Introduction

Escherichia coli (E. coli) infectionin dairy cowsis considered as one of the most common causes of environmental mastitis in different countries. The bacterium is a ubiquitous inhabitant of soil, the digestive tract, fece and leads to mastitis in cattle by multiplying in contaminated bedding and entering the mammary glands via the end of the teat when the teat comes in contact with it (Persson et al., 2015). Once inside the mammary gland, the bacterium reproduction rapidly. Then, the immune system of the cow lysis the bacteria, which causes release of endotoxins into the cow circulatory system. Endotoxins (also known as lipopolysaccharide) are complexes of lipid and polysaccharide that are located in the outer membrane of Gram-negative bacteria such as E. coli. The lipid portion of the complex, once released into the blood stream, acts as an endotoxin (Persson et al., 2015). Of the cows that naturally acquire coliform mastitis, 32%–75% develop septicemia along with the characteristic clinical signsof E. coli endotoxemia, which include loose of appetite, mammary gland inflammation, watery and serum-like milk, high fever, diarrhea, dehydration, and recumbency (Katholm &Anderson, 1992). Toxemia results in dehydration and disseminated intravascular coagulation (DIC), which can lead to mortality of untreated animals in a few days (Blowey & Edmondson, 2010).

Anderson (1989) suggests that administration of antibiotics, anti-inflammatory agents, and fluids may be a beneficial treatment due to endotoxin-induced shock. However, the effect of such treatment protocols on the course of Gram-negative mastitis remains relatively poorly researched to date. A few studies have assessed on the effects of antibiotic-based treatment protocols, including that by Hagiwara et al. (2014). In the mentioned study, which was conducted in Japan, compared antibiotic-treated cows with acute E. coli mastitis that did and did not die: they showed that death was related to dysstasia, reduced antithrombin activity, reduced platelet counts, and enhance hematocrit values and non-esterified fatty acid concentrations. In this regard, it was also suggested that when cows with E. coli mastitis were treated with placebo and enrofloxacin, the enrofloxacin-treated cows had lower somatic cell counts in their milk than placebo-treated cows (Persson et al., 2015). However, this treatment did not increase survival compared with placebo. In addition, it was found that in cases of spontaneous and induced coliform mastitis, an application of the intra-mammary antibiotics that are impressive in vitro does not ameliorate the cure rate or alter the elimination of bacteria from the mammary gland (Pyorala, 2009; Hogan & Smith, 2003). However, other studies showed that both acute and mild E. coli mastitis can be accompanied by bacteremia; they because cows are physiologically immunosuppressed during the peripartum period, those with acute coliform mastitis may benefit from parenteral antimicrobial treatment (Cebra et al., 1996; Wenz et al., 2001).

To improve our understanding of the efficacy of antibiotic treatment in dairy cows with E. coli mastitis, we performed a randomized clinical trial. Thus, dairy cows with naturally occurring acute E. coli mastitis were treated with supportive measures together with ceftiofur hydrochloride and placebo, and their clinical recovery assessed 5 days after commencing treatment.

Materials and methods

Animals

This randomized clinical trial, conducted between January 2014 and December 2016, involved 36 herds of Holstein-Friesian dairy cows located in Tabriz (38° 07’ N and 46º 29’ E), East-Azerbaijan Province, Iran. During the trial, the ambient temperature extended from -10℃ to 38℃. Of note, the annual rainfall in this city varies from 226 mm to 250 mm. All animals were kept in open shed barns and fed hay, corn silage, soybean, and complementary minerals. They were milked three times a day and their mean production was 30 kg milk/day/cow.

All affected cows had clinical signs of acute mastitis, including a high fever (≥ 40°C), redness and inflammation of the mammary gland, watery and yellowish milk, diarrhea, dehydration, and recumbency. The history of the affected cows was recorded, which included the date of mastitis onset, the parity, the bed type, the diet formulation, and the date of recent parturition or pregnancy.

Sampling

All cows that were naturally affected by acute clinical E. coli mastitis (n = 100) during the study period were considered for inclusion in the trial. Of note, before a treatment, milk samples were also obtained from the affected quarters into sterile culture tubes, according to National Mastitis Council (NMC) guidelines and standards (Hogan, 2017). All samples were subjected to bacterial culture (Harmon et al., 1990) in the Tabriz University Microbiology Laboratory. After collecting milk samples and before the laboratory results were recorded, all affected cows were administered supportive therapy. Moreover, the cows with odd-numbered ear tags (group A) were administered ceftiofur hydrochloride (Maymó Labs., Spain). The cows with even-numbered ear tags (group B) received placebo instead of antibiotic.

Clinical treatment

After receiving the laboratory data, all cows (n=100) with acute E. coli mastitis were enrolled in the trial. The remaining cows (264 cases), which had clinical mastitis due to other causes (e.g., Streptococcus uberis, Streptococcus agalactiae, Staphylococcus aureus, and Klebsiella pneumoniae), were excluded from the trial.

Supportive therapy comprised intramuscular flunixin meglumine (Razak Labs. Co. Iran) 2.2 mg/kg bodyweight/day for 4 days and, on the first day of treatment, a single intravenous dose of hypertonic saline (Zoopha Parnian Pars, Iran) (NaCl, 7.2 %; 5 ml/kg bodyweight) and calcium borogluconate 40% (Nasr Company, Iran) (250–500 ml/cow). Antibiotic treatment comprised daily intramuscular injections of ceftiofur hydrochloride (1 mg/5 kg bodyweight) into the neck region for 4 days. Placebo treatment consisted of daily intramuscular injections of saline into the neck region for 4 days. The cows in both groups were allowed water ad libitum, and the affected quarters were milked out every 2 hours to reduce the toxic effects of E. coli mastitis. All animals were evaluated daily and heart rate, appetite, body temperature, and color change in the milk from the affected quarters were noted. Additionally, on the 5^th day of treatment, all surviving cows were examined clinically.

Statistical analyses

The cows with (Group A, n = 50 cows) and without (Group B, n = 50 cows) antibiotic treatment were compared in terms of primary and secondary outcomes using Chi-squared tests. All statistical tests were conducted using SPSS software (version 22, Inc., Chicago, IL, USA). Differences were considered significant when P < 0.05.

Results

In total, 364 cows developed acute mastitis during the study period. In 264 cases, mastitis

Discussion

E. coli mastitis in cattle can vary from a subclinical mastitis to an acute lethal disease characterized by significant clinical symptoms. Therefore, it can lead to a considerable economic loss in modern dairy farms. Since both subacute and acute E. coli mastitis can be accompanied by bacteremia (Cebra et al., 1996; Wenz et al., 2001), parenteral antimicrobial therapy, which is administrated simultaneously with fluid therapy, is likely to be beneficial for animals with acute E. coli mastitis. This notion is supported in particular by the fact that the postpartum period associated with physiological immunosuppression in dairy cows. However, it remains unclear how effective local antimicrobial agents are for treating E. coli mastitis (Suojala et al., 2013; Hogan & Smith, 2003). In particular, growing evidence show that when enrofloxacin is used to treat acute clinical E. coli mastitis, the treatment does not increase survival relative to placebo (Persson et al., 2015; Bargi et al., 2015).

Our study showed that parenteral use of an effective antibacterial agent such as ceftiofur hydrochloride effectively increased the clinical recovery of naturally affected cows: 82% of the antibiotic-treated cows recovered, as opposed to 4% in the placebo-treated group. This finding is in agreement with a previous study which proposed that ceftiofur therapy reduces the mortality rates of dairy cattle with severe clinical mastitis caused by coliform organisms (Erskine et al., 2002). Ceftiofur is a third-generation cephalosporin that is impressive against a wide range of Gram-negative and Gram-positive bacterial pathogens that cause mastitis (Truchetti et al., 2014). Our results also agree with those of Suojala et al. (2013), who proposed that acute E. coli mastitis due to bacteremia following the multiplication of bacteria in the mammary gland should be treated with fluoroquinolones or third- or fourth-generation cephalosporins.

In the current study, both antibiotic- and placebo-treated cows were treated with supportive therapy; namely, fluid therapy, anti-inflammatory treatment, and frequent milking. The low clinical recovery rate of our placebo-treated cows (4%) is not consistent with the findings of Morin et al. (1998) and Katholm (2003), who found that 50% of dairy cows with E. coli mastitis recover when given the supportive approach used herein. It is possible that cows in the latter studies were largely affected by mild acute E. coli mastitis.

In the present study, most of the cows that were affected with E. coli mastitis had ≥ 4th parity (48%) (Fig. 1). Moreover, the majority of cases occurred on farms that used organic bedding material such as dried manure solids (52%) or sawdust (39%). These observations are in agreement with the results of other researchers (Hogan et al., 2003; Ward et al., 2002). Since it is very difficult to treat cattle with E. coli mastitis, it is notable that dairy farms seek to prevent its occurrence by instituting proper environmental sanitation. Vaccination of cows with the E. coli J5 mastitis vaccine is also recommended (Hogan et al. 1992).

Conclusion

Our results suggest that supportive treatment alone does not effectively decrease the mortality rate of dairy cows with E. coli mastitis. Thus, cows with this disease should be treated with an effective antibiotic such as ceftiofur hydrochloride, along with intensive fluid therapy.

Ethical approval

This study was approved by the Ethics and Research Committee of the University of Tabriz (certificate no: 012/2016).

Acknowledgements

We would like to thank the Research Deputy of the University of Tabriz (Faculty of Veterinary Medicine) for financially supporting this research (No: D/27/2016).