Cytological and molecular detection of Leishmania infantum in different tissues of clinically normal and sick cats

Veterinary Parasitology 202 (2014) 217–225

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Cytological and molecular detection of Leishmania infantum in different tissues of clinically normal and sick cats Manolis K. Chatzis a,∗ , Margarita Andreadou b , Leonidas Leontides c , Dimitrios Kasabalis a , Mathios Mylonakis d , Alexandros F. Koutinas d , Timoleon Rallis d , John Ikonomopoulos b , Manolis N. Saridomichelakis a a

Clinic of Medicine, Faculty of Veterinary Science, University of Thessaly, 224 Trikalon Str., GR-43100, Karditsa, Greece Department of Anatomy and Physiology of Farm Animals, Faculty of Animal Science and Hydrobiology, Agricultural University of Athens, 75 Iera Odos, Votanikos, GR-11855, Athens, Greece c Laboratory of Epidemiology, Biostatistics and Animal Health Economics, Faculty of Veterinary Science, University of Thessaly, 224 Trikalon Str., GR-43100, Karditsa, Greece d Companion Animal Clinic (Medicine), School of Veterinary Medicine, Aristotle University of Thessaloniki, 11 Stavrou Voutyra Str., GR-54627, Thessaloniki, Greece b

a r t i c l e

i n f o

Article history: Received 1 December 2013 Received in revised form 2 February 2014 Accepted 16 February 2014

Keywords: Cat Cytology Greece Leishmania infantum PCR

a b s t r a c t Natural infection of domestic cats by Leishmania infantum (synonym: L. chagasi) has been demonstrated in several European, Latin American, and Asian countries, and the estimated prevalence of infection, based mainly on blood PCR, ranges from 0.3% up to 60.6%. In this study we aimed to: (a) estimate the prevalence of the infection by L. infantum in clinically normal cats (group A; n = 50) and in cats with various clinical signs (group B; n = 50), living in an endemic region, by both cytological examination of four different tissues (lymph node, skin, bone marrow, and conjunctiva) and by PCR in four different tissues (blood, skin biopsies, bone marrow, and conjunctiva); (b) compare the diagnostic sensitivity of the above methods and evaluate for possible associations between their results; and (c) investigate the possible associations between infection by L. infantum and signalment, living conditions, season of sampling, and health status of the cats. The prevalence of the infection in the study population was 41% and did not differ (P = 0.839) between group A (42%) and B (40%) cats. Lymph node, skin, bone marrow and conjunctiva cytology was always negative. Therefore, the diagnosis of the infection was based only on PCR in blood, skin biopsy, bone marrow and conjunctiva, which was positive in 13%, 18.2%, 16% and 3.1% of the cats, respectively. PCR was positive in only one of the four tissues in 80.5% of the infected cats. The results differed (P = 0.014) among the four tissues and were less frequently positive in conjunctiva compared to skin biopsies and bone marrow (P = 0.007 for both comparisons), thus highlighting the need for multiple tissue PCR testing in order to minimize false-negative results. More PCR-positive cats were found when sampling was performed during the period of sandfly activity (odds ratio: 2.44; P = 0.022). Also, in group B cats, the likelihood of PCR-positivity was higher (odds ratio: 3.93; P = 0.042) among those presenting at least one systemic clinical sign that had been previously reported in cats with leishmaniosis. © 2014 Elsevier B.V. All rights reserved.

∗ Corresponding author. Tel.: +30 2441066095; fax: +30 2441066053. E-mail address: [email protected] (M.K. Chatzis). http://dx.doi.org/10.1016/j.vetpar.2014.02.044 0304-4017/© 2014 Elsevier B.V. All rights reserved.

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1. Introduction Natural infection of domestic cats by Leishmania infantum (synonym: L. chagasi) has been demonstrated in several European (Poli et al., 2002; Mancianti, 2004; Ayllon et al., 2008; Sherry et al., 2011; Ayllón et al., 2012), Latin American (da Silva et al., 2008; Coelho et al., 2010; Coelho et al., 2011; Vides et al., 2011) and Asian countries (Hatam et al., 2010). Although dogs are considered the main domestic and peridomestic reservoir of the parasite (Baneth et al., 2008), cats may be implicated in the transmission cycle as additional primary or secondary reservoirs (Mancianti, 2004; Maroli et al., 2007; Maia and Campino, 2011). Cross-sectional surveys of feline infection by L. infantum may be classified into either seroepidemiological studies, which investigate the prevalence of the infection indirectly through detection of Leishmania-specific antibodies, or studies that directly asses the prevalence of the infection through either the detection of parasitic DNA (PCR, realtime PCR) or of the parasite itself (cytology, histopathology, immunohistochemistry, culture). In general, the latter diagnostic methods resulted in a relatively lower frequency (ranging from 0 to 9.9%) of positive results (Simões-Mattos et al., 2004; Bresciani et al., 2010; Costa et al., 2010; Navarro et al., 2010; Coelho et al., 2011; Sobrinho et al., 2012) compared to the molecular tests. Interestingly, peripheral blood has been the only tissue examined in PCR-based surveys and positive results were obtained at a highly variable frequency, ranging from 0.3% to 60.6%, among cats living in regions where canine and/or human infection by L. infantum is endemic (Pennisi, 2002; Martín-Sánchez et al., 2007; Ayllon et al., 2008; Maia et al., 2008; Paludo et al., 2008; Tabar et al., 2008; Maia et al., 2010; Millán et al., 2011; Sherry et al., 2011; Ayllón et al., 2012; Vilhena et al., 2013). To the best of our knowledge, there is but a single study from Italy, published as an abstract (Pennisi et al., 2012), where multiple tissues (blood, lymph node, and conjunctiva) were examined by real-time PCR; in that study, the prevalence of the infection was higher when the results from all three tissues were interpreted in parallel. The aims of the present study were: (a) to examine a population of clinically normal cats and of cats with various cutaneous and/or systemic clinical signs, living in an endemic region, for infection by L. infantum by cytological examination of four different tissues (lymph node, skin, bone marrow, and conjunctiva) and by PCR in four different tissues (blood, skin biopsies, bone marrow, and conjunctiva); (b) to compare the diagnostic sensitivity of the above methods and to evaluate for possible associations between their results; and (c) to investigate the possible associations between infection by L. infantum and signalment, living conditions, season of sampling, and health status of the cats. 2. Materials and methods 2.1. Study population A total of 100 cats living in Thessaly (n = 77) or Macedonia (n = 23) prefectures, Greece, where canine leishmaniosis due to L. infantum is endemic (Leontides et al.,

2002; Athanasiou et al., 2012), were sampled between January 2009 and September 2011. All cats admitted to six sampling centers (two University Clinics and four private Veterinary Clinics) were eligible for the study, provided that: (a) their owners gave a written informed consent; b) they were at least 1-year old; (c) they had not been diagnosed or treated for leishmaniosis in the past; (d) they had not received drugs with known antiLeishmania activity (including amphotericin-B for the last 12 months and ketoconazole, metronidazole-spiramycin and fluoroquinolones for the last 3 months) for treatment of fungal or bacterial infections; and (e) they had not received immune-modulating medication (including longacting methylprednisolone acetate for the last 6 weeks and short-acting oral glucocorticoids for the last 2 weeks). Handling of these animals was in accordance with European Communities Council Directive 86/609/EEC and state laws and the experimental protocol had been approved by State Authorities (license No 3698/31-10-08). Signalment and historical data, collected using a standardized form, included breed (purebred or common European), age, sex, length of haircoat (short or long), living conditions (indoors, outdoors or both), living area (urban, semi-urban or rural), presence of lush vegetation within a radius of 100 m from their residency, reduced appetite or diarrhea on the day before examination and vomiting during the last week before examination. A thorough physical examination was performed with special attention being paid to the cutaneous (ulcers, nodules, papules, crusts, hemorrhagic cysts, scales, alopecia-hypotrichosis, blepharitis), ocular (conjunctivitis, keratitis, uveitis, panopthalmitis), and systemic (fever, peripheral lymphadenomegaly, hepatomegaly, splenomegaly, icterus) signs that have been reported in clinical cases of feline leishmaniosis (Hervás et al., 1999; Poli et al., 2002; Pennisi et al., 2004; SimõesMattos et al., 2004; Grevot et al., 2005; Leiva et al., 2005; Vita et al., 2005; Coelho et al., 2010; Navarro et al., 2010; Vides et al., 2011; Sobrinho et al., 2012). The cats were subsequently assigned to two groups: group A cats (n = 50) were clinically normal, whereas group B cats (n = 50) presented various dermatological, ocular and/or systemic clinical signs. In group B cats, additional laboratory examinations and imaging studies were performed, as needed, in order to reach a definitive diagnosis.

2.2. Tissue sampling For those cats less than 8 years old that did not present systemic clinical signs, chemical restraint was induced by an intramuscular administration of a ketamine (5 mg/kg body weight; Ketaset® , Fort Dodge Animal Health, Iowa, USA), midazolam (0.2 mg/kg body weight; Dormicum® , Roche Hellas, Athens, Greece) and dexmedetomidine (20 ␮g/kg body weight; Dexdomitor® , Zoetis, New Jersey, USA) combination. In older cats, and/or those with evidence of systemic disease, the dose of dexmedetomidine was reduced (5 ␮g/kg body weight) and butorphanol (0.2–0.4 mg/kg body weight; Butomidor® , Richter Pharma, Austria) was added to the drug combination.

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Blood samples collected by jugular venipuncture, were immediately transferred into EDTA-anticoagulated tubes and were stored at −20 ◦ C until tested by PCR. Lymph node samples were obtained with the fineneedle non-aspiration technique from either a popliteal or a prescapular lymph node, depending on which one was larger and/or more easily palpable. After sampling, the 21G needle was attached to a syringe containing 3 ml of air, the aspirate was expelled onto a glass slide and a cytology smear was prepared with the squash technique (Saridomichelakis et al., 2005; Hodges, 2013). An 8 mm punch biopsy sample was obtained from the skin overlying the iliac crest of all cats, after hair clipping with scissors and local anesthesia with 2% lidocaine (Xylocaine® ; AstraZeneca, New South Wales, Australia). Using the same technique, one (n = 18) or two (n = 2) additional biopsy samples were obtained from the lesional skin of all 20 group B cats that presented cutaneous lesions, even if similar lesions have not been previously reported in cats with leishmaniosis. Skin biopsy samples were bisected, perpendicularly to the epidermis, with a scalpel blade, excess blood was blotted onto a sterile gauge, and touch imprint cytology smears were prepared from their cut surfaces. Immediately afterwards, the biopsies were placed into sterile tubes containing buffer solution and stored at −20 ◦ C until tested by PCR. Bone marrow samples were obtained from the iliac crest, immediately after skin biopsy and before suturing the surgical trauma, after puncturing with an 18G disposable needle (Perfectus® , Medax Medical Devices, Italy) and aspirating with a 10 ml syringe containing 0.2 ml of sterile EDTA solution. Cytology smears were immediately prepared with the squash technique and the remaining samples were transferred into EDTA-anticoagulated tubes and stored at −20 ◦ C until tested by PCR. Lower conjunctiva swab samples were obtained with sterile cotton swabs from both eyes. Swabs from the left eye were rolled onto a glass slide to prepare cytology smears, whereas those from the right eye were placed into sterile tubes and stored at −20 ◦ C until tested by PCR.

2.3. Cytological examination Lymph node, skin, bone marrow, and conjunctiva cytology smears were stained with Diff-Quik® (Merck, Germany) and monolayer areas with sufficient cellularity were microscopically examined for Leishmania amastigotes, under 1000× magnification. Microscopic examination of lymph node, skin, and conjunctiva smears was terminated after 10 min of continuous observation and the results were expressed as the number of Leishmania amastigotes observed by that time. For bone marrow smears, microscopic examination included 1000 oil immersion fields and the results were expressed using a logarithmic scale from 0 to +6 (Chulay and Bryceson, 1983; Saridomichelakis et al., 2005). All cytological examinations were performed by the first author who was blinded to the results of PCR.

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2.4. PCR DNA isolation from blood, skin biopsy, bone marrow, and conjunctiva samples was performed by the High Pure PCR Template Preparation Kit® (Roche, Germany) according to the manufacturer’s instructions. The quality of the DNA extracts regarding purity and integrity was evaluated by measuring optical density at 260/280 nm and submerged gel electrophoresis. A PCR assay, originally described by (Piarroux et al., 1995), which has been modified, calibrated, and thoroughly evaluated in our laboratory (Andreadou et al., 2012), was used for the detection of Leishmania DNA. Reactions were performed in a final volume of 50 ␮l containing 5 ␮l of DNA template, 1X PCR Buffer [Tris–HCl, KCl, (NH4 )2 SO4 , MgCl2 ], 3 mM MgCl2 , 200 ␮M each dNTPs (Fermentas, Lithuania), 1.25 U Taq Polymerase (Invitrogen, California, U.S.A.) and 0.2 ␮M of each oligonucleotide primer (T2: 5 -CGGCTTCGCACCATGCGGTG-3 , B4: 5 -ACATCCCTGCCCACATACGC-3 ). The temperature profile consisted of an initial denaturation step at 95 ◦ C for 5 min, followed by 40 cycles of 30 s at 95 ◦ C, 30 s at 62 ◦ C and 20 s at 72 ◦ C. Thermal processing was completed with a final extension step of 6 min at 72 ◦ C. The amplification product of the specific PCR assay is a 216 bp DNA fragment of a repetitive sequence of L. infantum (Genebank accession number: L42486.1). To detect possible false-positive and false-negative reactions, 20% of the samples tested each time served as negative and positive controls. The former were negative samples (confirmed by culture, serology and PCR) of the same tissues as those examined, whereas the latter were negative samples spiked with DNA of L. infantum promastigotes isolated in pure culture. Control samples were processed identically to those from the 100 cats with regard to collection, DNA isolation, and preparation for PCR. The presence of PCR inhibitors in the samples was assessed by a PCR assay targeting a housekeeping gene (actin; data not shown). PCR amplification products were analyzed by submerged gel electrophoresis with a 100-bp DNA ladder (Fermentas, Lithuania) in 2% (w/v) agarose gels stained with ethidium bromide. The specificity of DNA amplification was confirmed by nucleotide sequence analysis of PCR products that was performed on both strands using the BigDye® Terminator Cycle Sequencing Kit and PRISM® 377 DNA Sequencer (Applied Biosystems, Foster City, California, USA). Results were analyzed and amplification products were compared to deposited sequences in the GenBank database using the Basic Local Alignment Search Tool (BLAST) of the National Center for Biotechnology Information (NCBI).

2.5. Infection status of the cats A cat was considered to be infected by L. infantum when amastigotes were detected in at least one of the four cytology smears and/or when at least one of the four tissues was PCR-positive.

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2.6. Statistical analysis The agreement of the results of PCR among the four different tissues (blood, skin biopsies, bone marrow, conjunctiva) was evaluated by Cochran’s Q test, followed by pairwise comparisons by McNemar’s test for symmetry. Possible associations between the infection status of the cats and those data that may represent risk factors for infection by L. infantum were examined, after considering all 100 cats as a single group, by either Pearson’s 2 test or Fisher’s exact test (breed, sex, type of haircoat, living conditions, season of sampling) or t-test (age). The possible associations between the infection status of the cats and those data that may represent a consequence of the infection, namely the health status of the cats (i.e. assignment to group A or to group B) and the presence of skin lesions, ocular and/or systemic clinical signs that have been previously reported in cats with leishmaniosis (group B cats only), were examined by either Pearson’s 2 test or Fisher’s exact test. For all comparisons, significance was attained at P < 0.05. The initially fitted multivariable models were subsequently reduced by backward elimination until all variables in the model were significant (P < 0.05). Analyses were done in Stata 13. 3. Results The study population included eight purebred (six Siamese, one Birman, and one Persian) and 92 common European cats with an age range of 1–24 years (median 3.75 years). Fifty of the cats were males and 50 females, whereas 89 were shorthaired and 11 longhaired. The number of cats that lived exclusively indoors, exclusively outdoors or both indoors and outdoors was 40, 43, and 17, respectively. The living area was characterized as urban (40%), semi-urban (57%) or rural (3%), and in 15 cases the owners reported the presence of lush vegetation within a radius of 100 m from their residency. Sampling was performed during the sandfly season (from April to November) in 62 cats and during the rest of the year in the remaining 38. Of the 50 group B cats, 18 (36%) presented at least one of the cutaneous lesions that have been previously reported in cats with leishmaniosis, including alopecia-hypotrichosis (n = 13; 26%), crusts (n = 8; 16%), ulcers (n = 7; 14%), papules (n = 6; 12%), blepharitis (n = 3; 6%), and scales (n = 2; 4%). Conjunctivitis was present in one (2%) group B cat and it was the only of the ocular signs that have been previously reported in cats with leishmaniosis. Finally, 28 (56%) group B cats presented at least one of the systemic signs that have been previously reported in cats with leishmaniosis, namely anorexia (n = 18; 36%), vomiting (n = 10; 20%), diarrhea (n = 6; 12%), peripheral lymphadenomegaly (n = 5; 10%), hepatomegaly (n = 4; 8%), icterus (n = 3; 6%), and splenomegaly (n = 1; 2%). In total, at least one of the cutaneous and/or ocular and/or systemic signs that have been previously reported in cats with leishmaniosis was present in 40/50 (80%) group B cats. Many of these cats presented additional signs and their final diagnoses included allergic skin disease (n = 13; 26%), urinary tract infection (n = 7; 14%), liver disease such as cholangiohepatitis and lipidosis (n = 6; 12%), feline immunodeficiency virus infection (n = 6; 12%), cutaneous neoplasia (n = 5; 10%), diabetes mellitus

(n = 4; 8%), bacterial dermatitis (n = 3; 6%), dermatophytosis due to Microsporum canis (n = 3; 6%), idiopathic form of feline lower urinary tract disease (n = 3; 6%), flea infestation (n = 2; 4%), otitis externa and/or media (n = 2; 4%), pleural effusion (n = 2; 4%), toxacarosis (n = 2; 4%), acute renal failure (n = 2; 4%), feline leukemia virus (FeLV) infection (n = 2; 4%), polycentric lymphoma in FeLV-negative cats (n = 2; 4%), chronic weight loss of unknown cause (n = 2; 4%), and otodectic mange, cutaneous drug eruption, cutaneous nevi, thermal burn, chronic periodontal disease, upper respiratory syndrome, non-cardiogenic pulmonary edema, mediastinal neoplasia, Horner’s syndrome, small bowel obstruction, ascites, chronic kidney disease, mammary neoplasia and pyometra (n = 1 cat each; 2%). A total of 19 (10 from group A and 9 from group B cats) lymph node smears were subjectively considered of low cellularity; in some, no lymphoid cells were present. On the contrary, all skin, bone marrow and conjunctiva cytology smears were considered of adequate cellularity. Cytological examination of lymph node, skin, bone marrow and conjunctiva smears was negative for Leishmania amastigotes in all cats. One biopsy from the skin overlying the iliac crest and four conjunctiva swabs, all from group B cats, were lost during transportation to the laboratory. For the remaining samples (Table 1), PCR positivity rates ranged from 2.2% (conjunctiva samples from group B cats) up to 26.5% (iliac crest skin and lesional skin biopsy samples from group B cats). In the majority (80.5%) of infected cats only one tissue was PCR-positive (Table 2), and the PCR amplification products examined showed >99% sequence homology to L. infantum. Given the negative results of cytology, the infection status of the cats was determined based solely on the results of PCR. In total 41% of the cats, including 21/50 (42%) group A and 20/50 (40%) group B cats, were infected by L. infantum. There was no agreement among the four different tissues regarding the results of PCR (P = 0.014). In the pairwise comparisons, significantly more positive results were obtained with skin biopsy and bone marrow samples compared to conjunctiva samples (P = 0.007 for both comparisons). In the univariable analysis of the possible risk factors for infection that are related to the signalment, the living conditions of the cats, and the season of sampling, only the latter was significantly associated with the infection status of the cats (P = 0.022, Table 3; odds ratio = 2.44, 95% CI: 1.014–5.881). In the univariable analysis, there was no association between the health status of the cats (i.e. assignment to group A or group B) and their infection status (P = 0.839) but the latter was positively associated with the presence of at least one systemic sign that has been previously reported in cats with leishmaniosis (P = 0.042; odds ratio = 3.93, 95% CI: 1.132–13.602) (Table 4). 4. Discussion Cytological examination of various tissues, including lymph nodes, skin and bone marrow, has been successfully used to demonstrate Leishmania amastigotes in cats with

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Table 1 Positive results of PCR for detection of L. infantum DNA in four different tissue samples collected from 50 clinically normal cats (group A) and 50 cats with various cutaneous, ocular and/or systemic clinical signs (group B). All cats were living in prefectures of Greece where canine leishmaniosis due to L. infantum is endemic. Tissue(s)

Group A

Group B

All cats

Blood Skin (iliac crest) Skin (lesional) Skin (iliac crest + lesional) Bone marrow Conjunctiva All tissues

10/50 (20%) 5/50 (10%) – 5/50 (10%) 9/50 (18%) 2/50 (4%) 21/50 (42%)

3/50 (6%) 10/49 (20.4%) 3/20 (15%) 13/49 (26.5%) 7/50 (14%) 1/46 (2.2%) 20/50 (40%)

13/100 (13%) 15/99 (15.2%) 3/20 (15%) 18/99 (18.2%) 16/100 (16%) 3/96 (3.1%) 41/100 (41%)

Table 2 Distribution of the PCR-positive results for L. infantum among four different tissue samples collected from 21 infected clinically normal cats (group A) and 20 infected cats with various cutaneous, ocular and/or systemic clinical signs (group B). Tissue(s)

Group A

Group B

All cats

PCR-positive in only one tissue sample Blood Skin Bone marrow Conjunctiva

8/21 (38.1%) 2/21 (9.5%) 5/21 (23.8%) 1/21 (4.8%)

1/20 (5%) 11/20 (55%) 4/20 (20%) 1/20 (5%)

9/41 (22%) 13/41 (31.7%) 9/41 (22%) 2/41 (4.9%)

PCR-positive in two tissue samples Blood + skin Blood + bone marrow Skin + bone marrow Bone marrow + conjunctiva

1/21 (4.8%) 1/21 (4.8%) 2/21 (9.5%) 1/21 (4.8%)

– 1/20 (5%) 1/20 (5%) –

1/41 (2.4%) 2/41 (4.9%) 3/41 (7.3%) 1/41 (2.4%)

PCR-positive in three tissue samples Blood + skin + bone marrow

–

1/20 (5%)

1/41 (2.4%)

Table 3 Univariable associations between the infection status of 100 cats living in prefectures of Greece where canine leishmaniosis due to L. infantum is endemic and possible risk factors for infection by L. infantum (signalment, living conditions, and season of sampling). Variables

Breed (purebred/common European) Age in years (mean ± standard deviation) Sex (male/female) Length of haircoat (short/long) Living conditions (indoors/outdoors/both) Living area (urban/semi-urban/rural) Lush vegetation within 100 m (yes/no) Sampling during the sandfly season (yes/no)

Infection status Infected

Non-infected

P value

4/37 4.9 ± 4.9 17/24 37/4 15/21/5 17/23/1 7/34 31/10

4/55 4.9 ± 4.1 33/26 52/7 25/22/12 23/34/2 8/51 31/28

0.713 0.981 0.222 1 0.327 0.942 0.777 0.022

Table 4 Univariable associations between the infection status of 50 cats living in prefectures of Greece where canine leishmaniosis due to L. infantum is endemic and the presence of clinical signs that have been previously reported in cats with leishmaniosis. Clinical signs

Alopecia-hypotrichosis Crusts Ulcers Papules Blepharitis Scales At least one cutaneous lesion Conjunctivitis Anorexia Vomiting Diarrhea Peripheral lymphadenomegaly Hepatomegaly Icterus Splenomegaly At least one systemic sign At least one cutaneous, ocular and/or systemic sign

Infection status Infected

Non-infected

P value

5 2 3 2 1 0 6 0 10 6 3 2 3 3 0 15 18

8 6 4 4 2 2 12 1 8 4 3 3 1 0 1 13 22

1 0.450 1 1 1 0.510 0.556 1 0.134 0.171 0.672 1 0.289 0.058 1 0.042 0.279

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clinical signs of leishmaniosis (Hervás et al., 1999; Pennisi, 2002; Poli et al., 2002; Pennisi et al., 2004; Savani et al., 2004; Leiva et al., 2005; Rüfenacht et al., 2005; Serrano et al., 2008; Marcos et al., 2009; Coelho et al., 2010; Vides et al., 2011). On the contrary, when cytology has been used as a diagnostic tool in cross-sectional epidemiological studies, positive results are rarely reported and the test underestimates the true prevalence of the infection (Simões-Mattos et al., 2004; Bresciani et al., 2010; Costa et al., 2010; Sobrinho et al., 2012), as it was also witnessed in the present study. In general, the sensitivity of cytology for the detection of Leishmania amastigotes depends on the parasitic density of the tissue that has been sampled, the quality of the smear, the skills of the observer and the number of oil immersion fields that are examined (Saridomichelakis et al., 2005). In the present study, a suboptimal quality of the smears can only partially account for the low sensitivity of lymph node cytology. For the remaining tissues and notably for bone marrow, where 1000 oil immersion fields were examined, the 0% sensitivity of cytology should be attributed to a low parasitic density. In a similar way, the diagnostic sensitivity of lymph node and bone marrow cytology has been shown to be low in dogs that are asymptomatically infected by L. infantum but high in dogs with the disease, in parallel with their parasitic density, which is generally low and high, respectively (Saridomichelakis et al., 2005; Saridomichelakis, 2009). In conclusion, although cytology is diagnostically valuable in cats that are suspect of leishmaniosis, it cannot be recommended as a sensitive diagnostic test in epidemiological surveys. Before PCR is employed for either clinical diagnostic purposes or within the context of epidemiological studies, the technical specifications of the assay must be carefully assessed. Such assessment involves a large number of interacting factors which makes a thorough evaluation of previously published PCR protocols an absolute prerequisite before their use (Saiki et al., 1988; Erlich, 1989). In the present study, the detection of Leishmania DNA was performed with a PCR assay that has been previously validated with regard to low detection limit, sensitivity, specificity, repeatability, and reproducibility (Andreadou et al., 2012). The specific assay was applied in full compliance with ISO 17025 standards for molecular detection of Leishmania DNA, including quality assurance of protocols, procedures and consumables, ring-trial testing, accreditation of the equipment, and routine evaluation of technical personnel. To ensure the accuracy of the results, vigorous precautions were taken to avoid the carry-over effect (contamination by amplicons from previous reactions) and positive results were confirmed through sequence analysis of the amplification product. Finally, to avoid false-negative results, fragmentation of DNA products and presence of PCR inhibitors were excluded in all samples prior to testing. With the theoretical exception of skin biopsies, that may be accidentally obtained from the site of recent parasite inoculation (Leontides et al., 2002), a positive PCR in the remaining tissues (blood, bone marrow, conjunctiva) denotes systemic dissemination of L. infantum. With this in mind, the lack of agreement among the four different tissues and the low number of infected cats where

two or three samples were PCR-positive, may be explained by a compartmentalization of the infection and/or a tissue parasitic density lower than the limit of detection of the assay. At least in dogs, parasitic burden differs among tissues due to the tropism of the parasite and organ-specific immunity (Solano-Gallego et al., 2001; Reithinger et al., 2002; Sanchez et al., 2004; Saridomichelakis, 2009). In the present study, given the precautions taken to avoid and detect false-negative PCR results, tissue parasitic density bellow the limit of detection is the only reasonable explanation for those cats where PCR was positive in the blood but negative in bone marrow, since the latter is inevitably contaminated by the former during the sample collection process. Nevertheless, these results clearly indicate that samples from multiple tissues should be examined by PCR in order to avoid underestimating of the true prevalence of the infection. Investigations in dogs infected by L. infantum have come to various, sometimes conflicting, conclusions regarding the relative sensitivity of PCR among the four tissues that were examined in the present study. Blood has been reported to be more, less or equally sensitive than skin samples (Manna et al., 2004; Manna et al., 2006; Manna et al., 2008; de Amorim et al., 2010; Leite et al., 2010; Chitimia et al., 2011), less or equally sensitive than bone marrow (Reithinger et al., 2000; Lachaud et al., 2001; Francino et al., 2006; Martínez et al., 2011) and less sensitive than conjunctiva (Strauss-Ayali et al., 2004; Ferreira et al., 2008; Leite et al., 2010; de Almeida Ferreira et al., 2012; Lombardo et al., 2012) for the molecular diagnosis of the infection. Also, skin samples have been reported to be more sensitive than bone marrow (Solano-Gallego et al., 2001; Michalsky et al., 2007) and more or less sensitive than conjunctiva (Solano-Gallego et al., 2001; Strauss-Ayali et al., 2004; Leite et al., 2010) and bone marrow has been reported to be less sensitive than conjunctiva (Solano-Gallego et al., 2001; de Almeida Ferreira et al., 2012). In a feline study, that has been published as an abstract (Pennisi et al., 2012), a higher percentage of cats (11/66–16.7%) was found to be positive by real-time PCR in conjunctiva compared to blood (16/203–7.8%) and lymph node (18/154–11.7%) samples. On the contrary, in the present study conjunctiva samples were of inferior diagnostic value and their sensitivity was significantly lower compared to that of skin biopsy and bone marrow samples. This is unfortunate because, among all the samples we have tested, conjunctiva were obtained less invasively and future studies should examine if their diagnostic yield can be increased in cats, as is the case in dogs (Lombardo et al., 2012), after PCR testing of samples obtained from both eyes. Nevertheless, the relatively common detection of L. infantum DNA in the blood and the skin (13% and 18.2% of the cats, respectively) may be considered as an indication in favor of their capability to transmit the parasite to sandfly vectors (Maroli et al., 2007; Saridomichelakis et al., 2007; Saridomichelakis, 2009). Previous blood PCR-based epidemiological surveys have shown no consistent risk factors for feline infection by L. infantum related to the signalment or the living conditions (Pennisi, 2002; Tabar et al., 2008; Sherry et al., 2011; Pennisi et al., 2012). Especially regarding age, the results have been conflicting with different studies

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showing a lower prevalence of infection in cats younger than 1 year (Pennisi, 2002), or younger than 3 years (Pennisi et al., 2012), or older than 5 years (Tabar et al., 2008). In the present study, where multiple different tissues were examined and only cats older than 1 year (i.e. cats that had sustained at least one whole period of sandfly activity) were included, there was no association between their age and infection status; furthermore, 15/34 (44.1%) of the 1–2 year-old cats were found to be infected (data not shown). These results are similar to our previous observations in clinically normal dogs living in the same endemic regions (Leontides et al., 2002) and they show that cats living in these regions can become infected by L. infantum at a relatively young age. An important finding of this study is the increased prevalence of infection when sampling was performed during the sandfly season. This may indicate that in some cats a transient disseminated infection occurs during the transmission period and/or that after the end of this period the parasitic burden is reduced and becomes lower than the detection limit of the assay (Maia et al., 2010; Maia and Campino, 2011; Pennisi et al., 2013). Future longitudinal studies are needed to further investigate this hypothesis. Cats are generally considered to be resistant to L. infantum (Mancianti, 2004; Pennisi et al., 2013) which may explain the lack of association between infection and clinical status in the present as well as in previous studies (Sherry et al., 2011). However, this is not always the case because some infected cats are susceptible and they develop clinical signs and/or laboratory abnormalities of leishmaniosis (Pennisi et al., 2013). The positive association between infection demonstrated by PCR-positivity and the presence of at least one systemic sign compatible with feline leishmaniosis may indicate a pathogenetic role of L. infantum in some group B infected cats. Since alternative diagnoses can explain the clinical signs of these patients, it is practically impossible to determine the true contribution, if any, of L. infantum in their clinical manifestations. In the face of concurrent disease conditions, the only way to investigate such a role is through the demonstration of the parasite in the diseased tissue(s) and organ(s), the presence of granulomatous or pyogranulomatous inflammation, and, most importantly, the response to anti-Leishmania treatment (Saridomichelakis, 2009) and this should be the subject of future studies. 5. Conclusions In conclusion, the present study demonstrates a relatively high prevalence of infection by L. infantum (41%) in cats living in endemic regions regardless of their clinical status (i.e. clinically normal cats or cats with cutaneous, ocular and/or systemic clinical signs). Lymph node, skin, bone marrow and conjunctiva swab smear cytology was always negative and the diagnosis of the infection was based on PCR performed on blood, skin biopsy, bone marrow and conjunctiva. The results of PCR differed among the four tissues, being infrequently positive in conjunctiva compared to skin biopsies and bone marrow, which clearly indicates the need for multiple tissue sampling for PCR in order to avoid underestimating the prevalence of the

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infection. The latter was found to be higher when sampling had been performed during the period of sandfly activity and when group B cats presented at least one systemic clinical sign that has been previously reported in cats with leishmaniosis. Conflict of interest None of the authors has a financial or personal relationship with other people or organizations that could inappropriately influence the content of this paper. Funding This research has been co-financed by the European Union (European Social Fund-ESF) and Greek national funds through the Operational Program “Education and Lifelong Learning” of the National Strategic Reference Framework (NSRF)-Research Funding Program: Heracleitus II Investing in knowledge society through the European Social Fund. The funding source was not involved in the study design, collection, analysis and interpretation of data, writing of the report or the decision to submit the article for publication. Acknowledgements Authors want to thank Drs. Kosmas Apostolidis, Dimitris Gougoulis, Dimitris Mpalomenos, Theodoros Petanidis, and Sofia Zoi for helping in the enrolment of cats into the study, Dr. Apostolos Galatos (Dip ECVAA) for his advice on the protocol for the chemical restrain of the cats and Dr. Ioannis Papas for his help during DNA isolation. References Andreadou, M., Liandris, E., Kasampalidis, I.N., Taka, S., Antoniou, M., Ntais, P., Vaiopoulou, A., Theodoropoulos, G., Gazouli, M., Ikonomopoulos, J., 2012. Evaluation of the performance of selected in-house and commercially available PCR and real-time PCR assays for the detection of Leishmania DNA in canine clinical samples. Exp. Parasitol. 131, 419–424. Athanasiou, L.V., Kontos, V.I., Saridomichelakis, M.N., Rallis, T.S., Diakou, A., 2012. A cross-sectional sero-epidemiological study of canine leishmaniasis in Greek mainland. Acta Trop. 122, 291–295. Ayllón, T., Diniz, P.P., Breitschwerdt, E.B., Villaescusa, A., Rodríguez-Franco, F., Sainz, A., 2012. Vector-borne diseases in client-owned and stray cats from Madrid Spain. Vector Borne Zoonotic Dis. 12, 143–150. Ayllon, T., Tesouro, M.A., Amusategui, I., Villaescusa, A., Rodriguez-Franco, F., Sainz, A., 2008. Serologic and molecular evaluation of Leishmania infantum in cats from Central Spain. Ann. N. Y. Acad. Sci. 1149, 361–364. Baneth, G., Koutinas, A.F., Solano-Gallego, L., Bourdeau, P., Ferrer, L., 2008. Canine leishmaniosis – new concepts and insights on an expanding zoonosis: part one. Trends Parasitol. 24, 324–330. Bresciani, K.D., Serrano, A.C., Matos, L.V., Savani, E.S., D’Auria, S.R., Perri, S.H., Bonello, F.L., Coelho, W.M., Aoki, C.G., Costa, A.J., 2010. Occurrence de Leishmania spp. in domestic cats from Arac¸atuba. SP. Rev. Bras. Parasitol. Vet. 19, 127–129. ˜ C.I., Sánchez-Velasco, D., Lizana, V., Del Río, L., Chitimia, L., Munoz-García, Murcia, L., Fisa, R., Riera, C., Giménez-Font, P., Jiménez-Montalbán, P., Martínez-Ramírez, A., Meseguer-Meseguer, J.M., García-Bacete, I., Sánchez-Isarria, M.A., Sanchis-Monsonís, G., García-Martínez, J.D., Vicente, V., Segovia, M., Berriatua, E., 2011. Cryptic Leishmaniosis by Leishmania infantum, a feature of canines only? A study of natural infection in wild rabbits, humans and dogs in southeastern Spain. Vet. Parasitol. 181, 12–16.

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