Economic consequences of an outbreak of ovine keratoconjunctivitis in a flock of Texel crossbred sheep kept on tropical pasturesABSTRACT
The aim of this study was to evaluate the impact of ovine infectious keratoconjunctivitis in a flock of Texel crossbred sheep on tropical pastures and the evolution of the disease under different oxytetracycline-based treatment protocols. Clinical signs observed in ewe lambs and ewes were conjunctivitis, photophobia, and epiphora, progressing to superficial to deep ulcerative keratitis. Experimental protocols are based on injectable oxytetracycline and flunixin, depending on the severity of the disease. A cost analysis of the disease control measure was conducted, considering the costs of sanitary inputs. To identify the etiological agent, samples of ocular secretion were collected from the affected animals. The etiologic agent identified was Mycoplasma conjunctivae. A total of 101 clinical observations were made on 23 ewe lambs, and 371 clinical observations were made on 65 ewes. The protocol of three consecutive injections of oxytetracycline every 72 hours was the most used for lambs and ewes during the outbreak. The cost of sanitary input for ewe lambs and ewes was US$ 73.62 and US$ 551.57, respectively. The recommendations described in this study are fundamental to reducing the impact of the disease on pasture sheep flocks and can be applied on farms to control outbreaks of infectious ovine keratoconjunctivitis.
Keywords:
bioeconomy; eye disease; health protocol; oxytetracycline
RESUMO
Objetivou-se avaliar o impacto da ceratoconjuntivite infecciosa ovina em um rebanho de ovelhas mestiças Texel em pastagens tropicais e a evolução da doença sob diferentes protocolos de tratamento à base de oxitetraciclina. Os sinais clínicos observados em borregas e ovelhas foram conjuntivite, fotofobia e epífora, progredindo para ceratite ulcerativa de superficial à profunda. Após a separação, foram definidos protocolos experimentais baseados em oxitetraciclina e flunixina injetáveis. Foi realizada uma análise de custos das medidas de controle da doença, levando em consideração os custos dos insumos sanitários. Para identificar o agente etiológico, amostras de secreção ocular foram coletadas dos animais afetados. O agente etiológico identificado foi o Mycoplasma conjunctivae. Foram feitas 101 observações clínicas em 23 borregas e 371 observações clínicas em 65 ovelhas. O protocolo de três injeções consecutivas de oxitetraciclina a cada 72 horas foi o mais comumente utilizado para borregas e ovelhas durante o surto. Os custos dos insumos sanitários para borregas e ovelhas foram de US$ 73,62 e US$ 551,57, respectivamente. As recomendações descritas neste estudo são fundamentais para reduzir o impacto da doença em rebanhos ovinos mantidos em pastos tropicais e podem ser aplicadas em fazendas para controlar surtos de ceratoconjuntivite infecciosa ovina.
Palavras-chave:
bioeconomia; doenças oculares; protocolo sanitário; oxitetraciclina
INTRODUCTION
Ovine infectious keratoconjunctivitis (OIKC) is a common disease worldwide, especially in ruminants (Almeida Neto et al., 2004). This is a very contagious disease characterized by an acute inflammatory response of the conjunctiva, hyperemia of the sclera, and neoformation of fibrovascular tissue (pannus). The clinical signs include epiphora, blepharospasm, photophobia, purulent ocular secretions, and, in more cases, corneal opacity and ulceration.
The etiological agent of OIKC remains unclear, as a wide variety of microorganisms have been associated with the disease. There are some primary pathogens and others are secondary invaders, so it is difficult to attribute a primary etiological cause to a single agent, since supposed agents have also been isolated from the eyes of healthy sheep. Mycoplasma conjunctivae and Chlamydophila spp. have been identified in OICK outbreaks, but M. conjunctivae seem to cause more severe disease compared to that caused by Chlamydophila spp. (Bezerra et al., 2019).
A serological survey conducted in 2003 in New Zealand for M. conjunctivae infection in sheep and goats, showed a 71% prevalence of antibodies to M. conjunctivae in 45 flocks tested from seven different farms, suggesting that this is the most common causative agent of OIKC in this country. Other bacteria, such as Staphylococcus aureus, Brahmella ovis, Escherichia coli, and Acholeplasma spp. have been in the conjunctival flora of both healthy and OIKC animals and Mycoplasma conjunctiva e have been identified as concomitant agents in cases of OIKC (Ter Laak et al., 1998). However, Moraxella bovis, which is associated with keratoconjunctivitis in cattle, does not appear to be associated with keratoconjunctivitis in sheep (Bezerra et al., 2019).
The source of infection is the infected animal, and OIKC can be transmitted directly or indirectly by flies, pastures, or dust contaminated by the tears of infected sheep. Initially, there is an acute inflammatory process of the conjunctiva, followed by scleral hyperemia, pannus, and corneal opacity (Almeida Neto et al., 2004; Bezerra et al., 2019).
Ovine keratoconjunctivitis is not a fatal disease, but its economic impact is significant (Conceição and Turnes 2003). The economic losses resulting from the disease are primarily the cost of medication, time, and management required for treatment, as well as weight loss, increased pregnancy toxemia, and trampling of lambs by blind ewes (Chaves et al., 2008). The variability of clinical signs in the expression of the disease makes it difficult to assess the extent of economic losses (Almeida Neto et al., 2004).
The aim of this study was to evaluate the impact of OIKC on a flock of crossbred Texel sheep grazing on Brachiaria brizantha cv. Marandu pasture and the evolution of the disease during the outbreak under different treatment protocols based on oxytetracycline.
ETICHAL ASPECTS
The sheep affected by the OIKC outbreak belonged to an experimental flock at the Federal University of Mato Grosso do Sul, approved by the Ethical Committee for the Use of Animals at the Federal University of Mato Grosso do Sul (UFMS), protocol no. 1.198/2021.
MATERIAL AND METHODS
The flock was evaluated at the Sheep Farming Sector of the farm school, part of the Faculty of Veterinary Medicine and Animal Science of the Federal University of Mato Grosso do Sul (20° 26’ 34.31” S, 54° 50’ 27.86’’ O, 530.7m de altitude).
The outbreak occurred between March and December 2023, and clinical signs were observed in two different categories: ewe lambs and ewes. Ewe lambs and ewes were kept on Brachiaria brizantha cv. Marandu pastures during the outbreak period. At the beginning of the outbreak, in March 2023, the initial body weight of ewe lambs and ewes were 25.83kg ±2.67 and 58.48±1.67, respectively. The average initial age of ewe lambs was 90 days and the ewes' age ranged two to five years old.
The beginning of the outbreak was observed with the introduction of a flock of sheep from different locations. After seven days, clinical signs compatible with keratoconjunctivitis, such as acute conjunctival inflammation and moderate hyperemia, were observed in this flock. The introduced animals did not stay in the same paddocks as the experimental flock, but, some ewe lambs and ewes had direct contact with the introduced flock. After this contact, the ewes and ewe lambs were returned to their respective paddocks. Seven days after contact, the ewe lambs and ewes began to show signs of keratoconjunctivitis, with simple conjunctivitis, photophobia and epiphora, progressing to superficial to deep ulcerative keratitis.
After the outbreaks began, ewes and ewe lambs were classified and grouped according to ocular lesion scores, which refer to the degree of clinical conjunctival involvement (Table 1). The protocols were defined according to the number of oxytetracycline-based injections (20g of oxytetracycline/100mL of solution). Protocol A (one dose of intramuscular injection); protocol B (two doses of intramuscular injection every 72 hours); protocol C (three doses of intramuscular injection every 72 hours); Protocol D (four doses of intramuscular injection every 72 hours); Protocol E (five doses of intramuscular injection every 72 hours). In cases of moderate to severe chemosis, an anti-inflammatory based on flunixin was used (83mg flunixin megluminate/mL). Regardless of the protocol applied, two doses of intramuscular injection were applied for two consecutive days, and the interval between the protocols applied were 10 days.
The drugs used in these protocols are licensed by the Ministry of Agriculture in accordance with the regulations of Decree No. 5,053, of April 22, 2004 (Brasil, 2004) and Normative Instruction No. 5, of April 23, 2019 (Brasil, 2019).
To analyze the cost of the OICK control measure, the market prices of health inputs (antibiotic and anti-inflammatory) during the outbreak period were observed for ewes and ewe lambs with scores of 2 and 3. The economic cost were calculated considering the cost of sanitary inputs for the two categories evaluated, presented in their respective protocols (A, B, C, D and E), as the following equation: Cost of antibiotic use for ewes and ewe lambs: total intake * US$ of sanitary input; Cost of anti-inflammatory use for ewes and ewe lambs: total intake *US$ of sanitary input.
The cost of health inputs for ewes and ewe lambs was obtained from the average prices practiced by the livestock market in the region in 2023, based on the values obtained from livestock companies located at Campo Grande, MS. The average exchange rate during the experimental period was US$ 1.00 = R$ 4.98 (Brazilian currency), and the values were: antibiotic = US$3.04 (50mL); anti-inflammatory = US$ 12.17 (50mL), with the cost (US$/mL) of the antibiotic being US$ 0.07 and the anti-inflammatory US$ 0.24.
To identify the etiologic agent of OIKC, samples of ocular secretions were collected from the affected animals using a swab and stored in saline, followed by DNA extraction, according to the method described by Araújo et al. (2009). The amount and purity of the extracted DNA was monitored in the Nanodrop® (Applied Biosystems) and its integrity verified by 1% agarose gel electrophoresis.
The pathogen was characterized by PCR analysis using the primers Mycu-F (5'-GTG GGG AGC AAA YAG GAT TAG A-3') and Mycu-R (5'-GGC ATG ATT TGA CGT CRT-3') developed by Kameziha et al. (2009) to amplify a 425bp fragment of the 16S rRNA Region. PCR mixes of 25µL were prepared containing PCR buffer, 1.5mM of MgCl2, 50µM of each dNTP 15pmol of each primer, 1.65U of Taq DNA polymerase, and approximately 100ng of extracted DNA. The thermocycling conditions consisted of an initial denaturation at 94°C for 3 minutes, followed by 32 cycles consisting of denaturation at 94°C for 1 minute, followed by annealing at 55°C for 30 seconds and extension at 72°C for 60 seconds, followed by a final extension was carried out at 72°C for 5 minutes. PCR products were analyzed by 1% agarose gel electrophoresis. Positive samples with good integrity were selected for sequencing.
The selected PCR products were separated into two 5µL aliquots and purified using ExoSAP-IT® (Applied Biosystems) according to the manufacturer's instructions. The fragments were sequenced in both directions using the Sanger method on the SeqStudio® platform (Applied Biosystems). The sequencing consensus sequences were assembled using the DNABaser software and analyzed using the Blastn tool to confirm the identity of the agent. The sequences obtained were deposited in GenBank under the accession number PV091351.
RESULTS
The sequence obtained showed 99.49% identity with Mycoplasma conjunctivae sequences deposited in GenBank under the number PV091351.
From March to December 2023, 101 clinical observations were made on 23 ewe lambs. Similarly, 371 observations of clinical signs were made in 65 ewes. Animals with disease scores of 2 and 3 were considered for evaluation. Data on the protocols used and the clinical evolution throughout the outbreak are presented in Tables 2 and 3 for ewe lambs and ewes, respectively.
Treatment protocols and clinical evolution of ewe lambs during an outbreak of ovine infectious keratoconjunctivitis
Treatment protocols and clinical evolution of ewes during an outbreak of infectious ovine keratoconjunctivitis
The results showed that ewe lambs consumed more antibiotics from July (203.36mL) to August (218.06mL), which corresponded to the highest clinical observations. Protocols A, B, and C began in May, followed by protocols B, C, and D in June. However, the highest antibiotic costs were in May and July, when Protocol D began. After the implementation of E protocol, the number of animals with OIKC decreased, but the cost of antibiotics per animal (US$/animal) increased (Table 4).
Anti-inflammatory drugs for ewe lambs’ consumption and cost were higher in the months of March and May, corresponding to the periods with the highest number of diseased ewe lambs and relapses (Table 4). For ewes, the highest antibiotic consumption was in April with 85 clinical observations submitted and in July with 54 clinical observations. The highest antibiotic cost can be observed with the introduction of protocol E. In August, the same scheme was repeated with protocols B, C, D and E. Antibiotic costs per animal (US$/animal) were higher as protocols with more than three doses every 72 hours were introduced (Table 5).
DISCUSSION
The observation of the manifestations parallel with the identification of the etiologic agent, it was possible to conclude that the identification of Mycoplasma conjunctivae in sheep with keratoconjunctivitis symptoms provides important data for the epidemiology of the disease in tropical climates. The occurrence of M. conjunctivae has been widely described, with several studies reporting a significant role for this pathogen in the etiology of OIKC (Almeida Neto et al., 2004; Willians et al., 2019). However, these reports are either experimental studies or refer to cases with a small number of animals affected by the disease (Dagnall, 1994; Van Halderen et al., 1994; Ter Laak et al., 1998; Almeida Neto et al., 2004; Fernández-Aguilar et al., 2017). Regarding the disease in sheep flocks kept on tropical pastures, there are an inexpressive number of clinical case reports (Gregory et al., 2003), specifically reports of OIKC outbreaks associated with M. conjunctivae. The presence of this etiologic pathogen and the rapid evolution of the disease have demonstrated the facility which the organism can be transmitted between animals, as well as its high degree of pathogenicity, that can cause high rates of morbidity over extended periods of time.
At the beginning of the outbreak, 14 ewe lambs presented clinical signs equivalent to OIKC with a score of 3. Due to the animal’s young age and to the weaning period, treatment was initiated with A and B protocols. The protocols were efficient in reducing the number of affected animals. However, due to the high the OIKC degree of severity, the protocol was repeated. Based on the general susceptibility of Mycoplasma spp. to oxytetracycline, the disease was considered under control (Gregory et al., 2003). Nevertheless, it is known that even with effective therapy to eliminate clinical signs, treated animals can continue to host the etiological agent for a long period of time (Almeida Neto, et al., 2004). The peak of the outbreak was in May with 21 ewes’ lambs presenting clinical symptoms. Due to the large number of affected animals and the fact that almost 43% of them presented a score of 3, 13 lambs were included in the C protocol. Although, in the subsequent month, clinical manifestations decreased significantly, the number of relapses and lambs with a score of 3 remained high.
In July, two OIKC control strategies were carried out simultaneously: the separation of clinically healthy ewe lambs and the beginning of D protocol. Thus, it was possible to control the severity of the disease and the number of clinical symptoms for this category.
At the onset of the outbreaks, approximately 74% of sheep were in advanced stages of OIKC, and it was observed that over 98% of sheep had been sickened. In the lamb category, the disease was expected to be under control (Grogory et al., 2003). However, the peak of the outbreak for the sheep category occurred in April, with 100% of animals showing clinical signs of OIKC in grades 2 and 3, confirming the theory of effective therapy associated with the etiological agent remaining in the host for long periods. (Almeida Neto et al., 2004). Protocols A and C were introduced during this period due to the large number of affected animals, a high number of clinical relapses were observed, with more than 50% of sheep presenting a disease score of 3. In addition to the applied protocols, the first discarded in this category was performed.
Implementing the new protocols was effective in reducing the number of diseased ewes and the number of ewes with the OIKC score 3 decreased. In August, there was a decrease in the number of diseased animals and a 30% decrease in the number of relapses. The E protocol showed a low efficiency in control of the OIKC and an increase of ewes culled was observed.
The identification of the etiologic agent, the clinical symptoms, and the high morbidity of OIKC described in this study for both categories are consistent with the previous research (Fernández-Aguilar et al., 2017; Willians et al., 2019). There are few epidemiologic studies on the incidence of OIKC in tropical pastures, and observed reports do not provide complete information. According to Willians et al. (2019), farmers have classified keratoconjunctivitis as a difficult disease to treat because outbreaks can be prolonged and difficult to control, affecting animal health and welfare. Although there is clinical evidence of the importance of OIKC and its impact on sheep flocks, the literature has not described established biosecurity protocols to reduce the spread of the disease among flocks that have been affected for long periods of time and also considering the different categories that is composed the flock.
The total cost of 101 clinical observations on ewe lambs was US$ 73.62, considering the use of antibiotics and anti-inflammatory drugs. The number of doses combined with the increased body weight of the ewe lambs were the factors responsible for the increases in the cost. However, it’s important to highlight that the introduction of these treatments has been fundamental in reducing the impact of OIKC on the ewe lamb category.
The total cost of 371 clinical observations of ewes was US$ 551.5, including antibiotic and anti-inflammatory consumption. In conclusion, the medication costs for the ewe category were higher due to the number and weight of animals in this category. The reduction in the number of diseased ewes and clinical relapses resulting from the introduction of these protocols, combined with culling, has been fundamental.
Despite the higher cost, the introduction of Protocol C was effective in reducing the number of affected ewe lambs, the severity of the disease and the overall control of the OIKC outbreak. In the ewe category, Protocols C and E had the highest costs, with both protocols having a higher number of doses administered to ewes. The introduction of Protocol C in the ewe category decreased the number of clinical observations. Protocol E was fundamental in controlling the severity of the disease.
Clinical signs of the OIKC were different, however, it was possible to assess the extent of the economic losses resulting from the outbreak of keratoconjunctivitis in this flock. The economic losses due to the disease corresponded to the cost of medication, time and management required to apply the protocols. Previous studies have reported production losses in sheep affected by OICK (Almeida Neto et al., 2004), but the body weight did not decrease in either category in this study. Nevertheless, due to the long prevalence of the OICK outbreak and in order to reduce contamination in the herd, it was not possible to perform the breeding season, which affected the herd lamb production duet to the decreases in reproductive efficiency in the respective period.
CONCLUSIONS
Infectious keratoconjunctivitis in sheep negatively affected Texel crossbred ewes and ewe lambs grazing on Brachiaria brizantha cv. Marandu pastures, increasing medication use, labor demand, and recovery time, with consequent financial costs to the flock. Mycoplasma conjunctivae infection was characterized by persistence in affected animals. Treatment with three doses of oxytetracycline administered at 72-hour intervals reduced the number of affected animals, clinical recurrence, and disease severity. In persistent cases with severe clinical signs, protocols D and E demonstrated improved therapeutic response under tropical grazing conditions.
ACKNOWLEDGEMENT
The authors are grateful to the Federal University of Mato Grosso do Sul, the Coordination for the Improvement of Higher Education Personnel - Brazil (CAPES), National Council for Scientific and Technological Development (CNPq), Foundation to Support the Development of Education, Science and Technology in the State of Mato Grosso do Sul (FUNDECT).
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