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Isolation and Diagnosis of Mycoplasma by Conventional and Molecular Methods from Pneumonia in Feedlot Calves

*Ahmed A. Mhawesh1; **Ahmed Sadoon Hassain2; ***Mayyahi Mohammed T.

Jaber3

*College of Biotechnology/ Al-Nahrain University/ Baghdad/Iraq

**Department of Animal Production, College of Agriculture, University of Misan, Maysan, Iraq

***Forensic DNA Research and Training Center, Al-Nahrain University, Baghdad, Iraq

Abstract:

The current study aimed to isolate and diagnose Mycoplasma from pneumonia in feedlot calves in Mosul, using traditional methods such as culture and modern molecular techniques. (180) different respiratory samples were collected, represented by nasal and tracheal swabs and lung samples for calves infected with the same lung.

The results of isolation varied in the method of transplantation between different types of samples. The nasal swabs were the most efficient in detecting Mycoplasma as the proportion of positive samples reached (58)% Tracheal swabs followed, with an isolation rate of 55.3%.Then the samples of the affected lungs with the same lung by (35)%, the results of the Polymerase Chain Reaction technique were the same on the isolates that we obtained and it appeared that the isolates of the genus Mycoplasma ratios in the proportions (100, 90.9, 97.1)% of the nasal and tracheal and affected lung isolates respectively, Whereas, the initiator of the type Mycoplasma ovipneumoniae gave positive results and proportions (52.3, 63.3, 64.7)% for the previously mentioned samples, respectively.The results of the detection of Mycoplasma and its diagnosis in samples of infected lungs with the same lung immediately after the extraction of DNA from them showed that the sum of the samples that gave a positive result for the genus of Mycoplasma by serial polymerase reaction technique was (17) samples at a rate of (56.6)%, of which 14 )%82.4(samples were positive for the type M. ovipneumoniae.

Keywords: Pneumonia, Calves, M. ovipneumoniae, PCRand 16S rRNA gene.

Introduction

Pneumonia in calves is considered an important and widespread disease in many countries in the world because it affects all ages leading to large economic losses.

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Some studies have indicated that the incidence of respiratory diseases may reach (69.34%) of the total of various diseases that It affects calves, and pneumonia cases constitute (75%) of these respiratory diseases. Feedlot calves are more sensitive to infection with this disease due to the high rates of injury and mortality caused by them, especially if they coincide with poor environmental conditions, as well as the observed decrease in weight The affected animal and the rapid spread of the disease between herds For animals, leading to higher costs of breeding and efforts to treat and control this disease.(1) (2)

Respiratory infections among flocks of animals result from the extensive invasion of various types of microorganisms, which include many bacteria, Bacteria, Viruses, Yeasts, and Mycoplasma. (3)(4)Mycoplasma is a common cause of many uppers and lowers respiratory infections, especially in young calves.(5)And due to its inability to pigment in dye and the difficulty of its development in the agricultural media that is used in microbial isolation laboratories, the diagnosis of the true cause of the injury may fail in some cases, as it requires a long time for its growth, and for its need for song materials such as serum and glucose that must be added to the agricultural medium. And that the presence of many bacterial cohabitants in the respiratory system may affect the diagnosis of the causes of atypical pneumonia.

Diagnosis of Mycoplasma infection was previously based on traditional serological methods, including Complement Fixation Test (CFT), Microimmunoflorescent (MIF), Enzyme Linked Immuno Sorbent Assay (ELISA) and other serological tests. (6) (7) Despite the use of many methods in the detection of Mycoplasma causing pneumonia in calves, it was recently directed as a result of scientific progress in the field of molecular biological and DNA dependence in accurate and rapid diagnosis, so it was possible to diagnose it from various samples through what is known as the polymerization reaction technique Polymerase Chain Reaction (PCR) of various types

(8) as it was widely used in the world to diagnose these injuries, taking into account the election of the appropriate sample for this examination (9).

Materials and methods of work Sample collection

One hundred eightydifferent respiratory samples were collected and were as follows:

- (60) nasal swabs taken from calves that were clinically suffering from signs of

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themselves, by inserting the cotton swabs into the nasal cavity and beginning Tracheal swabs and cotton swabs were then placed into glass tube containers on the stock of Mycoplasma development (9). As for the lung samples, it was (60) lung specimens those showed lesions and signs of infection with the same lung.

Samples culturing:

Nasal and tracheal swabs were planted directly in the Mycoplasma broth and placed in the incubator for 14 days at a temperature of 35° C with the necessary moisture provided. Pieces of infected lungs were taken from the lesion site and placed directly into the Mycoplasma broth and incubated as before.

Diagnostic tests for the Mycoplasma bacterium:

1- Formal tests include:

The morphological and colonial properties were studied by growth on Mycoplasma Agar Medium after the end of the incubation period using an anatomical microscope. Swabs from Mycoplasma implanted culture media, dyed a Giemsa, and examined the optical microscope using the oil lens to observe Mycoplasma forms.

2- Dennis stain:

The Dennis stain was adopted to dye the colonies, as the surface of the agar dish containing the colonies was submerged in (1) ml and washed with distilled water then added (1) ml of (95%) of the ethyl alcohol to the minors and left the Dennis stain working solution for one minute and then remove the excess alcohol and repeat the process Washing with distilled water and colonized the Mycoplasma colonies in terms of color.

3- Molecular tests for samples:

The sequence of the amplicon initiator used to diagnose the genus Mycoplasma genus for the 16S rRNA gene segment, according to (Botes et al., 2005)(10):- Mycoplasma genus – specific primers:

Forward(5' GGG AGC AAA CAC GAT AGA TAC CCT 3') Reverse(5' TGC ACC ATC TGT CAC TCT GTT AAC CTC 3')

The sequence of the amplicon initiator used to diagnose the specialized Mycoplasma type according to (Mc Auliffe et al., 2003) (11) :

- species – specific primers for Mycoplasma ovipneumoniae : Forward (5' TGA ACG GAA TAT GTT AGC TT 3')

Reverse (5' GAC TTC ATC CTG CAC TCT GT 3')

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DNA extraction from directly infected lung samples:

This procedure has been done in an accordance toBotes and et al., 2005)

(10)technique, as shown in Table () 1

Table 1: Components of the polymerase chain reaction mixture.

The final volume is 20 µl Ingredients

1µl Forward primer (10 picomol\µl)

1µl Reverse primer (10 picomol\µl)

5µl Template DNA 250 ng\µl

13µl D.D.W.

1X Accu power® profiTag PCR pre Mix

Results

-Culture characteristics

A study of the formal characteristics and colonial characteristics of the Mycoplasma showed a medium (PPLO agar), as spherical colonies with a center surrounded by white aura appeared to be colorless, similar to the appearance of fried eggs, as shown in Figure.) 1(

Figure: 1 Mycoplasmacolonies growing on a medium (PPLO Medium). The appearance of the fried egg.

- Dennis stain

The results of the use of the Dennis stain showed the appearance of developing Mycoplasma colonies on the colorless medium of PPLO agar due to their ability to reduce the methylene blue present in the Denis tincture composition and the appearance of colorless colonies as shown in Figure.) 2(

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Figure: 2 Dennis stain. It is noted that the colonies of Mycoplasma retain the stain of Dennis, as the center of the colony appeared in a dark blue color surrounded by a light blue contour.

-Bacterial isolation ratios

The results of the study showed that after all confirmatory tests, the percentage of Mycoplasma isolation from the total samples was (49.4%). As shown in Table.) 2( Table (2): Results of isolation of Mycoplasma by implantation method from samples used in the study.

Percentage Positive

samples Total No.

of samples Sample

type

58 35

60 Nasal swab

55.3 33

60 Tracheal

swab

35 21

60 lung

49.4 89

180 Total

Above represent the results of the polymerase chain reaction test on the different samples under study

a-Nasal swabs

The results of the serial polymerization reaction on the isolates showed a significant match with the results of the bacterial implantation of the Mycoplasma , as (34) isolates belonging to the genus of Mycoplasma of a total of (35) isolates from the nasal swabs (97.1%), and (22) isolates were dependent on the type of Mycoplasma myasthenia. Pulmonary rate (64.7%).

b-Tracheal swabs.

Bacterial isolates from tracheal smears were (30) isolates of which belong to the genus Mycoplasma of a total of (33) bacterial isolates, i.e. (90.9%), and from these isolates (19) isolates were dependent on the type of Mycoplasma lung at a rate of

( )%63.3 .

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c-Lung swabs

All bacterial isolates (21) were isolated from infected lung samples of the genus Mycoplasma , i.e. (100%). Of these isolates, (11) were isolated from the Mycoplasma lung type, at a rate of.)%52.3(

Table (3) shows the results of the PCR interactions test using the different prefixes that were applied to the obtained isolates by the implant method. The emergence of positive results in the PCR test for the proven samples is positive with the implant method, evidence of confirmation and support for the positive of the implant results.

The molecular weight of the samples using the initiator of the genus Mycoplasma reached (500) base pairs (Figure 15), while the packages gave a molecular weight of (600) base pairs.Using the initiator of Mycoplasma ovipneumoniae (3 and 4).

Figure (3): Results of DNA replication using Mycoplasma genus initiator for selected and carried samples on agarose gel at a concentration of 2%.

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M: Marker volumetric index (100 - 2000) base pair; Lanes (1-3) and (5-9): The samples that gave positive results at the molecular weight 500 represent; Lanes (4):

Represents samples that gave negative results; C + ve: represents positive control

sample; .C –ve: represents the negative control samples.

Figure (4): Results of DNA replication of the selected samples using the initiator of the type Lung Mycoplasma at the molecular weight of 600 base pairs and the stage on the agarose gel at a concentration of 2%.

M: the volumetric standard marker.)1000 - 100(

Lanes (1-5): Represents samples that gave a positive result for the type of lung Mycoplasma

C + ve: positive control sample.

C –ve: negative control sample.

Table (3): Results of sequential polymerase reaction tests using different prefixes that were applied to isolates after they were obtained by culture method

Percentage Positive

samples for M.

ovipneumoniae Percentage

Positive isolates for Mycoplasma No. of

positive culture Sample

type

64.7 22

97.1 34

35 Nasal

swab

63.3 19

90.9 30

33 Tracheal

swab

600 500 400 300

200

100

M +ve 1 2 3 4 5 - ve

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52.3 11

100 21

lung 21

61.2 52

95.5 85

Total 89

A serial polymerase reaction test was conducted on (30) pulmonary samples, of which (21) were positive for Mycoplasma isolation and (9) samples were randomly selected from a total of (39) negative pulmonary samples for Mycoplasma transplantation. The results showed that (16) pulmonary samples were positive for the Mycoplasma genus. Of the total of (21) lung samples, positive for bacterial isolation, at a rate of (76.2%), while negative samples for bacterial culture (9) samples, one sample waspositive for the genus Mycoplasma, at a rate of11.1)%).

As for testing to determine the type of Mycoplasma from the lungs directly, it was (13) pulmonary specimens belonging to the type of lung Mycoplasma from the total positive samples for the genus of Mycoplasma by PCR technology at a rate of (81.3%). (Table 4).

Table (4): Results of the chain polymerase reaction technique using the different prefixes that were applied to the lungs from which DNA was directly extracted.

Percentage Positive samples

for

M.ovipneumoniae Percentage

Positive isolates for Mycoplasma No. of

samples Samples

Type

81.3 13

76.2 16

21 Positive

Mycoplasma by culture

method

100.0 1

11.1 1

9 Negative

Mycoplasma by culture

method

82.3 14

56.6 17

Total 30

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Comparing the efficiency of the diagnostic methods used for the various samples The diagnostic ratios with the dependence of the bacterial culture and the chain reaction polymerase technique were similar at the probability level (P≤ .05) and through statistical analysis with the approval of the Z test for the marginal characteristics (Table 5).

Table (5): Comparing diagnostic methods with the approval of different types of samples

Percentage of positive sample

Test technique No. of

sample Sample type

No.

a58.0 culture

60 Nasal swab

1 Tracheal swab 60 culture a55.3

b35.0 culture

60 lung

a97.1 PCR

35 Nasal swab isolates

2

a90.9 PCR

33 Tracheal swab

isolates

a100.0 PCR

21 Lung isolates

a35.0 culture

60 Pneumonia samples

6 Pneumonia samples 30 PCR 56.6 b

*

-The percentages of independent comparisons that have different characters, differ statistically at the level (P≤ .05).

- Samples marked with an asterisk (*) represent samples to which PCR was applied immediately after DNA was extracted.

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Discussion

This study examined the isolation and diagnosis of Mycoplasma from pneumonia in feedlot calves in the city of Mosul, because this bacterium is of great importance in causing respiratory diseases in calves(7), which in turn leads to economic damage represented by declines, poor growth and low economic returns.

Therefore, various types of samples, such as nasal swabs, endotracheal swabs and infected lung samples, were used to isolate this bacterium, to find the best possible way to use it in transplanting, developing, and isolating Mycoplasma.

The DNA was extracted from the isolates obtained from the transplantation method, and extracted from the samples of the infected lungs, depending on the method (9) (12). And obtaining an appropriate amount of genomics DNA for use in a polymerase chain reaction (PCR). Since the amount of DNA used as a DNA template in PCR reactions is 50 ng of extracted DNA, so the concentration of the extract:

Polymerase chain reaction generally does not require this amount of DNA produced.It requires only a small amount or a single strip of DNA to be included in the reactions.(13). The low rate of DNA extracted directly from the tissue of the affected lungs may be due to the possibility of substances inhibiting the technique of chain polymerase reaction.(12).

The results of the test technique of chain polymerase reaction on isolates obtained from the implant method gave evidence of the presence of the genus Mycoplasma in these samples at a rate of (97.1, 90.9, 100%) for nasal and tracheal isolates and lungs, respectively, while the initiator for the Mycoplasma myogenic type was given. Pulmonary results were positive (64.7, 63.3, 52.3) for the mentioned samples, respectively. The presence of DNA in the samples that gave a positive result by the chain reaction polymerization technique and gave a negative result in transplantation of Mycoplasma is believed to be due to the presence of contamination with other bacteria or because of giving antibiotics before slaughter.(12).

The results showed the emergence of one package for positive samples to test the polymerase chain reaction, as this package represents the location of the DNA of the tested sample with the specialized initiator elected to isolate the Mycoplasma represented by the number of bases installed in the source followed, and from the presence of samples packages with the volumetric guide (marker) the molecular weight of this is calculated Firmness with comparison to negative control samples that

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did not show any duplication, and the failure of the beam to appear in the sample after replication indicates that it is a negative result (that is, free from Mycoplasma).

Depending on the results of the PCR test, lung Mycoplasma was isolated in this study from the highest nasal isolates (64.7%), then from endotracheal isolates and by (63.3%) and finally from the lung isolates and by.)%52.3(

The isolation ratios diagnosed with the polymerase chain reaction technique from the nasal smears in this study came somewhat less than what was mentioned by several previous studies, including the study conducted by the researcher (Alley et al., 2002)(14) on (63) animals suffering from pneumonia signs and the isolation rate for a species Mycoplasma lung Mycoplasma (79%) and the study conducted by the researcher (Gilbert, 2002) (15) in which the total isolation rate (77.02%) for the genus of Mycoplasma was the percentage of Mycoplasma lung myopathy of the total number (83%) and (35%) for the type Mycoplasmaarginini,While it was differingfrom the percentages mentioned in other studies, which were less than the percentage mentioned in our study, where the researcher recorded in the United States an isolation rate (40%) for the genus of Mycoplasma and was (55%) for the type of Mycoplasma lung, and the isolation rate in calves that were suffering from clinical signs For the same lung in a study (13.3%) for the genus of Mycoplasma, (47.77%) for the Mycoplasma lung type, and (13.5%) for the type Mycoplasmaarginini (1.9%) were related to other types of Mycoplasma.(16). As for the isolation ratios from tracheal smears, it was very close to the isolation ratios from nasal smears (63.3%), because both types of smears were from the same animal, and this indicates that Mycoplasma infection starts from the nasal cavity and extends towards the lower respiratory canal.and therefore, we conclude from these results the possibility of relying on nasal swabs in the diagnosis of Mycoplasma as an alternative to endotracheal swabs that are difficult to collect due to animal resistance when taking the swab. Previous studies did not indicate the isolation of Mycoplasma from endotracheal swabs due to the difficulty in obtaining these swabs as we previously explained, so we were unable to compare the isolation rates from these swabs with previous studies except in one study conducted on goats in Norway by the researcher (Lenfant et al., 2008)(17) ,as the isolation rate reached (20%) in the transplantation method, and the percentage of examination by direct chain polymerization reaction technique on the samples

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(18.02%) for the genus Mycoplasma genus, of which (65.5%) was for the type of Mycoplasma lung atrophy.

In this study, Mycoplasma was detected and diagnosed from samples of infected lungs of different degrees of pneumonia after DNA was directly extracted from them, and the number of these samples was (30) pulmonary samples, of which (21) samples gave positive results for the isolation of Mycoplasma and (9) samples were randomly selected Of the total of (39) pulmonary samples that gave a negative result for Mycoplasma transplantation, the results showed that the total of samples that gave a positive result for the Mycoplasma genus by PCR test were (17) samples of them and at a rate of (56.6%), of which (14) samples were positive for Mycoplasmaovipneumoniae and by a ratio )%82.4( .

When comparing the results of the serial polymerase reaction test on positive and negative samples for transplantation, (16) samples gave a positive result for Mycoplasma from a total of (21) positive lung samples for transplantation, and (13) were positive samples for the M.ovipneumoniae type (81.3%) and (76.2%) ), While one sample was positive for Mycoplasma from the negative samples for transplantation, which number (9) samples, at the rate of (11.1%), which was the same as that of M. ovipneumoniae .

The technique of serial polymerase interaction that was conducted directly on the pulmonary specimens was of high sensitivity, and this can be inferred from the diagnosis of M. ovipneumoniae isolation from the negative samples with bacterial implantation, and this is consistent with what many researchers suggested using this test directly on the samples to detect Mycoplasma infection As a result of the specificity and high sensitivity of this examination, the researcher (Walker et al., 1991)(18)indicated a diagnosis of Mycoplasma from infected lung samples with a high rate of (83.01%) for the genus of Mycoplasma and (68%) for the type M.

ovipneumoniae .

The researcher (Vasconcelos, 2004)(19)pointed to the diagnostic rate of lymphocytic Mycoplasma from 92 infected calves lung samples by applying the polymerase chain reaction directly to them and the ratio was (73.02%) for the type M.

ovipneumoniae . Some previous studies mentioned average diagnostic ratios for Mycoplasma from the lungs, as in a study conducted by researchers in India, they recorded (46.2%) for the genus of Mycoplasma and (61.1%) for the M.

(13)

ovipneumoniae .(20). While a single study indicated a low diagnostic ratio of (16.6%) of the total lungs examined, the researcher attributed this to the possibility of inhibiting substances for the technique of chain polymerase interaction.(12).

By comparing the percentages of positive results for Mycoplasma infection according to the type of sample used, nasal swabs were recorded as the most efficient types of samples in giving positive results and in both test methods (implantation and PCR), as (35) positive results were recorded out of a total of (60) samples with a ratio (58%) by implantation, while tracheal swabs showed a positive result (55.3%) for the same animal. While the samples of infected lungs recorded the lowest percentage of positive results, as (21) positive samples were given in the transplantation method and (21) positive samples were received by PCR method and by a percentage ,% )100 ,35( respectively, for implant screening and PCR on isolates.

The researchers ’recommendations regarding electing the sample type have varied, as the researcher mentioned when using the PCR test to compare the appropriate sample type, which included infected lung samples, nasal swabs and some body fluids, as it was found that the best were nasal smears, where they can be obtained easily from an Infected animal. This is also consistent with the researcher's findings (Noubit et al. 2007)(21). The researcher also indicated by (Nicholas, 1998)(22) that the adoption of this technique increases the sensitivity and speed of detection of this bacterium from nasal swabs, thus reducing the time required to use appropriate treatment. The researcher (Nicholas, 2002)(23) added that the selection of methods for detecting Mycoplasma is responsible for the results that differ between the different studies. While (Ongor et al., 2011) (24)recommended the approval of nasal swabs in the isolation of Mycoplasma and this is identical to what we obtained from the results of our current study.

We conclude from our study that samples and swabs that were positive for the genus of Mycoplasma by PCR test and were negative for the M. ovipneumoniae type indicate the presence of other types of the genus of Mycoplasma which may have been probably M. arginini or other types of Mycoplasma spores that cause damage to the respiratory system in its pathology.(12).

Conflict of interests

The authors declare no conflict of interest.

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Vet. Microbiol. 2005; 111: 159–169.

11- McAuliffe, L., Ellis R.J., Ayling R.D. and Nicholas R.A.J. Differentiation of MYCOPLASMA species by 165 Ribosomal DNA PCR and denaturing gradient gelelectro-phoresis finger printing. J. of Clin. Microbiol. 2003; 41:4844-4847.

12- Kilic, A., Kalender, H., eroksuz, H., Muz, A., and Tasdemir, B. Identification by culture, PCR and immunohistochemistry of Mycoplasma s and their molecular typing in calves and lamb lungs with pneumonia in Eastern Turkey. Trop. Anim.

Health Prod. 2013; 45: 1525-1531.

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1st ed. Springer. Dordrecht, Netherlands.2005; PP: 178-187.

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19- Vasconcelos, J.R.C. Detection of Mycoplasmaovipneumoniae in pneumonic lungs from infected calves. Aust. J. Eexp. Biol. Med. Sci.2004; 42: 373-384.

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and Thiaucourt F. A PCR for the detection of MYCOPLASMA s belonging to the Mycoplasma mycoides cluster: Application to the diagnosis of contagious agalactia. Science Direct. Molecular and Cellular Probes. 2007; 21: 391-399.

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