Cuban Strains Research Articles

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.

Molecular evolution of coxsackievirus A24v in Cuba over 23-years, 1986\u20132009

Coxsackievirus A24 variant (CVA24v) is a major causative agent of acute hemorrhagic conjunctivitis outbreaks worldwide, yet the evolutionary and transmission dynamics of the virus remain unclear. To address this, we analyzed and compared the 3C and partial VP1 gene regions of CVA24v isolates obtained from five outbreaks in Cuba between 1986 and 2009 and strains isolated worldwide. Here we show that Cuban strains were homologous to those isolated in Africa, the Americas and Asia during the same time period. Two genotypes of CVA24v (GIII and GIV) were repeatedly introduced into Cuba and they arose about two years before the epidemic was detected. The two genotypes co-evolved with a population size that is stable over time. However, nucleotide substitution rates peaked during pandemics with 4.39 × 10−3 and 5.80 × 10−3 substitutions per site per year for the 3C and VP1 region, respectively. The phylogeographic analysis identified 25 and 19 viral transmission routes based on 3C and VP1 regions, respectively. Pandemic viruses usually originated in Asia, and both China and Brazil were the major hub for the global dispersal of the virus. Together, these data provide novel insight into the epidemiological dynamics of this virus and possibly other pandemic viruses.

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 5ʹUTR 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.

Genetic characterization of bovine coronavirus in Vietnam.

A maximum clade credibility tree constructed using the full-length spike (S) and hemagglutinin-esterase genes revealed that Vietnamese Bovine coronavirus (BCoV) strains belong to a single cluster (C1); therefore, they might share a common origin with Cuban and Chinese BCoV strains. The omega values of cluster 1 (C1) and cluster 2 (C2) were 0.15734 and 0.11613, respectively, and naive empirical bayes analysis identified two amino acid positions (179 and 501) in the S protein in C1 and three amino acid positions (113, 501, and 525) in that of C2 that underwent positive selection (p > 99%). The evolutionary rate of C1 was estimated to be 7.6206 × 10−4 substitutions/site/year, and the most recent common ancestor (tMRCA) of Vietnamese BCoVs was estimated to date back to 1962 (95% HPD 1950–1973). The effective population sizes of C1 and C2 underwent a rapid reduction after 2000 and 2004, respectively.Electronic supplementary materialThe online version of this article (10.1007/s11262-019-01647-1) contains supplementary material, which is available to authorized users.

Survival of Neisseria meningitidis outside of the host: environmental effects and differences among strains.

Neisseria meningitidis is a gram-negative bacterium that lives as a commensal in the human nasopharynx. Meningococci are generally non-invasive, but can invade the nasopharyngeal epithelia and enter the bloodstream causing life-threatening illnesses. It is generally thought that meningococci do not survive for long outside the host, and that transmission requires relatively close contact between hosts. There are some reports, however, that meningococci can survive drying on surfaces, including glass, plastic and cloth. Our examination of N. meningitidis strains dried on glass showed differences in survival of isolates belonging to serogroups B, C and W135, including persistence of Cuban, New Zealand, and Norwegian epidemic strains up to 8 days, depending on temperature and humidity. Survival of a New Zealand epidemic strain isolate NZ98/254 under ambient conditions in the laboratory was greatest in winter suggesting that environmental factors impacted survival. For most isolates, including NZ98/254, survival under controlled conditions at 30 °C was greater at 22% than 30% relative humidity. There were also some differences in survival between carriage and invasive strains. The results suggest that N. meningitidis could be transmitted through contact with surfaces outside the host, potentially including contact through shared drinking vessels.

The Synonymy of the Toxic Dinoflagellates Prorocentrum mexicanum and P.rhathymum and the Description of P.steidingerae sp. nov. (Prorocentrales, Dinophyceae).

Prorocentrum mexicanum and P.rhathymum are toxicologically important dinoflagellates, but their relationship is not well defined. We investigated strains from Puerto Rico and Brazil by light and scanning electron microscopies. We provide molecular data from a strain isolated near the type locality of P.rhathymum, and the first morphological and molecular data from the South Atlantic Ocean. The rRNA gene (rDNA) sequences of the Puerto Rican and Brazilian strains were identical, and their morphologies fit the description of P.rhathymum. In the molecular phylogenies, the globally distributed populations under the names P.mexicanum and P.rhathymum are intermixed and branched together, except for several divergent strains from Florida and Cuba. We examined the original descriptions and iconotypes of the species Prorocentrum maximum, P.brochii, P.mexicanum, and P.rhathymum. We conclude that P.maximum sensu Schiller's figure 41a corresponds to the earlier description of this species; the split of P.mexicanum and P.rhathymum was based on a misidentification because P.mexicanum sensu Cortés-Altamirano & Sierra-Beltrán corresponds to P.texanum var. cuspidatum; and P.rhathymum is a junior synonym of P.mexicanum. Several Floridian and Cuban strains correspond to a new species, which we describe as Prorocentrum steidingerae sp. nov.

Antimicrobial Susceptibility Patterns of Recent Cuban Mycoplasma genitalium Isolates Determined by a Modified Cell-Culture-Based Method.

Isolation of Mycoplasma genitalium from clinical specimens remains difficult and few strains are available for antimicrobial susceptibility testing. We describe the antimicrobial susceptibility of M. genitalium strains grown in Vero cell culture with first- and second- line antibiotics, using a modified cell-culture-based method. Macrolide- and -fluoroquinolone resistance determinants were detected by sequencing of the 23S and parC genes, respectively. Seven strains were examined, including three new, genetically distinct M. genitalium strains isolated from endocervical and urethral swab specimens from Cuban patients together with four reference strains isolated from specimens collected from men in Denmark, Sweden and Australia. Azithromycin was the most active drug against two of the Cuban M. genitalium strains with MICs values of 0.008 mg/liter, however, one strain was macrolide resistant with an MIC of >8 mg/liter, and the A2059G resistant genotype. Ciprofloxacin was the least active antimicrobial drug and moxifloxacin was the most active fluoroquinolone against the new clinical strains, although an MIC of 1 mg/l was found for two strains. However, no relevant parC mutations were detected. MICs for tetracyclines were 0.5–4 mg/liter. Although the number of Cuban strains was low, the results suggest that a single-dose azithromycin treatment could be ineffective, and that a second-line treatment with moxifloxacin, should become an option in Cuba. To our knowledge, this is the first report of isolation and antibiotic susceptibility testing of M. genitalium strains from the Latin-American region, and the first detection of macrolide resistance in such strains.

New genetic variants of influenza A(H1N1)pdm09 detected in Cuba during 2011–2013

Influenza A(H1N1)pdm09 virus has evolved continually since its emergence in 2009. For influenza virus strains, genetic changes occurring in HA1 domain of the hemagglutinin cause the emergence of new variants. The aim of our study is to establish genetic associations between 35 A(H1N1)pdm09 viruses circulating in Cuba in 2011–2012 and 2012–2013 seasons, and A/California/07/2009 strain recommended by WHO as the H1N1 component of the influenza vaccine. The phylogenetic analysis revealed the circulation of clades 3, 6A, 6B, 6C and 7. Mutations were detected in the antigenic site or in the receptor-binding domains of HA1 segment, including S174P, S179N, K180Q, S202T, S220T and R222K. Substitutions S174P, S179N, K180Q and R222K were detected in Cuban strains for the first time.

First dengue haemorrhagic fever epidemic in the Americas, 1981: insights into the causative agent.

Historical records describe a disease in North America that clinically resembled dengue haemorrhagic fever during the latter part of the slave-trading period. However, the dengue epidemic that occurred in Cuba in 1981 was the first laboratory-confirmed and clinically diagnosed outbreak of dengue haemorrhagic fever in the Americas. At that time, the presumed source of the dengue type 2 strain isolated during this epidemic was considered controversial, partly because of the limited sequence data and partly because the origin of the virus appeared to be southern Asia. Here, we present a molecular characterisation at the whole-genome level of the original strains isolated at different time points during the epidemic. Phylogenetic trees constructed using Bayesian methods indicated that 1981 Cuban strains group within the Asian 2 genotype. In addition, the study revealed that viral evolution occurred during the epidemic - a fact that could be related to the increasing severity from month to month. Moreover, the Cuban strains exhibited particular amino acid substitutions that differentiate them from the New Guinea C prototype strain as well as from dengue type 2 strains isolated globally.

Molecular and phylogenetic analysis of influenza A H1N1 pandemic viruses in Cuba, May 2009 to August 2010

The influenza A(H1N1)pdm09 virus was detected in Cuba in May 2009. The introduction of a new virus with increased transmissibility into a population makes surveillance of the pandemic strain to the molecular level necessary. The aim of the present study was the molecular and phylogenetic analysis of pandemic influenza A(H1N1)pdm09 strains that circulated in Cuba between May 2009 and August 2010. Seventy clinical samples were included in the study. Nucleotide sequences from the hemagglutinin HA1 region segment were obtained directly from clinical samples. Genetic distances were calculated using MEGA v.5.05. A phylogenetic tree was constructed using MrBayes v.3.1.2 software. Potential N-glycosylation sites were predicted using NetNGlyc server 1.0. The 48 Cuban sequences of influenza A(H1N1)pdm09 obtained were similar to the A/California/07/2009 (H1N1) vaccine strain. Most of the Cuban strains belonged to clade 7. Cuban viruses showed amino acid changes, some of them located at three antigenic sites: Ca, Sa, and Sb. Two dominant mutations were detected: P83S (100%) and S203T (85.7%). Glycosylation site analysis revealed the gain of one site at position 162 in 13 sequences. The findings in this study contribute to our understanding of the progress of the influenza A(H1N1)pdm09 virus, since this virus is at the starting point of its evolution in humans.

Molecular and phylogenetic analysis of bovine coronavirus based on the spike glycoprotein gene

Bovine coronavirus has been associated with diarrhoea in newborn calves, winter dysentery in adult cattle and respiratory tract infections in calves and feedlot cattle. In Cuba, the presence of BCoV was first reported in 2006. Since then, sporadic outbreaks have continued to occur. This study was aimed at deepening the knowledge of the evolution, molecular markers of virulence and epidemiology of BCoV in Cuba. A total of 30 samples collected between 2009 and 2011 were used for PCR amplification and direct sequencing of partial or full S gene. Sequence comparison and phylogenetic studies were conducted using partial or complete S gene sequences as phylogenetic markers. All Cuban bovine coronavirus sequences were located in a single cluster supported by 100% bootstrap and 1.00 posterior probability values. The Cuban bovine coronavirus sequences were also clustered with the USA BCoV strains corresponding to the GenBank accession numbers EF424621 and EF424623, suggesting a common origin for these viruses. This phylogenetic cluster was also the only group of sequences in which no recombination events were detected. Of the 45 amino acid changes found in the Cuban strains, four were unique.

Molecular Epidemiologic Analysis ofEnterococcus faecalisIsolates in Cuba by Multilocus Sequence Typing

We carried out the first study of Enterococcus faecalis clinical isolates in Cuba by multilocus sequence typing linking the molecular typing data with the presence of virulence determinants and the antibiotic resistance genes. A total of 23 E. faecalis isolates recovered from several clinic sources and geographic areas of Cuba during a period between 2000 and 2005 were typed by multilocus sequence typing. Thirteen sequence types (STs) including five novel STs were identified, and the ST 64 (clonal complex [CC] 8), ST 6 (CC2), ST 21(CC21), and ST 16 (CC58) were found in more than one strain. Sixty-seven percent of STs corresponded to STs reported previously in Spain, Poland, and The Netherlands, and other STs (ST115, ST64, ST6, and ST40) were genetically close to those detected in the United States. Prevalence of both antimicrobial resistance genes [aac(6')-aph(2''), aph(3'), ant(6), ant(3'')(9), aph(2'')-Id, aph(2'')-Ic, erm(B), erm(A), erm(C), mef(A), tet(M), and tet(L)] and virulence genes (agg, gelE, cylA, esp, ccf, and efaAfs) were examined by polymerase chain reaction. Aminoglycoside resistance genes aac(6')-Ie-aph(2'')-Ia, aph(3'), ant(6), ant(3'')(9) were more frequently detected in ST6, ST16, ST23, ST64, and ST115. The multidrug resistance was distributed to all STs detected, except for ST117 and singleton ST225. The presence of cyl gene was specifically linked to the ST64 and ST16. Presence of the esp, gel, and agg genes was not specific to any particular ST. This research provided the first insight into the population structure of E. faecalis in Cuba, that is, most Cuban strains were related to European strains, whereas others to U.S. strains. The CC2, CC21, and CC8, three of the biggest CCs in the world, were evidently circulating in Cuba, associated with multidrug resistance and virulence traits.

Prevalence of vacA, cagA and babA2 genes in Cuban Helicobacter pylori isolates

To investigate the prevalence of vacuolating cytotoxin (vacA), cytotoxin associated gene A (cagA) and blood adhesion binding antigen (babA2) genotypes of Helicobacter pylori (H pylori) isolates from Cuban dyspeptic patients. DNA was extracted from H pylori-positive cultures taken from 130 dyspeptic patients. Genotyping was performed by PCR, using specific primers for vacA (s1, s2, m1, m2), cagA and babA2 genes. Endoscopic observations and histological examinations were used to determine patient pathologies. vacA alleles s1, s2, m1 and m2 were detected in 96 (73.8%), 34 (26.2%), 75 (57.7%) and 52 isolates (40%), respectively, while the cagA gene was detected in 95 isolates (73.2%). One hundred and seven isolates (82.3%) were babA2-positive. A significant correlation was observed between vacAs1m1 and cagA and between vacAs1m1 and babA2 genotypes (P < 0.001 and P < 0.05, respectively) and between babA2 genotype and cagA status (P < 0.05); but, no correlation was observed between vacAs1 and babA2 genotypes. Eighty five (65.4%) and 73 (56.2%) strains were type 1 (vacAs1-cagA-positive) and "triple-positive" (vacAs1-cagA-babA2-positive), respectively, and their presence was significantly associated with duodenal ulcer (P < 0.01 and P < 0.001, respectively). The distribution of the main virulence factors in the Cuban strains in this study resembled that of the Western-type strains, and the more virulent H pylori isolates were significantly associated with duodenal ulcer, ulcer disease being the worst pathology observed in the group studied.

Metschnikowia cubensis sp. nov., a yeast species isolated from flowers in Cuba

A novel yeast species is described from 19 strains isolated from flowers and insects in three provinces of Cuba. The species is so far known only from Cuba. Characteristic asci and ascospores as well as phylogenetic analysis of the rDNA sequence place the novel species in the genus Metschnikowia. The novel species belongs to the New World subclade of large-spored species of Metschnikowia. Mating tests with other members of the subclade resulted in the formation of sterile asci without ascospores, showing that the Cuban strains represent a distinct biological species. Intraspecies matings lead to the production of fertile asci containing large needle-shaped ascospores. The novel species was further distinguished from its close relatives by rDNA sequences and PCR fingerprinting using primers derived from mini- and microsatellites. We propose the name Metschnikowia cubensis sp. nov. and designate MUCL 45753(T) (=CRGF 279(T) =CBS 10832(T), h(+)) as the type strain and MUCL 45751 (=CRGF 278 =CBS 10833, h(-)) as the allotype.

The number and organization of the rRNA genes of several strains of Mycobacterium simiae

The type strain of Mycobacterium simiae and four Cuban strains, each representing a group of variants sharing a characteristic pattern of glycopeptidolipids, were investigated. Each of the five strains was found to have a single rRNA ( rrn) operon per genome. Each rrn operon was found to be located downstream from murA. Unusually for slow-growing mycobacteria, three transcription start points were identified for each operon. Gene sequences were established extending from near to the 3′-ends of murA, the intergenic regions and the 5′-ends of the 16S rDNAs. Characteristic strain differences were identified.

Detection and genetic relationship of dengue virus sequences in seventeen-year-old paraffin-embedded samples from Cuba.

This study describes the use of the reverse transcriptase-polymerase chain reaction (RT-PCR) to generate dengue 2 amplicons from paraffin-embedded autopsy tissues collected in Cuba 17 years ago. The presumptive diagnoses had been made only by clinical evolution without serologic confirmation. This study confirms once again that dengue 2 virus was directly associated with the fatal cases in children and illustrates the potential of the RT-PCR for retrospective diagnosis of dengue cases 17 years after death. A close similarity in the genomic sequences of the dengue 2 RNA detected in tissue samples from fatal cases and those dengue 2 Cuban strains that had been previously investigated confirms the appropriate genomic classification of the etiologic agent associated with the 1981 dengue hemorrhagic fever Cuban epidemic.

Broadly reactive antibodies against a gp120 V3 loop multi-epitope polypeptide neutralize different isolates of human immunodeficiency virus type 1 (HIV-1)

A gene encoding for a novel multi-epitope polypeptide (TAB4) was synthesized and expressed in Escherichia coli. The protein was composed of 15 amino acid fragments derived from the V3 loop of HIV-1 isolates MN, IIIB, RF, JY1, BRVA and LR150, joined by five-amino-acid linkers. Immunogenicity of TAB4 in rabbits was studied, and the antibody response against individual peptides investigated. TAB4 was shown to be immunogenic in Complete Freund's Adjuvant in a dose-dependent manner, and was able to elicit a humoral response against all V3 epitopes included on the protein. Sera from some of the animals were able to neutralize the replication of viral strain MN, and in one case IIIB, with moderate titers. Some sera also neutralized several Cuban clinical strains, isolated in peripheral blood mononuclear cells, after one round of amplification in MT4 cells.

Partial Nucleotide and Amino Acid Sequences of the Envelope and the Envelope/Nonstructural Protein-1 Gene Junction of Four Dengue-2 Virus Strains Isolated during the 1981 Cuban Epidemic

In 1981, an epidemic of dengue hemorrhagic fever (DHF) caused by dengue-2 virus occurred in Cuba. This was the first DHF epidemic reported in the Western Hemisphere. In this study, we have analyzed four dengue-2 Cuban strains for two short genomic fragments: one on the envelope (E) glycoprotein and one at the E/nonstructural protein-1 (NS1) gene junction. The E segment of these 1981 Cuban isolates were more closely related to older dengue-2 virus strains such as New Guinea C 1944, Thailand 1964, Sri Lanka 1968, and Burma 1976 than to more recent isolates of this virus from Jamaica and Vietnam. More than 9% of the divergence with strains isolated from Jamaica and Vietnam was observed at the E/NS1 gene junction. One nucleotide change was observed between the first strain isolated during the epidemic and the rest of the Cuban strains. This mutation induced a nonconserved amino acid change from phenylalanine to leucine at position 43 that was not observed in any of the other strains with which it was compared.

Serotype-specific outbreak of group B meningococcal disease in Iquique, Chile

From 1979 to August 1987, there have been 178 cases of meningococcal disease in Iquique, Chile, a city of about 140,000. The attack rate for the last 5 years has been in excess of 20/100,000 per year, more than 20 times greater than for the country overall. The mortality rate was 6%. The disease occurred in patients with ages from 4 months to 60 years, but 89% of cases were in patients less than 21 years. The largest number of cases were in the age group 5-9 years (n = 54), but the highest incidence occurred in children less than 1 year of age (72.8/100,000 per year). The male/female ratio was 1.2. Cases occurred all year round with little seasonal variation. Of the 178 cases, 173 were biologically confirmed. Serogroup analysis of strains from 135 patients revealed A = 1, B = 124, C = 10. Forty-four group B strains from 1985-7 were serotyped: 15:P1.3 = 36, 15:NT = 4, 4:P1.3 = 2, NT:NT = 2. Ten of 11 of the outbreak strains tested were sulfadiazine-resistant. This is the first recognized outbreak caused by a Gp B:15 strain in South America. It shares many of the characteristics of outbreaks caused by closely related strains in Europe, such as a predilection for older children and adolescents, sulfadiazine-resistance, and sustained high attack rates. The Iquique strain (B:15:P1.3) belongs to the same genetic clone (ET-5 complex) as the Norway (B:15:P1.16) and the Cuban (B:4:P1.15) strains.