Linking morbillivirus exposure to individual habitat use of common bottlenose dolphins (Tursiops truncatus) between geographically different sites.

Dolphin morbillivirus (DMV) is a virulent pathogen that causes high mortality outbreaks in delphinids globally and is spread via contact among individuals. Broadly ranging nearshore and open-ocean delphinids are likely reservoir populations that transmit DMV to estuarine populations. We assessed the seroprevalence of DMV antibodies and determined the habitat use of common bottlenose dolphins, Tursiops truncatus truncatus, from two estuarine sites, Barataria Bay and Mississippi Sound, in the northern Gulf of Mexico. We predicted that risk to DMV exposure in estuarine dolphins is driven by spatial overlap in habitat use with reservoir populations. Serum was collected from live-captured dolphins and tested for DMV antibodies. Habitat use of sampled individuals was determined by analysing satellite-tracked movements and stable isotope values. DMV seroprevalences were high among dolphins at Barataria Bay (37%) and Mississippi Sound (44%), but varied differently within sites. Ranging patterns of Barataria Bay dolphins were categorized into two groups: Interior and Island-associated. DMV seroprevalences were absent in Interior dolphins (0%) but high in Island-associated dolphins (45%). Ranging patterns of Mississippi Sound dolphins were categorized into three groups: Interior, Island-east and Island-west. DMV seroprevalences were detected across Mississippi Sound (Interior: 60%; Island-east: 20%; and Island-west: 43%). At both sites, dolphins in habitats with greater marine influence had enriched δ13 C values, and Barataria Bay dolphins with positive DMV titres had carbon isotope values indicative of marine habitats. Positive titres for DMV antibodies were more common in the lower versus upper parts of Barataria Bay but evenly distributed across Mississippi Sound. A dolphin's risk of exposure to DMV is influenced by how individual ranging patterns interact with environmental geography. Barataria Bay's partially enclosed geography likely limits the nearshore or open-ocean delphinids that carry DMV from interacting with dolphins that use interior, estuarine habitats, decreasing their exposure to DMV. Mississippi Sound's relatively open geography allows for greater spatial overlap and mixing among estuarine, nearshore and/or open-ocean cetaceans. The spread of DMV, and likely other diseases, is affected by the combination of individual movements, habitat use and the environment.

Exposure of the Gulf of St. Lawrence grey seal Halichoerus grypus population to potentially zoonotic infectious agents.

The population of grey seals Halichoerus grypus in Canadian waters is currently used as a commercial source of meat for human consumption. As with domestic livestock, it is important to understand the occurrence in these seals of infectious agents that may be of public health significance and thus ensure appropriate measures are in place to avoid zoonotic transmission. This study examined the prevalence of antibodies against Brucella spp., Erysipelothrix rhusiopathiae, 6 serovars of Leptospira interrogans, and Toxoplasma gondii in 59 grey seals and determined by polymerase chain reaction (PCR) the presence of these potentially zoonotic agents in specific organs and tissues of seropositive animals. The presence of encysted Trichinella spp. larvae was also investigated by digestion of tongue, diaphragm and other muscle samples, but none were detected. Seroprevalence against Brucella spp. and E. rhusiopathiae was low (5 and 3%, respectively). All 59 seals tested had antibodies against L. interrogans, but no carrier of this bacterium was detected by PCR. Seroprevalence against T. gondii was 53%, and DNA of this protozoan was detected by PCR in 11/30 (37%) seropositive animals. Standard sanitary measures mandatory for commercialization of meat products for human consumption should greatly reduce the potential for exposure to these infectious agents. However, special consideration should be given to freezing seal meat for at least 3 d to ensure destruction of tissue cysts of T. gondii.

Lymphoplasmacytic Meningoencephalitis and Neuronal Necrosis Associated With Parvoviral Infection in Cats.

Neurologic manifestations other than cerebellar hypoplasia are rarely associated with feline panleukopenia virus (FPV) infection in cats. Here the authors describe lymphoplasmacytic meningoencephalitis and neuronal necrosis in 2 cats autopsied after exhibiting ataxia and nystagmus. Gross changes consisted of cerebellar herniation through the foramen magnum, with flattening of cerebrocortical gyri and narrowing of sulci. Histologically, lymphoplasmacytic meningoencephalitis, extensive neuronal necrosis, and neuroaxonal degeneration with digestion chambers were present in the telencephalon and brain stem in both cats. Frozen brain tissue of both cats was positive for parvoviral antigen via fluorescent antibody testing, and formalin-fixed, paraffin-embedded tissue sections of brain were immunoreactive for parvovirus antigen and positive for parvoviral DNA on in situ hybridization. Frozen brain tissue from 1 case was positive for parvovirus NS1 and VP2 genes using conventional polymerase chain reaction, and subsequent DNA sequencing and phylogenetic analysis revealed that the viral strain was a FPV. Reverse transcription quantitative polymerase chain reaction on formalin-fixed, paraffin-embedded brain tissue revealed high levels of parvovirus in both cases, supporting an acute and active viral infection. Although rare, FPV infection should be considered in cases of lymphoplasmacytic meningoencephalitis and neuronal necrosis in cats.

Evaluation of Targeted Next-Generation Sequencing for Detection of Bovine Pathogens in Clinical Samples.

The laboratory diagnosis of infectious diseases, especially those caused by mixed infections, is challenging. Routinely, it requires submission of multiple samples to separate laboratories. Advances in next-generation sequencing (NGS) have provided the opportunity for development of a comprehensive method to identify infectious agents. This study describes the use of target-specific primers for PCR-mediated amplification with the NGS technology in which pathogen genomic regions of interest are enriched and selectively sequenced from clinical samples. In the study, 198 primers were designed to target 43 common bovine and small-ruminant bacterial, fungal, viral, and parasitic pathogens, and a bioinformatics tool was specifically constructed for the detection of targeted pathogens. The primers were confirmed to detect the intended pathogens by testing reference strains and isolates. The method was then validated using 60 clinical samples (including tissues, feces, and milk) that were also tested with other routine diagnostic techniques. The detection limits of the targeted NGS method were evaluated using 10 representative pathogens that were also tested by quantitative PCR (qPCR), and the NGS method was able to detect the organisms from samples with qPCR threshold cycle (CT ) values in the 30s. The method was successful for the detection of multiple pathogens in the clinical samples, including some additional pathogens missed by the routine techniques because the specific tests needed for the particular organisms were not performed. The results demonstrate the feasibility of the approach and indicate that it is possible to incorporate NGS as a diagnostic tool in a cost-effective manner into a veterinary diagnostic laboratory.

SURVEY FOR INFECTIOUS DISEASE IN THE SOUTH AMERICAN FUR SEAL (ARCTOCEPHALUS AUSTRALIS) POPULATION AT PUNTA SAN JUAN, PERU.

The Peruvian population of the South American fur seal ( Arctocephalus australis ) is a distinct evolutionarily significant unit that is endangered. One of the largest rookeries for this species in Peru is located within the Punta San Juan marine protected area (15°22'S, 75°12'W). To better understand the current health status of this population, exposure to 10 pinniped pathogens was evaluated in adult female fur seals (n=29) via serology and polymerase chain reaction (PCR) techniques in November 2010. The results suggest this population is naïve to canine and phocine distemper viruses (serum neutralization test), five Leptospira interrogans serovars (microscopic agglutination test), and Brucella canis (card test). Indirect fluorescent antibody testing for Toxoplasma gondii , Neospora caninum , and Sarcocystis neurona was also uniformly negative. PCR testing of nasal swabs using previously described Mycoplasma spp. primers was positive in 37.9% (11/29) of samples. One animal was positive via card test for Brucella abortus , whereas 53.7% (15/28) were positive or suspect using a marine Brucella competitive enzyme-linked immunosorbent assay. Antibody to phocine herpesvirus-1 (PHV-1) was identified in 85.7% (24/28) of the sampled population by serum neutralization testing. Overall, exposure to Mycoplasma spp., Brucella spp., and PHV-1 was observed, but results demonstrated low to no exposure to many key pinniped pathogens. The expansion of human populations, agriculture, and industry along the Peruvian coast may lead to increased pathogen exposure from human, domestic, and wild animal sources. The naïve nature of this key population of South American fur seals raises concerns about potential risk for disease outbreaks.

Retrospective study of etiologic agents associated with nonsuppurative meningoencephalitis in stranded cetaceans in the canary islands.

Nineteen natural cases of etiologically undetermined encephalitides in free-ranging cetaceans were studied retrospectively. Histological examination of the brains revealed variable degrees of nonsuppurative encephalitis or meningoencephalitis, characterized predominantly by perivascular lymphohistiocytic infiltrates. A PCR assay was used on brain and other available tissues to detect the presence of morbillivirus, herpesvirus, West Nile virus, Toxoplasma gondii, and Brucella spp. In addition, immunohistochemical (IHC) staining was performed on selected tissues to determine the presence of morbilliviral antigens. Six animals (5 striped dolphins and 1 common dolphin) showed IHC and/or molecular evidence of morbilliviral antigens and/or genomes, mainly in brain tissue. Conventional nested PCR detected herpesviral DNA in brain tissue samples from two striped dolphins. There was no evidence of West Nile virus, T. gondii, or Brucella spp. in any of the brain tissue samples examined. The information presented here increases the number of confirmed morbillivirus-positive cases within the Canarian archipelago from two previously reported cases to eight. Furthermore, a new nested-PCR method for the detection of morbillivirus is described here. Regarding herpesvirus, the phylogenetic analysis performed in the current study provides valuable information about a possible pathogenic branch of cetacean alphaherpesviruses that might be responsible for some fatal cases worldwide.

Review of historical unusual mortality events (UMEs) in the Gulf of Mexico (1990-2009): providing context for the multi-year northern Gulf of Mexico cetacean UME declared in 2010.

An unusual mortality event (UME) was declared for cetaceans in the northern Gulf of Mexico (GoM) for Franklin County, Florida, west through Louisiana, USA, beginning in February 2010 and was ongoing as of September 2014. The 'Deepwater Horizon' (DWH) oil spill began on 20 April 2010 in the GoM, raising questions regarding the potential role of the oil spill in the UME. The present study reviews cetacean mortality events that occurred in the GoM prior to 2010 (n = 11), including causes, durations, and some specific test results, to provide a historical context for the current event. The average duration of GoM cetacean UMEs prior to 2010 was 6 mo, and the longest was 17 mo (2005-2006). The highest number of cetacean mortalities recorded during a previous GoM event was 344 (in 1990). In most previous events, dolphin morbillivirus or brevetoxicosis was confirmed or suspected as a causal factor. In contrast, the current northern GoM UME has lasted more than 48 mo and has had more than 1000 reported mortalities within the currently defined spatial and temporal boundaries of the event. Initial results from the current UME do not support either morbillivirus or brevetoxin as primary causes of this event. This review is the first summary of cetacean UMEs in the GoM and provides evidence that the most common causes of previous UMEs are unlikely to be associated with the current UME.

Clinicoimmunopathologic findings in Atlantic bottlenose dolphins Tursiops truncatus with positive cetacean morbillivirus antibody titers.

Sera from free-ranging Atlantic bottlenose dolphins Tursiops truncatus inhabiting the Indian River Lagoon (IRL), Florida were tested for antibodies to cetacean morbilliviruses from 2003 to 2007 as part of a multidisciplinary study of individual and population health. A suite of clinicoimmunopathologic variables were evaluated in morbillivirus-seropositive dolphins (n = 14) and seronegative healthy dolphins (n = 49). Several important differences were found. Serum alkaline phosphatase, creatine phosphokinase, chloride, albumin and albumin/globulin ratios were significantly lower in seropositive dolphins. Innate immunity appeared to be upregulated with significant increases in lysozyme concentration and marginally significant increases in monocytic phagocytosis. Adaptive immunity was also impacted in dolphins with positive morbillivirus antibody titers. Mitogen-induced T lymphocyte proliferation responses were significantly reduced in dolphins with positive morbillivirus antibody titers, and marginally significant decreases were found for absolute numbers of CD4+ lymphocytes. The findings suggest impairment of cell-mediated adaptive immunity, similar to the immunologic pattern reported with acute morbillivirus infection in other species. In contrast, dolphins with positive morbillivirus antibody titers appeared to have at least a partially upregulated humoral immune response with significantly higher levels of gamma globulins than healthy dolphins, which may represent an antibody response to morbillivirus infection or other pathogens. These data suggest that subclinical dolphin morbillivirus infection in IRL dolphins may produce clinicoimmunopathologic perturbations that impact overall health.

Comparison of the immune response following subcutaneous versus intranasal modified-live virus booster vaccination against bovine respiratory disease in pre-weaning beef calves that had received primary vaccination by the intranasal route.

This study was performed to elucidate whether the route of booster vaccination affects the immune response against respiratory vaccine viruses in pre-weaning beef calves that receive primary intranasal (IN) vaccination during the first month of life. The objective was to compare the serum neutralizing antibody (SNA) titers to BHV1, BRSV, and BPI3V, cytokine mRNA expression and mucosal BHV1- and BRSV-specific IgA in nasal secretions following administration of IN or subcutaneous (SC) modified-live virus (MLV) booster vaccines 60 days after primary IN vaccination in young beef calves. Twenty-one beef calves were administered 2 mL of an IN MLV vaccine containing BHV1, BRSV, and BPI3V (Inforce3®) between one and five weeks of age. Sixty days after primary vaccination, calves were randomly assigned to one of two groups: IN-MLV (n = 11): Calves received 2 mL of the same IN MLV vaccine used for primary vaccination and 2 mL of a SC MLV vaccine containing BVDV1 & 2 (Bovi- Shield GOLD® BVD). SC-MLV (n = 10): Calves were administered 2 mL of a MLV vaccine containing, BHV1, BRSV, BPI3V, and BVDV1 & 2 (Bovi-Shield GOLD® 5). Blood and nasal secretion samples were collected on days -61 (primary vaccination), -28, -14, 0 (booster vaccination), 14, 21, 28, 42 and 60 for determination of SNA titers, cytokine gene expression analysis and nasal virus-specific IgA concentrations. Statistical analysis was performed using a repeated measures analysis through PROC GLIMMIX of SAS®. Booster vaccination by neither IN nor SC routes induced a significant increase in SNA titers against BHV1, BRSV, and BPI3V. Subcutaneous booster vaccination induced significantly greater BRSV-specific SNA titers (on day 42) and IgA concentration in nasal secretions (on days 21 and 42) compared to calves receiving IN booster vaccination. Both IN and SC booster vaccination were able to stimulate the production of BHV1-specific IgA in nasal secretions. In summary, booster vaccination of young beef calves using either SC or IN route two months after IN MLV primary vaccination resulted in comparable SNA titers, cytokine gene expression profile and virus-specific IgA concentration in nasal secretions. Only a few differences in the systemic and mucosal immune response against BHV1 and BRSV were observed. Subcutaneous booster vaccination induced significantly greater BRSV-specific SNA and secretory IgA titers compared to IN booster vaccination.

Immune response and onset of protection from Bovine viral diarrhea virus 2 infection induced by modified-live virus vaccination concurrent with injectable trace minerals administration in newly received beef calves.

Strategies to improve the onset of protective immunity induced by vaccination against respiratory pathogens may have a significant impact on health of newly received beef calves. The objective was to determine if the use of injectable trace minerals (ITM; Se, Zn, Cu, and Mn) concurrent with a modified-live virus (MLV) vaccine enhances the immune response and onset of protection in beef calves challenged with BVDV2 five days after vaccination. Forty-five calves were randomly assigned to one of three groups (15/group): VAC + ITM, received MLV-vaccine and ITM (Multimin®90) subcutaneously (SC); VAC + SAL, received the same vaccine and saline SC; or UNVAC, unvaccinated. Five days after vaccination (d.0), calves were challenged with BVDV2 strain 890. Health status was evaluated and blood samples were collected for leukocyte counts, BVDV1 and 2 serum neutralizing antibodies (SNA), BVDV-PCR, and percentage of CD4+, CD8+, WC1+ and CD25+ T-cells. VAC + ITM had lower health scores than UNVAC (d.8 and 9). VAC + ITM had higher BVDV1 & 2 SNA titers than VAC + SAL and UNVAC on d.21 and 28. Lymphocyte counts decreased in UNVAC but not in VAC + ITM or VAC + SAL (d.3 to 11). CD4+ T-cells significantly decreased in UNVAC and VAC + SAL (d.3). VAC + ITM had higher percentage of CD4+ T-cells than UNVAC (d.3 and 7). VAC + ITM had lower percentage of activated CD4+ and CD8+ T-cells than UNVAC (d.7). In summary, vaccination induced a rapid protection against BVDV2 infection. Administration of ITM was associated with increased SNA response to BVDV1 & 2, enhanced health status, mitigation of CD4+ T-cells decrease, and reduction of T-cell activation in calves challenged with BVDV2 five days after immunization. These results support the strategic use of ITM concurrent with vaccination, especially when a rapid protection is needed in newly received beef calves.

Detection methods and characterization of bovine viral diarrhea virus in aborted fetuses and neonatal calves over a 22-year period.

Detection of bovine viral diarrhea virus (BVDV) in aborted fetus samples is often difficult due to tissue autolysis and inappropriate sampling. Studies assessing different methods for BVDV identification in fetal specimens are scarce. The present study evaluated the agreement between different diagnostic techniques to detect BVDV infections in specimens from a large number of bovine aborted fetuses and neonatal deaths over a period of 22 years. Additionally, genetic, serological, and pathological analyses were conducted in order to characterize BVDV strains of fetal origin. Samples from 95 selected cases from 1997 to 2018 were analyzed by antigen-capture ELISA (AgELISA), nested RT-PCR (RT-nPCR), and real-time RT-PCR (RT-qPCR). In addition, amplification and sequencing of the 5'UTR region were performed for phylogenetic purposes. Virus neutralization tests against the BVDV-1a, BVDV-1b, and BVDV-2b subtypes were conducted on 60 fetal fluids of the selected cases. Furthermore, the frequency and severity of histopathological lesions were evaluated in BVDV-positive cases. This study demonstrated that RT-nPCR and RT-qPCR were more suitable than AgELISA for BVDV detection in fetal specimens. However, the agreement between the two RT-PCR methods was moderate. The BVDV-1b subtype was more frequently detected than the BVDV-1a and BVDV-2b subtypes. Neutralizing antibodies to any of the three subtypes evaluated were present in 94% of the fetal fluids. Microscopically, half of the BVDV-positive cases showed a mild non-suppurative inflammatory response. These results emphasize the need to consider different methods for a diagnostic approach of BVDV associated to reproductive losses.

Real-time RT-PCR assays for the rapid and differential detection of dolphin and porpoise morbilliviruses.

Real-time RT-PCR (rtRT-PCR) assays for identifying and differentiating infections caused by dolphin morbillivirus (DMV) and porpoise morbillivirus (PMV) were developed by targeting the hypervariable C-terminal domain of the nucleocapsid (N) gene. Total DMV and PMV RNA extracted from infected Vero cells expressing the canine signaling lymphocyte-activation molecule (SLAM) produced positive cycle threshold (C(T)) values after the 17th and 25th cycles, respectively. The assays were then validated using infected cetacean tissue RNA. The assays were specific for either DMV or PMV and did not cross-react with canine distemper virus (CDV), phocid distemper virus (PDV), rinderpest virus (RPV), peste des petits ruminants virus (PPRV) and measles virus (MV). The glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was targeted as control for RNA quality, and a consensus GAPDH probe that reacted with 11 different marine mammal species, generating positive C(T) values ranging from the 21st to the 37th cycle was used. The rtRT-PCR assays have advantages over conventional assays in that they are rapid, easier to scale up, and are less prone to cross-contamination and have improved the limit of detection and specificity.

Lung pathology and infectious agents in fatal feedlot pneumonias and relationship with mortality, disease onset, and treatments.

This study charted 237 fatal cases of bovine respiratory disease (BRD) observed from May 2002 to May 2003 in a single Oklahoma feed yard. Postmortem lung samples were used for agent identification and histopathology. Late in the study, 94 skin samples (ear notches) were tested for Bovine viral diarrhea virus (BVDV) by immunohistochemistry (IHC). Bovine respiratory disease morbidity was 14.7%, and the mortality rate of all causes was 1.3%, with more than half (53.8%) attributed to BRD (0.7% total of all causes). The agents isolated were the following: Mannheimia haemolytica (25.0%), Pasteurella multocida (24.5%), Histophilus somni (10.0%), Arcanobacterium pyogenes (35.0%), Salmonella spp. (0.5%), and Mycoplasma spp. (71.4%). Viruses recovered by cell culture were BVDV-1a noncytopathic (NCP; 2.7%), BVDV-1a cytopathic (CP) vaccine strain (1.8%), BVDV-1b NCP (2.7%), BVDV-2a NCP (3.2%), BVDV-2b CP (0.5%), and Bovine herpesvirus 1 (2.3%). Gel-based polymerase chain reaction (PCR) assays were 4.6% positive for Bovine respiratory syncytial virus and 10.8% positive for Bovine coronavirus. Bovine viral diarrhea virus IHC testing was positive in 5.3% of the animals. The mean values were determined for the treatment data: fatal disease onset (32.65 days), treatment interval (29.15 days), number of antibiotic treatments (2.65), number of different antibiotics (1.89), and day of death (61.81 days). Lesions included the following: 1) duration: acute (21%), subacute (15%), chronic (40.2%), healing (2.8%), normal (18.1%), and autolyzed (2.8%); 2) type of pneumonia: lobar bronchopneumonia (LBP; 27.1%), LBP with pleuritis (49.1%), interstitial pneumonia (5.1%), bronchointerstitial pneumonia (1.4%), septic (0.9%), embolic foci (0.5%), other (2.8%), normal (10.3%), and autolyzed (2.8%); and 3) bronchiolar lesions: bronchiolitis obliterans (39.7%), bronchiolar necrosis (26.6%), bronchiolitis obliterans/bronchiolar necrosis (1.4%), other bronchiolar lesions (6.5%), and bronchiolar lesion negative (25.7%). Statistically significant relationships were present among the agents, lesions, and the animal treatment, disease onset, and mortality data. Clinical illnesses observed in this study were lengthier than those reported 16-20 years ago, based on fatal disease onset, treatment interval, and day of death.

Prevalence of and risk factors for serum antibodies against Leptospira serovars in US veterinarians.

OBJECTIVE: To determine the seroprevalence of antibodies against Leptospira serovars among veterinarians and identify risk factors for seropositivity in veterinary care settings. DESIGN: Seroepidemiologic survey. STUDY POPULATION: Veterinarians attending the 2006 AVMA Annual Convention. PROCEDURES: Blood samples were collected from 511 veterinarians, and serum was harvested for a microcapsule agglutination test (MAT) to detect antibodies against 6 serovars of Leptospira. Aggregate data analysis was performed to determine the ratio of the odds of a given exposure (eg, types of animals treated or biosafety practices) in seropositive individuals to the odds in seronegative individuals. RESULTS: Evidence of previous leptospiral infection was detected in 2.5% of veterinarians. Most veterinarians reported multiple potential exposures to Leptospira spp and other pathogens in the previous 12 months, including unintentional needlestick injuries (379/511 [74.2%]), animal bites (345/511 [67.5%]), and animal scratches (451/511 [88.3%]). Treatment of a dog with an influenza-like illness within the past year was associated with seropositivity for antibodies against Leptospira spp. CONCLUSIONS AND CLINICAL RELEVANCE: Veterinarians are at risk for leptospirosis and should take measures to decrease potential exposure to infectious agents in general. Diagnostic tests for leptospirosis should be considered when veterinarians have febrile illnesses of unknown origin.

Exposure to novel parainfluenza virus and clinical relevance in 2 bottlenose dolphin (Tursiops truncatus) populations.

Parainfluenza virus (PIV) is a leading cause of respiratory infections in humans. A novel virus closely related to human and bovine parainfluenza viruses types 3 (HPIV-3 and BPIV-3), named Tursiops truncatus parainfluenza virus type 1 (TtPIV-1), was isolated from a dolphin with respiratory disease. We developed a dolphin-specific ELISA to measure acute- and convalescent-phase PIV antibodies in dolphins during 1999-2006 with hemograms similar to that of the positive control. PIV seroconversion occurred concurrently with an abnormal hemogram in 22 animals, of which 7 (31.8%) had respiratory signs. Seroprevalence surveys were conducted on 114 healthy bottlenose dolphins in Florida and California. When the most conservative interpretation of positive was used, 11.4% of healthy dolphins were antibody positive, 29.8% were negative, and 58.8% were inconclusive. PIV appears to be a common marine mammal virus that may be of human health interest because of the similarity of TtPIV-1 to BPIV-3 and HPIV-3.

Characterization of a parainfluenza virus isolated from a bottlenose dolphin (Tursiops truncatus).

A novel member of the parainfluenza virus family was identified in a bottlenose dolphin with respiratory disease. The case animal was a 19-year old male Atlantic bottlenose dolphin (Tursiops truncatus) that presented with signs of respiratory illness, including raspy, foul-odored breaths and cream-colored exudate from the blowhole. Focally extensive pyogranulomatous bronchointerstitial pneumonia with moderate numbers of intralesional yeast organisms was identified on histopathological examination. Other significant microscopic findings included multifocal erosive and ulcerative tracheitis and laryngitis consisting of active laryngeal lymphatic tissue and dilated glands with eosinophilic fluid. The cause of death was attributed to respiratory disease of unknown etiology. In addition to the postmortem isolation of Candida glabrata and mixed bacteria from lung tissue, a virus was isolated from two antemortem affected lung aspirates collected over a 2-month period and two postmortem samples (mediastinal lymph node and left lung tissue homogenate). The morphology of the virions on negative staining and transmission electron microscopy was consistent with that of paramyxoviruses. Two genomic fragments, comprising 532 and 419 nucleotides from the open reading frames that code for the viral polymerase and fusion protein, respectively, were amplified by polymerase chain reaction using degenerate primers. Phylogenetic analyses of the two viral RNA segments showed that the isolate comprised a novel virus strain, tentatively named T. truncatus parainfluenza virus type 1 (TtPIV-1). The virus is monophyletic with, but genetically distinct from, the various bovine parainfluenza virus type 3 strains.

Effect of age at the time of vaccination on antibody titers and feedlot performance in beef calves.

OBJECTIVE: To compare antibody responses, feedlot morbidity and mortality rates, feedlot performance, and carcass value for calves vaccinated with 1 of 2 vaccination strategies and for unvaccinated control calves. DESIGN: Randomized controlled clinical trial. ANIMALS: 451 beef steers and heifers. PROCEDURES: Calves were vaccinated with a modified-live infectious bovine rhinotracheitis virus (IBRV), bovine viral diarrhea virus types 1 (BVDV1) and 2 (BVDV2), parainfluenza type 3 virus, and bovine respiratory syncytial virus vaccine and Mannheimia haemolytica and Pasteurella multocida bacterin-toxoid at approximately 67 and 190 days of age (group 1; n = 151) or at approximately 167 and 190 days of age (group 2; 150) or were not vaccinated (control; 150). Serum antibody titers were measured at approximately 2, 67, 167, 190, and 232 days of age. Morbidity and mortality rates, feedlot performance, and carcass value were recorded for 361 calves shipped to feedlots. RESULTS: Percentages of calves seroconverting to IBRV, BVDV1, and BVDV2 were significantly higher for groups 1 and 2 than for the control group. Mean treatment costs were significantly lower for vaccinated than for control calves, and mean mortality rate was significantly higher for control calves than for group 1 calves. Feedlot performance and carcass value did not vary significantly among groups. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that vaccination of beef calves with a 5-antigen modified-live virus vaccine at 67 and 190 days of age was as effective in terms of immunologic responses as was vaccination at 167 and 190 days of age.

Multi-site evaluation of the LN34 pan-lyssavirus real-time RT-PCR assay for post-mortem rabies diagnostics.

Rabies is a fatal zoonotic disease that requires fast, accurate diagnosis to prevent disease in an exposed individual. The current gold standard for post-mortem diagnosis of human and animal rabies is the direct fluorescent antibody (DFA) test. While the DFA test has proven sensitive and reliable, it requires high quality antibody conjugates, a skilled technician, a fluorescence microscope and diagnostic specimen of sufficient quality. The LN34 pan-lyssavirus real-time RT-PCR assay represents a strong candidate for rabies post-mortem diagnostics due to its ability to detect RNA across the diverse Lyssavirus genus, its high sensitivity, its potential for use with deteriorated tissues, and its simple, easy to implement design. Here, we present data from a multi-site evaluation of the LN34 assay in 14 laboratories. A total of 2,978 samples (1,049 DFA positive) from Africa, the Americas, Asia, Europe, and the Middle East were tested. The LN34 assay exhibited low variability in repeatability and reproducibility studies and was capable of detecting viral RNA in fresh, frozen, archived, deteriorated and formalin-fixed brain tissue. The LN34 assay displayed high diagnostic specificity (99.68%) and sensitivity (99.90%) when compared to the DFA test, and no DFA positive samples were negative by the LN34 assay. The LN34 assay produced definitive findings for 80 samples that were inconclusive or untestable by DFA; 29 were positive. Five samples were inconclusive by the LN34 assay, and only one sample was inconclusive by both tests. Furthermore, use of the LN34 assay led to the identification of one false negative and 11 false positive DFA results. Together, these results demonstrate the reliability and robustness of the LN34 assay and support a role for the LN34 assay in improving rabies diagnostics and surveillance.

Occurrence of canine parvovirus type 2c in the United States.

Canine parvovirus (CPV) type 2 (CPV-2) emerged around 1978 as a major pathogen of dogs worldwide. In the mid-1980s, the original CPV-2 had evolved and was completely replaced by 2 variants, CPV-2a and CPV-2b. In 2000, a new variant of CPV (named CPV-2c) was detected in Italy and now cocirculates with types 2a and 2b in that country. The CPV-2c has also been reported from single outbreaks in Vietnam and Spain. This study was conducted to determine if CPV-2c occurs in the United States. Thirty-three fecal samples were collected from dogs in 16 states between April 2006 and April 2007 and were tested for CPV using real-time polymerase chain reaction (PCR). Positive samples were further tested using conventional PCR and minor-groove binding TaqMan PCR assays to determine the viral type and to differentiate vaccine strains from field strains. Twenty-seven samples were positive for CPV, 7 of which were CPV-2c from 5 states: Arizona, California, Georgia, Oklahoma, and Texas. Of the 7 isolates, 4 differed from European CPV-2c isolates by 2 additional single-nucleotide mutations at positions 4076 and 4104, the latter of which produces a ThrAla change at residue 440 located near a major antigenic site. The coast-to-coast geographic distribution of the states in which CPV-2c was detected strongly suggests that this new CPV variant is probably widespread in the United States. The continuous evolution of CPV requires that monoclonal antibody-based and nucleic acid-based diagnostic assays should be periodically checked for sensitivity on prevalent CPV strains.

Infectious disease monitoring of the endangered Hawaiian monk seal.

As part of conservation efforts between 1997 and 2001, more than 25% (332 animals) of the endangered Hawaiian monk seal (Monachus schauinslandi) population was sampled in the northwestern Hawaiian Islands. Serum samples were tested for antibodies to viruses, bacteria, and parasites known to cause morbidity and mortality in other marine mammal species. Antibodies were found to phocine herpesvirus-1 by using an enzyme-linked immunosorbent assay, but seropositive results were not confirmed by virus neutralization test. Antibodies to Leptospira bratislava, L. hardjo, L. icterohaemorrhagiae, and L. pomona were detected in seals from several sites with the microagglutination test. Antibodies to Brucella spp. were detected using 10 conventional serologic tests, but because of inconsistencies in test results and laboratories used, and the lack of validation by culture, the Brucella serology should be interpreted with caution. Antibodies to B. canis were not detected by card test. Chlamydophila abortus antibodies were detected by complement fixation (CF) test, and prevalence increased significantly as a function of age; the low sensitivity and specificity associated with the CF make interpretation of results difficult. Antibodies to Toxoplasma gondii and Dirofilaria immitis were rarely found. There was no serologic evidence of exposure to four morbilliviruses, influenza A virus, canine adenovirus, caliciviruses, or other selected viruses. Continuous surveillance provides a means to detect the introduction or emergence of these or other infectious diseases, but it is dependent on the development or improvement of diagnostic tools. Continued and improved surveillance are both needed as part of future conservation efforts of Hawaiian monk seals.