Comparison of Natural Resistance-associated Macrophage Protein (NRAMP)1 Expression between Cows with High and Low Milk Somatic Cells Counts

Studies using natural resistance-associated macrophage protein (NRAMP) identification indicated that cattle could be selected for immunity. Several studies performed on intracellular organisms such as Mycobacterium, Salmonella, Brucella and Leishmania in human and mouse revealed that resistance against these bacteria was dependent on high activity of NRAMP1 in macrophages. However, hardly any researches have been done on Staphylococcus aureus in bovine mastitis, which is an intracellular organism and the main cause of bovine mastitis. The objectives of this study were to establish reverse transcriptase polymerase chain reaction (RT–PCR) methods, through which NRAMP1 mRNA expression could be compared and analyzed between mastitis-resistant and -susceptible cows. NRAMP1 gene and its expression were investigated using 20 cows (Holstein Friesian) in Korea. Cows were evenly split into two groups, with and without histories of clinical mastitis. Equivalent numbers of cows were randomly selected from each group. Monocytes were isolated from the bovine peripheral blood of each selected cows and activated with lipopolysaccharide (LPS). mRNA was separated from the monocytes and cDNA of NRAMP1 was synthesized and amplified using RT-PCR with amplification of β-actin as a control. The difference in NRAMP1 expressions of mastitis-resistant (n=10) and -susceptible (n=10) Holstein cows was analyzed. Results demonstrate that resistant cows produced more NRAMP1 mRNA than the susceptible ones, and ratios of NRAMP1:β-actin expression were higher in resistant cows with or without LPS activation. Therefore, this study could be applied to select bovine mastitis resistant cows before infection based on the expression of NRAMP1. (Asian-Aust. J. Anim. Sci. 2003. Vol 16, No. 12 : 1830-1836)


INTRODUCTION
Selection for disease resistance has been advocated as a mechanism to control mastitis.Studies using Bovine Leukocyte Antigen (BoLA) typing or natural resistanceassociated macrophage protein (NRAMP) identification indicated that cattle could be selected for immunity (Lewin et al., 1999).The objective of both BoLA typing and NRAMP analysis was to reduce the pecuniary loss through the early detection of individuals with high resistance to the disease.Monocytes, macrophages, and neutrophils play important roles in the nonspecific immune responses of mammary mucosa during the early infection of mastitis and are essential for the eradication of pathogens and antigen processing.They are more bactericidal and/or bacteriostatic when the expression of NRAMP is induced by pathogens.However, they also offer refuge to intracellular pathogens such as Staphylococcus aureus thus causing difficulty in disease treatment (Sanchez et al., 1993).Therefore, studies on NRAMP1 of these cells, which participated in the phagolysosome activity against intracellular organisms was in need.
The NRAMP family, whose genetic sequences are highly conserved in humans, mice, sheep, porcine, and cattle, plays a key role on the homeostasis of iron and other metals as divalent metal transporters, among which NRAMP2 acts as an iron uptake protein in both the duodenum and peripheral tissues.NRAMP1, on the other hand, functions as a divalent-metal efflux pump in the phagosomal membrane of macrophages and neutrophils, and its mutation cause susceptibility to several intracellular pathogens.It is rapidly recruited into the membrane of maturing phagosomes, and is suggested to modulate microbial replication by altering the bacteriostatic and bactericidal properties of the fused phagosome.Mammalian and bacterial NRAMPs would compete for the same essential substrates within the microenvironment of the phagosome, in addition to the interface of host-pathogen interactions.
Several studies have been performed on intracellular organisms such as Mycobacterium, Salmonella, Brucella, and Leishmania in human and mouse, which revealed the high activity of NRAMP1 (Belouch et al., 1995).However, hardly any have been done on Stapylococcus aureus in bovine mastitis, which is an intracellular organism and the main cause of bovine mastitis, although some studies on the genotyping of S. aureus associated with mastitis of cows had been presented (Joo et al., 2001).In addition, few studies have analyzed the expression of NRAMP1 in mammalian tissues.The objectives of this study were to establish reverse transcriptase polymerase chain reaction (RT-PCR) methods, through which NRAMP1 mRNA expression could be compared and analyzed between mastitis-resistant and -susceptible cows.

Experimental animals
Holstein Friesian cows raised by the National Livestock Research Institute, the Rural Development Administration, Korea, were used (Table 1).Mastitis infection frequency, frequency of medical treatments, and treatment conditions recorded over a four-year period since 1997 were used to select animals.Animals were divided into two groups: resistant group, with no history of medical treatment for mastitis, and susceptible group, which had more than three treatments for bovine mastitis.Milk somatic cell counts (SCC) were determined using Foss 300 and Foss system 4000 (Foss Electric Co., Denmark).SCC of the resistant group averaged below 200 thousands/ml, which of the susceptible group was higher during the four-year period (Table 2).Furthermore, to verify the mastitic pathogen, bacteria were isolated when SCC was higher than 500 thousands/ml in whole milk during the lactating period.To establish the NRAMP1 mRNA detection method, we used five each bovine mastitis-resistant and -susceptible cows, which were randomly picked from each group.In addition, to analyze the expression capacity of NRAMP1 through the established method, 10 cows from each group were used.

Isolation and identification of mastitis pathogens from milk
The method of Joo et al. (2001) was applied for the isolation of pathogens from mastitis-susceptible cows.In brief, each milk sample of quarters of mastitis-susceptible cows was cultured on 5% sheep blood agar (KOMED, Korea) and incubated at 37°C for 48 h.Bacterial colonies presumptively identified as staphylococci by colony characteristics, catalase reaction, and hemolytic patterns were speciated following the National Mastitis Council protocols (Harmon et al., 1990).Those isolated were analyzed further using the API staph system (Biomerieux, France) and coagulase test.

Separation of monocytes from bovine peripheral blood and sensitization
Method used for leukocyte separation was based on that proposed by Barta (1993).Ten ml each blood samples were collected from both groups in heparinized vacutainer (Becton Dickinson, USA), into which 3 ml RPMI 1640 (Sigma, USA) medium was added.The collected samples were then transported to the laboratory.Final volumes of the samples were adjusted to 20 ml with RPMI 1640.Diluted blood samples were centrifuged at 330 g and 20°C for 30 min.Obtained buffy coat was diluted with Hank's Balance Salt Solution (Gibco BRL, USA) and centrifuged at 250 g for 10 min.Pellets were resuspended with RPMI 1640 and washed twice.A total 10 ml of RPMI 1640 with 10% fetal bovine serum (Gibco) was added and incubated in a 5% CO 2 incubator (Napco, USA) for 1 h.The cells attached to the bottom were collected.Monocytes were separated from the leukocyte based on their ability to attach to plastics.To activate NRAMP1 in the monocyte, 20 ng/ml S. enterica serovar Typhimurium lipopolysaccharide (LPS; Sigma) was added and incubated in a 5% CO 2 incubator for 6 h at 37°C.Controls were treated with RPMI 1640 only.

Pure separation of RNA
The method of Favaloro et al. (1980) was applied for the purification of RNA.Monocytes obtained were collected and centrifuged at 2,500 g for 10 min.Supernatant was aspirated, into which 100 µl of 3 M sodium acetate (pH 5.2) was added and mixed.Subsequently 500 µl cold ethanol was added and mixed well.After the mixture was centrifuged at 5,000 g for 10 min, the obtained pellet was dried at room temperature.The above procedure was repeated twice, the pellet was dissolved with 50 µl of 0.02 M EDTA (pH 8.0; Sigma) and an equal volume of 4chloroform:1-1-butanol (4:1) (Gibco) was added.The mixture was then shaken and centrifuged.Collected supernatant was treated with 4 M sodium acetate (pH 7.0) and centrifuged.Obtained pellet was dissolved with 0.2% SDS (Bio-rad, USA) and 0.05 M EDTA (pH 8.0).RNA was isolated using cold ethanol.RNA purity and quality were determined using a spectrophotometer (Pharmacia, LKB Mod.80-2, USA) at 260/280 nm.
Amplified cDNA fragments were fractionated through 1.5% agarose gel (Sigma) electrophoresis in 0.5X TBE (45 mM Tris-borate, 1 mM EDTA), at 50 V/cm for 30 min.Gels were stained with ethidium bromide (Sigma) and imaged with Advanced American Biotechnology Software Restriction Fragment Length Polymorphism (AAB software, USA).Based on the image analysis, band concentration was calculated in terms of optical density (OD) and normalized with β-actin.
Twenty µl of the NRAMP1-PCR product was cleaved with 20 U restriction enzyme Msp I (Promega) according to the manufacturer's instruction, and was subsequently precipitated in 2.0 M ammonium acetate and 2.5 volumes of ethanol.The pellet was dissolved in 12 µl of TE buffer, mixed with gel loading buffer, and separated on a 3% agarose gel in 0.5X TBE at 50 V/cm for 30 min.The gel was then stained and photographed.

Statistical analysis
NRAMP1 mRNA expression were subjected to SAS (ver 6.12 SAS institute, NC) for t-test to compare mastitissusceptible and -resistant cows, and paired t-test was used to analyze the difference between before and after activation with LPS (p<0.05).

Identification of mastitis pathogens from mastitissusceptible cows
S. aureus and S. epidermidis were the major pathogens isolated from milk sample of mastitis-susceptible cows.Especially, S. aureus were identified from more than one quarter of each dairy cow with high SCC (>500 thousands cellls/ml).

Detection of NRAMP1 mRNA by RT-PCR
NRAMP1-specific primers designed for RT-PCR revealed the existence of NRAMP1 specific 221 bp through electrophoretic analysis.Detected gene products digested with Msp I also showed NRAMP1-specific band pattern (Figure 1).Based on this result, a method which could measure the capacity of NRAMP1 mRNA expression in bovine monocytes was established.

Production of NRAMP1 mRNA derived from activated monocytes with LPS
To detect the individual bovine capacity for NRAMP1 expression, monocytes from peripheral blood were sensitized with LPS for 6 h.The mean OD values of preand post-activations of NRAMP1 expression were 0.3 and 0.98, respectively.Results showed that the degree of NRAMP1 gene expression after sensitization with LPS was significantly elevated than that of pre-sensitization (p<0.05),although the increased amounts were different among individuals within resistant or susceptible groups.

Comparison of NRAMP1 mRNA expression between bovine mastitis-resistant and -susceptible cows
NRAMP1 mRNA expression data (Table 3) showed that OD values of mastitis-resistant cows varied from 0.275 to 1.850 (1.080 average).On the other hand, OD values of susceptible cows varied from 0.05 to 1.575 with a mean value of 0.432, which were more than twofold lower than that of the resistant ones.The t-test indicated that the high NRAMP1 gene activity was more resistant to mastitis (p<0.05).
The NRAMP1 mRNA expression was compared between bovine mastitis-resistant and -susceptible cows and also between post-and pre-activations of both cow types (Figure 2).The average ratios of NRAMP1:β-actin expression for each group are as follows: those of resistant and susceptible groups after activation were 1.046±0.254and 0.503±0.122,and before sensitization were 0.454±0.185and 0.152±0.053,respectively.The ratios of NRAMP1:β-actin expression were different between resistant and susceptible cows with or without LPS activation (p<0.05),whereas no difference was observed in β-actin expression between the two groups regardless of activation.

DISCUSSION
Bovine mastitis, one of the most significant diseases in livestock industries, has been a subject of many researchers' studies as milk has been because it has caused much pecuniary loss to farmers (Kalorey et al., 2001;Shem et al., 2001;Dhaka et al., 2002;Mukhopadhyaya and Mehta, 2002;Shem et al., 2002;Mukherjee and Dash, 2003).Endeavors to select individuals genetically resistant to mastitis were taken as a line in the chain of these studies,   The primers used in this study amplified the exact size of NRAMP1, and digested product was also revealed expected products.Digested sizes of the two fragments were 158 and 65 bp, which were similar to those of humans (Belouchi et al., 1995;Kishi et al., 1996).According to our results, mastitis-resistant cows showed higher level of NRAMP1 expression than susceptible ones.It suggests that individuals with high NRAMP1 gene activity are not predisposed to mastitis, and cope with the infection of mastitis-causing pathogens effectively, because elevated NRAMP1 expression was confirmed especially after LPS treatment.Therefore, detection of NRAMP1 gene activity with RT-PCR could be a good indicator for the early detection of mastitis-resistant cows.
In porcine alveolar macrophages, peripheral blood mononuclear cells, and peripheral blood polymorphonuclear neutrophils, NRAMP1s were expressed in response to LPS (Zhang et al., 2000).Up-regulation of NRAMP1 occurred after a 6 h stimulation, and the induction was highly sensitive to LPS; as little as 10 ng/ml of LPS induced a level of expression close to the maximum induced by 1 µg/ml.This was similar with our study, in which mononuclear cells were sensitized with 20 ng/ml of LPS for a 6 h incubation period.
Several hypotheses have been made on the sites where NRAMP functions, including the phagosome, lysosome, cell membrane, and nuclear membrane.One report described that large quantity of NRAMP1 existed in phagolysosomes of macrophage and neutrophil, which reinforced the bactericidal activities about intracellular pathogens (Cellier et al., 1994;Hu et al., 1995;Govoni et al., 1995).Antibodies raised against NRAMP1 demonstrated that the protein was localized to late endosomal and lysosomal compartments, but not to early endosomes and outer macrophage membranes (Gruenheid et al., 1997;Blackwell et al., 2000).These results have important implications for any direct role that NRAMP1 might have in determining intracellular pathogenic survival.
Although it is certain that NRAMP1 plays a key role during intracellular pathogenic infection, some controversial results have also been reported.Vidal et al. (1995) analyzed NRAMP1 sequences of 27 mice and discovered that Salmonella-susceptible mouse had mutation at the second domain, in which Asp replaced Gly; that is, mutation of NRAMP1 had an effect on the susceptibility.Whereas the bactericidal activity of macrophage against Mycoplasma and Salmonella decreased in the NRAMP1 mutant, no effects were observed on Listeria monocytogenes and Legionella pneumophila (Swanson et al., 1983;Sanchez et al., 1988;Roach et al., 1991;Malo et al., 1994).Furthermore, the bactericidal activity of bovine macrophages regulated by NRAMP1 was superior against S. dublin, B. abortus, and M. bovis, but not against S. Typhimurium (Qureshi et al., 1996).In addition, recent studies failed to show evidence of association between NRAMP1 and tuberculosis resistance or susceptibility in cattle (Barthel et al., 2000) and higher NRAMP1 expression in healthy cattle than in M. bovis-infected bovines (Estrada-Chavez et al., 2001).These conflicting results might be due to little available information on NRAMP1 and its varying interactions with different intracellular pathogens.From this point of view, the present study is able to present a correlation of NRAMP1 and resistance against intracellular pathogens such as S. aureus in which few studies have been undertaken.
According to Brown et al. (1997), who studied the mRNA expressions of NRAMP1, TNF-α, and iNOS against Mycobacterium, much higher expression of NRAMP1 mRNA was observed in bacille Calmette-Guerin (BCG)resistant (Bcg R ) mouse than in BCG-susceptible (Bcg S ) mouse after sensitization with LPS, IFN-γ, and corticosterone.The continuation of expression was also observed upto 50 h in Bcg R mouse, but dipped below 20% in Bcg S mouse after 20 h.This result corresponded to our study that revealed the high expression of NRAMP1 gene in mastitis-resistant cows.
Some evidences were presented that NRAMP1 played a role in autoimmune diseases as well as infectious disease susceptibility (Blackwell et al., 2000).In human NRAMP1, Z-DNA-forming polymorphic repeats in the promoter region, which influence gene expression as functional polymorphism, were discovered (Searle et al., 1999).Four alleles and their different frequencies have been observed.NRAMP1 expression was enhanced or reduced by these alleles, whose response was highly dependent on the presence of external stimuli including LPS.Understanding the function of NRAMP1 in macrophages is an important prerequisite to understanding its complementary role in the infectious disease of cows.Therefore, further study should be carried out on bovine NRAMP1 function to control diseases, particularly major mastitis-causing intracellular pathogenic infection caused by S. aureus.
In conclusion, we established RT-PCR methods through which the aspect of NRAMP1 mRNA expression could be detected and the difference between mastitis-resistant andsusceptible cows analyzed.Results revealed that monocytes of resistant cows produced more NRAMP1 mRNA than susceptible ones in response to LPS challenge in vitro, and the average ratio of NRAMP1:β-actin expression was also higher in resistant group than the other group regardless of LPS stimulation.We estimate that the resistant group will show far better bactericidal and resistant capacities due to the enhanced phagolysosome activity when intracellular mastitic pathogens such as S. aureus invade the udder or alveolar cells.

Figure 1 .
Figure 1.Electrophoretic analysis of RT-PCR products of NRAMP1 gene after digestion with Msp I (lane M: moleculer maker (pGEM) lanes 1-5: RT-PCR products of NRAMP1 gene after digestion with Msp I, lane 6: No digestion).

Table 2 .
Somatic cell counts in raw milk during four-year period (×1 thousand cells/ml)

Table 3 .
Comparison of NRAMP1 m RNA expressions of monocytes activated with lipopolysaccharide in bovine mastitis-resistant and 1 Optical density: OD after activation -OD before activation . 2 Resistant to mastitis. 3Susceptible to mastitis.p<0.05 (p=0.01491).