Prevalence and Economic Impacts of Retained Placenta and Subclinical Mastitis in Tanzanian Dairy Cows

Background: Three dairy farms were investigated for occurrence of retained placenta and subclinical mastitis, and the economic impacts attributable to the conditions. Reproductive performance data from 1169 calvings of dairy cows for five consecutive years spanning from 2014 to 2018 were used in this study. A total of 167 cows were screened with the California Mastitis Test (CMT), and udder quarters with CMT score of ≥ 3 (scale 1–5) were milk sampled for culture and final bacteriological identification. Results: Overall prevalences of subclinical mastitis and retained fetal membranes were 35.93% and 10.35%, respectively. The prevalence of bacterial isolates at animal level was 38.92%. Individual bacteria prevalence rates were 66.15% for Staphylococcus aureus, 13.85% for Escherichia coli, 6.15% for Bacillus spp., 6.15% for Streptococcus agalactiae and 7.69% for none typed microorganisms. Overall loss from subclinical mastitis was estimated at 10,603,800/= Tanzanian shillings (Tshs) equivalent to USD $ 4,700 with major losses (81.75%) attributable to the production loss followed by treatment expenditure (18.25%). Average loss attributed to mastitis per quarter was estimated at 49,320/= Tshs (USD $21.5). Retained placenta resulted in an increase (P<0.05) in the period from parturition to conception (148.7 vs. 96.0 days), number of services per conception (2.9 vs. 1.9) and calving interval (434.7 vs. 374.0 days). Conclusion: These findings point out to the economic importance of subclinical mastitis and retained fetal membranes in the dairy industry. This calls for improved milking hygiene, proper attention to health of mammary glands, regular screening tests and application of biosecurity measures as intervention strategies to lower the prevalence of subclinical mastitis and minimize the spread of pathogens in dairy farms. Since retained fetal membrane is an important problem causing great economic losses, appropriate control measures have to be instituted including cow comfort, proper immunization and


INTRODUCTION
Reproductive performance is one of the major factors affecting profitability of a dairy herd. As a result reproductive and production disorders of dairy cattle significantly reduce their productivity thereby presenting a great concern to dairy producers across the world [1]. Poor reproductive performance due to post-partum reproductive disorders can reduce the number of born calves and milk production; and increase the cost of nutrition, therapy and artificial insemination. Aberrations of the post-partum period which include retained fetal membranes, cystic ovaries, metritis/endometritis, uterine prolapse, and pyometra are major causes of infertility during this period [2].
Normally fetal membranes drop within a short time post-partum (within 8 hours of parturition). If the membranes are retained up to 12 hrs they are referred to as delayed removal and if retained for more than 24 hours of parturition then the phrase 'Retention of placenta' is used [2,3]. Retention of fetal membranes is the most common condition occurring in cattle following parturition [4]. The incidence of retained placenta varies from 4.0 to 16.1% and can be much higher in problem herds [5,6]. Placental retention is usually accompanied by delayed involution of the uterus [7], reduced pregnancy rate [8], increased interval from calving to service, increased number of services per conception [9][10][11] and reduced milk yield [10,12]. The condition also delays the postpartum resumption of cyclic ovarian function and prolongs the interval from calving to first ovulation [3,10,13,14]. There are a number of predisposing factors to retention of fetal membranes, including mechanical, nutritional, managemental and infectious factors [5,14,15]. Manual removal, administration of intra-uterine and/or systemic antibiotics, injection of oxytocin, PGF2α and β2-receptor blockers are commonly used methods for treatment of fetal membrane retention [2] Mastitis, an inflammation of the mammary gland, is a highly prevalent disease in dairy cattle worldwide with a major economic impact in the dairy value chain (16)(17)(18). It is the most important and expensive disease in the dairy industry resulting into severe economic losses from milk production quality and quantity. It leads to reduced milk production, veterinary intervention costs, milk discards due to bacterial or antibiotic contamination, premature culling and occasionally deaths [19][20][21]. Depending on the visibility of effects of inflammation of the mammary gland, which is attributable to the degree of inflammation, mastitis is classified as clinical or subclinical [22]. Clinical mastitis includes gross abnormality in milk, physical abnormalities of udder and abnormality of cow with systemic involvement while subclinical mastitis does not produce visible effects on udder or milk quality but has important effects on milk composition, mainly an increase in somatic cell counts [23,24] [25][26][27]. However, CMT has been recognized as a highly sensitive test to detect bovine subclinical mastitis [24,25].
The occurrence and economic losses associated with bovine reproductive and productive disorders (including mastitis) are well documented in developed countries [16,28]. Such information is scanty in developing countries, including Tanzania. Therefore, the objective of this study was to determine the occurrence and economic impact of retained fetal membranes and subclinical mastitis among dairy cattle kept in the tropical environment under the semi-intensive production system.

Study Animals
In the study, two types of dairy cattle were involved. Farm A had Friesian cattle, full time grazed on planted temperate pastures species comprising mainly of Lolium perenne, Lolium multiflurium and Infolium repens. Milking cows were supplemented with homemade concentrate composed of maize bran and rice polishings (60-70%), sunflower seed cake or cotton seed cake (25%), mineral supplements (2%) and salt (1%) during milking. The amount of concentrate offered depended on the quantity (kg) of milk produced from each cow. Lactating cows were machinery milked and all animals had ad libitum water supply.
The farm practiced both artificial insemination and natural breeding. Animals are routinely vaccinated against common diseases, regularly dewormed and dipped against external parasites. Farms B and C had crossbred dairy cattle which were allowed to graze on natural pastures (Hyperrhenia spp, Panicum maximum, Cenchrus ciliaris and Brachiaria spp) for about 8 hours and fed on hay (Chloris gayana and Pennisetum purpureum) after returning to the housing pallor in the afternoon and during night hours. Milking cows were supplemented with homemade concentrates. All farms practiced machinery-milking twice daily and used both artificial insemination and natural breeding methods.

Milk Production Data
Trends in milk production for a minimum of 15 days' milk yields before and after the treatment were recorded to gather data on incidence rate and economic losses due to subclinical mastitis. For calculating the economic losses due to the disease, information on average daily milk yield, prevailing price of milk in the location, reduction in milk yield during the illness period, number of days of illness, discarded milk during illness period and veterinary expenses was collected from the sample farms. The loss of milk during the period of mastitis was calculated by the difference between the average milk yield potential of the dairy cows and average milk yield during disease. The milk yield potential of the dairy animals was taken as the average of 14 day's milk yield before the date of occurrence of disease and 14 days after the cure of animals. The quantity of milk that was reduced due to mastitis was considered as milk loss. The reduction in milk yield was recorded in liters. The cost of mastitis treatment consists of veterinary fees, drug cost and farmer's extra labour cost [29]. However, the farms involved in this study employ their own veterinarian with fixed salary, thus, making it a fixed cost, so we did not have to take the veterinary fees into account in our calculations. Similarly, the extra labour of the farmer was regarded as a fixed cost as this activity is part of the job of the farm employees with fixed salary.
Screening for subclinical mastitis A cross sectional study was conducted that involved screening a total of 668 mammary quarters from 167 lactating cows. The animals were first examined for presence of mastitis based on final cardinal signs of udder inflammation as previously described by Bachaya et al. [30]. At the time of each examination, information about breed, age, parity and stage of lactation were recorded. Before examination, the udder was thoroughly washed, dried with a clean towel and the teats were sprayed with 70% ethanol. Presence of subclinical mastitis was checked using California Mastitis Test (CMT) protocol as previously described [31]. Briefly, CMT score ranging from 0 to 4 was used, where 0 is there was no significant difference in the prevalence between the three farms ( Fig. 1).
Lower retained placenta occurrences were recorded in in Mazimbu and Magadu herds while highest occurrence was observed Kitulo herd (Fig. 3). The retention of fetal membrane greatly influenced the reproductive performance of animals (Table 4). Open days was longer (P < 0.05) in cows exhibiting retained placenta as compared to normal cows by about 53 days. Calving interval was longer (P < 0.05) in cows exhibiting retained placenta as compared to normal cows by about 61 days. Number of service per conception for cows exhibiting retained placenta was higher (P < 0.05) as compared to normal cows (2.9 vs. 1.7). Mastitis is a multi-etiologic disease of the mammary gland characterized mainly by reduction in milk production and considered an economically important disease in the dairy subsector in developed and developing nations. The results from this study show a high prevalence of subclinical mastitis; a finding that was comparable to what was previously reported both in the country [33][34][35] and elsewhere [36][37][38][39]. The overall high prevalence could be attributed to the invisible and silent nature of subclinical mastitis which is usually given little attention by farms when it comes to treatment, and the lack of mastitis control program which has been proved to decrease the prevalence of mastitis in developed countries [40]. However, the prevalence of subclinical mastitis reported in this study is lower than those reported in Uganda [41], Rwanda [31,42,43], Kenya [44], Ethiopia [39] and Vietnam [45]. It has been pointed out by previous authors that subclinical mastitis is a complex disease and discrepancies in the reported incidences in different studies could be attributed to differences in animal breeds, management systems, parity, teat morphology, and milking hygiene [46,47].
The bacterial pathogens isolated in the present study were dominated by Staphylococcus aureus ( Table 2). The prevalence rate of S. aureus (66.15%) in the current study agrees with previous findings reported in Tanzania [34,35,48], Rwanda [31], Algeria [49], Italy [50] and Brazil [51]. However, the prevalence of S. aureus in mastitic cows reported here is higher than 25.7% previously reported in Tanzania [52], 40% in Morocco [53], 24.1-48.75% in Ethiopia [39,54] and 30.6% in Kenya [55]. The high rate of isolation of S. aureus may be attributed to the fact that S. aureus easily localize the skin of the udder, and has the capacity to penetrate into the tissue, producing deep seated foci protected by a tissue barrier [56]. This high frequency of staphylococcal mastitis is considered to be due to poor milking hygiene and lack of proper attention to the health of the mammary gland in general. Thus, hygiene at milking is of paramount importance in control of these infections because they are spread during the milking process causing intramammary infections.
The findings of the current study have shown a low prevalence (13.85%) of E. coli, the coliform environmental bacteria associated with wet and muddy conditions. These findings corroborate with those previously reported in Tanzania [57] and Rwanda [31]. E. coli have been reported to be of more importance in clinical mastitis than subclinical mastitis, despite the environmental factors [58]. This confirms that the prevalence of coliform bacteria in this study would be low, as few samples were of clinical mastitis. Although environmental Streptococcus agalactiae (6.5%) were ranked third in the current study, the microorganism have been reported as the most prominent bacteria in cow mastitis in some countries [26,45]. The prevalence rate of S. agalactiae reported in the current study agrees with previous findings observed elsewhere [35,59]. Bacillus spp., were present in 6.15% of cases in the current work. Bacillus spp. has also been identified as important pathogens in both CM and SCM in previous investigations [60,61]. Nonetheless, these results contradict other studies showing that mastitis caused by Bacillus spp. is rare in dairy cows [36,62]. The presence of Bacillus spp. could be explained by the poor hygienic conditions of milkers as the bacteria are widely distributed in dairy environment, including on teat skin, milkers' skin and gloves, and farm floors, which represent reservoirs of bacteria associated with intramammary infections [61,63]. with previous studies [20,64,65]. Subclinical mastitis is an invisible problem that can cause financial losses to producers through the reduction of milk production, lowered milk quality, discarded milk, veterinary fees, drug costs and extra labor to farmer [16,17,66,67]. Estimation of the financial impact observed in this study could be used to advocate for the implementation of prevention methods that reduce the impact of subclinical mastitis in Tanzanian dairies.
The incidence of retained fetal membranes observed in the present study ranged from 4.0 to 16.4% (average of 10.35%). This finding is in agreement with other studies conducted in the country [68] and in other countries [2,6,8,69]. Some other authors have however reported higher incidences than the observation made in the current study [10,14,70]).
Nevertheless, significantly lower incidences of RFM have also been reported in Bangladesh crossbred and local cattle [71] and Ethiopian dairy cattle [72,73]. The differences in the incidences of RFM reported by different authors could be attributable to various factors, such as age, breed, heredity, environment, hormonal status, nutrition, and immunity.
Higher incidences of retained fetal membranes cause considerable economic losses to the farm [74]. Since the direct costs associated with RFM are difficult to derive because of a multitude of influences [75, economic losses are normally measured through establishing indirect costs to farmers resulting from periparturient disease, decreased milk production, extension of calving interval, reduced pregnancy rates, and increased risk of culling [76].
In this study, the mean number of services per conception in normal control group was 1.9 ± 0.5, whereas the corresponding value for the retained fetal membrane group was 2.9 ± 1.0, which differed significantly (P < 0.05), and corroborated with findings reported elsewhere [8, 10, 11, 14, ]. Open days was longer (P < 0.05) in cows exhibiting retained placenta as compared to normal cows by about 507 days (Table 4). This is because more than 50% of animals that calved normally had days open within the three months postpartum, while more than 70% of cows with retained placenta conceived after 150 days.
This finding concurs with the results reported by other researchers [8,10]. Calving interval was significantly longer (P < 0.05) in cows exhibiting retained placenta as compared to normal cows by about 60 days. A similar trend has been reported by other authors [3,14,10]. Retention of placenta is associated with secondary uterine infections, which may be related to probability of subsequent conception [77]. Cows with retained fetal membranes had longer intervals from calving to first service and to conception and required more services per conception and lower pregnancy rate and conception to first service. Declarations inappropriately influenced them in writing this article.

AUTHORS' CONTRIBUTIONS
IPK conceptualized the study, performed data collection, laboratory and statistical analysis, interpreted the results, and prepared the manuscript. EVGK assisted with refining the study design, laboratory analysis and implementation of the study and critically reviewed the manuscript. All authors read and approved the final manuscript.

AVAILABILITY OF DATA AND MATERIALS
All data generated or analysed during this study are included in this published article.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE
Ethical clearance to conduct this research was obtained from the ethical committee of College of Veterinary Medicine and Biomedical Sciences of Sokoine University of Agriculture. Verbal consent was obtained from the farm managers to collect dairy cattle information and biological samples.

CONSENT FOR PUBLICATION
Not applicable.

Figure 1
The status of study animals in relation to prevalence of mastitis in study farms Annual trends of retained placenta observed in the study farms in a five years period (2014 to 2018).