Altitude-Dependent Prevalence of Canine Granulocytic Anaplasmosis in Romania

Abstract

Canine granulocytic anaplasmosis (CGA) is an important tick-borne disease with worldwide distribution. The importance of this disease resides in the ability of Anaplasma phagocytophilum to infect humans and several animal species. The aim of the study was to evaluate the prevalence rate of CGA in different altitudinal areas of Romania. A total of 357 canine blood samples were collected during 2010–2013 from eight counties. To assess the influence of the altitude on A. phagocytophilum prevalence, the samples were collected from four different altitude areas (coastal 0–5 meters; lowland 6–100 meters; hilly areas 200–300 meters; low mountain areas >500 meters). These samples were evaluated for the presence of A. phagocytophilum DNA by amplifying part of the Ankyrin repeat protein (AnkA) gene. A higher prevalence was obtained for coastal compared with remaining areas, suggesting an influence of altitude on the CGA. Moreover, the results suggest an influence of climate and rainfall. In the present research work, we highlight the risk of granulocytic anaplasmosis in Central and Southern Romania, with a greater risk associated to Southern lowland region, especially in coastal areas. The importance of these results resides in the zoonotic potential of the canine A. phagocytophilum strains. In conclusion, the altitude and precipitation level may be risk factors for A. phagocytophilum infection in dogs and other hosts, including humans.

Introduction

Granulocytic anaplasmosis is an important tick-borne disease with a worldwide distribution, especially in the northern hemisphere (Stuen et al. 2013, Saintz et al. 2015). The etiologic agent is Anaplasma phagocytophilum (Chen et al. 1994), a Gram-negative bacterium, which belongs to the Anaplasmataceae family (Dumler et al. 2001).

Anaplasma phagocytophilum is well known as the agent of tick-borne fever in ruminants, equine granulocytic anaplasmosis in horses, canine granulocytic anaplasmosis (CGA) in domestic carnivores, and human granulocytic anaplasmosis (Dumler et al. 2001). The pathogen is maintained in nature through reservoir hosts, such as wild ruminants (Silaghi et al. 2011) and probably small mammals (Bown et al. 2008). Among these, the pathogen was detected in high rate in wild boars (Sus scrofa) (Michalik et al. 2012), red foxes (Vulpes vulpes) (Ebani et al. 2011), European brown bears (Ursus arctos) (Vichová et al. 2010), reptiles (Nieto et al. 2010), and birds (de la Fuente et al. 2005). However, the reservoir host status of these species is still under debate (Stuen et al. 2013).

The main vectors for A. phagocytophilum are the tick species belonging to the Ixodes persulcatus complex (Strle 2004), and in Europe, Ixodes ricinus is most commonly involved (Saintz et al. 2015). CGA was described for the first time in a dog originating from California in 1982 (Madewell and Gribble 1982). Since then, serological and molecular proof of infection in dogs and cats were reported in the Northern hemisphere, especially in United States, almost all European countries, and Northern Asia (Carrade et al. 2009, Stuen et al. 2013, Saintz et al. 2015). The majority of dogs naturally infected with A. phagocytophilum remain clinically healthy, but the screenings performed in asymptomatic dogs reveal a wide distribution of the pathogen (Beall et al. 2008).

In Romania, the agent of CGA was molecularly confirmed only in two hosts, dogs (Hamel et al. 2012) and wild boars (S. scrofa) (Kiss et al. 2014). Dogs have been suggested as important sentinels for tick-borne diseases due to free-roaming behavior of some of them, but also their increasing population and global traveling (Day 2011). Moreover, in case of A. phagocytophilum due to the persistent infection, dogs may be important sentinel species (Egenvall et al. 2000). Due to the genetic relatedness of canine and humans strains (Scharf et al. 2011), dogs could serve as sentinel species for monitoring the risk of human disease. Dogs spend majority of their life in close association with humans and they are coexposed to similar environments. Dogs can also provide a means by which infected ticks can be carried into the domestic environment (Hamer et al. 2009). However, no large studies regarding the epidemiology of CGA in Romania are available. With this in view, and considering also the recently suggested influence of altitude on A. phagocytophilum prevalence in questing ticks (Matei et al. 2015), we aim to evaluate the prevalence of A. phagocytophilum in dogs from different altitudinal areas in Romania.

Materials and Methods

Blood samples from 357 dogs were collected in 2011 and 2013 from the cephalic vein into sterile tubes filled with absolute ethanol. The identification data such as location, owner name, breed, age, and sex of the dog were recorded. The structure of the dog population is presented in Table 1. To compare the influence of altitude on the prevalence of CGA, the collection sites were chosen from four altitude intervals: coastal 0 to 5 meters (I), lowland 6–100 meters (zone II), hilly 200 to 400 meters (zone III), and low mountain >500 meters (zone IV) (Fig. 1). The climate of the studied region is diverse, with temperate steppe-continental influenced by the Black Sea in coastal regions (area I), medium temperate continental (III), and temperate continental with Mediterranean influences (II), or alpine influences (IV). All climatic data (mean annual and seasonal temperature, mean annual and seasonal rainfall and mean annual moisture) were downloaded from WorldClim database with the highest resolution provided, of 30 arc-seconds (one pixel equals cca. 0.6 km²) matching the sample locations of the study (Supplementary Table S1; Supplementary Data are available online at www.liebertpub.com/vbz).

FIG. 1.

Collection sites and altitudinal areas. Zone I–coastal area (●), 0–5 meters; zone II—lowland (●), 6–100 meters; zone III—hilly areas (■), 200–300 meters; zone IV—low mountain areas (▲), >500 meters.

Table 1.

Structure of Dog Population Taken in Study

Zone	Location	No.	F	M	Y	A	M.B	P.B	N	E	ALT
I	Chilia Veche	67	25	42	15	52	64	3	45.4140	29.2815	5
Total	—	67	25	42	15	52	64	3	—	—	—
II	Bechet	26	11	15	5	21	25	1	43.7864	23.9599	39
II	Călăraşi	25	10	15	4	21	22	3	43.7966	24.0519	56
II	Nanov	51	23	28	13	38	40	11	43.9886	25.2893	49
Total	—	102	44	58	22	80	87	15	—	—	—
III	Cugir	6	3	3	1	5	4	2	45.8613	23.3631	270
III	Noslac	31	11	20	3	28	25	6	46.3971	23.9350	308
III	Geoagiu	33	12	21	7	26	26	7	45.9172	23.1990	210
III	Orăştie	29	12	17	7	22	27	2	45.8488	23.1801	214
Total	—	99	38	61	18	81	82	17	—	—	—
IV	Cuca	46	18	28	17	29	43	3	45.0200	24.5835	544
IV	Goleşti	43	9	34	9	34	34	9	45.1297	24.4562	547
Total	—	89	27	62	26	63	77	12	—	—	—
Total	—	357	134	223	81	276	314	43	—	—	—

Structure of dog population according to locations, sex (F, females, M, males), age (Y under 1 year old; A-adult), breed (M.B, mixed breed; P.B, pure breed), and altitudinal zone (I coastal area 0–5 meters; II lowland, 6–100 meters; III hilly areas, 200–300 meters; IV low mountain areas, >500 meters).

ALT, altitude.

Genomic DNA was extracted from blood clots using a modified phenol–chloroform method according to described protocol (Albrechtová et al. 2011). For each extraction procedure, negative controls consisting of reaction mixes without DNA (PCR water instead of blood) were used, to check for possible cross-contamination. Isolated genomic DNA from a representative number of samples was assessed quantitatively by NanoDrop ND-1000 spectrophotometer analyzer (NanoDrop Technologies, Inc., Wilmington, DE).

PCR was initially performed using a group-specific set of primers that amplify a 345 bp fragment of the 16S rRNA gene. The primer sets EHR16SD (5′-GGTACCYACAGAAGA AGTCC-3′) and EHR16SR (5′-TAGCACTCATCGTTTACAGC-3′) amplify various species of Anaplasmataceae family (Parola et al. 2000). For all the positive samples, a second PCR was carried out using A. phagocytophilum-specific primers LA1/LA6 (forward primer: 5′-GAGAGATGCTTATGGTAAGAC-3′, and a reverse primer: 5′-CGTTCAGCCATCATTGTG AC-3′), amplifying a 444-bp fragment of ankA gene (Walls et al. 2000). The PCR was carried out using the T100™ Thermal Cycler (Bio-Rad) in 25 μL reaction mixture following the previously described optimal condition (Matei et al. 2015). In each PCR set (48 samples), a positive and a negative control were included to assess the specificity of the reaction and the possible presence of contaminants. Positive controls consisted of DNA isolated from I. ricinus ticks positive for A. phagocytophilum (GenBank acc. no. KP164412–KP164415). The reaction mix without DNA was used as negative control.

The total infection prevalence of A. phagocytophilum (95% CI), the infection prevalence based on age and sex of dogs, the infection prevalence in each location, and by altitudinal interval was assessed using the Fisher's exact test. The relationship between local climate conditions and A. phagocytophilum prevalence was tested using Pearson Rank correlation. Maps showing the collection sites and the altitudinal zone were generated using QGIS 2.6 software.

Results

The overall prevalence of A. phagocytophilum in dogs was 5.32% (19/357; 95% CI 3.32–8.33). The highest prevalence rate was obtained in Chilia Veche followed by Bechet (from the coastal and lowland areas, respectively) (Table 2). In the remaining localities, the prevalence rates were similar and all with values below 4% (Table 2). The prevalence in coastal area was significantly higher than the prevalence recorded in other regions (0.0198, p < 0.05), among which there were no significant differences recorded. According to our results, the sex, age, and breed are not risk factors in CGA, as the differences in prevalence between these categories were not statistically significant (Table 2). A risk factor that is suggested by our results is the geographical origin of the dogs. The difference in prevalence based on altitudinal zones have shown a statistical significance (Table 2), caused by the differences in climatic conditions. Analyzing together the prevalence in different localities and altitudinal intervals, the highest prevalence was recorded at a low altitude in the coastal areas, near to Danube (several meters), respectively, for Chilia Veche (Danube Delta), where the climate is steppe-continental influenced by the Black Sea (Table 2). We found a strong negative correlation between A. phagocytophilum prevalence and local median precipitation (Pearson Rank correlation, rs = −0.757, n = 10, R ² = 0.573) and a weak positive correlation between the prevalence and temperature (Pearson Rank correlation, rs = 0.6228, n = 10, R ² = 0.3879). We found no correlation between seasonal temperatures or local humidity/moisture and A. phagocytophilum prevalence. The results suggest the influence of altitude correlated with temperature and rainfall on the prevalence of A. phagocytophilum.

Table 2.

Prevalence of Anaplasma phagocytophilum in Dogs in Different Locations in Relation to Sex, Age, Breed, and Altitude

Category	Prevalence%	95% CI	Differences
Locality (altitude region)
Bechet (II)	11.5	2.45–30.2	^a
Călăraşi (II)	—	—
Chilia Veche (I)	14.93	7.40–25.74
Cuca (IV)	2.17	0.06–11.5
Cugir (III)	—	—
Geoagiu (III)	3.03	0.8–15.8
Goleşti (IV)	2.33	0.06–12.3
Nanov (II)	1.96	0.05–10.5
Noslac (III)	3.23	0.08–16.7
Orăştie (III)	3.45	0.09–17.8
Sex
Female	6.7	3.1–12.4	n.s.
Male	4.5	2.2–8.1
Age
Young	4.9	1.4–12.2	n.s.
Adult	5.4	3.1–8.8
Breed
Mixed breed	5.1	3.0–8.3	n.s.
Pure breed	7.0	1.5–19.1
Altitude region
I	14.93	7.4–25.7	^a
II	3.92	1.1–9.7
III	3.03	0.6–8.6
IV	2.25	2.3–7.9
Average	5.32	3.32–8.33	—

Altitudinal zone (I costal area 0–5 meters; II lowland, 6–100 meters; III hilly areas, 200–300 meters; IV low mountain areas, >500 meters).

Significant difference was considered for a value of p < 0.05, the prevalence obtained in Chilia Veche (I) was significantly higher than in Nanov (II) (4.8711, p < 0.05), Goleşti (III) (3.8893, p < 0.05), and in Cuca (4.2574, p < 0.05). The prevalence obtained in coastal area (I) was higher than the prevalence recorded in other regions (0.0198, p < 0.05).

n.s., no statistically significant differences

Discussion

This is the first study performed on the altitudinal distribution of CGA. However, in Romania, CGA was previously described in three stray dogs in 2012, and all came from a restricted area (Hamel et al. 2012). In the same year, a serological study was published, reporting a seroprevalence of A. phagocytophilum infection of 5.5% (Mircean et al. 2012), however, the method used could detect also antibodies against Anaplasma platys (Mircean et al. 2012).

The prevalence of CGA in dogs in Europe varies between 0.5% in Poland (Zygner et al. 2009) and 6.3% in Germany (Jensen et al. 2007). The prevalence of A. phagocytophilum infection in dogs obtained in our study (5.32%) falls within the European range (Carrade et al. 2009, Saintz et al. 2015).

The variability in prevalence of CGA and the risk factors were discussed in several studies. Similar to our results, neither the breed, nor the sex or age has been confirmed as risk factors for A. phagocytophilum infection (Saintz et al. 2015). However, other risk factors such as the presence and abundance of the vector and reservoir hosts, and the prevalence of infection in either, could be involved in the variability of infection in dogs. For example, the observed seasonality of infection in dogs was found to be correlated with the seasonality of the vectors (Carrade et al. 2009). Moreover, the variability in prevalence of A. phagocytophilum in vectors may be influenced by the availability of reservoir hosts, which in turn is also influenced by land structure, habitat, and climate (Stuen et al. 2013).

Our results indicate an influence of altitude on the prevalence of A. phagocytophilum infection in dogs. In a recent study, the presence of A. phagocytophilum in vectors was negatively influenced by the altitude (Matei et al. 2015). It was suggested that this influence was exercised by several factors, such as the vector abundance and the differences in host communities depending on altitude (Wagner 1976, Gilbert 2010, Matei et al. 2015). Additionally to altitude, we found a strong negative correlation between A. phagocytophilum prevalence and local mean precipitation and a weak positive correlation between the prevalence and temperature. Up to our knowledge, there is no large-scale study regarding the influence of climate on A. phagocytophilum prevalence or distribution. While the positive influence of temperature on A. phagocytophilum prevalence may be explained by influencing spread and abundance of the vector (Gray et al. 2009, Medlock et al. 2013), it is hard to explain the negative correlation between the rainfall and A. phagocytophilum prevalence obtained in our study. We presume that differences in reservoir or receptive host distribution may be involved, but these remain to be confirmed. Further research on the topic is required to explain how these factors are acting on A. phagocytophilum prevalence.

Conclusions

Altitude is influencing the prevalence of A. phagocytophilum dogs in Romania, throughout different climate exposure (rainfall and temperature). To establish the risk factors for dogs and other hosts, including humans, several geographical and climatic factors, such as altitude and precipitation level, should be considered.

Footnotes

Acknowledgments

The authors would like to thank their colleague A.D. Sándor for all his advice during the preparation of this article. The research was supported from grant PN-II-RU-TE-2014-4-0919: TE/298/2015. This article was published under the frame of EurNegVec COST Action TD1303.

Author Disclosure Statement

No competing financial interests exist.

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