Antibodies against Leptospira interrogans in California sea lion pups from Gulf of California

One hundred and twenty-five serum samples from California sea lion (Zalophus californianus californianus) pups, and one from an adult female from eight reproductive rookeries located in seven islands in the Gulf of California (Mexico), were collected during the 1994-96 reproductive seasons. These were tested for antibodies to 19 serovars of Leptospira interrogans using a Microscopic Agglutination Test (MAT). Forty-one samples (32%) had antibody levels from 1:20 to 1:320 to one or more serovars. The most frequently detected serotypes were Leptospira interrogans hardjo (n = 13), cynopteri (8), ballum (6), and szwajizak (5). Serovars with the highest prevalence were Leptospira interrogans hardjo and serjoe (1:320), ballum (1:160), and cynopteri, girppotyphosa, and tarassovi (1:80). Based on these results, exposure of sea lions to L. interrogans serovar hardjo seems to be relatively common among colonies located in the islands of the Gulf of California in contrast with those located on the Pacific coast, where the most frequently detected serovar is L. interrogans serovar pomona.     

AN INDIRECT ENZYME-LINKED IMMUNOSORBENT ASSAY FOR THE DETECTION OF LEPTOSPIRA INTERROGANS SEROVAR POMONA ANTIBODIES IN BOVINE SERA

An indirect ELISA was developed and initially evaluated for the detection of bovine antibodies to Leptospira interrogans serovar pomona. The antigen used in this ELISA was extracted from a serovar pomona culture supernatant by a combination of centrifugation, digestion with proteinase K and ultra-centrifugation. The antigen showed little cross-reaction with immune rabbit sera to L. interrogans serovars copenhageni, grippotyphosa, hardjo and sejroe and, Leptospira biflexa serovar patoc. Some cross-reaction was observed with immune rabbit serum to L. interrogans serovar canicola. The relative sensitivity of the ELISA was 94.76% confidence interval =± 3.32%) when estimated with bovine sera (n=172) with serovar pomona microscopic agglutination test (MAT) titers of 100. The relative specificity of the ELISA was 99.28% (95% confidence interval = 1.40%) when estimated with bovine sera (n=139) with MAT titers of <100 to L. interrogans serovars canicola, copenhageni, grippotyphosa, hardjo, pomona and sejroe. Thirty six of 258 field sera (13.95%) with serovar pomona MAT titers of <100, gave positive reactions in the ELISA.     

Evidence of Leptospira interrogans infection in California sea lion pups from the Gulf of California

Forty-two urine and 96 blood and serum samples were obtained from California sea lion (Zalophus californianus) pups from the Gulf of California during the 2000 reproductive season. Antibody prevalence to 13 serovars of Leptospira interrogans was determined by microagglutination tests (MAT); presence of pathogenic leptospires was detected by polymerase chain reaction (PCR). Samples with antibody titers > or = 1:25 or 115 bp fragments on ethidium bromidestained 1.5% agarose gels were considered positive. Antibody prevalence was 54% overall with highest prevalence against serovar cynopteri (50% of all positive reactions). Highest antibody titers (1:50) were detected against serovars cynopteri and pomona. Polymerase chain reaction products were observed in two of 42 urine samples, six of 96 blood samples, and one of 96 serum samples. Presence of PCR products in blood and serum was demonstrated in pups that were seronegative. Kruskall-Wallis tests and corresponding post hoc Tukey tests (alpha = 0.05) showed that prevalence of leptospirosis was significantly different among all rookeries. The high seroprevalence (54%), low antibody titers (maximum 1:50), absence of pups showing clinical signs indicative of the disease, and lack of recent reports of increased mortality of sea lions in the Gulf of California are suggestive of the presence of enzootic host-adapted serovars. Crowding in rookeries as well as the presence of bats and rodents on some of the islands may explain infection by L. interrogans (sensu lato) and some of the differences in seroprevalence among reproductive rookeries.     

Leptospirosis in northern elephant seals (Mirounga angustirostris) stranded along the California coast

Leptospirosis was identified in six northern elephant seals (Mirounga angustirostris) that were stranded in 1995 along the coast of California (USA). Histologic lesions in all seals included tubulointerstitial nephritis with tubular degeneration and necrosis. Infection was confirmed through identification of spirochetes using an immunohistochemical stain for Leptospira sp. antigens. One affected seal had an elevated titer to Leptospira interrogans serovar pomona. Four of the six seals developed leptospirosis during rehabilitation, and two seals had evidence of exposure in the wild. Potential sources of infection during rehabilitation include other elephant seals, California sea lions (Zalophus californianus), Pacific harbor seals (Phoca vitulina richardsii), or free-ranging wildlife. These results indicate that northern elephant seals are susceptible to leptospirosis and can develop disease both in the natural environment and in a rehabilitation setting.     

The impact of Leptospira seropositivity on reproductive performance in sows in southern Viet Nam

A serologic survey was conducted among sows in the Mekong delta in southern Viet Nam to investigate associations between leptospiral seropositivity and reproductive performance. Data were collected from a total of 339 sows in lactation or gestation, from four large-scale state farms on three occasions. The seroprevalence for Leptospira interrogans serovar (sv) autumnalis was 32%, for L. interrogans sv bratislava 29%, for L. kirschneri sv grippotyphosa 13%, for L. interrogans sv icterohaemorrhagiae 27%, for L. interrogans sv pomona 5%, and for L. borgpetersenii sv tarassovi 13%. The reproductive parameters number of days from weaning to service (WSI), number of piglets born, number of piglets born dead, and number of piglets born weak, were evaluated. Seropositivity for sv tarassovi was associated with 0.8 more dead piglets per litter (P = 0.06), and sv grippotyphosa with a 1 day longer WSI (P = 0.06). There were no significant associations between reproductive performance and sv autumnalis, sv bratislava, sv pomona, and sv icterohaemorrhagiae. It is concluded that seropositivity for Leptospira can be associated with impaired reproductive performance even in areas where a high degree of immunity among sows is expected.     

Serologic evaluation of New Zealand sea lions for exposure to Brucella and Leptospira spp

A serologic survey of anti-Brucella and antileptospiral antibodies was conducted on 147 adult, female New Zealand sea lions (Phocarctos hookeri). Most sea lions (n=138) were sampled at Sandy Bay, Enderby Island, Auckland Islands (50°30'S, 166°17'E), January 2000-March 2005. Nine were sampled at Otago, New Zealand (46°0'S, 170°40'E); four in April 2008 and five in March 2009. Serum from one of the Enderby Island females was weakly positive for antibodies to Brucella abortus using the competitive enzyme-linked immunosorbent assay, and one female had a low titer for Leptospira interrogans serovar pomona using the microscope agglutination test. All serum samples from Otago animals were negative. Brucellosis and leptospirosis are therefore considered unlikely to play a major role in population dynamics of these populations, and the low antibody prevalence of these agents suggests that they are an unlikely source of infection for humans, wildlife, or domestic species on mainland New Zealand.     

Serological evidence of the persistence of infection with Leptospira interrogans serovar Hardjo in cattle in Mongolia

Serum samples were collected randomly from Mongol breed cattle in three geographically distinct provinces of Mongolia. Antibodies specific to Leptospira interrogans serovar Hardjo were detected by microscopic agglutination test (MAT) at titres of 100 or more in 80.4% (86/107) of the samples from Dornod Province, but in only 28.9% (13/45) in Arkhangai and 23.5% (12/51) in Khuvsgul Provinces, respectively. Treatment of 9 serum samples from Dornod, positive to serovar Hardjo in MAT and negative in the homologous immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA), with 2-mercaptoethanol (2-ME) indicated that those animals were recently infected.     

Sensitivity of pathogenic and free-living Leptospira spp. to UV radiation and mitomycin C.

The habitats for the two major Leptospira spp. differ. The main habitat of L. biflexa is soil and water, whereas L. interrogans primarily resides in the renal tubules of animals. We investigated whether these two species, along with L. illini (species incertae sedis), differ with respect to their sensitivity to UV radiation. The doses of UV resulting in 37, 10, and 1% survival were determined for representative serovars from each species. L. interrogans serovar pomona was 3.0 to 4.8 times more sensitive to UV than the other Leptospira species under the 37, 10, and 1% survival parameters. In comparison to other bacteria, L. interrogans serovar pomona is among the most sensitive to UV. In a qualitative UV sensitivity assay, L. interrogans serovars were found to be in general more sensitive than L. biflexa serovars. All three species were found to have a photoreactivation DNA repair mechanism. Since organisms that are resistant to UV are often resistant to the DNA cross-linking agent mitomycin C, we tested the relative sensitivity of several Leptospira serovars to this compound. With few exceptions, L. biflexa and L. illini serovars were considerably more resistant to mitomycin C than the L. interrogans serovars. The mitomycin C sensitivity assay could be a useful addition to current characterization tests used to differentiate the Leptospira species.     

Comparison of antibodies to Leptospira in white-tailed deer (Odocoileus virginianus) and cattle in Ohio

A survey was conducted to determine the prevalence of leptospiral antibodies in sera from 248 white-tailed deer (Odocoileus virginianus) in Ohio. The sera were collected at check stations during the hunting season in 1983. The microscopic agglutination microtiter test was used to determine the presence of antibodies to Leptospira interrogans serovars pomona, icterohemorrhagiae, canicola, hardjo, and grippotyphosa. Eighteen of 248 (7.3%) serum samples had antibody titers (greater than or equal to 1:100) to at least one of the five serovars tested, with three of these samples reacting to more than one serovar. Prevalence did not differ significantly between sex or age groups. The serovar antigens reacting most frequently with serum antibodies were grippotyphosa (10 of 22, 45.5%) and pomona (eight of 22, 36.4%). Sera agglutinating with pomona antigen had higher titers (ranging from 1:200 to 1:6,400) than did sera agglutinating with the other serovars. These results were compared to results obtained from cattle tested at the Ohio Department of Agriculture Laboratories during 1983. There was a significant relationship between pomona infections detected in deer and cattle (P less than 0.05), but not with grippotyphosa.     

Antigens recognized by the human immune response to vaccination with a bivalent hardjo/pomona leptospiral vaccine

Serum from volunteer subjects vaccinated with a bivalent whole cell vaccine of Leptospira interrogans serovar hardjo/serovar pomona grown in protein-free medium, was tested by the microscopic agglutination test (MAT), enzyme-immunoassay (EIA) and immunoblotting. Specific IgM antibodies to either serovars hardjo or pomona were detected in some subjects as early as 6 days after vaccination with peak antibody levels occurring 13-68 days after vaccination. Whereas all subjects produced specific IgM to both serovars, not all produced specific IgG to both serovars. Immunoblotting with hardjo sonicate revealed that all subjects produced IgM antibodies reacting with the 15, 23 and 28 kDa components of hardjo lipopolysaccharide (LPS), and most produced IgM antibodies that reacted with the 34.5 kDa flagellar doublet. In contrast, not all sera immunoblotted against pomona sonicate reacted with the 29 and 35 kDa components of pomona LPS. However all subjects produced antibodies reacting with a diffuse 14.4-27 kDa band. These antibodies appeared early in the immune response. Serum from the one vaccinated subject tested protected hamsters from acute lethal infection with serovar pomona.     

Sensitivity of pathogenic and free-living Leptospira spp. to UV radiation and mitomycin C

The habitats for the two major Leptospira spp. differ. The main habitat of L. biflexa is soil and water, whereas L. interrogans primarily resides in the renal tubules of animals. We investigated whether these two species, along with L. illini (species incertae sedis), differ with respect to their sensitivity to UV radiation. The doses of UV resulting in 37, 10, and 1% survival were determined for representative serovars from each species. L. interrogans serovar pomona was 3.0 to 4.8 times more sensitive to UV than the other Leptospira species under the 37, 10, and 1% survival parameters. In comparison to other bacteria, L. interrogans serovar pomona is among the most sensitive to UV. In a qualitative UV sensitivity assay, L. interrogans serovars were found to be in general more sensitive than L. biflexa serovars. All three species were found to have a photoreactivation DNA repair mechanism. Since organisms that are resistant to UV are often resistant to the DNA cross-linking agent mitomycin C, we tested the relative sensitivity of several Leptospira serovars to this compound. With few exceptions, L. biflexa and L. illini serovars were considerably more resistant to mitomycin C than the L. interrogans serovars. The mitomycin C sensitivity assay could be a useful addition to current characterization tests used to differentiate the Leptospira species.     

Serologic survey for selected microbial pathogens in bison from Kansas.

A serologic survey was conducted on an American bison (Bison bison) herd in Kansas for antibodies against Brucella spp., Leptospira interrogans serovar canicola, pomona, grippotyphosa, icterohaemorrhagiae, and hardjo, Anaplasma spp., bluetongue virus, infectious bovine rhinotracheitis virus and bovine viral diarrhea virus. There was an increase in prevalence of bluetongue antibodies from 38% in 1987 to 100% in 1989 in animals greater than or equal to 24-mo-old. Prevalences of antibodies against the other livestock pathogens were either negative or at levels associated with previous vaccination.     

Antigens recognized by the human immune response to vaccination with a bivalent hardjo/pomona leptospiral vaccine

Abstract Serum from volunteer subjects vaccinated with a bivalent whole cell vaccine of Leptospira interrogans serovar hardjo /serovar pomona grown in protein-free medium, was tested by the microcospic agglutination test (MAT), enzyme-immunoassay and immunoblotting. Specific IgM antiboidies to either serevars hardjo or pomona were detected in some subjects as early as 6 days after vaccinated with peak antibody levels occurring 13–68 after vaccination. Whereas all subjects produced specific IgM to both serovars, not all produced specific IgG to both serovars, Immunoblotting with hardjo sonicate revealed that all subjects produced IgM antibodies reacting with the 15, 23 and 28 kDa components of hardjo lipopolysaccharide (LPS), and most produced IgM antibodies that reacted with the 34.5 kDa flagellar doublet. In contrast, not all sera immunoblotted against pomona sonicate reacted with the 29 and 35 kDa components of pomona LPS. However all subjects produced antibodies reacting with a diffuse 14.4–27 kDa band. These antibodies appeared early in the immune response. Serum from the one vaccinated subject tested protected hamsters from acute lethal infection with serovar pomona .     

Serologic survey for selected infectious disease agents in raccoons from Illinois

The determination of serologic titers to infectious organisms is a valuable tool for quantitating exposure to disease organisms. Raccoons (Procyon lotor) were live-trapped from September 1989 to October 1993 and samples collected from two distinct locations in west-central Illinois (USA); a state recreational facility (Park) and privately owned farming property (Farm). Sera were submitted for testing Leptospira interrogans (serovars bratislava, canicola, grippotyphosa, hardjo, icterohemmorhagiae, and pomona), canine distemper virus (CDV), pseudorabies virus (PV), and Toxoplasma gondii. Two-hundred and twenty-two (48%) of 459 raccoons were seropositive for L. interrogans. Eighty-five (23%) out of 368 raccoons were seropositive for canine distemper virus. Eighty-two (17%) of 479 raccoons raccoons were seropositive for pseudorabies virus. One hundred and eight-four (49%) of 379 raccoons were seropositive for T. gondii. A significant difference (P < 0.05) in seroprevalence for L. interrogans between the park (43%) and farm (52%) areas was found. A correlation between increasing age and seroprevalence was found for L. interrogans, CDV, PV, and T. gondii. Furthermore, there was a significant difference in seroprevalence for T. gondii during the spring trapping seasons (73%), when compared with the fall (33%). This type of information on exposure to infectious agents is important for developing control programs to manage raccoon-human and raccoon-domestic animals interactions.     

Identification of two novel coccidian species shed by California sea lions (Zalophus californianus)

Routine fecal examination revealed novel coccidian oocysts in asymptomatic California sea lions (Zalophus californianus) in a rehabilitation facility. Coccidian oocysts were observed in fecal samples collected from 15 of 410 California sea lions admitted to The Marine Mammal Center between April 2007 and October 2009. Phylogenetic analysis using the full ITS-1 region, partial small subunit 18S rDNA sequence, and the Apicomplexa rpoB region identified 2 distinct sequence clades, referred to as Coccidia A and Coccidia B, and placed them in the Sarcocystidae, grouped with the tissue-cyst-forming coccidia. Both sequence clades resolved as individual taxa at ITS-1 and rpoB and were most closely related to Neospora caninum. Coccidia A was identified in 11 and Coccidia B in 4 of 12 sea lion oocyst samples successfully sequenced (3 of those sea lions were co-infected with both parasites). Shedding of Coccidia A oocysts was not associated with age class, sex, or stranding location, but yearlings represented the majority of shedders (8/15). This is the first study to use molecular phylogenetics to identify and describe coccidian parasites shed by a marine mammal.     

Prevalence of Leptospira and Brucella antibodies in wild boars (Sus scrofa) in Tuscany, Italy

Five hundred sixty-two blood samples were collected from wild boars (Sus scrofa) shot in six districts of Tuscany, central Italy, between 1997 and 2000. Sera were examined for antibodies specific for Leptospira interrogans by microagglutination test and Brucella spp. by the Rose Bengal test and indirect enzyme-linked immunosorbent assay. Thirty-four (6.0%) samples tested positive for anti-Leptospira antibodies, 29 (5.1%) sera were positive for anti-L. interrogans serovar bratislava antibodies (titres ranging from 1:100-1:400), and 5 (0.9%) sera were positive for anti-L. interrogans serovar icterohaemorrhagiae antibodies (titres 1:100). All the examined sera were negative for anti-Brucella antibodies.     

Assay for measuring relative potency of leptospiral bacterins containing serovar pomona

An enzyme-linked immunosorbent assay (ELISA) was developed for the quantitation of leptospiral antigen in bacterins containing Leptospira interrogans serovar pomona type kennewicki. A monoclonal antibody (MAb), 2D7, which is directed against a surface antigen on whole cells of L. interrogans serovar pomona type kennewicki, was used in the assay. The capture of antigen in bacterins by a polyclonal antiserum was followed by the addition of the 2D7 ascites fluid, an anti-mouse conjugate and substrate. Biologicals evaluated with this system included preparations containing type kennewicki antigen (homologous) and those not containing type kennewicki antigen (heterologous). Heterologous bacterins gave optical density (OD) values comparable to those of blank wells. Homologous bacterins yielded OD values equal to or greater than those of the National Veterinary Services Laboratories (NVSL) reference pomona bacterin. The relative potencies (RP) of 84 licensed commercial Leptospira pomona bacterin serials were evaluated against the NVSL reference pomona bacterin using the NVSL Relative Potency computer program. Random samples of 1, 2, 3 and 5 ml dose products were selected for evaluation with this system. All products tested passed the hamster potency assay required for leptospiral bacterins. This ELISA system enables detection of antigen in bacterins containing L. interrogans serovar pomona type kennewicki and demonstrates the potential for in vitro testing of leptospiral bacterins.     

Demographics and spatio‐temporal signature of the biotoxin domoic acid in California sea lion (Zalophus californianus) stranding records

California sea lions (Zalophus californianus) in otherwise good nutritional condition have been consistently affected by the marine biotoxin domoic acid since the late 1990s. In this study we evaluated the temporal and spatial stranding patterns of suspected and confirmed cases of domoic acid intoxicated sea lions from 1998 to 2006, using records of strandings along the California coast obtained from members of the California Marine Mammal Stranding Network. The majority of domoic acid cases were adult females (47%–82% of the total annual domoic acid cases), a contrast to strandings that were not related to domoic acid, which were generally dominated by juveniles and pups. Exposure to this biotoxin led to a 6.67‐fold increase in adult female strandings in 2000, and a 5.44‐fold increase in adult female deaths in 2006, relative to strandings and deaths of adult female not affected by domoic acid. Domoic acid cases have occurred annually since 1998 (except for 1999) between April and August, with clusters centered primarily at Pismo Beach (San Luis Obispo County), as well as at other beaches in San Luis Obispo, Monterey, Santa Cruz, Santa Barbara, Orange, and San Diego counties. The larger ecological and population level implications of increased domoic acid strandings and deaths, particularly among adult female sea lions, warrant further attention and need to be investigated.     

Monoclonal antibodies reacting with serogroup and serovar specific epitopes on different lipopolysaccharide subunits of Leptospira interrogans serovar pomona

Monoclonal antibodies with two kinds of specificities, produced against Leptospira interrogans serovar pomona, were studied by agglutination and immunoblotting. Antibodies reacted either exclusively with serovar pomona or with all members of the Pomona serogroup, but none of the antibodies reacted with representative serovars of other serogroups. Both antibodies recognized epitopes on purified lipopolysaccharide (LPS) from serovar pomona. In immunoblotting experiments the serogroup specific antibody recognized both the major LPS bands of 21 kDa and 26 kDa whereas the serovar specific antibodies reacted only with the 26 kDa band, thus localizing serovar specificity in the 26 kDa band and serogroup specific epitopes on at least two different LPS subunits.     

Monoclonal antibodies reacting with serogroup and serovar specific epitopes on different lipopolysaccharide subunits of Leptospira interrogans serovar pomona

Abstract Monoclonal antibodies with two kinds of specificities, produced against Leptospira interrogans serovar pomona , were studied by agglutination and immunoblotting. Antibodies reacted either exclusively with serovar pomona or with all members of the Pomona serogroup, but none of the antibodies reacted with representative serovars of other serogroups. Both antibodies recognized epitopes on purified lipopolysaccharide (LPS) from serovar pomona . In immunoblotting experiments the serogroup specific antibody recognized both the major LPS bands of 21 kDa and 26 kDa whereas the serovar specific antibodies reacted only with the 26 kDa band, thus localizing serovar specificity in the 26 kDa band and serogroup specific epitopes on at least two different LPS subunits.