Determination of Contagious Agalactia in Sheep and Goats and Investigation of Antibiotic Susceptibility of Mycoplasma spp. Positive Isolates

Contagious agalactia (CA), which has been recognized for nearly 200 years, is one of the primary diseases affecting sheep and goats. It is characterized by arthritis, mastitis and keratoconjunctivitis. The high morbidity and mortality rates associated with this disease lead to significant economic losses in livestock farming and it is there fore included in List B of the diseases notifiable to the OIE (World Organisation for Animal Health) (OIE, 2004; Önat et al., 2011). The causative agent of CA is Mycoplasma agalactiae (Ma) (Bergonier et al., 1997; Bidhendi et al., 2011; Khezri et al., 2012).
               
However, M. capricolum subsp. capricolum (Mcc), M. mycoides subsp. Capri (Mmc) and M. putrefaciens also produce clinical symptoms similar to CA (Corrales et al., 2007; Madanat et al., 2001). Mycoplasma causes a number of important diasease viz., contagious caprine pleura pneumonia, contagious bovine pleura pneumonia, chronic respiratory disease, contagious agalactia, arthritis and mastitis in poultry and livestock (Manimaran and Singh, 2018). Globally, it has been reported in 31 countries on four continents since 1996, with the Oceanian Islands being a notable exception, although earlier occurrences were detected in Australia and New Zealand (Bergonier et al., 1997). Disease occurrence in herds is almost always associated with the entry of CA carriers into a flock, or contact with infected herds (Corrales et al., 2007). These asymptomatic carriers canbe present in both sick and healthy herds and play an important role in disease transmission. Recent studies have suggested there are a large number of Mycoplasma carriers in herds (Mercier et al., 2007; Tardy et al., 2007); Ma and Mmc have also been detected in the outer ear canal in chronically infected carriers. This indicates a need for diagnostic methods that are effective in identifying all carriers in a herd. Mastitis usually affects lactating females; arthritis, keratoconjunctivitis and respiratory problems are foundin non-lactating females, young animals and males. Methods used to investigate Mycoplasma antigenic structures or identify Mycoplasma infected animals include indirect hemagglutination testing, complement fixation, agar gel immunodiffusion and enzyme-linked immunosorbent assay (ELISA). Although serological testing allows a large number of animals to be assessed simultaneously, a detectable increase in antibodies occurs approximately two weeks after infection (Önat et al., 2011). However, the sensitivity and specificity of serological tests are adversely influenced because of effects on species specific antigens caused by variations in Mycoplasma antigenic properties (Razin et al., 1998).

Animals
 
The study material consisted of ear swab and blood serum samples obtained from healthy (clinically asymptomatic) and sick animals, the latter of which showed one or more symptoms of CA. Samples were taken from five sheep and five goat herds located in Elazýð and Malatya provinces in eastern Turkey. Thirty animals were sampled per herd, comprising a total of 150 sheep (90 healthy and 60 sick) and 150 goats (60 healthy and 90 sick). Samples were collected in April, May and June, when the disease is most prevalent. All samples were taken to the laboratory, where ELISA was used to determine any presence of Ma in ear swabs, Mycoplasma spp. cultures and blood serum samples. This study was carried out in Elazýð Veterinary Control Institute Microbiology Department between 2015-2016.
 
Isolation of Mycoplasma spp.
 
The samples were examined according to the method reported by Thiaucourt et al., (1992). Mycoplasmas were isolated using a modified Hayflick broth [pleuropneumonialike organism (PPLO) broth, 21 g/L], 20% inactive horse serum, 10% fresh yeast extract, 0.2% glucose and 0.4% sodium pyruvate, in both a liquid (crystal violet-free, 0.04% ampicillin) dilution and solid broth (PPLO agar) medium: both were prepared simultaneously. The solid medium was prepared by adding 1% noble agar (Difco; Detroit, MI, USA) rather than liquid medium. Samples were added to five tubes (ten-fold dilution: 10^-1, 10^-2, 10^-3, 10^-4, 10^-5) and incubated at 37°C in 5% CO₂ for 7-10 d. Fluid media were inspected daily for turbidity: mycoplasmas produce a homogenous and slight turbidity in broth. Reproduction was repeated from the last tube in which solid growth was observed. Contamination was indicated by intense turbidity in the broths, so 1 mL of contaminated broth was removed and filtered through a 0.45 µm syringe filter. Solid media were also inspected daily for Mycoplasma colony formation, using microscopic examination of the agar to determine if there was a sufficiently enlarged typical Mycoplasma colony. Agars with no growth were incubated for 14 d before discarding. Where growth on the agar was observed, an agar fragment containing a single colony was cut with the help of an inoculation loop. The agar block was then inverted onto the agar plate and incubated by carefully moving it across the agar surface. This procedure was repeated three times. After the last incubation stage, the growing colonies were examined using a small stereo microscope (´40). Detection of colonies of Mycoplasma subtypes, which were usually round with a fried-egg shape with a central button, was evaluated as a positive result (Fig 1). Isolates were identified using biochemical tests (digitonin sensitivity, film and spot formation). Digitonin sensitivity was the first test performed on clone disolates, to separate mycoplasmas from acholeplasmas. The final test detected the presence of ubiquitous pollutants that may exceed the quantity of mycoplasmas of interest (OIE, 2008). Colonies isolated from the media were inoculated into stock media and stored at -70°C.


 
Antibiogram tests
 
Mycoplasma inocula were prepared for minimum inhibitory concentration (MIC) testing according to the method of Hannan (2000). An amount of 1 mL of frozen inoculum (- 70°C) was extracted, to which 4 mL of mycoplasma broth was added; the pH was adjusted to 7.6. The diluted cultures were incubated for 2 h at 36°C until a certain acid and alkali reaction was observed. Then, 1 mL of mycoplasma culture was added to 9 mL of mycoplasma broth and vortexed for five seconds. Ten-fold dilutions (in 0.9 mL volumes) of the vortexed suspension (from 10^-2 to 10^-9) were prepared. A total of 0.1 mL of sterile mycoplasma broth was added horizontally from well 1 to well 8 of a 96-well U-bottom microdilution plate. A total of 0.2 mL sterile mycoplasma broth was added to well 12 (sterility control). Then, 0.1 mL of each mycoplasma dilution was added, from well 8 to well 1. The microdilution plate was covered with adhesive film and incubated at 36±1°C until color changes were completed. A dilution of 10^-5 was the lowest to show a color change, thus indicating that 0.1 mL of the undiluted culture contained 10⁵ cfu (10⁵ cfu/mL^-1).
       
Isolated identified bacteria were subjected to antibiotic susceptibility tests according to the method of Schultz et al., (2012). The material used in the antibiogram testing comprised positive isolates obtained from sheep and goat ear swab cultures. Firstly, 1.5 mL of each mycoplasma inocula in stock media and 13.5 mL of sterile PPLO broth [pH 6.8; containing 0.1% phenol red (pH color indicator) and 0.5% arginine] were diluted. Then, diluted broth was distributed to each well (100 µL per well) in 96-well microtiter plates (Sensititre®BOPO-6F, Thermo Fisher Scientific, Waltham, MA, USA) which contained freeze-dried antibiotics in their base. The plates were sealed tightly using a transparent film and incubated at 35±1°C for 4-5 d to prevent humidification. The plates were checked daily and all color changes were recorded (Fig 2).


 
Serological tests
 
Blood samples from all 300 animals used in the study were centrifuged at 3000 rpm for 10 min to determine the presence of Ma antibodies. The serum samples obtained were processed according to the specific Ma procedure indicated for the ELISA kit (IDEXX ELISA kit), according to which the wash solution (20x) was diluted with 1:20 distilled water before use. The conjugate was diluted (1:100) in dilution buffer N1. All reagents were allowed to stand at 18-26°C before use. The microplate location for each sample was determined. Dilution buffer N4 (190 µL) was dispensed into each well. Then, 10 µL of undiluted ready-to-use negative control was placed in one well, 10 µL of ready-to-use positive control in two wells and 10 µL of undiluted samples into each of the remaining wells. A microplate mixer was used to homogenize the well contents. After covering the microplates with aluminum foil and incubating at 37°C for 1 h±5 min, each well was washed three times with washing solution (300 µL) and the liquid content in all wells was then aspirated. Diluted conjugate (100 µL) was dispensed into each well and covered with microplates using aluminum foil and incubated for 30 min at 37°C (±3°C). After incubation, each well was washed three times with washing solution (300 µL). A total of 100 µL of tetramethylbenzidine (TMB) substrate was added to each well and incubated for 20 min at 18-26°C in a dark environment. Finally, 100 µL of stop solution was dispensed into each well. The microplate samples were mixed by hand using slight rotating wrist movements and the optical densities of the samples and controls were measured in the microplate reader at a wavelength of 450 nm and evaluated.

In recent years, in almost all countries globally, including Turkey, CA causes serious problems in sheep and goat herds, with symptoms including contagious pleuropneumonia, mastitis and arthritis. Mycoplasmosis is a respiratory tract infection of small ruminants which causes high morbidity and mortality levels and is therefore an established causeof severe economic losses in sheep and goat husbandry (Shaha et al., 2017); studies are currently being carried out in Turkey in order to mitigate these problems. In the present study, following inoculation of the ear swabs obtained for bacteriological examination, the suspected colonies were observed at the end of the incubation period to determine their morphological and other characteristics; a total of 87 (29%) of the 300 samples were evaluated as positive for Myoplasma spp. (Table 1). Generally, Mycoplasma colonies were observed under a stereoscopic microscope (x40) as being small, with a typical fried-egg shape, central button and rounded edges (Fig 1). Of the 87 positive culture samples, 33 (11%) were derived from sheep and 54 (18%) from goat ear swabs. The bacteriological examination showed that, of the 150 ear swab samples from each of Elaz1 and Malatya provinces, 7 (4.66%) and 80 (53.33%), respectively, were Mycoplasma spp. positive (Table 1).


       
Önat et al., (2011) used bacteriological and serological tests to study a goat herd displaying CA symptoms in Yeniþehir, Bursa and evaluated agent scattering after tylosin treatment. Only one of 10 tylosin-treated goats was found to be actively scattering agent with its milk and so the study concluded that tylosin treatment reduced post-lactation Ma scattering in milk.
       
The material used inthe antibiogram test comprised 87 positive isolates obtained from cultures derived from sheep and goat ear swabs. Color change was used to determine MIC values. Antibiotic susceptibility testing revealed that the most effective antibiotics were tulathromycin (which is macrolide-derived) and tiamulin (pleuromutilin-derived), while the least effective was neomycin. Table 2 shows the MIC values of Mycoplasma spp. isolates. In addition, it was determined that all isolates were resistant to all concentrations of penicillin/ampicillin and trimethoprim/sulfamethoxazole (a sulfonamide derivative).


       
Schultz et al., (2012) examined the susceptibility of M. hyosynoviae-positive isolates to 18 antibiotic agents and used an antibiogram test to show that clindamycin, a lincosamide-derived antibiotic, had the highest activity; the macrolide-derived antibiotics tylosin, tilmicosin and tulathromycin have been reported to have similar effects. The same authors also found that all the isolates were resistant to penicillin and penicillin-derived antibiotics, such as ampicillin and to ceftiofur, trimethoprim/sulfamethoxazole and sulfadimethoxine.
       
In the present study, the antibiotic sensibility tests determined that the two most powerful antibiotics were tulathromycin and tiamulin and the least powerful was trimethoprim/sulfamethoxazole. In a study carried out by Shaha et al., (2017), a total of 54 species-specific polymerase chain reaction (PCR) approved isolates were subjected to antibiogram assays. Disc diffusion and broth microdilution was used to test five diffuse antimicrobial agents (ceftiofur, enrofloxacin, gentamicin, oxytetracycline and tylosin). All the isolates were found to be resistant to tylocin, oxytetracycline and ceftiofur sodium antibiotics, which are favored by clinicians in treating contagious caprine pleuropneumonia.               
 
The agent cannot be detected during the incubation period in serological diagnosis of mycoplasma infections. However, diagnosis becomes possible at 10-14 d after clinical symptoms appear, due to an increased antibody titer. In the present study, seropositivity was observed in 10 of 300 blood serum samples, suggesting that the disease was in the peracute period with no detectable antibody response. Roy et al., (2010), using slide agglutination tests with Mmc colored antigens, reported seropositivity in 85 of 200 (42.5%) blood serum samples obtained from goats of different age and sex in the Anand, Navsari and Valsad districts of Gujarat. Other reports suggest that control of the disease requires regular screening tests and therapeutic and prophylactic measures. ELISA techniques are generally recommended for detecting antibodies because they enable a large number of animals to be analyzed at the same time. However, systematic vaccination of animals in areas where the disease is endemic prevents detection of infected herds, as it is not possible to differentiate between vaccine- and nonvaccine-derived antibodies (Corrales et al., 2007); for this reason, culture and PCR techniques should be used. Important problems in diagnosis arise from the particularly difficult and slow growth of mycoplasmas in culture, the inability to diagnose disease until 10-14 d after serological testing and the occurrence of false positives (Göçmen et al., 2015). In another study, the molecular mechanisms behind the resistance of Ma to macrolides and lincomycine were investigated. Compared with previous research, the authors found that MIC results from the studied Ma isolates showed increased tylosin resistance and reported that changes in L2 ribosomal protein played a role in decreasing Ma sensitivity. It was also determined that these mutations can be used as molecular markers that provide an interpretive breakpoint of antimicrobial resistance in Ma (Prats-van der Ham et al., 2017). The absence of effective antibiotic treatments or vaccination programs against infections resulting from these immunogenic factors causes significant losses in the dairy and meat industry (Çetinkaya et al., 2006-2008).
       
CA is caused by different Mycoplasma species, depending on the geographical region; studies suggest Mycoplasma species are endemic to specific regions. This is supported by the serological results in the present study; the bacteriological methods detected 87 Mycoplasma spp.- positive isolates, while serological techniques detected 10 (3.33%) seropositive isolates in animal blood serum samples. These results suggest that either the disease is in a peracute period, with no detectable antibody response, or that it is caused by a Mycoplasma species other than Ma.

In this study, the presence of Mycoplasma spp. in sheep and goat herds were investigated using bacteriological methods; antiogram testing showed tulatromycin and tiamulin to be the most effective antibiotics. Therefore, based on the in vitro studies, tulatromycin and tiamulin were determined to be the most effective drugs for treating Mycoplasma spp. infections in Malatya and Elazýð provinces. Antimicrobial susceptibility in mycoplasmas, which are resistant to many antibiotics, is determined using antibiogram tests. In this study, it was concluded that antibiogram tests are able to determine the most effective and appropriate antibiotics for treatment. This prevents disease agents from gaining resistance to antibiotics through avoiding in appropriate antibiotic use.