Anti-cancer Parasporin Toxins are Associated with Different Environments: Discovery of Two Novel Parasporin 5-like Genes.

Cry toxins are primarily a family of insecticidal toxins produced by the bacterium Bacillus thuringiensis (Bt). However, some Cry toxins, called parasporins (PSs), are non-insecticidal and have been shown to differentially kill human cancer cells. Based on amino acid homology, there are currently six different classes of parasporins (PS1-6). It is not known what role parasporins play in nature, nor if certain PSs are associated with Bt found in particular environments. Herein, we present ten parasporin-containing isolates of Bt from the Caribbean island of Trinidad. Genes coding for PS1 and PS6 were found in isolates associated mainly with artificial aquatic environments (e.g., barrels with rain water), while Bt possessing two novel PS5-like genes (ps5-1 and ps5-2), were isolated from manure collected directly from the rectum of cattle. The amino acid sequences inferred from the two PS5-like genes were 51 % homologous to each other, while being only 41 or 45 % similar to PS5Aa1/Cry64Aa, the only reported member of the parasporin five class. The low level of amino acid homology between the two PS5-like genes and PS5Aa1 indicate that the two PS5-like genes may represent a new class of parasporins, or greatly expand the level of diversity within the current parasporin 5 class.

Parasporins from a Caribbean Island: evidence for a globally dispersed Bacillus thuringiensis strain.

Parasporins represent a new functional class of Cry (crystal protein) toxins produced by the bacterium Bacillus thuringiensis (Bt). Unlike Cry toxins that demonstrate activity mainly against some insect cells, parasporins are characterized as being non-hemolytic, yet capable of preferentially killing some human cancer cells. Globally, six different parasporin types, PS1-PS6, based on protein sequence homology, have been identified in only four countries (Japan, Vietnam, India, and Canada). Herein we report the results of a screening study of 160 Bt isolates collected from the Caribbean island of Trinidad. One isolate (strain 64-1-94) was shown to kill human cancer cells and to contain one ps6 and two ps1 parasporin genes. The two ps1 genes were located approximately 6 kb apart from each other, sharing a similar spatial arrangement, and high sequence homology, with two plasmid-located ps1 genes, ps1Aa6 and ps1Ad1, recently isolated from a Japanese strain. Evidence is also presented that a parasporin gene reported previously for a Canadian strain, ps1Aa2, is most likely derived from a recombination event between these same two genes found in the Trinidadian and Japanese strains. Notably, all three strains share a ps6 parasporin gene, presumably located on a separate plasmid. These data suggest that the global population of ps1 genes may be have originated from a single pair of parasporin genes. Given the large geographical distance between the collection sites, which are located on both continental land masses and islands at sea, ps1 genes are able to retain a remarkable level of homology not easily explained.

Characterization of a novel Bacillus thuringiensis phenotype possessing multiple appendages attached to a parasporal body.

Bacillus thuringiensis is a bacterium best known for its production of crystal-like bodies comprised of one or more Cry-proteins, which can be toxic to insects, nematodes or cancer cells. Although strains of B. thuringiensis have occasionally been observed with filamentous appendages attached to their spores, appendages in association with their parasporal bodies are extremely rare. Herein we report the characterization of Bt1-88, a bacterial strain isolated from the Caribbean that produces a spore-crystal complex containing six long appendages, each comprised of numerous thinner filaments approximately 10 nm in diameter and 2.5 µm in length. Each of the multi-filament appendages was attached to a single, small parasporal body located at one end of the bacterial spore. Biochemical tests, 16S rDNA gene sequencing, and the identification of two Cry proteins by partial protein sequencing (putatively Cry1A and Cry2A), unambiguously identified Bt1-88 as a strain of B. thuringiensis. Bt1-88 represents the second reported strain of B. thuringiensis possessing a parasporal body/appendage phenotype characterized by one or more long appendages, comprised of numerous filaments in association with a parasporal body. This finding suggests that Bt1-88 is a member of a new phenotypic class of B. thuringiensis, in which the parasporal body may perform a novel structural role through its association with multi-filament appendages.

An exploratory study of methicillin-resistant Staphylococcus aureus and SCCmec elements obtained from a community setting along the Texas border with Mexico.

An exploratory study of methicillin-resistant Staphylococcus aureus (MRSA) and SCCmec elements in bacteria along the Mexican border of south Texas was performed. Between September and December of 2008, 375 swabs of anterior nares were self-collected by students attending the University of Texas-Pan American (UTPA) and cultured for MRSA. Fifty seven bacterial isolates were kept for further analysis that included suspected MRSA and other SCCmec-containing bacteria. Isolates were examined for the presence of nuc, mecA, lukS-PV, and spa genes using PCR. SCCmec and spa typing were also performed. Seven S. aureus isolates were found of which six were classified as MRSA. SCCmec typing showed five of the six MRSA strains to be type IV, while one MRSA strain, and most of the non-S. aureus strains, were untypeable, producing results that were indicative of mixed SCCmec types. Five of the six MRSA strains contained known spa types (two of which corresponded to USA300 and one to USA600), while one strain had a novel spa type. Only one isolate, a USA300 MRSA, was positive for lukS-PV. Easy access by the Texas border community to antibiotics in Mexico without a prescription, and the strong partition in SCCmec types between MRSA and non-S. aureus bacteria suggest that this border region of Texas may be uniquely suited for the study of emerging SCCmec types, their horizontal transfer, and perhaps other aspects of antibiotic resistance in bacteria.

Comparison of polymerase chain reaction and bacterial culture for Salmonella detection in the Muscovy duck in Trinidad and Tobago.

OBJECTIVES: The purpose of this study was to investigate the presence and serovar identity of Salmonella, at the national level, in farmed Muscovy ducks (Cairina moschata) in Trinidad and Tobago, and to compare the relative benefits of bacterial culture to those of polymerase chain reaction (PCR) for use in the routine detection and surveillance of Salmonella in these ducks. METHODS: From March-September 2003, 110 fecal samples were collected from 82 farms across the islands of Trinidad and Tobago. Salmonella was isolated from fresh and frozen samples and the serotype of each was determined through bacterial culture. An in-house, nested PCR that detects all pathogenic Salmonella species was utilized in analyzing the samples. RESULTS: Five samples were positive for Salmonella by bacterial culture, whereas 44 were positive by the nested PCR. Serovars isolated were Kiambu, Orion, Uganda, and two isolates from Group E1 whose H antigens could not be fully characterized. Of the samples, 87 (79%) gave equivalent PCR results for both enrichment broths-28 were positive for both and 59 were negative for both). However, 16 samples were positive for one broth, but not for the other, with the majority (14 of the 16) resulting positive for Selenite broth. PCR results for seven samples were inconclusive due to ambiguous band size or multiple bands near the expected band size. CONCLUSIONS: In Trinidad and Tobago, the Muscovy duck does not appear to be a significant source of S. typhimurium or S. enteritidis, but it does harbor other Salmonella species. In-house, nested PCR represents a simple, relatively inexpensive and potentially more sensitive method than bacterial culture for the routine surveillance of pathogenic Salmonella in the Muscovy duck.

Comparison of polymerase chain reaction and bacterial culture for Salmonella detection in the Muscovy duck in Trinidad and Tobago / Comparación de la reacción en cadena de la polimerasa y el cultivo bacteriano como métodos de detección de Salmonella en patos criollos en Trinidad y Tobago

OBJECTIVES: The purpose of this study was to investigate the presence and serovar identity of Salmonella, at the national level, in farmed Muscovy ducks (Cairina moschata) in Trinidad and Tobago, and to compare the relative benefits of bacterial culture to those of polymerase chain reaction (PCR) for use in the routine detection and surveillance of Salmonella in these ducks. METHODS: From March-September 2003, 110 fecal samples were collected from 82 farms across the islands of Trinidad and Tobago. Salmonella was isolated from fresh and frozen samples and the serotype of each was determined through bacterial culture. An in-house, nested PCR that detects all pathogenic Salmonella species was utilized in analyzing the samples. RESULTS: Five samples were positive for Salmonella by bacterial culture, whereas 44 were positive by the nested PCR. Serovars isolated were Kiambu, Orion, Uganda, and two isolates from Group E1 whose H antigens could not be fully characterized. Of the samples, 87 (79 percent) gave equivalent PCR results for both enrichment broths-28 were positive for both and 59 were negative for both). However, 16 samples were positive for one broth, but not for the other, with the majority (14 of the 16) resulting positive for Selenite broth. PCR results for seven samples were inconclusive due to ambiguous band size or multiple bands near the expected band size. CONCLUSIONS: In Trinidad and Tobago, the Muscovy duck does not appear to be a significant source of S. typhimurium or S. enteritidis, but it does harbor other Salmonella species. In-house, nested PCR represents a simple, relatively inexpensive and potentially more sensitive method than bacterial culture for the routine surveillance of pathogenic Salmonella in the Muscovy duck.

OBJETIVOS: Investigar la presencia de Salmonella en patos criollos (Cairina moschata) criados en Trinidad y Tobago e identificar los serotipos circulantes en el país, así como comparar los beneficios relativos del cultivo bacteriano con respecto a la reacción en cadena de la polimerasa (RCP) en la detección y la vigilancia cotidianas de la Salmonella en estos patos. MÉTODOS: Entre marzo y septiembre de 2003 se tomaron 110 muestras de heces fecales de 82 granjas distribuidas por las islas de Trinidad y Tobago. Se aisló Salmonella de muestras frescas y congeladas y se determinaron los serotipos mediante el cultivo bacteriano. Se utilizó un sistema autóctono de RCP anidada que detecta todas las especies patógenas de Salmonella en las muestras. RESULTADOS: Cinco muestras resultaron positivas para Salmonella mediante el cultivo bacteriano, mientras que 44 fueron positivas mediante la RCP anidada. Se asilaron los serotipos Kiambu, Orion, Uganda y dos aislamientos del grupo E1, cuyos antígenos H no se pudieron caracterizar totalmente. Hubo coincidencia en 87 (79 por ciento) de las muestras analizadas por RCP en ambos caldos de enriquecimiento (28 positivas y 59 negativas). Sin embargo, 16 muestras positivas en un caldo resultaron negativas en el otro; la mayoría de ellas (14 de 16) resultaron positivas en caldo selenito. Siete muestras resultaron indefinidas mediante la RCP debido a tallas ambiguas de las bandas o a múltiples bandas cerca de la talla esperada. CONCLUSIONES: El pato criollo no parece ser una fuente importante de infección por S. typhimurium y S. enteritidis en Trinidad y Tobago, aunque hospeda otras especies de Salmonella. El sistema autóctono de RCP anidada constituye un método simple, relativamente económico y posiblemente más sensible que el cultivo bacteriano en la vigilancia cotidiana de especies patógenas de Salmonella en el pato criollo.

In-house polymerase chain reaction for affordable and sustainable Chlamydia trachomatis detection in Trinidad and Tobago.

OBJECTIVES: To provide a preliminary assessment of in-house polymerase chain reaction (PCR) as an alternative to the more costly commercial test for detection of asymptomatic infection by Chlamydia trachomatis and to provide much needed demographic data on infection indicators within the Trinidad and Tobago public health care system. METHODS: An inexpensive in-house nested-PCR with an Internal Amplification Control was used to detect C. trachomatis and Neisseria gonorrhoeae in urine samples collected from 273 apparently healthy, pregnant women from March-September 2004 in Trinidad, West Indies. Demographic information on participants was collected and subjected to statistical analyses. RESULTS: C. trachomatis was detected in 57/273 (21 per cent) samples, of which 5 (2 per cent) were also positive for N. gonorrhoeae. Infection correlated well with certain demographic parameters, with the highest incidence of C. trachomatis infection found among pregnant women that were single or of African descent. CONCLUSIONS: Given the lack of commercial tests in Trinidad, in-house PCR is an inexpensive alternative that can be used to detect asymptomatic infections of C. trachomatis and to provide demographic information needed for interventions by the public health care system.

In-house polymerase chain reaction for affordable and sustainable Chlamydia trachomatis detection in Trinidad and Tobago / Sistema autóctono para la detección asequible y sustentable de Chlamydia trachomatis por la reacción en cadena de la polimerasa en Trinidad y Tobago

OBJECTIVES: To provide a preliminary assessment of in-house polymerase chain reaction (PCR) as an alternative to the more costly commercial test for detection of asymptomatic infection by Chlamydia trachomatis and to provide much needed demographic data on infection indicators within the Trinidad and Tobago public health care system. METHODS: An inexpensive in-house nested-PCR with an Internal Amplification Control was used to detect C. trachomatis and Neisseria gonorrhoeae in urine samples collected from 273 apparently healthy, pregnant women from March-September 2004 in Trinidad, West Indies. Demographic information on participants was collected and subjected to statistical analyses. RESULTS: C. trachomatis was detected in 57/273 (21 percent) samples, of which 5 (2 percent) were also positive for N. gonorrhoeae. Infection correlated well with certain demographic parameters, with the highest incidence of C. trachomatis infection found among pregnant women that were single or of African descent. CONCLUSIONS: Given the lack of commercial tests in Trinidad, in-house PCR is an inexpensive alternative that can be used to detect asymptomatic infections of C. trachomatis and to provide demographic information needed for interventions by the public health care system.

OBJETIVOS: Hacer una evaluación preliminar de un sistema autóctono para la detección de la infección asintomática por Chlamydia trachomatis mediante la reacción en cadena de la polimerasa (RCP), como alternativa a los costosos sistemas comerciales, y ofrecer datos demográficos muy necesarios relacionados con los indicadores de esta infección en el sistema de salud pública de Trinidad y Tobago. MÉTODOS: Se empleó un sistema autóctono y económico de RCP anidada con control interno de la amplificación para la detección de C. trachomatis y Neisseria gonorrhoeae en muestras de orina de 273 mujeres embarazadas asintomáticas, entre marzo y septiembre de 2004 en Trinidad y Tobago, Indias Occidentales. Se obtuvo la información demográfica de las participantes y se sometió a análisis estadístico. RESULTADOS: Se detectó C. trachomatis en 57/273 (21 por ciento) muestras, de las cuales 5 (2 por ciento) fueron también positivas para N. gonorrhoeae. La infección se correlacionó bien con algunos parámetros demográficos; la mayor incidencia de la infección por C. trachomatis se observó en las mujeres embarazadas solteras o de ascendencia africana. CONCLUSIONES: Debido al déficit de sistemas de diagnóstico comerciales en Trinidad, la RCP autóctona es una alternativa económica que puede emplearse para detectar la infección asintomática por C. trachomatis y obtener la información demográfica necesaria para que el sistema de salud pública implemente intervenciones.

In-house polymerase chain reaction for affordable and sustainable Chlamydia trachomatis detection in Trinidad and Tobago.

OBJECTIVES: To provide a preliminary assessment of in-house polymerase chain reaction (PCR) as an alternative to the more costly commercial test for detection of asymptomatic infection by Chlamydia trachomatis and to provide much needed demographic data on infection indicators within the Trinidad and Tobago public health care system. METHODS: An inexpensive in-house nested-PCR with an Internal Amplification Control was used to detect C. trachomatis and Neisseria gonorrhoeae in urine samples collected from 273 apparently healthy, pregnant women from March-September 2004 in Trinidad, West Indies. Demographic information on participants was collected and subjected to statistical analyses. RESULTS: C. trachomatis was detected in 57/273 (21%) samples, of which 5 (2%) were also positive for N. gonorrhoeae. Infection correlated well with certain demographic parameters, with the highest incidence of C. trachomatis infection found among pregnant women that were single or of African descent. CONCLUSIONS: Given the lack of commercial tests in Trinidad, in-house PCR is an inexpensive alternative that can be used to detect asymptomatic infections of C. trachomatis and to provide demographic information needed for interventions by the public health care system.

A Bacillus thuringiensis isolation method utilizing a novel stain, low selection and high throughput produced atypical results.

BACKGROUND: Bacillus thuringiensis is a bacterium known for producing protein crystals with insecticidal properties. These toxins are widely sought after for controlling agricultural pests due to both their specificity and their applicability in transgenic plants. There is great interest in isolating strains with improved or novel toxin characteristics, however isolating B. thuringiensis from the environment is time consuming and yields relatively few isolates of interest. New approaches to B. thuringiensis isolation have been, and continue to be sought. In this report, candidate B. thuringiensis isolates were recovered from environmental samples using a combination of a novel stain, high throughput and reduced selection. Isolates were further characterized by SDS-PAGE, light microscopy, PCR, probe hybridization, and with selected isolates, DNA sequencing, bioassay or Electron Microscopy. RESULTS: Based on SDS-PAGE patterns and the presence of cry genes or a crystal, 79 candidate, non-clonal isolates of B. thuringiensis were identified from 84 samples and over 10,000 colonies. Although only 16/79 (20%) of the isolates showed DNA homology by Probe Hybridization or PCR to common cry genes, initial characterization revealed a surprisingly rich library that included a putative nematocidal gene, a novel filamentous structure associated with a crystal, a spore with spikes originating from a very small parasporal body and isolates with unusually small crystals. When compared to reports of other screens, this screen was also atypical in that only 3/79 isolates (3.8%) produced a bipyramidal crystal and 24/79 (30%) of the isolates' spores possessed an attached, dark-staining body. CONCLUSION: Results suggest that the screening methodology adopted in this study might deliver a vastly richer and potentially more useful library of B. thuringiensis isolates as compared to that obtained with commonly reported methodologies, and that by extension, methodologies fundamentally different from current methods should also be explored.

A nested-PCR with an Internal Amplification Control for the detection and differentiation of Bartonella henselae and B. clarridgeiae: an examination of cats in Trinidad.

BACKGROUND: Bartonella species are bacterial blood parasites of animals capable of causing disease in both animals and man. Cat-Scratch Disease (CSD) in humans is caused mainly by Bartonella henselae and is acquired from the cat, which serves as a reservoir for the bacteria. A second species, B. clarridgeiae is also implicated in the disease. Diagnosis of Bartonellosis by culture requires a week or more of incubation on enriched media containing blood, and recovery is often complicated by faster growing contaminating bacteria and fungi. PCR has been explored as an alternative to culture for both the detection and species identification of Bartonella, however sensitivity problems have been reported and false negative reactions due to blood inhibitors have not generally been addressed in test design. METHODS: A novel, nested-PCR was designed for the detection of Bartonella henselae and B. clarridgeiae based on the strategy of targeting species-specific size differences in the 16S-23S rDNA intergenic regions. An Internal Amplification Control was used for detecting PCR inhibition. The nested-PCR was utilized in a study on 103 blood samples from pet and stray cats in Trinidad. RESULTS: None of the samples were positive by primary PCR, but the Nested-PCR detected Bartonella in 32/103 (31%) cats where 16 were infected with only B. henselae, 13 with only B. clarridgeiae and 3 with both species. Of 22 stray cats housed at an animal shelter, 13 (59%) were positive for either or both species, supporting the reported increased incidence of Bartonella among feral cats. CONCLUSION: The usefulness of a single PCR for the detection of Bartonella henselae and B. clarridgeiae in the blood of cats is questionable. A nested-PCR offers increased sensitivity over a primary PCR and should be evaluated with currently used methods for the routine detection and speciation of Bartonella henselae and B. clarridgeiae. In Trinidad, B. henselae and B. clarridgeiae are the predominant species in cats and infection appears highest with stray cats, however B. clarridgeiae may be present at levels similar to that of B. henselae in the pet population.

A nested-PCR with an Internal Amplification Control for the detection and differentiation of Bartonella henselae and B. clarridgeiae: an examination of cats in Trinidad

BACKGROUND: Bartonella species are bacterial blood parasites of animals capable of causing disease in both animals and man. Cat-Scratch Disease (CSD) in humans is caused mainly by Bartonella henselae and is acquired from the cat, which serves as a reservoir for the bacteria. A second species, B. clarridgeiae is also implicated in the disease. Diagnosis of Bartonellosis by culture requires a week or more of incubation on enriched media containing blood, and recovery is often complicated by faster growing contaminating bacteria and fungi. PCR has been explored as an alternative to culture for both the detection and species identification of Bartonella, however sensitivity problems have been reported and false negative reactions due to blood inhibitors have not generally been addressed in test design. METHODS: A novel, nested-PCR was designed for the detection of Bartonella henselae and B. clarridgeiae based on the strategy of targeting species-specific size differences in the 16S-23S rDNA intergenic regions. An Internal Amplification Control was used for detecting PCR inhibition. The nested-PCR was utilized in a study on 103 blood samples from pet and stray cats in Trinidad. RESULTS: None of the samples were positive by primary PCR, but the Nested-PCR detected Bartonella in 32/103 (31%) cats where 16 were infected with only B. henselae, 13 with only B. clarridgeiae and 3 with both species. Of 22 stray cats housed at an animal shelter, 13 (59%) were positive for either or both species, supporting the reported increased incidence of Bartonella among feral cats. CONCLUSION: The usefulness of a single PCR for the detection of Bartonella henselae and B. clarridgeiae in the blood of cats is questionable. A nested-PCR offers increased sensitivity over a primary PCR and should be evaluated with currently used methods for the routine detection and speciation of Bartonella henselae and B. clarridgeiae. In Trinidad, B. henselae and B. clarridgeiae are the predominant species in cats and infection appears highest with stray cats, however B. clarridgeiae may be present at levels similar to that of B. henselae in the pet population.

A Bacillus thuringiensis isolate possessing a spore-associated filament.

We report on a novel bacterium, isolated during a screen for environmental isolates of Bacillus thuringiensis, that possesses a novel filamentous structure. Nucleotide sequence from the isolate's 16S rRNA gene places the bacterium unambiguously within the Bacillus thuringiensis/Bacillus cereus group. Phase-contrast and electron microscopy indicate the presence of both a parasporal body and a long filament which are retained after sporulation. The filament is shown to consistently arise from the end of the exosporium and next to the parasporal body. Upon spore germination, the parasporal body/filament complex is retained on the cell wall of the resulting bacterium.

A field evaluation of PCR for the routine detection of Babesia equi in horses.

We report on a study that evaluated the usefulness of PCR for the routine detection of Babesia equi in horses. The blood from a total of 105 horses comprising both sick and apparently healthy animals were examined for the presence of B. equi using both Wright-Giemsa-stained blood smears and PCR. Microscopic analysis of Giemsa-stained blood smears revealed 10/105 animals positive for Babesia, compared to 16/105 for the primary PCR and 36/105 for the nested PCR. Three of the 10 samples positive by Wright-Giemsa-stain were negative by PCR for B. equi. However, evidence is presented that these samples contained B. caballi and not B. equi. The Wright-Giemsa-stain was shown to identify Babesia in mostly clinically ill animals while the nested PCR detected the organism in a large number of apparently healthy animals. The results of this study suggest that the nested PCR is superior to both Wright-Giemsa-stained and primary PCR methods, and should be considered for the routine detection of B. equi in both healthy and clinically ill horses.

Molecular characterization of rabies virus isolates from Trinidad

Bovine rabies continues to be a serious problem facing the cattle industry in South and Central America. Although Trinidad played an important role in originally demonstrating the link between bats and bovine rabies, relatively little is known about rabies in Trinidad, an island 7miles off the coast of Venezuela. In order to obtain a more complete understanding of bovine rabies in the region, we report herein on a study undertaken in Trinidad to characterize isolates of rabies virus obtained from infected cattle. A portion of the nucleotide sequence of the nucleoprotein gene from six rabies virus isolates collected from bovine rabies from the years 1997, 1998 and 2000 was determined and compared both to themselves and the nucleotide sequence of other South American isolates. Results indicate that there are at least two independently evolving variants of rabies virus in Trinidad. The nucleotide sequence of either variant failed to match completely the sequence of South American isolates. However, the lack of South American isolates from coastal regions facing Trinidad leaves undetermined the question of South American influence on rabies in Trinidad. The results of this study helps complete the picture of bovine rabies in the South American region and provide basic information required locally for the creation of an effective rabies control and eradication strategy.

Molecular characterization of rabies virus isolates from Trinidad.

Bovine rabies continues to be a serious problem facing the cattle industry in South and Central America. Although Trinidad played an important role in originally demonstrating the link between bats and bovine rabies, relatively little is known about rabies in Trinidad, an island 7miles off the coast of Venezuela. In order to obtain a more complete understanding of bovine rabies in the region, we report herein on a study undertaken in Trinidad to characterize isolates of rabies virus obtained from infected cattle. A portion of the nucleotide sequence of the nucleoprotein gene from six rabies virus isolates collected from bovine rabies from the years 1997, 1998 and 2000 was determined and compared both to themselves and the nucleotide sequence of other South American isolates. Results indicate that there are at least two independently evolving variants of rabies virus in Trinidad. The nucleotide sequence of either variant failed to match completely the sequence of South American isolates. However, the lack of South American isolates from coastal regions facing Trinidad leaves undetermined the question of South American influence on rabies in Trinidad. The results of this study helps complete the picture of bovine rabies in the South American region and provide basic information required locally for the creation of an effective rabies control and eradication strategy.