Tracking viral particles in the intestinal contents of the American bullfrog, Lithobates catesbeianus, by Transmission Electron Microscopy

Pesquisa de partículas virais em conteúdo intestinal de rãs-touro americanas, Lithobates catesbeianus, por meio da microscopia eletrônica de transmissão

VETERINARY MEDICINE

A.M. Antonucci^I; M.H. Catroxo^II; M. Hipolito^II; R.M. Takemoto^I; N.A. Melo^II; F.M. França^III; P.C. Teixeira^III; C.M. Ferreira^III

^IUniversidade Estadual de Maringá - Maringá, PR

^IIInstituto Biológico do Estado de São Paulo - São Paulo, SP

^IIIInstituto de Pesca, APTA, SAA. - São Paulo, SP

ABSTRACT

Feces are an important viral agent elimination route for infected carrier animals and in aquatic organisms these pathogenic agents can very rapidly propagate due to the habitation environment. The objective of this work is to track viral particles in the intestinal contents of bullfrogs (Lithobates catesbeianus) from five commercial frog farms in the region of Vale do Paraíba, in the State of São Paulo, Brazil, using negative contrast transmission electron microscopy (TEM). The Coronaviridae, Paramyxoviridae, Parvoviridae and Herpesviridae families were observed and photographed in specimens. This work emphasizes the importance of adopting sanitary measures in commercial farms and confirms that observing feces by TEM is an efficient and rapid diagnostic tool for detecting viral agents.

Keywords: amphibians, herpesvirus, coronavirus, paramixovirus, parvovirus

RESUMO

Sabendo-se que as fezes são uma importante via de eliminação de agentes virais pelos animais portadores e que, por estarem na água, os agentes patogênicos podem se propagar mais rapidamente, objetivou-se a pesquisa de vírus em conteúdo intestinal de rãs-touro (Lithobates catesbeianus) de cinco ranários comerciais na região do Vale do Paraíba, no estado de São Paulo, pela técnica de microscopia eletrônica de transmissão. As famílias Coronaviridae, Paramixoviridae, Parvoviridae e Herpesviridae foram observadas e fotografadas. Este trabalho ressalta a importância da adoção de medidas sanitárias nas criações, além da confirmação de que a observação de fezes pela microscopia eletrônica de transmissão é uma eficiente ferramenta de diagnóstico rápido para agentes virais.

Palavras-chave: anfíbios, herpesvírus, coronavírus, paramixovírus, parvovírus

INTRODUCTION

Viruses that infect amphibians have been described previously mainly in reports concerning zoo or wild animals (Hipolito et al., 2003). The renal adenocarcinoma known as Lucke tumor, a neoplasia associated with infection by the Herpesviridae family and low temperatures, was initially described in Rana pipiens in North America (Williams et al., 1996). In anurans, such as Xenopus laevis and R. pipiens, the occurrence of lymphosarcoma associated with infection by the Iridovirus group has been described. Associations have already been established between wild amphibians, which may act as viral reservoirs, and the viruses that cause western equine and Japanese B encephalitis (Hipolito et al., 2003).

In Brazil, frog raising is a very popular activity and this type of culture has occurred since the 1970s, with Brazilian production at 639 tons in 2006 (Dias et al., 2009). The American bullfrog, recently reclassified as Lithobates catesbeianus (Frost et al., 2006), is the main species used in frog farms and has an excellent capacity to adapt to different climates, as well as the distinct physical and feeding management used in the intensive culture systems (Ferreira et al., 2002).

In Brazilian frog farms, there is a high incidence of animal mortality cases without a complete clarification of the cause. In some Brazilian farms, viral particles similar to the Iridovirus group have been detected in the liver of bullfrogs. The swelling of the tadpoles of this species has been associated with a group of polyhedral cytoplasmic deoxyribo viruses: the tadpole edema virus (TEV) and frog virus 3 (FV3) (Mazzoni et al., 2009).

Viruses from the Togavirus group were observed in liquid from L. catesbeianus with asciti, and infection by viruses from the Paramyxoviridae family were described in the liver of tadpoles that had died from an unknown cause (Hipolito et al., 2003). This same author described the presence of particles similar to the Herpesviridae family in hepatic injuries of frogs that had died from intoxication problems and in skeletal muscular nodules (Hipolito and Bach, 2002). The Ranavirus belongs to the Iridovirus group and is a viral agent that is primarily reported in amphibians. Currently the illness caused by this virus is of interest to the World Organization for Animal Health (OIE) due to its rapid propagation and high lethality. The diagnosis of these viral agents is determined by PCR and electron microscopic techniques, and by antibodies using the ELISA method and immuno-electron microscopy (Zupanovic et al., 1998).

It is known that feces are an important route for viral agent transmission, along with contaminated food and water. However, to be a viable biological tool, the laboratory diagnosis must allow the identification of these agents in the applicable fecal material (Gregori et al., 2004). The objective of this work was to identify, through transmission electron microscopy, the possible viral groups isolated in the intestinal contents of Lithobates catesbeianus following intensive cultivation.

MATERIALS AND METHODS

The present study was completed in five frog farms in different municipalities in the Vale do Paraíba region (an altitude of 500 to 670m), São Paulo, Brazil. These frog farms were denominated A, B, C, D and E and this region was chosen due to the presence of a large number of active frog farms.

Nine post-metamorphosis and asymptomatic bullfrogs were selected from each of the properties, a total of 45 individuals, between March and June (autumn in the Southern hemisphere).

The frogs were taken to the Aquatic Organisms Pathology Laboratory from the Fish Institute in São Paulo, where they were conditioned in vivarium boxes adapted for amphibians (Bueno-Guimarães, 1999) and remained in the same location for a maximum of four days. Water exchange and forced feeding of the animals with extruded ration I containing 40% crude protein was completed daily.

Due to the difficulty of collecting feces from living animals due to possible leaching of the feces by the water, tissue contamination and other substances from the animal itself, it was decided that the direct dissection of the large intestine would be performed (Mouriño and Stéfani, 2006).

Before euthanizing the animals, they were anesthetized by hypothermia, inspected to detect any injury and subsequently desensitized and sacrificed through medullary sectioning and bleeding at the height of the atlas bone in the cervical region. The necropsy was carried out under stereoscopic microscopy through an incision made with scissors at the cloaca, completely exposing the coelomic cavity and its viscera. The intestines were separated and dissected with scissors. The contents of the large intestine were removed with a spatula, placed in a polypropylene microtube, and duly identified. For each necropsied frog, the bench and the material used were sanitized with isopropyl alcohol (70%) to minimize the contamination risk.

After collection the fecal samples were immediately cooled to 5^oC for a maximum of 24 hours until being taken to the Electron Microscopy Laboratory of the Biological Institute of São Paulo for processing and analysis. Intestine fragments which presented lesions macroscopically similar to enteritis were collected with the use of scissors to observe the viral particles associated with these organs.

The samples were processed for observation of the viral particles using negative contrast transmission electron microscopy. In this procedure, the samples are suspended in a 0.1 M phosphate buffer (pH 7.0), placed on metal grates covered with collodion and carbon film, drained with a paper filter, negatively contrasted with 2% ammonium molybdate (pH 5.0) and subsequently observed in a Philips EM 208 transmission electron microscope.

In order to extend the viral particle investigation in the intestinal contents of bullfrogs, two wild frogs were collected and analyzed in the same way. These two animals were bullfrogs that had fled from the frog farm and were living free in nature without any type of zootechnical, sanitary or food management. The American bullfrog (L. catesbeianus) is an alien species, introduced in Brazil in 1935 for zootechnical purposes. Nowadays it is the only species used in Brazilian commercial rearing. This husbandry is regulated and the release of these species is prohibited by the Agricultural and Environmental Department (IBAMA). We deduce that the species captured in lagoons resulted from accidental escapes, probably from this farm or another one in the same region.

RESULTS

In this study we identified that 42 out of 45 samples were positive for virus (91.3%), where two of the virus-negative frogs were from farm A and one was from farm B. The viral groups that were found in the intestinal content samples were Paramyxoviridae in 40 of the animals, Coronaviridae in 11, Herpesviridae in 7 and Parvoviridae in 1. (Table 1) indicates the prevalence of the encountered viral groups.

Three animals that presented Paramyxoviridae infection (Fig. 1) in the intestinal contents also presented intestinal lesion injuries similar to enteritis. The presence of viral particles similar to the Paramyxoviridae and the Herpesviridae families were observed in the analysis of these fragments. Frog farm E presented five animals with intestinal injuries, whereas the presence of Herpesvirus infection was observed in only one animal (Fig. 2) and the presence of Coronavirus in another (Fig. 3) was associated with the presence of Paramyxovirus.

One of the wild animals presented only Paramyxovirus infection in its intestinal contents and infection with the Paramyxoviridae and Herpesviridae families in the intestinal injury fragment. The second animal presented infection with the Paramyxoviridae and Herpesviridae families in its intestinal contents and the collected injury (Table 2).

DISCUSSION

Johnson and Wellehan (2005) reported that amphibians are a diverse group of species; much work remains to be done to elucidate the amphibians' viruses. Viral diseases may play an important role in wild and captive amphibian populations. The survival of a virus depends on its infectious capability. The elimination of viruses from an organism occurs via saliva, nasal secretion, urine, semen and feces (Kreutz, 2001). The lack of therapeutic processes or immunization for viruses confers an enormous importance on prophylactic measures, making viruses one of the most important pathogenic agents among aquatic organisms (Pavanelli et al., 2002). Besides being one of the sources of elimination of viral agents from the host, it has already been determined that enteric viruses are frequently associated with food contamination, probably due to manipulation errors (Schmid et al., 1977).

In amphibians, skin neoplasms are widely distributed and have already been described in Brazil, such as the occurrence of papillomas in bullfrogs commercially cultivated, but a concrete etiology has not been established to suggest a viral origin (Souza et al., 1985).

Historically, the common development of secondary bacterial infections and misinterpretation of postmortem bacterial invasion were quite likely significant factors in the under diagnosis of viral diseases of amphibians. Now, however, viruses are frequently isolated from captive and wild amphibians and many of them are temperature dependent (Densmore and Green, 2007).

Infection by the Paramyxoviridae family is widely distributed among several animal species, such as bovine, swine, equine, birds (Catroxo et al., 2000) and amphibians (Hipolito et al., 2003), and in excrement from snails of the Helix aspersa species (Hipolito, et al. 2002). These viruses are characterized by the presence of pleomorphic particles measuring between 150 and 200 nm in diameter and envelopes covered by spicules. These viral particles were also observed in the liver from bullfrog tadpoles that died of unknown causes (Hipolito et al., 2003). This viral group is well-known and causes serious illnesses in several animal species, such as the serious enteric and respiratory syndrome that affects domestic and wild birds called Newcastle disease (Oliveira Júnior et al., 2005).

The Coronaviridae is spread among mammals and is associated with enteritis and diarrhea, which are considered large etiological complexity syndromes (Gregori et al., 2000) and are some of the main morbidity and mortality causes in animals. In frogs we were not able to define diarrhea, but we identified watery intestinal contents. In bovines, infection with Coronaviridae is involved in the etiology of severe enteritis with the presence of diarrhea (Brandão et al., 2007). Coronaviridae are single stranded RNA viruses, pleomorphic, rounded or elongated, covered in bulbous features and, therefore, have the appearance of a crown, measuring an average of 140 nm in diameter and a total viral size of 160-175 nm (Catroxo et al., 2000). These viruses are also associated with diarrhea in wild birds (Catroxo et al., 2000).

The Herpes viral group is one of the most known and studied, and is widely distributed in production animal populations and in the human population. Genetic studies attribute the co-evolution of this viral group with its hosts where latent infections occur, without resulting in generally serious illnesses with high mortality, thus favoring the dissemination of the Herpesviridae (Franco and Roehe, 2007). However, Herpesviridae subfamilies are considered the main pathogens in bovines, goats and sheep, and seriously harm the reproduction of these animals, which generates economic damages to livestock industries (Dias et al., 2008). In bovines, the Herpesviridae family is associated with neurological infections, such as meningitis, and severe respiratory problems. Thus, various studies describe the pathology associated with Herpesviridae infection and possible treatments, and immunization of production animals to combat this viral group (Flores et al., 2009).

Herpesvirus are DNA viruses found in various well-known illnesses, such as Aujesky's disease in swine (Pongillupi et al., 2008), and are easily identified in cellular cultures due to their cytopathic effects (Flores et al., 2009). Advanced genetic studies and PCR identification (Pongillupi et al., 2007), as well as serological research for the Herpesviridae are also widespread. In wild amphibians the Herpesviridae family is the only one of the observed groups which is associated with a determined pathological process, such as the development of Lucke tumors (Williams et al., 1996). In bullfrogs from frog farms Hipolito et al., 2003 identified Herpesvirus particles in the liver and ascitic liquid from dead animals.

Infection by Parvovirus is widespread among domestic animals such as dogs and causes severe gastroenteritis, mainly in puppies, with the development of a highly characteristic clinical profile (Strottmann et al., 2008). These DNA viruses can be identified through serology, which is widely used for diagnosing these agents. Hemagglutination and PCR are used for differential diagnosis, since this infection has an endemic characteristic in the populations' possible hosts (Strottmann et al., 2008).

The morphological features observed were also reported in other studies such as: birds (Catroxo et al., 2012), frogs (Hipolito et al. 2002; 2003), mammals (Catroxo et al., 2010) and ferrets (Gregori et al., 2010).

Some animals can be asymptomatic carriers of viruses and, upon impairment of immunity, can initiate a viral process. The inability to confirm whether the presence of the virus was related to the animal's cause of death makes studying the pathology of such virus infections more difficult (Catroxo et al., 2003).

As confirmed by Flores (2007), the viral groups discussed here affect many animals, not only domestic and wild animals, but also endothermic and ectothermic (Franco and Roehe, 2007). Therefore, the use of biosecurity principles is the best solution in animal cultivation to prevent sanitary problems and serious economic impacts (Bordin et al., 2005) because the viruses are present among all animal groups. The confinement of large numbers of unfamiliar animals and non-implementation of basic cleanliness procedures can generate serious problems and economic damages, making the enterprises of aquatic organism cultivation impractical (Hipolito et al., 2002). The adoption of these concepts that involve biosecurity is unquestionably indispensable, and some HACCP procedures (Hazard Analysis and Critical Control Points) and GMPs (Good Management Practice) can be suggested to improve the productive performance of the commercial cultivation of frogs.

CONCLUSION

The use of the negative contrast technique for viral particle detection in intestinal contents should be disseminated and used for the quick diagnosis of virus infection.

ACKNOWLEDGMENTS

To the Electron Microscopy Laboratory of the Biological Institute for the use of their services, and CAPES from the Fisheries Institute of São Paulo for the stipend granted.

Recebido em 4 de fevereiro de 2013

Aceito em 5 de setembro de 2013

E-mail: [e-mail]

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Tracking viral particles in the intestinal contents of the American bullfrog, Lithobates catesbeianus, by Transmission Electron Microscopy

Pesquisa de partículas virais em conteúdo intestinal de rãs-touro americanas, Lithobates catesbeianus, por meio da microscopia eletrônica de transmissão

Publication Dates

• Publication in this collection
  15 May 2014
• Date of issue
  Apr 2014

History

• Received
  04 Feb 2013
• Accepted
  05 Sept 2013