Bacterial mastitis in large ruminants is a globally important challenge of the dairy industry, causing considerable economic losses due to a frequently sub-clinical course resulting in persistence of pathogens within the herd [1]. Subsequently, there is a drop in the productivity, and qualitative and quantitative alterations of milk with degradation of organoleptic characteristics and preservability [2]. It is estimated that two-thirds of the total production loss of milk is attributable to subclinical mastitis [34]. In addition, infected animals become asymptomatic carriers of pathogens that spread in the herd. To date, most mastitis studies have been conducted in cows [56]; however, recent reports indicate that domesticated dairy buffaloes are similarly affected with mastitis [7]. Numerous bacteria, such as contagious (Staphylococcus aureus, Streptococcus agalactiae, Mycoplasma spp.), environmental (Streptococcus uberis and Streptococcus dysgalactiae, Escherichia coli, Enterobacteriaceae, Klebsiella, Pseudomonas spp.), and opportunistic species (Coagulase-negative staphylococci; CNS) have been reported in mastitis of these ruminants [89]. Different bacteria are likely to cause different clinical forms of mastitis; nevertheless, research in this area considering the Mediterranean buffalo remains scanty and is limited to identification of few individual bacterial species [1011]. Mastitis susceptibility is chiefly related to intrinsic immune defense system of the mammary gland [12]. Leucocytes, together with epithelial cells shed from the udder, are naturally present in milk and are also known as the somatic cells (SCs). Along with a bacteriological examination, somatic cell count (SCC) is commonly used as an indicator of udder health and milk quality. Buffaloes are considered healthy in terms of dairy production if each mammary quarter produces bacteriologically negative milk with an SCC below 300 × 10³ cells/mL, as reported in annex III, section IX of the Regulation 853/2004 of the European Parliament, and of the Council of 29 April 2004 (Ref. L139/55 Official Journal of the European Union). However, buffaloes producing bacteriologically positive milk with an SCC higher than 300 × 10³ cells/mL are defined as suffering from subclinical mastitis [29]. It is well known that tissues harboring the target antigens of chronic inflammation are infiltrated mainly by lymphocytes, plasma cells and macrophages. In some chronic inflammatory disease, these cells organize themselves, leading to the de novo formation of lymphoid structures called tertiary lymphoid structures (TLSs) [13141516]. TLSs have a complex microarchitecture and are morphologically and functionally similar to secondary lymphoid organs (SLOs). Although TLSs are usually found in the background of chronic inflammation, autoimmunity and cancer [17181920], their functional significance remains unclear. For effective control of subclinical mastitis, to reduce the risk of chronic infections, and to target antimicrobial therapy, it is essential to identify etiological agents and to evaluate their interaction with the mammary gland immune defense system [5].

Thus, the present study was undertaken to examine clinically healthy buffaloes sent to slaughter, with the aim to: 1) effectuate microbiological screening of sampled mammary gland tissue, 2) identify and characterize subclinical-mastitis by evaluating the macro- and microscopic mammary gland changes, and the possible role played by TLSs to determine the chronic course of mastitis, 3) immuno-characterize the cell infiltrates, and 4) compare the degree of tissue inflammation with SCC.

Mastitis is a major problem in dairy cows and, as shown in the current study, also in buffaloes. Most remarkably, all 50 investigated buffalo udders (100%) were found bacteriologically positive for Pseudomonas spp. and Staphylococcous spp.; in addition, 86% were positive for Streptococcus spp., 76% for E. coli and 46% for Enterobacter spp. Among the udders positive for Staphylococcus spp. and Streptococcus spp., 28% were identified as S. aureus and 33% as S. viridans. These results confirm that mastitis in the group of buffaloes examined by us were mostly caused by environmental mastitis pathogens. Thus, our findings suggest a similar pathogenesis in buffaloes; that is, once established in the udder, environmental pathogens can persist for extended periods causing continuous infection and chronic inflammatory changes with fibrosis in the mammary tissue [1521]. This causes an increased milk SCC and can lead to reduced milk production and early culling.

Histological and immunofluorescence analysis of the udders affected by chronic sub-clinical mastitis (92%) showed a predominance of CD8+ lymphocytes mainly localized in close contact with the ductal epithelium and between epithelial cells, preferably in the middle, the low gland and in the teat regions. This finding indicates that, similar to that suggested in cows, the CD8+ lymphocytes play an important role in buffalo udders in the local immune response as cytotoxic cell scavengers, removing old or damaged secretory cells, thereby reducing susceptibility of the gland to infections [22]. The correlation of the infiltration grade with SCC values supports this hypothesis. Furthermore, CD8+ lymphocytes are thought to control or modulate the immune response to bacterial infections and have an important function in the chronic status of coliform intra-mammary infections [23]. Contrarily, several studies have demonstrated that the CD8+ lymphocytes activated during bacterial infections down-regulate the host immune response [1224]. Evaluation of lacteal secretions from mammary glands of dairy cows infected with S. aureus revealed a subpopulation of activated CD8+ lymphocytes capable of altering or suppressing the proliferative responses of CD4+ lymphocytes [2526]. Such hypo-responsiveness of lymphocyte interaction mediated in part by CD8+ lymphocyte activation by lecithins and bacterial antigens has been demonstrated in bovine mammary glands infected with S. aureus; thus, it is suggestive that CD8+ lymphocytes activated by antigens, such those of S. aureus, may enhance infectious mastitis and may contribute to its persistence [27].

In dairy cows, composition of the lymphocyte subpopulations vary during the lactation cycle [252728], where CD8+ lymphocytes are predominant in lactating udders, while CD4+ lymphocytes are over-expressed during the dry period. During the lactation cycle, CD8+ lymphocytes begin to increase in the peri-partum [29], reach a peak in the early lactation period, and decrease during late lactation [28].

The predominance of CD8+ lymphocytes observed in our study conducted on late lactating udders, confirms the presence of chronic mastitis.

Studies report observing a peculiar feature in 48% of bubaline mastitis; the sparse inflammatory infiltrates (lymphocytes, plasma cells, and macrophages) organize themselves in newly formed follicular aggregates, exhibiting distinctive histological features of TLSs. TLSs were previously described in urothelial cancer of buffalo [30] and in the endometrium of pregnant dairy cows [31]. In human medicine, they are known to be present in some organ specific disorders [32] such as infectious and neoplastic diseases [203334]. In our samples, these aggregates showed varying levels of organization (including formation of HEVs), which are characteristic for TLSs [35]. The differences in bystander-infiltrating lymphocytes and TLSs is not yet clearly defined but seems to reside in the degree of internal organization of the TLSs. However, the globular structuration with distinct B and T cell compartments, the presence of a network of fibroblast reticular cells as well as follicular dendritic cells and, not in the least, the presence of HEV, are highly indicative features for TLSs [19]. TLSs also shares numerous similarities with SLO. However, the order of events and the molecular mechanisms responsible for postnatal TLS development are significantly different from those regulating lymph node development, and partially different from those of the spleen that are present before birth [19]. The absence of a defined capsule and of an independent vascular network (it is not apparent if lymphatic vessels are afferent and/or efferent) represent the main morphological distinctive elements. The pathophysiological significance of TLSs is still unclear. They are considered as ectopic and local dynamic structures developing from scattered inflammatory infiltrates that aggregate and eventually organize into lymphofollicular structures. TLS formation is now recognized as a common feature of many chronic inflammatory diseases and might have a role in maintaining the immune responses against persistent antigens [1436]. During chronic inflammation, a cross talk between stromal cells and recruited immune cells can be established, which is also a necessary requirement for the formation of TLSs. Stromal cells, stimulated by inflammatory cytokines produced by the immune cells, could acquire lymphoid tissue-like characteristic able to provide local structure and survival mechanism to the incoming immune cells [37]. It has been suggested that the stimulus that triggers TLS formation in infected tissues is the causative agent itself [17].

In our study, the presence of TLSs increases progressively from low to severe infiltration grade, and the values are statistically significant both in the low/middle mammary gland and in the teat region, thereby confirming that TLSs are found in highly infiltrated tissues [17]. In the udders examined by us, TLSs were present without apparent correlation to specific bacteria types, except for a significant increase in the presence of Streptococcus uberis infection. It is thus reasonable to suggest that environmental mastitis pathogens stimulate the adaptive immune responses, leading to chronic inflammation in which TLSs develop. In our samples, TLSs were mainly present in the low and middle mammary gland and the teat regions. The presence of TLSs mostly in the teat region is suggestive of their role as the first line of immune defense in the ductal area, which is the entry point of germs. Previous studies in sub-clinically affected or healthy cows without mastitis reported complete absence of CD4+ and CD8+ lymphocytes in the teat region [338]. The presence of T-lymphocyte infiltrates and TLSs, especially in the teat region, as shown in this study (for the first time) in buffalo chronic mastitis, can be a further indication that tertiary lymphoid structures (TLSs) play a protective function by inducing and maintaining the immune response against infectious agents near their entry point [17]. In fact, it can be envisaged that when the immune system fails to mount an efficient response against an infectious agent, the efficacy of response is likely to increase if the crucial cells of the immune response are located at or near the site of antigen deposition. TLSs can therefore represent the attempt of stromal cells to respond locally and efficiently to the increased request of survival factors for the incoming lymphocytes [37]. Further studies are required to improve the knowledge on TLSs and their potential role in the pathogenesis of chronic mastitis in attempting to reduce or to prevent this serious problem of the buffalo dairy industry.