John F. Timoney

Pathogenesis of Salmonella enteritidis infection in laying chickens. I. Studies on egg transmission, clinical signs, fecal shedding, and serologic responses.

Laying hens were inoculated orally, intracloacally (IC), or intravenously (IV) with Salmonella enteritidis phage type 8 isolates from a human (E700-87) eggs (Y-8P2), or the ovary of a hen (27A). Oral or IV inoculation of 2 x 10(8) to 4 x 10(8) colony-forming units (CFU) of E700-87 caused depression, anorexia, reduced egg production, diarrhea, and some mortality. Lower doses resulted in milder clinical signs. S. enteritidis was cultured from the shells of a few eggs but not from egg contents. Fecal shedding persisted for up to 6 weeks in some birds. Isolate Y-8P2 (10(6) CFU) also caused anorexia, diarrhea, and a drop in egg production. Hens inoculated orally or IC were less severely affected than those inoculated IV. Fecal shedding was intermittent and lasted up to 18 days. Eggshells from the IC-inoculated birds had the highest rate of contamination, and S. enteritidis was isolated from the albumen of 11 and yolk of three of 726 eggs. Oral inoculation of 10(6) CFU of isolate 27A resulted in a bacteremic infection with seeding of the liver, spleen, peritoneum, ovule, and oviduct. However, the birds remained clinically normal with normal egg production. S. enteritidis was cultured from the yolk and albumen of a small number of eggs until 11 days postinfection. Antigen prepared from S. enteritidis detected antibody in more sera than did commercially available S. pullorum antigen in agglutination tests.

Cloning and molecular characterization of an immunogenic LigA protein of Leptospira interrogans.

ABSTRACT A clone expressing a novel immunoreactive leptospiral immunoglobulin-like protein A of 130 kDa (LigA) from Leptospira interrogans serovar pomona type kennewicki was isolated by screening a genomic DNA library with serum from a mare that had recently aborted due to leptospiral infection. LigA is encoded by an open reading frame of 3,675 bp, and the deduced amino acid sequence consists of a series of 90-amino-acid tandem repeats. A search of the NCBI database found that homology of the LigA repeat region was limited to an immunoglobulin-like domain of the bacterial intimin binding protein of Escherichia coli, the cell adhesion domain of Clostridium acetobutylicum, and the invasin of Yersinia pestis. Secondary structure prediction analysis indicates that LigA consists mostly of beta sheets with a few alpha-helical regions. No LigA was detectable by immunoblot analysis of lysates of the leptospires grown in vitro at 30°C or when cultures were shifted to 37°C. Strikingly, immunohistochemistry on kidney from leptospira-infected hamsters demonstrated LigA expression. These findings suggest that LigA is specifically induced only in vivo. Sera from horses, which aborted as a result of natural Leptospira infection, strongly recognize LigA. LigA is the first leptospiral protein described to have 12 tandem repeats and is also the first to be expressed only during infection. Thus, LigA may have value in serodiagnosis or as a protective immunogen in novel vaccines.

LfhA, a Novel Factor H-Binding Protein of Leptospira interrogans

ABSTRACT The early phase of leptospiral infection is characterized by the presence of live organisms in the blood. Pathogenic Leptospira interrogans is resistant to the alternative pathway of complement mediated-killing, while nonpathogenic members of the genus are not. Consistent with that observation, only pathogenic leptospires bound factor H, a host fluid-phase regulator of the alternative complement pathway. Ligand affinity blot analyses revealed that pathogenic L. interrogans produces at least two factor H-binding proteins. Through screening of a lambda phage expression library, we identified one of these as the novel membrane protein LfhA. Ligand affinity assays and surface plasmon resonance analyses of recombinant LfhA revealed specific binding of both factor H and factor H-related protein 1. Serological examination of infected humans and horses demonstrated that LfhA is expressed by L. interrogans during mammalian infection. LfhA may therefore contribute to the resistance of pathogenic leptospires to complement-mediated killing during leptospiremic phases of the disease.

A Clonal Outbreak of Acute Fatal Hemorrhagic Pneumonia in Intensively Housed (Shelter) Dogs Caused by Streptococcus equi subsp. zooepidemicus

An outbreak of acute, fatal, hemorrhagic pneumonia was observed in more than 1,000 mixed breed dogs in a single animal shelter. The Department of Anatomic Pathology at the University of California at Davis School of Veterinary Medicine performed necropsies on dogs that were found moribund in acute respiratory distress or found dead with evidence of nasal bleeding. All dogs had hemothorax and an acute, fibrinosuppurative pneumonia. Large numbers of gram-positive cocci were observed within the lungs of all dogs and within septic thromboemboli of remote organs in about 50% of cases. Bacterial cultures from the dogs and their environment revealed widespread beta-hemolytic Streptococus equi subspecies zooepidemicus (Lancefield Group C). Extensive diagnostic testing failed to reveal the consistent presence of copathogens in individual cases. The clinical, epidemiologic, molecular biologic, and pathologic data indicate that a single clone of S. zooepidemicus was the cause of an acutely fatal respiratory infection in these dogs.

Temperature-Regulated Protein Synthesis by Leptospira interrogans

ABSTRACT 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 withL. 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.

In vivo pathogenicity and resistance to phagocytosis of Streptococcus equi strains with different levels of capsule expression.

The glossy non-encapsulated strain of Steptococcus equi, NCTC 9682, was compared with the matt strain Hidaka/95/2 which expresses a medium sized capsule and with the mucoid CF32 which expresses a large sized capsule in phagocytosis assays and for virulence in inoculated horses. The three strains, NCTC 9682, Hidaka /95/2 and CF32 produced 2.0, 3.1, and 5.3 mg/g wet cells respectively after 3 h incubation, but similar amounts of M-like proteins, cytotoxin and mitogen. NCTC 9682 showed no resistance to phagocytosis by equine neutrophils regardless of the presence of opsonin while strains Hidaka /95/2 and CF32 showed almost complete resistance to phagocytosis. Furthermore, NCTC 9682 produced no clinical disease although it infected the guttural pouch and caused seroconversion. Typical strangles with guttural pouch invasion was observed in all horses infected with encapsulated strains.

LruA and LruB, Novel Lipoproteins of Pathogenic Leptospira interrogans Associated with Equine Recurrent Uveitis

ABSTRACT Recurrent uveitis as a sequela to Leptospira infection is the most common infectious cause of blindness and impaired vision of horses worldwide. Leptospiral proteins expressed during prolonged survival in the eyes of horses with lesions of chronic uveitis were identified by screening a phage library of Leptospira interrogans DNA fragments with eye fluids from uveitic horses. Inserts of reactive phages encoded several known leptospiral proteins and two novel putative lipoproteins, LruA and LruB. LruA was intrinsically labeled during incubation of L. interrogans in medium containing [14C]palmitic acid, confirming that it is a lipoprotein. lruA and lruB were detected by Southern blotting in infectious Leptospira interrogans but not in nonpathogenic Leptospira biflexa. Fractionation data from cultured Leptospira indicate that LruA and LruB are localized in the inner membrane. Uveitic eye fluids contained significantly higher levels of immunoglobulin A (IgA) and IgG specific for each protein than did companion sera, indicating strong local antibody responses. Moreover, LruA- and LruB-specific antisera reacted with equine ocular components, suggesting an immunopathogenic role in leptospiral uveitis.

Molecular Characterization of Thermoinduced Immunogenic Proteins Q1p42 and Hsp15 of Leptospira interrogans

ABSTRACT 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 qlp42 andhsp15 in pathogenic serovars of L. interrogansbut 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.

DETECTION OF STREPTOCOCCUS EQUI IN EQUINE NASAL SWABS AND WASHES BY DNA AMPLIFICATION

Streptococcus equi, the causative agent of equine strangles, is shed in nasal discharges and pus from lymph nodes of affected animals. Routine laboratory detection of the bacterium involves culture of nasal swabs, nasal washes and pus from abscesses and is often difficult because of background contamination, small numbers of the organism, or the presence of S zooepidemicus and other 3-haemolytic streptococci. Completion of culture and identification usually takes two to three days, an excessively lengthy interval given the highly contagious nature of strangles and the need to quickly identify shedding horses so that they may be isolated in the early stages of an outbreak. The recent elucidation of the DNA sequence of SeM, the gene for the protective, type-specific M-like protein unique to S equi has provided information for the development of a test for S equi DNA (Timoney and others 1997) based on the polymerase chain reaction (PCR). PCR takes advantage of the ability of DNA polymerase to synthesise many copies of as few as one DNA template molecule by repetitive cycles of strand separation, primer extension and subsequent strand annealing. Primers are short pieces of DNA made from the upper end of the coding strand and from the lower end of the noncoding strand of the DNA of interest. The resulting amplified fragment has a characteristic size. This short communication describes the application of PCR to the detection of S equi in nasal swabs and washes from horses from five different outbreaks of strangles in horses in Kentucky in the spring of 1996. Nasal swabs were collected from affected and exposed in-contact horses one to five days after clinical diagnosis of strangles on farms A, B, C and D. Some horses were sampled more than once over the following three weeks. Nasal washes were also collected from 13 horses on a UK farm, 15 and 85 days after exposure to two horses with clinical strangles. All 13 horses developed strangles within 17 days of this exposure. Nasal washes were collected by instilling 50 ml phosphate buffered saline (pH 7-2) via an 8 mm diameter latex tube (Fisher Scientific) inserted 15 cm into the nostril and collecting the fluid that drained out. The fluid was centrifuged at 3000 g and the pelleted debris separated for culture and PCR. Swabs and nasal wash pellets were cultured on Columbia CNA horse blood agar (Difco Laboratories) and incubated for 18 hours at 37°C. 0-haemolytic colonies were subcultured and their fermentation behaviour tested in lactose, sorbitol and trehalose broths. Mucoid P-haemolytic colonies that did not ferment any of these sugars were identified as S equi. DNA for PCR from the nasal swabs and washes was prepared as follows. Swab tips were placed in 300

Host-Inducible Immunogenic Sphingomyelinase-Like Protein, Lk73.5, of Leptospira interrogans

ABSTRACT Leptospira interrogans causes a variety of clinical syndromes in animals and humans. Although much information has accumulated on the importance of leptospiral lipopolysaccharide in protective antibody responses, relatively little is known about proteins that participate in immune responses. Identification of those proteins induced only in the host is particularly difficult. Using a novel double-antibody screen designed to identify clones in a gene library of L. interrogans serovar Pomona expressing host-inducible proteins, we have characterized a gene (lk75.3) encoding a sphingomyelinase-like preprotein of 648 amino acids with cytotoxic activity for equine pulmonary endothelial cells and weak hemolytic activity for equine and rabbit erythrocytes. lk73.5 was found as a single gene copy in all serovars of L. interrogans but not in other Leptospira spp. except L. inadai. The open reading frame (ORF) for Lk73.5 is followed by another partially homologous sequence containing an ORF (sph-like 2) for a 28.7-kDa peptide. Lk73.5 and Sph-like 2 share 95.1 and 97.7% amino acid identity with putative sphingomyelinases Sph2 and Sph1 (N terminus) from L. interrogans serovar Lai (S.-X. Ren, G. Fu, X.-G. Jiangk, R. Zeng, Y.-G. Miao, H. Xu, Y.-X. Zhang, H. Xiong, G. Lu, L.-F. Lu, H.-Q. Jiang, J. Jia, Y.-F. Tu, J.-X. Jiang, W.-Y. Gu, Y.-Q. Zhang, Z. Cai, H.-H. Sheng, H.-F. Yin, Y. Zhang, G.-F. Zhu, M. Wank, H.-L. Huangk, Z. Qian, S.-Y. Wang, Wei Ma, Z.-J. Yao, Y. Shen, B.-Q. Qiang, Q.-C. Xia, X.-K. Guo, A. Danchinq, I. S. Girons, R. L. Somerville, Y.-M. Wen, M.-H. Shik, Z. Chen, J.-G. Xuk, and G.-P. Zhao, Nature 422:88-893, 2003). Substantial homologies to sphingomyelinases from other leptospiras and other bacteria are also present. Lk73.5 was not detected in leptospiras cultured at 30 or 37°C. The recombinant protein reacted strongly with sera from recently infected mares but not with sera from horses vaccinated with commercial pentavalent bacterin. The host-inducible immunogenic Lk73.5 should have value in distinguishing vaccine from infection immune response.