Alejandro Llanes PUBLICACIONES

Animals 2020, 10(2), 350

Detection and characterization of Leptospira infection and exposure in rats on the Carib-bean island of Saint Kitts.

Sreekumari Rajeev, Kanae Shio-kawa, Alejandro Llanes, Mala-vika Rajeev, Carlos M Restrepo, Raymond Chin, Eymi Cedeño Cedeño, Esteban Ellis.


In this study, we detected and characterized Leptospira infection and exposure in rats on the Caribbean island of Saint Kitts for the first time. We detected Leptospira infection in 17/29 (59%), 14/29 (48)%, and 11/29 (38)% of rats by RT-PCR, culture, and immunoflu-orescence assay, respectively. Whole genome sequencing (WGS) and analysis and serogrouping of 17 Leptospira strains isolated from rats revealed their close relationship with L. interrogans serogroup Icterohaemorrhagiae (n = 10) and L. borgpetersenii serogroup Ballum (n = 7). WGS, serogrouping, and additional PCR tests on rat kidneys confirmed mixed infections with L. interrogans and L. borgpetersenii in the kidneys of three rats. Microscopic agglutination test (MAT) was positive for 25/29 (87%) of the rats tested, and the response was restricted to serovars Icterohaem-orrhagiae {24/29(83%)}, Mankarso {4/29(14%)}, Copenhageni

{4/29(14%)}, Grippotyphosa {2/29(7%)}, and Wolffi {1/29(3%)}. Interestingly, there was no agglutinating antibody response to serovar Ballum. We observed a similar pattern in the serologic response using Leptospira isolates obtained from this study with each of the rat sera, with strong response to L. interrogans isolates but minimal reactivity to L. borgpetersenii isolates. Our findings suggest the use of multiple complementary diagnostic tests for Leptospira surveillance and diagnosis to improve the accuracy of the data.




Microbiology Resource Announcements (ASM) 2020 9, 17

Complete Genome Sequence of a Virulent Leptospira interrogans Serovar Copenha-geni Strain, Assembled with a Combination of Nanopore and Illumina Reads

Alejandro Llanes, Dhani Prakoso, Carlos Mario Restrepo, Sreeku-mari Rajeev.


Here, we present the complete genome sequence of a highly virulent Leptospira interrogans serovar Copenhageni strain isolated from a dog with severe leptospirosis. In this work, a gapless genome draft was assembled with a combination of Nanopore and Illumina data of relatively low coverage.




Pathogens 2020, 9, 277

First Whole Genome Sequence of Anaplasma platys, an Obligate Intracellular Rickettsial Pathogen of Dogs

Alejandro Llanes, Sreekumari Rajeev.


We have assembled the first genome draft of Anaplasma platys, an obligate intracellular rickettsia, and the only known bacterial pathogen infecting canine platelets. A. platys is a not-yet-cultivated bacterium that causes infectious cyclic canine thrombocytopenia, a potentially fatal disease in dogs. Despite its global distribution and veterinary relevance, no genome sequence has been published so far for this pathogen. Here, we used a strategy based on metagenome assembly to generate a draft of the A. platys genome us-

ing the blood of an infected dog. The assembled draft is similar to other Anaplasma genomes in size, gene content, and synteny. Notable differences are the apparent absence of rbfA, a gene encoding a 30S ribosome-binding factor acting as a cold-shock protein, as well as two genes involved in biotin metabolism. We also observed differences associated with expanded gene families, including those encoding outer membrane proteins, a type IV secre-tion system, ankyrin repeat-containing proteins, and proteins with predicted intrinsically disordered regions. Several of these families have members highly divergent in sequence, likely to be associated with survival and interactions within the host and the vector. The sequence of the A. platys genome can benefit future studies regarding invasion, survival, and pathogenesis of Ana-plasma species, while paving the way for the better design of treatment and prevention strategies against these neglected intracellular pathogens.




International Journal of Molecular Sciences 2020, 21, 4546

Betacoronavirus Genomes:

How Genomic Information has been Used to Deal with Past Outbreaks and the COVID-19 Pandemic.

Alejandro Llanes, Carlos M. Restrepo, Zuleima Caballero, Sreekumari Rajeev , Melissa A. Kennedy y Ricardo Lleonart.


In the 21st century, three highly pathogenic betacoronaviruses have emerged, with an alarming rate of human morbidity and case fatality. Genomic information has been widely used to under-stand the pathogenesis, animal origin and mode of transmission of betacoronaviruses in the aftermath of the 2002-03 severe acute respiratory syndrome (SARS) and 2012 Middle East respiratory syndrome (MERS) outbreaks. Furthermore, genome sequencing and bioinformatic analysis have had an unprecedented relevance in the battle against the 2019-20 coronavirus disease 2019 (CO-VID-19) pandemic, the newest and most devastating outbreak caused by a coronavirus in the history of mankind, allowing the follow up of disease spread and transmission dynamics in near real time. Here, we review how genomic information has been used to tackle outbreaks caused by emerging, highly pathogenic, betacoronavirus strains, emphasizing on SARS-CoV, MERS-CoV and SARS-CoV-2..




Acta Tropica 2019;190:166-170

Peridomestic small Indian mongoose: An invasive species posing as potential

zoonotic risk for leptospirosis in the Caribbean

Kanae Shiokawa, Alejandro Llanes, Antreas Hindoyan, Luis Cruz-Martinez, Shamara Welcome, Sreekumari Rajeeva.


In this study, we investigated Leptospira infection and exposure in small Indian mongoose (Herpestes auropunctatus), an invasive animal species, in two diffe-rent sites in the Caribbean island of Saint Kitts. Overall a low seroprevalence (12/148; 8.1%: 95%CI: 3.7–12.5) was observed. Agglutinating antibodies to serovars Mankarso (3.4%), Copenhageni (2.7%), Icterohemorrhagiae (1.4%), Bratislava (1.4%), Canicola (1.4%), Autumnalis (0.7%), Alexi (0.7%), Pomona (0.7%) and Grippotyphosa (0.7%) was observed on the microscopic agglutination test. The seroprevalence observed in mongooses trapped from peridomestic sites was significantly higher compared to the arid and less inhabited site (p=0.0268). The real time PCR targeting lipL32 gene was positive for 9 out of 146 mongooses. Bacterial culture of kidneys resulted in two Leptospira isolates. Whole genome sequencing and analysis suggested that these isolates are closely related to L. interrogans serovar Copenhageni.

We observed mild to severe chronic renal lesions in 20.2% of mongooses in the absence of an antibody response or active infection. Our findings emphasize the need to investigate other infectious etiologies or atypical outcomes and potential chronic long-term impact of Leptospira infection in animals and people living in an endemic area. In addition, our data reinforces the need for including locally prevalent Leptospira isolates rather than representative members of a serogroup in the microscopic agglutination test panel in epidemiologic and diagnostic investigations. In conclusion, mongoose inhabiting the island are exposed to and harbor pathogenic Leptospira and hence may play a role in the transmission. The invasive nature of the species highlights their presence as a potential risk factor for this widespread zoonotic disease.





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