Leishmania infection in cats and feline leishmaniosis: An updated review with a proposal of a diagnosis algorithm and prevention guidelines

Abstract

Leishmaniosis is a vector-borne disease caused by protozoans of the genus Leishmania, which are transmitted to vertebrates, including cats, through the bites of female phlebotomine sand flies. An increasing number of epidemiological and experimental studies concerning Leishmania infection in cats, as well as case reports of clinical leishmaniosis in these felids, have been published in recent years. In the present study, a comprehensive review was made by sourcing the National Library of Medicine resources to provide updated data on epidemiology, immunopathogenesis, diagnosis, treatment, and prevention of feline leishmaniosis. Cats were found infected with Leishmania parasites worldwide, and feline leishmaniosis appears as an emergent disease mostly reported in countries surrounding the Mediterranean Sea and in Brazil. Cats with impaired immunocompetence seem to have a higher risk to develop clinical disease. The main clinical and clinicopathological findings are dermatological lesions and hypergammaglobulinemia, respectively. Diagnosis of feline leishmaniosis remains a challenge for veterinarians, in part due to the lack of diagnosis support systems. For this reason, a diagnostic algorithm for clinical decision support is herein proposed. No evidence-based treatment protocols are currently available, and these remain empirically based. Control measures are limited and scarce. Thus, a set of prevention guidelines are herein suggested.

Graphical abstract

Highlights

• •A comprehensive review on epidemiology, immunopathogenesis, diagnosis, treatment, and prevention of feline leishmaniosis.
• •An algorithm for assisting medical diagnosis of leishmaniosis in cats is suggested.
• •Guidelines for the prevention of Leishmania infection in cats are provided.
• •Dermatological lesions are the most common clinical manifestations.
• •Most cats with clinical leishmaniosis present hypergammaglobulinemia.

1. Introduction

Leishmaniosis is a disease that affects humans and both domestic and wild animals worldwide and is caused by protozoans of the genus Leishmania. The infection typically occurs through the bites of female phlebotomine sand flies of the genera Phlebotomus in the Old World and Lutzomyia in the New World (WHO, 2010).

In contrast to dogs, cats have been considered for several years as accidental hosts resistant to leishmaniosis. Nevertheless, this felid now appears as a relevant piece within the ecological system in which Leishmania parasites are maintained indefinitely (Asfaram et al., 2019). Feline Leishmania infection has frequently been reported in endemic areas of South America, Southern Europe and Western Asia, and the number of reported cases of feline leishmaniosis has been increasing in recent years (Pereira et al., 2019b; Baneth et al., 2020; da Costa-Val et al., 2020; Fernandez-Gallego et al., 2020).

The present review aimed to provide updated information concerning the epidemiology of Leishmania infection in cats and clinical management of feline leishmaniosis (FeL) with emphasis on immunopathogenesis, diagnosis, treatment, prognosis, and prevention, as well as the development of an algorithm to assist diagnosis and delineate strategic guidelines to prevent feline infection.

2. Search strategy, eligibility, and review

A comprehensive literature search was performed on 10 March 2021 by sourcing National Library of Medicine (NLM) resources through PubMed (https://pubmed.ncbi.nlm.nih.gov/) using the following Boolean string: (“leishmania” [MeSH Terms] OR “leishmania”[All Fields] OR “leishmanias” [All Fields] OR “leishmaniae” [All Fields] OR (“leishmaniasis” [MeSH Terms] OR “leishmaniasis” [All Fields] OR “leishmaniosis” [All Fields] OR “leishmaniases” [All Fields])) AND (“cat” [All Fields] OR (“felis” [MeSH Terms] OR “felis” [All Fields]) OR (“felidae” [MeSH Terms] OR “felidae” [All Fields] OR “felid” [All Fields] OR “felids” [All Fields]) OR (“cats” [MeSH Terms] OR “cats” [All Fields] OR “felines” [All Fields] OR “felidae” [MeSH Terms] OR “felidae” [All Fields] OR “feline” [All Fields])). Search results were saved as a comma-separated value (CSV) file, subsequently imported into Microsoft® Excel®. Study eligibility was manually assessed by two independent investigators in a blinded manner. Only available original research articles concerning Leishmania infection in cats were retained, including those published in languages other than English (Fig. 1). Except for the epidemiological section (which included data from all Leishmania spp. in felids belonging to the genus Felis), the present review refers exclusively to infection of domestic cats (Felis catus) by L. donovani (sensu lato). Although this complex is formally comprised of L. donovani (sensu stricto), L. chagasi and L. infantum, for the remainder of this review, L. infantum will be used to refer strictly to feline infection by L. donovani (s.l.).

Fig. 1.

Flow diagram of study searching and selection process

3. Aetiology, distribution, and risk factors

To date, six species belonging to the subgenus Leishmania and one to the subgenus Viannia have been identified in domestic cats (F. catus) through DNA or isoenzyme-based typing methods (Fig. 2):

• (i)L. (L.) amazonensis in Brazil (De Souza et al., 2005; Carneiro et al., 2020);
• (ii)L. (L.) infantum in Brazil (Schubach et al., 2004; De Souza et al., 2005; da Silva et al., 2008; Vides et al., 2011; Sobrinho et al., 2012; de Morais et al., 2013; Benassi et al., 2017; Metzdorf et al., 2017; Marcondes et al., 2018; Rocha et al., 2019; Berenguer et al., 2020; da Costa-Val et al., 2020), southern Europe (Ayllón et al., 2008, 2012; Maia et al., 2008, 2010, 2014, 2015b; Tabar et al., 2008; Millán et al., 2011; Chatzis et al., 2014a; Persichetti et al., 2016, 2018; Attipa et al., 2017a; Diakou et al., 2017; Otranto et al., 2017; Colella et al., 2019; Pereira et al., 2019c, 2020; Ebani et al., 2020), western Europe (Ozon et al., 1998; Pratlong et al., 2004; Pocholle et al., 2012; Richter et al., 2014) and western Asia (Hatam et al., 2010; Dincer et al., 2015; Akhtardanesh et al., 2017; Attipa et al., 2017b; Mohebali et al., 2017; Karakuş et al., 2019; Asgari et al., 2020; Baneth et al., 2020);
• (iii)L. (L.) major in Portugal (Pereira et al., 2020) and Turkey (Paşa et al., 2015);
• (iv)L. (L.) mexicana in the USA (Craig et al., 1986; Trainor et al., 2010; Minard et al., 2017) and Venezuela (Rivas et al., 2018);
• (v)L. (L.) tropica in western Asia (Paşa et al., 2015; Can et al., 2016; Akhtardanesh et al., 2017);
• (vi)L. (L.) venezuelensis in Venezuela (Bonfante-Garrido et al., 1991); and
• (vii)L. (V.) braziliensis in Brazil (Schubach et al., 2004; da Costa-Val et al., 2020) and French Guiana (Rougeron et al., 2011).

Fig. 2.

Worldwide distribution of Leishmania infection in cats (Felis spp.)

Besides, DNA of L. infantum and putative L. major/L. donovani (s.l.) hybrid parasites were detected in wild cats (Felis silvestris) in Spain (Del Río et al., 2014) and in a domestic cat in mainland Portugal (Pereira et al., 2020), respectively.

The proportion of cats infected with or exposed to Leishmania has been assessed in several epidemiological studies through parasitological, serological, or molecular methods (Table 1 and Table 2). However, reported values vary greatly (from 0 to > 70%) and appear to be influenced by local endemicity, sampling bias and heterogeneity/performance of diagnostic methodologies (manly cut-off, target gene and sample used for testing).

Table 1.

Epidemiological studies on the frequency of Leishmania infection in cats (Felis spp.) in the Old World

Country	Study	Sampling year	Species (origin)	No. tested	Method (test, cut-off/target gene)	Sample	% Positive (species)a
Albania	Silaghi et al. (2014)	2008–2010	F. catus (stray)	146	Serological (IFAT, 1:64)	Serum	0.7 (L. infantum)
					Molecular (qPCR, kDNA)	Whole blood	0
Angola	Lopes et al. (2017)	2014–2016	F. catus (domestic)	102	Serological (DAT, 1:100)	Serum	0
Bosnia and Herzegovina	Colella et al. (2019)	2017	F. catus (domestic)	5	Serological (IFAT)	Serum	0
					Molecular (qPCR, kDNA)	Whole blood	20.0 (Leishmania spp.)
				1b	Molecular (PCR, kDNA)	Whole blood	100 (L. infantum)
					Molecular (qPCR, ITS2)	Whole blood	100 (L. infantum)
Cyprus	Attipa et al., 2017a, Attipa et al., 2017b	2014	F. catus (domestic/shelter)	164	Serological (ELISA, 32 EU)	Serum	4.4 (L. infantum)
				174	Molecular (qPCR, kDNA)	Whole blood	2.3 (L. infantum)
Egypt	Michael et al. (1982)	na	F. catus (stray)	80	Serological (IHA)	Serum	3.8 (Leishmania spp.)
	Morsy et al. (1988)	na	F. catus (stray)	28	Serological (IHA)	Serum	3.6 (Leishmania spp.)
	Morsy & Abou el Seoud (1994)	na	F. catus (domestic/stray)	60	Serological (IHA, 1:32)	Serum	10.0 (Leishmania spp.)
Germany	Schäfer et al. (2021)	2012–2020	F. catus (domestic)	624	Serological (IFAT, 1:64)	Serum	4.0 (Leishmania spp.)
Greece	Chatzis et al. (2014a, b)	2009–2011	F. catus (domestic)	100	Parasitological (cytology)	Bone marrow	0
						Lymph node	0
						Skin	0
					Serological (ELISA, 0.145)	Serum	1.0 (Leishmania spp.)
					Serological (IFAT, 1:10)	Serum	10.0 (Leishmania spp.)
					Molecular (PCR, kDNA)	Bone marrow	16.0 (L. infantum)
						Whole blood	13.0 (L. infantum)
				99	Molecular (PCR, kDNA)	Skin	13.1 (L. infantum)
				96		Conjunctival swab	3.1 (L. infantum)
	Diakou et al. (2017)	2015	F. catus (stray)	148	Serological (IFAT, 1:80)	Serum	6.1 (L. infantum)
					Molecular (nPCR, SSU)	Whole blood	6.1 (L. infantum)
	Diakou et al. (2009)	na	F. catus (stray)	284	Serological (ELISA)	Serum	3.9 (Leishmania spp.)
	Morelli et al. (2020)	na	F. catus	153	Serological (IFAT, 1:80)	Serum	2.0 (L. infantum)
Iran	Mohebali et al. (2017)	2013–2015	F. catus (stray)	103	Serological (DAT, 1:320)	Serum	3.9 (L. infantum)
				4b	Parasitological (cytology)	Liver	25.0 (L. infantum)
						Spleen	25.0 (L. infantum)
				4b	Parasitological (culture)	Liver	0
						Spleen	0
				1b	Molecular (nPCR, ITS2)	Liver	100 (L. infantum)
						Spleen	100 (L. infantum)
	Akhtardanesh et al. (2020)	2016	F. catus (stray)	180	Molecular (nPCR, kDNA)	Whole blood	13.9 (L. infantum)
	Asgari et al. (2020)	2016–2018	F. catus (stray)	174	Serological (DAT, 1:100)	Serum	17.2 (L. infantum)
					Serological (ELISA)	Serum	27.6 (L. infantum)
					Molecular (nPCR, kDNA)	Buffy coat	20.7 (L. infantum)
	Sarkari et al. (2009)	na	F. catus (stray)	40	Serological (DAT, 1:20)	Serum	20.0 (L. infantum)
					Serological (IFAT, 1:10)	Serum	25.0 (L. infantum)
	Hatam et al. (2010)	na	F. catus (stray)	40	Parasitological (cytology)	Liver	2.5 (Leishmania spp.)
						Spleen	2.5 (Leishmania spp.)
					Parasitological (culture)	Liver	7.5 (Leishmania spp.)
						Spleen	2.5 (Leishmania spp.)
					Molecular (PCR, kDNA	Liver	7.5 (L. infantum)
						Spleen	5.0 (L. infantum)
	Fatollahzadeh et al. (2016)	na	F. catus (stray)	65	Parasitological (cytology)	Liver	0
						Spleen	0
					Parasitological (culture)	Liver	0
						Spleen	0
					Serological (DAT, 1:320)	Serum	23.1 (L. infantum)
					Molecular (PCR, kDNA)	Spleen	0
	Akhtardanesh et al. (2017)	na	F. catus (stray)	60	Serological (ELISA)	Serum	6.7 (L. infantum)
					Molecular (nPCR, 7SL RNA)	Whole blood	16.7 (L. infantum)
							1.7 (L. tropica)
Iraq	Otranto et al. (2019)	2008	F. catus (stray)	207	Molecular (qPCR, kDNA)	Whole blood	0
Israel	Nasereddin et al. (2008)	1999–2000	F. catus (domestic/stray)	104	Serological (ELISA)	Serum	6.7 (L. infantum)
	Baneth et al. (2020)	2018	F. catus (shelter)	67	Serological (ELISA, 0.4)	Serum	75.0 (L. infantum)
					Molecular (qPCR, kDNA)	Whole blood	16.0 (L. infantum)
					Molecular (HRMPCR, ITS1)	Whole blood	0
Italy	Vita et al. (2005)	2002–2004	F. catus (domestic/stray)	203	Serological (IFAT, 1:40)	Serum	16.3 (L. infantum)
				11b	Molecular (PCR)	Lymph node	100 (L. infantum)
						Whole blood	45.5 (L. infantum)
	Spada et al. (2013)	2008–2010	F. catus (stray)	233	Serological (IFAT, 1:40)	Serum	25.3 (L. infantum)
					Molecular (qPCR, kDNA)	Whole blood	0
	Morganti et al. (2019)	2010–2016	F. catus (shelter/stray)	286	Serological (IFAT, 1:40)	Serum	9.1 (L. infantum)
					Molecular (nPCR, SSU)	Buffy coat	0
						Conjunctival swab	15.7 (L. infantum)
	Dedola et al. (2018)	2011–2013	F. catus (domestic)	90	Serological (IFAT, 1:40)	Serum	10.0 (L. infantum)
					Molecular (nPCR, ITS)	Whole blood	5.5 (L. infantum)
	Veronesi et al. (2016)	2011–2014	F. silvestris (wild)	21	Molecular (qPCR, COII)	Spleen	0
	Persichetti et al. (2016)	2012–2013	F. catus (domestic)	42	Serological (IFAT, 1:80)	Serum	2.4 (L. infantum)
					Molecular (qPCR, kDNA)	Whole blood	42.8 (L. infantum)
	Persichetti et al. (2018)	2012–2013	F. catus (domestic)	197	Parasitological (cytology)	Whole blood	0
					Serological (IFAT, 1:80)	Serum	9.6 (L. infantum)
					Molecular (qPCR, kDNA)	Conjunctival swab	1.5 (L. infantum)
				181	Molecular (qPCR, kDNA)	Lymph node	1.7 (L. infantum)
				143	Molecular (qPCR, kDNA)	Urine	2.1 (L. infantum)
				197	Molecular (qPCR, kDNA)	Oral swab	1.5 (L. infantum)
						Whole blood	2.0 (L. infantum)
	Spada et al. (2016)	2014	F. catus (stray)	90	Serological (IFAT, 1:40)	Serum	30.0 (L. infantum)
					Molecular (qPCR, kDNA)	Conjunctival swab	0
						Lymph node	1.1 (L. infantum)
						Whole blood	1.1 (L. infantum)
	Brianti et al. (2017)	2015	F. catus (domestic)	159	Serological (IFAT, 1:80)	Serum	9.4 (L. infantum)
					Molecular (qPCR, kDNA)	Conjunctival swab	3.8 (L. infantum)
						Whole blood	7.5 (L. infantum)
	Otranto et al. (2017)	2015–2016	F. catus (domestic)	330	Serological (IFAT, 1:40)	Serum	25.7 (L. infantum)
					Molecular (qPCR, kDNA)	Conjunctival swab	1.8 (L. infantum)
						Whole blood	2.1 (L. infantum)
	Abbate et al. (2019)	2015–2017	F. silvestris (wild)	11	Molecular (qPCR, kDNA)	Lymph node/skin/spleen	0
	Priolo et al. (2019)	2016–2017	F. catus (domestic/stray)	66	Serological (ELISA)	Serum	17.0 (L. infantum)
					Serological (IFAT, 1:80)	Serum	14.0 (L. infantum)
					Molecular (qPCR, kDNA)	Whole blood	4.0 (L. infantum)
	Spada et al. (2020)	2016–2018	F. catus (stray)	102	Serology (IFAT, 1:80)	Serum	4.9 (L. infantum)
				117	Molecular (qPCR, kDNA)	Conjunctival swab	0
				115	Molecular (qPCR, kDNA)	Lymph node	4.3 (L. infantum)
				109	Molecular (qPCR, kDNA)	Whole blood	0
	Urbani et al. (2020)	2017	F. catus (domestic)	152	Serological (IFAT, 1:80)	Serum	11.8 (L. infantum)
				150	Molecular (qPCR, kDNA)	Conjunctival swab	0
						Hair	0.7 (L. infantum)
				146	Molecular (qPCR, kDNA)	Whole blood	0
	Iatta et al. (2019)	2017–2018	F. catus (domestic)	2,659	Serological (IFAT, 1:80)	Serum	3.3 (L. infantum)
					Molecular (qPCR, kDNA)	Whole blood	0.8 (L. infantum)
	Ebani et al. (2020)	2018–2019	F. catus (stray)	85	Serological (IFAT)	Serum	2.4 (Leishmania spp.)
					Molecular (PCR, SSU)	Bloodc	5.9 (Leishmania spp.)
	Persichetti et al. (2017)	2013	na	76	Serological (ELISA, 40 EU)	Serum	2.6 (L. infantum)
					Serological (IFAT, 1:80)	Serum	17.1 (L. infantum)
					Serological (WB)	Serum	18.4 (L. infantum)
				21b	Serological (ELISA, 40 EU)	Serum	100 (L. infantum)
					Serological (IFAT, 1:80)	Serum	95.2 (L. infantum)
					Serological (WB)	Serum	100 (L. infantum)
	Poli et al. (2002)	na	F. catus (domestic)	110	Serological (IFAT, 1:80)	Serum	0.9 (Leishmania spp.)
	Morelli et al. (2019)	na	F. catus (domestic)	167	Serological (IFAT, 1:80)	Serum	3.0 (L. infantum)
	Morelli et al. (2020)	na	F. catus	116	Serological (IFAT, 1:80)	Serum	4.3 (L. infantum)
Portugal	Duarte et al. (2010)	2003–2005	F. catus (stray)	180	Serology (IFAT, 1:40)	Serum	0.6 (L. infantum)
	Maia et al. (2008)	2004	F. catus (stray)	20	Serological (IFAT, 1:64)	Serum	0
				23	Molecular (PCR, ITS1)	Blood on filter paper	30.4 (Leishmania spp.)
					Molecular (PCR, kDNA)	Blood on filter paper	30.4 (Leishmania spp.)
				4b	Molecular (PCR–RFLP, ITS1)	Blood on filter paper	100 (L. infantum)
	Cardoso et al. (2010)	2004–2008	F. catus (domestic)	316	Serological (DAT, 1:100)	Serum	1.9 (L. infantum)
					Serological (ELISA)	Serum	2.8 (L. infantum)
	Maia et al. (2010)	2007–2008	F. catus (domestic/stray)	76	Serological (IFAT, 1:64)	Serum	1.3 (Leishmania spp.)
				138	Molecular (PCR, kDNA)	Whole blood	20.3 (L. infantum)
	Maia et al., 2015a, Maia et al., 2015b	2011–2014	F. catus (domestic/stray)	271	Serological (DAT, 1:100)	Serum	3.7 (L. infantum)
	Maia et al. (2014)	2012–2013	F. catus (domestic/stray)	649	Molecular (nPCR, SSU)	Whole blood	9.9 (Leishmania spp.)
	Pereira et al. (2019a, b, 2020)	2017–2018	F. catus (domestic/shelter/stray)	373	Serological (IFAT, 1:64)	Serum	1.6 (Leishmania spp.)
				465	Molecular (nPCR, SSU)	Buffy coat	5.4 (Leishmania spp.)
				25b	Molecular (nPCR, cytB)	Buffy coat	12.0 (L. donovani s.l.)
							4.0 (L. major)
							4.0 (L. major/L. donovani s.l.)f
					Molecular (PCR, g6pdh)	Buffy coat	4.0 (L. donovani s.l.)
					Molecular (nPCR, hsp70)	Buffy coat	12.0 (L. donovani s.l.)
							4.0 (L. major/L. donovani s.l.)f
					Molecular (nPCR, ITS)	Buffy coat	12.0 (L. donovani s.l.)
							4.0 (L. major)
	Neves et al. (2020)	2018–2019	F. catus (domestic)	141	Serological (DAT, 1:100)	Serum	0
	Vilhena et al. (2013)	na	F. catus (domestic)	320	Molecular (qPCR, kDNA)	Whole blood	0.3 (L. infantum)
Portugal/Spain	Mesa-Sanchez et al. (2020)	2015–2020	F. catus (domestic)g	173	Molecular (nPCR, SSU)	Whole blood	0
Qatar	Lima et al. (2019)	2016–2018	F. catus (domestic/stray)	79	Molecular (qPCR, kDNA)	Whole blood/on dried spot	1.3 (Leishmania spp.)
Saudi Arabia	Morsy et al. (1999)	na	F. margarita (wild)	10	Parasitological (cytology)	Liver	20.0 (Leishmania spp.)
						Spleen	40.0 (Leishmania spp.)
					Serological (IHA, 1:64)	Serum	40.0 (Leishmania spp.)
Spain	Del Río et al. (2014)	2001–2006	Felis silvestris (wild)	4	Molecular (qPCR, kDNA)	Liver and/or spleen	25.0 (L. infantum)
				1b	Molecular (PCR, ITS2)	Liver and/or spleen	100 (L. infantum)
	Martín-Sánchez et al. (2007)	2003–2004	F. catus (domestic)	183	Serological (IFAT, 1:40)	Serum	28.3 (Leishmania spp.)
					Molecular (PCR-ELISA, kDNA)	Whole blood	25.7 (L. infantum)
				7b	Parasitological (culture)	Leucoconcentrate	0
					Parasitological (cytology)	Leucoconcentrate	42.9 (Leishmania spp.)
	Ayllón et al. (2008)	2005–2006	F. catus (domestic)	233	Serological (IFAT, 1:100)	Serum	1.3 (L. infantum)
					Molecular (PCR, kDNA)	Whole blood	0.4 (L. infantum)
	Ayllón et al. (2012)	2005–2008	F. catus (domestic/stray)	680	Serological (IFAT, 1:50)	Serum	3.7 (L. infantum)
					Molecular (PCR, kDNA)	Whole blood	0.6 (L. infantum)
	Tabar et al. (2008)	2006	F. catus (domestic)	100	Molecular (qPCR, kDNA)	Whole blood	3.0 (L. infantum)
	Sherry et al. (2011)	2008	F. catus (shelter)	105	Serological (ELISA)	Serum	13.2 (L. infantum)
				104	Molecular (qPCR, kDNA)	Whole blood	8.7 (L. infantum)
	Millán et al. (2011)	2008–2009	F. catus (stray)	83	Serological (WB)	Serum	15.7 (L. infantum)
				73	Molecular (PCR, kDNA)	Blood and/or spleen	25.6 (L. infantum)
				14b	Molecular (PCR–RFLP, kDNA)	Blood and/or spleen	100 (L. infantum)
	Miró et al. (2014)	2012–2013	F. catus (stray)	346	Serological (IFAT, 1:100)	Serum	3.2 (L. infantum)
				57d	Molecular (nested PCR, ITS1)	Whole blood	0
					Molecular (nested PCR, SSU)	Whole blood	0
	Risueño et al. (2018)	2013–2015	F. silvestris (wild)	2	Molecular (qPCR, kDNA)	Skin	50.0 (L. infantum)
						Other organse	0
	Marenzoni et al. (2018)	2014–2015	F. catus (domestic)	31g	Molecular (PCR, kDNA)	Whole blood	0
	Montoya et al. (2018a)	2014–2017	F. catus (stray)	249	Serological (IFAT, 1:100)	Serum	4.8 (L. infantum)
					Molecular (PCR, ITS)	Skin/whole blood	0
	Priolo et al. (2019)	2016–2017	F. catus (domestic/stray)	113	Serological (ELISA)	Serum	7.0 (L. infantum)
					Serological (IFAT, 1:80)	Serum	19.0 (L. infantum)
					Molecular (qPCR, kDNA)	Whole blood	5.0 (L. infantum)
	Villanueva-Saz et al. (2021)	2020	F. catus (stray)	114	Serological (ELISA, 13 EU)	Serum	16.7 (L. infantum)
	Solano-Gallego et al. (2007)	na	F. catus (domestic/stray)	445	Serological (ELISA-IgG, 53 EU)	Serum	5.3 (L. infantum)
		na			Serological (ELISA-Prot A, 44 EU)	Serum	6.3 (L. infantum)
	Alcover et al. (2020)	na	F. catus (wild)	1	Molecular (qPCR, kDNA)	Liver	100 (Leishmania spp.)
		na				Skin	100 (Leishmania spp.)
		na				Spleen	100 (Leishmania spp.)
	Miró et al. (2011)	na	F. catus (breeding)	20	Serological (IFAT, 1:100)	Serum	15.0 (L. infantum)
	Moreno et al. (2014)	na	F. catus (stray)	43	Serological (IFAT, 1:50)	Serum	4.3 (L. infantum)
	Montoya et al. (2018b)	na	F. catus (stray)		Serological (IFAT, 1:100)	Serum	0
Thailand	Sukmee et al. (2008)	2006	F. catus	15	Serological (DAT; 1:100)	Serum	60.0 (Leishmania spp.)
				9b	Molecular (PCR, ITS1)	Whole blood	0
					Molecular (PCR, kDNA)	Whole blood	0
	Junsiri et al. (2017)	2013	F. catus (domestic)	250	Serological (ELISA, 0.2)	Serum	5.6 (L. infantum)
					Molecular (PCR, kDNA)	Whole blood	0
	Kongkaew et al. (2007)	na	F. catus	5	Serological (DAT, 1:100)	Serum	20.0 (Leishmania spp.)
				1b	Molecular (PCR)	Whole blood	0
Turkey	Dincer et al. (2015)	2013	F. catus (domestic/shelter)	22	Molecular (nPCR, kDNA)	Whole blood	4.5 (L. infantum)
	Karakuş et al. (2019)	2014	F. catus (stray)	5	Molecular (nPCR, SSU)	Conjunctival swab	0
		2015		8	Molecular (qPCR, ITS1)	Conjunctival swab	12.5 (L. infantum)
		2016		6	Molecular (qPCR, ITS1)	Conjunctival swab	0
	Dincer et al. (2016)	2015	F. catus (domestic/shelter)	50	Molecular (nPCR, kDNA)	na	0
	Dinçer et al. (2012)	na	F. catus (domestic)	1	Serological (IFAT)	Serum	0
					Molecular (PCR)	na	0
	Paşa et al. (2015)	na	F. catus (domestic)	147	Molecular (qPCR, ITS1)	Whole blood	2.7 (L. major)
							8.8 (L. tropica)
					Molecular (qPCR, hsp70)	Whole blood	2.0 (L. major)
							2.7 (L. tropica)
							2.7 (Leishmania spp.)
	Can et al. (2016)	na	F. catus (shelter)	1,101	Serological (ELISA)	Serum	10.8
					Serological (IFAT, 1:40)	Serum	15.2
					Molecular (qPCR, ITS1)	Whole blood	0.1 (L. tropica)
					Molecular (nPCR, kDNA)	Whole blood	0.1 (L. infantum)
							0.5 (L. tropica)
UK	Persichetti et al. (2017)	2013	F. catus	64	Serological (ELISA, 40 EU)	Serum	1.6 (L. infantum)
					Serological (IFAT, 1:80)	Serum	0
					Serological (WB)	Serum	3.1 (L. infantum)
Uzbekistan	Kovalenko et al. (2011)	na	F. catus	1	Serological (ELISA)	Serum	0

Abbreviations: COII, cytochrome oxidase II; cytB, cytochrome b; DAT, direct agglutination test; ELISA, enzyme-linked immunosorbent assay; EU, ELISA units; F., Felis; g6pdh, glucose-6-phosphate dehydrogenase; _HRMPCR, high resolution melt PCR; hsp70, heat-shock protein 70; IFAT, immunofluorescence antibody test; IgG, Immunoglobulin G; IHA, indirect hemagglutination; ITS, internal transcriber spacers; ITS1, internal transcriber spacer 1; ITS2, internal transcriber spacer 2; kDNA, kinetoplast minicircle DNA; L., Leishmania; na, not available; nPCR, nested PCR; PCR, one-step PCR (polymerase chain reaction); Prot A, Protein A; qPCR, real-time PCR; RFLP, restriction fragment length polymorphism; s.l., sensu lato; SSU, small subunit ribosomal DNA; WB, western blot.

^aSpecies defined according to the original study.

^bPreviously identified as positive by another test.

^cDNA extracted from the sediment obtained after centrifugation of the blood samples.

^dSeropositive for L.infantum and/or for feline retrovirus (feline leukemia virus and/or feline immunodeficiency virus).

^eNot specified.

^fPutative hybrid.

^gCats eligible for blood donation.

Table 2.

Epidemiological studies on the frequency of Leishmania infection in cats (Felis spp.) in the New World

Country	Study	Sampling year	Species (origin)	No. tested	Method (test, cut-off/target gene)	Sample	% Positive (species)a
Brazil	De Matos et al. (2018)	2004–2014	F. catus	679	Serological (ELISA)	Serum	43.4 (Leishmania spp.)
					Serological (IFAT, 1:40)	Serum	15.8 (Leishmania spp.)
	Figueiredo et al. (2009)	2005	F. catus (domestic)	43	Serological (ELISA)	Serum	2.4 (Leishmania spp.)
					Serological (IFAT, 1:40)	Serum	0
	Coelho et al. (2011a)	2007–2009	F. catus	70	Serological (ELISA)	Serum	4.2 (Leishmania spp.)
					Serological (IFAT, 1:40)	Serum	0.0 (Leishmania spp.)
	Vides et al. (2011)	2008–2009	F. catus	55	Parasitological (cytology)	Bone marrow	12.7 (Leishmania spp.)
						Liver	3.6 (Leishmania spp.)
						Lymph node	5.5 (Leishmania spp.)
						Spleen	7.3 (Leishmania spp.)
					Parasitological (IHC)	Skin	16.4 (Leishmania spp.)
					Serological (ELISA, 0.277)	Serum	25.4 (Leishmania spp.)
					Serological (IFAT, 1:40)	Serum	10.9 (Leishmania spp.
				3	Molecular (qPCR, gp63)	Whole blood	100 (L. chagasi)
	Cardia et al. (2013)	2010	F. catus (shelter/stray)	386	Serological (IFAT, 1:40)	Serum	0.5 (Leishmania spp.)
	Silva et al. (2014)	2010	F. catus (domestic/shelter)	153	Serological (ELISA)	Serum	3.9 (L. infantum)
	De Sousa Oliveira et al. (2015)	2012	F. catus	52	Molecular (PCR, kDNA)	Conjunctival swab	13.5 (Leishmania spp.)
	de Sousa et al. (2014)	2013	F. catus (domestic/stray)	151	Serological (IFAT, 1:40)	Serum	6.6 (L. infantum)
	Metzdorf et al. (2017)	2013–2014	F. catus (domestic/shelter)	100	Parasitological (cytology)	Bone marrow	4.0 (Leishmania spp.)
						Lymph node	4.0 (Leishmania spp.)
						Whole blood	4.0 (Leishmania spp.)
					Molecular (PCR-RFLP, kDNA)	Bone marrow	6.0 (L. infantum)
						Lymph node	3.0 (L. infantum)
						Whole blood	4.0 (L. infantum)
	Leonel et al. (2020)	2014	F. catus (shelter)	94	Serological (ELISA)	Serum	31.9 (Leishmania spp.)
					Serological (IFAT, 1:40)	Serum	29.8 (Leishmania spp.)
					Molecular (PCR, kDNA)	Conjunctival swab	0
						Whole blood	0
	Marcondes et al. (2018)	2014–2015	F. catus (domestic/shelter)	50b	Parasitological (cytology)	Bone marrow	14.0 (Leishmania spp.)
					Molecular (qPCR, kDNA)	Bone marrow	86.0 (L. infantum)
						Whole blood	72.0 (L. infantum)
	Rocha et al. (2019)	2016–2017	F. catus (domestic)	105	Serological (IFAT, 1:40)	Serum	30.5 (L. infantum)
					Molecular (PCR, CH1)	Whole blood	2.9 (L. infantum)
					Molecular (PCR, ITS1)	Whole blood	5.7 (L. infantum)
	Pedrassani et al. (2019)	2017	F. catus (domestic)	30	Serological (IFAT, 1:80)	Serum	6.6 (L. infantum)
					Molecular (PCR, kDNA)	Whole blood	0
	Berenguer et al. (2020)	2017	F. catus (domestic)	128	Molecular (PCR, kDNA)	Conjunctival swab	0
						Whole blood	0.8 (L. infantum)
				3c	Parasitological (cytology)	Lymph node	33.3 (Leishmania spp.)
					Molecular (PCR, kDNA)	Lymph node	33.3 (L. infantum)
	Bezerra et al. (2019)	2017–2018	F. catus (domestic)	91	Serological (IFAT, 1:40)	Serum	15.4 (Leishmania spp.)
					Molecular (PCR, kDNA)	Whole blood	0
	da Silva et al. (2008)	na	F. catus (domestic)	8	Serological (IFAT, 1:40)	Serum	25.0 (Leishmania spp.)
				3	Molecular (multiplex PCR, kDNA)	Whole blood	66.7 (Leishmania spp.)
				2b	Molecular (DB)	Whole blood	100 (L. chagasi)
	Bresciani et al. (2010)	na	F. catus (domestic)	283	Parasitological (cytology)	Lymph node	0.7 (Leishmania spp.)
					Serological (IFAT, 1:40)	Serum	0
	da Silveira Neto et al. (2011)	na	F. catus (shelter)	130	Serological (CAG-ELISA, 0.449)	Serum	23.0 (Leishmania spp.)
					Serological (FML-ELISA, 0.215)	Serum	13.3 (Leishmania spp.)
					Serological (rK39-ELISA, 0.347)	Serum	15.9 (Leishmania spp.)
	Coelho et al. (2011b)	na	F. catus (domestic)	52	Parasitological (cytology)	Bone marrow	0
						Lymph node	3.8 (Leishmania spp.)
						Spleen	0
					Molecular (PCR, kDNA)	Bone marrow	0
						Lymph node	3.8 (L. chagasi)
						Spleen	1.9 (L. chagasi)
	Sobrinho et al. (2012)	na	F. catus (shelter/stray)	302	Parasitological (Cytology)	Bone marrow	7.0 (Leishmania spp.)
						Lymph node	7.9 (Leishmania spp.)
					Serological (ELISA, 0.301)	Serum	13.0 (Leishmania spp.)
					Serological (IFAT, 1:40)	Serum	4.6 (Leishmania spp.)
				5b	Molecular (qPCR, gp63)	Whole blood	100 (L. infantum)
	de Morais et al. (2013)	na	F. catus (domestic)	5	Molecular (qPCR, kDNA)	Whole blood	80.0 (L. infantum)
					Molecular (PCR, kDNA)	Whole blood	80.0 (L. infantum)
	Braga et al. (2014a)	na	F. catus (domestic)	50	Serological (IFAT, 1:40)	Serum	4.0 (Leishmania spp.)
Braga et al. (2014b)	na	F. catus	100	Parasitological (culture)	Whole blood	2.0 (Leishmania spp.)
				Serological (IFAT, 1:40)	Serum	15.0 (Leishmania spp.)
				Molecular, PCR, kDNA)	Whole blood	0
Oliveira et al. (2015)	na	F. catus (domestic)	443	Serological (DAT, 1:40)	Serum	5.6 (Leishmania spp.)
				Serological (IFAT, 1:40)	Serum	4.1 (Leishmania spp.)
Benassi et al. (2017)	na	F. catus (domestic/stray)	108	Molecular (PCR, kDNA)	Conjunctival swab	1.9 (Leishmania spp.)
					Whole blood	0
				Molecular (qPCR, kDNA)	Conjunctival swab	1.9 (Leishmania spp.)
					Whole blood	0
			2b	Molecular (PCR, ITS1)	Conjunctival swab	50.0 (L. infantum)
Coura et al. (2018)	na	F. catus (shelter)	100	Parasitological (cytology)	Bone marrow	0
				Parasitological (culture)	Bone marrow	0
				Serological (IFAT, 1:40)	Serum	54.0 (Leishmania spp.)
			54b	Molecular (PCR, kDNA)	Bone marrow/skin	0
da Costa-Val et al. (2020)	na	F. catus (domestic)	64	Serological (ELISA, 0.955)	Serum	29.8 (Leishmania spp.)
			64	Molecular (PCR, kDNA)	Conjunctival swab	6.3 (Leishmania spp.)
			64	Molecular (PCR, kDNA)	Oral swab	4.7 (Leishmania spp.)
			8b	Molecular (PCR-RFLP, ITS1)	Conjunctival swab	12.5 (L. infantum)
					Oral swab	37.5 (L. infantum)
						12.5 (L. braziliensis)
Honduras	Mccown & Grzeszak (2010)	na	F. catus (stray)	12	Serological (IFAT, 1:32)	Serum	25.0 (L. donovani)
Mexico	Longoni et al. (2012)	2008–2009	F. catus (stray)	95	Serological (ELISA-H)	Serum	5.3 (L. braziliensis)
							13.7 (L. infantum)
							1.1 (L. mexicana)
					Serological (ELISA-SODe)	Serum	11.6 (L. baziliensis)
							22.1 (L. infantum)
							10.5 (L. mexicana)
					Serological (WB)	Serum	10.5 (L. baziliensis)
							20.0 (L. infantum)
							10.5 (L. mexicana)
Venezuela	Viettri et al. (2018)	na	na	5	Molecular (nested PCR, ITS1)	Blood on filter paper	20.0 (Leishmania spp.)
					Molecular (nPCR, SSU rDNA)	Blood on filter paper	20.0 (Leishmania spp.)
	Rivas et al. (2018)		F. catus (domestic/stray)	6	Parasitological (cytology)	Skin lesions	66.7 (Leishmania spp.)
				5	Parastiological (histology)	Skin lesions	80.0 (Leishmania spp.)
				5	Parasitological (IHC)	Skin lesions	100 (Leishmania spp.)
				30	Serological (ELISA, 15.3 EU)	Serum	6.7 (L. braziliensis)
					Serological (ELISA, 15.3 EU)	Serum	6.7 (L. infantum)
					Serological (WB)	Serum	33.3 (L. braziliensis)
					Serological (WB)	Serum	33.3 (L. infantum)
				31	Molecular (qPCR, kDNA)	Whole blood	9.7 (Leishmania spp.)
				5	Molecular (qPCR, kDNA)	Skin lesions	100 (Leishmania spp.)
					Molecular (qPCR, ITS1)	Skin lesions	40.0 (L. mexicana)
				2b	Molecular (PCR-RFLP, ITS1)	Skin lesions	50.0 (L. mexicana)
	Paniz Mondolfi et al. (2019)	na	na	12	Molecular (nPCR, cytB)	Skin lesions	83.3 (L. mexicana)
							16.7 (Leishmania spp.)

Abbreviations: CAG, crude antigen; CH1, chitinase; cytB, cytochrome b; DAT, direct agglutination test; DB, dot blot; ELISA, enzyme-linked immunosorbent assay; EU, ELISA units; F., Felis; FML, fucose-mannose ligand; gp63, metalloprotease gp63; H, total parasite extract; IFAT, immunofluorescence antibody test; IHC, immunohistochemistry; ITS1, internal transcriber spacer 1; kDNA, kinetoplast minicircle DNA; L., Leishmania; na, not available; nPCR, nested PCR; PCR, one-step PCR (polymerase chain reaction); qPCR, real-time PCR; RFLP, restriction fragment length polymorphism; rK39, recombinant K39; SODe, superoxide dismutase excreted; SSU, small subunit ribosomal DNA; WB, western blot.

^aSpecies defined according to the original study.

^bPreviously identified as positive by another test.

^cCats with lymphadenomegaly.

Specific antibodies or Leishmania DNA have been mostly detected in domestic cats living in endemic areas of South America, the Mediterranean Region and western Asia. Some studies also suggest that wild cats from Spain (Del Río et al., 2014; Risueño et al., 2018) and sand cats (Felis margarita) from Saudi Arabia (Morsy et al., 1999) are frequently exposed to Leishmania infection.

In non-endemic countries, as seen in dogs, feline Leishmania infection has been particularly associated with cats travelling to or rehomed from southern Europe and Brazil (Rüfenacht et al., 2005; Richter et al., 2014; Maia & Cardoso, 2015; Schäfer et al., 2021). Also, antibodies to Leishmania were detected in three domestic cats living in the UK, but in all cases, the travel and clinical history were unknown (Persichetti et al., 2017).

Although blood transfusion is regarded as a probable non-vector-borne transmission pathway of Leishmania in cats, no feline infection cases by this parasite (screened by PCR) were identified among eligible blood donors (Marenzoni et al., 2018; Mesa-Sanchez et al., 2020).

Several factors have been highlighted as possibly associated with Leishmania infection in cats based on univariate analysis, including old age (Akhtardanesh et al., 2017; Junsiri et al., 2017; Morganti et al., 2019; Asgari et al., 2020), male sex (Cardoso et al., 2010; Sobrinho et al., 2012; Montoya et al., 2018a; Asgari et al., 2020; Latrofa et al., 2020), non-neutered status (Otranto et al., 2017; Latrofa et al., 2020), presence of clinical or clinicopathological abnormalities (such as crusting skin lesions, leukopaenia, increase in alanine aminotransferase (ALT) levels, lymphadenomegaly, lymphocytosis and neutrophilia) (Ayllón et al., 2008; Sherry et al., 2011; Sobrinho et al., 2012; Spada et al., 2013; Akhtardanesh et al., 2017; Otranto et al., 2017; Latrofa et al., 2020), concomitant infections (such as feline coronavirus (FCoV), feline immunodeficiency virus (FIV), feline leukemia virus and Toxoplasma gondii) (Sherry et al., 2011; Sobrinho et al., 2012; Spada et al., 2013, 2016; Montoya et al., 2018a), geographical area/local environment (such as altitude and rural areas) (Nasereddin et al., 2008; Cardoso et al., 2010; Asgari et al., 2020), lifestyle (such as access to the outdoors) (Rocha et al., 2019) and cohabitation with dogs (Rocha et al., 2019; Morelli et al., 2020). Epidemiological studies using logistic regression models (a powerful analytic research tool that avoids confounding effects) have evidenced that adult cats (Iatta et al., 2019; Akhtardanesh et al., 2020), males (Iatta et al., 2019; Akhtardanesh et al., 2020), non-neutered (Iatta et al., 2019), or with concomitant infections by FeLV (Martín-Sánchez et al., 2007; Sherry et al., 2011; Spada et al., 2013; Akhtardanesh et al., 2020), FIV (Iatta et al., 2019; Akhtardanesh et al., 2020), “Candidatus Mycoplasma turicensis” or Hepatozoon spp. (Attipa et al., 2017b) have an increased risk for Leishmania infection.

4. Immunopathogenesis

In dogs, several studies have provided evidence demonstrating that the course of L. infantum infection is directly linked to the immune response. Development of progressive disease in susceptible dogs is typically characterised by high antibody levels and an impaired ability to mount a strong and effective cell-mediated response characterised by the expression of interferon-gamma (IFN-γ), tumour necrosis factor-alpha (TNF-α), and interleukin (IL)-2 (reviewed by Maia & Campino, 2018). However, very limited data are available on the pathogenesis of leishmaniosis in cats. Experimental studies involving intravenous/intraperitoneal inoculation of axenic promastigotes suggest that cats are hypothetically less susceptible to developing disease by L. infantum when compared to dogs, despite also presenting a long-lasting parasitaemia (Kirkpatrick et al., 1984; Akhtardanesh et al., 2018). Recently, Priolo et al. (2019) demonstrated that cats naturally exposed to L. infantum infection produce IFN-γ following ex vivo blood stimulation with parasite antigens, as reported in dogs (Solano-Gallego et al., 2016). This finding is important to highlight that Leishmania parasites can elicit a protective cell-mediated immune response in cats. The only study assessing the role of the complement system in feline L. infantum infection showed that, contrary to humans and dogs, catʼs proteins are consumed by parasites in the lectin pathway, which hypothetically may justify their low predisposition to develop clinical disease (Tirado et al., 2021).

5. Clinical presentation and clinicopathological findings

Feline leishmaniosis caused by L. infantum is mostly reported in adult (median age: 7 years; range: 2–21 years) domestic short-hair cats living in or travelling to endemic countries of southern Europe and Brazil. The disease has a chronic course and may be manifested by a plethora of clinical signs and/or clinicopathological abnormalities, which are summarised in Table 3 and Table 4, respectively. About one-third of cats with leishmaniosis showed concomitant infections/diseases including FIV (Hervás et al., 2001; Poli et al., 2002; Pennisi et al., 2004; Grevot et al., 2005; Pocholle et al., 2012; Pimenta et al., 2015; Fernandez-Gallego et al., 2020), FeLV (Poli et al., 2002; Grevot et al., 2005; Pereira et al., 2019c), FCoV (Pennisi et al., 2004; Savani et al., 2004), T. gondii (Pennisi et al., 2004), Bartonella henselae (Pennisi et al., 2004), diabetes mellitus (Leiva et al., 2005), pemphigus foliaceus (Rüfenacht et al., 2005), neoplasia (Grevot et al., 2005; Pocholle et al., 2012; Maia et al., 2015b) and/or were under immunosuppressive therapies at the time of diagnosis (Fernandez-Gallego et al., 2020).

Table 3.

Frequency of clinical signs in domestic cats (Felis catus) with clinical leishmaniosis caused by Leishmania infantum

Historical or physical signs	Frequency (%)a	Reference
Dermatological
Nodules	38	Poli et al. (2002); Savani et al. (2004); Rüfenacht et al. (2005); Richter et al. (2014); Pimenta et al. (2015); Basso et al. (2016); Attipa et al., 2017a, Attipa et al., 2017b; Leal et al. (2018); Brianti et al. (2019); Headley et al. (2019); Pereira Mondolfi et al. (2019); Fernandez-Gallego et al. (2020); Silva et al. (2020)
Erosive/ulcerative skin disease	37	Ozon et al. (1998); Hervás et al. (1999, 2001); Pennisi et al. (2004); Grevot et al. (2005); Rüfenacht et al. (2005); Coelho et al. (2010); Pocholle et al. (2012); Maia et al. (2015); Basso et al. (2016); Brianti et al. (2019); Headley et al. (2019); Fernandez-Gallego et al. (2020); Silva et al. (2020)
Scaling/crusting	21	Ozon et al. (1998); Hervás et al. (1999); Pennisi et al. (2004); Rüfenacht et al. (2005); Coelho et al. (2010); da Silva et al. (2010); Headley et al. (2019); Fernandez-Gallego et al. (2020)
Alopecia	12	Hervás et al. (1999); Pennisi et al. (2004); Rüfenacht et al. (2005); Fernandez-Gallego et al. (2020)
Onychogryphosis	6	da Silva et al. (2010); Headley et al. (2019)
Bloody cyst	4	Pennisi et al. (2004)
Depigmentation	4	Rüfenacht et al. (2005); Pocholle et al. (2012)
Pruritus	4	Rüfenacht et al. (2005); Pocholle et al. (2012)
Pustule/papule	4	Rüfenacht et al. (2005); Pocholle et al. (2012)
Footpad hyperkeratosis	2	Fernandez-Gallego et al. (2020)
General/miscellaneous
Lymphadenomegaly	27	Hervás et al. (1999, 2001); Poli et al. (2002); Pennisi et al. (2004); Savani et al. (2004); Maroli et al. (2007); da Silva et al. (2010); Brianti et al. (2019); Fernandez-Gallego et al. (2020); Silva et al. (2020)
Lethargy/depression	25	Poli et al. (2002); Pennisi et al. (2004); Leiva et al. (2005); Rüfenacht et al. (2005); Marcos et al. (2009); Pocholle et al. (2012); Richter et al. (2014); Fernandez-Gallego et al. (2020)
Anorexia/inappetence	21	Pennisi et al. (2004); Rüfenacht et al. (2005); Marcos et al. (2009); da Silva et al. (2010); Fernandez-Gallego et al. (2020)
Weight loss	21	Ozon et al. (1998); Hervás et al. (1999); Pennisi et al. (2004); Savani et al. (2004); da Silva et al. (2010); Fernandez-Gallego et al. (2020); Silva et al. (2020)
Hyperthermia	12	Leiva et al. (2005); Basso et al. (2016); Headley et al. (2019); Fernandez-Gallego et al. (2020)
Hepatomegaly	4	Pennisi et al. (2004); Leiva et al. (2005)
Splenomegaly	4	Poli et al. (2002); Leal et al. (2018)
Bruising	2	Maia et al. (2015)
Mastitis	2	Pereira Mondolfi et al. (2019)
Ocular
Uveitis	27	Hervás et al. (2001); Pennisi et al. (2004); Verneuil (2013); Richter et al. (2014); Pimenta et al. (2015); Leal et al. (2018); Pereira Mondolfi et al. (2019); Fernandez-Gallego et al. (2020)
Corneal oedema	10	Hervás et al. (2001); Pimenta et al. (2015); Fernandez-Gallego et al. (2020)
Conjunctivitis	8	Migliazzo et al. (2015); Brianti et al. (2019); Fernandez-Gallego et al. (2020)
Chorioretinitis	4	Pennisi et al. (2004); Fernandez-Gallego et al. (2020)
Corneal opacification	4	Hervás et al. (2001); Pimenta et al. (2015)
Glaucoma	4	Leiva et al. (2005); Richter et al. (2014)
Keratitis	4	Richter et al. (2014); Fernandez-Gallego et al. (2020)
Blepharitis	2	Brianti et al. (2019)
Chemosis	2	Fernandez-Gallego et al. (2020)
Masse	2	Hervás et al. (2001)
Gastrointestinal/abdominal
Stomatitis	21	Hervás et al. (2001); Leiva et al. (2005); Maroli et al. (2007); Verneuil (2013); Maia et al. (2015); Migliazzo et al. (2015); Fernandez-Gallego et al. (2020)
Glossitis	4	Fernandez-Gallego et al. (2020)
Jaundice	4	Hervás et al. (1999); Fernandez-Gallego et al. (2020)
Vomiting	4	Hervás et al. (1999); Fernandez-Gallego et al. (2020)
Abdominal distension	2	Leiva et al. (2005)
Diarrhoea	2	Fernandez-Gallego et al. (2020)
Cardiorespiratory
Dispnoea/tachypnoea	12	da Silva et al. (2010); Basso et al. (2016); Leal et al. (2018); Headley et al. (2019); Silva et al. (2020)
Pallor	10	Hervás et al. (2001); Pennisi et al. (2004); Marcos et al. (2009); Maia et al. (2015); Richter et al. (2014)
Abnormal respiratory sounds	4	Leal et al. (2018); Altuzarra et al. (2020)
Nasal discharge	4	Migliazzo et al. (2015); Altuzarra et al. (2020)
Sneezing	2	Leal et al. (2018)
Musculoskeletal
Muscle atrophy	2	da Silva et al. (2010)
Neurological
Ataxia	2	Fernandez-Gallego et al. (2020)
Urogenital
Vaginal bleeding	2	Maia et al. (2015)

^an = 52.

Table 4.

Frequency of clinicopathological abnormalities in domestic cats (Felis catus) with leishmaniosis caused by Leishmania infantum

Parameter	Frequency (%)a	Reference
Hemogram
Anaemia	31	Hervás et al. (1999); Pennisi et al. (2004); Marcos et al. (2009); Richter et al. (2014); Pereira Mondolfi et al. (2019); Fernandez-Gallego et al. (2020); Pimenta et al. (2015)
Neutrophilia	19	Poli et al. (2002); Leiva et al. (2005); da Silva et al. (2010); Verneuil (2013); Fernandez-Gallego et al. (2020); Silva et al. (2020)
Thrombocytopaenia	17	Pennisi et al. (2004); Marcos et al. (2009); Richter et al. (2014); Pimenta et al. (2015); Basso et al. (2016); Pereira Mondolfi et al. (2019)
Leukocytosis	10	Ozon et al. (1998); da Silva et al. (2010); Fernandez-Gallego et al. (2020)
Leukopaenia	10	Pennisi et al. (2004); Rüfenacht et al. (2005); Richter et al. (2014)
Eosinophilia	7	Ozon et al. (1998); Hervás et al. (1999); Marcos et al. (2009); Altuzarra et al. (2020)
Neutropaenia	5	Fernandez-Gallego et al. (2020)
Lymphopaenia	2	Rüfenacht et al. (2005)
Monocytosis	2	Leiva et al. (2005)
Blood chemistry
Hyperproteinaemia	36	Ozon et al. (1998); Hervás et al. (1999); Poli et al. (2002); Pennisi et al. (2004); Pimenta et al. (2015); Attipa et al., 2017a, Attipa et al., 2017b; Leal et al. (2018); Brianti et al. (2019); Pereira Mondolfi et al. (2019); Fernandez-Gallego et al. (2020)
Hyperglobulinaemia	31	Pennisi et al. (2004); Leiva et al. (2005); Richter et al. (2014); Pimenta et al. (2015); Brianti et al. (2019); Altuzarra et al. (2020)
Azotemia	21	Pennisi et al. (2004); Leiva et al. (2005); Marcos et al. (2009); da Silva et al. (2010); Leal et al. (2018); Fernandez-Gallego et al. (2020)
Hypoalbuminaemia	10	Hervás et al. (1999; Rüfenacht et al. (2005); Richter et al. (2014); Fernandez-Gallego et al. (2020)
Hyperglycaemia	8	Leiva et al. (2005); Richter et al. (2014); Fernandez-Gallego et al. (2020)
Bilirrubinaemia	5	Fernandez-Gallego et al. (2020)
Hyperphosphataemia	3	Fernandez-Gallego et al. (2020)
Hypophosphataemia	3	Fernandez-Gallego et al. (2020)
Increased alanine aminotransferase	3	Fernandez-Gallego et al. (2020)
Increased aspartate transaminase	3	da Silva et al. (2010)
Increased creatinine kinase	3	Fernandez-Gallego et al. (2020)
Protein electrophoresis
Hypergammaglobulinaemia	84	Ozon et al. (1998); Hervás et al. (1999); Poli et al. (2002); Pennisi et al. (2004); Leiva et al. (2005); Marcos et al. (2009); Richter et al. (2014); Basso et al. (2016); Leal et al. (2018); Brianti et al. (2019); Pereira Mondolfi et al. (2019); Altuzarra et al. (2020); Fernandez-Gallego et al. (2020)
Increased α2 globulins	13	Basso et al. (2016); Fernandez-Gallego et al. (2020)
Hyperbetaglobulinaemia	3	Hervás et al. (1999)
Urinalysis
Proteinuria	25	Marcos et al. (2009); Leal et al. (2018); Fernandez-Gallego et al. (2020)
Bilirrubinuria	4	Marcos et al. (2009)
Glycosuria	4	Leiva et al. (2005)

^aHemogram, n = 42; Blood chemistry, n = 39; Serum protein electrophoresis, n = 32; Urianalysis, n = 24.

Dermatological disorders were found in about 75% of reported clinical cases. Although uncommon, they may occur in the apparent absence of other obvious signs of disease (Fernandez-Gallego et al., 2020). Nodular dermatitis seems to be the main cutaneous lesion associated with FeL and is typically found on the eyelids (Hervás et al., 2001; Richter et al., 2014; Pimenta et al., 2015; Leal et al., 2018; Pereira et al., 2019c; Fernandez-Gallego et al., 2020; Silva et al., 2020). Erosive/ulcerative dermatitis is another clinical finding suggestive of FeL and has been identified on the head (Hervás et al., 2001; Grevot et al., 2005; Coelho et al., 2010; Pocholle et al., 2012; Maia et al., 2015b; Basso et al., 2016; Brianti et al., 2019; Headley et al., 2019; Fernandez-Gallego et al., 2020), extremities (Rüfenacht et al., 2005; Coelho et al., 2010; Basso et al., 2016; Fernandez-Gallego et al., 2020; Silva et al., 2020), trunk (Pocholle et al., 2012; Fernandez-Gallego et al., 2020), and over bony prominences (Hervás et al., 1999). Although less frequent, some cats with clinical leishmaniosis showed onychogryphosis (da Silva et al., 2010; Headley et al., 2019), a rather specific sign of canine leishmaniosis (CanL) (Maia & Campino, 2018). Generalised or focal lymphadenopathy appears as a common finding in FeL (Hervás et al., 1999; 2001; Poli et al., 2002; Savani et al., 2004; Pennisi et al., 2004; Maroli et al., 2007; da Silva et al., 2010; Brianti et al., 2019; Fernandez-Gallego et al., 2020; Silva et al., 2020) as well as non-specific signs including lethargy/depression (Poli et al., 2002; Pennisi et al., 2004; Leiva et al., 2005; Rüfenacht et al., 2005; Marcos et al., 2009; Pocholle et al., 2012; Richter et al., 2014; Fernandez-Gallego et al., 2020), anorexia/inappetence (Pennisi et al., 2004; Rüfenacht et al., 2005; Marcos et al., 2009; da Silva et al., 2010; Fernandez-Gallego et al., 2020), and weight loss (Ozon et al., 1998; Hervás et al., 1999; Pennisi et al., 2004; Savani et al., 2004; da Silva et al., 2010; Fernandez-Gallego et al., 2020; Silva et al., 2020).

Approximately one-fourth of cats with clinical leishmaniosis showed uveitis (Hervás et al., 2001; Pennisi et al., 2004; Verneuil, 2013; Richter et al., 2014; Pimenta et al., 2015; Leal et al., 2018; Pereira et al., 2019c; Fernandez-Gallego et al., 2020); stomatitis (Hervás et al., 2001; Leiva et al., 2005; Maroli et al., 2007; Verneuil, 2013; Maia et al., 2015b; Migliazzo et al., 2015; Fernandez-Gallego et al., 2020) and/or cardiorespiratory signs such as dyspnoea/tachypnoea, pallor, abnormal respiratory sounds, nasal discharge and sneezing (Hervás et al., 2001; Pennisi et al., 2004; Marcos et al., 2009; da Silva et al., 2010; Richter et al., 2014; Migliazzo et al., 2015; Maia et al., 2015b; Basso et al., 2016; Leal et al., 2018; Headley et al., 2019; Altuzarra et al., 2020; Silva et al., 2020). Musculoskeletal (i.e. muscle atrophy; da Silva et al., 2010), neurological (i.e. ataxia; Fernandez-Gallego et al., 2020), and urogenital (i.e. vaginal bleeding; Maia et al., 2015b) signs were also occasionally described, but in some cases, they appear to be secondary to concomitant diseases (Maia et al., 2015b; Fernandez-Gallego et al., 2020). Other clinical manifestations rarely found and which may represent a further diagnostic challenge to veterinarians include: depigmentation (Rüfenacht et al., 2005; Pocholle et al., 2012), cutaneous bloody cyst (Pennisi et al., 2004), pruritus (Rüfenacht et al., 2005; Pocholle et al., 2012), footpad hyperkeratosis (Fernandez-Gallego et al., 2020), hepatomegaly (Pennisi et al., 2004; Leiva et al., 2005), splenomegaly (Poli et al., 2002; Leal et al., 2018), bruising (Maia et al., 2015b), mastitis (Pereira et al., 2019c), chorioretinitis (Pennisi et al., 2004; Fernandez-Gallego et al., 2020), corneal opacification (Hervás et al., 2001; Pimenta et al., 2015), glaucoma (Leiva et al., 2005; Richter et al., 2014), blepharitis (Brianti et al., 2019), chemosis (Fernandez-Gallego et al., 2020), ocular masses (Hervás et al., 2001), glossitis (Fernandez-Gallego et al., 2020), jaundice (Hervás et al., 1999; Fernandez-Gallego et al., 2020), abdominal distension (Leiva et al., 2005), and vomiting/diarrhoea (Hervás et al., 1999; Fernandez-Gallego et al., 2020).

Most consistent laboratory abnormalities found in FeL cases include anaemia (generally of the normochromic, normocytic type) (Hervás et al., 1999; Pennisi et al., 2004; Marcos et al., 2009; Richter et al., 2014; Pimenta et al., 2015; Pereira et al., 2019c; Fernandez-Gallego et al., 2020) and hyperproteinaemia with hypergammaglobulinaemia (Ozon et al., 1998; Hervás et al., 1999; Poli et al., 2002; Pennisi et al., 2004; Leiva et al., 2005; Marcos et al., 2009; Richter et al., 2014; Basso et al., 2016; Leal et al., 2018; Brianti et al., 2019; Pereira et al., 2019c; Altuzarra et al., 2020; Fernandez-Gallego et al., 2020). The latter was detected in more than 80% of sick cats and should be investigated as a possible biomarker of FeL. Leukocytosis (Ozon et al., 1998; da Silva et al., 2010; Fernandez-Gallego et al., 2020) and leukopaenia (Pennisi et al., 2004; Rüfenacht et al., 2005; Richter et al., 2014) are inconsistent findings, whereas thrombocytopenia (Pennisi et al., 2004; Marcos et al., 2009; Richter et al., 2014; Pimenta et al., 2015; Basso et al., 2016; Pereira et al., 2019c) and azotaemia (Pennisi et al., 2004; Leiva et al., 2005; Marcos et al., 2009; da Silva et al., 2010; Leal et al., 2018; Fernandez-Gallego et al., 2020) have been frequently reported. About a quarter of the sick cats presented proteinuria (Marcos et al., 2009; Leal et al., 2018; Fernandez-Gallego et al., 2020), suggesting a possible association between FeL and kidney disease as described in dogs. Recently, Chatzis et al. (2020) observed that cats infected with Leishmania parasites had higher concentrations of inorganic phosphorus than non-infected cats, reinforcing this assumption. Mild increases of liver enzyme activities are also described (Fernandez-Gallego et al., 2020), but less frequently than in cases of CanL (Maia & Campino, 2018).

6. Diagnosis

Clinical presentation combined with epidemiological context may lead to suspicion of FeL, but for a definitive diagnosis, Leishmania-specific laboratory tests are required (Table 5). These include direct tests (cytology, histology, immunohistochemistry, culture, and PCR), demonstrating the presence of the parasite or its components, and indirect tests (serology) assessing the hostʼs response to infection.

Table 5.

Common laboratory tests performed for diagnostics of Leishmania infection in domestic cats (Felis catus)

Type/test	Aim	Confirmation of clinical disease	Confirmation of subclinical disease	Preferential sample	Advantages	Disadvantages	Observations
Parasitological
Cytology	Detection of parasites	+++	+	‣Bone-marrow (FNB); ‣Lymph node (FNB); ‣Nodular lesions (FNB); ‣Erosive/ulcerative skin lesions (scraping)	‣Does not require specific laboratory equipment; ‣Low cost; ‣Rapid; ‣High specificity	‣Requires experienced observers; ‣Strictly qualitative; ‣Not suitable for identification at the species level	‣Amastigotes can be found in both intracellular and extracellular areas
Histopathology	Detection of parasites	+++	+	‣Skin/ocular lesions; ‣Bone marrow; ‣Lymph-nodes; ‣Spleen	‣Preserves structure and maintains tissue pathology; ‣High specificity; ‣Good sensitivity using IHC	‣Invasive; ‣Requires experienced observers; ‣Requires specific laboratory equipment; ‣More laborious and time-consuming; ‣IHC is not widely available; ‣Only qualitative; ‣Not suitable for identification at the species level
Parasite culture	Isolation of viable parasites	++	+	‣Biopsy lesions; ‣Bone marrow; ‣Lymph nodes	‣Provides parasites for further analysis; ‣Confirms active infection; ‣High specificity	‣Labour-intensive; ‣Restricted to specialised reference laboratories; ‣Up to more than 30 days to provide a result; ‣Only qualitative; ‣Not suitable for identification at the species level	‣Aseptic sampling should be ensured; ‣Biopsy sample must be homogenised in saline or culture medium under sterile conditions
Molecular
PCR	Detection of parasite DNA	+++	+++	‣Biopsy lesions; ‣Bone marrow; ‣Lymph nodes	‣Allows identification at the species level; ‣High sensitivity and specificity	‣Transient infection cannot be excluded; ‣Requires specific laboratory equipment; ‣Requires vigilance against false-positive results; ‣Only qualitative; ‣Expensive	‣Protocols targeting multicopy genes are preferable for diagnosis; ‣Nested PCR has more sensitivity than conventional PCR
qPCR	Detection of parasite DNA	+++	+++	‣Biopsy lesions ‣Bone marrow ‣Lymph nodes	‣Allows identification at the species level; ‣High sensitivity and specificity; ‣Quantification of parasite load; ‣Reduced cross-contamination probability; ‣Valuable for treatment follow-up; ‣Qualitative/quantitative	‣Transient infection cannot be excluded; ‣Standardised methods to parasite load quantification may not be offered by some laboratories; ‣Expensive	‣Protocols targeting multicopy genes are preferable for diagnosis
Serological
ELISA	Detection of specific antibodies	+++	++	‣Serum; ‣Plasma	‣Valuable for treatment follow-up; ‣Relatively low cost; ‣Qualitative/quantitative	‣Possible cross-reactivity; ‣Difficult to assess results at threshold of positivity; ‣Not suitable for unambiguous identification at the species level	‣Established cut-off (40 EU)
IFAT	Detection of specific antibodies	++	+++	‣Serum; ‣Plasma	‣Valuable for treatment follow-up; ‣Relatively low cost; ‣Qualitative/quantitative	‣Requires experienced observers; ‣Subjective interpretation; ‣Possible cross-reactivity; ‣Not suitable for unambiguous identification at the species level	‣Reference method for the serodiagnosis of human and canine leishmanioses; ‣Established cut-off (1:80)
Western blot	Detection of specific antibodies	+++	+++	‣Serum; ‣Plasma	‣High sensitivity and specificity	‣Labour-intensive; ‣Expensive; ‣Not available in routine practice	‣Marker for positivity: 18 kDA band

Abbreviations: ELISA, enzyme-linked immunosorbent assay; EU, ELISA units; FNB, fine needle biopsy; IFAT, immunofluorescence antibody test; IHC, immunohistochemistry, KDa, kilodaltons; PCR, conventional/nested polymerase chain reaction; qPCR, real-time polymerase chain reaction; WB, western blot. +++, recommended test; ++ suitable test; +, limited test.

Cytology is strongly advised in cats presenting erosive/ulcerative skin disease, nodular lesions and/or lymphadenomegaly (Hervás et al., 1999; Poli et al., 2002; Savani et al., 2004; Coelho et al., 2010; Richter et al., 2014; Maia et al., 2015b; Pimenta et al., 2015; Basso et al., 2016; Attipa et al., 2017a; Leal et al., 2018; Brianti et al., 2019; Headley et al., 2019; Pereira et al., 2019c; Silva et al., 2020). Material for diagnosis can be obtained by fine-needle biopsy (with or without aspiration), scraping or imprinting. The presence of Leishmania parasites has been demonstrated in cytological examinations of feline nodular lesions (Poli et al., 2002; Savani et al., 2004; Richter et al., 2014; Pimenta et al., 2015; Basso et al., 2016; Attipa et al., 2017a; Leal et al., 2018; Brianti et al., 2019; Pereira et al., 2019c; Fernandez-Gallego et al., 2020; Silva et al., 2020), erosive/ulcerative lesions (Maia et al., 2015b; Headley et al., 2019; Fernandez-Gallego et al., 2020; Silva et al., 2020), whole-blood (Marcos et al., 2009; Metzdorf et al., 2017), buffy coat/leucoconcentrate (Martín-Sánchez et al., 2007; Marcos et al., 2009), lymph nodes (Hervás et al., 1999; Poli et al., 2002; Pennisi et al., 2004; Bresciani et al., 2010; Coelho et al., 2010, 2011b; Vides et al., 2011; Sobrinho et al., 2012; Metzdorf et al., 2017; Berenguer et al., 2020; Fernandez-Gallego et al., 2020; Silva et al., 2020), bone marrow (Pennisi et al., 2004; Marcos et al., 2009; Vides et al., 2011; Sobrinho et al., 2012; Metzdorf et al., 2017; Marcondes et al., 2018; Fernandez-Gallego et al., 2020), liver (Vides et al., 2011; Mohebali et al., 2017; Fernandez-Gallego et al., 2020), spleen (Vides et al., 2011; Mohebali et al., 2017; Fernandez-Gallego et al., 2020), nasal exudate (Migliazzo et al., 2015), corneal impression (Pimenta et al., 2015), and inflammatory breast fluid (Pereira et al., 2019c). Cytological preparations consistent with FeL typically have a cell composition characteristic of pyogranulomatous, granulomatous or lymphoplasmacytic inflammation (Poli et al., 2002; Headley et al., 2019; Pereira et al., 2019c). Similar patterns are reported in histological studies on feline paraffin-embedded specimens (Poli et al., 2002; Navarro et al., 2010; Migliazzo et al., 2015; Di Mattia et al., 2018; Leal et al., 2018; Altuzarra et al., 2020). Nevertheless, compared with cytology, histology has the main advantage of providing a more detailed diagnostic information on the tissue architecture, which allows understanding if parasites are indeed associated with lesions (Paltrinieri et al., 2016). Immunohistochemistry may be further performed to confirm the presence of Leishmania organisms in biological samples obtained from cats (Poli et al., 2002; Navarro et al., 2010; Migliazzo et al., 2015). Based on histological and immunohistochemical examinations, it has been observed that this parasite may invade several feline organs/tissues such as skin (Ozon et al., 1998; Poli et al., 2002; Grevot et al., 2005; Rüfenacht et al., 2005; Attipa et al., 2017a; Rivas et al., 2018; Fernandez-Gallego et al., 2020; Silva et al., 2020), nasal and oral mucosa (Pennisi et al., 2004; Migliazzo et al., 2015; Leal et al., 2018), eyes (Hervás et al., 2001; Fernandez-Gallego et al., 2020), nasopharynx (Leal et al., 2018), stomach (Hervás et al., 1999), liver (Hervás et al., 1999; Silva et al., 2020), kidneys (Ozon et al., 1998), spleen (Hervás et al., 1999; Grevot et al., 2005; Marcos et al., 2009; Maia et al., 2015b; Fernandez-Gallego et al., 2020; Silva et al., 2020), bone marrow (Ozon et al., 1998; Pimenta et al., 2015; Silva et al., 2020), and lymph nodes (Hervás et al., 1999), and may also be associated with neoplasia (Grevot et al., 2005; Rüfenacht et al., 2005; Pocholle et al., 2012; Maia et al., 2015b; Altuzarra et al., 2020).

Parasite culture is an accurate test allowing conclusive diagnosis of an active infection. However, this test is not suitable for rapid diagnosis and is restricted to specialised laboratories. Parasite culture is a starting point for parasite identification and characterisation by isoenzyme electrophoresis (Pratlong et al., 2004). Viable parasites have been isolated from whole blood (Pocholle et al., 2012), nodular lesions (Poli et al., 2002; Basso et al., 2016), liver (Maia et al., 2015b; Silva et al., 2020), spleen (Maia et al., 2015b; Silva et al., 2020), lymph nodes (Pennisi et al., 2004; Maroli et al., 2007; Maia et al., 2015b; Basso et al., 2016; Silva et al., 2020), and bone marrow (Silva et al., 2020) of cats with leishmaniosis.

Polymerase chain reaction (PCR)-based tests have allowed the detection of Leishmania DNA in several feline samples, including whole blood (Marcos et al., 2009; Pocholle et al., 2012; Pimenta et al., 2015; Basso et al., 2016; Attipa et al., 2017a; Brianti et al., 2019; Fernandez-Gallego et al., 2020; Silva et al., 2020), buffy coat (Pereira et al., 2019c), conjunctival and oral swabs (Migliazzo et al., 2015; Brianti et al., 2019; da Costa-Val et al., 2020), hair (Urbani et al., 2020), skin (Rüfenacht et al., 2005; da Silva et al., 2010; Richter et al., 2014; Maia et al., 2015b; Basso et al., 2016; Fernandez-Gallego et al., 2020; Silva et al., 2020), nasal tissue (Leal et al., 2018), liver (Maia et al., 2015b; Silva et al., 2020), spleen (Savani et al., 2004; Coelho et al., 2010; da Silva et al., 2010; Maia et al., 2015b; Pimenta et al., 2015; Fernandez-Gallego et al., 2020; Silva et al., 2020), kidneys (da Silva et al., 2010), lymph nodes (Poli et al., 2002; Pennisi et al., 2004; Coelho et al., 2010; da Silva et al., 2010; Maia et al., 2015b; Migliazzo et al., 2015; Pimenta et al., 2015; Silva et al., 2020), bone marrow (da Silva et al., 2010; Richter et al., 2014; Pimenta et al., 2015; Fernandez-Gallego et al., 2020; Silva et al., 2020), and inflammatory breast fluid (Pereira et al., 2019c). Conventional PCR, nested PCR, and real-time PCR (qPCR) targeting kinetoplast minicircle DNA (kDNA) or the small subunit ribosomal DNA (SSU rDNA) multicopy genes have been widely used in routine veterinary practise for FeL diagnosis (Pimenta et al., 2015; Brianti et al., 2019; Pereira et al., 2019c) as well as in epidemiological studies concerning Leishmania infection in cats (Maia et al., 2014; Vilhena et al., 2013; Pereira et al., 2020). Nevertheless, two-step PCR when used to amplify stretches of multicopy genes has increased the sensitivity of detection, and should be preferred for sample testing under suboptimal conditions (i.e. where the parasite load tends to be low) such as when whole blood is used (Pereira et al., 2020). On the other hand, quantitative PCR (qPCR) may further provide information about the amount of parasite DNA present in the sample (Galluzzi et al., 2018). This aspect is particularly relevant for monitoring the efficacy of anti-Leishmania treatments (Pocholle et al., 2012; Basso et al., 2016). However, it is important to highlight that a PCR-positive result may only reflect a transient infection and, for this reason, should be carefully interpreted in a clinical context. PCR products may be subsequently analysed by restriction enzyme digestion (i.e. restriction fragment length polymorphism) and/or DNA sequencing for parasite species identification (Metzdorf et al., 2017; Pereira et al., 2020).

The most common serological tests used to detect anti-Leishmania antibodies in cats are based on enzyme-linked immunosorbent assay (ELISA) and immunofluorescent antibody test (IFAT). The latter is considered as the reference test for the serodiagnosis of canine and human leishmaniosis (OIE, 2018; WHO, 2010). Persichetti et al. (2017) established 1:80 serum dilution as IFAT cut-off for FeL serodiagnosis, and demonstrated that this test helps to detect subclinical or early Leishmania infections in cats. More recently, Iatta et al. (2020) validated IFAT as an accurate test to assess the exposure of cats to L. infantum, reporting positive and negative predictive values of 80.7% and 89.9%, respectively. Compared to IFAT, ELISA (cut-off 40 ELISA units) presents a better performance for the serodiagnosis of clinical FeL (Persichetti et al., 2017). Western blot analysis is mainly intended for research and is rarely available in routine practice. However, this test seems to offer the best diagnostic performance (considering an 18 kDa band as a marker for positivity) to detect antibodies against L. infantum in cats (Persichetti et al., 2017). Direct agglutination test has also occasionally been used in both clinical and epidemiological contexts for serological diagnosis of FeL (Pimenta et al., 2015; Asgari et al., 2020). Some authors have considered a cut-off value of 1:100 to distinguish infected from uninfected cats (Kongkaew et al., 2007; Cardoso et al., 2010; Maia et al., 2015a; Lopes et al., 2017; Asgari et al., 2020; Neves et al., 2020). Indirect hemagglutination was exclusively performed in epidemiological studies in domestic cats in Egypt (Michael et al., 1982; Morsy et al., 1988; Morsy & Abou el Seoud, 1994).

Cats with clinical leishmaniosis tend to present high antibody levels (Richter et al., 2014; Maia et al., 2015b; Pimenta et al., 2015; Basso et al., 2016), and specific treatment frequently leads to the reduction of anti-Leishmania antibodies (Pennisi et al., 2004; Richter et al., 2014; Basso et al., 2016; Pereira et al., 2019c). In some cases, an increase of antibody titres was associated with clinical relapse. Nevertheless, it is essential to emphasise that a positive serological result formally only reflects exposure to pathogens and should be interpreted in a clinical context (Paltrinieri et al., 2016).

In conclusion, the diagnosis of FeL can be a real challenge for veterinarians and is seldom considered during the differential diagnosis. Therefore, the algorithm illustrated in Fig. 3 is proposed for clinically healthy cats used as blood donors or for breeding purposes, and for cats with suspected leishmaniosis.

Fig. 3.

Proposed diagnostic algorithm for clinically healthy cats used as blood donors or for breeding, and cats with suspected leishmaniosis

7. Treatment and prognosis

Treatment should be considered only after confirmation of disease (see Section 6). Although several treatment regimens have been empirically used for FeL (Table 6), no controlled studies on their efficacy and safety have yet been performed. Long-term administration of allopurinol as monotherapy is the most common regimen prescribed for FeL (Pennisi et al., 2004; Leiva et al., 2005; Rüfenacht et al., 2005; Marcos et al., 2009; Pocholle et al., 2012; Richter et al., 2014; Maia et al., 2015b; Migliazzo et al., 2015; Pimenta et al., 2015; Basso et al., 2016; Attipa et al., 2017a; Leal et al., 2018; Brianti et al., 2019; Pereira et al., 2019c; Altuzarra et al., 2020; Fernandez-Gallego et al., 2020). This drug is generally well-tolerated, but possible cases of cutaneous adverse reactions (Leal et al., 2018; Brianti et al., 2019), coprostasis (Maia et al., 2015b), and elevated liver enzymes (Rüfenacht et al., 2005) have been sporadically reported. Favourable results (i.e. clinical cure or improvement of clinical status) with allopurinol as monotherapy have been commonly obtained (Pennisi et al., 2004; Leiva et al., 2005; Rüfenacht et al., 2005; Pocholle et al., 2012; Richter et al., 2014; Migliazzo et al., 2015; Pimenta et al., 2015; Attipa et al., 2017a; Fernandez-Gallego et al., 2020; Altuzarra et al., 2020). Nevertheless, relapse after discontinuation or low-dose administration (Pennisi et al., 2004; Leiva et al., 2005; Brianti et al., 2019; Pereira et al., 2019c) and no or poor response to allopurinol therapy have been occasionally reported, even in cats with no apparent history of concomitant infections or immunosuppressive therapies (Rüfenacht et al., 2005; Marcos et al., 2009; Basso et al., 2016; Fernandez-Gallego et al., 2020). Therefore, the combination of meglumine antimoniate and allopurinol has been proposed for FeL treatment, appearing to be more effective (Basso et al., 2016; Pereira et al., 2019c), but acute kidney injury has already been reported (Leal et al., 2018). Although controversial, this drug is suspected of inducing nephrotoxicity in dogs (reviewed by Roura et al., 2021). Thus, its use in cats with altered renal function should be carefully considered. Meglumine antimoniate plus ketoconazole was used in a cat with cutaneous and systemic signs of FeL, resulting in apparent clinical cure (Hervás et al., 1999). Miltefosine was recently adopted as an alternative to meglumine antimoniate in an azotemic cat, resulting in rapid clinical improvement (Leal et al., 2018). In this case, transient vomiting episodes were reported in the first week of treatment but were managed using antiemetics (i.e. maropitant). Nevertheless, Fernandez-Gallego et al. (2020) recently reported a case of FeL with concomitant FIV infection not responsive to miltefosine plus allopurinol (combination therapy). Pennisi et al. (2004) reported treatment failure in a seropositive cat for FIV, T. gondii and B. henselae suffering from leishmaniosis. In this case, three distinct regimens were used (i.e. metronidazole plus spiramycin, fluconazole and itraconazole) (Pennisi et al., 2004). In another cat with leishmaniosis associated with an invasive squamous cell carcinoma, domperidone was used after unsuccessful allopurinol monotherapy, but clinical signs remained after one month of treatment (Maia et al., 2015b). The dietary supplement active hexose correlated compound (AHCC) was recently suggested as a possible alternative maintenance therapy to allopurinol (Leal et al., 2018). Surgical removal of lesions was also reported as an additional therapeutic approach (Hervás et al., 2001; Rüfenacht et al., 2005; Basso et al., 2016).

Table 6.

Treatment regimens used for feline leishmaniosis

Type	Drug (regimen and dose)	Outcome	Adverse reactionsa	Issues to consider	Reference
Monotherapy
	Allopurinol (10–30 mg/kg or 100 mg/cat PO q12–24 h; for long-term)	Variable (no response to clinical cure)	Increased liver enzymes; coprostasisb; toxidermia	Secondary xanthine urolithiasis has been reported in dogs	Pennisi et al. (2004); Rüfenacht et al. (2005); Marcos et al. (2009); Pocholle et al. (2012); Richter et al. (2014); Maia et al. (2015); Migliazzo et al. (2015); Pimenta et al. (2015); Basso et al. (2016); Leal et al. (2018); Attipa et al., 2017a, Attipa et al., 2017b; Brianti et al. (2019); Pereira Mondolfi et al. (2019); Altuzarra et al. (2020); Fernandez-Gallego et al. (2020)
	Domperidone (0.5 mg/kg PO q24 h for 1 month)	No improvement	Not reported	Immunomodulatory drug used on prevention and treatment of CanL	Maia et al. (2015)
	Fluconazole (5 mg/kg PO q24 h for 2 months)	No response	Not reported	May be hepatotoxic	Pennisi et al. (2004)
	Itraconazole (50 mg/cat PO q24 h for 2 months)	No response	Not reported	Hepatotoxic drug; may lead to suppression of adrenal function	Pennisi et al. (2004)
	Meglumine antimoniate (50 mg/kg SC q24 h for 25 days)	Not applicable	AKI - suspected	Treatment stopped due to AKI development; painful to administer; may be nephrotoxic (controversial)	Leal et al. (2018)
	Meglumine antimoniate (300 mg/cat SC q24 h for 4 months)	Resolution of clinical signs	See previous line	See previous line	Fernandez-Gallego et al. (2020)
Combination therapy
	Meglumine antimoniate (50 mg/kg SC q24 h for 30 days) plus allopurinol (10 mg/kg PO q12–24 h for long-term)	Variable (partial resolution of clinical signs to clinical cure)	See meglumine antimoniate and allopurinol monotherapy	Proposed for FeL refractory cases	Pimenta et al. (2015); Basso et al. (2016); Pereira Mondolfi et al. (2019); Fernandez-Gallego et al. (2020)
	Meglumine antimoniate (5 mg/kg SC q24 h) plus ketoconazole (10 mg/kg q24 h); 3 cycles of 4 weeks, 10 days apart	Resolution of lesions	Not reported; see meglumine antimoniate monotherapy	According to BSAVA (2020) ketoconazole is not recommended for cats	Hervás et al. (1999)
	Metronidazole (25 mg/kg PO q24 h for 35 days) plus spiramycin (150,000 IU/kg PO q24 h for 35 days)	No response	Not reported		Pennisi et al. (2004)
	Miltefosine (2 mg/kg PO q24 h for 28 days) plus N-AHCC (½ tablet once daily for long-term)	Resolution of clinical signs	Transient vomiting associated with miltefosine administration	Miltefosine licenced formulations for CanL contain propylene glycol which can hypothetically induce Heinz body haemolytic anaemia in cats (Pennisi and Persichetti, 2018)	Leal et al. (2018)
	Miltefosine (2 mg/kg PO q24 h for 28 days) plus allopurinol (10 mg/kg PO q12 for long-term)	No response	See previous line	See previous line	Fernandez-Gallego et al. (2020)

Abbreviations: AHCC, active hexose correlated compounds; AKI, acute kidney injury; CanL, canine leishmanosis; FeL, feline leishmaniosis; IU, internacional unit; PO, per os; SC, subcutaneous.

^aReported during treatment of cats with clinical leishmaniosis.

^bAssociated with high doses (50 mg/kg q24 h).

Like in dogs, Leishmania parasites may persist in treated cats (Pocholle et al., 2012; Pimenta et al., 2015; Attipa et al., 2017a), suggesting that treatment may lead to clinical cure but may not eliminate the infection.

Overall, FeL has a good prognosis even in cases with underlying viral infections (i.e. FIV or FeLV) (Hervás et al., 1999; Pennisi et al., 2004; Rüfenacht et al., 2005; Richter et al., 2014; Migliazzo et al., 2015; Pimenta et al., 2015; Basso et al., 2016; Attipa et al., 2017a; Leal et al., 2018; Pereira et al., 2019c; Altuzarra et al., 2020; Fernandez-Gallego et al., 2020). On the other hand, panleukopaenia, acute kidney injury and lack of treatment seem to be critical factors associated with poor prognosis (Ozon et al., 1998; Hervás et al., 1999; Poli et al., 2002; Pennisi et al., 2004; Pimenta et al., 2015; Fernandez-Gallego et al., 2020).

8. Prophylaxis and control

No vaccines or drugs preventing leishmaniosis are currently available for use in cats, and most repellents avoiding infection in dogs are toxic to these felids. In endemic areas, cats are frequently exposed to phlebotomine sand fly bites, and this is associated with an increased risk of Leishmania infection (Pereira et al., 2019b). Chemoprophylaxis may be achieved by using a matrix collar containing 10% imidacloprid and 4.5% flumethrin. This formulation showed to be safe and effective in reducing infection risk by L. infantum in cats (Brianti et al., 2017). Nevertheless, keeping cats indoors from dusk to dawn during the period of vector activity (April to November in Mediterranean areas, see Alten et al., 2016), as well as using physical barriers such as nets (i.e. mesh size 1,240 holes/in²) on windows and doors (Faiman et al., 2009) may eschew exposure to phlebotomine sand fly bites, thereby minimising the risk of Leishmania infection. Spraying with residual insecticides on walls and roofs of human houses and animal shelters has been proposed as an additional measure for preventing CanL (Maroli et al., 2010). However, their use in environments with cats should be carefully considered since most of these products contain compounds (i.e. pyrethrins or pyrethroids) that can induce feline toxicosis. Isoxazolines, namely afoxolaner and fluralaner, have been regarded as a new promising class of drugs for controlling CanL and human leishmaniosis in endemic areas (Miglianico et al., 2018; Bongiorno et al., 2020; Queiroga et al., 2020). A spot-on formulation of fluralaner (112.5–500 mg) is licensed for ectoparasite (i.e. ticks, fleas and mites) control in cats. This systemic insecticide induced long-term mortality of Lutzomyia longipalpis and Phlebotomus perniciosus (vectors of L. infantum in the New and Old Worlds, respectively) after feeding on treated dogs (Bongiorno et al., 2020; Queiroga et al., 2020). Similar results are expected to be observed in cats. Although studies are undoubtedly needed, this drug may also hypothetically represent an affordable indirect method for reducing Leishmania infection in cats in endemic areas. The detection and treatment of cats with leishmaniosis is also likely a beneficial control measure, as they may serve as a source of infection to phlebotomine sand fly vectors (Maroli et al., 2007; da Silva et al., 2010; Mendonça et al., 2020). In the absence of evidence indicating otherwise, Leishmania-infected cats should not be used for breeding or as blood donors due to the potential risk of transmission through blood transfusion and venereal/congenital infection, as reported in dogs (Owens et al., 2001; Naucke & Lorentz, 2012).

In summary, and according to the current knowledge, the following prophylactic measures are proposed to prevent and control &feline infection:

• •In endemic areas, keeping cats indoors from dusk to dawn during the phlebotomine sand fly season should be encouraged.
• •Use of physical barriers on houses and animal shelters located in endemic areas with high vector density.
• •Use of a matrix collar containing 10% imidacloprid and 4.5% flumethrin as well topical solutions containing 112.5–500 mg of fluralaner in cats living in or travelling to (cover the time of travel) endemic areas during the known transmission season.
• •After the return from endemic areas, cats should be clinically evaluated and tested.
• •Cats eligible for breeding and blood transfusion should be periodically tested.
• •Infected cats should not be used for breeding or as blood donors.
• •Cats with leishmaniosis should be treated and periodically monitored.

9. Public health considerations

Zoonotic visceral leishmaniosis (ZVL) caused by L. infantum is a life-threatening human disease endemic in the Mediterranean Basin, the Middle East, western Asia, and Brazil (WHO, 2010). Domestic dogs are considered the primary source of human infection, which typically occurs via the bites of female phlebotomine sand flies (WHO, 2010). Nevertheless, during the last years, cats have been deserved attention due to their potential enrolment in ZVL epidemiology, appearing now as possible primary or secondary reservoir hosts (Asfaram et al., 2019). This hypothesis arises by the following reasons (Maroli et al., 2007; da Silva et al., 2010; GfK, 2016; Pereira et al., 2019b; 2019c; 2020; Carneiro et al., 2020; Fernandez-Gallego et al., 2020; Mendonça et al., 2020):

• •Cats are frequently exposed to the bites of competent vectors.
• •Cats are naturally susceptible to L. infantum infection.
• •Feline infection often runs a subclinical course.
• •Parasites are frequently found in the skin and blood of infected cats.
• •Naturally infected cats are infectious to competent vectors.
• •Naturally infected cats may be the source of infection to other mammals through competent vectors.
• •Strains of feline origin seem to be indistinguishable from those isolated from dogs, humans, and competent vectors.
• •Cats are among the most popular animals owned as a pet.
• •Cats are often present in domestic/peridomestic areas where transmission cycles occur.

10. Conclusions

During the last years, several studies concerning Leishmania infection in cats were conducted. Feline leishmaniosis has also gained importance appearing nowadays as an emergent disease. Nevertheless, its immunopathogenesis is poorly known. This protozoonosis is manifested by a broad spectrum of clinical signs and clinicopathological abnormalities, which, associated with the lack of standardised protocols, make its diagnosis further challenging for veterinarians. In this review, a diagnostic algorithm for FeL is proposed for clinical decision support. Treatment options currently available are empirical and suboptimal. The main form to prevent disease is to avoid infection. However, in contrast to dogs, very limited options are currently available to keep infective sand flies away from cats. Thus, a set of prevention guidelines are herein suggested.

Funding

The Global Health and Tropical Medicine centre is funded by the Fundação para a Ciência e a Tecnologia, I.P. (FCT) (GHTM-UID/Multi/04413/2013), Portugal. AP was supported by the Portuguese Ministry of Science, Technology and Higher Education (via FCT) through a PhD grant (SFRH/BD/116516/2016).

CRediT author statement

André Pereira: Conceptualisation, Methodology, Validation, Formal analysis, Investigation, Writing – Original Draft, Writing - Review & Editing. Carla Maia: Conceptualisation, Methodology, Validation, Writing - Review & Editing, Supervision. The authors read and approved the final manuscript.

Declaration of competing interests

The authors declare that they have no competing interests.