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Revista mexicana de ciencias pecuarias

versão On-line ISSN 2448-6698versão impressa ISSN 2007-1124

Rev. mex. de cienc. pecuarias vol.13 no.3 Mérida Jul./Set. 2022  Epub 22-Ago-2022

https://doi.org/10.22319/rmcp.v13i3.5873 

Articles

Detection of bluetongue virus in sheep by real-time RT-PCR in different production systems in San Martin, Peru

Alicia María López Floresa  * 

Roni David Cruz Vasqueza 

Víctor Humberto Puicón Niño de Guzmána 

Alicia Bartra Reáteguia 

Orlando Ríos Ramíreza 

Fredy Fabián Domíngueza 

a Universidad Nacional de San Martin. Laboratorio de Sanidad Animal de la Escuela Profesional de Medicina Veterinaria, San Martin, Perú.


Abstract

The present study aimed to determine the prevalence of Bluetongue Virus (BTV) in sheep, by the real-time Reverse Transcription-polymerase chain reaction (RT-PCR) technique. Three hundred sixty-six sheep from the ten provinces of the Peru region were evaluated. The methodology used was the collection of blood samples from the jugular vein of the sheep, then the process of extraction and purification of RNA was carried out with the QIAmp® kit, then the reverse transcription to obtain the cDNA, and finally perform the real-time RT-PCR, for which the SuperScript III platinium One-step qRT-PCR kit was used, with the primers and probes being directed to segment 10 of the NS3 gene of BTV. The results of the real-time RT-PCR test revealed two positive sheep with a value of cycle threshold (Ct) of 35.21 and 35.57, with a prevalence of 0.54 % of BTV-positive sheep in the extensive production system, with environmental conditions that favor the development of the Culicoides vector. It is concluded that, by means of the real-time RT-PCR technique, the presence of BTV in this region of Peru is confirmed, which makes future studies necessary to determine the detection of other potential serotypes of BTV in the Peruvian Amazon in order to improve the control strategies of the disease.

Key words RNA; Gene; Molecular diagnosis; Ruminants; Amazonia

Resumen

El presente estudio tuvo como objetivo determinar la prevalencia del Virus de la Lengua Azul (VLA) en ovinos, por la técnica reacción en cadena de la polimerasa con Transcripción Reversa (RT- PCR) en tiempo real. Se evaluaron 366 ovinos procedentes de las diez provincias de la región del Perú. La metodología empleada fue la toma de muestras sanguínea de la vena yugular del ovino, seguidamente se realizó el proceso de extracción y purificación de ARN con el kit QIAmp®, luego la transcripción reversa para obtener el ADNc, y finalmente realizar la RT- PCR en tiempo real, para lo cual se utilizó el kit SuperScript III platinium One- step qRT-PCR, siendo los iniciadores y sondas dirigidos al segmento 10 del gen NS3 del VLA. Los resultados de la prueba de RT-PCR en tiempo real revelaron dos ovinos positivos con valor de ciclo umbral (Ct) de 35.21 y 35.57, siendo la prevalencia de 0.54 % de ovinos positivos a VLA en sistema de crianza extensiva, con las condiciones del ambiente que favorecen el desarrollo del vector Culicoide. Se concluye que mediante la técnica RT-PCR en tiempo real, se confirma la presencia del VLA en esta región del Perú, lo que hace necesario futuros estudios para determinar la detección de otros serotipos potenciales de VLA en la Amazonia peruana, con la finalidad de mejorar las estrategias de control de la enfermedad.

Palabras clave ARN; Gen; Diagnóstico molecular; Rumiantes; Amazonia

Introduction

Bluetongue virus [BTV; in Spanish: virus de la lengua azul (VLA)], genus Orbivirus1, without envelope, the genome has 10 segments of double-stranded RNA, seven structural and five non-structural proteins2. VP7 is a viral capsid protein3. VP2 of the surface of the virion4. In addition, serotypes 9, 13, 185 and 276, with serotype 28 being recently identified7.

Bluetongue is a disease, non-contagious and transmitted through the bite of Culicoides insects8. Of the 1,357 species of Culicoides in the world9, only about 30 have been reported as vectors10. Vectors, when feeding on an animal with BTV, will be infected all their lives11, they can infect animals with variations in clinical signs12.

Sheep manifest seromucous nasal discharge, facial edema, thrombo-hemorrhagic fever, coronitis and ulceration in the lips and hard palate13; the viremia is usually detected between 3 and 5 days after infection, reaching febrile peaks on day 714. However, these clinical signs (e.g., fever in sheep and cattle, salivation in cattle, facial edema in sheep) were observed in infected and non-infected herds, these signs are not indicative of the disease15. Likewise, macroscopic lesions are pulmonary edema, cranio-ventral pulmonary consolidation, swollen and cyanotic tongue; and hemorrhagic lesions in rumen mucosa16. Cattle, goats, buffaloes and other wild ruminants act as reservoirs of the virus, being animals that may not show clinical signs17,18,19, however, there are reports of mortality in cattle with BTV serotype 820; other clinical signs such as amaurosis, which is the inability to stand and the absence of the sucking reflex in infected calves21. Therefore, the different serotypes of BTV in small and large ruminants indicate its enzootic expansion22. Likewise, the transmission of the virus, host-vector is complex, with a variety of ecological drivers23.

The epidemiology of BTV is listed in the OIE Terrestrial Animal Health Code24. It has generated outbreaks in Israel in 2006, generating losses of $ 2.5 million25, as well as an outbreak in Western Europe26. Thus, it has had an annual cost of $ 3 billion, it has become one of the diseases of economic importance27.

Currently, this disease is found worldwide, except in Antarctica28. It has been reported in Brazil in sheep16; in Sudan, in reproductive disorders in cattle29; in Japan30 and India, in a transplacental transmission of BTV-1, in the middle stage of gestation in sheep31. In Australia, BTV originated from Southeast Asia32. While in the United Kingdom, wild animals in zoos are susceptible to arboviruses, acting as native hosts of Culicoides33.

In Peru, BTV has been reported in sheep, camelids and wild animals in tropical areas34,35,36. Preliminary studies were conducted in 1984 and 1987, finding 88, 41 and 56 % of sheep from three regions (north, central and south)37 that were seropositive for BTV and 21 % of seroreactor alpacas from the southern region38. In studies in Tayassu tajacu in Madre de Dios, 7.5 % of samples with BTV-specific antibodies are reported and 29.2 % had antibodies to the BTV serogroup34. Recently, Navarro et al35 confirmed the presence of the virus in sheep of extensive farming, in addition to identifying the Culicoides that are vectors of the disease.

However, the San Martín region, in its tropical climate, is an optimal environment for the vector and facilitates its infection routes in domestic and wild hosts; mentioned by Felippe-Bauer39, who reported Culicoides species.

In relation to the diagnosis, the seroneutralization test is specific, but its disadvantage is the low sensitivity and cost40. However, competitive ELISA is suggested in llamas, wild ruminants41, sheep and goats42. Currently, the tests recommended by the OIE for BTV are RT-PCR, agar gel immunodiffusion assay and competitive ELISA43,44.

With the real-time RT-PCR being for the rapid detection of BTV directed to Seg-1/VP145,46, Seg-2/VP2 and Seg-10/NS347, it is a widely used method48, in the detection of all BTV serotypes49, based on the TaqMan fluorescence probe50,51,52,53 to detect BTV in samples of infected sheep54. Therefore, the objective of the study was to detect BTV in sheep by real-time RT-PCR, considering the influence of environmental characteristics and presence of the disease.

Material and methods

Place of study

The study was conducted in the San Martín region, Peru. Blood samples were collected from sheep in stable health condition, in the 10 provinces of the San Martín region (Table 1), in the period from August to December 2018.

Table 1 Number of sheep sampled by province suitable for the development of the vector 

Provinces No. of samples
Moyobamba 9
Rioja 11
El Dorado 35
Lamas 41
Bellavista 74
Picota 41
Mariscal Cáceres 15
Huallaga 24
Tocache 30
San Martín 86
Total 366

Sample collection

Three hundred sixty-six blood samples were collected from the jugular vein of the randomly selected sheep, which were from 42 farms. For the collection, vacuum vacutainer needles and tubes with EDTA anticoagulant were used. The samples were transported in a refrigerated container with cooling gel for their processing in the Laboratory of Molecular Biology and Genetics of the Professional School of Agronomy - Faculty of Agricultural Sciences, National University of San Martín.

According to the 2012 Census55, the population is 7,656 sheep in the entire department of San Martín. The sample was calculated based on this population:

n=Z2pqNE2n-1+ Z2pq

Where: n is the sample size; Z= is the confidence level 95%= 1.96; p= is the probability of success 50%/100= 0.5; q= is the probability of failure 50%/100= 0.5; E= is the error level 5%/100= 0.05; N= is the population size= 7656.

n= (1.96)2 (0.5) (0.5) (7656)

(0.05)2(7656-1) + (1.96)2(0.5) (0.5)

n=366

Sample subpopulation formula.

nh=NhN(n)

Nh= subpopulation or group; N= total population; n= total sample; nh= sample of the groups.

RNA extraction

For the processing of the blood sample, it was centrifuged at 800 xg for 10 min, then the plasma was extracted, and sterile PBS was added, the tube was inverted several times to mix, centrifuge again at 800 xg for 10 min to separate the red blood cells from the PBS. The QIAmp® kit, Qiagen brand, was used for RNA extraction. The RNA extraction step was performed according to the procedures specified by the manufacturer. The final product obtained is RNA in Buffer, to finally continue with the real-time RT-PCR, which targets segment 10 of BTV (NS3 Gene).

Real-time Reverse transcription - Polymerase chain reaction

Real-time RT-PCR is a test of choice for diagnosis. The method described here is recommended by the OIE and Hofmann et al56, to detect segment 10 of the NS3 Gene of BTV. To obtain the cDNA by real-time reverse transcription and PCR, the SuperScript III platinium One-step qRT-PCR kit, Invitrogen brand, was used, being the sequences of the primers for the detection of segment 10 of the NS3 gene of BTV. The primary solutions of the primer were diluted to a concentration of 20 pmol/μl, the nucleotide sequences of the primers: VLA_IVI_F 5’-TGG-AYA-AAG-CRA-TGT-CAA-A-3’, VLA_IVI_R 5’-ACR-TCA-TCA-CGA-AAC-GCT-TC-3’57. The probe solution for the NS3 gene of BTV was diluted to a concentration of 5 pmol/μl, the sequence of the probe: VLA_IVI_P 5’FAM-ARG-CTG-CAT-TCG-CAT-CGT-ACG- C-3’ BHQ1.

Zero point five microliters of each primary primer at a concentration of 20 pmol/μL were added to each well, the plate must be kept on ice. Then 2 μL of RNA samples, both from the target sample and from the positive and negative controls, are added to the corresponding wells of the plate following the distribution. The denaturing temperature was 95 °C for 5 min, and they were kept on ice for another 3 min. A volume of the primary mixture57 of the RT-PCR was prepared, following the manufacturer’s instructions. The probe was included in the primary mixture to obtain a final concentration of 0.2 pmol/μL per sample. Twenty microliters of primary mixture were distributed in each well of the plate located in the real-time thermocycler programmed for reverse transcription and amplification, detection by fluorescence of cDNA.

Temperature conditions

The reaction was carried out in a light cycler 480 System Roche Applied Biosystems. Following the following thermal profile: Reverse transcription 48 °C for 30 min, reverse transcriptase inactivation or initial denaturation 95 °C for 2 min, followed by 50 cycles, amplification 95 °C for 15 sec, 56 °C for 30 sec, 72 °C for 30 sec57.

Results and discussion

Real-time RT-PCR analysis

Real-time RT-PCR results indicated that 0.54 % (2/366) of the samples were BTV-positive. For the first individual, with code (E8), positive for BTV, it had a cycle threshold (Ct) value of about 35.21, with a dissociation temperature™ value of 84 °C for BTV. For the sample with code (G12) - C+VLA 1/10, it had a Ct of 28.22 and 31.63 for the sample with code (H12) - C+VLA 1/100. No amplification was observed in the negative control. The high viral load and amplification of a specific product were evidenced (Figure 1).

Figure 1 Results of the real-time RT-PCR test for the first positive individual (E8), in the amplification curve, it is observed with a Ct value of 35.21. Opticon Monitor software v.3.0 

For the second individual (E6) positive for BTV, it had a Ct value of about 35.57, with a dissociation temperature™ value of 84 °C. For the sample (G12)- C+VLA 1/10, it had a Ct of 28.81 and 32.72 for the sample (H12)- C+VLA 1/100. No amplification was observed in the negative control. The high viral load and amplification of a specific product were evidenced (Figure 2).

Figure 2 Results of the real-time RT-PCR test for the second positive individual (E6), in the amplification curve, a Ct value of 35.57 is observed. Opticon Monitor Software v.3.0 

A panoramic review in South America, using serological studies for the detection of antibodies conducted on cattle, goats, sheep and buffaloes, indicates varied minimum and maximum prevalences in the following countries: in Argentina (0-95 %)58, Brazil (1.22-89.69 %)59,60, Chile (0-19.6 %)61,62, Colombia (51.8-56 %)63,64, Ecuador (10 %)65, Guyana (0-56 %)66, Suriname (82-91 %)66 and Venezuela (74.8-94.7 %)67, in Peru, preliminary studies in 1984 and 1987 reported a seroprevalence of 87.5, 41 and 55.5 % of BTV in sheep from the north, center and south of the country’s highlands37. In the present research, the viral genome of BTV was detected, with the prevalence being 0.54 % in sheep sera by real-time RT-PCR, which confirms the epidemiological presence of this virus in this region. The areas where the positive animals were detected are very close to water tributaries, such as the Sisa River and the Ishangayacu Ravine. With environmental sensitivities being as a key component of the capacity of the vector68.

It is also possible that BTV infection in the sampled sheep was subclinical, as the animals were under normal conditions. This is explained, according to Maclachlan et al69, because the development of the clinical signs of the disease depends on whether the infection is endemic or not; as a consequence, animals have antibodies, but rarely show clinical signs. This is possibly because in sheep the period of viremia rarely persists for more than 14 d, unlike in cattle, whose viremia can be up to 90 to 120 d.

As mentioned by Navarro36, Peru is one of the countries that is predisposed to present BTV disease, since it has several ecosystems conducive to the development of Culicoides. It is considered necessary to determine the seroprevalence of BTV in livestock areas, identify the different serotypes, map the location of the different species of Culicoides spp., in the different geographical areas and altitudes, and determine the endemic areas.

Environment of BTV-positive sheep

An assessment of the surroundings of the farms was carried out to observe the habitat of the Culicoides vector. It was found that BTV-positive animals are near water tributaries, which contributes to the development of the vector (Table 2). On the other hand, the positive sheep were of the Pelibuey breed, however, the predisposition due to breed is not determinant. In addition, extensive farming of sheep allows their farming together with cattle, chickens, dogs and horses. In the surveys conducted, the owners of extensive, intensive and semi-intensive farms do not deworm their animals, nor do they fumigate for the presence of flies or mosquitoes.

Table 2 Environment of BTV-positive sheep 

Province District Location of the farm Farming system Environment of the farm Breed Body condition
Bellavista San
Pablo
Angélica
Sector
Extensive Left bank of
the Sisa River
Pelibuey 3
Bellavista San
Pablo
Hamlet of
San
Ignacio
Extensive Left bank of
the
Ishangayacu
Ravine
Pelibuey 2.5

Epidemiological studies indicate that BTV exists in a large area in the world, between 40° north latitude and 35° south latitude, with tropical, subtropical and temperate ecosystems70, these characteristics coincide with the San Martín region.

The first studies of the presence of the BTV-transmitting vector in this region were conducted by Felippe-Bauer et al39, who identified five species of Culicoides that are vectors of the virus. Similarly, Navarro et al35, of the 7,930 mosquitoes captured, 94.8 % were identified as Culicoides insignis, and the presence of BTV in sheep in the Pucallpa region is also confirmed36.

On the other hand, the Culicoides vector usually develops in areas where there are certain types of drivers such as land use, trade, animal husbandry and the presence of wild animals as a reservoir of BTV; the latter is reinforced by the work carried out by Rivera et al34, who found 7.5 % of white-lipped peccaries (Tayassu pecari) positive for BTV in the Madre de Dios region.

The present research work is the first to be developed in the entire San Martín region, where the results using real-time RT-PCR show the presence of BTV. Felippe-Bauer et al39 mention having found the Culicoides vector of the virus. Therefore, it is required to detect other existing serotypes of BTV in domestic and wild animals of the region that are susceptible or reservoirs of the disease.

Farming systems

Regarding the farming system, most producers opt for an extensive sheep farming in the San Martín Region (Table 3).

Table 3 Production systems 

Provinces Farming systems
Intensive Extensive Semi
Intensive
Moyobamba 0 9 0
Rioja 0 11 0
El Dorado 0 35 0
Lamas 0 27 14
Bellavista 0 74 0
Picota 0 29 12
Mariscal Cáceres 0 15 0
Huallaga 0 19 5
Tocache 0 20 10
San Martín 33 15 38
    Total 366

Conclusions and implications

It is concluded that BTV has a low prevalence in sheep in the San Martín Region of Peru, however, future studies are needed to determine morbidity and the detection of other potential BTV serotypes in the country, to better elucidate the management of vectors and control strategies of the disease.

Acknowledgements

This research work received financial support from the Research and Development Institute of the UNSM-T, competition for undergraduate thesis projects, period 2018, with Resolution No. 611 -2018 - UNSM/CU - R/ NLU.

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Received: November 19, 2020; Accepted: November 08, 2021

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