An Investigation of Contagious Agalactia Disease of Sheep and Goats in Isparta and Afyonkarahisar in Turkey

Mycoplasma have a very wide variety of hosts, namely small ruminants (Marenda et al., 2005; Manimaran and Singh, 2018), cattle (Goswami et al., 2019; Waseem et al., 2020) Poultry (Manimaran and Singh, 2018; Rajkumar et al., 2019).
 
Contagious agalactia occurs in many parts of the world, especially in the Mediterranean and in Turkey (Amores et al., 2011; Birben, 2020; Ozturkler and Otlu, 2020) Basin.
       
It is on the list of mandatorily notifiable diseases of the International Office of Epizooties (OIE). Contagious agalactia causes high morbidity in sheep and goat breeding and serious economic losses with the treatment costs of the disease (OIE Terrestrial Manuel, 2018; Tardy et al., 2012).
       
Mycoplasma agalactiae (Ma), Mycoplasma capricolum subsp. Capricolum (Mcc), Mycoplasma mycoides subsp. capri (Mmc) and Mycoplasma putrefaciens (Mp) which are strains of contagious agalactia have been described several times in cattle as symptomatic or asymptomatic (Amores et al., 2011; Gomez-Martin et al., 2013; Heller et al., 2015).
       
The most prominent etiological agent of contagious agalactia disease is M. agalactiae. Clinical manifestations of contagious agalactia are arthritis, mastitis, keratoconjunctivitis, sporadic or epidemic abortions, emaciation, respiratory system symptoms ranging from cough to dyspnea (Gomez-Martin et al., 2013). With the birth of lambs in the spring, the sudden spread of the disease in lactating ewes has been shown and newborns have been reported to be infected with infected colostrum or milk (OIE Terrestrial Manuel, 2018). Epidemiologically, early diagnosis of the disease is important (Pooladgar et al., 2015; Razin et al., 1998). Using immunomagnetic PCR methods to detect M. agalactiae in milk samples gives faster results than using the culture enrichment procedure (Cillara et al., 2015).
       
A multiplex PCR assay capable of simultaneously detecting M. agalactiae, Mcc and Mmc has been described. In addition, a PCR based on the lpdA gene and a restriction fragment length polymorphism PCR (RFLP-PCR) method has been described to distinguish between Mmc and Mcc (Gil et al., 2003).
       
The aim of this study was to investigate the presence of the disease for the first time by using both cultural and molecular methods on the field strains that cause contagious agalatiae in Isparta and Afyonkarahisar provinces in Turkey.

Animals and samples
 
For this investigation, 45,500 goats and sheep were scanned from 220 ovine farms in the Isparta and Afyonkarahisar regions. Clinical and bacteriological examinations were performed on a total of 202 animals, 144 goats and 58 sheep, from 21 small ruminant farms with mastitis, keratoconjunctivitis, limping, swelling/arthritis in the joints, abortus, runny nose and pneumonia symptoms for the isolation and identification of Mycoplasma species. This study was carried out during 2018-2019.
       
A total of 289 samples were taken, including 91 milk samples (68 goats and 23 sheep), 101 eye swabs (47 goats and 54 sheep), 61 ear swabs (59 goats and 2 sheep), 28 nose swabs (13 goats and 15 sheep) and 8 joint fluid samples (8 sheep).
 
Bacterial isolation and identification of Mycoplasma spp.
 
Eye, nose and ear swabs taken from animals with suspected disease were collected in Hayflik’s transport medium, joint fluid was drawn into a sterile syringe and milk samples were filled into sterile tubes to reach the laboratory in a cold chain as soon as possible. 10^-1 dilutions of the suspicious samples were prepared in a liquid medium and/or cultivated directly into the Mycoplasma agar. Petri dishes were incubated at 37^oC in a 5-10% CO₂ setup. Solid media were examined for growth with a stereo microscope (X5-50 magnification) every 2-3 days. Negative petri dishes were destroyed after 14 days of incubation. The morphology of observed colonies was examined, the part containing the colony was cut as a block and transferred into a liquid medium. Isolates were identified by using biochemical tests (film and spot formation, glucose fermentation test) and the growth inhibition test (Poveda, 1998).
 
PCR
 
This test was carried out at the Ministry of Agriculture and Forestry of the Republic of Turkey, Istanbul Pendik Veterinary Control Institute Mycoplasma Reference Diagnostic Laboratory. M. agalactiae PCR was performed for 3 isolates obtained using the primer sequences indicated in Table 1.
 

At the end of the incubation period, a total of 289 samples were examined under a stereo microscope for typical ‘fried egg’ colony formation. Mycoplasma spp. was determined in only 3 (1.03) out of 289 samples. Mycoplasma spp. could not be isolated from any samples other than 3 goat nasal swabs (Table 2).
 

       
The results of the biochemical tests revealed that film and spot formation was negative and the glucose fermentation test indicated that two colonies gave positive results and one colony gave negative results. Colony morphologies were examined and as a result of biochemical tests and growth inhibition tests, Mycoplasma spp. 2 of the 3 (1.03%) isolates evaluated as M. ovipneumoniae (0.67%) and 1 isolate were identified as M. arginine (0.36%). In addition, M. agalactiae PCR was applied to 3 isolated samples and the isolates were found negative for agalactiae (Table 3).
 

       
Valsala et al., (2017) examined a total of 244 goat lung tissue samples with pneumonia collected for 10 years (2002-2013) and according to the results of biochemical tests, M. arginine (13/244, 5.3%) was the most common isolate. In the phylogeny analysis performed in the same study, they reported that the sequences of all these M. arginine isolates were >98% identical compared to the standard M. arginine (ATCC 23243) strains and that M. agalactiae was isolated in only one sample.
       
In a thesis study, Göçmen (2014) investigated the existence of Mycoplasma spp. in sheep and goats in the Marmara Region of Turkey for which  a total of 339 samples, including 162 milk samples, 147 eye swabs, 15 joint fluid samples, 11 nasal swabs and 4 lung tissue samples were examined by bacteriological and molecular methods. As a result of the biochemical tests and growth inhibition tests applied in the bacteriological examination, Mycoplasma spp. was identified in 25 of 29 (8.5%) isolates evaluated as M. agalactiae (86.20%). She reported that only 1 isolate each was identified from the M. ovipneumoniae and M. arginine (6.89%) cases according to the biochemical test results.
       
Ozturkler and Otlu (2020) conducted a study in which Mycoplasma spp. were investigated by cultural and molecular methods in the lung tissue of 250 sheep with pneumonia and 30 healthy sheep purchased from slaughterhouses and butchers in Kars province of Turkey. Mycoplasma was isolated in only 26 (10.4%) of 250 sheep lung tissues with pneumonia. They reported that M. ovipneumoniae isolated from Mycoplasma spp.’s was encountered the most with 12 (46.15%) samples, followed by M. arginine with 4 (15.38%) samples.
       
In Egypt, Halium et al., (2019) carried out a study for Mycoplasma spp. in sheep and goats. They examined a total of 335 samples, including 142 nasal swabs, 167 lung tissues with pneumonia, 18 tracheal bifurcations and 8 bronchial fluids. They reported that a total of 24 Mycoplasma were isolated and they confirmed by PCR that 10 of these 24 isolates were M. arginine and 4 were M. ovipneumoniae.
       
Zhao et al., (2021) isolated M. ovipneumoniae in 4 out of 6 lung tissues in a study they conducted on 6 sheep lungs and a total of 824 nasal swab samples from animals that had died from pneumonia in 4 different regions in China between 2018 and 2020. They confirmed by PCR that 336 (40.78%) of 824 nasal swab specimens tested in the same study were M. ovipneumoniae. They also reported that they detected M. arginine in this study and that M. arginine is usually found together with M. ovipneumoniae, but it is not pathogenic. Similarly, in our study, M. arginine was identified in 1 sample while M. ovipneumoniae was identified in 2 samples.

Literature reviews indicate that the disease still continues to be seen in our country. The isolation of M. ovipneumoniae and M. arginine from animals with clinically suspected contagious agalaxia made us think that the effects of these factors on the disease should be investigated. Controlling animal movements, implementing proper milking hygiene, prioritizing the hygiene of milking tools and equipment as well as the hygiene of the operation, checking the herd with serological methods at regular intervals, removing sick or suspected animals from the herd, implementing planned vaccination programs and parasite control can be counted among measures that can be taken against contagious agalaxia. In addition to M. agalactiae causing the disease, the incidence of M. ovipneumoniae and M. arginine agents in our country should be investigated and these factors should be taken into consideration in vaccine development studies. It has been concluded that it would be beneficial to include the disease in the list of notifiable diseases to determine the true prevalence of the disease.

This study was summarized in the Masters’ Thesis titled “Investigation of the Presence of Contagious Agalaxia Disease in Sheep and Goats in the Region of Isparta and Afyonkarahisar” and was supported by the Afyon Kocatepe University Scientific Research Projects Coordination Unit (Project no: 2019-053).
     
This study was produced from “Investigation of the Presence of Contagious Agalaxia Disease in Sheep and Goats in the Region of Isparta and Afyonkarahisar” master’s thesis and was presented in XIV. National Veterinary Microbiology Congress (With International Participation) Konya, Turkey, 13-16 October 2020.
       
Molecular studies of this research were carried out in the Ministry of Agriculture and Forestry, Pendik Institute of Veterinary Control, Mycoplasma Reference Laboratory.

The necessary permission for milk, joint fluid, eye, ear and nose swab samples taken from animals during this study was approved by Afyon Kocatepe University Animal Experiments Local Ethics Committee (HADYEK) with the decision dated 14/02/2018 and numbered 49533702/01.