diarea vs leokosit

8/13/2019 Diarea vs Leokosit

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Advance Publication

The Journal of Veterinary Medical Science

Accepted Date: 13 Jun 2011

J-STAGE Advance Published Date: 27 Jun 2011


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Note1

Internal Medicine2

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Title:Relationship between Diarrhea and Peripheral Leukocyte Population in4

Neonatal Japanese Black Calves5

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Hiromichi OHTSUKA1)

, Machiko MUKAI2)

, Erika TANAMI2)

, Mayumi7

TOKITA1)

, Yayoi HASHIBA1)

, Masayuki KOHIRUIMAKI2)

, Ken-ichi8

SHIBANO3)

Kiyoshi MIURA4)

and Stephen MORRIS5)

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Running head; LEUKOCYTE POPULATION IN NEONATAL CALVES11

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1)School of Veterinary Medicine, Kitasato University, Towada, Aomori 034-8628,13

2)Kohiruimaki Animal Medical Service, Tohoku, Aomori 039-2683, 3)Hyogo Prefecture14

Federation of Agricultural Mutual Aid Association, Tanba, Hyogo 669-3309,4)

Tanko15

Agricultural Mutual Relief Association, Oshu, Iwate, 023-0023, Japan,5)

College of16

Sciences, Massey University, Palmaerston North 4442, New Zealand.17

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Correspondenceto HIROMICHI OHTSUKA19

Tel: +008-0176-23-437120

Fax: +008- 0176-23-870321

E-mail address:[email protected]

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AbstractNeonatal Japanese Black (JB) calves show a high incidence of diarrhea. The26

objective of this study was to analyze the immune cell populations of neonatal JB27

calves in detail and examine its correlation with the incidence of diarrhea immediately28

after birth.Understanding the immune cell populations is helpful in clinic in order to29determine the condition of immune system thus for prevention of diseases. Blood30

samples were obtained from JB calves on the day of birth. The peripheral leukocyte31

populations were analyzed separately for calves that had diarrhea within 2 weeks after32

birth (diarrhea group; n=26) and for calves without diarrhea (control group; n=74).The33numbers of the peripheral blood CD3

+TcR1-N12

+and CD8

+T cells were significantly34

lower in the diarrhea group compared to the control group. These findings suggest that35

the congenital lower peripheral T cell and CD8+ T cell results in a high risk of36

diarrhea in the neonatal JB calves.37

Keywords: Diarrhea, Japanese Black calves, Leukocyte population38

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Neonatal calves are exposed to a large number of infectious microorganisms,50

including viruses, bacteria, and parasites, immediately after birth. There is a high51

incidence of infectious diseases in calves, resulting in swelling of joints, diarrhea,52

pneumonia, or meningitis in their early lives. Neonates showed substantially lower53

percentages of IFN- producing CD4+ and CD8

+cells, and a negative correlation54

between gestational age and IFN-producing CD4+, IL-2 producing CD8

+, and IL-1055

producing CD4+ cells [3]. In the former study peripheral CD4

+ and CD8

+ T cell56

percentages in Holstein calves were the highest in the fetus and these percentages57

decreased after birth and became lower than adult levels by 120 days of age [21]. T58

cell percentage was the highest at birth and it decreased to the adult level by 5 months59

of age [8]. It was known that significant decrease in T cells is observed healthy60

newborns, this incidence has been examined due to low reactivity to hrIL-2 of blood61

mononuclear cells of preterm neonates as compared with adult [10]. The lower number62

of T cells may account for low cytokine production in response to bacterial infection in63

neonates. Immune cells are less functional and ineffective in mounting an immune64

response in the first week of life, and this immune immaturity seems to be one of the65

reasons of high susceptibility to infections in neonatal calves.66

Japanese Black (JB) calves with weak calves syndrome (WCS), such as low birth67

weight, sickness, depression, weakness, reluctant standing, and poor suckling, are68

frequently observed within a week after birth [12]. We previously reported lower69

peripheral CD8+T cells and T cells in JB calves with WCS during the first month of70

birth [13]. In human, T cell repertoire is established during the last fetal development71

prior to the birth, thus thymectomy does not cause immediate immunodeficiency after72

birth [4, 5]. Neonatal immune responses have been extensively studied in murine73

models, and these studies showed lower cell proliferation and cytokine responses to74


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stimuli at birth than in adulthood [1, 15, 16]. There is a paucity of research, which75

evaluates immune status in fetal JB calves at birth. Therefore, in order to clarify whether76

lower peripheral leukocyte population already existed before the onset of diarrhea in77

neonatal JB calves, peripheral leukocyte populations were analyzed on the day of birth.78

One hundred JB calves housed on one farm in Aomori, Japan were examined in this79

study. Artificial suckling was applied to all calves 3 to 5 days after birth. Twenty-six80

calves developed diarrhea within 2 weeks after birth (diarrhea group) and the other81

seventy-four calves did not show diarrhea during this period (control group). Ten calves82

in the diarrhea group and five calves in the control group were the first calves of their83

dams. Ten out of 26 calves in the diarrhea group, and 41 out of 74 calves in the control84

group were male. Depression, dehydration, and white watery feces were observed in the85

diarrhea group during the sick period within 2 weeks after birth, and these animals were86

treated with transfusion of Ringer and glucosesolutionsand injection of antibiotics. All87

diseased calves recovered within 3 to 5 days after treatment. We observed unexpected88

fever of some calves in this group, but watery diarrhea did not reoccurred after89

treatment. The watery diarrhea was not observed in all calves after 2 weeks. There was90

no case of dystocia, and the peripheral leukocyte populations of all calves were91

examined on the day of birth after consuming sufficient colostrum. Body weights of all92

calves were recorded on the day of birth.93

Blood samples were collected into one tube with dipotassium-EDTA. The total94

number of the white blood cells (WBC) was determined using a blood cell counter95

(CelltacMEK-6358, NIHON KOHDEN, Tokyo, Japan).96

Leukocytes surface receptors were determined using single or two-color staining of97

peripheral blood mononuclear cells using specific antibodies listed in Table 1.98


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Leukocytes were isolated from 2 mL of blood after lysing red blood cell by adding 4 mL99

of 0.83% ammonium chloride solution. After hemolysis and washing with PBS (pH =100

7.2), WBC were resuspended in cold PBS. WBC (1x106) were incubated with101

monoclonal antibodies at 4 oC for 60 min. After washing with PBS, the cells were102

incubated with fluorescein isothiocyanate-conjugated goat anti-mouse IgM and103

phycoerythrin-conjugated goat anti-mouse IgG (ICN Biomedicals, Costa Mesa,104

California, USA) at 4 oC for 30 min.After washing the labeled WBC with PBS, the flow105

cytometric analysis was performed using a FACScan flow cytometer (BD Biosciences106

Immunocytometry Systems, San Jose, CA, USA). Data from 10000 events per sample107

were analyzed using Cell Quest software (BD).108

Statistical analysis was performed for each parameter in order to find the differences109

between the two groups using Mann-Whitney test and correlation among the data of110

each parameter using Pearsons test. The differences between groups were considered111

significant at P < 0.05.112

Body weight of male calves (30.7 1.1 kg) and female calves (26.5 0.7 kg) in the113

diarrhea group was significantly lower than that of male calves (33.5 0.8 kg) and114

female calves (29.6 0.7 kg) in the control group, respectively (P< 0.05). The number115

of WBC was lower in the diarrhea group (90.0 6.9 102/l) than the control group116

(110.7 4.4 102/l). The calves in the diarrhea group had lower numbers of117

CD3+TcR1-N12

+, CD8

+ T cells, and CD14

+ cells (Table 2). There was no significant118

difference in CD4+ T cells. A significant positive correlation was observed between119

body weight and peripheral CD3+TcR1-N12

+T cell (R=0.39, p


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We reported that peripheral T cells and CD8+T cells remained low in JB calves123

with WCS in the first month after birth [13], but it was not clear whether these T cells124

were lower on the day of birth. In the present study, lower numbers of peripheral T125

cell and CD8+T cell were observed at birth in the calves, which developed diarrhea126

within 2 weeks after birth. Lower number of T cells seemed to be associated with the127

incidence of diarrhea in JB calves.128

T cells display tissue-specific accumulation in response to infection. Neonatal T129

cells are functionally different from the majority of adult T cells and display a130

distinct TCR repertoire and accessory molecule profiles. T cells obtained from cord131

blood had weak cytolytic activity in tumor cell killing assay [11]. Cytotoxic CD8+T132

cells are the main cells that kill virus infected cells, and a decrease in this subset of cells133

would diminish the ability to control viral infection, as reported in other animal models134

[17]. Infants with a severe clinical respiratory syncytial virus infection exhibited135

significantly lower CD8+T cell and CD8

+/CD25

+(activated) T cell counts in the acute136

phase of illness [6], and the number of peripheral CD8+T cells of infants with severe137

disease were significantly lower compared with those of infants with milder forms of138

illness [2]. T cell or CD8+T cell represent important effector cells in immune system,139

and lower number of these T cells in the diarrhea group might indicate that those140

neonates are more immune-suppressed.141

The calves in the diarrhea group had significantly lighter body weight and higher142

percentage of primiparous dams. Neonatal calves largely depend on the dams for their143

physical development prior to calving. All muscle tissue, nerve fibers, and energy144

reserves present in the calf are dependent on the nutritional status of the dam, and thus it145

is highly likely that weak calves were born to dams with undernourished condition [9].146


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In human, T cell responses are immature at birth compared to adults [15], and the147

premature neonates demonstrated a significantly lower number of T cells [7, 14]. In148

addition, development of peripheral T cell or B cells after birth was suppressed in JB149

calves that were born from dams with insufficient nutrition during the last gestation or150

calves born from primiparous dams [19]. Thymic hypoplasia associated with decreased151

immune function was reported in calves with stillbirth/perinatal WCS, and it was152

suggested that intrauterine growth retardation was caused by lack of growth factors153

during the fetal period [18]. Taken together, results of the present study suggest a154

disturbance of T cell development during the last gestation since this period can155

influence a peripheral leukocyte population in newborn JB calves.156

The principal observation in the present study was lower peripheral T cell and157

CD8+T cell in JB calves with diarrhea. This reduced immune condition could aggravate158

infectious diseases, and may explain some of the immunologic abnormalities159

accompanied by diarrhea. The reason for no difference in CD4+ T cell between two160

groups in this study is not clear. Children born very preterm had significantly lower161

CD4+ T cell percentages compared with the healthy controls [20]. Potent immune162

activation in early life of JB calves might contribute to protection from bacterial163

infection. It is still unclear what are ideal immune conditions for keeping JB calves164

healthy. Further studies are needed in this field.165

ACKNOWLEGEMENTS. The authors are grateful to Dr. Ogata of Yamagata166

Agricultural Mutual Relief Association for support in this study, and indebted to all farm167

owners and farm personnel in the enrolled farms for their cooperation. This research168

was supported in part by a Grant for Encouragement from Kitasato University.169

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Table1. Monoclonal antibodies against bovine leukocyte antigens used for flow cytometric analysis

Antibodies Clone Isotype Specificity Source

CD3 MM1A IgG1 Pan T cell VMRD1

CD4 CACT138A IgG1 Helper/inducer VMRDCD8 BAT82A IgG1 Cytotoxic VMRD

TcR1-N12 CACT61A IgM T cell VMRD

MHC class II CAT82A IgG1 B cell / Monocyte VMRD

CD14 MY4 IgG2b Monocyte Coulter1VMRD=VMRD, Inc. (pullman, WA, USA)

2Coulter=Coulter Immunology Hailed (Florida, USA)


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Table 2. Peripheral leukocytes numbers in two groups

Cell Type diarrhea group control group P value

CD3+TcR1-N12

-(cell/l) 546.152.5 664.057.1 0.482

CD3+TcR1-N12

+(cell/l) 425.365.0 721.862.5 0.011

CD4+ (cell/l) 125.624.8 131.010.4 0.832

CD8+

(cell/l) 122.019.9 199.916.0 0.001

CD4+/CD8

+rate 0.960.16 0.640.05 0.292

MHC class-+CD14

-(cell/l) 119.123.9 144.311.5 0.545

CD14+

(cell/l) 340.755.2 531.852.1 0.088

Values are expressed as the meanS.E.

diarea vs leokosit

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