FACULDADES METROPOLITANAS UNIDAS

MEDICINA VETERINÁRIA

Infection of Corallus caninus by Chlamydophila psittaci

Fernando de Castro Figliolini

Trabalho de graduação em Medicina Veterinária

Das Faculdades Metropolitanas Unidas

Orientadora: Professora Dra. Terezinha Knöbl

SÃO PAULO

2008

1

ABSTRACT: The occurrence of regurgitation syndrome in four animals belonging to the Corallus caninus species. Three serpents died after a period of four to sixteen months after the clinical manifestations were observed. One of the dead serpents was sent to necropsy and complementary exams. Stool samples from both serpents were submitted to PCR reaction. PCR showed positive results to Chamydophila psittaci. After the diagnosis, the serpent was submitted to treatment with doxycycline and showed negative results to PCR exams. During treatment, side effects were observed, and this animal was submitted to a twelve month follow-up after treatment.

Key words: Chlamydophila, Corallus, zoonosis, reptiles, regurgitation syndrome.

Chlamydophila is a mandatory, intracellular, negative Gram bacterium that causes a series of diseases in vertebrates. The Chlamydiales order includes four families, and the Chlamydiaceae family includes two genera Chlamydia (C.) and Chlamydophila

(Cp.), which in turn includes nine species: C. trachomatis, C. suis, C. muridarum, Cp. psittaci, Cp. pneumoniae, Cp. felis, Cp. percorum, Cp. abortus, and Cp. caviae

(Everett et al, 1999) With a dimorphous life cycle, it is characterized by two cellular stages: Elementary Bodies (EB) that are responsible for the cellular invasion, and

Reticulate Bodies (RB) that are responsible for the agent multiplication. Agents in the

Chlamydiaceae family can be considered as pathogens in reptiles such as the Greek

Toirtoises (Testudo graeca), Chamelions (Chamaeleo dileps), Green Tortoises

(Chelonia mydas), Vipers (Bitis arietans), Crocodiles (Crocodylus niloticus), Pythons

(Python molurus bivittatus), Iguanas (Iguana iguana), and Emerald tree boas

(Corallus caninus). The lesions associated to these infections in reptiles vary and include granulomatous inflammation, necrotic enteritis, necrotic myocarditis, and proliferative pneumonia. It is important to have a better understanding of the epidemiology of this agent in reptiles because it is a zoonosis, also to meet the 2 growing market demand for such animals, and to preserve endangered species as well.

This paper was carried out after a follow—up in a group of Carollus caninus with a syndrome of regurgitation that is a very common symptom found in the species, mainly in animals captured in the wilderness and that is characterized by vomits between three and five days after the ingestion of food and chronic evolution leading to the animal’s death. This clinical condition is usually associated to a difficult adaptation of the said species to captivity and the improper management, mainly related to temperature, moisture, and food intake (frequency and volume). The regurgitation syndrome was associated in this paper to the presence of

Chlamydophila as suggested by Lock et al., (2003).

Two Corallus caninus subjects, female 1 (FI) and male 1 (MI) belonged to a private collection since January, 2003. According to their owner, these animals did not present the syndrome of regurgitation and in the rare occasions when they vomited such event was always related to high temperatures and to the ingestion of oversize prays. December, 2005, two new animals were brought to the collection, female 2

(FII) and male 2 (MII), both apparently healthy and in good body shape, and due to the lack of room, animal FII was placed in a terrarium together with FI and MI. Animal

MII was placed in a different terrarium. According to the owner information, specimens FII and MII had been acquired from the same source that kept both together in a same terrarium.

The animals newly introduced to the collection already showed signs of the syndrome of regurgitation from the first ingestion of pray, even after they acclimatized into the new environment with controlled moisture and temperature. 3

Since the introduction of the new specimens, animals FI and MI started to present the same although lighter symptoms (Table 1) probably because they had already been acclimatized to the captivity.

The four animals that were studied showed unaltered appetite and they all performed food capture and ingestion every time food was offered. All the animals with regurgitation syndrome were fed with prays up to 5% of live weight and in intervals of

10 days minimum.

May, 2006, the stool of the four animals was submitted to parasitological exam and the results showed nematodes (Oxyurideos) in animals FII and MII and Coccids in animals FI and MI Also in May, blood was extracted from the caudal vein of specimens FII and MI for the performance of hemogram and the presence of hematozoa (Hemogreganina) was detected in both samples inside the red blood cells. Specimen FII was treated with Febendazol to control nematodes.

Five months after the arrival of the new animals, MII died two days after a vomit episode. Two months after, FI also died but because of an accidental trauma.

Both remaining animals, FII and MI continued suffering of temporary vomit episodes for nearly 16 months, and they were more frequent in FII than in MI. Specimen FII was diagnosed positive for Chlamydophila psitacci on 05/07/07, and MI on 06/28/20.

Animals FII and MI were diagnosed positive for Chlamydophila psitacci after stool tests run according to PCR reaction (Unigen Tecnologia do DNA LTDA) proved positive.

The present DNA in the feces samples was purified by passing through silica column with commercial kit. The PCR reaction used primers of specific oligonucleotídes for amplification of a portion of 264 pb of gene MOMP (major to outer membrane protein) of C psittaci. The employed method reaches sensitivity to detect a minimum 4 of 6 to 60 copies of the bacterium DNA. To confirm the specificity of the PCR reaction, the amplified product it was digested with the restriction enzyme EcoRV, what originate bands of 37 pb and 227 pb.

On 05/22/2007, specimen FII was found dead on the terrarium ground with stomach exposition by oral cavity (Fig.1) and with the presence of vomit on the side, a pray ingested four days before. This animal was frozen for further necropsy (Fig. 2) that was performed in FMU veterinary hospital.

During macroscopic evaluation a reasonable amount of fatty bodies was observed, a rupture of the esophagus caudal segment, and a discrete hepatomegaly. The microscopic evaluation found steatosis (Fig. 3) in the liver, and enteritis in the intestines with the predominance of mononuclear cells and necrosis of the vilosities

The other organs such as the stomach, the spleen, the myocardium, and lungs showed no hystopathological alterations worthy of notice.

On 08/16/07 the treatment of the remainder MI animal started with the application of a dose of 10mg/kg of doxycycline for forty days (Mader, et al 2006). The drug was introduced through an orogastric probe and 10 to 15 ml of water in an attempt to decrease its nephrotoxic potential was added. During MI treatment period, polyuria, hematuria, and weight loss occurred, and in the thirty ninth day it vomited a brownish malformed mass although it had not been fed during the treatment period. At the end of the treatment on September, 2007, MI presented two episodes of vomit, one in

September and one in December, 2007, and during the following months the animal presented steady weight gain and a regularization of the gastrointestinal activity; a follow-up was carried with this animal until November, 2008 and no further episodes of vomit occurred, and the animal was fed with prays up to 15% of its live weight. 5

The Corallus caninus are tree-living animals of night habits, and there are few records about their feeding habits, but there is suspicion that they feed on tree-living mammals, and they certainly feed on birds, a chance of a possible form of contact with C. psittaci. Another important point to be mentioned is the fact that many of these animals are captured during the harvesting of the palm açai (Euterpe oleraceae) and the animals are found on the canopy of the palm trees where many psitacids gather to feed from the fruit; and because they feed on such birds, or are in contact with their feces, there is such possibility of infection. In addition, because these animals are captured illegally and kept in groups under improper conditions, under stress and many times in contact with other species such as psitacids that have high value in the illegal market, there is an increase in the number of infected animals and a greater possibility of occurrence of the clinical disease due to the lower immunity caused by stress.

The preferred form of diagnosis for Chlamydophilosis in reptiles is the PCR reaction, which is mentioned by Soldati et al, (2004); Boldetti ei al, (2002); Jacobson et al,

(2004) among others. Another possible solution is through the isolation of an embryonated egg mentioned by Vanrompay et al, (1994). Immunoperoxidase test is also mentioned by Jacobson et al (2002) as a diagnosis method. Histopathological findings are of great value for the identification of this agent, in addition to the identification of already mentioned lesions the presence of granulomas has been associated to this infection by a few authors such as Jacobson et al, (1989); Lock et al, (2003).

Lock et al, (2003) mentioned the association between the regurgitation syndrome and the infection by Chlamydophila in Corallus caninus in a paper they produced. A high frequency of animals that during the histopathological evaluation present 6 evidence of gastroenteritis and some also present granulomas, a later Electron microscopic evaluation of granulomas showed organisms consistent with

Chlamydophila sp. Through their findings, the authors suggest that the agent must have contributed to the episode of regurgitation and to the death of the animals, and that probably other isolated bacteria and fungi identified in the histological examination were possible secondary opportunistic agents.

Jacobson et al., (1989) report a case with six vipers (Bitis arietans) that suffered of regurgitation episodes that evolved to death and that were associated to infection by

Chlamydophila. Many were the histopathological findings including granulomatous inflammation, myocardial necrosis, and basophilic inclusions in different sizes in the cytoplasm of the macrophages.

The syndrome of regurgitation in C. caninus and in other serpents can, in addition to

Chlamydophila infection be related to other factors; this paper reports the main factors.

Cryptosporidiosis presents as main lesion the irritation of the gastric mucosa in serpents, and of the intestinal mucosa in lizards causing vomit and regurgitation

(Klingenberg et al., 2007).

Inclusion bodies disease of boid snakes is a viral disease where regurgitation, lack of coordination, disorientation, and paresis are the main clinical signals (Raymond et al., 2001).

Infections caused by helmintic parasites and coccids can also incite vomits and regurgitation because of the lesions caused on the gastrointestinal mucosa. 7

Errors in management are also known causes that incite vomits and regurgitation in serpents. The Corallus caninus, and mainly those captured in the wild and that come to market because of animal trafficking, are animals difficult to adapt to captivity, very demanding in terms of ideal control of moisture and temperature, frequency and size of pray, and the need of certain privacy.

Vanrompay et al., (1994) report pneumonia in Moorish tortoise (Testudo graeca) associated with avian serovar A C. psittaci.

Green sea turtles (Chelonia mydas) that received a positive diagnosis for chlamydiosis, show that the main lesions found were mainly located in the heart with myocardial degeneration and presence of necrosis as the most common presence.

Using electronic microscopy, it was possible to observe populations of bodies matching the Chlamydiaceae family in the cytoplasm of macrophages from the samples obtained in the necrotic areas of the myocardium (Homer et al., 1994).

The Chlamydophila are probably the most important infectious ethiology in granulomatous inflamation in reptiles (Soldati et al., 2004).

Huchzermeyer et al., (1994) report the presence of Chlamydophila as the ethiology for hepatitis in crocodiles (Crocodylus niloticus) born in captivity,

Although hepatic lipidosis in reptiles is traditionally associated to over feeding, obesity, and lack of exercises, other factors may influence accumulation of liver fat.

Just like in other animals, humans included, the excessive deposition of fat on the liver may be the consequence of several causes, including toxins, anoxia, carbohydrate and fatty acids metabolic deficiency (Mader et al., 2006) . Bolukbas et al., (2005) suggest an association between human subjects of the masculine gender and non-alcoholic steatohepatitis (NASH) and the persistent infection by C. 8 pneumoniae as a probable cause. Wang et al., (2007) demonstrated in a model using hepatocites and C. trachomatis that infections by Chlamydia can stimulate the host cells and absorb fatty acids and later promote intracellular replication of the agent.

Lock et al., (2003) report the treatment of more than fifty C. caninus that suffered from frequent regurgitations. He deployed an intramuscular dose of 10mg/kg of oxytetracicline every 3-5 days for 6 weeks. The animals showed a reduction on vomit frequency and were able to digest small pray. Some of these animals died and the other were transferred to another site were a follow-up of their evolution was not possible.

Huchzermeyer et al., (1994) used an intramuscular oxytetracicline based dose of

75mg/kg in a treatment for infected crocodiles that later was added to their food on the same daily dose for 10 days, they reports that as of the third day mortality rate of the animals decreased, and that on the following months no more deaths were reported in the lot.

Chlamydophila should be included as a differential diagnosis in the event of frequent vomits in serpents in general. The presence of granulomatous inflamation, necrotic enteritis, gastroenteritis, pneumonia, and hepatitis may be associated to infection by

Chlamydophila in reptiles. The relationship between steatosis found in animal FII and the presence of C. psittaci has not been made clear, but as suggested by Bolukbas et al., (2005), Wang et al., (2007) Chlamydophilas may infect hepatocites altering the metabolism of lipids.

Stool samples and PCR reaction in the delivery of diagnosis seems to be practical and efficient in the identification of Chlamydophila genera in serpents and can be a 9 good means of control for this infection. The treatment with doxycycline, despite its long term duration and side effects, presented a good result releasing the animal of the infections and of its clinical condition.

This report reinforces the association of the infection by Chlamydophila and the syndrome of regurgitation in C. caninus and in other serpents. Its diagnosis, treatment, and possible associated pathologies can possibly be an addition to already existing papers and for a better understanding of the pathologies of reptiles, and to help their preservation.

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Table 1. Period of clinical signals after the introduction of FII and MII

Animal Group Period (months) Meals Interval (days) Vomits

MI* adapted to captivity 18 28 5,2 4

FI adapted to captivity 5 10 4,3 3

MII recently added 4 9 2,8 5

FII* recently added 16 46 3,6 13

MI treated 12 14 3,5 2

*animals with positive diagnosis for Chlamydophila psittaci by PCR examination

Figure 1. FII and exposition of the stomach through the oral cavity.

Figure 2. FII after the replacement of the stomach on its anatomical position. 13

Figure 3. Liver presenting steatosis.

Figure 4. Enteritis with presence of mononuclear cells and necrosis of vilosities.

Coradas pela tecnica de Hematoxilina-Eosina com aumento de 200 x

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SHORT COMUNICATION

Infection of Corallus caninus by Chlamydophila psittaci

Fernando C. Figliolini¹ ³, Terezinha Knobl¹, Alexandre Rego¹, Antonio F.F.

Neto², ¹ Faculdades Metropolitanas Unidas, Rua Tagua, 150, Liberdade, CEP 01.508-010,

São Paulo / SP – Brasil; ² Unigen Tecnologia do DNA LTDA, Rua Doutor Zuquim, 1720, cj.

62, Santana - São Paulo / SP - Brasil - CEP 02035-022; ³ Corresponding author: (email: [email protected]).