To the best of our knowledge, the present study is the first attempt to establish RIs for hematologic analytes in water buffalo (Bubalus bubalis) in South America and in accordance with the recently published ASVCP QALS committee guidelines for the determination of RIs in veterinary species (Arnold et al. 2019) comparing the different ranges used in cattle.
The data gave us a unique opportunity to compare hemogram results for healthy animals. The variations between the datasets were matched for sex, age, and natural infection with Trypanosoma spp. For the RI analysis, animals with Trypanosoma spp. positivity by blood smears were excluded. The stages of lactation or gestation could not be compared, we did not included animals with gestation status, which could affect, leukocyte counts except for a few weeks peripartum, although without influencing hemoglobin concentration (Sanci et al. 2017; Patel et al. 2016; Jain et al. 1982).
Most textbooks and previous studies reporting RIs in water buffaloes in South America do not specify in a representative sample according, the biological, clinical, or geographic characteristics for the establishment of RIs. In addition, a limitation of some studies is that they only provide a statistical description of data without a statistical representative sample of a country that cannot represent RIs for the population.
Previous investigations have measured hematologic values for water buffalo in various countries in the Eastern Hemisphere, including Australia, Egypt, India, and Italy (Abd Ellah et al. 2014; Torres-Chable et al. 2017). Most hematologic measurements vary between countries.
All hematologic means inferred from Colombian data differ significantly from the corresponding means reported from other countries (Londoño R, Sánchez M, y Prada Sanmiguel 2012). One reason for these changes could be geographic location. Previous studies have shown that hematological variables are influenced by geography (11). Alternatively, the differences could be attributed to the age and sex of the animals as a factor of influence for hematological analytes (29).
Additionally, the different reference populations did not necessarily contain animals of the same breed or lactation status. Previous studies have demonstrated that hematologic measurements can vary according to the lactation (Dhillod et al. 2017) and reproductive status (29) of the species under investigation. Other factors that could account for the observed differences are different management practices, the undefined health and nutritional status of the studied animals, temporal variations, differences in the analytical methods used to collect blood, and differences in the analyzers used to measure hematological variables. Reticulocytes were not detected in any water buffaloes analyzed in this study in other studies in bovines. Fibrinogen showed lower values compared with previous studies (32).
The need for buffalo-specific RIs is increasingly being discussed in veterinary medicine. In several countries, cattle values are normally used to analyze the physiological and health statuses of water buffaloes, but in this study, we found several differences according to the species; for example, RDWc was found to have high ranges, indicating the relationship with a larger amount of red blood cells, as in other studies of buffaloes (Hussain et al. 2018). Additionally, a higher concentration of WBC counts was found to be associated with a high presence of neutrophils in the studied population. All these values were compared with the parameters given by the analyzer used for bovines and cattle.
Males showed higher RBC, WBC, lymphocyte, and thrombocyte values, and females showed higher MCV and neutrophil parameter values. Several differences were found in the age groups: calves showed more hemoconcentration, with high ranges of PCV, MCV and RBC counts, compared with young animals and adults. White cell lines for calves showed more concentration derivates of a high range of lymphocytes, and thrombocyte ranges were higher than in the other age groups. This can be explained by the fact that hematopoietic activity is higher in young animals (Euler et al. 2013) and by the loss of hormonal production in older animals (Bórnez, Linares, y Vergara 2009).
Young animals showed less total protein, fewer neutrophils and thrombocytes, and higher WBC and lymphocyte counts than the adult population, suggesting that exposure to several microorganisms helps produce more lymphocytes in the WBC count, improves resistance in an infective environment, and produces antibodies (32). The results are consistent with other research on water buffaloes, where the values are related and show the depletion of the cell population when the animals get older (Brito et al. 2021).
Trypanosomiasis is caused by the genus Trypanosoma spp. and is a vector-borne disease that commonly affects water buffalo and other bovines in tropical and subtropical regions. Although buffaloes that displayed signs of illness or had evidence of parasitic infections were omitted from our study, Trypanosoma spp. infections are often asymptomatic, and parasitemia cannot always be detected by microscopic blood smear examination (16). Therefore, a subset of animals in our sample population had Trypanosoma spp. infections. Differences in Trypanosoma spp. status could be another reason why the hematologic values reported in our study differed from those in other studies. This could also explain the observed differences between the farms used in our study.
The amounts of neutrophils in the blood were found to decrease with the presence of infection by the agent, according to studies on Trypanosoma spp. Neutrophils are rapidly recruited at the site of the bite by the vector, and cytokine expression produces inflammatory diseases (il1b, il6, il10) and neutrophil chemokines (cxcl1, cxcl5) that are subsequently transiently upregulated at the site of inoculation of the parasite. However, evidence shows that a second influx of neutrophils occurs that coincides with the retention and expansion of the parasite, which contradicts evidence that neutrophils do not significantly contribute to the control of dermal parasites and instead cause higher levels of systemic parasitemia during the onset of infection (Caljon et al. 2018).
Regarding eosinophils, parasites are known to increase the level of eosinophils in the blood in acute parasitemia (Staumont-Sallé, Capron, y Delaporte 2017). In the case of our study, it was found that all infected animals presented a decrease in the presence of said cell, an opposite event to that found in the article, where it is mentioned that in acute processes, a high content of these cells in blood is presented (33). This is possibly due to the state of the disease. As the infection is subclinical, it possibly generates another cellular displacement. It may also be an indication of chronicity, associated in turn with an increased presence of lymphocytes, which is associated with chronicity in the case of Trypanosoma cruzi (Gonzalez, Cuéllar, y J. Puerta 2018).
The concentration of cells of the red line showed a high decrease in blood vessels, a process associated with parasitemia in animals, and this association has been found in all cases of Trypanosoma infection in human and animal species (9,16,33,40).
Acute, chronic, and recovery phases of anemia have been described in Trypanosoma spp. infections(Shiflett et al. 2007). In the acute phase, death is caused by marked pancytopenia and normochromic macrocytic anemia, and in the chronic phase, anemia is usually normochromic normocytic with little fluctuation of the parasite in the blood. In the recovery phase, erythrocyte levels begin to increase, and macrocytosis is detected (Mbaya, Kumshe, y Okwudiri 2012). In this study, there was no increase in cell size in the infected animals, so it fits the hypothesis of the presence of a chronic infection during sampling. This leads us to believe that the initial infection generally occurs in young animals, and those calves that do not die are usually carriers of the parasite.
Due to mechanical damage to erythrocytes, Trypanosoma spp. causes irreversible recovery anemias to the red cell, its flagellum, and alters and damages the membrane, allowing the exit of its components and the loss of function of hemoglobin, as observed in its interaction with cells in experimental infection (Biéler et al. 2012).
Trypanosoma spp. infection usually causes microangiopathy, known as platelet aggregation, due to the presence of intact Trypanosoma fragments or parasites in the blood vessel. This can cause the release of platelet autoantibodies that, in turn, promote the release of procoagulants, causing fibrin and micro clot deposits (41). This alteration may be the cause of the clinical presentation with edema, hypoproteinemia, and prostration, with generalized coagulopathy mentioned by some authors (9,16,33).
In summary, we measured hematologic variables in healthy water buffaloes from several farms according to age, sex, and natural infection for this haemotropical parasite and provided RIs and various other statistical data for each hematologic variable. The information obtained in this study will be particularly helpful to veterinarians and other individuals involved in buffalo breeding.