International Journal of Biotechnology and Biochemistry ISSN 0973-2691 Volume 12, Number 2 (2016) pp. 123-130 © Research India Publications http://www.ripublication.com

Molecular Detection Of Bovine Associated With Cutaneous In Some Breeds Ofnigerian

Jeremiah, O.T.1, Fagbohun, O.A.2 and Babalola O. J.1

1Department of Veterinary Medicine, University of Ibadan, Ibadan, Oyo State 2Department of Veterinary Microbiology and Parasitology, University of Ibadan, Ibadan, Oyo State

Abstract

Bovine Papillomaviruses (BPVs) cause cutaneous papillomatosis, a common disease of the that manifests as non-threatening, non – invasive (warts), seen mostly in young cattle. Various methods have been used in the identification of bovine papillomatosis associated with skin warts worldwide such as clinical signs, immunohistochemistry, histopathology and electron microscopy. Herein, we describethe molecular detection of papillomatosis in some Nigerian cattle(n=31) through partial amplification of the L1 gene using polymerase chain reaction.Skin biopsies were obtained by both plucking and excision techniques from twenty bulls and eleven cows of White Fulani (17), Red Bororo (4), Sokoto Gudali (9) and a cross between White Fulani and Red Bororobreeds at an International cattle market in Ibadan, Nigeria.White Fulani breed had more positive cases of bovine papillomatosis than other breeds clinically and when polymerase chain reaction technique was performed.Six out of the thirty one skin samples were positive for the presence of the DNA of bovine papillomaviruses across the breeds of cattle. Amplification of the viral DNA from the positive samples produced 450bp bands on agarose gel electrophoresis.Thus, this report provides new information on the molecular diagnosis of papillomaviruses associated with this cutaneous benign tumour in some Nigerian native cattle.Therefore, polymerase chain reaction can be used as a rapid confirmatory diagnostic tool in cases of bovine papillomatosis.

Key words: Bovine Papillomatosis, Cattle, Polymerase chain reaction.

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INTRODUCTION Papillomaviruses (PVs) aresmall, non-enveloped, doublestrandedDNA viruses with circular genomes that replicate intranuclearly in infected cells. (Alfieri et. al.,2008 ; Flores et. al., 2010).Papillomaviruses have high affinity for stratified squamous epithelia of warm blooded animals including cattle. (Egawa, 2005). Papillomatosis occurs in all animal species throughout the world (Radostits et al., 2007), and are highly species-specific (Borzacchiello and Roperto 2008).However, the only reported cases of cross-species infection had occurred in horses and other equids (Campo, 2002).The familycomprises 16 genera which were classified based on genomic DNAhomology, using the late L1 structural gene (Alfieri et. al.,2008 ; Flores et. al., 2010). Papillomatosis affecting cattle is a frequent infectious of the skin, seen mostly in young cattle, that manifests as self limiting and caused by bovine papillomavirus(BPV) (Zuckerman et al; 2000;Hamadet.al,2011).Thirteen types of the bovine papillomaviruses (BPVs), have been characterized in cattle; namely BPV-1 to BPV-13, which are divided into three genera, asDeltapapillomavirus(BPV-1,-2 and-13), (BPV-5 and- 8) and (BPV-3,-4,-6,-9,-10,-11 and-12), and an as yet unclassified PV genus (BPV-7) (Bernard et al., 2010; Hatama et al., 2011; Zhu et al., 2011; Freita et al, 2011, Lunardi et. al.,2013). Bovine papillomaviruses exhibit site specificity or site preference (Jarret et al., 1984). BPVs-1,-2,-3,-5,-6,-8,-9 and-10 have been shown to cause skin related papillomatosisin animals(Jarret et al., 1984; Campo et al., 1994; Borzacchielloand Roperto,2008; Hamadet.al,2011). Diagnosis of bovine papillomatosis is not always a hard task as the lesions can always be seen on the skin manifesting as firm fibropappilomatous growths usually hairless and greyish in colour (Khan et.al.,2010; Terziev, et al., 2015). Confirmatory diagnosis of warts can be achieved by histopathological examination of wart lesions, which shows marked parakeratotic hyperkeratosis with long, thick, hair-like, cornified surface projections and papillate, epidermal hyperplasia, with patchy areas of erosion, ulceration, and neutrophil infiltration(Khan et.al.,2010; Salib and Farghali, 2011).Identification of the virus by electron microscopy reveals the presence of viral particles (about 40nm in diameter) in the nucleus of cells in the stratum corneum, stratum spinosum, and stratum granulosum of the skin (Misdorp, 2002;Terziev, et al., 2015).Bovine papillomatosis can also be confirmed with immunohistochemical examination of the basal layer of the affected epithelial and connective tissue using against specific antigen of BPV-1, Proliferative Cell Nuclear Antigen (PCNA) and Ki-67 protein(Ozsoy et. al., 2011). There are several skin diseases of cattle inNigeria. Most common among such skin infections are dermatophilosis, lumpy skin disease and cutaneous papillomatosis known as warts (Radostits et al., 2007). Cutaneous papillomatosis is widespreadamong indigenous and sometimesimported beef and dairy cattle in Nigeria.There is shortageof information on the use of polymerase chain reaction as a confirmatorytool in the diagnosis of bovine papillomatosis. The present study reports for the first time, the moleculardetectionof bovine papillomavirusDNA from cutaneous warts in someindigenous breeds ofcattle in Nigeriausing polymerase chain reaction technique.

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MATERIALS AND METHODS Bovine papilloma lesions Thirty-one bovine skinbiopsies were collected from some breeds of cattle showing the classicalpresentation of cutaneous papillomatosis. Such cattle breeds included Sokoto Gudali, Red Bororo, White Fulani(Bunaaji) and their crosses of both sexes. Two main techniques were employed to obtain the skin biopsy samples from each animal.The techniques included bothexcision and plucking methods which have been described elsewhere (Campo, 2006, Borzacchiello and Roperto, 2008). The sampling took placein an International cattle market in Ibadan, Oyo State, Nigeria, located on Longitude 7° 31’30.01’’ and Latitude 3° 54’54.82’’ (Google Earth Software, USA;2007). The samples were obtained over a period of two months from cattle that originated from Niger, Kwara and Oyo States(Nigeria) and their ages,sexes and sources of origin were documented. The ages of all the cattle sampled were estimated using dentition technique as described by Lasisi et al.(2002).The samples were kept in microcentrifuge tubes filled with 500 µL of virus transport medium(Copan Diagnostics Inc, USA) and stored at-200C until tested.

Preparation of DNA samples A fragment of approximately 5 mg of each papilloma collected was macerated in virus transport medium using a manual tissue homogenizer and subjected to DNA extraction using DNeasy® blood and tissue kit (Qiagen, Valencia, CA) according to the manufacturer’s instruction. Extracted DNAswere kept in sterile Eppendorf tubes and stored at-20°C.

Polymerase chain reaction The primers employed for the PCR designated Primer B1 and Primer B2 with the 5'- GCMCAGGGWCATAAYAATGG-3' and 5'-CGTCCMARRGGAWACTGATC-3', respectively (Ogawa et.al., 2004; Flores et. al., 2010).Polymerase chain reactions were performed in a 20-μlreaction mixture, using approximately 2.5 μl of total DNA, 1 μl of 0.1μM ofeach primer, 12.5 μl of PCR Master Mix (2X)-(DreamTaqbuffer, 0.4 mM of each deoxyribonucleotidetriphosphate, and4 mM MgCl2)and 3μl of nuclease free water. The PCR was done in a 9700 PCR Thermal Cycler (Applied Biosystems, USA) with the following conditions:initial denaturation(94°C/2 min/1 cycle), followed by 35 cycles (denaturation(95°C/20secs); annealing at 48.5°C/min ;72°C/90 secs; and a final extension of 7 min at 72°C).Five microliters ofeach PCR products was subjected to electrophoretic fractionation in a 2.0 % agarose gelcontaining1μgethidium bromide and visualized under ultraviolet light.

Statistical analysis All data obtained were subject to descriptive statistics.

RESULTS White Fulani breed and bulls were encountered more frequently than other breedsandcows respectively in the study.Out of the 31 cattle showing the classical

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lesions of papillomatosis, White Fulani breed of cattle (54.84%) were encountered more among these cattle breeds during the course of the study(Table 1).

Table 1: Breed susceptibility of cattle showing clinicalpapillomatosis

Cattle breeds Red Sokoto White White Ful. Total number showing classical Bororo Gudali Fulani X Red of all cattle papillomatosis Bororo breeds lesions sampled Number (%) 4(12.90%) 9(29.03%) 17(54.84%) 1(3.23%) 31

Bull calves(64.51%) were encountered more in the course of the study thantheir heifer counterparts(35.48%). Among the White Fulani breed, 11 bull calves were found to be clinically showing cutaneous papillomatosis lesions while only one crossed bull calf of White Fulani and Red Bororo breeds with the clinical skin lesions, was sampled (Table 2).

Table 2: Sex Susceptibility of cattle breeds ampled to Papillomatosis

Cattle breeds showing Red Sokoto White White Ful. Sex (%) classical papillomatosis Bororo Gudali Fulani X Red distribution among lesions Bororo cattle breeds Sex (%) M=3 M=5 M=11 M=1 M=20(64.51)

F=1 F=4 F=6 F=0 F=11(35.48)

In the field, the diagnosis of cutaneous papillomatosis (also known as warts) in cattle is by the observablefirm fibropappilomatous growth on the skin of affected animals. The confirmation of the tentative diagnosis is often by histochemical reactions. To develop a better and faster method of confirmatory diagnosis for the skin infection in some breeds of Nigerian cattle, we attempted for the first time, to test the ability of PCR to detect the presence of papillomavirus in the skin lesions of several breeds of Nigerian cattle showing the clinical skin disease.Six samples(19.35%) were confirmed positive for bovine papillomatosis while twenty five samples(80.65%) were confirmed negative for bovine papillomatosis after amplification with the set of primers B1 and B2. Figure 1 shows the results obtained.

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Figure 1: Agarose gel electrophoresis of partial BPV L1 gene of some Nigerian breeds of cattle.Lane M=100-bp molecular marker.White Fulani, White Fulani X Red Bororo, Red Bororo, Sokoto Gudali, White Fulani and Sokoto Gudali breeds of cattle (lanes A,B,C,D,E and F, respectively).Lane –ve was the negative control.5 µl of each PCR amplicon was usedfor agarose gel for electrophoresis. The 450 bp PCR amplicons are shown.

DISCUSSION Despite the fact that, bovine papillomavirus which induces exophytic of cutaneous epithelia in cattle is perhaps the most extensively studied animal papillomavirus, there is a sheer lack of information on the susceptibility of various Nigerian breeds of cattle to the virus. That White Fulani cattle were the most susceptible to the classical papillomatosis is worth noting.This finding is in agreement with the work of Dim, et. al.,(2012) which reported that the Bunaaji/ White Fulani cattle were the most populous and most widely distributed breed in Nigeria.This is because this breed is more heat tolerant(Hansen, 2004) and has lower mortality rate than other indigenous breeds in Nigeria.Although the White Fulani cattlehave been said to be more resistant to diseases than other breeds especially the Red Bororo breed (Boutrais,2007), our finding here that these cattle were found to be most susceptible

128 Jeremiah, O.T. et al to cutaneous papillomatosis seems to be an exception.This may be due to the thin skin possessed by this breed of cattle(Hansen, 2004). Also sex susceptibility to cutaneous papillomatosis among the Nigerian cattle breeds in the present study is very exciting. More males were found with the clinical skin condition among the cattle sampled. This finding is contrary to the work of Salib and Farghali, (2011) which showed that bovine papillomatosis has a higher prevalence in females than in males. This variation could be explained by the fact that more farmers are willing to sell off most of the bull calves with clinical papillomatosis due to their poor aesthetic appearance and the projected lower economic value in terms of production compared to the heifers when they reach adulthood. Working with various breeds of Nigerian cattle has been done elsewhere (Lasisi and Isehunwa, 2011) in which the blood glucose, erythrocyte indices and serum of three breeds of Nigerian cattle were studied across various ages. Therefore, this present study is in agreement with such earlier work. That only a very small fraction of the samples (obtained from Nigerian cattle breeds showing clinical cutaneous lesions) from the total samples were confirmed positive for the papilloma virus using the selected set of primers is very noteworthy. This finding is in line with the work of Flores et. al.,(2010) in which 12 out of 49 Brazilian cattle samples (24%) collected were amplified with MY09/MY11 (Primer B). This shows that Primer B is very useful in the detection of bovine papillomaviruses, associated with cutaneous warts, in Nigeria.The high number of negative samples using Primer B, despite being obtained from clinically affected cattle, is worth noting. This is in line with the work of Freitas et. al.,(2013) in which some samples showed the presence of virus, but with no virus expression. This difference might be related to genetic reassortment as it relates to viral infections. Hence, further scientific work needs to be focused on primer design that will be efficient in the detection of bovine papillomatosis in Nigerian cattle.

CONCLUSION Conventional polymerase chain reaction is a reliable diagnostic method that can be used to confirm cutaneous-associated papillomatosis in clinically affected cattle across all breeds in Nigeria. This technique has been used in diagnosing bovine trypanosomiasis in Burkina Faso (Solano et al, 1999), lumpy skin disease in Egypt, ( El-Kholy et al., 2008) and also, bovine babesiosis in South Africa (Mtshali and Mtshali, 2013).

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