Comparison of whole genome sequencing to restriction endonuclease analysis and gel diffusion precipitin test of Pasteurella multocida

The gel diffusion precipitin test (GDPT) and restriction endonuclease analysis (REA) have commonly been used in serotyping and further identifying isolates of Pasteurella multocida. GDPT has shown problems with repeatability and cross reactions. The hindrance with REA has been the subjectivity of the test. Whole genome sequencing (WGS) has become a new option in diagnostic testing due to reduced cost and quicker turnaround time. WGS has shown with other organisms to be a useful diagnostic tool and this study examined its use on P. multocida. This study compared WGS to REA and GDPT on 166 isolates of P. multocida to determine if WGS produced similar results and could be used in place of REA and/or GDPT. The isolates used represented the sixteen reference serotypes (1-16), isolates with REA profiles matching the fowl cholera vaccine strain, and ten different animal species. Isolates originated from across the United States and from Chile. This study found that identical REA profiles clustered together in the phylogenetic tree. It was also discovered that REA profiles that differed by only a few bands also appeared closely related on the tree. The GDPT results were more diverse but it was common to see a single serotype show up repeatedly within clusters. Several errors were also found when examining the REA profiles, such as two assigned numbers for one profile or profiles misidentified. WGS was able to confirm these errors and compensate for the subjectivity in analysis of REA. It also provided epidemiological information that is not available with GDPT. From the data produced in this study it appears that WGS could be used in place or in conjunction with REA and GDPT

Isolation and characterization of saprophytic and pathogenic strains of Leptospira from water sources in the Midwestern United States

The genus Leptospira is a diverse and unique group of bacteria comprising multiple saprophytic and pathogenic species, which survive and persist in suitable moist environments. Pathogenic species cause human and animal leptospirosis, a global and neglected zoonotic disease. Disease transmission occurs by exposure to contaminated water and moist soil environments or by contact with domestic animals and wildlife acting as reservoir hosts that shed Leptospira via urine. Here, we describe the unexpected diversity of saprophytic and pathogenic species of Leptospira isolated from water in the Midwestern United States. Samples were collected by volunteers in 11 counties in Iowa from water sources, including puddles, sewage, creeks, ponds, lakes, and rivers, during the summer of 2021. One hundred and five water samples were tested by culture for the presence of saprophytic and pathogenic species and by lipL32 qPCR specific for the detection of pathogens; 82 (78.1%) were culture positive and five (4.8%) were positive by lipL32 qPCR. Whole genome sequencing of isolates cultured from water samples identified 10 species of saprophytes, namely L. montravelensis, L. kemamanensis, L. bandrabouensis, L. bourretii, L. bouyouniensis, L. chreensis, L. ellinghausenii, L. terpstrae, L. yanagawae, and L. abararensis, as well as three novel saprophytic species. Whole genome sequencing also identified two novel pathogenic species. The remaining cultures comprised mixed populations of saprophytic species and six comprised a mixture of saprophytic and pathogenic species. One of these mixed cultures was enriched to select for a clonal isolate of pathogenic Leptospira, strain WS101.C1, which was classified as L. interrogans serogroup Djasiman serovar Djasiman. Cumulatively, 9.5% (10/105) of water samples were positive for pathogenic Leptospira. This study emphasizes the diversity of Leptospira present in water sources in the Midwestern United States and provides unique opportunities to explore the geographic diversity and evolution of this genus. The identification of known and novel pathogenic species circulating in local water sources highlights their potential usefulness as diagnostic antigens, as well as the role of water in the transmission of infection to human and animal populations. Integrating knowledge on human, animal, and environmental health is essential to control and predict risk for zoonoses

Mongooses (\u3ci\u3eUrva auropunctata\u3c/i\u3e) as reservoir hosts of leptospira species in the United States Virgin Islands, 2019–2020

During 2019–2020, the Virgin Islands Department of Health investigated potential animal reservoirs of Leptospira spp., the bacteria that cause leptospirosis. In this cross-sectional study, we investigated Leptospira spp. exposure and carriage in the small Indian mongoose (Urva auropunctata, syn: Herpestes auropunctatus), an invasive animal species. This study was conducted across the three main islands of the U.S. Virgin Islands (USVI), which are St. Croix, St. Thomas, and St. John. We used the microscopic agglutination test (MAT), fluorescent antibody test (FAT), real-time polymerase chain reaction (lipl32 rt-PCR), and bacterial culture to evaluate serum and kidney specimens and compared the sensitivity, specificity, positive predictive value, and negative predictive value of these laboratory meth-ods. Mongooses (n = 274) were live-trapped at 31 field sites in ten regions across USVI and humanely euthanized for Leptospira spp. testing. Bacterial isolates were sequenced and evaluated for species and phylogenetic analysis using the ppk gene. Anti-Leptospira spp. antibodies were detected in 34% (87/256) of mongooses. Reactions were observed with the following serogroups: Sejroe, Icterohaemorrhagiae, Pyrogenes, Mini, Cynopteri, Australis, Hebdomadis, Autumnalis, Mankarso, Pomona, and Ballum. Of the kidney specimens exam-ined, 5.8% (16/270) were FAT-positive, 10% (27/274) were culture-positive, and 12.4% (34/ 274) were positive by rt-PCR. Of the Leptospira spp. isolated from mongooses, 25 were L. borgpetersenii, one was L. interrogans, and one was L. kirschneri. Positive predictive values of FAT and rt-PCR testing for predicting successful isolation of Leptospira by culture were 88% and 65%, respectively. The isolation and identification of Leptospira spp. in mongooses highlights the potential role of mongooses as a wildlife reservoir of leptospirosis; mongooses could be a source of Leptospira spp. infections for other wildlife, domestic animals, and humans

Comparison of whole genome sequencing to restriction endonuclease analysis and gel diffusion precipitin test of Pasteurella multocida

The gel diffusion precipitin test (GDPT) and restriction endonuclease analysis (REA) have commonly been used in serotyping and further identifying isolates of Pasteurella multocida. GDPT has shown problems with repeatability and cross reactions. The hindrance with REA has been the subjectivity of the test. Whole genome sequencing (WGS) has become a new option in diagnostic testing due to reduced cost and quicker turnaround time. WGS has shown with other organisms to be a useful diagnostic tool and this study examined its use on P. multocida. This study compared WGS to REA and GDPT on 166 isolates of P. multocida to determine if WGS produced similar results and could be used in place of REA and/or GDPT. The isolates used represented the sixteen reference serotypes (1-16), isolates with REA profiles matching the fowl cholera vaccine strain, and ten different animal species. Isolates originated from across the United States and from Chile. This study found that identical REA profiles clustered together in the phylogenetic tree. It was also discovered that REA profiles that differed by only a few bands also appeared closely related on the tree. The GDPT results were more diverse but it was common to see a single serotype show up repeatedly within clusters. Several errors were also found when examining the REA profiles, such as two assigned numbers for one profile or profiles misidentified. WGS was able to confirm these errors and compensate for the subjectivity in analysis of REA. It also provided epidemiological information that is not available with GDPT. From the data produced in this study it appears that WGS could be used in place or in conjunction with REA and GDPT.</p

Bovine Leptospirosis Due to Persistent Renal Carriage of Leptospira borgpetersenii Serovar Tarassovi

Leptospirosis is a global zoonotic disease that causes significant morbidity and mortality in human and animal populations. Leptospira interrogans is a leading cause of human disease, and L. borgpetersenii is a leading cause of animal disease. Cattle are reservoir hosts of L. borgpetersenii serovar Hardjo, which is transmitted via urine, semen, and uterine discharges resulting in abortion and poor reproductive performance. Bovine bacterin vaccines can only protect against those serovars included in vaccine formulations and typically include serovar Hardjo among others. Genotyping and serotyping represent two different and unique methods for classifying leptospires that do not always correlate well; comprehensive characterization using either method requires recovery of isolates from infected animals. In this study, we report for the first time, isolation of L. borgpetersenii serovar Tarassovi from the urine of a dairy cow in the U.S. The classification of the isolate, designated strain MN900, was confirmed by whole-genome sequencing, serotyping with reference antisera and monoclonal antibodies, Matrix Assisted Laser Desorption/Ionization (MALDI), and immunoblotting with reference antisera. Strain MN900 was excreted in urine samples for 18 weeks even as the cow was seronegative for serovar Tarassovi. Strain MN900 has an unusual morphology since it is not as motile as other leptospires and lacks hooked ends. Serovar Tarassovi is not included in U.S. bacterin vaccines. These results demonstrate the importance of culture and concomitant genotyping and serotyping to accurately classify leptospires, and as required to design efficacious vaccine and diagnostic strategies to not only limit animal disease but reduce zoonotic risk

Data_Sheet_1_Isolation and characterization of saprophytic and pathogenic strains of Leptospira from water sources in the Midwestern United States.PDF

The genus Leptospira is a diverse and unique group of bacteria comprising multiple saprophytic and pathogenic species, which survive and persist in suitable moist environments. Pathogenic species cause human and animal leptospirosis, a global and neglected zoonotic disease. Disease transmission occurs by exposure to contaminated water and moist soil environments or by contact with domestic animals and wildlife acting as reservoir hosts that shed Leptospira via urine. Here, we describe the unexpected diversity of saprophytic and pathogenic species of Leptospira isolated from water in the Midwestern United States. Samples were collected by volunteers in 11 counties in Iowa from water sources, including puddles, sewage, creeks, ponds, lakes, and rivers, during the summer of 2021. One hundred and five water samples were tested by culture for the presence of saprophytic and pathogenic species and by lipL32 qPCR specific for the detection of pathogens; 82 (78.1%) were culture positive and five (4.8%) were positive by lipL32 qPCR. Whole genome sequencing of isolates cultured from water samples identified 10 species of saprophytes, namely L. montravelensis, L. kemamanensis, L. bandrabouensis, L. bourretii, L. bouyouniensis, L. chreensis, L. ellinghausenii, L. terpstrae, L. yanagawae, and L. abararensis, as well as three novel saprophytic species. Whole genome sequencing also identified two novel pathogenic species. The remaining cultures comprised mixed populations of saprophytic species and six comprised a mixture of saprophytic and pathogenic species. One of these mixed cultures was enriched to select for a clonal isolate of pathogenic Leptospira, strain WS101.C1, which was classified as L. interrogans serogroup Djasiman serovar Djasiman. Cumulatively, 9.5% (10/105) of water samples were positive for pathogenic Leptospira. This study emphasizes the diversity of Leptospira present in water sources in the Midwestern United States and provides unique opportunities to explore the geographic diversity and evolution of this genus. The identification of known and novel pathogenic species circulating in local water sources highlights their potential usefulness as diagnostic antigens, as well as the role of water in the transmission of infection to human and animal populations. Integrating knowledge on human, animal, and environmental health is essential to control and predict risk for zoonoses.</p

Table_1_Isolation and characterization of saprophytic and pathogenic strains of Leptospira from water sources in the Midwestern United States.XLSX

The genus Leptospira is a diverse and unique group of bacteria comprising multiple saprophytic and pathogenic species, which survive and persist in suitable moist environments. Pathogenic species cause human and animal leptospirosis, a global and neglected zoonotic disease. Disease transmission occurs by exposure to contaminated water and moist soil environments or by contact with domestic animals and wildlife acting as reservoir hosts that shed Leptospira via urine. Here, we describe the unexpected diversity of saprophytic and pathogenic species of Leptospira isolated from water in the Midwestern United States. Samples were collected by volunteers in 11 counties in Iowa from water sources, including puddles, sewage, creeks, ponds, lakes, and rivers, during the summer of 2021. One hundred and five water samples were tested by culture for the presence of saprophytic and pathogenic species and by lipL32 qPCR specific for the detection of pathogens; 82 (78.1%) were culture positive and five (4.8%) were positive by lipL32 qPCR. Whole genome sequencing of isolates cultured from water samples identified 10 species of saprophytes, namely L. montravelensis, L. kemamanensis, L. bandrabouensis, L. bourretii, L. bouyouniensis, L. chreensis, L. ellinghausenii, L. terpstrae, L. yanagawae, and L. abararensis, as well as three novel saprophytic species. Whole genome sequencing also identified two novel pathogenic species. The remaining cultures comprised mixed populations of saprophytic species and six comprised a mixture of saprophytic and pathogenic species. One of these mixed cultures was enriched to select for a clonal isolate of pathogenic Leptospira, strain WS101.C1, which was classified as L. interrogans serogroup Djasiman serovar Djasiman. Cumulatively, 9.5% (10/105) of water samples were positive for pathogenic Leptospira. This study emphasizes the diversity of Leptospira present in water sources in the Midwestern United States and provides unique opportunities to explore the geographic diversity and evolution of this genus. The identification of known and novel pathogenic species circulating in local water sources highlights their potential usefulness as diagnostic antigens, as well as the role of water in the transmission of infection to human and animal populations. Integrating knowledge on human, animal, and environmental health is essential to control and predict risk for zoonoses.</p

Image_3_Isolation and characterization of saprophytic and pathogenic strains of Leptospira from water sources in the Midwestern United States.TIF

The genus Leptospira is a diverse and unique group of bacteria comprising multiple saprophytic and pathogenic species, which survive and persist in suitable moist environments. Pathogenic species cause human and animal leptospirosis, a global and neglected zoonotic disease. Disease transmission occurs by exposure to contaminated water and moist soil environments or by contact with domestic animals and wildlife acting as reservoir hosts that shed Leptospira via urine. Here, we describe the unexpected diversity of saprophytic and pathogenic species of Leptospira isolated from water in the Midwestern United States. Samples were collected by volunteers in 11 counties in Iowa from water sources, including puddles, sewage, creeks, ponds, lakes, and rivers, during the summer of 2021. One hundred and five water samples were tested by culture for the presence of saprophytic and pathogenic species and by lipL32 qPCR specific for the detection of pathogens; 82 (78.1%) were culture positive and five (4.8%) were positive by lipL32 qPCR. Whole genome sequencing of isolates cultured from water samples identified 10 species of saprophytes, namely L. montravelensis, L. kemamanensis, L. bandrabouensis, L. bourretii, L. bouyouniensis, L. chreensis, L. ellinghausenii, L. terpstrae, L. yanagawae, and L. abararensis, as well as three novel saprophytic species. Whole genome sequencing also identified two novel pathogenic species. The remaining cultures comprised mixed populations of saprophytic species and six comprised a mixture of saprophytic and pathogenic species. One of these mixed cultures was enriched to select for a clonal isolate of pathogenic Leptospira, strain WS101.C1, which was classified as L. interrogans serogroup Djasiman serovar Djasiman. Cumulatively, 9.5% (10/105) of water samples were positive for pathogenic Leptospira. This study emphasizes the diversity of Leptospira present in water sources in the Midwestern United States and provides unique opportunities to explore the geographic diversity and evolution of this genus. The identification of known and novel pathogenic species circulating in local water sources highlights their potential usefulness as diagnostic antigens, as well as the role of water in the transmission of infection to human and animal populations. Integrating knowledge on human, animal, and environmental health is essential to control and predict risk for zoonoses.</p

Image_1_Isolation and characterization of saprophytic and pathogenic strains of Leptospira from water sources in the Midwestern United States.TIFF

The genus Leptospira is a diverse and unique group of bacteria comprising multiple saprophytic and pathogenic species, which survive and persist in suitable moist environments. Pathogenic species cause human and animal leptospirosis, a global and neglected zoonotic disease. Disease transmission occurs by exposure to contaminated water and moist soil environments or by contact with domestic animals and wildlife acting as reservoir hosts that shed Leptospira via urine. Here, we describe the unexpected diversity of saprophytic and pathogenic species of Leptospira isolated from water in the Midwestern United States. Samples were collected by volunteers in 11 counties in Iowa from water sources, including puddles, sewage, creeks, ponds, lakes, and rivers, during the summer of 2021. One hundred and five water samples were tested by culture for the presence of saprophytic and pathogenic species and by lipL32 qPCR specific for the detection of pathogens; 82 (78.1%) were culture positive and five (4.8%) were positive by lipL32 qPCR. Whole genome sequencing of isolates cultured from water samples identified 10 species of saprophytes, namely L. montravelensis, L. kemamanensis, L. bandrabouensis, L. bourretii, L. bouyouniensis, L. chreensis, L. ellinghausenii, L. terpstrae, L. yanagawae, and L. abararensis, as well as three novel saprophytic species. Whole genome sequencing also identified two novel pathogenic species. The remaining cultures comprised mixed populations of saprophytic species and six comprised a mixture of saprophytic and pathogenic species. One of these mixed cultures was enriched to select for a clonal isolate of pathogenic Leptospira, strain WS101.C1, which was classified as L. interrogans serogroup Djasiman serovar Djasiman. Cumulatively, 9.5% (10/105) of water samples were positive for pathogenic Leptospira. This study emphasizes the diversity of Leptospira present in water sources in the Midwestern United States and provides unique opportunities to explore the geographic diversity and evolution of this genus. The identification of known and novel pathogenic species circulating in local water sources highlights their potential usefulness as diagnostic antigens, as well as the role of water in the transmission of infection to human and animal populations. Integrating knowledge on human, animal, and environmental health is essential to control and predict risk for zoonoses.</p

Data_Sheet_2_Isolation and characterization of saprophytic and pathogenic strains of Leptospira from water sources in the Midwestern United States.PDF

The genus Leptospira is a diverse and unique group of bacteria comprising multiple saprophytic and pathogenic species, which survive and persist in suitable moist environments. Pathogenic species cause human and animal leptospirosis, a global and neglected zoonotic disease. Disease transmission occurs by exposure to contaminated water and moist soil environments or by contact with domestic animals and wildlife acting as reservoir hosts that shed Leptospira via urine. Here, we describe the unexpected diversity of saprophytic and pathogenic species of Leptospira isolated from water in the Midwestern United States. Samples were collected by volunteers in 11 counties in Iowa from water sources, including puddles, sewage, creeks, ponds, lakes, and rivers, during the summer of 2021. One hundred and five water samples were tested by culture for the presence of saprophytic and pathogenic species and by lipL32 qPCR specific for the detection of pathogens; 82 (78.1%) were culture positive and five (4.8%) were positive by lipL32 qPCR. Whole genome sequencing of isolates cultured from water samples identified 10 species of saprophytes, namely L. montravelensis, L. kemamanensis, L. bandrabouensis, L. bourretii, L. bouyouniensis, L. chreensis, L. ellinghausenii, L. terpstrae, L. yanagawae, and L. abararensis, as well as three novel saprophytic species. Whole genome sequencing also identified two novel pathogenic species. The remaining cultures comprised mixed populations of saprophytic species and six comprised a mixture of saprophytic and pathogenic species. One of these mixed cultures was enriched to select for a clonal isolate of pathogenic Leptospira, strain WS101.C1, which was classified as L. interrogans serogroup Djasiman serovar Djasiman. Cumulatively, 9.5% (10/105) of water samples were positive for pathogenic Leptospira. This study emphasizes the diversity of Leptospira present in water sources in the Midwestern United States and provides unique opportunities to explore the geographic diversity and evolution of this genus. The identification of known and novel pathogenic species circulating in local water sources highlights their potential usefulness as diagnostic antigens, as well as the role of water in the transmission of infection to human and animal populations. Integrating knowledge on human, animal, and environmental health is essential to control and predict risk for zoonoses.</p