Advance Publication

The Journal of Veterinary Medical Science

Accepted Date: 13 Jun 2011 J-STAGE Advance Published Date: 27 Jun 2011

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
1) 2)

Note Internal Medicine

Title: Relationship between Diarrhea and Peripheral Leukocyte Population in Neonatal Japanese Black Calves

Hiromichi OHTSUKA1), Machiko MUKAI2), Erika TANAMI2), Mayumi TOKITA1), Yayoi HASHIBA1), Masayuki KOHIRUIMAKI 2), Ken-ichi SHIBANO3) Kiyoshi MIURA4) and Stephen MORRIS5)

Running head; LEUKOCYTE POPULATION IN NEONATAL CALVES

School of Veterinary Medicine, Kitasato University, Towada, Aomori 034-8628,

4)

Kohiruimaki Animal Medical Service, Tohoku, Aomori 039-2683, 3)Hyogo Prefecture Tanko
5)

Federation of Agricultural Mutual Aid Association, Tanba, Hyogo 669-3309, Agricultural Mutual Relief Association, Oshu, Iwate, 023-0023, Japan, Sciences, Massey University, Palmaerston North 4442, New Zealand.

College of

Correspondence to HIROMICHI OHTSUKA Tel: +008-0176-23-4371 Fax: +008- 0176-23-8703 E-mail address:otsuka@vmas.kitasato-u.ac.jp

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49

Abstract Neonatal Japanese Black (JB) calves show a high incidence of diarrhea. The objective of this study was to analyze the immune cell populations of neonatal JB calves in detail and examine its correlation with the incidence of diarrhea immediately after birth. Understanding the immune cell populations is helpful in clinic in order to determine the condition of immune system thus for prevention of diseases. Blood samples were obtained from JB calves on the day of birth. The peripheral leukocyte populations were analyzed separately for calves that had diarrhea within 2 weeks after birth (diarrhea group; n=26) and for calves without diarrhea (control group; n=74). The numbers of the peripheral blood CD3+TcR1-N12+ and CD8+ T cells were significantly lower in the diarrhea group compared to the control group. These findings suggest that the congenital lower peripheral T cell and CD8+ T cell results in a high risk of diarrhea in the neonatal JB calves. Keywords: Diarrhea, Japanese Black calves, Leukocyte population

50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74

Neonatal calves are exposed to a large number of infectious microorganisms, including viruses, bacteria, and parasites, immediately after birth. There is a high incidence of infectious diseases in calves, resulting in swelling of joints, diarrhea, pneumonia, or meningitis in their early lives. Neonates showed substantially lower percentages of IFN- producing CD4+ and CD8+ cells, and a negative correlation between gestational age and IFN- producing CD4+, IL-2 producing CD8+, and IL-10 producing CD4+ cells [3]. In the former study peripheral CD4+ and CD8+ T cell percentages in Holstein calves were the highest in the fetus and these percentages decreased after birth and became lower than adult levels by 120 days of age [21]. T cell percentage was the highest at birth and it decreased to the adult level by 5 months of age [8]. It was known that significant decrease in T cells is observed healthy newborns, this incidence has been examined due to low reactivity to hrIL-2 of blood mononuclear cells of preterm neonates as compared with adult [10]. The lower number of T cells may account for low cytokine production in response to bacterial infection in neonates. Immune cells are less functional and ineffective in mounting an immune response in the first week of life, and this immune immaturity seems to be one of the reasons of high susceptibility to infections in neonatal calves. Japanese Black (JB) calves with weak calves syndrome (WCS), such as low birth weight, sickness, depression, weakness, reluctant standing, and poor suckling, are frequently observed within a week after birth [12]. We previously reported lower peripheral CD8+ T cells and T cells in JB calves with WCS during the first month of birth [13]. In human, T cell repertoire is established during the last fetal development prior to the birth, thus thymectomy does not cause immediate immunodeficiency after birth [4, 5]. Neonatal immune responses have been extensively studied in murine models, and these studies showed lower cell proliferation and cytokine responses to

75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98

stimuli at birth than in adulthood [1, 15, 16]. There is a paucity of research, which evaluates immune status in fetal JB calves at birth. Therefore, in order to clarify whether lower peripheral leukocyte population already existed before the onset of diarrhea in neonatal JB calves, peripheral leukocyte populations were analyzed on the day of birth. One hundred JB calves housed on one farm in Aomori, Japan were examined in this study. Artificial suckling was applied to all calves 3 to 5 days after birth. Twenty-six calves developed diarrhea within 2 weeks after birth (diarrhea group) and the other seventy-four calves did not show diarrhea during this period (control group). Ten calves in the diarrhea group and five calves in the control group were the first calves of their dams. Ten out of 26 calves in the diarrhea group, and 41 out of 74 calves in the control group were male. Depression, dehydration, and white watery feces were observed in the diarrhea group during the sick period within 2 weeks after birth, and these animals were treated with transfusion of Ringer and glucose solutions and injection of antibiotics. All diseased calves recovered within 3 to 5 days after treatment. We observed unexpected fever of some calves in this group, but watery diarrhea did not reoccurred after treatment. The watery diarrhea was not observed in all calves after 2 weeks. There was no case of dystocia, and the peripheral leukocyte populations of all calves were examined on the day of birth after consuming sufficient colostrum. Body weights of all calves were recorded on the day of birth. Blood samples were collected into one tube with dipotassium-EDTA. The total number of the white blood cells (WBC) was determined using a blood cell counter (Celltac MEK-6358, NIHON KOHDEN, Tokyo, Japan). Leukocytes surface receptors were determined using single or two-color staining of peripheral blood mononuclear cells using specific antibodies listed in Table 1.

99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122

Leukocytes were isolated from 2 mL of blood after lysing red blood cell by adding 4 mL of 0.83% ammonium chloride solution. After hemolysis and washing with PBS (pH = 7.2), WBC were resuspended in cold PBS. WBC (1x106) were incubated with monoclonal antibodies at 4 oC for 60 min. After washing with PBS, the cells were incubated with fluorescein isothiocyanate-conjugated goat anti-mouse IgM and phycoerythrin-conjugated goat anti-mouse IgG (ICN Biomedicals, Costa Mesa, California, USA) at 4 oC for 30 min. After washing the labeled WBC with PBS, the flow cytometric analysis was performed using a FACScan flow cytometer (BD Biosciences Immunocytometry Systems, San Jose, CA, USA). Data from 10000 events per sample were analyzed using Cell Quest software (BD). Statistical analysis was performed for each parameter in order to find the differences between the two groups using Mann-Whitney test and correlation among the data of each parameter using Pearsons test. The differences between groups were considered significant at P < 0.05. Body weight of male calves (30.7 1.1 kg) and female calves (26.5 0.7 kg) in the diarrhea group was significantly lower than that of male calves (33.5 0.8 kg) and female calves (29.6 0.7 kg) in the control group, respectively (P < 0.05). The number of WBC was lower in the diarrhea group (90.0 6.9 102/l) than the control group (110.7 4.4 102/l). The calves in the diarrhea group had lower numbers of CD3+TcR1-N12+, CD8+ T cells, and CD14+ cells (Table 2). There was no significant difference in CD4+ T cells. A significant positive correlation was observed between body weight and peripheral CD3+TcR1-N12+ T cell (R=0.39, p<0.05) and CD8+T cell (R=0.36, p<0.05) numbers in this study. There were no correlations between the numbers of other leukocyte subsets and body weight.

123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146

We reported that peripheral T cells and CD8+ T cells remained low in JB calves with WCS in the first month after birth [13], but it was not clear whether these T cells were lower on the day of birth. In the present study, lower numbers of peripheral T cell and CD8+ T cell were observed at birth in the calves, which developed diarrhea within 2 weeks after birth. Lower number of T cells seemed to be associated with the incidence of diarrhea in JB calves. T cells display tissue-specific accumulation in response to infection. Neonatal T cells are functionally different from the majority of adult T cells and display a distinct TCR repertoire and accessory molecule profiles. T cells obtained from cord blood had weak cytolytic activity in tumor cell killing assay [11]. Cytotoxic CD8+ T cells are the main cells that kill virus infected cells, and a decrease in this subset of cells would diminish the ability to control viral infection, as reported in other animal models [17]. Infants with a severe clinical respiratory syncytial virus infection exhibited significantly lower CD8+ T cell and CD8+/CD25+ (activated) T cell counts in the acute phase of illness [6], and the number of peripheral CD8+ T cells of infants with severe disease were significantly lower compared with those of infants with milder forms of illness [2]. T cell or CD8+ T cell represent important effector cells in immune system, and lower number of these T cells in the diarrhea group might indicate that those neonates are more immune-suppressed. The calves in the diarrhea group had significantly lighter body weight and higher percentage of primiparous dams. Neonatal calves largely depend on the dams for their physical development prior to calving. All muscle tissue, nerve fibers, and energy reserves present in the calf are dependent on the nutritional status of the dam, and thus it is highly likely that weak calves were born to dams with undernourished condition [9].

147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170

In human, T cell responses are immature at birth compared to adults [15], and the premature neonates demonstrated a significantly lower number of T cells [7, 14]. In addition, development of peripheral T cell or B cells after birth was suppressed in JB calves that were born from dams with insufficient nutrition during the last gestation or calves born from primiparous dams [19]. Thymic hypoplasia associated with decreased immune function was reported in calves with stillbirth/perinatal WCS, and it was suggested that intrauterine growth retardation was caused by lack of growth factors during the fetal period [18]. Taken together, results of the present study suggest a disturbance of T cell development during the last gestation since this period can influence a peripheral leukocyte population in newborn JB calves. The principal observation in the present study was lower peripheral T cell and CD8+ T cell in JB calves with diarrhea. This reduced immune condition could aggravate infectious diseases, and may explain some of the immunologic abnormalities accompanied by diarrhea. The reason for no difference in CD4+ T cell between two groups in this study is not clear. Children born very preterm had significantly lower CD4+ T cell percentages compared with the healthy controls [20]. Potent immune activation in early life of JB calves might contribute to protection from bacterial infection. It is still unclear what are ideal immune conditions for keeping JB calves healthy. Further studies are needed in this field. ACKNOWLEGEMENTS. The authors are grateful to Dr. Ogata of Yamagata Agricultural Mutual Relief Association for support in this study, and indebted to all farm owners and farm personnel in the enrolled farms for their cooperation. This research was supported in part by a Grant for Encouragement from Kitasato University.

171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200

REFERENCES [1] Adkins, B., Leclerc, C. and Marshall-Clarke, S. 2004. Neonatal adaptive immunity comes of age. Nat. Rev. Immunol. 4:553564. [2] De Weerd, W., Twilhaar, W.N. and Kampen, J.L.L. 1998. T cell subset analysis in peripheral blood of children with RSV bronchiolitis. Scand. J. Infect. Dis. 30:7780. [3] Gasparoni, A., Ciardelli, L., Avanzini, A., Castellazzi, A.M., Carini, R., Rondini, G. and Chirico, G. 2003. Age-related changes in intracellular TH1/TH2 cytokine production, immunoproliferative T lymphocyte response and natural killer cell activity in newborns, children and adults. Biol. Neonate. 84:297-303. [4] Haynes, B.F. and Heinly, C.S. 1995. Early human T cell development: analysis of the human thymus at the time of initial entry of hematopoietic stem cells into the fetal thymic microenvironment. J. Exp. Med. 181:1445-1458. [5] Haynes, B.F., Sempowski, G.D., Wells, A.F. and Hale, L.P. 2000. The human thymus during aging. Immunol Res. 22:253-261. [6] Isaacs, D., Bangham, C. R. M. and McMichael. A. J. 1987. Cell-mediated cytotoxic response to respiratory syncytial virus in infants with bronchiolitis. Lancet 330:769771. [7] Jureti, E., Uzarevi, B., Petrovecki, M. and Jureti, A. 2000. Two-color flow cytometric analysis of preterm and term newborn lymphocytes. Immunobiology 202:421-428. [8] Kampen, A.H., Olsen, I., Tollersrud, T., Storset, A.K. and Lund, A. 2006. Lymphocyte subpopulations and neutrophil function in calves during the first 6 months of life. Vet. Immunol. Immunopathol. 113:53-63. [9] Kume, S., Toharmat, T. and Kobayashi, N. 1998. Effect of restricted feed intake of dams and heat stress on mineral status of newborn calves. J. Dairy Sci. 81:1581-1590. [10] Miceli Sopo, S., Pesaresi, M.A., Celestini, E., Piastra, M., Stabile, A.M., Rumi, C., Bussa, S. and Stabile, A. 1992. Interleukin 2 proliferative response by cord blood mononuclear cells of term and preterm neonates. Allergol. Immunopathol. 20:91-95.

201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233

[11] Morita, C.T., Parker, C.M., Brenner, M.B. and Band, H. 1994 TCR usage and functional capabilities of human gamma delta T cells at birth. J. Immunol. 153:3979-3988. [12] Ogata, Y., Nakao, T., Takahashi, K., Abe, H., Misawa, T., Urushiyama, Y. and Sakai, J. 1999. Intrauterine growth retardation as a cause of perinatal mortality in Japanese black beef calves. Zentralbl Veterinarmed A. 46:327-334. [13] Ohtsuka, H., Fukunaga, N., Hara, H., Fukuda, S., Hayashi, T., Hoshi, F., Yoshino, T., Koiwa, M. and Kawamura, S. 2003. Changes in peripheral leukocyte populations of weak calf syndrome of Japanese black calves. J. Vet. Med. Sci. 65:793-796. [14] Peoples, J.D., Cheung, S., Nesin, M., Lin, H., Tatad, A.M., Hoang, D., Perlman, J.M. and Cunningham-Rundles, S. 2009. Neonatal cord blood subsets and cytokine response to bacterial antigens. Am. J. Perinatol. 26:647-657. [15] Schaub, B., Liu, J., Schleich, I., Hppler, S., Sattler, C. and von Mutius, E. 2008. Impairment of T helper and T regulatory cell responses at birth. Allergy 63:1438-1447. [16] Siegrist, C.A. 2001. Neonatal and early life vaccinology. Vaccine 19:33313346. [17] Sopper, S., Sauer, U., Hemm, S., Demuth, M., Mller, J., Stahl-Hennig, C., Hunsmann, G., ter Meulen, V. and Drries, R. 1998. Protective role of the virus-specific immune response for development of severe neurologic signs in simian immunodeficiency virus-infected macaques. J. Virol. 72:99409947. [18] Takasu, M., Shirota, K., Ohba, Y., Nishii, N., Murase, T., Miyazawa, K. and Kitagawa, H. 2008. Thymic hypoplasia in Japanese black calves with stillbirth/perinatal weak calf syndrome. J. Vet. Med. Sci. 70:1173-1177. [19] Tanami, E., Ohtsuka, H., Mukai, M., Kohiruimaki, M., Ogata, M. and Kawamura, S. 2009. Effect of nutritive condition in the dam during the later pregnant period to peripheral leukocyte population in Japanese Black calves after birth. J. Jpn. Vet. Med. Assoc. 62:623-629. [20] Wang-Rodriguez, J., Fry, E., Fiebig, E., Lee, T., Busch, M., Mannino, F. and Lane, T.A. 2000. Immune response to blood transfusion in very-low-birth weight infants. Transfusion 40:25-34. [21] Wilson, R.A., Zolnai, A., Rudas, P. and Frenyo, L.V. 1996. T-cell subsets in blood and lymphoid tissues obtained from fetal calves, maturing calves, and adult bovine. Vet. Immunol. Immunopathol. 53:49-60. 9

Table1. Monoclonal antibodies against bovine leukocyte antigens used for flow cytometric analysis Antibodies Clone Isotype Specificity Source 1 VMRD CD3 MM1A IgG1 Pan T cell CD4 CACT138A IgG1 Helper/inducer VMRD CD8 BAT82A IgG1 Cytotoxic VMRD T cell TcR1-N12 CACT61A IgM VMRD MHC class II CAT82A IgG1 B cell / Monocyte VMRD Coulter2 CD14 MY4 IgG2b Monocyte
1 2

VMRD=VMRD, Inc. (pullman, WA, USA) Coulter=Coulter Immunology Hailed (Florida, USA)

Table 2. Peripheral leukocytes numbers in two groups Cell Type diarrhea group 546.152.5 CD3+TcR1-N12- (cell/l) + + 425.365.0 CD3 TcR1-N12 (cell/l) + 125.624.8 CD4 (cell/l) + 122.019.9 CD8 (cell/l) + + 0.960.16 CD4 /CD8 rate 119.123.9 MHC class-+CD14- (cell/l) 340.755.2 CD14+ (cell/l) Values are expressed as the meanS.E.

control group 664.057.1 721.862.5 131.010.4 199.916.0 0.640.05 144.311.5 531.852.1

P value 0.482 0.011 0.832 0.001 0.292 0.545 0.088