Diagnostics for Viral Pathogens in Veterinary Diagnostic Laboratories.

Laboratory testing is one part of clinical diagnosis, and quick and reliable testing results provide important data to support treatment decision and develop control strategies. Clinical viral testing has been shifting from traditional virus isolation and electron microscopy to molecular polymerase chain reaction and point-of-care antigen tests. This shift in diagnostic methodology also means change from looking for infectious virions or viral particles to hunting viral antigens and genomes. With technological development, it is predicted that metagenomic sequencing will be commonly used in veterinary clinical diagnosis for unveiling the whole picture of microbes involved in diseases in the future.

Microbial and clinical epidemiology of invasive fungal rhinosinusitis in hospitalized COVID-19 patients, the divergent causative agents.

Since COVID-19 spread worldwide, invasive fungal rhinosinusitis (IFRS) has emerged in immunocompromised patients as a new clinical challenge. In this study, clinical specimens of 89 COVID-19 patients who presented clinical and radiological evidence suggestive of IFRS were examined by direct microscopy, histopathology, and culture, and the isolated colonies were identified through DNA sequence analysis. Fungal elements were microscopically observed in 84.27% of the patients. Males (53.9%) and patients over 40 (95.5%) were more commonly affected than others. Headache (94.4%) and retro-orbital pain (87.6%) were the most common symptoms, followed by ptosis/proptosis/eyelid swelling (52.8%), and 74 patients underwent surgery and debridement. The most common predisposing factors were steroid therapy (n = 83, 93.3%), diabetes mellitus (n = 63, 70.8%), and hypertension (n = 42, 47.2%). The culture was positive for 60.67% of the confirmed cases, and Mucorales were the most prevalent (48.14%) causative fungal agents. Different species of Aspergillus (29.63%) and Fusarium (3.7%) and a mix of two filamentous fungi (16.67%) were other causative agents. For 21 patients, no growth was seen in culture despite a positive result on microscopic examinations. In PCR-sequencing of 53 isolates, divergent fungal taxons, including 8 genera and 17 species, were identified as followed: Rhizopus oryzae (n = 22), Aspergillus flavus (n = 10), A. fumigatus (n = 4), A. niger (n = 3), R. microsporus (n = 2), Mucor circinelloides, Lichtheimia ramosa, Apophysomyces variabilis, A. tubingensis, A. alliaceus, A. nidulans, A. calidoustus, Fusarium fujikuroi/proliferatum, F. oxysporum, F. solani, Lomentospora prolificans, and Candida albicans (each n = 1). In conclusion, a diverse set of species involved in COVID-19-associated IFRS was observed in this study. Our data encourage specialist physicians to consider the possibility of involving various species in IFRS in immunocompromised and COVID-19 patients. In light of utilizing molecular identification approaches, the current knowledge of microbial epidemiology of invasive fungal infections, especially IFRS, may change dramatically.

Invasive fungal rhinosinusitis (IFRS) may infect people with diabetes, cancer, or COVID-19. In this study, various types of fungi were identified from COVID-19-associated-IFRS, encouraging physicians to consider specific treatments.

Detection of anti-feline infectious peritonitis virus activity of a Chinese herb extract using geneLEAD VIII, a fully automated nucleic acid extraction/quantitative PCR testing system.

The geneLEAD VIII is a fully-automated nucleic acid extraction/quantitative PCR equipment developed by Precision System Science Co., Ltd., (PSS). To take advantage of its capability, we developed a quantitative assay system to measure growth of animal viruses. The system was used to assay one of the Chinese herbal extracts whose anti-malarial activities were previously reported and demonstrated its dose-dependent anti-viral activity against feline infectious peritonitis virus (FIPV), a feline coronavirus causing the fatal diseases in cats, and relatively low cell toxicity. The assay developed in this study is useful to screen antiviral drugs and the anti-FIPV activity of the herbal extract identified have a potential to lead to development of new drugs against FIPV and other coronaviruses, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Disseminated Mycobacterium genavense infection in a guinea pig (Cavia porcellus): a case report.

BACKGROUND: Mycobacteria are found in many environmental conditions and infect a variety of species, including rodents and rabbits. Guinea pigs are used experimentally as a model for Mycobacterium tuberculosis, but natural mycobacteriosis in guinea pigs has not been reported. CASE PRESENTATION: A 1.5-year-old female guinea pig was found acutely deceased with no premonitory illness. On gross post-mortem examination, multifocal to coalescing, raised, firm, pale tan nodules with discrete, irregular margins were noted over the surfaces of all lung lobes. Histopathology revealed nodules composed of clustered foamy macrophages and multinucleated giant cells containing numerous bacterial rods. Similar bacteria-laden macrophages were noted within sections of the liver, heart, palpebral conjunctiva, duodenum, and cecum. Polymerase chain reaction was performed on tissues collected during post-mortem examination. The 16S rRNA gene product was sequenced and was identical to the Mycobacterium genavense type strain. CONCLUSIONS: To the best of the author's knowledge, this report details the first documented case of Mycobacterium genvaense infection in a guinea pig and a follow up investigation of close-contact animals. Given their experimental susceptibility and this clinical case report, mycobacteriosis should be considered as a differential in guinea pigs exhibiting weight loss in the absence of other clinical signs. With the potential for zoonotic transmission in immunosuppressed individuals, precautions should be taken to safeguard human health in cases of guinea pigs with suspected M. genavense infection.

Characterization of viral, bacterial, and parasitic causes of disease in small-scale chicken flocks in the Mekong Delta of Vietnam.

In the Mekong Delta region of Vietnam, small-scale chicken farming is common. However, high levels of disease or mortality in such flocks impair economic development and challenge the livelihoods of many rural households. We investigated 61 diseased small-scale flocks (122 chickens) for evidence of infection with 5 bacteria, 4 viruses, and helminths. Serological profiles (ELISA) were also determined against 6 of these pathogens. The aims of this study were the following: (1) to investigate the prevalence of different pathogens and to compare the probability of detection of bacterial pathogens using PCR and culture; (2) to investigate the relationship between detection of organisms in birds' tissues and the observed morbidity and mortality, as well as their antibody profile; and (3) to characterize risk factors for infection with specific viral or bacterial pathogens. We used PCR to test for viral (viruses causing infectious bronchitis [IB], highly pathogenic avian influenza [HPAI], Newcastle disease, and infectious bursal disease [IBD]) and bacterial pathogens (Mycoplasma gallisepticum, Pasteurella multocida, Avibacterium paragallinarum, and Ornithobacterium rhinotracheale [ORT]). The latter two were also investigated in respiratory tissues by conventional culture. Colisepticemic Escherichia coli was investigated by liver or spleen culture. In 49 of 61 (80.3%) flocks, at least one bacterial or viral pathogen was detected, and in 29 (47.5%) flocks, more than one pathogen was detected. A. paragallinarum was detected in 62.3% flocks, followed by M. gallisepticum (26.2%), viruses causing IBD (24.6%) and IB (21.3%), septicemic E. coli (14.8%), ORT (13.1%), and HPAI viruses (4.9%). Of all flocks, 67.2% flocks were colonized by helminths. Mortality was highest among flocks infected with HPAI (100%, interquartile range [IQR]: 81.6-100%) and lowest with flocks infected with ORT (5.3%, IQR: 1.1-9.0%). The results indicated slight agreement (kappa ≤ 0.167) between detection by PCR and culture for both A. paragallinarum and ORT, as well as between the presence of cestodes and ORT infection (kappa = 0.317). Control of A. paragallinarum, viruses causing HPAI, IBD, and IB, M. gallisepticum, and gastrointestinal helminths should be a priority in small-scale flocks.

Development of a droplet digital PCR for detection and quantification of porcine epidemic diarrhea virus.

Porcine epidemic diarrhea, a disease caused by porcine epidemic diarrhea virus (PEDV), results in large economic losses to the global swine industry. To manage this disease effectively, it is essential to detect PEDV early and accurately. We developed a sensitive and accurate droplet digital PCR (ddPCR) assay to detect PEDV. The optimal primer-to-probe concentration and melting temperature were identified as 300:200 nM and 59.2°C, respectively. The specificity of the ddPCR assay was confirmed by negative test results for common swine pathogens. The detection limit for the ddPCR was 0.26 copies/µL, which is a 5.7-fold increase in sensitivity compared to that of real-time PCR (rtPCR). Both ddPCR and rtPCR assays exhibited good linearity, although ddPCR provided higher sensitivity for clinical detection compared to that of rtPCR. Our ddPCR methodology provides a promising tool for evaluating the PEDV viral load when used for clinical testing, particularly for detecting samples with low-copy viral loads.