Laboratory characterization of invasive Haemophilus influenzae isolates from Nunavut, Canada, 2000-2012.

BACKGROUND: With invasive Haemophilus influenzae serotype b (Hib) disease controlled by vaccination with conjugate Hib vaccines, there is concern that invasive disease due to non-serotype b strains may emerge. OBJECTIVE: This study characterized invasive H. influenzae (Hi) isolates from Nunavut, Canada, in the post-Hib vaccine era. METHODS: Invasive H. influenzae isolates were identified by conventional methods at local hospitals; and further characterized at the provincial and federal public health laboratories, including detection of serotype antigens and genes, multi-locus sequence typing and antibiotic susceptibility. RESULTS: Of the 89 invasive H. influenzae cases identified from 2000 to 2012, 71 case isolates were available for study. There were 43 serotype a (Hia), 12 Hib, 2 Hic, 1 Hid, 1 Hie, 2 Hif and 10 were non-typeable (NT). All 43 Hia were biotype II, sequence type (ST)-23. Three related STs were found among the Hib isolates: ST-95 (n=9), ST-635 (n=2) and ST-44 (n=1). Both Hif belonged to ST-124 and the 2 Hic were typed as ST-9. The remaining Hid (ST-1288) and Hie (ST-18) belonged to 2 separate clones. Of the 10 NT strains, 3 were typed as ST-23 and the remaining 7 isolates each belonged to a unique ST. Eight Hib and 1 NT-Hi were found to be resistant to ampicillin due to ß-lactamase production. No resistance to other antibiotics was detected. CONCLUSION: During the period of 2000-2012, Hia was the predominant serotype causing invasive disease in Nunavut. This presents a public health concern due to an emerging clone of Hia as a cause of invasive H. influenzae disease and the lack of published guidelines for the prophylaxis of contacts. The clonal nature of Hia could be the result of spread within an isolated population, and/or unique characteristics of this strain to cause invasive disease. Further study of Hia in other populations may provide important information on this emerging pathogen. No antibiotic resistance was detected among Hia isolates; a small proportion of Hib and NT-Hi isolates demonstrated resistance to ampicillin due to ß-lactamase production.

Use of capillary electrophoresis and fluorescent labeled peptides to detect the abnormal prion protein in the blood of animals that are infected with a transmissible spongiform encephalopathy.

Transmissible spongiform encephalopathies in humans and in animals are fatal neuro-degenerative diseases with long incubation times. The putative cause of these diseases is a normal host protein, the prion protein, that becomes altered. This abnormal prion protein is found mostly in the brains of infected individuals in later stages of the disease, but also can be found in lymphoid and other tissues in lower amounts. In order to eradicate this disease in animals, it is important to develop a system that can concentrate the abnormal prion protein and an assay that is very sensitive. The sensitivity that can be achieved with capillary electrophoresis makes it possible to detect the abnormal protein in blood. A peptide from the carboxyl terminal region, amino acid positions 218-232, was labeled with fluorescein during the synthesis of the peptide at the amino terminus. Antibodies that have been produced to this peptide were affinity purified and used in a capillary electrophoresis immunoassay. The amount of fluorescein labeled peptide in the capillary was 50 amol. Blood was obtained from normal sheep and elk, from sheep infected with scrapie and elk infected with chronic wasting disease. Buffy coats and plasma were prepared by a conventional method. After treatment with proteinase K, which destroys the normal protein but not the altered one, the blood fractions were extracted and tested in the capillary electrophoresis immunoassay for the abnormal prion protein. The abnormal prion protein was detected in fractions from blood from infected animals but not from normal animals. This assay makes a pre-clinical assay possible for these diseases and could be adapted to test for the abnormal prion protein in process materials that are used for manufacture of pharmaceuticals and products for human consumption.

Polymerase chain reaction identification of Mycobacterium avium in formalin-fixed, paraffin-embedded animal tissues.

A PCR procedure previously developed for identification of Mycobacterium bovis in formalin-fixed tissues was used to identify mycobacteria of the M. avium complex. Tissues were examined from 100 culture-positive cases of M. avium complex infection, including 86 in which the subspecies was not identified and 14 that had been identified as M. avium subsp. paratuberculosis. Each sample was tested with 5 primer sets, 16S ribosomal RNA (rRNA), IS900, IS901, IS1245, and a heat shock protein (hspX), that detect 1 or both M. avium subspecies. The success rate of PCR detection varied with the primers used and the animal species tested. Among the 86 cases with no M. avium subspecies designation, primers for the 16S rRNA gene were clearly the most efficient because they produced amplicons from all samples that reacted with any other primer set. The overall detection rate in this group of samples was 71%: highest in avian tissues (89%) followed by swine (72%) and ruminants (57%) None of the avian or swine tissues reacted with primers for IS900 or hspX, which identify M. a. paratuberculosis. In contrast, 7 of the 12 ruminant samples that were 16S rRNA positive reacted with 1 or both of these primers. All of the 14 cases shown by culture to be M. a. paratuberculosis infections were positive with IS900 primers, whereas only 11 were positive for 16S rRNA. These results indicate that 16S rRNA primers are the most useful for PCR identification of M. avium in formalin-fixed tissues of nonruminant species. However, IS900 primers should also be used when ruminant tissues are examined because these primers provide the greatest sensitivity for detection of M. a. paratuberculosis infections.

Immunohistochemical diagnosis of chronic wasting disease in preclinically affected elk from a captive herd.

An immunohistochemical (IHC) method was used to test brain tissues from 17 elk in a captive herd in which chronic wasting disease (CWD) had previously occurred. The IHC technique detects the protease-resistant prion protein (PrP-res), which is considered a disease-specific marker for transmissible spongiform encephalopathies (TSE), regardless of the species affected. Of the 17 elk tested, 10 were positive by IHC. Only 2 of these 10 animals had shown clinical signs and histologic lesions of CWD, and an additional animal had histologic lesions only. The most consistently IHC-positive tissue was medulla oblongata, especially the obex. These results show that the PrP-res IHC test on brain tissue, specifically medulla oblongata at the obex, should be considered an essential component of any surveillance study intended to determine the incidence of CWD in captive or free-ranging cervids.

Clinical, serologic, and histopathologic characterization of experimental Borna disease in ponies.

Borna disease was originally described as an equine neurologic syndrome over 200 years ago, although the infectious etiology of the disorder was unproven until the early 20th century. Borna disease virus (BDV) was finally isolated from horses dying of the disorder, and that virus has been used to experimentally reproduce Borna disease in several species of laboratory animals. However, BDV has never been inoculated back into horses to experimentally and etiologically confirm the classic clinical, pathologic, and serologic characteristics of the disease in that species. Three ponies were intracerebrally inoculated with different amounts of BDV and were evaluated clinically, serologically, and neurohistopathologically. All 3 animals developed the clinical signs characteristically described for naturally occurring Borna disease, including ataxia, torticollis, postural unawareness, rhythmic repetitive motor activities, muscle fasciculation, and cutaneous hyperesthesia and hypoesthesia over several body surfaces. Two ponies died after rapid onset of these signs 28-30 days postinoculation. The third animal made a nearly complete clinical recovery. Seroconversion occurred only after the onset of signs and to a marked degree only in the convalescent animal. Virus was recovered postmortem from 2 of the 3 ponies, and a BDV-specific nucleic acid sequence was detectable in all 3 animals using a reverse transcription-polymerase chain reaction procedure. Gross neural lesions were absent, but histopathologically there was generalized intense mononuclear perivascular cuffing, glial nodule formation, and astrocytosis in all 3 brains. Confirming a diagnosis of Borna disease is difficult and perhaps best accomplished using a combination of the clinical, serologic, and histopathologic indicators of this unusual disease supported by positive reverse transcription-polymerase chain reaction findings.

Immunohistochemical detection of prion protein in sheep with scrapie.

Prion protein (PrP), which is involved in the pathogenesis of scrapie, occurs in 2 forms. The form extracted from scrapie brain is protease resistant (PrP-res), whereas PrP from normal brain is protease sensitive (PrP-sen). This study examined whether PrP-res could be detected in brains of sheep with scrapie by immunohistochemistry (IHC). A suitable IHC procedure was developed using brain tissue from hamsters that had been inoculated with the transmissible mink encephalopathy agent. Tissue samples were fixed in PLP (periodate, lysine, paraformaldehyde) that contained paraformaldehyde at a concentration of 0.125%. Before application of the IHC technique, tissue sections were deparaffinized and treated with formic acid to simultaneously enhance PrP-res immunoreactivity and degrade PrP-sen. Primary antibody was obtained from a rabbit immunized to PrP-res extracted from brains of mice with experimentally induced scrapie. Brain from 21 sheep with histopathologically confirmed scrapie were examined by IHC. In all 21 brains, PrP-res was widely distributed throughout the brain stem. Staining was particularly intense in neuronal cell bodies and around blood vessels. The IHC technique successfully detected PrP-res in brain samples that had been frozen or that were severely autolyzed before fixation in PLP. Brains from 11 scrapie-suspect sheep that were not considered histologically positive were also examined by IHC. PrP-res was found in 4 of these brains. Sections of brains from 14 clinically normal sheep did not have detectable PrP-res. Results of this study indicate that IHC detection of PrP-res is equivalent, and perhaps superior, to histopathology for the diagnosis of scrapie in sheep. Furthermore, IHC is applicable to tissues that have autolytic changes or processing artifacts that prevent satisfactory histopathologic evaluation for lesions of scrapie.

Resistance of cattle to scrapie by the oral route.

Early epidemiological information indicated that bovine spongiform encephalopathy (BSE) originated from scrapie in sheep. The question arose if scrapie in North America would induce a BSE-like disease in cattle. Six years ago, we reported that brain tissue from sheep with scrapie caused a neurologic disease when injected directly into the brains of cattle, but the disease induced was different from BSE as it occurs in the United Kingdom and Europe. Here, we report that cattle fed raw brain or meat and bone meal and tallow prepared from sheep with scrapie remained normal for 8 years after exposure. This work indicates that cattle are highly resistant to North American scrapie by the oral route.

Relationship of sulfamethazine in swine diets and resultant tissue concentrations, using Tishler and gas liquid chromatographic methods.

Sulfamethazine (110 mg/kg of feed) was fed to 4 groups of pigs for 10 days. Each group was then fed withdrawal rations containing 0, 1.1, 11.0, or 110.0 mg/kg of feed for 10 days before slaughter. A 5th group was fed a sulfamethazine-free diet only. Fat, kidney, liver, and muscle from each pig were analyzed by several laboratories, using the Tishler Bratton-Marshall (BM) and gas liquid chromatographic (GLC) methods. Mean background (BG) amounts of sulfamethazine, as measured in control tissues, were essentially negligible using GLC; however, BM BG values (mean +/- SD), expressed as milligrams of sulfamethazine per kilogram of edible animal tissue (ppm) were: fat 0.019 +/- 0.003, kidney 0.048 +/- 0.012, liver 0.053 +/- 0.034, and muscle 0.018 +/- 0.017. Regression of tissue sulfamethazine on feed sulfamethazine, as measured by GLC and BM adjusted for BM-BG values, yielded essentially linear plots. Values obtained by GLC and corrected BM were highly correlated, but the variability was higher, using GLC. The linear model predicted that the dietary sulfamethazine required to produce 0.1 ppm in liver as measured by the BM, BM-BG, and GLC were 1.6, 2.8, and 3.4 ppm, respectively. The concentrations necessary to produce 0.1 ppm in muscle were 5.3, 6.2, and 5.5 ppm. The thin-layer chromatographic method of screening swine plasma was a good predictor of tissue sulfamethazine values.

An outbreak of type C botulism in 12 horses and a mule.

A USDA Early Response Team investigated deaths of several horses and a mule in northern Arizona at the request of local animal health officials. Thirteen animals (12 horses and 1 mule) housed at 5 facilities in a 7.4 square mile area died between August 1998 and January 1999. Clinical signs consisted of muscular weakness that rapidly progressed to lateral recumbency. Ten animals had paresis of the tongue, throat, or lips. Affected animals appeared alert and were interested in eating and drinking, even while recumbent. All 13 animals were euthanatized. Clostridium botulinum type C was isolated from feces or intestinal contents from 3 affected horses. Preformed toxin was detected in samples of soil and bird droppings collected from a nearby horse burial site. It was hypothesized that the outbreak was a result of birds, presumably ravens, feeding at the burial site and at horse facilities in the area that transferred toxin to the affected animals.

Improvements in a competition assay to detect scrapie prion protein by capillary electrophoresis.

Scrapie in sheep and goats is the prototype of transmissible spongiform encephalopathies found in humans and animals. A feature of these diseases is the accumulation of rod-shaped fibrils in the brain that form from an aggregated protein. This protein is a protease-resistant form of a normal host cell protein. When the aggregated protein is denatured in SDS and beta-mercaptoethanol, a monomer form (prion protein) with a molecular mass of 27 kDa is observed. Free zone capillary electrophoresis and peptides labeled with fluorescein were used to detect the prion protein through competition for a labeled peptide in immune complex formation. The separation of the immune complexes from the unbound peptide using 200 mM Tricine (pH 8.0) was faster and was better resolved than that obtained with phosphate or borate buffer systems. The amount of immune complex formation was dependent on the amount of antibody in the assay. The amount of bound labeled peptide and unbound labeled peptide could be measured directly by calculating the area of each respective peak. As increasing amounts of unlabeled peptide were added to the assay, a concentration dependent reduction in the immune complex peak was observed. The assay could detect less than 10.0 fmol of unlabeled peptide. There was a quantitative difference in the competition of preparations from scrapie infected sheep brain and normal sheep brain.

Surveillance for lesions of bovine spongiform encephalopathy in U.S. cattle.

The appearance of bovine spongiform encephalopathy (BSE) as a new disease of cattle in 1985-1987 increased worldwide interest in various aspects of human and animal spongiform encephalopathies. In the United States, a part of the surveillance effort has been directed toward prospective examination of bovine brain specimens for lesions of BSE. One focus area has been to obtain specimens from cattle that (1) are two years of age or older, (2) have documented signs of neurologic disease, and (3) have received protein supplement as a substantial part of the i.v. ration. Another focus area has been to examine rabies-suspect cases that were rabies-negative. A third area has been to obtain the results of bovine neuropathology examinations being conducted at other state and regional laboratories. Specimens have been obtained by direct submission and by referral from other public health and veterinary diagnostic laboratories. Many of the cases have been classified as having (1) inflammatory lesions such as listeriosis, pseudorabies, brain abscesses and inflammation of undetermined cause, (2) degenerative lesions such as polio-encephalomalacia, lead poisoning, Wallerian degeneration, siderocalcinosis, and lipofuscinosis, (3) neoplastic lesions such as meningioma and Schwannoma, and (4) no significant findings. Other case results were reported as inflammation or no significant findings. Of the 459 cases reported here none has contained lesions with the characteristics and distribution typical of BSE.

Preliminary observations on the experimental transmission of scrapie to elk (Cervus elaphus nelsoni) by intracerebral inoculation.

To determine the transmissibility of scrapie to Rocky Mountain elk (Cervus elaphus nelsoni), six elk calves were inoculated intracerebrally with brain suspension from sheep naturally affected with scrapie. One elk developed a brain abscess and was euthanatized at 7 weeks postinoculation (PI), and two others died at 6 and 15 months PI because of physical injuries. At 25 and 35 months PI, two other elk died after brief terminal neurologic episodes. Necropsy of these revealed moderate weight loss but no other gross lesions. Microscopically, characteristic lesions of spongiform encephalopathy were seen throughout the brains and the spinal cords, and in both cases these tissues were positive for PrP(res) by immunohistochemistry. Brains of both animals were positive for PrP(res) by western blot and for scrapie-associated fibrils (SAFs) by negative stain electron microscopy. PrP(res) and SAFs were not detected in the three elk that died or were euthanatized because of coincidental causes. Over 3.5 years after initiation of this experiment, the one remaining inoculated elk and two uninoculated (control) elk are alive and apparently healthy. These preliminary findings demonstrate that 1) sheep scrapie agent can be transmitted to elk by intracerebral inoculation; 2) the infection can result in severe, widely distributed spongiform change and accumulations of PrP(res) in the central nervous system (CNS); and 3) based on the examination of a limited number of CNS sections from two cases, this condition cannot be distinguished from chronic wasting disease with currently available diagnostic techniques.

Intracerebral transmission of scrapie to cattle.

To determine if sheep scrapie agent(s) in the United States would induce a disease in cattle resembling bovine spongiform encephalopathy, 18 newborn calves were inoculated intracerebrally with a pooled suspension of brain from 9 sheep with scrapie. Half of the calves were euthanatized 1 year after inoculation. All calves kept longer than 1 year became severely lethargic and demonstrated clinical signs of motor neuron dysfunction that were manifest as progressive stiffness, posterior paresis, general weakness, and permanent recumbency. The incubation period was 14-18 months, and the clinical course was 1-5 months. The brain from each calf was examined for lesions and for protease-resistant prion protein. Lesions were subtle, but a disease-specific isoform of the prion protein was present in the brain of all calves. Neither signs nor lesions were characteristic of those for bovine spongiform encephalopathy.

Homozygosity for prion protein alleles encoding glutamine-171 renders sheep susceptible to natural scrapie.

Natural scrapie has been viewed both as a recessive trait and as a contagious disease modulated by a host locus. To address this conundrum, we determined the structure of the sheep prion protein (PrP) gene, which contains three exons and extends over 20 kb of DNA. In the United States 86.4% of scrapie cases occur in Suffolk sheep, and within this breed 49 +/- 6% (+/- S.D., n = 69) of healthy animals carry one or more PrP alleles encoding Arg (R)-171. Four scrapie-affected sheep were homozygous for wild-type PrP open reading frames encoding the alternative Gln (Q)-171 allele. Analysis of additional cases revealed that all were Q/Q-171 homozygotes (n = 31), yielding a probability of 0.000004 that PrP genotype is unrelated to susceptibility. These data imply that homozygosity for Q-171 codons is necessary but not sufficient for the development of natural scrapie, echo reports of recessive manifestation, and parallel over-representation of PRNP codon 129 homozygotes in Creutzfeldt-Jakob disease of humans. Whereas progress has been substantial regarding experimental scrapie in rodents, the occurrence and spread of disease in flocks of sheep has remained enigmatic. Appreciation of the relationship between codon 171 genotype and susceptibility may help define the molecular basis of natural scrapie.