John F. Timoney

Leptospirosis is a major problem of dairy farms in Tamilnadu, India, resulting in abortions, stillbirths and infertility. Serologic and genetic analyses of samples from cattle, humans and rodents were performed in order to estimate infection prevalence and identify leptospiral species. Five hundred and fifteen sera and 76 urine samples were collected from dairy cattle on 25 farms including a farm that practiced rat control. Sera and kidney samples were also collected from field rats (Rattus norvegicus) in the vicinity of these farms. In addition, sera were collected from farm workers. Serum antibody was measured by the microscopic agglutination test. Leptospires isolated from blood, kidney, and urine were characterized as to serovar. Genomospecies were predicted using random amplified polymorphic DNA (RAPD) profiling. SecY gene sequencing was performed as a tool for tracing of source. Seroprevalence of 87.%, 51.% and 76.5% for cattle, rats and humans, respectively, was observed on endemic farms. Prevalences on a non-endemic farm were lower. Antibodies to Autumnalis, Javanica, Icterohaemorrhagiae and Pomona predominated in both cattle and rats. Thirteen isolates from rat kidneys were identified as serogroup Javanica, serovar Javanica. RAPD comparisons and secY gene sequencing identified these isolates as Leptospira borgpetersenii. These results altogether indicated that L. borgpetersenii was the dominant species in these areas with serovar Javanica apparently derived from rats which provided an important source of infection in cattle resulting a high incidence of infertility, abortion and.still-birth in the Cauvery river valley, Tiruchirappalli, Tamilnadu.

Leptospira interrogans is a mammalian pathogen which must adapt to a range of new environmental conditions including temperature change when it infects new hosts. In vitro studies of organisms cultured at 30°C and shifted to 37°C for 5 to 7 days have confirmed that synthesis of several proteins involved in equine infection is regulated in response to temperature change (J. E. Nally, J. F. Timoney, and B. Stevenson, Infect. Immun. 69:400–404, 2001). In order to specifically identify antigenic proteins upregulated at 37°C, groups of three ponies were immunized with organisms shifted to 37°C for 5 to 7 days or with organisms maintained at 30°C. A lambda ZAP II genomic DNA library was screened with the pool of antisera to organisms shifted to 37°C. Clones reactive with this pool but unreactive with the pool of pony antisera to organisms cultured at 30°C were selected for further analysis. Sequence analysis of the first two clones identified open reading frames for proteins designated Qlp42 and Hsp15. Qlp42 is predicted to be an outer membrane lipoprotein. Its synthesis was upregulated when cultures were shifted from 30 to 37°C and downregulated when cultures were shifted from 37 to 30°C. Although the predicted molecular mass of Qlp42 is 39.8 kDa for the mature protein, Qlp42-specific equine antiserum was reactive with two bands of 30 and 29.5 kDa. Hsp15 is a stress response protein and a member of the Hsp20/α-crystallin family. PCR detected homologues of qlp 42 and hsp 15 in pathogenic serovars of L. interrogans but not in the nonpathogenic Leptospira biflexa . Enzyme-linked immunosorbent assays of antibody in convalescent sera from mares naturally infected with L. interrogans suggest that Qlp42 is expressed during leptospiral infection.

Leptospira interrogans is an important mammalian pathogen. Transmission from an environmental source requires adaptations to a range of new environmental conditions in the organs and tissues of the infected host. Since many pathogenic bacteria utilize temperature to discern their environment and regulate the synthesis of appropriate proteins, we investigated the effects of temperature on protein synthesis in L. interrogans . Bacteria were grown for several days after culture temperatures were shifted from 30 to 37°C. Triton X-114 cellular fractionation identified several proteins of the cytoplasm, periplasm, and outer membrane for which synthesis was dependent on the culture temperature. Synthesis of a cytoplasmic protein of 20 kDa was switched off at 37°C, whereas synthesis of a 66-kDa periplasmic protein was increased at the higher temperature. Increased synthesis of a 25-kDa outer membrane protein was observed when the organisms were shifted from 30 to 37°C. A 36-kDa protein synthesized at 30 but not at 37°C was identified as LipL36, an outer membrane lipoprotein. In contrast, expression of another lipoprotein, LipL41, was the same at either temperature. Immunoblotting with convalescent equine sera revealed that some proteins exhibiting thermoregulation of synthesis elicited antibody responses during infection. Our results show that sera from horses which aborted as a result of naturally acquired infection with L. interrogans serovar pomona type kennewicki recognize periplasmic and outer membrane proteins which are differentially synthesized in response to temperature and which therefore may be important in the host-pathogen interaction during infection.

The nasal turbinates of 5 young horses were studied by light and scanning electron‐microscopy. Stratified cuboidal epithelium lined the rostral part of the dorsal and ventral nasal turbinates of the vestibular region. The polyangular microvillus cells of this region were separated by linear depressions. The mid and caudal parts of the dorsal and ventral nasal turbinates and the rostral part of the ethmoturbinates were lined by pseudostratified columnar ciliated respiratory epithelium. Numerous cilia with dilated blebs on the ciliated cells concealed adjacent non‐ciliated supporting cells and goblet cells. The olfactory zone consisting of the olfactory vesicle and a dense network of olfactory cilia localized to the caudal part of the ethmoturbinates. The three regions were delineated from each other by transitional zones.

Sarcocystis neuronais the etiologic agent of equine protozoal myeloencephalitis (EPM). Based on an analysis of 25,000 equine serum and cerebrospinal fluid (CSF) samples, including samples from horses with neurologic signs typical of EPM or with histologically or parasitologically confirmed EPM, four major immunoblot band patterns have been identified. Twenty-three serum and CSF samples representing each of the four immunoblot patterns were selected from 220 samples from horses with neurologic signs resembling EPM and examined for inhibitory effects on the infectivity ofS. neuronaby an in vitro neutralization assay. A high correlation between immunoblot band pattern and neutralizing activity was detected. Two proteins, Sn14 and Sn16 (14 and 16 kDa, respectively), appeared to be important for in vitro infection. A combination of the results of surface protein labeling, immunoprecipitation, Western blotting, and trypsin digestion suggests that these molecules are surface proteins and may be useful components of a vaccine againstS. neuronainfection. AlthoughS. neuronais an obligate intracellular parasite, it is potentially a target for specific antibodies which may lyse merozoites via complement or inhibit their attachment and penetration to host cells.