It is known that cats can sporadically suffer from leptospirosis [12,16,17], but while the clinical presentation of the disease is rare, it may be missed [2]. Asymptomatic cats can shed the bacteria through urine [9,15], and unlike infected dogs, antibody titres against Leptospira spp. detected in sub-clinically infected animals are low in the species [6-7,20]. Cats infected by Leptospira spp. may become an incidental or reservoir host. The incidental host develops an acute disease state with mild to moderate clinical signs, leptospiraemia (1-7 days/sub-acute state), leptospires urinary shedding (10 day-about a month/late acute state) and antibodies titres. Reservoir cats usually exhibit a chronic state of infection and have no clinical signs of disease or leptospiraemia, but they do have long-term and intermittent leptospires urinary shedding and low or absent antibodies titres [21].
APPs analysis has been studied in cats with infectious diseases [27-28, 52-53] and its use is highly recommended to determine the active state of disease. TAC as an antioxidant biomarker can contribute to the morbidity of many diseases and is likely to become, along with APPs, an important component in the active state detection of diseases, including leptospirosis in cats.
A PCA statistical approach is based on the fact that cats naturally infected by Leptospira spp. may be grouped into different patterns that may be associated with different serum concentrations of APPs and TAC as antioxidant biomarkers. The information obtained could facilitate the understanding of the pathogenesis of Leptospira spp. and infection course in cats.
PC1, accounting for 35.23% of the data variance, was significantly associated with the APPs profile or inflammatory pattern. The supplementary qualitative variable PCR (negative/positive) was significant in the PC1 (R2 0.22, P-value 0.006), suggesting a direct relationship between the presence of the bacteria in blood or urine and the APPs responses.
Patterns of inflammation described in preliminary works were determined and confirmed by our PCA analysis in PCR positive cats (blood or urine). They include a positive correlation found between SAA and Hp (-0.72 and -0.67 respectively), albumin and PON1 (0.76 and 0.66 respectively) [30,54], and negative correlation between SAA-Hp and albumin-PON1 [54-55]. It is well known that albumin falls gradually with extensive inflammation, with the reduction in concentration being more noticeable in chronic inflammatory disease [56]. Serum PON1activity was associated with a negative APP and did not play a role as a biomarker of oxidative status in PC1 or PC2, in contrast to previous studies [27,57].
Likewise, based on Leptospira spp. pathogenesis, it is possible to assume that PCR positive animals had a sub-acute infection status (Leptospira spp. present in blood/ early stage of the infection) or were mostly reservoirs (Leptospira spp. present in urine/intermittent urinary shedding). Based on the PC1 results, animals of group 2 reflected an active infection, compared to those infected with anti-leptospiral antibodies (group 1), which did not reflect an active state of infection. The difference in inflammatory patterns between the two groups of infected cats its very likely due to differences in bacterial load and the infecting serovar involved, among others. In diseases such as FIV, the relationship between antigenic load and high serum levels of APPs, and more specifically SAA, has been shown [58]. Likewise, Leptospira serovars involved in the infection have also been reported to influence the inflammatory response in other species. In dogs, it has been shown that serogroup Pomona [59] and Icterohaemorrhagiae [2] trigger the strongest inflammatory responses and have the worst prognosis. In cats, this remains poorly understood [21]. In our study, the infecting serovar or serogroup was not detected in the PCR positive cats as they had no anti-leptospiral antibodies.
PC2, accounting for 24.9% of the data variance, was significantly associated with the oxidative status pattern. A positive correlation was found between antibody titre and serum concentration of TAC (0.85 and 0.72, respectively) with PC2; thus, is very likely that as antibody titres against Leptospira spp. increased, endogenous antioxidant synthesis increased as well. Likewise, the supplementary qualitative variable MAT (+/-) was significant in the PC2 (R2 0.62, P-value < 0.05).
Cats with low anti-leptospiral antibodies titre (ranging from 1:20 1:40), probably at a resolution stage of infection, had increases in serum concentrations of TAC, most likely, to counteract the oxidative state associated with the inflammation. In human and companion animals, it has been reported that high serum concentrations of TAC are due to counteracting the increases in oxidants, while decreases are attributed to a persistent state of oxidative stress [27,38, 60].
Seropositive cats seemed to be at risk of developing oxidative stress as antioxidant response measured by the TEC1 assay was above the cut-off point, unlike DNA positive animals. Considering that one of the 8 seropositive cats, also tested positive for FIV infection, it is likely that in this cat the TAC serum values were also increased by the co-infection with the virus.
Antibody titres, in which the antioxidants become exhausted, showing a decrease in serum concentration of TAC and leading to an oxidative status [30], are not yet established. The little information available for cats has used serum concentrations of TAC to assess the antioxidant components globally. Some authors have reported a decrease in serum concentrations of TAC associated with oxidative stress [27, 30, 55, 61].
Despite the positive correlation found in our study between antibody titres against Leptospira spp. and serum concentrations of TAC with PC2, it is not possible to establish whether the variation in the serum concentration of TAC obtained in the two groups of animals naturally infected by Leptospira spp. (Group 1 and 2) is due to the inflammatory response caused by Leptospira spp. infection or whether this is due to the differences in the available assays for measuring TAC [57].
Infectious diseases other than VIF/ViLEF were not ruled out. As mentioned above, diseases such as feline infectious peritonitis (FIP) [27,28], FIV [34] and haemoplasmas [53] have also shown an increase in some of these biomarkers. As well as non-infectious inflammatory diseases [32, 43, 54]. In the present study, Cats health status was based on physical exam findings and biochemistry, so these results need to be interpreted with caution.
One of the limitations of our study is the small sample size (total and by groups), and the lack of follow up of the serum values of the biomarkers measured, due to the nature of the animals in the study.
Further studies should be undertaken to elucidate, the Leptospira serovars role
in cats and their involvement in the inflammatory response through serum concentrations of APPs and TAC as oxidative stress markers by using different assays and enzymes implied in the antioxidant response. To the authors' knowledge, this is the first report measuring the serum concentration of APPs and TAC in cats naturally infected by pathogenic leptospires.
Finally, based on PCA analysis results and the PON-1 arrangement on the inflammatory PC1, further experimental studies are needed to estimate its importance as a negative APP in cats.