Introduction

Mycoplasmas are in the class Mollicutes, are small prokaryotic cells have the ability to self-replication. They require enriched media for growth and although most Mycoplasmas are facultative anaerobes, some grow optimally in an atmosphere of 5-10% CO₂. Many species are important veterinary pathogens and settled the mucous membrane of respiratory, genital tract as well as red blood cells causing respiratory infection, mastitis, conjunctivitis, arthritis and infrequently abortion (1-4). The Dog (Canis lupus familiaris) is a domestic mammal of the family Canidae (order Carnivora). It is a subspecies of the gray wolf (Canis lupus) and is linked to foxes and jackals. The dog is one of the two most abundant and most popular domestic animals in the world (the cat is the another). For more than 12,000 years it has lived with humans as a hunting companion, protector, object of scorn or adoration, and friend (5-8). The first notarized occurrence of Mycoplasma in dogs was in 1934 (9). Dogs were sensitive to multiple Mycoplasma species, despite of not all Mycoplasma infection caused clinical disease (10,11). Mycoplasma in dogs is believed to form portion of the normal bacterial flora of the upper airways (12,13), but resent study assessing the microbiome of healthy dogs showed that Mycoplasma occurred in reducing frequently from the nasal cavity to the lower airways. This could indicate that colonization of the lower respiratory tract by Mycoplasma may result from the upper airways (14-19). Moreover, the task of Mycoplasma as an important etiological cause of canine infectious respiratory disease complex (CIRDC), recently isolated Mycoplasma cynos and Mycoplasma canis from CIRDC in dogs (20-25).

 The existing study aimed to isolate and identify Mycoplasma from the upper respiratory tract and conjunctival infections of household dogs using the Conventional cultural methods and describe its isolation rate.

Materials and methods

Ethical approve

The committee of scientific ethics gave the ethical approval to conduct this scientific work in the College of Veterinary Medicine according to the approval certificate numbered UM.VET.2022.018.

Samples

Three hundred swabs were taken from nasal 100, oropharyngeal 100, and conjunctiva 100 of both diseased that showed clinical signs of respiratory infections, and healthy household dogs of different breeds, ages and sex from many veterinary clinics in Mosul city during the period of 1/2/2022 to 15/6/2022. The swabs were placed in a Mycoplasma broth medium and aseptically carried immediately to the laboratory of Microbiology, College of Veterinary Medicine, University of Mosul.

Mycoplasma broth medium

3.7 gm of brain heart infusion broth suspended in 100 ml of distilled water, then boiled and autoclaved at 121ºC for 15min. The medium was cooled at 45ºC and added the prepared supplement to the medium (3,4). The yeast extract with a serum prepared as 0.5 gm of yeast extract powder suspended in 40 ml of distilled water, boiled, then cooled and accurate pH=8, and filtered. For prepared yeast extract-serum, added 20 ml of filtered bovine serum to 10 ml of filtered yeast extract in percentage as 2:1, which were added 100 ml of prepared medium. 3 ml of filtered penicillin solution which contain100,000 un/ml at pH=7.8 added to 100 ml of the prepared medium for inhibiting the growth of contaminated gram-positive bacteria. 3 ml of filtered sulfadiazine-trimethoprim solution was added to 100 ml of the prepared medium to inhibit the growth of contaminated Gram-negative bacteria. 1 ml of nystatin solution was added to 100 ml of the prepared medium to inhibit the growth of yeast. 1 ml of clotrimazole solution was added to 100 ml of the prepared medium to inhibit fungi.

Mycoplasma agar medium

4.9 gm of brain heart infusion agar suspended in 100 ml of distilled water, boiled and autoclaved at 121ºC for 15 min. then cooled at 45ºC and added the supplement as in Mycoplasma broth medium (3,4).

Culture

The swabs were placed in Mycoplasma broth medium and transported immediately to the laboratory of Microbiology, and incubated at 37ºC with 5% CO₂ for 4-14 days in a candle jar. When turbidity appeared, an inoculum of each broth was cultured on Mycoplasma agar medium and incubated at 37ºC with 5% CO₂ for 7-14 days in a candle jar. The culture was checked daily for the first 7 days and weekly thereafter.

Staining with modified dienes stain

The stock solution prepared from Methylene blue 2.5 gm, Azure II 1.25 gm, Maltose 10 gm, Na₂CO₃ 0.25 gm, distal water 100 ml.while the working solution prepared from diluting stock solution in distal water by adding three volumes of distal water to one volume of stock solution. For staining the colonies, submerged the agar dish surface which contain the colonies of Mycoplasma in 1ml of working solution of Dienes stain, washed with distal water then decolorized with 1ml of 95% of ethyl alcohol for 1min., removed the excess alcohol, washed with distal water, and examined the color of colonies of Mycoplasma (4).

Results

Colony morphology

The growing colonies had an umbonate appearance when illuminated obliquely and small, circle colonies with center surrounded by white to colorless halo identical to a fried egg appearance in transmitted light. According to the colonies s appearance, there were two types of colony morphology on agar plate, in the first type the colonies were transparent (Figure1a), while the other type the colonies appeared as opaque brown color (Figure 1b).

Figure 1: Two types of colony morphology.

Dienes stain

The results of staining using the modified Dienes stain on Mycoplasma colonies grown on mycoplasma agar, which showed light blue center circumscribed with dark blue halo (Figure 2).

Figure 2: a: staining petri dish b: staining colonies

Infection and isolation rates

Mycoplasma isolation rate from total swabs were 56.6%, while high rate indicated from nasal swabs 68%, followed by oropharyngeal swabs and conjunctival swabs 58 and 44% respectively. The results of infections indicated that respiratory infections were predominant in household dogs in comparison with conjunctival infections in all examined dogs. The young ages were more susceptible to both respiratory and conjunctival infections 58.3 and 16.7% respectively. According to the sex, upper respiratory and conjunctival infections appeared more frequently in males 66.6 and 20% respectively. The conjunctival infections were obvious only in Husky 16.7% and Belgium dogs 25% (Table 1).

Table 1: Respiratory and conjunctival infections in diseased household dogs

The results of Mycoplasma isolation rate indicated that the high isolation rate of Mycoplasma was from upper respiratory tract infections in diseased dogs more than one-year age 100%, while it was the opposite situation with conjunctival infections 66.6%, these according to age. But according to sex, the respiratory swabs from diseased female dogs were more perfect for Mycoplasma isolation than in males since all female swabs 100% were positive for Mycoplasma culturing, whereas the conjunctival infections were more dominant and higher isolation rate in male swabs 50%. According to the breed, the existing study revealed a high Mycoplasma isolation rate in respiratory swabs of Terrier 100% and German dogs 93.75%, while the conjunctival swabs were positive for Mycoplasma culture in Husky and Belgium dogs 100% for each one, also the healthy dogs indicated positive culture of Mycoplasma but in very low rate (Table 2).

Table 2: The isolation rate of Mycoplasma from diseased and healthy household dogs

Discussion

Dogs are sensitive to infection by several Mycoplasma species, and can be identified from the respiratory tract of healthy and diseased dogs despite not all Mycoplasma infections causing clinical disease (10,26-28). The growing colonies in the existing study were identical of Mycoplasma when grown on Mycoplasma agar, and revealed the fried egg appearance as reported in scientific references (1,3), which confirmed that the in vitro culture was the standard method for isolation and identification of Mycoplasma, however the slow growing of these bacteria make this method time-consuming even for particular laboratories (3,28,29).

Mycoplasma isolation rate from total swabs in our study 56.6% which was far away from data recorded by Randolph et al., (30) who recorded 27% and Barreto et al., (31) who recorded 29.96%. The differences may be attributed to the way of samples collection, conditions used for isolation, case history and samples collection period (3). In addition to many risk factors related to dogs themselves, such as multi-dog householding, interest in dog training, and doggy day attention (32). The higher isolation rate of Mycoplasma was from nasal swabs 68% followed by oropharyngeal swabs 58% and conjunctival swabs 44%, whereas different results were confirmed by previous studies Barreto et al. (31) and Schulz et al., (33) who indicated the highest isolation rate from oropharyngeal swabs 42.86%, and agreed with him in the lowest rate of isolation from conjunctival swabs 18.30%. The high isolation rate from oropharyngeal swabs supports that Mycoplasma are normal commensals of the upper respiratory tract and when stressful conditions occurred, the Mycoplasma convert to pathogens and the clinical respiratory cases appear (34,35), and what proves that is the low isolation rates from the same swab types of normal healthy dogs (31).The high upper respiratory tract infections in current study was recorded in the young (less than one -year aged) dogs 58.3%, While they occurred less frequently in the more-one year dogs and more frequent in diseased males 66.6% than diseased females 8.3% and mainly in Terrier and German dogs 100%, although the Mycoplasma isolation rate was higher from the swabs of the more-one year female dogs. These results were agreed with Chalker et al.,(36) who confirmed that there was a considerable relation between Mycoplasma cynos infection and young age et al.,(37) who recorded high isolation rate of Mycoplasma from upper respiratory tract infections in age near 1.5year, also they recorded high isolation rate of Mycoplasma from upper respiratory tract infections in female more than male, and in Toy dog breed more than another breed. in common, the likelihood of upper respiratory tract infections is improved in dogs that are Mycoplasma from conjunctival infections were recorded in diseased Husky and Belgium 100%, while other researchers recorded low isolation rate of Mycoplasma from conjunctival infections (31,38-40), may be due to these are more common breeding of these species recently to be used for a variety of purposes.

Conclusion

The results of our study indicated high isolation rate of Mycoplasma in diseased household dogs with upper respiratory tract and conjunctival infections by cultural method, and also recorded Mycoplasma with high isolation rate in diseased Terrier and German with upper respiratory tract infection, and in diseased Husky and Belgium with conjunctival infections.

Acknowledgments

The author would like to thank the dean of College Veterinary Medicine, the head of Department of Microbiology, University of Mosul.

Conflict of interest

The author declares that there is no conflict of interest regarding the publication of this manuscript.