Accepted Manuscript

Title: Neonatal Anaplasma platys Infection in Puppies: Further Evidence for

Possible Vertical Transmission

Author: I.A. Matei, S. Stuen, D. Modrý, A. Degan, G. D'Amico, A.D. Mihalca

PII: S1090-0233(16)30215-5
DOI: http://dx.doi.org/doi: 10.1016/j.tvjl.2016.12.005
Reference: YTVJL 4920

To appear in: The Veterinary Journal

Accepted date: 7-12-2016

Please cite this article as: I.A. Matei, S. Stuen, D. Modrý, A. Degan, G. D'Amico, A.D. Mihalca,
Neonatal Anaplasma platys Infection in Puppies: Further Evidence for Possible Vertical
Transmission, The Veterinary Journal (2016), http://dx.doi.org/doi: 10.1016/j.tvjl.2016.12.005.

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 1 Neonatal Anaplasma platys infection in puppies: Further evidence for possible vertical
2 transmission
3
4
5 I.A. Matei a, S. Stuen b, D. Modrý c,d,e, A. Degan a, G. D’Amico a,*, A.D. Mihalca a
6
a
7 Department of Parasitology and Parasitic Diseases, University of Agricultural Sciences and
8 Veterinary Medicine, Cluj-Napoca, Romania
b
9 Department of Production Animal Clinical Sciences, Section of Small Ruminant Research,
10 Norwegian University of Life Sciences (NMBU), Sandnes, Norway
c
11 Department of Pathology and Parasitology, University of Veterinary and Pharmaceutical
12 Sciences, Brno, Czech Republic
d
13 CEITEC VFU, University of Veterinary and Pharmaceutical Sciences, Brno, Czech Republic.
e
14 Institute of Parasitology, Biology Centre of Academy of Sciences of the Czech Republic, České
15 Budějovice, Czech Republic
16
17
18
19
20 * Corresponding author. Tel.: +40 755 369153.
21 E-mail address: gianluca.damico@usamvcluj.ro (G. D’Amico).

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22 Highlights

23  Puppies born from some infected bitches are positive for Anaplasma platys by PCR

24 without evidence of tick infestation.

25  Vertical transmission of Anaplasma platys may occur in dogs, either transplacentally or

26 in the perinatal period.

27

28 Abstract

29 Anaplasma platys, the aetiological agent of infectious canine cyclic thrombocytopaenia,

30 infects platelets of dogs, usually causing mild or asymptomatic disease. Although A. platys is

31 transmitted by ticks, as for other Anaplasma species, alternative modes of transmission may be

32 involved. The aim of the present study was to evaluate the occurrence of A. platys infection in

33 litters of puppies, which could suggest possible vertical transmission. Twelve litters, together

34 with the respective bitches, were included in the study for the detection of A. platys DNA by

35 PCR, followed by sequencing. Five puppies, from 2/4 litters < 28 days of age, tested positive for

36 A. platys DNA. No puppies from eight litters 1-3 months of age tested positive for A. platys

37 DNA. The identical sequences (16S rRNA and gltA partial gene), the absence of ticks on puppies

38 at the time of collection and the young age of the five infected puppies suggest vertical

39 transmission of A. platys. This mode of transmission might contribute to the maintenance and

40 spread of the pathogen in canine populations.

41

42 Keywords: Anaplasma platys; Canine; Vertical transmission

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43 Vertical transmission of tick-borne pathogens in mammalian hosts plays an important

44 role in the maintenance and spread of infectious agents, especially when the pathogen has a low

45 virulence and is not associated with mortality of the reservoir host (Jasik et al., 2015).

46 Anaplasma platys causes infectious canine cyclic thrombocytopaenia and is probably transmitted

47 by the brown dog tick, Rhipicephalus sanguineus sensu lato (Harvey et al., 1978; Sanogo et al.,

48 2003). As with other tick-borne pathogens, A. platys might also be transmitted by vertical

49 transmission, as was recently suggested in dog foetuses in the first period of gestation (Latrofa et

50 al., 2016); however, evidence of vertical transmission has not been shown in puppies previously.

51

52 We evaluated the presence of A. platys DNA in 12 litters of young puppies and their

53 dams in January 2016 in Northern Kenya. We divided the litters into two age groups (< 28 days

54 and 28-90 days of age) to determine if infections were likely to be prenatal or postnatal. The

55 presence of ticks on each dam and pup, and in the environment around shelters, was recorded for

56 each dog (see Appendix: Supplementary Table 1). Blood was collected from the cephalic vein

57 using sterile syringes and then transferred into tubes containing ethanol for storage. This study

58 was approved by the bioethics committee of the University of Agricultural Sciences and

59 Veterinary Medicine of Cluj-Napoca, Romania, with the registration number 23 from 21st

60 September 2015, following the EU 2010/63 and National directives Order 28/31-08-2011 and

61 National Law 206/2004. Our study was part of an international collaborative project focused on

62 preventive veterinary medicine (Mt Kulal Dogs’ Preventive Vaccination) coordinated by

63 Vétérinaires Sans Frontières (VSF) Czech Republic.

64

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65 Genomic DNA was extracted from ethanol-preserved blood using commercial kits

66 (Isolate II Genomic DNA Kit, Bioline). PCR was performed using primers amplifying fragments

67 of the 16S rRNA and gltA genes (Sanago et al., 2003; Matei et al., 2016). Positive and negative

68 controls were included to assess the specificity of the reaction and the possible presence of

69 contaminants. Products were visualised by agarose gel electrophoresis and sequences (Macrogen

70 Europe, Amsterdam) were compared with those available in GenBank by Basic Local

71 Alignments Tool (BLAST) analysis1.

72

73 Of 44 dogs included in the study, seven (two dams and 5/11 puppies from their two

74 litters) tested positive for A. platys DNA (see Appendix: Supplementary Table 1). No infected

75 dams had litters that tested negative and no puppies that tested positive came from dams that

76 tested negative. The PCR positive puppies were < 28 days old had not been treated for

77 ectoparasites. However, no ticks were found on either the dams or puppies at the time of

78 examination. DNA sequencing confirmed that puppies and their respective dams were likely to

79 be infected by the same A. platys variants; gltA sequences obtained from each dam and her

80 respective litter were identical and differed between litters only by one nucleotide. Both

81 sequences had 100% similarity with A. platys strains from Italy and Spain (GenBank DQ525687,

82 AY530807).

83

84 Our results strongly support vertical transmission of A. platys from infected bitches to

85 their puppies, either transplacentally or in the perinatal period. We cannot completely exclude

86 the possibility of transmission by tick bite; however, this seems to be unlikely, since the isolates

87 within litters were identical and no ticks were found on any dog. Since the PCR-positive puppies
1
See: http://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed 13 June 2016).

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 88 had no ticks at the time of sampling and considering that the feeding period of an adult R.

89 sanguineus sensu lato is at least 2 weeks under natural conditions (Dantas-Torres 2010), the

90 attachment of infected ticks would have had to occur in the first or second week of life in order

91 to be detached by the time of blood sampling. Due to the foraging behaviour of this tick (Dantas-

92 Torres 2010) and the absence of ticks inside or around the shelters, the attachment of an infected

93 tick is improbable, suggesting an alternative, vertical method of transmission. Our findings are

94 also supported by previous studies indicating transplacental transmission of A. phagocytophilum

95 in experimental infections in a sheep (Reppert et al., 2013) and a cow (Pusterla et al., 1997).

96

97 Vertical transmission in mammalian hosts can occur during pregnancy, at parturition or

98 the postpartum period. Anaplasma platys is an obligate intracellular pathogen and its presence

99 and replication has only been observed in platelets (Harvey et al., 1978). An extracellular phase

100 in mammalian blood has not been described, suggesting that a transfer of infected platelets is

101 required for vertical transmission. To our knowledge, platelets are not found in milk, so trans-

102 mammary transmission seems unlikely. Intrapartum transmission is also unlikely, because

103 persistently infected dogs display low levels of parasitaemia (Harvey et al., 1978). Therefore,

104 transplacental transmission appears to be most probable, supported by previous findings (Latrofa

105 et al., 2016). Although the placenta should be a barrier against infection, in a canine

106 endotheliochorial placenta, transmission could occur by extravasation of blood at the maternal-

107 foetal interface and the subsequent phagocytosis of infected platelets by trophoblast cells from

108 the marginal hematophagous zone, in a manner similar to the passage of maternal red cells in the

109 iron transport process (Oliveira et al., 2012).

110

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111 Whilst our results strongly suggest the vertical transmission of A. platys infection, the

112 field conditions of the study prevented serial sampling of animals, and we could not perform

113 quantitative PCR or serological testing for A. platys antigen. Therefore, additional confirmatory

114 studies are required to confirm our hypothesis of vertical transmission.

115

116 Acknowledgements

117 The study was conducted under EurNegVec COST Action TD1303 (grant number PN-II-

118 RU-TE-2014-4-0919: TE/298/2015) and supported in part by the Ministry of Education, Youth

119 and Sports of the Czech Republic (project COST CZ LD14048). We would like to express our

120 gratitude to all the pastoral communities from the study area, especially to our local helpers and

121 chiefs of the villages for kindly supporting our work. We would like to thank VSF Germany and

122 VSF Czech Republic for their unconditional logistical support. We would like to give special

123 thanks to Dragoș Bȋrţoiu, Alexandra Dregan, Szidonia Ballai, Andreea Ilişan and Ana

124 Zdrafcovici for their valuable help during the field work. Special thanks go also to the Prof. Paul

125 W.N. Kanyari and to Shukri Lasapicho.

126

127 Appendix: Supplementary material

128 Supplementary data associated with this article can be found, in the online version, at doi:

129 …

130

131 Conflict of interest statement

132 None of the authors of this paper have a financial or personal relationship with other

133 people or organisations that could inappropriately influence or bias the content of the paper.

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134

135 References

136 Dantas-Torres, F., 2010. Biology and ecology of the brown dog tick, Rhipicephalus sanguineus.
137 Parasites and Vectors 3, 26-37.
138
139 Harvey, J.W., Simpson, C.F., Gaskin, J.M., 1978. Cyclic thrombocytopenia induced by a
140 Rickettsia-like agent in dogs. Journal of Infectious Diseases 137, 182-188.
141
142 Jasik, K.P., Okła, H., Słodki, J., Rozwadowska, B., Słodki, A., Rupik, W., 2015. Congenital tick
143 borne diseases: Is this an alternative route of transmission of tick-borne pathogens in
144 mammals? Vector-Borne and Zoonotic Diseases 15, 637-644.
145
146 Latrofa, M.S., Dantas-Torres, F., de Caprariis, D., Cantacessi, C., Capelli, G., Lia, R.P.,
147 Breitschwerdt, E.B., Otranto, D., 2016. Vertical transmission of Anaplasma platys and
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160 Oliveira, C.M.D., Rodrigues, M.N., Miglino, M.A., 2012. Iron transportation across the placenta.
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