Emerging Zoonotic Diseases among Pastoral Communities of Caia and Búzi Districts, Sofala, Mozambique: Evidence of Antibodies against Brucella, Leptospira, Rickettsia, and Crimean-Congo Hemorrhagic Fever Virus

Emerging zoonotic diseases are an increasing threat to public health. There is little data on the seroprevalence of zoonotic diseases among pastoralists in the country. We aim to carry out a cross-sectional study on the prevalence of major zoonotic diseases among pastoral communities in the Caia and Búzi districts. Methods: Between January and December 2018, a questionnaire was used to solicit socio-demographic data from consenting pastoralists with the collection of blood samples in the Caia and Búzi districts of the Sofala province. All samples were tested using ELISA commercial reagents for the detection of IgM antibodies against Brucella and Leptospira. Likewise, IgM and IgG antibodies against Rickettsia and CCHFV were determined using ELISA kits. Results: A total of 218 samples were tested, of which 43.5% (95/218) were from the district of Caia and 56.4% (123/218) from the Búzi district. Results from both districts showed that the seroprevalence of IgM antibodies against Brucella and Leptospira was 2.7% (6/218) and 30.3% (67/218), respectively. Positivity rates for IgM and IgG anti-Rickettsia and CCHFV were 8.7% (19/218), 2.7% (6/218), 4.1% (9/218), and 0.9% (2/218), respectively. Conclusions: Results from our study showed evidence of antibodies due to exposure to Brucella, Leptospira, Rickettsia, and CCHFV with antibodies against Leptospira and Rickettsia being the most prevalent. Hence, laboratory diagnosis of zoonotic diseases is essential in the early detection of outbreaks, the identification of silent transmission, and the etiology of non-febrile illness in a pastoral community. There is a need to develop public health interventions that will reduce the risk of transmission.

Transposable Prophages in Leptospira: An Ancient, Now Diverse, Group Predominant in Causative Agents of Weil's Disease.

The virome associated with the corkscrew shaped bacterium Leptospira, responsible for Weil's disease, is scarcely known, and genetic tools available for these bacteria remain limited. To reduce these two issues, potential transposable prophages were searched in Leptospiraceae genomes. The 236 predicted transposable prophages were particularly abundant in the most pathogenic leptospiral clade, being potentially involved in the acquisition of virulent traits. According to genomic similarities and phylogenies, these prophages are distantly related to known transposable phages and are organized into six groups, one of them encompassing prophages with unusual TA-TA ends. Interestingly, structural and transposition proteins reconstruct different relationships between groups, suggesting ancestral recombinations. Based on the baseplate phylogeny, two large clades emerge, with specific gene-contents and high sequence divergence reflecting their ancient origin. Despite their high divergence, the size and overall genomic organization of all prophages are very conserved, a testimony to the highly constrained nature of their genomes. Finally, similarities between these prophages and the three known non-transposable phages infecting L. biflexa, suggest gene transfer between different Caudovirales inside their leptospiral host, and the possibility to use some of the transposable prophages in that model strain.

Isolation, Purification, and Characterization of Leptophages.

To date, only three bacteriophages of leptospires-leptophages-are known. Nonetheless, numerous prophages have been found in the genus, especially in the genomes of pathogenic species. Thus, some laboratories attempt to isolate leptophage particles from environmental samples or following mitomycin C induction of bacterial cultures. Here, we propose multiple procedures to isolate, purify, and characterize bacteriophages, based on protocols used for LE3 and LE4 characterization.

Characterization of LE3 and LE4, the only lytic phages known to infect the spirochete Leptospira.

Leptospira is a phylogenetically unique group of bacteria, and includes the causative agents of leptospirosis, the most globally prevalent zoonosis. Bacteriophages in Leptospira are largely unexplored. To date, a genomic sequence is available for only one temperate leptophage called LE1. Here, we sequenced and analysed the first genomes of the lytic phages LE3 and LE4 that can infect the saprophyte Leptospira biflexa using the lipopolysaccharide O-antigen as receptor. Bioinformatics analysis showed that the 48-kb LE3 and LE4 genomes are similar and contain 62% genes whose function cannot be predicted. Mass spectrometry led to the identification of 21 and 23 phage proteins in LE3 and LE4, respectively. However we did not identify significant similarities with other phage genomes. A search for prophages close to LE4 in the Leptospira genomes allowed for the identification of a related plasmid in L. interrogans and a prophage-like region in the draft genome of a clinical isolate of L. mayottensis. Long-read whole genome sequencing of the L. mayottensis revealed that the genome contained a LE4 phage-like circular plasmid. Further isolation and genomic comparison of leptophages should reveal their role in the genetic evolution of Leptospira.

High Prevalence of Intermediate Leptospira spp. DNA in Febrile Humans from Urban and Rural Ecuador.

Leptospira spp., which comprise 3 clusters (pathogenic, saprophytic, and intermediate) that vary in pathogenicity, infect >1 million persons worldwide each year. The disease burden of the intermediate leptospires is unclear. To increase knowledge of this cluster, we used new molecular approaches to characterize Leptospira spp. in 464 samples from febrile patients in rural, semiurban, and urban communities in Ecuador; in 20 samples from nonfebrile persons in the rural community; and in 206 samples from animals in the semiurban community. We observed a higher percentage of leptospiral DNA-positive samples from febrile persons in rural (64%) versus urban (21%) and semiurban (25%) communities; no leptospires were detected in nonfebrile persons. The percentage of intermediate cluster strains in humans (96%) was higher than that of pathogenic cluster strains (4%); strains in animal samples belonged to intermediate (49%) and pathogenic (51%) clusters. Intermediate cluster strains may be causing a substantial amount of fever in coastal Ecuador.

Quantitative tracking of isotope flows in proteomes of microbial communities.

Stable isotope probing (SIP) has been used to track nutrient flows in microbial communities, but existing protein-based SIP methods capable of quantifying the degree of label incorporation into peptides and proteins have been demonstrated only by targeting usually less than 100 proteins per sample. Our method automatically (i) identifies the sequence of and (ii) quantifies the degree of heavy atom enrichment for thousands of proteins from microbial community proteome samples. These features make our method suitable for comparing isotopic differences between closely related protein sequences, and for detecting labeling patterns in low-abundance proteins or proteins derived from rare community members. The proteomic SIP method was validated using proteome samples of known stable isotope incorporation levels at 0.4%, ∼50%, and ∼98%. The method was then used to monitor incorporation of (15)N into established and regrowing microbial biofilms. The results indicate organism-specific migration patterns from established communities into regrowing communities and provide insights into metabolism during biofilm formation. The proteomic SIP method can be extended to many systems to track fluxes of (13)C or (15)N in microbial communities.

Comparação entre a imunidade induzida em bovinos vacinados com bacterinas polivalentes comerciais e uma monovalente experimental / Comparison between immunity induced in cattle vaccinated with commercial polyvalent bacterins and one experimental monovalent

O presente estudo avaliou a indução da produção de anticorpos contra Leptospira spp.por dez bacterinas, sendo nove polivalentes e uma monovalente experimental para a sorovariedade Hardjo amostra Norma. A concentração celular foi controlada e utilizou-se adjuvante de emulsão óleo em água. Um ensaio imunoenzimático (ELISA) indireto foi desenvolvido utilizando-se conjugado anti-IgG total para mensurar os níveis de anticorpos da classe IgG conferido pelas bacterinas utilizando três amostras diferentes: Hardjoprajitino, Norma e Hardjo-bovis. Paralelamente foi utilizado também o Teste de Soroaglutinação Microscópica (SAM) para mensurar os níveis de anticorpos contra as mesmas amostras. Encontraram-se títulos variáveis entre as bacterinas de acordo com o teste ELISA. Os títulos no SAM foram de pouca intensidade e de curta duração indicando a necessidade de controle celular para uma posterior padronização destes produtos. Com base nos resultados encontrados no presente estudo, a bacterina monovalente foi a que apresentou melhor desempenho.

The study evaluated the induction of antibody production against ten bacterins, nine polyvalent and one experimental monovalent to serovar Hardjo strain Norma. An indirect enzyme linked immunosorbent assay (ELISA) was developed using anti-IgG conjugate to measure total levels of IgG class antibodies conferred by bacterins using three different strains: Hardjoprajiitino, Norma and Hardjo-bovis. Microscopic Agglutination Test (MAT) was also used to measure immunoglobulin levels of the same strains. Variable ELISA titers were induced by the tested bacterins. The MAT titers found showed lower intensity and shorter duration, indicating the need to cellular control in further standardization of these vaccines. Based on results of this study, the monovalent bacterin showed best performance.

Detección de anticuerpos IgM a Leptospira en humanos en riesgo ocupacional en Villavicencio, Meta / Detection of IgM antibodies to Leptospira in human population with occupational risk factors in Villavicencio, Meta

humana y los factores de riesgo ocupacional asociados en Villavicencio, Meta. Materiales ymétodos. Se aplicó un modelo epidemiológico transversal con selección por convenienciade grupos e individuos dentro de grupos. Se obtuvieron muestras de sangre por puncióncubital de 273 personas correspondientes a 8 grupos de riesgo. Se obtuvo información defactores de riesgo mediante entrevista. Se determinaron anticuerpos IgM mediante ELISAindirecta. Se utilizó una prueba de χ2 mediante el programa SPSS 11.0 para Windows.Resultados. La seroprevalencia general fue 19%. Por grupos fue para trabajadores dematadero 7%, veterinarios y auxiliares de clínica de pequeños animales 17%, estudiantes deúltimo año de MVZ 17%, ordeñadores 21%, trabajadores de arrozales 23%, trabajadores degranjas porcícolas 35%, trabajadores de piscícolas 48%. Se encontraron 3 factores asociados,el estrato rural p = 0.0005 ICCR 1.50 a 3.83, tenencia de mascota canina p = 0.046 ICCR1.03 a 3.26 y contacto con roedores en el trabajo p = 0.000037 ICCR = 1.73 a 4.75.Conclusiones. Se encontró una alta seroprevalencia general de infectados por Leptospira,siendo los grupos de más alta prevalencia los trabajadores piscícolas y de granjas porcícolas.Se reconoce una vez más el carácter ocupacional de la infección posiblemente por falta demedidas de higiene y protección laboral.

Complete nucleotide sequence of the LE1 prophage from the spirochete Leptospira biflexa and characterization of its replication and partition functions.

The first and, to date, only extrachromosomal circular replicon identified in the spirochete Leptospira is the LE1 prophage from Leptospira biflexa. The 74-kb LE1 genome has a GC content of 36%, which is similar to the GC content of Leptospira spp. Most of the 79 predicted open reading frames (ORFs) showed no similarities to known ORFs. However 21 ORFs appeared to be organized in clusters that could code for head and tail structural proteins and immunity repressor proteins. In addition, the pattern of gene expression showed that several LE1 genes are expressed specifically either in LE1 prophage or in L. biflexa late after infection. Since the LE1 prophage replicates autonomously as a circular replicon in L. biflexa, we were able to engineer an L. biflexa-Escherichia coli shuttle vector from a 5.3-kb DNA fragment of LE1 (Saint Girons et al., J. Bacteriol. 182:5700-5705, 2000), opening this genus to genetic manipulation. In this study, base compositional asymmetry confirms the location of the LE1 replication region and suggests that LE1 replicates via a bidirectional Theta-like replication mechanism from this unique origin. By subcloning experiments, the replication region can be narrowed down to a 1-kb region. This minimal replication region consists of a rep encoding a protein of 180 amino acids. Upstream from rep, putative partitioning genes, called parA and parB, were found to be similar to the par loci in Borrelia plasmids. A significant increase of plasmid stability in L. biflexa can be seen only when both parA and parB are present. These results enable the construction of new shuttle vectors for studying the genetics of Leptospira spp. This study will also contribute to a better knowledge of phages unrelated to lambdoid phages.

First evidence for a restriction-modification system in Leptospira sp.

The LE1 leptophage exhibited a host range restricted to the saprophytic Leptospira biflexa [Saint Girons et al., Res. Microbiol. 141 (1990) 1131-1133] and mainly to the Patoc 1 strain (hereafter called PFRA) kept in the Paris, France collection. Results of titration of LE1 lysates indicated the presence of a host-controlled modification and restriction system within PUSA (Patoc 1 strain maintained in the Morgantown, WV, USA collection) that was absent in PFRA. Because genomic DNA of PITAL (Patoc 1 strain maintained in Trieste, Italy) appeared smeared in pulsed field gel electrophoresis (PFGE), this strain is likely to contain nucleases that are activated upon DNA isolation. Moreover, comparative NotI digestions of PUSA and PFRA DNAs, as visualized by PFGE, indicated that PUSA belonged to a different serovar than PFRA. Finally, 16S ribosomal sequence analysis indicated that PUSA belonged to the saprophytic Leptospira meyeri species, while PITAL and PFRA appertained to L. biflexa. The evolutionary significance and the importance of the restriction and modification enzymes or non-specific nucleases within strains for genetic experiments are discussed.

The LE1 bacteriophage replicates as a plasmid within Leptospira biflexa: construction of an L. biflexa-Escherichia coli shuttle vector.

We have discovered that LE1, one of the plaque-forming phages previously described as lytic for the Leptospira biflexa saprophytic spirochete (I. Saint Girons, D. Margarita, P. Amouriaux, and G. Baranton, Res. Microbiol. 141:1131-1138, 1990), was indeed temperate. LE1 was found to be unusual, as Southern blot analysis indicated that it is one of the few phages to replicate in the prophage state as a circular plasmid. The unavailability of such small endogenous replicons has hindered genetic experimentation in Leptospira. We have developed a shuttle vector with DNA derived from LE1. Random LE1 DNA fragments were cloned into a pGEM 7Zf(+) derivative devoid of most of the bla gene but carrying a kanamycin resistance marker from the gram-positive bacterium Enterococcus (Streptococcus) faecalis. These constructs were transformed into L. biflexa strain Patoc 1 by electroporation, giving rise to kanamycin-resistant transformants. A 2.2-kb fragment from LE1 was responsible for replication of the vector in L. biflexa. However, a larger region including an intact parA gene homologue was necessary for the stability of the shuttle vector. Direct repeats and AT-rich regions characterized the LE1 origin of replication. Our data indicate that the replicon derived from the LE1 leptophage, together with the kanamycin resistance gene, is a promising tool with which to develop the genetics of Leptospira species.