Seroprevalence Rates of Tick-Borne Pathogens in Cats from Southern Bulgaria

Introduction

The most common feline tick-borne pathogens (FTBPs) are Borrelia spp., Ehrlichia spp., Anaplasma spp., Babesia spp., and Hepatozoon spp. (Day 2016). Among Borrelia spp. the most common is Borrelia burgdorferi sensu lato (s.l.), among Ehrlichia spp.—Ehrlichia canis and E. canis-like agent, and among Anaplasma spp.—Anaplasma phagocytophilum and Anaplasma platys (Lappin 2018). B. burgdorferi and A. phagocytophilum are transmitted by ticks Ixodes spp. and E. canis by Rhipicephalus sanguineus (Lappin 2018).

There are few studies about feline ticks and which are the most common: in cats from Belgium—Ixodes ricinus and Ixodes hexagonus (Claerebout et al. 2013), in Italy—R. sanguineus, Rhipicephalus pusillus, Ixodes ventalloi, and I. ricinus (Persichetti et al. 2016, Otranto et al. 2017a, 2017b). Investigations carried out in United Kingdom found the presence of I. ricinus, I. hexagonus, Ixodes trianguliceps, Haemaphysalis punctata, and Dermacentor reticulatus (Abdullah et al. 2016, Davies et al. 2017, Duplan et al. 2018).

The aim of the present study was to investigate the prevalence rates of the FTBPs—B. burgdorferi, E. canis, and A. phagocytophilum in stray cats from Southern Bulgaria. Similar studies on FTBPs in Bulgaria have not been conducted.

Materials and Methods

One hundred stray female cats (n = 100) in Trap-Neuter-Return (TNR) program were enrolled from three districts of Southern Bulgaria—Burgas (n = 30), Stara Zagora (n = 20), and Sofia (n = 50). Animals showed no clinical signs at sampling time point (spring of 2019), and they were aged from 1 to 10 years based on the clinical expertise of veterinarians participating in the TNR program. The investigated districts were located among plains (∼42.51 N and 42.69 N Latitude, 23.31 E and 27.46 E Longitude), and the climate is subtropical/continental (mean annual temperature 13–15°C, precipitations about 650 mm/m²).

Feline blood samples (up to 1.5 mL per individual) were taken by puncture of the cephalic vein. Immediately after blood collection, fresh anticoagulated whole blood samples were tested for B. burgdorferi s.l., E. canis, and A. phagocytophilum antibodies using a commercial ELISA rapid test (SNAP^® 4Dx^® Plus Test, IDEXX Laboratories, Inc., Westbrook, ME), according to the manufacturer's instructions. The SNAP 4Dx Plus is a test for dogs and screens for seven vector-borne pathogens (A. phagocytophilum, A. platys, B. burgdorferi, D. immitis, E. canis, E. chaffeensis, and E. ewingii). The SNAP 4Dx Plus has also been used in cats for A. phagocytophilum, B. burgdorferi, and E. canis (Lappin et al. 2015, Ravicini et al. 2016).

This study was approved by the Ethics Committee in Animal Experimentation and Animal Welfare at Trakia University of Stara Zagora and was conducted according to the ethical principles of animal experimentation, adopted by the Bulgarian Ministry of Agriculture, Food, and Forestry.

The results obtained from the present study do not allow detailed statistical analysis. Therefore, basic statistical indicators such as confidence interval (CI), standard deviation, and so on were applied. Statistical analysis was performed by Excel 2007 (Microsoft, Redmond, WA) and SPSS Statistics 19.0 (IBM Corp., Armonk, NY). A p-value <0.05 was considered statistically significant.

Results and Discussion

The overall FTBP seroprevalence was 3% (Table 1). The seropositivity varied narrowly on investigated districts. Antibodies against B. burgdorferi were found in one region (Sofia district) in two percent (1/50) of the investigated cats. In two out of the three regions—Stara Zagora and Sofia—antibodies against E. canis were detected in 2.8% (2/70) of the stray cats. Antibodies against A. phagocytophilum were not found in all three regions (Burgas, Stara Zagora, and Sofia) in all feline blood samples.

There are few studies on seroprevalence of B. burgdorferi infection in cats. Feline B. burgdorferi infection could be subclinical or with nonspecific clinical signs—fever, lameness, fatigue, or anorexia (Shaw et al. 2001). Magnarelli et al. reported overall seropositivity of 47% of 93 serum samples—56% (5/9) in nine ill cats and 46% (39/84) in 84 healthy cats (Magnarelli et al. 2005). Hegarty et al. announced seropositivity of 5.5% in 715 feline serum samples from veterinary hospitals located in the United States, Canada, and the Caribbean (Hegarty et al. 2015). Ravicini et al. performed SNAP 4Dx test (IDEXX) in 116 feline sera (Ravicini et al. 2016). They found that one healthy 3-month-old kitten was positive for B. burgdorferi antibodies in serum and negative results for Ehrlichia species and Anaplasma species. In 2015, Pantchev et al. reported 2.4% (95% CI: 0.7–6.0%) seropositivity of B. burgdorferi in dogs from central-southern Bulgaria with a similar test (Pantchev et al. 2015). In 2018/2019, we reported 23.2% (95% CI: 16.8–30.7%) B. burgdorferi seropositivity in South Bulgarian horses (Tsachev et al. 2018) and 15.1% (95% CI: 10.4–21.0%) in horses from Northern Bulgaria (Tsachev et al. 2019). For the period 2008 to 2018 in Bulgaria the registered cases of human Lyme borreliosis were 7.23 cases per 100 thousand populations (NCIPD 2020). The lower seroprevalence (antibodies to C6 peptide) in cats compared to dogs and horses could be explained by the different tick exposure and the presence of different registered acaricides for tick prophylaxis.

Fever is the most common clinical presentation of feline ehrlichiosis. In 2002 Breitschwerdt et al. found E. canis partial 16S ribosomal DNA (rDNA: 382 nucleotide positions) in cats, which were identical to a canine isolate of E. canis (GenBank accession no. AF373613) (Unver et al. 2001, Breitschwerdt et al. 2002). Afterward DNA and antibodies against E. canis were established in cats from Spain (Ayllon et al. 2012), Brazil (Braga et al. 2014), and Italy (Persichetti et al. 2016). Also seropositivity of E. canis in cats has been found in Spain—10.6% (13/122) (Aguirre et al. 2004), 17.9% (42/235) (Ortuno et al. 2005), 11.3% (19/168) (Solano-Gallego et al. 2006), and 9.9% (67/680) (Ayllon et al. 2012); in Italy—1% (2/203) (Vita et al. 2005); and in Brazil—5.5% (11/200) (Braga Mdo et al. 2012). A study from central-southern Bulgaria performed with a similar test in dogs revealed a seroprevalence of 21% (95% CI: 15.1–27.9%) (Pantchev et al. 2015). Data reported in 2018 presented 3.9% (95% CI: 1.4–8.2%) seroprevalence of Ehrlichia spp. in horses from Southeastern Bulgaria (Tsachev et al. 2018) and 0.5% (95% CI: 0.0–2.9%) in horses from Northern Bulgaria (Tsachev et al. 2019). The varying data could be influenced by geographic location, climate characteristics, and vectors' variation. Furthermore, the reasons for these results could be the same as those mentioned above for the serological evidence for infection with B. burgdorferi, likely to be explained by availability of a certain tick vector.

Fever, anorexia, and fatigue are the most common clinical signs in feline anaplasmosis (Lappin et al. 2004, Savidge et al. 2016). A. phagocytophilum infection in cats was established in many countries—Sweden (Bjoersdorff et al. 1999, Elfving et al. 2015), United States (Lappin et al. 2004), Spain (Solano-Gallego et al. 2006, Ayllon et al. 2012), Germany (Hamel et al. 2012), and Italy (Ebani and Bertelloni 2014, Persichetti et al. 2016). Variation in feline seropositivity (low and middle) for A. phagocytophilum was found in United States—38% (35/93) (Magnarelli et al. 2005), 4.3% (20/460) (Billeter et al. 2007), and 1.8% (13/715) (Hegarty et al. 2015); in Spain—1.8% (3/168) (Solano-Gallego et al. 2006) and 8.4% (57/680) (Ayllon et al. 2012); in Germany—16.2% (53/326) (Hamel et al. 2012); in Italy—4.5% (25/560) (Ebani and Bertelloni 2014) and 33.3% (14/42) (Persichetti et al. 2016); and in Sweden—22.1% (19/90) (Elfving et al. 2015). In humans, granulocytic anaplasmosis are of concern in the southern districts of the country (Christova and Dumler 1999, Christova et al. 2008, Pishmisheva et al. 2017), but A. phagocytophilum in cats was not found in any of the three studied districts in the present study.

This study has some limitations that need to be addressed. The study included a relatively small number of cats; therefore, the results should be cautiously interpreted. In addition, determining anti-FTBP IgG and IgM seroprevalence rate is important in assessing past and recent infections. However, in immunocompromised cats ELISA tests have limited the diagnostic value. In these cases, RT-PCR is required to establish an active infection. Despite these limitations, this is the first study on B. burgdorferi and E. canis prevalence in Bulgarian stray cats that offers new insights into FTBP epidemiology in the Balkan Peninsula.

Conclusion

This study provides the first evidence for B. burgdorferi and E. canis in stray cats from Southern Bulgaria. Results from this study support the need for serological and molecular FTBP prevalence studies in Bulgaria and in the other countries from Balkan peninsula.