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ANIMAL PRODUCTION |
School of Veterinary and Biomedical Sciences, James Cook University, Townsville 4811, Australia
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Abstract |
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production, and tetanus-toxoid specific antibody levels were determined. Total leukocyte and eosinophil numbers showed a transient decrease at 72 h (immediately after transport; P< 0.05) and returned to baseline values by 6 d after transport. Lymphocyte numbers and antibody titers were unaffected by transportation. The PHA-stimulated lymphocyte proliferation decreased (P < 0.05) at 72 h and returned to baseline levels 6 d after transport. This study demonstrated that transportation of mature Bos indicus steers caused transient decreases in leukocyte numbers and lymphocyte function, although all measures recovered by 6 d after transport. Therefore, Bos indicus cattle may be vulnerable to infection during this period.
Key Words: Bos indicus • Interferon- • Lymphocyte Functions • Transportation Stress
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Introduction |
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; Dixit et al., 2001) and thereby may increase susceptibility to infectious diseases (Mormede et al., 1982; Grandin, 1997). On a cellular level, it was shown that stress in Bos taurus cattle, including transportation, resulted in leukocytosis, with associated neutrophilia, lymphopenia, and eosinopenia (Kent and Ewbank, 1986; Mackenzie et al., 1997), and impaired leukocyte function, with decreased response to mitogen-stimulated lymphocyte proliferation and impaired antibody production (Kelley et al., 1981; Blecha et al., 1984).
Transport-induced immunosuppression is of particular concern for animals transported to feedlots and those shipped to international markets, and it has been linked to increased incidences of "shipping fever," resulting in productivity losses (Grandin, 1997; Fazio and Ferlazzo, 2003). To date, researchers investigating transport stress in cattle have primarily employed a Bos taurus calf model, whereas the effects of transporting mature, tropical Bos indicus breeds have largely been ignored.
Bos indicus breeds, which dominate the tropical livestock industry in Australia, were introduced because they are better able to withstand the environmental stressors of a harsh environment. Although the transportation of Bos indicus animals is conducted in accordance with protocols developed for Bos taurus animals to improve animal welfare during transport, no previous studies have investigated whether the transportation of Bos indicus animals has a detrimental effect on their immune status.
The present study was designed to analyze the effects of transport stress on the immunological competence of mature Bos indicus steers. We investigated the relationships among transportation stress, leukocyte numbers, lymphocyte function, and antibody levels of cattle transported by road for 72 h.
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Materials and Methods |
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Two days before transportation, animals were weighed, and blood samples were collected (–48 h). Twelve hours before the initiation of transport, feed and water were withdrawn. Animals were trucked for 72 h in a rigid truck (8-ton tare) and loaded into the truck at a density of 0.86 m2 per animal in accordance with the Australian Code of Practice for the transportation of cattle. Over the 72-h transportation period, steers were transported a total of 5,760 km on sealed roads. The cattle spent a total of 72 h in motion with no rest stops. Immediately following transport (72 h), steers were unloaded and sampled before being returned to the original pasture. Animals had unlimited access simultaneously to feed and water immediately following the collection of the 72-h samples. Final samples were collected at 216 h (i.e., 6 d following the cessation of transport).
For the collection of blood samples, each animal was captured in a head bail, and a halter was placed over the animal’s head to restrain it while blood samples were collected. Body weight was then recorded. The mean daily temperature-humidity indices recorded at – 48, 72, and 216 h were 69, 72, and 71, respectively. All experimental procedures were reviewed and approved by the Institutional Ethics Committee (JCU No. A730_02).
Sample Collection
At each sample time, 55 mL of blood was obtained by jugular venipuncture using sterile Vacutainers (Becton Dickinson, Melbourne, Australia). A 10-mL volume of blood collected into an EDTA tube was used for enumeration of leukocytes; 40 mL of heparinized blood were used to carry out lymphocyte assays; and serum for antibody titers was obtained from 5 mL of clotted blood.
Leukocyte Numbers
Total and differential cell counts were determined using an automated counter calibrated for cattle blood (Sysmex Se-9000; Dade Behring, Sydney, Australia).
Lymphocyte Proliferation Assay
Lymphocyte proliferation assays were performed on isolated mononuclear cells as follows. Whole blood was diluted 1:1 with RPMI-1640 medium (Invitrogen, Melbourne, Australia) supplemented with penicillin (100 U/mL; Invitrogen) and streptomycin (0.1 mg/mL; Invitrogen). This whole blood suspension was overlaid onto 3 mL of Ficollpaque (Amersham Pharmacia Biotech, Sydney, Australia) and centrifuged for 20 min at 500 x g. The interface was removed, and red blood cells were lysed with Tris-ammonium chloride (0.1 to 0.8%, vol/vol). Samples were centrifuged for an additional 10 min and resuspended in the RPMI medium. Cells were washed twice by adding RPMI medium followed by centrifugation at 500 x g for 10 min. Mononuclear cells were resuspended at a concentration of 106 cells/mL in RPMI. Triplicate wells of 96-well plates (Nunc, Medos, Melbourne, Australia) were seeded with 100 µL of cell suspension and were either stimulated with phytohemagglutinin (PHA) by adding PHA to wells (10 µg/mL; Sigma Aldrich, Sydney, Australia) or remained unstimulated. Into all wells, 100 µL of RPMI-1640 culture medium supplemented with penicillin, streptomycin, L-glutamine (2 mM; Sigma Aldrich), HEPES buffer (20 mM; Invitrogen), and fetal bovine serum (10% final concentration; Invitrogen) were added, and plates were incubated at 37° C with 5% CO2 in a humidified incubator. Plates were harvested at 24-h intervals from 72 to 144 h. At 4 h before harvest, 10 µL of 3H-thymidine (1.25 µCi/mL) was added to each well. Plates were harvested, and cells were trapped onto glass fiber filter mats (Perkin Elmer, Melbourne, Australia) using an automated cell harvester (Canberra Packard, Sydney, Australia) and air-dried overnight. Levels of 3H-thymidine incorporation were evaluated using a Microplate Scintillation Counter (Canberra Packard). Results were expressed as counts per minute, and stimulation indices were determined (mean test counts per minute/mean control counts per minute). Of the four stimulation indices obtained from 72 to 144 h for each steer, the maximum stimulation index was used to compare lymphocyte proliferation within the group.
Interferon- Production
One milliliter of whole blood was dispensed into duplicate wells of a 24-well microtiter plate (Nunc) and either stimulated with PHA (10 µg/mL) or left unstimulated. Plates were incubated at 37° C for 24 and 48 h. Following incubation, whole blood was centrifuged at 500 x g for 20 min, and the supernatant fraction was aspirated into 1.5-mL Eppendorf tubes and stored at –70° C for future use. Levels of interferon- (IFN-) were determined using a commercially available BOVI-GAM ELISA kit (Pfizer Animal Health, Melbourne, Australia).
Antibody Levels
An optimized indirect ELISA was used to determine the levels of tetanus toxoid-specific plasma IgG. Briefly, 96-well microtiter plates were coated overnight with a solution of 40 mL of tetanus toxoid coating buffer/mL (TropBio, Townsville, Australia). Serum samples diluted 1:100 with ELISA diluent were added to triplicate wells, incubated for 1 h, and washed with a Tween-PBS solution using an automated microplate washer (Wellwash 4 MK 2; Labsystems, Sydney, Australia). Antibovine IgG conjugate (Sigma-Aldrich, Castle Hill, Australia) was prepared (1:200) with ELISA diluent, incubated for 1 h, and washed as described previously. Then, 2,2'-azino-di (3-ethylbenzthiazoline) sulfonic acid was added to each plate, and the plates were incubated for 1 h before being assessed at 450 nm using a spectrophotometer (Multiskan EX with Genesis software; Lab-systems).
Statistical Analyses
Data were analyzed using a one-way ANOVA, with sample time as a fixed effect using SPSS 11 software (SPSS, Chicago, IL). Multiple comparison tests were undertaken using the LSD test, where the level of significance was set at P < 0.05.
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Results |
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. Following 72 h of road transportation, a significant but transient leukopenia was evident (Figure 1a; P < 0.05). There was a significant decrease in total eosinophil counts following 72 h of transport; however, these levels returned to baseline by 6 d (Figure 1b; P < 0.05). Although there was a decrease in absolute lymphocyte numbers following transportation, these were not significant (Figure 1c). There were no differences in total neutrophil counts after transport. Mitogen-stimulated lymphocyte proliferation was decreased following transportation (P < 0.05) and returned to baseline levels 6 d after transport (Figure 1d). Although INF- production (Figure 1e) showed a trend similar to lymphocyte proliferation, no significant differences in IFN- level were observed. Tetanus toxoid-specific IgG levels were not affected by transportation (Figure 1f).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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; Kent and Ewbank, 1986; Murata and Hirose, 1990). Until recently, it has been assumed that the behavioral, physiological, and immunological changes experienced by Bos taurus breeds mirror those experienced by the more tropically adapted Bos indicus breeds. A review compiled by Grandin (1997), however, stated that genetic factors, including temperament, influence the degree to which animals respond to stress. Fazio and Ferlazzo (2003) highlighted that significant differences in temperament are evident both within and between breeds. Consequently, data obtained using Bos taurus models are not necessarily applicable to Bos indicus animals. To our knowledge, this is the first study to assess the effects of transport-induced stress on leukocyte numbers and lymphocyte function in Bos indicus cattle.
In transportation studies using a Bos taurus calf model, leukocytosis associated with neutrophilia has been reported (Blecha et al., 1984; Kent and Ewbank, 1986; Murata and Hirose, 1991). Such responses may be indicative of a physiological or pathological change, but also may indicate an epinephrine-induced response (Jain, 1993). In the present study, a transient decrease in total leukocyte numbers associated with eosinopenia was observed. When assessing such responses, the duration and distances covered may contribute to alteration in leukocyte numbers. It is difficult to compare this study with published literature because of a lack of uniformity and variation in the methodology used in these studies.
Decreased neutrophil and lymphocyte numbers following transportation also have been documented in previous work using Bos taurus calves (Colditz and Hennessy, 2001). Transport-associated increases in plasma cortisol concentrations have been well-documented (Tyler and Cummins, 2003; Yagi et al., 2004). It also has been demonstrated that ACTH secretion from peripheral blood mononuclear cells increased following transportation (Dixit et al., 2001). Because these cells also express receptors for ACTH, this could lead to immunosuppression. Researchers using dexamethasone to induce a stress response pharmacologically have found that in addition to a suppression of lymphocyte numbers, lymphocyte subpopulations also were affected (Anderson et al., 1999).
The cause of stress-induced lymphopenia could be due to either lympholysis or a redistribution of cells (Jain, 1993), but it also could be due to a combination of both lympholysis and redistribution of cells. Although previous studies on Bos taurus animals have demonstrated significant suppression of lymphocyte numbers after transport (Kent and Ewbank, 1986; Murata and Hirose, 1991), such changes were not observed in the present study. Despite these differences in lymphocyte responses, lymphocyte numbers might not necessarily reflect greater resilience of Bos indicus animals compared with Bos taurus animals following transport-induced stress. Therefore, lymphocyte functional assays in terms of PHA-stimulated lymphocyte proliferation and IFN- production were assessed.
Lymphocyte functional assays indicated that although lymphocyte numbers remained unchanged, lymphocyte function was suppressed after transport. In previous studies evaluating the immunocompetence of cattle following transport and disease challenge, animals were most susceptible to disease in the few days immediately following transport (Mormede et al., 1982). Although none of the animals in this study succumbed to infection after transport, PHA-stimulated lymphocyte proliferation showed a significant decrease following transport, indicating that animals may experience impaired cellular immunity during this period. The IFN- levels were decreased after transport, but these changes were not significant. Previous studies using dexamethasone to induce a stress response also have shown that although it was possible for PHA-stimulated lymphocyte proliferation to be significantly suppressed, IFN- levels could remain unchanged (Anderson et al., 1999). Impaired lymphocyte proliferation and cytokine production could potentially influence immune responses to infectious agents after transport. Future studies using specific antigens as immunogens delivered immediately following transportation could help to determine the immunocompetence of transported Bos indicus animals.
The level of tetanus toxoid-specific antibody remained unaffected by transportation, which is supported by the results of previous studies (Kelley et al., 1981). However, Kelley et al. (1981) did not determine the relationship between transport stress and the capacity of Bos indicus animals to produce antibodies. Mackenzie et al. (1997) reported that calves immunized with anti-keyhole limpet hemocyanin showed increased IgG, IgM, and IgA responses following a weaning/transportation regimen, suggesting that transportation influenced the humoral immune status of Bos taurus calves. Future studies could assess the relationship between transport stress and the capacity of cattle to produce antibodies against an antigen when immunized during the stress period, along with specific cell function. The assessment of these characteristics would evaluate the interaction of adaptive immune responses with transport-associated stress in Bos indicus animals.
In conclusion, this study demonstrated that transportation of Bos indicus animals might affect their immune status. Nonetheless, a more detailed investigation of both innate and adaptive immune responses in Bos indicus cattle following transportation is required to determine whether specific protocols for transportation and handling of these animals should be considered to minimize the effect of stress on the immune system.
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Footnotes |
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2 Current address: Dept. of Primary Industries, Livestock Systems, Rutherglen, Victoria, Australia.
3 Correspondence—phone: 61 7 47814168; fax: 61 7 47816174; e-mail: lee.fitzpatrick{at}jcu.edu.au.
Received for publication October 24, 2004. Accepted for publication July 25, 2005.
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  J. L. Salak-Johnson and J. J. McGlone Making sense of apparently conflicting data: Stress and immunity in swine and cattle J Anim Sci, March 1, 2007; 85(13_suppl): E81 - E88. [Abstract] [Full Text] [PDF]
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