In Vivo and In Vitro Characterization of the Recently Emergent PRRSV 1-4-4 L1C Variant (L1C.5) in Comparison with Other PRRSV-2 Lineage 1 Isolates.

The recently emerged PRRSV 1-4-4 L1C variant (L1C.5) was in vivo and in vitro characterized in this study in comparison with three other contemporary 1-4-4 isolates (L1C.1, L1A, and L1H) and one 1-7-4 L1A isolate. Seventy-two 3-week-old PRRSV-naive pigs were divided into six groups with twelve pigs/group. Forty-eight pigs (eight/group) were for inoculation, and 24 pigs (four/group) served as contact pigs. Pigs in pen A of each room were inoculated with the corresponding virus or negative media. At two days post inoculation (DPI), contact pigs were added to pen B adjacent to pen A in each room. Pigs were necropsied at 10 and 28 DPI. Compared to other virus-inoculated groups, the L1C.5-inoculated pigs exhibited more severe anorexia and lethargy, higher mortality, a higher fraction of pigs with fever (>40 °C), higher average temperature at several DPIs, and higher viremia levels at 2 DPI. A higher percentage of the contact pigs in the L1C.5 group became viremic at two days post contact, implying the higher transmissibility of this virus strain. It was also found that some PRRSV isolates caused brain infection in inoculation pigs and/or contact pigs. The complete genome sequences and growth characteristics in ZMAC cells of five PRRSV-2 isolates were further compared. Collectively, this study confirms that the PRRSV 1-4-4 L1C variant (L1C.5) is highly virulent with potential higher transmissibility, but the genetic determinants of virulence remain to be elucidated.

Feed batch sequencing to decrease the risk of porcine epidemic diarrhea virus (PEDV) cross-contamination during feed manufacturing.

Feed has been identified as a vector of transmission for porcine epidemic diarrhea virus (PEDV). The objective of this study was to determine if feed batch sequencing methods could minimize PEDV cross-contamination. Porcine epidemic diarrhea virus-free swine feed was manufactured to represent the negative control. A 50 kg feed batch was mixed in a pilot scale feed mill for 5 min, sampled, then discharged for 10 min into a bucket elevator and sampled again upon exit. Next, a pathogenic PEDV isolate was used to inoculate 49.5 kg of PEDV-free feed to form the positive control. The positive control was mixed, conveyed and sampled similar to the negative control. Subsequently, 4 sequence batches (sequence 1 to 4) were formed by adding a 50 kg batch of PEDV-negative feed to the mixer after the prior batch was mixed and conveyed; all sequences were mixed, conveyed, and sampled similar to the negative and positive control batches. None of the equipment was cleaned between batches within a replicate. This entire process was replicated 3 times with cleaning the feed mill between replicates. Feed was then analyzed for PEDV RNA by real-time reverse transcriptase semiquantitative polymerase chain reaction (rRT-PCR) as measured by cycle threshold (Ct) and for infectivity by bioassay. Sequence 1 feed had higher (P ˂ 0.05) rRT-PCR Ct values than the positive batch and sequence 2 feed had higher (P ˂ 0.05) Ct values than sequence 1, regardless of sampled location. Feed sampled from the mixer from sequence 2, 3, and 4 was rRT-PCR negative whereas feed sampled from the bucket elevator was rRT-PCR negative from sequence 3 and 4. Bioassay was conducted using 66 mixed sex 10-d-old pigs confirmed negative for PEDV allocated to 22 different rooms. Pigs were initially 10-d old. Control pigs remained PEDV negative for the study. All pigs from the mixer positive batch (9/9) and bucket elevator positive batch (3/3) were rRT-PCR positive on fecal swabs by the end of the study. One replicate of pigs from mixer sequence 1 was rRT-PCR positive (3/3) by 7 dpi. One replicate of mixer pigs from sequence 2 was rRT-PCR positive (3/3) by 7 dpi although no detectable PEDV RNA was found in the feed. The results demonstrate sequenced batches had reduced quantities of PEDV RNA although sequenced feed without detectible PEDV RNA by rRT-PCR can be infectious. Therefore, a sequencing protocol can reduce but not eliminate the risk of producing infectious PEDV carryover from the first sequenced batch of feed.

Characterizing the rapid spread of porcine epidemic diarrhea virus (PEDV) through an animal food manufacturing facility.

New regulatory and consumer demands highlight the importance of animal feed as a part of our national food safety system. Porcine epidemic diarrhea virus (PEDV) is the first viral pathogen confirmed to be widely transmissible in animal food. Because the potential for viral contamination in animal food is not well characterized, the objectives of this study were to 1) observe the magnitude of virus contamination in an animal food manufacturing facility, and 2) investigate a proposed method, feed sequencing, to decrease virus decontamination on animal food-contact surfaces. A U.S. virulent PEDV isolate was used to inoculate 50 kg swine feed, which was mixed, conveyed, and discharged into bags using pilot-scale feed manufacturing equipment. Surfaces were swabbed and analyzed for the presence of PEDV RNA by quantitative real-time polymerase chain reaction (qPCR). Environmental swabs indicated complete contamination of animal food-contact surfaces (0/40 vs. 48/48, positive baseline samples/total baseline samples, positive subsequent samples/total subsequent samples, respectively; P < 0.05) and near complete contamination of non-animal food-contact surfaces (0/24 vs. 16/18, positive baseline samples/total baseline samples, positive subsequent samples/total subsequent samples, respectively; P < 0.05). Flushing animal food-contact surfaces with low-risk feed is commonly used to reduce cross-contamination in animal feed manufacturing. Thus, four subsequent 50 kg batches of virus-free swine feed were manufactured using the same system to test its impact on decontaminating animal food-contact surfaces. Even after 4 subsequent sequences, animal food-contact surfaces retained viral RNA (28/33 positive samples/total samples), with conveying system being more contaminated than the mixer. A bioassay to test infectivity of dust from animal food-contact surfaces failed to produce infectivity. This study demonstrates the potential widespread viral contamination of surfaces in an animal food manufacturing facility and the difficulty of removing contamination using conventional feed sequencing, which underscores the importance for preventing viruses from entering and contaminating such facilities.

Elimination of Porcine Epidemic Diarrhea Virus in an Animal Feed Manufacturing Facility.

Porcine Epidemic Diarrhea Virus (PEDV) was the first virus of wide scale concern to be linked to possible transmission by livestock feed or ingredients. Measures to exclude pathogens, prevent cross-contamination, and actively reduce the pathogenic load of feed and ingredients are being developed. However, research thus far has focused on the role of chemicals or thermal treatment to reduce the RNA in the actual feedstuffs, and has not addressed potential residual contamination within the manufacturing facility that may lead to continuous contamination of finished feeds. The purpose of this experiment was to evaluate the use of a standardized protocol to sanitize an animal feed manufacturing facility contaminated with PEDV. Environmental swabs were collected throughout the facility during the manufacturing of a swine diet inoculated with PEDV. To monitor facility contamination of the virus, swabs were collected at: 1) baseline prior to inoculation, 2) after production of the inoculated feed, 3) after application of a quaternary ammonium-glutaraldehyde blend cleaner, 4) after application of a sodium hypochlorite sanitizing solution, and 5) after facility heat-up to 60°C for 48 hours. Decontamination step, surface, type, zone and their interactions were all found to impact the quantity of detectable PEDV RNA (P < 0.05). As expected, all samples collected from equipment surfaces contained PEDV RNA after production of the contaminated feed. Additionally, the majority of samples collected from non-direct feed contact surfaces were also positive for PEDV RNA after the production of the contaminated feed, emphasizing the potential role dust plays in cross-contamination of pathogen throughout a manufacturing facility. Application of the cleaner, sanitizer, and heat were effective at reducing PEDV genomic material (P < 0.05), but did not completely eliminate it.

Evaluation of the minimum infectious dose of porcine epidemic diarrhea virus in virus-inoculated feed.

OBJECTIVE To determine the minimum infectious dose of porcine epidemic diarrhea virus (PEDV) in virus-inoculated feed. ANIMALS 30 crossbred 10-day-old pigs. PROCEDURES Tissue culture PEDV was diluted to form 8 serial 10-fold dilutions. An aliquot of stock virus (5.6 × 10(5) TCID50/mL) and each serial PEDV dilution were mixed into 4.5-kg batches of feed to create 9 PEDV-inoculated feed doses; 1 virus-negative dose of culture medium in feed was also created. Pigs were challenge exposed via oral administration of PEDV-inoculated feed, and fecal swab specimens were collected. All pigs were euthanized 7 days after challenge exposure; fresh tissues were collected and used for PCR assay, histologic examination, and immunohistochemical analysis. RESULTS The PCR cycle threshold (Ct) decreased by approximately 10 when PEDV was added to feed, compared with results for equivalent PEDV diluted in tissue culture medium. Pigs became infected with PEDV when challenge exposed with the 4 highest concentrations (lowest concentration to cause infection, 5.6 × 10(1) TCID50/g; Ct = 27 in tissue culture medium and 37 in feed). CONCLUSIONS AND CLINICAL RELEVANCE In this study, PEDV in feed with detectable Ct values of 27 to 37 was infective. The Ct was 37 for the lowest infective PEDV dose in feed, which may be above the limit of detection established for PEDV PCR assays used by some diagnostic laboratories. Overall, results indicated 5.6 × 10(1) TCID50/g was the minimum PEDV dose in feed that can lead to infection in 10-day-old pigs under the conditions of this study.

Pyogranulomatous panophthalmitis with systemic coronavirus disease in a domestic ferret (Mustela putorius furo).

A 15-month-old spayed female ferret (Mustela putorius furo) presented for lethargy and weight loss of 2 weeks duration. Upon physical examination, a 2-mm-diameter focal area of opacity was noted in the left cornea. In addition, the ferret was quiet, in poor body condition, and dehydrated. A complete blood count and plasma biochemistry revealed a severe nonregenerative anemia, azotemia, hyperproteinemia, hypoalbuminemia, and mild hyperphosphatemia and hyperchloremia. Urinalysis revealed hyposthenuria. Whole body radiographs showed multifocal thoracic nodular disease, splenomegaly, and renomegaly. Abdominal ultrasonography confirmed bilaterally enlarged kidneys, hypoechoic liver and spleen, and a caudal abdominal hypoechoic mobile nodule. The ferret was humanely euthanized, and a postmortem examination with subsequent histopathology showed multifocal necrotizing pyogranulomas in the lung, spleen, kidneys, mesenteric lymph nodes, and serosa of the duodenum. Pyogranulomatous panophthalmitis was diagnosed in the left eye. The multisystemic granulomatous lesions were suggestive of ferret systemic coronavirus (FRSCV). The presence of coronavirus in the left eye was confirmed by positive immunohistochemistry. Reverse transcriptase polymerase chain reaction (RT-PCR) on formalin fixed paraffin embedded tissue from the lung, spleen, and kidney was negative for FRSCV and positive for ferret enteric coronavirus (FRECV). Systemic coronavirus disease in ferrets closely resembles feline infectious peritonitis (FIP) in domestic cats, which can manifest with anterior uveitis, chorioretinitis, optic neuritis, and retinal detachment. To the authors' knowledge, this is the first report of ocular lesions in a ferret with systemic coronavirus disease, suggesting that ferrets presented with similar ocular lesions should also be evaluated for evidence of coronavirus infection.

Canine intestinal histoplasmosis containing hyphal forms.

A 12-year-old intact male Miniature Schnauzer dog with chronic diarrhea that was unresponsive to empirical treatment was presented to a referring veterinarian. A laparotomy was performed, and formalin-fixed biopsies of duodenum, jejunum, and colon were sent to Oklahoma Animal Disease Diagnostic Laboratory for evaluation. Histologic examination revealed a severe, diffuse, granulomatous enteritis and colitis with intralesional yeast and hyphal forms. Grocott methenamine silver stains revealed short, aseptate hyphae co-mingled with 2-8 µm, oval to round yeast organisms consistent with Histoplasma capsulatum. The atypical presentation of both yeast and hyphal forms prompted identification of the organism. Direct sequencing of a polymerase chain reaction product from paraffin-embedded intestinal samples confirmed the presence of Ajellomyces capsulatus with a homology over 99% to several sequences in GenBank. Ajellomyces capsulatus is the holomorphic name for H. capsulatum. Therefore, the mycelial form of a dimorphic fungus such as H. capsulatum can coexist with yeast cells within lesions of histoplasmosis. Following diagnosis, the dog was treated with itraconazole for 6 months and has improved.

Pulmonary adenocarcinoma with osseous metastasis and secondary paresis in a blue and gold macaw (Ara ararauna).

A 16-yr-old female blue and gold macaw (Ara ararauna) was presented with an acute history of lethargy, inappetance, ataxia, and paralysis. The bird had rapidly progressed from a normal state to complete inability to perch or ambulate within a 48-hr period. Neurologic examination revealed bilateral hind limb paresis with upper motor neuron signs present in both legs and the vent. Radiographs identified multiple nodular soft-tissue opacities within the cranial coelomic cavity and a single nodule superimposed with the thoracic spine. The bird was euthanized and submitted for necropsy, which revealed a primary pulmonary adenocarcinoma with multiple sites of osseous metastasis, including the vertebrae, and subsequent spinal cord compression. This is the first report of pulmonary adenocarcinoma in this species, although reports of similar tumors in other psittacines have been published. This report, along with others previously published, suggests that vertebral metastasis of primary pulmonary tumors may be more common in psittacine species than previously recognized and, as such, should be considered as a differential diagnosis in psittacine birds exhibiting signs of neurologic dysfunction attributed to a spinal cord lesion.