Serum amyloid A as an marker of cow֨ s mastitis caused by Streptococcus sp.

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Abstract

The aim of the study was to evaluate the concentrations of amyloid A in serum (SAA) and in milk (MAA) of cows with mastitis caused by Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis and healthy cows. The blood and milk samples were obtained from Holstein-Friesian cows with clinical signs of mastitis from two tie-stall housing systems herds in the Lublin region in Poland. A total of 80 milk and serum samples from 30 cows with mastitis and 10 healthy cows were selected for study. In the quarter milk samples from cows with mastitis Streptococcus strains were isolated: Strep. agalactiae (7 cows), Strep. dysgalactiae (9 cows) and Strep. uberis (14 cows). The present study indicates that amyloid A concentration was significantly higher in milk of cows with mastitis compared to control cows (1134.25 ng/mL and 324.50 ng/mL, P < 0.001). The highest concentration of amyloid A was found in milk of cows with mastitis caused by Strep. agalactiae and Strep. uberis whereas lowest in the milk of cows with mastitis caused by Strep. dysgalactiae (3882.50 ng/mL, 2587.75 ng/mL and 812.00 ng/mL, respectively). No statistically significant difference in amyloid A concentration in serum was revealed between all unhealthy cows and control group (2140.00 ng/mL and 2510.00 ng/mL, P > 0.05). There was also no statistically significant difference between the level of amyloid A in serum and in milk of cows with mastitis caused by Strep. agalactiae and Strep. uberis. Whereas, in the case of Strep. dysgalactiae, like in the group of healthy cows, the level of amyloid A was significantly higher in serum compared to this in milk (2100 ng/mL and 812.00 ng/mL, P < 0.001; 2510.00 ng/mL and 324.50 ng/mL, P < 0.001; respectively).

Introduction

Acute phase response (APR) is one of the most important mechanisms restoring homeostasis in organism after different disorders. APR comprises many physiologic reactions - fever, leucocytosis, hormonal and metabolic alterations, changes in serum protein levels [1]. Acute phase proteins (APPs) are a group of proteins whose plasma concentrations increase during the systemic response following local tissues inflammation [2]. One of the most important of APPs is serum amyloid A (SAA) [1,[3], [4], [5], [6], [7]]. The term „amyloid” originally referred to material found in plants and presumed to be carbohydrate („amylon” – starch”), only later studies showed that it is a protein. SAA are present, but at generally quite low levels, in the blood of healthy humans and animals. However, their levels can rise as much as 1000-fold within the initial 24 h after APR onset, largely reflecting de novo synthesis in the liver. Consistent with the APR definitione, blood levels of SAA fall rapidly and then returning to very low levels as the event resolves [8].

Several isotypes of SAA in animals are found; types 1, 2 and 3 represent positive APPs. SAA1 and SAA2 isoforms mRNA transcription take places mainly in hepatocytes, whereas extrahepatic SAA isoform 3 mRNA expression occurs in the mammary gland. Transcription site of the amyloid A isoform gene (MAA, SAA3, AA3, M-SAA3) contained in milk is mammary epithelial cells (MEC). MAA (milk amyloid A) has at the N-end of the sequence four TFLK amino acids, regardless of species (horse, cow, sheep) that are absent in the isoform of the liver synthesis protein [9]. In cattle, MAA contains 83 % of the same amino acid sequence as SAA. Serum amyloid A and milk amyloid A are stimulated by the release of cytokines such as Interleukin-1α (IL-1α), Interleukin-1β (IL-1β), Interleukin-6 (IL-6) and tumor necrosis factor (TNF –α) [9,10]. IL-6 is most effective early in APR but the combined activity of all factors gives the highest level of transcription [[11], [12], [13]]. Hagihara et al. [13] found that an antibody to the IL-6 receptor blocked the APR whereas an IL-1 receptor antagonist or anti-TNF antibody only partially inhibited it. In turn, Morrow et al. [14] showed that the level of murine hepatic mRNA for SAA rose 500-fold after inducing by intraperitoneal administration of bacterial lipopolysaccharide (LPS) and that SAA synthesis could rise to comprise 2.5 % of total mouse hepatic protein synthesis. Pathogen-associated molecular patterns on cell surfaces (PAMPS) can be recognized by Toll-like receptors on innate immune cells such as macrophages leading to inflammatory cytokine secretion [15].

SAA exhibits significant immunological activity by inducing the synthesis of several cytokines and being chemotactic for neutrophils and mast cells, inhibition effect on fever, inhibition effect on the oxidative burst of neutrophilic granulocytes and platelet activation, and induction of calcium mobilization by monocytes [4,16]. It exerts many of its immunological activities by binding and activating cell-surface receptors, including Toll-like receptor TLR2 and TLR4, formyl peptide receptor-like 1 (FPRL1), class B scavenger receptor CD36, and the ATP receptor P2 × 7 [4].

Mastitis (intramammary infection) is the most costly disease affecting dairy cattle worldwide. The economic losses in the case of mastitis involve: declined milk production, therapy and discarded milk costs, increased additional workload, replacement cost due to culling of severely affected animals [17,18]. Many microorganisms can infect the udder in cows, but Streptococcus species have been reported as the most common aetiological agents of mastitis [[19], [20], [21], [22]]. Contagious pathogens, such as Strep. agalactiae and Strep. dysgalactiae, are appearing inside the cow udder and disseminate from infected to uninfected mammary glands during milking (by milking machines and milkers’ hands) [17,23]. Strep. agalactiae is able to affect pre-milking heifers, as well as older cows and is considered one of the major causes of economic losses in dairy herds without a control program [24]. In contrast, environmental pathogens, such as Strep. uberis, easily colonize, grow and multiply in the environment [21,25]. They are found in many extra-mammary sources like feces, bedding, water, forage, soil [26]. Substantial amount of bacteria cells in the environment influences the larger incidence rate of mastitis [27].

The aim of the study was to evaluate the concentrations of amyloid A in serum (SAA) and in milk (MAA) of cows with mastitis caused by Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis and healthy cows.

Section snippets

Materials and methods

The blood and milk samples were obtained from Holstein-Friesian cows with clinical signs of mastitis from two tie-stall housing systems herds in the Lublin region in Poland, from May to September. The study was performed in accordance with national animal protection regulations (Animal Experimentation Act dated 15 January 2015) which agreement with European legislation about ethics in animal experiments. The milk samples from cows with mastitis were collected from the quarter with inflammation

Results

A total of 80 milk and serum samples from 30 cows with mastitis and 10 healthy cows were selected for study. In the quarter milk samples from 30 cows with mastitis Streptococcus strains were isolated: Strep. agalactiae (7 cows), Strep. dysgalactiae (9 cows) and Strep. uberis (14 cows). One sample of milk from infected quarter and one sample of serum from each cows with mastitis were qualified for evaluation of level of amyloid A. In the milk obtained from all quarters of 10 healthy cows, growth

Discussion

In the present study, the concentration of amyloid A was investigated in serum and in milk obtained from cows suffering from mastitis caused by Streptococcus sp. For many years, Strep. agalactiae and Strep. dysgalactiae were described as the main reason of mastitis in cows. However, current studies indicate that the prevalence of these two pathogens in intramammary infections is definitely lower, most probably due to the advance of hygiene in milk production and dry cow therapy. Whereas

Conclusion

The present study showed that evaluation of the concentration of serum amyloid A in milk may provide important information about inflammatory status in mammary gland in cows. The level of this protein was significantly higher in milk of cows with mastitis compared to control group. These findings may have important implications for diagnosis of mastitis in cows because MAA may be used in the future as a novel prognostic marker in milk of intramammary infection. Statistically significant

Declaration of Competing Interest

The authors declare that there is no conflict of interests regarding the publication of this article.

Acknowledgements

This work has been possible thanks to the support of research project Miniatura 1 no. DEC-2017/01/X/NZ4/01769. Mariola Bochniarz was funded by National Science Center, Poland.

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