Highly pathogenic avian influenza A(H7N3) virus in poultry workers, Mexico, 2012.

We identified 2 poultry workers with conjunctivitis caused by highly pathogenic avian influenza A(H7N3) viruses in Jalisco, Mexico. Genomic and antigenic analyses of 1 isolate indicated relatedness to poultry and wild bird subtype H7N3 viruses from North America. This isolate had a multibasic cleavage site that might have been derived from recombination with host rRNA.

Isolation of a virulent Newcastle disease virus from confiscated LaSota vaccine.

Vials of Newcastle disease vaccine labeled as LaSota were confiscated by the Arizona Division of Customs and Border Protection officials. Two different avian type 1 paramyxoviruses were isolated from all three vials of vaccine submitted to the National Veterinary Services Laboratories. The LaSota strain of avian paramyxovirus type 1 virus was isolated from all three vials and analyzed by nucleotide sequence analysis. A virulent Newcastle disease virus was also present in all three vials, but in low concentration. The virulence of the Newcastle disease virus was characterized by the intracerebral chicken pathogenicity index chicken inoculation assay but could not be determined by nucleotide sequence analysis from the virus isolated from embryonating chicken eggs. The intracerebral chicken pathogenicity index value for the isolated Newcastle disease virus was 1.55. Strains of Newcastle disease virus with intracerebral pathogenicity indexes significantly above 1.0 have been found to selectively kill many types of cancer cells while not affecting normal nonneoplastic cells and are considered to be a viable option for cancer treatment in humans by alternative medical researchers; however, the treatment is not approved for use in the United States by the Food and Drug Administration. Customs and Border Protection officials have been notified of an increased risk of Newcastle disease virus entering the United States for use as a nonapproved cancer treatment. Illegal importation of Newcastle disease vaccine for vaccination of backyard poultry is also a threat. This case report emphasizes the importance of conducting chicken inoculation for complete virus pathotyping and demonstrates the need for stringent security procedures at U.S. borders to detect known livestock pathogens that may be smuggled in for use in animal agriculture and reasons unrelated to animal agriculture.

A pre-pandemic outbreak of triple-reassortant swine influenza virus infection among university students, South Dakota, 2008.

BACKGROUND: After identifying a student with triple-reassortant swine influenza virus (SIV) infection and pig exposure at a livestock event, we investigated whether others were infected and if human-to-human transmission occurred. METHODS: We conducted a cohort study and serosurvey among persons exposed to (1) event pigs, (2) other pigs, (3) the index case, and (4) persons without pig or index case exposure. Confirmed cases had respiratory specimens positive for SIV within 2 weeks of the index case's illness. Probable and suspected cases had illness and (1) exposure to any pig or (2) contact with a confirmed case preceding illness. Probable cases were seropositive. Suspected cases did not give serum samples. RESULTS: Of 99 event pig-exposed students, 72 (73%) participated in the investigation, and 42 (42%) provided serum samples, of whom 17 (40%) were seropositive and 5 (12%) met case criteria. Of 9 students exposed to other pigs, 2 (22%) were seropositive. Of 8 index case-exposed persons and 10 without exposures, none were seropositive. Pig-exposed persons were more likely to be seropositive than persons without pig exposure (37% vs 0%, P < .01). CONCLUSIONS: We identified an outbreak of human SIV infection likely associated with a livestock event; there was no evidence of human-to-human transmission.

Biosafety principles and practices for the veterinary diagnostic laboratory.

Good biosafety and biocontainment programs and practices are critical components of the successful operation of any veterinary diagnostic laboratory. In this chapter we provide information and guidance on critical biosafety management program elements, facility requirements, protective equipment, and procedures necessary to ensure that the laboratory worker and the environment are adequately protected in the challenging work environment of the veterinary diagnostic laboratory in general and provide specific guidance for those laboratories employing molecular diagnostic techniques.

Pathogenicity of a Venezuelan equine encephalomyelitis serotype IE virus isolate for ponies.

The enzootic or endemic strains of Venezuelan equine encephalomyelitis (VEE) virus (ID, IE, IF, and II-VI) are considered avirulent. In 1993 and 1996, outbreaks of encephalitis occurred in the horse populations in the Chiapas and Oaxaca provinces of Mexico, respectively. In both instances, enzootic VEE virus subserotype IE was isolated from brain tissues of dead horses. The present study investigated the pathogenicity of the Chiapas viral isolate (NVSL VEE IE 93-42124) in ponies. Three ponies were inoculated intradermally with 4, 5, and 6 logs, respectively, of the NVSL VEE IE 93-42124 viral isolate. All ponies showed fluctuations in body temperature, encephalitis, and other signs of infection with VEE virus. Virus was isolated only from the blood of ponies from day 1 to day 3 postinfection. Microscopic examination of hematoxylin and eosin-stained tissue sections showed mild to moderate nonsuppurative encephalitis, perivascular cuffing by mononuclear cells, gliosis, and meningoencephalitis. Antibody (IgM) to VEE virus IE was unable to differentiate between various subserotypes of VEE I viruses (serotypes IAB, IC, ID, and IF). Virus neutralizing antibody titers to heterologous VEE I viruses were 10-100-fold less than those for NVSL VEE IE 93-42124 virus and Mena II, a human isolate of VEE IE virus. The study confirmed that NVSL VEE IE 93-42124 virus, which was isolated from a brain of a horse during an outbreak of VEE in Chiapas, Mexico, was pathogenic for ponies.

Serologic survey of cattle in the northeastern and north central United States, Virginia, Alaska, and Hawaii for antibodies to Cache Valley and antigenically related viruses (Bunyamwera serogroup virus).

Bovine sera from northeastern states (Connecticut, Delaware, Maine, Maryland, Massachusetts, New York, Pennsylvania, Vermont, and West Virginia), north central states (Indiana, Illinois, Iowa, Kentucky, Michigan, Minnesota, North Dakota, Ohio, South Dakota, and Wisconsin), Virginia, Alaska, and Hawaii were examined for the presence of neutralizing antibodies to Cache Valley (CV), Lokern (LK), Main Drain (MD), Northway (NW), and Tensaw (TS) viruses. Microneutralization tests were performed using Vero cells. Ninety percent inhibition of the virus at a 1:10 serum dilution was considered positive for the presence of specific antibody. Sera having antibody to more than one virus were titrated from 1:10 to 1:640. The results indicated that 4-28% of the cattle per region had specific antibodies to CV virus. Neutralizing antibodies to NW, LK, and TS viruses were also detected, indicating possible exposure to these Bunyamwera serogroup viruses along with CV virus. Antibody titers measured against NW virus were very similar to those against CV virus. Antibodies to MD virus were present in low levels in bovine sera from Illinois, Maryland, and Ohio. Cattle from Alaska had only antibodies to NW virus. Antibodies to Bunyamwera serogroup viruses were not observed in sera from Hawaii.

Nested multiplex RT-PCR for detection and differentiation of West Nile virus and eastern equine encephalomyelitis virus in brain tissues.

A traditional nested reverse transcription-polymerase chain reaction (RT-PCR) assay specific for eastern equine encephalomyelitis (EEE) virus was designed to multiplex with a previously described West Nile (WN) virus nested RT-PCR assay. Differentiation of EEE and WN was based on base pair size of the amplified product. One hundred fifty-seven mammalian and avian brain tissues were tested by EEE/WN nested multiplex RT-PCR, EEE nested RT-PCR, and WN nested RT-PCR, and results were compared with other diagnostic test results from the same animals. Serological and virus isolation testing confirmed the results of the multiplex PCR assay. When compared with cell culture virus isolation, the multiplex assay was shown to be more sensitive in detecting the presence of EEE or WN virus in brain tissues. The multiplex assay was shown to be sensitive and specific for North American EEE and WN and provided a rapid means of identifying both viruses in brain tissues. No apparent sacrifice in sensitivity was observed in the multiplex procedure compared with the individual EEE and WN nested RT-PCR assays. Data collected from an additional 485 multiplex RT-PCR tests conducted during the summer and fall of 2002 further support the validity of the procedure.

Vesicular stomatitis.

Vesicular stomatitis is an infrequent yet important vesicular disease of cattle, horses, and swine. Periodic outbreaks of this disease in the United States have caused economic losses in cattle herds because of decreased production, movement restrictions, and trade embargoes. Vesicular stomatitis causes clinical signs indistinguishable from those of foot-and-mouth disease. It is of utmost importance that appropriate samples are collected from clinical cases of vesicular disease in cattle and swine so a rapid laboratory diagnosis can be made.

Emergence of Porcine epidemic diarrhea virus in the United States: clinical signs, lesions, and viral genomic sequences.

During the 10 days commencing April 29, 2013, the Iowa State University Veterinary Diagnostic Laboratory received the first 4 of many submissions from swine farms experiencing explosive epidemics of diarrhea and vomiting affecting all ages, with 90-95% mortality in suckling pigs. Histology revealed severe atrophy of villi in all segments of the small intestines with occasional villus-epithelial syncytial cells, but testing for rotaviruses and Transmissible gastroenteritis virus (Alphacoronavirus 1) were negative. Negative-staining electron microscopy of feces revealed coronavirus-like particles and a pan-coronavirus polymerase chain reaction (PCR) designed to amplify a conserved region of the polymerase gene for all members in the family Coronaviridae produced expected 251-bp amplicons. Subsequent sequencing and analysis revealed 99.6-100% identity among the PCR amplicons from the 4 farms and 97-99% identity to the corresponding portion of the polymerase gene of Porcine epidemic diarrhea virus (PEDV) strains, with the highest identity (99%) to strains from China in 2012. Findings were corroborated at National Veterinary Services Laboratories using 2 nested S-gene and 1 nested N-gene PCR tests where the sequenced amplicons also had the highest identity with 2012 China strains. Whole genome sequence for the virus from 2 farms in 2 different states using next-generation sequencing technique was compared to PEDV sequences available in GenBank. The 2013 U.S. PEDV had 96.6-99.5% identity with all known PEDV strains and the highest identity (>99.0%) to some of the 2011-2012 Chinese strains. The nearly simultaneous outbreaks of disease, and high degree of homology (99.6-100%) between the PEDV strains from the 4 unrelated farms, suggests a common source of virus.

Comparison of Q fever serology methods in cattle, goats, and sheep.

Coxiella burnetii is an obligate intracellular bacterium that is responsible for the zoonotic disease Q fever. The distribution of this agent is worldwide except for New Zealand, and infection can be asymptomatic in both human beings and animals. Chronic exposures can produce abortions, stillbirths, and infertility issues in animals and endocarditis in human beings. A commercial enzyme-linked immunosorbent assay (ELISA) kit marketed in the European Union was purchased to compare C. burnetii antibody detection methods. The current study examined the agreement of ELISA and complement fixation results in over 668 diagnostic ruminant sera submitted to the National Veterinary Services Laboratories for Q fever serologic testing. The majority of combined sera (548) were negative on both tests. Fifty-seven of the combined sera were positive on both tests. There were 45 combined sera with low complement fixation titers at 1:10 and negative ELISA results. The results were surprising given the expectations that ELISA methods, by nature, amplify detection of antibody-antigen interactions leading to higher sensitivity. Potential mechanisms for these discrepant results are discussed.

Isolation of Equine rhinitis A virus from a horse semen sample.

Semen from an apparently healthy 4-year-old American Quarter Horse was submitted to the National Veterinary Services Laboratories for Equine arteritis virus isolation. Visual inspection of the semen sample upon arrival noted it was unusually yellow in color. The semen sample was inoculated onto cell monolayers, and cytopathic effect was observed 5 days postinoculation. The resultant isolate tested negative for Equine arteritis virus, and was subsequently identified as Equine rhinitis A virus. Equine rhinitis A virus has been isolated from horse urine, but has not been described in stallion semen. The present study documents the isolation of Equine rhinitis A virus from stallion semen that was likely contaminated with urine at the time of collection.

Differential diagnostic test technology: sensitivity and specificity, an OIE validation perspective.

Companion differential diagnostic technology is extremely useful when utilized with marker vaccines for disease control and eradication. Examples of this technology include enzyme-linked immunoassays and particle concentration fluorescence immunoassays. The predictive values of such assays are dependent upon adequate validation of their diagnostic sensitivities and specificities. The World Organization for Animal Health (OIE) has put forth official guidelines for performing complete validation of diagnostic tests. This article presents an overview of these important guidelines.