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CpG-ODN enhances mammary gland defense during mastitis induced by Escherichia coli infection in goats
Veterinary Immunology and Immunopathology 120 (2007) 168–176 www.elsevier.com/locate/vetimm
CpG-ODN enhances mammary gland defense during mastitis induced by Escherichia coli infection in goats Yu-Min Zhu a,b, Jin-Feng Miao a, Yuan-Shu Zhang a, Zhen Li b, Si-Xiang Zou a,*, Yue -E Deng a b
a College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China Animal and Veterinary Science Research Institute Shanghai Academy of Agriculture Sciences, Shanghai 201106, China
Received 9 March 2007; received in revised form 13 July 2007; accepted 2 August 2007
Abstract Seven healthy native goats in early lactation, weighing 30–40 kg, were used in this study. The right mammary gland of the seven does were infused with CpG-ODN at a dosage of 100 mg kg1 body weight on the day 5 postpartum (PP). The left glands were used as controls and infused with sterile phosphate-buffered saline (PBS). On day 8 PP, the same dosage of CpG-ODN or PBS was again infused. On day 9 PP, the mammary glands (both right and left) of the seven does were infused with 6 106 colony-forming units (CFU) Escherichia coli and, at 0, 8, 16, 24, 48 and 72 h postinfection (PI), milk samples were collected from all glands. Goats were euthanized at 72 h PI and the mammary tissue harvested. Infusion with 6 106 CFU ml1 E. coli induced acute mastitis. Histopathological evaluations showed that polymorphonuclear neutrophils (PMNs) were still present in alveoli at 72 h PI, but PMNs in the CpG-ODN-treated glands has disappeared. Bacteria counts in milk peaked at 16 h PI and CpG-ODN induced a significant decrease in viable bacteria from 16 h PI until the end of the experiment. This study showed that CpG-ODN promoted the expression of its specific receptor (TLR-9 mRNA) in mammary tissue, stimulated IL-6 production, reduced bacteria counts in milk, attenuated the impact of inflammation mediators on cells and significantly shortened the inflammation course. These results suggest that the CpGODN improved mammary gland defense and, thereby, had a beneficial effects against mastitis caused by E. coli infection in goats. # 2007 Elsevier B.V. All rights reserved. Keywords: CpG-ODN; Goat; Mastitis; Escherichia coli; TLR-9
1. Introduction CpG-ODN is an unmethylated, specific DNA motif containing cytosine phosphate-guanosine dinucleotides (CpG). It induces immune cells to secrete a number of
Abbreviations: CpG, cytosine phosphate-guanosine dinucleotides; IMI, intramammary infection; NAGase, N-acetyl-b-D-glucosaminidase; PMNs, polymorphonuclear leukocytes; CFU, colonyforming units; Mab, monoclonal antibody; PI, postinfection. * Corresponding author at: College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China. Tel.: +86 21 62200389. E-mail address: [email protected] (Y.-M. Zhu). 0165-2427/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2007.08.003
cytokines, thereby upregulating specific and nonspecific immunity. It has been well documented that CpG-ODN not only stimulates B-lymphocytes and innate immune cells, such as macrophages, dendritic cells and natural killer cells, in mammals, but also results in the activation and secretion of cytokines, including interleukin-1b (IL-1b), IL-6, IL-12, IL-18, tumor necrosis factor-a (TNF-a), interferon-a (IFN-a) and interferon (IFN-g) (Krieg et al., 2002). The immunomodulatory use of CpG-ODN is currently being investigated for anti-tumor, anti-infectious, anti-asthmatic and anti-inflammatory applications and for adjuvant activity in immunotherapy (Krieg et al., 2002). In recent years, the protection provided by
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CpG-ODN against infection has been demonstrated. For example, pre-treatment of mice with CpG-ODN provided protection against lethal challenge by the bacteria, Listeria monocytogenes (Krieg et al., 1998) and Francisella tularensis (Klinman et al., 1999), and parasitic protozoans, such as Leishmania spp. (Zimmermann et al., 1998) and Plasmodium yoelii (Gramzinski et al., 2001). Recently, Gomis et al. (2003) demonstrated for the first time that CpG-oligonucleotides (CpG-DNA) were effective in controlling extracellular Escherichia coli infections in poultry in a dose-dependent manner. E. coli is the main causative agent of clinical mastitis in ewes and 2–12% of E. coli has been isolated (Bergonier and Berthelot, 2003). Clinical mastitis in ewes induced by E. coli infection may occur at every stage of lactation (Gonzalez et al., 1990). Despite the frequent occurrence of the disease worldwide, effective therapy has not yet been established unequivocally. Traditional mastitis control measures, such as dry cow antibiotic therapy, teat dipping and selective culling, are relatively ineffective in controlling E. coli mastitis (Gonzalez et al., 1990). Additionally, Hill (1991) showed that vaccination provided little prophylactic benefit against naturally occurring intramammary infection (IMI). Therefore, with increasing knowledge of immunobiology of the mammary gland, research into mastitis is focusing on augmenting the innate immune response as a means of increasing resistance of the mammary gland to invading pathogens. A mouse model of infectious mastitis was first described by Chandler (1970). This model has been used to assess the pathophysiology of E. coli IMI (Cooray and Jonsson, 1990). In 2006, we established a reliable and sensitive mastitis model in rats infected with E. coli and found, for the first time, that CpG-DNA induced a more rapid migration of PMNs from blood to mammary gland at the initial stage of E. coli infection, decreased bacterial counts in the mammary gland and attenuated the impact of inflammation mediators on the cell (Zhu et al., 2007). Pathophysiological differences most likely exist between mouse/rat and caprine mammary glands; therefore, the present work aims to extensively characterize the immunostimulation effect of CpG-ODN on the mammary gland of goats, which may help to develop new modalities for mastitis prophylaxis and treatment. 2. Materials and methods 2.1. CpG-ODN CpG-ODN was kindly provided by HongFei Zhu of the Chinese Academy of Agricultural Sciences
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(CAAS). CpG-ODN was purified from the plasmid pCpG, which was cloned with the synthesized CpG sequence and the pUC18 vector. The CpG-ODN was diluted in sterile pyrogen-free phosphate-buffered saline (PBS) and adjusted to a concentration of 2 mg ml1. 2.2. Bacteria E. coli (CMCC, 25922) was purchased from the Chinese Medical Culture Collection (CMCC). Bacterial inocula were prepared from freeze-dried cultures grown in nutrient broth at 37 8C overnight. The bacteria were harvested and suspended in 0.01 mol l1, pH 7.2 PBS. The bacteria were then washed three times in PBS by centrifugation at 5000 g for 10 min at 4 8C and diluted in PBS. The number of colony-forming units (CFU) was determined by serial dilution and plate counting. The cultures were further diluted in PBS to 2 106 CFU ml1. 2.3. Animals and samplings Animal studies were approved by the Animal Ethics Committee. Seven, 30–40 kg lactating female goats were provided with food and water ad libitum. The goats were kept under observation for 14 days in thoroughly cleaned premise before the start of the experiment. All the animals were free from sub-clinical mastitis and no bacteria or fungi were isolated from preinoculation milk samples. The right mammary glands of the seven does were infused with 100 mg kg1 CpGODN on day 5 postpartum (PP). Left mammary glands served as controls and were infused with sterile PBS. On day 8 PP, repeat dosages of CpG-ODN or PBS were infused, respectively. On day 9 PP, after morning milking, the right and left glands of the does were infused with 6 106 CFU ml1 E. coli. Milk samples from both glands were aseptically collected just before inoculation of the bacteria (defined as 0 h) and at 8, 16, 24, 48 and 72 h postinfection (PI). Rectal temperatures were recorded at 0, 1, 2, 3, 4, 5, 6, 7, 8, 16, 24, 48 and 72 h PI. At each sampling, both halves of the udder were emptied as completely as possible by hand milking. Kids were withheld from does for the first 8 h of infection during which time the goats were housed in a group pen with access to water and lucerne chaff. Subsequently, does and kids were reunited and returned to pasture. The goats were mustered each morning for sample collection, then returned to pasture. Does were euthanized at 72 h PI and the mammary tissue harvested. A portion of each mammary gland was
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immediately snap-frozen in liquid nitrogen and stored at 80 8C until RNA extraction. One portion of each mammary gland was fixed in Bouin’s fluid and embedded in paraffin. The remainder of the mammary glands were aseptically collected and stored at 20 8C. 2.4. Preparation and assay of milk and mammary gland 2.4.1. Mammary tissue preparation and microscopic observations Sections of 5 mm thickness were stained with hematoxylin and eosin and the stained tissue sections examined by light microscopy. Parameters evaluated were gland morphology and inflammatory cell infiltrate. 2.4.2. Preparation and assay of milk samples Milk samples were diluted in sterile PBS prior to plating on brain–heart infusion agar containing 5% sheep blood. After a 24-h incubation at 37 8C, bacterial colonies were counted and the colony-forming units (CFU) per ml of milk were calculated. The remaining milk samples were centrifuged at 44 000 g for 30 min at 4 8C and the fat layer removed with a spatula. The skim milk was decanted into a clean tube and centrifuged again for 30 min, as described above; the translucent whey fractions were collected and then frozen at 20 8C until use. Whey protein concentration was determined using the Bradford method. TNF-a and IL-6 levels in milk whey were measured by radioimmunoassay. Commercial kits were purchased from the Institute of Radiation of Science & Technology Development Center of the General Hospital of the People’s Liberation Army. The assay was conducted following the manufacturer’s protocol. The level of serum albumin in milk whey was measured using commercial kits purchased from the Nanjing Jiancheng Bioengineering Institute (NJBI) following manufacturer’s protocols. The activity of N-acetyl-b-D-glucosaminidase (NAGase) in milk whey was determined using commercial kits purchased from NJBI and following the manufacturer’s protocols. The optical density of paranitrophenol during the reaction (at 37 8C) of the 4-methylumbelliferyl-N-acetyl-b-glucosaminide substrate with NAGase contained in the analyzed samples was measured spectrophotometrically in triplicate at a wavelength of 400 nm. One unit of NAGase activity represents the amount of paranitrophenol released from 1 l of milk in 15 min at 37 8C.
2.4.3. RNA extraction and RT-PCR for the Toll-like receptor (TLR-9) 2.4.3.1. RNA extraction and primer. Total RNA was extracted from the tissue samples with acid guanidinium thiocyanate–phenol–chloroform (Chomczynski and Sacchi, 2006). RNA concentration was quantified by measuring absorbance at 260 nm. Ratios of absorption (260/280 nm) of all preparations were between 1.8 and 2.0. Aliquots of the RNA samples were subjected to electrophoresis through a 1.4% agarose– formaldehyde gel to verify their integrity. The nucleotide sequences of TLR-9 primers were as follows: TLR-9 (forward, 50 -TATGTGCCGCGCTTTT-30 ; reverse, 50 -TTCGCGGAACCAGTCTTT-30 ); the amplified product was 384 bp. b-Actin (forward, 50 -GCACCACACCTTCTACAA-CGAGC-30 ; reverse, 50 TCCTTGATGTCACGGACGATTTC-30 ); the amplified product was 765 bp. 2.4.3.2. Reverse transcription (RT) and polymerase chain reaction (PCR). Two mg of total RNA were reverse transcribed by incubation at 37 8C for 1 h in a 25 ml mixture consisting of 200 U of M-MLV reverse transcriptase, 10 U Raze inhibitor, 0.8 mM oligo(dT18) primer, 0.8 mM dNTP, 10 mM Tris–HCl (pH 8.3), 0.6 mM MgCl2, 15 mM KCl and 2 mM DDT. RNA samples were denatured at 70 8C for 5 min and placed on ice for 5 min together with the oligo-(dT18) primer and dNTP before reverse transcription (RT). The RT reaction was terminated by heating at 95 8C for 5 min and then quickly cooled on ice. Three ml of RT reaction mixture was used for PCR in a final volume of 25 ml containing 1.25 U Taq DNA polymerase, 1 mM Tris–HCl (pH 9.0), 5 mM KCl, 0.01% Triton X-100, 0.4 mM dNTP, 2 mM MgCl2, 0.8 mM sense and antisense primers for TLR-9, 0.04 pM sense and antisense primers for b-actin and 1.5 ml DMSO. After denaturation at 94 8C for 5 min, 30 amplification cycles with denaturation at 94 8C for 30 s, annealing at 57 8C for 60 s and an extension at 72 8C for 60 s, with a final incubation at 72 8C for 10 min were performed. 2.4.3.3. Quantitation of PCR products and statistical analysis. Twenty ml of PCR product was separated by electrophoresis on 2.0% agarose gels stained with ethidium bromide and photographed with a digital camera. The net intensities of individual bands were measured using Kodak Digital Science 1D software (Eastman-Kodak, Rochester, NY, USA). The ratios of net intensities of target genes to b-actin were used to
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represent the relative levels of target gene expression. The average of three repeats was used for statistical analysis.
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CpG-ODN-treated glands. PMNs accumulated in the alveoli of control glands; however, the infiltration of PMNs in CpG-ODN-treated glands disappeared (Fig. 1).
2.5. Statistical analysis 3.2. E. coli enumeration Analysis of variance was performed using SPSS software (SPSS11.5 for Windows). Data were expressed as means standard error (S.E.M.), except that data from bacterial counts were converted to log10 to maintain a normal distribution. Differences were considered significant at P < 0.05 by t-test for independent samples or ANOVA with STATISTICA for Windows 5.0 (StatSoft, Tulsa, OK, USA). 3. Results 3.1. Clinical symptoms, rectal temperature and histological observations Acute clinical mastitis developed in all treated and control glands at 3 h PI. The does were depressed and swelling was observed in infected glands. The does lay down and did not drink any water from 4 to 6 h PI. Milk from control glands contained some flakes or clots, whereas milk from CpG-ODN-treated glands appeared normal. The temperature of goats with CpGODN-treated glands was lower than PBS controls throughout the experiment and the extent of swelling and firmness of the treated glands was milder than controls. The rectal temperature started to increase at 1 h PI, peaked at 6 h PI (41.0 8C), then decreased gradually and returned to normal at 24 h PI (38.2 8C). Histopathological evaluation indicated that the lumen of control mammary glands were smaller than
The numbers of E. coli in mammary tissue increased significantly and peaked at 5.82 0.52(log10 CFU ml1) in the control group and at 4.73 0.32(log10 CFU ml1) in the CpG-ODN-treated group at 16 h PI, then slowly declined. The bacterial concentration from the CpG-ODN-treated group decreased by 18.40% (P < 0.05) at 16 h, 19.15% (P < 0.05) at 24 h, 26.42% (P < 0.05) at 48 h and 44.74% (P < 0.05) at 72 h PI compared to controls (Fig. 2). 3.3. IL-6 and TNF-a in milk whey IL-6 in milk from both control and CpG-ODNtreated glands increased after infection. Peak levels of IL-6 from controls (5.73 1.05 pg mg1) and CpG-ODN-treated group (8.99 1.02 pg mg1) was recorded at 16 h PI (P < 0.05). Compared to controls, IL-6 in milk from the CpG-ODN-treated glands increased by 66.05% (P < 0.05) preinfection. IL-6 decreased gradually after 16 h PI and, in CpG-ODNtreated glands, it decreased by 33.78% (P < 0.05) at 24 h PI compared to controls (Fig. 3). E. coli infection induced an increase in TNF-a in milk from both control and CpG-ODN-treated glands. Maximal concentrations of TNF-a—0.42 0.06 and 0.37 0.03 ng mg1 for the control and CpG-ODNtreated group, respectively, was observed at 16 h PI. Compared to controls, TNF-a in milk from CpG-ODNtreated glands increased by 37.25% at 8 h PI, but did not
Fig. 1. Histology of mammary gland 72 h postinfusion with Escherichia coli (250). One gland of each udder was infused with either CpG-ODN or PBS on day 5 postpartum. On day 8, a second, identical infusion was administered to the respective glands. On day 9, each gland was infused with 6 106 CFU E. coli. (A) Control mammary gland. Only small numbers of PMNs are present in alveoli. (B) CpG-treated mammary gland. No PMNs are present in alveoli.
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Fig. 2. E. coli counts in milk from PBS control and CpG-ODN-treated mammary glands following inoculation with 6 106 CFU E. coli. One gland of each udder was infused with either CpG-ODN or PBS on day 5 postpartum. On day 8, a second, identical infusion was administered to the respective gland. On day 9, each gland was infused with 6 106 CFU E. coli. Data are geometric mean log10 colony-forming unit (S.E.M.) per ml of milk. *,#Significant difference (P < 0.05) compared with pre-infection values in PBS control and CpG-ODN-treated glands, respectively. aSignificant difference (P < 0.05) of CpG-ODN-treated glands compared with control at the same time-point.
reach a significant difference level. TNF-a from CpGODN-treated glands decreased gradually after 16 h PI and decreased by 35.07% (P < 0.05) at 24 h PI compared to controls (Fig. 4).
Fig. 3. IL-6 in whey from PBS control and CpG-ODN-treated mammary glands. One gland of each udder was infused with either CpGODN or PBS on day 5 postpartum. On day 8, a second, identical infusion was administered to the respective glands. On day 9, each gland was infused with 6 106 CFU E. coli. Data are expressed as means of IL-6 per milligram of protein in whey (S.E.M.). *,#Significant difference (P < 0.05) compared with pre-infection values in PBS control and CpG-ODN-treated glands, respectively. aSignificant difference (P < 0.05) of CpG-ODN-treated glands compared with control at the same time-point.
Fig. 4. TNF-a in whey from PBS control and CpG-ODN-treated glands. One gland of each udder was infused with either CpG-ODN or PBS on day 5 postpartum. On day 8, a second, identical infusion was administered to the respective glands. On day 9, each gland was infused with 6 106 CFU E. coli. Data are expressed as means of TNF-a per milligram of protein in whey (S.E.M.). *,#Significant difference (P < 0.05) compared with pre-infection values in PBS control and CpG-ODN-treated glands, respectively. aSignificant difference (P < 0.05) of CpG-ODN-treated glands compared with control at the same time-point.
3.4. NAGase and serum albumin in milk whey Concentrations of NAGase in milk from control and CpG-ODN-treated glands were statistically elevated
Fig. 5. NAGase in whey from PBS control and CpG-ODN-treated mammary glands. One gland of each udder was infused with either CpGODN or PBS on day 5 postpartum. On day 8, a second, identical infusion was administered to the respective glands. On day 9, each gland was infused with 6 106 CFU E. coli. Data are expressed as means of NAGase per gram of protein in whey (S.E.M.). *,#Significant difference (P < 0.05) compared with pre-infection values in PBS control and CpG-ODN-treated group, respectively. aSignificant difference (P < 0.05) of CpG-ODN-treated group compared with control at the same time-point. One unit of NAGase activity represents the amount of paranitrophenol released from 1 l of whey in 15 min at 37 8C.
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elevated and increased by 65.83% (P < 0.05) during the initial stage of infection at 8 h PI (Fig. 5). E. coli infection induced an increase in serum albumin in milk from both control and CpG-ODNtreated glands. Maximal concentrations of 0.41 0.07 and 0.47 0.05 mg mg1 from controls and CpGODN-treated glands, respectively, was observed at 16 h PI. Compared to controls, serum albumin in milk from CpG-ODN-treated glands was rapidly elevated during the initial stage of infection by 56.16% (P < 0.05) at 8 h PI. Serum albumin from CpG-ODN-treated glands decreased gradually after 16 h PI and decreased by 56.16% (P < 0.05) at 24 h PI compared to controls (Fig. 6). Fig. 6. Serum albumin in whey from PBS control and CpG-ODNtreated glands. One gland of each udder was infused with either CpGODN or PBS on day 5 postpartum. On day 8, a second, identical infusion was administered to the respective glands. On day 9, each gland was infused with 6 106 CFU E. coli. Data are expressed as means of serum albumin per milligram of protein in whey (S.E.M.). *,# Significant difference (P < 0.05) compared with pre-infection values in PBS control and CpG-ODN-treated glands, respectively. a Significant difference (P < 0.05) of CpG-ODN-treated glands compared with control at the same time-point.
after infection with E. coli and reached a peak of 10.68 1.23 (P < 0.05) and 11.12 1.565 U g1 (P < 0.05) in controls and CpG-ODN-treated glands, respectively, at 16 h PI. Compared to controls, NAGase in milk from CpG-ODN-treated group was rapidly
Fig. 7. TLR-9 mRNA at 72 h PI from PBS control and CpG-ODNtreated mammary glands following inoculation with 6 106 CFU E. coli. One gland of each udder was infused with either CpG-ODN or PBS on day 5 postpartum. On day 8, a second, identical infusion was administered to the respective glands. On day 9, each gland was infused with 6 106 CFU E. coli. Data are expressed as means SEM. *Significant difference (P < 0.05) compared with preinfection values in PBS control and CpG-ODN-treated group, respectively.
3.5. mRNA expression of TLR-9 in mammary glands As shown in Fig. 7, the expression of TLR-9 mRNA in the CpG-ODN-treated group was significantly higher than in controls at 72 h PI (P < 0.05). 4. Discussion The immune system has evolved two general mechanisms for combating infectious diseases. The first involves a rapid innate immune response characterized by the production of immunostimulatory cytokines and polyreactive antibodies. This early response helps limit spread of the pathogen prior to the development of antigen-specific sterilizing immunity. CpG motifs present in bacterial DNA induce such an innate immune response (Krieg et al., 2002). Recent reports demonstrate that CpG motifs improve host resistance to infection by a variety of bacterial, viral and parasitic pathogens (Krieg et al., 1998; Zimmermann et al., 1998; Klinman, 1998). In the present study, we examined whether administering CpG-ODN could stimulate the innate immune system of the mammary gland in goats, thereby protecting against mastitis induced by E. coli infection. Infusion of 6 106 CFU ml1 E. coli into the caprine mammary gland induced clinical mastitis. The CpGODN-treated and control glands became swollen and firm. Milk from CpG-ODN-treated glands appeared normal; the milk from controls contained flakes and/or clots. Rectal temperature of goats with CpG-ODNtreated glands was lower than controls throughout the experiment and the extent of swelling/hardness of the glands was milder than in controls. Histopathological evaluation indicated that the lumen of mammary glands in controls were shrunken and smaller than in the
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CpG-ODN-treated group. PMNs were present in alveoli from controls, whereas PMNs in CpG-ODN-treated group had disappeared at 72 h PI. Consistent with the clinical and histological examinations, the numbers of E. coli in mammary tissue from the CpG-ODN-treated glands were significantly reduced compared to controls. Bacterial growth in each inoculated quarter was used to quantify the severity of the disease as bacterial growth is directly related to the variation in clinical signs of experimental mastitis (Lohuis et al., 1990). This result suggests that, in agreement with the result in rat (Zhu et al., 2007), CpG-ODN decreased the growth of E. coli, alleviated the severity of mastitis and shortened the duration of inflammation. In vivo and in vitro studies showed that CpG-ODN could activate monocytes in humans (Klinman et al., 2002), splenocytes (Bjersing et al., 2004), peripheral blood mononuclear cells and macrophages in mice (Zhou et al., 2004), a macrophage cell line (HD11) in chickens (Xie et al., 2003), peripheral blood mononuclear cells in swine (Kamstrup et al., 2001) and peripheral blood mononuclear cells and macrophages in cattle (Zhang et al., 2003; Brown and Corral, 2002) to secrete IL-6. Study of Blum and Dosogene (2000) showed that IL-6 could promote the transition from neutrophil to mononuclear cell infiltrate in the alveolus of mammary tissue. This transition possibly weakened the detrimental effect of PMN, which is beneficial to immune defense. Li (1999) demonstrated that mice without IL-6 could not control infection by a virus or bacterium. Recently, Wu et al. (2004) found that IL-6 could be employed as an effective immunoadjuvant. Kehrli and Harp (2001) also demonstrated that cytokines IL-1b, IL-6, IL-8 and PMN migration and infiltration together determine the outcome of mastitis infection. In our study, IL-6 in milk was elevated significantly and peaked at 16 h PI, which coincides with the result in rat (Zhu et al., 2007). Although there have been no reports on the secretion of IL-6 in caprine immunocytes, the present study found that IL-6 in milk from the CpG-ODNtreated group increased by 66.05% (P < 0.05) preinfection compared to controls. The results of our previous study in rat also showed that CpG-ODN induced a significantly higher level of IL-6 in the mammary tissue at 16 h after inoculation of E. coli (Zhu et al., 2007). IL-6 from CpG-ODN-treated glands decreased by 33.78% (P < 0.05) at 24 h PI compared to control. The present study confirms that CpG-ODN facilitates a rapid response to microbial infection by inducing the production of IL-6 and accelerating the regression of inflammation.
Numerous studies have demonstrated that CpGODN could induce macrophages in mice (Zhou et al., 2004), peripheral blood mononuclear cells in pigs (Kamstrup et al., 2001) and macrophages in cattle (Brown and Corral, 2002) to secrete TNF-a. TNF-a play an important role in E. coli mastitis by inducing plasma haptoglobin, recruiting and activating neutrophils and elevating intra-mammary and systemic nitrite and nitrate (Blum and Dosogene, 2000). Although there have been no reports on TNF-a secretion of immunocytes in goat, the results of this study show that TNF-a in milk from control and CpG-ODN-treated group peaked at 16 h PI simultaneously. Compared with controls, TNF-a in milk from the CpG-ODN-treated group increased by 37.25% at 8 h PI, but did not reach a significant difference level. As seen from the different modes of action seen in the present study, CpG-ODN induced the rapid release of TNF-a, peaking 8 h earlier than control, which peaked at 16 h (Zhu et al., 2007). TNF-a from the CpG-ODN-treated group decreased by 35.07% (P < 0.05) at 24 h PI compared to controls, suggesting that CpG-ODN expedited the regression of inflammation. The lysosomal enzyme NAGase is found in milk, serum and other body fluids. Milk NAGase increases during mastitis and has been identified as a possible indicator of secretory cell damage and, thus, the severity of clinical mastitis (Kitchm et al., 1984). In the present study, NAGase in milk from the controls and CpGODN-treated groups increased significantly and both peaked at 16 h PI. NAGase in the CpG-ODN-treated group increased by 65.83% (P < 0.01) at 8 h PI. Previous studies show that elevation of NAGase is induced by leakage of NAGase from damaged secretory epithelial cells (Mattila et al., 1986). Wilson et al. (1991) also found that NAGase originates from white blood cells that increase in milk as a result of inflammation. Studies have shown that a significant correlation is present between the somatic cell count and NAGase in sheep (r = 0.85) (Leitner et al., 2001) and cattle (r = 0.88) (Williams et al., 1991). In the present study, NAGase in milk from the CpG-ODNtreated group was rapidly elevated and increased by 65.83% (P < 0.05) during the initial stage of infection at 8 h PI compared to controls, suggesting that CpGODN induces the migration of PMNs to the mammary gland. Contrary to the present study, NAGase in mammary tissue of rat from the CpG-ODN-treated group did not changed significantly, whereas it increased significantly in control (Zhu et al., 2007), suggesting that CpG-ODN alleviated the severity of mastitis in rat.
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IMI increases the permeability of microcirculatory vessels by secreting various chemical mediators, such as histamine, prostaglandin, kinins and free oxygen radicals, from inflammatory cells (Honkanen-Buzalski and Sandhom, 1981). The concentration of serum albumin in milk is considered to be an indicator of increased permeability of the vascular and blood–milk barrier induced by inflammatory mediators (Morkoc et al., 1993). In the present study, serum albumin in milk from CpG-ODN-treated glands was significantly elevated at 8 h PI and then significantly decreased at 24 h PI compared to controls, suggesting that CpGODN could induce rapid inflammation at the initial stage of infection and shorten the duration of inflammation. TLR-9 has been identified as a receptor for bacterial DNA containing a specific sequence pattern, including unmethylated CpG dinucleotides (Hemmi et al., 2000). Evidence from mice and humans indicates that TLR-9 specifically recognizes CpG-ODN. TLR-9-deficient mice fail to respond to CpG-ODN and transfection of cells with the human TLR9 gene confers the ability to respond to CpG-ODN (Takeshita et al., 2001). Quantitative real-time PCR assays have confirmed TLR9 expression within the ovine jejunum, Peyer’s patches and mesenteric lymph nodes (Menzies and Ingham, 2006). There have been no reports, however, confirming the expression of TLR-9 mRNA within caprine mammary tissue. This study confirmed that TLR-9mRNA expression within caprine mammary glands at 72 h PI was significantly higher in CpGODN-treated glands than in controls, which suggests that CpG-ODN may promote the expression of TLR9mRNA. TLR-9 signaling is mediated through the adaptor protein MyD88. This signaling pathway initiates the IRAK/TRAF6 signal cascade and ultimately results in activation of transcription factors, including NF-kB. NF-kB is crucial for inducing the expression of various pro-inflammatory cytokines and mediators, including IL-6 and TNF-a (Schnare et al., 2001), which is consistent with the higher levels of IL-6 and TNF-a in CpG-ODN-treated glands compared to controls. Similarly to rats (Zhu et al., 2007), the present study confirmed that CpG-ODN could promote a specific receptor for TLR-9mRNA expression to initiate a protective effect against E. coli mastitis in goats. 5. Conclusions CpG-ODN induced higher levels of TLR-9 mRNA in mammary tissue, stimulated the production of IL-6 in milk, prompted the release of the TNF-a, reduced
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E. coli counts in milk, attenuated the damage of inflammatory mediators and shortened the course of inflammation. Thus, this study demonstrated that CpGODN stimulated defenses in the mammary gland, with a reduction in symptoms and bacterial shedding after experimental infection with E. coli. Acknowledgement This work was supported by the National Nature Science Foundation of China (project no. 30671533). References Bergonier, D., Berthelot, X., 2003. New advances in epizootiology and control of ewe mastitis. Live Prod. Sci. 79, 1–16. Bjersing, J.L., Tarkowski, A., Collins, L.V., 2004. Anti-proliferative effects of phosphodiester oligodeoxynucleotides. Immunobiology 209, 637–645. Blum, J.W., Dosogene, H., 2000. Hoeben Tumor necrosis factor-a and nitrite/nitrate responses during acute mastitis induced by Escherichia coli infection and endotoxin in dairy cows. Domest. Anim. Endocrine 19, 2230–2235. Brown, W.C., Corral, R.S., 2002. Stimulation of B lymphocytes, macrophages, and dendritic cells by protozoan DNA. Microb. Infect. 4 (9), 969–974. Chandler, R.L., 1970. Experimental bacterial mastitis in the mouse. J. Med. Microbiol. 3, 273–282. Cooray, R., Jonsson, P., 1990. Modulation of resistance to mastitis pathogens by pretreatment of mice with T-2 toxin. Food Chem. Toxicol. 28, 687–692. Chomczynski, P., Sacchi, N., 2006. The single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction: twenty-something years on. Nat. Prot. 581–585. Gomis, S., Babiuk, L., Godson, D.L., Allan, B., Thrush, T., 2003. Protection of chickens against Escherichia coli infections by DNA containing CpG motifs. Infect. Immun. 71, 857–863. Gonzalez, N.R., Jasper, D.E., Krunlund, N.C., Farver, T.B., Cullor, J.S., Bushnell, R.B., Dellinger, J.D., 1990. Clinical mastitis in two California dairy herds participating in contagious mastitis control program. J. Dairy Sci. 73, 648–660. Gramzinski, R.A., Doolan, D.L., Sedegah, M., Davis, H.L., Krieg, A.M., Hoffman, S.L., 2001. Interleukin-12- and gammainterferondependent protection against malaria conferred by CpG oligodeoxynucleotide in mice. Infect. Immun. 69 (3), 643–1649. Hemmi, H., Takeuchi, O., Kawai, T., Kaisho, T., Sato, S., Sanjo, H., Matsumoto, M., Hoshino, K., Wagner, H., Takeda, K., Akira, S.A., 2000. Toll-like receptor recognizes bacterial DNA. Nature 408, 740–745. Hill, A.W., 1991. Vaccination of cows with rough Escherichia coli mutants fails to protect against experimental intramammary bacterial challenge. Vet. Res. Commun. 15, 7. Honkanen-Buzalski, T., Sandhom, M., 1981. Trypsin inhibitors in mastitic milk and colostrums: correlation between trypsin inhibitor capacity, bovine serum albumin and somatic cell content. J. Dairy Res. 48, 213–233. Kamstrup, S., Verthelyi, D., Dennis, M., Klinman, 2001. Response of porcine peripheral blood mononuclear cells to CpG-containing oligodeoxynucleotides. Vet. Microbiol. 78, 353–362.
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endotoxin-induced intramammary inflammation. J. Dairy Sci. 76, 2579–2588. Schnare, M., Barton, G.M., Holt, A.C., Takeda, K., Akira, S., Medzhitov, R., 2001. Toll-like receptors control activation of adaptive immune response. Nat. Immunol. 2 (10), 947–950. Takeshita, F., Leifer, C.A., Gursel, I., Iashii, K.L., Takeshita, S., Gursel, M., Klinman, D.M., 2001. Cutting edge: role of Toll-like receptor 9 in CpG-DNA induced activation of human cells. J. Immunol. 167 (7), 3555–3558. Williams, D.J., Marschke, R.J., Nottingham, S.M., Kitchen, B.J., 1991. Effects of stage of lactation, number of lactations and dry period on N-acetyl-beta-D-glucosaminidase and somatic cell count in bovine milk. Aust. J. Dairy Technol. 46, 43–45. Wilson, D.J., Herer, P.S., Sears, P.M., 1991. N-Acetyl-b-D-glucosaminidase, etiologic agent and duration of clinical signs for sequential episodes of chronic clinical mastitis in dairy cows. J. Dairy Sci. 74, 1539–1543. Wu, M., Rong, G., Meng, M.J., 2004. Regulating effects of porcine interleukin-6 gene and CpG motifs on immune responses to porcine trivalent vaccines in mice. Res. Vet. Sci. 77, 49–57. Xie, H., Raybourne, R.B., Babu, U.S., Lillehoj, H.S., Heckert, R.A., 2003. CpG-induced immunomodulation and intracellular bacterial killing in a chicken macrophage cell line. Dev. Comp. Immunol. 27, 823–834. Zhang, Y., Guy, H.P., Jeffrey, R.A., Howard, C.J., Hope, J.C., Brown, W.C., 2003. CpG ODN 2006 and IL-12 are comparable for priming Th1 lymphocyte and IgG responses in cattle immunized with a rickettsial outer membrane protein in alum. Vaccine 21, 3307–3318. Zhou, H., Zheng, J., Wang, L.X., Ding, G.F., Luo, P., Lu, Y.L., Pan, W.D., Wang, M.H., 2004. Chloroquine protects mice from challenge with CpG ODN and LPS by decreasing proinflammatory cytokine release. Int. Immunopharmacol. 4, 223– 234. Zhu, Y.M., Fan, H.J., Miao J.F., Zou, S.X., 2007. Protective effect of CpG-DNA against mastitis induced by Escherichia coli infection in a rat model. Vet. J., doi:10.1016/j.tvjl.2007.01.027. Zimmermann, S., Egeter, O., Hausmann, S., Lipford, G.B., Rocken, M., Wagner, H., Heeg, K., 1998. CpG oligodeoxynucleotides trigger protective and curative Th1 responses in lethal murine leishmaniasis. J. Immunol. 160 (8), 3627–3630.
CpG-ODN enhances mammary gland defense during mastitis induced by Escherichia coli infection in goats Yu-Min Zhu a,b, Jin-Feng Miao a, Yuan-Shu Zhang a, Zhen Li b, Si-Xiang Zou a,*, Yue -E Deng a b
a College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China Animal and Veterinary Science Research Institute Shanghai Academy of Agriculture Sciences, Shanghai 201106, China
Received 9 March 2007; received in revised form 13 July 2007; accepted 2 August 2007
Abstract Seven healthy native goats in early lactation, weighing 30–40 kg, were used in this study. The right mammary gland of the seven does were infused with CpG-ODN at a dosage of 100 mg kg1 body weight on the day 5 postpartum (PP). The left glands were used as controls and infused with sterile phosphate-buffered saline (PBS). On day 8 PP, the same dosage of CpG-ODN or PBS was again infused. On day 9 PP, the mammary glands (both right and left) of the seven does were infused with 6 106 colony-forming units (CFU) Escherichia coli and, at 0, 8, 16, 24, 48 and 72 h postinfection (PI), milk samples were collected from all glands. Goats were euthanized at 72 h PI and the mammary tissue harvested. Infusion with 6 106 CFU ml1 E. coli induced acute mastitis. Histopathological evaluations showed that polymorphonuclear neutrophils (PMNs) were still present in alveoli at 72 h PI, but PMNs in the CpG-ODN-treated glands has disappeared. Bacteria counts in milk peaked at 16 h PI and CpG-ODN induced a significant decrease in viable bacteria from 16 h PI until the end of the experiment. This study showed that CpG-ODN promoted the expression of its specific receptor (TLR-9 mRNA) in mammary tissue, stimulated IL-6 production, reduced bacteria counts in milk, attenuated the impact of inflammation mediators on cells and significantly shortened the inflammation course. These results suggest that the CpGODN improved mammary gland defense and, thereby, had a beneficial effects against mastitis caused by E. coli infection in goats. # 2007 Elsevier B.V. All rights reserved. Keywords: CpG-ODN; Goat; Mastitis; Escherichia coli; TLR-9
1. Introduction CpG-ODN is an unmethylated, specific DNA motif containing cytosine phosphate-guanosine dinucleotides (CpG). It induces immune cells to secrete a number of
Abbreviations: CpG, cytosine phosphate-guanosine dinucleotides; IMI, intramammary infection; NAGase, N-acetyl-b-D-glucosaminidase; PMNs, polymorphonuclear leukocytes; CFU, colonyforming units; Mab, monoclonal antibody; PI, postinfection. * Corresponding author at: College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China. Tel.: +86 21 62200389. E-mail address: [email protected] (Y.-M. Zhu). 0165-2427/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2007.08.003
cytokines, thereby upregulating specific and nonspecific immunity. It has been well documented that CpG-ODN not only stimulates B-lymphocytes and innate immune cells, such as macrophages, dendritic cells and natural killer cells, in mammals, but also results in the activation and secretion of cytokines, including interleukin-1b (IL-1b), IL-6, IL-12, IL-18, tumor necrosis factor-a (TNF-a), interferon-a (IFN-a) and interferon (IFN-g) (Krieg et al., 2002). The immunomodulatory use of CpG-ODN is currently being investigated for anti-tumor, anti-infectious, anti-asthmatic and anti-inflammatory applications and for adjuvant activity in immunotherapy (Krieg et al., 2002). In recent years, the protection provided by
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CpG-ODN against infection has been demonstrated. For example, pre-treatment of mice with CpG-ODN provided protection against lethal challenge by the bacteria, Listeria monocytogenes (Krieg et al., 1998) and Francisella tularensis (Klinman et al., 1999), and parasitic protozoans, such as Leishmania spp. (Zimmermann et al., 1998) and Plasmodium yoelii (Gramzinski et al., 2001). Recently, Gomis et al. (2003) demonstrated for the first time that CpG-oligonucleotides (CpG-DNA) were effective in controlling extracellular Escherichia coli infections in poultry in a dose-dependent manner. E. coli is the main causative agent of clinical mastitis in ewes and 2–12% of E. coli has been isolated (Bergonier and Berthelot, 2003). Clinical mastitis in ewes induced by E. coli infection may occur at every stage of lactation (Gonzalez et al., 1990). Despite the frequent occurrence of the disease worldwide, effective therapy has not yet been established unequivocally. Traditional mastitis control measures, such as dry cow antibiotic therapy, teat dipping and selective culling, are relatively ineffective in controlling E. coli mastitis (Gonzalez et al., 1990). Additionally, Hill (1991) showed that vaccination provided little prophylactic benefit against naturally occurring intramammary infection (IMI). Therefore, with increasing knowledge of immunobiology of the mammary gland, research into mastitis is focusing on augmenting the innate immune response as a means of increasing resistance of the mammary gland to invading pathogens. A mouse model of infectious mastitis was first described by Chandler (1970). This model has been used to assess the pathophysiology of E. coli IMI (Cooray and Jonsson, 1990). In 2006, we established a reliable and sensitive mastitis model in rats infected with E. coli and found, for the first time, that CpG-DNA induced a more rapid migration of PMNs from blood to mammary gland at the initial stage of E. coli infection, decreased bacterial counts in the mammary gland and attenuated the impact of inflammation mediators on the cell (Zhu et al., 2007). Pathophysiological differences most likely exist between mouse/rat and caprine mammary glands; therefore, the present work aims to extensively characterize the immunostimulation effect of CpG-ODN on the mammary gland of goats, which may help to develop new modalities for mastitis prophylaxis and treatment. 2. Materials and methods 2.1. CpG-ODN CpG-ODN was kindly provided by HongFei Zhu of the Chinese Academy of Agricultural Sciences
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(CAAS). CpG-ODN was purified from the plasmid pCpG, which was cloned with the synthesized CpG sequence and the pUC18 vector. The CpG-ODN was diluted in sterile pyrogen-free phosphate-buffered saline (PBS) and adjusted to a concentration of 2 mg ml1. 2.2. Bacteria E. coli (CMCC, 25922) was purchased from the Chinese Medical Culture Collection (CMCC). Bacterial inocula were prepared from freeze-dried cultures grown in nutrient broth at 37 8C overnight. The bacteria were harvested and suspended in 0.01 mol l1, pH 7.2 PBS. The bacteria were then washed three times in PBS by centrifugation at 5000 g for 10 min at 4 8C and diluted in PBS. The number of colony-forming units (CFU) was determined by serial dilution and plate counting. The cultures were further diluted in PBS to 2 106 CFU ml1. 2.3. Animals and samplings Animal studies were approved by the Animal Ethics Committee. Seven, 30–40 kg lactating female goats were provided with food and water ad libitum. The goats were kept under observation for 14 days in thoroughly cleaned premise before the start of the experiment. All the animals were free from sub-clinical mastitis and no bacteria or fungi were isolated from preinoculation milk samples. The right mammary glands of the seven does were infused with 100 mg kg1 CpGODN on day 5 postpartum (PP). Left mammary glands served as controls and were infused with sterile PBS. On day 8 PP, repeat dosages of CpG-ODN or PBS were infused, respectively. On day 9 PP, after morning milking, the right and left glands of the does were infused with 6 106 CFU ml1 E. coli. Milk samples from both glands were aseptically collected just before inoculation of the bacteria (defined as 0 h) and at 8, 16, 24, 48 and 72 h postinfection (PI). Rectal temperatures were recorded at 0, 1, 2, 3, 4, 5, 6, 7, 8, 16, 24, 48 and 72 h PI. At each sampling, both halves of the udder were emptied as completely as possible by hand milking. Kids were withheld from does for the first 8 h of infection during which time the goats were housed in a group pen with access to water and lucerne chaff. Subsequently, does and kids were reunited and returned to pasture. The goats were mustered each morning for sample collection, then returned to pasture. Does were euthanized at 72 h PI and the mammary tissue harvested. A portion of each mammary gland was
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immediately snap-frozen in liquid nitrogen and stored at 80 8C until RNA extraction. One portion of each mammary gland was fixed in Bouin’s fluid and embedded in paraffin. The remainder of the mammary glands were aseptically collected and stored at 20 8C. 2.4. Preparation and assay of milk and mammary gland 2.4.1. Mammary tissue preparation and microscopic observations Sections of 5 mm thickness were stained with hematoxylin and eosin and the stained tissue sections examined by light microscopy. Parameters evaluated were gland morphology and inflammatory cell infiltrate. 2.4.2. Preparation and assay of milk samples Milk samples were diluted in sterile PBS prior to plating on brain–heart infusion agar containing 5% sheep blood. After a 24-h incubation at 37 8C, bacterial colonies were counted and the colony-forming units (CFU) per ml of milk were calculated. The remaining milk samples were centrifuged at 44 000 g for 30 min at 4 8C and the fat layer removed with a spatula. The skim milk was decanted into a clean tube and centrifuged again for 30 min, as described above; the translucent whey fractions were collected and then frozen at 20 8C until use. Whey protein concentration was determined using the Bradford method. TNF-a and IL-6 levels in milk whey were measured by radioimmunoassay. Commercial kits were purchased from the Institute of Radiation of Science & Technology Development Center of the General Hospital of the People’s Liberation Army. The assay was conducted following the manufacturer’s protocol. The level of serum albumin in milk whey was measured using commercial kits purchased from the Nanjing Jiancheng Bioengineering Institute (NJBI) following manufacturer’s protocols. The activity of N-acetyl-b-D-glucosaminidase (NAGase) in milk whey was determined using commercial kits purchased from NJBI and following the manufacturer’s protocols. The optical density of paranitrophenol during the reaction (at 37 8C) of the 4-methylumbelliferyl-N-acetyl-b-glucosaminide substrate with NAGase contained in the analyzed samples was measured spectrophotometrically in triplicate at a wavelength of 400 nm. One unit of NAGase activity represents the amount of paranitrophenol released from 1 l of milk in 15 min at 37 8C.
2.4.3. RNA extraction and RT-PCR for the Toll-like receptor (TLR-9) 2.4.3.1. RNA extraction and primer. Total RNA was extracted from the tissue samples with acid guanidinium thiocyanate–phenol–chloroform (Chomczynski and Sacchi, 2006). RNA concentration was quantified by measuring absorbance at 260 nm. Ratios of absorption (260/280 nm) of all preparations were between 1.8 and 2.0. Aliquots of the RNA samples were subjected to electrophoresis through a 1.4% agarose– formaldehyde gel to verify their integrity. The nucleotide sequences of TLR-9 primers were as follows: TLR-9 (forward, 50 -TATGTGCCGCGCTTTT-30 ; reverse, 50 -TTCGCGGAACCAGTCTTT-30 ); the amplified product was 384 bp. b-Actin (forward, 50 -GCACCACACCTTCTACAA-CGAGC-30 ; reverse, 50 TCCTTGATGTCACGGACGATTTC-30 ); the amplified product was 765 bp. 2.4.3.2. Reverse transcription (RT) and polymerase chain reaction (PCR). Two mg of total RNA were reverse transcribed by incubation at 37 8C for 1 h in a 25 ml mixture consisting of 200 U of M-MLV reverse transcriptase, 10 U Raze inhibitor, 0.8 mM oligo(dT18) primer, 0.8 mM dNTP, 10 mM Tris–HCl (pH 8.3), 0.6 mM MgCl2, 15 mM KCl and 2 mM DDT. RNA samples were denatured at 70 8C for 5 min and placed on ice for 5 min together with the oligo-(dT18) primer and dNTP before reverse transcription (RT). The RT reaction was terminated by heating at 95 8C for 5 min and then quickly cooled on ice. Three ml of RT reaction mixture was used for PCR in a final volume of 25 ml containing 1.25 U Taq DNA polymerase, 1 mM Tris–HCl (pH 9.0), 5 mM KCl, 0.01% Triton X-100, 0.4 mM dNTP, 2 mM MgCl2, 0.8 mM sense and antisense primers for TLR-9, 0.04 pM sense and antisense primers for b-actin and 1.5 ml DMSO. After denaturation at 94 8C for 5 min, 30 amplification cycles with denaturation at 94 8C for 30 s, annealing at 57 8C for 60 s and an extension at 72 8C for 60 s, with a final incubation at 72 8C for 10 min were performed. 2.4.3.3. Quantitation of PCR products and statistical analysis. Twenty ml of PCR product was separated by electrophoresis on 2.0% agarose gels stained with ethidium bromide and photographed with a digital camera. The net intensities of individual bands were measured using Kodak Digital Science 1D software (Eastman-Kodak, Rochester, NY, USA). The ratios of net intensities of target genes to b-actin were used to
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represent the relative levels of target gene expression. The average of three repeats was used for statistical analysis.
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CpG-ODN-treated glands. PMNs accumulated in the alveoli of control glands; however, the infiltration of PMNs in CpG-ODN-treated glands disappeared (Fig. 1).
2.5. Statistical analysis 3.2. E. coli enumeration Analysis of variance was performed using SPSS software (SPSS11.5 for Windows). Data were expressed as means standard error (S.E.M.), except that data from bacterial counts were converted to log10 to maintain a normal distribution. Differences were considered significant at P < 0.05 by t-test for independent samples or ANOVA with STATISTICA for Windows 5.0 (StatSoft, Tulsa, OK, USA). 3. Results 3.1. Clinical symptoms, rectal temperature and histological observations Acute clinical mastitis developed in all treated and control glands at 3 h PI. The does were depressed and swelling was observed in infected glands. The does lay down and did not drink any water from 4 to 6 h PI. Milk from control glands contained some flakes or clots, whereas milk from CpG-ODN-treated glands appeared normal. The temperature of goats with CpGODN-treated glands was lower than PBS controls throughout the experiment and the extent of swelling and firmness of the treated glands was milder than controls. The rectal temperature started to increase at 1 h PI, peaked at 6 h PI (41.0 8C), then decreased gradually and returned to normal at 24 h PI (38.2 8C). Histopathological evaluation indicated that the lumen of control mammary glands were smaller than
The numbers of E. coli in mammary tissue increased significantly and peaked at 5.82 0.52(log10 CFU ml1) in the control group and at 4.73 0.32(log10 CFU ml1) in the CpG-ODN-treated group at 16 h PI, then slowly declined. The bacterial concentration from the CpG-ODN-treated group decreased by 18.40% (P < 0.05) at 16 h, 19.15% (P < 0.05) at 24 h, 26.42% (P < 0.05) at 48 h and 44.74% (P < 0.05) at 72 h PI compared to controls (Fig. 2). 3.3. IL-6 and TNF-a in milk whey IL-6 in milk from both control and CpG-ODNtreated glands increased after infection. Peak levels of IL-6 from controls (5.73 1.05 pg mg1) and CpG-ODN-treated group (8.99 1.02 pg mg1) was recorded at 16 h PI (P < 0.05). Compared to controls, IL-6 in milk from the CpG-ODN-treated glands increased by 66.05% (P < 0.05) preinfection. IL-6 decreased gradually after 16 h PI and, in CpG-ODNtreated glands, it decreased by 33.78% (P < 0.05) at 24 h PI compared to controls (Fig. 3). E. coli infection induced an increase in TNF-a in milk from both control and CpG-ODN-treated glands. Maximal concentrations of TNF-a—0.42 0.06 and 0.37 0.03 ng mg1 for the control and CpG-ODNtreated group, respectively, was observed at 16 h PI. Compared to controls, TNF-a in milk from CpG-ODNtreated glands increased by 37.25% at 8 h PI, but did not
Fig. 1. Histology of mammary gland 72 h postinfusion with Escherichia coli (250). One gland of each udder was infused with either CpG-ODN or PBS on day 5 postpartum. On day 8, a second, identical infusion was administered to the respective glands. On day 9, each gland was infused with 6 106 CFU E. coli. (A) Control mammary gland. Only small numbers of PMNs are present in alveoli. (B) CpG-treated mammary gland. No PMNs are present in alveoli.
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Fig. 2. E. coli counts in milk from PBS control and CpG-ODN-treated mammary glands following inoculation with 6 106 CFU E. coli. One gland of each udder was infused with either CpG-ODN or PBS on day 5 postpartum. On day 8, a second, identical infusion was administered to the respective gland. On day 9, each gland was infused with 6 106 CFU E. coli. Data are geometric mean log10 colony-forming unit (S.E.M.) per ml of milk. *,#Significant difference (P < 0.05) compared with pre-infection values in PBS control and CpG-ODN-treated glands, respectively. aSignificant difference (P < 0.05) of CpG-ODN-treated glands compared with control at the same time-point.
reach a significant difference level. TNF-a from CpGODN-treated glands decreased gradually after 16 h PI and decreased by 35.07% (P < 0.05) at 24 h PI compared to controls (Fig. 4).
Fig. 3. IL-6 in whey from PBS control and CpG-ODN-treated mammary glands. One gland of each udder was infused with either CpGODN or PBS on day 5 postpartum. On day 8, a second, identical infusion was administered to the respective glands. On day 9, each gland was infused with 6 106 CFU E. coli. Data are expressed as means of IL-6 per milligram of protein in whey (S.E.M.). *,#Significant difference (P < 0.05) compared with pre-infection values in PBS control and CpG-ODN-treated glands, respectively. aSignificant difference (P < 0.05) of CpG-ODN-treated glands compared with control at the same time-point.
Fig. 4. TNF-a in whey from PBS control and CpG-ODN-treated glands. One gland of each udder was infused with either CpG-ODN or PBS on day 5 postpartum. On day 8, a second, identical infusion was administered to the respective glands. On day 9, each gland was infused with 6 106 CFU E. coli. Data are expressed as means of TNF-a per milligram of protein in whey (S.E.M.). *,#Significant difference (P < 0.05) compared with pre-infection values in PBS control and CpG-ODN-treated glands, respectively. aSignificant difference (P < 0.05) of CpG-ODN-treated glands compared with control at the same time-point.
3.4. NAGase and serum albumin in milk whey Concentrations of NAGase in milk from control and CpG-ODN-treated glands were statistically elevated
Fig. 5. NAGase in whey from PBS control and CpG-ODN-treated mammary glands. One gland of each udder was infused with either CpGODN or PBS on day 5 postpartum. On day 8, a second, identical infusion was administered to the respective glands. On day 9, each gland was infused with 6 106 CFU E. coli. Data are expressed as means of NAGase per gram of protein in whey (S.E.M.). *,#Significant difference (P < 0.05) compared with pre-infection values in PBS control and CpG-ODN-treated group, respectively. aSignificant difference (P < 0.05) of CpG-ODN-treated group compared with control at the same time-point. One unit of NAGase activity represents the amount of paranitrophenol released from 1 l of whey in 15 min at 37 8C.
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elevated and increased by 65.83% (P < 0.05) during the initial stage of infection at 8 h PI (Fig. 5). E. coli infection induced an increase in serum albumin in milk from both control and CpG-ODNtreated glands. Maximal concentrations of 0.41 0.07 and 0.47 0.05 mg mg1 from controls and CpGODN-treated glands, respectively, was observed at 16 h PI. Compared to controls, serum albumin in milk from CpG-ODN-treated glands was rapidly elevated during the initial stage of infection by 56.16% (P < 0.05) at 8 h PI. Serum albumin from CpG-ODN-treated glands decreased gradually after 16 h PI and decreased by 56.16% (P < 0.05) at 24 h PI compared to controls (Fig. 6). Fig. 6. Serum albumin in whey from PBS control and CpG-ODNtreated glands. One gland of each udder was infused with either CpGODN or PBS on day 5 postpartum. On day 8, a second, identical infusion was administered to the respective glands. On day 9, each gland was infused with 6 106 CFU E. coli. Data are expressed as means of serum albumin per milligram of protein in whey (S.E.M.). *,# Significant difference (P < 0.05) compared with pre-infection values in PBS control and CpG-ODN-treated glands, respectively. a Significant difference (P < 0.05) of CpG-ODN-treated glands compared with control at the same time-point.
after infection with E. coli and reached a peak of 10.68 1.23 (P < 0.05) and 11.12 1.565 U g1 (P < 0.05) in controls and CpG-ODN-treated glands, respectively, at 16 h PI. Compared to controls, NAGase in milk from CpG-ODN-treated group was rapidly
Fig. 7. TLR-9 mRNA at 72 h PI from PBS control and CpG-ODNtreated mammary glands following inoculation with 6 106 CFU E. coli. One gland of each udder was infused with either CpG-ODN or PBS on day 5 postpartum. On day 8, a second, identical infusion was administered to the respective glands. On day 9, each gland was infused with 6 106 CFU E. coli. Data are expressed as means SEM. *Significant difference (P < 0.05) compared with preinfection values in PBS control and CpG-ODN-treated group, respectively.
3.5. mRNA expression of TLR-9 in mammary glands As shown in Fig. 7, the expression of TLR-9 mRNA in the CpG-ODN-treated group was significantly higher than in controls at 72 h PI (P < 0.05). 4. Discussion The immune system has evolved two general mechanisms for combating infectious diseases. The first involves a rapid innate immune response characterized by the production of immunostimulatory cytokines and polyreactive antibodies. This early response helps limit spread of the pathogen prior to the development of antigen-specific sterilizing immunity. CpG motifs present in bacterial DNA induce such an innate immune response (Krieg et al., 2002). Recent reports demonstrate that CpG motifs improve host resistance to infection by a variety of bacterial, viral and parasitic pathogens (Krieg et al., 1998; Zimmermann et al., 1998; Klinman, 1998). In the present study, we examined whether administering CpG-ODN could stimulate the innate immune system of the mammary gland in goats, thereby protecting against mastitis induced by E. coli infection. Infusion of 6 106 CFU ml1 E. coli into the caprine mammary gland induced clinical mastitis. The CpGODN-treated and control glands became swollen and firm. Milk from CpG-ODN-treated glands appeared normal; the milk from controls contained flakes and/or clots. Rectal temperature of goats with CpG-ODNtreated glands was lower than controls throughout the experiment and the extent of swelling/hardness of the glands was milder than in controls. Histopathological evaluation indicated that the lumen of mammary glands in controls were shrunken and smaller than in the
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CpG-ODN-treated group. PMNs were present in alveoli from controls, whereas PMNs in CpG-ODN-treated group had disappeared at 72 h PI. Consistent with the clinical and histological examinations, the numbers of E. coli in mammary tissue from the CpG-ODN-treated glands were significantly reduced compared to controls. Bacterial growth in each inoculated quarter was used to quantify the severity of the disease as bacterial growth is directly related to the variation in clinical signs of experimental mastitis (Lohuis et al., 1990). This result suggests that, in agreement with the result in rat (Zhu et al., 2007), CpG-ODN decreased the growth of E. coli, alleviated the severity of mastitis and shortened the duration of inflammation. In vivo and in vitro studies showed that CpG-ODN could activate monocytes in humans (Klinman et al., 2002), splenocytes (Bjersing et al., 2004), peripheral blood mononuclear cells and macrophages in mice (Zhou et al., 2004), a macrophage cell line (HD11) in chickens (Xie et al., 2003), peripheral blood mononuclear cells in swine (Kamstrup et al., 2001) and peripheral blood mononuclear cells and macrophages in cattle (Zhang et al., 2003; Brown and Corral, 2002) to secrete IL-6. Study of Blum and Dosogene (2000) showed that IL-6 could promote the transition from neutrophil to mononuclear cell infiltrate in the alveolus of mammary tissue. This transition possibly weakened the detrimental effect of PMN, which is beneficial to immune defense. Li (1999) demonstrated that mice without IL-6 could not control infection by a virus or bacterium. Recently, Wu et al. (2004) found that IL-6 could be employed as an effective immunoadjuvant. Kehrli and Harp (2001) also demonstrated that cytokines IL-1b, IL-6, IL-8 and PMN migration and infiltration together determine the outcome of mastitis infection. In our study, IL-6 in milk was elevated significantly and peaked at 16 h PI, which coincides with the result in rat (Zhu et al., 2007). Although there have been no reports on the secretion of IL-6 in caprine immunocytes, the present study found that IL-6 in milk from the CpG-ODNtreated group increased by 66.05% (P < 0.05) preinfection compared to controls. The results of our previous study in rat also showed that CpG-ODN induced a significantly higher level of IL-6 in the mammary tissue at 16 h after inoculation of E. coli (Zhu et al., 2007). IL-6 from CpG-ODN-treated glands decreased by 33.78% (P < 0.05) at 24 h PI compared to control. The present study confirms that CpG-ODN facilitates a rapid response to microbial infection by inducing the production of IL-6 and accelerating the regression of inflammation.
Numerous studies have demonstrated that CpGODN could induce macrophages in mice (Zhou et al., 2004), peripheral blood mononuclear cells in pigs (Kamstrup et al., 2001) and macrophages in cattle (Brown and Corral, 2002) to secrete TNF-a. TNF-a play an important role in E. coli mastitis by inducing plasma haptoglobin, recruiting and activating neutrophils and elevating intra-mammary and systemic nitrite and nitrate (Blum and Dosogene, 2000). Although there have been no reports on TNF-a secretion of immunocytes in goat, the results of this study show that TNF-a in milk from control and CpG-ODN-treated group peaked at 16 h PI simultaneously. Compared with controls, TNF-a in milk from the CpG-ODN-treated group increased by 37.25% at 8 h PI, but did not reach a significant difference level. As seen from the different modes of action seen in the present study, CpG-ODN induced the rapid release of TNF-a, peaking 8 h earlier than control, which peaked at 16 h (Zhu et al., 2007). TNF-a from the CpG-ODN-treated group decreased by 35.07% (P < 0.05) at 24 h PI compared to controls, suggesting that CpG-ODN expedited the regression of inflammation. The lysosomal enzyme NAGase is found in milk, serum and other body fluids. Milk NAGase increases during mastitis and has been identified as a possible indicator of secretory cell damage and, thus, the severity of clinical mastitis (Kitchm et al., 1984). In the present study, NAGase in milk from the controls and CpGODN-treated groups increased significantly and both peaked at 16 h PI. NAGase in the CpG-ODN-treated group increased by 65.83% (P < 0.01) at 8 h PI. Previous studies show that elevation of NAGase is induced by leakage of NAGase from damaged secretory epithelial cells (Mattila et al., 1986). Wilson et al. (1991) also found that NAGase originates from white blood cells that increase in milk as a result of inflammation. Studies have shown that a significant correlation is present between the somatic cell count and NAGase in sheep (r = 0.85) (Leitner et al., 2001) and cattle (r = 0.88) (Williams et al., 1991). In the present study, NAGase in milk from the CpG-ODNtreated group was rapidly elevated and increased by 65.83% (P < 0.05) during the initial stage of infection at 8 h PI compared to controls, suggesting that CpGODN induces the migration of PMNs to the mammary gland. Contrary to the present study, NAGase in mammary tissue of rat from the CpG-ODN-treated group did not changed significantly, whereas it increased significantly in control (Zhu et al., 2007), suggesting that CpG-ODN alleviated the severity of mastitis in rat.
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IMI increases the permeability of microcirculatory vessels by secreting various chemical mediators, such as histamine, prostaglandin, kinins and free oxygen radicals, from inflammatory cells (Honkanen-Buzalski and Sandhom, 1981). The concentration of serum albumin in milk is considered to be an indicator of increased permeability of the vascular and blood–milk barrier induced by inflammatory mediators (Morkoc et al., 1993). In the present study, serum albumin in milk from CpG-ODN-treated glands was significantly elevated at 8 h PI and then significantly decreased at 24 h PI compared to controls, suggesting that CpGODN could induce rapid inflammation at the initial stage of infection and shorten the duration of inflammation. TLR-9 has been identified as a receptor for bacterial DNA containing a specific sequence pattern, including unmethylated CpG dinucleotides (Hemmi et al., 2000). Evidence from mice and humans indicates that TLR-9 specifically recognizes CpG-ODN. TLR-9-deficient mice fail to respond to CpG-ODN and transfection of cells with the human TLR9 gene confers the ability to respond to CpG-ODN (Takeshita et al., 2001). Quantitative real-time PCR assays have confirmed TLR9 expression within the ovine jejunum, Peyer’s patches and mesenteric lymph nodes (Menzies and Ingham, 2006). There have been no reports, however, confirming the expression of TLR-9 mRNA within caprine mammary tissue. This study confirmed that TLR-9mRNA expression within caprine mammary glands at 72 h PI was significantly higher in CpGODN-treated glands than in controls, which suggests that CpG-ODN may promote the expression of TLR9mRNA. TLR-9 signaling is mediated through the adaptor protein MyD88. This signaling pathway initiates the IRAK/TRAF6 signal cascade and ultimately results in activation of transcription factors, including NF-kB. NF-kB is crucial for inducing the expression of various pro-inflammatory cytokines and mediators, including IL-6 and TNF-a (Schnare et al., 2001), which is consistent with the higher levels of IL-6 and TNF-a in CpG-ODN-treated glands compared to controls. Similarly to rats (Zhu et al., 2007), the present study confirmed that CpG-ODN could promote a specific receptor for TLR-9mRNA expression to initiate a protective effect against E. coli mastitis in goats. 5. Conclusions CpG-ODN induced higher levels of TLR-9 mRNA in mammary tissue, stimulated the production of IL-6 in milk, prompted the release of the TNF-a, reduced
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