Clinical, diagnostic and epidemiological implications of Hepatozoon spp., Babesia spp. and Leishmania infantum infection in cats and dogs in a Mediterranean periurban setting

Hepatozoon spp., Babesia spp. and Leishmania infantum are common parasites of dogs in Mediterranean countries and are less frequent in cats, particularly Babesia spp. and L. infantum. Moreover, there is limited information on coinfections between these parasites and on L. infantum’s distribution in blood, skin and lymphoid tissue in cats. We used PCR and DNA sequencing to investigate the prevalence of these parasites and the aetiology of Hepatozoon spp. and Babesia spp., in blood, skin, spleen and lymph node samples from up to 212 stray cats and 82 abandoned dogs in southeast Spain. All except 2 dogs were healthy; instead, 112 cats had clinical signs. The estimated PCR prevalences (95% confidence interval) were 25% (19–31%) Hepatozoon felis in cats, 13% (6–21%) Hepatozoon canis in dogs, 1% (0–4%) Babesia vogeli in dogs, 0% Babesia spp. in cats and 21% (15–26%) and 44% (33–55%) L. infantum in cats and dogs, respectively, and infections were not associated with each other. Leishmania infantum prevalence in lymphoid tissue was significantly higher in dogs than in cats (p < 0.001), and dogs had higher parasite loads than cats (p = 0.012). Moreover, L. infantum prevalence was significantly higher in the skin and lymphoid tissue compared to blood in infected, asymptomatic animals but it was similar in cats with clinical signs, which also had higher parasite loads compared to infected, asymptomatic cats (p < 0.05). The study highlights significant differences between sympatric dogs and cats with respect to the parasite infections investigated, as well as the need to examine both lymphoid tissue and skin samples to maximise the sensitivity of L. infantum infection diagnosis.

wild animal species may become infected and parasite reservoirs (Cardoso et al. 2021;Jiménez et al. 2014;Molina et al. 2012). Xenodiagnostic experiments have demonstrated the cat's ability to transmit L. infantum to vectors (Maroli et al. 2007;Mendonça et al. 2020). Feline leishmaniosis (FeL) is being increasingly reported worldwide (Abramo et al. 2021), and it is similarly associated with a wide range of clinical signs, with a predominance for cutaneous lesions and lymphadenomegaly (Pennisi et al. 2015). Leishmania infantum was recently detected in a wild cat and other wildlife species in southern Spain, and an association was found between infections with L. infantum and Hepatozoon spp. and Babesia spp. in wild carnivores (Ortuño et al. 2021). The latter tick-borne apicomplexan protozoan parasites of vertebrates display a higher degree of host specificity and a comparatively low zoonotic potential (Springer et al. 2020). Babesia spp. infects erythrocytes and Hepatozoon spp. infects leukocytes. The severity of babesiosis and hepatozoonosis depends on the host species and may range from subclinical infections to severe illness Irwin 2010;Solano-Gallego & Baneth 2011).
The present study builds on the previously described work on leishmaniosis, hepatozoonosis and babesiosis in wildlife in southern Spain with the following aims: (i) estimating L. infantum prevalence in domestic stray cats by polymerase chain reaction (PCR) analysis of blood, skin and lymphoid tissue samples, (ii) assessing the aetiology and prevalence of Hepatozoon spp. and Babesia spp. in stray cats and abandoned dogs by PCR and DNA sequence analysis and (iii) investigating the statistical relationship between infections by the three parasites. We expected to provide clinical, diagnostic and epidemiological information on these infections in dogs and cats and on the extent to which Hepatozoon spp. and Babesia spp. species are shared between domestic animals and wildlife.

Study population and experimental design
The study was performed on blood, skin, spleen and/or lymph node DNA from samples from 294 animals, including 212 stray cats and 82 abandoned dogs from the city of Murcia metropolitan area in southeast Spain (37° 59′ 10″ N, 1° 07′ 49″ W). Abandoned dogs were animals whose owners refused to keep them and were either left in the street or taken to the municipal kennel. Those left in the streets are quickly removed by the local authorities and taken to the municipal kennel. In contrast, stray cats were animals born and raised in the street that had never been under human care.
The number of DNA samples analysed included 445 samples from cats (123 blood, 147 skin, 153 spleen and 22 lymph node DNA samples) and 287 samples from dogs (76 blood, 65 skin, 82 spleen and 64 lymph node DNA samples) ( Table 1). The samples were part of a larger number collected between 2010 and 2016 for previous studies of randomly selected dogs and cats euthanised by the local authorities as part of a zoonosis control program. Samples here analysed included every one of those collected between 2010 and 2016 that had not been exhausted in previous studies. Leishmania infantum results from dogs were reported elsewhere (Ortuño et al. 2017;Risueño et al. 2012) and were used in the present study to investigate coinfections with Hepatozoon spp. and Babesia spp. and to compare dog and cat L. infantum infections.
Animals were clinically examined pre-mortem, weighed and aged, and their body condition was estimated post-mortem. Age was assessed based on dentition ("How to determine a cat's or dog's age," 1996), and cats were grouped accordingly into four categories: kittens (up to 1 year), young adults (2-6 years), mature adults (7-10 years) and senior (more than 10 years) (Quimby et al. 2021). For dogs, we considered a mean life expectancy of 11 years (Teng et al. 2022), and they were accordingly grouped as puppies (up to 9 months), young adults (from 9 months to 3 years), mature adults (from 4 to 8 years) and senior (more than 8 years) (Creevy et al. 2019). Similarly, three weight categories (small., medium and big) were considered: < 10 kg, 10-20 kg and > 20 kg for dogs and ≤ 3 kg, 3-4.5 kg and > 4.5 kg for cats. Animals were considered to be in low body condition if they had minimal or absent fat covering the ribs and the abdominal fat pad and had prominent ribs, lumbar vertebrae and pelvic bones (WSAVA 2020). Finally, dogs and cats were classified as pure or mongrel according to breed.
The studies had been approved by the University of Murcia's ethical committee.
Leishmania infantum DNA detection was carried out using a real-time PCR (rtPCR) assay with a Taqman probe, targeting kinetoplast minicircle DNA (kDNA). The protocols used were those of Mary et al. (2004) for dog samples and those of Dantas-Torres et al. (2017) for cat samples. The latter has a lower reaction volume and shorter running time . Samples were tested in duplicate using 250 µg of template DNA per reaction and were deemed rtPCR-positive for cycle thresholds (CT) ≤ 38 in one or both reactions (Mackay 2007).

Hepatozoon and Babesia DNA sequencing and phylogenetic analyses
Hepatozoon spp.-and Babesia spp.-positive PCR products were sequenced, and the BLAST tool (http:// blast. ncbi. nlm. nih. gov/ Blast. cgi) was used to compare with reference sequences in GenBank. They are now available in the GenBank database under the accession numbers MZ424831-MZ424837.
Phylogenetic trees were constructed using the MEGA X software (Kumar et al. 2018), and tree topology was inferred using the Neighbour-Joining method. Tree branches showing bootstrap values > 70% were considered confidential. Homologous sequences of Sarcocystis cymruensis (MG564723) and Plasmodium falciparum (accession number MF155937) were used as outgroups in Hepatozoon spp. and Babesia spp. phylograms, respectively.

Statistical analysis
Prevalence was defined as the percentage of PCR-positives and was used to calculate 95% confidence intervals (95% CI). Fisher's exact tests were used to compare the proportion of PCR-positives across independent demographic and clinical variables. Median CT of L. infantum-PCRpositive animals were similarly compared using the nonparametric Kruskal-Wallis test. For some comparisons, animals that were PCR-positive in more than one sample type were assigned the CT of the sample with the lowest value. The degree of agreement between PCR results in different biological samples from the same animals was analysed with the kappa statistic, considering > 0.81, 0.61-0.80, 0.41-60, 0.21-0.40, 0-0.20 and 0 as substantial, moderate, fair, slight and poor agreement, respectively (Thrusfield 2018). Associations were considered statistically significant for p < 0.05 and marginally significant for p < 0.10. The R software (http:// cran.r-proje ct. org/) was used for all analyses.
The prevalence of Hepatozoon spp. infection in cats and dogs did not vary significantly according to gender, age, breed, size or the presence of clinical signs and was significantly higher in the summer in cats (p < 0.05) and in the

Leishmania infantum prevalence in blood, skin and lymphoid tissue samples
The overall PCR prevalence (95% CI) of L. infantum was 21% (15-26%) in cats and 44% (33-55%) in dogs (Table 1), and the median (range) CTs in PCR-positives were 35 (13-38) in cats and 32 (12-38) in dogs (p < 0.05). Prevalence overall, in the skin and in tissue (both lymphoid tissue and skin) samples, was higher in cats without clinical signs compared to those with clinical signs (p < 0.05). In blood samples, however, the prevalence was marginally higher in cats with clinical signs than in those without (p = 0.06). Moreover, overall prevalence was also marginally higher in big cats compared to other cats, and it was not associated with the animal's gender, age or breed, and it was significantly higher in those sampled in winter compared to other times of the year (p < 0.01). Furthermore, L. infantum PCR prevalence and CTs in positive animals differed significantly depending on the sample type and between species for the same sample type. In cats, PCR prevalence (95% CI) was 6% (2-10%) in blood, 14% (0-28%) in lymph nodes, 16% (10-22%) in the skin and 18% (12-24%) in the spleen (Table 1). In dogs, it was 4% (0-8%) in blood, 12% (4-20%) in the skin, 22% (12-32%) in lymph nodes and 33% (23-43%) in the spleen. Prevalence was higher in the skin and lymphoid tissue compared to blood in dogs and healthy cats, in lymphoid tissue in dogs compared to cats and in the skin in healthy cats compared to cats with clinical signs (p < 0.05) (Table 1). Moreover, CTs among lymphoid tissue-positive dogs were lower (indicating greater parasite load) than in lymphoid tissue-positive cats, in cats with clinical signs compared to those without and in mature adults compared to younger cats (p < 0.05).
All cats and dogs positive in blood were positive in skin, spleen and lymph node samples, except one dog that was positive in blood but not in the skin (Table 2). In contrast, results from skin, spleen and lymphoid tissue samples did not always coincide, particularly in cats, for which 7 animals were positive in the skin and negative in the spleen and vice versa (Table 2). In dogs, 2 and 4 animals were positive in the skin and negative in the spleen and lymph node, respectively, 9 and 12 animals were positive in the spleen and lymph node, respectively and negative in the skin and 5 were positive in the spleen and negative in lymph node and the opposite was the case for 7 animals (Table 2). Consequently, significant kappa coefficients reflected only fair agreement between PCR results in blood and skin and lymphoid tissue samples (k = 0.21-0.30), and either fair or moderate agreement between the skin and lymphoid tissues and between spleen and lymph nodes (k = 0.31-0.46) ( Table 2).

Relationship between Hepatozoon spp., Babesia spp. and L. infantum infections
There were no significant associations between parasitic infections in either dogs or cats. Mixed Hepatozoon spp. and L. infantum infections were detected in 12/205 (6%) of the animals, specifically in 6/82 (7%) of dogs and 6/123 (5%) of cats, and no animal was infected with all three parasites.

Characterisation and phylogenetic analyses of Hepatozoon spp. and Babesia spp. DNA sequences
Three different Hepatozoon spp. sequences differing from one to three nucleotides were obtained from 11 PCR amplicons from dogs and were 100% identical to H. canis reference sequences (MN628317, MN628318, MN628320) and were named HC16, HC17 and HC18. Similarly, three Hepatozoon spp. sequences differing from one to three nucleotides from 31 PCR amplicons from cats were 99.7-100% identical to H. felis (MG386483, MG386484) and coded  foxes from France and in dogs from Algeria and India (71% bootstrap) (Fig. 1). Isolates HC17 and HC18 clustered with H. canis from dogs in Spain, Tunisia, Taiwan, India and Israel, cats and golden jackals from Israel, foxes from Murcia (HC2) and a beech marten from Andalucía (HC3), Spain (70% bootstrap) (Fig. 1).
Hepatozoon felis isolate HF4 clustered with H. felis from domestic cats in Spain, Israel, Italy and Angola (86% bootstrap) and a wild cat from Andalucía, Spain (HF1). Isolates HF5 and HF6 grouped with H. felis found in cats from Italy and Angola and in wild cats from Bosnia and Herzegovina, and also with Hepatozoon ingwe from an African leopard from South Africa, and separate from isolate HF2 found in wild cats from Murcia (Fig. 1).
In the phylogenetic analyses, B. vogeli from the dog (isolate BVo1) clustered with those in dogs from France, Malta, Colombia, Algeria and China, cats from Qatar, China and Brazil and foxes from France (99% bootstrap) (Fig. 2).

Discussion
The Hepatozoon spp. DNA sequences in abandoned dogs and stray cats were in most cases identical to those in foxes, beech martens and wild cats in southern Spain (Ortuño et al. 2021), suggesting a common transmission cycle, as previously proposed for L. infantum (Ortuño et al. 2019). In contrast to wild carnivores in Spain (Ortuño et al. 2021) and cats in Cyprus (Attipa et al. 2017), Hepatozoon spp. infection was not associated with L. infantum. Potential synergism between leishmaniosis and other vector-borne infections in dogs and cats has been postulated (Baxarias et al. 2018;Cringoli et al. 2002;Mekuzas et al. 2009;Toepp et al. 2019), but the pathogenesis of apicomplexan parasites and Leishmania coinfections have not been established.
Hepatozoon canis and B. vogeli are transmitted by Rhipicephalus sanguineus, the most common tick in dogs in Spain (Checa et al. 2019;Estrada-Peña et al. 2017). The almost absence of Babesia spp. in dogs and cats suggests a low circulation of these parasites in the studied area and not because of low exposure to R. sanguineus since H. canis was found in 13% of dogs. Other articles have similarly reported low prevalences of Babesia canis, B. vogeli and Babesia gibsoni in dogs in Spain (Baxarias et al. 2018;Movilla et al. 2017;Tabar et al. 2009), whereas Babesia vulpes in dogs and foxes was more prevalent in northwest Spain and Portugal Checa et al. 2019;García 2006;Miró et al. 2015). Feline babesiosis is rare worldwide and mostly restricted to South Africa (Jacobson et al. 2000). Vilhena et al. (2013) found 11% of B. vogeli PCR-positive cats in Portugal, and Babesia spp.-positive cats have been sporadically reported elsewhere in Europe and Asia (Penzhorn & Oosthuizen 2020).
The prevalence of H. canis in dogs and H. felis in cats reported here is higher than in most previous studies in the Iberian Peninsula, where it ranged from 0.7 to 3.3% for H. canis and 1.4 to 16% for H. felis (Díaz-Regañón et al. 2017;Maia et al. 2015;Movilla et al. 2017;Ortuño et al. 2008;Tabar et al. 2009;Vilhena et al. 2013). Albeit, Criado-Fornelio et al. (2006) and Dordio et al. (2021) reported 20% and 27% of H. canis and H. felis prevalence, respectively. The prevalence of these two apicomplexan species in other European countries was similarly variable (Cimpan et al. 2020;Licari et al. 2017;Otranto et al. 2017). In contrast to other studies from Spain and other countries in Europe (Criado-Fornelio et al. 2009;Díaz-Regañón et al. 2017;Giannelli et al. 2017), we did not detect H. canis or Hepatozoon silvestris in cats.
The risk of infection with Hepatozoon spp. depends on exposure to ticks and is conditioned by the animal's lifestyle, use of ectoparasiticides and contact with wildlife (Pacifico et al. 2020). It is likely that most of the animals in this study, particularly stray cats, had never received ectoparasitic treatments and shared habitat with wildlife species and infected ticks. However, H. felis transmission routes are not known yet ), but ticks are suspected vectors. Congenital transmission may also occur, like for H. canis in dogs Murata et al. 1993). Predation was described for Hepatozoon americanum in dogs feeding on rodents and rabbits infected with a quiescent cystozoite form of the parasite (Johnson et al. 2009(Johnson et al. , 2008. Stray cats may engage more in predation than dogs and may ingest arthropod vectors when grooming. The time of the year when samples were collected could be an important factor in conditioning prevalence, and, in contrast to cats, no dogs were examined in the summer. Winter Hepatozoon spp. infections might be accounted for by transmission at this time of the year since R. sanguineus in Spain may also be active in the winter (Estrada-Peña et al. 2017) and/or infection becoming chronic for a long time (Baneth et al. 1998). In Israel, Hepatozoon spp. were most prevalent in cats during the winter (Baneth et al. 1998), but it was not seasonal in dogs (Baneth & Weigler 1997). In Iran, the prevalence of Hepatozoon spp. in dogs was highest in the summer (Barati & Razmi 2018).
Leishmania infantum was also strongly seasonal, and winter peaks in both dogs and cats would be associated Fig. 1 Phylogenetic tree of Hepatozoon spp. sequences from dogs and cats in this study (diamond-shaped) together with homologous sequences from GenBank. The evolutionary history was inferred by using the Neighbour-Joining method and the Kimura 2-parameter model. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Bootstrap values below 70% are not shown. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. All positions with less than 95% site coverage were eliminated, i.e., fewer than 5% alignment gaps, missing data, and ambiguous bases were allowed at any position (partial deletion option). There were a total of 334 positions in the final dataset. Evolutionary analyses were conducted in MEGA X ◂ MT588817 with leishmaniosis' long incubation period (Hernández et al. 2015;Oliva et al. 2006;Pennisi et al. 2012). Compared to other studies in the Iberian Peninsula, the prevalence of L. infantum in cats was slightly lower than the 26% to 30% found by Maia et al. (2008), Martín-Sánchez et al. (2007) and Millán et al. (2011) and higher than the 0% to 20% found in other studies (Alcover et al. 2021;Maia et al. 2010;Miró et al. 2014;Montoya et al. 2018;Sherry et al. 2011;Tabar et al. 2008). Prevalence estimations may differ depending on the number and lifestyle of cats sampled, the sample used for diagnosis, the time of the year and the animal's clinical status and coinfections. Elevated parasitaemia is most typical in animals suffering clinical leishmaniosis, whilst in infected but healthy dogs, parasitaemia is less common than infection in the skin and lymphoid tissue (Chitimia et al. 2011), and according to our results, this would also apply to cats. Other studies comparing L. infantum prevalence in different cat samples have reported variable results. In stray and colony cats in Italy, PCR prevalence in blood was lower than in conjunctival swabs (Morganti et al. 2019) and marginally lower than in lymph node samples (Spada et al. 2020). In contrast, Chatzis et al. (2014) reported similar PCR prevalence in blood and tissue in healthy cats and a higher prevalence in tissues compared to blood in cats with skin lesions. It could be concluded that samples from skin and various lymphoid tissues, taken at different times of the year, are required for an accurate estimation of L. infantum infection prevalence in cat and dog populations. The greater prevalence of infection in lymphoid tissue in dogs compared to cats and the similar prevalence in skin samples suggest that parasite visceralitation is lower in cats than in dogs and is compatible with cutaneous leishmaniosis being the most common clinical manifestation of the disease in cats (Solano-Gallego et al. 2007). However, in the present study, L. infantum infection in cats was not associated with clinical signs typical of FeL (Abramo et al. 2021;Pennisi et al. 2015). Still, none of the clinical signs detected is specific to FeL and may be due to numerous causes; for example, open skin wounds may be the result of fighting, which is common in stray cats. The differences between cats and dogs in infection prevalence and the parasite's body distribution are remarkable. They are consistent with cats' lower risk of leishmaniosis and having mostly cutaneous lesions, and they support the dogs' role as a primary reservoir of infection. Notwithstanding this, the cats' role in L. infantum transmission requires further investigation given that they are able to transmit the parasite to the vector, the prevalence may be relatively high, and cat populations are large. An objective assessment may only be possible in natural environments in the absence of dogs or other reservoirs of infection (Zanet et al. 2014).
Our L. infantum and Hepatozoon spp. studies support a common domestic and sylvatic transmission cycle for these parasites in southeast Spain (Ortuño et al. 2019(Ortuño et al. , 2021, as well as the need to consider cats as a potentially important element in leishmaniosis epidemiology. It also highlights the importance of controlling stray cats and abandoned dog populations to reduce parasite prevalence, their impact on pets and the risk of zoonotic transmission.

Conclusions
Hepatozoon spp. and L. infantum but not Babesia spp. are common parasites of stray cats and abandoned dogs in south-east Spain, and Hepatozoon spp. transmission cycles are likely to be shared with wildlife. A better demographic and sanitary control of this vulnerable population is required. Compared to dogs, cats have a lower probability of L. infantum infection and the parasite becoming latent in lymphoid tissue, and when it does, parasite concentration is lower. Differences between cats and dogs in L. infantum infection prevalence and body distribution have important epidemiological, diagnostic and clinical implications.
Funding Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. The study was funded by the Fig. 2 Phylogenetic tree of Babesia spp. sequences from dogs and cats in this study (diamond-shaped) together with homologous sequences from GenBank. The evolutionary history was inferred by using the Neighbour-Joining method and the Kimura 2-parameter model. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Bootstrap values below 70% are not shown. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. All positions containing gaps and missing data were eliminated (complete deletion option). There were a total of 275 positions in the final dataset. Evolutionary analyses were conducted in MEGA X ◂ Universidad de Murcia and the Hebrew University of Jerusalem. María Ortuño held a "Contrato Predoctoral FPU" from the Universidad de Murcia and was also beneficiary of an "Erasmus + Movilidad Internacional" grant to do some of the present work at the Koret School of Veterinary Medicine, Hebrew University of Jerusalem.

Data availability
The datasets supporting the conclusions of this article are included within the article. Raw data are available from the corresponding author upon reasonable request.

Conflict of interests The authors declare no competing interests.
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