The Extent Response of Bacteria Isolated from Cases of Acute, Subacute and Chronic Mastitis in Iraqi Camels to some Antibiotics used in Treatment

Our study was carried out on (75) dairy camels, aged (7-22) years, from Iraqi camels for each of the governorates (Babylon-Karbala-Al-Qadisiyah) with (25) camels from each governorate for the time period 1/6/2021 until 1/12/2022 to isolate and diagnosis the bacteria causing acute, subacute and chronic mastitis in camels, examining their sensitivity to common antibiotics used in treatment. The results of the study showed that the number of camels infected with mastitis for all types was (51) camels with a rate of (68%) out of a total of (75) milking camels, and the number of healthy camels was (24) with a rate of (32%). The number and percentage of subacute mastitis was (26) with a rate of (35%), acute mastitis (10) with a rate of (13%), and chronic mastitis (15) with a rate of (20%). As for the results of mastitis on the basis of inflammation tests for each quarter of the udder, the highest rate was in two quarters, where the percentage of infected camels was (35%), while the lowest percentage of infection was in four quarters or the whole udder with a rate of (18%). Results of bacteriological culture of milk samples of camels infected with mastitis, which numbered (306) samples. The highest percentage of isolates recorded was of C.N. Staphylococci by (35.62%), while the percentage of Staph.sciuri, Staphylococcus aureus and Staph.epidermidies respectively was (16.66%), (13) and (10.13%). The percentage of Streptococcus agalactia, Pasteurella haemolytica, Arcanobacterium pyogenes and E. coli respectively (10.45%), (6.2%), (5%) and (2.94%). The results of the sensitivity and antibiotic resistance examination, we show that there is statistical significant difference at the level of p ≥ 0.05 between the groups of the ages of camels.


Introduction
Camels have adapted to live in hot desert conditions that are characterized by a lack of water and food.They can live and endure thirst and not drink water for a period of (10) days [1,2]; However, they have the ability to produce 11 liters of milk / day [2], so the camel plays an important role in the survival and livelihood of (Bedouin) desert dwellers [3], camels are considered milk-producing animals and dominant in arid lands [4]; Also, their milk is dependent as a staple food for subsistence and may be the only milk available in places where other dairy animals cannot be maintained [5,6].Milk is always consumed in pastoral conditions either fresh or with varying degrees of acidity, in the raw state without processing Heat, in this case, can pose a threat to the health of the consumer, therefore attention must be paid to the health of camels in general and the health of their udders in 1259 (2023) 012081 IOP Publishing doi:10.1088/1755-1315/1259/1/012081 2 particular [7,8,9].The period of lactation in camels' ranges from 9 to 18 months; the total lactation productivity ranges from 800 to 3600 liters, with an average daily production ranging from 2.8-11 liters / day [10,11,12,13,14]; since camels are milk-producing animals, so the health of the udder is necessary to maintain the efficiency of produced milk, bacterial infection is one of the main causes of mastitis in domestic animals, including camels.Because of the camel's body physiology and its endurance, many authors believed in the past that mastitis is uncommon in camels [15,16], but in the past recent years, a number of researches have been conducted on the function, physiology, and diseases of the udder in camels in different countries; it was noted camel breeding that infected mastitis caused by many bacteria, some of which are resistant to antibiotic treatment [17,18].Mastitis disease is one of the main diseases that affect lactating camels, and it causes multiple serious effects on the animal and in turn affects human health [19,20] Clinical mastitis in camels can be detected by examining the udder or milk or both.[21,22] ; However, detecting subclinical mastitis is difficult and depends on performing various tests aimed at detecting the cause of the inflammation [23]; A previous study, in which they attempted to evaluate somatic cell contents (SCC), California mastitis test (CMT), adenosine triphosphate (ATP), N-acetyl-D-glucosamimidase (NAGase) and serum albumin as indirect diagnostic tools for infection and non-infectious quadrants Infected from the mammary gland of a camel [24]; There was a problem in interpreting the results because the basal levels of cells and their physiological changes in camels were and still are not defined [25,26].Some studies indicated that among the bacterial inflammatory causes of udders in camels are Staphylococcus aureus, Streptococcus spp.Micrococcus spp.Streptococcus agalactiae, Staphylococcus aureus, Pasteurella haemolytica, Escherichia coli, Corynebacterium spp.[27,28,29] Accordingly, this study was conducted with the aim of determining the bacterial causes of mastitis in Iraqi camels in some of AL-Furat Al-Awsat governorates (Babylon -Karbala -Al-Qadisiyah) and their sensitivity to treatment with different antibiotics used in treatment.

Clinical Examination of the Camel and the Time Period for the Research
(75) milking she camels were examined randomly from natural pasture camels and camels that were brought for treatment in veterinary clinics for each of the governorates (Al-Qadisiyah -Babil -Karbala) and laboratories in Mosel university Ethical approval No. um.VET.2021.5 for the period from 1/6/2221 to 1/12/2022.The clinical examination was carried out on method (19) for diagnosis of mastitis and its type for dairy camels.The examination was carried out by (25) camels from each governorate, at the age of (7-22 years).The research animals were divided into four groups based on the clinical signs that apparent form of mastitis, which included examination by hand and viewing it by looking to know the temperature of the udder -are there nodules -the softness or hardness of the udder -examining the lymph nodes of the udder and then milking some drops by hand for visual observation of the quality of milk and whether; There is blood or an abscess.The groups were divided into: the first three groups, which included: types of mastitis (acute -subacute -chronic), while the fourth group, which did not bear any pathological symptoms and when examining its milk models and cultivating them in the culture media, the result was free of germs.

Collection of Milk Samples
(450) milk samples were collected from four quarters of the udder after washing and cleaning the udder with soap and water well, and wiping the nipple with a piece of cotton soaked with ethyl alcohol at a concentration of (70%).Before collecting the sample a few first jets of milk are discarded.The volume of the milk sample from each camel was (20) ml.The samples were kept in sterile glass tubes and placed immediately in an ice box and transported to the laboratory for bacterial culture, examination and isolation of the bacteria causing mastitis [30].

California Mastitis Test (CMT)
After diagnosing mastitis with a clinical examination, the California mastitis test CMT is conducted according to method [25] to confirm whether or not mastitis is present by withdrawing the first drops of milk from each nipple and then placing them in a plastic dish with four cavities.Each cavity is numbered with the nipple number from which it is taken drops of milk, then an equal amount of the reagent chemical is added to the milk; The dish is stirred to mix the milk with the reagent chemical, then we notice the changes in the milk and read the result.In natural milk which take from a healthy camel, it is liquid and flows easily; As for camels infected with mastitis, clumps or small parts of sediment milk form.After reading the CMT result, the examination for mastitis is evaluated as shown in table number (1).
Table 1.Shows how to read the results of the California Mastitis test (CMT).

Evaluation of test Changes in milk negative
Liquid and no precipitate 1 Little precipitate and vanishes by stirring 2 Visible precipitate -no agglomeration 3 Be agglomeration 4 A clear agglomeration sticks to the bowl

Isolation and Diagnosis Bacteria
Bacteriological examinations were carried out by following the method [31]; bacterial culture of CMT-positive milk samples on culture media: blood agar and nutrient media they were incubated aerobic for (24-48) hours at 37 °C [32,33].Bacterial isolates were identified on basis of the morphological shape of the bacterial colonies, staining with Gram stain, hemolysis characteristics and catalase test.To identify the bacterial isolates, adopted a method by [34].Staphylococcus and Micrococci were identified by growth on mannitol salt agar, coagulation production, catalase and oxidase tests; Gram-stain negative isolates were cultured on MacConcky agar then tested using the trisaccharide TSI, Indole, methyl red, voges-proskauer, citrate test, urea and oxidase reaction.

Bacterial Sensitivity Test
We used the disc diffusion method to test the sensitivity of bacteria to antibiotics to find out the extent of their resistance by following [35], single antibiotic tablets produced by a company that included OXOID were used (Ampicillin, Tetracycline, Cephalexin, Erythromycin, Gentamicin, Methicillin, Cloxacillin, Amikacin, Maxipime, Trimethoprim mesulfamethoxa zole, Vancomycin, Nitrofurantoin).

Statistical Analysis
The spss program was used to analyze the data and extract the percentage of infection and the significant statistical difference between the camel groups used in the research [36].

Results
The results of our study showed according to the age of the groups of milking camels that were included in our study, which amounted to (75) camels with ages between

No. of sample percentage
As for the results of examining sensitivity and resistance to antibiotics; we used in our research (Ampicillin , Tetracycline , Cephalexin , Erythromycin , Gentamicin , Cloxacillin , Amikacin , Maxipime , Trimethopri mesulfamethoxa zole , Vancomycin , Nitrofurantoin ) they are shown in Figure No. (7).

Figure 7.
Shows the results of antibiotic sensitivity pattern of organisms that isolated from mastitis in camels.

Discussion
The results of our study showed according to the age of the groups of milking camels that were included in our study, which amounted to (75) camels with ages between (7-22) years, as shown in Figure No. (1).The highest group of ages for all camels infected and uninfected with all types of mastitis at the ages (15-19 years) with a rate of (36%), while the lowest ages of group examined was (20-22 years) with a rate of (16%), in the second degree the group of ages (8)(9)(10)(11)(12)(13)(14) years with a rate of (31%), then the group of ages (≤ 7) with a rate of (17%); These percentages correspond to (4-9) in terms of the ages of the milking camels and the time period for milk production; there was no statistical significant difference at the level of p≤ 0.05 between the groups of the ages of camels and the results of examining the milk samples; which numbered (450) milk samples collected from four quarters of the udder of all the research camels (infected with mastitis and not infected) showed that the number (51) by (68%) camels infected with mastitis, where the number of samples of milk collected from them ( 306) samples, while the number of uninfected camels was (24), with a rate of (32%); the total number of milk samples is (144) samples, as shown in Figure No. (2).The research result corresponds to the percentage of infected camels with [17,18,37,38] they say about the possibility of she camels to develop mastitis, the percentage of infection and the factors that lead to infection while it is not corresponds with [15,16] because they believed mastitis is uncommon in she camels ; as a result of its physiology and endurance of harsh desert conditions.As for the results of mastitis for the infected camels, which numbered (51) camels, with a rate of (68%) for all the types which dealt in our research.According to the groups of age of camels, the highest age affected for all types of mastitis was (15-19 years) with a rate of (39%) while the lowest category affected ages was (≤ 7) with a rate of (12%); so there was statistical significant difference at the level of p≤ 0.05 between the groups of the ages of infected camels; as shown in Figure (3).This result is consistent with the results of the study [6,37,38]; they say for reasoning for this result is related to the immunity of the animal; in the older animals it gets lower immunity as a result of the multiple times of pregnancy and childbirth than in young ages.The results of the California Mastitis Test CMT to diagnoses the types of mastitis of camels in the three governorates.We saw infected with sub-acute mastitis (+++) in number (26) at rate of (35%); infected with chronic mastitis in number (15) with a rate of (20%) and infected with acute mastitis (++) in number (10) at rate of (13%) while the result was (-) with a number of ( 24) at a rate of (32%) indicating camels without mastitis, as shown in Figure No. (4); this agrees with [26,37,39] In terms of compatibility ratio of inflammation and types of mastitis.As for the results of mastitis based on inflammation tests for each quarter of the udder, the highest rate was in two quarters, where the percentage of affected camels was (35%); in the second degree infection in one quarter by (25%);in the third degree infection of three quarters with a rate of (22%) ;while the lowest percentage ; the infection in four quarters or in the whole udder, at a rate of (18%), as shown in Figure No. (5), corresponding to [18,26,39]; They mentioned that the infection rate is high with one quarter or two quarters of the udder because of the shape of the udder in camels, which differs from what it is in cows.Thus, when the animal sits and stands, it is affected two quarters at most, and rarely affected and exposed to bruises of all four quarters.As for our reasoning for these percentages through our study, Shepherds and owners of camels mentioned the causes of mastitis in camels, due to the poor milking method, the remnants of hot ash near the tent and the stings of snakes and scorpions in the desert.(7); It shows us that there are differences in resistance and sensitivity to antibiotics according to the type of bacteria isolated and according to the type of antibiotic used in treatment.To explain this situation through our research and asking camel owners and breeders, we found One of the important reasons for the resistance of bacteria isolated from mastitis is the lack of sufficient knowledge by these people about the treatment of their animals and misuse of antibiotics by owners and breeders, whether in terms of adherence to treatment, its focus, the number of treatment times, or the multiplicity of types of antibiotics used in our research for the same case ; agrees with [41,42].Through our study, we concluded that attention should be paid to the health condition of udder to the milking camel"s , as it suffers from mastitis, such as cows, goats, and sheep; So it must bacterial isolation of mastitis cases must be carried out, the type of germ causing the condition must be known, and a sensitivity test should be performed to give appropriate treatment for each cases to avoid bacterial resistance to antibiotics; And the work of mobile veterinary health teams to examine the milking camel and give appropriate treatments to it.Also, mobile educational teams should be set up to explain the importance of camels; their health in general; the health of the udder of the milking camel and its impact on animal productivity, and then its impact on the health of the consumer if it is not treated.

Conclusion
Through our study, we concluded:  The health status of the udders of dairy camels should be taken care of because they suffer from mastitis like cows, goats, and sheep. Bacterial isolation of camels infected with mastitis should be carried out to know the type of bacteria causing the infection. -Sensitivity test to antibiotics for isolated bacteria should be done to give the appropriate treatment for each case to avoid the occurrence of antibiotic resistance. -Mobile veterinary teams should be formed to explain the importance of the health of camels on animal's productivity and then its impact on the health of the consumer if it is not treated.

Figure 1 .
Figure 1.Shows the division of camel groups into four types according to the age.

4 Figure
Figure No.(1) shows the total number of camels used in the research according to the groups of age, which was divided into four groups: (7 ≥) years,(8)(9)(10)(11)(12)(13)(14) years,(15)(16)(17)(18)(19) years, and(20)(21)(22) years.The results of examining the milk samples; which numbered (450) milk samples collected from four quarters of the udder of all the research camels (infected with mastitis and not infected) showed that the number (51) infected with mastitis, where the number of samples of milk collected from them ( 306) samples, while the number of uninfected camels was(24);the total number of milk samples is (144) samples, as shown in Figure No. (2).

Figure 2 .
Figure 2. Shows the totel number , percentage of camels and milk samples that used in research , the number , percentage of healthy and infected camels.

Figure No. ( 2 )
Figure No. (2) shows the number of camels and total samples of milk used in the research, the number and percentage of camels infected with mastitis, and the number and percentage of uninfected camels.As for the results of mastitis for the infected camels, which numbered (51) animals for all the types which dealt in our research, according to the groups of age of camels; the highest age affected for all types of mastitis was (15-19 years) ; while the lowest category affected ages was (≤ 7) as shown in Figure(3).

Figure 3 .
Figure 3. Shows the number and percentage of camels infected with mastitis of all types according to groups of age.The results of the California Mastitis Test CMT to diagnoses the types of mastitis of camels in the three governorates.We saw infected with sub-acute mastitis, chronic mastitis and infected with acute mastitis as shown in Figure No. (4).

Figure 4 .
Figure 4. Shows the types of mastitis in affected camels and the percentage of infection for each type.As for the results of mastitis based on inflammation tests for each quarter of the udder, the highest rate was in two quarters; in the second degree infection in one quarter; in the third degree infection of three quarters; while the lowest percentage the infection in four quarters or in the whole udder as shown in Figure No. (5).

Figure 5 .
Figure 5. Shows the number and percentage of mastitis infection according to the number of the quarters of udder infected with mastitis.The results of bacteriological culture of milk samples of infected camels with mastitis, which numbered (306) samples.The isolates were recorded of C.N. Staphylococci, Staph.sciuri,Staphylococcus aureus, Staph.epidermidies,Streptococcus agalactia, Pasteurella haemolytica, Arcanobacterium pyogenes and E. coli as shown in Figure (6).

Figure 6 .
Figure 6.Shows the common isolated microorganisms from Mastitis.