible to experimental challenge exposure in preliminary studies conducted by our research group. Mean anti-lkt antibody titer remained low for the control calves and was 248 (95% CI, 181 to 339) at the time of challenge exposure (day 21). Calves vaccinated with the 5-way MLV vaccine containing M haemolytica toxoid had a significantly (P < 0.001) higher mean antibody titer (5,254; 95% CI, 3,867 to 7,138) before challenge exposure. After challenge exposure with B trehalosi, mean antibody titers increased in both groups of calves, although the mean antibody titer for the calves vaccinated with the 5-way MLV vaccine containing
Table 1—Lung lesion scores, mortality rate, and clinical BRD rate for 36 calves vaccinated by SC administration of a commercial 5-way MLV vaccine (control group) and 38 calves vaccinated by SC administration of a 5-way MLV vaccine containing Mannheimia haemolytica toxoid (5-way toxoid) and challenge exposed with Bibersteinia trehalosi 21 days later. Group
Lung lesion score (%)*
Control 5-way toxoid
43 (35–50) 30‡ (23–57)
Mortality rate (%)
Calves with clinical score ≥ 2 (%)†
33.3 (19.8–50.3) 13.2§ (5.5–28.3)
72.2 (55.3–84.5) 34.2 ll(20.8–50.7)
Values reported are least squares mean (95% CI). *Represents the percentage of consolidated lung for all lung lobes. †Clinical score was assessed by use of a scale of 0 to 3 (0, healthy calf; 1, nonspecific illness; 2, acute BRD; and 3, severe BRD). ‡,§,llWithin a column, value differs significantly (‡P = 0.012; §P = 0.049; llP = 0.002) from the value for the control group. Table 2—Serum anti-lkt antibody titers for 36 calves vaccinated (day 0) by SC administration of a commercial 5-way MLV vaccine (control group) and 38 calves vaccinated (day 0) by SC administration of a 5-way MLV vaccine containing M haemolytica toxoid (5-way toxoid) and challenge exposed with B trehalosi on day 21. Group Control 5-way toxoid
Day –1 367 (269–501) 417 (307–566)
Day 20 248 (181–339) 5,254† (3,867–7,138)
Day 22 to 27* 2,714 (1,911–3,852) 7,566† (5,506–10,393)
Values reported are least squares mean (95% CI). *Calves were observed for 6 days after challenge exposure; titers were determined in samples obtained from calves on the day they died or were euthanized because of illness attributable to B trelahosi (12 control calves and 5 calves vaccinated with the 5-way MLV vaccine containing M haemolytica toxoid) or in samples obtained from the surviving calves (24 control calves and 33 calves vaccinated with the 5-way MLV vaccine containing M haemolytica toxoid), which were euthanized on day 27. †Within a column, value differs significantly (P < 0.001) from the value for the control group. AJVR, Vol 75, No. 8, August 2014
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M haemolytica toxoid remained significantly (P < 0.001) higher (7,566; 95% CI, 5,506 to 10,393), compared with the mean antibody titer for the control calves (2,714; 95% CI, 1,911 to 3,852). Nasopharyngeal swab specimens obtained on days –1 and 20 had negative results for bacterial culture of B trehalosi and M haemolytica. Lung swab specimens obtained at necropsy yielded B trehalosi for 25 of 36 (69.4%) control calves and 24 of 38 (63.2%) calves vaccinated with the 5-way MLV vaccine containing M haemolytica toxoid. None of the calves in either group had specimens that yielded H somni. One control calf had specimens that yielded T pyogenes and M haemolytica, and 1 calf vaccinated with the 5-way MLV vaccine containing M haemolytica toxoid had specimens that yielded P multocida. This indicated that the lung lesion scores were most likely attributable to infection with B trehalosi. Discussion Bibersteinia trehalosi is a pathogen of cattle that causes acute pneumonia and septicemia in a wide range of age groups. The acute nature of B trehalosi infections and the sometimes poor response to antimicrobial treatment9 suggest the need for a vaccine to control infection. Currently, there are no vaccines with label claims for prevention of diseases attributable to B trehalosi in cattle. Several vaccines have had variable results for controlling respiratory tract disease attributable to B trehalosi, but most of those studies3,10,22–25 were conducted in domestic and bighorn sheep. A decrease in survival rate and an increase in incidence of respiratory tract disease were reported3 in lambs of bighorn ewes vaccinated IM with 2 mL of a commercial vaccine labeled for cattle that included P multocida bacterin and M haemolytica leukotoxoid.o In another study23 in a large commercial sheep flock, 2 doses of an experimental vaccine containing M haemolytica strains A1, A2, A6, A7, and A9 and B trehalosi strains T3,T4, T10, and T15p administered SC in the neck did not improve average daily gain, reduce severity of respiratory tract disease, or reduce the recovery of either pathogen during postmortem examination. In a third study,24 a 2-mL dose of an M haemolytica–P multocida bacterin leukotoxoidq did not reduce pharyngeal colonization by M haemolytica or B trehalosi in pack goats, despite good induction of lkt-neutralizing antibodies. There have been other reports that use of experimental vaccines reduced disease attributable to B trehalosi. An unidentified vaccine or vaccines against B trehalosi improved disease control in sheep.10 In another study,20 a protease produced by a B trehalosi isolate was found to be immunogenic, and serum antibodies were associated with a reduction in lung lesions in bighorn sheep challenge exposed with B trehalosi. In a separate study,25 bighorn sheep vaccinated several times with culture supernatants from M haemolytica A2 and B trehalosi as well as with an M haemolytica A1 vaccineo had a reduction in the severity and incidence of pneumonia and an increase in survival rate. The sheep in that study25 also had high titers of lkt-neutralizing and bacterial surface-specific antibodies. In a study26 in which investigators evaluated use of an iron-regulated protein–based B trehalosi vac774
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cine in sheep, there was good efficacy in reducing the mortality rate after challenge exposure. However, to our knowledge, no vaccines have been tested for efficacy in cattle. The vaccination trial reported here was conducted to evaluate the efficacy of a 5-way MLV vaccine containing M haemolytica toxoid with inactivated lkt. Investigators have found that this vaccine induces good protection in cattle in the face of challenge exposure with M haemolytica.27,28 Although it has been suggested that cattle isolates of B trehalosi typically are nonhemolytic and less likely to produce lkt, evaluation of a large number of isolates from across the United States suggests that the prevalence of lkt genes in B trehalosi is much higher.r The fact that B trehalosi possesses several other antigens related to those expressed by M haemolytica suggests that cross protection by a vaccine is plausible. Apparent failures in sheep and wild ungulates may have been attributable to a variety of reasons, such as differences in the sample of animals used as indicators of protection, prevalence of disease in the herd or flock at the time of the study, and other stressors (especially handling of wild ungulates) that could reduce efficacy of vaccines. Although similarities exist genetically with the lktA gene, there are significant differences among Pasteurellaceae, including B trehalosi, in the lktA gene as well as in lktB, lktC, and lktD genes29 and periplasmic iron-regulated proteins.30 This may also explain some of the differences in protection among vaccines. In the present study, the 5-way MLV vaccine containing an M haemolytica toxoid reduced lung lesion scores, mortality rate, and clinical signs of disease in calves challenge exposed with a virulent isolate of B trehalosi. Serologic testing confirmed that calves vaccinated with the 5-way MLV vaccine containing M haemolytica toxoid had high antibody titers against M haemolytica lkt. The PFGE analysis of the strain of B trehalosi used for the challenge exposure confirmed the presence of lktB and lktD genes. In an unpublished study,r 18 of 40 B trehalosi isolates from 14 states obtained from 1999 to 2010 contained both lktB and lktD genes.This suggests that at least in situations whereby lkt is produced by B trehalosi, an M haemolytica vaccine with inactivated lkt may protect calves against infection. Field reports support the use of M haemolytica toxoid in protecting cattle and controlling outbreaks of pneumonia attributable to B trehalosi in cattle.9 Results of the present study supported that a 5-way MLV vaccine containing M haemolytica toxoid can help prevent pneumonia caused by B trehalosi in cattle. a. b. c. d. e. f. g. h. i.
Blanchard PC, California Animal Health and Food Safety Lab, University of California-Davis, Tulare, Calif: Personal communication, 2012. Central States Testing, Elizabethtown, Ky. PROC MIXED, SAS, version 9.1.3, SAS Institute Inc, Cary, NC. Bovi-Shield GOLD 5, Zoetis Inc, Kalamazoo, Mich. Bovi-Shield GOLD One Shot, Zoetis Inc, Kalamazoo, Mich. California Animal Health and Food Safety Lab, University of California-Davis, Tulare, Calif. Biolog Microbial ID Systems, Biolog Inc, Hayward, Calif. HP proprietary medium used to culture fastidious respiratory pathogens, Zoetis Inc, Kalamazoo, Mich. Lidocaine hydrochloride injection 2%, Sparhane Laboratories, Lenexa, Kan. AJVR, Vol 75, No. 8, August 2014
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j. k. l. m. n. o. p. q. r.
Large animal tracheal wash kit, Semi-Flex, product No. 64900, Har-Vet, Spring Valley, Wis. Zoetis Inc, Kalamazoo, Mich. Horseradish peroxidase–conjugated goat anti-bovine IgG (H+L), Jackson Laboratory, Bar Harbor, Me. ABTS peroxidase substrate, Kirkegaard and Perry Laboratories Inc, Gaithersburg, Md. Dryswab laryngeal swab, MW128, MWE Medical Wire, Corsham, England. PresponseHM, Boehringer Ingelheim, St Joseph, Mo. OviPast, Merck, Milton Keynes, England. Pulmoguard PH-M, Agri Laboratories Ltd, St Joseph, Mo. Murray RW, Global Discovery, Zoetis Inc, Kalamazoo, Mich: Personal communication, 2013.
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
Donachie W. Pasteurellosis. In: Aiken ID, ed. Diseases of sheep. 4th ed. Oxford, England: Blackwell, 2007;224–231. Blackall PJ, Bjoesen AM, Christensen H, et al. Reclassification of [Pasteurella] trehalosi as Bibersteinia trehalosi. Int J Syst Evol Microbiol 2007;57:666–674. Cassirer EF, Rudolph KM, Fowler P, et al. Evaluation of ewe vaccination as a tool for increasing bighorn lamb survival following pasteurellosis epizootics. J Wildl Dis 2001;37:49–57. Dyer NW, Ward AC, Weiser GC, et al. Seasonal incidence and antibiotic susceptibility patterns of Pasteurellaceae isolated from American bison (Bison bison). Can J Vet Res 2001;65:7–14. Ward ACS, Dyer NW, Fenwick BW. Pasteurellaceae isolated from tonsillar samples of commercially reared American Bison (Bison bison). Can J Vet Res 1999;63:161–165. Allan EM, Wiseman A, Gibbs HA, et al. Pasteurella species isolated from the bovine respiratory tract and their antimicrobial sensitivity patterns. Vet Rec 1985;117:629–631. Ball HJ, Connolly M, Cassidy J. Pasteurella haemolytica serotypes isolated in Northern Ireland during 1989–1991. Br Vet J 1993;149:561–570. Quirie M, Donachie W, Gilmour JL. Serotypes of Pasteurella haemolytica from cattle. Vet Rec 1986;119:93–94. Cortese VS, Braun DA, Crouch D, et al. Case report—peracute to acute fatal pneumonia in cattle caused by Bibersteinia trehalosi. Bovine Pract 2012;46:138–142. Collins RL. Bibersteinia trehalosi in cattle—another component of the bovine respiratory disease complex? Cattle Pract 2011;19:9–12. California Animal Health and Food Safety Laboratory System 2004 annual report. Available at: www.cahfs.ucdavis.edu/ local-assets/annual_reports/2004ar.pdf. Accessed Feb 25, 2014. California Animal Health and Food Safety Laboratory System 2005 annual report. Available at: www.cahfs.ucdavis.edu/ local-assets/annual_reports/2005ar.pdf. Accessed Feb 25, 2014. California Animal Health and Food Safety Laboratory System 2006 annual report. Available at: www.cahfs.ucdavis.edu/ local-assets/annual_reports/2006ar.pdf. Accessed Feb 25, 2014. California Animal Health and Food Safety Laboratory System 2007 annual report. Available at: www.cahfs.ucdavis.edu/ local-assets/annual_reports/2007ar.pdf. Accessed Feb 25, 2014. California Animal Health and Food Safety Laboratory System 2008 annual report. Available at: www.cahfs.ucdavis.edu/ local-assets/annual_reports 2008ar.pdf. Accessed Feb 25, 2014.
16. California Animal Health and Food Safety Laboratory System 2009 annual report. Available at: www.cahfs.ucdavis.edu/ local-assets/annual_reports 2009ar.pdf. Accessed Feb 25, 2014. 17. Villard L, Gauthier D, Lacheretz A, et al. Serological and molecular comparison of Mannheimia haemolytica and Pasteurella trehalosi strains isolated from wild and domestic ruminants in the French Alps. Vet J 2006;171:545–550. 18. Villard L, Gauthier D, Maurin F, et al. Serotypes A1 and A2 of Mannheimia haemolytica are susceptible to genotypic, capsular and phenotypic variations in contrast to T3 and T4 serotypes of Bibersteinia (Pasteurella) trehalosi. FEMS Microbiol Lett 2008;280:42–49. 19. Sneath PH, Stevens M. Actinobacillus rossii sp. nov., Actinobacillus seminis sp. nov., nom. rev., Pasteurella bettii sp. nov., Pasteurella lymphangitidis sp. nov., Pasteurella mairi sp. nov., and Pasteurella trehalosi sp. nov. Int J Syst Bacteriol 1990;40:148–153. 20. McNeil HJ, Shewen PE, Lo RYC, et al. Novel protease produced by a Pasteurella trehalosi serotype 10 isolate from a pneumonic bighorn sheep: characteristics and potential relevance to protection. Vet Microbiol 2003;93:145–152. 21. Shewen PE, Wilkie BN. Pasteurella haemolytica cytotoxin: production by recognized serotypes and neutralization by typespecific rabbit antisera. Am J Vet Res 1983;44:715–719. 22. McNeil HJ, Shewen PE, Lo RYC, et al. Mannheimia haemolytica serotype 1 and Pasteurella trehalosi serotype 10 culture supernatants contain fibrinogen-binding proteins. Vet Immunol Immunopathol 2002;90:107–110. 23. Goodwin-Ray KA, Stevenson MA, Heurer C. Effect of vaccinating lambs against pneumonic pasteurellosis under New Zealand field conditions on their weight gain and pneumonic lung lesions at slaughter. Vet Rec 2008;162:9–11. 24. Ward ACS, Weiser GC, DeLong WJ, et al. Characterization of Pasteurella spp isolated from healthy domestic pack goats and evaluation of the effects of a commercial Pasteurella vaccine. Am J Vet Res 2002;63:119–123. 25. Subramaniam R, Shanthalingam S, Bavananthasivam J, et al. A multivalent Mannheimia-Bibersteinia vaccine protects bighorn sheep against Mannheimia haemolytica challenge. Clin Vaccine Immunol 2011;18:1689–1694. 26. Hodgson JC, Moon GM, Quirie M, et al. Biochemical signs of endotoxaemia in lambs challenged with T10 strain of Pasteurella haemolytica and the effect of vaccination on the host response, in Proceedings. Sheep Vet Soc 1993;201–204. 27. Srinand S, Maheswaran SK, Ames TR, et al. Evaluation of efficacy of three commercial vaccines against experimental bovine pneumonic pasteurellosis. Vet Microbiol 1996;52:81–89. 28. Confer AW, Fulton RW, Clinkenbeard KD, et al. Duration of serum antibody responses following vaccination and revaccination of cattle with non-living commercial Pasteurella haemolytica vaccines. Vaccine 1998;16:1962–1970. 29. Davies RL, Campbell S, Whittam TS. Mosaic structure and molecular evolution of the leukotoxin operon (lktCABD) in Mannheimia (Pasteurella) haemolytica, Mannheimia glucosida, and Pasteurella trehalosi. J Bacteriol 2002;184:266–277. 30. Tabatabai LB, Frank GH. Conservation of expression and n-terminal sequences of the Pasteurella haemolytica 31-kilodalton and Pasteurella trehalosi 29-kilodalton periplasmic iron-regulated proteins. Clin Diagn Lab Immunol 1999;6:617– 620.
Appendix appears on the next page
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Appendix Clinical evaluation scoring used for all calves from 2 days before challenge exposure until the end of the study.* Score
Classification
Description
0
Healthy calf
1
Nonspecific illness
2
Acute BRD
3
Severe BRD
Calf is alert and active; stands, moves, and responds to stimuli quickly and steadily; and has continuous interest in its surroundings. Clinical signs not necessarily specific for acute BRD infection; clinical signs may include nasal discharge, and the calf may hold its head low and be mildly lethargic. Clinical signs are moderate and specific for acute BRD and may include a marked increase in respiratory effort. Calf is lethargic and has signs of depression and a reduced interest in its surroundings, walks slowly, and may stagger. Calf is slow to respond to stimuli and is frequently recumbent. Repeated coughing and nasal discharge are not common but may be evident. Clinical signs may include acute respiratory distress (with open-mouth breathing or grunting) and marked lethargy and signs of depression. Calf is gaunt and recumbent, has little or no response to stimuli, and has difficulty when attempting to stand or move.
*Calves with a score of 3 were euthanized for humane reasons; surviving calves were euthanized 6 days after challenge exposure.
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Objective—To determine the efficacy of a multivalent modified-live virus (MLV) vaccine containing a Mannheimia haemolytica toxoid to reduce pneumonia and mortality rate when administered to calves challenge exposed with virulent Bibersteinia trehalosi. Animals—74 Holstein calves. Procedures—Calves were assigned to 2 treatment groups. Calves in the control group (n = 36) were vaccinated by SC administration of 2 mL of a commercial 5-way MLV vaccine, and calves in the other group (38) were vaccinated by SC administration of a 2-mL dose of a 5-way MLV vaccine containing M haemolytica toxoid (day 0). On day 21, calves were transtracheally administered B trehalosi. Serum was obtained for analysis of antibody titers against M haemolytica leukotoxin. Nasopharyngeal swab specimens were collected from calves 1 day before vaccination (day –1) and challenge exposure (day 20) and cultured to detect bacterial respiratory pathogens. Clinical scores, rectal temperature, and death attributable to the challenge-exposure organism were recorded for 6 days after challenge exposure. Remaining calves were euthanized at the end of the study. Necropsy was performed on all calves, and lung lesion scores were recorded. Results—Calves vaccinated with the MLV vaccine containing M haemolytica toxoid had significantly lower lung lesion scores, mortality rate, and clinical scores for respiratory disease, compared with results for control calves. Conclusions and Clinical Relevance—Administration of a multivalent MLV vaccine containing M haemolytica toxoid protected calves against challenge exposure with virulent B trehalosi by reducing the mortality rate, lung lesion scores, and clinical scores for respiratory disease. (Am J Vet Res 2014;75:770–776)
B
ibersteinia trehalosi (previously known as Pasteurella trehalosi and Pasteurella haemolytica type T) is a gram-negative bacterial respiratory tract pathogen that causes pneumonia (as well as septicemia) in domestic sheep, goats, and bighorn sheep.1–3 The organism is the most common pathogen found in tonsils of clinically normal American Bison.4,5 It has been identified as a major cause of pneumonia and septicemia in cattle.6–8 Bibersteinia trehalosi can cause peracute bacterial pneumonia in adult dairy cattle, feedlot cattle, and young calves.9 Clinical signs range from pneumonia to sudden death. Lungs often have exudative fibrinous pneumonia, bronchiolitis, and alveolitis.9,10 Received February 25, 2014. Accepted April 9, 2014. From Global Biological Research (Bowersock, Leyh) and Global Development and Operations (Sobecki, Terrill, Martinon, Meinert), Zoetis Inc, Building 300 Dock, 333 Portage St, Kalamazoo, MI 49007. Supported by Zoetis Inc. Presented in abstract form at the Bovine Respiratory Disease Symposium 2014: New Approaches to Bovine Respiratory Disease Prevention, Management and Diagnosis, Denver, July 2014. Address correspondence to Dr. Bowersock (terry.l.bowersock@zoetis. com). 770
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AHDRCC BRD CI lkt MLV OD PBST PFGE
ABBREVIATIONS
Animal Health Developmental Research Culture Collection of Zoetis Inc Bovine respiratory disease Confidence interval Leukotoxin Modified-live virus Optical density PBS solution and 0.05% Tween 20 Pulsed-field gel electrophoresis
Bibersteinia trehalosi has been identified in healthy as well as pneumonic or septicemic cattle in the United Kingdom since 2003.10 Increasingly, diagnostic laboratories are isolating B trehalosi from clinically affected cattle.6–8 A report10 from the United Kingdom indicated that B trehalosi was the only pathogen in 18 of 65 (28%) cattle with pneumonia. From 2004 to 2009, B trehalosi was identified in 22 to 45 dairy cattle with pneumonia/y,11–16 and from 2008 to 2011, 149 cases of respiratory disease attributable to B trehalosi were identified at a diagnostic laboratory in California.a In the past, field isolates of B trehalosi were likely to be classified within the Mannheimia haemolytica comAJVR, Vol 75, No. 8, August 2014
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plex, which may have obscured the role of B trehalosi in disease in cattle.2 Some of the increase in apparent prevalence is the result of previous misdiagnosis of B trehalosi, which is phenotypically similar to M haemolytica. The use of PFGE has been beneficial in the assessment of the source of respiratory Pasteurellaceae in epidemiological investigations of wild and domestic ruminants and has confirmed the role of B trehalosi as a primary pathogen.17,18 The name B trehalosi was proposed by scientists following molecular evaluation of this group of organisms and the determination that the organism differs sufficiently from other Pasteurella spp and thus deserves a group to itself on the basis of DNA-DNA relatedness19 and on the basis of amplified fragment length polymorphism and 16s rRNA sequencing.2 Although genetically different to the extent that it is now a separate genus and species, B trehalosi shares some pathogenic characteristics with M haemolytica. The gene lktA is key to expression of lkt, which is a major virulence factor of M haemolytica; lktA has been found in B trehalosi.20 There is evidence that rabbit antisera to all serotypes of M haemolytica 1 through 12 (including what is currently referred to as B trehalosi) cytotoxin (lkt) cross neutralizes toxin from M haemolytica A1.21 Fibrinogen-binding proteins have been found in supernatants of both M haemolytica and B trehalosi, and evaluation of western blots has revealed that the fibrinogen molecules are of a similar size.22 A protease has been identified in B trehalosi that is similar to a protease in M haemolytica. For both organisms, prechallenge sera neutralizes protease activity, which results in lower lung lesion scores and suggests an immune response to protease is important in protection against both B trehalosi and M haemolytica.20 In the study2 that was intrinsic to the name change of this organism to B trehalosi, 9 of 43 isolates evaluated were isolated from cattle with pneumonia. Antimicrobial treatment cannot reliably control the peracute-acute nature of infections attributable to B trehalosi9; therefore, a vaccine is needed to aid in control of this bacterial pathogen in domestic as well as wild ruminants. The purpose of the study reported here was to evaluate the efficacy of a commercial vaccine containing multivalent MLV plus an M haemolytica toxoid for protection of calves against pneumonia after challenge exposure with B trehalosi. Materials and Methods Animals—Young (1 to 2 days old) top grade Holstein bull calves (n = 74) were purchased from sale barns and transported to a commercial farm. Requirements were that calves weigh > 45.5 kg and be vigorous and healthy. At the time of purchase, all calves had negative results for persistent infection with bovine viral disease virus, as determined by use of an antigencapture ELISA performed by personnel at a commercial laboratory.b Calves were raised on the commercial farm until they were 9 to 10 weeks old, when they were moved to a Zoetis Research farm in Richland, Mich. Before transport to the research farm, calves were judged to be healthy and had negative results for M haemolytica, Pasteurella multocida, and B trehalosi on nasopharynAJVR, Vol 75, No. 8, August 2014
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geal swab specimens. In addition, serum antibody titers against M haemolytica lkt and P multocida outer membrane proteins were tested with ELISAs by use of cutoff values that had been used to indicate cattle would be susceptible to challenge exposure with Pasteurellaceae in preliminary studies conducted by our research group. At the research farm, calves were housed in groups in rooms (14.94 X 8.23 X 3.05 m). Calves were housed such that they could not directly or indirectly contact calves in other rooms for the duration of the study. Animal identification and assignment per room were provided to the investigators. Calves then were randomly allocated to treatments within each room in accordance with a generalized randomized block design with the blocking factor based on room. The random treatment allocation plan was generated with a computer programc that used a random number generator function. There were 26 calves in one room, 24 calves in a second room and 24 calves in a third room; the 2 latter rooms had 12 calves of each treatment group, whereas the other room had 12 calves of one treatment group and 14 calves of the other treatment group. Animals remained in their assigned room for the duration of the study. The study was approved by the Animal Use and Care Committee of Zoetis Inc. Study design—Before assignment to study groups, all calves were clinically assessed (Appendix). Each calf had a clinical score of 0 and rectal temperature < 39.4°C and was free of B trehalosi and M haemolytica. Calves (n = 36) in one group were injected SC with a single 2-mL dose of a commercially available 5-way MLV vaccine containing bovine herpesvirus 1, bovine viral diarrhea virus types 1 and 2, parainfluenza virus type 3, and bovine respiratory syncytial virus,d and calves in the other group (38) were injected SC with a single 2-mL dose of a 5-way MLV vaccine containing the same 5 viral agents and M haemolytica toxoid.e Day of vaccination was designated as day 0. Twenty-one days after vaccination, calves were challenge-exposed with B trehalosi strain AHDRCC 40499. The challenge organism was acquired at a diagnostic laboratoryf from a calf with pneumonia. The challenge organism was identified by use of biochemical methods including a commercial microbial identification systemg and 16sRNA; subsequently, identity of a subset was confirmed by use of matrix-assisted laser desorption ionization–time-of-flight (ie, MALDI-TOF) and to possess lkt genes by use of PFGE. Challenge exposure—Calves were challenge exposed by transtracheal injection with 120 mL of a logarithmic-phase broth culture of B trehalosi AHDRCC 40499. Culture of B trehalosi was initiated by inoculating 50 mL of warmed HP mediumh into a 250-mL baffled flask that contained a loop (approx 100 µL) of frozen stock. The flask was incubated at 37°C with shaking (150 revolutions/min) for 15 to 18 hours. An aliquot (2 mL) of the culture was added to each 100 mL of HP medium in an appropriately sized baffled flask, which was incubated at 37°C with shaking (150 revolutions/min) until the OD (measured at 650 nm) approxi771
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mated that for a solution containing 1.0 X 109 CFUs/ mL. The flask was immediately placed on ice, and the contents were used as a stock solution that was diluted in PBS solution to achieve a final challenge-exposure dose of 6.4 X 1010 CFUs in 120 mL. Diluted challengeexposure material was maintained on ice until administered. Aliquots of the challenge-exposure material as well as the stock solution were retained in the laboratory and used to perform plate counts to determine the actual dose used for challenge exposure. Challenge exposure was conducted in the rooms used to house the calves. Briefly, calves were restrained in a standing position. Hair over the tracheal region of each calf was clipped, and the area was disinfected with alcohol wipes. Then, 4 mL of lidocaine solutioni was infused SC into the tissues over the trachea (diameter of infiltrated area, 1 to 2 cm). A 16-gauge trocharj was directed caudally through the skin and into the trachea at the location of the local anesthesia. A cannula was inserted through the trochar and advanced to the bifurcation of the trachea; it was then retracted 2 to 4 cm to ensure the tip was located in the distal aspect of the trachea and not in a major bronchus. A total of 120 mL of diluted challenge-exposure material was then infused through the cannula, which was followed by flushing with 60 mL of cold HP medium. The cannula was then removed. Clinical monitoring—Calves were observed, clinical scores of disease were assigned, and rectal temperatures were recorded daily after challenge exposure by individuals who were unaware of the treatment group of each calf. Calves that died or were euthanized because of clinical disease after challenge exposure were submitted for necropsy. All remaining calves were euthanized by captive bolt 6 days after challenge exposure (ie, day 27) and submitted for necropsy. Sample collection—Blood samples were collected from each calf on days –1, 20, and day of death or euthanasia (day 27 for most calves in the study). Samples were allowed to clot at room temperature (22.2°C). Serum was harvested, divided into 2 aliquots, and submitted for testing or stored frozen. Serum titers against M haemolytica lkt—Serum samples were assayed for antibodies against M haemolytica lkt by use of a sandwich ELISA developed at Zoetis Inc. Each well of a 96-well plate was coated with 100 µL of capture antibody by use of 0.43 mg of murine monoclonal antibodyk against M haemolytica lkt/ mL in 0.01M borate buffer. The plate was sealed and incubated for 16 to 32 hours at 4°C. The contents of each well were decanted, and 200 µL of 1% casein in PBST was added to each well. The plate was sealed and incubated for 1 to 4 hours at 37°C. The buffer was then decanted, and 100 µL of M haemolytica lkt supernatantk diluted 1:500 in 1% casein in PBST was added to each well. The plate was sealed and incubated for a mean ± SD of 60 ± 10 minutes at 37°C. The plate was decanted, and 100 µL of calf serum, control samples, and blanks was added to each well. The plate was then sealed and incubated for another 60 ± 10 minutes at 37°C. Serum samples from each calf and positive and 772
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negative control sera were prepared as serial 1:2 dilutions in 1% casein in PBST. Briefly, samples from each calf were plated in duplicate at dilutions of 1:100 to 1:3,200, the positive internal assay control sample was plated in duplicate at dilutions of 1:1,600 to 1:51,200, and the negative internal assay control sample was plated in duplicate at dilutions of 1:100 to 1:3,200. Each positive and negative internal control sample was also included (dilution of 1:800) in 5 wells of each plate and as a full dilution series (1:1,600 to 1:51,200) to duplicate wells in a separate plate. At the end of the incubation period, each well of every plate was washed 3 times with PBST (300 µL/well). The PBST was decanted, and 100 µL of goat anti-bovine IgGl diluted 1:5,000 in 1% casein was added to each well. The plate was sealed and incubated for 60 ± 10 minutes at 37°C. The plate then was washed 3 times with PBST (300 µL/well). The PBST was decanted, and 100 µL of substrate was added to each well. Substrate was prepared with equal parts of 2 components of a peroxidase substrate.m The plate was protected from light and incubated at 27 ± 2°C for approximately 10 minutes. The OD of plates was determined at 405 and 490 nm by use of a spectrophotometer; the OD for 490 nm was subtracted from the OD for 405 nm. The assay was considered valid and the plate value determined when the positive control sample was between 0.8 and 1.2 net absorbance units, the negative control sample was < 0.3 net absorbance units, and the blank was < 0.12 net absorbance units. The cutoff for the positive control sample was determined as the mean absorbance for the positive control sample divided by 5. A sample titer was calculated as the last titer point at which the mean absorption value for a sample minus the positive cutoff value was greater than zero. Nasopharyngeal swab specimensn were collected on days –1 (before vaccination) and 20 (before challenge exposure). Samples were submitted for standard bacterial culture to detect B trehalosi, P multocida, M haemolytica, Histophilus somni, and Trueperella pyogenes. Swab specimens and tissues were collected from the lungs of calves on the day of necropsy. Lung swab specimens were cultured to detect B trehalosi, P multocida, M haemolytica, H somni, and T pyogenes. Determination of lung lesion scores—Lung lesion scores (total percentage of consolidated lung in each lobe) were determined by an individual who was unaware of the treatment group of each calf. Percentage consolidation in each lung lobe was determined manually and was weighted by use of the following ratios of individual lung lobe to total lung mass: cranial part of the left cranial lobe, 5%; caudal part of the left cranial lobe, 6%; left caudal lobe, 32%; cranial part of the right cranial lobe, 6%; caudal part of the right cranial lobe, 5%; right medial lobe, 7%; right caudal lobe, 35%; and accessory lobe, 4%. Weighted lung lobe values were summed for all lobes to yield the percentage consolidation of the lungs for each calf. Data analysis—Arc-sin square root transformation was applied to the percentage consolidation of lung values, which was followed by analysis with a general linear mixed modelc that had treatment as a fixed efAJVR, Vol 75, No. 8, August 2014
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fect and block (room) as a random effect. When there was a significant (P ≤ 0.05) overall treatment effect, linear combinations of parameter estimates were used in a priori contrasts to make comparisons between treatments. Back-transformed least squares means, SEMs, and 95% CIs were calculated from least squares parameter estimates obtained from the analyses. For mortality data, a calf was described as having survived (euthanized at the end of the study on day 27) or as having died or been euthanized (died or euthanized before day 27) on the basis of data collected on the final disposition form and summarized by treatment. When death or euthanasia of a calf was related to the challenge exposure (ie, before day 27), then death was coded as a binary variable (0 = survived; 1 = died or was euthanized) and analyzed with a generalized mixed model proceduren by use of a binomial error and logit link. The model included treatment as a fixed effect and block (room) as a random effect. Least squares means and back-transformed least squares means and 95% CIs were reported. When there was a significant (P ≤ 0.05) treatment effect, comparison of mortality rates was performed. Death or euthanasia related to challenge exposure was summarized by use of the prevented fraction with 95% confidence limits. For clinical observation data, the number of calves with a clinical score ≥ 2 (acute or severe BRD) after challenge exposure was summarized. Results Clinical signs of respiratory tract disease, mortality rate, and lung lesion scores—Calves developed typical clinical signs of respiratory tract disease after challenge exposure. Acute BRD (clinical score ≥ 2 on
at least 1 day after challenge exposure) was observed during the 6 days after challenge exposure in significantly (P = 0.002) more control calves (26/36 [72.2%]) than in calves vaccinated with the 5-way MLV vaccine containing M haemolytica toxoid (13/38 [34.2%]; Table 1). Control calves had a significantly (P = 0.049) higher mortality rate (12/36 [33.3%]), compared with the mortality rate for the calves vaccinated with the 5-way MLV vaccine containing M haemolytica toxoid (5/38 [13.2%]). Mean percentage consolidated lung for the control calves (43%) was significantly (P = 0.012) higher than the percentage for the calves vaccinated with the 5-way MLV vaccine containing M haemolytica toxoid (30%). There were no significant differences in mean rectal temperature between groups during the study. Serum testing and bacterial culture of nasopharyngeal swab specimens and lung tissues—All of the calves had serum anti-lkt antibody titers ≤ 1:3,200 at the start of the study (Table 2). This titer had been found to be the break point at which animals were susceptible to experimental challenge exposure in preliminary studies conducted by our research group. Mean anti-lkt antibody titer remained low for the control calves and was 248 (95% CI, 181 to 339) at the time of challenge exposure (day 21). Calves vaccinated with the 5-way MLV vaccine containing M haemolytica toxoid had a significantly (P < 0.001) higher mean antibody titer (5,254; 95% CI, 3,867 to 7,138) before challenge exposure. After challenge exposure with B trehalosi, mean antibody titers increased in both groups of calves, although the mean antibody titer for the calves vaccinated with the 5-way MLV vaccine containing
Table 1—Lung lesion scores, mortality rate, and clinical BRD rate for 36 calves vaccinated by SC administration of a commercial 5-way MLV vaccine (control group) and 38 calves vaccinated by SC administration of a 5-way MLV vaccine containing Mannheimia haemolytica toxoid (5-way toxoid) and challenge exposed with Bibersteinia trehalosi 21 days later. Group
Lung lesion score (%)*
Control 5-way toxoid
43 (35–50) 30‡ (23–57)
Mortality rate (%)
Calves with clinical score ≥ 2 (%)†
33.3 (19.8–50.3) 13.2§ (5.5–28.3)
72.2 (55.3–84.5) 34.2 ll(20.8–50.7)
Values reported are least squares mean (95% CI). *Represents the percentage of consolidated lung for all lung lobes. †Clinical score was assessed by use of a scale of 0 to 3 (0, healthy calf; 1, nonspecific illness; 2, acute BRD; and 3, severe BRD). ‡,§,llWithin a column, value differs significantly (‡P = 0.012; §P = 0.049; llP = 0.002) from the value for the control group. Table 2—Serum anti-lkt antibody titers for 36 calves vaccinated (day 0) by SC administration of a commercial 5-way MLV vaccine (control group) and 38 calves vaccinated (day 0) by SC administration of a 5-way MLV vaccine containing M haemolytica toxoid (5-way toxoid) and challenge exposed with B trehalosi on day 21. Group Control 5-way toxoid
Day –1 367 (269–501) 417 (307–566)
Day 20 248 (181–339) 5,254† (3,867–7,138)
Day 22 to 27* 2,714 (1,911–3,852) 7,566† (5,506–10,393)
Values reported are least squares mean (95% CI). *Calves were observed for 6 days after challenge exposure; titers were determined in samples obtained from calves on the day they died or were euthanized because of illness attributable to B trelahosi (12 control calves and 5 calves vaccinated with the 5-way MLV vaccine containing M haemolytica toxoid) or in samples obtained from the surviving calves (24 control calves and 33 calves vaccinated with the 5-way MLV vaccine containing M haemolytica toxoid), which were euthanized on day 27. †Within a column, value differs significantly (P < 0.001) from the value for the control group. AJVR, Vol 75, No. 8, August 2014
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M haemolytica toxoid remained significantly (P < 0.001) higher (7,566; 95% CI, 5,506 to 10,393), compared with the mean antibody titer for the control calves (2,714; 95% CI, 1,911 to 3,852). Nasopharyngeal swab specimens obtained on days –1 and 20 had negative results for bacterial culture of B trehalosi and M haemolytica. Lung swab specimens obtained at necropsy yielded B trehalosi for 25 of 36 (69.4%) control calves and 24 of 38 (63.2%) calves vaccinated with the 5-way MLV vaccine containing M haemolytica toxoid. None of the calves in either group had specimens that yielded H somni. One control calf had specimens that yielded T pyogenes and M haemolytica, and 1 calf vaccinated with the 5-way MLV vaccine containing M haemolytica toxoid had specimens that yielded P multocida. This indicated that the lung lesion scores were most likely attributable to infection with B trehalosi. Discussion Bibersteinia trehalosi is a pathogen of cattle that causes acute pneumonia and septicemia in a wide range of age groups. The acute nature of B trehalosi infections and the sometimes poor response to antimicrobial treatment9 suggest the need for a vaccine to control infection. Currently, there are no vaccines with label claims for prevention of diseases attributable to B trehalosi in cattle. Several vaccines have had variable results for controlling respiratory tract disease attributable to B trehalosi, but most of those studies3,10,22–25 were conducted in domestic and bighorn sheep. A decrease in survival rate and an increase in incidence of respiratory tract disease were reported3 in lambs of bighorn ewes vaccinated IM with 2 mL of a commercial vaccine labeled for cattle that included P multocida bacterin and M haemolytica leukotoxoid.o In another study23 in a large commercial sheep flock, 2 doses of an experimental vaccine containing M haemolytica strains A1, A2, A6, A7, and A9 and B trehalosi strains T3,T4, T10, and T15p administered SC in the neck did not improve average daily gain, reduce severity of respiratory tract disease, or reduce the recovery of either pathogen during postmortem examination. In a third study,24 a 2-mL dose of an M haemolytica–P multocida bacterin leukotoxoidq did not reduce pharyngeal colonization by M haemolytica or B trehalosi in pack goats, despite good induction of lkt-neutralizing antibodies. There have been other reports that use of experimental vaccines reduced disease attributable to B trehalosi. An unidentified vaccine or vaccines against B trehalosi improved disease control in sheep.10 In another study,20 a protease produced by a B trehalosi isolate was found to be immunogenic, and serum antibodies were associated with a reduction in lung lesions in bighorn sheep challenge exposed with B trehalosi. In a separate study,25 bighorn sheep vaccinated several times with culture supernatants from M haemolytica A2 and B trehalosi as well as with an M haemolytica A1 vaccineo had a reduction in the severity and incidence of pneumonia and an increase in survival rate. The sheep in that study25 also had high titers of lkt-neutralizing and bacterial surface-specific antibodies. In a study26 in which investigators evaluated use of an iron-regulated protein–based B trehalosi vac774
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cine in sheep, there was good efficacy in reducing the mortality rate after challenge exposure. However, to our knowledge, no vaccines have been tested for efficacy in cattle. The vaccination trial reported here was conducted to evaluate the efficacy of a 5-way MLV vaccine containing M haemolytica toxoid with inactivated lkt. Investigators have found that this vaccine induces good protection in cattle in the face of challenge exposure with M haemolytica.27,28 Although it has been suggested that cattle isolates of B trehalosi typically are nonhemolytic and less likely to produce lkt, evaluation of a large number of isolates from across the United States suggests that the prevalence of lkt genes in B trehalosi is much higher.r The fact that B trehalosi possesses several other antigens related to those expressed by M haemolytica suggests that cross protection by a vaccine is plausible. Apparent failures in sheep and wild ungulates may have been attributable to a variety of reasons, such as differences in the sample of animals used as indicators of protection, prevalence of disease in the herd or flock at the time of the study, and other stressors (especially handling of wild ungulates) that could reduce efficacy of vaccines. Although similarities exist genetically with the lktA gene, there are significant differences among Pasteurellaceae, including B trehalosi, in the lktA gene as well as in lktB, lktC, and lktD genes29 and periplasmic iron-regulated proteins.30 This may also explain some of the differences in protection among vaccines. In the present study, the 5-way MLV vaccine containing an M haemolytica toxoid reduced lung lesion scores, mortality rate, and clinical signs of disease in calves challenge exposed with a virulent isolate of B trehalosi. Serologic testing confirmed that calves vaccinated with the 5-way MLV vaccine containing M haemolytica toxoid had high antibody titers against M haemolytica lkt. The PFGE analysis of the strain of B trehalosi used for the challenge exposure confirmed the presence of lktB and lktD genes. In an unpublished study,r 18 of 40 B trehalosi isolates from 14 states obtained from 1999 to 2010 contained both lktB and lktD genes.This suggests that at least in situations whereby lkt is produced by B trehalosi, an M haemolytica vaccine with inactivated lkt may protect calves against infection. Field reports support the use of M haemolytica toxoid in protecting cattle and controlling outbreaks of pneumonia attributable to B trehalosi in cattle.9 Results of the present study supported that a 5-way MLV vaccine containing M haemolytica toxoid can help prevent pneumonia caused by B trehalosi in cattle. a. b. c. d. e. f. g. h. i.
Blanchard PC, California Animal Health and Food Safety Lab, University of California-Davis, Tulare, Calif: Personal communication, 2012. Central States Testing, Elizabethtown, Ky. PROC MIXED, SAS, version 9.1.3, SAS Institute Inc, Cary, NC. Bovi-Shield GOLD 5, Zoetis Inc, Kalamazoo, Mich. Bovi-Shield GOLD One Shot, Zoetis Inc, Kalamazoo, Mich. California Animal Health and Food Safety Lab, University of California-Davis, Tulare, Calif. Biolog Microbial ID Systems, Biolog Inc, Hayward, Calif. HP proprietary medium used to culture fastidious respiratory pathogens, Zoetis Inc, Kalamazoo, Mich. Lidocaine hydrochloride injection 2%, Sparhane Laboratories, Lenexa, Kan. AJVR, Vol 75, No. 8, August 2014
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j. k. l. m. n. o. p. q. r.
Large animal tracheal wash kit, Semi-Flex, product No. 64900, Har-Vet, Spring Valley, Wis. Zoetis Inc, Kalamazoo, Mich. Horseradish peroxidase–conjugated goat anti-bovine IgG (H+L), Jackson Laboratory, Bar Harbor, Me. ABTS peroxidase substrate, Kirkegaard and Perry Laboratories Inc, Gaithersburg, Md. Dryswab laryngeal swab, MW128, MWE Medical Wire, Corsham, England. PresponseHM, Boehringer Ingelheim, St Joseph, Mo. OviPast, Merck, Milton Keynes, England. Pulmoguard PH-M, Agri Laboratories Ltd, St Joseph, Mo. Murray RW, Global Discovery, Zoetis Inc, Kalamazoo, Mich: Personal communication, 2013.
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
Donachie W. Pasteurellosis. In: Aiken ID, ed. Diseases of sheep. 4th ed. Oxford, England: Blackwell, 2007;224–231. Blackall PJ, Bjoesen AM, Christensen H, et al. Reclassification of [Pasteurella] trehalosi as Bibersteinia trehalosi. Int J Syst Evol Microbiol 2007;57:666–674. Cassirer EF, Rudolph KM, Fowler P, et al. Evaluation of ewe vaccination as a tool for increasing bighorn lamb survival following pasteurellosis epizootics. J Wildl Dis 2001;37:49–57. Dyer NW, Ward AC, Weiser GC, et al. Seasonal incidence and antibiotic susceptibility patterns of Pasteurellaceae isolated from American bison (Bison bison). Can J Vet Res 2001;65:7–14. Ward ACS, Dyer NW, Fenwick BW. Pasteurellaceae isolated from tonsillar samples of commercially reared American Bison (Bison bison). Can J Vet Res 1999;63:161–165. Allan EM, Wiseman A, Gibbs HA, et al. Pasteurella species isolated from the bovine respiratory tract and their antimicrobial sensitivity patterns. Vet Rec 1985;117:629–631. Ball HJ, Connolly M, Cassidy J. Pasteurella haemolytica serotypes isolated in Northern Ireland during 1989–1991. Br Vet J 1993;149:561–570. Quirie M, Donachie W, Gilmour JL. Serotypes of Pasteurella haemolytica from cattle. Vet Rec 1986;119:93–94. Cortese VS, Braun DA, Crouch D, et al. Case report—peracute to acute fatal pneumonia in cattle caused by Bibersteinia trehalosi. Bovine Pract 2012;46:138–142. Collins RL. Bibersteinia trehalosi in cattle—another component of the bovine respiratory disease complex? Cattle Pract 2011;19:9–12. California Animal Health and Food Safety Laboratory System 2004 annual report. Available at: www.cahfs.ucdavis.edu/ local-assets/annual_reports/2004ar.pdf. Accessed Feb 25, 2014. California Animal Health and Food Safety Laboratory System 2005 annual report. Available at: www.cahfs.ucdavis.edu/ local-assets/annual_reports/2005ar.pdf. Accessed Feb 25, 2014. California Animal Health and Food Safety Laboratory System 2006 annual report. Available at: www.cahfs.ucdavis.edu/ local-assets/annual_reports/2006ar.pdf. Accessed Feb 25, 2014. California Animal Health and Food Safety Laboratory System 2007 annual report. Available at: www.cahfs.ucdavis.edu/ local-assets/annual_reports/2007ar.pdf. Accessed Feb 25, 2014. California Animal Health and Food Safety Laboratory System 2008 annual report. Available at: www.cahfs.ucdavis.edu/ local-assets/annual_reports 2008ar.pdf. Accessed Feb 25, 2014.
16. California Animal Health and Food Safety Laboratory System 2009 annual report. Available at: www.cahfs.ucdavis.edu/ local-assets/annual_reports 2009ar.pdf. Accessed Feb 25, 2014. 17. Villard L, Gauthier D, Lacheretz A, et al. Serological and molecular comparison of Mannheimia haemolytica and Pasteurella trehalosi strains isolated from wild and domestic ruminants in the French Alps. Vet J 2006;171:545–550. 18. Villard L, Gauthier D, Maurin F, et al. Serotypes A1 and A2 of Mannheimia haemolytica are susceptible to genotypic, capsular and phenotypic variations in contrast to T3 and T4 serotypes of Bibersteinia (Pasteurella) trehalosi. FEMS Microbiol Lett 2008;280:42–49. 19. Sneath PH, Stevens M. Actinobacillus rossii sp. nov., Actinobacillus seminis sp. nov., nom. rev., Pasteurella bettii sp. nov., Pasteurella lymphangitidis sp. nov., Pasteurella mairi sp. nov., and Pasteurella trehalosi sp. nov. Int J Syst Bacteriol 1990;40:148–153. 20. McNeil HJ, Shewen PE, Lo RYC, et al. Novel protease produced by a Pasteurella trehalosi serotype 10 isolate from a pneumonic bighorn sheep: characteristics and potential relevance to protection. Vet Microbiol 2003;93:145–152. 21. Shewen PE, Wilkie BN. Pasteurella haemolytica cytotoxin: production by recognized serotypes and neutralization by typespecific rabbit antisera. Am J Vet Res 1983;44:715–719. 22. McNeil HJ, Shewen PE, Lo RYC, et al. Mannheimia haemolytica serotype 1 and Pasteurella trehalosi serotype 10 culture supernatants contain fibrinogen-binding proteins. Vet Immunol Immunopathol 2002;90:107–110. 23. Goodwin-Ray KA, Stevenson MA, Heurer C. Effect of vaccinating lambs against pneumonic pasteurellosis under New Zealand field conditions on their weight gain and pneumonic lung lesions at slaughter. Vet Rec 2008;162:9–11. 24. Ward ACS, Weiser GC, DeLong WJ, et al. Characterization of Pasteurella spp isolated from healthy domestic pack goats and evaluation of the effects of a commercial Pasteurella vaccine. Am J Vet Res 2002;63:119–123. 25. Subramaniam R, Shanthalingam S, Bavananthasivam J, et al. A multivalent Mannheimia-Bibersteinia vaccine protects bighorn sheep against Mannheimia haemolytica challenge. Clin Vaccine Immunol 2011;18:1689–1694. 26. Hodgson JC, Moon GM, Quirie M, et al. Biochemical signs of endotoxaemia in lambs challenged with T10 strain of Pasteurella haemolytica and the effect of vaccination on the host response, in Proceedings. Sheep Vet Soc 1993;201–204. 27. Srinand S, Maheswaran SK, Ames TR, et al. Evaluation of efficacy of three commercial vaccines against experimental bovine pneumonic pasteurellosis. Vet Microbiol 1996;52:81–89. 28. Confer AW, Fulton RW, Clinkenbeard KD, et al. Duration of serum antibody responses following vaccination and revaccination of cattle with non-living commercial Pasteurella haemolytica vaccines. Vaccine 1998;16:1962–1970. 29. Davies RL, Campbell S, Whittam TS. Mosaic structure and molecular evolution of the leukotoxin operon (lktCABD) in Mannheimia (Pasteurella) haemolytica, Mannheimia glucosida, and Pasteurella trehalosi. J Bacteriol 2002;184:266–277. 30. Tabatabai LB, Frank GH. Conservation of expression and n-terminal sequences of the Pasteurella haemolytica 31-kilodalton and Pasteurella trehalosi 29-kilodalton periplasmic iron-regulated proteins. Clin Diagn Lab Immunol 1999;6:617– 620.
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Appendix Clinical evaluation scoring used for all calves from 2 days before challenge exposure until the end of the study.* Score
Classification
Description
0
Healthy calf
1
Nonspecific illness
2
Acute BRD
3
Severe BRD
Calf is alert and active; stands, moves, and responds to stimuli quickly and steadily; and has continuous interest in its surroundings. Clinical signs not necessarily specific for acute BRD infection; clinical signs may include nasal discharge, and the calf may hold its head low and be mildly lethargic. Clinical signs are moderate and specific for acute BRD and may include a marked increase in respiratory effort. Calf is lethargic and has signs of depression and a reduced interest in its surroundings, walks slowly, and may stagger. Calf is slow to respond to stimuli and is frequently recumbent. Repeated coughing and nasal discharge are not common but may be evident. Clinical signs may include acute respiratory distress (with open-mouth breathing or grunting) and marked lethargy and signs of depression. Calf is gaunt and recumbent, has little or no response to stimuli, and has difficulty when attempting to stand or move.
*Calves with a score of 3 were euthanized for humane reasons; surviving calves were euthanized 6 days after challenge exposure.
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