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Treatment of Bovine papillomavirus-induced teat warts in a cow by using
Podophyllin magistral formula and autologous vaccine applications together
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B- 911
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*Corresponding author’s e-mail: drmkalex@yahoo.com
1Department of Virology, Faculty of Veterinary Medicine, Burdur, Turkey.
2Department of Virology, Faculty of Veterinary Medicine, Selcuk University, Konya, Turkey.
3Department of Internal Diseases, Faculty of Veterinary Medicine, Mehmet Akif Ersoy University, Burdur, Turkey. Indian J. Anim. Res.,
Print ISSN:0367-6722 / Online ISSN:0976-0555
Treatment of Bovine papillomavirus-induced teat warts in a cow by using
Podophyllin magistral formula and autologous vaccine applications together
M. Kale1*, H.S. Saltik1, S. Hasircioglu1, Y. Yildirim1, S. Yavru2, N. Mamak3 and K. Atli1
Department of Virology, Faculty of Veterinary Medicine, Mehmet Akif Ersoy University, Burdur, Turkey.
Received: 08-02-2018 Accepted: 30-09-2018 DOI: 10.18805/ijar.B-911
ABSTRACT
In this study, a 32-month-old Holstein cow that had a calving and common Bovine papillomavirus (BPV)-induced wart on its teats was treated. For this purpose, BPV antigen presence in blood serum sample from the animal was studied by ELISA and was revealed as positive. Magistral drug containing podophyllin active substance was applied topically on teat warts of the animal. Applications were performed for all teat warts once in three days during 27 days. Eight hours after applying the drug, teats were washed with sterile water. Warts on teat were proved to have regressed and dried out on the day 27th. The same animal was given papilloma autologous vaccine prepared from warts coming off from the teat on the day 28th. As a result of the vaccination, teats showed full recovery from warts. Finally, it is recommended that using podophyllin formulations topical and autologous vaccine applications together in treating teat warts caused by BPV.
Key words: Bovine papillomavirus, Cow, Teat, Treatment, Wart.
Bovine papillomaviruses (BPV) are usually known as the agent of warts seen in cattle. BPVs takes its place as DNA viruses in Papillomaviridae family (Lunardi et al. 2013). This virus is highly resistant to disinfectant and environmental factors. Thirteen types of BPV have been identified in cattle (De Villiers et al. 2004). BPV type 1-11 can cause teat warts in dairy cattle (Dagalp et al. 2017; Ogawa et al. 2004). These types might cause teat warts as single or various combinations. Especially, as BPV-5 is white colored not pointed and in the shape of benign nodules, they are called as rice-grain shaped warts. BPV-6 is long, filiform leaf warts and they might cause pointed warts when they appear together with BPV-1 and BPV-2. When Lindhorm et al. (1984) studied papilloma agents in tissues taken from teats and teats of 1657 dairy cattle in a slaughterhouse; they detected 28.4% as 1, 88.5% 5 and 92.3% BPV-6. According to their comments, BPV-4 could not be detected in samples and was not related with teat papillomas. Jarrett (1985) stated that economical losses might happen since BPV type 6 causes mastitis and defects in milk ducts following the infection in teats. Particularly as a result of teat papillomas caused by BPV-6, cessation of milking, suckling problem calves and these papillomas rupture, there happens infection with environmental contamination effect and mastitis and economical losses might be seen. These papillomas do not generally regress and that’s why animals need to be kept out of the herd (Campo, 2002). Because warts grow, bleed and injure teats and bacteria or other viruses (pseudocowpox,
bovine herpes mammillitis virus etc.) step in, there is a growing liability to mastitis (Kahrs, 2001; Murphy et al. 1999). Besides, warts caused by the virus prevent teats from getting into milking machines. In this case, teat dries up in time and milk cannot be obtained.
For skin and mucosa in fections caused by papillomaviruses in humans, podophyllin obtained from the root of Podophyllum peltatum plant is used. It is especially used for Verruca vulgaris and Condyloma accuminata cases developed in humans (Durusoy and Gozel Ulusal, 2007; Kutlubay et al. 2011). Podophyllin is a herbal (American or Indian) resin that stops mitosis in metaphase and causes cells to die (Gross and Von Krogh, 1997).
In the study, for BPV-caused teat warts seen commonly among dairy cattle, a new and alternative treatment application has been developed and applied in field.
A 32-month-old Holstein cow that had calving and common warts on its teats provided us with research material. This animal was located in a foundation that had an open breeding system in Akdere district of Central Burdur. The amount and running of milk from the teats of this animal was low and manual milking had to be performed since teats could not get into milking machines. BPV antigen presence was searched in blood serum sample taken from Vena jugularis of this animal. For this purpose, Qualitative BPV ELISA kit (My Bio Source Inc., Cat. No: MBS109004, San
2 INDIAN JOURNAL OF ANIMAL RESEARCH Diego, USA) was used. Test was applied according to the
procedure stated by the commercial firm. The studied serum sample was detected as BPV antigen positive. The magistral drug containing podophyllin active substance (Podophyllum Resin) (Galenik Ecza ve Kimyevi Maddeler Deposu, 10 g, Menderes, İzmir, Turkey) formulated by us for teat warts was prepared fluidly. For this purpose, podophyllin and potassium iodine was smashed in 96% of alcohol in a porcelain pound and homogenized. Later, this mixture was kept in room temperature during usage in brown, light-proof, sterile glass bottles. The magistral drug containing podophyllin active substance was applied topically for warts on left and right teats by sterile swabs once in three days. Eight hours after drug application, all teats were washed with sterilized distilled water. It applied that ligation using thread for some large warts on teats. Warts were removed away from the parts which were about to drop. Autogenous vaccine was prepared according to the laboratory protocol previously stated by the researcher called Hunt (1984). Accordingly, 5 grams of live papilloma tissue was smashed in a sterile pound pouring some sterile sand to cover the surface of the tissue and adding 1/10 phosphate buffer solution (PBS). Later, this mixture was drained into a sterilized beaker covered by muslin cloth. The obtained supernatant was transferred into a sterile glass tube and centrifuged at 4ºC at 3000 rpm for 30 minutes. After centrifugation, the obtained supernatant was transferred into another 10 ml. sterile tube and 0.04 ml. of 40% formaldehyde was added onto it. It was incubated in the stove at 37ºC for 24 hours with the tube inlet open. At the end of this period, the fluid in the tube was passed from 0.22 µm diameter injector filters and drained into sterile, dark colored glass bottles in a sterile environment. The fluid drained for sterility control was planted in McConkey agar, left for incubation in 25°C stoves at 37°C for 48 hours and planted in Sabarous agar, left for incubation at 37°C for 5 days. At the end of this period, crystal penicilline (200.000 IU/10 ml) and streptomycine (250 mg/10 ml) was added in vaccines not having bacteria and fungi growth. The sterilized vaccine was inoculated as 6ml. to the animal subcutaneously without breaking the cold chain. The application was repeated every ten days. Three vaccination applications were performed in total. The animal had no antibiotics or immune modulator application before and after vaccination.
Magistral drug containing podophyllin active substance was applied for once in three days for 27 days for teat warts and warts regressed and dried out from day 27. For the same animal, papilloma autogenous vaccine prepared from warts previously removed from teats was applied from the day 28th. At the end of the vaccination, teats got rid of warts on the day 60th (Fig 1-6).
In our country, many researchers have been performed for diagnosis and treatment on cattle skin papillomatosis (Atasever et al. 2005; Cihan et al. 2004; Kýrmýzýgul et al. 2010; Tan et al. 2012).
Fig 1: Administration of magistral drug containing the active
substance Podophyllin ® (Right front-back teat-1st day).
Fig 2: Administration of magistral drug containing the active
substance Podophyllin ® (Left front-back teat-1st day).
Fig 3: Administration of magistral drug containing the active
substance Podophyllin ® (Right front-back teat-27th day).
Fig 4: Administration of magistral drug containing the active
In our country, free-lance vets either ignore cattle teat warts or apply temporary treatment attempts. In general, topical applications include electrocautery, cauterization with silver nitrate, curettage, ligature with thread and cryotherapy. These kind of applications might cause teat injuries, mastitis cases due to other infection agents correspondingly and dry teats. There is also a study supporting this case (Risvanli and Kalkan, 2001). BPV-caused warts (especially on udder and teats) are commonly seen in dairy cattle farms in Burdur. This case is one of the most unwanted annoying problems in an imals owned by dair y cattle br eeders (Per son al communication, Burdur Association of Breeding Cattle Breeders). Autogenous vaccines are the most effective method in treatment of BPV-caused warts. BPV is highly difficult to grow in monolayer cell cultures prepared in vitro since it replicates in laminated squamous epithelia cells. Therefore, for diagnosis, viral DNA detection techniques such as clinical manifestation and polymerase chain reaction (PCR) and molecular hybridization are used (Howley and Lowey, 2007). This virus has a long replication stage including vegetative viral DNA synthesis, production of viral capsid proteins and collection of virions (Howley and Lowey, 2007). BPV is not a good immunogenic for mammal organisms and cannot create any warning signals about virus presence for immune system (except local cellular immunity
Fig 5: Administration of autogenous vaccine(Right front-back
teat- 60th day).
Fig 6: Administration of autogenous vaccine(Left front-back
60th day).
formation) because it cannot create inflammation. Therefore, by using BPV vaccines prepared for prophylactic and therapeutic purposes, among the basic targets are the neutralisation antibody development against the virus, cellular immunity warning, destruction of infected cells producing early proteins and introducing the virus with keratinocytes (Campo, 2006; Nicholls and Stanley, 2000). Usually, warts regress and remove. However, some warts are permanent and hard. It is believed that persistent papillomatosis is caused by immunosuppression (Knowles et al. 1996; Nasir and Campo, 2008). Cimtay et al. (2003) comparatively studied the treatment effectiveness of autologous, strain specific and herd specific vaccine applications in cattle with papilloma. They detected all three applications effective against cattle papillomatosis. Many researchers (Lesnik et al. 1999; Ocal et al. 2001) also r epor ted autologous vaccin es successful in cattle papillomatosis. New alternative and supportive treatments are needed as this vaccination is subject to permission at the moment (Kale, 2016). In treatment of BPV-caused warts in dairy cattle in veterinary medicine, autohemotherapy (Rakesh et al. 2013), baypamun (Turk et al. 2005), levamisole (Cihan et al. 2004; Paksoy et al. 2015), theranecron (Paksoy et al. 2015), ivermectin (Kýrmýzýgul et al. 2010), homeopathy (Marins et al. 2006) and Newcastle virus La Sota strain applications (Avki et al. 2004; Oswaldo et al. 2011) are carried out commonly. In human medicine, in topical tr eatmen t of Human Papillomavir us (HPV) war ts podophyllin, podophyllotoxin, imiquimod, 5-flourouracil, cidofovir, cantharidin, salicylic acid and dinitrochlo-robenzene are used (Kutlubay et al. 2011; Sarýkaya and Iscimen, 2010). One of the most preferred of these is podophyllin. Podophyllin (=Podofillum, Podofilino, Podophyllinum, podophyllum rhizome, wild mandrake) is a topical, cytotoxin agent. Podophyllin resin shows an antimitotic effect. Wart treatments with this substance (anogenital warts, condyloma accuminata) are mostly applied in human medicine. Podophyllin was approved in 1945 in the USA by Food and Drug Agency (FDA). In Turkey, the usage of topical podophyllin preparate in treatment of human venereal, general, granuloma inguinale
and plantar warts was approved on April 3rd 2008 (Pekcan,
2014). Mitosis of podophyllin in epithelia cells was stated to have remained at metaphase stage. Podophyllin is reported to be more effective on warts with less keratin storage such as plantar warts rather than those with intense storage. Lytic effect cannot go beyond epidermal cells, remains intact in basal layer and has minimal effect on corium. Topical application of podophyllin substance does not cause scar formation and undergoes systemic absorption. When this substance was applied on dispersible, bleeding and newly biopsy-conducted warts, the absorbed amount increased (Pekcan, 2014). No study was found on the usage of magistral drug prepared with this substance in veterinary field.
4 INDIAN JOURNAL OF ANIMAL RESEARCH
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The reason for the application of magistral drug containing podophyllin active substance primarily on teat warts was to detect whether this formulation alone was effective in treatment or not. The magistral podophyllin formulation we used in our study did not develop acute inflammatory reactions and necrosis on teats before and after treatment and milking process continued. Hence we would like to state that topical podophyllin applications in dairy cattle are quite reliable. Besides, autogenous vaccine applications accelerated the healing period and prevented warts from recurring. At the end of this study, it came to the conclusion that in treatment of BPV-caused warts, using our magistral podophyllin formulations together with topical and autologous vaccine applications would be useful. On the
basis of this case, we will continue working on a larger animal population.
ACKNOWLEDGEMENT
We owe a great debt of gratitude to our post graduate student Chemist Ahmet Ak and Chemist Technician Ramazan Sencan for th eir assistance in preparing podophyllin magistral formulation. Summary of the research
was presented as a poster in 12th Veterinarians’ Microbiology
Congress (International participation) in Cappadocia,
Nevşehir on August 30th-September 2nd, 2016 and printed in
the congress book. This study received the printing permit
dated and numbered as April 18th 2017 / May 15th 2017 and
49423435-325.99-E.969432 / 49423435-325.99-E.1210046 from Ministry of Food, Agriculture and Livestock General Directorate of Food and Control.
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