Evaluation of haematological parameters in haemolytic anaemia caused by tick‐borne pathogens in grazing cattle

Abstract Background No tick‐borne pathogens (TBPs) causing haemolytic anaemia in cattle have been reported, except Theileria orientalis and complete blood count (CBC) profile is the only haematological parameter to determine the severity of regenerative haemolytic anaemia. Objectives To identify the causative agents of TBP‐induced haemolytic anaemia and determine haematological parameters that indicate haemolytic anaemia in grazing cattle. Methods Eighty‐two Korean indigenous cattle (Hanwoo) were divided into two groups: grazing (n = 67) and indoor (n = 15) groups. CBC and serum biochemistry were performed. PCR was conducted using whole blood‐extracted DNA to investigate the prevalence of TBPs. Results TBP‐induced haemolytic anaemia was observed in the grazing group. In grazing cattle, co‐infection (43.3%, 29/67) was most frequently detected, followed by T. orientalis (37.6%, 25/67) and Anaplasma phagocytophilum infections (1.5%, 1/67). In indoor cattle, only co‐infection (20%, 3/15) was identified. Grazing cattle exhibited regenerative haemolytic anaemia with marked monocytosis, mild neutropenia, and thrombocytopenia. According to grazing frequency, the 1st‐time grazing group had more severe anaemia than the 2nd‐time grazing group. Elevations in indirect bilirubin and L‐lactate due to haemolytic anaemia were identified, and correlations with the respective markers were determined in co‐infected grazing cattle. Conclusions Quantitative evaluation of haematocrit, mean corpuscular volume, and reticulocytes (markers of regenerative haemolytic anaemia in cattle) was performed for the first time. Our results show that, in addition to T. orientalis, A. phagocytophilum is strongly associated with anaemia. The correlation between haemolytic anaemia severity and haematological parameters (indirect bilirubin, reticulocytes, and L‐lactate) was confirmed.

anaplasmosis is caused by A. marginale, A. centrale, A. phagocytophilum, and A. bovis, causing extravascular haemolytic anaemia (Kocan et al., 2000;Kuttler, 1984).Theileria parasites are divided into two species based on their ability to transform host cells.Theileria annulata and T. parva are distributed in East and Central Africa and considered the most pathogenic species in cattle.However, other Theileria species, such as T. mutans, T. velifera and T. orientalis, that do not transform host cells, usually cause a less severe disease in cattle (Sivakumar et al., 2014).Bovine theileriosis in the Republic of Korea (ROK) is mainly associated with T. orientalis infection, and its prevalence is increasing (Alkathiri et al., 2023;Park et al., 2017).
Anaemia in cattle can be regenerative or non-regenerative and regenerative anaemia is further divided into haemorrhagic and haemolytic anaemia (Jones & Allison, 2007;Schalm et al., 2010).Bovine regenerative anaemia can be diagnosed by increased polychromatinisation of red blood cells (RBCs) on a blood smear or by identification of basophilic stippling.The most accurate way to diagnose regenerative anaemia in cattle is through reticulocytosis.Haemolytic anaemia in cattle may be caused by protozoan infections (Theileria/Babesia spp.), bacterial infections (Anaplasma spp., Mycoplasma spp., Clostridium spp., and Leptospira spp.), toxicosis (copper, onion, Brassica spp., and red maple leaves) or immune-mediated haemolytic anaemia (Chase et al., 2017;Jones & Allison, 2007;Schalm et al., 2010).
Oxygen delivery is a product of cardiac output and arteriovenous oxygen content.When anaemia occurs, haemoglobin (Hb) decreases, causing hypoxia in peripheral tissues and a compensatory increase in cardiac output (Anand & Florea, 2001;Metivier et al., 2000).Anaemia should be treated carefully because hypoxia occurs in peripheral tissues and the heart is overloaded.Anaemia -induced hypoxia of peripheral tissues can lead to an increase in organic acids, such as lactate, resulting in metabolic acidosis (Constable et al., 2016;Lee et al., 2015).The severity of metabolic acidosis; an increase in the anion gap; and most importantly, an increase in lactate may be used to assess the severity of anaemia (Constable et al., 2016;Hardy, 2004;Magdesian et al., 2006).
In haemolytic anaemia, clinicopathological changes accompany RBC destruction.During intravascular haemolysis, RBCs within blood vessels are destroyed, and Hb and lactate dehydrogenase (LDH) are released into the blood.Haptoglobin (Hp) binds to Hb, and unbound Hb is transported to the kidneys where it is broken down, causing indirect bilirubinemia (Russell & Roussel, 2007;Smith et al., 2020;Stockham & Scott, 2008a).Destruction of RBCs by macrophages in the spleen, an increase in indirect bilirubin (a metabolite of Hb) and splenomegaly have been reported in extravasation haemolysis (Smith et al., 2020;Stockham & Scott, 2008a;Zachary & McGavin, 2017).
Only few studies showing the presence or increase in reticulocytes as evidence of regenerative anaemia have been presented, with no quantitative count of reticulocytes.Moreover, no studies have correlated an increase in indirect bilirubin with the severity of haemolytic anaemia in cattle.Therefore, this study aimed to identify the causative agents of tick-borne haemolytic anaemia in grazing cattle and determine the severity of anaplasmosis and theileriosis, which are considered benign in the ROK.In addition, we examined the changes in complete blood count (CBC), reticulocytes, indirect bilirubin and L-lactate, which are involved in regenerative anaemia, and their correlation with anaemia severity in cattle with tick-borne haemolytic anaemia.All samples were placed on ice and immediately transported to the laboratory.On arrival, L-lactate was measured using a portable lactate meter (Statstrip Xpress lactate meter; Nova Biomedical, Waltham, MA, USA), and the serum was separated by centrifugation at 3000 g for 10 min.

Animal and sampling
Cattle in the indoor group were usually pastured in the past; however, but these animals were housed in a barn for the past year.The grazing and indoor cattle were used as the experimental and control groups, respectively.

CBC and serum biochemical profiles
CBC was performed using an automated haemocytometer, IDEXX Pro-

DNA extraction, PCR and sequencing analysis
Genomic DNA was extracted from each blood sample (200 µL) using the DNeasy Blood Kit (Qiagen, Valencia, California, USA) according to the manufacturer's instructions and stored at −80

Statistical analysis
Statistical analyses were performed using SPSS 29.0 software package (SPSS, Chicago, Illinois, USA).The results of blood tests from indoor and grazing cattle were analysed using two-tailed independent t-test and the Mann−Whitney U test according to results of the normality test, and data sorted by infection status of TBPs were analysed using one-way ANOVA and Kruskal-Wallis test.Data are presented as mean ± standard deviation, and statistical significance was set at p < 0.05.

Comparison of CBC and serum biochemistry results of indoor and grazing cattle
Among the RBC profiles, RBC (p = 0.000), HCT (p = 0.000), Hb (p = 0.000), and MCHC (p = 0.020) values were significantly lower in grazing cattle than in indoor cattle (Table 1).Platelet counts were also significantly lower in grazing cattle (p = 0.002) than in indoor cattle (Table 1).MCV and reticulocyte levels in grazing cattle greatly increased compared to than in indoor cattle; however, the differences were not statistically significant (Table 1).
Although the total bilirubin values in grazing cattle increased threefold compared to those in indoor cattle, the difference was not statistically significant.L-Lactate levels were significantly higher in grazing cattle than in indoor cattle (Table 1).

Comparison of CBC and serum biochemistry results between indoor and grazing cattle according to tick-borne infection
Haematological analyses of indoor and grazing cattle according to tickborne infection were compared.Grazing cattle with A. phagocytophilum infection showed the most significant differences in haematological parameters (Table 3).In indoor cattle, no significant differences were observed in the haematological results between the 'no infection' and 'co-infection' groups (Table 3).In contrast, by Scheff's post-hoc test, in grazing cattle, RBC and HCT values significantly decreased com-pared to those in the 'no infection' , T. orientalis-infected and co-infection groups.Although no statistically significant differences were observed in the MCV and reticulocyte values, both increased in grazing cattle with co-infection.Moreover, significant neutropenia was observed in grazing cattle with co-infection compared to that in the 'no infection' and T. orientalis-infected groups (Table 3).Monocytes greatly increased in this group compared to that in the non-infected group; however, the difference was not statistically significant.In addition, grazing cattle showed no significant differences in other parameters.

Differences in TBP infections according to grazing frequency
We compared the prevalence of TBPs according to grazing frequency in grazing cattle.The overall prevalence of TBPs was 80.8% (95% CI: 70.1−91.5)in 1st-time grazing cattle and 86.7% (95% CI: 69.5−100) in 2nd-time grazing cattle (Table 4).Regarding infection rates, coinfections were most frequently detected in 1st-time grazing cattle, followed by T. orientalis and A. phagocytophilum infections.Whereas in 2nd-time grazing cattle, T. orientalis infection was the most common, followed by co-infection (Table 4).Interestingly, A. phagocytophilum infection markedly reduced in 2nd-time grazing cattle compared to that in 1st-time grazing cattle (Table 4).

Correlation analysis between non-infected and co-infected 1st-time grazing cattle
Correlation analysis was performed for four parameters: HCT, reticulocytes, indirect bilirubin, and L-lactate.These showed the greatest changes between non-infected and co-infected 1st-time grazing cattle.
Compared to non-infected cattle, a negative correlation was observed between HCT and reticulocytes (r = −0.657;p < 0.05) and between HCT and L-lactate (r = −0.574;p < 0.05) in grazing cattle with co-infection.Furthermore, in these cattle, a strong negative correlation (r = −0.839;p < 0.05) was found between HCT and indirect bilirubin (Figure 3).

DISCUSSION
TBPs have been reported to cause serious health problems in ruminants, leading to substantial economic losses to the livestock industry worldwide.Among these, T. orientalis and Anaplasma spp.are the most F I G U R E 2 HCT, reticulocyte, indirect bilirubin and L-lactate levels (mean ± SD) according to infection status of TBPs between 1st-and 2nd-time grazing cattle.*p < 0.05, **p < 0.01.
orientalis rather than a single infection.According to our results, T. orientalis was mainly found in grazing cattle but not in indoor cattle.This can be explained by the limited number of indoor cattle.Furthermore, coinfection with T. orientalis and A. phagocytophilum was more frequent in grazing cattle than in indoor cattle.Currently, the transmission route of these pathogens cannot be ascertained in indoor cattle because these cattle were housed in a barn.Our results suggest that tick exposure cannot be completely ruled out in indoor cattle.
T. orientalis has been reported to cause anaemia in cattle (Choi et al., 2016).However, in this study, cattle infected with T. orientalis alone did not have severe anaemia but mild anaemia (Choi et al., 2016;Kim et al., 2017;Park et al., 2019).This result is somewhat different from previous studies reported in other countries (Forshaw et al., 2020;Lawrence et al., 2018;McFadden et al., 2017).Based on the sequence analysis of the major piroplasm surface protein (MPSP) gene, T. orientalis is divided into at least 11 different genotypes globally (Sivakumar et al., 2014).
Of the 11 genotypes, type 2 (Ikeda) is known to be associated with severe disease (Kamau et al., 2011).Since sequencing analysis was not performed for MPSP-positive samples, the exact genotypes were not determined.This may explain the different results between studies.

F I G U R E 3
Correlation analysis of HCT, reticulocyte, indirect bilirubin, and L-lactate between non-infected and co-infected in 1st-time grazing cattle.
Anaemia was more severe in grazing cattle co-infected with T. orientalis and A. phagocytophilum than those infected with T. orientalis alone.
Although we cannot draw an exact conclusion because only one animal was infected with A. phagocytophilum, this animal exhibited severe anaemia.These results indicated that A. phagocytophilum infection is strongly associated with anaemia.To the best of our knowledge, this is the first study to report anaemia caused by A. phagocytophilum infection in cattle.Although the incidence of A. phagocytophilum infection is increasing in the ROK, its pathogenicity remains unclear.Thus, further studies are required to determine the association between anaemia and A. phagocytophilum.
Anaemia is generally classified based on MCV and MCHC values (Jain et al., 1990;Jones & Allison, 2007;Schalm et al., 2010;Stockham & Scott, 2008b).In grazing cattle with co-infection, the MCV value increased; however, MCHC values were not significantly different from that in the 'no infection' group.In this study, most cattle with anaemia had macrocytic normochromic anaemia, whereas grazing cattle with co-infection had macrocytic hypochromic anaemia.The more severe the haemolytic anaemia, the higher the number of immature RBCs.This is consistent with the increase in reticulocytes observed in this study, although there was no significant difference between the 'no infection' and co-infection groups.In cattle, regenerative anaemia can be confirmed by the presence of polychromasia or reticulocytes on a blood smear as well as basophilic stippling or Howell-Jolly bodies (Jain et al., 1990;Jones & Allison, 2007;Schalm et al., 2010;Stockham & Scott, 2008b).In particular, reticulocytosis is considered a sensitive indicator of regenerative anaemia in cattle.Several studies have reported that during regenerative anaemia, reticulocytes are known to increase six-to eight-fold in dogs and three-to five-fold in cats; however, the increase in reticulocytes is not high in cattle (Stockham & Scott, 2008b).Nevertheless, in this study, the number of reticulocytes greatly increased in cattle with co-infection, compared to those with only T. orientalis infection.Our findings suggest that in grazing cattle with co-infection, the relative increase in reticulocytes may be caused by a decrease in normal RBCs.Therefore, the corrected reticulocyte percentage (CRP) is required to determine whether the increase in reticulocytes is due to regenerative anaemia (Schalm et al., 2010;Stockham & Scott, 2008b).The mean CRP (11.8 ± 3.37 × 10 3 cells/µL) in the six grazing cattle with co-infection showing significantly increased reticulocyte count was very high compared to that of the indoor group (0.9 ± 0.3 × 10 3 cells/µL).Consequently, it can be assumed that the calculated CRP levels increased due to regenerative anaemia.Therefore, reticulocyte production index in cattle should be investigated in future studies.
Once haemolysis occurs, RBCs are lysed, and Hb and LDH are released into the blood, resulting in elevated serum LDH and haemoglobinemia (Schalm et al., 2010;Stockham & Scott, 2008a;Stockham & Scott, 2008b;Zachary & McGavin, 2017).Owing to the cytotoxic nature of free Hb, it binds to Hp and travels to the spleen, where it is phagocytosed by macrophages (Schaer et al., 2014).Thus, a decrease in Hp is observed.However, in this study, Hp slightly increased in grazing cattle with co-infection compared to that in cattle with no infection, and the concentration was similar to that of indoor cattle.As a result, the amount of Hp in these cattle was not sufficient to bind to Hb, and the remaining (free) Hb was transported to the kidneys, where it was broken down.In degraded Hb, haeme is converted to bilirubin, which is bound to albumin and transported to the liver (Schalm et al., 2010;Stockham & Scott, 2008a;Stockham & Scott, 2008b;Zachary & McGavin, 2017).This leads to an increase in indirect bilirubin levels, which is consistent with the current results.
In this study, mild neutropenia and monocytosis were observed in grazing cattle with co-infection.A. phagocytophilum infects granulocytes, particularly neutrophils (Atif, 2015), and the infection is generally characterised by leukopenia and thrombocytopenia.Grazing cattle with co-infection exhibited significant neutropenia and leukopenia compared to those infected with only T. orientalis.In particular, the band form of neutrophils was observed in cattle with anaemia, supporting the idea that neutrophil destruction occurs without reducing the pool of stored neutrophils.As neutrophils and monocytes share the same precursor cells, monocytes tend to increase as neutrophils decrease (Schalm et al., 2010;Stockham & Scott, 2008b).Moreover, thrombocytopenia occurred more frequently in grazing cattle with coinfection than in grazing cattle with only T. orientalis infection.This can be explained by the presence of A. phagocytophilum.
We found that the prevalence of TBPs in 1st-and 2nd-time grazing cattle differed according to grazing frequency.Interestingly, the infection rate of A. phagocytophilum markedly reduced in 2nd-time grazing cattle compared to that in 1st-time grazing cattle.These results show that grazing frequency affects A. phagocytophilum infection.When put out to pasture for the first time, cattle appear to be highly susceptible to A. phagocytophilum.This may be due to the antibodies produced after A. phagocytophilum infection during 1st-time grazing, which then act during 2nd-time grazing.In contrast, a previous study showed that antibodies against T. orientalis can persist for 3−5 years without reinfection in cattle that have recovered from infection (Delves & Roitt, 1998).
Moreover, haemolytic anaemia was more severe in the 1st-time grazing group than in the 2nd-time grazing group.Contrary to our expectations, our findings demonstrated that severe anaemia was caused by co-infection with T. orientalis and A. phagocytophilum compared to that of single infection with T. orientalis, indicating that A. phagocytophilum infection is significantly associated with severe anaemia.These results highlight the importance of A. phagocytophilum in grazing cattle.
More severe haemolytic anaemia may require blood transfusion, and the primary measure used in clinical practice is the HCT, with an HCT of 14% or less recognised as a panic value (Hardy, 2004;Metivier et al., 2000).It has also been reported that a venous blood oxygen tension of 25 mmHg or less, a lactate concentration of 4 mmol/L or more, and the degree of metabolic acidosis can be used as cofactors for transfusion requirements in large animals (Hardy, 2004).Because anaemia-induced metabolic acidosis is caused by accumulation of lactate in response to tissue hypoxia, we measured whole blood L-lactate rather than venous blood pH or anion gap in this study.However, in our study, L-lactate levels were higher in the anaemic population than in the control group.More importantly, we found a negative correlation between L-lactate and HCT levels (r = 0.574, p < 0.05).L-lactate test may be an adequate adjunct test to assess anaemia severity.Further research is needed to identify L-lactate as a panic value predictor of cattle that require blood transfusion.
This study was approved by the Institutional Animal Care and Use Committee of the National Institute of Animal Science, ROK (JBNU IACUC No. NON2023-123).All experimental procedures involving animals were conducted in strict accordance with relevant guidelines and regulations.Blood was collected from 82 Korean indigenous cattle [grazing (n = 67) and indoor (n = 15)], which were raised on one farm in the ROK (Figure1).Pregnant Korean beef cows aged 22−48 months were selected.The grazing group was fed forages of orchard grass and tall fescue, and the indoor group was fed concentrate and roughage with a dry matter intake of 5.5 kg/day (10% crude protein).Blood samples (10 mL) were taken from the jugular vein and divided into F I G U R E 1 Map showing the region where blood samples were collected in the Republic of Korea.EDTA-supplemented tubes (BD Vacutainer ® , Franklin Lakes, NJ, USA) and serum separation tubes (Vacutte serum tubes ® , Greiner Bio-One, Austria).

TA B L E 3
Comparison of complete blood counts and serum biochemistry profiles between indoor and grazing cattle according to TBPs infection.

Table 2
Prevalence of TBPs detected in Korean native cattle according to grazing type.
In addition, single infections by T. orientalis or A. phagocytophilum were not detected in indoor cattle, whereas each single infection of T. orientalis and A. phagocytophilum was found in 25 (37.6%, 25/67) and one (1.5%,1/67)grazing cattle, respectively.Overall, marked differences in the infection rate of TBPs were observed between indoor and grazing cattle.TA B L E 2 Difference in TBP occurrence according to the grazing frequency in grazing cattle.