Rhodococcus equi (R. equi) is a Gram-positive bacterium recognized for causing pneumonia in foals, representing a common cause of economic losses in the equine industry (1). As a facultative intracellular bacterium, it relies on its ability to survive within macrophages (2). This phenomenon is associated with the presence of a plasmid encoding virulence genes, among which vapA is considered the most crucial (3–5). The expression of the virulence-associated protein A (VapA) is markedly enhanced under iron-restricted conditions (6). Foals commonly develop R. equi between 1 and 6 months of age. However, exposure to this pathogen has been reported during the first week of life (7, 8). Enhanced intracellular replication of R. equi in bronchoalveolar macrophages of young foals, accompanied by age- and macrophage-specific cytokine expression patterns, may contribute to the pathogen’s pulmonary tropism and the age-dependent susceptibility to infection (9).

The primary manifestation of R. equi infection is a respiratory disorder, characterized by the development of pyogranulomatous lesions in the lungs (10). The first clinical signs may include sporadic or intermittent cough, pyrexia, lethargy, respiratory distress, reduced appetite, and weight loss (11). However, extrapulmonary infections and immunomediated inflammatory disorders are also common (12–15). Diagnosis typically relies on clinical presentation, hematological abnormalities, as well as radiographic and ultrasonographic evidence of pulmonary lesions (16). Additionally, cytological evidence of intracellular Gram-positive or acid-fast coccobacilli and/or detection of virulent R. equi in tracheal aspirates can be pursued (17, 18).

Several reports agree that the primary risk factor for pneumonia is the concentration of virulent R. equi in the environment (19–21) and the foal’s exposure to this pathogen (22, 23). Differences in the host’s innate and adaptive immune responses (24) and genetic variations (25) are also considered significant factors contributing to the disease’s development. Despite considerable advances in understanding this pathogen, controlling and preventing R. equi pneumonia in equine farms remains a challenge. Consequently, many efforts are focused on elucidating predictors of the disease before its clinical onset (26–28). Ultrasound diagnosis has been used as a screening method to identify affected animals early on (29), although it has been found that this diagnostic method is more useful for assessing the severity of the disease than as a screening tool (30). Something similar happens with radiographic diagnosis, where its most evident utility is in locating deep abscesses or determining the prognosis in affected animals (31). Acute-phase inflammation proteins have been explored as predictive indicators of the disease, as well as the significance of leukocyte count (27, 32, 33). However, their relationship with other blood parameters as predisposing factors is still unknown.

This study aims to assess whether hematological and biochemical blood parameter levels, as well as the colostrum quality in newborn foals, may anticipate the subsequent development of R. equi pneumonia. These parameters could potentially serve as biomarkers for the early detection of the infection.

This study highlights differences in urea, serum creatinine, and gamma GT at birth between foals that develop R. equi pneumonia and those that remain healthy. Interestingly, neonatal gamma GT has been identified as an early risk factor for the future development of the infection.

Our research was performed on foals less than 24 h of life. It has been proposed that lung infection is the result of inhalation of virulent R. equi by aerosols from contaminated soil (34), with intestinal colonization observed within the first week of life and R. equi being isolated in the feces of 3-day-old foals (35). Also, increased mRNA expression of different interleukins has been found in bronchoalveolar macrophages in foals 1 to 3 days old (9). This background justifies that the collection of samples was performed during the first day after the delivery, since it is suggested that the most likely time of infection would be soon after birth (22).

Urea is a product generated in the liver because of protein metabolism. The lower values observed in the foals that developed the disease in this study can also be explained by hepatic immaturity. Another possibility is that these foals had a diet lower in protein due to poorer milk quality from their mothers. However, this seems unlikely since there were no differences in colostrum quality, and the samples were taken within the first 24 h of life, which is not enough time for metabolic alterations attributable to diet to develop. In human neonates, changes in urea concentrations are due to factors other than dietary and renal ones (36). Interestingly, in human medicine, it has also been observed that premature neonates with functional immaturity are unable to increase plasma urea concentrations despite the administration of protein-rich diets, assuming that this is due to hepatic functional immaturity (37). This fact could explain why foals suffering from the disease had lower urea concentrations than healthy ones and could also be related to the immaturity of other systems, in this case, the respiratory immune system.

In this study, serum creatinine was higher in foals that developed R. equi. However, the mean value observed in those animals was within the range considered physiological in the neonatal foal (1.5–2.9 mg/dL) (38). Foals present a higher upper limit value than adult horses (39). Similarly, the serum concentration of gamma GT was higher in the sick group but also within the reference range. In neonatal foals, the reference ranges for hematological and biochemical parameters are highly variable and difficult to interpret. As both hypercreatinemia and pathological increased gamma GT levels have been linked to placental insufficiency, fetal stress, and immaturity (40, 41), is it likely that a certain level of immaturity might be present in foals that developed pneumonia in this study. Additionally, the importance of the adaptive immune system in human neonates has been highlighted in the development of respiratory diseases (42, 43). The association of gamma GT as a risk variable for R. equi pneumonia in this study could be considered a potential marker useful for identifying foals at risk, allowing for the necessary preventive measures to be taken to avoid the development of the infection.

Virulent R. equi replicates within macrophages, beginning its replication within 6–12 h, affecting macrophage viability within 48 h (3). Although macrophages do not prevent intracellular bacterial replication, R. equi infection induces an immune response in macrophages characterized by cytokine secretion (44). Studies in rats, pigs, and primates have shown that alveolar macrophages from young animals are less efficient, both in phagocytosis and in their ability to kill bacteria, particularly in the first weeks of life (45–47). Considering these findings and the research in horses, alveolar macrophages in newborn foals would be less bactericidal than in older foals (2). In our research, sick foals showed higher creatinine and gamma GT levels, indicating a certain degree of immaturity, which may induce a greater disease susceptibility. Indeed, several cases of pneumonia and other infections caused by R. equi have been detected in people (48–50), and this pathogen is always associated with states of immunosuppression (51–53).

Although in previous studies, WBC count and fibrinogen concentration have proven to be influential parameters in identifying foals affected with R. equi, no significant differences between groups were observed in this study evaluating foals less than 24 h of life. Low leukocyte and segmented neutrophil counts have been considered an early indicator of this disease when blood samples were obtained from foals aged 1 and 4 weeks (8), as well as a value of more than 13.000 leukocytes/μL in foals aged 3–5 weeks of age showed a sensitivity and specificity of 95.2 and 61.2%, respectively, to detect foals infected with R. equi pneumonia (33). Similarly, foals aged 3 to 19 weeks have also been analyzed hematologically with an interval of 4 weeks, where a total leukocyte count ≥13,000/μL and neutrophil count ≥10,000/μL were found with a sensitivity of 59 and 50% and a specificity of 37 and 55%, respectively (34).

All the studies mentioned in the previous paragraph were carried out in older foals and the samples were measured several times. This fact contrasts with our study, where blood samples were analyzed once within the first 24 h of life, which probably represents a more immature immune system. It has been observed that neutrophils have a reduced phagocytic capacity during the first 3 weeks and a decreased bactericidal capacity during the first 3 months of life (39). Both chemotaxis and neutrophil phagocytosis are low at birth, increasing significantly after colostrum ingestion (54). This suggests that newborn foal phagocytic cells are functionally mature but with limited chemotactic and phagocytic functions. According to this, age could be an important factor in the variability of the leukocyte count, especially the neutrophil count, so these values could not be considered reliable indicators to predict R. equi pneumonia within the first hours of life. Evidence suggests that foal neutrophils undergo numerical expansion and functional maturation in response to microbial exposure and other environmental stimuli (19, 55, 56), a phenomenon similarly observed in other species. The absence of significant differences in neutrophil counts may reflect insufficient postnatal time for activation or adaptation to environmental cues, as samples were obtained within the first 24 h of life. Furthermore, it seems evident that respiratory tract macrophages are involved in the initiation and early stages of this disease’s development, while neutrophils would be involved at a later stage, in the development of pulmonary abscesses (24, 57).

Similar to WBC count, serum fibrinogen concentration at birth was similar between healthy foals and those who became ill. While fibrinogen concentrations of 400 mg/dL are often considered the physiological upper limit in adult horses, it appears that in foals, the physiological values of fibrinogen vary most dramatically over the first weeks of life (27). Like our findings, previous studies have shown no differences in this parameter between foals aged 1 and 4 weeks (8). Another study observed an increase in plasma fibrinogen concentrations early in the development of R. equi pneumonia. Additionally, in foals older than 3 weeks of life, the sensitivity and specificity of fibrinogen concentration (≥600 mg/dL) as a predictive value were 59 and 33%, respectively (32), which means relatively low-reliability values. However, a more recent study analyzed weekly blood concentrations of fibrinogen in foals from the first 12 h of life to the seventh week, where it did observe statistically significant differences between concentrations of healthy foals versus preclinically/subclinically infected foals, suggesting fibrinogen is most likely to have a predictive value between the first and fourth week of infection, regardless of age (27). This background could explain our results, since considering that the incubation period of R. equi is 3 days to 4 weeks, depending on the dose of infection, and that the response time of equine fibrinogen occurs on average within 24–72 h, it is very unlikely that significant differences will be observed between the two groups studied within the first 24 h of life.

Serum concentration of total plasma proteins and albumin did not differ between the two groups studied, which was expected, since, to the author’s knowledge, they have not been directly related to the development of pneumonia by R. equi. However, we included these parameters as a measurement of total proteins and albumin are routinely assessed in the evaluation of neonatal foals to assess the degree of dehydration and/or failure of passive transfer of immunity, together with other markers such as urine density and IgGs. Additionally, these parameters were taken into account along with the parameters of the physical examination to determine if the foals were healthy at birth.

In this research, iron serum concentration did not change between healthy and sick foals, and values remained within the reference ranges (262–488 μg/dL) (57). This parameter was included in the study as iron is used by R. equi for the intracellular multiplication (6), which occurs around the first 6–12 h in culture, reaching a fivefold increase of bacteria after 48 h (3, 58). Accordingly, several studies propose the occurrence of an early infection of this pathogen after birth (7, 9). Additionally, in sick neonatal foals, plasma iron concentration does not seem to be an early indicator of systemic inflammation (57) unlike in adult horses, in which a decrease in serum iron levels is shown in acute inflammation (59). Based on this, we suggest monitoring iron concentrations to assess their relationship with the onset of clinical signs.

It has been hypothesized that failure of passive transfer immunity would increase the risks of infections and that foals with fewer colostrum antibodies are more susceptible to R. equi infection (60). Nevertheless, this study shows no difference in colostrum Brix values that suckled foals, nor in serum IgG concentration between healthy foals and those with pneumonia. These results are consistent with previous studies (16, 38). All these results indicate that the protection provided by colostrum and IgG levels are not strongly associated with the development of R. equi, a disease that is likely induced by a multifactorial cause (61). Indeed, a limitation of this study was that we only recruited one blood sample per foal, therefore, there was not a follow-up from birth to the onset of clinical signs of pneumonia. However, we aimed to identify changes at birth that may predict future development of the disease, as most studies that look for early indicators of R. equi pneumonia are carried out in foals older than 1 week. Additionally, although the sample size was high (207 foals), the number of sick foals was low (25 foals) and may limit the results obtained. In addition, due to the retrospective nature of this study, the sample size varied for each parameter analyzed. This was mainly caused by missing data and inconsistencies in the parameters originally recorded. Such heterogeneity in sample sizes may affect the statistical power of certain analyses, particularly those with fewer measurements, such as the fibrinogen.

To the author’s knowledge, this work is the first to assess predictive markers of R. equi pneumonia in neonatal foals in less than 24 h. Our results show differences in urea, serum concentrations of creatinine, and gamma GT between foals that became ill and those that remained healthy, and remarkably, these values remained in range. Since these findings could be related to the immaturity of macrophages and a more diminished innate immune response, it would be interesting to conduct studies that sought to relate neonatal immaturity to the risk of suffering pneumonia by R. equi in the future. In addition, findings in this report may indicate that special attention should be paid to neonatal foals presenting changes in these three parameters, mainly in endemic farms for R. equi. Interestingly, neonatal gamma GT may serve as a possible risk factor for developing the infection.