Nested PCR effective to detect low viral loads of SARS-CoV-2 in animal samples.

SARS-CoV-2 emerged from an animal source and was then transmitted to humans, causing the COVID-19 pandemic. Since a wide range of animals are susceptible to SARS-CoV-2 infection, the zoonotic potential of SARS-CoV-2 increases with every new animal infected. The molecular gold standard assay for SARS-CoV-2 detection is real-time RT-PCR, where the Ct obtained is proportional to the amount of nucleic acid and can be a semi-quantitative measure of the viral load. However, since the use of real-time RT-PCR assays in animal samples is low due to the high costs, the use of validated nested PCR assays will help to monitor large-scale animal samplings, by reducing the costs of detection. In the present study, 140 samples from dogs and cats (15 SARS-CoV-2-positive samples with Ct values from 27 to 33, and 125 negative samples), previously analyzed by real-time RT-PCR, were analyzed by nested PCR. To increase the number of positive samples to determine the sensitivity of the assay, 40 human samples obtained during COVID-19 diagnosis in 2020 were included. The specificity of the primers was analyzed against samples positive to canine coronavirus (CCV) and feline infectious peritonitis virus (FIPV). To calculate the limit of detection (LoD) of the nested PCR, the viral load was estimated extrapolating the Ct value obtained by real-time RT-PCR. The Ct values obtained were considered as semi-quantitative and were able to distinguish between high, moderate and low viral loads. The Kappa value or "agreement" between assays and reliability of the nested PCR were also determined. Eleven of the animal samples analyzed by nested PCR targeting the N gene were detected as positive, while 129 were detected as negative to the virus, with Ct values ranging between17 and 31.5. All the samples from humans analyzed by nested PCR were positive. These results indicate that the assay has a sensitivity of near 95 % and a specificity of 100 %. No unspecific reactions analyzed by nested PCR were observed with the samples positive to CCV and FIPV. The samples detected as positive to SARS-CoV-2 by nested PCR were those that presented a Ct between17 and 31.5. The LoD of the nested PCR was estimated close to 50 copies/µL of viral load, corresponding with a Ct of 31.5. The Kappa value between assays was excellent (k = 0.829). The results obtained demonstrate that nested PCR is useful to detect SARS-CoV-2 low viral loads at a lower cost than with real-time RT-PCR.

Serological evidence of SARS-CoV-2 infection in pets naturally exposed during the COVID-19 outbreak in Argentina.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of coronavirus disease 2019 (COVID-19), has rapidly spread worldwide. The monitoring of animals has shown that certain species may be susceptible to be infected with the virus. The present study aimed to evaluate the presence of SARS-CoV-2 antibodies by ELISA and virus neutralization (VN) in pets from owners previously confirmed as COVID-19-positive in Argentina. Serum samples of 38 pets (seven cats and 31 dogs) were obtained for SARS-CoV-2 antibody detection. Three out of the seven cats and 14 out of the 31 dogs were positive for SARS-CoV-2 by ELISA, and one cat and six dogs showed the presence of neutralizing antibodies in which the cat and two of the six dogs showed high titers. Another dog from which three serum samples had been obtained within eight months from the diagnosis of its owner showed the presence of antibodies at different times by both ELISA and VN. However, the results showed that the antibodies decreased slightly from the first to the third sample. Our results provide evidence that SARS-CoV-2 infection in pets living with COVID-19-positive humans from Argentina during the outbreak of SARS-CoV-2 can be detected by serology assay.

Lotmaria passim (Kinetoplastea: Trypanosomatidae) in honey bees from Argentina.

Lotmaria passim (Kinetoplastea) is considered the most prevalent as well as the most virulent trypanosomatid associated to the European honey bee Apis mellifera. We used qPCR to screen for the presence of this parasite in 57 samples from ten Argentinian provinces, and were able to detect its presence throughout most of the country with 41% of the samples testing positive. In a retrospective analysis, we detected L. passim in 73% of honey bee samples from 2006 showing that this flagellate has been widely present in Argentina for at least ~15 years. Additionally, three primer sets for L. passim detection were compared, with the pair that produced smallest PCR product having the best detection capability. Finally, we also found L. passim DNA in 100% (n = 6) of samples of the mite Varroa destructor. The role of this ectoparasite in the lifecycle of Lotmaria, if any, remains unrevealed.

First detection and molecular analysis of SARS-CoV-2 from a naturally infected cat from Argentina.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of coronavirus disease 2019 (COVID-19), has rapidly spread worldwide. Studies of transmission of the virus carried out in animals have suggested that certain animals may be susceptible to infection with SARS-CoV-2. The aim of the present study was to investigate the infection of SARS-CoV-2 in pets (18 cats and 20 dogs) from owners previously confirmed as COVID-19-positive. Oropharyngeal and rectal swabs were taken and analyzed by real-time RT-PCR assays, while blood samples were taken for antibody detection. Of the total pets analyzed, one cat was found reactive to SARS-CoV-2 by real-time RT-PCR of an oropharyngeal and a rectal swab. This cat presented only sneezing as a clinical sign. Serological analysis confirmed the presence of antibodies in the serum sample from this cat, as well as in the serum from another cat non-reactive to real-time RT-PCR. Complete sequence and phylogenetic analysis allowed determining that the SARS-CoV-2 genome belonged to the B.1.499 lineage. This lineage has been reported in different provinces of Argentina, mainly in the Metropolitan Area of Buenos Aires. This study notifies the first detection of the natural infection and molecular analysis of SARS-CoV-2 in a cat from Argentina whose owner where COVID-19-positive. Although there is currently no evidence that cats can spread COVID-19, results suggest that health authorities should test pets with COVID-19-positive owners.

Analysis of the pX region of bovine leukemia virus in different clinical stages of Enzootic Bovine Leukemia in Argentine Holstein cattle.

Bovine leukemia virus (BLV) infection in cattle causes Enzootic Bovine Leukemia (EBL). About 30% of infected cattle develop persistent lymphocytosis (PL), a 0.1-5% develops tumors, and a 70% remains asymptomatic in an aleukemic stage (AL). Regulatory genes of BLV (Tax, Rex, R3 and G4) are located in a region known as pX(BLV). The variability of those genes had been postulated with the progression of the disease. The aim of this work was to compare the wild-type proviral pX(BLV) region at different stages of BLV natural infected cattle from Argentine Holstein. Pairs of primers were designed to amplify the proviral pX region of 12 cattle by PCR, and products were then sequenced, aligned and compared both with each other and with the reference sequence. Results show a divergence percentage from 0 to 6.1 for the Tax gene, from 0 to 9.4% for the Rex gene, from 0 to 12.1% for the R3 gene and finally from 0 to 6.5% for the G4 gene. Results obtained with hierarchical clustering showed two clusters well differentiated, where the members of each cluster are cattle that had tumor, PL and AL, not allowing differentiate those two cluster by clinical stage.