Dept. of Animal Medicine,

Fac. Vet. Med., AssiutUniversity,

SOME CLINICAL, DIAGNOSTIC

AND EPIDEMIOLOGICAL STUDIES

ON CHLAMYDOPHILA INFECTION IN CAMELS

(With 3 Tables and One Figure)

By

U. ABD EL-HAKIM

(Received at 2/3/2008)

بعض الدراسات الإکلينيکية والتشخيصية والوبائية للإصابة

 بالکلاميدوفيلا فى الجمال

اسامه عبد الحکيم

للتعرف على صورة )أو صور( الإصابة بالکلاميدوفيلا (الاسم الجديد للکلاميديا بعد إعادة التصنيف) فى الجمال و لمعرفة الدور الوبائى الذى يمکن أن تقوم به الجمال فى نقل البکتيريا المسببة لهذه الإصابة إلى حيوانات أخرى تم دراسة هذه الإصابة من الناحية الإکلينيکية والتشخيصية والوبائية فى الجمال والأغنام المحتکة بهذه الجمال. فى هذه الدراسة تم استخدام 30 جمل  و300 من الأغنام من 6 مزارع مختلفة تعيش فيها الأغنام مع الجمال (فى المنطقة الغربية من المملکة العربية السعوديه), 3 من هذه المزارع کانت تعانى من إجهاضات فى الأغنام. الدراسة التشخيصية شملت استخدام نوعين من الاختبارات, اختبار الإليزا واختبار التفاعل التبلمرى المتسلسل. تم إجراء هذين الاختبارين على عينات من السيرم والحليب من الجمال والأغنام 3 مرات متتالية يفصل بين کل مرة وأخرى شهر. نتائج الفحص الإکلينيکى أوضحت أن الجمال المصابة بالکلاميدوفيلا تعانى فقط من بعض الأعراض التنفسية بينما بعض الأغنام المصابة أجهضت والبعض ظهرت عليه أعراض تنفسية تتراوح بين خفيفة وشديدة بينما لم يتم تسجيل أى حالات إجهاض فى الجمال المصابة. الدراسة التشخيصية أثبتت إصابة الجمال بثلاثة أنواع من الکلاميدوفيلا (کلاميدوفيلا أبورتس – کلاميدوفيلا بيکوريم – کلاميدوفيلا نيمونى) بينما تم التعرف على الکلاميدوفيلا أبورتس والکلاميدوفيلا بيکوريم فقط فى الأغنام المصابة. إختبار الإليزا الذى تم استخدامه لم يتمکن من التفريق بين الأنواع المختلفة للکلاميدوفيلا بينما باستخدام التفاعل التبلمرى المتسلسل تم التعرف على هذه الأنواع الثلاثة والتفريق بينها. البيانات التى تم جمعها والتاريخ المرضى للمزارع التى تم إجراء البحث على بعض حيواناتها ونتائج هذه الدراسة (ومنها التحول السيرولوجى الذى حدث فى 16 غنم والتعرف على الحامض النووى للکلاميدوفيلا فى 25 غنم کانت سلبية وذلک بعد إدخال جملين مصابين بالکلاميدوفيلا إلى المزرعة التى لم تکن تعانى من أى حالات إجهاض أو أى صورة من صور الإصابة بالکلاميدوفيلا) تثبت أن الجمال تلعب دور هام فى وبائية هذا المرض. أثبت التفاعل التبلمرى المتسلسل أنه أشد حساسية من اختبار الإليزا حيث أنه أعطى نتائج إيجابية مع عدد 18 جمل و142 غنم بينما 11 جمل و 109 غنم فقط کانت إيجابية مع الإليزا وفى نفس الوقت لم يسجل التفاعل التبلمرى المتسلسل أى نتيجة إيجابية مع الحيوانات المحصنة بينما کانت کل الحيوانات المحصنة إيجابية لاختبار الإليزا. من خلال النتنائج التى تم الحصول عليها من هذا العمل أستطيع أن أسجل أن الجمال تصاب بالکلاميدوفيلا لکن معظم الجمال المصابة تبدو سليمة ظاهريا لهذا يمکن أن تلعب دورا هاما فى نقل هذه الإصابة إلى الحيوانات المحتکة معها. کما يمکن تسجيل التفاعل التبلمرى المتسلسل کاختبار حساس وسريع ويمکن استخدامه لتشخيص جميع صور الإصابة بالکلاميدوفيلا والتفريق بين أنواعها المختلفة فى الحيوانات المصابة وتزيد الحاجة اليه فى الحيوانات الحاملة للميکروب وتبدو سليمة ظاهريا بينما اختبار الإليزا يمکن استخدامه فى إجراء المسح السيرولوجى فى المزارع التى تعانى من الإجهاضات.

SUMMARY

To investigate form (or forms) of Chlamydophila infection in camels and to determine the role that can be played by camels in transmission of this organism to other animals, this infection was studied from clinical, diagnostic and epidemiological aspects in camels and contact sheep. In this study, 30 camel and 300 sheep from different 6 farms where camels and sheep found together in the same place in Western region of KSA were used, three of these farms suffered from abortion in sheep. Diagnostic study included two assays, enzyme linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR). These tests performed on serum and milk samples of camels and sheep three successive times one month apart between each two examinations. Results of clinical examination showed that some camels infected with Chlamydophila suffered only from respiratory signs while some sheep aborted and other suffered from respiratory signs varied from mild to severe, no abortion recorded in infected camels. Diagnostic study proved infection of camels with three spp. of Chlamydophila (C. abortus – C. pecorum – C. pneumoniae) while only C. abortus and C. pecorum were recorded in sheep. ELISA used in this study couldn’t differentiate between different spp. of Chlamydophila while PCR could identify and differentiate between three Chlamydophila Spp. Collected data and disease history of the used farms with results of this study (seroconversion of 16 sheep and detection of Chlamydophila nucleic acid in 25 sheep after introducing of Chlamydophila infected newly purchased two camels to the farm without any previous history of abortion and did not suffered from any form of Chlamydophil, all these animals were negative for Chlamydophila antibodies and nucleic acid at the beginning of the study) proved that camels plays a very important role in transmission and epidemiology of Chlamydophila infection. PCR assay proved to be more sensitive than ELISA where it gave positive results with 18 camels and 142 sheep while ELISA was positive in 11 camels and 109 sheep only. At the same time, PCR didn’t give any positive results with vaccinated sheep while all vaccinated animals were positive with ELISA. From results of this work I can record that camels could infected with Chlamydophila but most infected camels appear healthy so they can play a very important role in transmission of this infection to contact animals. Also PCR could be recorded as fast and sensitive technique and can be used for diagnosis of all forms of Chlamydophila infection, its importance increase in apparently healthy carrier animals while ELISA could be used in serological screening of animals in farms suffered from abortion.

Key words: Chlamydophila, ELISA, PCR, Camel, Sheep, Abortion

INTRODUCTION

The family Chlamydiaceae consists of obligate, intracellular gram-negative bacteria that cause a broad range of disease in both humans and animals, which include abortion, pneumonia, gastroenteritis, polyarthritis and conjunctivitis (Twomey et al., 2006; Yang et al., 2006; Harley et al., 2007; Skilton et al., 2007). The family Chlamydiaceae, which previously contained the single genus Chlamydia, has recently undergone reclassification into two genera, Chlamydia and Chlamydophila, and nine species (Everett et al., 1999). The most economically important animal pathogen of small ruminants is Chlamydophila abortus (previously classified as Chlamydia psittaci serotype 1), which causes abortion in sheep and goats (Philips and Clarkson, 1998; Rodolakis et al., 1998; Entrican et al., 2001).

Intracellular bacteria of the order Chlamydiales were firstassociated with diseases of cattle when McNuttand Waller (1940) isolated such organisms from feedlot cattle with sporadic bovineencephalomyelitis. When chicken embryo and cell culturemethods for Chlamydiales became widely used, around 1955, anumber of studies worldwide documented chlamydial agents inmany acute diseases of animals. Chlamydial strains from ruminant abortion were identified asserotype 1, biotype 1, immunotype 1 chlamydiae (Perez-Martenez and Storz, 1985). Recently, a reclassification asChlamydophila abortus    (C. abortus) was proposed (Everett et al., 1999; Schachter et al., 2001). C. abortus has also been associated with mastitis (Jee et al., 2004).

Another chlamydial agent has been associated worldwide withclinically severe chlamydial disease manifestations otherthan abortion (Jee et al., 2004). This chlamydialstrain was diagnosed as serotype 2, biotype 2, immunotype 2 chlamydiae (Kaltenboeck et al., 1993) andwas classified as a separate chlamydial species (Fukushi and Hirai, 1992). Recently,reclassification of this agent as Chlamydophila pecorum wasproposed (Everett et al., 1999; Schachter et al., 2001).

Numerous studies confirmed the disease potential of C. abortusand C. pecorum by experimentally reproducing the acute and severediseases (Jones, 1997; Jones et al., 1998). Shewen summarized in (1980) the status of our understandingof chlamydial infections in animals,some animals may experience severe or even fatal disease asa result of chlamydial exposure. A well balanced host-parasiterelationship represents the common nature of chlamydial infection.The long-lasting inapparent or ‘latent’ state hasbeen documented in several species: birds, cattle, guinea pigs,sheep and humans. Under circumstances of stress, ‘carrier’animals may shed the organisms in large numbers or may in factlapse into clinical disease.

Ovine enzootic abortion (OEA) resulting from infection of sheep and goats with C. abortus is of major economic importance worldwide (Longbottom et al., 2002). C. abortus (formerly Chlamydia psittaci serotype 1) is the most common infectious bacteria in small ruminants, some studies reported that 39% of examined abortions in sheep and 23% in goats caused by this agent. In newly infected flock, up to 30% of ewes may abort in the last trimester of gestation or give birth to a weak or dead lambs (Gerber et al., 2007). Approximately 71% of aborted cows and 58% of aborted goats had IgG against C. abortus in their sera (Wang et al., 2001).

In the United Kingdom, chlamydial abortion accounts for about 50% of all diagnosed abortions, resulting in losses estimated to be in excess of £20 million each year. C. abortus can also cause abortion in cattle and represents a significant zoonotic risk to pregnant women (Longbottom et al., 2002).

Over the last 50 years the serological diagnosis of chlamydial infections has been based mainly on complement fixation test (CFT), which lacks both sensitivity and specificity because of cross-reactive antibodies to other gram-negative bacteria, including another common chlamydial pathogen (Longbottom et al., 2002). Jones et al., (1997) used 5 different serological tests for the detection of antibodies against Chlamydophila and suggested that further improvements in Chlamydial diagnosis must come because none of these tests was sufficiently satisfactory.

Despite improvement in diagnostic techniques, our understanding about the prevalenceand pathogenetic significance of these infections has not substantiallychanged since Shewen's review in 1980. The major impedimenthas been the cumbersome nature and insensitivity of diagnosticprocedures, particularly of the complement fixation test fordetermination of seroprevalence of chlamydial infection in animals(Kaltenboeck et al., 1997; Jee et al., 2004).

If low-level clinically inapparent infections represent thenorm and such infections occasionally aggregate into clinicalmanifestations. In this case, our detection methods simplywould not be sensitive and specific enough to detect such lowlevels of chlamydial infections. Recently,a highly sensitive PCR method suitable for large-throughputroutine detection, quantification, and differentiation of ChlamydophilaDNA was established (DeGraves et al., 2003a). A 53% prevalence of C. abortusand C. pecorum infection was detected, supporting the notionof continuous low-level infection (DeGraves et al., 2003b).  

ELISA also was used for diagnosis of chlamydia (Salti-montesanto et al., 1997; Buendia et al., 2001) but it couldn’t differentiate ovine C. abortus and C. pecorum (Wang et al., 2001), but was shown to be more sensitive than CFT particularly where abortion had occurred and can be used as screening test for chlamydial abortion (Anderson et al., 1995; Griffiths et al., 1996; Donn et al., 1997; Gerber et al., 2007). However, ELISA was identified as being more sensitive and specific than other serological tests (Buendia et al., 2001; Longbottom et al., 2002; McCauley et al., 2007; Vretou et al., 2007). McCauley et al.,(2007) reported 60%and 70% sensitivity of CFT and ELISA respectivelyin diagnosis of Chlamydophila infections, so, they recommended ELISA as an alternative to CFT but they also concluded that the search for more specific and sensitive assay for diagnosis of Chlamydophila should be continue.  

Prevention and control of OEA can be achieved by application of live vaccine. Results of works of Borel et al., (2005) and Gerber et al., (2007) showed that by using serology, no distinction can be made between vaccinated and naturally infected animals. As a result, confirmation of negative OEA status in vaccinated animals can not be determined by serology. ELISA also couldn’t differentiate between different Chlamydophila species (Siarkou et al., 2002).

PCR was attempted as recent technique for diagnosis of Chlamydophila infections (Ciervo et al., 2003; Ongor et al., 2004; Yang et al., 2006; Gullsby et al., 2007; Branley et al., 2008). PCR has the potential to detect a single organism (Laroucau et al., 2001; Amin, 2003a; Masala et al., 2007). Some study groups reported positivity rates of up to 100% detection of Chlamydophila pneumoniae by PCR (Apfalter et al., 2001).

Unfortunately, little is still known about the ability of C. abortus to persist in animal (Gerber et al., 2007). The prevalence of Chlamydophilainfections in camels and the rates of acquisition and transmissionhave not been studied in detail. Therefore, the present study was designed to study Chlamydophila infections in camels and to investigate presence and cause of persistence of Chlamydophila species in farms containing camels and sheep. Comparison between ELISA and PCR as a diagnostic tools for Chlamydophila infections was an another objective of this study.

MATERIALS and METHODS

Animals

30 camels and 300 contact sheep found in different 6 farms were used in this work (5 of these sheep were vaccinated against Chlamydophila), three of these farms had a previous history of abortion but none of the used animals were suffered from abortion before this investigation, After planning and the beginning of the study, 2 newly purchased camels were added to farm (that hadn`t history of abortion). These 2 camels were added to the study. 

Serum

 For ELISA, blood was collected from the jugular vein with a     7-ml blood collection tube (Vacutainer tubeswith Hemogard closures; Becton Dickinson and Co., FranklinLakes,N.J.). The serum was separated by centrifugation at 1,300 xg for 15 min and stored at –80°C in 2-ml microcentrifugetubes with screw caps till use.

Milk

For DNA extraction, 600 µl of milkwas mixed with 600 µl of 6 M guanidine-HCl, 10 mM urea,10 mM Tris-HCl, and 20% Triton X-100 (vol/vol), pH 4.4, in a2.0-ml microcentrifuge tube with a screw cap.

ELISA

Antigen equivalent to 0.7 µgof EB protein per well, diluted to 100 µl in coating buffer(15 mM Na₂CO₃ and 35 mM NaHCO₃ [pH 9.6]), was added per wellto white C-bottom 96-well microtiter plates (White MaxiSorp;Fisher Scientific Co.). Plates were incubated overnight at 4°C,the coating solution was aspirated, and wells were washed fivetimes with wash buffer (0.1 M Tris-HCl [pH 7.5], 0.15 M NaCl,and 0.1% Tween 20). Wells were blocked by adding 200 µlof assay diluent (0.1 M Tris-HCl [pH 7.5], 0.15 M NaCl, 0.1%Tween 20, and 10% normal rabbit serum) for 1 h at room temperature.The assay diluent was removed, and 100 µl of serum samplediluted 1:100 with the assay diluent was added to each well of the plate except wells A1,A2 and B1,B2 which used for positive and negative controls. Incubation for 1.5 h atroom temperature. After five washes, 100 µl of alkalinephosphatase (AP)-conjugated antibodies against immunoglobulinA (IgA), IgG, or IgM (Bethyl Laboratories, Inc., Montgomery,Tex.) per well was added, diluted in assay diluent (IgM, 1:300; IgG, 1:600; and  IgA, 1:500), and incubated for 1 h at room temperature. Plateswere washed five times, 150 µl of freshly prepared APsubstrate buffer solution (BM Chemiluminescence ELISA SubstrateAP; Roche Applied Science) per well was added, and the luminescenceemitted was read with a microplate reader (Labsystems, A Thermo Bioanalysis Company, Research technology Devision, Helsinki, FINLAND).After 10 min of shaking. Luminescencedata were calculated and evaluated as follow:-

1-Calculation of negative control mean (NCX)

   (NCX) = A1 + A2

                        2

2-Calculation of positive control mean (PCX)

  (PCX) = B1 + B2

                       2

3-Caculation of blocking % for tested samples

    Blocking % = NCX – optical density of tested sample X 100

                                                   NCX

4-Caculation of blocking % for positive control

    Blocking % = NCX – PCX X 100

                                NCX

5-Interpritation of the result

-          Serum with blocking % less than 45% were considered negative for Chlamydophila antibodies.

-          Serum with blocking % equal or greater than 45% but less than 55% considered suspected and retested.

-          Serum with blocking % of 55% and greater considered positive for Chlamydophila antibodies.

ELISA was performed according to Longbottom et al., (2002) and manufacturer’s (IDEXX Laboratories,Inc. Switzerland) directions.

POLYMERASE CHAIN REACTION (PCR)

(a) DNA extraction. Isolation of milk nucleic acid for PCR was performedwith a High Pure PCR Template Preparation kit (Roche AppliedScience) according to the manufacturer's instructions. 120 µl of proteinase K (20 mg/ml in double distilledH₂O) was added to milk samplesand samples were incubated for 30 min at 72°C with shakingat 600 rpm. 300µl of isopropanol and 300µl of chloroform were added to milk samples. After brief agitation,the sample solution was transferred to the DNA-binding glassfiber filter device, except for the lipophilic chloroform bottomphase of the milk samples. Samples were filtered by centrifugationat 3,000 x g for 3 min, followed by the addition of 500 µlof inhibitor removal buffer and centrifugation at 3,000 x gfor 3 min. Samples were washed twice with 500-µl washbuffer and were centrifuged at 3,000 x g for 3 min. Traces ofwash buffer were removed by centrifugation at 13,000 x g for10 s, and 20 µl of elution buffer (10 mM Tris-HCl [pH8.4], and 0.01 mM EDTA) prewarmed to 72°C was added to eachsample filter inserted into the collection tube. The glass fiberfilter devices were incubated for 2 min at 72°C with shakingat 600 rpm, and elution buffer was recovered by centrifugationat 13,000 x g for 1 min. After a second elution step with 20µl of buffer, the eluted DNA stock (typically 35 µlper specimen) was stored at –80°C.

(b) Primer design. Primer sets specific for each of the three species     (C abortus, C pecorum and C pneumoniae) were designed based on the DNA sequences published by Greco et al., (2005) and Liu et al., (2007). These primers supplied to Ministry of Agriculture, KSA from the Johns HopkinsGenetic Core Laboratory. Data of oligonucleotide primers used in this study are summarized in Table (1)

Table 1: Sequences and specifications of used olignucleotide primers.

( c ) PCR. PCR mixtureoverlaid with 1 drop of mineral oil. The final mixture contained25 pmol of each primer, 0.25 mM deoxynucleosides triphosphates(dNTPs), PCR buffer, and 2 U of AmpliTaq Gold DNA polymerase (Perkin-Elmer,Branchburg, N.J.). 2.5 mM MgCl₂ was usedasrecommended by the manufacturer of the polymerase (Perkin-Elmer).PCR was used with a DNA thermal cycler(480; Perkin-Elmer Cetus, Norwalk, Conn.). Cycling times were75 s at 95°C (to activate a small fraction of the heat-activatedDNA polymerase), followed by 60 cycles of denaturation at 94°Cfor 45 s, annealing beginning at 62°C and ending at 52°C for 45 s, and extension at 72°C for 1 min. The annealing temperaturewas lowered 1°C every four cycles until it reached 52°C; thisannealing temperature was kept until the end of the cycling process.Progressive release of the heat-activated DNA polymerase occurredduring the thermal cycling process. The DNA polymerase was graduallyactivated at each cycle during denaturation to extend its activityto 60 cycles of DNA amplification.

(d) Electrophoresis: PCR products (20 µl) were separatedby electrophoresis in 1.2% polyacrylamide gels (7 by 10 cm) at80 mA for 30 min with Tris-borate-EDTA buffer (pH 8.3) and visualizedwith ethidium bromide (0.5 µg/ml). Jee et al., (2004).

PCR performed according to Degraves et al., (2003a); Degraves et al., (2003b); Ongor et al., (2004)

RESULTS

Clinical study

Camel:

a-      Rise of body temperature was reported in 3 camels (infected with C. pneumoniae).

b-      Nasal discharge only was reported in 6 camels (infected with C. pecorum)

c-      Rise of body temperature, nasal discharge and cough were reported in 2 camels (infected with C. pneumoniae)

d-      7 infected camels were clinically normal.

Sheep:

a-      Abortion was recorded in 21 sheep (infected with C. abortus and/or C. pecorum).

b-      Sight respiratory signs in the form of nasal discharge and/or weak cough were recorded in 49 infected sheep (infected with   C. abortus and/or C. pecorum).

c-      Severe respiratory signs in the form of strong cough and pneumonia were recorded in 39 infected sheep (infected with    C. abortus and/or C. pecorum).

d-      4 infected sheep were died from sever pneumonia during performing this study.

ELISA

a-   First: 9 camels and 97 sheep were positive for Chlamydophila antibodies.

b-      Second: 11 camels and 97 sheep were positive for Chlamydophila antibodies

c-      Third: 11 camels and 109 sheep (seroconversion of 16 sheep from farm without previous history of abortion was recorded after contact with new purchased two camels) were positive for Chlamydophila antibodies.4 sheep were died from severe pneumonia therefore, they excluded from 3^rd ELISA.

d-      All sheep with a history of previous vaccination against Chlamydophila (5 sheep) were positive with ELISA.

PCR

a-      First: nucleic acid of Chlamydophila was detected in 16 camel and 121 sheep

b-      Second: nucleic acid of Chlamydophila was detected in 18 camel (Two newly purchased camels were added to the farm and examined) and 135 sheep (14 of them were from farm without previous history of abortion and gave negative result with 1^st PCR but gave positive results after contact with positive newly purchased camels).

c-      Third:  nucleic acid of Chlamydophila was detected in 18 camel and 142 sheep (number of positive sheep increased by increase the time of contact with positive newly purchased camels), 25 of these positive sheep were from farm without previous history of abortion. 4 sheep were died from severe pneumonia therefore, they excluded from 3^rd PCR.

d-      The nucleic acid detected positive camels were belong three Chlamydophila species (C. abortus, C. pecorum and                  C. pneumoniae)

Results of laboratory investigation are summarized in Tables (2), (3) and results of PCR are shown in Figure (1).

Table 2: Results of laboratory investigation:

Table 3: different Chlamydophila Species recorded with PCR

C.ab= Chlamydophila abortus     C.pe=Chlamydophila pecorum      C.pn=Chlamydophila pneumoniae

Fig. 1: Agarose gel electrophoresis analysis of amplified PCR products in camels and sheep.

                Lane1 (DNA size marker). Lanes 2,4 (C.Pneumoniae in camels). Lane 3 (negative result). Lane 5 (C.Abortus in sheep). Lane 6 (C.Pecorum in sheep). Lane 7 (C.Abortus in camels). Lane 8 (C.pecorum in camels).

DISCUSSION

Large number of camels examined in the present study and gave positive result with ELISA and/or PCR was apparently healthy without any abnormal clinical signs. Monthly PCR testing revealed persistent recurring infection with C. pecorum and C. abortus despite the absence of any clinical illness (Jaegr et al., 2007; Miyashita et al., 2007; Reinhold et al., 2007). Both carriers and ill animals can shedthe bacterium from many sites, including via nasal and faecalsecretions (Jager et al., 2007). Inapparent infection with Chlamydophila was recorded (Griffiths et al., 1996; Reinhold et al., 2007).

No abortion recorded in any camels examined in this study even these animals that proved to have C. abortus and/or C. pecorum. Some of these infected camels showed only respiratory signs. Twomey et al., (2006) studied role of Chlamydia species in upper respiratory tract infections and detected C. abortus and C. pecorum as a main cause of bovine upper respiratory tract disease outbreak.Some infected camels showed only rise of body temperature. Reinhold et al., (2007) recorded only higher temperature in Chlamydophila infected calves. Tibary et al., (2006) investigated the causes of reproductive losses in camels and coudn`t record any infection with Chlamydophila as a cause of abortion in camels.

Abortion was recorded in some infected sheep in this work while other infected sheep showed respiratory signs end fatally in 4 sheep.     C. abortus caused abortion in sheep and considered one of the most important cause of abortion in sheep (Tsakos et al., 2001; Longbottom et al., 2002; Ongor et al., 2004; Bagadonas et al., 2007; Masala et al., 2007; Michalopolou et al., 2007; Reitt et al., 2007). Respiratory signs and pneumonia had been recorded in some Chlamydophila infected animals (Jager et al., 2007).

In this study I used ELISA and PCR for detection of Chlamydophila in camels and contact sheep. McCauley and co workers   (2007) recommended ELISA as an alternative to CFT but they added that the search for more specific assay should be continue. PCR has gained increasing importance as a tool for directly demonstrating the presence of Chlamydophila in a clinical samples (Michalopolou et al., 2007; Ortega et al., 2007). Oktem et al., (2007) reported that utilization of Just one diagnostic technique such as serological tests or PCR-based detection methods during Chlamydophila outbreaks can result in some of the positive samples being missed. After obtaining of results of this work I can add that PCR alone is enough for sensitive diagnosis of Chlamydophila as all positive samples with ELISA was positive with PCR in addition to samples give positive results with PCR while it were negative with ELISA.

In this study milk samples were used for detection of Chlamydophila nucleic acid in PCR assay and proved to be good sample for diagnosis. PCR can potentially be used for different clinical samples (Messmer et al., 1997; Madico et al., 2000) and Ongor et al., (2004) used milk for isolation of Chlamydophila nucleic acid.

In thepresent investigation, highly sensitive PCRto study the prevalenceof Chlamydophila sp. infection in camels was used. The results of this investigation have the potentialto shift the focus from Chlamydophila infection as a rare, severedisease to Chlamydophila infection as a pervasive, low-levelinfection in camels without apparent disease (silent epidemic)or with only a subtle expression of disease, one that impactsherd health and fertility but is difficult to recognize in individualanimals.The same observation was recorded by Reinhold et al., (2007) in cattle.

PCR used in this investigation could differentiate between C. abortus, C. pecorum and C. pneumoniae. Molecularamplification techniques based on genomic sequences have beenused for the differentiation of Chlamyophila species (Messmer et al., 1997; Madico et al., 2000).

Results of PCR assay was obtained within few hours in this work. PCR technique is fast, simple, inexpensive, and easy to perform and requiresminimal sample manipulation, which may reduce the chances forcontamination (Madico et al., 2000)

In the present work, ELISA failed to detect any of inapparent infection. Classic methods for the detection of Chlamydophila agents andof antibodies against these agents have indicated that these methodsdemonstrated acute Chlamydophila - induced diseases with generally high, Chlamydophila seroprevalence. However, it was impossible to consistently detect low levels of these organisms and its antibodies by these classic methods (Jee et al., 2004).

The prevalence of C. pecorum in camels in this studywas more than C. abortus and C. Pneumoniae. Jee et al., (2004) in their study recorded the same observation in calves (except for C. pneumoniae which not recorded in calves) and added that the prevalence of C. pecorum was approximately five times as high as that of C. abortus

18 out of 32 examined camels (56.25%) were positive for 3 different Chlamydophila species. Study of Bagdonas et al., (2007) in Lithuania revealed that 54.5% of animals were positive for Chlamydophila.

The results obtained from this study confirm the previous observation of Borel et al., (2005) and Gerber et al., (2007) that ELISA can not be used to distinguish between animals vaccinated with the live attenuated vaccine and naturally infected animals.

Results of this work indicated that PCR is more sensitive and specific than ELISA. Wang et al., (2001) reported that only 22.7% from aborted cows and 33.3% from aborted sheep that gave positive result with PCR was positive with ELISA. PCR assay provides a simple, sensitive, rapid and reliable means for the detection of the Chlamydophila  (Madico et al., 2000; Amin, 2003b) and is considered to be more suitable for the detection of Chlamydophila (Yang et al., 2006; Reitt et al., 2007). PCR is a useful method for sensitive and early detection and identification of Chlamydophila. During endemic infections, the sensitivity of diagnostic tests and rapid diagnosis is particularly important (Oktem et al., 2007; Ortega et al., 2007).

PCR used in this investigation could differentiate between different species of Chlamydophila (C. abortus, C. pecorum and C. pneumoniae) while ELISA failed to differentiate between them. Wang et al., (2001) recorded certain degree of cross reactivity between C. abortus and C. pecorum and therefore they could not differentiate between them by ELISA.

Seroconversion was recorded in 12 sheep (from farm without previous history of abortion) by the 3^rd ELISA and after introducing of newly purchased two camels to the farm. These two camels proved to be infected by Chlamydophila by 2^nd ELISA and 2^nd PCR. At the same time 2^nd and 3^rd PCR detect nucleic acid of Chlamydophila in 17 and 24 sheep respectively in this farm. The same observation has been recorded by Twomey et al., (2006) in cattle who added that adult cows were the most likely source of infection to calves after contact with them. This result also clearly proved that PCR is a suitable technique for diagnosis of early detection of Chlamydophila infection. PCR is useful method for early detection and identification of Chlamydophila (Oktem et al., 2007)    

Results of the present investigation proved that camels infected with C. abortus and/or C. pecorum (even in absence of any clinical signs) are the source of infection of contact sheep. Epidemiological observations and laboratory testing of Twomey et al., (2006) indicated that adult dairy cows were the most likely source of infection with Chlamydophila species to calves. Results of Salwa and co workers (2007) suggested that there is some transmission of Chlamydophila species occurring between domestic and wild ruminant.

From results of this study, I can conclude that camels could be infected with different species of Chlamydophila but most infected camels were apparently healthy, so, they can play a very important role in transmission of infection to contact sheep and persistence of infection for a long time. Also, results of this work proved that C. abortus and C. pecorum which causes abortion in small and large ruminants couldn’t  cause abortion in camels. PCR seems to be more sensitive than ELISA in diagnosis of Chlamydophila infections and has the priority to differentiate between different species of Chlamydophila as well as differentiate between infected and vaccinated animals while ELISA failed in both differentiations. Therefore, these results recommend using of PCR in diagnosis of any forms of Chlamydophila infections specially in silent or latent infection while ELISA can be used in serological screening in areas suffered from abortions.

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