- Email: [email protected]
Evaluation of the natural vertical transmission of Theileria orientalis
Veterinary Parasitology 263 (2018) 1–4
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short communication
Evaluation of the natural vertical transmission of Theileria orientalis a,b,⁎
c
c
a
Hirohisa Mekata , Tomoya Minamino , Yoko Mikurino , Mari Yamamoto , Ayako Yoshida Nariaki Nonakab,d, Yoichiro Horiib,c
T b,d
,
a
Organization for Promotion of Tenure Track, University of Miyazaki, 1-1 Gakuen-Kibanadai-Nishi, Miyazaki 8892192, Japan Center for Animal Disease Control, University of Miyazaki, 1-1 Gakuen-Kibanadai-Nishi, Miyazaki 8892192, Japan Divisions of Research & Education for Livestock and Veterinary Clinic, Honkawa Ranch, Takase, Hita 8770056, Japan d Laboratory of Veterinary Parasitic Diseases, Department of Veterinary Sciences, Faculty of Agriculture, University of Miyazaki, 1-1 Gakuen-Kibanadai-Nishi, Miyazaki 8892192, Japan b c
A R T I C LE I N FO
A B S T R A C T
Keywords: Parasite load PCR T. orientalis Vertical transmission
Bovine theileriosis, caused by Theileria orientalis, is endemic from East Asia to Oceania. Even though the disease is mainly transmitted by Haemaphysalis ticks, the T. orientalis parasite can also be transmitted vertically. To develop proper control measures, the frequency of each transmission route must be elucidated. However, the frequency of vertical transmission, including transplacental transmission, of T. orientalis in naturally infected cattle is still controversial. This study aimed to clarify the frequency of the vertical transmission of T. orientalis in naturally infected cattle. Blood samples were collected from 204 T. orientalis-infected dams and their 211 newborn calves (including 7 sets of twins) within the first 24 h as well as 30 days after birth. Furthermore, 31 and 24 calves born to T. orientalis-infected and uninfected dams, respectively, were continuously surveyed for infection until 5 months of age. A total of 5 (2.4%) dams were diagnosed with mild anemia, whereas most of the dams were asymptomatic based on hematological examination and clinical signs. PCR analysis was performed on whole blood to determine the presence of T. orientalis in calves, and no calves were PCR positive 0 and 30 days after birth. However, 9.6% and 0% of the calves born to T. orientalis-infected and uninfected dams, respectively, tested positive at 3 and 5 months of age. The sampled calves were fed in-house, and the survey was conducted during the cold season; thus, horizontal transmission through blood-sucking insects rarely occurred. Therefore, the vertical transmission of T. orientalis took as long as 3 months to become detectable by PCR and occurred in approximately 10% of field cattle.
1. Introduction
maff.go.jp/j/chikusan/sinko/shiryo/houboku/houboku.html; in Japanese). Without parasite control, this approach will result in an increasing incidence of Theileria infection in cattle. Transplacental transmission has been detected for T. orientalis (Baek et al., 2003) and a number of other Theileria species, such as T. equi (Chhabra et al., 2012) and T. annulata (Sudan et al., 2015), by PCR analysis. However, the frequency of vertical transmission, including transplacental transmission, of T. orientalis in the field remains controversial. A Japanese study reported that 10% (3/30) of newborn calves were infected via this route (Sato et al., 2012). In contrast, studies conducted in Australia and New Zealand have reported that vertical transmission may occur but at a very low rate in the field (Lawrence et al., 2016; Swilks et al., 2017). The purpose of this study was to investigate the frequency of vertical transmission of T. orientalis from dams to calves under routine conditions. This study may aid in the development of control measures for bovine theileriosis.
Theileria orientalis is a tick-borne protozoan parasite and an etiological agent of benign theileriosis in cattle. This parasite is distributed from East Asia to Oceania and is an economically important pathogen (Eamens et al., 2013; Liu et al., 2011; Yokoyama et al., 2012). The Theileria infection is often asymptomatic but may cause signs, including fever, anemia and abortion, in cattle under a stressful situation. No effective drugs or vaccines are currently available for the control of theileriosis. Once cattle are infected, they appear to harbor the parasite for an extended period of time (Kubota et al., 1996). Therefore, screening for T. orientalis infection in cattle before pasturing and keeping ticks free of Theileria on pasture land are important strategies to minimize parasite transmission and expansion. However, the Japanese government promotes cattle grazing on pasture land to reduce rearing costs and workloads and to utilize abandoned farmlands (http://www.
⁎
Corresponding author at: Organization for Promotion of Tenure Track, University of Miyazaki, 1-1 Gakuen-Kibanadai-Nishi, Miyazaki 8892192, Japan. E-mail address: [email protected] (H. Mekata).
https://doi.org/10.1016/j.vetpar.2018.09.017 Received 7 May 2018; Received in revised form 26 September 2018; Accepted 29 September 2018 0304-4017/ © 2018 Elsevier B.V. All rights reserved.
Veterinary Parasitology 263 (2018) 1–4
H. Mekata et al.
2. Materials and methods
dams immediately after delivery using the Wizard Genomic DNA Purification Kit (Promega, Fitchburg, USA) and from the 10-fold diluted blood of PCR-positive calves after 3 and 5 months of age using the magLead system (Precision System Science, Chiba, Japan) according to the manufacturer’s instructions. The DNA concentration was determined using a NanoDrop 8000 spectrophotometer (Thermo Fisher Scientific, Waltham, USA), and samples were diluted to 20 ng/μL. Quantitative real-time PCR was performed using a LightCycler 96 system (Roche Diagnostics, Indianapolis, USA). A probe qPCR mix (TaKaRa Bio) and PrimeTime qPCR assays (Integrated DNA Technologies, Coralville, USA), including target gene-specific primers and probes, were used to quantify the copies number of T. orientalis MPSP and bovine β-actin exon 5 genes. The forward primer for β-actin exon 5 was modified from a previous report to detect only the exon sequence (Toussaint et al., 2007). The following primer and probe sequences were used: MPSP F primer: 5’-GCAAACAAGGATTTGCACGC-3’, MPSP R primer: 5’-TGTGAGACTCAATGCGCCTAGA-3’, MPSP probe: 5’FAM/TCGACAAGT/Zen/TCTCACCAC/IBFQ-3’ (Bogema et al., 2015); β-actin F primer: 5’-AAGTACTCCGTGTGGATTGGC-3’, β-actin R primer: 5’-CGGACTCATCGTACTCCTGCTT-3’, β-actin probe: 5’-FAM/TCGCTG TCC/ZEN/ACCTTCCAGCAGATGT/IBFQ-3’. To identify the T. orientalis subtype, the MPSP forward and reverse primer and the subtype specific probes (Ikeda and Chitose) were used in the PCR-positive calves and their dams. The subtype specific probes were as follow: T. orientalis Ikeda: FAM-CATGAACAG/Zen/TGCTTGGC/IBFQ-3’, T. orientalis Chitose: FAM-TCCTCRGCG/Zen/CTGTTCT/IBFQ-3’ (Bogema et al., 2015). Standard curves were generated using the pGEM-T Easy Vector (Promega, Madison, USA) containing the corresponding MPSP or βactin insert. The pGEM-MPSP and pGEM-actin plasmids were doubledigested by restriction enzymes (PstI and SacI) for linearizing, and tenfold serial dilutions prepared with EASY Dilution Solution for RealTime PCR (TaKaRa Bio), ranging from 1 × 102 to 1 × 105 gene copies/ μL, were used for standard curves. The T. orientalis parasite load was expressed as the number of MPSP copies divided by the number of βactin copies and multiplied by 1000. All quantitative real-time PCR tests were conducted in duplicate wells.
2.1. Sampling farm This study was conducted at a dairy and beef cattle production farm in Kyushu, Japan. This farm stopped grazing cattle on a piece of pasture land located away from the farm 7 years ago. Since then, the incidence of bovine theileriosis has sharply decreased, and ticks have been confirmed to be absent on this farm. However, our preliminary survey found that many cattle imported from Australia were infected with T. orientalis without exhibiting any signs, and less than 10% of the cattle born to imported dams were also infected. Therefore, we strongly suspected that vertical or in-house horizontal transmission of T. orientalis occurred. 2.2. Sample information The cattle used in this study were mainly Holstein and barely Brown Swiss cattle. Blood samples were collected from dams 3 months prior to delivery and placed into tubes containing ethylenediaminetetraacetic acid (EDTA) to screen for T. orientalis infection using the direct PCR method described below. After molecular diagnosis, blood samples were obtained from 204 PCR-positive dams and 211 calves (7 sampled dams had twins) within 24 h of birth. Blood samples were also corrected from the calves at 30 days of age. Additionally, 31 and 24 calves born to direct PCR-positive and PCR-negative dams, respectively, were sampled at 3 and 5 months of age to conduct a follow-up survey. 2.3. Blood test Blood counts were performed on the blood samples collected from the dams immediately after delivery. Numbers of red blood cells (RBCs) and hematocrit (Ht) values were assessed using an automated veterinary hematology analyzer (MEK-6550 Celltac α, Nihon Kohden, Tokyo, Japan). All blood samples were kept refrigerated, and blood counts were determined within five days of obtaining each blood sample. Anemia was diagnosed if both the number of RBCs and the Ht value were less than 5.1 × 106 cells/μL and 22%, respectively (George et al., 2010). Blood smears of anemic dams were stained with Diff-Quik (Sysmex, Kobe, Japan) and examined under a light microscope to detect piroplasms.
2.6. Statistical analysis The D’Agostino-Pearson normality test was used to verify the normal distribution of the parasite load, the number of RBCs and Ht value. The parasite load, the number of RBCs and Ht value were distributed lognormal, normal and non-normal, respectively. Therefore, Student’s t-test, Pearson correlation coefficient and nonparametric Spearman correlation analysis were used to compare the mean parasite load with one another, the parasite load with the number of RBCs and the parasite load with the Ht value, respectively. These analyses were performed using GraphPad Prism 6 software (GraphPad Software, La Jolla, USA). P < 0.05 was considered statistically significant in this study.
2.4. Blood direct PCR Blood samples taken from dams 3 months prior to delivery, those from the newborn calves and those from the calves in the follow-up survey were diluted 10-fold with distilled water. After freezing and thawing, the samples were assessed using blood direct PCR to detect the T. orientalis p23 gene. Amplification of this gene was performed in a reaction mixture containing 10 μL of 2× Ampdirect Plus (Shimadzu, Kyoto, Japan), which is superior for amplification from crude samples, 0.1 μL of BIOTAQ TM HS DNA polymerase (Bioline, London, UK), specific primers at 0.5 μM (p23-F: 5’-GTACACACCTTGAAATCTGGC-3’ and p23-R: 5’-CAAGAGAGGCAACAACAACGA-3’) (Ota et al., 2009), 2 μL of 10x loading buffer (TaKaRa Bio, Kusatsu, Japan), 2 μL of the diluted blood sample and PCR-grade water for a final volume of 20 μL. Amplification was performed under the following conditions: an initial denaturation step at 95 °C for 10 min, followed by 40 cycles of denaturation at 94 °C for 30 s and annealing and extension at 66 °C for 1 min. Initially, sequence analysis was performed to confirm the specificity of the PCR products. Afterward, the size of each PCR product was confirmed by gel electrophoresis.
3. Results and discussion Based on the hematological examination, 5 (2.4%) dams were diagnosed with anemia. The dams diagnosed as anemic had a significantly higher mean parasite load than the dams without anemia (1,914.2 vs. 142.2, Student’s t-test; P < 0.01), and the parasite load was significantly correlated with the number of RBCs and the Ht value (Fig. 1). Therefore, T. orientalis infection was considered one of the reasons for the anemia found in cattle on this farm. Pregnancy and calving are high-risk factors for developing clinical theileriosis (Izzo et al., 2010). However, piroplasms were microscopically found in RBCs of anemic dams with very low levels of parasitemia (0.005–0.04%). Therefore, most of the sampled dams were thought to be asymptomatic carriers in the chronic phase of infection. To determine the rate of vertical transmission, we investigated the
2.5. DNA extraction and quantitative real-time PCR Genomic DNA was extracted from the blood of the PCR-positive 2
Veterinary Parasitology 263 (2018) 1–4
H. Mekata et al.
Fig. 1. Correlations between parasite load and the number of red blood cells and hematocrit value. Correlations between parasite load and (a) the number of red blood cells (RBCs) and (b) the hematocrit (Ht) value in Theileria orientalis-infected dams on the day of delivery. The solid line is a linear regression line between the logarithmic values of the parasite load and (a) the numbers of RBCs and (b) Ht values. The dotted line shows the cut-off values for anemia: < 5.1 × 106 cells/μL (a) and < 22% (b) (George et al., 2010). Correlations were analyzed by the Pearson correlation coefficient (a) and nonparametric Spearman correlation (b). Table 1 Changes in the Theileria orientalis positivity rate in calves born from T. orientalisinfected dams. Infection status of the dam
T. orientalis (-)
a
T. orientalis subtype
ID 136
ID 155
ID 163
T. orientalis positivity rate in calves Dam at birth
T. orientalis (+)
Table 2 The subtypes of Theileria orientalis in vertically infected calves and their dams.
0% (0/ 211) 0% (0/ 26)
30 days old 0% (0/ 211) 0% (0/26)
3 months old
a
Calf (age)
Dam
Calf
Dam
Calf
5 months old
a
9.6% (3 /31)
9.6% (3 /31)
0% (0/24)
0% (0/24)
Ikeda Chitose
+ +
3
5
+ +
+ +
+ –
3
5
+ –
+ –
+ +
3
5
+ –
+ –
against T. orientalis might control the parasitemia levels, keeping them undetectable for more than one month. Previous reports have suggested that T. orientalis is transmitted by some biting arthropods (Fujisaki et al., 1993). However, the follow-up survey was conducted from December to March, when blood-sucking arachnids are inactive. The sampled calves were raised without pasturing, and ticks were confirmed to be absent from the farm. For these reasons, in-house horizontal transmission seems unlikely in this study. However, it is difficult to completely rule out this route of infection. We also cannot exclude the possibility that T. orientalis was transmitted through the colostrum of infected dams. But, no study has confirmed the transmission of T. orientalis by this route. A previous study reported that there was no relationship between the infection status of dams at calving and that of their calves at 4 months of age (Lawrence et al., 2016). This study may have been affected by the environment where 88% of 4-month-old calves were infected with T. orientalis. The environment like this might mask the true relationship. The research was conducted under conditions involving limited horizontal transmission of T. orientalis by blood-sucking insects or via the iatrogenic route. Although infection with T. orientalis was not detected in calves born to infected dams 0–30 days postdelivery, 9.6% of the calves became PCR positive at three months of age. These results suggest that the vertical transmission of T. orientalis can take three months to become detectable by PCR and that the frequency of vertical transmission may be higher than previously reported (Lawrence et al., 2016; Swilks et al., 2017). Further investigations are required to identify the exact vertical transmission routes of this parasite and to elucidate the underlying mechanisms to establish effective methods for preventing vertical infections.
Same calves were PCR positive at 3 and 5 months old.
211 samples collected within 24 h from newborn calves born to T. orientalis PCR-positive dams. All samples were confirmed to be PCR negative. In addition, all samples collected at 30 days after birth were confirmed to be PCR negative. It was reported that the transplacental transmission of T. orientalis infection would be rarely observed in chronically infected cattle (Swilks et al., 2017). Detection of T. orientalis in calves older than 1 month has also been reported (Lawrence et al., 2016) and therefore we conducted a follow-up survey. A total of 31 and 24 calves born to dams with and without T. orientalis infection, respectively, were tested for infection at the ages of 3 and 5 months. No calves born to dams without T. orientalis infection became PCR positive. However, 3 (9.6%) calves born to dams with T. orientalis infection tested positive at 3 and 5 months of age (Table 1). Although significant difference was not obtained due to the small sample size, the mean parasite load of the dams whose offspring became positive was higher than that of the dams whose offspring remained negative (148.9 vs. 87.7, Student's t-test; P = 0.40). The subtypes of T. orientalis found in the 3 positive calves (Dam and calf ID: 136, 155 and 163) and 30 dams (1 sampled dam had twins) were identified. T. orientalis Ikeda and Chitose subtype were vertically transferred to 13.0% (3/23) and 5.5% (1/18) of the calves, respectively. The infection subtype in two dam and calf pairs (ID 136 and 155) were consistent between each other (Table 2). Although another dam (ID 163) was coinfected with both the Ikeda and Chitose subtypes, its calf was only infected with the Ikeda subtype. A previous study reported that T. orientalis could become detectable by PCR after 5 months under extremely low mechanical transmission (Hammer et al., 2016). Most of the sampled dams were thought to be asymptomatic carriers in this study. Therefore, the vertically transferred parasite load might be low and thus a longer interval of time to become detectable. In addition, maternal antibodies against other Theileria species ingested with colostrum were demonstrated to be present for more than 2 months (Kumar et al., 2008; Toye et al., 2013). Therefore, maternal antibodies
Acknowledgment This study was supportedby the Honkawa Ranch Research Grant, Japan [grant number HRRG-17-B-02].
3
Veterinary Parasitology 263 (2018) 1–4
H. Mekata et al.
References
Liu, A.H., Guan, G.Q., Liu, J.L., Liu, Z.J., Leblanc, N., Li, Y.Q., Gao, J.L., Ma, M.L., Niu, Q.L., Ren, Q.Y., Bai, Q., Yin, H., Luo, J.X., 2011. Polymorphism analysis of Chinese Theileria sergenti using allele-specific polymerase chain reaction of the major piroplasm surface protein gene. J. Parasitol. 97, 116–121. Ota, N., Mizuno, D., Kuboki, N., Igarashi, I., Nakamura, Y., Yamashina, H., Hanzaike, T., Fujii, K., Onoe, S., Hata, H., Kondo, S., Matsui, S., Koga, M., Matsumoto, K., Inokuma, H., Yokoyama, N., 2009. Epidemiological survey of Theileria orientalis infection in grazing cattle in the eastern part of Hokkaido, Japan. J. Vet. Med. Sci. 71, 937–944. Sato, Y., Maeno, K., Saito, A., Nakase, A., Yokoyama, N., Inokuma, H., 2012. Vertical transmission of Theileria orientalis in dairy Holstein cattle (in Japanese). J. Hokkaido Vet. Med. Assoc. 56, 615–618. Sudan, V., Singh, S.K., Jaiswal, A.K., Parashar, R., Shanker, D., 2015. First molecular evidence of the transplacental transmission of Theileria annulata. Trop. Anim. Health Prod. 47, 1213–1215. Swilks, E., Fell, S.A., Hammer, J.F., Sales, N., Krebs, G.L., Jenkins, C., 2017. Transplacental transmission of Theileria orientalis occurs at a low rate in field-affected cattle: infection in utero does not appear to be a major cause of abortion. Parasites Vectors 10, 227. Toussaint, J.F., Sailleau, C., Breard, E., Zientara, S., De Clercq, K., 2007. Bluetongue virus detection by two real-time RT-qPCRs targeting two different genomic segments. J. Virol. Methods 140, 115–123. Toye, P., Handel, I., Gray, J., Kiara, H., Thumbi, S., Jennings, A., van Wyk, I.C., Ndila, M., Hanotte, O., Coetzer, K., Woolhouse, M., Bronsvoort, M., 2013. Maternal antibody uptake, duration and influence on survival and growth rate in a cohort of indigenous calves in a smallholder farming system in western Kenya. Vet. Immunol. Immunopathol. 155, 129–134. Yokoyama, N., Sivakumar, T., Ota, N., Igarashi, I., Nakamura, Y., Yamashina, H., Matsui, S., Fukumoto, N., Hata, H., Kondo, S., Oshiro, M., Zakimi, S., Kuroda, Y., Kojima, N., Matsumoto, K., Inokuma, H., 2012. Genetic diversity of Theileria orientalis in tick vectors detected in Hokkaido and Okinawa, Japan. Infect. Genet. Evol. 12, 1669–1675.
Baek, B.K., Soo, K.B., Kim, J.H., Hur, J., Lee, B.O., Jung, J.M., Onuma, M., Oluoch, A.O., Kim, C.-H., Kakoma, I., 2003. Verification by polymerase chain reaction of vertical transmission of Theileria sergenti in cows. Can. J. Vet. Res. 67, 278–282. Bogema, D.R., Deutscher, A.T., Fell, S., Collins, D., Eamens, G.J., Jenkins, C., 2015. Development and validation of a quantitative PCR assay using multiplexed hydrolysis probes for detection and quantification of Theileria orientalis isolates and differentiation of clinically relevant subtypes. J. Clin. Microbiol. 53, 941–950. Chhabra, S., Ranjan, R., Uppal, S.K., Singla, L.D., 2012. Transplacental transmission of Babesia equi (Theileria equi) from carrier mares to foals. J. Parasit. Dis. 36, 31–33. Eamens, G.J., Gonsalves, J.R., Jenkins, C., Collins, D., Bailey, G., 2013. Theileria orientalis MPSP types in Australian cattle herds associated with outbreaks of clinical disease and their association with clinical pathology findings. Vet. Parasitol. 191, 209–217. Fujisaki, K., Kamio, T., Kawazu, S., Shimizu, S., Simura, K., 1993. Theileria sergenti: experimental transmission by the long-nosed cattle louse, Linognathus vituli. Ann. Trop. Med. Parasitol. 87, 217–218. George, J.W., Snipes, J., Lane, V.M., 2010. Comparison of bovine hematology reference intervals from 1957 to 2006. Vet. Clin. Pathol. 39, 138–148. Hammer, J.F., Jenkins, C., Bogema, D., Emery, D., 2016. Mechanical transfer of Theileria orientalis: possible roles of biting arthropods, colostrum and husbandry practices in disease transmission. Parasit. Vectors 9, 34. Izzo, M.M., Poe, I., Horadagoda, N., De Vos, A.J., House, J.K., 2010. Haemolytic anaemia in cattle in NSW associated with Theileria infections. Aust. Vet. J. 88, 45–51. Kubota, S., Sugimoto, C., Onuma, M., 1996. Population dynamics of Theileria sergenti in persistently infected cattle and vector ticks analysed by a polymerase chain reaction. Parasitology 112, 437–442. Kumar, S., Kumar, R., Gupta, A.K., Dwivedi, S.K., 2008. Passive transfer of Theileria equi antibodies to neonate foals of immune tolerant mares. Vet. Parasitol. 151, 80–85. Lawrence, K.E., Gedye, K., McFadden, A.M.J., Pulford, D.J., Pomroy, W.E., 2016. An observational study of the vertical transmission of Theileria orientalis (Ikeda) in a New Zealand pastoral dairy herd. Vet. Parasitol. 218, 59–65.
4
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short communication
Evaluation of the natural vertical transmission of Theileria orientalis a,b,⁎
c
c
a
Hirohisa Mekata , Tomoya Minamino , Yoko Mikurino , Mari Yamamoto , Ayako Yoshida Nariaki Nonakab,d, Yoichiro Horiib,c
T b,d
,
a
Organization for Promotion of Tenure Track, University of Miyazaki, 1-1 Gakuen-Kibanadai-Nishi, Miyazaki 8892192, Japan Center for Animal Disease Control, University of Miyazaki, 1-1 Gakuen-Kibanadai-Nishi, Miyazaki 8892192, Japan Divisions of Research & Education for Livestock and Veterinary Clinic, Honkawa Ranch, Takase, Hita 8770056, Japan d Laboratory of Veterinary Parasitic Diseases, Department of Veterinary Sciences, Faculty of Agriculture, University of Miyazaki, 1-1 Gakuen-Kibanadai-Nishi, Miyazaki 8892192, Japan b c
A R T I C LE I N FO
A B S T R A C T
Keywords: Parasite load PCR T. orientalis Vertical transmission
Bovine theileriosis, caused by Theileria orientalis, is endemic from East Asia to Oceania. Even though the disease is mainly transmitted by Haemaphysalis ticks, the T. orientalis parasite can also be transmitted vertically. To develop proper control measures, the frequency of each transmission route must be elucidated. However, the frequency of vertical transmission, including transplacental transmission, of T. orientalis in naturally infected cattle is still controversial. This study aimed to clarify the frequency of the vertical transmission of T. orientalis in naturally infected cattle. Blood samples were collected from 204 T. orientalis-infected dams and their 211 newborn calves (including 7 sets of twins) within the first 24 h as well as 30 days after birth. Furthermore, 31 and 24 calves born to T. orientalis-infected and uninfected dams, respectively, were continuously surveyed for infection until 5 months of age. A total of 5 (2.4%) dams were diagnosed with mild anemia, whereas most of the dams were asymptomatic based on hematological examination and clinical signs. PCR analysis was performed on whole blood to determine the presence of T. orientalis in calves, and no calves were PCR positive 0 and 30 days after birth. However, 9.6% and 0% of the calves born to T. orientalis-infected and uninfected dams, respectively, tested positive at 3 and 5 months of age. The sampled calves were fed in-house, and the survey was conducted during the cold season; thus, horizontal transmission through blood-sucking insects rarely occurred. Therefore, the vertical transmission of T. orientalis took as long as 3 months to become detectable by PCR and occurred in approximately 10% of field cattle.
1. Introduction
maff.go.jp/j/chikusan/sinko/shiryo/houboku/houboku.html; in Japanese). Without parasite control, this approach will result in an increasing incidence of Theileria infection in cattle. Transplacental transmission has been detected for T. orientalis (Baek et al., 2003) and a number of other Theileria species, such as T. equi (Chhabra et al., 2012) and T. annulata (Sudan et al., 2015), by PCR analysis. However, the frequency of vertical transmission, including transplacental transmission, of T. orientalis in the field remains controversial. A Japanese study reported that 10% (3/30) of newborn calves were infected via this route (Sato et al., 2012). In contrast, studies conducted in Australia and New Zealand have reported that vertical transmission may occur but at a very low rate in the field (Lawrence et al., 2016; Swilks et al., 2017). The purpose of this study was to investigate the frequency of vertical transmission of T. orientalis from dams to calves under routine conditions. This study may aid in the development of control measures for bovine theileriosis.
Theileria orientalis is a tick-borne protozoan parasite and an etiological agent of benign theileriosis in cattle. This parasite is distributed from East Asia to Oceania and is an economically important pathogen (Eamens et al., 2013; Liu et al., 2011; Yokoyama et al., 2012). The Theileria infection is often asymptomatic but may cause signs, including fever, anemia and abortion, in cattle under a stressful situation. No effective drugs or vaccines are currently available for the control of theileriosis. Once cattle are infected, they appear to harbor the parasite for an extended period of time (Kubota et al., 1996). Therefore, screening for T. orientalis infection in cattle before pasturing and keeping ticks free of Theileria on pasture land are important strategies to minimize parasite transmission and expansion. However, the Japanese government promotes cattle grazing on pasture land to reduce rearing costs and workloads and to utilize abandoned farmlands (http://www.
⁎
Corresponding author at: Organization for Promotion of Tenure Track, University of Miyazaki, 1-1 Gakuen-Kibanadai-Nishi, Miyazaki 8892192, Japan. E-mail address: [email protected] (H. Mekata).
https://doi.org/10.1016/j.vetpar.2018.09.017 Received 7 May 2018; Received in revised form 26 September 2018; Accepted 29 September 2018 0304-4017/ © 2018 Elsevier B.V. All rights reserved.
Veterinary Parasitology 263 (2018) 1–4
H. Mekata et al.
2. Materials and methods
dams immediately after delivery using the Wizard Genomic DNA Purification Kit (Promega, Fitchburg, USA) and from the 10-fold diluted blood of PCR-positive calves after 3 and 5 months of age using the magLead system (Precision System Science, Chiba, Japan) according to the manufacturer’s instructions. The DNA concentration was determined using a NanoDrop 8000 spectrophotometer (Thermo Fisher Scientific, Waltham, USA), and samples were diluted to 20 ng/μL. Quantitative real-time PCR was performed using a LightCycler 96 system (Roche Diagnostics, Indianapolis, USA). A probe qPCR mix (TaKaRa Bio) and PrimeTime qPCR assays (Integrated DNA Technologies, Coralville, USA), including target gene-specific primers and probes, were used to quantify the copies number of T. orientalis MPSP and bovine β-actin exon 5 genes. The forward primer for β-actin exon 5 was modified from a previous report to detect only the exon sequence (Toussaint et al., 2007). The following primer and probe sequences were used: MPSP F primer: 5’-GCAAACAAGGATTTGCACGC-3’, MPSP R primer: 5’-TGTGAGACTCAATGCGCCTAGA-3’, MPSP probe: 5’FAM/TCGACAAGT/Zen/TCTCACCAC/IBFQ-3’ (Bogema et al., 2015); β-actin F primer: 5’-AAGTACTCCGTGTGGATTGGC-3’, β-actin R primer: 5’-CGGACTCATCGTACTCCTGCTT-3’, β-actin probe: 5’-FAM/TCGCTG TCC/ZEN/ACCTTCCAGCAGATGT/IBFQ-3’. To identify the T. orientalis subtype, the MPSP forward and reverse primer and the subtype specific probes (Ikeda and Chitose) were used in the PCR-positive calves and their dams. The subtype specific probes were as follow: T. orientalis Ikeda: FAM-CATGAACAG/Zen/TGCTTGGC/IBFQ-3’, T. orientalis Chitose: FAM-TCCTCRGCG/Zen/CTGTTCT/IBFQ-3’ (Bogema et al., 2015). Standard curves were generated using the pGEM-T Easy Vector (Promega, Madison, USA) containing the corresponding MPSP or βactin insert. The pGEM-MPSP and pGEM-actin plasmids were doubledigested by restriction enzymes (PstI and SacI) for linearizing, and tenfold serial dilutions prepared with EASY Dilution Solution for RealTime PCR (TaKaRa Bio), ranging from 1 × 102 to 1 × 105 gene copies/ μL, were used for standard curves. The T. orientalis parasite load was expressed as the number of MPSP copies divided by the number of βactin copies and multiplied by 1000. All quantitative real-time PCR tests were conducted in duplicate wells.
2.1. Sampling farm This study was conducted at a dairy and beef cattle production farm in Kyushu, Japan. This farm stopped grazing cattle on a piece of pasture land located away from the farm 7 years ago. Since then, the incidence of bovine theileriosis has sharply decreased, and ticks have been confirmed to be absent on this farm. However, our preliminary survey found that many cattle imported from Australia were infected with T. orientalis without exhibiting any signs, and less than 10% of the cattle born to imported dams were also infected. Therefore, we strongly suspected that vertical or in-house horizontal transmission of T. orientalis occurred. 2.2. Sample information The cattle used in this study were mainly Holstein and barely Brown Swiss cattle. Blood samples were collected from dams 3 months prior to delivery and placed into tubes containing ethylenediaminetetraacetic acid (EDTA) to screen for T. orientalis infection using the direct PCR method described below. After molecular diagnosis, blood samples were obtained from 204 PCR-positive dams and 211 calves (7 sampled dams had twins) within 24 h of birth. Blood samples were also corrected from the calves at 30 days of age. Additionally, 31 and 24 calves born to direct PCR-positive and PCR-negative dams, respectively, were sampled at 3 and 5 months of age to conduct a follow-up survey. 2.3. Blood test Blood counts were performed on the blood samples collected from the dams immediately after delivery. Numbers of red blood cells (RBCs) and hematocrit (Ht) values were assessed using an automated veterinary hematology analyzer (MEK-6550 Celltac α, Nihon Kohden, Tokyo, Japan). All blood samples were kept refrigerated, and blood counts were determined within five days of obtaining each blood sample. Anemia was diagnosed if both the number of RBCs and the Ht value were less than 5.1 × 106 cells/μL and 22%, respectively (George et al., 2010). Blood smears of anemic dams were stained with Diff-Quik (Sysmex, Kobe, Japan) and examined under a light microscope to detect piroplasms.
2.6. Statistical analysis The D’Agostino-Pearson normality test was used to verify the normal distribution of the parasite load, the number of RBCs and Ht value. The parasite load, the number of RBCs and Ht value were distributed lognormal, normal and non-normal, respectively. Therefore, Student’s t-test, Pearson correlation coefficient and nonparametric Spearman correlation analysis were used to compare the mean parasite load with one another, the parasite load with the number of RBCs and the parasite load with the Ht value, respectively. These analyses were performed using GraphPad Prism 6 software (GraphPad Software, La Jolla, USA). P < 0.05 was considered statistically significant in this study.
2.4. Blood direct PCR Blood samples taken from dams 3 months prior to delivery, those from the newborn calves and those from the calves in the follow-up survey were diluted 10-fold with distilled water. After freezing and thawing, the samples were assessed using blood direct PCR to detect the T. orientalis p23 gene. Amplification of this gene was performed in a reaction mixture containing 10 μL of 2× Ampdirect Plus (Shimadzu, Kyoto, Japan), which is superior for amplification from crude samples, 0.1 μL of BIOTAQ TM HS DNA polymerase (Bioline, London, UK), specific primers at 0.5 μM (p23-F: 5’-GTACACACCTTGAAATCTGGC-3’ and p23-R: 5’-CAAGAGAGGCAACAACAACGA-3’) (Ota et al., 2009), 2 μL of 10x loading buffer (TaKaRa Bio, Kusatsu, Japan), 2 μL of the diluted blood sample and PCR-grade water for a final volume of 20 μL. Amplification was performed under the following conditions: an initial denaturation step at 95 °C for 10 min, followed by 40 cycles of denaturation at 94 °C for 30 s and annealing and extension at 66 °C for 1 min. Initially, sequence analysis was performed to confirm the specificity of the PCR products. Afterward, the size of each PCR product was confirmed by gel electrophoresis.
3. Results and discussion Based on the hematological examination, 5 (2.4%) dams were diagnosed with anemia. The dams diagnosed as anemic had a significantly higher mean parasite load than the dams without anemia (1,914.2 vs. 142.2, Student’s t-test; P < 0.01), and the parasite load was significantly correlated with the number of RBCs and the Ht value (Fig. 1). Therefore, T. orientalis infection was considered one of the reasons for the anemia found in cattle on this farm. Pregnancy and calving are high-risk factors for developing clinical theileriosis (Izzo et al., 2010). However, piroplasms were microscopically found in RBCs of anemic dams with very low levels of parasitemia (0.005–0.04%). Therefore, most of the sampled dams were thought to be asymptomatic carriers in the chronic phase of infection. To determine the rate of vertical transmission, we investigated the
2.5. DNA extraction and quantitative real-time PCR Genomic DNA was extracted from the blood of the PCR-positive 2
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Fig. 1. Correlations between parasite load and the number of red blood cells and hematocrit value. Correlations between parasite load and (a) the number of red blood cells (RBCs) and (b) the hematocrit (Ht) value in Theileria orientalis-infected dams on the day of delivery. The solid line is a linear regression line between the logarithmic values of the parasite load and (a) the numbers of RBCs and (b) Ht values. The dotted line shows the cut-off values for anemia: < 5.1 × 106 cells/μL (a) and < 22% (b) (George et al., 2010). Correlations were analyzed by the Pearson correlation coefficient (a) and nonparametric Spearman correlation (b). Table 1 Changes in the Theileria orientalis positivity rate in calves born from T. orientalisinfected dams. Infection status of the dam
T. orientalis (-)
a
T. orientalis subtype
ID 136
ID 155
ID 163
T. orientalis positivity rate in calves Dam at birth
T. orientalis (+)
Table 2 The subtypes of Theileria orientalis in vertically infected calves and their dams.
0% (0/ 211) 0% (0/ 26)
30 days old 0% (0/ 211) 0% (0/26)
3 months old
a
Calf (age)
Dam
Calf
Dam
Calf
5 months old
a
9.6% (3 /31)
9.6% (3 /31)
0% (0/24)
0% (0/24)
Ikeda Chitose
+ +
3
5
+ +
+ +
+ –
3
5
+ –
+ –
+ +
3
5
+ –
+ –
against T. orientalis might control the parasitemia levels, keeping them undetectable for more than one month. Previous reports have suggested that T. orientalis is transmitted by some biting arthropods (Fujisaki et al., 1993). However, the follow-up survey was conducted from December to March, when blood-sucking arachnids are inactive. The sampled calves were raised without pasturing, and ticks were confirmed to be absent from the farm. For these reasons, in-house horizontal transmission seems unlikely in this study. However, it is difficult to completely rule out this route of infection. We also cannot exclude the possibility that T. orientalis was transmitted through the colostrum of infected dams. But, no study has confirmed the transmission of T. orientalis by this route. A previous study reported that there was no relationship between the infection status of dams at calving and that of their calves at 4 months of age (Lawrence et al., 2016). This study may have been affected by the environment where 88% of 4-month-old calves were infected with T. orientalis. The environment like this might mask the true relationship. The research was conducted under conditions involving limited horizontal transmission of T. orientalis by blood-sucking insects or via the iatrogenic route. Although infection with T. orientalis was not detected in calves born to infected dams 0–30 days postdelivery, 9.6% of the calves became PCR positive at three months of age. These results suggest that the vertical transmission of T. orientalis can take three months to become detectable by PCR and that the frequency of vertical transmission may be higher than previously reported (Lawrence et al., 2016; Swilks et al., 2017). Further investigations are required to identify the exact vertical transmission routes of this parasite and to elucidate the underlying mechanisms to establish effective methods for preventing vertical infections.
Same calves were PCR positive at 3 and 5 months old.
211 samples collected within 24 h from newborn calves born to T. orientalis PCR-positive dams. All samples were confirmed to be PCR negative. In addition, all samples collected at 30 days after birth were confirmed to be PCR negative. It was reported that the transplacental transmission of T. orientalis infection would be rarely observed in chronically infected cattle (Swilks et al., 2017). Detection of T. orientalis in calves older than 1 month has also been reported (Lawrence et al., 2016) and therefore we conducted a follow-up survey. A total of 31 and 24 calves born to dams with and without T. orientalis infection, respectively, were tested for infection at the ages of 3 and 5 months. No calves born to dams without T. orientalis infection became PCR positive. However, 3 (9.6%) calves born to dams with T. orientalis infection tested positive at 3 and 5 months of age (Table 1). Although significant difference was not obtained due to the small sample size, the mean parasite load of the dams whose offspring became positive was higher than that of the dams whose offspring remained negative (148.9 vs. 87.7, Student's t-test; P = 0.40). The subtypes of T. orientalis found in the 3 positive calves (Dam and calf ID: 136, 155 and 163) and 30 dams (1 sampled dam had twins) were identified. T. orientalis Ikeda and Chitose subtype were vertically transferred to 13.0% (3/23) and 5.5% (1/18) of the calves, respectively. The infection subtype in two dam and calf pairs (ID 136 and 155) were consistent between each other (Table 2). Although another dam (ID 163) was coinfected with both the Ikeda and Chitose subtypes, its calf was only infected with the Ikeda subtype. A previous study reported that T. orientalis could become detectable by PCR after 5 months under extremely low mechanical transmission (Hammer et al., 2016). Most of the sampled dams were thought to be asymptomatic carriers in this study. Therefore, the vertically transferred parasite load might be low and thus a longer interval of time to become detectable. In addition, maternal antibodies against other Theileria species ingested with colostrum were demonstrated to be present for more than 2 months (Kumar et al., 2008; Toye et al., 2013). Therefore, maternal antibodies
Acknowledgment This study was supportedby the Honkawa Ranch Research Grant, Japan [grant number HRRG-17-B-02].
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