Cryptosporidiosis. A global challenge.

During the last 30 years, our concept of cryptosporidiosis has changed from that of a rare, largely asymptomatic disease, to an important cause of diarrhea in animals and humans worldwide. Significant disease first appeared in cattle. Subsequently, the zoonotic danger of the organism was recognized in HIV-infected persons and young children. Cryptosporidium are now ubiquitous and disease has been described in over 79 host species. Cryptosporidiosis has become a major cause of calfhood diarrhea worldwide. In humans it accounts for up to 20% of all cases of childhood diarrhea in developing countries and is a potentially fatal complication of AIDS. Waterborne contamination is a growing concern as a source of widespread outbreaks of disease. Factors that have contributed to the emergence of cryptosporidiosis in animals include biological features of the organism, the lack of an effective treatment or preventative, increased environmental contamination, and trends in livestock production. In humans the zoonotic nature of infection and an increased at-risk population have contributed to disease. Genetic characterization of Cryptosporidium, improved detection methods, and a better understanding of the factors that predispose to disease are important contributions to understanding the epidemiology of cryptosporidiosis.

In situ hybridizations of Eperythrozoon suis visualized by electron microscopy.

Eperythrozoon suis is an extracellular red blood cell parasite that causes icteroanemia and poor growth performance in feeder pigs and has been associated with anemias in baby pigs and reproductive failures in sows. At present, few efficient tests are available for the diagnosis and study of E. suis infection in swine. This report discusses how a recently developed recombinant DNA probe (KSU-2) specific to E. suis DNA was utilized in in situ DNA hybridizations that couple biotinylated TaqI digested products of KSU-2 DNA with an immunogold detection system allowing the visualization by transmission electron microscopy of the gold particles within eperythrozoon organisms. Specific labelling by the probe of eperythrozoon organisms demonstrated a pattern that changed with the various stages of the eperythrozoon life cycle.

Detection of Eperythrozoon suis DNA from swine blood by whole organism DNA hybridizations.

A procedure is described for isolating Eperythrozoon suis DNA of sufficient quantity and purity to serve as a probe in whole-organism DNA hybridizations for detecting parasitized swine. The E. suis organisms were isolated from the blood of infected swine; the DNA was recovered and digested with restriction endonucleases and resolved on agarose gels. In DNA hybridizations using recovered E. suis DNA, blood samples from parasitized swine could be differentiated from uninfected, control samples. A high salt lysate recovery technique was used in sampling swine whole blood for E. suis DNA and found to offer many advantages in the collection and recovery process.

Partial characterization of the leukotoxin of Pasteurella haemolytica-like bacteria isolated from swine enteritis.

Pasteurella haemolytica-like (PHL) strains isolated from diarrheic pigs are known to produce a leukotoxin that is lethal to ruminant leukocytes. In the present study, 12 PHL strains were screened for leukotoxin production using a tetrazolium dye-reduction assay. Sterile culture supernatant from strain 6213A, the maximum leukotoxin producer, was used as the crude leukotoxin for characterization studies. The leukotoxin was inactivated by heat at 60 degrees C and by trypsin, protease, and amylase. Toxicity was retained over a pH range of 3.0-11.0. The leukotoxin was lethal to polymorphoneutrophils (PMNs) of cattle, sheep, goat, and swine. Chromosomal DNA of all 12 PHL strains hybridized with a 3.9 kb Pasteurella haemolytica A1 leukotoxin probe, indicating similarities between the leukotoxin genes of P. haemolytica and PHL strains.

Biochemical and biological characterizations and ribotyping of Actinomyces pyogenes and Actinomyces pyogenes-like organisms from liver abscesses in cattle.

Actinomyces pyogenes is the second most frequently encountered pathogen, next only to Fusobacterium necrophorum, in liver abscesses of feedlot cattle. Ninety-one isolates, presumptively identified as A. pyogenes, isolated from liver abscesses of cattle were studied. Biochemical characteristics determined by the API 20 Strep kit were similar to those reported previously for A. pyogenes isolated from other infections, except that 18% of isolates hydrolyzed esculin. Nine isolates that resembled A. pyogenes in morphology and in certain biochemical characteristics, but fermented mannitol and/or raffinose, were called A. pyogenes-like (APL) organisms. The five antimicrobial agents, bacitracin, chlortetracycline, oxytetracycline, tylosin, and virginiamycin were inhibitory to all strains of A. pyogenes and APLs. Generally, APL organisms had higher mean hemolytic and leukotoxic activities than A. pyogenes. All isolates of A. pyogenes and APLs produced proteases and neuraminidases. Ribotyping with endonucleases, including BstEII, ClaI, EcoRI, EcoRV, HaeIII, MboI, PvuII, SalI, and SmaI alone or in combinations, showed considerable genetic heterogeneity in both A. pyogenes and APLs. No specific ribopattern characteristic of each group was observed with any of the endonuclease used. The origin of A. pyogenes and APLs and the relative importance of APLs in causing liver abscesses in feedlot cattle are not known.

Genetic reassortment of bluetongue virus serotype 11 strains in the bovine.

Reassortants of bluetongue virus Serotype 11 (BTV-11) were isolated from a yearling heifer experimentally infected with two electrophoretically different strains (UC-2 and UC-8) by subcutaneous inoculation. Viruses were recovered by direct titration of sonicated blood samples onto Vero cell monolayers, which were overlaid with agarose and later plaque purified. The parental electropherotype of UC-8 was identified as the predominant virus strain during the infection; UC-2 was not isolated. UC-2 infectivity was shown by reassortants which contained genome segments that were identical in migration pattern to the parental UC-2 electropherotype. The observations demonstrate that segmental reassortment can occur during mixed infections in the bovine, between strains of the same BTV serotype.

Further characterization of Pasteurella haemolytica-like bacteria isolated from swine enteritis.

DNA-DNA hybridization studies were conducted on six Pasteurella haemolytica-like (PHL) organisms recovered from cases of swine enteritis. Chromosomal-enriched fractions of PHL organisms served as the source of DNA for Southern blots or as whole-chromosomal DNA probes. Under stringent hybridization conditions, chromosomal DNA probes of a prototype PHL (strain 6213A) organism distinguished other PHL organisms from Pasteurella haemolytica types A1 and T3, Pasteurella multiocida types A:1 and A:3, Escherichia coli, Pseudomonas aeruginosa, Actinobacillus pleuropneumoniae type 1, and Salmonella cholerasuis. The guanine-cytosine content of the DNA of three PHL strains was 41.2 to 42.8 mol % as calculated from the thermal denaturation midpoint temperatures. The PHL strains are Gram-negative, nonmotile, beta-hemolytic, pleomorphic, oxidase-positive, urease- and indole-negative, fermentative rods with the key characteristics of the species Pasteurella haemolytica. None of the PHL strains reacted with the type-specific antisera of P. haemolytica types 1 through 12 as tested by an agglutination procedure. These swine strains differed in their biochemical differentiation from P. haemolytica types A1 and T3 in that all produced acid from M-inositol and failed to grow on MacConkey agar. Acid production from trehalose and L-arabinose was variable with PHL strains. Leukotoxicity of PHL strains was evaluated by a colorimetric micro-titration assay. Sterile culture supernatants of three of five PHL strains were toxic to bovine neutrophils. Results of these studies suggest that the PHL organisms may belong to a new group of organisms under the genus Pasteurella.

Evaluation of a 5'-nuclease (TaqMan) assay with the thin agar layer oxyrase method for the detection of Yersinia enterocolitica in ground pork samples.

A 5'-nuclease (TaqMan) assay was evaluated for its capability to recover and detect stressed Yersinia enterocolitica. Sensitivity studies of a 5'-nuclease assay for detecting Y. enterocolitica 0:8 in a pure culture system and spiked ground pork samples demonstrated that the assay has reliable sensitivity with a detection limit of 3 to 4 log CFU/ml or CFU/g. The PCR 5'-nuclease (TaqMan) assay was evaluated with the Thin Agar Layer Oxyrase method (TALO, overlaying 14 ml of Trypticase soy agar with a 1:30 dilution of "Oxyrase for Agar" onto a prepoured pathogen-specific, selective medium), and it was compared against the selective medium cefsulodin-irgasan-novobiocin (CIN) for recovering and detecting Y. enterocolitica from inoculated nonfrozen and frozen (-15 degrees C, 2 days) ground pork samples. The TALO method showed more sensitivity (detection limit, 2 log CFU/ml), and it has greater recovery capability (0.5 to 1 log CFU/ml) than CIN (P < 0.05). The 5'-nuclease assay provided rapid detection processing (5 versus 24 h after an 18-h enrichment). The sensitivity per PCR was calculated to as low as 0 to 1 log CFU per PCR reaction; however, in the method's current developmental stage, target pathogens should be enriched to 3 to 4 log CFU/ml or CFU/g to show consistent results. In a survey of 100 ground pork samples using TALO, CIN, and PCR methods, no Y. enterocolitica was recovered. A combined cultivation and an automated PCR TaqMan could be used as a presumptive screening test for detecting Y. enterocolitica in food samples.

Evaluation of a 5'-nuclease (TaqMan) assay for the detection of virulent strains of Yersinia enterocolitica in raw meat and tofu samples.

Culture methods for detecting virulent Yersinia enterocolitica require selective enrichment and a series of confirmatory tests that are time-consuming, costly, and laborious. The objective of this study was to evaluate a fluorogenic 5'-nuclease assay for detecting the enterotoxin yst gene of virulent Y. enterocolitica in pure cultures, inoculated ground pork samples, and naturally contaminated food samples. These results were then compared with "gold standard" methods recommended by the U.S. Food and Drug Administration in the Bacteriological Analytical Manual for detecting pathogenic Y. enterocolitica. The 5'-nuclease assay was able to identify the organism in 100% of the repetitions when 10(2) CFU/ml or more organisms were present in pure cultures and 10(3) CFU/g or more organisms were present in ground pork. Similar recovery efficiency on cefsulodin-irgasan-novobiocin (CIN) agar plates was only evident when 10(5) CFU/ml or more organisms were present in pure culture and 10(6) CFU/g or more organisms were present in inoculated ground pork. The 5'-nuclease assay indicated a contamination rate of 35.5% (94/265) in various meats and tofu, whereas the CIN plating method indicated a contamination rate of 28.3% (75/265). This resulted in 100% sensitivity and 64.5% specificity for the 5'-nuclease assay when compared with the standard culture recovery method. Only 75% (60/80) of the Yersinia spp. isolated on CIN was identified as containing a virulence plasmid by autoagglutination and crystal violet binding tests. These results indicate that the true rate of contamination of virulent Y. enterocolitica in pork and other processed meats and foods is being underestimated using current detection methods. This study demonstrates the potential of the 5'-nuclease assay for rapidly and specifically detecting virulent Y. enterocolitica in processed foods with the added advantage of being an automated detection system with high-throughput capability.

Comparison of an improved polymerase chain reaction protocol and the indirect hemagglutination assay in the detection of Eperythrozoon suis infection.

The ability of an improved polymerase chain reaction (PCR) protocol to detect Eperythrozoon suis DNA in the blood of experimentally infected nonsplenectomized pigs was evaluated. The protocol utilizes previously described E. suis-specific primers and a proprietary DNA-releasing reagent in a 2-step amplification cycle followed by visualization of the 492-bp amplification product on agarose gels. This PCR protocol successfully amplified E. suis DNA in blood from all postinfection samples and from the preinfection samples of 2 pigs, indicating preexisting natural infections. Results of the indirect hemagglutination test on serum samples from these pigs revealed that only 1 pig developed detectable antibody titers to E. suis infection during the 43-day study; that pig was determined by PCR to have been infected naturally with E. suis prior to experimental inoculation. These results confirm previous reports of poor antibody response of young pigs to E. suis infection and demonstrate the potential of PCR as a valuable tool for the diagnosis and study of E. suis infection in pigs.

Detection of Eperythrozoon suis using the polymerase chain reaction.

Eperythrozoon suis is an extracellular red blood cell parasite that causes icteroanemia in acutely ill pigs and a variety of syndromes in chronically infected pigs. Current techniques to detect E. suis infection are limited by variability of parasitemias and antibody responses in infected animals. The polymerase chain reaction (PCR) was investigated to determine its potential as a means of detecting E. suis infection in pigs. With DNA samples extracted from either purified E. suis organisms or E. suis-infected pig blood, PCR produced an amplification product 492 base pairs in length. This amplification product hybridized successfully with the fragment of the DNA probe from which the primer sequences had been selected. Sensitivity studies indicated that the PCR protocol was capable of amplifying total genomic E. suis DNA in quantities as low as 450 pg. When PCR was used with DNA from blood samples from a splenectomized pig that had been infected with E. suis, amplification products were detectable as early as 24 hours postinfection. This preliminary analysis indicates that PCR shows promise as a means of efficiently detecting E. suis infection in pigs.

Experimental infections and natural outbreaks of eperythrozoonosis in pigs identified by PCR-DNA hybridizations.

Eperythrozoon-specific DNA amplification reactions and subsequent hybridizations with an eperythrozoon DNA probe (KSU-2) were used in experimental infection studies to identify Eperythrozoon suis DNA in the blood of splenectomized and nonsplenectomized pigs. The results indicate that E. suis DNA is present in nonsplenectomized pigs at levels that can be amplified by polymerase chain reaction (PCR) and identified in DNA hybridizations within 24 hours after infection. The ability of the E. suis PCR/hybridization assay to detect eperythrozoonosis was further demonstrated in blood samples collected from pigs in 2 separate natural outbreaks in Oklahoma. Results from these initial samplings indicate that pigs infected with E. suis from geographically distinct locations can be identified using the eperythrozoon-specific PCR/hybridization assay, which offers many advantages over conventional laboratory procedures for diagnosing eperythrozoonosis in pigs.

Characteristic differences in reverse transcription-polymerase chain reaction products of ovine, bovine, and human respiratory syncytial viruses.

In reverse transcription-polymerase chain reactions (RT-PCR) and DNA hybridizations using primers and an oligonucleotide probe to the fusion (F) protein mRNA of bovine respiratory syncytial virus (BRSV), all the BRSV isolates and a goat isolate could be distinguished from prototype isolates of human respiratory syncytial viruses (HRSV) and ovine (sheep and bighorn sheep) respiratory syncytial viruses (RSV). However, RT-PCR amplifications with primers to sequences of the HRSV F protein mRNA resulted in amplified products of approximately 243 bp if mRNA templates of subgroup A HRSV strains were present and slightly larger amplified products with subgroup B HRSV strains. No amplified products were observed in HRSV-primed RT-PCR with BRSV or goat or ovine RSV mRNA templates. Although the ovine RSV isolates were antigenically cross-reactive with the goat RSV, HRSV and BRSV isolates, they were no amplified with either HRSV- or BRSV-specific primers in RT-PCR. These results confirm previous immunological comparisons suggesting that some ovine RSV isolates should be considered as distinct respiratory syncytial viruses.

Prevalence of Salmonella in raw meat used in diets of racing greyhounds.

One hundred twelve samples of commercial raw meat used in greyhound diets were collected and cultured for Salmonella using standard procedures. Fifty (44.64%) of these samples were positive for Salmonella. Salmonella typhimurium was the most frequently isolated serovar (48%), followed by S. newport (12.76%), S. agona (8.51%), and S. muenster (6.38%). The remaining 10 serovars recovered in this study represented 27.59% of the total Salmonella isolates. In addition, the meat samples were screened for Salmonella using a commercial DNA probe. Of the 106 samples tested, 70 (66.03%) were positive for Salmonella, which indicated that the DNA probe assay was more sensitive than the culture method for screening of Salmonella in raw meat. Antimicrobial susceptibility testing revealed that most of the Salmonella isolates were sensitive to a variety of antimicrobials, particularly amikacin and apramycin, and resistant to some others, such as clindamycin, erythromycin, penicillin, and sulfadimethoxine. The cumulative percentages of susceptibility (MIC50 and MIC90) of the Salmonella isolates were also determined. Most isolates were susceptible (MIC90) to low concentrations of gentamicin (2.0 micrograms/ml), imipenem (< or = 0.25 microgram/ml), and ciprofloxacin (< or = 0.5 microgram/ml). Marked resistance was found with the other antimicrobial agents. However, the high MIC values found for these isolates would not be achievable in vivo with the normal recommended doses of antimicrobial agents, so their use would not be beneficial. Numerous plasmid patterns were found in 17 randomly selected Salmonella isolates. Eight of the 17 isolates had 2-7 plasmids ranging from 2.4 to 15 kilobases in size. Eight isolates also exhibited large plasmids in the range of 50-60 and 95-105 kilobases.(ABSTRACT TRUNCATED AT 250 WORDS)

Application of polymerase chain reaction for the correlation of Salmonella serovars recovered from greyhound feces with their diet.

The polymerase chain reaction was employed to correlate Salmonella serovars isolated from fecal material of greyhounds suffering from gastroenteritis with those isolated from the diet fed to the greyhounds prior to onset of diarrhea. Kennels around the Abilene, Kansas, area were contacted and supplied with materials needed to collect a portion of the diet each day. With the onset of diarrhea, the kennels were instructed to ship the fecal material and diet from the previous 10 days to the laboratory for testing. Forty-one fecal samples and corresponding diets were screened for Salmonella, Clostridium perfringens, Campylobacter jejuni, Staphylococcus aureus, Staphylococcus intermedius, and pathogenic (piliated) Escherichia coli by direct culture using standard procedures. The fecal material was also screened for coronavirus and parvovirus using electron microscopy. Thirty-five "normal" fecal samples were screened for all of the above mentioned microorganisms as a control. In addition, the fecal material was screened for E. coli verotoxins I and II and clostridial enterotoxins. A total of 61 Salmonella isolates were recovered from the 41 samples of feces and diet submitted for testing; 31 were recovered from the feces and 30 from the diet. Four Salmonella isolates were recovered from the normal fecal samples. Results obtained by PCR, plasmid profiles, antigenic analysis, and antibiogram profiles indicated that 16 of the 31 isolates recovered from the fecal material were the same strain as that recovered from the diet.

Multiplex polymerase chain reaction for simultaneous detection of Lawsonia intracellularis, Serpulina hyodysenteriae, and salmonellae in porcine intestinal specimens.

Proliferative enteritis, swine dysentery, and porcine salmonellosis are the most common enteric bacterial diseases affecting pigs in the growing and finishing stages of production. Currently, diagnoses of these diseases by standard cultural techniques of intestinal specimens can be laborious, time consuming, and expensive (swine dysentery, porcine salmonellosis) or impossible (proliferative enteritis). Amplification by polymerase chain reaction (PCR) of DNA sequences specific for each bacterial agent is a highly sensitive and specific method that overcomes the limitations associated with standard detection methods. A multiplex PCR (M-PCR) assay was developed for simultaneous detection and identification of the etiologic agents associated with proliferative enteritis, swine dysentery, and porcine salmonellosis in a single reaction using total DNA obtained directly from intestinal specimens. Purified DNA obtained from pure cultures of each bacterial agent alone or mixed in different combinations and concentrations and total DNA from intestinal specimens were amplified using the Lawsonia intracellularis-, Serpulina hyodysenteriae-, and salmonellae-specific M-PCR assay. Intestinal specimens consisted of feces and mucosal scrapings obtained from field cases of each disease alone or in combinations and feces obtained from pigs challenged with S. hyodysenteriae. The banding pattern of the amplified PCR products, after agarose gel electrophoresis and staining, indicated the presence of individual or combinations of etiologic agents in each specimen. Results from this study indicated that simultaneous amplification of L. intracellularis-, S. hyodysenteriae-, and salmonellae-specific DNA sequences by M-PCR can be used for specific detection and identification of three major enteric bacterial pathogens associated with proliferative enteritis, swine dysentery, and porcine salmonellosis occurring alone or in combinations. Also, the M-PCR assay can be done using DNA obtained directly from intestinal specimens submitted for diagnostic investigation.

Pleuropneumonia caused by Actinobacillus pleuropneumoniae biotype 2 in growing and finishing pigs.

Actinobacillus pleuropneumoniae biotype 2 was isolated in pure culture or as the predominant isolate from the lungs of 9 growing and finishing pigs with pleuropneumonia. Gross and microscopic lesions resembled those caused by A. pleuropneumoniae biotype 1 serotypes (Nos. 1, 5, and 7) traditionally seen in the United States. The overall mortality rate for growing and finishing pigs on this 1,200-sow farrow-to-finish farm ranged from 0.37% to 0.84% per month from July 1990 to February 1991, and mortality due to respiratory disease ranged from 0.17% to 0.52% per month for the same period. This Actinobacillus species did not require V factor (no satellitism on blood agar with a Staphylococcus streak), was strongly beta-hemolytic, and demonstrated restriction fragment length polymorphisms in hybridization studies with A. suis, A. lignieresii, and A. equuli. Biochemically, the isolate most closely resembled A. pleuropneumoniae, and a DNA fragment considered specific for A. pleuropneumoniae biotypes 1 and 2 was demonstrated using polymerase chain reaction. Necrohemorrhagic pleuropneumonia similar to that caused by A. pleuropneumoniae biotype 1 was reproduced experimentally in 2 4-week-old pigs inoculated intratracheally with broth cultures of the A. pleuropneumoniae biotype 2. This study demonstrated the presence of A. pleuropneumoniae biotype 2 in the United States.

Differentiation of Cryptosporidium parvum, C. muris, and C. baileyi by PCR-RFLP analysis of the 18S rRNA gene.

We have developed a combined polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) assay for the detection and differentiation of Cryptosporidium parvum, Cryptosporidium muris, and Cryptosporidium baileyi. The assay utilizes PCR to amplify an approximately 1750 basepair product of the Cryptosporidium 18S rRNA gene. Following double digestion with restriction endonucleases Dral and Vsp1, this PCR product yields DNA fragments that can be resolved electrophoretically into RFLP profiles that are distinct for C. parvum, C. muris, and C. baileyi. Previous PCR-restriction analyses could not simultaneously differentiate all three species. Future application of this technique could include predicting the disease-causing potential of oocyst-contaminated environmental specimens and helping to determine the source of oocyst contamination.

Enhanced karyotype resolution of Cryptosporidium parvum by contour-clamped homogeneous electric fields.

Previous karyotype analysis with contour-clamped homogeneous electric fields (CHEF) indicated that the Cryptosporidium parvum karyotype comprises a minimum of five chromosomes all less than 1.6 Mb in size. In this study we were able to improve the resolution of the karyotype using a modified CHEF gel electrophoretic procedure that allows for the routine identification of seven C. parvum chromosomes ranging in size from about 0.945 to 2.2 Mb. Our data also suggest that an eighth chromosome is present and comigrates with another chromosome at about 1.3 Mb.

Experimental cryptosporidiosis in adult and neonatal rabbits.

Neonatal and adult New Zealand White rabbits were infected experimentally with Cryptosporidium parvum. No histologic evidence of infection was found in adult rabbits. However, increased levels of anti-cryptosporidial serum IgG were present, and multiple antigens were detected by serum on immunoblots. In neonates, variably severe, transient infection was present from Days 3 to 21 postinoculation. Serum IgG was initially elevated, decreased until Day 10 postinoculation, then progressively increased for the remainder of the study. A prominent 15 kilodalton antigen was detected on immunoblots using serum obtained on Days 14 until 28 postinoculation. Neonate anti-cryptosporidial fecal IgA were slightly elevated on Days 14 and 21 postinoculation.