Published 3 July 2019 [618270]

Published 3 July 2019
DOI: 10.1645/18-137Contents and archives available through www.bioone.org or www.jstor.org
Journal of Parasitology
journal homepage: www.journalofparasitology.org
CLINICAL OCCURRENCE OF CANINE BABESIOSIS IN THE COASTAL AREA OF THE
BLACK SEA (DOBROGEA) IN SOUTHEASTERN ROMANIA AND ASSOCIATEDEPIDEMIOLOGICAL IMPLICATIONS
Laurent xiu Leica, Ioan Liviu Mitrea, and Mariana Ionita
Department of Parasitology and Parasitic Diseases and Animal Biology, Faculty of Veterinary Medicine, University of Agronomic Sciences and Veteri nary
Medicine of Bucharest, 105 Splaiul Independentei, 050097, Bucharest, Romania.
Correspondence should be sent to Ioan Liviu Mitrea at: [anonimizat]
KEY WORDS ABSTRACT
Dogs
Babesiosis
Clinico-Pathology
Epidemiology
Southeastern RomaniaCanine babesiosis (CB) is a significant tick-borne disease caused by intra-erytrocytic protozoa of the
genus Babesia (Apicomplexa: Piroplasmida) and is currently emerging or re-emerging in many
European countries. Despite the increasing incidence of clinical CB, little is known of theepidemiology of this disease in Romania. Therefore, the present study was conducted to provide
epidemiological data on CB in the coastal area of the Black Sea (Dobrogea), southeastern Romania.
For this, 306 owned dogs presented during 2016–2018 at a veterinary clinic in the area and clinicallysuspected for CB were tested for the presence of intra-erytrocytic piroplasms using Giemsa-stained
thin blood smears. Overall, 27.8% (95% CI: 22.83–33.16) of dogs were positive for large piroplasms.
The Babesia spp.–positive dogs were 51 males and 34 females aged from 3 mo to 13 yr (mean 4.2 yr;
SD¼3.6). A higher rate of infection was registered in Bichon Maltese (n ¼13) and mixed-breed (n
¼28) dogs. Most cases (76/85; 89.4%) were diagnosed from March to June, with only a few cases
registered in November and December (1 case each). Infected dogs displayed different clinicalpresentations, varying from mild (38.8%) to moderate (29.4%) and severe disease (29.4%). Based onclinical- pathological changes, 38 (44.7%), 34 (40.0%), and 13 (15.3%) dogs were diagnosed with
uncomplicated babesiosis, complicated babesiosis with a single organ dysfunction, and complicated
babesiosis with multiple-organ-dysfunction syndrome (MODS), respectively. The recovery rate(81.6%, 70.1%, and 38.4%, respectively) was significantly different in these groups. The age of dogs
appeared to be a risk factor for both severe disease (mean age of 5.8 yr) and MODS (mean age 6.8
yr). These findings document the common occurrence of CB in Dobrogea, southeastern Romania,and suggest that appropriate control measures need to be taken.
Canine babesiosis (CB), which is caused by large and small
intra-erythrocytic piroplasms of the genus Babesia (Apicomplexa:
Piroplasmida), is a significant tick-borne disease, currentlyemerging in many European countries (Irwin, 2009; Halos etal., 2014; Solano-Gallego et al., 2016). At present, there are 4Babesia species known to infect dogs in Europe: The 2 large
Babesia species, Babesia canis and Babesia vogeli , are transmitted
by the tick species Dermacentor reticulatus and Rhipicephalus
sanguineus , respectively. For the 2 small Babesia species, Babesia
gibsoni and Babesia microti –like, the tick vectors (most likely R.
sanguineus and Ixodes hexagonus , respectively) still have to be
confirmed (Camacho et al., 2003; Uilenberg, 2006; Solano-Gallego et al., 2016).
Both the geographical distribution and prevalence of CB in
Europe vary but generally correlate with the occurrence of localR. sanguineus ,D. reticulatus , and/or I. hexagonus tick popula-
tions. Whereas R. sanguineus is predominantly occurring insouthern European countries, D. reticulatus can be found in
various ecologically appropriate areas of most western, southern,and eastern European countries (Halos et al., 2014). Data on theoccurrence of I. hexagonus are generally rather scattered and
often related to feline tick-borne infections (Nitschke, 2014).
According to the occurring tick spectrum, many cases of CB
due either to B. canis orB. vogeli have been reported in various
European countries, including France (Bourdoiseau, 2006; Rene ´-
Martellet et al., 2015), Poland (Welc-Faleciak et al., 2009), Spain(Solano-Gallego et al., 2016), Austria (Halos et al., 2014), Italy(Furlanello et al., 2005), Hungary (Fo ¨ldva´ri et al., 2005),
Switzerland (Schaarschmidt et al., 2013), and Germany (Beelitzet al., 2012). More recently, outbreaks of autochthonous CB duetoB. canis have been reported in the Netherlands (Matjila et al.,
2005), in Norway (Øines et al., 2010), and in Belgium (Halos etal., 2014), indicating a further spread of the infection towardsnorthern Europe.Journal of Parasitology 2019 105(4) 491–496
/C211American Society of Parasitologists 2019
491

In Romania, particularly in SE Romania, the tick spectrum is
broader and includes generally—in addition to the aforemen-
tioned genera Ixodes ,Dermacentor ,a n d Rhipicephalus —also
Haemaphysalis spp. and Hyalomma spp. (Ionita et al., 2010;
Mihalca et al., 2012). However these 2 latter tick genera are not
involved in CB transmission, and more recent surveys also
revealed D. reticulatus and R. sanguineus as the most common
dog-infesting ticks in SE Romania (Ionita et al., 2016).
Furthermore, several studies show a steadily increasing abun-
dance of the tick population over the last decades in this area
(Ionita, 2004; Ionita et al., 2010, 2012). Accordingly, this increasecoincides with the rising number of reported autochthonous CB
cases. Recent studies have revealed B. canis prevalence ranging
from 19.8 to 44.8% (Hamel et al., 2012; Imre et al., 2013).
Despite the increasing number of clinical CB cases, there is a
significant lack of knowledge on the epidemiological pattern of
this disease in Romania. Therefore, the aim of this study was to
provide further data on the clinical pathology and the epidemi-ology of CB in a tick-endemic area in the coastal area of the Black
Sea.
MATERIALS AND METHODS
Study area
The study was carried out in a veterinary clinic in the Dobrogea
area (coastal area of the Black Sea) from January 2016 to June
2018. Dobrogea is a historical region of Romania located between
the lower Danube River and the Black Sea. It is characterized by
hilly relief, with an average altitude of about 200–300 m and a
temperate continental climate. The Black Sea exerts an influenceover the region’s climate, particularly within 40–60 km of the
coast. The average annual temperatures range from 11 C inland
and along the Danube to 11.8 C along the coast. The average
annual precipitation varies from 400 to 500 mm, with an average
relative humidity varying from 80–85% (spring) to 65–70%
(summer) (Posea et al., 2005).
Dogs
In total, 306 dogs of different breed, sex (205 males and 101
females), and age (from 2-mo-old to 13-yr-old dogs) presented at
a veterinary clinic were included in this study, according to the
following criteria: (1) dogs exhibiting clinical signs compatible for
babesiosis, i.e., anorexia, lethargy, hyperthermia, pale mucous
membrane, hemolytic anemia, icterus, and hemoglobinuria(Irwin, 2009); (2) tick-infestation history; and/or (3) presence of
ticks on the dog’s body during a routine physical examination.
All data on breed, age, tick-infestation history, lifestyle, and
outdoor access were recorded during the consultation. Each dog
was subjected to a physical and clinical examination, and all
detectable ticks were collected for subsequent species identifica-
tion.
Hematology and biochemistry
Peripheral blood samples were collected for testing for the
presence of intra-erythrocytic piroplasms on Giemsa-stained thin
blood smear.
Briefly, thin air-dried blood smears were stained using a Dia-
Quick Panoptic kit (Reagents Kft., Budapest, Hungary) and
examined by light microscopy ( 31,000) for intra-erythrocyticparasites (Ionita et al., 2012). Additionally, selected hematology
(blood cell count) and biochemistry (glucose [Glu], amylase[AMY], glutamic oxaloacetic transaminase [GOT], glutamate
pyruvate transaminase [GPT], creatinine [CRE], blood urea
nitrogen [BUN], total bilirubin [TBIL], Ca, lactate dehidrogenase
[LDH], alkaline phosphatase ALP], and albumin [Alb]) param-
eters were determined using automatic devices, i.e., an Abacus Vet
Jr. hemo-analyzer (Diatron MI ZRT, Budapest, Hungary) and
Spotchem EZ SP-4430 (Arkray Europe, B.V., Amstelveen, the
Netherlands), respectively.
Clinical classification
Depending on the clinical presentation and/or severity of
anemia, the clinical cases were classified as follows: mild (packed
cell volume [PCV] /C210.35 L/L), moderate (0.20 /C20PCV,0.35 L/
L), or severe (PCV ,0.20 L/L) (Jacobson and Clark, 1994;
Tvedten, 1994).
Additionally, based on detectable clinico-pathological changes,
babesiosis was defined as:
(1) uncomplicated babesiosis; (2) complicated babesiosis with a
single-organ dysfunction; and (3) complicated babesiosis withmultiple-organ-dysfunction syndrome (MODS).
Uncomplicated babesiosis was defined on the basis of the
presence of varying degrees of hemolytic anemia with associated
clinical signs such as fever, depression, anorexia, and pale mucous
membranes (Irwin, 2009).
In addition to the hemolytic anemia, the following abnormal-
ities were used to define complicated babesiosis: acute renal
failure (ARF) (creatinine .1.5 mg/dl and PCV ,0.55 L/L),
icterus and hepatopathy (at least 2 liver enzymes elevated:
alkaline phosphatase [AP] .280 U/L; alanine aminotransferase
[ALT].60 U/L; !-glutamyl transpherase [GGT] .10 U/L, or a
single enzyme elevated: AP .560 U/L; ALT .120 U/L; GGT .
20 U/L), pancreatitis (amylase .900 U/L); clinical signs of acute
respiratory distress syndrome (ARDS); and cerebral signs.
MODS was considered when at least two of the above
complications were present (Jacobson and Clark, 1994; Lobetti,
2000; Jacobson, 2006).
RESULTS
The microscopic analysis of the blood smears revealed that 85/
306 (27.8%; 95% CI: 22.83–33.16) of the dogs were positive for
intra-erythrocytic large piroplasms (Table I). The Babesia spp.–
positive group consisted of 51 males and 34 females; these
included 57 pure-bred vs. 28 mixed-breed dogs with an age
ranging from 3 mo to 13 yr (mean 4.2 yr; SD ¼3.6). Bichon
Maltese and mixed-breed dogs appeared in higher numbers (13
and 28, respectively), and the remainder was of other different
breeds, with lower numbers.
Most cases (76/85; 89.4%) were diagnosed from March to June
(Fig. 1), while only a few cases were registered in other months ofthe year, including 1 case each in November and December,
respectively.
Altogether, 249 ixodid ticks, including 181 adults and 68
nymph stages, were collected from dogs. Of these, 196 were
identified as R. sanguineus sensu lato (68 nymphs, 52 males, 76
female ticks), 38 as D. reticulatus (14 males, 24 females), and 15 as
Ixodes ricinus (4 males, 11 female ticks).492 THE JOURNAL OF PARASITOLOGY , VOL. 105, NO. 4, AUGUST 2019

Babesia -infected dogs displayed different clinical presentation,
varying from mild (38.8%) to moderate (29.4%) and severe
disease (29.4%). Based on the aforementioned clinico-patholog-
ical definition of babesiosis, 38 (44.7%), 34 (40.0%), and 13(15.3%) of the dogs were diagnosed with uncomplicatedbabesiosis, complicated babesiosis with a single-organ dysfunc-tion, and complicated babesiosis with MODS, respectively.
All dogs with clinical babesiosis were treated with imidocarb-
dipropionate (Imizol t; Schering Plough Animal Health; Intervet
Inc./Merck Animal Health, Friesoythe, Germany), according tothe recommendations by the manufacturer. Additional supportivetherapy (intravenous fluid therapy, antipyretics, blood transfu-sion) was administered according to the clinical presentation.Altogether, 60 (70.6%) dogs were successfully treated, while 18
(21.2%) dogs died, and for 7 dogs, the outcome was not known.
A single imidocarb-dipropionate intramuscular injection was
followed by rapid clinical improvement in the majority ofuncomplicated CB cases; fever was usually reduced or even
absent by the following day, and the color of the urine returned to
normal in hemoglobinuric animals within 2 days.
Dogs with uncomplicated babesiosis demonstrated a higher
recovery rate (81.6%) compared to those with complicated
babesiosis with a single- or multiple-organ dysfunction, in which
the recovery rate varied from 70.1 to 38.4%, respectively (TableII). The cause of death was either babesiosis-associated compli-
cations (15/18; 83.3%) or a late diagnosis (3/18; 16.7%).
Table I. Epidemiological data on canine babesiosis from the coastal area of the Black Sea (Dobrogea), southeastern Romania.
VariableNo. of
investigated dogsDogs positive for babesiosis
No. % [95% CI]Clinical presentation (%)
Mild Moderate Severe
Age
,1 yr 34 14 41.2 9 4 1
1–3 yr 76 33 43.4 13 12 8
4–6 yr 73 16 21.9 4 3 9
7–9 yr 71 14 16.9 6 4 4.10 yr 52 8 15.4 1 2 5
Total 306 85 27.8 [23.02–33.15] 33 (38.8) 25 (29.4) 27 (31.8)
Gender
Male 205 51 24.9 [21.02–21.7] 20 (39.2) 12 (23.5) 19 (37.3)
Female 101 34 33.7 [27.15–25.7] 13 (38.2) 13 (38.2) 8 (23.6)
Total 306 85 27.8 33 25 27
Figure 1. Seasonal dynamics of 85 babesiosis cases in dogs from the coastal area of the Black Sea (Dobrogea), southeastern Romania. Bars: open,
2016; closed, 2017; stippled, 2018; left hash, total.LEICA ET AL.—EPIDEMIOLOGY OF CANINE BABESIOSIS IN ROMANIA 493

Age appeared to be a risk factor for both severe disease (mean
age of 5.8 yr) and MODS (mean age of 6.8 yr) (Table II).
DISCUSSION
The findings of the present study indicate that dogs from the
investigated area are potentially at risk of babesiosis. Thepresence of the vector tick, D. reticulatus , among the tick species
found infesting the investigated dogs confirms the occurrence of
autochthonous CB cases in this area.
The risks for CB in Dobrogea appear to be a result of favorable
ecological factors, such as mild temperature and humidity, whichinfluence the abundance of tick populations, as previouslyreported by other studies (Leschnik et al., 2008). Additionally,host availability, including domestic and wild animals, on which
ticks can feed, may also have a potential for both maintaining and
spreading infected ticks from endemic to non-endemic areas.However, ongoing extended epidemiological studies will allow abetter characterization of the epidemiology of CB in Romania.
The overall incidence of CB of 27.8% found in this study in
dogs presenting with clinical signs is in line with previous studies
in southeastern Romania, reporting prevalences ranging from
26.1 to 30.5% in the Bucharest area (Anghel et al., 2016, 2017),but higher when compared with western Romania, in the Banatarea, where a prevalence of 10.7% had been reported (Ilie et al.,2010). The increasing abundance of D. reticulatus in the last
decades in SE Romania suggests a gradually increasing risk for
tick-borne pathogens, including CB (Ionita et al., 2016).
In Dobrogea, babesiosis was usually seen during the warm
season (Fig. 1). The annual dynamic of CB in Dobrogea showed aparticular pattern, with a high peak in spring, starting fromMarch until to June, which decreased in summer months. Lowernumbers of cases were diagnosed in autumn, and sporadic cases
were diagnosed during the cold season. It is known that the
annual rate of CB incidence is influenced by the climatic factorson which the ticks’ activity relies. The relatively mild climate andthe proximity to the Black Sea provide ideal ecological conditions
and habitats for ticks. The mild temperature and humidity in the
spring months are the most important factors favoring outbreaksof CB in this area. However, CB may occur 1–2 wk after mild
summer and winter days, as registered in this study. In line with
our findings are reports from neighboring countries, such asHungary and other European countries (Ma ´the´et al., 2006;
Leschnik et al., 2008).
It is known that the clinical presentation of Babesia infection
can be highly variable (Irwin, 2009). The observed clinical picture
of CB in this study, varying from mild, uncomplicated babesiosis
to severe disease and multi-organ failure with a risk of death, isconsistent with other clinical reports due to B. canis (Solano-
Gallego et al., 2016). Dogs with mild and moderate uncompli-
cated babesiosis typically present with a febrile illness withapparent anemia. However, these may progress, sometimesthrough neglect, to become severe cases in which anemia has
become life-threatening, or severe acid-base abnormalities with
secondary multiple-organ failure and complications, such as ARFand hepatopathy, may occur (Jacobson and Clark, 1994). It has
been demonstrated that early treatment prevents massive intra-
vascular hemolysis and its potential complications (Furlanello etal., 2005). As evidenced by the findings of the present study, indogs affected with babesiosis, prognosis was good if early
diagnosis and treatment occurred, but severely affected (14 dogs),
late diagnosis (3 dogs), or untreated (1 dog) animals died.
Apart from the Babesia species involved, other factors are
reported to affect the severity of disease, such as age, immunecompetence, or concurrent infection (Irwin, 2009). In this study,
the age was significantly associated with the severity of diseases
and the mortality rate in CB.
An important and fundamental step in controlling CB consists
of a prevention of tick bites (Pfister and Armstrong, 2016). Thereare several ways to do this, including the regular checking of the
dogs for ticks by the owner/veterinarian and the regular
administration of acaricidal products. There are several suchTable II. Recovery and mortality rates of 85 dogs with clinical babesiosis, according to the pathological changes and clinical presentation.
Clinical group nAge (yr) Recovery rate Mortality rate
Unknown
outcome (n) Range Mean [SD*] n % n %
Uncomplicated babesiosis
Mild 20 0.4–10 3.6 [3.24] 18 90.0 0 0 2
Moderate 13 0.2–13 3.4 [3.48] 11 84.6 0 0 2Severe 5 2–10 5.4 [3.84] 2 40.0 3 60.0 0
Total 38 0.2–13 3.75 [3.37] 31 81.6 3 7.9 4
Single-organ dysfunction
Mild 13 0.25–8 2.7 [2.72] 12 92.3 0 1
Moderate 11 0.2–8 3.4 [3.9] 8 72.3 1 9.1 2Severe 10 0.75–10 5.3 [3.5] 4 40.0 6 60.0 0Total 34 0.20–10 3.68 [3.45] 24 70.6 7 20.6 3
Multiple-organ dysfunctions
Mild 0 — — 0 — 0 — 0
Moderate 1 2.0 2.0 1 100.0 0 0Severe 12 3.0–12.0 7.25 [3.24] 4 33.3 8 66.7 0Total 13 2.0–12.0 6.8 [3.63] 5 38.5 8 61.50 0
One-way ANOVA p¼0.014 Fisher’s exact test p¼0.002 Fisher’s exact test p¼0.002
*S D¼standard deviation.494 THE JOURNAL OF PARASITOLOGY , VOL. 105, NO. 4, AUGUST 2019

products licensed and on the market with varying periods of
efficacy: sprays (2–3 days), spot-on pipettes (3–5 wk), long-lasting
acaricide-impregnated collars (6–8 mo), and the new oralchewable tablets (1–3 mo) (Solano-Gallego et al., 2016).
Therefore, in tick-endemic areas, with high risks for CB, an
acaricidal prophylaxis should be considered in order to protectanimals from serious outbreaks. In fact, acaricidal prophylaxisshould cover the entire period when ticks are active, according to
local seasonality of ticks and the lifestyle of dogs.
ACKNOWLEDGMENTS
The authors thank Prof. Peter Holmes, Glasgow University, for
reviewing the manuscript.
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