Risk factors and haemato-biochemical parameters associated with Theileria equi infection in equids in Central Italy

doi:10.21203/rs.3.rs-1606077/v2

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Risk factors and haemato-biochemical parameters associated with Theileria equi infection in equids in Central Italy

https://doi.org/10.21203/rs.3.rs-1606077/v2

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Background: Equine piroplasmosis is a tick-borne disease affecting equids caused by protozoa belonging to two genus named Theileria and Babesia, causing losses in the horse industry and constraint to the international movement. In endemic areas, clinical diagnosis is difficult because signs are not pathognomonic. Identifying the associated risks factors and the most suitable laboratory analyses are required to prevent the disease, confirm the diagnosis and assess therapy.

Methods: A total of 438 horse and donkey samples were collected by practitioners during their clinical activities. The anamnestic information were collected by a standardized form. The samples were screened for piroplasmosis and other infectious diseases in differential diagnosis and haemato-biochemical parameters, and thus assigned to three groups: confirmed cases of T. equi, unconfirmed suspects and controls. Statistical analysis was performed to detect differences among groups regarding anamnestic data and haemato-biochemical parameters.

Results: Among anamnestic variables, only age (> 6 years) increases the probability of infection compared to the controls, while ectoparasitic treatments seem to have a protective effect. A decrease in platelet count was the only haemato-biochemical parameter associated with T. equi infection.

Conclusions: This study confirms the diagnosis of T. equi infection as an issue, for similarities regarding anamnesis and laboratory findings with other diseases. Moreover, it focuses the attention on prevention measures and good management practices that can reduce the spread of the disease, together with regular laboratory checks, useful to early detect the parasite and assess and monitor the therapy.

Theileria equi

Babesia caballi

equine

Italy

risk factors

haemato-biochemical parameters

Equine piroplasmosis (EP) is a tick-borne disease of equids caused protozoan parasites belonging to two genus named Theileria and Babesia. EP is responsible for significant sanitary and economical losses in the horse industry due to impaired performance and also to the constraint to the international movement [1, 2]. EP distribution is correlate to that of its vectors, ticks of genera Dermacentor, Hyalomma, and Rhipicephalus [3], being thus endemic in tropical and temperate areas, including Central-Southern regions of Italy [4].

Several studies reported risk factors and management practices associated with the presence of the parasites, as access to pasture, living on soils favouring the abundance of ticks, lack of hygiene, antiparasitic treatments and vaccination [4, 5, 6, 7]. Adjustment to the rearing condition can mitigate the effects of these factors, reducing the risk to be infected.

The clinical diagnosis of EP in endemic areas presents several issues and therefore can be arduous. Although peracute, acute and chronic forms are reported, the most frequent manifestation of the infection by both parasites is the unapparent carrier status with non-pathognomonic signs of disease [8, 9]. Even in symptomatic cases, the signs do not allow a differential diagnosis between the two parasites which is crucial for the therapy setting, therefore the laboratory diagnosis is necessary to detect haemato-biochemical alterations and/or presence of parasites [5]. Within this clinical framework, the choice of treating a positive subject must be carefully considered, especially for animals having an economic or personal worth. In fact, severe side effects such as hepatotoxicity and nephrotoxicity are reported using imidocarb dipropionate in the T. equi sterilization protocol, that requires a dose of 340 mg/100 kg body weight, four times with an inter-dose interval of 72 h [10 11, 12].

Laboratory diagnostic methods are employed with two main purposes: to detect directly or indirectly the presence of the parasites, and to evaluate the health status of the infected subjects, in order to assess and monitor therapy. In the first group are included stained blood smears, serological tests, such as indirect fluorescent antibody test (IFAT), ELISA, and PCR methods [13], using specific protocols, as described and recommended by World Organization for Animal Health (WOAH)^a. The reliability of these methods and the results' interpretation could be affected or complicated by intrinsic characteristics of the methods, i.e., lack of sensitivity during chronic infections or in the presence of particular parasite strains (ELISA), cross-reactivity (IFAT); or to the host-parasite interaction, i.e., antibody persistence and carrier status with segregation of the parasite in capillaries and bone marrow [9]. The haemato-biochemical investigations are usually represented by complete blood cells count (CBC) and clinical biochemistry. The parameters altered during T. equi infections have been widely studied, both in horses and donkeys, with the aim to identify those parameters with alterations associated with infection to the parasites responsible for EP.

Several studies on horses are available for many region of the world, as Africa, South America, Middle East and Europe [13–20], with very variable results on whose parameters are associated with T. equi infections. However, parameters such as haematocrit, RBC and haemoglobin content are reflective of the red blood cell (RBC) damage while as, those reflecting the liver functionality, includes aspartate amino transferase (AST), creatine phosphokinase (CK) and seroprotein electrophoresis can be useful to evaluate the suitability of a chemoterapy, its efficacy and the restoration of the healthy status. Very few studies instead involve donkeys, available only from Iran, Italy and the Balkan region [21–23], and also with discordant results: for example, [21] reported all the hematological parameters and the biochemical parameters as associated with EP, while [22] only four hematological parameters resulted significant, Only one study involves both horses and donkeys [24] in Egypt, and another study in Italy investigated, beside biochemical parameters, also interleukines in infected horses [25].

The scope of this study was to confirm, in the Italian situation, data available on the practices and the usefulness of the haemato-biochemical parameters in the diagnosis and assessment of therapeutic protocols by the practitioners for the treatment and prognosis of EP. In order to pursue this goal, we used a case control study approach for the a comparison of possible risks factors and haemato-biochemical alterations among animals with acute EP infection in healthy horses (controls) and horses with clinical signs compatible with the presence of EP, but not confirmed as infected.

Characteristic of the study area

The sampling was carried out on the equine population of Lazio and Tuscany regions, Central Italy, between May 2013 and November 2014. Figure 1 reports the localization of the farms involved in the study.

The study area has a cover management factor between 0.01 and 0,07, excluding the urbanized area [26]. The soils of the area of interest are variegated and belong to different soil type as those of the Apennines with temperate climate, while soils of the central and southern Apennines, soils of the hills of Central and Southern Italy contains marine deposits and limestone, soils of the hills of Central and Southern Italy contains volcanic deposits and limestone and soils of the plains and low hills of Central and Southern Italy. For a more in-depth description of the different soils the authors refers to [27].

Regarding meteorological parameters, in Tuscany during the period 2009–2018, the mean minimum temperature ranged from 8.8°C to 9.9°C, while the mean maximum temperature ranged from 16.5°C to 18.8°C. Rainfall ranged from 592 mm to 1154 mm, while evapotranspiration ranged from 804 mm to 992 mm. In Latium, during the same observation period, the mean minimum temperature ranged from 8.7°C to 9.8°C, while the mean maximum temperature ranged from 17.6°C to 20.1°C. Rainfall ranged from 538 mm to 1100 mm, while evapotranspiration ranged from 821 mm to 1023 mm [28].

No ticks abundance studies are available for the area of study, but several maps of presence are available, reporting the presence of the following ticks species: Dermacentor reticulatus, Rhipicephalus sangineus group, Hyalomma marginatum and Ixodes ricinus [29].

Sampling and anamnestic form

Sixty practitioners joined the project and recruited the animals (suspect cases and controls) during their clinical activity. The eligibility criteria for the suspect case for piroplasmosis were initially: temperature > 39°C and at least one sign among anaemia, haematuria, petechial haemorrhages and jaundice, observed during the clinical examination. Controls were selected either in farms with similar characteristics to those where the suspected cases came from, or by the contemporary sampling of two asymptomatic subjects together with the suspect case within the same premise. Since the expected number of cases could not be forecast, a ratio of 10:1 between controls and suspect cases has been pre-set, being this ratio assuring the highest statistical power to the study [30].

Blood samples were obtained by jugular puncture from each animal, collected in nine ml tubes with and without EDTA. After the sampling the tubes were kept refrigerated until delivered to laboratories where sera were separated by centrifugation and frozen until assayed while EDTA tubes were kept refrigerated until tested. Two tubes of each type were requested, as one tube without EDTA was employed for serological analysis while the second for biochemical tests and electrophoresis. Importantly, blood samples collected in EDTA tubes was used for testing the haematological parameters and also for DNA extraction and subsequently for carrying out PCR assays. Contextually to the sampling, an anamnestic data form was administered to the owners of all subjects (suspects and controls), who gave prior consent for the procedure. Information requested by the form are listed in Table 1.

Table 1

Information requested by the anamnestic data collection form administrated to the owners and available choices.
Variable	Available choices
Individual anamnesis
Species	Horse, Mule, Donkey
Sex	Male, Gelding, Female
Age	Year of birth
Fur colour	Light, Dark
Breed	Autochthonous^a, Saddler, Trotter, Galloper, Other
Attitude	Meat production, Equestrian/Horse show, Work, Reproduction
Presence of ticks (at the sampling)	No, Yes
Recent Introduction	No, Yes
Management practices
Housing	Inner, Access to outer
	Single, Group
Vaccination	No, Yes
External parasiticide treatment	No, Yes
Deworming	No, Yes
Clinical signs	Anaemia, Ataxia, Abdominal cramping, Fever, Haematuria, Jaundice, Lack of appetite, Lethargy, Lymphadenomegaly, Oedema, Petechial haemorrhages, Tachycardia, Tachypnea, Weakness, Weight loss
^aItalian autochthonous breeds: Avelignese; Bardigiano; Cavallo Agricolo Italiano; Lipizzano; Maremmano; Murgese; Pony Esperia; Salernitano; Siciliano; Tolfetano

Three levels of potential bias were considered: selection, recall and interviewer bias. Since practitioners were involved in the recruitment of suspect cases, a selection bias could have occurred, being some categories of horses (equestrian and sportive) kept more under veterinary control than those bred for meat production. In order to minimize this bias, all practitioners that had ever submitted an equine sample to our laboratory in the previous years were invited to a presentation of the study, explaining aims and protocols and during which the collaboration in sampling all categories of equids was requested. To reduce the possibility of collecting different anamnestic data between case and controls (recall bias) and minimize interviewer interferences (interviewer bias), the anamnestic form was designed in a structured scheme.

At the end of the sampling, a total of 438 samples were collected, 300 from suspect cases (282 horses and 18 donkeys; 34 males, 139 geldings, 123 females and four missing gender data) and 138 from controls (118 horses and 20 donkeys; 25 males, 44 geldings and 69 females).

Laboratory analyses

A panel of serological, biomolecular and haemato-biochemical tests was performed on all samples (Table 2).

Table 2

Serological and haemato-biochemical analyses performed on the samples collected during the study. Reference intervals were set as reported by Blood et al., 1994 [56], Aguggini et al., 2006 [57] and Riond et al. (2009) [58]. Value within round brackets are the reference values for percentage of WBC components.
Babesia caballi‍ ‍	Babesia caballi Antibody Test Kit, cELISA VMRD®
Babesia caballi‍ ‍	Real time PCR (Bhoora et al. 2010) [31]
Theileria equi	Babesia equi Antibody Test Kit, cElisa VMRD®
Theileria equi	Real time PCR (Kim et al. 2009) [32]
Leptospira spp.	Microscopic Agglutination Test [36]
Leptospira spp.	Real time PCR [37]
Anaplasma phagocitophilum	Indirect Immunofluorescent Test
Anaplasma phagocitophilum	Nested PCR (Massung et al., 1998) [38]
Equine Infectious Anaemia	CTB-ELISA (Nardini et al. 2016) [39]
Complete blood count (10³/µL)	WBC	6.00–10.0	HGB	11.0–15.0
	Neutrophils	2.70–7.60 (47–76%)	HCT	33.0–42.0
	Lymphocytes	1.50–4.50 (25–45%)	MCV	37.0–59.0
	Monocytes	0.00–0.80 (0–8%)	MCH	13.0–20.0
	Eosinophils	0.00–0.40 (0–4%)	MCHC	31.3–39.0
	Basophils	0.00–0.1 (0–1%)	RDW	0.0–27.0
	RBC	6.00–9.90	PLT	100–350
Blood Protein Electrophoresis	Albumin	2.10–3.00 g/dL	Beta 2	0.40–0.70 g/dL
	Alpha 1	0.70–1.20 g/dL	Gamma	1.70–2.50 g/dL
	Beta 1	0.30–0.70 g/dL	A/G ratio	0.93–1.65
Biochemical analyses	Total protein		5.50–7.70 g/dL
	AST		200–400 U/L
	CK		< 500 U/L
	Total bilirubine		0.20–6.00 mg/dL
	Direct bilirubine		< 0.40 mg/dL
WBC: total white blood cells, RBC: total red blood cells count, HGB: haemoglobin level, HCT: hematocrit, MCV: mean corpuscular volume, MCH: mean corpuscular haemoglobin, MCHC: mean corpuscular haemoglobin concentration, RDW: red cell distribution width, PLT: platelets, TP: total protein, AST: aspartate aminotransferase, CK: creatinphosphokinase.

Molecular detection of equine piroplasms in blood

Two in house real-time PCRs were employed on all samples. The one for B. caballi amplifies a 95-base-pair (bp) fragment in the V4 hyper variable region of 18S rRNA gene of B. caballi with primers F: Bc-18SF402; R and Bc-18SR496 reported by [31], used at a concentration of 0.9 µM and TaqMan 5120 MGB TM probe (FAM MGB, Bc-18SP) employed at 0.25 mM. The thermal profile is: 10 min at 95°C for polymerase activation and 45 cycles consisting of 20 s at 95°C and one min at 60°C. Reaction volume contains 20 µl of mix solution and five µl of DNA eluate.

The real-time PCR for T. equi amplifies an 81-bp fragment outside the V4 hyper variable region of 18S rRNA gene. Primers (F: Be18SF; R: Be18SR) at 0.9 µM and TaqMan probe (VIC-TAMRA, Be 18SP) at 0.25 mM were employed as reported by [32]. The thermal profile used was: 10 min at 95°C and 45 cycles constituted by 20 sec at 95°C and one min at 55°C. Reaction volume contains 20 µl of mix solution and five µl of eluate. Together with the primers and probe, the mix solution contains 12.5 µl of TaqMan 2X PCR Master Mix (Applied Biosystems, ThermoFisher Scientific, Waltham, MA USA) and a volume of UltraPure™ DEPC-Treated Water (Applied Biosystems, ThermoFisher Scientific, Waltham, MA USA) to reach the final reaction volume of 20 µl..

Confirmation of specificity of real time PCR by sequencing

The specificity of the rtPCR results was verified by sequencing the amplicons of a random sample of the PCR positive group (19) using a nested PCR protocol as described by [33]. The nested-PCR amplify the hypervariable V4 region of the 18 rRNA gene of both protozoa. The amplicons were sequenced using an automated sequencer (3500 Genetic Analyzer, A. Biosystems, Foster City, CA,USA) and the nucleotide sequences obtained were analysed using the Genetic Analyzer Sequencing v5.4 (A. Biosystems, Foster City,CA, USA). Sequence identity was verified using the Basic LocalAlignment Search Tool (BLAST) and by comparing those obtained with B. caballi and T. equi sequences present in NCBI GenBank(http://www.ncbi.nlm.nih.gov). Sequences presenting an identityand query coverage ≥ 98% were considered as homologous to those deposited in GenBank for the two piroplasms.

Moreover, as [34] reported that samples positive with other PCRs resulted negative in the EMA-1 PCR for T. equi, to verify if this also occurred in the present set of samples, the protocol described by [35] that targets a 268-bp fragment of the T. equi EMA-1 gene was employed on RT-PCR positive samples.

PCR products were visualised in 1.5% agarose gel electrophoresis by staining with GelRed dye (Biotium, Hayward, CA, USA) and once recovered they were purified using the QIAquick® PCR Purification kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions

and sequenced using reagents (BigDye Terminators) and instrumentation (3500 Genetic Analyzer) from Applied Biosystems, (Foster City, CA, USA).

Serological screening for equine piroplasms

Two c-ELISA assays were performed according to the manufacturer's instructions: B. caballi Antibody test kit (VMRD®, Inc, WA, USA) uses a recombinant rhoptry-associated protein 1 (rap-1) as antigen, while B. equi Antibody test kit (VMRD®, Inc, WA, USA) employs a recombinant Equi Merozoite Antigen 1 (ema-1).

Screening for other pathogens

Leptospirosis, anaplasmosis, and equine infectious anaemia are included in the differential laboratory diagnosis in case of jaundice and anaemia. In order to exclude these diseases, the following tests were performed: micro agglutination test and real-time PCR against leptospirosis [36–37]; IFAT (A. phagocytophilum IFA IgG Antibody Kit; Fuller Laboratories®, CA, USA) and nested PCR [38] for anaplasmosis, and ELISA for equine infectious anaemia [39].

Haemato-biochemical analysis

A CBC was performed by the automated counter Cell-Dyn 3700 (Abbott, Lake Forest, Illinois, USA) on EDTA samples. Serum samples were analysed by the automated analyser Olympus AU400 (Olympus, Tokyo, Japan). The electrophoresis of serum protein was performed by the G26 system (Interlab, Rome, Italy). The parameters investigated are listed in Table 2.

Groups definition

The first step of data analysis was a verification on the suspect case forms, excluding those subject not fitting the case definition, as not reporting one of the signs included. After laboratory analysis, signs that affect laboratory parameters, such as anaemia and jaundice, were verified by CBC and total and direct bilirubin values respectively and subjects with not confirmed signs were excluded. Being signs often not confirmed by laboratory results, the actual presence of haematuria and petechial haemorrhages was brought into question and a modified suspect case definition was set as: temperature > 39°C and presence of at least one laboratory confirmed sign among anaemia and jaundice. Considering the long persistence of antibodies against EP in infected animals, in particular in the case of T. equi infection, the serological positivity although in presence of compatible signs was not considered sufficient to define an acute case of piroplasmosis, thus, a suspect case as confirmed only if real-time PCR was positive. Suspect cases resulted positive to biomolecular assay for T. equi were classified as confirmed cases (Group A), while controls resulted negative to biomolecular assay for T. equi were classified as confirmed healthy controls (Group B). Finally, suspect cases resulted negative to PCR were gathered as non-confirmed suspects (Group C).

Statistical analysis

Anamnestic data were considered as exposure variables and were reported as absolute frequencies and percentages (%). Age variable (in years) was categorized in three classes following the quantile distribution: from 0 to 6 years, from 7 to 12, and over 13 years. Univariate logistic regressions were performed using groups (A vs B and A vs C) as outcome and anamnestic data as independent variables. The significant variables in univariate regressions were combined in multivariable logistic analysis as independent variables. ORs with 95% confidence intervals (CI) were estimated.

Haemato-biochemical parameters were reported as medians and interquartile ranges (IQR = 75°-25° percentile). Differences among groups for each parameter were evaluated by Kruskal-Wallis H-test (H), followed, when significant, by Bonferroni correction for multiple comparisons. A P value < 0.05 was considered statistically significant. SPSS v.27 (IBM SPSS Statistics) were used for all statistical analyses.

Laboratory analyses

Molecular detection of equine piroplasms in blood

On the total of 130 controls, 16 horses and 1 donkey resulted positive to T. equi PCR, while on a total of 300 suspect cases 64 horses and 5 donkeys resulted positive to T. equi PCR. Concerning B. caballi biomolecular results, on a total of 130 controls 1 horse and 1 donkey resulted positive, while on a total of 300 suspect cases 10 horses resulted positive.

Confirmation of specificity of real time PCR by sequencing

All the 19 samples sequenced to confirm the specificity of PCR assays resulted belonging to the pathogen detected by the PCRs. In Table 3a summary of the results of sequencing, with relative deposited accession number are presented. A phylogenetic description of the strains circulating in the area, including also the sample of this study, is presented in Manna et al. (2018) [40]

Table 3

Results of sequencing for confirmation of specificity of PCR assays.
Specie	Reason for exclusion from the study	Number of samples	Target sequenced	Accession Number
T.equi	None. Samples included in the study	8	Hypervariable V4 region of the 18 rRNA gene	KU923651 KU923642 KU923652 KU923653 KU923643 KU923644 KU923640 KU923649
	None. Samples included in the study	6	T. equi EMA-1 gene	KU923598 KU923611 (same sample of KU923651) KU923601 (same sample of KU923642) KU923599 (same sample of KU923652) KU923600 (same sample of KU923653) KU923587 (same sample of KU923643)
	No fever reported	4	Hypervariable V4 region of the 18 rRNA gene	KU923619 KU923632 KU923641 KU923650
	No fever reported	1	T. equi EMA-1 gene	KU923610 (same sample of KU923650)
	Jaundice not reported and anaemia not confirmed by analysis	1	Hypervariable V4 region of the 18 rRNA gene	KU923631
	Jaundice not reported and anaemia not confirmed by analysis	1	T. equi EMA-1 gene	KU923614
	Anaemia not reported and jaundice not confirmed by analysis	1	Hypervariable V4 region of the 18 rRNA gene	KU923648
	Anaemia not reported and jaundice not confirmed by analysis	1	T. equi EMA-1 gene	KU923612 (same sample of KU923648)
B.caballi	Only positive sample for B.caballi meeting all the requisites for inclusion	1	Hypervariable V4 region of the 18 rRNA gene	KU923665
B.caballi	Anaemia not reported and jaundice not confirmed by analysis	2	Hypervariable V4 region of the 18 rRNA gene	KU923667 KU923667

Serological screening for equine piroplasms

On the total of 130 controls, 23 horses and 5 donkeys resulted positive to T. equi ELISA, while on a total of 300 suspect cases, 90 horses and 7 donkeys resulted positive. Concerning B. caballi serological results on a total of 130 controls 3 donkeys resulted positive, while on a total of 300 suspect cases 6 horses resulted positive.

Considering the combined results of serological and biomolecular results for T. equi, all the PCR positive subjects among the controls were also serologically positive, while among suspects only 48 horses and three donkeys were positive to both techniques. For B. caballi only one donkey among positive controls and 1 horses among positive suspects were also serologically positive.

Considering the contemporary positivity for both parasites, six positive subjects for B. caballi PCR were also positive to T. equi PCR (all the controls and four out of ten suspects).

Screening for other pathogens

Regarding the positivity to other diseases, among samples belonging to group A one horse was serologically positive for Leptospira spp., and another one to IFAT for Anaplasma phagocytophilum; group B included five samples serologically positive for Leptospira spp and three positive to IFAT for A. phagocytophilum, and finally in group C two samples were also serologically positive to A. phagocytophilum. A further seven and 21 subjects were serologically positive to Leptospira spp. and A. phagocytophilum respectively, but they were excluded for one of the reasons reported in Fig. 2. No DNA of both Leptospira spp. and A. phagocytophilum was detected. Moreover, all samples were negative to equine infectious anaemia.

Haemato-biochemical analysis

Medians and interquartile ranges (IQR = 75°-25° percentile) of each haemato-biochemical parameter for horses were reported in Table 4. The descriptive analysis of donkeys haemato-biochemical parameters are reported in Additional file 1: Table S1.

Table 4

Results of the logistic univariable analysis on individual anamnesis and management parameters collected with the anamnestic form.
Individual anamnesis		Groups			Logistic univariable analysis
Variable	Outcome	Cases (A)	Control (B)	Not confirmed suspects (C)	A vs B		A vs C
Variable	Outcome	[N = 19]	[N = 121]	[N = 27]	OR	95% CI	OR	95% CI
Species	Horse	16 (84%)	102 (84.3%)	23 (85.2%)
Species	Donkey	3 (16%)	19 (15.7%)	4 (14.8%)	1.00	0.27–3.79	1.01	0.21–5.49
Sex	Male	2 (11%)	24 (19.8%)	0
	Gelding	11 (58%)	38 (31.4%)	15 (55.6%)	3.47	0.71–17.05
	Female	6 (32%)	59 (48.8%)	11 (40.7%)	1.22	0.23–6.47
	Missing	0	0	1 (3.7%)
Age (y)	0–6	2 (11%)	56 (46.3%)	2 (7.4%)
	7–12	9 (47%)	35 (28.9%)	4 (14.8%)	7.20^*	1.46–35.28	2.25	0.23–22.14
	> 13	8 (42%)	30 (24.8%)	20 (74.1%)	7.46^*	1.49–37.41	0.4	0.04–3.34
	Missing	0	0	1 (3.7%)
Fur colour	Light	11 (58%)	48 (39.7%)	18 (66.7%)
	Dark	8 (42%)	71 (58.7%)	9 (33.3%)	0.49	0.18–1.31	1.45	0.43–4.88
	Missing	0	2 (1.7%)	0
Breed	Autochthonous	2 (11%)	2 (1.7%)	3 (11.1%)
	Saddler	10 (53%)	65 (52%)	18 (66.7%)	0.15	0.02–1.22	0.83	0.12–5.85
	Trotter	1 (5%)	10 (8%)	0	0.10	0.01–1.71
	Galloper	2 (11%)	24 (19.8%)	0	0.08^*	0.01–0.95
	Other	1 (5%)	1 (0.8%)	2 (7.4%)	1	0.03–29.81	0.75	0.04–14.97
	Missing	3 (16%)	19 (15.7%)	4 (14.8%)
Ticks	No	12 (63%)	54 (43.2%)	15 (55.6%)
	Yes	7 (37%)	65 (52%)	10 (37%)	0.48	0.17–1.31	0.87	0.26–2.99
	Missing	0	2 (1.6%)	2 (7.4%)
Management		Groups			Logistic univariable analysis
Variable	Outcome	Cases (A)	Control (B)	Not confirmed suspects	A vs B		A vs C
Variable	Outcome	[N = 19]	[N = 121]	[N = 27]	OR	(95% CI)	OR	(95% CI)
Recent Introduction	No	16 (84%)	119 (98.4%)	27 (100%)
	Yes	3 (16%)	1 (0.8%)	0	22.31^**	2.19-227.63
	Missing	0	1 (0.8%)	0
Housing	Inner	7 (37%)	86 (71.1%)	15 (55.6%)
	Outer	12 (63%)	35 (33.9%)	11 (40.7%)	4.21^**	1.53–11.58	2.33	0.69–7.87
	Missing	0	0	1 (3.7%)
	Single	10 (53%)	78 (64.5%)	9 (33.3%)
	Group	5 (26%)	31 (25.6%)	4 (14.8%)	1.67	0.55–5.10	1.50	0.31–7.18
	Missing	4 (21%)	12 (9.9%)	14 (51.9%)
Vaccination	No	3 (16%)	1 (0.8%)	6 (22.2%)
	Yes	15 (79%)	119 (98.4%)	21 (77.8%)	0.04^**	0.00-0.43	1.42	0.31–6.64
	Missing	1 (5%)	1 (0.8%)	0
Ectopar. treatments	No	15 (79%)	64 (52.9%)	19 (70.4%)
	Yes	2 (11%)	57 (47.1%)	7 (25.9%)	0.15^*	0.03–0.68	0.36	0.065-2.00
	Missing	2 (11%)	0	1 (3.7%)
Deworming	No	1 (5%)	1 (0.8%)	1 (3.7%)
	Yes	16 (84%)	120 (99.2%)	26 (96.3%)	0.13	0.01–2.23	0.62	0.04–10.54
	Missing	2 (11%)	0	0
Deworming periodicity	Regular	13 (68%)	95 (79%)	3 (11%)
	Sporadic	4 (21%)	25 (20%)	3 (11%)	1.17	0.35–3.89	3.38	0.54–21.11
	Missing	2 (11%)	1 (1%)	21 (78%)
p < 0.05, *p < 0.01. Significant OR and 95% CI highlighted in bold

Groups definition

The selection pathway is displayed in the flow-chart of Fig. 2 prepared with [41]. From an initial 300 suspect cases and 138 controls at the first step, the form verification, 66 suspect subjects were excluded for not reporting fever as signs in the suspect case form, and a further 23 suspects for not reporting neither anaemia nor jaundice.

After the laboratory analysis, a total of 164 suspect subjects were excluded for having anaemia (27), jaundice (57) or both (80) signs reported on the form but not confirmed by laboratory analysis. Moreover, the only remaining positive subject to B. caballi, was excluded from the analysis. Out of 138 controls, 17 were excluded for being positive to one of the PCRs for EP.

Finally, Group A, confirmed cases, was composed by 19 T. equi PCR-positive animals, 16 horses and three donkeys. Percentages of confirmed cases showing each sign present on the submission form are displayed in Fig. 3, compared to those reported by Risso et al. (2022) [42] on horses, Laus et al. (2015) [22] that involves donkeys, Al Saad et al. (2010) [15], Zobba et al. (2008) [16], and Hailat et al. (1997) [17], all focused on horses. One hundred and two horses and 19 donkeys composed Group B, and 23 horses and four donkeys were included in group C.

Statistical analysis

Results of the univariate analysis on the individual and management characteristics are presented in Table 4. Anamnestic variables that influenced the probability to belong to group A versus B were age, breed, recent introduction, housing, vaccination and ectoparasitic treatment. Age (OR = 7.20 [95% CI 1.46–35.28] for 7–12 years and OR = 7.46 [95% CI 1.49–37.41] for ≥ 13 years) recent introduction (OR = 22.31 [95% CI 2.19-227.63]) and outer housing (OR = 4.21 [95% CI 1.53–11.58]) variables were significant risk factors, instead galloper breed (OR = 0.08 [95% CI 0.01–0.95]), vaccination (OR = 0.04 [95% CI 0.00-0.43]) and ectoparasitic treatment (OR = 0.15 [95% CI 0.03–0.68]) were significant protective factors. No risk differences were detected between group A and group B in respect to other variables.

According to multivariate analysis results, age variable was a significant risk factor (OR = 11.80 for 7–12 years [95% CI 2.18–63.83] and OR = 18.70 for ≥ 13 years [95% CI 3.39–103]), while ectoparasitic treatment (OR = 0.06 [95% CI 0.01–0.31]) was a significant protective factor.

Regarding horses' haemato-biochemical parameters, results of statistical analysis are reported in Table 5. Three sets of parameters could be identified. The first set includes the parameters that did not vary significantly among group A (confirmed cases) and group B (control group) such as AST, GGT, WBC, segmented and band neutrophils, lymphocytes (both as absolute and percentage values), MCV, total proteins, particularly alpha and beta seroprotein fractions. The second set gathers the parameters that are significantly different between group A and B (control group) even though not between group A and group C (not confirmed suspects). These are total and direct bilirubin, monocytes, basophils, MCH and gamma fraction, that have median values higher in group A compared to group B. CK, eosinophils (both as absolute and percentage values), RBC, HGB, HCT, RDW, albumin, and A/G ratio have median values lower in group A compared to group B. Ultimately, the platelet count is the only parameter that shows a significant difference between groups A and both the other groups (Fig. 4).

Table 5

Comparisons of haemato-biochemical parameters among horses groups: confirmed cases (A), controls (B), and not-confirmed suspects (C).
Haematobiochemical parameters	Cases (A)	Control (B)	Not confirmed suspects (C)	H test ^a, p value	Multiple comparisons
	[N = 16]	[N = 102]	[N = 23]
	Median (IQR)	Median (IQR)	Median (IQR)
AST	251 (121)	300 (98)	296 (186)	H = 2.69, P = 0.260
Direct bilirubine	0.77 (0.35)	0.48 (0.13)	0.58 (0.44)	H = 23.72, P < 0.001	A vs B*, B vs C
Total Bilirubine	5.55 (6.66)	1.79 (0.82)	1.85 (4.92)	H = 16.53, P < 0.001	A vs B***
CK	171 (158)	258 (136)	188 (116)	H = 20.48, P < 0.001	A vs B, B vs C
GGT	13 (6.5)	12 (6)	15 (15)	H = 1.79, P = 0.408
WBC	7.96 (2.78)	6.89 (2.19)	7.42 (3.13)	H = 2.15, P = 0.341
Neutrophils	4.98 (1.94)	4.20 (1.39)	5.05 (3.04)	H = 6.05, P = 0.049
Segmented neutrophils (%)	70 (13)	62 (9)	70 (15)	H = 13.23, P = 0.001	B vs C***
Band cells (%)	0 (0)	0 (0)	0 (0)	H = 3.97, P = 0.137
Lymphocytes	1.67 (1.08)	2.01 (0.87)	1.29 (1.09)	H = 18.56, P < 0.001	B vs C***
Lymphocytes (%)	25 (11)	33.5 (10.5)	20 (12)	H = 19.38, P < 0.001	B vs C***
Monocytes	0.93 (1.46)	0.32 (0.17)	0.42 (0.44)	H = 21.07, P < 0.001	A vs B***
Monocytes (%)	4 (1)	0 (1)	0 (5)	H = 19.31, P < 0.001	A vs B***
Eosinophils	0.09 (0.13)	0.19 (0.19)	0.20 (0.24)	H = 13.74, P = 0.001	A vs B***
Eosinophils (%)	1 (2)	3 (3)	2 (5)	H = 9.66, P = 0.008	A vs B***
Basophils	0.04 (0.06)	0.02 (0.02)	0.02 (0.02)	H = 8.33, P = 0.015	A vs B*
Basophil (%)	0 (0)	0 (0)	0 (0)	H = 2.79, P = 0.247
RBC	4.71 (1.69)	7.5 (1.36)	5.415 (0.92)	H = 56.63, P < 0.001	A vs B*, B vs C*
HGB	8.33 (2.80)	12.6 (1.6)	9.73 (2.49)	H = 52.04, P < 0.001	A vs B*, B vs C*
HCT	23.4 (8.6)	36.9 (5)	28.4 (6.9)	H = 49.49, P < 0.001	A vs B*, B vs C*
MCV	50.05 (2.25)	48.6 (5.2)	52.9 (5.9)	H = 21.14, P < 0.001	B vs C***
MCH	17.35 (1.05)	16.65 (1.2)	18.4 (2.1)	H = 32.59, P < 0.001	A vs B, B vs C**
MCHC	34.8 (0.75)	34.3 (1)	33.75 (0.8)	H = 20.23, P < 0.001	A vs B, B vs C, A vs C***
RDW	22.7 (2)	25.1 (2.1)	23.2 (1.7)	H = 29.36, P < 0.001	A vs B, B vs C*
PLT	41 (41.25)	112 (42.4)	120 (53.1)	H = 22.5, P < 0.001	A vs B*, A vs C*
Total protein	6.2 (0.65)	6.3 (0.5)	6.4 (0.6)	H = 0.74, P = 0.690
Albumin	2.7 (0.28)	3.05 (0.21)	2.81 (0.33)	H = 33.78, P < 0.001	A vs B*, B vs C*
Alfa 1	0.09 (0.02)	0.09 (0.02)	0.1 (0.03)	H = 2.67, P = 0.263
Alfa 2	0.73 (0.19)	0.83 (0.1)	0.82 (0.23)	H = 5.89, P = 0.052
Beta	0.76 (0.19)	0.7 (0.19)	0.71 (0.2)	H = 1.80, P = 0.406
Gamma	1.92 (0.63)	1.52 (0.47)	1.78 (0.69)	H = 17.43, P < 0.001	A vs B*, B vs C
A/G ratio	0.77 (0.23)	0.96 (0.15)	0.77 (0.24)	H = 36.46, P < 0.001	A vs B*, B vs C*
^aH test: Kruskal-Wallis test, df = 2; p < 0.05, p < 0.01, **p < 0.001. IQR: interquartile range. Significant P highlighted in bold. AST: aspartate aminotransferase, CK: creatinphosphokinase, WBC: total white blood cells, RBC: total red blood cells count, HGB: haemoglobin level, HCT: hematocrit, MCV: mean corpuscular volume, MCH: mean corpuscular haemoglobin, MCHC: mean corpuscular haemoglobin concentration, RDW: red cell distribution width, PLT: platelets, A/G ratio: Albumin/Globulin ratio

EP is widely present in many countries of the world, so much so Schnittger et al. (2012) [43] estimated that around 90% of the horses live in endemic areas. Thus, EP should always to be considered in the differential diagnosis, together with other diseases such as leptospirosis, equine infectious anaemia and anaplasmosis, in case of any anaemic/haemolytic clinical forms, as presence of anaemia, jaundice, or petechiae. This study was set with the aim of investigating if individual and management characteristics could influence the probability of being infected. Furthermore, it was assessed the possible association of some haemato-biochemical alterations with confirmed cases of T. equi infection, comparing them to healthy subjects and to those with compatible signs but not confirmed as T. equi infected. The original purpose was to investigate both EP agents but, since only one case of B. caballi was detected among the eligible subjects, the study was focused on T. equi.

Symptoms are not often reported in details in the studies on EP. Comparing the present study with the few others for which clinical data are reported or easily computable (Fig. 3) a variability in the presence of symptoms emerges, with the predominance (presence in ≥ 50% of confirmed cases) of anaemia, jaundice, lack of appetite, lethargy, petechial haemorrhages, weakness, and weight loss [15–18, 22, 42].

Sex and species seem to have no influence on the presence of T. equi, while age resulted a risk factor for T. equi, with a cumulative age-dependent increasing presence of the pathogen. Positive cases increase in the groups 7–12 and > 13 years of age compared to the 0–6 years group. This result was expected and in agreement with previous studies [44], even though it disagrees both with the results of a study by the authors of the same regions [4], reporting that OR decreased with age, and with Steinman et al. (2012) [45] that, in Israel, observed no significant differences within the age classes considered. This evidence could be due to the cumulative risk of being in contact with an infected tick through the life, to the inefficacy of the treatment, that can be variable depending on the strain involved [46] and to the fact that T. equi is not self-cleared as B. caballi but leads to a chronic infection status [5].

Breed analysis highlighted a lower risk (OR = 0.08, [95% CI 0.01–0.95]) to be infected in the galopper breed compared to others. It could be a proxy related to different management practices, according to breed and attitude, i.e., more appropriate rearing conditions of pure breed horses and of those with economical value, that resulted less exposed to ticks [45, 47]. Because of the sampling was carried out almost only amongst animals belonging to saddler breed and with equestrian attitude, despite authors recommendations to practitioners, no conclusions can be certainly inferred.

The assessment of the fur brightness was included in the study as well as previously considered by Katiyatiya et al. (2015) [48] in the bovine, assuming that dark colour furs could result in a higher tick load and, consequently, in an increased risk of infection. The present study detected no differences between dark and light colours, however, being it at a preliminary stage further studies will be needed to clarify a possible association between T. equi infection and fur colours.

T. equi confirmed cases were approximately four times more likely to be found amongst animals having external access compared to controls, reinforcing the evidence that access to the outside, including not only a paddock near the stable but also pastures and free-ranging animals, in which the infection risk could be higher for a greater exposure to ticks or for a lack of grooming practices that improve tick removal, is a significant variable, in agreement with previous studies [45, 49, 50].

Vaccinations against other equine diseases and ectoparasitic treatment seems to have a protective effect on T. equi infection. While for the latter variable the association with a lower presence of infection is expectable, the correlation with vaccination is not clear; it is the opinion of the authors that this evidence could be explained with prophylactic treatments being a proxy for good management practices that prevent the infection. This hypothesis is reinforced also by data provided by [6], that reported the vaccination for tetanus as a protective factor, even though the study is based only on serological assays; and [13] reporting the regular deworming/vaccination practices as protective factors and an unorganized management of the farm as a risk factor.

Several studies on haemato-biochemical parameters associated with EP are available for both horses and donkeys. In the present study, the authors performed a comparison of haemato-biochemical parameters among the three groups. Due to the low number of donkeys, the statistical analysis was performed only on horses, while only a descriptive analysis of donkeys haemato-biochemical parameters is presented in SM. AST value resulted lower in group A, even if not statistically significant (p = 0.06). This finding differs from previous studies [15, 17, 19, 25] reporting an increase, even if not constant, of this enzyme in acute cases as well as for muscle enzyme CK. Both total and direct bilirubin values resulted higher in cases compared to controls, as well as reported by several other studies [15–19, 22, 51], except for [14] whereas, in the unconfirmed suspects, bilirubin values resulted between those of the other two groups. However, only direct bilirubin values were over the reference value, and the reason proposed by [16] is a centrilobular necrosis of the liver caused by reduced blood flow.

A decrease in RBC count, HGB and HCT confirmed T. equi associated findings, caused by the haemolysis of the red blood cells [15–19, 22, 45–47]. The decrease in platelet count is widely reported [15–20, 23–25], and it is the only parameter that, according to the present data, differs between both cases and controls rather than between cases and non-confirmed suspect (Fig. 4). This statistical evidence, though, seems to lack any biological explanation justifying it, if compared to other similar disease as B. caballi. In other species, such as dogs with babesiosis, the platelet count decrease is supposed to be caused by sequestration in the spleen, systemic disseminated intravascular coagulation, or immune-mediated depletion [52]. A possible explanation could be that the patogenicity of T. equi, being higher than that of B. caballi, could results in a more consistent decrease of platelets. An increase in MCH was detected in cases in the present study although variations in both directions are reported in donkeys andhorses [18, 24]. Variation in MCV, as an increased value, is reported only by Davitkov et al. (2017) [23] and Mahmoud et al. (2016) [24] and as a decreased value only in two out of 16 cases by Pasolini et al. (2018) [25]. In the present study, MCV resulted different only between controls and unconfirmed suspects. MCHC is reported to vary in affected donkeys [22, 23] whereas in the present study resulted not significantly different in horses, as well as for others [15–17, 19–20, 24]. An increase of WBC count was not observed in the present study despite being reported in several others [15–17, 19–20, 23, 42] as well as a decrease in total protein [15, 17]. This is the first report describing a decrease in eosinophils, basophils and RDW associated to T. equi infection, in contrast with Mahmoud et al. (2016) [24] but in agreement with previous studies in cattle with theileriosis [53].

Hypoalbuminemia and hyperglobulinemia were observed, with a consequent decrease in the ratio of the two parameters. Usually, albumin decreases as a result of liver dysfunction due to the disease. A decrease in albumin was reported also in dogs with babesiosis [54], but this is the first finding in infected equids, following Zobba et al. (2008) [16] that described, without inferential analysis, an albumin decrease in 15% of the positive subjects whereas globulin was reported as normal in all of them; and Pasolini et al. (2018) [25] that reported a decreased value in two of 16 positive horses. Finally, it has to be highlighted that some haemato-biochemical parameters, in particular the total bilirubin, CK, percentage of monocytes, eosinophils, basophils, MCH, RDW, albumin, and gamma fraction, despite being associated to T. equi, are still within the reference values. The same observation has been reported in most of the studies previously mentioned in the discussion, meaning that, in evaluating the laboratory results, these parameters are not useful for practitioners, that have to refer only to meaningful parameters such as direct bilirubin, RBC, HGB, HCT and A/G. Limitations of this study have been already partially discussed in ‘material and methods’ section, as they influence the case definition and data analysis. Interviewer bias brought the major constraint to the study, in fact, often, symptoms reported by the practitioners were not confirmed by the laboratory results, suggesting that the data provided were manipulated to result eligible for the study. The reason could be the following: the Istituto Zooprofilattico Sperimentale del Lazio e della Toscana “M. Aleandri” offered free analysis for the subject enrolled in the study to incentive the collaboration of the equine practitioners, and some of them could have taken advantage of this offer, submitting forms reporting non-observed signs. The double check between signs and laboratory findings aimed to overcome this limitation, excluding subjects with insubstantial data.

The aim of the study, as already mentioned, was to provide valid tools for the clinical activities to the practitioners even if, after discussing the statistical and the construct validity, external validity should be discussed, i.e., whether the study results can be generalized to different settings and times [55], as other species, breeds and attitudes, and different level of prevalence. Moreover, a similar investigation should be performed also for B. caballi and other diseases in the differential diagnosis of anaemic/haemolytic clinical form to provide more data in order to assess a more precise diagnosis.

The main conclusion highlighted by the study is that any investigated haemato-biochemical parameters, except platelet count, seem to be irrevocably useful to discriminate T. equi from other diseases. The present study confirms that the diagnosis of T. equi could be an issue, for its similarities with other diseases, but it still represents a starting point for further investigations, highlighting unsolved critical points and providing the practitioners with further data for the interpretation of laboratory results. Moreover, this study focuses the attention on prevention practices, as external antiparasitic treatment, the control of access to pastures and good management practices in general, that together with regular laboratory controls, even in asymptomatic animals, can reduce the spread of the disease.

A/G: Albumin globulin ratio; AST: aspartate amino transferase; CBC: complete blood cell count CK: creatine phosphokinase; EDTA: ethylenediaminetetraacetic acid; ELISA: enzyme-linked immunosorbent assay; EP: equine piroplasmosis; GGT: gamma-glutamyltransferase; HCT: haematocrit; HGB: haemoglobin; IFAT: indirect fluorescent antibody test; MCH: mean corpuscular haemoglobin; MCHC: mean corpuscular haemoglobin concentration; MCV: mean corpuscular volume; OR: Odds Ratio; PCR: polymerase chain reaction; PLT: platelets count; RBC: red blood cells; RDW: red blood cells Distribution Width; WAHO: World Animal Health Organization; WBC: total white blood cells.

Acknowledgements

Not applicable.

Funding

This study was conducted within the IZSLT 16/11 research project funded by the Italian Ministry of Health.

Availability of data and material

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

RN and MTS concept and designed the study; RN, AC, FR, OL, GB, MRV and AA performed laboratory analysis; RN and FI performed statistical analyses; RN and FI drafted the work; RN, FI, FR, PS, OL, GB and MTS revised the manuscript.

Ethics approval and consent to participate

This article does not contain any studies on animals performed by any of the authors. Veterinary practitioners collected blood samples during their clinical activities for diagnostic purposes.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Endnotes

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No competing interests reported.

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Risk factors and haemato-biochemical parameters associated with Theileria equi infection in equids in Central Italy

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Abstract

Figures

Background

Methods

Characteristic of the study area

Sampling and anamnestic form

Laboratory analyses

Molecular detection of equine piroplasms in blood

Confirmation of specificity of real time PCR by sequencing

Serological screening for equine piroplasms

Screening for other pathogens

Haemato-biochemical analysis

Groups definition

Statistical analysis

Results

Laboratory analyses

Molecular detection of equine piroplasms in blood

Confirmation of specificity of real time PCR by sequencing

Serological screening for equine piroplasms

Screening for other pathogens

Haemato-biochemical analysis

Groups definition

Statistical analysis

Discussion

Conclusions

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

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