Disease phenotype of classical sheep scrapie is changed upon experimental passage through white-tailed deer.

Prion agents occur in strains that are encoded by the structure of the misfolded prion protein (PrPSc). Prion strains can influence disease phenotype and the potential for interspecies transmission. Little is known about the potential transmission of prions between sheep and deer. Previously, the classical US scrapie isolate (No.13-7) had a 100% attack rate in white-tailed deer after oronasal challenge. The purpose of this study was to test the susceptibility of sheep to challenge with the scrapie agent after passage through white-tailed deer (WTD scrapie). Lambs of various prion protein genotypes were oronasally challenged with WTD scrapie. Sheep were euthanized and necropsied upon development of clinical signs or at the end of the experiment (72 months post-inoculation). Enzyme immunoassay, western blot, and immunohistochemistry demonstrated PrPSc in 4 of 10 sheep with the fastest incubation occurring in VRQ/VRQ sheep, which contrasts the original No.13-7 inoculum with a faster incubation in ARQ/ARQ sheep. Shorter incubation periods in VRQ/VRQ sheep than ARQ/ARQ sheep after passage through deer was suggestive of a phenotype change, so comparisons were made in ovinized mice and with sheep with known strains of classical sheep scrapie: No. 13-7 and x-124 (that has a more rapid incubation in VRQ/VRQ sheep). After mouse bioassay, the WTD scrapie and x-124 isolates have similar incubation periods and PrPSc conformational stability that are markedly different than the original No. 13-7 inoculum. Furthermore, brain tissues of sheep with WTD scrapie and x-124 scrapie have similar patterns of immunoreactivity that are distinct from sheep with No. 13-7 scrapie. Multiple lines of evidence suggest a phenotype switch when No. 13-7 scrapie prions are passaged through deer. This represents one example of interspecies transmission of prions resulting in the emergence or selection of new strain properties that could confound disease eradication and control efforts.

Characterization of Classical Sheep Scrapie in White-tailed Deer after Experimental Oronasal Exposure.

BACKGROUND: Classic scrapie is a prion disease of sheep and goats that is associated with accumulation of abnormal prion protein (PrPSc) in the central nervous and lymphoid tissues. Chronic wasting disease (CWD) is the prion disease of cervids. This study was conducted to determine the susceptibility of white-tailed deer (WTD) to the classic scrapie agent. METHODS: We inoculated WTD (n = 5) by means of a concurrent oral/intranasal exposure with the classic scrapie agent from sheep or oronasally with the classic scrapie agent from goats (n = 6). RESULTS: All deer exposed to the agent of classic scrapie from sheep accumulated PrPSc. PrPSc was detected in lymphoid tissues at preclinical time points, and necropsies in deer 28 months after inoculation showed clinical signs, spongiform lesions, and widespread PrPSc in neural and lymphoid tissues. Western blots on samples from the brainstem, cerebellum, and lymph nodes of scrapie-infected WTD have a molecular profile similar to CWD and distinct from samples from the cerebral cortex, retina, or the original classic scrapie inoculum. There was no evidence of PrPSc in any of the WTD inoculated with classic scrapie prions from goats. CONCLUSIONS: WTD are susceptible to the agent of classic scrapie from sheep, and differentiation from CWD may be difficult.

Transmission of Raccoon-Passaged Chronic Wasting Disease Agent to White-Tailed Deer.

The transmission characteristics of prion diseases are influenced by host prion protein sequence and, therefore, the host species. Chronic wasting disease (CWD), a prion disease of cervids, has widespread geographical distribution throughout North America and occurs in both wild and farmed populations. CWD prions contaminate the environment through scattered excrement and decomposing carcasses. Fresh carcasses with CWD prions are accessible by free-ranging mesopredators such as raccoons and may provide a route of exposure. Previous studies demonstrated the susceptibility of raccoons to CWD from white-tailed deer. In this study, we demonstrate that white-tailed deer replicate raccoon-passaged CWD prions which results in clinical disease similar to intraspecies CWD transmission. Six white-tailed deer were oronasally inoculated with brain homogenate from a raccoon with CWD. All six deer developed clinical disease, had widespread lymphoid distribution of misfolded CWD prions (PrPSc), and had neuropathologic lesions with PrPSc accumulation in the brain. The presence of PrPSc was confirmed by immunohistochemistry, enzyme-linked immunoassay, and western blot. The western blot migration pattern of raccoon-passaged CWD was different from white-tailed deer CWD. Transmission of raccoon CWD back to white-tailed deer resulted in an interposed molecular phenotype that was measurably different from white-tailed deer CWD.

Increased Attack Rates and Decreased Incubation Periods in Raccoons with Chronic Wasting Disease Passaged through Meadow Voles.

Chronic wasting disease (CWD) is a naturally-occurring neurodegenerative disease of cervids. Raccoons (Procyon lotor) and meadow voles (Microtus pennsylvanicus) have previously been shown to be susceptible to the CWD agent. To investigate the potential for transmission of the agent of CWD from white-tailed deer to voles and subsequently to raccoons, we intracranially inoculated raccoons with brain homogenate from a CWD-affected white-tailed deer (CWDWtd) or derivatives of this isolate after it had been passaged through voles 1 or 5 times. We found that passage of the CWDWtd isolate through voles led to a change in the biologic behavior of the CWD agent, including increased attack rates and decreased incubation periods in raccoons. A better understanding of the dynamics of cross-species transmission of CWD prions can provide insights into how these infectious proteins evolve in new hosts.

Experimental Oronasal Transmission of Chronic Wasting Disease Agent from White-Tailed Deer to Suffolk Sheep.

Chronic wasting disease (CWD) is a fatal prion disease of cervids. We examined host range of CWD by oronasally inoculating Suffolk sheep with brain homogenate from a CWD-positive white-tailed deer. Sixty months after inoculation, 1/7 sheep had immunoreactivity against the misfolded form of prion protein in lymphoid tissue. Results were confirmed by mouse bioassay.

Transmission of the atypical/Nor98 scrapie agent to Suffolk sheep with VRQ/ARQ, ARQ/ARQ, and ARQ/ARR genotypes.

Scrapie is a transmissible spongiform encephalopathy that occurs in sheep. Atypical/Nor98 scrapie occurs in sheep that tend to be resistant to classical scrapie and it is thought to occur spontaneously. The purpose of this study was to test the transmission of the Atypical/Nor98 scrapie agent in three genotypes of Suffolk sheep and characterize the distribution of misfolded prion protein (PrPSc). Ten sheep were intracranially inoculated with brain homogenate from a sheep with Atypical/Nor98 scrapie. All sheep with the ARQ/ARQ and ARQ/ARR genotypes developed Atypical/Nor98 scrapie confirmed by immunohistochemistry, and one sheep with the VRQ/ARQ genotype had detectable PrPSc consistent with Atypical/Nor98 scrapie at the experimental endpoint of 8 years. Sheep with mild early accumulations of PrPSc in the cerebellum had concomitant retinal PrPSc. Accordingly, large amounts of retinal PrPSc were identified in clinically affected sheep and sheep with dense accumulations of PrPSc in the cerebellum.

Experimental inoculation of CD11c+ B1 lymphocytes, CD68+ macrophages, or platelet-rich plasma from scrapie-infected sheep into susceptible sheep results in variable infectivity.

Many studies have demonstrated prion infectivity in whole blood and blood components in a variety of transmissible spongiform encephalopathies of livestock and rodents, and variant Creutzfeldt-Jakob disease in humans, as well as an association between pathogenic prion protein (PrPSc) and different immune cells (e.g. follicular dendritic cells, T and B lymphocytes, monocytes and tingible body macrophages). To further investigate the role of various blood components in prion disease transmission, we intracranially inoculated genetically susceptible VRQ/ARQ and ARQ/ARQ sheep with inocula composed of CD11c+ B1 lymphocytes, CD68 +macrophages, or platelet-rich plasma derived from clinically ill sheep infected with the US no. 13-7 scrapie agent. At the completion of the study, we found that VRQ/ARQ and ARQ/ARQ sheep inoculated with CD11c+ B1 lymphocytes and CD68+ macrophages developed scrapie with detectable levels of PrPSc in the central nervous system and lymphoreticular system, while those inoculated with platelet-rich plasma did not develop disease and did not have detectable PrPSc by immunohistochemistry or enzyme immunoassay. This study complements and expands on earlier findings that white blood cells harbour prion infectivity, and reports CD11c+ B1 lymphocytes and CD68+ macrophages as additional targets for possible preclinical detection of prion infection in blood.

Evaluation of Antemortem Diagnostic Techniques in Goats Naturally Infected With Scrapie.

Scrapie is a naturally occurring transmissible spongiform encephalopathy (TSE) that affects sheep and goats. Sheep and goats can be infected with scrapie as lambs or kids via contact with the placenta or placental fluids, or from ingestion of prions shed in the environment and/or bodily fluids (e.g., saliva, urine, and feces). Like other TSEs, scrapie is generally not diagnosed before extensive and irreversible brain damage has occurred. Therefore, a reliable method to screen animals may facilitate diagnosis. Additionally, while natural scrapie in sheep has been widely described, naturally acquired goat scrapie is less well-characterized. The purpose of this study was to better understand natural goat scrapie in regard to disease phenotype (i.e., incubation period, clinical signs, neuroanatomical deposition patterns of PrPSc, and molecular profile as detected by Western blot) and to evaluate the efficacy of antemortem tests to detect scrapie-positive animals in a herd of goats. Briefly, 28 scrapie-exposed goats were removed from a farm depopulated due to previous diagnoses of scrapie on the premises and observed daily for 30 months. Over the course of the observation period, antemortem biopsies of recto-anal mucosa-associated lymphoid tissue (RAMALT) were taken and tested using immunohistochemistry and real-time quaking-induced conversion (RT-QuIC), and retinal thickness was measured in vivo using optical coherence tomography (OCT). Following the observation period, immunohistochemistry and Western blot were performed to assess neuroanatomical deposition patterns of PrPSc and molecular profile. Our results demonstrate that antemortem rectal biopsy was 77% effective in identifying goats naturally infected with scrapie and that a positive antemortem rectal biopsy was associated with the presence of clinical signs of neurologic disease and a positive dam status. We report that changes in retinal thickness are not detectable over the course of the observation period in goats naturally infected with scrapie. Finally, our results indicate that the accumulation of PrPSc in central nervous system (CNS) and non-CNS tissues is consistent with previous reports of scrapie in sheep and goats.

Experimental Study Using Multiple Strains of Prion Disease in Cattle Reveals an Inverse Relationship between Incubation Time and Misfolded Prion Accumulation, Neuroinflammation, and Autophagy.

Proteinopathies result from aberrant folding and accumulation of specific proteins. Currently, there is a lack of knowledge about the factors that influence disease progression, making this a key challenge for the development of therapies for proteinopathies. Because of the similarities between transmissible spongiform encephalopathies (TSEs) and other protein misfolding diseases, TSEs can be used to understand other proteinopathies. Bovine spongiform encephalopathy (BSE) is a TSE that occurs in cattle and can be subdivided into three strains: classic BSE and atypical BSEs (H and L types) that have shorter incubation periods. The NACHT, LRR, and PYD domains-containing protein 3 inflammasome is a critical component of the innate immune system that leads to release of IL-1ß. Macroautophagy is an intracellular mechanism that plays an essential role in protein clearance. In this study, the retina was used as a model to investigate the relationship between disease incubation period, prion protein accumulation, neuroinflammation, and changes in macroautophagy. We demonstrate that atypical BSEs present with increased prion protein accumulation, neuroinflammation, and decreased autophagy. This work suggests a relationship between disease time course, neuroinflammation, and the autophagic stress response, and may help identify novel therapeutic biomarkers that can delay or prevent the progression of proteinopathies.

In Situ Temporospatial Characterization of the Glial Response to Prion Infection.

Mammalian transmissible spongiform encephalopathies (TSEs) display marked activation of astrocytes and microglia that precedes neuronal loss. Investigation of clinical parallels between TSEs and other neurodegenerative protein misfolding diseases, such as Alzheimer's disease, has revealed similar patterns of neuroinflammatory responses to the accumulation of self-propagating amyloids. The contribution of glial activation to the progression of protein misfolding diseases is incompletely understood, with evidence for mediation of both protective and deleterious effects. Glial populations are heterogeneously distributed throughout the brain and capable of dynamic transitions along a spectrum of functional activation states between pro- and antiinflammatory polarization extremes. Using a murine model of Rocky Mountain Laboratory scrapie, the neuroinflammatory response to prion infection was characterized by evaluating glial activation across 15 brain regions over time and correlating it to traditional markers of prion neuropathology, including vacuolation and PrPSc deposition. Quantitative immunohistochemistry was used to evaluate glial expression of iNOS and Arg1, markers of classical and alternative glial activation, respectively. The results indicate progressive upregulation of iNOS in microglia and a mixed astrocytic profile featuring iNOS expression in white matter tracts and detection of Arg1-positive populations throughout the brain. These data establish a temporospatial lesion profile for this prion infection model and demonstrate evidence of multiple glial activation states.

Sheep Are Susceptible to the Bovine Adapted Transmissible Mink Encephalopathy Agent by Intracranial Inoculation and Have Evidence of Infectivity in Lymphoid Tissues.

Transmissible mink encephalopathy (TME) is a food borne prion disease. Epidemiological and experimental evidence suggests similarities between the agents of TME and L-BSE. This experiment demonstrates the susceptibility of four different genotypes of sheep to the bovine adapted TME agent by intracranial inoculation. The four genotypes of sheep used in this experiment had polymorphisms corresponding to codons 136, 154, and 171 of the prion gene: V136R154Q171/VRQ, VRQ/ARQ, ARQ/ARQ, and ARQ/ARR. All intracranially inoculated sheep without comorbidities (15/15) developed clinical signs and had detectable PrPSc by immunohistochemistry, western blot, and enzyme immunoassay (EIA). The mean incubation periods in sheep with bovine adapted TME correlated with their relative genotypic susceptibility. There was peripheral distribution of PrPSc in the trigeminal ganglion and neuromuscular spindles; however, unlike classical scrapie and C-BSE in sheep, sheep inoculated with the bovine TME agent did not have immunohistochemically detectable PrPSc in the lymphoid tissue. To rule out the presence of infectivity, the lymph nodes of two sheep genotypes, VRQ/VRQ, and ARQ/ARQ, were bioassayed in transgenic mice expressing ovine prion protein. Mice intracranially inoculated with retropharyngeal lymph node from a VRQ/VRQ sheep were EIA positive (3/17) indicating that sheep inoculated with the bovine TME agent harbor infectivity in their lymph nodes despite a lack of detection with conventional immunoassays. Western blot analysis demonstrated similarities in the migration patterns between bovine TME in sheep, the bovine adapted TME inoculum, and L-BSE. Overall, these results demonstrate that sheep are susceptible to the bovine adapted TME agent, and the tissue distribution of PrPSc in sheep with bovine TME is distinct from classical scrapie.

Raccoons accumulate PrPSc after intracranial inoculation of the agents of chronic wasting disease or transmissible mink encephalopathy but not atypical scrapie.

Prion diseases are neurodegenerative diseases characterized by the accumulation of misfolded prion protein (PrPSc) in the brain and other tissues. Animal prion diseases include scrapie in sheep, chronic wasting disease (CWD) in cervids, and transmissible mink encephalopathy (TME) in ranch-raised mink. We investigated the susceptibility of raccoons to various prion disease agents and compared the clinicopathologic features of the resulting disease. Raccoon kits were inoculated intracranially with the agents of raccoon-passaged TME (TMERac), bovine-passaged TME (TMEBov), hamster-adapted drowsy (TMEDY) or hyper TME (TMEHY), CWD from white-tailed deer (CWDWtd) or elk (CWDElk), or atypical (Nor98) scrapie. Raccoons were euthanized when they developed clinical signs of prion disease or at study endpoint (<82 mo post-inoculation). Brain was examined for the presence of spongiform change, and disease-associated PrPSc was detected using an enzyme immunoassay, western blot, and immunohistochemistry. All raccoons inoculated with the agents of TMERac and TMEBov developed clinical disease at ~6.6 mo post-inoculation, with widespread PrPSc accumulation in central nervous system tissues. PrPSc was detected in the brain of 1 of 4 raccoons in each of the CWDWtd-, CWDElk-, and TMEHY-inoculated groups. None of the raccoons inoculated with TMEDY or atypical scrapie agents developed clinical disease or detectable PrPSc accumulation. Our results indicate that raccoons are highly susceptible to infection with raccoon- and bovine-passaged TME agents, whereas CWD isolates from white-tailed deer or elk and hamster-adapted TMEHY transmit poorly. Raccoons appear to be resistant to infection with hamster-adapted TMEDY and atypical scrapie agents.

Pathologic and biochemical characterization of PrPSc from elk with PRNP polymorphisms at codon 132 after experimental infection with the chronic wasting disease agent.

BACKGROUND: The Rocky Mountain elk (Cervus elaphus nelsoni) prion protein gene (PRNP) is polymorphic at codon 132, with leucine (L132) and methionine (M132) allelic variants present in the population. In elk experimentally inoculated with the chronic wasting disease (CWD) agent, different incubation periods are associated with PRNP genotype: LL132 elk survive the longest, LM132 elk are intermediate, and MM132 elk the shortest. The purpose of this study was to investigate potential mechanisms underlying variations in incubation period in elk of different prion protein genotypes. Elk calves of three PRNP genotypes (n = 2 MM132, n = 2 LM132, n = 4 LL132) were orally inoculated with brain homogenate from elk clinically affected with CWD. RESULTS: Elk with longer incubation periods accumulated relatively less PrPSc in the brain than elk with shorter incubation periods. PrPSc accumulation in LM132 and MM132 elk was primarily neuropil-associated while glial-associated immunoreactivity was prominent in LL132 elk. The fibril stability of PrPSc from MM132 and LM132 elk were similar to each other and less stable than that from LL132 elk. Real-time quaking induced conversion assays (RT-QuIC) revealed differences in the ability of PrPSc seed from elk of different genotypes to convert recombinant 132 M or 132 L substrate. CONCLUSIONS: This study provides further evidence of the importance of PRNP genotype in the pathogenesis of CWD of elk. The longer incubation periods observed in LL132 elk are associated with PrPSc that is more stable and relatively less abundant at the time of clinical disease. The biochemical properties of PrPSc from MM132 and LM132 elk are similar to each other and different to PrPSc from LL132 elk. The shorter incubation periods in MM132 compared to LM132 elk may be the result of genotype-dependent differences in the efficiency of propagation of PrPSc moieties present in the inoculum. A better understanding of the mechanisms by which the polymorphisms at codon 132 in elk PRNP influence disease pathogenesis will help to improve control of CWD in captive and free-ranging elk populations.

Experimental Transmission of the Chronic Wasting Disease Agent to Swine after Oral or Intracranial Inoculation.

Chronic wasting disease (CWD) is a naturally occurring, fatal neurodegenerative disease of cervids. The potential for swine to serve as hosts for the agent of CWD is unknown. The purpose of this study was to investigate the susceptibility of swine to the CWD agent following experimental oral or intracranial inoculation. Crossbred piglets were assigned to three groups, intracranially inoculated (n = 20), orally inoculated (n = 19), and noninoculated (n = 9). At approximately the age at which commercial pigs reach market weight, half of the pigs in each group were culled ("market weight" groups). The remaining pigs ("aged" groups) were allowed to incubate for up to 73 months postinoculation (mpi). Tissues collected at necropsy were examined for disease-associated prion protein (PrPSc) by Western blotting (WB), antigen capture enzyme immunoassay (EIA), immunohistochemistry (IHC), and in vitro real-time quaking-induced conversion (RT-QuIC). Brain samples from selected pigs were also bioassayed in mice expressing porcine prion protein. Four intracranially inoculated aged pigs and one orally inoculated aged pig were positive by EIA, IHC, and/or WB. By RT-QuIC, PrPSc was detected in lymphoid and/or brain tissue from one or more pigs in each inoculated group. The bioassay was positive in four out of five pigs assayed. This study demonstrates that pigs can support low-level amplification of CWD prions, although the species barrier to CWD infection is relatively high. However, detection of infectivity in orally inoculated pigs with a mouse bioassay raises the possibility that naturally exposed pigs could act as a reservoir of CWD infectivity.IMPORTANCE We challenged domestic swine with the chronic wasting disease agent by inoculation directly into the brain (intracranially) or by oral gavage (orally). Disease-associated prion protein (PrPSc) was detected in brain and lymphoid tissues from intracranially and orally inoculated pigs as early as 8 months of age (6 months postinoculation). Only one pig developed clinical neurologic signs suggestive of prion disease. The amount of PrPSc in the brains and lymphoid tissues of positive pigs was small, especially in orally inoculated pigs. Regardless, positive results obtained with orally inoculated pigs suggest that it may be possible for swine to serve as a reservoir for prion disease under natural conditions.

Horizontal Transmission of Chronic Wasting Disease in Reindeer.

We challenged reindeer by the intracranial route with the agent of chronic wasting disease sourced from white-tailed deer, mule deer, or elk and tested for horizontal transmission to naive reindeer. Reindeer were susceptible to chronic wasting disease regardless of source species. Horizontal transmission occurred through direct contact or indirectly through the environment.

A Comparison of Classical and H-Type Bovine Spongiform Encephalopathy Associated with E211K Prion Protein Polymorphism in Wild-Type and EK211 Cattle Following Intracranial Inoculation.

In 2006, a case of H-type bovine spongiform encephalopathy (BSE-H) was diagnosed in a cow that was associated with a heritable polymorphism in the bovine prion protein gene (PRNP) resulting in a lysine for glutamate amino acid substitution at codon 211 (called E211K) of the prion protein. Although the prevalence of this polymorphism is low, cattle carrying the K211 allele may be predisposed to rapid onset of BSE-H when exposed or to the potential development of a genetic BSE. This study was conducted to better understand the relationship between the K211 polymorphism and its effect on BSE phenotype. BSE-H from the US 2006 case was inoculated intracranially (IC) in one PRNP wild-type (EE211) calf and one EK211 calf. In addition, one wild-type calf and one EK211 calf were inoculated IC with brain homogenate from a US 2003 classical BSE case. All cattle developed clinical disease. The survival time of the E211K BSE-H inoculated EK211 calf (10 months) was shorter than the wild-type calf (18 months). This genotype effect was not observed in classical BSE inoculated cattle (both 26 months). Significant changes in retinal function were observed in H-type BSE challenged cattle only. Cattle challenged with the same inoculum showed similar severity and neuroanatomical distribution of vacuolation and disease-associated prion protein deposition in the brain, though differences in neuropathology were observed between E211K BSE-H and classical BSE inoculated animals. Western blot results for brain tissue from challenged animals were consistent with the inoculum strains. This study demonstrates that the phenotype of E211K BSE-H remains stable when transmitted to cattle without the K211 polymorphism, and exhibits a number of features that differ from classical BSE in both wild-type and heterozygous EK211 animals.

Phenotype shift from atypical scrapie to CH1641 following experimental transmission in sheep.

The interactions of host and infecting strain in ovine transmissible spongiform encephalopathies are known to be complex, and have a profound effect on the resulting phenotype of disease. In contrast to classical scrapie, the pathology in naturally-occurring cases of atypical scrapie appears more consistent, regardless of genotype, and is preserved on transmission within sheep homologous for the prion protein (PRNP) gene. However, the stability of transmissible spongiform encephalopathy phenotypes on passage across and within species is not absolute, and there are reports in the literature where experimental transmissions of particular isolates have resulted in a phenotype consistent with a different strain. In this study, intracerebral inoculation of atypical scrapie between two genotypes both associated with susceptibility to atypical forms of disease resulted in one sheep displaying an altered phenotype with clinical, pathological, biochemical and murine bioassay characteristics all consistent with the classical scrapie strain CH1641, and distinct from the atypical scrapie donor, while the second sheep did not succumb to challenge. One of two sheep orally challenged with the same inoculum developed atypical scrapie indistinguishable from the donor. This study adds to the range of transmissible spongiform encephalopathy phenotype changes that have been reported following various different experimental donor-recipient combinations. While these circumstances may not arise through natural exposure to disease in the field, there is the potential for iatrogenic exposure should current disease surveillance and feed controls be relaxed. Future sheep to sheep transmission of atypical scrapie might lead to instances of disease with an alternative phenotype and onward transmission potential which may have adverse implications for both public health and animal disease control policies.

Estimation of nasal shedding and seroprevalence of organisms known to be associated with bovine respiratory disease in Australian live export cattle.

The prevalence of organisms known to be associated with bovine respiratory disease (BRD) was investigated in cattle prior to export. A quantitative reverse transcription polymerase chain reaction assay was used to detect nucleic acids from the following viruses and bacteria in nasal swab samples: Bovine coronavirus (BoCV; Betacoronavirus 1), Bovine herpesvirus 1 (BoHV-1), Bovine viral diarrhea virus 1 (BVDV-1), Bovine respiratory syncytial virus (BRSV), Bovine parainfluenza virus 3 (BPIV-3), Histophilus somni, Mycoplasma bovis, Mannheimia haemolytica, and Pasteurella multocida. Between 2010 and 2012, nasal swabs were collected from 1,484 apparently healthy cattle destined for export to the Middle East and Russian Federation. In addition, whole blood samples from 334 animals were tested for antibodies to BoHV-1, BRSV, BVDV-1, and BPIV-3 using enzyme-linked immunosorbent assay. The nasal prevalence of BoCV at the individual animal level was 40.1%. The nasal and seroprevalence of BoHV-1, BRSV, BVDV-1, and BPIV-3 was 1.0% and 39%, 1.2% and 46%, 3.0% and 56%, and 1.4% and 87%, respectively. The nasal prevalence of H. somni, M. bovis, M. haemolytica, and P. multocida was 42%, 4.8%, 13.4%, and 26%, respectively. Significant differences in nasal and seroprevalence were detected between groups of animals from different geographical locations. The results of the current study provide baseline data on the prevalence of organisms associated with BRD in Australian live export cattle in the preassembly period. This data could be used to develop strategies for BRD prevention and control prior to loading.

Mortality of live export cattle on long-haul voyages: pathologic changes and pathogens.

The cause of death in 215 cattle on 20 long-haul live export voyages from Australia to the Middle East, Russia, and China was investigated between 2010 and 2012 using gross, histologic, and/or molecular pathology techniques. A quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay was used to detect nucleic acids from viruses and bacteria known to be associated with respiratory disease in cattle: Bovine coronavirus (Betacoronavirus 1), Bovine herpesvirus 1, Bovine viral diarrhea virus 1 and 2, Bovine respiratory syncytial virus, Bovine parainfluenza virus 3, Histophilus somni, Mycoplasma bovis, Mannheimia haemolytica, and Pasteurella multocida. The most commonly diagnosed cause of death was respiratory disease (107/180, 59.4%), followed by lameness (n = 22, 12.2%), ketosis (n = 12, 6.7%), septicemia (n = 11, 6.1%), and enteric disease (n = 10, 5.6%). Two thirds (130/195) of animals from which lung samples were collected had histologic changes and/or positive qRT-PCR results indicative of infectious lung disease: 93 out of 130 (72%) had evidence of bacterial infection, 4 (3%) had viral infection, and 29 (22%) had mixed bacterial and viral infections, and for 4 (3%) the causative organism could not be identified. Bovine coronavirus was detected in up to 13% of cattle tested, and this finding is likely to have important implications for the management and treatment of respiratory disease in live export cattle. Results from the current study indicate that although overall mortality during live export voyages is low, further research into risk factors for developing respiratory disease is required.