Distribution of the misfolded isoform of the prion protein in peripheral tissues and spinal cord of Rocky Mountain elk (Cervus elaphus nelsoni) with naturally occurring chronic wasting disease.

Chronic wasting disease (CWD) is an infectious transmissible spongiform encephalopathy of cervids associated with the presence of a misfolded prion protein (PrPCWD). Progression of PrPCWD distribution has been described using immunohistochemistry and histologic changes in a single section of brain stem at the level of the obex resulting in scores from 0 (early) to 10 (terminal) in elk with naturally occurring CWD. Here we describe the spread and distribution of PrPCWD in peripheral tissues and spinal cord in 16 wild and 17 farmed Rocky Mountain elk (Cervus elaphus nelsoni) with naturally occurring CWD and correlate these findings with obex scores. Spinal cord and approximately 110 peripheral tissues were collected, processed, stained with hematoxylin and eosin, and immunolabeled with the anti-prion protein monoclonal antibody F99/97.6.1. The medial retropharyngeal and tracheobronchial lymph nodes were the first tissues to accumulate PrPCWD, followed by other lymphoid tissues, myenteric plexus, spinal cord, and finally tissues outside of the lymphatic and neural systems. However, the only significant histological lesion observed was mild spongiform encephalopathy in the dorsal column of the lower spinal cord in elk with an obex score of ≥9. Initial exposure to CWD prions may be through the respiratory system and spread appears to occur primarily via the autonomic nervous system. Therefore, we suggest using obex scores as a proxy for stage of disease progression and verifying with key peripheral tissues.

Evidence of leptospirosis in the kidneys and serum of feral swine (Sus scrofa) in the United States.

Leptospirosis is the most widespread zoonosis in humans worldwide. In the United States, widespread detection of antibodies to leptospirosis have been identified in feral swine (Sus scrofa) with the highest detection of serovars, Bratislava, Icterohaemorrhagiae, and Pomona. Over the past few years, feral swine populations have expanded their geographical range and distribution in the United States with reports in at least 39 of 50 states. Since feral swine serve as reservoirs for serovars that can infect humans, it is important to understand the risk of transmission. In order to learn more about the probability that feral swine shed infectious leptospires, we collected kidneys and paired serum when possible from 677 feral swine in 124 counties of 29 states. These counties had previously been identified as antibody positive for Leptospira interrogans serovars Bratislava, Canicola, Grippotyphosa, Hardjo, Icterohaemorrhagiae or Pomona. Although exposure to these same six serovars of leptospirosis continued to be high (53% overall) in the counties we sampled, we detected leptospiral DNA in only 3·4% of feral swine kidneys tested. Based on these results, it appears that although feral swine can serve as a source of infection to humans, especially in those who are more likely to encounter them directly such as wildlife biologists, veterinarians, and hunters, the risk may be relatively low. However, further studies to examine the relationship between leptospiral shedding in the urine and kidneys in addition to culturing the organism are recommended in order to better understand the risk associated with feral swine.

Clinical and pathologic responses of American crows (Corvus brachyrhynchos) and fish crows (C ossifragus) to experimental West Nile virus infection.

West Nile virus (WNV)-associated disease has a range of clinical manifestations among avian taxa, the reasons for which are not known. Species susceptibility varies within the avian family Corvidae, with estimated mortality rates ranging from 50 to 100%. We examined and compared virologic, immunologic, pathologic, and clinical responses in 2 corvid species, the American crow (Corvus brachyrhynchos) and the fish crow (C ossifragus), following experimental WNV inoculation. Unlike fish crows, which remained clinically normal throughout the study, American crows succumbed to WNV infection subsequent to dehydration, electrolyte and pH imbalances, and delayed or depressed humoral immune responses concurrent with marked, widespread virus replication. Viral titers were approximately 3,000 times greater in blood and 30,000 to 50,000 times greater in other tissues (eg, pancreas and small intestine) in American crows versus fish crows. Histologic lesion patterns and antigen deposition supported the differing clinical outcomes, with greater severity and distribution of lesions and WNV antigen in American crows. Both crow species had multiorgan necrosis and inflammation, although lesions were more frequent, severe, and widespread in American crows, in which the most commonly affected tissues were small intestine, spleen, and liver. American crows also had inflammation of vessels and nerves in multiple tissues, including heart, kidney, and the gastrointestinal tract. WNV antigen was most commonly observed within monocytes, macrophages, and other cells of the reticuloendothelial system of affected tissues. Collectively, the data support that WNV-infected American crows experience uncontrolled systemic infection leading to multiorgan failure and rapid death.

Detection of the abnormal isoform of the prion protein associated with chronic wasting disease in the optic pathways of the brain and retina of Rocky Mountain elk (Cervus elaphus nelsoni).

Eyes and nuclei of the visual pathways in the brain were examined in 30 Rocky Mountain elk (Cervus elaphus nelsoni) representing 3 genotypes of the prion protein gene PRNP (codon 132: MM, ML, or LL). Tissues were examined for the presence of the abnormal isoform of the prion protein associated with chronic wasting disease (PrP(CWD)). Nuclei and axonal tracts from a single section of brain stem at the level of the dorsal motor nucleus of the vagus nerve were scored for intensity and distribution of PrP(CWD) immunoreactivity and degree of spongiform degeneration. This obex scoring ranged from 0 (elk with no PrP(CWD) in the brain stem) to 10 (representing elk in terminal stage of disease). PrP(CWD) was detected in the retina of 16 of 18 (89%) elk with an obex score of > 7. PrP(CWD) was not detected in the retina of the 3 chronic wasting disease-negative elk and 9 elk with an obex score of < 6. PrP(CWD) was found in the nuclei of the visual pathways in the brain before it was found in the retina. Within the retina, PrP(CWD) was first found in the inner plexiform layer, followed by the outer plexiform layer. Intracytoplasmic accumulation of PrP(CWD) was found in a few neurons in the ganglion cell layer in the PRNP 132ML elk but was a prominent feature in the PRNP 132LL elk. Small aggregates of PrP(CWD) were present on the inner surface of the outer limiting membrane in PRNP 132LL elk but not in PRNP 132MM or 132ML elk. This study demonstrates PrP(CWD) accumulation in nuclei of the visual pathways of the brain, followed by PrP(CWD) in the retina.

Identification of Brucella spp. in feral swine (Sus scrofa) at abattoirs in Texas, USA.

Various tissues, nasal swabs, urine and blood samples were collected from 376 feral swine at two federally inspected abattoirs in Texas during six separate sampling periods in 2015. Samples were tested for Brucella spp. by culture and serology. Brucella spp. were cultured from 13.0% of feral swine, and antibodies were detected in 9.8%. Only 32.7% of culture-positive feral swine were also antibody positive, and 43.2% of antibody-positive feral swine were culture positive. Approximately, the same number of males (14.0%) and females (12.1%) were culture positive, and slightly more males (10.5%) than females (8.7%) were antibody positive. Our results indicate that serology likely underestimates the prevalence of feral swine infected, and that those who come in contact with feral swine should be aware of the symptoms of infection with Brucella spp. to ensure prompt treatment.

Comparison of two automated immunohistochemical procedures for the diagnosis of scrapie in domestic sheep and chronic wasting disease in North American white-tailed deer (Odocoileus virginianus) and mule deer (Odocoileus hemionus).

Two commercially available automated immunohistochemistry platforms, Ventana NexES and DakoCytomation Autostainer Universal Staining System, were compared for diagnosing sheep scrapie and cervid chronic wasting disease. Both automated platforms used the same antiprion protein monoclonal primary antibodies, but different platform-specific linker and amplification reagents and procedures. Duplicate sections of brainstem (at the level of the obex) and lymphoid tissue (retropharyngeal lymph node or tonsil) from the same tissue block were immunostained for the comparison. Examination of 1,020 tissues from 796 sheep revealed 100% concordance of results between the Ventana NexES and DakoCytomation platforms for diagnosing sheep scrapie from lymphoid tissue (103/103 positive; 405/405 negative) and brainstem (120/120 positive; 392/392 negative). Similarly, examination of 1,008 tissues from 504 white-tailed deer revealed 100% concordance between the Ventana NexES and DakoCytomation platforms for diagnosing chronic wasting disease from lymphoid tissue (104/104 positive; 400/400 negative) and brainstem (104/104 positive; 400/400 negative). Examination of 1,152 tissues from 482 mule deer revealed a concordance of 98.6% in lymphoid tissue and 99.9% in brainstem between the Ventana NexES and DakoCytomation platforms for diagnosing chronic wasting disease. The results indicate equivalence or near equivalence between the DakoCytomation and Ventana NexES autostainer platforms for identification of the disease-associated prion protein (PrPd)-positive and PrPd-negative brain and lymphoid tissues in sheep, white-tailed deer, and mule deer.

Widespread detection of highly pathogenic H5 influenza viruses in wild birds from the Pacific Flyway of the United States.

A novel highly pathogenic avian influenza virus belonging to the H5 clade 2.3.4.4 variant viruses was detected in North America in late 2014. Motivated by the identification of these viruses in domestic poultry in Canada, an intensive study was initiated to conduct highly pathogenic avian influenza surveillance in wild birds in the Pacific Flyway of the United States. A total of 4,729 hunter-harvested wild birds were sampled and highly pathogenic avian influenza virus was detected in 1.3% (n = 63). Three H5 clade 2.3.4.4 subtypes were isolated from wild birds, H5N2, H5N8, and H5N1, representing the wholly Eurasian lineage H5N8 and two novel reassortant viruses. Testing of 150 additional wild birds during avian morbidity and mortality investigations in Washington yielded 10 (6.7%) additional highly pathogenic avian influenza isolates (H5N8 = 3 and H5N2 = 7). The geographically widespread detection of these viruses in apparently healthy wild waterfowl suggest that the H5 clade 2.3.4.4 variant viruses may behave similarly in this taxonomic group whereby many waterfowl species are susceptible to infection but do not demonstrate obvious clinical disease. Despite these findings in wild waterfowl, mortality has been documented for some wild bird species and losses in US domestic poultry during the first half of 2015 were unprecedented.

Experimental exposure of llamas (Lama glama) to Brucella abortus: humoral antibody response.

Positive antibody reactions to brucella were observed in the sera of four llamas receiving Brucella abortus Strain 19 subcutaneously at 2-3 weeks post-exposure (PE) using five of eight conventional brucella serologic tests and an ISU-ELISA. Positive brucella antibody reactions were detected in sera of four llamas exposed by intraocular instillation (IOI) of 1.02x10(8) (high dose) B. abortus Strain 2308 at 16-35 days PE using seven of eight serologic tests or an ISU-ELISA. Brucella antibody was also detected in sera of four llamas exposed by IOI of 9x10(5) (low dose) B. abortus using each of four agglutination tests, Complement Fixation test, PCFIA, the rivanol test and the ISU-ELISA at 16-35 days PE. Positive reactions were observed using the Card test, BAPA, SPT, STT, the rivanol test, the PCFIA, and the ISU-ELISA on sera collected on days 42-70 PE, except on one llama, given the low dose; that llama was negative on the PCFIA on day 42. Positive or suspicious reactions were not detected in sera of controls, receiving saline subcutaneously, using the routine tests, with the exception of the CFT. The B. abortus Strain 2308 was isolated from tissues of seven of eight llamas exposed to virulent B. abortus Strain 2308.

Seroconversion and abortion in cattle experimentally infected with Brucella sp. isolated from a Pacific harbor seal (Phoca vitulina richardsi).

Previously unrecognized Brucella species have been isolated from a number of marine mammals, including harbor seals (Phoca vitulina richardsi) in the Puget Sound area of the state of Washington. Because of the presence of dairy herds in proximity to the harbor seal populations, a study was conducted to determine the effects of the harbor seal Brucella isolate in experimentally inoculated cattle. Six pregnant cattle were exposed by intravenous injection (n = 3) or intraconjunctival inoculation (n = 3). Two pregnant cows were intravenously injected with saline and served as controls. All of the cows receiving the Brucella seroconverted on 1 or more tests commonly used for the detection of Brucella abortus infection. Two of the cattle receiving the intravenous inoculation aborted, and brucellae were demonstrated in the fetuses and dams immediately following abortion. The remaining 4 Brucella-inoculated animals and their fetuses were culture negative for the organism at 14 weeks postinoculation. Results of this study indicate the marine mammal Brucella is capable of producing seroconversion and abortion in cattle but is less pathogenic in that species than B. abortus.

Seminal vesiculitis and orchitis caused by Brucella abortus biovar 1 in young bison bulls from South Dakota.

Specimens of blood, lymph nodes, spleens, and genitalia were collected at slaughter from seven 3- and 4-year-old male bison that had recently become seropositive for brucellosis. The animals were from a captive herd of approximately 3,500 bison located in central South Dakota. Brucella abortus biovar 1 was isolated from 2 or more specimens from each of 6 bison. Severe necrotizing and pyogranulomatous orchitis was present in 1 testicle from 1 bull, and 4 animals had mild to marked seminal vesiculitis. Immunohistochemical staining labeled organisms in seminal vesicles and the testicle with orchitis. Ultrastructurally, intact bacilli were present in cytoplasmic vacuoles of some macrophages; other macrophages contained intracytoplasmic aggregates of calcified coccobacilli.

Experimental and serologic observations on avian pneumovirus (APV/turkey/Colorado/97) infection in turkeys.

An avian pneumovirus (APV) was isolated from commercial turkeys in Colorado (APV/Colorado) showing clinical signs of a respiratory disease. The results of virus neutralization and indirect fluorescent antibody tests showed that the APV/Colorado was partially related to APV subgroup A but was unrelated to APV subgroup B. Turkeys experimentally inoculated with the APV/Colorado were observed for signs, lesions, seroconversion, and virus shedding. Thirty-six 7-wk-old turkeys were distributed into three groups. Eighteen turkeys were inoculated oculonasally with APV/Colorado, six were placed in contact at 1 day postinoculation (DPI), and 12 served as noninoculated controls. Tracheal swabs and blood samples were collected at 3, 5, 7, 10, 14, and 21 DPI. Tissues were collected from three inoculated and two control turkeys on aforementioned days for pathologic examination and APV isolation. Inoculated turkeys developed respiratory disease, yielded APV at 3, 5, and 7 DPI, and seroconverted at 10 DPI. Contact turkeys yielded APV at 7 and 10 DPI. No gross lesions were observed in the turbinates, infraorbital sinuses, and trachea. However, microscopic examination revealed acute rhinitis, sinusitis, and tracheitis manifested by congestion, edema, lymphocytic and heterophilic infiltration, and loss of ciliated epithelia. The inflammatory lesions were seen at 3 DPI and became extensive at 5 and 7 DPI. Active regenerative changes in the epithelia were seen at 10 and 14 DPI. Serologic survey for the presence of antibodies in commercial turkeys (24,504 sera from 18 states) and chickens (3,517 sera from 12 states) to APV/Colorado showed seropositive turkeys in Minnesota, North Dakota, and South Dakota and no seropositive chickens. This report is the first on the isolation of an APV and APV infection in the United States.

Biosafety and antibody responses of adult bison bulls after vaccination with Brucella abortus strain RB51.

OBJECTIVE: To evaluate clearance, antibody responses, potential shedding, and histologic lesions in reproductive tissues of adult bison bulls after vaccination with Brucella abortus strain RB51. ANIMALS: 61 two- and 3-year-old bison bulls. PROCEDURE: 12 bison bulls were vaccinated s.c. with B abortus strain RB51, 3 were inoculated s.c. with 0.15 M NaCl, and antibody responses were evaluated. Various specimens were obtained to evaluate bacterial shedding. Four vaccinates and 1 control were necropsied 10, 20, and 30 weeks after vaccination. In a separate experiment, bison bulls were vaccinated s.c. with 0.15 M NaCl, or by hand or ballistically with strain RB51. Antibody responses were monitored 6 weeks after vaccination and during necropsy 13 weeks after vaccination. Tissue specimens obtained during necropsy from both studies were evaluated bacteriologically and histologically. RESULTS: Strain RB51 was recovered at various times from semen of 3 of 12 vaccinated bison bulls in experiment 1. During necropsy, strain RB51 was recovered 10 and 20, but not 30, weeks after vaccination. In experiment 2, strain RB51 was recovered from lymphoid tissues of hand- and ballistic-vaccinated bison bulls during necropsy. In both experiments, microscopic lesions in testes, epididymis, and seminal vesicles were minimal and did not differ between strain RB51-vaccinated and saline-inoculated bison bulls. CONCLUSIONS AND CLINICAL RELEVANCE: Strain RB51 does not induce relevant inflammatory lesions in reproductive tissues of adult bison bulls. Shedding of strain RB51 in semen may be transient in some bison bulls; however, the importance of this observation is unknown.

Safety of Brucella abortus strain RB51 in black bears.

In two studies conducted from October 1999 to March 2000 and December 2000 to April 2001, adult black bears (Ursus americanus) were orally inoculated with 1.4-3.1 x 10(10) colony-forming units (CFU) of Brucella abortus strain RB51 (SRB51, n=12) or 2 ml of 0.15 M NaCl solution (saline, n=11). We did not detect a difference (P>0.05) in antibody titers to SRB51 in serum obtained before vaccination, at 8 wk after vaccination, or at necropsy at 21 or 23 wk after vaccination between SRB51-vaccinated and nonvaccinated bears. The SRB51 vaccine strain was recovered from tissues obtained at necropsy from one of six SRB51-vaccinated bears in study 1, but none of the six SRB51-vaccinated bears in study 2. Vaccination of black bears with SRB51 did not appear to influence (P>0.05) reproductive performance.

Pathology of brucellosis in bison from Yellowstone National Park.

Between February 1995 and June 1999, specimens from seven aborted bison (Bison bison) fetuses or stillborn calves and their placentas, two additional placentas, three dead neonates, one 2-wk-old calf, and 35 juvenile and adult female bison from Yellowstone National Park (USA) were submitted for bacteriologic and histopathologic examination. One adult animal with a retained placenta had recently aborted. Serum samples from the 35 juvenile and adult bison were tested for Brucella spp. antibodies. Twenty-six bison, including the cow with the retained placenta, were seropositive, one was suspect, and eight were seronegative. Brucella abortus biovar 1 was isolated from three aborted fetuses and associated placentas, an additional placenta, the 2-wk-old calf, and 11 of the seropositive female bison including the animal that had recently aborted. Brucella abortus biovar 2 was isolated from one additional seropositive adult female bison. Brucella abortus was recovered from numerous tissue sites from the aborted fetuses, placentas and 2-wk-old calf. In the juvenile and adult bison, the organism was more frequently isolated from supramammary (83%), retropharyngeal (67%), and iliac (58%) lymph nodes than from other tissues cultured. Cultures from the seronegative and suspect bison were negative for B. abortus. Lesions in the B. abortus-infected, aborted placentas and fetuses consisted of necropurulent placentitis and mild bronchointerstitial pneumonia. The infected 2-wk-old calf had bronchointerstitial pneumonia, focal splenic infarction, and purulent nephritis. The recently-aborting bison cow had purulent endometritis and necropurulent placentitis. Immunohistochemical staining of tissues from the culture-positive aborted fetuses, placentas, 2-wk-old calf, and recently-aborting cow disclosed large numbers of B. abortus in placental trophoblasts and exudate, and fetal and calf lung. A similar study with the same tissue collection and culture protocol was done using six seropositive cattle from a B. abortus-infected herd in July and August, 1997. Results of the bison and cattle studies were similar.

Experimental Brucella abortus induced abortion in a llama: pathologic effects.

Brucella abortus infection has not been documented in llamas. This report describes the abortion of the only pregnant animal in a group of 12. The llama was infected by inoculating 1 x 10(8) viable B. abortus organisms into the conjunctival sac. Forty-three days postinfection, the llama aborted a fetus of approximately 8 months gestational age. Brucella organisms were isolated from the placenta and all fetal specimens examined. These organisms were also isolated from the dam's mammary gland and numerous lymph nodes when the llama was necropsied 42 days later. Microscopically, there was a moderate, multifocal, lymphocytic and histiocytic, subacute placentitis with marked loss of trophoblastic epithelial cells. The superficial chorioallantoic stroma contained abundant necrotic and mineralized debris as well as numerous swollen capillaries protruding multifocally from the denuded surface. Immunohistochemistry revealed that these capillaries, as well as sloughed and intact trophoblasts, were expanded by large numbers of Brucella organisms. Brucellar antigen was also detected in occasional macrophages in the fetal kidney and lung. Ultrastructurally, bacteria labeled by an antibody-based colloidal gold procedure were located within degenerate capillaries, within necrotic leukocytes, and extracellularly in the placental stroma.