Molecular and serological detection of Theileria equi, Babesia caballi and Anaplasma phagocytophilum in horses and ticks in Maranhão, Brazil

Detecção molecular e serológica de Theileria equi, Babesia caballi e Anaplasma phagocytophilum em equinos e carrapatos no Maranhão

ABSTRACT:

Equine piroplasmosis is a tick-borne disease caused by the intraeytrhocytic protozoans Babesia caballi and Theileria equi. It has been reported as a main equine parasitic disease. In addition, Anaplasma phagocytophilum, the causative agent of granulocytic ehrlichiosis, causes a seasonal disease in horses. Both diseases, can be detrimental to animal health. In this sense, blood samples and ticks were collected from 97 horses raised in the microregion of Baixada Maranhense, Maranhão State, Brazil. Serum samples were subjected to Indirect Fluorescence Antibody Test (IFAT) and blood samples and ticks to Polymerase Chain Reaction (PCR) to evaluate the infection by Theileria equi, Babesia caballi and Anaplasma phagocytophilum. The overall seroprevalence was 38.14%, 18.55% and 11.34% for T. equi, B. caballi and A. phagocytophilum, respectively. The results of PCR from blood samples showed 13.40% and 3.09% positive samples to T. equi and B. caballi, respectively. A total of 170 tick specimens were collected and identified as Dermacentor nitens, Amblyomma cajennense sensu lato and Rhipicephalus (Boophilus) microplus. It was detected 2.35% (4/170) and 0.59% (1/170) positive tick samples by PCR for T. equi and B. caballi, respectively. All samples were negative to A. phagocytophilum. No statically difference (p>0.05) was observed when gender, age, use of ectoparasiticide and tick presence were analyzed. A BLASTn analysis of the sequenced samples indicated 97 to 100% similarity with T. equi 18S rRNA gene sequences in GenBank and 98 to 100% with B. caballi. Genetic analysis classified the obtained sequences as T. equi and B. caballi cluster, respectively. It can be concluded that these pathogens occur and are circulating in the studied area.

INDEX TERMS: Vector-borne disease; Theileria equi; Babesia caballi; Anaplasma phagocytophilum; horses; ticks; Brazil

RESUMO:

A piroplasmose equina é uma doença transmitida por carrapatos causada pelos protozoários intraeritrocitários Babesia caballi e Theileria equi. É relatada como uma doença parasitária comum em equinos. Além disso, Anaplasma phagocytophilum, o agente causal da ehrlichiose granulocítica, causa uma doença sazonal em equinos. Ambas as doenças, podem ser prejudiciais para a saúde animal. Nesse sentido, amostras de sangue e carrapatos foram coletadas de 97 cavalos criados na microrregião da Baixada Maranhense, estado do Maranhão, Brasil. As amostras de soro foram submetidas ao Teste de Imunofluorescência Indireta (RIFI) e amostras de sangue e os carrapatos a Reação da Polimerase em Cadeia (PCR) para avaliar a infecção por Theileria equi, Babesia caballi e Anaplasma phagocytophilum. A prevalência foi de 38,14%, 18,55% e 11,34% para T. equi, B. caballi e A. phagocytophilum, respectivamente. Os resultados da PCR para as amostras de sangue demonstraram 13,40% e 3,09% de positividade para T. equi e B. caballi, respectivamente. Um total de 170 specimens de carrapatos foi coletado e foram identificados Dermacentor nitens, Amblyomma cajennense sensu lato and Rhipicephalus (Boophilus) microplus. Obteve-se 2,35% (4/170) e 0,59% (1/170) positivos por PCR para T. equi e B. caballi, respectivamente. Todas as amostras foram negativas para A. phagocytophilum. Não houve diferença estatística significativa (p>0.05) em relação ao sexo, idade, uso de ectoparasiticida e presença de carrapatos. A análise BLASTn das amostras sequenciadas para gene 18S rRNA indicaram 97 a 100% de similaridade com T. equi e 98-100% com B. caballi no GenBank. Análises genéticas classificaram as sequencias obtidas no mesmo clado que T. equi e B. caballi, respectivamente. Podemos concluir que estes patógenos estão circulando na área de estudo.

TERMOS DE INDEXACÃO: Doença transmitida por vetores; Theileria equi; Babesia caballi; Anaplasma phagocytophilum; cavalos; carrapatos; Brasil

Introduction

Equine piroplasmosis is a tick-borne disease caused by the intraeytrhocytic protozoans Babesia caballi and Theileria equi (Mehlhorn & Scheinh 1988, De Waal 1992, De Waal & Van Heerden 1994, 2004). It has been reported as a main equine parasitic disease due to direct (loose of performance and mortality) and indirect damage (threat to horse industry) caused to animal health (Nogueira et al. 2005). It has great impact on international movement of equines for importation purpose or sports practices since positive or seropositive animals are not allowed to enter in countries free (Friedhoff et al. 1990, Knowles 1996). Once infected the animals remain carriers for long periods and act as source of infection to ticks (De Waal 1992, Laus et al. 2015). It is endemic in tropical and subtropical regions, and some temperate zones of the world (Shkap et al. 1998, Steinman et al. 2012). Mixed infections by B. caballi and T. equi are common in endemic areas (Scoles & Ueti 2015).

Anaplasma phagocytophilum, the causative agent of granulocytic ehrlichiosis, causes a seasonal disease. Members of the A. phagocytophilum complex have been recognized as worldwide tick-borne agents for several species of wild and domestic mammals. Besides, bacteria within this complex have recently emerged as zoonotic agents (Passamonti et al. 2010). In Brazil there are few reports about the infection as well as its natural vector.

The northeastern of Brazil has the second biggest horse herd. In the State of Maranhão, the microregion of Baixada Maranhense is characterized by an extensive group of lakes and lagoons with dry and rainy periods (Pinheiro et al. 2005). In this region calls the attention a native horse named “baixadeiro horse” that is used to work and therefore assumes a great local social economic importance.

In Maranhão State limited information are available regarding the infection by these three agents in horses; therefore the purpose of the present survey was to determine the relative frequency of B. caballi, T. equi and A. phagocytophilum by testing horses by Indirect Immunofluorescence Test (IFAT) and molecular methods (Polymerase Chain Reaction-PCR). Ticks were also analysed.

Materials and Methods

Animals and sampling. The study was conducted from may/2012 to may/2013 in the microregion of Baixada Maranhense, in the municipalities of Santa Helena (02° 13’ 51” S 45° 18’ 00” O), Pinheiro (02° 31’ 15” S 45° 04’ 58” O) and Viana (03° 13’ 12” S 45° 00’ 14” O) in the Maranhão State northeastern Brazil. Ninety seven horses were randomly selected from three municipalities of Baixada Maranhense. Data from each horse including age and gender were recorded.

Blood samples (10mL) from each horse were collected from the jugular vein and placed into serum and EDTA tube. The serum tubes were centrifugated and sera were separated. Both sera and blood EDTA tubes were stored at -20°C until the time for serological and molecular analysis.

The horses were searched for ticks (approximately one minute per horse) which were removed manually and preserved in 70% ethanol for identification and PCR assays. Ticks were identified by the use of identification keys (Aragão & Fonsceca 1961, Barros-Battesti et al. 2006).

Indirect fluorescent antibody test (IFAT). Samples were screened for IgG antibodies against Babesia caballi, Theileria equi and Anaplasma phagocytophilum using a commercially available IFAT according to the manufacturer’s instructions (Fuller Laboratories USA).

Initially samples were scored and it was considered positive sera that reacted at the dilution of 1:80. Seropositive samples were analyzed to determine the titration end point and antibodies titers were summarized in the following groups: 1:60, 1:160, 1:320, 1:640, 1:1280. Positive and negative serum controls, provided by the kit, were added to each slide.

Polymerase Chain Reaction (PCR) and sequencing. Genomic DNA was extracted from the blood samples by using QIAmp DNA Minikit (QIAGEN Hilden Germany) according to the manufacturer’s instructions and stored at -20°C until use. Sampled ticks were submitted to DNA genomic extraction used guanidine isothiocyanate phenol technique, as described by Chomekzynski (1993) and modified by Sangioni et al. (2005).

For the detection of B. caballi and T. equi the extracted material was subjected to a PCR using primers previously described by Criado-Fornelio et al. (2003): BT-F1=5-GGTTGATCCTGCCAGTAGT-3, BT-R1=5-GCCTGCTGCCTTCCTTA-3 e BT-R2 = 5-TTGCGACCATACTCCCCCCA-3 that amplifies a region of 395bp for Babesia spp. and 410bp for Theleiria spp. of 18S rRNA gene.

For the detection of A. phagocytophilum DNA the extracted material was subjected to PCR primers MSP3F=5-CCAGCGTTTAGCAAGATAAGAG-3 and MSP3R=5-GCCCAGTAACAACATCATAAGC-3 that amplifies a fragment of 334bp of the p44 gene (Zeidner et al. 2000).

The PCR products were detected by electrophoresis on 2% agarose gel (100mL TAE 0.5%, 2g agarose Ultra Pure^TM Agarose Invitrogem^TM) stained with ethidium bromide distinct bands of B. caballi T. equi and A. phagocytophilum were visualized by UV transilluminator.

Positive products were selected purified and subjected for sequence confirmation in an automatic sequencer (ABI 3730xL - Applied Biosystems Foster City CA USA) according to Otto et al. (2008). The obtained sequences for each gene were analyzed and edited in ChromasPRO 1.5 (Technelysium Queensland Australia). Comparisons with sequences deposited in GenBank were done using BLASTn.

The sequences identified were aligned using software program ClustalW available in the MEGA 5.2. A phylogenetic tree was constructed with Neighbor-Joining at MEGA 5.2 program (Tamura el al. 2011) using the evolutive model Kimura-2-parameteres (Kimura 1980) with 1000 repetitions and exclusion of gaps.

Data analysis. Statistical associations of seropositivity to tick-borne pathogens with potential risk factors with the variables gender, age, use of ectoparasiticides and presence of ticks in the moment of the collection were performed. Data were tested by means of the chi-square or Fischer`s exact test, when necessary. The Odds Ratio (OR) was calculated for each variable with 95% confidence limits (p<0.05). All analyses were performed using the Epi Info software, version 6.04d (CDC, Atlanta, GA, USA).

Results

Blood samples were obtained from 97 horses from Baixada Maranhense microregion. By means of IFAT, 37 (38.14%) samples reacted to Theileria equi, and 18 (18.55%) for Babesia caballi with titers of 1:80 to 1:1280 for both protozoans. 11 (11.34%) samples reacted to Anaplasma phagocytophilum, titers varying from 1:80 to 1:320 (Table 1).

Table 1.
Frequency of IFAT IgG antibody titers against Theileria equi, Babesia caballi and Anaplasma phagocytophilum in naturally infected horses (n=97) raised in the microregion of Baixada Maranhense, Maranhão State, Brazil

	Titers of IgG antibodies					Seroreactive animals
	1:80	1:160	1:320	1:640	1:1280
Anti-T. equi	9	4	4	10	10	37
Anti-B. caballi	4	2	2	8	2	18
Anti- A. phagocytophilum	2	6	3	-	-	11

There was no statistical difference (p>0.05) among the studied variables (gender, age, use of ectoparasiticide and presence of ticks) in relation to T. equi, B. caballi and A. phagocytophilum infection (Table 2, 3 and 4). However horses aged from 5 to 8 years and 9 to 12 years (p<0.05) had more chance to get the infection by T. equi (Table 2).

Table 2.
Distribution of the variables gender, age, use of ectoparasiticide and presence of ticks in horses seropositive for Theileria equi in the microregion of Baixada Maranhense, Maranhão State, Brazil

Variable		Reactive T. equi		Non reactive T. equi		Total	OR	IC	Value-P
		N	%	N	%
Gender	M	9	26.47	25	73.53	34	0.45	0.16-1.22	0.1285628(a)
	F	28	44.44	35	55.56	63
Age (years)	<1	2	15.38	11	84.62	13
	1-4	5	18.52	22	81.48	27	0.80	0.09-6.00	0.5917915(b)
	5-8	17	53.12	15	46.88	32	0.16	0.02-0.98	0.0465580(a)*
	9- 2	10	62.50	6	37.50	16	0.11	0.01-0.84	0.0290414(a)*
	>13	3	33.33	6	66.67	9	0.36	0.03-3.96	0.3157895(b
Use of ectoparasiticide	Yes	20	37.74	33	62.26	53	0.96	0.39-2.38	0.9052469(a)
	No	17	38.64	27	61.36	44
Presence of ticks	Yes	32	37.65	53	63.35	85	0.85	0.22-3.40	0.5116261(a)
	No	5	41.67	7	58.33	12

• M = male, F = female, (a) = Chi-Square Test, (b) = Fisher’s Exact Test, OR = Odds Ratio, IC = Confidence Interval; * Significative difference.

Table 3.
Distribution of the variables gender, age, use of ectoparasiticide and presence of ticks in horses seropositive for Babesia caballi in the microregion of Baixada Maranhense, Maranhão State, Brazil

Variable		Reactive B. caballi		Non reactive B. caballi		Total	OR	IC	Value-P
		N	%	N	%
Gender	M	4	11.76	30	88.24	34	0.47	0.12-1.72	0.3219866(a)
	F	14	22.22	49	77.78	63
Age (years)	<1	-	-	-	-	13
	1-4	-	-	-	-	27
	5-8	11	34.38	21	65.62	32
	9-12	4	25	12	75	16	1.57	0.35-7.50	0.7411815(a)
	>13	3	33.34	6	66.66	9	1.05	0.18-6.63	0.6403036(b)
Use of ectoparasiticide	Yes	13	24.53	40	75.47	53	2.53	0.74-9.09	0.1620887(a)
	No	5	11.36	39	88.64	44
Presence of ticks	Yes	16	18.82	69	81.18	85	1.16	0.20-8.51	0.6092381(b)
	No	2	16.67	10	83.33	12

• M = male, F = female, (a) = Chi-Square Test, (b) = Fisher’s Exact Test, OR = Odds Ratio, IC = Confidence Interval.

Table 4.
Distribution of the variables gender, age, use of ectoparasiticide and presence of ticks in horses seropositive for Anaplasma phagocytophilum in the microregion of Baixada Maranhense, Maranhão State, Brazil

Variable		Reactive A. phagocytophilum		Non reactive A. phagocytophilum		Total	OR	IC	Value-P
		N	%	N	%
Gender	M	4	11.76	30	88.24	34	1.07	0.24-4.53	0.5836453(a)
	F	7	11.11	56	88.89	63
Age (years)	< 1	3	23.07	10	76.93	13
	1-4	2	7.40	25	92.6	27	3.75	0.41-39.15	0.1838549(a)
	5 a 8	3	9.37	29	90.63	32	2.90	0.38-22.67	0.2229695(a)
	9 a 12	2	12.50	14	87.50	16	2.10	0.22-22.83	0.3961686(a)
	>13	1	11.11	8	88.89	9	2.40	0.16-73.20	0.4496241(a)
Use of ectoparasiticide	Yes	6	11.32	47	88.68	53	1.00	0.24-4.14	0.6200802(a)
	No	5	11.36	39	88.64	44
Presence of ticks	Yes	10	8.5	75	91.5	85	1.47	0.16-33.56	0.5919983(a)
	No	1	8.33	11	91.67	12

• M = male, F = female, (a) = Fisher’s Exact Test, OR = Odds ratio, IC = Confidence interval.

The results of PCR and sequencing of DNA showed 13.40% (13/97) of horses were positive to T. equi and 3.09% (3/97) for B. caballi. All samples were negative to A. phagocytophilum.

A total of 170 ticks specimens were collected and the following species were identified: Dermacentor nitens (151), Amblyomma cajennense sensu lato (13) and Rhipicephalus (Boophilus) microplus (6). We obtained 2.35% (4/170) and 0.59% (1/170) positive by PCR for T. equi and B. caballi, respectively. All tick specimens were negative for A. phagocytophilum. The positive tick species for T. equi were R. (B.) microplus and D. nitens. Only D. nitens was positive to B. caballi.

Similarity analysis from the sequences obtained showed identity of 97% to 100% with T. equi (access GenBank JQ390047, KJ573372, KF559357). The phylogenetic dendogram formed by the sequences derivate from the products based on gene 18S rRNA demonstrated that the sequences obtained in this study are phylogenetically near T. equi. The 15 analyzed sequences formed a group along with T. equi (KJ573372, KJ573374, JQ390047 and KF559357) and Babesia equi (KM046918 and KM046922) (Fig.1). Partial sequence of the 18S rRNA gene from Theileria sp generated was deposited into GenBank with an accession number (KX165362­-KX165376).

Fig.1.
Phylogenetic tree of Theileria sp. using 18S rRNA gene sequences constructed by neighbor-joining method with Kimura two-parameter as evolution model and based on the nucleotide sequences. The GenBank accession codes are presented in parenthesis. The numbers at nodes are the bootstrap values obtained from 1,000 re-samplings. Bootstrap values below 60% are not present. DN = Dermacentor nitens.

Similarity analysis from the sequences obtained showed identity 98% to 100% with B. caballi (access AB734392 and KJ549665). To analyze the sequences of gene 18S rRNA of Babesia spp. we noted that in the sequences of this study are phylogenetically near B. caballi and aligned forming a group with the sequences of B. caballi Brazil (KJ549665) and B. caballi Mongolia (AB734392) with bootstrap support 76%, as shown in Fig.2. Partial sequence of the 18S rRNA gene from Babesia sp. generated was deposited into GenBank with an accession number (KX165377- KX165380).

Fig.2.
Phylogenetic tree of Babesia sp. using 18S rRNA gene sequences. Neighbor-joining analysis with 2-Kimura-parameters evolutionary model was performed. The GenBank accession codes are presented in parenthesis. The numbers at nodes are the bootstrap values obtained from 1,000 re-samplings. Bootstrap values bellow 60% are not present. DN = Dermacentor nitens.

Discussion

Serological studies related to Theileria equi and Babesia caballi infections have demonstrated that these agents are widely distributed in Brazil with prevalence ranging from 7 to 90% (Heim et al. 2007, Kerber et al. 2009, García-Bocanegra et al. 2013). Furthermore, other researchers in Brazil pointed out that the main tick species infesting horses in Brazil are Dermacentor nitens, Amblyomma cajennense s.l. and R. (B.) microplus, same tick species identified in this work. In the American continent D. nitens is the only known vector of B. caballi (Roby & Anthony 1963) and in Brazil the prevalence of this protozoan coincides with the distribution of this tick species. Studies performed by Guerra & Brito (2004) reported a high prevalence of D. nitens in horses in Maranhão State.

This work detected R. (B.) microplus tick infected with T. equi. R. (B.) microplus which is a monoxenous tick and has cattle as it’s the main host. However, horses may become alternative hosts in environments with there is contact between cattle and horses. It can be suggested that, at least, in Brazil, R. (B.) microplus, main tick in cattle and in many areas in equines, plays an important role in the transmission of T. equi (Torres et al. 2012). In addition, the vector competence of R. (B.) microplus has been demonstrated by the transtadial and intrastadial survival of T. equi in this tick specie (Ueti et al. 2008). Evidenciating the importance of this species of tick in the maintenance of the enzootic cycle of T. equi.

In the present study a greater seroprevalence was observed for T. equi then for B. caballi. Special attention should be given to the fact that a greater presence of T. equi was detected, because this agent is considered more pathogenic (Posada-Guzmán et al. 2015). These data differs from the one reported by Kerber et al. (2009) that studied the infection by both agents and the association with tick infestation in São Paulo State. They found out higher prevalence for B. caballi. Tick species D. nitens, A. cajennense and R. (B.) microplus were identified in 95%, 50% and 4% in horses, respectively. Moreover the infestations by D. nitens were more associated to T. equi.

Some factors can be associated to infection as proved by García-Bocanegra et al. (2013) in Spain. They reported different seroprevalence to B. caballi in mules (32.1%), donkeys (17%) and horses (7.9%). While for T. equi the results were much higher. The risk related to a higher seroprevalence to T. equi increases with age, presence of ticks and vaccination against other disease. On the other hand, the risk factors to B. caballi were animal species, presence of ticks and presence of shelters. Our results showed no differences among gender, age and presence of ticks. But as observed by García-Bocanegra et al. (2013) the risk of infection by T. equi increases with age, the same observed in the data presented here. According to Malekifard et al. (2014) the absence of differences in age and sex groups may be due to high number of tick in the area and continuous exposure of young and old horses to infected ticks.

The diagnoses of Equine Piroplasmosis can be done by direct and indirect methods; however, molecular assays appears as useful tools to identify the infection (Figueroa et al. 1993, Bashiruddin et al. 1999, Nicolaiewsky et al. 2001, Peckle et al 2013, Malekifard et al. 2014, Dória et al. 2016, Mahmoud et al. 2016). Therefore, sensitive and specific tests for Equine Piroplasmosis diagnosis are required to prevent introduction of causative agents into countries that are regarded free of the infection or disease (Mahmoud et al. 2016).The results presented here indicate that PCR is a sensitive assay and proves that the causative agents of Equine Piroplasmosis are circulating in the studied area. Studies performed in Brazil using PCR or IFAT have also detected both agents in horses, as the one reported in Minas Gerais (Heim et al. 2007), Rio Grande do Sul (Torres et al. 2012), Rio de Janeiro (Peckle et al. 2013) and São Paulo (Dória et al. 2016). Antibodies against Anaplasma phagocytophilum were detected in low levels however all blood and tick samples were negative by PCR. So the occurrence of this pathogen and its vector remains scarce. The finding of this pathogen is important since it represents a potential zoonotic risk. According to Van Andel et al. (1998) the capability of horses to produce anti-A. phagocytophilum antibodies that can be detected by commercial tests for up to six months enables this animal species to be used as sentinels in preventing human outbreaks.

Recently in Brazil, Rolim et al. (2015) investigated the rate of anti-A. phagocytophilum antibodies in horses from the Cavalry Squadron and Regiment of Mounted Police and found out 12% (11/91) animals with titer≥1:80. Animals aged from five to 14 years presented the highest rate of positive reactions but antibodies were detected in all ages. There was no statistical difference between males and females. These authors did not search for ticks although they stated that the circulation of the parasite among animals was not dependent of tick infestations. The data presented here did not show difference in gender and age among sampled animals as well as tick infestation.

Previously studies in Brazil reported 65% (Salvagni et al. 2010) and 3% (Parra et al. 2009) using serologic methods. However these authors obtained negative results when PCR was used for the detection of the pathogen as also observed in the present study and by Dória et al. (2016) when sports and traction horses were examined. Although Passamonti et al. (2010) detected the infection using serologic and molecular methods in horses. No bacterial infection was detected from ticks collected in the horses.

On the other hand, M’ghirbi et al. (2012) using serologic and molecular methods to detect A. phagocytophilum in horses and ticks in Tunisia found out 67% (40/60) positive by IFAT and 13% by PCR, with non-significant regional and gender differences, but a significant breed difference. 3 specimens of Hyalomma marginatum, which was the predominant tick species (130/154) were positive.

According to Passamonti et al. (2010) a positive serologic test against a PCR-negative result could possibly correspond to the dated past infection since A. phagocytophilum infection is characterized by limited and short-last bacteraemia. Based on this assumption it can be supposed that the horses of the present study had contact with the pathogen in the past.

Conclusions

The results pointed out the occurrence and circulation of Babesia caballi, Theileria equi and Anaplasma phagocytophilum in the Baixada Maranhense region.

This appears to be the first report of Anaplasma phagocytophilum in horses in Maranhão State

Acknowledgments

To Prof. Daniel Moura Aguiar, from Laboratório de Laboratório de Virologia e Rickettsioses do Hospital Veterinário, Universidade Federal de Mato Grosso (UFMT), for providing the positive control of A. phagocytophilum. The authors would like to thanks Plataforma Genômica-Sequenciamento de DNA-RPT01A, PDTIS/Fiocruz. This investigation was suported by Fundação de Amparo à Pesquisa e ao Desenvolvimento Científico e Tecnológico do Maranhão (FAPEMA). We also thank FAPEMA for the scholarship for Arannadia Barbosa Silva

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Publication Dates

• Publication in this collection
  Dec 2017

History

• Received
  03 Jan 2017
• Accepted
  30 May 2017