Microfluidic PCR and network analysis reveals complex tick-borne pathogen interactions in the tropics.

BACKGROUND: Ixodid ticks, particularly Rhipicephalus sanguineus s.l., are important vectors of various disease-causing agents in dogs and humans in Cuba. However, our understading of interactions among tick-borne pathogens (TBPs) in infected dogs or the vector R. sanguineus s.l. remains limited. This study integrates microfluidic-based high-throughput real-time PCR data, Yule's Q statistic, and network analysis to elucidate pathogen-pathogen interactions in dogs and ticks in tropical western Cuba. METHODS: A cross-sectional study involving 46 client-owned dogs was conducted. Blood samples were collected from these dogs, and ticks infesting the same dogs were morphologically and molecularly identified. Nucleic acids were extracted from both canine blood and tick samples. Microfluidic-based high-throughput real-time PCR was employed to detect 25 bacterial species, 10 parasite species, 6 bacterial genera, and 4 parasite taxa, as well as to confirm the identity of the collected ticks. Validation was performed through end-point PCR assays and DNA sequencing analysis. Yule's Q statistic and network analysis were used to analyse the associations between different TBP species based on binary presence-absence data. RESULTS: The study revealed a high prevalence of TBPs in both dogs and R. sanguineus s.l., the only tick species found on the dogs. Hepatozoon canis and Ehrlichia canis were among the most common pathogens detected. Co-infections were observed, notably between E. canis and H. canis. Significant correlations were found between the presence of Anaplasma platys and H. canis in both dogs and ticks. A complex co-occurrence network among haemoparasite species was identified, highlighting potential facilitative and inhibitory roles. Notably, H. canis was found as a highly interconnected node, exhibiting significant positive associations with various taxa, including A. platys, and E. canis, suggesting facilitative interactions among these pathogens. Phylogenetic analysis showed genetic diversity in the detected TBPs. CONCLUSIONS: Overall, this research enhances our understanding of TBPs in Cuba, providing insights into their prevalence, associations, and genetic diversity, with implications for disease surveillance and management.

First molecular evidence of Mycoplasma haemocanis and 'Candidatus Mycoplasma haematoparvum' infections and its association with epidemiological factors in dogs from Cuba.

Haemotrophic mycoplasmas (haemoplasmas) are unculturable, epicellular, cell wall-less gram-negative bacteria distributed worldwide, which infect several mammalian species. In dogs, Mycoplasma haemocanis and 'Candidatus Mycoplasma haematoparvum' have been reported as causative agents of infectious anaemia, especially in splenectomised or immunocompromised animals. The present cross-sectional study aims to assess the prevalence, risk factors, and molecular characterization of canine haemoplasmas in Cuba. A total of 391 dog blood samples and 247 tick samples were tested for the presence of canine haemoplasmas by species-specific quantitative TaqMan® real-time PCR assays. Overall, 17.9% (70/391; 95% CI: 14.1-21.7) blood samples were PCR-positive for at least one canine haemoplasmas species, where 15.1% (59/391; 95% CI: 11.5-18.7) for Mycoplasma haemocanis, 4.4% (17/391; 95% CI: 2.3-6.4) for 'Candidatus Mycoplasma haematoparvum', and 1.5% (6/391; 95% CI: 0.3-2.8) were co-infected. All collected ticks were identified morphologically as Rhipicephalus sanguineus sensu lato, and none of the tested tick samples was found PCR-positive for the presence of Mycoplasma haemocanis and 'Candidatus Mycoplasma haematoparvum'. Risk factors for canine haemoplasmas species infection included the presence of tick infestation, crossbreeding and living in kennels, while no association was found with the occurrence of anaemia. Phylogenetic analyses based on the 16S rRNA gene sequences of Mycoplasma haemocanis and 'Candidatus Mycoplasma haematoparvum' revealed >99% identity to other isolates distributed worldwide, indicating low genetic variability amongst these canine haemoplasmas species. To the best of the authors´ knowledge, this is the first molecular evidence of Mycoplasma haemocanis and 'Candidatus Mycoplasma haematoparvum' infections in dogs from 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.

Unraveling the Global Phylodynamic and Phylogeographic Expansion of Mycoplasma gallisepticum: Understanding the Origin and Expansion of This Pathogen in Ecuador.

Mycoplasma gallisepticum (MG) is among the most significant problems in the poultry industry worldwide, representing a serious threat to international trade. Despite the fact that the mgc2 gene has been widely used for diagnostic and molecular characterization purposes, there is a lack of evidence supporting the reliability of this gene as a marker for molecular epidemiology approaches. Therefore, the current study aimed to assess the accuracy of the mgc2 gene for phylogenetic, phylodynamic, and phylogeographic evaluations. Furthermore, the global phylodynamic expansion of MG is described, and the origin and extension of the outbreak caused by MG in Ecuador were tracked and characterized. The results obtained strongly supported the use of the mgc2 gene as a reliable phylogenetic marker and accurate estimator for the temporal and phylogeographic structure reconstruction of MG. The phylodynamic analysis denoted the failures in the current policies to control MG and highlighted the imperative need to implement more sensitive methodologies of diagnosis and more efficient vaccines. Framed in Ecuador, the present study provides the first piece of evidence of the circulation of virulent field MG strains in Ecuadorian commercial poultry. The findings derived from the current study provide novel and significant insights into the origin, diversification, and evolutionary process of MG globally.

Ticks and Tick-Borne Diseases in Cuba, Half a Century of Scientific Research.

Ticks and the vast array of pathogens they transmit, including bacteria, viruses, protozoa, and helminths, constitute a growing burden for human and animal health worldwide. In Cuba, the major tropical island in the Caribbean, ticks are an important cause of vector-borne diseases affecting livestock production, pet animal health and, to a lesser extent, human health. The higher number of tick species in the country belong to the Argasidae family and, probably less known, is the presence of an autochthonous tick species in the island, Ixodes capromydis. Herein, we provide a comprehensive review of the ticks and tick-borne pathogens (TBPs) affecting animal and human health in Cuba. The review covers research results including ecophysiology of ticks, the epidemiology of TBPs, and the diagnostic tools used currently in the country for the surveillance of TBPs. We also introduce the programs implemented in the country for tick control and the biotechnology research applied to the development of anti-tick vaccines.