Dept. of Animal Medicine,

Fac. Vet. Med. Assiut University.

FOOT AND MOUTH DISEASE IN CAMELS : ROLE OF CAMELS IN THE EPIZOOTIOLOGY AND TRANSMISSION OF FOOT AND MOUTH

DISEASE IN EGYPT

(With 2 Tables and One Figure)

By

U. Abd El-Hakim

(Received at 21/9/2005)

مرض الحمى القلاعية فى الجمال

دور الجمال فى وبائية وانتقال المرض فى مصر

أسامه عبد الحکيم

لدراسة مرض الحمى القلاعية فى الجمال ومعرفة الدور الذى تلعبه الجمال فى وبائية ونقل هذا المرض للأبقار فى مصر تم استخدام 50 جمل و50 بقرة على احتکاک بهذه الجمال. تم فحص هذه الحيوانات من الناحية الإکلينيکية والفيروسية بالإضافة إلى استخدام البيولوجيا الجزيئية (التفاعل التبلمرى المتسلسل). تم إجراء العزل الفيروسى واختبار التفاعل التبلمرى المتسلسل مرتين متتاليتين يفصل بينهما شهر. الفحص الإکلينيکى فى بداية التجربة أوضح ظهور أعراض فى صورة إفرازات لعابية على 3 جمال بينما کانت باقى الجمال وکل الأبقار سليمة ظاهريا. بعد 30 يوم من احتکاک الأبقار مع الجمال ظهرت بعض الأعراض المرضية المتمثلة فى إفرازات لعابية وأنفيةمع ارتفاع فى درجة الحرارة وعرج فى عدد 7 أبقار بينما کانت باقى الحيوانات سليمة ظاهريا. فى العزل الفيروسى الأول, تم عزل فيروس مرض الحمى القلاعية من عدد 14 جمل وکانت کل الأبقار سلبية للعزل الفيروسى بينما تم عزل فيروس مرض الحمى القلاعية من 17 جمل بالإضافة إلى 25 بقرة فى العزل الفيروسى الثانى. تم التعرف على الحامض النووى لفيروس مرض الحمى القلاعية فى 19 جمل فى الوقت الذى کانت کل الأبقار سلبية فى اختبار التفاعل التبلمرى المتسلسل الأول. بعد إجراء اختبار التفاعل التبلمرى المتسلسل الثانى, تم التعرف على الحامض النووى لفيروس مرض الحمى القلاعية فى 24 جمل و27 بقرة. أوضح التفاعل التبلمرى المتسلسل وجود عترتين   (OوA) من فيروس مرض الحمى القلاعية فى الجمال والأبقار کما أظهر وجود تشابه جينى بين فيروس مرض الحمى القلاعية فى الجمال والأبقار. نتائج هذا العمل أثبتت أن التفاعل التبلمرى المتسلسل أشد حساسية من العزل الفيروسى فى تشخيص مرض الحمى القلاعية فى الجمال والأبقار فى مصر بالإضافة إلى أنه أمکن استخدامه فى تصنيف الفيروس وتحديد عترته کما أمکن استخدامه فى دراسة العلاقة الجينية بين فيروس مرض الحمى القلاعية فى الجمال والأبقار مما أعطى الفرصة لتتبع ومعرفة مصدر العدوى فى هذه الدراسة. أوضح هذا العمل أن مرض الحمى القلاعية موجود فى الجمال فى مصر وينتقل بينها وإن کانت معظم الجمال المصابة تبدو سليمة ظاهريا وهذا عامل خطير جدا من الناحية الوبائية. أثبتت هذه الدراسة الدور الهام الذى تلعبه الجمال فى وبائية ونقل مرض الحمى القلاعية للأبقار فى مصر. هذه أول دراسة تتناول الدور الذى تلعبه الجمال فى نقل مرض الحمى القلاعية للأبقار کما أنها المرة الأولى التى يستخدم فيها التفاعل التبلمرى المتسلسل فى تشخيص وتصنيف فيروس مرض الحمى القلاعية فى الجمال ودراسة العلاقة الجينية بينه وبين الفيروس فى الأبقار بالإضافة إلى أنها المرة الأولى التى يتم فيها تسجيل العترة A لفيروس مرض الحمى القلاعية فى الجمال فى مصر. 

SUMMARY

To study FMD in camels and investigate the role played by camels in epizootiology and transmission of the disease to cattle, 50 camel and 50 cattle (in contact with camels) were used. All these animals examined clinically virologicaly (virus isolation, VI) in addition to examination using molecular biology based technique (reverse transcription polymerase chain reaction, RT-PCR). VI and RT-PCR were performed two times one month apart. Clinical examination at the beginning of this work showed 3 camels suffered from excessive salivation while remainmder of camels and all cattle were apparently healthy. After 30 day from contact between camels and cattle, salivation, nasal discharge in addition to rise of body temperature and lameness were recorded in 7 cattle and other animals were apparently healthy. In the 1^st VI, foot and mouth disease virus (FMDV) was isolated from 14 camels while all cattle were negative for VI. FMDV was isolated from 17 camel in addition to 25 cattle in the 2^nd VI. FMD viral RNA was identified in 19 camel while all examined cattle were negative for 1^st RT-PCR. After performing 2^nd RT-PCR, FMD viral RNA was observed in 24 camel and 27 cattle. 2 serotypes (O and A) of FMDV were detected in camels and cattle and genetic relationship between FMDV in camels and cattle has been proved after using RT-PCR. Results of this work proved that     RT-PCR is more sensitive than VI in diagnosis of FMD in camels and cattle in Egypt, in addition, this technique could be used in serotyping of FMDV and could be used in studying the genetic relationship between FMDV in camels and cattle. Therefore, RT-PCR enabled us to fellow up and know the source of infection in this study. This work proved that FMDV is present and transmite between camels in Egypt inspite of absence of any clinical signs in most infected camels and this is very dangerous factor from epizootiological point of view. Important role played by camels in epizootiology and transmission of FMD to cattle in Egypt has been proved in this study. This is the 1^st study concerning the role of camels in transmission of FMD to cattle and the 1^st to use        RT-PC R in diagnosis, typing of FMDV in camels and study its genetic relationship with FMDV in cattle, in addition to the 1^st recording of FMDV serotype A in camels in Egypt.

Key words: Foot and mouth disease (FMD), foot and mouth disease virus (FMDV), camels, cattle, Virus isolation (VI), Reverse transcription polymerase chain reaction (RT-PCR), Egypt

INTRODUCTION

            Foot and mouth disease virus (FMDV) causes a highly contagious viral disease of even-toed ungulates and is one of the most important economic disease of livestock. The disease was  reported in more than 33 species of a  domesticated or wild species including camels (Fondevila et al., 1995; Barnett and Cox, 1999; Bronsvoort et al., 2004; Deem et al., 2004; Ishimaru et al., 2004).It has been reported in many Arabic counteries, still present in Africa and considered endemic disease in the Middle East (Hafez et al., 1993; Hafez et al., 1994; Callens et al., 1998; Marquardt and Haas.,1998). The disease was initially described in the 16^th century and was the first animal pathogen identified as a virus which is RNA virus belongs Picornaviridae family, genus Aphthovirus, 7 immunologically distinct serotypes of the virus have been identified (Callens and De Clerck, 1997; Grubman and Baxt, 2004; Musser, 2004).

            Since the clinical signs can be very mild or absent, Callens et al., (1998) and Wee et al. (2004) emphasized the danger of carrier animals acting as a maintenance host and asymptomatic transmitter. It is clear that once an individual in a herd become infected with FMDV, herd infectivity is not static (Jones et al., 2004). Therefore, early diagnosis of FMD rely on early virus detection  from animals in the preclinical phase of infection or in carrier animals will be critical in limiting the number of infectious animals capable of transmitting the virus to other herds (Carpenter et al., 2004). FMDV spreads extremely fast and therefore there is a need for rapid and robust diagnostic system that is crucial for disease control (Marquardt and Haas,1998; Rasmussen et al., 2003). An important FMD risk reduction factor is early recognition of the disease at the source of the commodity (Sutmoller and Casas Olascoaha, 2003).

At present, identification and typing of FMDV largely relies on serological tests. However, these tests have many disadvantages and do not provide complete information on the epizootiological tracing of viruses (Nunez et al., 1998; Lomakina et al., 2004). At the same time, positive serology dose not indicate that the virus is still present. Therefore, reverse transcription polymerase chain reaction (RT-PCR) or virus isolation (VI) are necessary to demonstrate the presence of the virus (Callens et al., 1998).

Isolation of FMDV has to be attempted, but usually take several days. RT-PCR ia an additional method that can be used to diagnose FMD (Marquardt and Hass 1998).

The application of molecular diagnostic methods offers good alternative procedure for developing and optimizing a sensitive method for the detection of FMDV (Barlic-Maganja et al., 2004; Sangare et al., 2004). RT-PCR specifically detected FMDV, provides fast results and can handle large number of samples. Therefore, it is seen as a valuable tool for FMDV diagnosis (Reid et al., 2002). RT-PCR is being 500 fold more sensitive than conventional indirect ELISA and constitutes a simple, rapid and efficient alternative method for diagnosis of FMD (Rodriguez et al., 1994 Pattnaik et al., 1997). This assay also exceeded sensitivity of viral isolation. In many instances the assay detected viral RNA in the mouth and nose 24 to 96 hours before the onset of clinical disease and it could be detected within 24 hours post infection (Callahan et al., 2002; Zhang et al., 2004).

The increased international commercialization of camels has led to a need to obtain more information on the epidemiology of various disease agents that may affect these animals. Although some studies have been conducted with regard to their role in the epidemiology of many animal infectious conditions, the epidemiology of FMDV in camels has not been extensively studied. Unlike cattle, which are known to carry FMDV to extended period of time, little is known about the carrier abbility, if any, of camels (Fondevila et al., 1995).

In Egypt, Little is known about FMD in camels and there is no data concerned role of camels in epizotiology and trasmission of FMD to cattle. Therefore this study was planned to study FMD among camels and to study the role of camels, if any, in epizootiology and transmission of FMD to cattle. Selection of the most sensitive and reliable technique for detection and typing of FMDV in camels and cattle was another important aim of this work.

MATERIALS and METHODS

Animals: 50 camel and 50 cattle in contact with these camels were used in the study, some camels (3) were suffered from clinical signs of FMD while other camels and all cattle were apparently healthy at the beginning of the work.

Nasal swabs: 2 nasal swabs were collected one month apart from each examined camel and cattle for extraction of viral RNA.

Mouth swabs: 2 mouth swabs were collected one month apart from each examined animal for extraction of viral RNA.

Probang materials: Probang material was collected twice one month apart from each examined animal for virus isolation.

1-Clinica examination:

Clinical examination of all investigated animals was performed three times weekly, any abnormal clinical signs were reported.

2-RT-PCR:

(a) Sample preparation

Nose and mouth swabs were solubilised in 1ml phosphate buffer saline buffer

(b) Synthetic oligonucleotide primers

The primers for FMD serotypes (Pharmacia Biotech.) were designed by comparison of published sequences of FMDVand based on VPI gene of FMDV (Suryanarayana et al., 1999; Chen et al., 2004; Oem et al., 2004).Four primers were used in the present study. More specifications of these primers are listed in table (1)

(c) RNA extraction

Total RNA was extracted with TRIzol Reagent (Gibco BRL). 100 ul sample (nasal or mouth swab extracted in phosphate buffer saline) was mixed with 900 ul TRIzol Reagent.The samples were incubated for 5 minutes at room temperature, addition of equal amount of chloroform, precipitation of RNA with isopropanol and washing of RNA with 75% ethanol. The purified RNA was resuspended in 20ul Rnase free-water and redissolved by incubating for 10 min at 55 °C.

(d) Reverse transcription 

First strand cDNA synthesis was performed for 15 min at 37°C in a 10ul reaction mix containing the followings:- (1)50mM Tris-Hcl (pH 8.3), (2)75mM KCl, (3)3mM MgCl2, (4)10mM dithiothreitol, 0.125 mM (each) dNTP, (5)5uM (each) downstream primer, (6) 100 units Moloney murine leukemia virus reverse transcriptase (GibcoBRL) and (7)0.5 ug RNA. This was followed by heating for 5min at 95°C, in order to denature RNA-cDNA hybrid. Samples were chilled and stored at -20°C.

(e) PCR amplification

A PCR mix (10ul) consists of the following: 10mMTris-Hcl (pH8.8), 50mM KCl, 1.5mM MgCl2, 0.1% Triton X-100, 0.05mM (each) dNTP, 0.6um (each) primer, 0.25 units Taq polymerase (Promega) and 2% cDNA reaction mix. Amplification and reverse transcription was performed on a gene Amp CR system 9600 (Perkin Elmer) using program as follow: (1) 1 min at 95°C (2) 15 sec at 95°C (3) 1 min at 60°C (4) 6 min at 60°C, repeating steps (2) and (3) for 35 cycles.

(f) Gel electrophoresis   

The samples were electrophoresed at 100 V for 30 min in TAE buffer on 1.5% agarose gels, stained with ethidium bromide (0.6ug/ml). DNA molecular weight marker type 100bp DNA ladder (Gibco BRL) was applied to identify the size of the PCR products.Negative control for RT-PCR were performed by adding of distilled water instead of the primers. RT-PCR was performed as described by Vangrysperre and De Clercq (1996); Marquardt et al., 1996 and Marquardt and Haas (1998).

Table 1: Specification of used primers.

3-Virus isolation

Virus isolation was performed according to Sakamoto et al., (2002). In brief, the probang materials were inoculated to bovine kidney (BK) and bovine thyroid cell cultures. Cytopathic effect (CPE) was observed at two days post inoculation. 

RESULTS

1-Clinical examination:

(a) At the beginning of the study: 3 camels showed salivation only while other camels and all cattle were clinicaly healthy.

(b) After contact between camels and cattle: salivation, nasal discharge in addition to rise of body temperature and lameness were recorded in 7 cattle and other animals were clinicaly healthy.

2-RT-PCR:

(a) First examination:  FMD viral nucleic acid (RNA) was detected in 19 camel  while no positive result was reported among examined cattle with using of mouth swabs for extraction of viral RNA while 18 camel and no cattle were positive for RT-PCR when nasal swabs used for extraction of viral RNA.

(b) Second examination: FMD viral RNA was detected in 24 camel (including all 19 camel that gave positive result in the first examination) and 27 cattle when we used mouth swabs for extraction of viral RNA  while 22 camel and 24 cattle were positive for RT-PCR with using of nasal swabs for extraction of viral RNA     

(c) The positive samples in both camels and cattle were belong  2 FMD viral strains (O and C). Strain O was reported in 2o camel and 18 cattle, while strain A was recorded in 4 camels and 9 cattle.

(d) The similarity of size and location of nucleic acid band in both camels and cattle were observed and indicated the genetic identity between FMDV in both animals.

   -Results of RT-PCR are showen in Fig.1

3- Virus isolation (VI):

(a) First examination: FMD virus was isolated from 14 camel wherase all probang materials collected  from cattle were negative for FMD viral isolation

(b) Second examination: FMD virus was isolated from 17 camel and 25 cattle.

Table 2: Results of laboratory investigation.

     VI= Virus isolation          RT-PCR= Reverse transcription polymerase chain reaction

     (O)= Serotype O of FMDV                             (A)= Serotype A of FMDV

Fig. 1: Results of reverse transcription polymerase chain reaction        (RT-PCR) in camels (A) and cattle (B).

(A) Agarose gel electrophoresis of PCR product in camels. Lane 1 (100 base pair DNA ladder), Lane 2 (FMD virus serotype O), Lane 3 (FMD virus srotype A),Lane 4 (FMD virus serotype O), Lane 5 (negative result), Lane 6 (negative control) and lane 7 (FMD virus serotype A).

(B) Agarose gel electrophoresis of PCR product in cattle. Lane 1 (100 base pair DNA ladder), Lane 2 (negative result, Lane 3 (FMD virus serotype A), Lane 4 (FMD virus serotype O), Lane 5 (FMD virus serotype A), Lane 6 (negative control) and lane 7 (negative result).

DISCUSSION

Results of the present work proved that FMD is present and transmitted among camels in Egypt. Hedger (1976); Palling et al. (1979) Hedger et al. (1980); Hafez et al. (1993) reported that camels known to be quite susceptible to FMDV where the virus has been detected for months to years after infection. This contrasts with that of Fondevila et al., (1995) who mentioned that their study clearly indicate that, although camels can be infected with FMDV by direct contact, it is not a very susceptible animal. The results of these authors could be explained by the fact that they study FMD in one species only of camels (Lama glama) that found in North America.

The disease has a wide spectrum of clinical signs. There is, however, general agreement that the disease in naturally acquired infections often takes on a milder form in small ruminants and camels than in cattle, and in many cases may be vague (Barnett and Cox, 1999). In our study, most infected camels were apparently healthy and showed no clinical signs. 

Our present investigation  proved that camels (even apparently healthy one) plays an important role in transmission of FMD to contact cattle. It is clear that serologically positive contact animal without clinical signs present a danger for the transmission of FMDV (Callens et al., 1998; Carpenter et al., 2004)

            Mouth and nasal swabs were used in extraction of FMD viral RNA in our work. Callens et al. (1998) and Zhang et al. (2004) concluded that FMDV was most often found in saliva followed by nasal secretion and sera. Nasal swabs are suitable samples to detect viral genomes at asymptomatic stage of the disease (Marquardt and Hass, 1998). Results of the present study proved that mouth swabs are more suitable for extraction of FMD viral RNA. Most success was obtained with the saliva samples for detection of FMD viral RNA. In subclinically infected, like in carriers, FMDV was only intermittently recovered. It is therefore essential to take several different kinds of samples such as mouth swabs and nose swabs (Callens et al., 1998).

The results of this study clearly indicate that RT-PCR assay is more sensitive than VI in diagnosis of FMD in both camels and cattle as FMD viral RNA was detected in 51 animal (24 camel and 27 cattle) while FMDV was isolated from 41 animal (17 camel and 25 cattle) only. RT-PCR is a robust, reliable and sensitive test in diagnosis of FMD. Its sensitivity is 500-1000 times higher than VI and  serological tests so, it is efficient alternative for the diagnosis and characterization of FMD and it is presented for highly sensitive and specific detection of FMDV (Rodriguez et al., 1992; Rodriguez et al., 1994; Suryanarayana et al., 1999; Callahan et al., 2003; Moonen et al., 2003 and Saiz et al., 2003).

In our study we used RT-PCR in diagnosis and serotyping of FMDV. By using this technique, 2 FMDV serotypes (O and A) were detected. RT-PCR had been shown to identify and differentiate all seven serotypes of FMDV because it can be used for the specific detection and identification of viral sequences that correlate with established FMDV serotype (Rodriguez et al., 1992; Callens and De Clerck., 1997; Reid et al., 1999; Reid et al., 2000; Callahan et al., 2002; Reid et al., 2002; Rasmussen et al., 2003 and Barlic-Maganja et al., 2004).

Persistence and the carrier state of FMD have been reported (Barnett et al., 2004).Only three camels (out of 24 camels proved to be infected with FMDV) showed clinical signs while the rest of animals (21) were apparently healthy. These animals could be classified as persistently infected or carrier camels as the FMDV was detected in these animals two successive times one month apart. No clinical signs reported in animals infected with FMDV in the study of Bouma et al (2004) who considered these animals  persistently infected with FMDV without suffering from any clinical signs. The same observation has been rercorded by Musser (2004) who concluded that FMD infected animals can become inapparent carriers of FMDV.  

The detection of FMDV in persistently infected or carriers among exposed ruminats is of great importance (Zhang and Alexandersen, 2003). In the present work, RT-PCR was used in detection of apparently healthy persistently infected or carrier animals. RT PCR assay may be suitable for detection of FMDV carrier animals (Zhang and Alexandersen, 2003; Rasmussen et al., 2003). 

RT-PCR provides fast results. Therefore it is seen as a valuable tool for diagnosis of FMDV (Marquardt et al., 1995; Marquardt et al., 1996; Reid et al., 2001; Reid et al., 2002). In the present study we obtained results of RT-PCR within sex hours from collection of the samples. The same time was recorded in the study of  Marquardt and Haas (1998) and Zhang et al. (2004) who added that the rapidity by which results are obtained and sensitivity by which viral genomes are detected are the greatest advantages of this technique. Therefore, a RT-PCR was set up with the aim of being able to detect all virus isolates irrespective of their antigenic characteristics.

            In the present investigation we did not use RT-PCR in detection and serotyping FMDV only but also we used it in establishment a genetic relationship between FMDV in infected or persistently infected carrier camels and FMDV in contact cattle. Our results reported the genetic similarity between FMDV in camels and cattle. Therefore, this study proved the role of camels in transmission of FMDV to cattle. RT-PCR enables fast determination of genetic character of FMDV and could be used in detection of probable origin of the causative virus and the source of infection (Nunez et al., 1998; Scherbakov et al., 1998)  

            Results of virological and molecular biology investigations confirmed that RT-PCR more sensitive than virus isolation in diagnosis of FMD, so, we recommend using of this recent technique for diagnosis and typing of FMDV specially in imported camels (even those apparently healthy) before permission of its enterance into Egypt. These results also proved that camels are susceptible to FMD infection, and that they play a major role in transmitting the virus to domestic livestock. This is the first experiment to clearly show the role of camels in transmission of FMD to cattle and the 1^st to use RT-PC R in diagnosis, typing of FMDV in camels and study its genetic relationship with FMDV in cattle, in addition to the 1^st recording of FMDV serotype A in camels in Egypt

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