Advances in the Epidemiological Surveillance of Tick-Borne Pathogens.

Ticks are obligate blood-feeding ectoparasites of mammals, birds, and reptiles, which are globally important vectors of pathogens that impact both human and animal health [...].

Can Domestic Cats Act as Reservoirs of Cytauxzoon felis?

Cytauxzoonosis is a worldwide tick-borne disease of domestic and wild felids, caused by infection of the haemoparasites belonging to the genus Cytauxzoon (Piroplasmida, Apicomplexa), which are apicomplexan protozoans closely related to those of the genera Babesia and Theileria [...].

Emerging tick-borne spotted fever group rickettsioses in the Balkans.

The impact of tick-borne pathogens (TBPs) on human health has increased in the last decades, since the incidence of emerging and re-emerging infectious and zoonotic tick-borne diseases has increased worldwide. Tick-borne rickettsiae of the Spotted Fever group (SFGR) are considered as emerging pathogens that can infect humans and cause a variety of non-specific clinical symptoms. Here, we report nine cases of atypical tick-borne diseases (9/460; 1.95%) that occurred over a period of four months (from 15 April 2021 to 16 August 2021) in Serbia, from which five cases were classified as confirmed SFGR infection, two cases as probable SFGR infection and two cases as suspected SFGR infection. Within cases of confirmed SFGR infection, R. helvetica was detected as the causative agent in two cases. The most common clinical finding was non-expanding persistent circular redness, followed by eschar and enlargement of regional lymph nodes, and pain at lesion site. Rickettsia outer membrane protein B (ompB) and citrate synthase (gltA) gene fragments were amplified from clinical samples and ticks attached to patients and IgG reacting with Rickettsia conorii antigen were detected in sera samples of patients, which are highly suggestive of exposure to SFGR. Surveillance and monitoring of rickettsial diseases in Serbia should continue and extended to new areas due to the increasing trend of clinical infections caused by SFGR in the country.

Unexpected TBEV Seropositivity in Serbian Patients Who Recovered from Viral Meningitis and Encephalitis.

The tick-borne encephalitis virus (TBEV) causes a life-threatening disease named Tick-borne encephalitis (TBE). The clinical symptoms associated with TBE range from non-specific to severe inflammation of the central nervous system and are very similar to the clinical presentation of other viral meningitis/encephalitis. In consequence, TBE is often misclassified by clinical physicians, mainly in the non-identified high-risk areas where none or only a few TBE cases have been reported. Considering this situation, we hypothesized that among persons from northern Serbia who recovered from viral meningitis or encephalitis, there would be evidence of TBEV infection. To test this hypothesis, in this observational study, we evaluated the seroreactivity against TBEV antigens in patients from northern Serbia who were hospitalized due to viral meningitis and/or viral encephalitis of unknown etiology. Three cases of seroreactivity to TBEV antigens were discovered among convalescent patients who recovered from viral meningitis and/or encephalitis and accepted to participate in the study (n = 15). The clinical and laboratory findings of these patients overlap with that of seronegative convalescent patients. Although TBE has been a notifiable disease in Serbia since 2004, there is no active TBE surveillance program for the serologic or molecular screening of TBEV infection in humans in the country. This study highlights the necessity to increase the awareness of TBE among physicians and perform active and systematic screening of TBEV antibodies among patients with viral meningitis and/or encephalitis.

Ticks and Tick-Borne Diseases in Central America and the Caribbean: A One Health Perspective.

Ticks have complex life cycles which involve blood-feeding stages found on wild and domestic animals, with humans as accidental hosts. At each blood-feeding stage, ticks can transmit and/or acquire pathogens from their hosts. Therefore, the circulation of tick-borne pathogens (TBPs), especially the zoonotic ones, should be studied in a multi-layered manner, including all components of the chain of infections, following the 'One Health' tenets. The implementation of such an approach requires coordination among major stakeholders (such as veterinarians, physicians, acarologists, and researchers) for the identification of exposure and infection risks and application of effective prevention measures. In this review, we summarize our current knowledge on the epidemiology of tick-borne diseases in Central America and the Caribbean and the challenges associated with the implementation of 'One Health' surveillance and control programs in the region.

A One Health approach to study the circulation of tick-borne pathogens: A preliminary study.

Tick-borne pathogens (TBPs) have complex life cycles involving tick vectors and vertebrate hosts. However, there is limited empirical evidence on the zoonotic circulation of TBPs. In this study, we used a One Health approach to study the possible circulation of TBPs in ticks, animals and humans within a rural household in the foothills of the Fruska Gora mountain, northern Serbia. The presence of TBP DNA was assessed using microfluidic PCR (25 bacterial species, 7 parasite species, 5 bacterial genera, 3 parasite genera) in animal, human and tick samples and the presence of tick-borne encephalitis virus (TBEV) RNA was screened for using RT-qPCR on tick samples. In addition, Lyme borreliosis serology was assessed in patients sera. Rhipicephalus sanguineus and Ixodes ricinus ticks were identified on dogs and Haemaphysalis punctata was identified on house walls. Rickettsia helvetica was the most common pathogen detected in pooled R. sanguineus and I. ricinus tick samples, followed by Hepatozoon canis. None of the H. punctata tick samples tested positive for the presence of TBPs. Anaplasma phagocytophilum and Rickettsia monacensis were the most frequent pathogens detected in dogs, followed by Rickettsia felis, whereas Anaplasma bovis was the only pathogen found in one of the goats tested. None of the human blood samples collected from family members tested positive for the presence of TBPs. Although microfluidic PCR did not detect Borrelia sp. in any of the tested tick or blood samples, a family member with a history of Lyme disease was seropositive for Borrelia burgdorferi sensu lato (s.l.). We conclude that, despite the presence of TBPs in tick and vertebrate reservoirs, there is no evidence of infection with TBPs across various components of the epidemiological chain in a rural Fruska Gora household.

Tick-Borne Encephalitis Virus Seropositivity among Tick Infested Individuals in Serbia.

Tick-borne encephalitis (TBE), caused by the TBE virus (TBEV), is a life-threatening disease with clinical symptoms ranging from non-specific to severe inflammation of the central nervous system. Despite TBE is a notifiable disease in Serbia since 2004, there is no active TBE surveillance program for the serologic or molecular screening of TBEV infection in humans in the country. This prospective cohort study aimed to assess the TBEV exposure among tick-infested individuals in Serbia during the year 2020. A total of 113 individuals exposed to tick bites were recruited for the study and screened for anti-TBEV antibodies using a commercial indirect fluorescent antibody test (IFA) test. Blood samples from 50 healthy donors not exposed to tick bites were included as a control group. Most of the enrolled patients reported infestations with one tick, being I. ricinus the most frequent tick found in the participants. The TBEV seroprevalence was higher (13.27%, 15 total 113) in tick-infested individuals than in healthy donors (4%, 2 total 50), although the difference was not significant. Notably, male individuals exposed to tick bites showed five times higher relative risk (RR) of being TBEV-seropositive than healthy donors of the same gender (RR= 5.1, CI = 1.6-19; p = 0.007). None of the seropositive individuals developed clinical manifestations of TBE, but the first clinical-stage of Lyme borreliosis (i.e., erythema migrans) was detected in seven of them. Potential TBEV foci were identified in rural areas, mostly in proximity or within the Fruska Gora mountain. We conclude that the Serbian population is at high risk of TBEV exposure. Further epidemiological studies should focus on potential TBEV foci identified in this study. The implementation of active surveillance for TBEV might contribute to evaluating the potential negative impact of TBE in Serbia.

Shared Odds of Borrelia and Rabies Virus Exposure in Serbia.

Lyme borreliosis (LB) is the most common tick-borne disease in Serbia and other European countries. Rabies is a fatal zoonosis distributed worldwide and is caused by the rabies virus. Professionals at risk of rabies-including veterinarians, hunters, communal service workers, and forestry workers-overlap with some professions at a higher risk of exposure to tick bites and tick-borne pathogen infections. We hypothesized that individuals identified by the public health system as at risk of rabies virus infection, and consequently vaccinated against rabies virus, also share a higher likelihood of Borrelia exposure. To test our hypothesis, a case-control study was carried out during 2019 in Serbia to determine the seroprevalence of anti-Borrelia antibodies in two case groups (individuals at risk and vaccinated against rabies virus) and a control group (individuals without risk of rabies). Individuals vaccinated against rabies following either "pre-exposure protocol" (PrEP, n = 58) or "post-exposure protocol" (PEP, n = 42) were considered as rabies risk groups and healthy blood donors (n = 30) as the control group. The results showed higher Borrelia seroprevalence in PrEP (17.2%; 10/58) and PEP (19.0%; 8/42) groups compared with the control group (6.67%; 2/30). Furthermore, odds ratio (OR) analysis showed that risk of rabies (in either the PrEP (OR = 2.91) or PEP (OR = 3.29) groups) is associated with increased odds of being seropositive to Borrelia. However, the difference in Borrelia seroprevalence between groups was not statistically significant (Chi-square (χ²) test p > 0.05). The shared odds of LB and rabies exposure found in this study suggest that, in countries where both diseases occur, the common citizen can be at risk of both diseases when in a risky habitat. These findings are important to guide physicians in targeting high-risk groups, and diagnose LB, and to guide decision-makers in targeting control and prevention measures for both infections in risk areas.

Humans infested with Ixodes ricinus are exposed to a diverse array of tick-borne pathogens in Serbia.

Tick-borne pathogens (TBPs) pose a major threat to human health in Europe and the whole northern hemisphere. Despite a high prevalence of TBPs in Ixodes ricinus ticks, knowledge on the incidence of tick-borne diseases in humans infested by this tick species is limited. This study was conducted in the year 2019 on patients who presented themselves to the Pasteur Institute Novi Sad with tick infestations. Ticks (n = 31) feeding on human (n = 30) and blood samples from the same individuals were collected by physicians and a microfluidic real-time high-throughput PCR system was used to test the genomic DNA of the samples for the presence of 27 bacterial and eight parasitic microorganisms in Serbia. Except for one Rhipicephalus sanguineus s.l. adult male tick, all ticks infesting humans were morphologically identified as I. ricinus. A high proportion of ticks (74 %, 23/31) were infected with at least one of the tested TB microorganisms, being Rickettsia helvetica (54 %, 17/31) the most common pathogen, but Borrelia afzelii (9 %, 3/31), Anaplasma phagocytophilum (6 %, 2/31), Borrelia miyamotoi (6 %, 2/31), and Francisella like-endosymbiont (6 %, 2/31), Borrelia valaisiana (3 %, 1/31), Borrelia lusitaniae (3 %, 1/31), Rickettsia felis (3 %, 1/31) and Rickettsia aeschlimannii (3 %, 1/31) were also identified. Despite the high infection rate of TBPs in ticks, only two human blood samples (6 %, 2/30) tested positive for the presence of TBPs, one patient (code H12, 67 years old female) was diagnosed with Borrelia spp. and the other patient was diagnosed (code H17, 71 years old female) with R. felis infection. The tick infesting patient H12 tested positive for B. afzelii, and R. helvetica and the tick infesting patient H17 tested positive for R. felis. Upon clinical examination, both patients were diagnosed with erythema migrans. No additional discomfort was reported by the patient and no additional pathology was observed by the physician. We concluded that humans bitten by I. ricinus in Serbia are exposed to a diverse array of TBPs with clinical impact in the Serbian cohort studied.

Molecular detection and characterization of Hepatozoon canis in stray dogs from Cuba.

Canine hepatozoonosis caused by Hepatozoon canis is a worldwide distributed tick-borne disease of domestic and wild canids that is transmitted by ingestion of Rhipicephalus sanguineus sensu lato (s.l.) ticks. The present study was aimed to determine the prevalence of Hepatozoon infections in 80 stray dogs from Havana Province in Cuba, and to confirm the species identity and phylogenetic relationships of the causative agent. Samples were screened by microscopical examination of thin blood smears for the presence of Hepatozoon spp. gamonts and by genus-specific SYBR green-based real-time PCR assay targeting the 18S rRNA gene. Direct microscopy examination revealed Hepatozoon gamonts in the peripheral blood of 8 dogs (10.0%; 95% CI: 4.80-18.0%), while 38 animals (47.5%; 95% CI: 36.8-58.4%) were PCR-positive, including all microscopically positive dogs. Hence, the agreement between the two detection methods was 'poor' (κ = 0.20). Hematological parameters did not differ significantly between PCR-positive and PCR-negative dogs (p > 0.05). The DNA sequences of the 18S rRNA gene of the Hepatozoon spp. from Cuban dogs showed a nucleotide identity >99% with those of 18S rRNA sequences of Hepatozoon canis isolates from Czech Republic, Brazil and Spain. Phylogenetic analysis revealed that obtained sequences clustered within the Hepatozoon canis clade, different from the Hepatozoon felis or Hepatozoon americanum clades. The present study represents the first molecular characterization of Hepatozoon canis in stray dogs within Cuba.

Molecular Diagnosis, Prevalence and Importance of Zoonotic Vector-Borne Pathogens in Cuban Shelter Dogs-A Preliminary Study.

The present study aimed to determine the prevalence of zoonotic vector-borne pathogens, including Anaplasma platys, Anaplasma phagocytophilum, Borrelia burgdorferi sensu lato, Ehrlichia canis and Rickettsia spp. in shelter dogs from Cuba. Blood samples were collected from 100 shelter dogs and examined by molecular methods. Overall, 85 (85%; 95% CI: 77.88-92.12) dogs tested positive for at least one vector-borne pathogen using species-specific qPCR assays. Among the positive samples, E. canis was the most prevalent 62% (95% CI: 52.32-71.68), followed by A. platys 40% (95% CI: 30.23-49.77) and Rickettsia felis 27% (95% CI: 18.15-35.85), whereas 36% (95% CI: 26.43-45.57) showed co-infections. All samples were negative for A. phagocytophilum and B. burgdorferi s.l. The presence of 248 Rhipicephalus sanguineus ticks collected from the dogs was not statistically associated with the occurrence of infections. Thrombocytopenia was the most frequent haematological alteration found in PCR-positive dogs; it was statistically associated with the presence of E. canis, as well as co-infections (p < 0.05). The phylogenetic analyses of A. platys and E. canis based on 16S rRNA, groEL and gltA genes showed a low genetic diversity between Cuban strains. The present study demonstrates the high prevalence of vector-borne pathogens with zoonotic potential in shelter dogs from Cuba.

Efficient Transovarial Transmission of Babesia Spp. in Rhipicephalus microplus Ticks Fed on Water Buffalo (Bubalus bubalis).

Water buffaloes can be infected by tick-borne pathogens (TBPs) in endemic areas where cattle and buffalo coexist. Among TBPs affecting buffaloes is the Apicomplexan hemoparasites Babesia bovis and B. bigemina, transmitted by Rhipicephalus microplus ticks. However, little empirical evidence exists on whether buffalo can support TBPs' infection and transmission. A cohort study was designed to measure the infestation levels of R. microplus in buffaloes as well as the ability of buffalo-fed ticks to transmit B. bovis and B. bigemina to their offspring. Tick infestation of different life stages was quantified in cattle and buffalo kept in field conditions in western Cuba. Engorged adult female ticks were allowed to lay eggs in controlled conditions of humidity and temperature, and reproductive parameters were measured and analyzed. Hosts and tick larvae were tested for the presence of Babesia spp. using species-specific qPCR assays. Tick infestation was not observed in adult buffaloes. However, buffalo and cattle calves were equally infested, although the larval survival rate was higher in cattle calves than in buffalo calves. All larval pools (31) obtained from the adult female ticks were positive for B. bovis, whereas only 68% (21/31) was positive for B. bigemina. Among the 10 larval pools negative for B. bigemina, three proceeded from adult females fed on Babesia-negative buffaloes. The other seven pools were from Babesia-positive animals, three from cattle and four from buffalo calves. Babesia infection levels in tick larvae, quantified by qPCR, were similar in female ticks fed on buffalo and bovine calves. We conclude that water buffalo can sustain tick vector populations and support Babesia infection in levels high enough as to be infective for ticks. Our results also validated the hypothesis that adult female ticks fed on buffalo can transmit the pathogens B. bovis and B. bigemina to their offspring. Nevertheless, further laboratory studies are needed to address the question of whether the transovarial transmission of Babesia occurs in the following settings: (1) When adult females are infected previous to the feeding on the buffalo or/and (2) when the adult females acquire the infection while feeding on the buffalo.

Epidemiology and genetic diversity of Anaplasma marginale in Zamora-Chinchipe, Ecuador.

Bovine anaplasmosis, caused by the tick-borne pathogen Anaplasma marginale, is a hemolytic disease that constitutes a major constraint to cattle production in tropical and subtropical regions including Ecuador. However, the epidemiological situation of this hemoparasitosis in Ecuador is poorly characterized. The present study was aimed to determine the prevalence and genetic diversity of A. marginale in cattle of Ecuador. A cross-sectional study was carried out covering several farms from six out nine cantons of the Zamora-Chinchipe province. A total of 185 cattle were randomly selected and blood samples were collected from the animals. The studied group of animals included six breeds, three age groups, and both sexes. The molecular diagnostic was performed based on a nPCR assay targeting the A. marginale msp5 gene. Anaplasma marginale prevalence was 63.8 % and the bacteria were detected in all the cantons studied. Thirteen representative strains were selected and genetically characterized based on the msp1α gene. Genetic diversity analysis revealed that different strains circulate in the bovine herds studied. The results suggest that cattle movement may contribute to the circulation of common strains in the area. The results demonstrate a high prevalence of A. marginale in the region which should be considered by the sanitary authorities. The epidemiological surveillance for this disease should increase to anticipate acute disease outbreaks with high mortality. Bovine anaplasmosis outbreaks can cause economic losses and the death of several animals; therefore, measures for the prevention and control of this disease are required.

Development and application of a multiplex TaqMan® real-time qPCR assay for the simultaneous detection of Anaplasma marginale and Theileria annulata and molecular characterization of Anaplasma marginale from cattle in Western Cuba.

Anaplasmosis and theileriosis are considered the most important tick-borne diseases for livestock production worldwide, causing significant economic losses in tropical and subtropical regions. The present study was aimed to develop a multiplex TaqMan® qPCR assay to simultaneously detect Anaplasma marginale and Theileria annulata and to applied it to investigate naturally infected cattle in Cuba. The assay was highly specific, sensible, and efficient; it was more sensitive than a well-established nested PCR and detected 1 DNA copy of each target. Consistent repeatability and reproducibility within and between multiplex qPCR runs was shown. A total of 223 blood samples collected in western Cuba were analyzed for haemoparasites infection in cattle. The multiplex qPCR assay detected A. marginale in 213 samples (95.5%; CI: 95%; 91.9%-97.5%), but all samples were negative for T. annulata. Additionally, the genetic diversity of A. marginale was assessed using 16S rRNA, MSP1a and MSP4 nucleotide and protein sequences. The MSP1a tandem repeats ranged from three to five, and twelve different MSP1a tandem repeats of A. marginale were found, which presented genotypes C, E, and G in the 5'UTR microsatellite region. Phylogenetic analysis using the msp4 gene showed that Cuban strains were closely related to others previously reported in Mexico, Brazil and Asian countries. The multiplex qPCR described here proved to be a rapid, specific and cost-effective mean for the simultaneous detection of A. marginale and T. annulata. Further epidemiological studies using this assay will improve the surveillance of the associated diseases in regions where they are endemic.

Prevalence, Geographic Distribution, Risk Factors and Co-Infections of Feline Gammaherpesvirus Infections in Domestic Cats in Switzerland.

Recently, a gammaherpesvirus was described in domestic cats (FcaGHV1). The goal of the present study was to investigate the presence of FcaGHV1 in Swiss domestic cats and analyze potential risk factors. Blood samples from 881 cats presented to veterinarians in all Swiss cantons and from 91 stray cats and neoplastic tissue samples from 17 cats with lymphoma were evaluated. FcaGHV1 was detected by real-time PCR targeting the glycoprotein B gene, followed by sequencing. Blood samples were also tested for feline hemoplasmas, feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV). The molecular prevalence of FcaGHV1 was 6.0% (95% confidence interval (CI), 4.5-7.8%) in cats presented to veterinarians and 5.5% (95% CI, 1.8-12.4%) in stray cats. FcaGHV1 PCR-positive cats originated from 19/26 Swiss cantons. Factors significantly associated with FcaGHV1 detection included male sex, age >3 years, nonpedigree status and co-infection with FIV and hemoplasmas. Moreover, FeLV viremia tended to be associated with FcaGHV1 detection. High FcaGHV1 blood loads were found more frequently in FeLV-viremic cats and less frequently in hemoplasma-infected cats than in uninfected cats. Clinical information was unavailable for most of the 881 cats, but leukemia, carcinoma and cardiomyopathy were reported in FcaGHV1-positive cats. None of the tissue samples from the 17 cats with lymphoma tested positive for FcaGHV1. Sequence analyses revealed homogeneity among the Swiss isolates and >99.7% identity to published FcaGHV1 sequences. In conclusion, FcaGHV1 is present in Switzerland with a similar prevalence in cats presented to veterinarians and in stray cats. The pathogenic potential of FcaGHV1 needs further evaluation.

First molecular evidence of bovine hemoplasma species (Mycoplasma spp.) in water buffalo and dairy cattle herds in Cuba.

BACKGROUND: Hemotropic mycoplasmas (aka hemoplasmas) are small bacteria which cause infectious anemia in several mammalian species including humans. Information on hemoplasma infections in Cuban bovines remains scarce and no studies applying molecular methods have been performed so far. The aim of the present study was to utilize real-time PCR and sequence analysis to investigate dairy cattle and buffalo from Cuba for the presence of bovine hemoplasma species. RESULTS: A total of 80 blood samples from 39 buffalo and 41 dairy cattle were investigated for the presence of Mycoplasma wenyonii and "Candidatus Mycoplasma haemobos" using two species-specific real-time TaqMan PCR assays. PCR results revealed overall 53 (66.2%; 95% CI: 55.3-75.7%) positive animals for M. wenyonii and 33 (41.2%; 95% CI: 31.1-52.2%) for "Ca. M. haemobos"; the latter were all co-infections with M. wenyonii. The sample prevalences were similar in cattle and buffalo. Based on the sequence analysis of the nearly full-length 16S rRNA gene from two cattle and two buffalo, the presence of M. wenyonii and "Ca. M. haemobos" was confirmed. Statistical analysis revealed that buffalo and cattle one year of age or older were more frequently infected with M. wenyonii or "Ca. M. haemobos" than younger animals. PCR-positivity was not associated with anemia; however, the infection stage was unknown (acute infection versus chronic carriers). CONCLUSIONS: The high occurrence of bovine hemoplasma infections in buffalo and dairy cattle may have a significant impact on Cuban livestock production. To the best of our knowledge, this is the first molecular evidence of bovine hemoplasma species infection in dairy cattle and buffalo from Cuba and the Caribbean.

First molecular evidence of Babesia caballi and Theileria equi infections in horses in Cuba.

Equine piroplasmosis is a disease of Equidae, including horses, donkeys, mules, and zebras, caused by either Theileria equi or Babesia caballi. This disease represents a serious problem for the horse industry and its control is critical for the international trade of horses. The objective of the present study was to detect B. caballi and T. equi infections in horses reared in western Cuba. Blood samples from 100 horses were tested for the presence of piroplasms by using Giemsa-stained blood smears and nested PCR (nPCR) assays targeting merozoite antigen genes of B. caballi (bc48) and T. equi (ema-1). All animals were inspected for the detection of tick infestation and tick specimens were collected for species identification. Erythrocyte inclusions were observed in 13 (13%) of the analyzed samples. nPCR analysis showed that 25 (25%) samples were positive for B. caballi, 73 (73%) for T. equi, and 20 (20%) showed dual infections. Only one tick species was found infesting horses, Dermacentor nitens. In addition, three nearly full-length sequences of T. equi 18S rRNA gene were obtained and subjected to phylogenetic analyses. This study reports a high prevalence of T. equi and B. caballi single and coinfections in horses in western Cuba. Molecular analysis of the 18S rRNA gene of T. equi suggested that different genotypes of this hemoparasite circulate in Cuba. To the best of our knowledge, this is the first report describing the molecular detection of B. caballi and T. equi in horses in Cuba.