Case report Sublingual epidermoid cyst: a case report
2009

Karthikeya Patil
Department of Oral Medicine and Radiology
JSS Dental College and Hospital
SS Nagar
570015MysoreIndia

V G Mahima
Department of Oral Medicine and Radiology
JSS Dental College and Hospital
SS Nagar
570015MysoreIndia

Suchetha N Malleshi firstname.lastname@example.com
Department of Oral Medicine and Radiology
JSS Dental College and Hospital
SS Nagar
570015MysoreIndia

Case report Sublingual epidermoid cyst: a case report

Cases Journal
28848200910.4076/1757-1626-2-8848Received: 1 August 2009 Accepted: 21 August 2009 Published: 9 September 2009* Corresponding author This article is available from:
Of all the epidermoid cysts encountered throughout the body, only 7% occurs in the head and neck area, with the oral cavity accounting for only 1.6%. Intraorally this benign slow growing and painless entity is usually located in the submandibular, sublingual and submental region. They can cause symptoms of dysphagia and dyspnoea and have a malignant transformation potential. Surgical excision is the treatment of choice. Described here is a case of gigantic sublingual epidermoid cyst.

Introduction

Epidermoid cysts are benign pathologies that can occur anywhere in the body, predominantly seen in areas where embryonic elements fuse together. 1 Most cases have been reported in the ovaries and the testicles (80%), with head and neck accounting for 7% [1,2]. Dermoid and epidermoid cysts in the mouth are uncommon and comprise less than 0.01% of all the oral cysts [2,3,4]. Majority of them occur in sublingual region, but there are rare case reports of occurrence in other sites.

Case presentation

A 28-year-old male of Indian origin, who was moderately built and nourished presented for treatment of mobile lower front teeth. However, the patient had also noticed a swelling in the floor of the mouth of five months duration. Initially pea sized, the swelling had constantly and gradually increased in size. The swelling nevertheless did not cause any pain, discomfort, dysphagia nor speech or masticatory difficulties to the patient.

Extra orally there was no clue of the swelling. Clinical intra oral examination revealed periodontally compromised mandibular anteriors. The floor of the mouth revealed a solitary, well circumscribed, distinct, dome shaped sessile midline swelling extending from the lingual aspect of the mucogingival junction of mandibular anterior teeth up to the mandibular molars bilaterally. The mucosa over the swelling appeared normal without any secondary changes. Tongue was slightly raised but the morphology of the swelling did not vary with tongue moment (Figure 1). On palpation the swelling was soft to firm, non tender, smooth, fluctuant and was not associated with any discharge. Although submandibular and sublingual gland orifices could not be assessed because they were masked by the swelling, bilateral milking of the glands produced thick, mucous saliva. Mandibular cross sectional radiograph did not disclose any calcifications. The buccal and lingual cortical plates were normal with no indication of expansion or decortication.

Aspiration from two different sites using an 18 gauge needle yielded scanty white cheesy material. Complete hemogram and ESR were within normal limits.

Ultrasonography showed a well defined submucosal oval mass in floor of mouth in the midline measuring approximately 4.0 × 3.0 cms in diameter with internal echoes. The underlying tongue musculature was normal.

FNAC showed few squamous cells with mild hyperchromatic nuclei visible in the background of sheets of macrophages, few lymphocytes, abundant keratin flakes and anucleate squames.

Excision of the swelling under local anesthesia yielded a yellowish white smooth surfaced oval mass of tissue measuring approximately 3 × 2 × 2 cms which was soft in consistency and cystic in nature ( Figure 2). The mass upon sectioning was filled with a cheesy material. Post operative healing period was uneventful ( Figure 3).

The histopathologic investigation showed cystic cavity filled with keratin flakes with the epithelial lining discontinuous and missing in certain areas. Connective tissue showed lymphocytes and multinucleated giant cells with deeper tissue fibrosis and prominent blood vessels (Figures 4 and 5). Hence, it was conclusively diagnosed as epidermoid cyst.

Discussion

Epidermoid and dermoid cysts are rare, benign lesions found throughout the body, with 7% occurring in the head and neck area, 1.6% of which occurs in the oral cavity. Of all the oral cysts dermoid cysts account for only 0.01% [2,3,4] Roser was the first to designate dermoid cysts in the floor of the mouth as epidermoid tumours [5].    Even though the expression "dermoid cyst" characterizes a distinct entity, the word "dermoid" has been used to designate true dermoid cysts, epidermoid cysts, and teratoid cysts [2,3,6].

Based on the histopathological picture Meyer divided the floor of the mouth cysts into following types: [1,2,4].

1. Epidermoid cystswhere in the cystic cavity is lined with epithelium without skin appendages.

2. Dermoid cystshere the epithelial lined cystic cavity encloses skin appendages such as hair, hair follicles, sebaceous, and sweat glands.

3. Teratoid cystsin this entity, the cystic cavity in addition to skin appendages also encloses mesodermal derivatives such as bone, muscle, gastrointestinal and respiratory tissue.

All these three cysts owing to their squamous epithelium lining may enclose cheesy keratinaceous material within their lumen. Hence the fundamental difference between the dermoid and the epidermoid is the presence of skin appendages within the wall of the former and the lack of the same in the latter [3,6].

Epidermoid cysts may be categorized as congenital or acquired based on their origin although there is no disparity between the two either clinically or histologically [2,4,7]. Ambiguity about their exact pathogenesis exists and dysontogenetic, traumatic, and thyroglossal anomaly theories have been postulated [1,2,4,6]. Most congenital dermoid and epidermoid cysts perhaps begin due to an embryologic accident during the early stages of development but hardly get perceived until their size causes annoyance [3]. The origin of epidermoid cysts is believed to be from entrapment of epithelial remnants during midline closure of the bilateral first and second branchial arches [1,4,5,7]. It has also been opined that ectodermal differentiation of multipotential cells, most probably pinched off at the point of anterior neuropore closure may give rise to these cysts [3]. On the other hand, they may also crop up from the tuberculum impar of His [4,5].

Traumatic or iatrogenic inclusion of epithelial cells or the blockage of a sebaceous gland duct have been postulated as the pathogenesis of acquired cyst [4,7]. However, some authors have also stated that midline cysts may represent a diverse form of thyroglossal duct cyst [4,6,7].

They may be found in any age group but show preponderance between 15-35 years of age with no gender predilection [1,4,5]. Although floor of the mouth in the midline is most favored site, occasional occurrence involving the buccal mucosa, tongue, lips, uvula, temporomandibular joint dermal graft, intradiploic, intracranial, and intraosseous location within the mandible and maxilla also have been cited in literature [4,8,9]. These lesions show variation in size and weight from few millimeters to centimeters and a gram to several hundred gram respectively [3,5]. Symptoms of dysphagia, dyspnoea and dysphonia may occur due to upward displacement of tongue by these sublingual swellings [4,5]. Further more growth in a inferior direction may give rise to appearance of characteristic "double chin" [1,4,8]. These well encapsulated lesions typically feel "dough like" on palpation, although they may be fluctuant and cyst like based on consistency of the luminal contents, that may range from a cheesy, sebaceous to liquefied substance [1,5,6].

Fine needle aspiration cytology, ultrasound, CT and MR imaging provide essential information on the cyst location that allows optimal preoperative planning. Ultrasonographic findings comprise solid and cystic structures within a heterogeneous mass [3]. On CT scans, the dermoids appear as moderately thin walled, unilocular masses filled with a homogeneous, hypoattenuating fluid substance with numerous hypoattenuating fat nodules giving the pathognomonic "sack-of-marbles" appearance [3]. On MR imaging dermoid cysts give variable signal intensity on T1-weighted images and are usually hyperintense on T2-weighted images [3,9]. Fine needle aspiration cytology has been advocated as an essential investigation. Although not equivalent to CT and MRI, it is safe, economical and dependable technique and is therefore useful for analysis of sublingual lesions [1,6].

The differential diagnosis for sublingual dermoids should comprise ranula, unilateral or bilateral blockage of Wharton's ducts, lipoma, thyroglossal duct cyst, cystic hygroma, branchial cleft cysts, acute infection or cellulitis of the floor of the mouth, infections of submaxillary and sublingual salivary glands, floor of the mouth and adjacent salivary glands benign and malignant tumors, heterotopic gastrointestinal cyst and duplication foregut cyst [1,2,4,6].

Treatment comprises total surgical excision [1,2,[4][5][6]. Caution should be taken not to rupture the cyst, as cystic contents may act as irritants to fibrovascular tissues, causing postoperative inflammation [3]. Recurrences are unusual after absolute surgical excision [1,3]. Reports of malignant transformation of sublingual dermoid and epidermoid to squamous carcinoma and basal cell carcinoma are present [1,2]. A 5% rate of malignant transformation of the teratoid variety of oral dermoid cysts has also been quoted in literature [3][4][5].

Conclusion

Epidermoid cyst of the oral cavity is an uncommon entity. Ample understanding and vigilance about this slow growing painless mass is essential not only because of the symptoms it produces but also due to its malignant potential.

Consent

All reasonable attempts to obtain patient's permission/ consent have been made, however the authors opine that there is no reason to think that the patient or their family would object to publication. The identity of the patient has not been revealed either in the text or in the photographs.

Figure 1 .
1Pre operative photo showing dome shaped sublingual swelling.

Figure 2 .
2Photograph of excised cyst.

Figure 3 .
3Post operative photograph.

Figure 4 .
4Low power photomicrograph showing cystic cavity with keratin flakes.

Figure 5 .
5High power view of keratin lining cystic cavity.
(page number not for citation purposes)
(page number not for citation purposes) Cases Journal 2009, 2:8848 http://casesjournal.com/casesjournal/article/view/8848
Competing interestsThe authors declare that they have no competing interests.Authors' contributions
Epidermoid cyst in the floor of the mouth: report of a case. H Koca, T Seckin, A Sipahi, A Kaznac, Quintessence Int. 38Koca H, Seckin T, Sipahi A, Kaznac A: Epidermoid cyst in the floor of the mouth: report of a case. Quintessence Int 2007, 38:473-477.

Epidermoid cyst of the buccal mucosa: A case report. F Ozan, H B Polat, S Ay, F Goze, Dent Pract. 3J ContempOzan F, Polat HB, Ay S, Goze F: Epidermoid cyst of the buccal mucosa: A case report. J Contemp, Dent Pract 2007, 3:90-96.

Midline submental epidermoid cyst: A Rare Case. A Pancholi, S Raniga, P A Vohra, V Vaidya, Internet J Otorhinolaryngol. 2Pancholi A, Raniga S, Vohra PA, Vaidya V: Midline submental epidermoid cyst: A Rare Case. Internet J Otorhinolaryngol 2006, 2.

Sublingual epidermoid cyst: a case report. T Kandogan, M Koc, E Vardar, E Selek, O Sezgin, J Med Case Reports. 187Kandogan T, Koc M, Vardar E, Selek E, Sezgin O: Sublingual epidermoid cyst: a case report. J Med Case Reports 2007, 1:87.

Epidermoid cyst of the floor of the mouth. case report. M V Damle, D K Irani, N L Hiranandani, Bombay Hosp J. 44Damle MV, Irani DK, Hiranandani NL: Epidermoid cyst of the floor of the mouth. case report. Bombay Hosp J 2002, 44.

Case report of a dermoid cyst of the floor of the mouth. T E Seah, W Sufyan, B Singh, Anna Acad Med. 33Seah TE, Sufyan W, Singh B: Case report of a dermoid cyst of the floor of the mouth. Anna Acad Med Singapore 2004, 33:77S-79S.

. N Hemaraju, S K Nanda, S B Mediker, Sublingual dermoid cyst. Indian Journal of Otolaryngology and Head and Neck Surgery. 3Hemaraju N, Nanda SK, Mediker SB: Sublingual dermoid cyst. Indian Journal of Otolaryngology and Head and Neck Surgery 2004, 3:218- 220.

From Developmental cysts of head and neck. In Cysts of Oral and Maxillofacial Region. M Shear, P Speight, Blackwell Munksgaard. 4 th editionShear M, Speight P: From Developmental cysts of head and neck. In Cysts of Oral and Maxillofacial Region. 4 th edition. Singapore: Blackwell Munksgaard; 2007:181-183.

Skull Base: An Interdisciplinary Approach. S A Rutherford, P A Leach, A T King, 2Rutherford SA, Leach PA, King AT: Skull Base: An Interdisciplin- ary Approach 2006, 2:109-115.


Open Access RESEARCH Canine babesiosis in northern Portugal and molecular characterization of vector-borne co-infections
2010

Luís Cardoso
†1
Yael Yisaschar-Mekuzas
Filipa T Rodrigues
Álvaro Costa
João Machado
Duarte Diz-Lopes
Gad Baneth
Open Access RESEARCH Canine babesiosis in northern Portugal and molecular characterization of vector-borne co-infections

Parasites & Vectors
3272010BioMed Central Research
Background: Protozoa and bacteria transmitted by arthropods, including ticks and phlebotomine sand flies, may cause a wide range of canine vector-borne diseases. Dogs can be simultaneously or sequentially infected with multiple pathogens. Canine babesiosis caused by Babesia canis canis and Babesia canis vogeli is known to occur in Portugal. This study assessed, by means of blood smear examination, PCR and DNA nucleotide sequencing, the presence of Babesia spp. and co-infecting agents Leishmania, Anaplasma/Ehrlichia and Hepatozoon in 45 dogs from northern Portugal clinically suspected of babesiosis.Results: Forty-four dogs (98%) had infection with B. canis canis and one with B. canis vogeli.Co-infections were detected in nine animals (20%). Eight dogs were found infected with two vector-borne agents: six with B. canis canis and Leishmania infantum; one with B. canis canis and Ehrlichia canis; and one with B. canis canis and Hepatozoon canis. Another dog was infected with three vector-borne pathogens: B. canis vogeli, E. canis and L. infantum. Overall, L. infantum was found in seven (16%), E. canis in two (4%), and H. canis in one (2%) out of the 45 dogs with babesiosis.

Morphological appearance in the erythrocytes and especially molecular analysis have allowed the differentiation of several large (3-5 μm) and small (0.5-2.5 μm) piroplasms of dogs [3]. In Europe, the causative agents of canine babesiosis include large Babesia canis canis, a subspecies transmitted by the tick Dermacentor reticulatus which causes a mild to severe disease, and B. canis vogeli, transmitted by Rhipicephalus sanguineus [8][9][10][11]. Babesia canis vogeli, the less virulent subspecies of B. canis, is also present in tropical and subtropical areas of Africa [12], Asia [13], Australia [14], North and South America [15,16]. The most virulent of the three subspecies, B. canis rossi, has been reported only in central and southern Africa [4,17]. Furthermore, another large yet unnamed Babesia sp., genetically related to Babesia bigemina of cattle, has been reported in dogs with clinical signs of babesiosis in North Carolina [18,19].

Small Theileria annae, a Babesia microti-like piroplasm [20] endemic in northwestern Spain [21], and Babesia gibsoni have also been detected as agents of babesial disease in European dogs [22][23][24]. Outside Europe, T. annae has been detected in one dog from Mississipi [25]. Babesia gibsoni has a wider distribution, with infections reported primarily from Asia [13] but also from America [15,26] and Australia [27]. One other genetically distinct small piroplasm species is recognised to cause canine babesiosis: Babesia conradae in southern California [28]. Furthermore, DNA of large Babesia caballi [24] and small Theileria equi [24,29] and Theileria annulata [30], in Europe, and an unnamed Theileria sp., in South Africa [31], have been detected in dogs, but their role in canine piroplasmosis needs to be confirmed.

The geographical distribution of canine piroplasms is largely determined by the ecological ranges of their vector arthropods [32]. Epidemiological surveillance of disease occurrence and prevalence is required to map local risk and forecast vector-borne infection scenarios. Canine babesiosis caused by large piroplasms is known to occur in northeastern Portugal [33], and both B. canis canis and B. canis vogeli have recently been identified in some naturally infected dogs from this area [34]. In areas where canine vector-borne diseases (CVBD) are endemic, dogs can be simultaneously or sequentially infected with more than one vector-borne agent, depending on the presence and abundance of arthropod vectors [1]. Leishmania infantum [35] and the rickettsiae Anaplasma platys and Ehrlichia canis are also proven agents of CVBD in northern Portugal [36]. The sand fly season runs from May to October in the Douro subregion of northern Portugal: Phlebotomus perniciosus and Phlebotomus ariasi, vectors of Leishmania, are most abundant in July and September, respectively [37]. Ticks of the species D. reticulatus and R. sanguineus have been found to infest dogs in northeastern Portugal [38][39][40]. In addition to transmitting B. canis vogeli and E. canis, and presumably A. platys, R. sanguineus is also the main vector for protozoan Hepatozoon canis in Europe [41,42]. This study assessed the presence of Babesia spp. and the co-infecting pathogens Leishmania, Anaplasma/Ehrlichia and Hepatozoon in dogs from northern Portugal clinically suspected of babesiosis by means of blood smear examination, PCR and DNA nucleotide sequencing.

Methods

Animals and samples

Forty-five dogs from Alto Trás-os-Montes and Douro (northern Portugal) suspected of babesiosis, between October 2007 and March 2009, were included in this study. The two subregions of Alto Trás-os-Montes (north) and Douro (south) cover a total area of 12,282 sq. km and are bordered by Spain to the north and east. The terrain is hilly, and agriculture is the main source of occupation and income. No history of travel to southern Portugal or to Spain was obtained for any of the 45 dogs. After recording the dog's signalment, each animal was physically examined and blood samples were collected from the ear tip to prepare glass slide smears and to assess microhaematocrit (HCT). The thin smears were air-dried, fixed with methanol, Giemsa-stained and then examined under light microscopy (magnification 1000×; 100 fields) for detection of babesial piroplasms and other possible infective agents. Additional samples of peripheral or venous blood were spotted onto individual filter papers (7.5 cm × 2.5 cm; GB 002 Schleicher and Schuell, Dassel, Germany) allowed to air-dry and stored at -20°C until further use. Dogs were also examined for the presence of infesting ticks. The anti-babesial treatment administered was recorded and the disease outcome followed.

DNA extraction

Filter paper portions, corresponding to approximately 20 μl of spotted blood, were cut out by use of individual sterile scalpel blades and put into sterile tubes for DNA extraction [43]. DNA was extracted by adding 300 μl of lysis buffer [50 mM NaCl, 50 mM Tris, 10 mM EDTA (pH 8.0)], proteinase K to a final concentration of 250 μg/ml and Triton X-100 (20%) to a final concentration of 1%. Following a 2 h incubation at 56°C and an inactivation of proteinase K at 90°C for 10 min, 300 μl of phenol (75%), chloroform (24%) and isoamylalcohol (1%) mixture were added, vortexed and centrifuged (12,000 × g) for 4 min. The supernatant was collected and 300 μl of a mixture of phenol (50%), chloroform (48%) and isoamylalcohol (2%) were added, vortexed and centrifuged (12,000 × g) for 4 min. The supernatant was collected and 300 μl of a mix-ture of chloroform (96%) and isoamylalcohol (4%) were added, vortexed and centrifuged (12,000 × g) for 4 min. The supernatant was collected, and 1:10 volume of Naacetate (3 M) and one volume of ice cold 100% isopropanol (-20°C) were added and incubated over night at -20°C. Following centrifugation (14,000 × g) at 4°C for 30 min, the supernatant was discarded and the pellet was washed with 150 μl of ethanol (75%, -20°C) and centrifuged (13,000 × g 4°C) for 15 min. The supernatant was discarded and the pellet was air-dried. The DNA was then hydrated with 30 μl of double-distilled H 2 O for 1 h at 50°C.

PCR assays for Babesia, Anaplasma/Ehrlichia, Hepatozoon and Leishmania

Primers PIRO-A and PIRO-B (Table 1) were used to amplify an approximate 408 bp fragment of the 18S ribosomal RNA (rRNA) gene of Babesia spp. by PCR [44]. Amplification was done under the following conditions: 94°C for 1 min followed by 39 cycles of 94°C for 45 sec, 62°C for 45 sec and 72°C for 45 sec.

Primers EHR16SD and EHR16SR (Table 1) were used to amplify an approximate 345 bp fragment of the Ehrlichia and Anaplasma genera 16S rRNA gene [45]. PCR amplification was performed under the following conditions: 95°C for 5 min; 40 cycles of 94°C for 30 sec, 55°C for 30 sec and 72°C for 90 sec; then final extension at 72°C for 5 min [46].

PCR for the detection of Hepatozoon was performed using primers (125 nM each) HEP-F and HEP-R [47,48] ( Table 1). The following conditions were used to amplify a partial 666 bp fragment of the 18S rRNA gene sequence of Hepatozoon spp.: 95°C for 5 min; 35 cycles of 95°C for 20 sec, 57°C for 30 sec and 72°C for 90 sec; and 72°C for 5 min. PCR was performed using Syntezza PCR-Ready High Specificity (Syntezza Bioscience, Israel).

A 265 bp fragment within the internal transcribed spacer 1 (ITS1) region of the L. infantum rRNA operon was amplified by real-time PCR using the primers ITS-219F and ITS-219R (Table 1) and then evaluated by high resolution melt (HRM) analysis [49]. The PCR reaction was performed in a total volume of 20 μl containing 5 μl DNA, 40 nM of each primer, 10 μl Thermo-start PCR Master Mix (Thermo-start ABgene, Rochester, NY, USA), 0.6 μl 100-fold diluted SYTO9 (Invitrogen, Carlsbad, CA, USA), and sterile, DNase/RNase-free water (Sigma, St. Louis, MO, USA) using a Rotor-Gene 6000 real-time PCR machine (Corbett Life Science). Initial denaturation for 15 min at 95°C was followed by 40 cycles of denaturation at 5 sec at 95°C per cycle, annealing and extension for 30 sec at 57°C, and final extension for 1 sec at 76°C. This was followed by a conventional melting step from 60°C to 95°C at 1°C/sec, after which the temperature was slowly decreased from 90 to 50°C (1°C/sec) to allow re-annealing. In the final step, HRM analysis was carried out increasing the temperature from 75°C to 90°C at 0.4°C/ sec increments [49].

Positive B. canis, E. canis, H. canis and L. infantum DNA control samples from the blood of naturally infected dogs negative by PCR for other pathogens, and  negative DNA controls from colony-bred dogs negative by PCR for vector-borne pathogens were run with each corresponding PCR reaction.

Sequencing

DNA sequencing was performed at the Center for Genomics Technologies, Hebrew University of Jerusalem. Obtained DNA sequences were evaluated with Chro-masPro software version 1.33 and compared for similarity to sequences in GenBank, using the BLAST program hosted by NCBI, National Institutes of Health, USA http:/ /www.ncbi.nlm.nih.gov.

Statistical analysis

The Chi-squared or Fisher's exact tests were used to compare proportions. Differences between independent groups were analyzed with the Mann-Whitney U test [50]. Analyses were performed with SPSS 10.0 software for Windows, with a probability (p) value < 0.05 as statistically significant.

Results

The 45 dogs suspected of having babesiosis consisted of 24 males and 21 females. Age was not determined for seven dogs; in the remaining 38 animals it ranged from 2 months to 14 years (median value of 3.0 years [interquartile range: 1.1-4.8]). There were 28 dogs from nine defined breeds and 16 mongrels; breed was not determined for one dog. The most represented breeds were Podengo (n = 17) and the Brittany (n = 3). The clinical signs found on physical examination in the 38 dogs were: lethargy (n = 24; 63%), red urine (n = 19; 50%), hyperthermia (n = 18; 47%), anorexia (n = 17; 45%), pale mucous membranes (n = 17; 45%), hypothermia (n = 9; 24%), yellow mucous membranes (n = 5; 13%), vomiting (n = 4; 11%), abdominal pain (n = 3; 8%), ataxia (n = 2; 5%), uterine discharge (n = 2; 5%), cough (n = 1; 3%), gingival petechiae (n = 1; 3%) and ocular discharge (n = 1, 3%). Blood tests showed that 26 (79%) out of 33 dogs were anaemic, with a HCT value below the reference interval (37-55%). Data on physical examination were not determined in six and HCT was not evaluated in 12 out of the 45 dogs. Peripheral blood smear evaluation showed intraerythrocytic piroplasms morphologically compatible with B. canis (3-5 μm long and mainly occurring in pairs or single ring shapes) in 41 (91%) of the 45 clinically suspected dogs. Presence of other infective agents could not be confirmed by microscopy of blood smears. Those 41 dogs and the remaining four suspected animals were all found positive for Babesia spp. by PCR. Further sequence analysis revealed that 44 dogs (98%) were infected with B. canis canis (98-100% relatedness to the GenBank closest sequence) and one with B. canis vogeli (100% relatedness).

Results concerning the observation of large babesial parasites in smears, and PCR amplification with sequencing of Babesia and co-infecting Anaplasma/Ehrlichia, Hepatozoon or Leishmania agents are shown in Table 2. Thirty-six dogs were found infected only with B. canis canis, whereas co-infections were detected in nine dogs (20%). Eight dogs were found infected with two vectorborne agents: six dogs with B. canis canis and L. infantum; one dog with B. canis canis and E. canis; and one dog with B. canis canis and H. canis. Another dog was found infected with three vector-borne organisms: B. canis vogeli, E. canis and L. infantum. Overall, L. infantum was found in seven dogs (16%), E. canis in two dogs (4%), and H. canis was found in one (2%) out of the 45 dogs diagnosed with babesiosis.

Ticks were detected on 22 dogs and tick identification was performed in 10 of these animals. Dermacentor spp. found on four dogs infected with B. canis canis, and Ixodes spp. on four other dogs also infected with B. canis canis. Rhipicephalus sanguineus was present on the dog found co-infected with B. canis vogeli, E. canis and L. infantum. Another dog was co-infested with Dermacentor spp. and R. sanguineus and infected with B. canis canis. Ticks were not detected on 22 dogs and information from one animal was not determined. Twenty-two (50%) out of 44 dogs infected with B. canis were found infested by ticks; and seven (78%) out of nine dogs with co-infections had ticks. Table 3 provides information on gender, breed, age, clinical signs, HCT, detected vector-borne agents, presence of ticks and month of sampling for the nine dogs found co-infected. Differences between HCT values in a group of eight co-infected (B. canis canis and L. infantum, E. canis or H. canis) and another group of 25 dogs not found co-infected (infected solely with B. canis canis) were not statistically significant (Mann-Whitney U test [MWU]; p = 0.449); the differences in HCT between a group of five dogs co-infected with B. canis canis and L. infantum and those 25 dogs not found co-infected were also not significant (MWU; p = 0.504). The same was true for age differences between eight co-infected and 30 dogs not found co-infected (MWU; p = 0.971); and for differences between age in five dogs co-infected with B. canis canis and L. infantum and in those 30 dogs solely infected with B. canis canis (MWU; p = 0.409). No significant dif-ference, although close to significance, was observed when comparing the proportions of co-infected mongrel dogs (6/16; 37.5%) and that of co-infected defined breed animals (3/28; 10.7%) (Fisher's exact test; p = 0.053). Statistically significant differences were not found upon comparison of proportions of co-infected male (6/24; 25%) and co-infected female dogs (3/21; 14.3%); or coinfected dogs among those infested with ticks (7/22; 31.6%) and co-infected dogs among those with no ticks (2/22; 9.1%).

Of the 45 dogs diagnosed with babesiosis, four (9%) died. Despite treatment with imidocarb dipropionate, two dogs died (22%) out of the nine found co-infected ( Table 3). One of these animals was found infected with B. canis vogeli, E. canis and L. infantum; and the other one with B. canis canis and L. infantum. Two other dogs with babesiosis were subject to euthanasia as requested by their owners. Molecular analysis later revealed infection with B. canis canis in these two animals. Forty-one dogs -including seven co-infected with B. canis canis and L. infantum, E. canis or H. canis and 34 with single infection (only B. canis canis) -clinically recovered after treatment with imidocarb dipropionate.

Discussion

In this study, babesiosis in northern Portugal was found to be caused predominantly by infection with B. canis canis, with L. infantum as the most prevalent co-infecting agent. Although ticks were found only on approximately half the dogs with babesiosis, a considerable proportion of the co-infected dogs were infested by ticks (78%). In addition, canine babesiosis was diagnosed mainly from October to March, when climate conditions favour the activity of Dermacentor spp. ticks [38]. In agreement with previous studies [34,36], molecular confirmation of the presence of vector-borne pathogens in northern Portugal has been reestablished for B. canis vogeli and E. canis. In addition, to our best knowledge, this is the first report of molecular identification of H. canis in dogs from northern Portugal. Based on the observation of H. canis gamonts in neutrophils, it had been previously assumed that H. canis is the species involved in canine infection, but genetic characterization was not available at the species level [40].

Babesia canis canis was detected in 98% of the 45 cases of canine babesiosis. This could be due to a higher prevalence of infected dogs or tick vectors in the study area, in comparison to B. canis vogeli, or to its more virulent nature. Due to the severity of clinical presentation, as compared with the relatively milder signs induced by B. canis vogeli [7], dogs infected with B. canis canis would potentially be brought in more often for veterinary consultation [34]. No comparisons were done between the dogs found infected with each one of the two subspecies of B. canis, because there was only one animal found infected with B. canis vogeli. An investigation of 164 Italian dogs suspected of tick-borne disease found B. canis canis in 34 and B. canis vogeli in 11 different cases [51]. This same study showed that clinical cases with B. canis vogeli infection did not present a homogenous clinicopathological pattern as observed in the clinical cases of infection with B. canis canis. Furthermore, in these dogs from Italy, B. canis vogeli infections were found in three puppies (1-2 months) associated with severe haemolytic anaemia (fatal disease in one case) but with no reported concomitant disease; in one other young dog with chronic renal failure; and in four older dogs with leishmaniosis (n = 1), immunosuppression (n = 2) or post splenectomy (n = 1) [51]. Percentage values refer to species or subspecies relatedness to the GenBank closest sequences.

In the present study, co-infection with L. infantum was more prevalent (16%) than with E. canis (4%) or H. canis (2%) among the 45 dogs with babesiosis. Due to relatively lower parasite loads of Leishmania in the blood, compared with other tissues, use of blood to assess infection with Leishmania may have limited the sensitivity of detection; however, the use of highly sensitive quantitative real time PCR for Leishmania spp. in this study probably improved the prospects of detection, when compared with conventional PCR assays [49,52]. In the present study, large babesial piroplasms were detected in blood smears of nearly 90% of the clinically suspected dogs further confirmed as infected with B. canis canis or B. canis vogeli. Parasites were not detected in the smears of four dogs found infected with B. canis canis and diagnosed by PCR and sequencing. Microscopy may lack sensitivity in dogs clinically suspected of babesiosis, possibly due to low parasitaemia [2,7].

The  [37,39]. In this study, Dermacentor spp. were found on dogs infected with B. canis canis and R. sanguineus on one dog co-infected with B. canis vogeli and E. canis (and also L. infantum). History of travel outside this area, where canine leishmaniosis and babesiosis are endemic, was not obtained for any of the dogs. This situation supports the assumption that infections with Babesia, Leishmania and the other vector-borne agents were acquired locally.

The only dog found infected with B. canis vogeli in our study also had co-infection with E. canis and L. infantum. It is possible that chronic subclinical or acute infection with B. canis vogeli had been made clinically apparent by these co-infections. We had previously detected one clinical case in a dog from northern Portugal infected with B. canis vogeli concurrently with A. platys [36]. Babesia canis vogeli and E. canis share the same vector species, i.e. R. sanguineus ticks. The co-infected dog may have been exposed to arthropods infected with single pathogen species at different points in time or to vector(s) concurrently infected with multiple agents [1]. Co-infections with Leishmania and tick-borne organisms may affect the severity of CVBD and the variety of associated clinical signs [53]. In a study with beagle dogs naturally exposed to E. canis and L. infantum, the frequency of clinical signs (lymphadenomegaly, splenomegaly, epistaxis, onychogryposis, dermatits and weight loss) was significantly different between animals with dual infection and those with single infection [54]. However, the clinical signs of coinfections with two or more vector-borne organisms are often difficult to be specifically assigned to each one of the infecting agents [55]. In the present study, although a complete clinicopathological evaluation was not performed, especially blood cell counts, no significant differences among HCT values were found between the coinfected dogs and those with one single infection detected. Nevertheless, dogs with co-infections had a lower survival rate when compared to those with single infection. In fact, two dogs (22%) died out of the nine found co-infected: one with B. canis vogeli, E. canis and L. infantum, and the other one with B. canis canis and L. infantum infection. From the 36 dogs found infected only with B. canis canis, two (6%) were euthanized and the remaining 34 animals (94%) clinically recovered with the anti-babesial treatment.

Another study in rural and hunting dogs (n = 473), from northeastern Portugal, showed a 15% seroprevalence of antibodies to E. canis, and a 2% prevalence of Hepatozoon spp. in blood smears [40]. Six dogs were simultaneously found to be seropositive for E. canis and positive for Hepatozoon spp., but PCR did not detect Ehrlichia or Anaplasma in any of those animals. Nevertheless, E. canis DNA was sequenced from four other dogs, thus revealing a 0.9% prevalence of infection. No babesial piroplasms were found in blood smears from all the dogs included in the same study. The differences between these prevalence rates for E. canis, as detected by molecular methods, and piroplasms and those observed in the present study may be explained by a different sample population and the methods used. In fact, only 10% of the dogs studied by Figueiredo [40] were clinically suspected of bacterial or protozoal diseases, and infection with Babesia spp. was not assessed molecularly by this author, whereas all the dogs in the present study were positive to Babesia spp. and thus exposed to at least one species of tick-borne pathogen.

In this study, two littermates aged two months old were both found co-infected with B. canis canis and L. infantum. This finding could suggest the possibility of transplacental transmission of L. infantum [56] and/or B. canis canis [57]. However, both puppies were found infested with ticks (species not identified), which should be regarded as the most likely source of transmitting Babesia to them. Regarding infection with Leishmania, these animals were born in early October and transmission by phlebotomine sand flies should still be considered [37]. Data on physical examination were not available for one of the dogs. The other dog presented hyperthermia, pale mucous membranes and red urine, which could be attributed to B. canis canis infection. Both animals suffered from anaemia, and one of the dogs died. It is not clear whether infection with L. infantum contributed to the clinical abnormalities in these two puppies and whether they were suffering from pathological effects of infection with Leishmania. Other tests, including serological analysis for antibodies to Leishmania, complete blood count, serum biochemistry panel and urinalysis, could have been helpful in clarifying the clinical status of these two and of the other seven co-infected dogs as well [58]. In general, the incubation period of canine babesiosis is short (4-21 days) [3], while the incubation of canine leishmaniosis is much longer (2 months to several years) [59].

The trend of canine babesiosis seasonality found in the present study is further strengthened by results from an additional study (Diz-Lopes D,   mately 90% of the 45 cases of babesiosis in dogs from northern Portugal were diagnosed in October (18%), November (27%), December (20%), February (13%) and March (9%), i.e. autumn and winter months. In central Europe, the occurrence of canine babesiosis due to B. canis has been found to change in an annual seasonal pattern, although exact time of beginning and ending of Dermacentor spp. activity is strongly correlated with specific local climate conditions [60]. In fact, epidemiological and clinical surveillance studies are needed for mapping the risk of babesiosis and other CVBD in different geographical regions.

A study in urban and rural dogs (n = 651) from Hungary revealed a 6% seropositivity to B. canis [61]. Seroprevalence to B. canis was significantly different for German shepherd and Komondor dogs, suggesting a genetic predisposition to chronic subclinical infection (carrier state) with long-term maintenance of seropositivity. A higher prevalence of specific antibodies in three out of four Komondors, a local breed, was explained by an increased risk of them having unnoticed ticks attached to their heavy hair coat [61]. In the present study, B. canis canis was found in males and females, younger and older dogs, from nine defined breeds and particularly from mongrels. There was no clear distinction of age and sex between single-infected and co-infected dogs. When comparing the proportions of co-infected mongrel dogs (~38%) and that of co-infected defined breed animals (~11%), there was a quantitative but not significant difference. Mongrels, Podengo and Brittany dogs represented the larger part of those found affected by babesiosis. Rather than a genetic or breed predisposition, this situation probably reflects the fact that these dog breeds and crosses are popular and over-represented in northern Portugal. Furthermore, a considerable percentage of these dogs live outdoors and are used for hunting activities in the field, where they face a higher risk of contacting with infected arthropod vectors.

Theleria annae may cause severe illness in dogs, including renal failure, and is endemic in northwestern Spain [21], which borders part of the area where the present study was carried out. To our knowledge, there are no written reports of autochthonous canine T. annae infection in Portugal. Nevertheless, due to the increasing mobility of dogs and the existence of competent or presumptive vectors, piroplasms may spread into nonendemic areas [1,2].

Conclusions

In conclusion, this study confirmed the presence of B. canis canis and B. canis vogeli as agents of babesiosis in dogs from northern Portugal, with a large majority of the clinical cases found with the former piroplasm. A higher sensitivity of Babesia spp. detection was obtained by use of PCR assays, compared to microscopy of blood smears. Co-infections with some other vector-borne agents were also detected and molecularly characterized, namely L. infantum, E. canis and H. canis. Detection and identification of species and subspecies of pathogens, either in single or in co-infection, are necessary for the treatment, clinical management and prevention of CVBD.

1 :
1Primer sets for the PCR amplification and sequencing of vector-borne infective agents used in the study Agent Primers Reference(s) Babesia spp. PIRO-A: 5'-AAT ACC CAA TCC TGA CAC AGG G-3' PIRO-B: 5'-TTA AAT ACG AAT GCC CCC AAC-3' [44] Anaplasma spp./Ehrlichia spp. EHR16SD: 5'-GGT ACC YAC AGA AGA AGT CC-3' EHR16SR: 5'-TAG CAC TCA TCG TTT ACA GC-3' [45,46] Hepatozoon spp. HEP-F: 5'-ATA CAT GAG CAA AAT CTC AAC-3' HEP-R: 5'-CTT ATT ATT CCA TGC TGC AG-3' [47,48] Leishmania spp. ITS-219F: 5'-AGC TGG ATC ATT TTC CGA TG-3' ITS-219R: 5'-ATC GCG ACA CGT TAT GTG AG-3' [49]

Figure 1
1Monthly distribution of canine babesiosis and of co-infection cases in 45 dogs from northeastern Portugal. Light grey bars represent 36 cases of babesiosis with no co-infecting agents (Anaplasma/Ehrlichia, Hepatozoon or Leishmania). All 36 dogs infected solely with Babesia had B. canis canis infection. Dark grey bars represent nine cases of babesiosis with one or two concurrent infecting agents along with B. canis canis (n = 8) or B. canis vogeli (n = 1), respectively.

Table

Table 2 :
2Comparison of results from blood smear examination, PCR and sequence analysis in 45 dogs suspected of babesiosis.Blood smear
PCR and sequencing
Dogs (n)

Babesia spp.
Co-infection

Large piroplasms
B. canis canis (98-100%)
Negative
32

B. canis canis (99-100%)
L. infantum (99-100%)
6

B. canis canis (99%)
E. canis (100%)
1

H. canis (99%)
1

B. canis vogeli (99%)
E. canis (100%) and L. infantum
(99%)

1

Piroplasms not found
B. canis canis (100%)
Negative
4

arthropods described as vectors of the detected pathogens -D. reticulatus for B. canis canis; Phlebotomus spp. for L. infantum; and R. sanguineus for B. canis vogeli, E. canis, and H. canis -are present in northern Portugal

Table 3 :
3Signalment, clinical signs and vector-borne agents in nine co-infected dogs.Agents
Gender
Breed
Age
(months)

Clinical
signs

HCT (%)
Ticks
Month
Clinical
outcome
(imidocarb
treatment)

B. canis canis
and L.
infantum

F
Dalmatian
72
ND
ND
No
October
Recovered

M
M o n g r e l
0 2
H y p e r t h e r m i
a, PMM, RU

20
Yes
November
Died

M
Mongrel
02
ND
10
Yes
November
Recovered

F
Mongrel
36
ND
40
Yes
February
Recovered

M
M o n g r e l
3 6
H y p e r t h e r m i
a, RU

40
Yes
February
Recovered

M
Mongrel
ND
PMM, RU
15
Yes
March
Recovered

B. canis canis
and H. canis

F
P o d e n g o
4 7
H y p e r t h e r m i
a, PMM, RU

25
Yes
May
Recovered

B. canis canis
and E. canis

M
G e r m a n
pointer

78
Anorexia,
hyperthermia
, lethargy, RU

40
No
March
Recovered

B. canis
vogeli, E. canis
and L.
infantum

M
Podengo
36
Anorexia,
hypothermia,
lethargy,
YMM

03
Yes*
April
Died

F: female; HCT: haematocrit (normal range: 37-55%); M: male; ND: not determined; PMM: pale mucous membranes; RU: red urine; YMM: yellow
mucous membranes. *R. sanguineus.

Rodrigues FT: Babesiose canina -estudo clínico no Nordeste Transmontano [unpublished abstract]. V Congresso Veterinário Montenegro: 17-18 January 2009; Porto). A higher occurrence of disease was found during October and November (21 cases during each month) in 98 dogs from northeastern Portugal diagnosed with babesiosis by clinical examination and by observation of intraerythrocytic large piroplasms, from January 2005 to December 2008. Considerable numbers of canine babesiosis cases were also found from December to May, with monthly values ranging between 6% and 11%. Sixty-two per cent of all the cases were detected in hunting dogs, and 52% of all the affected animals were Podengo dogs (Diz-Lopes D, Rodrigues FT: Babesiose canina -estudo clínico no Nordeste Transmontano [unpublished abstract]. V Congresso Veterinário Montenegro: 17-18 January 2009; Porto). In the present study, it was found that approxi-
AcknowledgementsThe authors thank Dr. Joana Tuna and Dr. Lisete Vieira, from Clínica Veterinária Os Bichos, Dr. Dalit Talmi-Frank, from the Hebrew University, and the Board and the Staff of the Veterinary Teaching Hospital, University of Trás-os-Montes e Alto Douro, for their assistance. Publication of the thematic series has been sponsored by Bayer Animal Health GmbH.Competing interestsThe authors declare that they have no competing interests.Authors' contributionsConceived and design the study: LC, YYM and GB. Collected and characterized clinical samples: FTR, AC, JM and DDL. Performed PCR and genetic analysis: YYM. Analyzed data, drafted and revised the manuscript: LC and GB. All authors gave final approval of the version to be submitted.
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Canine babesiosis in northern Portugal and molecular characterization of vector-borne co-infections Parasites & Vectors. Cardoso, 327Cite this article asCite this article as: Cardoso et al., Canine babesiosis in northern Portugal and molecular characterization of vector-borne co-infections Parasites & Vec- tors 2010, 3:27