Duplex nested-PCR for detection of small ruminant lentiviruses

Marinho, Rebeca Cavalcante; Martins, Gabrielle Rosemblit; Souza, K. C. de; Sousa, A. L. M. de; Silva, Sabrina Tainah C.; Nobre, Juliana A.; Teixeira, María Francisca Simas

doi:10.1016/j.bjm.2018.04.013

Cited by 18 publications

(11 citation statements)

References 42 publications

(71 reference statements)

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“…Molecular diagnosis by PCR may add diagnostic value to serodiagnosis since seronegative animals may show PCR positive results due to low antibody production [ 12 , 13 ]. New molecular methods are being described focused on the design of universal primers, thereby increasing sensitivity to enable the identification and removal of animals with low viral load in vivo [ 14 , 15 , 16 , 17 ].…”

Section: Introductionmentioning

confidence: 99%

Accurate Diagnosis of Small Ruminant Lentivirus Infection Is Needed for Selection of Resistant Sheep through TMEM154 E35K Genotyping

Ramírez

Echeverría

Benito³

et al. 2021

Pathogens

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Small ruminant lentiviruses (SRLV) cause an incurable multiorganic disease widely spread in sheep and goats that disturbs animal welfare and production. In the absence of a vaccine, control measures have been traditionally based on early diagnosis and breeding with virus-inactivated colostrum with segregation of seropositive animals. However, antigenic heterogeneity, poor antibody production due to low viral load, and single strain design of most available ELISA, pose a threat to SRLV diagnosis. Genome-wide association studies have described TMEM154 E35K polymorphism as a good genetic marker for selection of resistant animals in some American and European breeds. In this study, a multitargeted serological and virological screening of more than 500 animals from four different breeds (latxa, raza Navarra, assaf, and churra) attending to SRLV infection status was performed. Then, animals were genotyped to characterize TMEM154 E35K polymorphism. ELISA procedures, individually considered, only identified a proportion of the seropositive animals, and PCR detected a fraction of seronegative animals, globally offering different animal classifications according to SRLV infection status. TMEM154 allele frequency differed substantially among breeds and a positive association between seroprevalence and TMEM154 genotype was found only in one breed. Selection based on TMEM154 may be suitable for specific ovine breeds or SRLV strains, however generalization to the whole SRLV genetic spectrum, ovine breeds, or epidemiological situation may need further validation.

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Section: Introductionmentioning

confidence: 99%

Accurate Diagnosis of Small Ruminant Lentivirus Infection Is Needed for Selection of Resistant Sheep through TMEM154 E35K Genotyping

Ramírez

Echeverría

Benito³

et al. 2021

Pathogens

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“…In general, serological methods may fail due to certain factors, such as the period of immunological window, low antibody titers, late seroconversion or intermittent seropositivity and seronegativity reactions (27) . On the other hand, molecular techniques, such as PCR, although of high cost, show promising results, allowing the detection of Lentivirus infection before seroconversion (24,28) .…”

Section: Resultsmentioning

confidence: 99%

Seropositivity for Maedi-Visna virus in sheep in Porto Acre city - Western Amazon, Brazil

Vinha

Silva

2020

Ciênc. anim. bras.

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Lentivirosis of small ruminants (LVPR) are chronic and degenerative infectious diseases, caused by Lentivirus, associated with numerous losses such as: drop in meat and milk production, predisposition to secondary infections, expenses with veterinary assistance and, even, early disposal of animals. In the northern region of Brazil, the epidemiological situation is poorly understood. Thus, this study aimed to determine the seropositivity of sheep for Lentivirus in Porto Acre city, Western Amazon, Brazil. 122 blood samples from sheep were collected and as a diagnostic method, agarose gel immunodiffusion was used, using the p28 protein of the capsid as antigen. The seropositivity of the sheep to the test was 8.2% (10/122). In 80% (4/5) of the investigated properties, the presence of seropositive animals was detected. It is worth noting that the acquisition of small ruminants from other states likely represented a risk to sheep health in the municipality of Porto Acre, Western Amazon, Brazil. It is concluded that there is a need for more systematic investigations on the prevalence of LVPR in the state of Acre.

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“…After collection, the tubes without anticoagulant were centrifuged in a centrifuge at room temperature at 1500g for 10 minutes to separate the blood serum, subsequently subjected to the Western Blot test [23]. The anticoagulant tubes were destined for the extraction of deoxyribonucleic acid (DNA) according to standard methodology [26] and submitted to Nested PCR (nPCR) [27]. At birth, before ingesting colostrum, without any contact between the mother and the young, 73 kids were subjected to the same blood collection procedure, with subsequent performance of the same diagnostic tests.…”

Section: Blood Collection and Diagnostic Testmentioning

confidence: 99%

“…Amplification by nPCR occurred at 94˚C for five minutes, 35 cycles of 94˚C for one minute, 56˚C for one minute and 72˚C for 45 seconds, followed by a final extension at 72˚C for seven minutes. The amplified samples and the controls (positive and negative), were submitted to electrophoresis in 2% agarose gel (Sigma-Aldrich1, USA), stained with ethidium bromide (Sigma-Aldrich1, USA) and visualized in ultraviolet transilluminator (UVP, Benchtop UV Transiluminator M-26) [27].…”

Section: Pro-viral Dna Extraction From Cell Supernatant and Npcrmentioning

confidence: 99%

Vertical transmissibility of small ruminant lentivirus

et al. 2020

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This study aimed to evaluate by means of Nested Polymerase Chain Reaction (nPCR), co-cultivation and sequencing, with genetic comparison between strains (mother/newborn), the occurrence of vertical transmission of Small Ruminant Lentiviruses (SRLV) from naturally occurring nannies infected for their offspring. For the detection of SRLV seropositive progenitors, blood was collected from 42 nannies in the final third of gestation in tubes with and without anticoagulant. The diagnostic tests used were Western Blot (WB) and nPCR. During the period of birth, the same blood collection procedure was performed on 73 newborns at zero hours of birth, with the same diagnostic tests. Seventeen blood samples from seven-day-old kids, proven positive for SRLV by nPCR, chosen at random, were subjected to coculture in goat synovial membrane (GSM) cells for 105 days. The pro-viral DNA extracted from the cell supernatant from the coculture was subjected to nPCR. For DNA sequencing from the nPCR products, nine positive samples were chosen at random, four nannies with their respective offspring, also positive. Each sample was performed in triplicate, thus generating 27 nPCR products of which only 19 were suitable for analysis. Among the 42 pregnant goats, in 50% (21/42) pro-viral DNA was detected by nPCR, while in the WB, only 7.14% (3/42) presented antibodies against SRLV. Regarding neonates, of the 73 kids, 34 (46.57%) were positive for the virus, using the nPCR technique, while in the serological test (WB), three positive animals (4.10%) were observed. The coculture of the 17 samples with a positive result in the nPCR was confirmed in viral isolation by amplification of the SRLV pro-viral DNA. When aligned, the pro-viral DNA sequences (nannies and their respective offspring) presented homology in relation to the standard strain CAEV Co. It was concluded that the transmission of SRLV through intrauterine route was potentially the source of infection in the newborn goats.

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Duplex nested-PCR for detection of small ruminant lentiviruses

Cited by 18 publications

References 42 publications

Accurate Diagnosis of Small Ruminant Lentivirus Infection Is Needed for Selection of Resistant Sheep through TMEM154 E35K Genotyping

Accurate Diagnosis of Small Ruminant Lentivirus Infection Is Needed for Selection of Resistant Sheep through TMEM154 E35K Genotyping

Seropositivity for Maedi-Visna virus in sheep in Porto Acre city - Western Amazon, Brazil

Vertical transmissibility of small ruminant lentivirus

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